Mechanisms of Karyotypic Diversification in Ancistrus (Siluriformes, Loricariidae): Inferences from Repetitive Sequence Analysis.

Ancistrus is a highly diverse neotropical fish genus that exhibits extensive chromosomal variability, encompassing karyotypic morphology, diploid chromosome number (2n = 34-54), and the evolution of various types of sex chromosome systems. Robertsonian rearrangements related to unstable chromosomal sites are here described. Here, the karyotypes of two Ancistrus species were comparatively analyzed using classical cytogenetic techniques, in addition to isolation, cloning, sequencing, molecular characterization, and fluorescence in situ hybridization of repetitive sequences (i.e., 18S and 5S rDNA; U1, U2, and U5 snDNA; and telomere sequences). The species analyzed here have different karyotypes: Ancistrus sp. 1 (2n = 38, XX/XY) and Ancistrus cirrhosus (2n = 34, no heteromorphic sex chromosomes). Comparative mapping showed different organizations for the analyzed repetitive sequences: 18S and U1 sequences occurred in a single site in all populations of the analyzed species, while 5S and U2 sequences could occur in single or multiple sites. A sequencing analysis confirmed the identities of the U1, U2, and U5 snDNA sequences. Additionally, a syntenic condition for U2-U5 snDNA was found in Ancistrus. In a comparative analysis, the sequences of rDNA and U snDNA showed inter- and intraspecific chromosomal diversification. The occurrence of Robertsonian rearrangements and other dispersal mechanisms of repetitive sequences are discussed.

Benchmarking second and third-generation sequencing platforms for microbial metagenomics.

Shotgun metagenomic sequencing is a common approach for studying the taxonomic diversity and metabolic potential of complex microbial communities. Current methods primarily use second generation short read sequencing, yet advances in third generation long read technologies provide opportunities to overcome some of the limitations of short read sequencing. Here, we compared seven platforms, encompassing second generation sequencers (Illumina HiSeq 300, MGI DNBSEQ-G400 and DNBSEQ-T7, ThermoFisher Ion GeneStudio S5 and Ion Proton P1) and third generation sequencers (Oxford Nanopore Technologies MinION R9 and Pacific Biosciences Sequel II). We constructed three uneven synthetic microbial communities composed of up to 87 genomic microbial strains DNAs per mock, spanning 29 bacterial and archaeal phyla, and representing the most complex and diverse synthetic communities used for sequencing technology comparisons. Our results demonstrate that third generation sequencing have advantages over second generation platforms in analyzing complex microbial communities, but require careful sequencing library preparation for optimal quantitative metagenomic analysis. Our sequencing data also provides a valuable resource for testing and benchmarking bioinformatics software for metagenomics.

Chromosomal Diversification in Pseudacanthicus Species (Loricariidae, Hypostominae) Revealed by Comparative Mapping of Repetitive Sequences.

Pseudacanthicus is a genus of Neotropical fish with eight valid species, in addition to numerous lineages not formally identified. It occurs along the Amazon and Tocantins River basins, in Suriname and in the Guiana shield. There are no karyotypic data in the literature for species of this genus. Here, the karyotypes of three Pseudacanthicus species (P. spinosus, P. leopardus and Pseudacanthicus sp.) were comparatively analyzed by classical cytogenetics and fluorescence in situ hybridization using 18S and 5S rDNA probes, U2 snDNA and telomeric sequences. The analyzed species presented 52 chromosomes and KF = 18 m + 34 sm. Constitutive heterochromatin occurred in blocks on a few chromosomes. The 18S rDNA occurred in a single pair; interestingly, P. leopardus presented only one locus of this sequence in its diploid genome. The 5S rDNA sequence occurred in only one pair in P. leopardus, and in multiple sites in Pseudacanthicus sp. and P. spinosus. The snDNA U2 occurred in only one pair in all analyzed species. Telomeric sequences did not show interstitial sites. Although Pseudacanthicus species share the same 2n and KF, repetitive sequence analysis revealed karyotypic diversity among these species. The occurrence of DNA double-strand breaks related to fragile sites, unequal crossing over and transpositions is proposed as the mechanism of karyotypic diversification, suggesting that the conservation of the karyotypic macrostructure is only apparent in this group of fish.

Chromosomal Diversification in Ancistrus Species (Siluriformes: Loricariidae) Inferred From Repetitive Sequence Analysis.

