Sample and library preparation approaches for the analysis of the virome of irrigation water.

BACKGROUND: The virome (i.e. community of mainly RNA and DNA eukaryotic viruses and bacteriophages) of waters is yet to be extensively explored. In particular, the virome of waters used for irrigation could therefore potentially carry viral pathogens that can contaminate fresh produce. One problem in obtaining viral sequences from irrigation waters is the relatively low amount of virus particles, as well as the presence of human, bacterial and protozoan cells. The present aimed study was to compare different processing, amplification, and sequencing approaches for virome characterization in irrigation waters. RESULTS: Our analyses considered percentages of viral reads, values for diversity indices and number of families found in sequencing results. The results obtained suggest that enrichment protocols using two (bezonase and microccocal nuclease) or four enzymes at once (bezonase, microccocal nuclease, DNAse and RNase), regardless of an Amicon filtration step, are more appropriate than separated enzymatic treatments for virome characterization in irrigation water. The NetoVIR protocol combined with the ScriptSeq v2 RNA-Seq Library (P0-L20 protocol) showed the highest percentages of RNA viruses and identified the higher number of families. CONCLUSION: Although virome characterization applied in irrigation waters is an important tool for protecting public health by informing on circulating human and zoonotic infections, optimized and standardized procedures should be followed to reduce the variability of results related to either the sample itself and the downstream bioinformatics analyses. Our results show that virome characterization can be an important tool in the discovery of pathogenic viruses in the environment and can be used to inform and optimize reference-based detection methods provided that appropriate and rigorous controls are included. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effects of daily consumption of the probiotic Bifidobacterium animalis subsp. lactis CECT 8145 on anthropometric adiposity biomarkers in abdominally obese subjects: a randomized controlled trial.

BACKGROUND: The effects of probiotic Bifidobacterium animalis subsp. lactis CECT 8145 (Ba8145) and those of its heat-killed form (h-k Ba8145) on human anthropometric adiposity biomarkers are unknown. OBJECTIVE: To assess the effect of Ba8145 and h-k Ba8145 ingestion on anthropometric adiposity biomarkers. DESIGN: Randomized, parallel, double-blind, placebo-controlled trial with abdominally obese individuals. Participants (n = 135) consumed 1 capsule/day containing 1010 colony forming unit (CFU) of Ba8145, 1010 CFU of h-k Ba8145, or placebo (maltodextrin) for 3 months. RESULTS: Ba8145 ingestion decreased waist circumference, waist circumference/height ratio, and Conicity index (P < 0.05) versus its baseline. Changes versus the placebo group reached significance (P < 0.05) after the h-k Ba8145 treatment. Ba8145 decreased the body mass index compared with baseline and placebo group (P < 0.05). The decrease in visceral fat area after Ba8145 treatments reached significance (P < 0.05) only after h-k Ba8145. When analyses by gender were performed, significance remained only for women. Diastolic blood pressure and HOMA index decreased (P < 0.05) after h-k Ba8145. Gut microbiome analyses showed an increase in Akkermansia spp. after Ba8145 treatment, particularly in the live form, which was inversely related to weight (P = 0.003). CONCLUSIONS: In abdominally obese individuals, consumption of Ba8145, both as viable and mainly as heat-killed cells, improves anthropometric adiposity biomarkers, particularly in women. An increase in the gut Akkermansia genus appears as a possible mechanism involved. Our results support Ba8145 probiotic as a complementary strategy in obesity management.

Yeast ß-glucans and microalgal extracts modulate the immune response and gut microbiome in Senegalese sole (Solea senegalensis).

One bottleneck to sustainability of fish aquaculture is the control of infectious diseases. Current trends include the preventive application of immunostimulants and prebiotics such as polysaccharides. The present study investigated how yeast ß-glucan (Y), microalgal polysaccharide-enriched extracts (MAe) and whole Phaeodactylum tricornutum cells (MA) modulated the gut microbiome and stimulated the immune system in Senegalese sole (Solea senegalensis) when administered by oral intubation. Blood, intestine and spleen samples were taken at 3 h, 24 h, 48 h and 7 days after treatment. The short-term response (within 48 h after treatment) consisted of up-regulation of il1b and irf7 expression in the gut of the Y treated group. In contrast, administration of MAe decreased expression of tnfa and the chemokine cxc10 in the gut and spleen. Both treatments down-regulated the expression of irf3 with respect to the control group. Lysozyme activity in plasma decreased at 48 h only in the MAe-treated soles. Medium-term response consisted of the up-regulation of clec and irf7 expression in the gut of the Y, MAe and MA groups and of il1b mRNAs in the spleen of the MA group compared to the control group. Microbiome analysis using 16S rDNA gene sequencing indicated that the intestine microbiome was dominated by bacteria of the Vibrio genus (>95%). All the treatments decreased the relative proportion of Vibrio in the microbiome and Y and MAe decreased and MA increased diversity. Quantitative PCR confirmed the load of bacteria of the Vibrio genus was significantly decreased and this was most pronounced in Y treated fish. These data indicate that orally administrated insoluble yeast ß-glucans acted locally in the gut modulating the immune response and controlling the Vibrio abundance. In contrast, the MAe slightly reduced the Vibrio load in the intestine and caused a transient systemic anti-inflammatory response. The results indicate that these polysaccharides are a promising source of prebiotics for the sole aquaculture industry.

