Sequencing and comparative analysis of fugu protocadherin clusters reveal diversity of protocadherin genes among teleosts

BACKGROUND: The synaptic cell adhesion molecules, protocadherins, are a vertebrate innovation that accompanied the emergence of the neural tube and the elaborate central nervous system. In mammals, the protocadherins are encoded by three closely-linked clusters (α, β and γ) of tandem genes and are hypothesized to provide a molecular code for specifying the remarkably-diverse neural connections in the central nervous system. Like mammals, the coelacanth, a lobe-finned fish, contains a single protocadherin locus, also arranged into α, β and γ clusters. Zebrafish, however, possesses two protocadherin loci that contain more than twice the number of genes as the coelacanth, but arranged only into α and γ clusters. To gain further insight into the evolutionary history of protocadherin clusters, we have sequenced and analyzed protocadherin clusters from the compact genome of the pufferfish, Fugu rubripes. RESULTS: Fugu contains two unlinked protocadherin loci, Pcdh1 and Pcdh2, that collectively consist of at least 77 genes. The fugu Pcdh1 locus has been subject to extensive degeneration, resulting in the complete loss of Pcdh1γ cluster. The fugu Pcdh genes have undergone lineage-specific regional gene conversion processes that have resulted in a remarkable regional sequence homogenization among paralogs in the same subcluster. Phylogenetic analyses show that most protocadherin genes are orthologous between fugu and zebrafish either individually or as paralog groups. Based on the inferred phylogenetic relationships of fugu and zebrafish genes, we have reconstructed the evolutionary history of protocadherin clusters in the teleost fish lineage. CONCLUSION: Our results demonstrate the exceptional evolutionary dynamism of protocadherin genes in vertebrates in general, and in teleost fishes in particular. Besides the 'fish-specific' whole genome duplication, the evolution of protocadherin genes in teleost fishes is influenced by lineage-specific gene losses, tandem gene duplications and regional sequence homogenization. The dynamic protocadherin clusters might have led to the diversification of neural circuitry among teleosts, and contributed to the behavioral and physiological diversity of teleosts

Sequencing and comparative analysis of fugu protocadherin clusters reveal diversity of protocadherin genes among teleosts

Abstract Background The synaptic cell adhesion molecules, protocadherins, are a vertebrate innovation that accompanied the emergence of the neural tube and the elaborate central nervous system. In mammals, the protocadherins are encoded by three closely-linked clusters (α, β and γ) of tandem genes and are hypothesized to provide a molecular code for specifying the remarkably-diverse neural connections in the central nervous system. Like mammals, the coelacanth, a lobe-finned fish, contains a single protocadherin locus, also arranged into α, β and γ clusters. Zebrafish, however, possesses two protocadherin loci that contain more than twice the number of genes as the coelacanth, but arranged only into α and γ clusters. To gain further insight into the evolutionary history of protocadherin clusters, we have sequenced and analyzed protocadherin clusters from the compact genome of the pufferfish, Fugu rubripes. Results Fugu contains two unlinked protocadherin loci, Pcdh1 and Pcdh2, that collectively consist of at least 77 genes. The fugu Pcdh1 locus has been subject to extensive degeneration, resulting in the complete loss of Pcdh1γ cluster. The fugu Pcdh genes have undergone lineage-specific regional gene conversion processes that have resulted in a remarkable regional sequence homogenization among paralogs in the same subcluster. Phylogenetic analyses show that most protocadherin genes are orthologous between fugu and zebrafish either individually or as paralog groups. Based on the inferred phylogenetic relationships of fugu and zebrafish genes, we have reconstructed the evolutionary history of protocadherin clusters in the teleost fish lineage. Conclusion Our results demonstrate the exceptional evolutionary dynamism of protocadherin genes in vertebrates in general, and in teleost fishes in particular. Besides the 'fish-specific' whole genome duplication, the evolution of protocadherin genes in teleost fishes is influenced by lineage-specific gene losses, tandem gene duplications and regional sequence homogenization. The dynamic protocadherin clusters might have led to the diversification of neural circuitry among teleosts, and contributed to the behavioral and physiological diversity of teleosts.</p

Treatment of HMBA differentiates hPSCs into mesoderm-ectoderm lineages.

