Clustering analysis of lipoprotein profiles to identify subtypes of hypertriglyceridemia in Miniature Schnauzers.

BACKGROUND: Hypertriglyceridemia (HTG) is prevalent in Miniature Schnauzers, predisposing them to life-threatening diseases. Varied responses to management strategies suggest the possibility of multiple subtypes. HYPOTHESIS/OBJECTIVE: To identify and characterize HTG subtypes in Miniature Schnauzers through cluster analysis of lipoprotein profiles. We hypothesize that multiple phenotypes of primary HTG exist in this breed. ANIMALS: Twenty Miniature Schnauzers with normal serum triglyceride concentration (NTG), 25 with primary HTG, and 5 with secondary HTG. METHODS: Cross-sectional study using archived samples. Lipoprotein profiles, generated using continuous lipoprotein density profiling, were clustered with hierarchical cluster analysis. Clinical data (age, sex, body condition score, and dietary fat content) was compared between clusters. RESULTS: Six clusters were identified. Dogs with primary HTG were dispersed among 4 clusters. One cluster showed the highest intensities for triglyceride-rich lipoprotein (TRL) and low-density lipoprotein (LDL) fractions and also included 4 dogs with secondary HTG. Two clusters had moderately high TRL fraction intensities and low-to-intermediate LDL intensities. The fourth cluster had high LDL but variable TRL fraction intensities with equal numbers of NTG and mild HTG dogs. The final 2 clusters comprised only NTG dogs with low TRL intensities and low-to-intermediate LDL intensities. The clusters did not appear to be driven by differences in the clinical data. CONCLUSIONS AND CLINICAL IMPORTANCE: The results of this study support a spectrum of lipoprotein phenotypes within Miniature Schnauzers that cannot be predicted by triglyceride concentration alone. Lipoprotein profiling might be useful to determine if subtypes have different origins, clinical consequences, and response to treatment.

Temporal Variability of the Dominant Fecal Microbiota in Healthy Adult Cats.

While shifts in gut microbiota have been studied in diseased states, the temporal variability of the microbiome in cats has not been widely studied. This study investigated the temporal variability of the feline dysbiosis index (DI) and the abundance of core bacterial groups in healthy adult cats. The secondary aim was to evaluate the relationship between the fecal abundance of Clostridium hiranonis and the fecal concentrations of unconjugated bile acids. A total of 142 fecal samples collected from 17 healthy cats were prospectively included: nine cats with weekly collection over 3 weeks (at least four time points), five cats with monthly collection over 2 months (three time points), and three cats with additional collections for up to 10 months. The DI remained stable within the reference intervals over two months for all cats (Friedman test, p > 0.2), and 100% of the DI values (n = 142) collected throughout the study period remained within the RI. While some temporal individual variation was observed for individual taxa, the magnitude was minimal compared to cats with chronic enteropathy and antibiotic exposure. Additionally, the abundance of Clostridium hiranonis was significantly correlated with the percentage of fecal primary bile acids, supporting its role as a bile acid converter in cats.

Analytical validation of an ELISA for the measurement of feline pancreas-specific lipase and re-evaluation of the reference interval and decision threshold for diagnosing pancreatitis.

BACKGROUND: The diagnosis of feline pancreatitis can be challenging. The clinical presentation often includes mild, nonspecific clinical signs, such as vomiting, anorexia, and weight loss. Measurement of feline pancreatic lipase immunoreactivity (fPLI) concentration in serum has been reported to be sensitive and specific for a diagnosis of pancreatitis in cats. However, analytical validation for a widely available commercial assay for the measurement of fPLI concentration has not been published. OBJECTIVE: We aimed to analytically validate the Spec fPL assay (IDEXX Laboratories, Westbrook, ME), a commercial ELISA for the measurement of fPLI concentration, and re-evaluate its reference interval and decision threshold for diagnosing pancreatitis in cats. METHODS: Dilutional linearity, accuracy, precision, and the effect of interfering substances were assessed. The upper limit of the reference interval was calculated based on the 95th percentile of results from clinically healthy cats (n = 107), and a decision threshold for diagnosing pancreatitis was calculated with an expected specificity of 99%. RESULTS: Analytical validation demonstrated good linearity, accuracy, and precision, as well as the absence of interference from lipemia, hemolysis, or icterus. The upper limit of the reference interval for Spec fPL was determined to be 4.4 µg/L, and the decision threshold (a theoretical cut-off) for diagnosing pancreatitis was determined to be 8.8 µg/L based on a desired specificity of 99%. CONCLUSIONS: The Spec fPL assay is analytically valid, and results suggest that a decision threshold of 8.8 µg/L would have high diagnostic specificity for excluding clinically healthy cats.