Endometrial receptivity revisited: endometrial transcriptome adjusted for tissue cellular heterogeneity.

STUDY QUESTION: Does cellular composition of the endometrial biopsy affect the gene expression profile of endometrial whole-tissue samples? SUMMARY ANSWER: The differences in epithelial and stromal cell proportions in endometrial biopsies modify the whole-tissue gene expression profiles and affect the results of differential expression analyses. WHAT IS ALREADY KNOWN: Each cell type has its unique gene expression profile. The proportions of epithelial and stromal cells vary in endometrial tissue during the menstrual cycle, along with individual and technical variation due to the method and tools used to obtain the tissue biopsy. STUDY DESIGN, SIZE, DURATION: Using cell-population specific transcriptome data and computational deconvolution approach, we estimated the epithelial and stromal cell proportions in whole-tissue biopsies taken during early secretory and mid-secretory phases. The estimated cellular proportions were used as covariates in whole-tissue differential gene expression analysis. Endometrial transcriptomes before and after deconvolution were compared and analysed in biological context. PARTICIPANTS/MATERIAL, SETTING, METHODS: Paired early- and mid-secretory endometrial biopsies were obtained from 35 healthy, regularly cycling, fertile volunteers, aged 23-36 years, and analysed by RNA sequencing. Differential gene expression analysis was performed using two approaches. In one of them, computational deconvolution was applied as an intermediate step to adjust for the proportions of epithelial and stromal cells in the endometrial biopsy. The results were then compared to conventional differential expression analysis. Ten paired endometrial samples were analysed with qPCR to validate the results. MAIN RESULTS AND THE ROLE OF CHANCE: The estimated average proportions of stromal and epithelial cells in early secretory phase were 65% and 35%, and during mid-secretory phase, 46% and 54%, respectively, correlating well with the results of histological evaluation (r = 0.88, P = 1.1 × 10-6). Endometrial tissue transcriptomic analysis showed that approximately 26% of transcripts (n = 946) differentially expressed in receptive endometrium in cell-type unadjusted analysis also remain differentially expressed after adjustment for biopsy cellular composition. However, the other 74% (n = 2645) become statistically non-significant after adjustment for biopsy cellular composition, underlining the impact of tissue heterogeneity on differential expression analysis. The results suggest new mechanisms involved in endometrial maturation, involving genes like LINC01320, SLC8A1 and GGTA1P, described for the first time in context of endometrial receptivity. LARGE-SCALE DATA: The RNA-seq data presented in this study is deposited in the Gene Expression Omnibus database with accession number GSE98386. LIMITATIONS REASONS FOR CAUTION: Only dominant endometrial cell types were considered in gene expression profile deconvolution; however, other less frequent endometrial cell types also contribute to the whole-tissue gene expression profile. WIDER IMPLICATIONS OF THE FINDINGS: The better understanding of molecular processes during transition from pre-receptive to receptive endometrium serves to improve the effectiveness and personalization of assisted reproduction protocols. Biopsy cellular composition should be taken into account in future endometrial 'omics' studies, where tissue heterogeneity could potentially influence the results. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by: Estonian Ministry of Education and Research (grant IUT34-16); Enterprise Estonia (EU48695); the EU-FP7 Eurostars program (NOTED, EU41564); the EU-FP7 Marie Curie Industry-Academia Partnerships and Pathways (SARM, EU324509); Horizon 2020 innovation program (WIDENLIFE, EU692065); MSCA-RISE-2015 project MOMENDO (No 691058) and the Miguel Servet Program Type I of Instituto de Salud Carlos III (CP13/00038); Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER): grants RYC-2016-21199 and ENDORE SAF2017-87526. Authors confirm no competing interests.

