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This study describes the discovery and characterization of raffinocyclicin, a novel plasmid-encoded circular bacteriocin, produced by the raw milk isolate Lactococcus raffinolactis APC 3967. This bacteriocin has a molecular mass of 6,092 Da and contains 61 amino acids with a three-amino acid leader peptide. It shows the highest identity to the circular bacteriocins bacicyclicin XIN-1 (42.62%), aureocyclicin 4185 (42.62%), and garvicin ML (41.53%). A broad inhibitory spectrum includes strains from Staphylococcus, Enterococcus, Streptococcus, Micrococcus, Lactobacillus, Leuconostoc, and Listeria, in addition to a pronounced inhibitory effect against Lactococcus and Clostridium. It displays low sensitivity to trypsin, most likely as a result of its circular nature. The raffinocyclicin gene cluster is composed of 10 genes: 6 core genes, genes encoding an accessory three-component ABC transporter (rafCDE), and a putative transcriptional regulator related to the MutR family. A lack of inhibitory activity in the cell-free supernatant combined with the pronounced activity of cell extracts suggests that the majority of raffinocyclicin is associated with the cell rather than being released to the extracellular environment. This is the first report of a bacteriocin produced by the L. raffinolactis species.IMPORTANCEThe present study aimed to characterize raffinocyclicin, a novel circular bacteriocin produced by the lactic acid bacteria Lactococcus raffinolactis APC 3967. Bacteriocins are generally cationic and hydrophobic peptides with antimicrobial activity, which present diverse biotechnological properties of interest for the food industry. Raffinocyclicin inhibits a wide range of bacteria, including foodborne pathogens, and is stable against different treatments which suggest its potential as a natural biopreservative. Whole-genome sequencing and the genetic analysis of the raffinocyclicin gene cluster showed that it is encoded by plasmid that could be used in the future to transfer the ability to produce the bacteriocin to other lactic acid bacteria for industrial applications. These results together highlight the potential of this novel antimicrobial as a biopreservative to be used by the food industry.
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Temporary rivers, forming the majority of river networks worldwide, are key biodiversity hotspots. Despite their great value for maintaining biodiversity and ecosystem functioning, they are often neglected in biomonitoring programs due to several challenges, such as their variable hydromorphology and the difficulty of establishing reference conditions given their dynamic nature, resulting in highly variable communities. Disconnected pools often form in temporary rivers when flow ceases, providing refuge for aquatic taxa. Given their importance for biodiversity conservation, revising and adapting biotic indices are needed. Here, we evaluate the performance of current biological indices designed for perennial rivers (macroinvertebrates, diatoms) and functional metrics (macroinvertebrates) in assessing biological quality of disconnected pools. We sampled 55 disconnected pools in Catalonia, NE Spain, covering local (e.g., physico-chemical variables, water chemistry) and regional (e.g., human influence, hydrological variables at the water body level) natural and anthropogenic gradients. Only a few macroinvertebrate biotic indices (e.g., family richness, EPT/EPT + OCH and OCH) showed strong responses to anthropogenic predictors and were unaffected by natural predictors at both local and regional scales, making them suitable for biomonitoring. Of the newly adopted functional metrics of macroinvertebrate communities tested, only two (i.e., functional redundancy of predators and response diversity based on the total community) responded strongly to anthropogenic predictors. The rest showed varying responses to the interactive effect of anthropogenic and natural predictors, requiring calibration efforts. Models assessing these metrics explained <40 % of the total variation, likely due to the interplay of colonization/extinction dynamics and density-dependent trophic interactions governing community assemblages in disconnected pools. Although some existing biological metrics could potentially be used to monitor the ecological status of disconnected pools, we call for further development of biomonitoring tools specifically designed for these habitats since they will become more widespread with global change.
