Impact of the Mediterranean Diet on the Gut Microbiome of a Well-Defined Cohort of Healthy Individuals.

A comprehensive understanding of gut microbiota in a clearly defined group of healthy individuals is essential when making meaningful comparisons with various diseases. The Mediterranean diet (MD), renowned for its potential health benefits, and the influence of adherence thereto on gut microbiota have become a focus of research. Our aim was to elucidate the impact of adherence to the MD on gut microbiota composition in a well-defined cohort. In this prospective study, healthy volunteers completed a questionnaire to provide demographic data, medical history, and dietary intake. Adherence was evaluated using the Med-DQI. The V4 region of the 16S rRNA gene was sequenced. Analysis of sequencing data and statistical analysis were performed using MOTHUR software and R. The study included 60 patients (51.7% females). Adherence correlated with alpha diversity, and higher values were recorded in good adherers. Good adherers had a higher abundance of Paraprevotella and Bacteroides (p < 0.001). Alpha diversity correlated inversely with fat intake and positively with non-starch polysaccharides (NSPs). Evenness correlated inversely with red meat intake and positively with NSPs. Predicted functional analysis highlighted metabolic pathway differences based on adherence to the MD. In conclusion, our study adds useful information on the relationship between the MD and the gut microbiome.

Diagnosis and treatment of lupus nephritis: a summary of the Consensus Document of the Spanish Group for the Study of Glomerular Diseases (GLOSEN).

Lupus nephritis (LN) is the most frequent serious manifestation of patients with systemic lupus erythematosus (SLE). Up to 60% of SLE patients develop LN, which has a significant impact on their quality of life and prognosis. Recent advances have improved the diagnostic approach to LN, and new drugs that block specific pathways and kidney damage progression have been developed. Several randomized and well-powered clinical trials have confirmed the efficacy of these agents in terms of proteinuria remission and preservation of kidney function in the medium and long term, with an acceptable safety profile and good tolerance. The combination of different therapies allows for reduction of the dose and duration of corticosteroids and other potentially toxic therapies and leads to an increase in the number of patients achieving complete remission of the disease. This consensus document carried out by the Spanish Group for the Study of Glomerular Diseases (GLOSEN) provides practical and updated recommendations, based on the best available evidence and clinical expertise of participating nephrologists.

Consensus document of the Spanish Group for the Study of the Glomerular Diseases (GLOSEN) for the diagnosis and treatment of lupus nephritis.

A significant number of patients with systemic lupus erythematosus (between 20% and 60% according to different reported series) develop lupus nephritis in the course of its evolution, which directly influences their quality of life and vital prognosis. In recent years, the greater knowledge about the pathogenesis of systemic lupus and lupus nephritis has allowed relevant advances in the diagnostic approach and treatment of these patients, achieving the development of drugs specifically aimed at blocking key pathogenic pathways of the disease. Encouragingly, these immunomodulatory agents have shown in well-powered, randomized clinical trials good clinical efficacy in the medium-term, defined as proteinuria remission and preservation of kidney function, with an acceptable safety profile and good patient tolerability. All this has made it possible to reduce the use of corticosteroids and other potentially more toxic therapies, as well as to increase the use of combined therapies. The present consensus document carried out by the Glomerular Diseases Working Group of the Spanish Society of Nephrology (GLOSEN), collects in a practical and summarized, but rigorous way, the best currently available evidence about the diagnosis, treatment, and follow-up of lupus nephritis patients, including cases of special situations, with the main objective of providing updated information and well-founded clinical recommendations to treating physicians, to improve the diagnostic and therapeutic approach to our patients.

Treatment with the senolytics dasatinib/quercetin reduces SARS-CoV-2-related mortality in mice.

The enormous societal impact of the ongoing COVID-19 pandemic has been particularly harsh for some social groups, such as the elderly. Recently, it has been suggested that senescent cells could play a central role in pathogenesis by exacerbating the pro-inflammatory immune response against SARS-CoV-2. Therefore, the selective clearance of senescent cells by senolytic drugs may be useful as a therapy to ameliorate the symptoms of COVID-19 in some cases. Using the established COVID-19 murine model K18-hACE2, we demonstrated that a combination of the senolytics dasatinib and quercetin (D/Q) significantly reduced SARS-CoV-2-related mortality, delayed its onset, and reduced the number of other clinical symptoms. The increase in senescent markers that we detected in the lungs in response to SARS-CoV-2 may be related to the post-COVID-19 sequelae described to date. These results place senescent cells as central targets for the treatment of COVID-19, and make D/Q a new and promising therapeutic tool.

