DNA Sequence Research Articles

Synthesis and Evaluations of Cleavable Triazene‐Modified Nucleotide for DNA Sequencing

Synthesis and Evaluations of Cleavable Triazene‐Modified Nucleotide for DNA Sequencing

Molecular diagnostics of familial hypercholesterolemia in Russia: yesterday, today and tomorrow

Familial hypercholesterolemia is a severe hereditary disease leading to the development of atherosclerosis and its complications in the form of angina pectoris, myocardial infarction, cerebral stroke, or even leading to sudden death. Since the description of the disease, the concept of it has undergone significant evolution. First, it became clear that the prevalence of this disease was significantly higher than originally thought (1:300 for heterozygous familial hypercholesterolemia and not as 1:500 as estimated earlier). Secondly, it has been established that it is not based on the pathology of the low-density lipoprotein receptor gene alone, but includes at least four monogenic forms (defects of the APOB, PCSK9, ARH genes) and may also have a multigenic nature. Thirdly, with the development of DNA analysis methods from the initially available Southern hybridization to next generation DNA sequencing, the exceptional molecular heterogeneity of familial hypercholesterolemia became obvious and, accordingly, the need to establish national spectra of mutations leading to the development of familial hypercholesterolemia was established. Researchers have moved from characterizing individual mutations to creating national registries and databases. Finally, research into the genetics of familial hypercholesterolemia has led to the emergence of new classes of cholesterol-lowering drugs. In Russia, molecular diagnostics of familial hypercholesterolemia has also undergone significant changes since the beginning of the study of familial hypercholesterolemia in 1987 and to the present, consideration of these changes formed the basis of this review.

Digitizing DNA Sequences Using Multiset-Based Nucleotide Frequencies for Machine Learning-Based Mutation Detection

Investigating algebraic structures in a non-conventional framework supplements mathematics for hard-nosed practical applications to the fields of theoretical biology and computer science. One such algebraic structure is multigroup whose underlying set is a multiset. The genome is the entire set of DNA instructions found within a cell which contains all the information needed for an individual to develop and function. DNA and RNA are the hereditary materials that play a vital role in the metabolism process of living things, especially protein synthesis. In genomic database DNA sequences are stored in the form of string or text data types. The only data that works with machine learning algorithms is numerical. Thus, it is necessary to transform DNA sequence strings to numerical values. This article is organized in the following manner. Firstly, we prove that standard genetic code is a multigroup and genome architecture of the whole population can be interpreted as the sum of multisets. Next, it is described how a numerical representation of DNA bases relates to its algebraic representation. We further employed Gated Recurrent Unit, Long Short-Term Memory, and Bidirectional Long Short-Term Memory to identify changes between the DNA sequences. Experimental results show that GRU with multiset-based numerical values for DNA bases offers 98% accuracy on testing data. This novel technique will aid in the detection of mutations in complex diseases.

Characterization and analysis of a Commiphora species germinated from an ancient seed suggests a possible connection to a species mentioned in the Bible

A seed recovered during archaeological excavations of a cave in the Judean desert was germinated, with radiocarbon analysis indicating an age of 993 CE– 1202 calCE. DNA sequencing and phylogenetic analysis identified the seedling as belonging to the angiosperm genus Commiphora Jacq., sister to three Southern African Commiphora species, but unique from all other species sampled to date. The germinated seedling was not closely related to Commiphora species commonly harvested for their fragrant oleoresins including Commiphora gileadensis (L.) C.Chr., candidate for the locally extinct “Judean Balsam” or “Balm of Gilead” of antiquity. GC-MS analysis revealed minimal fragrant compounds but abundance of those associated with multi-target bioactivity and a previously undescribed glycolipid compound series. Several hypotheses are offered to explain the origins, implications and ethnobotanical significance of this unknown Commiphora sp., to the best of our knowledge the first identified from an archaeological site in this region, including identification with a resin producing tree mentioned in Biblical sources and possible agricultural relationship with the historic Judean Balsam.