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Biodiversidad , Monitoreo del Ambiente , Invertebrados , Ríos , Ríos/química , España , Invertebrados/fisiología , Monitoreo del Ambiente/métodos , Animales , Monitoreo Biológico/métodos , EcosistemaRESUMEN
Many agricultural landscapes offer few resources for maintaining natural enemy populations and floral plantings have frequently been adopted to enhance biological pest control in crops. However, restored margins may harbour both pests and natural enemies. The aim was to compare the abundance of pests and natural enemies in three types of margins (unmanaged, sown herbaceous floral strips and shrubby hedgerows) as well as in adjacent melon fields. Besides, yield was compared among melon fields as way of testing the effect of the type of margin on biocontrol. The research was carried out during 2 years in twelve melon fields from four different locations in southern Spain. Arthropods were sampled periodically in margins and melon fields by visual inspections and Berlese extraction. Hedgerow and floral strips hosted higher numbers of both pests and predators than unmanaged margins. Besides, hedgerows had a similar or higher number of natural enemies than floral strips but lower number of pests. In just a few occasions, the type of margin had a significant effect on the abundance of pests and natural enemies in melon fields, but rarely there was consistency between the two growing seasons. No differences were found in yield. We hypothesised that the lack of association in the abundances of pests and natural enemies between margins and melon fields could be attributed to the overriding effects of the landscape and/or the internal population dynamics of arthropods in melon fields. Overall, shrubby hedgerows are more recommended than herbaceous floral strips.
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Cucurbitaceae , Control Biológico de Vectores , Animales , Cucurbitaceae/parasitología , Control Biológico de Vectores/métodos , España , Conducta Predatoria/fisiología , Productos Agrícolas/parasitología , Artrópodos/fisiología , Agricultura/métodosRESUMEN
The fading efficacy of antibiotics is a growing global health concern due to its life-threatening consequences and increased healthcare costs. Non-genetic mechanisms of antimicrobial resistance, such as those employed by Chlamydia pneumoniae and Chlamydia trachomatis, complicate treatment as these bacteria can enter a non-replicative, persistent state under stress, evading antibiotics and linking to inflammatory conditions. Understanding chlamydial persistence at the molecular level is challenging, and new models for studying Chlamydia-host interactions in vivo are urgently needed. Caenorhabditis elegans offers an alternative given its immune system and numerous orthologues of human genes. This study established C. elegans as an in vivo model for chlamydial infection. Both Chlamydia species reduced the worm's lifespan, their DNA being detectable at three- and six-days post-infection. Azithromycin at its MIC (25â¯nM) failed to prevent the infection-induced lifespan reduction, indicating a persister phenotype. In contrast, the methanolic extract of Schisandra chinensis berries showed anti-chlamydial activity both in vitro (in THP-1 macrophages) and in vivo, significantly extending the lifespan of infected C. elegans and reducing the bacterial load. Moreover, S. chinensis increased the transcriptional activity of SKN-1 in the worms, but was unable to impact the bacterial load or lifespan in a sek-1 defective C. elegans strain. In summary, this study validated C. elegans as a chlamydial infection model and showcased S. chinensis berries' in vivo anti-chlamydial potential, possibly through SEK/SKN-1 signaling modulation.
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Antibacterianos , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Infecciones por Chlamydia , Caenorhabditis elegans/microbiología , Caenorhabditis elegans/efectos de los fármacos , Animales , Humanos , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Infecciones por Chlamydia/microbiología , Infecciones por Chlamydia/tratamiento farmacológico , Antibacterianos/farmacología , Chlamydia trachomatis/efectos de los fármacos , Interacciones Huésped-Patógeno , Extractos Vegetales/farmacología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Células THP-1 , Azitromicina/farmacología , Longevidad/efectos de los fármacos , Chlamydophila pneumoniae/efectos de los fármacosRESUMEN
BACKGROUND: Here we report the reliability and test/re-test validity of a Castillan version of the PCL-5 (PCL5-C) in mental health nurses. METHODS: A sample of 52 consecutive nurses was recruited from two psychiatric hospitals and four psychiatrists units of general hospitals in Madrid, Spain. RESULTS: We detected high internal consistency for the study questionnaire at the test assessment (n = 52) and at retest 0.929 and 0.935, respectively, by total Cronbach's α. All of the items at test and re-test correlated with the total score. CONCLUSIONS: Reproducibility analysis showed excellent test/re-test reliability for the total score and each item. Based on our findings, we conclude that the PCL5-C is a valid and reliable questionnaire when applied among Spanish mental health nurses population.
