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El present treball té com a objectiu dotar de recursos al professorat de tecnologia per poder donar compliment al nou Decret d'Educació pel que fa a l'ensenyament de la programació i la robòtica als instituts de Catalunya. S'utilitzarà la marca LEGO per familiaritzar als estudiants en uns camps molt concrets de la tecnologia, com els engranatges, els sensors i els actuadors, analitzant les seves característiques i possibilitats. Al mateix temps s'iniciarà als estudiants en el món de la programació per blocs, que en aquest cas tindrà la finalitat de controlar els mecanismes construïts. Es proposaran diverses situacions d'aprenentatge en què s'utilitzaran els diferents components analitzats, de manera que els estudiants puguin relacionar el component teòric de l'aprenentatge amb una part pràctica on experimentaran el funcionament real dels mecanismes treballats. S'aposta per l'ús de material de la marca LEGO per dos motius: en primer lloc, la seva divisió LEGO Education disposa de sets molt específics i complets per l'ensenyament, fet que fa molt fàcil el treball amb el seu material a l'aula. En segon lloc, fer servir una marca tan coneguda pel seu vessant lúdic la converteix en una metodologia estimulant pels alumnes.

The small gate overvoltage margin is a key reliability concern of the GaN Schottky-type p-gate high electron mobility transistor (GaN SP-HEMT). Current evaluation of gate reliability in GaN SP-HEMTs relies on either the DC bias stress or pulse I-V method, neither of which resembles the gate voltage (VGS) overshoot waveform in practical converters. This work develops a new circuit method to characterize the gate robustness and reliability in GaN SP-HEMTs, which features a resonance-like VGS ringing with pulse width down to 20 ns and an inductive switching concurrently in the drain-source loop. Using this method, the gate's single-pulse failure boundary, i.e., dynamic gate breakdown voltage (BVDYN), is first obtained under the hard switching (HSW) and drain-source grounded (DSG) conditions. The gate's switching lifetime is then tested under the repetitive VGS ringing, and the number of switching cycles to failure (SCTF#) is fitted by Weibull or Lognormal distributions. The SCTF# shows a power law relation with the VGS peak value and little dependence on the switching frequency. More interestingly, the gate's BVDYN and lifetime are both higher in HSW than those in DSG, as well as at higher temperatures. Such findings, as well as the gate degradation behaviors in a prolonged overvoltage stress test, can be explained by the time-dependent Schottky breakdown mechanism. The gate leakage current is found to be the major precursor of gate degradation. At 125 oC and 100 kHz, the VGS limits for a 10-year lifetime are projected to be ∼6 V and ∼10 V under the DSG and HSW conditions, respectively. These results provide a new qualification method and reveal new physical insights for gate reliability and robustness in p-gate GaN HEMTs.

Background: Virginia is a large state in the USA, yet it remains unclear what percentage of the population has had natural COVID-19 infection and whether risk factors for infection have changed over time. Methods: Using a longitudinal cohort, from December 2021-July 2022 we performed follow up serology and a questionnaire on 784 individuals from across Virginia who had previously participated in a statewide COVID-19 seroepidemiology study in 2020. Children were also invited to participate and an additional 62 children also completed the study. Serology was performed using Roche nucleocapsid and spike serological assays. Results: The majority of participants were white (78.6%), over 50 years old (60.9%), and reported having received COVID-19 vaccine (93.4%). 28.6% had evidence of prior COVID-19 infection (nucleocapsid positive). Reweighted by region, age, and sex to match the Virginia census data, the seroprevalence of nucleocapsid antibodies was estimated to be 30.6% (95% CI: 24.7, 36.6). We estimated that 25–53% of COVID-19 infections were asymptomatic. Infection rates were lower in individuals > 60 years old and were higher in Blacks and Hispanics. Infection rates were also higher in those without health insurance, in those with greater numbers of household children, and in those that reported a close contact or having undergone quarantine for COVID-19. Participants from Southwest Virginia had lower seropositivity (16.2%, 95% CI 6.5, 26.0) than other geographic regions. Boosted vaccinees had lower infection rates than non-boosted vaccinees. Frequenting indoor bars was a risk factor for infection, while frequently wearing an N95 mask was protective, though the estimates of association were imprecise. Infection rates were higher in children than adults (56.5% vs. 28.6%). Infection in the parent was a risk factor for child infection. Spike antibody levels declined with time since last vaccination, particularly in those that were vaccinated but not previously infected

Objectives: To study if interictal epileptiform discharges (IEDs) are associated with language performance or pre-/perinatal factors in children with developmental language disorder (DLD). Methods: We recorded routine EEG in wake and sleep in 205 children aged 2.9–7.1 years with DLD, without neurologic diseases or intellectual disability. We examined the language performance of the children and collected data on pre-/perinatal factors. Results: Interictal epileptiform discharges were not associated with lower language performance. Children with so-called “rolandic”, i.e. centrotemporoparietal, IEDs had better language skills, but age explained this association. Most pre-/perinatal factors evaluated did not increase the risk of rolandic IEDs, except for maternal smoking (OR 4.4, 95% CI 1.4–14). We did not find electrical status epilepticus during slow-wave sleep (ESES)/spike-and-wave activation in sleep (SWAS) in any children. Conclusions: Interictal epileptiform discharges are not associated with lower language performance, and ESES/SWAS is not common in children with DLD. Significance: Routine EEGs do not bring additional information about language performance in children with DLD who do not have any neurologic diseases, seizures, intellectual disability, or regression of language development.

The current gold standard assay for detecting neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the conventional virus neutralization test (cVNT), which requires infectious virus and a biosafety level 3 laboratory. Here, we report the development of a SARS-CoV-2 surrogate virus neutralization test (sVNT) that, with Luminex technology, detects NAbs. The assay was designed to mimic the virus-host interaction and is based on antibody blockage between the human angiotensin-converting enzyme 2 (hACE2) receptor and the spike (S) protein of the Wuhan, Delta, and Omicron (B.1.1.529) variants of SARS-CoV-2. The sVNT proved to have a 100% correlation with a SARS-CoV-2 cVNT regarding qualitative results. Binding between the hACE2 receptor and the S1 domain of the B.1.1.529 lineage of the Omicron variant was not observed in the assay but between the receptor and an S1 + S2 trimer and the receptor binding domain (RBD) in a reduced manner, suggesting less efficient receptor binding for the B.1.1.529 Omicron variant. The results indicate that the SARS-CoV-2 sVNT is a suitable tool for both the research community and the public health service, as it may serve as an efficient diagnostic alternative to the cVNT.

The current gold standard assay for detecting neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the conventional virus neutralization test (cVNT), which requires infectious virus and a biosafety level 3 laboratory. Here, we report the development of a SARS-CoV-2 surrogate virus neutralization test (sVNT) that, with Luminex technology, detects NAbs. The assay was designed to mimic the virus-host interaction and is based on antibody blockage between the human angiotensin-converting enzyme 2 (hACE2) receptor and the spike (S) protein of the Wuhan, Delta, and Omicron (B.1.1.529) variants of SARS-CoV-2. The sVNT proved to have a 100% correlation with a SARS-CoV-2 cVNT regarding qualitative results. Binding between the hACE2 receptor and the S1 domain of the B.1.1.529 lineage of the Omicron variant was not observed in the assay but between the receptor and an S1 + S2 trimer and the receptor binding domain (RBD) in a reduced manner, suggesting less efficient receptor binding for the B.1.1.529 Omicron variant. The results indicate that the SARS-CoV-2 sVNT is a suitable tool for both the research community and the public health service, as it may serve as an efficient diagnostic alternative to the cVNT.