Documento de consenso del Grupo de Estudio de Enfermedades Glomerulares de la Sociedad Española de Nefrología (GLOSEN) para el diagnóstico y tratamiento de la nefritis lúpica / Consensus document of the Spanish Group for the Study of the Glomerular Diseases (GLOSEN) for the diagnosis and treatment of lupus nephritis

Un número importante de pacientes con lupus eritematoso sistémico (entre 20 a 60%, según diferentes series) desarrolla nefritis lúpica en el curso de su evolución, lo que influye directamente en su calidad de vida y pronóstico vital. En años recientes, el mayor conocimiento sobre la patogénesis del lupus sistémico y de la nefritis lúpica ha permitido avances relevantes en el abordaje diagnóstico y en el tratamiento de estos pacientes, lográndose desarrollar fármacos dirigidos específicamente a bloquear vías patogénicas claves de la enfermedad. Alentadoramente estos agentes inmunomoduladores han demostrado en ensayos clínicos aleatorizados, y bien ponderados, buena eficacia clínica a mediano plazo, definida como remisión de proteinuria y preservación de la función renal, con un aceptable perfil de seguridad y buena tolerabilidad del paciente. Todo esto ha permitido reducir el uso de corticoides y de otras terapias potencialmente más tóxicas, así como incrementar el uso de terapias combinadas. El presento documento de consenso realizado por el Grupo de Trabajo de Enfermedades Glomerulares de la Sociedad Española de Nefrología (GLOSEN) recoge de manera práctica y resumida, pero rigurosa, la mejor evidencia actual disponible acerca del diagnóstico, tratamiento y seguimiento del paciente con nefritis lúpica, incluyendo casos de situaciones especiales, con el objetivo principal de brindar información actualizada y recomendaciones clínicas bien fundamentadas a los médicos tratantes, para mejorar el enfoque diagnóstico y terapéutico a nuestro pacientes. (AU)

A significant number of patients with systemic lupus erythematosus (between 20% to 60% according to different reported series) develop lupus nephritis in the course of its evolution, which directly influences their quality of life and vital prognosis. In recent years, the greater knowledge about the pathogenesis of systemic lupus and lupus nephritis has allowed relevant advances in the diagnostic approach and treatment of these patients, achieving the development of drugs specifically aimed at blocking key pathogenic pathways of the disease. Encouragingly, these immunomodulatory agents have shown in well-powered, randomized clinical trials good clinical efficacy in the medium-term, defined as proteinuria remission and preservation of kidney function, with an acceptable safety profile and good patient tolerability. All this has made it possible to reduce the use of corticosteroids and other potentially more toxic therapies, as well as to increase the use of combined therapies. The present consensus document carried out by the Glomerular Diseases Working Group of the Spanish Society of Nephrology (GLOSEN), collects in a practical and summarized, but rigorous way, the best currently available evidence about the diagnosis, treatment, and follow-up of lupus nephritis patients, including cases of special situations, with the main objective of providing updated information and well-founded clinical recommendations to treating physicians, to improve the diagnostic and therapeutic approach to our patients. (AU)

Vaccine breakthrough infections with SARS-CoV-2 Alpha mirror mutations in Delta Plus, Iota, and Omicron.

Replication of SARS-CoV-2 in the human population is defined by distributions of mutants that are present at different frequencies within the infected host and can be detected by ultra-deep sequencing techniques. In this study, we examined the SARS-CoV-2 mutant spectra of amplicons from the spike-coding (S-coding) region of 5 nasopharyngeal isolates derived from patients with vaccine breakthrough. Interestingly, all patients became infected with the Alpha variant, but amino acid substitutions that correspond to the Delta Plus, Iota, and Omicron variants were present in the mutant spectra of the resident virus. Deep sequencing analysis of SARS-CoV-2 from patients with vaccine breakthrough revealed a rich reservoir of mutant types and may also identify tolerated substitutions that can be represented in epidemiologically dominant variants.