Bifidobacterium longum subsp infantis CECT7210-supplemented formula reduces diarrhea in healthy infants: a randomized controlled trial.

BackgroundIntestinal microbiota of breast-fed infants is plenty of beneficial bifidobacteria. We aimed to determine whether an infant formula supplemented with probiotic Bifidobacterium longum subsp. infantis CECT7210 (B. infantis IM1) is effective at reducing diarrhea incidence in healthy term infants.MethodsDouble-blinded, randomized, multicenter, controlled clinical trial, where formula-fed infants (<3 months) received an infant formula supplemented (Probiotic) or not (Control) with 107 cfu/g of B. infantis IM1 over 12 weeks. Diarrheas, growth, digestive symptoms, stool bifidobacteria, and microbiota were assessed.ResultsIn all, 97 (Control) and 93 (Probiotic) infants were randomized, and 78 (Control) and 73 (Probiotic) completed the 12 week-follow-up. In the overall study period, a median of 0.29±1.07 and 0.05±0.28 diarrhea events/infant was observed in the Control and Probiotic groups, respectively (P=0.059). This trend to less diarrhea episodes in the Probiotic group reached statistical significance at 8 weeks (0.12±0.47 vs. 0.0±0.0 events/infant, P=0.047). Constipation incidence was higher (odds ratio (OR) 2.67 (1.09-6.50)) and stool frequency lower (2.0±1.0 vs. 2.6±1.3 stools/day, P=0.038) in the Control group after 4 weeks. No differences were found at other time points nor in other digestive symptoms, growth, or formula intake.ConclusionA B. infantis IM1-supplemented infant formula may reduce diarrhea episodes, being safe, well tolerated, and associated with lower constipation prevalence.

Evidence that the endometrial microbiota has an effect on implantation success or failure.

BACKGROUND: Bacterial cells in the human body account for 1-3% of total body weight and are at least equal in number to human cells. Recent research has focused on understanding how the different bacterial communities in the body (eg, gut, respiratory, skin, and vaginal microbiomes) predispose to health and disease. The microbiota of the reproductive tract has been inferred from the vaginal bacterial communities, and the uterus has been classically considered a sterile cavity. However, while the vaginal microbiota has been investigated in depth, there is a paucity of consistent data regarding the existence of an endometrial microbiota and its possible impact in reproductive function. OBJECTIVE: This study sought to test the existence of an endometrial microbiota that differs from that in the vagina, assess its hormonal regulation, and analyze the impact of the endometrial microbial community on reproductive outcome in infertile patients undergoing in vitro fertilization. STUDY DESIGN: To identify the existence of an endometrial microbiota, paired samples of endometrial fluid and vaginal aspirates were obtained simultaneously from 13 fertile women in prereceptive and receptive phases within the same menstrual cycle (total samples analyzed n = 52). To investigate the hormonal regulation of the endometrial microbiota during the acquisition of endometrial receptivity, endometrial fluid was collected at prereceptive and receptive phases within the same cycle from 22 fertile women (n = 44). Finally, the reproductive impact of an altered endometrial microbiota in endometrial fluid was assessed by implantation, ongoing pregnancy, and live birth rates in 35 infertile patients undergoing in vitro fertilization (total samples n = 41) with a receptive endometrium diagnosed using the endometrial receptivity array. Genomic DNA was obtained either from endometrial fluid or vaginal aspirate and sequenced by 454 pyrosequencing of the V3-V5 region of the 16S ribosomal RNA (rRNA) gene; the resulting sequences were taxonomically assigned using QIIME. Data analysis was performed using R packages. The χ2 test, Student t test, and analysis of variance were used for statistical analyses. RESULTS: When bacterial communities from paired endometrial fluid and vaginal aspirate samples within the same subjects were interrogated, different bacterial communities were detected between the uterine cavity and the vagina of some subjects. Based on its composition, the microbiota in the endometrial fluid, comprising up to 191 operational taxonomic units, was defined as a Lactobacillus-dominated microbiota (>90% Lactobacillus spp.) or a non-Lactobacillus-dominated microbiota (<90% Lactobacillus spp. with >10% of other bacteria). Although the endometrial microbiota was not hormonally regulated during the acquisition of endometrial receptivity, the presence of a non-Lactobacillus-dominated microbiota in a receptive endometrium was associated with significant decreases in implantation [60.7% vs 23.1% (P = .02)], pregnancy [70.6% vs 33.3% (P = .03)], ongoing pregnancy [58.8% vs 13.3% (P = .02)], and live birth [58.8% vs 6.7% (P = .002)] rates. CONCLUSION: Our results demonstrate the existence of an endometrial microbiota that is highly stable during the acquisition of endometrial receptivity. However, pathological modification of its profile is associated with poor reproductive outcomes for in vitro fertilization patients. This finding adds a novel microbiological dimension to the reproductive process.