The current gold standard assay for detecting neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the conventional virus neutralization test (cVNT), which requires infectious virus and a biosafety level 3 laboratory. Here, we report the development of a SARS-CoV-2 surrogate virus neutralization test (sVNT) that, with Luminex technology, detects NAbs. The assay was designed to mimic the virus-host interaction and is based on antibody blockage between the human angiotensin-converting enzyme 2 (hACE2) receptor and the spike (S) protein of the Wuhan, Delta, and Omicron (B.1.1.529) variants of SARS-CoV-2. The sVNT proved to have a 100% correlation with a SARS-CoV-2 cVNT regarding qualitative results. Binding between the hACE2 receptor and the S1 domain of the B.1.1.529 lineage of the Omicron variant was not observed in the assay but between the receptor and an S1 + S2 trimer and the receptor binding domain (RBD) in a reduced manner, suggesting less efficient receptor binding for the B.1.1.529 Omicron variant. The results indicate that the SARS-CoV-2 sVNT is a suitable tool for both the research community and the public health service, as it may serve as an efficient diagnostic alternative to the cVNT.

Viral variants of concern continue to arise for SARS-CoV-2, potentially impacting both methods for detection and mechanisms of action. Here, we investigate the effect of an evolving spike positive charge in SARS-CoV-2 variants and subsequent interactions with heparan sulfate and the angiotensin converting enzyme 2 (ACE2) in the glycocalyx. We show that the positively charged Omicron variant evolved enhanced binding rates to the negatively charged glycocalyx. Moreover, we discover that while the Omicron spike-ACE2 affinity is comparable to that of the Delta variant, the Omicron spike interactions with heparan sulfate are significantly enhanced, giving rise to a ternary complex of spike-heparan sulfate-ACE2 with a large proportion of double-bound and triple-bound ACE2. Our findings suggest that SARS-CoV-2 variants evolve to be more dependent on heparan sulfate in viral attachment and infection. This discovery enables us to engineer a second-generation lateral-flow test strip that harnesses both heparin and ACE2 to reliably detect all variants of concern, including Omicron.

The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor-binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-RBD and prevent viral entry into host cells ("receptor traps"). Here we determine cryo-EM structures of our receptor traps in complex with stabilized Spike ectodomain. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high-affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high-affinity (0.53-4.2 nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron and Delta pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus.

The current gold standard assay for detecting neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the conventional virus neutralization test (cVNT), which requires infectious virus and a biosafety level 3 laboratory. Here, we report the development of a SARS-CoV-2 surrogate virus neutralization test (sVNT) that, with Luminex technology, detects NAbs. The assay was designed to mimic the virus-host interaction and is based on antibody blockage between the human angiotensin-converting enzyme 2 (hACE2) receptor and the spike (S) protein of the Wuhan, Delta, and Omicron (B.1.1.529) variants of SARS-CoV-2. The sVNT proved to have a 100% correlation with a SARS-CoV-2 cVNT regarding qualitative results. Binding between the hACE2 receptor and the S1 domain of the B.1.1.529 lineage of the Omicron variant was not observed in the assay but between the receptor and an S1 + S2 trimer and the receptor binding domain (RBD) in a reduced manner, suggesting less efficient receptor binding for the B.1.1.529 Omicron variant. The results indicate that the SARS-CoV-2 sVNT is a suitable tool for both the research community and the public health service, as it may serve as an efficient diagnostic alternative to the cVNT.

We analyse a mathematical model for the growth of thin filaments into a two dimensional medium. More exactly, we focus on a certain reaction/diffusion system, describing the interaction between three chemicals (an activator, an inhibitor and a growth factor), and including a fourth cell variable characterising irreversible incorporation to a filament. Such a model has been shown numerically to generate structures shaped like nets. We perform an asymptotical analysis of the behaviour of solutions, in the case when the system has parameters very large and very small, thereby allowing the onset of different time and space scales. In particular, we describe the motion of the tip of a filament, and the changes in the relevant chemical species nearby., Depto. de Análisis Matemático y Matemática Aplicada, Fac. de Ciencias Matemáticas, TRUE, pub

The recent emergence of the omicron variant of the SARS-CoV-2 virus with large numbers of mutations has raised concern about a potential new surge in infections. Here we use molecular dynamics to study the biophysics of the interface of the BA1 and BA2 omicron spike protein binding to (i) the ACE2 receptor protein, (ii) antibodies from all known binding regions, and (iii) the furin binding domain. Our simulations suggest that while there is a significant reduction of antibody (Ab) binding strength corresponding to escape, the omicron spikes pay a cost in terms of weaker receptor binding as measured by interfacial hydrogen bonds (H-bond). The furin cleavage domain (FCD) is the same or weaker binding than the delta variant, suggesting lower fusogenicity resulting in less viral load and disease intensity than the delta variant. IMPORTANCE The BA1 and BA2 and closely related BA2.12.2 and BA.5 omicron variants of SARS-CoV-2 dominate the current global infection landscape. Given the high number of mutations, particularly those which will lead to antibody escape, it is important to establish accurate methods that can guide developing health policy responses that identify at a fundamental level whether omicron and its variants are more threatening than its predecessors, especially delta. The importance of our work is to demonstrate that simple in silico simulations can predict biochemical binding details of the omicron spike protein that have epidemiological consequences, especially for binding to the cells and for fusing the viral membrane with the cells. In each case, we predicted weaker binding of the omicron spike, which agreed with subsequent experimental results. Future virology experiments will be needed to test these predictions further.

The current gold standard assay for detecting neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the conventional virus neutralization test (cVNT), which requires infectious virus and a biosafety level 3 laboratory. Here, we report the development of a SARS-CoV-2 surrogate virus neutralization test (sVNT) that, with Luminex technology, detects NAbs. The assay was designed to mimic the virus-host interaction and is based on antibody blockage between the human angiotensin-converting enzyme 2 (hACE2) receptor and the spike (S) protein of the Wuhan, Delta, and Omicron (B.1.1.529) variants of SARS-CoV-2. The sVNT proved to have a 100% correlation with a SARS-CoV-2 cVNT regarding qualitative results. Binding between the hACE2 receptor and the S1 domain of the B.1.1.529 lineage of the Omicron variant was not observed in the assay but between the receptor and an S1 + S2 trimer and the receptor binding domain (RBD) in a reduced manner, suggesting less efficient receptor binding for the B.1.1.529 Omicron variant. The results indicate that the SARS-CoV-2 sVNT is a suitable tool for both the research community and the public health service, as it may serve as an efficient diagnostic alternative to the cVNT.

The small gate overvoltage margin is a crucial concern in applications of GaN Schottky-type p-gate high electron mobility transistors (SP-HEMTs). The parasitic inductance of the gate loop can induce repetitive gate-voltage (VG) spikes during the device turn-on transients. However, the gate lifetime of the GaN SP-HEMTs under VG overshoot in power converters still remains unclear. We fill this gap by developing a new circuit method to measure the gate switching lifetime. The method features several capabilities: 1) LC-resonance-like VG overshoots with pulse width down to 20 ns and dVG/dt up to 2 V/ns; 2) adjustable power loop condition including the drain-source grounded (DSG) as well as the hard switching (HSW); and 3) repetitive switching test at an adjustable switching frequency (fSW). We use this method to test over 150 devices, and found that the gate lifetimes under a certain peak magnitude of VG overshoot (VG(PK)) can be fitted by both Weibull and Lognormal distributions. The gate lifetime is primarily determined by the number of switching cycles and is higher under the HSW than under the DSG conditions. Finally, the max VG(PK) for 10-year gate lifetime is predicted under different fSW in both DSG and HSW conditions. The results provide direct reference for GaN SP-HEMT’s converter applications and a new method for the device gate qualification.