Guanosine inhibits hepatitis C virus replication and increases indel frequencies, associated with altered intracellular nucleotide pools.

In the course of experiments aimed at deciphering the inhibition mechanism of mycophenolic acid and ribavirin in hepatitis C virus (HCV) infection, we observed an inhibitory effect of the nucleoside guanosine (Gua). Here, we report that Gua, and not the other standard nucleosides, inhibits HCV replication in human hepatoma cells. Gua did not directly inhibit the in vitro polymerase activity of NS5B, but it modified the intracellular levels of nucleoside di- and tri-phosphates (NDPs and NTPs), leading to deficient HCV RNA replication and reduction of infectious progeny virus production. Changes in the concentrations of NTPs or NDPs modified NS5B RNA polymerase activity in vitro, in particular de novo RNA synthesis and template switching. Furthermore, the Gua-mediated changes were associated with a significant increase in the number of indels in viral RNA, which may account for the reduction of the specific infectivity of the viral progeny, suggesting the presence of defective genomes. Thus, a proper NTP:NDP balance appears to be critical to ensure HCV polymerase fidelity and minimal production of defective genomes.

A proposal for the management of COVID-19-induced coagulopathy in adults.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the cause of the coronavirus disease 2019 (COVID-19) pandemic, which has a high case fatality rate. Most severely ill patients develop a special type of coagulopathy that had not been described before and that is now considered the main cause of death. For this reason, anticoagulant treatment has become one of the cornerstones of the treatment of this infection. However, the rate at which the evidence regarding the use of anticoagulants is generated is quite fast, and sometimes it is difficult to interpret and conflicting. After having performed an extensive review of the published literature, this proposal for the use of anticoagulant treatment is made, taking into account available resources in Mexico.

La infección por coronavirus 2 del síndrome respiratorio agudo grave (SARS-CoV-2) es la causante de la pandemia de enfermedad por coronavirus 2019 (COVID-19), con un índice de letalidad alto. La mayoría de los pacientes graves desarrollan un tipo especial de coagulopatía no descrito hasta ahora y la cual se considera ahora la principal causa de muerte. Por esta razón, el tratamiento anticoagulante se ha convertido en una de las piedras angulares del tratamiento de esta infección. Sin embargo, la velocidad con la que se genera la evidencia respecto al uso de anticoagulantes es muy rápida y, en ocasiones difícil de interpretar y contradictoria. Luego de hacer una revisión extensa de la literatura publicada, se hace esta propuesta para el uso del tratamiento anticoagulante tomando en cuenta los recursos disponibles en México.

Propuesta para manejo de la coagulopatía asociada a la COVID-19 en adultos / A proposal for the management of COVID-19-induced coagulopathy in adults

Resumen La infección por coronavirus 2 del síndrome respiratorio agudo grave (SARS-CoV-2) es la causante de la pandemia de enfermedad por coronavirus 2019 (COVID-19), con un índice de letalidad alto. La mayoría de los pacientes graves desarrollan un tipo especial de coagulopatía no descrito hasta ahora y la cual se considera ahora la principal causa de muerte. Por esta razón, el tratamiento anticoagulante se ha convertido en una de las piedras angulares del tratamiento de esta infección. Sin embargo, la velocidad con la que se genera la evidencia respecto al uso de anticoagulantes es muy rápida y, en ocasiones difícil de interpretar y contradictoria. Luego de hacer una revisión extensa de la literatura publicada, se hace esta propuesta para el uso del tratamiento anticoagulante tomando en cuenta los recursos disponibles en México.

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the cause of the coronavirus disease 2019 (COVID-19) pandemic, which has a high case fatality rate. Most severely ill patients develop a special type of coagulopathy that had not been described before and that is now considered the main cause of death. For this reason, anticoagulant treatment has become one of the cornerstones of the treatment of this infection. However, the rate at which the evidence regarding the use of anticoagulants is generated is quite fast, and sometimes it is difficult to interpret and conflicting. After having performed an extensive review of the published literature, this proposal for the use of anticoagulant treatment is made, taking into account available resources in Mexico.

Proposal for the management of COVID-19-associated coagulopathy in children.