Genomic analysis of the emergence and evolution of multidrug resistance during a Klebsiella pneumoniae outbreak including carbapenem and colistin resistance.

OBJECTIVES: To characterize at the genomic level the evolution of multiresistance during an outbreak of Klebsiella pneumoniae in a burns intensive care unit. The outbreak involved a DHA-1 ß-lactamase-producing strain that later acquired carbapenem and fosfomycin resistance, and in one case colistin resistance. METHODS: The genomes of two isolates were sequenced and compared with a previously sequenced genome. The role of hypermutability was investigated by measuring the mutation frequencies of the isolates and comparison with a collection of control strains. RESULTS: Sequence comparison identified four single-nucleotide variants and two transposon insertions. Analysis of the variants in the whole collection related carbapenem and fosfomycin resistance to a nonsense mutation in the ompK36 porin gene and colistin resistance to an IS1 insertion in the mgrB gene. The plasmid carrying the blaDHA-1 gene was unstable in the absence of antibiotics, and analysis of isolates that had lost the plasmid showed that the porin mutation alone was not sufficient to generate carbapenem resistance. The mutation frequencies were similar among all the strains analysed. CONCLUSIONS: Carbapenem resistance required production of the DHA-1 ß-lactamase and decreased permeability, but fosfomycin resistance depended only on permeability. Resistance to colistin might be related to an alteration in the regulation of the phoPQ system. Hypermutation is not related to the selection of porin mutants. Plasmid instability might be due to the high number of mobile elements and suggests a major role for antibiotic selection pressure in the emergence and evolution of this outbreak.

Declining prevalence of HIV-1 drug resistance in antiretroviral treatment-exposed individuals in Western Europe.

HIV-1 drug resistance represents a major obstacle to infection and disease control. This retrospective study analyzes trends and determinants of resistance in antiretroviral treatment (ART)-exposed individuals across 7 countries in Europe. Of 20 323 cases, 80% carried at least one resistance mutation: these declined from 81% in 1997 to 71% in 2008. Predicted extensive 3-class resistance was rare (3.2% considering the cumulative genotype) and peaked at 4.5% in 2005, decreasing thereafter. The proportion of cases exhausting available drug options dropped from 32% in 2000 to 1% in 2008. Reduced risk of resistance over calendar years was confirmed by multivariable analysis.

A young child formula supplemented with a synbiotic mixture of scGOS/lcFOS and Bifidobacterium breve M-16V improves the gut microbiota and iron status in healthy toddlers.

Early-life gut microbiota development depends on a highly synchronized microbial colonization process in which diet is a key regulator. Microbiota transition toward a more adult-like state in toddlerhood goes hand in hand with the transition from a milk-based diet to a family diet. Microbiota development during the first year of life has been extensively researched; however, studies during toddlerhood remain sparse. Young children's requirement for micronutrients, such as dietary iron, is higher than adults. However, their intake is usually sub-optimal based on regular dietary consumption. The Child Health and Residence Microbes (CHaRM) study, conducted as an adjunct to the GUMLi (Growing Up Milk "Lite") trial, was a double-blind randomized controlled trial to investigate the effects on body composition of toddler milk compared to unfortified standard cow's milk in healthy children between 1 and 2 years of age in Brisbane (Australia). In this trial, fortified milk with reduced protein content and added synbiotics [Bifidobacterium breve M-16V, short-chain galactooligosaccharides, and long-chain fructooligosaccharides (ratio 9:1)] and micronutrients were compared to standard unfortified cow's milk. In the present study, the effects of the intervention on the gut microbiota and its relationship with iron status in toddlers were investigated in a subset of 29 children (18 in the Active group and 11 in the Control group) who completed the CHaRM study. The toddler microbiota consisted mainly of members of the phyla Firmicutes, Bacteroidota, and Actinobacteriota. The abundance of the B. breve species was quantified and was found to be lower in the Control group than in the Active group. Analysis of blood iron markers showed an improved iron status in the Active group. We observed a positive correlation between Bifidobacterium abundance and blood iron status. PICRUSt, a predictive functionality algorithm based on 16S ribosomal gene sequencing, was used to correlate potential microbial functions with iron status measurements. This analysis showed that the abundance of predicted genes encoding for enterobactin, a class of siderophores specific to Enterobacteriaceae, is inversely correlated with the relative abundance of members of the genus Bifidobacterium. These findings suggest that healthy children who consume a young child formula fortified with synbiotics as part of a healthy diet have improved iron availability and absorption in the gut and an increased abundance of Bifidobacterium in their gut microbiome.