Cells have evolved proteins that detect foreign DNA, RNA, or proteins which then activate cellular pathways to combat bacterial or viral infection.1–4 Apolipoprotein B editing complex 3 (APOBEC3 or A3) is a host cytidine deaminase that deaminates cytidine to uridine in viral DNA in the cytoplasm causing hyper G→A mutation leading to destabilization and degradation of the viral genome.5–8 HIV viral infectivity factor (vif) has evolved to regulate A3 degradation of viral DNA.9 Vif hijacks host ubiquitination machinery to degrade A3s and prevent packaging of A3 into the viral particle.10,11 Vif hijacks the cotranscription factor core factor binding unit beta (CBF-beta), Elongin B (ELOB) and Elongin C (ELOC) to form the VCBC complex.12–15 VCBC binds A3s and Cullin 5 (Cul5) for ubiquitination and degradation of A3 thereby preventing packaging.12,16 Antigen binding fragments (Fabs) were generated against VCBC using a naïve B-cell Fab phage display library to isolate biological tools that are specific for the host-virus interaction.17 Two high affinity Fabs were found to bind at distinct epitopes on VCBC and produced distinct phenotypes when expressed in cells as single chain variable fragments (scFvs).17 The Fab 3C9 shields A3F from ubiquitination and restores packaging of A3F into the viral particle. The Fab 1D1 blocks binding of Cul5 and ubiquitination in vitro. The scFv 1D1 prevented ubiquitination of A3F but did not restore packaging. Affinity purification mass spectroscopy (AP-MS) in HEK293Ts with 3C9 scFv and 1D1 scFv showed different interactomes in the presence of vif. AP-MS with 1D1 scFv did not interact with CBF-beta, an important component of the VCBC complex. Spatial and temporal elucidation of proteins interacting with the complex will further determine events effecting viral infectivity.

En esta investigación nos proponemos avanzar sobre el estudio de la incorporación de pruebas para argumentar. Partimos de la cuestión sociocientífica del SARS-CoV-2 utilizando una noticia mediática acerca de su creación en el laboratorio. El objetivo fue describir la evolución de líneas argumentativas realizadas por cuatro profesores en un curso de una maestría profesionalizante en Educación en Biología. Analizamos los argumentos escritos en un cuestionario en dos momentos, por medio de un modelo de seis niveles epistémicos considerando el uso de datos y teorías para construir una conclusión sobre la noticia. Los resultados evidencian que no se incorporaron datos del nivel epistémico asociado a la estructura primaria en ninguno de los momentos. Sin embargo, se encontraron cambios respecto al nivel epistémico asociados a la estructura terciara y cuaternaria del virus., In this work, we investigate the incorporation of evidence in a line of argument. We start from the SARS-CoV-2 socio-scientific issue, based on a news story about the creation of the virus in a laboratory. The goal was to describe the evolution of the line of argument undergone by four teachers during a biology class at a professionalizing master's degree in Education in Biology. We analyzed the written arguments derived from a questionnaire at two different times. We used a six-level epistemic model that considers incorporation of data and theories into the conclusion about the veracity of the news. The results show that teachers did not incorporate data at the epistemic level associated with the virus primary structure at any time. Nevertheless, we found changes at the epistemic level associated with the tertiary and quaternary structure of the virus.

BackgroundThe 2019 novel coronavirus infectious disease (COVID-19) pandemic resulted in a surge of assays aimed at detecting severe acute respiratory syndrome (SARS) - coronavirus (CoV) - 2 infection and prior exposure. Although both molecular and antigen testing have clearly defined uses, the utility of serology remains uncertain and is presently not recommended for assessing immunity.MethodsWe conducted a pragmatic, observational study evaluating four commercially available emergency use authorized laboratory-based COVID-19 serology assays (Assays A-D). Remnant samples from hospitalized, and non-hospitalized SARS-CoV-2 PCR positive patients, as well as vaccinated and unvaccinated individuals were collected and tested. Positive percent agreement (PPA) and negative percent agreement (NPA) were calculated. Antibody concentrations were compared across the platforms and populations.ResultsA total of 588 remnant samples derived from 500 patients were tested. PPA at 5-12 weeks post-PCR positive results for Assays A-D was 98.3, 97.4, 99.2, and 95.8% respectively. NPA was 100% across all platforms. Mean antibody concentrations at 2-4 weeks post-PCR positive result were significantly higher in hospitalized versus non-hospitalized patients, respectively, for Assay A (131.8 [101.7] vs. 95.6 [100.3] AU/mL, P < 0.001), B (61.7 [62.4] vs. 38.1 [40.5] AU/mL, P < 0.001), and C (157.6 [105.3] vs. 133.3 [100.7] AU/mL, P < 0.001). For individuals receiving two vaccine doses mean antibody concentrations were respectively 169.6 (104.4), 27.3 (50.8), 189.6 (120.9), 21.19 (13.1) AU/mL for Assays A-D.ConclusionsOverall, PPA and NPA differed across the four assays. Assays A and C produced higher PPA and NPA and detected larger concentrations of antibodies following vaccination.

SARS-CoV-2 Spike is a key protein that mediates viral entry into cells and elicits antibody responses. Its importance in infection, diagnostics, and vaccinations has created a large demand for purified Spike for clinical and research applications. Spike is difficult to express, prompting modifications to the protein and expression platforms to improve yields. Alternatively, the Spike receptor-binding domain (RBD) is commonly expressed with higher titers, though it has lower sensitivity in serological assays. Here, we improve transient Spike expression in Chinese hamster ovary (CHO) cells. We demonstrate that Spike titers increase significantly over the expression period, maximizing at 14 mg L-1 on day 7. In comparison, RBD titers peak at 54 mg L-1 on day 3. Next, we develop eight Spike truncations (T1-T8) in pursuit of truncation with high expression and antibody binding. The truncations T1 and T4 express at 130 and 73 mg L-1 , respectively, which are higher than our RBD titers. Purified proteins were evaluated for binding to antibodies raised against full-length Spike. T1 has similar sensitivity as Spike against a monoclonal antibody and even outperforms Spike for a polyclonal antibody. These results suggest that T1 is a promising Spike alternative for use in various applications.

Episodic memory is a recollection of past personal experiences associated with particular times and places. This kind of memory is commonly subject to loss of contextual information or “semantization,” which gradually decouples the encoded memory items from their associated contexts while transforming them into semantic or gist-like representations. Novel extensions to the classical Remember/Know (R/K) behavioral paradigm attribute the loss of episodicity to multiple exposures of an item in different contexts. Despite recent advancements explaining semantization at a behavioral level, the underlying neural mechanisms remain poorly understood. In this study, we suggest and evaluate a novel hypothesis proposing that Bayesian–Hebbian synaptic plasticity mechanisms might cause semantization of episodic memory. We implement a cortical spiking neural network model with a Bayesian–Hebbian learning rule called Bayesian Confidence Propagation Neural Network (BCPNN), which captures the semantization phenomenon and offers a mechanistic explanation for it. Encoding items across multiple contexts leads to item-context decoupling akin to semantization. We compare BCPNN plasticity with the more commonly used spike-timing-dependent plasticity (STDP) learning rule in the same episodic memory task. Unlike BCPNN, STDP does not explain the decontextualization process. We further examine how selective plasticity modulation of isolated salient events may enhance preferential retention and resistance to semantization. Our model reproduces important features of episodicity on behavioral timescales under various biological constraints while also offering a novel neural and synaptic explanation for semantization, thereby casting new light on the interplay between episodic and semantic memory processes.

