Hamilton and Saarman et al. molecular phylogeny and patterns of diversification in syngnathid fishes.
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•Expanded taxonomic and genetic sampling reveals new patterns of syngnathid ...evolution.•Molecular patterns suggest convergence in traditional morphological characters.•Complex brood pouches, prehensile tails, and pygmy morphology evolved multiple times.•Biogeographic patterns reveal multiple drivers of evolutionary diversification.•Two subfamilies are formally recognized and further taxonomic revisions are required.
The family Syngnathidae is a large and diverse clade of morphologically unique bony fishes, with 57 genera and 300 described species of seahorses, pipefishes, pipehorses, and seadragons. They primarily inhabit shallow coastal waters in temperate and tropical oceans, and are characterized by a fused jaw, male brooding, and extraordinary crypsis. Phylogenetic relationships within the Syngnathidae remain poorly resolved due to lack of generic taxon sampling, few diagnostic morphological characters, and limited molecular data. The phylogenetic placement of the threatened, commercially exploited seahorses remains a topic of intense interest, with conflicting topologies based on morphology and predominantly mitochondrial genetic data. In this study, we integrate eight nuclear and mitochondrial markers and 17 morphological characters to investigate the phylogenetic structure of the family Syngnathidae at the generic level. We include 91 syngnathid species representing 48 of the 57 recognized genera, all major ocean basins, and a broad array of temperate and tropical habitats including rocky and coral reefs, sand and silt, mangroves, seagrass beds, estuaries, and rivers. Maximum likelihood and Bayesian analyses of 5160bp from eight loci produced high congruence among alternate topologies, defining well-supported and sometimes novel clades. We present a hypothesis that confirms a deep phylogenetic split between lineages with trunk- or tail-brood pouch placement, and provides significant new insights into the morphological evolution and biogeography of this highly derived fish clade. Based on the fundamental division between lineages - the tail brooding “Urophori” and the trunk brooding “Gastrophori” - we propose a revision of Syngnathidae classification into only two subfamilies: the Nerophinae and the Syngnathinae. We find support for distinct principal clades within the trunk-brooders and tail-brooders, the latter of which include seahorses, seadragons, independent lineages of pipehorses, and clades that originated in southern Australia and the Western Atlantic. We suggest the seahorse genus Hippocampus is of Indo-Pacific origin and its sister clade is an unexpected grouping of several morphologically disparate Indo-Pacific genera, including the Pacific pygmy pipehorses. Taxonomic revision is required for multiple genera, particularly to reflect deep evolutionary splits in nominal lineages from the Atlantic versus the Indo-Pacific.
Biomarkers of inflammation and altered coagulation are of increasing interest as predictors of chronic disease and mortality in HIV patients, as well as the use of risk stratification scores such as ...the Framingham index and the Veterans Aging Cohort Study (VACS) score.
Demographic and laboratory data for 252 HIV patients were assessed for their relationship with 5 biomarkers: hsCRP, D-dimer, Cystatin C, IL-6 and TNF-alpha. Analysis of variance was used to model the association between the number of elevated biomarkers patients had and their Framingham 10 year cardiovascular risk and VACS scores.
87% of patients were male and 75.7% were virally suppressed (HIV RNA <48 copies/ml). The median and interquartile ranges for each biomarker were: hsCRP 1.65 ug/mL (0.73, 3.89), D-dimer 0.17 ug/mL (0.09, 0.31), Cystatin C 0.87 mg/L (0.78, 1.01), IL-6 2.13 pg/mL (1.3, 3.59), TNF-alpha 4.65 pg/mL (3.5, 5.97). 62.6% of patients had more than one biomarker >75th percentile, while 18.6% had three or more elevated biomarkers. Increased age, cigarette smoking, CD4 counts of <200 cells/mm3, Framingham scores and VACS scores were most strongly associated with elevations in biomarkers. When biomarkers were used to predict the Framingham and VACS scores, those with a higher number of elevated biomarkers had higher mean VACS scores, with a similar but less robust finding for Framingham scores.
Despite viral suppression and immunological stability, biomarkers of inflammation and coagulation remain elevated in a significant number of patients with HIV and are associated with higher scores on risk stratification indices.
Over the last 10 years, there have been over 3300 genome-wide association studies (GWAS). Almost every GWAS study provides a Manhattan plot either as a main figure or in the supplement. Several ...software packages can generate a Manhattan plot, but they are all limited in the extent to which they can annotate gene-names, allele frequencies, and variants having high impact on gene function or provide any other added information or flexibility. Furthermore, in a conventional Manhattan plot, there is no way of distinguishing a locus identified due to a single variant with very significant p-value from a locus with multiple variants which appear to be in a haplotype block having very similar p-values.