<p>HES-3 cells were incubated with 1, 3, 5 or 10 mM HMBA for 7 PDs. RNAs prepared from the treated cells were analyzed by QRT-PCR to determine the expression of (A) pluripotent (NANOG and OCT3/4), (B) endodermal (AFP and GATA4), (C) mesodermal (Col2A1, IGF2, and ACTC1), and (D) ectodermal genes (MSX1, PAX6, and SOX1). Results were reproducible, and data from one experiment are presented. Error bars indicate the standard deviations between triplicates. *<i>P</i><0.05 versus control.</p

Treatment of HMBA increases expression of HEXIM1 and induces differentiation in HES-3 cells.

<p>(A) HES-3 cells were incubated with 1, 3, 5, or 10 mM HMBA for 7 PDs, followed by FACS analysis. Percentages of cells expressing pluripotent markers, including OCT3/4, PODXL, and Tra-1-60, were indicated (i.e. open histograms). Cells treated with 1% ethanol were used as the vehicle control. (B) HEXIM1 mRNA levels of the HMBA-treated HES-3 cells were measured by QRT-PCR. (C) HEXIM1 and OCT3/4 protein levels of the HMBA-treated HES-3 cells were examined by western blotting. Actin was used as a loading control.</p

High-level expression of HEXIM1 up-regulates the marker genes of three germ layers in hPSCs.

<p>HES-3 cells were transiently transfected with a HEXIM1 expression plasmid, followed by antibiotic selection for the HEXIM1-transfected cells. The differentiated and undifferentiated populations of the HEXIM1-transfected HES-3 cells were sorted by FACS analysis using an anti-TRA-1-60 antibody. The mRNAs prepared from the TRA-1-60 positive and negative HEXIM1-transfected HES-3 cells (i.e. HEXIM1 Tra pos. and HEXIM1 Tra neg, respectively) were analyzed by QRT-PCR to determine the expression of (A) pluripotent (NANOG and OCT3/4), (B) endodermal (AFP and GATA4), (C) mesodermal (Col2A1, IGF2, and ACTC1), and (D) ectodermal genes (MSX1, PAX6, and SOX1). Results were reproducible, and data from one experiment are presented. Error bars indicate the standard deviations between triplicates. *<i>P</i><0.05 versus control.</p

Rare Occurrence of Aristolochic Acid Mutational Signatures in Oro-Gastrointestinal Tract Cancers

Background: Aristolochic acids (AAs) are potent mutagens commonly found in herbal plant-based remedies widely used throughout Asian countries. Patients and Methods: To understand whether AA is involved in the tumorigenesis of the oro-gastrointestinal tract, we used whole-exome sequencing to profile 54 cases of four distinct types of oro-gastrointestinal tract cancer (OGITC) from Taiwan. Results: A diverse landscape of mutational signatures including those from DNA mismatch repair and reactive oxygen species was observed. APOBEC mutational signatures were observed in 60% of oral squamous cell carcinomas. Only one sample harbored AA mutational signatures, contradictory to prior reports of cancers from Taiwan. The metabolism of AA in the liver and urinary tract, transient exposure time, and high cell turnover rates at OGITC sites may explain our findings. Conclusion: AA signatures in OGITCs are rare and unlikely to be a major contributing factor in oro-gastrointestinal tract tumorigenesis

DataSheet1_A comprehensive next generation sequencing tissue assay for Asian-prevalent cancers—Analytical validation and performance evaluation with clinical samples.xlsx

Introduction: A well-validated diagnostic assay with curated biomarkers complements clinicopathological factors to facilitate early diagnosis and ensure timely treatment delivery. This study focuses on an Asian-centric cancer diagnostic assay designed and thoroughly validated against commercially available standard references and a cohort of over 200 clinical specimens spanning 12 diverse Asian-centric cancer types.Methods: The assay uses hybrid-capture probes capable of profiling DNA aberrations from 572 cancer-related genes and 91 RNA fusion partners. The panel can detect clinically-tractable biomarkers such as microsatellite instability (MSI) and tumor mutation burden (TMB).Results: Analytical evaluation demonstrated 100% specificity and 99.9% sensitivity within a ≥5% VAF limit of detection (LoD) for SNV/Indels. RNA-based fusion features an LoD of ≥5 copies per nanogram input when evaluated against commercial references. Excellent linearity and concordance were observed when benchmarking against orthogonal methods in identifying MSI status, TMB scores and RNA fusions. Actionable genetic alterations were identified in 65% of the clinical samples.Conclusion: These results demonstrate a molecular diagnostic assay that accurately detects genomic alterations and complex biomarkers. The data also supports an excellent performance of this assay for making critical diagnoses and well-informed therapeutic decisions in Asian prevalent cancers.</p