The ACE2 receptor accelerates but is not biochemically required for SARS-CoV-2 membrane fusion.

The SARS-CoV-2 coronavirus infects human cells via the ACE2 receptor. Structural evidence suggests that ACE2 may not just serve as an attachment factor but also conformationally activate the SARS-CoV-2 spike protein for membrane fusion. Here, we test that hypothesis directly, using DNA-lipid tethering as a synthetic attachment factor in place of ACE2. We find that SARS-CoV-2 pseudovirus and virus-like particles are capable of membrane fusion without ACE2 if activated with an appropriate protease. Thus, ACE2 is not biochemically required for SARS-CoV-2 membrane fusion. However, addition of soluble ACE2 speeds up the fusion reaction. On a per-spike level, ACE2 appears to promote activation for fusion and then subsequent inactivation if an appropriate protease is not present. Kinetic analysis suggests at least two rate-limiting steps for SARS-CoV-2 membrane fusion, one of which is ACE2 dependent and one of which is not. Since ACE2 serves as a high-affinity attachment factor on human cells, the possibility to replace it with other factors implies a flatter fitness landscape for host adaptation by SARS-CoV-2 and future related coronaviruses.

Single-Virus Fusion Measurements Reveal Multiple Mechanistically Equivalent Pathways for SARS-CoV-2 Entry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to cell surface receptors and is activated for membrane fusion and cell entry via proteolytic cleavage. Phenomenological data have shown that SARS-CoV-2 can be activated for entry at either the cell surface or in endosomes, but the relative roles in different cell types and mechanisms of entry have been debated. Here, we used single-virus fusion experiments and exogenously controlled proteases to probe activation directly. We found that plasma membrane and an appropriate protease are sufficient to support SARS-CoV-2 pseudovirus fusion. Furthermore, fusion kinetics of SARS-CoV-2 pseudoviruses are indistinguishable no matter which of a broad range of proteases is used to activate the virus. This suggests that the fusion mechanism is insensitive to protease identity or even whether activation occurs before or after receptor binding. These data support a model for opportunistic fusion by SARS-CoV-2 in which the subcellular location of entry likely depends on the differential activity of airway, cellsurface, and endosomal proteases, but all support infection. Inhibition of any single host protease may thus reduce infection in some cells but may be less clinically robust. IMPORTANCE SARS-CoV-2 can use multiple pathways to infect cells, as demonstrated recently when new viral variants switched dominant infection pathways. Here, we used single-virus fusion experiments together with biochemical reconstitution to show that these multiple pathways coexist simultaneously and specifically that the virus can be activated by different proteases in different cellular compartments with mechanistically identical effects. The consequences of this are that the virus is evolutionarily plastic and that therapies targeting viral entry should address multiple pathways at once to achieve optimal clinical effects.

Mechanistic dissection of antibody inhibition of influenza entry yields unexpected heterogeneity.

Neutralizing antibodies against influenza have generally been classified according to their recognition sites, with antibodies against the head domain of hemagglutinin thought to inhibit attachment and antibodies against the stalk region thought to inhibit fusion. Here, we report the development of a microfluidic assay to measure neutralization of viral entry that can clearly differentiate between effects on attachment and fusion. Testing multiple broadly neutralizing antibodies against the hemagglutinin stalk domain, we obtain a surprising result: some broadly neutralizing antibodies inhibit fusion only, while others inhibit both fusion and viral attachment. Antibodies binding the globular head domain primarily inhibit attachment but can also reduce the fusogenic capability of viral particles that nonetheless bind the receptor. These findings shed light on the unexpectedly heterogeneous mechanisms of antibody neutralization even within similar recognition sites. The assay we have developed also provides a tool to optimize vaccine design by permitting assessment of the elicited antibody response with greater mechanistic resolution.

Reconsidering the 'Decline' of Dental Student Empathy within the Course in Latin America.

INTRODUCTION: The controversy over the presence of empathic decline within the course in students of medicine, dentistry and health sciences in general, has not fully been studied. This controversy could be partially solved if massive studies of empathy levels are made in similar cultural, social and economic contexts. MATERIAL AND METHODS: Empathy levels within the course were studied in eighteen dental schools from six countries in Latin America (2013). The mean of the empathy levels were used to study the behavior between first and fifth academic years. The values of empathy levels within the course were observed by applying the Jefferson Scale of Physician Empathy, the Spanish version. All these studies were cross-sectional. The value of means observed, were subjected to regression studies and further adjustment curves were obtained and the coefficient of determination were calculated. RESULTS: Six different models of behavior were observed, which found that five of them suffer empathic decline within the course, but with different final results: in some the decline persists until the fifth academic year and in others, this decline 'recovers' persistently until the fifth academic year. The sixth model is characterized by a constant and persistent increase of levels of empathy within the course until the last academic year. DISCUSSION: There are six different models for the behavior of means of levels of empathy within the course evaluated by a common methodology in eighteen dental schools from six countries of Latin America. These findings support the existence of variability of empathic response and a comprehensive approach is needed to find the causes that give rise to this variability. CONCLUSION: In dental students of Latin America, there is variability in the behavior of the distribution in means between the academic years of the dentistry schools examined in this study.

