BackgroundHisto–blood group antigens and secretor status have been associated with susceptibility to Norovirus infections, which suggests that antibody prevalence and titer might correlate with these ...phenotypes MethodsPlasma samples (n=105) from Swedish blood donors that had been genotyped for secretor (FUT2) and Lewis (Le; FUT3) genotypes and phenotyped for ABO and Le blood groups were analyzed for immunoglobulin G antibody prevalence and titers to norovirus genogroup (GG) II.4 ResultsThe results showed that nonsecretors (se428se428) and Lea+b− individuals not only had significantly lower antibody titers than did secretors (P<.0001) and Lea−b+ individuals (P<.0002) but were also significantly more often antibody negative (P<.05). Antibody titers in secretors were not significantly different between individuals of different Le (FUT3) genotypes or different ABO phenotypes ConclusionsNonsecretors and Lea+b− individuals are significantly less prone to be infected with GGII noroviruses. This new information extends previous knowledge and supports the hypothesis that nonsecretors are relatively but not absolutely resistant to norovirus infections
•Laurel forest recovery does not depend only on initial vegetation conditions.•The early recovery stage was driven by aspect and distance from young-growth forests.•In middle stage, the recovery was ...driven by old-growth forest cover around each site.•The oldest-growth forests showed more endemic, threatened species and β-diversity.
Deforestation and forest fragmentation have serious negative consequences for biodiversity and species distribution, but many studies approach species distributions and speciation processes without taking in account the local history of human disturbances. Nevertheless, the complex recovery process after forest destruction is widely distributed around the world and takes place under a wide mosaic of biotic and abiotic factors that may be influencing the species distribution. In this study aerial pictures from 1951 and 2019 were used to assess forest recovery during about sixty years. We tested if the initial stage (forest completely destroyed or young growth forest in 1951) determines the successional process, evaluating the role of different spatial drivers (distance from remnant forest fragments and unfelled areas around each site) and mesoclimate conditions, by examining 40 disturbed sites and 18 old-growth forest sites distinguished in 1951 aerial photos. The final stage during this successional process was assessed using 2019 aerial photos, covering the same these sites. At each plot, plant species composition and forest structure were studied, which allowed us to obtain a maturity index. Our results reveal that the initial stage is not the only driver explaining forest structure and species composition at the end of the 60-year period analyzed. The role of each forest recovery driver varies depending on each stage. In the early successional stages, the slope aspect (exposure) was the most important factor, which is correlated with mesoclimatic conditions, followed by the distance from young-growth forest in 1951. However, in intermediate successional stages the cover of old-growth forest around each plot in 1951 was the main factor in the recovery. At the end of the study period (2019), the differences in species richness between young-growth and old-growth forest were not significant, although old-growth forests were characterized by high densities of endemics, threatened species and β-diversity, showing the species composition a strong dependence on forest structure. Our results reveal the important need to consider human disturbance history in forest research and how climate conditions and the lack of nearby remnants can negatively affect the forest recovery process, greatly increasing the time needed to return to old-growth laurel forest conditions, which requires specific management recommendations.
•SARS-CoV-2-S1/M-reactive-IFN-γ CD4+ and CD8+ T cells were detected in less than 30% recovered patients from severe COVID-19.•60% of individuals displayed detectable SARS-CoV-2-RBD-specific IgGs at ...2–5 months following COVID-19 diagnosis.•The presence of comorbidities may hamper persistence of SARS-CoV-2 -S1/M-reactive T cells in recovered COVID-19 patients.
There is an imperative need to determine the durability of adaptive immunity to SARS-CoV-2. We enumerated SARS-CoV-2-reactive CD4+ and CD8+ T cells targeting S1 and M proteins and measured RBD-specific serum IgG over a period of 2–6 months after symptoms onset in a cohort of subjects who had recovered from severe clinical forms of COVID-19.
We recruited 58 patients (38 males and 20 females; median age, 62.5 years), who had been hospitalized with bilateral pneumonia, 60% with one or more comorbidities. IgG antibodies binding to SARS-CoV-2 RBD were measured by ELISA. SARS-CoV-2-reactive CD69+-expressing-IFNγ-producing-CD4+ and CD8+ T cells were enumerated in heparinized whole blood by flow cytometry for ICS.