Cells have evolved proteins that detect foreign DNA, RNA, or proteins which then activate cellular pathways to combat bacterial or viral infection.1–4 Apolipoprotein B editing complex 3 (APOBEC3 or A3) is a host cytidine deaminase that deaminates cytidine to uridine in viral DNA in the cytoplasm causing hyper G→A mutation leading to destabilization and degradation of the viral genome.5–8 HIV viral infectivity factor (vif) has evolved to regulate A3 degradation of viral DNA.9 Vif hijacks host ubiquitination machinery to degrade A3s and prevent packaging of A3 into the viral particle.10,11 Vif hijacks the cotranscription factor core factor binding unit beta (CBF-beta), Elongin B (ELOB) and Elongin C (ELOC) to form the VCBC complex.12–15 VCBC binds A3s and Cullin 5 (Cul5) for ubiquitination and degradation of A3 thereby preventing packaging.12,16 Antigen binding fragments (Fabs) were generated against VCBC using a naïve B-cell Fab phage display library to isolate biological tools that are specific for the host-virus interaction.17 Two high affinity Fabs were found to bind at distinct epitopes on VCBC and produced distinct phenotypes when expressed in cells as single chain variable fragments (scFvs).17 The Fab 3C9 shields A3F from ubiquitination and restores packaging of A3F into the viral particle. The Fab 1D1 blocks binding of Cul5 and ubiquitination in vitro. The scFv 1D1 prevented ubiquitination of A3F but did not restore packaging. Affinity purification mass spectroscopy (AP-MS) in HEK293Ts with 3C9 scFv and 1D1 scFv showed different interactomes in the presence of vif. AP-MS with 1D1 scFv did not interact with CBF-beta, an important component of the VCBC complex. Spatial and temporal elucidation of proteins interacting with the complex will further determine events effecting viral infectivity.

BackgroundThe 2019 novel coronavirus infectious disease (COVID-19) pandemic resulted in a surge of assays aimed at detecting severe acute respiratory syndrome (SARS) - coronavirus (CoV) - 2 infection and prior exposure. Although both molecular and antigen testing have clearly defined uses, the utility of serology remains uncertain and is presently not recommended for assessing immunity.MethodsWe conducted a pragmatic, observational study evaluating four commercially available emergency use authorized laboratory-based COVID-19 serology assays (Assays A-D). Remnant samples from hospitalized, and non-hospitalized SARS-CoV-2 PCR positive patients, as well as vaccinated and unvaccinated individuals were collected and tested. Positive percent agreement (PPA) and negative percent agreement (NPA) were calculated. Antibody concentrations were compared across the platforms and populations.ResultsA total of 588 remnant samples derived from 500 patients were tested. PPA at 5-12 weeks post-PCR positive results for Assays A-D was 98.3, 97.4, 99.2, and 95.8% respectively. NPA was 100% across all platforms. Mean antibody concentrations at 2-4 weeks post-PCR positive result were significantly higher in hospitalized versus non-hospitalized patients, respectively, for Assay A (131.8 [101.7] vs. 95.6 [100.3] AU/mL, P < 0.001), B (61.7 [62.4] vs. 38.1 [40.5] AU/mL, P < 0.001), and C (157.6 [105.3] vs. 133.3 [100.7] AU/mL, P < 0.001). For individuals receiving two vaccine doses mean antibody concentrations were respectively 169.6 (104.4), 27.3 (50.8), 189.6 (120.9), 21.19 (13.1) AU/mL for Assays A-D.ConclusionsOverall, PPA and NPA differed across the four assays. Assays A and C produced higher PPA and NPA and detected larger concentrations of antibodies following vaccination.

SARS-CoV-2 Spike is a key protein that mediates viral entry into cells and elicits antibody responses. Its importance in infection, diagnostics, and vaccinations has created a large demand for purified Spike for clinical and research applications. Spike is difficult to express, prompting modifications to the protein and expression platforms to improve yields. Alternatively, the Spike receptor-binding domain (RBD) is commonly expressed with higher titers, though it has lower sensitivity in serological assays. Here, we improve transient Spike expression in Chinese hamster ovary (CHO) cells. We demonstrate that Spike titers increase significantly over the expression period, maximizing at 14 mg L-1 on day 7. In comparison, RBD titers peak at 54 mg L-1 on day 3. Next, we develop eight Spike truncations (T1-T8) in pursuit of truncation with high expression and antibody binding. The truncations T1 and T4 express at 130 and 73 mg L-1 , respectively, which are higher than our RBD titers. Purified proteins were evaluated for binding to antibodies raised against full-length Spike. T1 has similar sensitivity as Spike against a monoclonal antibody and even outperforms Spike for a polyclonal antibody. These results suggest that T1 is a promising Spike alternative for use in various applications.

Background: Since the beginning of the COVID-19 pandemic, new variants of significance to public health have emerged. Consequently, early detection of new mutations and variants through whole-genome sequencing remains crucial to assist health officials in employing appropriate public health measures. Methods: We utilized the ARTIC Network SARS-CoV-2 tiled amplicon approach and Nanopore sequencing to sequence 4,674 COVID-19 positive patient samples from Uppsala County, Sweden, between week 15 and 52 in 2021. Using this data, we mapped the circulating variants of concern (VOC) in the county over time and analysed the Spike (S) protein mutational dynamics in the Delta variant throughout 2021. Results: The distribution of the SARS-CoV-2 VOC matched the national VOC distribution in Sweden, in 2021. In the S protein of the Delta variant, we detected mutations attributable to variants under monitoring and variants of interest (e.g., E484Q, Q613H, Q677H, A222V and Y145H) and future VOC (e.g., T95I and Y144 deletion, which are signature mutations in the Omicron variant). We also frequently detected some less well-described S protein mutations in our Delta sequences, that might play a role in shaping future emerging variants. These include A262S, Q675K, I850L, Q1201H, V1228L and M1237I. Lastly, we observed that some of the Delta variant's signature mutations were underrepresented in our study due to artifacts of the used bioinformatics tools, approach and sequencing method. We therefore discuss some pitfalls and considerations when sequencing SARS-CoV-2 genomes. Conclusion: Our results suggest that genomic surveillance in a small, representative cohort can be used to make predictions about the circulating variants nationally. Moreover, we show that detection of transient mutations in currently circulating variants can give valuable clues to signature mutations of future VOC. Here we suggest six such mutations, that we detected frequently in the Delta variant during 2021. Last

Background: Since the beginning of the COVID-19 pandemic, new variants of significance to public health have emerged. Consequently, early detection of new mutations and variants through whole-genome sequencing remains crucial to assist health officials in employing appropriate public health measures. Methods: We utilized the ARTIC Network SARS-CoV-2 tiled amplicon approach and Nanopore sequencing to sequence 4,674 COVID-19 positive patient samples from Uppsala County, Sweden, between week 15 and 52 in 2021. Using this data, we mapped the circulating variants of concern (VOC) in the county over time and analysed the Spike (S) protein mutational dynamics in the Delta variant throughout 2021. Results: The distribution of the SARS-CoV-2 VOC matched the national VOC distribution in Sweden, in 2021. In the S protein of the Delta variant, we detected mutations attributable to variants under monitoring and variants of interest (e.g., E484Q, Q613H, Q677H, A222V and Y145H) and future VOC (e.g., T95I and Y144 deletion, which are signature mutations in the Omicron variant). We also frequently detected some less well-described S protein mutations in our Delta sequences, that might play a role in shaping future emerging variants. These include A262S, Q675K, I850L, Q1201H, V1228L and M1237I. Lastly, we observed that some of the Delta variant's signature mutations were underrepresented in our study due to artifacts of the used bioinformatics tools, approach and sequencing method. We therefore discuss some pitfalls and considerations when sequencing SARS-CoV-2 genomes. Conclusion: Our results suggest that genomic surveillance in a small, representative cohort can be used to make predictions about the circulating variants nationally. Moreover, we show that detection of transient mutations in currently circulating variants can give valuable clues to signature mutations of future VOC. Here we suggest six such mutations, that we detected frequently in the Delta variant during 2021. Last