SARS-CoV-2 infection (COVID-19) has become a pandemic with a high case fatality rate that mainly affects adults. Most severely ill adult patients develop a coagulopathy that was not described until recently, and which is currently considered a main cause of death. Everything indicates that a similar phenomenon also occurs in children with COVID-19. Anticoagulant treatment has become one of the therapeutic foundations for this infection; however, its implementation in children can be difficult since, until recently, it was not considered in the pediatric population. Evidence regarding the use of anticoagulants in COVID-19 is rapidly generated, changes constantly, it is often difficult to interpret, and can be contradictory. After an extensive review of the published literature, a proposal was generated that offers suggestions for anticoagulant treatment, considering available resources in Mexico.

La infección por SARS-CoV-2 (COVID-19) se ha constituido en una pandemia con alto índice de letalidad que afecta principalmente a los adultos. La mayor parte de los pacientes adultos graves desarrolla una coagulopatía que no estaba descrita, la cual actualmente se considera la principal causa de muerte. Todo indica que un fenómeno parecido ocurre también en el niño con COVID-19. El tratamiento anticoagulante se ha convertido en uno de los fundamentos terapéuticos de esta infección; sin embargo, su establecimiento en el niño puede ser difícil ya que, hasta hace poco, no estaba considerado en la población pediátrica. La evidencia respecto al uso de anticoagulantes en COVID-19 se genera con rapidez, cambia constantemente, con frecuencia es difícil de interpretar y puede ser contradictoria. Después de una extensa revisión de la literatura publicada, se generó una propuesta que ofrece sugerencias para el tratamiento anticoagulante en la que se consideran los recursos disponibles en México.

Mycophenolate Mofetil in C3 Glomerulopathy and Pathogenic Drivers of the Disease.

BACKGROUND AND OBJECTIVES: C3 glomerulopathy is a complement-mediated disease arising from abnormalities in complement genes and/or antibodies against complement components. Previous studies showed that treatment with corticosteroids plus mycophenolate mofetil (MMF) was associated with improved outcomes, although the genetic profile of these patients was not systematically analyzed. This study aims to analyze the main determinants of disease progression and response to this therapeutic regimen. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We conducted a retrospective, multicenter, observational cohort study in 35 nephrology departments belonging to the Spanish Group for the Study of Glomerular Diseases. Patients diagnosed with C3 glomerulopathy (n=81) or dense deposit disease (n=16) between January 1995 and March 2018 were enrolled. Multivariable and propensity score matching analyses were used to evaluate the association of clinical and genetic factors with response to treatment with corticosteroids and MMF as measured by proportion of patients with disease remission and kidney survival (status free of kidney failure). RESULTS: The study group comprised 97 patients (84% C3 glomerulopathy, 16% dense deposit disease). Forty-two patients were treated with corticosteroids plus MMF, and this treatment was associated with a higher rate of remission and lower probability of kidney failure (79% and 14%, respectively) compared with patients treated with other immunosuppressives (24% and 59%, respectively), or ecluzimab (33% and 67%, respectively), or conservative management (18% and 65%, respectively). The therapeutic superiority of corticosteroids plus MMF was observed both in patients with complement abnormalities and with autoantibodies. However, patients with pathogenic variants in complement genes only achieved partial remission, whereas complete remissions were common among patients with autoantibody-mediated forms. The main determinant of no remission was baseline proteinuria. Relapses occurred after treatment discontinuation in 33% of the patients who had achieved remission with corticosteroids plus MMF, and a longer treatment length of MMF was associated with a lower risk of relapse. CONCLUSIONS: The beneficial response to corticosteroids plus MMF treatment in C3 glomerulopathy appears independent of the pathogenic drivers analyzed in this study.

Proposal for the management of COVID-19-associated coagulopathy in children / Propuesta para el manejo de la coagulopatía asociada a COVID-19 en niños

Abstract SARS-CoV-2 infection (COVID-19) has become a pandemic with a high case fatality rate that mainly affects adults. Most severely ill adult patients develop a coagulopathy that was not described until recently, and which is currently considered a main cause of death. Everything indicates that a similar phenomenon also occurs in children with COVID-19. Anticoagulant treatment has become one of the therapeutic foundations for this infection; however, its implementation in children can be difficult since, until recently, it was not considered in the pediatric population. Evidence regarding the use of anticoagulants in COVID-19 is rapidly generated, changes constantly, it is often difficult to interpret, and can be contradictory. After an extensive review of the published literature, a proposal was generated that offers suggestions for anticoagulant treatment, considering available resources in Mexico.