Detectable SARS-CoV-2-S1/M-reactive CD69+-IFN-γ CD4+ and CD8+ T cells were displayed in 17 (29.3%) and 6 (10.3%) subjects respectively, at a median of 84 days after onset of symptoms (range, 58–191 days). Concurrent comorbidities increased the risk (OR, 3.15; 95% CI, 1.03–9.61; P = 0.04) of undetectable T–cell responses in models adjusted for age, sex and hospitalization ward. Twenty-one out of the 35 patients (60%) had detectable RBD-specific serum IgGs at a median of 118 days (range, 60–145 days) after symptoms onset. SARS-CoV-2 RBD-specific IgG serum levels were found to drop significantly over time.
A relatively limited number of subjects who developed severe forms of COVID-19 had detectable SARS-CoV-2-S1/M IFNγ CD4+ and CD8+ T cells at midterm after clinical diagnosis. Our data also indicated that serum levels of RBD-specific IgGs decline over time, becoming undetectable in some patients.
Basement membranes are extracellular structures of epithelia and endothelia that have collagen IV scaffolds of triple α-chain helical protomers that associate end-to-end, forming networks. The ...molecular mechanisms by which the noncollagenous C-terminal domains of α-chains direct the selection and assembly of the α1α2α1 and α3α4α5 hetero-oligomers found
remain obscure. Autoantibodies against the noncollagenous domains of the α3α4α5 hexamer or mutations therein cause Goodpasture's or Alport's syndromes, respectively. To gain further insight into oligomer-assembly mechanisms as well as into Goodpasture's and Alport's syndromes, crystal structures of non-collagenous domains produced by recombinant methods were determined. The spontaneous formation of canonical homohexamers (dimers of trimers) of these domains of the α1, α3 and α5 chains was shown and the components of the Goodpasture's disease epitopes were viewed. Crystal structures of the α2 and α4 non-collagenous domains generated by recombinant methods were also determined. These domains spontaneously form homo-oligomers that deviate from the canonical architectures since they have a higher number of subunits (dimers of tetramers and of hexamers, respectively). Six flexible structural motifs largely explain the architectural variations. These findings provide insight into noncollagenous domain folding, while supporting the
operation of extrinsic mechanisms for restricting the self-assembly of noncollagenous domains. Intriguingly, Alport's syndrome missense mutations concentrate within the core that nucleates the folding of the noncollagenous domain, suggesting that this syndrome, when owing to missense changes, is a folding disorder that is potentially amenable to pharmacochaperone therapy.
Noroviruses (NoVs) are the main etiologic agents of acute epidemic gastroenteritis and probiotic bacteria have been reported to exert a positive effect on viral diarrhea. The protruding (P) domain ...from NoVs VP1 capsid protein has the ability to assemble into the so-called P-particles, which retain the binding ability to host receptors. We purified the P-domains from NoVs genotypes GI.1 and GII.4 as 6X(His)-tagged proteins and determined that, similar to native domains, they were structured into P-particles that were functional in the recognition of the specific glycoconjugated receptors, as established by surface plasmon resonance experiments. We showed that several lactic acid bacteria (probiotic and non-probiotic) and a Gram-negative probiotic strain have the ability to bind P-particles on their surfaces irrespective of their probiotic status. The binding of P-particles (GI.1) to HT-29 cells in the presence of selected strains showed that bacteria can inhibit P-particle attachment in competitive exclusion experiments. However, pre-treatment of cells with bacteria or adding bacteria to cells with already attached P-particles enhanced the retention of the particles. Although direct viral binding and blocking of viral receptors have been postulated as mechanisms of protection against viral infection by probiotic bacteria, these results highlight the need for a careful evaluation of this hypothesis. The work presented here investigates for the first time the probiotic-NoVs-host interactions and points up the NoVs P-particles as useful tools to overcome the absence of in vitro cellular models to propagate these viruses.
•A European Network on Next-Generation Sequencing (ENNGS) has been established.•Recommendations for the implementation of viral metagenomic high-throughput sequencing (mHTS) in diagnostic ...laboratories are provided, focusing on the wet lab (Part I).•Recommendations for implementation and validation of the bioinformatic procedure of viral mHTS warrants separate recommendations (Part II).•In the current manuscript, practical recommendations on facility requirements, extraction of nucleic acid, viral enrichment, library preparation, controls and assay validation are provided.
Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols.
Noroviruses are the leading cause of sporadic cases and outbreaks of viral gastroenteritis. For more than 20 years, most norovirus infections have been caused by the pandemic genotype GII.4, yet ...recent studies have reported the emergence of recombinant strains in many countries. In the present study, 4,950 stool samples collected between January 2016 and April 2020 in Valencia, Spain, from patients with acute gastroenteritis were analyzed to investigate the etiological agent. Norovirus was the most frequently detected enteric virus, with a positivity rate of 9.5% (471/4,950). Among 224 norovirus strains characterized, 175 belonged to genogroup II (GII) and 49 belonged to GI. Using dual genotyping based on sequencing of the open reading frame 1 (ORF1)/ORF2 junction region, we detected 25 different capsid-polymerase-type associations. The most common GII capsid genotype was GII.4 Sydney 2012, followed by GII.2, GII.3, GII.6, and GII.17. A high prevalence of recombinant strains (90.4%) was observed among GII infections between 2018 and 2020. GII.4 SydneyP16 was the predominant genotype from 2019 to 2020. In addition, GII.P16 polymerase was found harbored within six different capsid genes. GI.4 and GI.3 were the predominant genotypes in genogroup I, in which recombinant strains were also found, such as GI.3P10, GI.3P13, and GI.5P4. Interestingly, applying the criterion of 2 times the standard deviation, we found that 12 sequences initially classified as GI.3 may represent two new tentative genotypes in genogroup I, designated GI.10 and GI.11. This study shows the extensive diversity of recombinant noroviruses circulating in Spain and highlights the role of recombination events in the spread of noroviruses. IMPORTANCE Human noroviruses are the most common cause of viral diarrhea. There are no approved vaccines to prevent their infections yet, which would be very useful to protect infants, small children, and the elderly in residential institutions. These viruses are extremely contagious and can be transmitted by contaminated food and water as well as directly from person to person. Molecular surveillance and epidemiology of norovirus infections allow the identification of the most common viral strains in different geographical areas over time. Noroviruses show wide genetic variability due to a high rate of mutations but also due to genomic recombinations, as we demonstrate in this study. We have detected 25 different viral capsid-polymerase gene associations among 224 norovirus strains characterized in Spain between January 2016 and April 2020, including two tentative new capsid genotypes in genogroup I.
Fucosyl-N-acetylglucosamine disaccharides are important core structures that form part of human mucosal and milk glyco-complexes. We have previously shown that AlfB and AlfC α-L-fucosidases from ...Lactobacillus casei are able to synthesize fucosyl-α-1,3--N-acetylglucosamine (Fuc-α1,3-GlcNAc) and fucosyl-α-1,6-N-acetylglucosamine (Fuc-α1,6-GlcNAc), respectively, in transglycosylation reactions. Here, these reactions were performed in a semipreparative scale, and the produced disaccharides were purified. The maximum yields obtained of Fuc-α1,3-GlcNAc and Fuc-α1,6-GlcNAc were 4.2 and 9.3 g/l, respectively. The purified fucosyl-disaccharides were then analyzed for their prebiotic effect in vitro using strains from the Lactobacillus casei/paracasei/rhamnosus group and from Bifidobacterium species. The results revealed that 6 out of 11 L. casei strains and 2 out of 6 L. rhamnosus strains tested were able to ferment Fuc-α1,3-GlcNAc, and L. casei BL87 and L. rhamnosus BL327 strains were also able to ferment Fuc-α1,6-GlcNAc. DNA hybridization experiments suggested that the metabolism of Fuc-α1,3-GlcNAc in those strains relies in an α-L-fucosidase homologous to AlfB. Bifidobacterium breve and Bibidobacterium pseudocatenolatum species also metabolized Fuc-α1,3-GlcNAc. Notably, L-fucose was excreted from all the Lactobacillus and Bifidobacterium strains fermenting fucosyl-disaccharides, except from strains L. rhamnosus BL358 and BL377, indicating that in these latest strains, L-fucose was catabolized. The fucosyl-disaccharides were also tested for their inhibitory potential of pathogen adhesion to human colon adenocarcinoma epithelial (HT29) cell line. Enteropathogenic Escherichia coli (EPEC) strains isolated from infantile gastroenteritis were used, and the results showed that both fucosyl-disaccharides inhibited adhesion to different extents of certain EPEC strains to HT29 cells in tissue culture.