Two chaperonin systems in bacterial genomes with distinct ecological roles.

Bacterial chaperonins are essential to cell viability and have a role in endosymbiosis, which leads to increased biological complexity. However, the extent to which chaperonins promote ecological innovation is unknown. We screened 622 bacterial genomes for genes encoding chaperonins, and found archaeal-like chaperonins in bacteria that inhabit archaeal ecological niches. We found that chaperonins encoded in pathogenic bacteria are the most functionally divergent. We identified the molecular basis of the dramatic structural changes in mitochondrial GROEL, a highly derived chaperonin gene. Our analysis suggests that chaperonins are important capacitors of evolutionary and ecological change.

Deep molecular characterization of HIV-1 dynamics under suppressive HAART.

In order to design strategies for eradication of HIV-1 from infected individuals, detailed insight into the HIV-1 reservoirs that persist in patients on suppressive antiretroviral therapy (ART) is required. In this regard, most studies have focused on integrated (proviral) HIV-1 DNA forms in cells circulating in blood. However, the majority of proviral DNA is replication-defective and archival, and as such, has limited ability to reveal the dynamics of the viral population that persists in patients on suppressive ART. In contrast, extrachromosomal (episomal) viral DNA is labile and as a consequence is a better surrogate for recent infection events and is able to inform on the extent to which residual replication contributes to viral reservoir maintenance. To gain insight into the diversity and compartmentalization of HIV-1 under suppressive ART, we extensively analyzed longitudinal peripheral blood mononuclear cells (PBMC) samples by deep sequencing of episomal and integrated HIV-1 DNA from patients undergoing raltegravir intensification. Reverse-transcriptase genes selectively amplified from episomal and proviral HIV-1 DNA were analyzed by deep sequencing 0, 2, 4, 12, 24 and 48 weeks after raltegravir intensification. We used maximum likelihood phylogenies and statistical tests (AMOVA and Slatkin-Maddison (SM)) in order to determine molecular compartmentalization. We observed low molecular variance (mean variability ≤0.042). Although phylogenies showed that both DNA forms were intermingled within the phylogenetic tree, we found a statistically significant compartmentalization between episomal and proviral DNA samples (P<10(-6) AMOVA test; P = 0.001 SM test), suggesting that they belong to different viral populations. In addition, longitudinal analysis of episomal and proviral DNA by phylogeny and AMOVA showed signs of non-chronological temporal compartmentalization (all comparisons P<10(-6)) suggesting that episomal and proviral DNA forms originated from different anatomical compartments. Collectively, this suggests the presence of a chronic viral reservoir in which there is stochastic release of infectious virus and in which there are limited rounds of de novo infection. This could be explained by the existence of different reservoirs with unique pharmacological accessibility properties, which will require strategies that improve drug penetration/retention within these reservoirs in order to minimise maintenance of the viral reservoir by de novo infection.

Back to the sea twice: identifying candidate plant genes for molecular evolution to marine life.

BACKGROUND: Seagrasses are a polyphyletic group of monocotyledonous angiosperms that have adapted to a completely submerged lifestyle in marine waters. Here, we exploit two collections of expressed sequence tags (ESTs) of two wide-spread and ecologically important seagrass species, the Mediterranean seagrass Posidonia oceanica (L.) Delile and the eelgrass Zostera marina L., which have independently evolved from aquatic ancestors. This replicated, yet independent evolutionary history facilitates the identification of traits that may have evolved in parallel and are possible instrumental candidates for adaptation to a marine habitat. RESULTS: In our study, we provide the first quantitative perspective on molecular adaptations in two seagrass species. By constructing orthologous gene clusters shared between two seagrasses (Z. marina and P. oceanica) and eight distantly related terrestrial angiosperm species, 51 genes could be identified with detection of positive selection along the seagrass branches of the phylogenetic tree. Characterization of these positively selected genes using KEGG pathways and the Gene Ontology uncovered that these genes are mostly involved in translation, metabolism, and photosynthesis. CONCLUSIONS: These results provide first insights into which seagrass genes have diverged from their terrestrial counterparts via an initial aquatic stage characteristic of the order and to the derived fully-marine stage characteristic of seagrasses. We discuss how adaptive changes in these processes may have contributed to the evolution towards an aquatic and marine existence.