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Lista de Verificación , Enfermería Psiquiátrica , Psicometría , Trastornos por Estrés Postraumático , Humanos , España , Reproducibilidad de los Resultados , Femenino , Trastornos por Estrés Postraumático/diagnóstico , Trastornos por Estrés Postraumático/psicología , Encuestas y Cuestionarios , Adulto , Masculino , Manual Diagnóstico y Estadístico de los Trastornos Mentales , Persona de Mediana EdadRESUMEN
Introducción: El programa de vacunación universal con la vacuna antineumocócica conjugada 13-valente (VNC13) se implantó en Andalucía en diciembre de 2016. Métodos: Estudio transversal de colonización nasofaríngea por Streptococcus pneumoniae. Se seleccionó a 397 niños sanos en centros de atención primaria de Sevilla durante los periodos 1/4/2018-28/2/2020 y 1/11/2021-28/2/2022 (periodo VNC13). Se utilizó una colección histórica de un estudio de colonización desarrollado en niños sanos y con infección respiratoria superior entre el 1/01/2006 y el 30/06/2008 (periodo VNC7) para comparar las distribuciones de serotipos/genotipos y las tasas de resistencias antibióticas. Resultados: Un total de 76 (19%) niños estaban colonizados con S. pneumoniae en el periodo VNC13 y se dispuso de 154 aislamientos del periodo VNC7. La colonización por serotipos incluidos en VNC13 disminuyó significativamente entre los periodos VNC13 y VNC7 (11 vs. 38%; p=0,0001); los serotipos 19F (8%), 3 (1%) y 6B (1%) fueron los únicos serotipos vacunales circulantes. Los serotipos 15B/C y 11A fueron los serotipos no VNC13 más prevalentes durante el periodo VNC13 (14% y 11%, respectivamente); este último se incrementó de forma significativa entre periodos de tiempo (p=0,04). El serotipo 11A solo se asoció en el periodo VNC13 con variantes resistentes a la ampicilina del clon Spain9V-ST156 (ST6521 y genéticamente relacionado ST14698), no detectados en el periodo anterior. Conclusiones: Hubo una circulación muy residual de los serotipos vacunales durante el periodo VNC13, con excepción del serotipo19F. El serotipo 11A se incrementó de forma significativa entre los periodos VNC13 y VNC7 por expansión clonal del genotipo resistente a la ampicilina ST6521.(AU)
Background: The 13-valent pneumococcal conjugate vaccine (PCV13) universal vaccination program was introduced in December 2016 in Andalusia. Methods: A cross-sectional study was conducted on the molecular epidemiology of pneumococcal nasopharyngeal colonization. A total of 397 healthy children were recruited from primary healthcare centres in Seville for the periods 1/4/2018 to 28/2/2020 and 1/11/2021 to 28/2/2022 (PCV13 period). Data from a previous carriage study conducted among healthy and sick children from 1/01/2006 to 30/06/2008 (PCV7 period) were used for comparison of serotype/genotype distributions and antibiotic resistance rates. Results: Overall, 76 (19%) children were colonized with S. pneumoniae during the PCV13 period and there were information available from 154 isolates collected during the PCV7 period. Colonization with PCV13 serotypes declined significantly in the PCV13 period compared with historical controls (11 vs. 38%, P=0.0001), being serotypes 19F (8%), 3 (1%) and 6B (1%) the only circulating vaccine types. Serotypes 15B/C and 11A were the most frequently identified non-PCV13 serotypes during the PCV13 period (14% and 11%, respectively); the later one increased significantly between time periods (P=0.04). Serotype 11A was exclusively associated in the PCV13 period with ampicillin-resistant variants of the Spain9V-ST156 clone (ST6521 and genetically related ST14698), not detected in the preceding period. Conclusions: There was a residual circulation of vaccine types following PCV13 introduction, apart from serotype 19F. Serotype 11A increased between PCV13 and PCV7 periods due to emergence and clonal expansion of ampicillin-resistant genotype ST6521.(AU)