Here we present a software tool written in R, which generates a transposed Manhattan plot along with additional features like variant consequence and minor allele frequency to annotate the plot and addresses these limitations. The software also gives flexibility on how and where the user wants to display the annotations. The software can be downloaded from CRAN repository and also from the GitHub project page.
We present a major step up to the existing conventional Manhattan plot generation tools. We hope this form of display along with the added annotations will bring more insight to the reader from this new Manhattan++ plot.
During embryonic germ layer development, cells communicate with each other and their environment to ensure proper lineage specification and tissue development. Connexin (Cx) proteins facilitate ...direct cell-cell communication through gap junction channels. While previous reports suggest that gap junctional intercellular communication may contribute to germ layer formation, there have been limited comprehensive expression analyses or genetic ablation studies on Cxs during human pluripotent stem cell (PSC) germ lineage specification. We screened the mRNA profile and protein expression patterns of select human Cx isoforms in undifferentiated human induced pluripotent stem cells (iPSCs), and after directed differentiation into the three embryonic germ lineages: ectoderm, definitive endoderm, and mesoderm. Transcript analyses by qPCR revealed upregulation of Cx45 and Cx62 in iPSC-derived ectoderm; Cx45 in mesoderm; and Cx30.3, Cx31, Cx32, Cx36, Cx37, and Cx40 in endoderm relative to control human iPSCs. Generated Cx43 (
) CRISPR-Cas9 knockout iPSCs successfully differentiated into cells of all three germ layers, suggesting that Cx43 is dispensable during directed iPSC lineage specification. Furthermore, qPCR screening of select Cx transcripts in our
iPSCs showed no significant Cx upregulation in response to the loss of Cx43 protein. Future studies will reveal possible compensation by additional Cxs, suggesting targets for future CRISPR-Cas9 ablation studies in human iPSC lineage specification.
Every single cell in the body communicates with nearby cells to locally organize activities with their neighbors and dysfunctional cell-cell communication can be detrimental during cell lineage ...commitment, tissue patterning and organ development. Pannexin channels (PANX1, PANX2, and PANX3) facilitate purinergic paracrine signaling through the passage of messenger molecules out of cells. PANX1 is widely expressed throughout the body and has recently been identified in human oocytes as well as 2 and 4-cell stage human embryos. Given its abundance across multiple adult tissues and its expression at the earliest stages of human development, we sought to understand whether PANX1 impacts human induced pluripotent stem cells (iPSCs) or plays a role in cell fate decisions. Western blot, immunofluorescence and flow cytometry reveal that PANX1 is expressed in iPSCs as well as all three germ lineages derived from these cells: ectoderm, endoderm, and mesoderm. PANX1 demonstrates differential glycosylation patterns and subcellular localization across the germ lineages. Using CRISPR-Cas9 gene ablation, we find that loss of PANX1 has no obvious impact on iPSC morphology, survival, or pluripotency gene expression. However,
gene knockout iPSCs exhibit apparent lineage specification bias under 3-dimensional spontaneous differentiation into the three germ lineages. Indeed, loss of PANX1 increases representation of endodermal and mesodermal populations in
knockout cells. Importantly,
knockout iPSCs are fully capable of differentiating toward each specific lineage when exposed to the appropriate external signaling pressures, suggesting that although PANX1 influences germ lineage specification, it is not essential to this process.
Liquid chromatography tandem mass spectrometry (LC-MS/MS) has been shown to be a viable tool for preclinical pharmacokinetic (PK) analysis of monoclonal antibody (mAb) therapeutics. This work ...describes free and total PK assays for the mAb PF-00547,659 in serum of ulcerative colitis patients in a First-In-Human study Vermeire, S. et al. Gut 2011, 60 (8), 1068–1075. The assay to measure free PF-00547,659 used immuno-enrichment with a biotinylated anti-idiotypic antibody and streptavidin magnetic beads. The total assay used enrichment by protein G magnetic beads. Following elution of PF-00547,659 from the beads, addition of an extended sequence stable isotope labeled peptide and trypsin digestion, a proteotypic peptide derived from the CDR region of the light chain of PF-00547,659 was quantified by LC-MS/MS. The free assay had a calibration range from 7.03 ng/mL to 450 ng/mL. The assay was precise and accurate with interbatch imprecision <16.5%, and interbatch inaccuracy <13.7% at all concentrations investigated during assay qualification. Results from LC-MS/MS methodologies are compared with historical immunoassay data originally acquired during the course of the clinical study. PK parameter estimates were highly correlated between the two analytical approaches. This work provides precedence that immunoaffinity LC-MS/MS can effectively be used to measure the serum concentrations of mAb therapeutics in clinical studies.