Detection of drug resistance mutations at low plasma HIV-1 RNA load in a European multicentre cohort study.

BACKGROUND AND OBJECTIVES: Guidelines indicate a plasma HIV-1 RNA load of 500-1000 copies/mL as the minimal threshold for antiretroviral drug resistance testing. Resistance testing at lower viral load levels may be useful to guide timely treatment switches, although data on the clinical utility of this remain limited. We report here the influence of viral load levels on the probability of detecting drug resistance mutations (DRMs) and other mutations by routine genotypic testing in a large multicentre European cohort, with a focus on tests performed at a viral load <1000 copies/mL. METHODS: A total of 16 511 HIV-1 reverse transcriptase and protease sequences from 11 492 treatment-experienced patients were identified, and linked to clinical data on viral load, CD4 T cell counts and antiretroviral treatment history. Test results from 3162 treatment-naive patients served as controls. Multivariable analysis was employed to identify predictors of reverse transcriptase and protease DRMs. RESULTS: Overall, 2500/16 511 (15.14%) test results were obtained at a viral load <1000 copies/mL. Individuals with viral load levels of 1000-10000 copies/mL showed the highest probability of drug resistance to any drug class. Independently from other measurable confounders, treatment-experienced patients showed a trend for DRMs and other mutations to decrease at viral load levels <500 copies/mL. CONCLUSIONS: Genotypic testing at low viral load may identify emerging antiretroviral drug resistance at an early stage, and thus might be successfully employed in guiding prompt management strategies that may reduce the accumulation of resistance and cross-resistance, viral adaptive changes, and larger viral load increases.

HLA-Cw*03-restricted CD8+ T-cell responses targeting the HIV-1 gag major homology region drive virus immune escape and fitness constraints compensated for by intracodon variation.

The potential importance of HLA-C-restricted CD8+ cytotoxic T lymphocytes (CTL) in HIV infection remains undetermined. We studied the dominant HLA-Cw*03-restricted CTL response to YVDRFFKTL(296-304) (YL9), within the conserved major homology region (MHR) of the Gag protein, in 80 HLA-Cw*03-positive individuals with chronic HIV infection to better define the efficacy of the YL9 HLA-C-restricted response. The HLA-Cw*03 allele is strongly associated with HIV sequence changes from Thr-303 to Val, Ile, or Ala at position 8 within the YL9 epitope (P=1.62×10(-10)). In vitro studies revealed that introduction of the changes T303I and T303A into the YL9 epitope both significantly reduced CTL recognition and substantially reduced the viral replicative capacity. However, subsequent selection of the Val-303 variant, via intracodon variation from Ile-303 (I303V) or Ala-303 (A303V), restored both viral fitness and CTL recognition, as supported by our in vivo data. These results illustrate that HLA-C-restricted CTL responses are capable of driving viral immune escape within Gag, but in contrast to what was previously described for HLA-B-restricted Gag escape mutants, the common Cw*03-Gag-303V variant selected resulted in no detectable benefit to the host.

Microbiota Variation Across Life Stages of European Field-Caught Anopheles atroparvus and During Laboratory Colonization: New Insights for Malaria Research.

The potential use of bacteria for developing novel vector control approaches has awakened new interests in the study of the microbiota associated with vector species. To set a baseline for future malaria research, a high-throughput sequencing of the bacterial 16S ribosomal gene V3-V4 region was used to profile the microbiota associated with late-instar larvae, newly emerged females, and wild-caught females of a sylvan Anopheles atroparvus population from a former malaria transmission area of Spain. Field-acquired microbiota was then assessed in non-blood-fed laboratory-reared females from the second, sixth, and 10th generations. Diversity analyses revealed that bacterial communities varied and clustered differently according to origin with sylvan larvae and newly emerged females distributing closer to laboratory-reared females than to their field counterparts. Inter-sample variation was mostly observed throughout the different developmental stages in the sylvan population. Larvae harbored the most diverse bacterial communities; wild-caught females, the poorest. In the transition from the sylvan environment to the first time point of laboratory breeding, a significant increase in diversity was observed, although this did decline under laboratory conditions. Despite diversity differences between wild-caught and laboratory-reared females, a substantial fraction of the bacterial communities was transferred through transstadial transmission and these persisted over 10 laboratory generations. Differentially abundant bacteria were mostly identified between breeding water and late-instar larvae, and in the transition from wild-caught to laboratory-reared females from the second generation. Our findings confirmed the key role of the breeding environment in shaping the microbiota of An. atroparvus. Gram-negative bacteria governed the microbiota of An. atroparvus with the prevalence of proteobacteria. Pantoea, Thorsellia, Serratia, Asaia, and Pseudomonas dominating the microbiota associated with wild-caught females, with the latter two governing the communities of laboratory-reared females. A core microbiota was identified with Pseudomonas and Serratia being the most abundant core genera shared by all sylvan and laboratory specimens. Overall, understanding the microbiota composition of An. atroparvus and how this varies throughout the mosquito life cycle and laboratory colonization paves the way when selecting potential bacterial candidates for use in microbiota-based intervention strategies against mosquito vectors, thereby improving our knowledge of laboratory-reared An. atroparvus mosquitoes for research purposes.