Resumen La infección por SARS-CoV-2 (COVID-19) se ha constituido en una pandemia con alto índice de letalidad que afecta principalmente a los adultos. La mayor parte de los pacientes adultos graves desarrolla una coagulopatía que no estaba descrita, la cual actualmente se considera la principal causa de muerte. Todo indica que un fenómeno parecido ocurre también en el niño con COVID-19. El tratamiento anticoagulante se ha convertido en uno de los fundamentos terapéuticos de esta infección; sin embargo, su establecimiento en el niño puede ser difícil ya que, hasta hace poco, no estaba considerado en la población pediátrica. La evidencia respecto al uso de anticoagulantes en COVID-19 se genera con rapidez, cambia constantemente, con frecuencia es difícil de interpretar y puede ser contradictoria. Después de una extensa revisión de la literatura publicada, se generó una propuesta que ofrece sugerencias para el tratamiento anticoagulante en la que se consideran los recursos disponibles en México.

Synergistic lethal mutagenesis of hepatitis C virus.

Lethal mutagenesis is an antiviral approach that consists in extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagenic agent, often a nucleotide analogue. One of its advantages is its broad spectrum nature that renders the strategy potentially effective against emergent RNA viral infections. Here we describe synergistic lethal mutagenesis of hepatitis C virus (HCV) by a combination of favipiravir (T-705) and ribavirin. Synergy has been documented over a broad range of analogue concentrations using the Chou-Talalay method as implemented in the CompuSyn graphics, with average dose reduction index (DRI) above 1 (68.02±101.6 for favipiravir, and 5.83±6.07 for ribavirin), and average combination indices (CI) below 1 (0.52±0.28). Furthermore, analogue concentrations that individually did not extinguish high fitness HCV in ten serial infections, when used in combination they extinguished high fitness HCV in one to two passages. Although both analogues display a preference for G→A and C→U transitions, deep sequencing analysis of mutant spectra indicated a different preference of the two analogues for the mutation sites, thus unveiling a new possible synergy mechanism in lethal mutagenesis. Prospects of synergy among mutagenic nucleotides as a strategy to confront emerging viral infections are discussed.

Lethal Mutagenesis of Rift Valley Fever Virus Induced by Favipiravir.

Rift Valley fever virus (RVFV) is an emerging, mosquito-borne, zoonotic pathogen with recurrent outbreaks taking a considerable toll in human deaths in many African countries, for which no effective treatment is available. In cell culture studies and with laboratory animal models, the nucleoside analogue favipiravir (T-705) has demonstrated great potential for the treatment of several seasonal, chronic, and emerging RNA virus infections in humans, suggesting applicability to control some viral outbreaks. Treatment with favipiravir was shown to reduce the infectivity of Rift Valley fever virus both in cell cultures and in experimental animal models, but the mechanism of this protective effect is not understood. In this work, we show that favipiravir at concentrations well below the toxicity threshold estimated for cells is able to extinguish RVFV from infected cell cultures. Nucleotide sequence analysis has documented RVFV mutagenesis associated with virus extinction, with a significant increase in G to A and C to U transition frequencies and a decrease of specific infectivity, hallmarks of lethal mutagenesis.

Viral fitness: history and relevance for viral pathogenesis and antiviral interventions.

The quasispecies dynamics of viral populations (continuous generation of variant genomes and competition among them) has as one of its frequent consequences variations in overall multiplication capacity, a major component of viral fitness. This parameter has multiple implications for viral pathogenesis and viral disease control, some of them unveiled thanks to deep sequencing of viral populations. Darwinian fitness is an old concept whose quantification dates back to the early developments of population genetics. It was later applied to viruses (mainly to RNA viruses) to quantify relative multiplication capacities of individual mutant clones or complex populations. The present article reviews the fitness concept and its relevance for the understanding of the adaptive dynamics of viruses in constant and changing environments. Many studies have addressed the fitness cost of escape mutations (to antibodies, cytotoxic T cells or inhibitors) as an influence on the efficacy of antiviral interventions. Here, we summarize the evidence that the basal fitness level can be a determinant of inhibitor resistance.