The mitochondrial genome of Heterosentis pseudobagri (Wang & Zhang, 1987) Pichelin & Cribb, 1999 reveals novel aspects of tRNA genes evolution in Acanthocephala.

BACKGROUND: Acanthocephala is a clade of obligate endoparasites whose mitochondrial genomes (mitogenomes) and evolution remain relatively poorly understood. Previous studies reported that atp8 is lacking from acanthocephalan mitogenomes, and that tRNA genes often have nonstandard structures. Heterosentis pseudobagri (Arhythmacanthidae) is an acanthocephalan fish endoparasite for which no molecular data are currently available, and biological information is unavailable in the English language. Furthermore, there are currently no mitogenomes available for Arhythmacanthidae. METHODS: We sequenced its mitogenome and transcriptome, and conducted comparative mitogenomic analyses with almost all available acanthocephalan mitogenomes. RESULTS: The mitogenome had all genes encoded on the same strand and unique gene order in the dataset. Among the 12 protein-coding genes, several genes were highly divergent and annotated with difficulty. Moreover, several tRNA genes could not be identified automatically, so we had to identify them manually via a detailed comparison with orthologues. As common in acanthocephalans, some tRNAs lacked either the TWC arm or the DHU arm, but in several cases, we annotated tRNA genes only on the basis of the conserved narrow central segment comprising the anticodon, while the flanking 5' and 3' ends did not exhibit any resemblance to orthologues and they could not be folded into a tRNA secondary structure. We corroborated that these are not sequencing artefacts by assembling the mitogenome from transcriptomic data. Although this phenomenon was not observed in previous studies, our comparative analyses revealed the existence of highly divergent tRNAs in multiple acanthocephalan lineages. CONCLUSIONS: These findings indicate either that multiple tRNA genes are non-functional or that (some) tRNA genes in (some) acanthocephalans might undergo extensive posttranscriptional tRNA processing which restores them to more conventional structures. It is necessary to sequence mitogenomes from yet unrepresented lineages and further explore the unusual patterns of tRNA evolution in Acanthocephala.

Chronic Inflammatory Enteropathy and Low-Grade Intestinal T-Cell Lymphoma Are Associated with Altered Microbial Tryptophan Catabolism in Cats.

Chronic inflammatory enteropathy (CIE) and low-grade intestinal T-cell lymphoma (LGITL) are common chronic enteropathies (CE) in cats. Enteric microbiota dysbiosis is implicated in the pathogenesis of CE; however, the mechanisms of host-microbiome interactions are poorly understood in cats. Microbial indole catabolites of tryptophan (MICT) are gut bacterial catabolites of tryptophan that are hypothesized to regulate intestinal inflammation and mucosal barrier function. MICTs are decreased in the sera of humans with inflammatory bowel disease and previous studies identified altered tryptophan metabolism in cats with CE. We sought to determine whether MICTs were decreased in cats with CE using archived serum samples from cats with CIE (n = 44) or LGITL (n = 31) and healthy controls (n = 26). Quantitative LC-MS/MS was used to measure serum concentrations of tryptophan, its endogenous catabolites (kynurenine, kynurenate, serotonin) and MICTs (indolepyruvate, indolealdehyde, indoleacrylate, indoleacetamide, indoleacetate, indolelactate, indolepropionate, tryptamine). Serum concentrations of tryptophan, indolepropionate, indoleacrylate, indolealdehyde, indolepyruvate, indolelactate were significantly decreased in the CIE and LGITL groups compared to those in healthy controls. Indolelactate concentrations were significantly lower in cats with LGITL compared to CIE (p = 0.006). Significant correlations were detected among serum MICTs and cobalamin, folate, fPLI, and fTLI. Our findings suggest that MICTs are promising biomarkers to investigate the role of gut bacteria in the pathobiology of chronic enteropathies in cats.

Mitochondrial phylogenomics of Acanthocephala: nucleotide alignments produce long-branch attraction artefacts.