Background: Since the beginning of the COVID-19 pandemic, new variants of significance to public health have emerged. Consequently, early detection of new mutations and variants through whole-genome sequencing remains crucial to assist health officials in employing appropriate public health measures. Methods: We utilized the ARTIC Network SARS-CoV-2 tiled amplicon approach and Nanopore sequencing to sequence 4,674 COVID-19 positive patient samples from Uppsala County, Sweden, between week 15 and 52 in 2021. Using this data, we mapped the circulating variants of concern (VOC) in the county over time and analysed the Spike (S) protein mutational dynamics in the Delta variant throughout 2021. Results: The distribution of the SARS-CoV-2 VOC matched the national VOC distribution in Sweden, in 2021. In the S protein of the Delta variant, we detected mutations attributable to variants under monitoring and variants of interest (e.g., E484Q, Q613H, Q677H, A222V and Y145H) and future VOC (e.g., T95I and Y144 deletion, which are signature mutations in the Omicron variant). We also frequently detected some less well-described S protein mutations in our Delta sequences, that might play a role in shaping future emerging variants. These include A262S, Q675K, I850L, Q1201H, V1228L and M1237I. Lastly, we observed that some of the Delta variant's signature mutations were underrepresented in our study due to artifacts of the used bioinformatics tools, approach and sequencing method. We therefore discuss some pitfalls and considerations when sequencing SARS-CoV-2 genomes. Conclusion: Our results suggest that genomic surveillance in a small, representative cohort can be used to make predictions about the circulating variants nationally. Moreover, we show that detection of transient mutations in currently circulating variants can give valuable clues to signature mutations of future VOC. Here we suggest six such mutations, that we detected frequently in the Delta variant during 2021. Last

Background: Since the beginning of the COVID-19 pandemic, new variants of significance to public health have emerged. Consequently, early detection of new mutations and variants through whole-genome sequencing remains crucial to assist health officials in employing appropriate public health measures. Methods: We utilized the ARTIC Network SARS-CoV-2 tiled amplicon approach and Nanopore sequencing to sequence 4,674 COVID-19 positive patient samples from Uppsala County, Sweden, between week 15 and 52 in 2021. Using this data, we mapped the circulating variants of concern (VOC) in the county over time and analysed the Spike (S) protein mutational dynamics in the Delta variant throughout 2021. Results: The distribution of the SARS-CoV-2 VOC matched the national VOC distribution in Sweden, in 2021. In the S protein of the Delta variant, we detected mutations attributable to variants under monitoring and variants of interest (e.g., E484Q, Q613H, Q677H, A222V and Y145H) and future VOC (e.g., T95I and Y144 deletion, which are signature mutations in the Omicron variant). We also frequently detected some less well-described S protein mutations in our Delta sequences, that might play a role in shaping future emerging variants. These include A262S, Q675K, I850L, Q1201H, V1228L and M1237I. Lastly, we observed that some of the Delta variant's signature mutations were underrepresented in our study due to artifacts of the used bioinformatics tools, approach and sequencing method. We therefore discuss some pitfalls and considerations when sequencing SARS-CoV-2 genomes. Conclusion: Our results suggest that genomic surveillance in a small, representative cohort can be used to make predictions about the circulating variants nationally. Moreover, we show that detection of transient mutations in currently circulating variants can give valuable clues to signature mutations of future VOC. Here we suggest six such mutations, that we detected frequently in the Delta variant during 2021. Last

Episodic memory is a recollection of past personal experiences associated with particular times and places. This kind of memory is commonly subject to loss of contextual information or “semantization,” which gradually decouples the encoded memory items from their associated contexts while transforming them into semantic or gist-like representations. Novel extensions to the classical Remember/Know (R/K) behavioral paradigm attribute the loss of episodicity to multiple exposures of an item in different contexts. Despite recent advancements explaining semantization at a behavioral level, the underlying neural mechanisms remain poorly understood. In this study, we suggest and evaluate a novel hypothesis proposing that Bayesian–Hebbian synaptic plasticity mechanisms might cause semantization of episodic memory. We implement a cortical spiking neural network model with a Bayesian–Hebbian learning rule called Bayesian Confidence Propagation Neural Network (BCPNN), which captures the semantization phenomenon and offers a mechanistic explanation for it. Encoding items across multiple contexts leads to item-context decoupling akin to semantization. We compare BCPNN plasticity with the more commonly used spike-timing-dependent plasticity (STDP) learning rule in the same episodic memory task. Unlike BCPNN, STDP does not explain the decontextualization process. We further examine how selective plasticity modulation of isolated salient events may enhance preferential retention and resistance to semantization. Our model reproduces important features of episodicity on behavioral timescales under various biological constraints while also offering a novel neural and synaptic explanation for semantization, thereby casting new light on the interplay between episodic and semantic memory processes., QC 20230524

COG-UK Mutation Explorer (COG-UK-ME, https://sars2.cyr.gla.ac.uk/cog-uk/-last accessed date 16 March 2022) is a web resource that displays knowledge and analyses on SARS-CoV-2 virus genome mutations and variants circulating in the UK, with a focus on the observed amino acid replacements that have an antigenic role in the context of the human humoral and cellular immune response. This analysis is based on more than 2 million genome sequences (as of March 2022) for UK SARS-CoV-2 data held in the CLIMB-COVID centralised data environment. COG-UK-ME curates these data and displays analyses that are cross-referenced to experimental data collated from the primary literature. The aim is to track mutations of immunological importance that are accumulating in current variants of concern and variants of interest that could alter the neutralising activity of monoclonal antibodies (mAbs), convalescent sera, and vaccines. Changes in epitopes recognised by T cells, including those where reduced T cell binding has been demonstrated, are reported. Mutations that have been shown to confer SARS-CoV-2 resistance to antiviral drugs are also included. Using visualisation tools, COG-UK-ME also allows users to identify the emergence of variants carrying mutations that could decrease the neutralising activity of both mAbs present in therapeutic cocktails, e.g. Ronapreve. COG-UK-ME tracks changes in the frequency of combinations of mutations and brings together the curated literature on the impact of those mutations on various functional aspects of the virus and therapeutics. Given the unpredictable nature of SARS-CoV-2 as exemplified by yet another variant of concern, Omicron, continued surveillance of SARS-CoV-2 remains imperative to monitor virus evolution linked to the efficacy of therapeutics.

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.

