Variable Subunit Research Articles

The Typhoid Toxin Produced by the Nontyphoidal Salmonella enterica Serotype Javiana Is Required for Induction of a DNA Damage Response In Vitro and Systemic Spread In Vivo.

ABSTRACTThe Salmonella cytolethal distending toxin (S-CDT), first described as the “typhoid toxin” in Salmonella enterica subsp. enterica serotype Typhi, induces DNA damage in eukaryotic cells. Recent studies have shown that more than 40 nontyphoidal Salmonella (NTS) serotypes carry genes that encode S-CDT, yet very little is known about the activity, function, and role of S-CDT in NTS. Here we show that deletion of genes encoding the binding subunit (pltB) and a bacteriophage muramidase predicted to play a role in toxin export (ttsA) does not abolish toxin activity in the S-CDT-positive NTS Salmonella enterica subsp. enterica serotype Javiana. However, S. Javiana strains harboring deletions of both pltB and its homolog artB, had a complete loss of S-CDT activity, suggesting that S. Javiana carries genes encoding two variants of the binding subunit. S-CDT-mediated DNA damage, as determined by phosphorylation of histone 2AX (H2AX), producing phosphorylated H2AX (γH2AX), was restricted to epithelial cells in S and G2/M phases of the cell cycle and did not result in apoptosis or cell death. Compared to mice infected with a ΔcdtB strain, mice infected with wild-type S. Javiana had significantly higher levels of S. Javiana in the liver, but not in the spleen, ileum, or cecum. Overall, we show that production of active S-CDT by NTS serotype S. Javiana requires different genes (cdtB, pltA, and either pltB or artB) for expression of biologically active toxin than those reported for S-CDT production by S. Typhi (cdtB, pltA, pltB, and ttsA). However, as in S. Typhi, NTS S-CDT influences the outcome of infection both in vitro and in vivo.

Folding and assembly defects of pyruvate dehydrogenase deficiency-related variants in the E1α subunit of the pyruvate dehydrogenase complex.

The pyruvate dehydrogenase complex (PDC) bridges glycolysis and the citric acid cycle. In human, PDC deficiency leads to severe neurodevelopmental delay and progressive neurodegeneration. The majority of cases are caused by variants in the gene encoding the PDC subunit E1α. The molecular effects of the variants, however, remain poorly understood. Using yeast as a eukaryotic model system, we have studied the substitutions A189V, M230V, and R322C in yeast E1α (corresponding to the pathogenic variants A169V, M210V, and R302C in human E1α) and evaluated how substitutions of single amino acid residues within different functional E1α regions affect PDC structure and activity. The E1α A189V substitution located in the heterodimer interface showed a more compact conformation with significant underrepresentation of E1 in PDC and impaired overall PDC activity. The E1α M230V substitution located in the tetramer and heterodimer interface showed a relatively more open conformation and was particularly affected by low thiamin pyrophosphate concentrations. The E1α R322C substitution located in the phosphorylation loop of E1α resulted in PDC lacking E3 subunits and abolished overall functional activity. Furthermore, we show for the E1α variant A189V that variant E1α accumulates in the Hsp60 chaperonin, but can be released upon ATP supplementation. Our studies suggest that pathogenic E1α variants may be associated with structural changes of PDC and impaired folding of E1α.

Implication of Genes for the N-Methyl-D-Aspartate (NMDA) Receptor in Substance Addictions.