Molecular study of a case with variant of RHCE*ce allele in haplotype dce resulting in weakened e antigen

To explore the Rh genotype for a female with RhD(-) blood type and its molecular basis. A 26-year-old female who had attended the outpatient clinic of the First Affiliated Hospital of Zhengzhou University in August 2019 was selected as the study subject. Peripheral blood samples were collected from the proband and her parents for Rh phenotyping with gel card method. PCR-sequence-based typing (PCR-SBT) and DNA sequencing were used to determine the RHD zygosity and Rh genotype of the proband and her parents. Homology modeling of Rh proteins was performed with bioinformatic software, and protein structural alterations caused by the variant was simulated by molecular dynamics. Serological tests showed that the proband and her father both had weakened e antigen of the Rh phenotype. PCR-SBT and DNA sequencing showed that the genotypes of the proband and her parents were dce/dCE, dce/DcE and dCE/DcE, respectively. And the genotypes of the RHD and RHCE of the proband were RHD*01N.01/RHD*01N.16, RHCE*01.01/RHCE*04, respectively. Protein simulation and molecular dynamics analysis revealed that the ce_16C variant resulted from RHCE*ce (c.48G>C) may alter the structure of intracellular and extracellular loops, mainly affecting the mobility of extracellular loops 2, 6 and intracellular loops 3, 4. Variant of the RHCE*ce allele c.48G>C probably underlay the weakened e antigen in this proband.

Integrated analyses reveal two molecularly and clinically distinct subtypes of H3 K27M-mutant diffuse midline gliomas with prognostic significance

H3 K27M-altered diffuse midline gliomas (DMGs) are highly malignant tumours that arise in the midline structures of the CNS. Most DMGs carry an H3 K27M-mutation in one of the genes encoding for histone H3. Recent studies suggested that epigenetic subgroups of DMGs can be distinguished based on alterations in the MAPK-signalling pathway, tumour localisation, mutant H3-gene, or overall survival (OS). However, as these parameters were studied individually, it is unclear how they collectively influence survival. Hence, we analysed dependencies between different parameters, to define novel epigenetic, clinically meaningful subgroups of DMGs. We collected a multifaceted cohort of 149 H3 K27M-mutant DMGs, also incorporating data of published cases. DMGs were included in the study if they could be clearly allocated to the spinal cord (n = 31; one patient with an additional sellar tumour), medulla (n = 20), pons (n = 64) or thalamus (n = 33), irrespective of further known characteristics. We then performed global genome-wide DNA methylation profiling and, for a subset, DNA sequencing and survival analyses. Unsupervised hierarchical clustering of DNA methylation data indicated two clusters of DMGs, i.e. subtypes DMG-A and DMG-B. These subtypes differed in mutational spectrum, tumour localisation, age at diagnosis and overall survival. DMG-A was enriched for DMGs with MAPK-mutations, medullary localisation and adult age. 13% of DMG-A had a methylated MGMT promoter. Contrarily, DMG-B was enriched for cases with TP53-mutations, PDGFRA-amplifications, pontine localisation and paediatric patients. In univariate analyses, the features enriched in DMG-B were associated with a poorer survival. However, all significant parameters tested were dependent on the cluster attribution, which had the largest effect on survival: DMG-A had a significantly better survival compared to DMG-B (p < 0.001). Hence, the subtype attribution based on two methylation clusters can be used to predict survival as it integrates different molecular and clinical parameters.

A modified dual preparatory method for improved isolation of nucleic acids from laser microdissected fresh-frozen human cancer tissue specimens

Abstract A central theme in cancer research is to increase our understanding of the cancer tissue microenvironment (TME), which is comprised of a complex and spatially heterogeneous ecosystem of malignant and non-malignant cells, both of which actively contribute to an intervening extracellular matrix. Laser microdissection (LMD) enables histology selective harvest of cellular subpopulations from the tissue microenvironment for their independent molecular investigation, such as by high-throughput DNA and RNA sequencing. Although enabling, LMD often requires a labor-intensive investment to harvest enough cells to achieve the necessary DNA and/or RNA input requirements for conventional next generation sequencing workflows. To increase efficiencies, we sought to use a commonplace dual preparatory (DP) procedure to isolate DNA and RNA from the same LMD harvested tissue samples. While the yield of DNA from the DP protocol was satisfactory, the RNA yield from the LMD harvested tissue samples was significantly poorer compared to a dedicated RNA preparation procedure. We determined that this low yield of RNA was due to incomplete partitioning of RNA in this widely used DP protocol. Here we describe a modified DP protocol that more equally partitions nucleic acids and results in significantly improved RNA yields from LMD harvested cells.