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Humanos , Masculino , Femenino , Niño , Epidemiología Molecular , Programas de Inmunización , Streptococcus pneumoniae/inmunología , Streptococcus pneumoniae/genética , Infecciones Neumocócicas , Ampicilina , España , Estudios Transversales , Portador SanoRESUMEN
BACKGROUND AND PURPOSE: There is a need for effective anti-COVID-19 treatments, mainly for individuals at risk of severe disease such as the elderly and the immunosuppressed. Drug repositioning has proved effective in identifying drugs that can find a new application for the control of coronavirus disease, in particular COVID-19. The purpose of the present study was to find synergistic antiviral combinations for COVID-19 based on lethal mutagenesis. EXPERIMENTAL APPROACH: The effect of combinations of remdesivir and ribavirin on the infectivity of SARS-CoV-2 in cell culture has been tested. Viral populations were monitored by ultra-deep sequencing, and the decrease of infectivity as a result of the treatment was measured. KEY RESULTS: Remdesivir and ribavirin exerted a synergistic inhibitory activity against SARS-CoV-2, quantified both by CompuSyn (Chou-Talalay method) and Synergy Finder (ZIP-score model). In serial passage experiments, virus extinction was readily achieved with remdesivir-ribavirin combinations at concentrations well below their cytotoxic 50 value, but not with the drugs used individually. Deep sequencing of treated viral populations showed that remdesivir, ribavirin, and their combinations evoked significant increases of the number of viral mutations and haplotypes, as well as modification of diversity indices that characterize viral quasi-species. CONCLUSION AND IMPLICATIONS: SARS-CoV-2 extinction can be achieved by synergistic combination treatments based on lethal mutagenesis. In addition, the results offer prospects of triple drug treatments for effective SARS-CoV-2 suppression.
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Adenosina Monofosfato , Alanina , Antivirales , Sinergismo Farmacológico , Ribavirina , SARS-CoV-2 , Alanina/análogos & derivados , Alanina/farmacología , Ribavirina/farmacología , Antivirales/farmacología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , SARS-CoV-2/efectos de los fármacos , Chlorocebus aethiops , Células Vero , Animales , Humanos , Tratamiento Farmacológico de COVID-19 , COVID-19/virologíaRESUMEN
Introduction: SARS-CoV-2 isolates of a given clade may contain low frequency genomes that encode amino acids or deletions which are typical of a different clade. Methods: Here we use high resolution ultra-deep sequencing to analyze SARS-CoV-2 mutant spectra. Results: In 6 out of 11 SARS-CoV-2 isolates from COVID-19 patients, the mutant spectrum of the spike (S)-coding region included two or more amino acids or deletions, that correspond to discordant viral clades. A similar observation is reported for laboratory populations of SARS-CoV-2 USA-WA1/2020, following a cell culture infection in the presence of remdesivir, ribavirin or their combinations. Moreover, some of the clade-discordant genome residues are found in the same haplotype within an amplicon. Discussion: We evaluate possible interpretations of these findings, and reviewed precedents for rapid selection of genomes with multiple mutations in RNA viruses. These considerations suggest that intra-host evolution may be sufficient to generate minority sequences which are closely related to sequences typical of other clades. The results provide a model for the origin of variants of concern during epidemic spreadâin particular Omicron lineagesâthat does not require prolonged infection, involvement of immunocompromised individuals, or participation of intermediate, non-human hosts.