Epidemiological studies suggest a positive association between coronary artery disease (CAD) and late-onset Alzheimer's disease (LOAD). This large-scale genetic study brings together 'big data' ...resources to examine the causal impact of genetic determinants of CAD on risk of LOAD. A two-sample Mendelian randomization approach was adopted to estimate the causal effect of CAD on risk of LOAD using summary data from 60,801 CAD cases from CARDIoGRAMplusC4D and 17,008 LOAD cases from the IGAP Consortium. Additional analyses assessed the independent relevance of genetic associations at the APOE locus for both CAD and LOAD. Higher genetically determined risk of CAD was associated with a slightly higher risk of LOAD (Odds Ratio (OR) per log-odds unit of CAD 95% CI: 1.07 1.01-1.15; p = 0.027). However, after exclusion of the APOE locus, the estimate of the causal effect of CAD for LOAD was attenuated and no longer significant (OR 0.94 0.88-1.01; p = 0.072). This Mendelian randomization study indicates that the APOE locus is the chief determinant of shared genetic architecture between CAD and LOAD, and suggests a lack of causal relevance of CAD for risk of LOAD after exclusion of APOE.
The administration of biological protein therapeutics can lead to an unwanted immune response resulting in the generation of anti-drug antibodies (ADA) with potentially harmful clinical consequences. ...Hence, to develop safe and efficacious biotherapeutics, the immunogenic potential needs to be examined during the development phase. Current assay technologies measuring ADAs are subject to interference by high circulating concentrations of the protein therapeutic, raising concerns about data reliability since protein therapeutic-free washout samples are not always available. Herein, we report the development and characterization of a magnetic bead based immunoprecipitation method followed by quantitative LC/MS to determine ADA in human and cynomolgus serum in the presence of high circulating concentrations of the protein therapeutic. Available ADA binding sites are saturated by the addition of excess therapeutic followed by magnetic bead based protein G isolation of IgG antibodies and their bound antigens before elution and digestion. Peptides of the target therapeutic proteins are then quantified by LC/MS using stable isotope labeled standards inferring the presence of total ADA. This approach complements established methodologies for the assessment of immunogenicity responses and currently supports clinical programs addressing the safety and tolerability of human growth hormone analogues.
Abstract As part of a 2010 conference entitled “Patients and Populations: Public Health in Medical Education,” faculty from four U.S. medical schools (Case Western Reserve University, Harvard Medical ...School, the University of Colorado School of Medicine, and the University of Vermont College of Medicine), collaborated on a workshop to help other medical educators develop scenario-based learning experiences as practical, engaging, and effective mechanisms for teaching public health principles to medical school students. This paper describes and compares four different medical schools' experiences using a similar pandemic exercise scenario, discusses lessons learned, and suggests a curricular framework for medical schools adding such exercises to their population health curriculum. Different strategies to create realistic scenarios and engage students, including use of professionals and stakeholders from the community, are described.
Advances in recombinant antibody technology and protein engineering have provided the opportunity to reduce antibodies to their smallest binding domain components and have concomitantly driven the ...requirement for devising strategies to increase serum half-life to optimise drug exposure, thereby increasing therapeutic efficacy. In this study, we adopted an immunization route to raise picomolar affinity shark immunoglobulin new antigen receptors (IgNARs) to target human serum albumin (HSA). From our model shark species, Squalus acanthias, a phage display library encompassing the variable binding domain of IgNAR (VNAR) was constructed, screened against target, and positive clones were characterized for affinity and specificity. N-terminal and C-terminal molecular fusions of our lead hit in complex with a naïve VNAR domain were expressed, purified and exhibited the retention of high affinity binding to HSA, but also cross-selectivity to mouse, rat and monkey serum albumin both in vitro and in vivo. Furthermore, the naïve VNAR had enhanced pharmacokinetic (PK) characteristics in both N- and C-terminal orientations and when tested as a three domain construct with naïve VNAR flanking the HSA binding domain at both the N and C termini. Molecules derived from this platform technology also demonstrated the potential for clinical utility by being available via the subcutaneous route of delivery. This study thus demonstrates the first in vivo functional efficacy of a VNAR binding domain with the ability to enhance PK properties and support delivery of multifunctional therapies.
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