Continental carbonates, such as travertines and tufas, formed from CO
2
-rich groundwater degassing as it emerges at the Earth’s surface, are often associated with major crustal-scale faults. The ...Carraclaca site, in the Lorca-Totana section of the Alhama de Murcia Fault, Spain, presents a complex geomorphological landscape controlled by active tectonics. The geology here records the interaction between Quaternary alluvial fans, travertines, and a pop-up structure developed in a transpressional section of the fault. The Alhama de Murcia Fault is an 80 km long left-lateral strike-slip fault that is one of the main seismogenic structures in the Iberian Peninsula. In this work, we examined the relation between travertine precipitation in the Carraclaca site and the tectonic activity of this fault zone through morphological and geochemical studies. The δ
13
C and δ
18
O isotopic signals indicate that the carbonate deposits are hydrothermal. In addition, the
87
Sr/
86
Sr ratios in the samples suggest subsurface fluid interaction with the Miocene sediments and the Alpujárride basement, located below the alluvial deposits. Tectonic activity in the Alhama de Murcia Fault might generate the opening of deep water circulation in the crust every time a seismic event occurs, giving rise to hydrothermally derived carbonates precipitation. Deep waters rise and reach the surface interacting with meteoric waters, resulting in travertine formation. Therefore, the Carraclaca carbonate deposits study can inform us about the seismogenic cycle of the fault in the Lorca-Totana section.
The survival of commensal bacteria in the human gut partially depends on their ability to metabolize host-derived molecules. The use of the glycosidic moiety of
-glycoproteins by bacteria has been ...reported, but the role of
-glycopeptides or glycoamino acids as the substrates for bacterial growth has not been evaluated. We have identified in
strain BL23 a gene cluster (
) involved in the catabolism of the glycoamino acid fucosyl-α-1,6-
-GlcNAc-Asn (6'FN-Asn), a constituent of the core-fucosylated structures of mammalian
-glycoproteins. The cluster consists of the genes
, encoding a major facilitator superfamily (MFS) permease and the α-l-fucosidase AlfC, and the divergently oriented
(aspartate 4-decarboxylase),
(transcriptional regulator),
(peptidase),
(glycosyl-asparaginase), and
(sugar kinase) genes. Knockout mutants showed that
,
,
,
, and
are necessary for efficient 6'FN-Asn utilization. The
genes are induced by 6'FN-Asn, but not by its glycan moiety, via the AlfR2 regulator. The constitutive expression of
genes in an
strain allowed the metabolism of a variety of 6'-fucosyl-glycans. However, GlcNAc-Asn did not support growth in this mutant background, indicating that the presence of a 6'-fucose moiety is crucial for substrate transport via AlfH. Within bacteria, 6'FN-Asn is defucosylated by AlfC, generating GlcNAc-Asn. This glycoamino acid is processed by the glycosylasparaginase AsnA2. GlcNAc-Asn hydrolysis generates aspartate and GlcNAc, which is used as a fermentable source by
These data establish the existence in a commensal bacterial species of an exclusive metabolic pathway likely to scavenge human milk and mucosal fucosylated
-glycopeptides in the gastrointestinal tract.
The gastrointestinal tract accommodates more than 10
microorganisms that have an enormous impact on human health. The mechanisms enabling commensal bacteria and administered probiotics to colonize the gut remain largely unknown. The ability to utilize host-derived carbon and energy resources available at the mucosal surfaces may provide these bacteria with a competitive advantage in the gut. Here, we have identified in the commensal species
a novel metabolic pathway for the utilization of the glycoamino acid fucosyl-α-1,6-
-GlcNAc-Asn, which is present in the core-fucosylated
-glycoproteins from mammalians. These results give insight into the molecular interactions between the host and commensal/probiotic bacteria and may help to devise new strategies to restore gut microbiota homeostasis in diseases associated with dysbiotic microbiota.
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