Effects of Whole-Grain and Sugar Content in Infant Cereals on Gut Microbiota at Weaning: A Randomized Trial.

The introduction of complementary foods during infancy marks an important step in the development of the infant gut microbiome. Infant cereals are popular weaning foods but consistent evidence on their effect on the intestinal microbiota, especially when differing in nutritional quality, is lacking. Fecal samples from 4-7-month-old Spanish infants who consumed infant cereals differing in whole grain and sugar content as first weaning foods were analyzed on changes in microbial composition by massively parallel sequencing of the 16S ribosomal RNA gene at baseline and after 7 weeks of intervention. Samples were obtained from a previous trial conducted in Spain demonstrating whole-grain cereal acceptability. In total, samples of 18 infants consuming 0% whole grain cereals with 24 g sugar (0-WG) and 25 infants consuming 50% whole grain cereals with 12 g sugar (50-WG) were analyzed. Microbial composition changed significantly over time (p = 0.001), per intervention group (p = 0.029) and per infant (p = 0.001). Abundance of genus Veillonella increased in both groups while Enterococcus decreased. Within the 0-WG group, phylum Actinobacteria decreased along with genus Bifidobacterium. In the 50-WG, we observed an increase in Lachnoclostridium and Bacteroides. In addition, 50-WG decreased Proteobacteria and Escherichia to levels lower than 0-WG. Although weaning itself appeared to be responsible for most changes, the increased presence of anaerobic fermenters together with inhibition of pathogenic Escherichia may indicate a supporting effect of infant cereals with 50% whole grains and a reduced sugar content over infant cereals manufactured with refined hydrolyzed flours on the infant microbiota. In fact, using a novel methodology for the identification of microbial signatures, we found two groups of microbial taxa predictive of infants consuming enriched whole-grain infant cereals with a high predictive value of about 93%.

Changes in Gut Microbiota Correlates with Response to Treatment with Probiotics in Patients with Atopic Dermatitis. A Post Hoc Analysis of a Clinical Trial.

Atopic dermatitis (AD) is a chronic recurrent inflammatory skin disease with a high impact on the comfort of those who are affected and long-term treated with corticosteroids with limited efficacy and a high prevalence of relapses. Because of the limited effectiveness of these treatments, new strategies for recovery from AD lesions are continually being explored. In this article, we describe the gut microbiome changes achieved in a recently published clinical trial with the probiotic formulation Bifidobacterium animalis subsp. lactis CECT 8145, Bifidobacterium longum CECT 7347, and Lacticaseibacillus casei CECT 9104 (formerly Lactobacillus casei CECT 9104), showing a significant improvement in SCORAD (scoring atopic dermatitis) index in children (4-17 years) with AD (Clinicaltrials.gov identifier: NCT02585986). The present gut microbiome post hoc study showed no significant changes in diversity (Shannon and Simpson indexes) after probiotic consumption. In the probiotic group, genera Bacteroides, Ruminococcus, and Bifidobacterium significantly increased their levels while Faecalibacterium decreased, compared to the placebo group. Faecalibacterium showed the highest presence and significant positive correlation with AD severity (SCORAD index), whereas Abyssivirga, Bifidobacterium, and Lactococcus were inversely correlated. The results suggest that the consumption of the probiotic formulation here assayed modulates the gut microbiome with significant changes in genera Bacteroides and Faecalibacterium. In turn, the improvement in SCORAD correlates with a decrease in Faecalibacterium and an increase in Bifidobacterium, among others.

Mutational dynamics of murine angiogenin duplicates.