Les betacoronavirus forment un genre de coronavirus dont certains membres peuvent infecter l’humain et provoquer un syndrome de détresse respiratoire aigüe pouvant mener à la mort. Depuis le début du XXIème siècle, plusieurs épidémies de betacoronavirus se sont répandues sur la planète. L’un de ces virus, le coronavirus du syndrome respiratoire aigu sévère (SARS-CoV) est responsable d’une épidémie qui a débuté en Novembre 2002 dans le Guangdon en Chine et ne s’est stoppée qu’en 2004. Il existe deux modes de transmission interhumaine. Le virus peut se retrouver dans des gouttelettes aérosolisées qui iront directement se déposer sur les muqueuses des voies respiratoires supérieures d’une personne non infectée et pouvant la contaminer. Ces gouttelettes peuvent aussi se déposer sur des surfaces que touchera une personne non infectée, en se touchant le visage, cette personne risque de se contaminer à son tour. Au vue des conséquences sanitaires, sociétales et économiques de ces virus, le développement de produits limitant leur transmission est essentiel. Dans ce travail, il est utilisé un modèle d’entrée des betacoronavirus dans des cellules de mammifères basé sur des virus de la leucémie murine exprimant à leur surface le spicule du SARS-CoV & du SARS-CoV-2. Différents détergents commerciaux et une matrice de carboxymethylcellulose contenant des nanoparticules de cuivre sont utilisés pour traiter des pseudoparticules virales avant leur mise en contact avec les cellules cibles. L’entrée de pseudoparticules virales permettra l’intégration dans le génome des cellules cibles d’un gène rapporteur. Les résultats indiquent que le détergent commercial Saber™ et les nanoparticules de cuivre possèdent un effet protecteur contre l’infection de manière dose dépendante. L’activité virucide des nanoparticules de cuivre semble être portée par le cuivre car cet effet est complètement annulé lorsque le produit est préalablement traité par un antioxydant ou un chélateur de cuivre. L’util, The betacoronaviruses form a genera of coronavirus of which certain members can infect the human and cause a severe acute respiratory syndrome, being able to lead to the death. Since the beginning of the XXIst century, several epidemics of betacoronavirus have spread across the planet. One of these viruses, the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is responsible for an epidemic that started in November 2002 in Guangdon, China, and only stopped in 2004. There are two modes of human-to-human transmission. The virus can be found in aerosolized droplets that will be deposited directly on the mucous membranes of the upper respiratory tract of uninfected people and can contaminate them. These droplets can also be deposited on surfaces touched by an uninfected person, and by touching its face, this person may be contaminated in turn. In view of the health, societal and economic consequences of these viruses, the development of products limiting their transmission is essential. In this work, a model of betacoronavirus entry into mammalian cells is used based on murine leukemia viruses expressing the SARS-CoV and SARS-CoV-2 Spikes on their surface. Different commercial detergents and a carboxymethylcellulose matrix containing copper nanoparticles are used to treat the viral pseudoparticles before they are placed in contact with target cells. Pseudoparticles entry allow a reporter gene to be integrated into the cell genome. Results indicate that the commercial detergent Saber™ and copper nanoparticles possess a protective effect against infection in a dose-dependent manner. The virucidal activity of the copper nanoparticles appears to be copper driven as this effect is abolished when the product is previously treated with an antioxidant or copper chelator. The combined use of Saber™ and copper nanoparticles has a stronger protective effect, superior to each treatment on its own. Taken together, these results show that copper nanoparticles would

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.

Background: Since the beginning of the COVID-19 pandemic, new variants of significance to public health have emerged. Consequently, early detection of new mutations and variants through whole-genome sequencing remains crucial to assist health officials in employing appropriate public health measures. Methods: We utilized the ARTIC Network SARS-CoV-2 tiled amplicon approach and Nanopore sequencing to sequence 4,674 COVID-19 positive patient samples from Uppsala County, Sweden, between week 15 and 52 in 2021. Using this data, we mapped the circulating variants of concern (VOC) in the county over time and analysed the Spike (S) protein mutational dynamics in the Delta variant throughout 2021. Results: The distribution of the SARS-CoV-2 VOC matched the national VOC distribution in Sweden, in 2021. In the S protein of the Delta variant, we detected mutations attributable to variants under monitoring and variants of interest (e.g., E484Q, Q613H, Q677H, A222V and Y145H) and future VOC (e.g., T95I and Y144 deletion, which are signature mutations in the Omicron variant). We also frequently detected some less well-described S protein mutations in our Delta sequences, that might play a role in shaping future emerging variants. These include A262S, Q675K, I850L, Q1201H, V1228L and M1237I. Lastly, we observed that some of the Delta variant's signature mutations were underrepresented in our study due to artifacts of the used bioinformatics tools, approach and sequencing method. We therefore discuss some pitfalls and considerations when sequencing SARS-CoV-2 genomes. Conclusion: Our results suggest that genomic surveillance in a small, representative cohort can be used to make predictions about the circulating variants nationally. Moreover, we show that detection of transient mutations in currently circulating variants can give valuable clues to signature mutations of future VOC. Here we suggest six such mutations, that we detected frequently in the Delta variant during 2021. Last

To control the coronavirus disease 2019 (COVID-19) pandemic and the emergence of different variants of concern (VoCs), novel vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed. In this study, we report the potent immunogenicity and efficacy induced in hamsters by a vaccine candidate based on a modified vaccinia virus Ankara (MVA) vector expressing a human codon optimized full-length SARS-CoV-2 spike (S) protein (MVA-S). Immunization with one or two doses of MVA-S elicited high titers of S- and receptor-binding domain (RBD)-binding IgG antibodies and neutralizing antibodies against parental SARS-CoV-2 and VoC alpha, beta, gamma, delta, and omicron. After SARS-CoV-2 challenge, MVA-S-vaccinated hamsters showed a significantly strong reduction of viral RNA and infectious virus in the lungs compared to the MVA-WT control group. Moreover, a marked reduction in lung histopathology was also observed in MVA-S-vaccinated hamsters. These results favor the use of MVA-S as a potential vaccine candidate for SARS-CoV-2 in clinical trials.

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 ultra-deep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of thirty 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.

Coronaviruses and influenza A viruses are notorious for crossing host species barriers and their potential emergence upon zoonotic introduction poses a substantial threat to public health. In addition to animal health implications, the close contact between companion animals and humans also has potential risks of zoonotic virus infection. High-quality serological assays are key to understand the prevalence of and immunity to virus infections. In this thesis, we focused on the development of robust and specific serological assays that can serve as a toolkit for rapid diagnosis of CoVs and IAVs infections. This will give more insight and knowledge on the occurrence of different CoVs and IAVs infections in companion animals and their association with clinical diseases. In Chapter 2, we set out to detect CoV infection in cats through profiling of antibody presence in cat serum samples. Recombinant CoV spike S1 proteins of different animal and human CoVs were used as antigens for screening of cat sera for the presence of antibodies against the respective proteins. Positive samples were further tested by virus neutralization assays. This investigation intends to extend our knowledge of CoV epidemiology, potential reservoirs, and cross-species transmission. Chapter 3 describes the development and validation of an S1-protein-based ELISA method for the detection of specific antibodies against ECoV. With this method, we are able to provide a consolidated diagnostic test to confirm ECoV outbreaks, as a complement to qRT-PCR analysis of equine feces samples. In addition, this method will be used for future estimation of ECoV prevalence and incidence in various equine (sub) populations. In Chapter 4, we performed a survey to follow and study the spread of SARS-CoV-2 in companion animals. A set of serological assays including ELISA and virus neutralization were developed and validated and next used to conduct the first seroprevalence study in the Netherlands. This will provide in

This research was funded by the Council of Health, Community of Madrid., The virological meaning of the different patterns of serology in COVID-19 has been little examined in clinical settings. Asymptomatic subjects with IgM-spike (S) and IgG-nucleocapsid (N) determinations by chemiluminescence were studied for SARS-CoV-2 shedding in respiratory secretions by transcription-mediated amplification (TMA). In subjects showing IgM-S positive and IgG-N negative, IgG-S was determined by lateral flow assay. A total of 712 individuals were tested: 30.0% presented IgM-S(+)/IgG-N(−), 25.8% had IgM-S(+)/IgG-N(+) and 44.2% had IgM-S(−)/IgG-N(+); the proportion with TMA(+) were comparable in these three groups: 12.1, 8.7 and 10.5%, respectively. In individuals with IgM-S(+)/IgG-N(−), IgG-S(+) was detected in 66.5%. The frequency of IgM-S(+)/IgG-S(−) in the total population was 10.0%, of whom 24.1% had TMA(+); the chances for TMA(+) in subjects with an IgM-S(+) alone pattern were 2.4%. Targeting of the same SARS-CoV-2 antigen seems to be better for the characterization of IgM/IgG patterns of response. IgM-S(+) alone reactivity is rare, and a small proportion is associated with viral shedding., Comunidad de Madrid, Fac. de Medicina, TRUE, pub