Metagenomic analysis of soil from landfill site reveals a diverse microbial community involved in plastic degradation

In this study, we have investigated microbial communities structure and function using high throughput amplicon sequencing and whole metagenomic sequencing of DNA extracted from different depths of a plastic-laden landfill site. With diverse taxonomic groups inhabiting the plastic-rich soil, our study demonstrates the remarkable adaptability of microbes to use this new substrate as a carbon source. FTIR spectroscopic analysis of soil indicated degradation of plastic as perceived from the carbonyl index of 0.16, 0.72, and 0.44 at 0.6, 0.9 and 1.2 m depth, respectively. Similarly, water contact angles of 108.7 degree, 99.7 degree, 62.7 degree, and 77.8 degree of plastic pieces collected at 0.3, 0.6, 0.9, and 1.2 m depths respectively showed increased wettability and hydrophilicity of the plastic. Amplicon analysis of 16S and 18 S rRNA revealed a high abundance of several plastic-degrading bacterial groups, including Pseudomonas, Rhizobiales, Micrococcaceae, Chaetomium, Methylocaldum, Micromonosporaceae, Rhodothermaceae and fungi, including Trichoderma, Aspergillus, Candida at 0.9 m. The co-existence of specific microbial groups at different depths of landfill site indicates importance of bacterial and fungal interactions for plastic. Whole metagenome analysis of soil sample at 0.9 m depth revealed a high abundance of genes encoding enzymes that participate in the biodegradation of PVC, polyethylene, PET, and polyurethane. Curation of the pathways related to the degradation of these materials provided a blueprint for plastic biodegradation in this ecosystem. Altogether, our study has highlighted the importance of microbial cooperation for the biodegradation of pollutants. Our metagenome-based investigation supports the current perception that consortia of fungi-bacteria are preferable to axenic cultures for effective bioremediation of the environment.

An accurate haplotyping method using multiplex pyrosequencing with AS-PCR to detect ABCB1 haplotypes associated with rivaroxaban-derived hemorrhagic events

In clinical practice, owing to the comprehensive genetic insights they offer, haplotypes have attracted greater attention than individual single nucleotide polymorphisms (SNPs). Due to the long distances across SNP locations, detecting the haplotype using genomic DNA is challenging. Current haplotyping methods are either expensive and labor-intensive (high-throughput DNA sequencing), or haplotyping a single clinical sample (computational approach) is impossible. Herein, we propose using mRNA as a haplotyping target to minimize the distance among SNPs and employing allele-specific PCR (AS-PCR) to pick up a desired haplotype, followed by multiplex pyrosequencing to type the alleles at the SNP location of interest. AS-PCR was improved by combining an additional 3′-phosphorylated modified probe to achieve the specific separation of two closely similar templates. Only the sample with more than two heterozygotes needs to be haplotyped; therefore, we propose a stratification strategy to screen the samples for further haplotyping. This method was evaluated by associating ABCB1 haplotypes with the rivaroxaban-derived side effect in a cohort of 505 patients with nephrotic syndrome, focusing on the SNPs of ABCB1: rs1236C > T, rs2677G > T/A, and rs3435C > T. We successfully identified five bleeding-related haplotypes: rs1236T-rs2677T-rs3435T, rs1236C-rs2677G-rs3435T, rs1236T-rs2677G-rs3435C, rs1236C-rs2677G-rs3435C, and rs1236T-rs2677T-rs3435C. We compared the results with those from the conventional computational algorithm PHASE and observed that PHASE results dismissed the impact of rs1236C-rs2677G-rs3435C and rs1236C-rs2677G-rs3435T on bleeding risk and erroneously suggested a false positive association of rs1236C-rs2677A-rs3435T with increased bleeding risk.

Molecular Profiling Defines Three Subtypes of Synovial Sarcoma.