BACKGROUND: Angiogenin (Ang) is a protein involved in angiogenesis by inducing the formation of blood vessels. The biomedical importance of this protein has come from findings linking mutations in Ang to cancer progression and neurodegenerative diseases. These findings highlight the evolutionary constrain on Ang amino acid sequence. However, previous studies comparing human Angiogenin with homologs from other phylogenetically related organisms have led to the conclusion that Ang presents a striking variability. Whether this variability has an adaptive value per se remains elusive. Understanding why many functional Ang paralogs have been preserved in mouse and rat and identifying functional divergence mutations at these copies may explain the relationship between mutations and function. In spite of the importance of testing this hypothesis from the evolutionarily and biomedical perspectives, this remains yet unaccomplished. Here we test the main mutational dynamics driving the evolution and function of Ang paralogs in mammals. RESULTS: We analysed the phylogenetic asymmetries between the different Ang gene copies in mouse and rat in the context of vertebrate Ang phylogeny. This analysis shows strong evidence in support of accelerated evolution in some Ang murine copies (mAng). This acceleration is not due to non-functionalisation because constraints on amino acid replacements remain strong. We identify many of the amino acid sites involved in signal localization and nucleotide binding by Ang to have evolved under diversifying selection. Compensatory effects of many of the mutations at these paralogs and their key structural location in or nearby important functional regions support a possible functional shift (functional divergence) in many Ang copies. Similarities between 3D-structural models for mAng copies suggest that their divergence is mainly functional. CONCLUSIONS: We identify the main evolutionary dynamics shaping the variability of Angiogenin in vertebrates and highlight the plasticity of this protein after gene duplication. Our results suggest functional divergence among mAng paralogs. This puts forward mAng as a good system candidate for testing functional plasticity of such an important protein while stresses caution when using mouse as a model to infer the consequences of mutations in the single Ang copy of humans.

Metabolic Response of Faecalibacterium prausnitzii to Cell-Free Supernatants from Lactic Acid Bacteria.

Interest in preventive or therapeutic strategies targeting gut microbiota is increasing. Such strategies may involve the direct replenishment of the gut microbiota with single strains or strain mixtures, or the manipulation of strain abundance through dietary intervention, including lactic acid bacteria. A few candidate species associated with health benefits have been identified, including Faecalibacterium prausnitzii. Given its growth requirements, modulation of this bacterium has not been extensively studied. In this investigation, we explored the capacity of cell-free supernatants of different Lactobacillus, Streptococcus, Lactococcus, and Bifidobacterium strains to stimulate the growth of F. prausnitzii A2-165. Modulation by four strains with the greatest capacity to stimulate growth or delay lysis, Lactococcus lactis subsp. lactis CNCM I-1631, Lactococcus lactis subsp. cremoris CNCM I-3558, Lactobacillus paracasei CNCM I-3689, and Streptococcus thermophilus CNCM I-3862, was further characterized by transcriptomics. The response of F. prausnitzii to cell-free supernatants from these four strains revealed several shared characteristics, in particular, upregulation of carbohydrate metabolism and cell wall-related genes and downregulation of replication and mobilome genes. Overall, this study suggests differential responses of F. prausnitzii to metabolites produced by different strains, providing protection against cell death, with an increase in peptidoglycan levels for cell wall formation, and reduced cell mobilome activity.

Influence of the Ovine Genital Tract Microbiota on the Species Artificial Insemination Outcome. A Pilot Study in Commercial Sheep Farms.

To date, there is a lack of research into the vaginal and sperm microbiome and its bearing on artificial insemination (AI) success in the ovine species. Using hypervariable regions V3-V4 of the 16S rRNA, we describe, for the first time, the combined effect of the ovine microbiome of both females (50 ewes belonging to five herds) and males (five AI rams from an AI center) on AI outcome. Differences in microbiota abundance between pregnant and non-pregnant ewes and between ewes carrying progesterone-releasing intravaginal devices (PRID) with or without antibiotic were tested at different taxonomic levels. The antibiotic treatment applied with the PRID only altered Streptobacillus genus abundance, which was significantly lower in ewes carrying PRID with antibiotic. Mageebacillus, Histophilus, Actinobacilllus and Sneathia genera were significantly less abundant in pregnant ewes. In addition, these genera were more abundant in two farms with higher AI failure. Species of these genera such as Actinobacillus seminis and Histophilus somni have been associated with reproductive disorders in the ovine species. These genera were not present in the sperm samples of AI rams, but were found in the foreskin samples of rams belonging to herd 2 (with high AI failure rate) indicating that their presence in ewes' vagina could be due to prior transmission by natural mating with rams reared in the herd.