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Rhipicephalus microplus, the cattle fever tick, is the most important ectoparasite impacting the livestock industry worldwide. Overreliance on chemical treatments for tick control has led to the emergence of acaricide-resistant ticks and environmental contamination. An immunological strategy based on vaccines offers an alternative approach to tick control. To develop novel tick vaccines, it is crucial to identify and evaluate antigens capable of generating protection in cattle. Chitinases are enzymes that degrade older chitin at the time of moulting, therefore allowing interstadial metamorphosis. In this study, 1 R. microplus chitinase was identified and its capacity to reduce fitness in ticks fed on immunized cattle was evaluated. First, the predicted amino acid sequence was determined in 4 isolates and their similarity was analysed by bioinformatics. Four peptides containing predicted B-cell epitopes were designed. The immunogenicity of each peptide was assessed by inoculating 2 cattle, 4 times at 21 days intervals, and the antibody response was verified by indirect ELISA. A challenge experiment was conducted with those peptides that were immunogenic. The chitinase gene was successfully amplified and sequenced, enabling comparison with reference strains. Notably, a 99.32% identity and 99.84% similarity were ascertained among the sequences. Furthermore, native protein recognition was demonstrated through western blot assays. Chitinase peptide 3 reduced the weight and oviposition of engorged ticks, as well as larvae viability, exhibiting a 71% efficacy. Therefore, chitinase 3 emerges as a viable vaccine candidate, holding promise for its integration into a multiantigenic vaccine against R. microplus.
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Since its introduction in the human population, SARS-CoV-2 has evolved into multiple clades, but the events in its intrahost diversification are not well understood. Here, we compare three-dimensional (3D) self-organized neural haplotype maps (SOMs) of SARS-CoV-2 from thirty individual nasopharyngeal diagnostic samples obtained within a 19-day interval in Madrid (Spain), at the time of transition between clades 19 and 20. SOMs have been trained with the haplotype repertoire present in the mutant spectra of the nsp12- and spike (S)-coding regions. Each SOM consisted of a dominant neuron (displaying the maximum frequency), surrounded by a low-frequency neuron cloud. The sequence of the master (dominant) neuron was either identical to that of the reference Wuhan-Hu-1 genome or differed from it at one nucleotide position. Six different deviant haplotype sequences were identified among the master neurons. Some of the substitutions in the neural clouds affected critical sites of the nsp12-nsp8-nsp7 polymerase complex and resulted in altered kinetics of RNA synthesis in an in vitro primer extension assay. Thus, the analysis has identified mutations that are relevant to modification of viral RNA synthesis, present in the mutant clouds of SARS-CoV-2 quasispecies. These mutations most likely occurred during intrahost diversification in several COVID-19 patients, during an initial stage of the pandemic, and within a brief time period.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/genética , Haplotipos , Proteínas no Estructurales Virales , ARN ViralRESUMEN
INTRODUCTION: The 13-valent pneumococcal conjugate vaccine (PCV13) universal vaccination programme was introduced in December 2016 in Andalusia. METHODS: A cross-sectional study was conducted on the molecular epidemiology of pneumococcal nasopharyngeal colonization. A total of 397 healthy children were recruited from primary healthcare centres in Seville for the periods 1/4/2018 to 28/2/2020 and 1/11/2021 to 28/2/2022 (PCV13 period). Data from a previous carriage study conducted among healthy and sick children from 1/01/2006 to 30/06/2008 (PCV7 period), were used for comparison of serotype/genotype distributions and antibiotic resistance rates. RESULTS: Overall, 76 (19%) children were colonized with S. pneumoniae during the PCV13 period and there were information available from 154 isolates collected during the PCV7 period. Colonization with PCV13 serotypes declined significantly in the PCV13 period compared with historical controls (11% vs 38%, pâ¯=â¯0.0001), being serotypes 19F (8%), 3 (1%) and 6B (1%) the only circulating vaccine types. Serotypes 15B/C and 11A were the most frequently identified non-PCV13 serotypes during the PCV13 period (14% and 11%, respectively); the later one increased significantly between time periods (pâ¯=â¯0.04). Serotype 11A was exclusively associated in the PCV13 period with ampicillin-resistant variants of the Spain9V-ST156 clone (ST6521 and genetically related ST14698), not detected in the preceding period. CONCLUSIONS: There was a residual circulation of vaccine types following PCV13 introduction, apart from serotype 19F. Serotype 11A increased between PCV13 and PCV7 periods due to emergence and clonal expansion of ampicillin-resistant genotype ST6521.