The basic functions of living things are predominantly carried out by uniquely adapted protein nanomachines. Molecular phenomenology is an approach to biology driven by the most basic questions about these species: what are their atomic structures, and how do they move? The past two centuries have seen great strides in biology, yet we are only beginning to understand its fundamental units of function. In the past decade, revolutionary advances in electron microscopy have enabled direct imaging of biological molecules in solution, opening what may be the final frontier of molecular phenomenology. My doctoral work has focused especially on applying single-particle electron microscopy to the dynamics of those protein machines that lie at the plasma membrane that divides cells from their environment.In the first chapter of this dissertation I introduce a previously unpublished method for modeling protein motions through multi-body refinement and principal component analysis with geodesic kernels. While multi-body analysis is not new, other methods have not considered the intrinsic nature of rigid-body motions as a non-linear manifold. As such, they have been forced to rely on ad-hoc parameter scaling, and are valid only over very small motions where manifold curvature can be neglected. The kernel approach, in contrast, eliminates the parameter scaling problem and is applicable to arbitrary rigid-body motions. The basic method may be of use outside cryo-EM of biological molecules, for example in 3D animation of articulated figures or controlling swarms of drones or satellites. In the second chapter, I and others apply molecular phenomenology to uncover the mechanisms of action underlying antibody modulation of the fusogenic activity of the SARS-CoV-2 Spike protein. This work was the first study demonstrating that Spike-reactive antibodies can either inhibit or enhance Spike-mediated membrane fusion leading to viral entry or pathological cell-cell fusion and formation of

SARS-CoV-2 can mutate and evade immunity, with consequences for efficacy of emerging vaccines and antibody therapeutics. Here, we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is a highly variable region of S and provide epidemiological, clinical, and molecular characterization of a prevalent, sentinel RBM mutation, N439K. We demonstrate N439K S protein has enhanced binding affinity to the hACE2 receptor, and N439K viruses have similar in vitro replication fitness and cause infections with similar clinical outcomes as compared to wild type. We show the N439K mutation confers resistance against several neutralizing monoclonal antibodies, including one authorized for emergency use by the US Food and Drug Administration (FDA), and reduces the activity of some polyclonal sera from persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.

The basic functions of living things are predominantly carried out by uniquely adapted protein nanomachines. Molecular phenomenology is an approach to biology driven by the most basic questions about these species: what are their atomic structures, and how do they move? The past two centuries have seen great strides in biology, yet we are only beginning to understand its fundamental units of function. In the past decade, revolutionary advances in electron microscopy have enabled direct imaging of biological molecules in solution, opening what may be the final frontier of molecular phenomenology. My doctoral work has focused especially on applying single-particle electron microscopy to the dynamics of those protein machines that lie at the plasma membrane that divides cells from their environment.In the first chapter of this dissertation I introduce a previously unpublished method for modeling protein motions through multi-body refinement and principal component analysis with geodesic kernels. While multi-body analysis is not new, other methods have not considered the intrinsic nature of rigid-body motions as a non-linear manifold. As such, they have been forced to rely on ad-hoc parameter scaling, and are valid only over very small motions where manifold curvature can be neglected. The kernel approach, in contrast, eliminates the parameter scaling problem and is applicable to arbitrary rigid-body motions. The basic method may be of use outside cryo-EM of biological molecules, for example in 3D animation of articulated figures or controlling swarms of drones or satellites. In the second chapter, I and others apply molecular phenomenology to uncover the mechanisms of action underlying antibody modulation of the fusogenic activity of the SARS-CoV-2 Spike protein. This work was the first study demonstrating that Spike-reactive antibodies can either inhibit or enhance Spike-mediated membrane fusion leading to viral entry or pathological cell-cell fusion and formation of

COVID-19 is one of the most important pandemics in recent history. It is an emerging infectious disease, probably of animal origin. Several domesticated and non-domesticated animals are naturally susceptible to SARS-CoV-2 infection, including Mustelidae, of which mink is the first species identified to suffer from this infection. We report herein the detection of the SARS-CoV-2 RNA in lymph node tissue from two feral American mink (Neovison vison) trapped in the wild in the Valencian Community (Eastern Spain) during invasive species trapping campaigns. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, is considered a pathogen of animal origin that is mainly transmitted from human to human. Several animal species can be naturally or experimentally infected by SARS-CoV-2, with compelling evidence that mink is highly susceptible to SARS-CoV-2 infection. Human-to-mink infection cases have been reported and there are also suggestions that mink-to-human infection occurs. Mink infections have been reported to date only on fur farms, except for one infected free- ranging wild mink near a Utah (USA) fur farm, which suggests a transmission pathway from farms to wild mink. We now report the detection of SARS-CoV-2 in 2 of 13 feral dark brown American mink (Neovison vison) trapped in the Valencian Community (Eastern Spain), during an invasive species trapping campaign. They were trapped in riverbeds in sparsely inhabited rural areas known to harbor self-sustained feral mink populations. The closest fur farm is about 20 km away. SARS-CoV-2 RNA was detected by two-step RT-PCR in these animals' mesenteric lymph nodes and was confirmed by sequencing a 397-nucleotide amplified region of the S gene, yielding identical sequences in both animals. A molecular phylogenetic analysis was run on this sequence, which was found to correspond to the consensus SARS-CoV-2 sequence from Wuhan. Our findings appear to represent the first example of SARS-CoV

Several vaccines with varying efficacies have been developed and are currently administered globally to minimize the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite having an RNA-dependent RNA polymerase with a proofreading activity, new variants of SARS-CoV-2 are on the rise periodically. Some of the mutations in these variants, especially mutations on the spike protein, aid the virus in transmission, infectivity and host immune evasion. Further, these mutations also reduce the effectiveness of some of the current vaccines and monoclonal antibodies (mAbs). In the present study, using the available 984,769 SARS-CoV-2 nucleotide sequences on the NCBI database from the end of 2019 till 28 July 2021, we have estimated the global prevalence of so-called 'adaptive mutations' and 'mutations identified in the prolonged infections', in the receptor-binding domain (RBD) of the spike (S) protein. Irrespective of the geographical region, in the case of the adaptive mutations, N501Y (48.38%) was found to be the dominant mutation followed by L452R (17.52%), T478K (14.31%), E484K (4.69%), S477N (3.29%), K417T (1.64%), N439K (0.7%) and S494P (0.7%). Other mutations were found to be less prevalent (less than 0.7%). Since the last two months, there has been a massive increase of L452R and T478K mutations (delta variant) in certain areas. In the case of prolonged infections' mutations (long-term SARS-CoV-2 infections), V483A (0.009%) was found to be dominant followed by Q493R (0.009%), while other mutations were found in less than 0.007% of the studied sequences. The data obtained in this study will aid in the development of better infection control policies, thereby curbing the spread of this virus.

Several vaccines with varying efficacies have been developed and are currently administered globally to minimize the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite having an RNA-dependent RNA polymerase with a proofreading activity, new variants of SARS-CoV-2 are on the rise periodically. Some of the mutations in these variants, especially mutations on the spike protein, aid the virus in transmission, infectivity and host immune evasion. Further, these mutations also reduce the effectiveness of some of the current vaccines and monoclonal antibodies (mAbs). In the present study, using the available 984,769 SARS-CoV-2 nucleotide sequences on the NCBI database from the end of 2019 till 28 July 2021, we have estimated the global prevalence of so-called 'adaptive mutations' and 'mutations identified in the prolonged infections', in the receptor-binding domain (RBD) of the spike (S) protein. Irrespective of the geographical region, in the case of the adaptive mutations, N501Y (48.38%) was found to be the dominant mutation followed by L452R (17.52%), T478K (14.31%), E484K (4.69%), S477N (3.29%), K417T (1.64%), N439K (0.7%) and S494P (0.7%). Other mutations were found to be less prevalent (less than 0.7%). Since the last two months, there has been a massive increase of L452R and T478K mutations (delta variant) in certain areas. In the case of prolonged infections' mutations (long-term SARS-CoV-2 infections), V483A (0.009%) was found to be dominant followed by Q493R (0.009%), while other mutations were found in less than 0.007% of the studied sequences. The data obtained in this study will aid in the development of better infection control policies, thereby curbing the spread of this virus.