The Ancistrus genus has extensive chromosomal diversity among species, including heteromorphic sex chromosomes occurrence. However, studies have been shown that chromosomal diversity may still be underestimated. Repetitive sequences represent a large part of eukaryotic genomes, associated with mechanisms of karyotypic diversification, including sex chromosomes evolution. This study analyzed the karyotype diversification of two Ancistrus species (Ancistrus sp. 1 and Ancistrus sp. 2) from the Amazon region by classical and molecular chromosomal markers. Conventional chromosome bands and fluorescence in situ hybridization using probes 18S and 5S rDNA, besides (CA)n, (CG)n, (GA)n, (CAC)n, (CAG)n, (CAT)n, (GAA)n, (GAC)n, (TAA)n, and (TTAGGG)n in tandem repeats were determined on the karyotypes. Ancistrus sp. 1 and Ancistrus sp. 2 presented karyotypes with 2n = 38 (20 m + 14sm+4st, XX/XY) and 2n = 34 (20 m + 14sm, without heteromorphic sex chromosomes), respectively. Robertsonian rearrangements can explain the diploid number difference. C-bands occurred in pericentromeric regions in some chromosomes, and a single 18S rDNA locus occurred in both species. The 5S rDNA showed variation in the number of loci between species karyotypes, suggesting the occurrence of unstable sites and rearrangements associated with these sequences in Ancistrus. The microsatellite mapping evidenced distinct patterns of organization between the two analyzed species, occurring mainly in the sex chromosomes in Ancistrus sp. 1, and in the centromeric and pericentromeric regions of chromosomes m/sm in Ancistrus sp. 2. These data shows the extensive chromosomal diversity of repetitive sequences in Ancistrus, which were involved in Robertsonian rearrangements and sex chromosomes differentiation.

Diet Supplementation with NUTRIOSE, a Resistant Dextrin, Increases the Abundance of Parabacteroides distasonis in the Human Gut.

SCOPE: An imbalance of the gut microbiota ("dysbiosis") is associated with numerous chronic diseases, and its modulation is a promising novel therapeutic approach. Dietary supplementation with soluble fiber is one of several proposed modulation strategies. This study aims at confirming the impact of the resistant dextrin NUTRIOSE (RD), a soluble fiber with demonstrated beneficial health effects, on the gut microbiota of healthy individuals. METHODS AND RESULTS: Fifty healthy women are enrolled and supplemented daily with either RD (n = 24) or a control product (n = 26) during 6 weeks. Characterization of the fecal metagenome with shotgun sequencing reveals that RD intake dramatically increases the abundance of the commensal bacterium Parabacteroides distasonis. Furthermore, presence in metagenomes of accessory genes from P. distasonis, coding for susCD (a starch-binding membrane protein complex) is associated with a greater increase of the species. This suggests that response to RD might be strain-dependent. CONCLUSION: Supplementation with RD can be used to specifically increase P. distasonis in gut microbiota of healthy women. The magnitude of the response may be associated with fiber-metabolizing capabilities of strains carried by subjects. Further research will seek to confirm that P. distasonis directly modulates the clinical effects observed in other studies.

Long-term diosmectite use does not alter the gut microbiota in adults with chronic diarrhea.

BACKGROUND: Diosmectite, a natural colloidal clay, has been used worldwide for a number of approved indications, including the treatment of chronic functional diarrhea. Here, we used high-resolution whole metagenome shotgun sequencing to assess the impact of a 5 weeks administration of diosmectite (3 g/sachet, 3 sachets/day) on the fecal microbiota of 35 adults with functional chronic diarrhea. RESULTS: Gut microbiota was not impacted by diosmectite administration. In particular, richness remained stable and no microbial species displayed a significant evolution. Segregating patients either by diosmectite response (non responder, early responder, late responder) or by nationality (Great-Britain or Netherlands) yielded the same results. CONCLUSION: We concluded that no microbiota-related physiological alterations are expected upon long-term treatment with diosmectite. TRIAL REGISTRATION: Clinicaltrials.gov NCT03045926.

Rifaximin-α reduces gut-derived inflammation and mucin degradation in cirrhosis and encephalopathy: RIFSYS randomised controlled trial.