Introdução: A controvérsia sobre o declínio da empatia relativamente ao curso em estudantes de Medicina, Odontologia e Ciências da Saúde em geral ainda não foi completamente elucidada. Esta controvérsia poderia ser parcialmente solucionada se fossem realizados estudos significativos sobre os níveis de empatia em contextos culturais, sociais e económicos similares. Material e Métodos: Foram estudados os níveis de empatia com o curso em dezoito escolas de Odontologia de seis países na América Latina (2013). As médias dos níveis de empatia foram usadas para estudar o comportamento entre académicos do primeiro e quinto anos. Os valores de níveis de empatia para com o curso foram observados aplicando a versão em espanhol da escala Jefferson de Empatia Médica. Todos estes estudos foram transversais. O valor das médias observadas sujeito a tratamento estatístico de regressão, obtendo-se as subsequentes curvas de ajustes e calculado o coeficiente de determinação. Resultados: Foram observados seis diferentes modelos de comportamento, sendo que em cinco deles se verificou declínio da empatia, com diferentes resultados finais: em alguns, o declínio persistiu até o quinto ano académico e, em outros, o declínio 'recuperou-se' persistentemente até ao quinto ano académico. O sexto modelo caracterizou-se por um constante e persistente aumento nos níveis de empatia com o curso até o último ano académico. Discussão: Existem seis modelos diferentes para o comportamento das médias dos níveis de empatia para com o curso, avaliados por uma metodologia comum em dezoito escolas de Odontologia de seis países na América Latina. Estes resultados suportam a existência de variabilidade de resposta empática, sendo necessária uma abordagem compreensiva para encontrar as suas causas. Conclusão: Em estudantes de Odontologia da América Latina verifica-se variabilidade no comportamento da distribuição de meios entre os anos acadêmicos das escolas de odontologia examinadas neste trabalho.

Mineralocorticoid receptor blockade reduced oxidative stress in renal transplant recipients: a double-blind, randomized pilot study.

BACKGROUND: Previous experimental studies from our laboratory have demonstrated that aldosterone plays a central role in renal ischemic processes. This study was designed to evaluate the effect of mineralocorticoid receptor blockade in renal transplant recipients from living donors. METHODS: 20 adult kidney transplant recipients from living donors were included in a double-blind, randomized, placebo-controlled clinical pilot study that compared spironolactone and placebo. Placebo or spironolactone (25 mg) was administered 1 day before and 3 days posttransplantation. Renal function and urinary kidney injury molecule-1, interleukin-18, and heat shock protein 72 as well as urinary hydrogen peroxide (H2O2) levels were quantified. RESULTS: No significant differences were seen between the groups studied regarding age, gender, indication for kidney transplantation, residual renal function, renal replacement therapy, or warm and cold ischemia periods. In contrast, spironolactone administration significantly reduced the oxidative stress assessed by the urinary H2O2 excretion, in spite of no differences in renal function or reduction in tubular injury biomarkers. CONCLUSIONS: The findings of this exploratory study strongly suggest that aldosterone promotes oxidative stress and that the administration of spironolactone reduces the production of urinary H2O2 as a result of lesser formation of surrogate reactive oxygen species secondary to the ischemia-reperfusion phenomenon.

Hsp72 is an early and sensitive biomarker to detect acute kidney injury.

This study was designed to assess whether heat shock protein Hsp72 is an early and sensitive biomarker of acute kidney injury (AKI) as well as to monitor a renoprotective strategy. Seventy-two Wistar rats were divided into six groups: sham-operated and rats subjected to 10, 20, 30, 45 and 60 min of bilateral ischemia (I) and 24 h of reperfusion (R). Different times of reperfusion (3, 6, 9, 12, 18, 24, 48, 72, 96 and 120 h) were also evaluated in 30 other rats subjected to 30 min of ischemia. Hsp72 messenger RNA (mRNA) and protein levels were determined in both kidney and urine. Hsp72-specificity as a biomarker to assess the success of a renoprotective intervention was evaluated in rats treated with different doses of spironolactone before I/R. Renal Hsp72 mRNA and protein, as well as urinary Hsp72 levels, gradually increased relative to the extent of renal injury induced by different periods of ischemia quantified by histomorphometry as a benchmark of kidney damage. Urinary Hsp72 increased significantly after 3 h and continued rising until 18 h, followed by restoration after 120 h of reperfusion in accord with histopathological findings. Spironolactone renoprotection was associated with normalization of urinary Hsp72 levels. Accordingly, urinary Hsp72 was significantly increased in patients with clinical AKI before serum creatinine elevation. Our results show that urinary Hsp72 is a useful biomarker for early detection and stratification of AKI. In addition, urinary Hsp72 levels are sensitive enough to monitor therapeutic interventions and the degree of tubular recovery following an I/R insult.