Synovial Sarcomas (SS) are characterized by the presence of the SS18::SSX fusion gene, which protein product induce chromatin changes through remodeling of the BAF complex. To elucidate the genomic events that drive phenotypic diversity in SS, we performed RNA and targeted DNA sequencing on 91 tumors from 55 patients. Our results were verified by proteomic analysis, public gene expression cohorts and single-cell RNA sequencing. Transcriptome profiling identified three distinct SS subtypes resembling the known histological subtypes: SS subtype I and was characterized by hyperproliferation, evasion of immune detection and a poor prognosis. SS subtype II and was dominated by a vascular-stromal component and had a significantly better outcome. SS Subtype III was characterized by biphasic differentiation, increased genomic complexity and immune suppression mediated by checkpoint inhibition, and poor prognosis despite good responses to neoadjuvant therapy. Chromosomal abnormalities were an independent significant risk factor for metastasis. KRT8 was identified as a key component for epithelial differentiation in biphasic tumors, potentially controlled by OVOL1 regulation. Our findings explain the histological grounds for SS classification and indicate that a significantly larger proportion of patients have high risk tumors (corresponding to SS subtype I) than previously believed.

Bifidobacterium longum subsp. infantis NKU FB3-14 attenuates loperamide-induced constipation through regulation of gut microbiota

Abstract Objectives Constipation is a prevalent gastrointestinal issue, and the efficacy of probiotics in alleviating constipation has been well proved. This study aimed to investigate the impact of Bifidobacterium longum subsp. infantis NKU FB3-14 on loperamide- induced constipation by focusing on improving intestinal barrier function and modulating gut microbiota composition Materials and Methods The constipated model mice induced by loperamide were treated with NKU FB3-14, and the laxative effect was assessed based on the fecal water content, first black stool time and gastrointestinal transit rate. Gastrointestinal regulatory peptides in serum, intestinal neurotransmitter and inflammatory cytokines in colon tissues were measured using ELISA kits. Changes of gut microbiota composition were analyzed through 16S ribosomal DNA sequencing identification. Additionally, HPLC detection was performed to quantify short chain fatty acids levels in feces Results Treatment with NKU FB3-14 led to increase the fecal water content, shorten the first black stool time and improve small intestine transit rate. Motilin and substance-P significantly decreased in the model group, and only motilin increased in FB3-14 group; somatostatin and vasoactive intestinal peptide were decreased in model mice and both increased in FB3-14 group; 5-HT levels in the colon tissue were upregulated following NKU FB3-14 treatment. Histological examination revealed thinner colonic mucosa in the model group along with significant in TNF-α, IL-1β and IL-17 levels form the colon tissues which were all relieved by NKU FB 3-14 treatment. Furthermore, NKU FB3-14 intervention resulted in reduced abundance of Desulfobacterota and Desulfovibrio while increasing the abundance of Ruminococcaceae and Eubacterium; higher level of butyric acid was observed in feces Conclusions In summary, our findings demonstrated that NKU FB3-14 treatment significantly enhanced the intestinal motility, regulated the expression levels of gastrointestinal regulatory peptides, prevented damage to colonic barriers as well as ameliorated gut microbiota imbalance associated with loperamide-induced constipation.

First record of the family Callianopsidae (Decapoda: Axiidea) and a new species of Vulcanocalliax from the Hikurangi Margin off Aotearoa New Zealand, with a key to species of Callianopsidae

IntroductionThe Aotearoa New Zealand ghost shrimp of the infraorders Axiidea and Gebiidea have never been comprehensively reviewed, with recent work uncovering a diverse regional fauna representing eight of the 14 known families.MethodsUsing standard morphological and DNA sequencing tools, the family Callianopsidae is, for the first time, recorded off New Zealand, represented by a new species of Vulcanocalliax.ResultsThe new species was found near hydrocarbon seeps on the Hikurangi Margin, on the eastern New Zealand continental slope, and is only the second species now known in this genus. The single congener, V. arutyunovi, is only known from a mud volcano in the Gulf of Cádiz, off the Iberian Peninsula. Vulcanocalliax sp. nov. was formerly reported as an unnamed host of a new endemic New Zealand rhizocephalan barnacle Parthenopea australis and is here formally described as Vulcanocalliax beervana sp. nov.DiscussionThe new species differs, e.g., in the shape of the anterior carapace margin (convexly rounded and without elevated postantennal shoulder present in V. arutyunovi), the ocular peduncle having a convex anterolateral margin (compared to a straight margin), and the uropodal exopod has a dorsomedian ridge, lacking the elevated anterior portion that is distinct in V. arutyunovi. This brings the number of described New Zealand ghost shrimp species to 18. A key to all known Callianopsidae is provided.ZOOBANKLSID urn:lsid:zoobank.org:pub:4EDE6CE1-B644-4FFB-94CA-C8725A7549DD.