BACKGROUND: Classification of the Acanthocephala, a clade of obligate endoparasites, remains unresolved because of insufficiently strong resolution of morphological characters and scarcity of molecular data with a sufficient resolution. Mitochondrial genomes may be a suitable candidate, but they are available for a small number of species and their suitability for the task has not been tested thoroughly. METHODS: Herein, we sequenced the first mitogenome for the large family Rhadinorhynchidae: Micracanthorhynchina dakusuiensis. These are also the first molecular data generated for this entire genus. We conducted a series of phylogenetic analyses using concatenated nucleotides (NUC) and amino acids (AAs) of all 12 protein-coding genes, three different algorithms, and the entire available acanthocephalan mitogenomic dataset. RESULTS: We found evidence for strong compositional heterogeneity in the dataset, and Micracanthorhynchina dakusuiensis exhibited a disproportionately long branch in all analyses. This caused a long-branch attraction artefact (LBA) of M. dakusuiensis resolved at the base of the Echinorhynchida clade when the NUC dataset was used in combination with standard phylogenetic algorithms, maximum likelihood (ML) and Bayesian inference (BI). Both the use of the AA dataset (BI-AAs and ML-AAs) and the CAT-GTR model designed for suppression of LBA (CAT-GTR-AAs and CAT-GTR-NUC) at least partially attenuated this LBA artefact. The results support Illiosentidae as the basal radiation of Echinorhynchida and Rhadinorhynchidae forming a clade with Echinorhynchidae and Pomporhynchidae. The questions of the monophyly of Rhadinorhynchidae and its sister lineage remain unresolved. The order Echinorhynchida was paraphyletic in all of our analyses. CONCLUSIONS: Future studies should take care to attenuate compositional heterogeneity-driven LBA artefacts when applying mitogenomic data to resolve the phylogeny of Acanthocephala.

Characterization and comparative analysis of the complete mitochondrial genome of Azygia hwangtsiyui Tsin, 1933 (Digenea), the first for a member of the family Azygiidae.

Azygia hwangtsiyui (Trematoda, Azygiidae), a neglected parasite of predatory fishes, is little-known in terms of its molecular epidemiology, population ecology and phylogenetic study. In the present study, the complete mitochondrial genome of A. hwangtsiyui was sequenced and characterized: it is a 13,973 bp circular DNA molecule and encodes 36 genes (12 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes) as well as two non-coding regions. The A+T content of the A. hwangtsiyui mitogenome is 59.6% and displays a remarkable bias in nucleotide composition with a negative AT skew (-0.437) and a positive GC skew (0.408). Phylogenetic analysis based on concatenated amino acid sequences of twelve protein-coding genes reveals that A. hwangtsiyui is placed in a separate clade, suggesting that it has no close relationship with any other trematode family. This is the first characterization of the A. hwangtsiyui mitogenome, and the first reported mitogenome of the family Azygiidae. These novel datasets of the A. hwangtsiyui mt genome represent a meaningful resource for the development of mitochondrial markers for the identification, diagnostics, taxonomy, homology and phylogenetic relationships of trematodes.

Characterization of the complete mitochondrial genome of Brentisentisyangtzensis Yu & Wu, 1989 (Acanthocephala, Illiosentidae).

The mitogenome of Brentisentisyangtzensis is 13,864 bp in length and has the circular structure typical of metazoans. It contains 36 genes: 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and 12 protein-encoding genes (PCGs). All genes are transcribed from the same strand. Thirteen overlapping regions were found in the mitochondrial genome. The overall A+T content of B.yangtzensis is 68.3% versus 31.7% of G+C content (A = 27.8%, T = 40.5%, C = 9.0%, G = 22.7%). B.yangtzenensis (Illiosentidae) and Leptorhynchoidesthecatus (Rhadinorhynchidae) form a sister clade, showing the relatively close relationship between the Illiosentidae and the Rhadinorhynchidae. The mitochondrial gene arrangements of acanthocephalan species are relatively conserved, with only a few translocations of tRNAs (trnS1, trnS2, trnV, and trnK) detected. An identical gene order was found both in a sister clade (Centrorhynchusaluconis and Plagiorhynchustransversus) and across different classes (B.yangtzensis (Palaeacanthocephala), Acanthosentischeni (Eoacanthocephala) and Macracanthorhynchushirudinaceus (Archiacanthocephala), Oncicolaluehei and L.thecatus (Palaeacanthocephala)). More studies and more sequences of acanthocephalan species are needed to gain a clear understanding of the phylogenetic relationships.

Multiple Aneurysms AnaTomy CHallenge 2018 (MATCH)-phase II: rupture risk assessment.