BACKGROUND & AIMS: Rifaximin-α is efficacious for the prevention of recurrent hepatic encephalopathy (HE), but its mechanism of action remains unclear. We postulated that rifaximin-α reduces gut microbiota-derived endotoxemia and systemic inflammation, a known driver of HE. METHODS: In a placebo-controlled, double-blind, mechanistic study, 38 patients with cirrhosis and HE were randomised 1:1 to receive either rifaximin-α (550 mg BID) or placebo for 90 days. PRIMARY OUTCOME: 50% reduction in neutrophil oxidative burst (OB) at 30 days. SECONDARY OUTCOMES: changes in psychometric hepatic encephalopathy score (PHES) and neurocognitive functioning, shotgun metagenomic sequencing of saliva and faeces, plasma and faecal metabolic profiling, whole blood bacterial DNA quantification, neutrophil toll-like receptor (TLR)-2/4/9 expression and plasma/faecal cytokine analysis. RESULTS: Patients were well-matched: median MELD (11 rifaximin-α vs. 10 placebo). Rifaximin-α did not lead to a 50% reduction in spontaneous neutrophil OB at 30 days compared to baseline (p = 0.48). However, HE grade normalised (p = 0.014) and PHES improved (p = 0.009) after 30 days on rifaximin-α. Rifaximin-α reduced circulating neutrophil TLR-4 expression on day 30 (p = 0.021) and plasma tumour necrosis factor-α (TNF-α) (p <0.001). Rifaximin-α suppressed oralisation of the gut, reducing levels of mucin-degrading sialidase-rich species, Streptococcus spp, Veillonella atypica and parvula, Akkermansia and Hungatella. Rifaximin-α promoted a TNF-α- and interleukin-17E-enriched intestinal microenvironment, augmenting antibacterial responses to invading pathobionts and promoting gut barrier repair. Those on rifaximin-α were less likely to develop infection (odds ratio 0.21; 95% CI 0.05-0.96). CONCLUSION: Rifaximin-α led to resolution of overt and covert HE, reduced the likelihood of infection, reduced oralisation of the gut and attenuated systemic inflammation. Rifaximin-α plays a role in gut barrier repair, which could be the mechanism by which it ameliorates bacterial translocation and systemic endotoxemia in cirrhosis. CLINICAL TRIAL NUMBER: ClinicalTrials.gov NCT02019784. LAY SUMMARY: In this clinical trial, we examined the underlying mechanism of action of an antibiotic called rifaximin-α which has been shown to be an effective treatment for a complication of chronic liver disease which effects the brain (termed encephalopathy). We show that rifaximin-α suppresses gut bacteria that translocate from the mouth to the intestine and cause the intestinal wall to become leaky by breaking down the protective mucus barrier. This suppression resolves encephalopathy and reduces inflammation in the blood, preventing the development of infection.

Comparative Cytogenetics Analysis Among Peckoltia Species (Siluriformes, Loricariidae): Insights on Karyotype Evolution and Biogeography in the Amazon Region.

Peckoltia is widely distributed genus in the Amazon and Orinoco basins and the Guiana Shield, containing 18 valid species, and distinct morphotypes still needing description in the scientific literature due to its great taxonomic complexity. This study performed a comparative chromosomal analysis of two undescribed Peckoltia species (Peckoltia sp. 3 Jarumã and Peckoltia sp. 4 Caripetuba) from the Brazilian Amazon using conventional chromosome bands methods and in situ localization of the repetitive DNA (5S and 18S rRNA and U1 snRNA genes and telomeric sequences). Both species presented 2n = 52 but differed in their karyotype formula, probably due to inversions or translocations. The nucleolus organizer regions (NORs) showed distal location on a probably homeologous submetacentric pair in both species, besides an extra signal in a subtelocentric chromosome in Peckoltia sp. 4 Caripetuba. Heterochromatin occurred in large blocks, with different distributions in the species. The mapping of the 18S and 5S rDNA, and U1 snDNA showed differences in locations and number of sites. No interstitial telomeric sites were detected using the (TTAGGG)n probes. Despite 2n conservationism in Peckoltia species, the results showed variation in karyotype formulas, chromosomal bands, and locations of repetitive sites, demonstrating great chromosomal diversity. A proposal for Peckoltia karyotype evolution was inferred in this study based on the diversity of location and number of chromosomal markers analyzed. A comparative analysis with other Peckoltia karyotypes described in the literature, their biogeography patterns, and molecular phylogeny led to the hypothesis that the derived karyotype was raised in the left bank of the Amazon River.

StrainFLAIR: strain-level profiling of metagenomic samples using variation graphs.