Phylogeny and taxonomic revision of Hygrophorus robustus (Hygrophoraceae, Agaricales) from southwestern China

Hygrophorus robustus was originally described from Yunnan province, China. This taxon is characterized by its robust basidiomata, salmon buff lamellae, tapered toward the base of the stipe, the odor of Tricholoma matsutake, the absence of cystidia and clamp connections, and a symbiotic association with Castanopsis delavayi. In this study, the phylogenetic position of this taxon is inferred by molecular phylogenetic analysis based on DNA sequences of internal transcribed spacer (ITS). The ITS result showed that it is nested within the genus Tricholoma and is affiliated with the North American T. grave. The morphological anatomy of this species also shows that its characteristics fit the concept of the genus Tricholoma, such as regular lamellar trama. Thus, H. robustus should be taxonomically transferred to Tricholoma, and the name of the new combination should be T. robustum. However, this name was used in the genus as Tricholoma robustum (Alb. &amp; Schwein.) Ricken. Thus, we propose a new name T. aurantiophyllum for H. robustus.

Evidence for compensatory evolution within pleiotropic regulatory elements.

Pleiotropy, measured as expression breadth across tissues, is one of the best predictors for protein sequence and expression conservation. In this study, we investigated its effect on the evolution of cis-regulatory elements (CREs). To this end, we carefully reanalyzed the Epigenomics Roadmap data for nine fetal tissues, assigning a measure of pleiotropic degree to nearly half a million CREs. To assess the functional conservation of CREs, we generated ATAC-seq and RNA-seq data from humans and macaques. We found that more pleiotropic CREs exhibit greater conservation in accessibility, and the mRNA expression levels of the associated genes are more conserved. This trend of higher conservation for higher degrees of pleiotropy persists when analyzing the transcription factor binding repertoire. In contrast, simple DNA sequence conservation of orthologous sites between species tends to be even lower for pleiotropic CREs than for species-specific CREs. Combining various lines of evidence, we propose that the lack of sequence conservation in functionally conserved pleiotropic CREs is due to within-element compensatory evolution. In summary, our findings suggest that pleiotropy is also a good predictor for the functional conservation of CREs, even though this is not reflected in the sequence conservation of pleiotropic CREs.

Prevalence of germline variants in Brazilian pancreatic carcinoma patients

We evaluated the prevalence of pathogenic/likely pathogenic germline variants (PGV) in Brazilian pancreatic adenocarcinoma (PC) patients, that represent a multiethnic population, in a cross-sectional study. We included 192 PC patients unselected for family history of cancer. We evaluated a panel of 113 cancer genes, through genomic DNA sequencing and 46 ancestry-informative markers, through multiplex PCR. The median age was 61 years; 63.5% of the patients presented disease clinical stages III or IV; 8.3% reported personal history of cancer; 4.7% and 16.1% reported first-degree relatives with PC or breast and/or prostate cancer, respectively. Although the main ancestry was European, there was considerable genetic composition admixture. Twelve patients (6.25%) were PGV carriers in PC predisposition genes (ATM, BRCA1, BRCA2, CDKN2A, MSH2, PALB2) and another 25 (13.0%) were PGV carriers in genes with a limited association or not previously associated with PC (ACD, BLM, BRIP1, CHEK2, ERCC4, FANCA, FANCE, FANCM, GALNT12, MITF, MRE11, MUTYH, POLE, RAD51B, RAD51C, RECQL4, SDHA, TERF2IP). The most frequently affected genes were CHEK2, ATM and FANC. In tumor samples from PGV carriers in ACD, BRIP1, MRE11, POLE, SDHA, TERF2IP, which were examined through exome sequencing, the main single base substitutions (SBS) mutational signature was SBS1+5+18, probably associated with age, tobacco smoking and reactive oxygen species. SBS3 associated with homologous repair deficiency was also represented, but on a lower scale. There was no difference in the frequency of PGV carriers between: (a) patients with or without first-degree relatives with cancer; and (b) patients with admixed ancestry versus those with predominantly European ancestry. Furthermore, there was no difference in overall survival between PGV carriers and non-carriers. Therefore, genetic testing should be offered to all Brazilian pancreatic cancer patients, regardless of their ancestry. Genes with limited or previously unrecognized associations with pancreatic cancer should be further investigated to clarify their role in cancer risk.