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Infecciones Neumocócicas , Niño , Humanos , Infecciones Neumocócicas/epidemiología , Infecciones Neumocócicas/prevención & control , Estudios Transversales , Epidemiología Molecular , España/epidemiología , Portador Sano/epidemiología , Streptococcus pneumoniae/genética , Ampicilina , Programas de InmunizaciónRESUMEN
Upon the emergence of SARS-CoV-2 in the human population, it was conjectured that for this coronavirus the dynamic intra-host heterogeneity typical of RNA viruses would be toned down. Nothing of this sort is observed. Here we review the main observations on the complexity and diverse composition of SARS-CoV-2 mutant spectra sampled from infected patients, within the framework of quasispecies dynamics. The analyses suggest that the information provided by myriads of genomic sequences within infected individuals may have a predictive value of the genomic sequences that acquire epidemiological relevance. Possibilities to reconcile the presence of broad mutant spectra in the large RNA coronavirus genome with its encoding a 3' to 5' exonuclease proofreading-repair activity are considered. Indeterminations in the behavior of individual viral genomes provide a benefit for the survival of the ensemble. We propose that this concept falls in the domain of "stochastic thinking," a notion that applies also to cellular processes, as a means for biological systems to face unexpected needs.
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COVID-19 , Virus ARN , SARS-CoV-2 , Humanos , COVID-19/virología , Genoma Viral , Cuasiespecies , Virus ARN/genética , SARS-CoV-2/genética , SARS-CoV-2/fisiologíaRESUMEN
MicroRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) are potential diagnostic and prognostic biomarkers. However, discrepancies in miRNA patterns and their validation are still frequent due to differences in sample origin, EV isolation, and miRNA sequencing methods. The aim of the present study is to find a reliable EV isolation method for miRNA sequencing, adequate for clinical application. To this aim, two comparative studies were performed in parallel with the same human plasma sample: (i) isolation and characterization of EVs obtained using three procedures: size exclusion chromatography (SEC), iodixanol gradient (GRAD), and its combination (SEC+GRAD) and (ii) evaluation of the yield of miRNA sequences obtained using NextSeq 500 (Illumina) and three miRNA library preparation protocols: NEBNext, NEXTFlex, and SMARTer smRNA-seq. The conclusion of comparison (i) is that recovery of the largest amount of EVs and reproducibility were attained with SEC, but GRAD and SEC+GRAD yielded purer EV preparations. The conclusion of (ii) is that the NEBNext library showed the highest reproducibility in the number of miRNAs recovered and the highest diversity of miRNAs. These results render the combination of GRAD EV isolation and NEBNext library preparation for miRNA retrieval as adequate for clinical applications using plasma samples.
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Vesículas Extracelulares , MicroARNs , Humanos , Reproducibilidad de los Resultados , MicroARNs/genética , Vesículas Extracelulares/genética , Cromatografía en Gel , PlasmaRESUMEN
The concept of a mild mutagen was coined to describe a minor mutagenic activity exhibited by some nucleoside analogues that potentiated their efficacy as antiretroviral agents. In the present study, we report the mild mutagen activity of sofosbuvir (SOF) for hepatitis C virus (HCV). Serial passages of HCV in human hepatoma cells, in the presence of SOF at a concentration well below its cytotoxic concentration 50 (CC50) led to pre-extinction populations whose mutant spectra exhibited a significant increase of CâU transitions, relative to populations passaged in the absence of SOF. This was reflected in an increase in several diversity indices that were used to characterize viral quasispecies. The mild mutagenic activity of SOF was largely absent when it was tested with isogenic HCV populations that displayed high replicative fitness. Thus, SOF can act as a mild mutagen for HCV, depending on HCV fitness. Possible mechanisms by which the SOF mutagenic activity may contribute to its antiviral efficacy are discussed.