Current studies are shifting from the use of single linear references to representation of multiple genomes organised in pangenome graphs or variation graphs. Meanwhile, in metagenomic samples, resolving strain-level abundances is a major step in microbiome studies, as associations between strain variants and phenotype are of great interest for diagnostic and therapeutic purposes. We developed StrainFLAIR with the aim of showing the feasibility of using variation graphs for indexing highly similar genomic sequences up to the strain level, and for characterizing a set of unknown sequenced genomes by querying this graph. On simulated data composed of mixtures of strains from the same bacterial species Escherichia coli, results show that StrainFLAIR was able to distinguish and estimate the abundances of close strains, as well as to highlight the presence of a new strain close to a referenced one and to estimate its abundance. On a real dataset composed of a mix of several bacterial species and several strains for the same species, results show that in a more complex configuration StrainFLAIR correctly estimates the abundance of each strain. Hence, results demonstrated how graph representation of multiple close genomes can be used as a reference to characterize a sample at the strain level.

Alterations in Gut Microbiome in Cirrhosis as Assessed by Quantitative Metagenomics: Relationship With Acute-on-Chronic Liver Failure and Prognosis.

BACKGROUND AND AIMS: Cirrhosis is associated with changes in gut microbiome composition. Although acute-on-chronic liver failure (ACLF) is the most severe clinical stage of cirrhosis, there is lack of information about gut microbiome alterations in ACLF using quantitative metagenomics. We investigated the gut microbiome in patients with cirrhosis encompassing the whole spectrum of disease (compensated, acutely decompensated without ACLF, and ACLF). A group of healthy subjects was used as control subjects. METHODS: Stool samples were collected prospectively in 182 patients with cirrhosis. DNA library construction and sequencing were performed using the Ion Proton Sequencer (ThermoFisher Scientific, Waltham, MA). Microbial genes were grouped into clusters, denoted as metagenomic species. RESULTS: Cirrhosis was associated with a remarkable reduction in gene and metagenomic species richness compared with healthy subjects. This loss of richness correlated with disease stages and was particularly marked in patients with ACLF and persisted after adjustment for antibiotic therapy. ACLF was associated with a significant increase of Enterococcus and Peptostreptococcus sp and a reduction of some autochthonous bacteria. Gut microbiome alterations correlated with model for end-stage liver disease and Child-Pugh scores and organ failure and was associated with some complications, particularly hepatic encephalopathy and infections. Interestingly, gut microbiome predicted 3-month survival with good stable predictors. Functional analysis showed that patients with cirrhosis had enriched pathways related to ethanol production, γ-aminobutyric acid metabolism, and endotoxin biosynthesis, among others. CONCLUSIONS: Cirrhosis is characterized by marked alterations in gut microbiome that parallel disease stages with maximal changes in ACLF. Altered gut microbiome was associated with complications of cirrhosis and survival. Gut microbiome may contribute to disease progression and poor prognosis. These results should be confirmed in future studies.

The intellectual disability PAK3 R67C mutation impacts cognitive functions and adult hippocampal neurogenesis.

The link between mutations associated with intellectual disability (ID) and the mechanisms underlying cognitive dysfunctions remains largely unknown. Here, we focused on PAK3, a serine/threonine kinase whose gene mutations cause X-linked ID. We generated a new mutant mouse model bearing the missense R67C mutation of the Pak3 gene (Pak3-R67C), known to cause moderate to severe ID in humans without other clinical signs and investigated hippocampal-dependent memory and adult hippocampal neurogenesis. Adult male Pak3-R67C mice exhibited selective impairments in long-term spatial memory and pattern separation function, suggestive of altered hippocampal neurogenesis. A delayed non-matching to place paradigm testing memory flexibility and proactive interference, reported here as being adult neurogenesis-dependent, revealed a hypersensitivity to high interference in Pak3-R67C mice. Analyzing adult hippocampal neurogenesis in Pak3-R67C mice reveals no alteration in the first steps of adult neurogenesis, but an accelerated death of a population of adult-born neurons during the critical period of 18-28 days after their birth. We then investigated the recruitment of hippocampal adult-born neurons after spatial memory recall. Post-recall activation of mature dentate granule cells in Pak3-R67C mice was unaffected, but a complete failure of activation of young DCX + newborn neurons was found, suggesting they were not recruited during the memory task. Decreased expression of the KCC2b chloride cotransporter and altered dendritic development indicate that young adult-born neurons are not fully functional in Pak3-R67C mice. We suggest that these defects in the dynamics and learning-associated recruitment of newborn hippocampal neurons may contribute to the selective cognitive deficits observed in this mouse model of ID.