Pipeline for specific subtype amplification and drug resistance detection in hepatitis C virus.

BACKGROUND: Despite the high sustained virological response rates achieved with current directly-acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 5-10% of treated patients do not respond to current antiviral therapies, and basal resistance to DAAs is increasingly detected among treatment-naïve infected individuals. Identification of amino acid substitutions (including those in minority variants) associated with treatment failure requires analytical designs that take into account the high diversification of HCV in more than 86 subtypes according to the ICTV website (June 2017). METHODS: The methodology has involved five sequential steps: (i) to design 280 oligonucleotide primers (some including a maximum of three degenerate positions), and of which 120 were tested to amplify NS3, NS5A-, and NS5B-coding regions in a subtype-specific manner, (ii) to define a reference sequence for each subtype, (iii) to perform experimental controls to define a cut-off value for detection of minority amino acids, (iv) to establish bioinformatics' tools to quantify amino acid replacements, and (v) to validate the procedure with patient samples. RESULTS: A robust ultra-deep sequencing procedure to analyze HCV circulating in serum samples from patients infected with virus that belongs to the ten most prevalent subtypes worldwide: 1a, 1b, 2a, 2b, 2c, 2j, 3a, 4d, 4e, 4f has been developed. Oligonucleotide primers are subtype-specific. A cut-off value of 1% mutant frequency has been established for individual mutations and haplotypes. CONCLUSION: The methodological pipeline described here is adequate to characterize in-depth mutant spectra of HCV populations, and it provides a tool to understand HCV diversification and treatment failures. The pipeline can be periodically extended in the event of HCV diversification into new genotypes or subtypes, and provides a framework applicable to other RNA viral pathogens, with potential to couple detection of drug-resistant mutations with treatment planning.

The effect of high-volume online haemodiafiltration on nutritional status and body composition: the ProtEin Stores prEservaTion (PESET) study.

Background: Compared with conventional haemodialysis (HD), online haemodiafiltration (OL-HDF) achieves a more efficient removal of uraemic toxins and reduces inflammation, which could favourably affect nutritional status. We evaluate the effect of OL-HDF on body composition and nutritional status in prevalent high-flux HD (HF-HD) patients. Methods: In all, 33 adults with chronic kidney disease (CKD) Stage 5 undergoing maintenance HF-HD were assigned to post-dilution OL-HDF (n = 17) or to remain on HF-HD (n = 16, control group) for 12 months. The primary outcome was the change in lean tissue mass (LTM), intracellular water (ICW) and body cell mass (BCM) assessed by multifrequency bioimpedance spectroscopy (BIS) at baseline and 4, 8 and 12 months. The rate of change in these parameters was estimated with linear mixed-effects models. Results: Compared with OL-HDF, patients assigned to HF-HD experienced a gradual reduction in LTM, ICW and BCM. These differences reached statistical significance at Month 12, with a relative difference of 7.31 kg [95% confidence interval (CI) 2.50-12.11; P = 0.003], 2.32 L (95% CI 0.63-4.01; P = 0.008) and 5.20 kg (95% CI 1.74-8.66; P = 0.004) for LTM, ICW and BCM, respectively. The normalized protein appearance increased in the OL-HDF group compared with the HF-HD group [0.26 g/kg/day (95% CI 0.05-0.47); P = 0.002], with a relative reduction in high-sensitive C-reactive protein [-13.31 mg/dL (95% CI -24.63 to -1.98); P = 0.02] at Month 12. Conclusions: OL-HDF for 1 year compared with HF-HD preserved muscle mass, increased protein intake and reduced the inflammatory state related to uraemia and dialysis, supporting the hypothesis that high convection volume can benefit nutritional status and prevent protein-energy wasting in HD patients.

Molecular and Functional Bases of Selection against a Mutation Bias in an RNA Virus.