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Hepatitis C Crónica , Hepatitis C , Humanos , Sofosbuvir/farmacología , Sofosbuvir/uso terapéutico , Hepacivirus/genética , Mutágenos/farmacología , Antivirales/farmacología , Antivirales/uso terapéutico , Hepatitis C/tratamiento farmacológico , Hepatitis C Crónica/tratamiento farmacológico , Genotipo , Ribavirina/uso terapéutico , Resultado del Tratamiento , Quimioterapia CombinadaRESUMEN
AIMS/HYPOTHESIS: Type 2 diabetes is associated with increased risk of cognitive decline although the pathogenic basis for this remains obscure. Deciphering diabetes-linked molecular mechanisms in cells of the cerebral cortex could uncover novel therapeutic targets. METHODS: Single-cell transcriptomic sequencing (scRNA-seq) was conducted on the cerebral cortex in a mouse model of type 2 diabetes (db/db mice) and in non-diabetic control mice in order to identify gene expression changes in distinct cell subpopulations and alterations in cell type composition. Immunohistochemistry and metabolic assessment were used to validate the findings from scRNA-seq and to investigate whether these cell-specific dysfunctions impact the neurovascular unit (NVU). Furthermore, the behavioural and cognitive alterations related to these dysfunctions in db/db mice were assessed via Morris water maze and novel object discrimination tests. Finally, results were validated in post-mortem sections and protein isolates from individuals with type 2 diabetes. RESULTS: Compared with non-diabetic control mice, the db/db mice demonstrated disrupted brain function as revealed by losses in episodic and spatial memory and this occurred concomitantly with dysfunctional NVU, neuronal circuitry and cerebral atrophy. scRNA-seq of db/db mouse cerebral cortex revealed cell population changes in neurons, glia and microglia linked to functional regulatory disruption including neuronal maturation and altered metabolism. These changes were validated through immunohistochemistry and protein expression analysis not just in the db/db mouse cerebral cortex but also in post-mortem sections and protein isolates from individuals with type 2 diabetes (74.3 ± 5.5 years) compared with non-diabetic control individuals (87.0 ± 8.5 years). Furthermore, metabolic and synaptic gene disruptions were evident in cortical NVU cell populations and associated with a decrease in vascular density. CONCLUSIONS/INTERPRETATION: Taken together, our data reveal disruption in the cellular and molecular architecture of the cerebral cortex induced by diabetes, which can explain, at least in part, the basis for progressive cognitive decline in individuals with type 2 diabetes. DATA AVAILABILITY: The single-cell sequencing data that supports this study are available at GEO accession GSE217665 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE217665 ).
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Disfunción Cognitiva , Diabetes Mellitus Tipo 2 , Ratones , Animales , Diabetes Mellitus Tipo 2/complicaciones , Disfunción Cognitiva/tratamiento farmacológico , Corteza Cerebral/metabolismo , Modelos Animales de EnfermedadRESUMEN
Introduction: Cellular epigenetic modifications occur in the course of viral infections. We previously documented that hepatitis C virus (HCV) infection of human hepatoma Huh-7.5 cells results in a core protein-mediated decrease of Aurora kinase B (AURKB) activity and phosphorylation of Serine 10 in histone H3 (H3Ser10ph) levels, with an affectation of inflammatory pathways. The possible role of HCV fitness in infection-derived cellular epigenetic modifications is not known. Methods: Here we approach this question using HCV populations that display a 2.3-fold increase in general fitness (infectious progeny production), and up to 45-fold increase of the exponential phase of intracellular viral growth rate, relative to the parental HCV population. Results: We show that infection resulted in a HCV fitness-dependent, average decrease of the levels of H3Ser10ph, AURKB, and histone H4 tri-methylated at Lysine 20 (H4K20m3) in the infected cell population. Remarkably, the decrease of H4K20m3, which is a hallmark of cellular transformation, was significant upon infection with high fitness HCV but not upon infection with basal fitness virus. Discussion: Here we propose two mechanisms âwhich are not mutually exclusiveâ to explain the effect of high viral fitness: an early advance in the number of infected cells, or larger number of replicating RNA molecules per cell. The implications of introducing HCV fitness as an influence in virus-host interactions, and for the course of liver disease, are warranted. Emphasis is made in the possibility that HCV-mediated hepatocellular carcinoma may be favoured by prolonged HCV infection of a human liver, a situation in which viral fitness is likely to increase.