PURPOSE: Assessing the rupture probability of intracranial aneurysms (IAs) remains challenging. Therefore, hemodynamic simulations are increasingly applied toward supporting physicians during treatment planning. However, due to several assumptions, the clinical acceptance of these methods remains limited. METHODS: To provide an overview of state-of-the-art blood flow simulation capabilities, the Multiple Aneurysms AnaTomy CHallenge 2018 (MATCH) was conducted. Seventeen research groups from all over the world performed segmentations and hemodynamic simulations to identify the ruptured aneurysm in a patient harboring five IAs. Although simulation setups revealed good similarity, clear differences exist with respect to the analysis of aneurysm shape and blood flow results. Most groups (12/71%) included morphological and hemodynamic parameters in their analysis, with aspect ratio and wall shear stress as the most popular candidates, respectively. RESULTS: The majority of groups (7/41%) selected the largest aneurysm as being the ruptured one. Four (24%) of the participating groups were able to correctly select the ruptured aneurysm, while three groups (18%) ranked the ruptured aneurysm as the second most probable. Successful selections were based on the integration of clinically relevant information such as the aneurysm site, as well as advanced rupture probability models considering multiple parameters. Additionally, flow characteristics such as the quantification of inflow jets and the identification of multiple vortices led to correct predictions. CONCLUSIONS: MATCH compares state-of-the-art image-based blood flow simulation approaches to assess the rupture risk of IAs. Furthermore, this challenge highlights the importance of multivariate analyses by combining clinically relevant metadata with advanced morphological and hemodynamic quantification.

[Graphene-based pipette tip solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry for determination of ten lipophilic shellfish toxins in shellfish].

A method for the simultaneous determination of ten lipophilic shellfish toxins was established based on graphene-based pipette tip solid-phase extraction (G-PT-SPE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The factors influencing the extraction efficiency, including the types of extractants, the amount of graphene, the types and volumes of washing and eluent solvents, were optimized in detail. Under the optimal conditions, the calibration curves showed linear relationships between the LC peak areas of the selected ion-pairs and the mass concentrations of the ten lipophilic shellfish toxins with correlation coefficients >0.99. The limits of detection (LODs) and the limits of quantification (LOQs) of the method were 0.1-1.1 µg/kg and 0.3-3.2 µg/kg, respectively. The recoveries of the ten lipophilic shellfish toxins spiked in blank oyster at three levels ranged from 72.0% to 101.2% with relative standard deviations <15%. The method is sensitive, simple, and effective, and is suitable for the determination of the lipophilic shellfish toxins in shellfish products.

Phylogeographic patterns and conservation implications of the endangered Chinese giant salamander.

Understanding genetic diversity patterns of endangered species is an important premise for biodiversity conservation. The critically endangered salamander Andrias davidianus, endemic to central and southern mainland in China, has suffered from sharp range and population size declines over the past three decades. However, the levels and patterns of genetic diversity of A. davidianus populations in wild remain poorly understood. Herein, we explore the levels and phylogeographic patterns of genetic diversity of wild-caught A. davidianus using larvae and adult collection with the aid of sequence variation in (a) the mitochondrial DNA (mtDNA) fragments (n = 320 individuals; 33 localities), (b) 19 whole mtDNA genomes, and (c) nuclear recombinase activating gene 2 (RAG2; n = 88 individuals; 19 localities). Phylogenetic analyses based on mtDNA datasets uncovered seven divergent mitochondrial clades (A-G), which likely originated in association with the uplifting of mountains during the Late Miocene, specific habitat requirements, barriers including mountains and drainages and lower dispersal ability. The distributions of clades were geographic partitioned and confined in neighboring regions. Furthermore, we discovered some mountains, rivers, and provinces harbored more than one clades. RAG2 analyses revealed no obvious geographic patterns among the five alleles detected. Our study depicts a relatively intact distribution map of A. davidianus clades in natural species range and provides important knowledge that can be used to improve monitoring programs and develop a conservation strategy for this critically endangered organism.

Multiple Aneurysms AnaTomy CHallenge 2018 (MATCH): Phase I: Segmentation.