The selective pressures acting on viruses that replicate under enhanced mutation rates are largely unknown. Here, we describe resistance of foot-and-mouth disease virus to the mutagen 5-fluorouracil (FU) through a single polymerase substitution that prevents an excess of A to G and U to C transitions evoked by FU on the wild-type foot-and-mouth disease virus, while maintaining the same level of mutant spectrum complexity. The polymerase substitution inflicts upon the virus a fitness loss during replication in absence of FU but confers a fitness gain in presence of FU. The compensation of mutational bias was documented by in vitro nucleotide incorporation assays, and it was associated with structural modifications at the N-terminal region and motif B of the viral polymerase. Predictions of the effect of mutations that increase the frequency of G and C in the viral genome and encoded polymerase suggest multiple points in the virus life cycle where the mutational bias in favor of G and C may be detrimental. Application of predictive algorithms suggests adverse effects of the FU-directed mutational bias on protein stability. The results reinforce modulation of nucleotide incorporation as a lethal mutagenesis-escape mechanism (that permits eluding virus extinction despite replication in the presence of a mutagenic agent) and suggest that mutational bias can be a target of selection during virus replication.

Lethal Mutagenesis of Hepatitis C Virus Induced by Favipiravir.

Lethal mutagenesis is an antiviral approach that consists in extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagen. Here we show that favipiravir (T-705) is a potent mutagenic agent for hepatitis C virus (HCV) during its replication in human hepatoma cells. T-705 leads to an excess of G → A and C → U transitions in the mutant spectrum of preextinction HCV populations. Infectivity decreased significantly in the presence of concentrations of T-705 which are 2- to 8-fold lower than its cytotoxic concentration 50 (CC50). Passaging the virus five times in the presence of 400 µM T-705 resulted in virus extinction. Since T-705 has undergone advanced clinical trials for approval for human use, the results open a new approach based on lethal mutagenesis to treat hepatitis C virus infections. If proven effective for HCV in vivo, this new anti-HCV agent may be useful in patient groups that fail current therapeutic regimens.

What is the optimal level of vitamin D in non-dialysis chronic kidney disease population?

AIM: To evaluate thresholds for serum 25(OH)D concentrations in relation to death, kidney progression and hospitalization in non-dialysis chronic kidney disease (CKD) population. METHODS: Four hundred and seventy non-dialysis 3-5 stage CKD patients participating in OSERCE-2 study, a prospective, multicenter, cohort study, were prospectively evaluated and categorized into 3 groups according to 25(OH)D levels at enrollment (less than 20 ng/mL, between 20 and 29 ng/mL, and at or above 30 ng/mL), considering 25(OH)D between 20 and 29 ng/mL as reference group. Association between 25(OH)D levels and death (primary outcome), and time to first hospitalization and renal progression (secondary outcomes) over a 3-year follow-up, were assessed by Kaplan-Meier survival curves and Cox-proportional hazard models. To identify 25(OH)D levels at highest risk for outcomes, receiver operating characteristic (ROC) curves were performed. RESULTS: Over 29 ± 12 mo of follow-up, 46 (10%) patients dead, 156 (33%) showed kidney progression, and 126 (27%) were hospitalized. After multivariate adjustment, 25(OH)D < 20 ng/mL was an independent predictor of all-cause mortality (HR = 2.33; 95%CI: 1.10-4.91; P = 0.027) and kidney progression (HR = 2.46; 95%CI: 1.63-3.71; P < 0.001), whereas the group with 25(OH)D at or above 30 ng/mL did not have a different hazard for outcomes from the reference group. Hospitalization outcomes were predicted by 25(OH) levels (HR = 0.98; 95%CI: 0.96-1.00; P = 0.027) in the unadjusted Cox proportional hazards model, but not after multivariate adjusting. ROC curves identified 25(OH)D levels at highest risk for death, kidney progression, and hospitalization, at 17.4 ng/mL [area under the curve (AUC) = 0.60; 95%CI: 0.52-0.69; P = 0.027], 18.6 ng/mL (AUC = 0.65; 95%CI: 0.60-0.71; P < 0.001), and 19.0 ng/mL (AUC = 0.56; 95%CI: 0.50-0.62; P = 0.048), respectively. CONCLUSION: 25(OH)D < 20 ng/mL was an independent predictor of death and progression in patients with stage 3-5 CKD, with no additional benefits when patients reached the levels at or above 30 ng/mL suggested as optimal by CKD guidelines.