Identification of the genetic determinants of shovel-shaped incisors and Carabelli’s cusp

Shovel-shaped incisors (SSI) and Carabelli’s cusp (CC) are noteworthy human dental non-metric traits which presence and degree of expression have been reported to cluster within distinct populations. Recent advances in developmental biology suggest that SSI and CC are likely under polygenic developmental control; therefore, genetic variation in multiple genes is likely to contribute to differential SSI and CC expression. The exact genetic mechanisms underlying variation in SSI and CC development, however, remain mostly unknown. This study aims to identify whether variation in the basal DNA sequences of six candidate genes, NKX2-3, SOSTDC1, BMP4, FGF3, FGF4, and WNT10A, has any impact on SSI and/or CC expression. Study methods involve collection of saliva samples and dental data from 36 participants. The Arizona State University Dental Anthropology System (ASUDAS) has been used to score SSI and CC expression. Next-generation sequencing (NGS) methods were utilized to sequence the entire gene region of the candidate genes. Spearman’s correlation test was used to score the relationship between the genotype and degree of trait expression of participants. Fifteen SNPs/INDELs belonging to SOSTDC1, FGF3, FGF4 and WNT10A were significantly associated with SSI and/or CC expression. No SNPs/INDELs were detected in the genes BMP4 and NKX2-3 that significantly contributes to observed phenotypes. FGF3, FGF4, SOSTDC1 and WNT10A were possibly involved in the formation of shoveling and Carabelli’s cusp. However, because of the small sample size, more studies are needed to confirm their role and rule out any potential role of NKX2-3 and BMP4 in the production of SSI and CC.

Hindered intermolecular stacking of anti-parallel telomeric G-quadruplexes.

Telomeric G-quadruplexes (G4s) are non-canonical DNA structures composed of TTAGGG repeats. They are extensively studied both as biomolecules key for genome stability and as promising building blocks and functional elements in synthetic biology and nanotechnology. This is why it is extremely important to understand how the interaction between G4s is affected by their topology. We used small-angle x-ray scattering to investigate the end-to-end stacking of antiparallel telomeric G-quadruplexes formed by the sequence AG3(T2AG3)3. To represent the experimental data, we developed a highly efficient coarse-grained fitting tool, which successfully described the samples as an equilibrium mixture of monomeric and dimeric G4 species. Our findings indicate that the antiparallel topology prevents the formation of long multimeric structures under self-crowding conditions, unlike the hybrid/parallel structures formed by the same DNA sequence. This result supports the idea that the stacking of monomeric G-quadruplexes is strongly affected by the presence of diagonal loops.

Exploring the impact of urban pollution on ciliate diversity along the Sapucaí River (Minas Gerais, Brazil) via DNA metabarcoding.

Protists are diverse single-celled eukaryotes found in various habitats. They exhibit a wide range of forms and functions, representing a significant portion of the eukaryotic tree of life, which also includes animals, plants, and fungi. Due to their high sensitivity to environmental changes, these organisms are widely used as biological indicators of organic pollution. We investigated the molecular diversity of ciliate protists at seven strategic points along the Sapucaí River (Itajubá, Minas Gerais State, Brazil), to assess the impact of urban pollution on the richness, abundance, and diversity indexes of these communities. For each sampling point, values of physicochemical parameters were also recorded. DNA sequences were obtained by high-throughput sequencing (HTS) and analyzed using the V4 18S-rRNA molecular marker, employing the DNA metabarcoding method. We recorded 125 ciliate taxonomic units (OTUs), with nearly 80% corresponding to the classes Spirotrichea, Oligohymenophorea, and Litostomatea. At the genus level, 54 OTUs (43.2%) were identified, spanning 28 genera. The composition of ciliates varied significantly along the river's course, from upstream to downstream of Itajubá city. Samples collected from the urban area displayed the lowest richness and diversity, corroborating the influence of the pollution gradient on these communities. The physicochemical parameters showed little variation among the samples and were not linked to the observed changes in ciliate communities, revealing that these organisms are strongly affected by environmental changes and respond more sensitively to these disturbances than physicochemical parameters, emphasizing their potential as bioindicators.