PURPOSE: Advanced morphology analysis and image-based hemodynamic simulations are increasingly used to assess the rupture risk of intracranial aneurysms (IAs). However, the accuracy of those results strongly depends on the quality of the vessel wall segmentation. METHODS: To evaluate state-of-the-art segmentation approaches, the Multiple Aneurysms AnaTomy CHallenge (MATCH) was announced. Participants carried out segmentation in three anonymized 3D DSA datasets (left and right anterior, posterior circulation) of a patient harboring five IAs. Qualitative and quantitative inter-group comparisons were carried out with respect to aneurysm volumes and ostia. Further, over- and undersegmentation were evaluated based on highly resolved 2D images. Finally, clinically relevant morphological parameters were calculated. RESULTS: Based on the contributions of 26 participating groups, the findings reveal that no consensus regarding segmentation software or underlying algorithms exists. Qualitative similarity of the aneurysm representations was obtained. However, inter-group differences occurred regarding the luminal surface quality, number of vessel branches considered, aneurysm volumes (up to 20%) and ostium surface areas (up to 30%). Further, a systematic oversegmentation of the 3D surfaces was observed with a difference of approximately 10% to the highly resolved 2D reference image. Particularly, the neck of the ruptured aneurysm was overrepresented by all groups except for one. Finally, morphology parameters (e.g., undulation and non-sphericity) varied up to 25%. CONCLUSIONS: MATCH provides an overview of segmentation methodologies for IAs and highlights the variability of surface reconstruction. Further, the study emphasizes the need for careful processing of initial segmentation results for a realistic assessment of clinically relevant morphological parameters.

Computational Fluid Dynamics Modeling of the Human Pulmonary Arteries with Experimental Validation.

Pulmonary hypertension (PH) is a chronic progressive disease characterized by elevated pulmonary arterial pressure, caused by an increase in pulmonary arterial impedance. Computational fluid dynamics (CFD) can be used to identify metrics representative of the stage of PH disease. However, experimental validation of CFD models is often not pursued due to the geometric complexity of the model or uncertainties in the reproduction of the required flow conditions. The goal of this work is to validate experimentally a CFD model of a pulmonary artery phantom using a particle image velocimetry (PIV) technique. Rapid prototyping was used for the construction of the patient-specific pulmonary geometry, derived from chest computed tomography angiography images. CFD simulations were performed with the pulmonary model with a Reynolds number matching those of the experiments. Flow rates, the velocity field, and shear stress distributions obtained with the CFD simulations were compared to their counterparts from the PIV flow visualization experiments. Computationally predicted flow rates were within 1% of the experimental measurements for three of the four branches of the CFD model. The mean velocities in four transversal planes of study were within 5.9 to 13.1% of the experimental mean velocities. Shear stresses were qualitatively similar between the two methods with some discrepancies in the regions of high velocity gradients. The fluid flow differences between the CFD model and the PIV phantom are attributed to experimental inaccuracies and the relative compliance of the phantom. This comparative analysis yielded valuable information on the accuracy of CFD predicted hemodynamics in pulmonary circulation models.

An Integrated Artificial Cilia Based Microfluidic Device for Micropumping and Micromixing Applications.

A multi-purpose microfluidic device that can be used for both micromixing and micropropulsion operations has always been in demand, as it would simplify the various process flows associated with the current micro-total analysis systems. In this aspect, we propose a biomimetic artificial cilia-based microfluidic device that can efficiently facilitate both mixing and propulsion sequentially at the micro-scale. A rectangular microfluidic device consists of four straight microchannels that were fabricated using the microfabrication technique. An array of artificial cilia was embedded within one of the channel's confinement through the aforementioned technique. A series of image processing and micro-particle image velocimetry technologies were employed to elucidate the micromixing and micropropulsion phenomena. Experiment results demonstrate that, with this proposed microfluidic device, a maximum micromixing efficiency and flow rate of 0.84 and 0.089 µL/min, respectively, can be achieved. In addition to its primary application as a targeted drug delivery system, where a drug needs to be homogeneously mixed with its carrier prior to its administration into the target body, this microfluidic device can be used as a micro-total analysis system for the handling of other biological specimens.

Photonic crystal-enhanced fluorescence through the extraction of dually polarized modes.

In this Letter, we demonstrate that fluorescence detection by using surface-bound fluorescent molecules can be enhanced by coupling the fluorescence emission with dually polarized resonant modes of a photonic crystal (PC) substrate. The PC was fabricated through nanoreplica molding by using a plastic substrate. It was designed such that the transverse magnetic (TM)- and transverse electric (TE)-polarized resonance coincided at a specific combination of the incident angle and the illumination wavelength. During excitation, the nonpolarized emission from cyanine-5 was simultaneously coupled with the TE and TM resonant modes and reradiated into the detection instrument, increasing the fluorescence collection efficiency and thus, enhancing the fluorescence detection.