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Carcinoma Hepatocelular , Hepatitis C , Humanos , Hepacivirus/genética , Replicación Viral , Carcinoma Hepatocelular/genética , Epigénesis GenéticaRESUMEN
Diabetes mellitus is a heterogeneous chronic metabolic disorder characterized by the presence of hyperglycemia, commonly preceded by a prediabetic state. The excess of blood glucose can damage multiple organs, including the brain. In fact, cognitive decline and dementia are increasingly being recognized as important comorbidities of diabetes. Despite the largely consistent link between diabetes and dementia, the underlying causes of neurodegeneration in diabetic patients remain to be elucidated. A common factor for almost all neurological disorders is neuroinflammation, a complex inflammatory process in the central nervous system for the most part orchestrated by microglial cells, the main representatives of the immune system in the brain. In this context, our research question aimed to understand how diabetes affects brain and/or retinal microglia physiology. We conducted a systematic search in PubMed and Web of Science to identify research items addressing the effects of diabetes on microglial phenotypic modulation, including critical neuroinflammatory mediators and their pathways. The literature search yielded 1327 records, including 18 patents. Based on the title and abstracts, 830 papers were screened from which 250 primary research papers met the eligibility criteria (original research articles with patients or with a strict diabetes model without comorbidities, that included direct data about microglia in the brain or retina), and 17 additional research papers were included through forward and backward citations, resulting in a total of 267 primary research articles included in the scoping systematic review. We reviewed all primary publications investigating the effects of diabetes and/or its main pathophysiological traits on microglia, including in vitro studies, preclinical models of diabetes and clinical studies on diabetic patients. Although a strict classification of microglia remains elusive given their capacity to adapt to the environment and their morphological, ultrastructural and molecular dynamism, diabetes modulates microglial phenotypic states, triggering specific responses that include upregulation of activity markers (such as Iba1, CD11b, CD68, MHC-II and F4/80), morphological shift to amoeboid shape, secretion of a wide variety of cytokines and chemokines, metabolic reprogramming and generalized increase of oxidative stress. Pathways commonly activated by diabetes-related conditions include NF-κB, NLRP3 inflammasome, fractalkine/CX3CR1, MAPKs, AGEs/RAGE and Akt/mTOR. Altogether, the detailed portrait of complex interactions between diabetes and microglia physiology presented here can be regarded as an important starting point for future research focused on the microglia-metabolism interface.
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Demencia , Diabetes Mellitus , Hiperglucemia , Humanos , Microglía , Sistema Nervioso CentralRESUMEN
We report that ribavirin exerts an inhibitory and mutagenic activity on SARS-CoV-2-infecting Vero cells, with a therapeutic index higher than 10. Deep sequencing analysis of the mutant spectrum of SARS-CoV-2 replicating in the absence or presence of ribavirin indicated an increase in the number of mutations, but not in deletions, and modification of diversity indices, expected from a mutagenic activity. Notably, the major mutation types enhanced by replication in the presence of ribavirin were AâG and UâC transitions, a pattern which is opposite to the dominance of GâA and CâU transitions previously described for most RNA viruses. Implications of the inhibitory activity of ribavirin, and the atypical mutational bias produced on SARS-CoV-2, for the search for synergistic anti-COVID-19 lethal mutagen combinations are discussed.