Toxoplasma gondii DNA in Tissues of Anadromous Arctic Charr, Salvelinus alpinus, Collected From Nunavik, Québec, Canada.

Toxoplasma gondii is a very common zoonotic parasite in humans and animals worldwide. Human seroprevalence is high in some regions of Canada's North and is thought to be associated with the consumption of traditionally prepared country foods, such as caribou, walrus, ringed seal and beluga. While numerous studies have reported on the prevalence of T. gondii in these animals, in the general absence of felid definitive hosts in the North there has been considerable debate regarding the source of infection, particularly in marine mammals. It has been proposed that fish could be involved in this transmission. The objectives of the present study were to perform a targeted survey to determine the prevalence of T. gondii DNA in various tissues of anadromous Arctic charr sampled in Nunavik, Québec, and to investigate the possible role of this commonly consumed fish in the transmission of infection to humans and marine mammals in Canada's North. A total of 126 individual Arctic charr were sampled from several sites in Nunavik, and various tissues were tested for the presence of T. gondii DNA using PCR. Overall, 12 out of 126 (9.5%) Arctic charr tested in the present study were PCR-positive, as confirmed by DNA sequencing. Brain tissue was most commonly found to be positive, followed by heart tissue, while none of the dorsal muscle samples tested were positive. Although the presence of T. gondii DNA in brain and heart tissues of Arctic charr is very intriguing, infection in these fish, and their possible role in the transmission of this parasite to humans and marine mammals, will need to be confirmed using mouse bioassays. Arctic charr are likely exposed to T. gondii through the ingestion of oocysts transported by surface water and ocean currents from more southerly regions where the definitive felid hosts are more abundant. If infection in Arctic charr can be confirmed, it is possible that these fish could play an important role in the transmission of toxoplasmosis to Inuit, either directly through the consumption of raw fish or indirectly through the infection of fish-eating marine mammals harvested as country foods.

Gut Dysbiosis in Patients With Fontan Circulation.

The process underlying Fontan pathophysiology is multifactorial and may include gut dysbiosis (GD). We investigated the presence of GD and elucidated its correlation with Fontan pathophysiology. Gut microbiomes of 155 consecutive patients with Fontan pathophysiology and 44 healthy individuals were analyzed using 16S rRNA sequencing of bacterial DNA extracted from fecal samples. GD was evaluated on the basis of α and ß diversities of the gut microbiome and was compared with natural log-transformed C-reactive protein, hemodynamics, von Willebrand factor antigen (a bacterial translocation marker), Mac-2 binding protein glycosylation isomer (a liver fibrosis indicator), peak oxygen uptake, and heart failure hospitalization. Patients with Fontan exhibited GD in terms of α and ß diversities as compared with controls (P<0.01). Reduced α diversity was associated with a failed hemodynamic phenotype, hypoxia, high natural log-transformed C-reactive protein levels, and elevated von Willebrand factor antigen and Mac-2 binding protein glycosylation isomer levels (P<0.05-0.01). In addition to elevated von Willebrand factor antigen and hypoxia, decreased α diversity was independently correlated with a high natural log-transformed C-reactive protein level (P<0.05), which was associated with liver imaging abnormalities and a heightened risk of heart failure hospitalization (P<0.01 for both). Patients with Fontan pathophysiology exhibited GD compared with healthy individuals, and GD was linked to failed hemodynamics and systemic inflammation with a poor prognosis. Therefore, GD may play a pivotal role in a failing Fontan status, including Fontan-associated liver disease, through GD-associated systemic inflammation.