Several vaccines with varying efficacies have been developed and are currently administered globally to minimize the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite having an RNA-dependent RNA polymerase with a proofreading activity, new variants of SARS-CoV-2 are on the rise periodically. Some of the mutations in these variants, especially mutations on the spike protein, aid the virus in transmission, infectivity and host immune evasion. Further, these mutations also reduce the effectiveness of some of the current vaccines and monoclonal antibodies (mAbs). In the present study, using the available 984,769 SARS-CoV-2 nucleotide sequences on the NCBI database from the end of 2019 till 28 July 2021, we have estimated the global prevalence of so-called 'adaptive mutations' and 'mutations identified in the prolonged infections', in the receptor-binding domain (RBD) of the spike (S) protein. Irrespective of the geographical region, in the case of the adaptive mutations, N501Y (48.38%) was found to be the dominant mutation followed by L452R (17.52%), T478K (14.31%), E484K (4.69%), S477N (3.29%), K417T (1.64%), N439K (0.7%) and S494P (0.7%). Other mutations were found to be less prevalent (less than 0.7%). Since the last two months, there has been a massive increase of L452R and T478K mutations (delta variant) in certain areas. In the case of prolonged infections' mutations (long-term SARS-CoV-2 infections), V483A (0.009%) was found to be dominant followed by Q493R (0.009%), while other mutations were found in less than 0.007% of the studied sequences. The data obtained in this study will aid in the development of better infection control policies, thereby curbing the spread of this virus.

Severe acute respiratory syndrome coronavirus 2 (SARS–CoV–2) has led to the coronavirus disease 2019 (COVID–19) pandemic, severely affecting public health and the global economy. Adaptive immunity plays a crucial role in fighting against SARS–CoV–2 infection and directly influences the clinical outcomes of patients. Clinical studies have indicated that patients with severe COVID–19 exhibit delayed and weak adaptive immune responses; however, the mechanism by which SARS–CoV–2 impedes adaptive immunity remains unclear. Here, by using an in vitro cell line, we report that the SARS–CoV–2 spike protein significantly inhibits DNA damage repair, which is required for effective V(D)J recombination in adaptive immunity. Mechanistically, we found that the spike protein localizes in the nucleus and inhibits DNA damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment to the damage site. Our findings reveal a potential molecular mechanism by which the spike protein might impede adaptive immunity and underscore the potential side effects of full-length spike-based vaccines., Retracted (DOI):https://doi.org/10.3390/v14051011

Severe acute respiratory syndrome coronavirus 2 (SARS–CoV–2) has led to the corona-virus disease 2019 (COVID–19) pandemic, severely affecting public health and the global economy. Adaptive immunity plays a crucial role in fighting against SARS–CoV–2 infection and directly influences the clinical outcomes of patients. Clinical studies have indicated that patients with severe COVID–19 exhibit delayed and weak adaptive immune responses; however, the mechanism by which SARS–CoV–2 impedes adaptive immunity remains unclear. Here, by using an in vitro cell line, we report that the SARS–CoV–2 spike protein significantly inhibits DNA damage repair, which is required for effective V(D)J recombination in adaptive immunity. Mechanistically, we found that the spike protein localizes in the nucleus and inhibits DNA damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment to the damage site. Our findings reveal a potential molecular mechanism by which the spike protein might impede adaptive immunity and underscore the potential side effects of full‐length spike‐based vaccines.

Severe acute respiratory syndrome coronavirus 2 (SARS–CoV–2) has led to the corona-virus disease 2019 (COVID–19) pandemic, severely affecting public health and the global economy. Adaptive immunity plays a crucial role in fighting against SARS–CoV–2 infection and directly influences the clinical outcomes of patients. Clinical studies have indicated that patients with severe COVID–19 exhibit delayed and weak adaptive immune responses; however, the mechanism by which SARS–CoV–2 impedes adaptive immunity remains unclear. Here, by using an in vitro cell line, we report that the SARS–CoV–2 spike protein significantly inhibits DNA damage repair, which is required for effective V(D)J recombination in adaptive immunity. Mechanistically, we found that the spike protein localizes in the nucleus and inhibits DNA damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment to the damage site. Our findings reveal a potential molecular mechanism by which the spike protein might impede adaptive immunity and underscore the potential side effects of full‐length spike‐based vaccines.

The basic functions of living things are predominantly carried out by uniquely adapted protein nanomachines. Molecular phenomenology is an approach to biology driven by the most basic questions about these species: what are their atomic structures, and how do they move? The past two centuries have seen great strides in biology, yet we are only beginning to understand its fundamental units of function. In the past decade, revolutionary advances in electron microscopy have enabled direct imaging of biological molecules in solution, opening what may be the final frontier of molecular phenomenology. My doctoral work has focused especially on applying single-particle electron microscopy to the dynamics of those protein machines that lie at the plasma membrane that divides cells from their environment.In the first chapter of this dissertation I introduce a previously unpublished method for modeling protein motions through multi-body refinement and principal component analysis with geodesic kernels. While multi-body analysis is not new, other methods have not considered the intrinsic nature of rigid-body motions as a non-linear manifold. As such, they have been forced to rely on ad-hoc parameter scaling, and are valid only over very small motions where manifold curvature can be neglected. The kernel approach, in contrast, eliminates the parameter scaling problem and is applicable to arbitrary rigid-body motions. The basic method may be of use outside cryo-EM of biological molecules, for example in 3D animation of articulated figures or controlling swarms of drones or satellites. In the second chapter, I and others apply molecular phenomenology to uncover the mechanisms of action underlying antibody modulation of the fusogenic activity of the SARS-CoV-2 Spike protein. This work was the first study demonstrating that Spike-reactive antibodies can either inhibit or enhance Spike-mediated membrane fusion leading to viral entry or pathological cell-cell fusion and formation of

Severe acute respiratory syndrome coronavirus 2 (SARS–CoV–2) has led to the corona-virus disease 2019 (COVID–19) pandemic, severely affecting public health and the global economy. Adaptive immunity plays a crucial role in fighting against SARS–CoV–2 infection and directly influences the clinical outcomes of patients. Clinical studies have indicated that patients with severe COVID–19 exhibit delayed and weak adaptive immune responses; however, the mechanism by which SARS–CoV–2 impedes adaptive immunity remains unclear. Here, by using an in vitro cell line, we report that the SARS–CoV–2 spike protein significantly inhibits DNA damage repair, which is required for effective V(D)J recombination in adaptive immunity. Mechanistically, we found that the spike protein localizes in the nucleus and inhibits DNA damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment to the damage site. Our findings reveal a potential molecular mechanism by which the spike protein might impede adaptive immunity and underscore the potential side effects of full‐length spike‐based vaccines.

Severe acute respiratory syndrome coronavirus 2 (SARS–CoV–2) has led to the corona-virus disease 2019 (COVID–19) pandemic, severely affecting public health and the global economy. Adaptive immunity plays a crucial role in fighting against SARS–CoV–2 infection and directly influences the clinical outcomes of patients. Clinical studies have indicated that patients with severe COVID–19 exhibit delayed and weak adaptive immune responses; however, the mechanism by which SARS–CoV–2 impedes adaptive immunity remains unclear. Here, by using an in vitro cell line, we report that the SARS–CoV–2 spike protein significantly inhibits DNA damage repair, which is required for effective V(D)J recombination in adaptive immunity. Mechanistically, we found that the spike protein localizes in the nucleus and inhibits DNA damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment to the damage site. Our findings reveal a potential molecular mechanism by which the spike protein might impede adaptive immunity and underscore the potential side effects of full‐length spike‐based vaccines.