Drug dependence is a chronic brain disease with harmful consequences for both individual users and society. Glutamate is a primary excitatory neurotransmitter in the brain, and both in vivo and in vitro experiments have implicated N-methyl-D-aspartate (NMDA) receptor, a glutamate receptor, as an element in various types of addiction. Recent findings from genetics-based approaches such as genome-wide linkage, candidate gene association, genome-wide association (GWA), and next-generation sequencing have demonstrated the significant association of NMDA receptor subunit genes such as GluN3A, GluN2B, and GluN2A with various addiction-related phenotypes. Of these genes, GluN3A has been the most studied, and it has been revealed to play crucial roles in the etiology of addictions. In this communication, we provide an updated view of the genetic effects of NMDA receptor subunit genes and their functions in the etiology of addictions based on the findings from investigation of both common and rare variants as well as SNP-SNP interactions. To better understand the molecular mechanisms underlying addiction-related behaviors and to promote the development of specific medicines for the prevention and treatment of addictions, current efforts aim not only to identify more causal variants in NMDA receptor subunits by using large independent samples but also to reveal the molecular functions of these variants in addictions.

Molecular Characterization of GABA-A Receptor Subunit Diversity within Major Peripheral Organs and Their Plasticity in Response to Early Life Psychosocial Stress.

Gamma aminobutyric acid (GABA) subtype A receptors (GABAARs) are integral membrane ion channels composed of five individual proteins or subunits. Up to 19 different GABAAR subunits (α1–6, β1–3, γ1–3, δ, ε, θ, π, and ρ1–3) have been identified, resulting in anatomically, physiologically, and pharmacologically distinct multiple receptor subtypes, and therefore GABA-mediated inhibition, across the central nervous system (CNS). Additionally, GABAAR-modulating drugs are important tools in clinical medicine, although their use is limited by adverse effects. While significant advances have been made in terms of characterizing the GABAAR system within the brain, relatively less is known about the molecular phenotypes within the peripheral nervous system of major organ systems. This represents a potentially missed therapeutic opportunity in terms of utilizing or repurposing clinically available GABAAR drugs, as well as promising research compounds discarded due to their poor CNS penetrance, for the treatment of peripheral disorders. In addition, a broader understanding of the peripheral GABAAR subtype repertoires will contribute to the design of therapies which minimize peripheral side-effects when treating CNS disorders. We have recently provided a high resolution molecular and function characterization of the GABAARs within the enteric nervous system of the mouse colon. In this study, the aim was to determine the constituent GABAAR subunit expression profiles of the mouse bladder, heart, liver, kidney, lung, and stomach, using reverse transcription polymerase chain reaction and western blotting with brain as control. The data indicate that while some subunits are expressed widely across various organs (α3–5), others are restricted to individual organs (γ2, only stomach). Furthermore, we demonstrate complex organ-specific developmental expression plasticity of the transporters which determine the chloride gradient within cells, and therefore whether GABAAR activation has a depolarizing or hyperpolarizing effect. Finally, we demonstrate that prior exposure to early life psychosocial stress induces significant changes in peripheral GABAAR subunit expression and chloride transporters, in an organ- and subunit-specific manner. Collectively, the data demonstrate the molecular diversity of the peripheral GABAAR system and how this changes dynamically in response to life experience. This provides a molecular platform for functional analyses of the GABA–GABAAR system in health, and in diseases affecting various peripheral organs.

Extending the phenotype associated with the CSNK2A1-related Okur-Chung syndrome-A clinical study of 11 individuals.

Variants in the Protein Kinase CK2 alpha subunit, encoding the CSNK2A1 gene, have previously been reported in children with an intellectual disability and dysmorphic facial features syndrome: now termed the Okur-Chung neurodevelopmental syndrome. More recently, through trio-based exome sequencing undertaken by the Deciphering Developmental Disorders Study (DDD study), a further 11 children with de novo CSNK2A1 variants have been identified. We have undertaken detailed phenotyping of these patients. Consistent with previously reported patients, patients in this series had apparent intellectual disability, swallowing difficulties, and hypotonia. While there are some shared facial characteristics, the gestalt is neither consistent nor readily recognized. Congenital heart abnormalities were identified in nearly 30% of the patients, representing a newly recognized CSNK2A1 clinical association. Based upon the clinical findings from this study and the previously reported patients, we suggest an initial approach to the management of patients with this recently described intellectual disability syndrome.

Identification of Inherited Retinal Disease-Associated Genetic Variants in 11 Candidate Genes.

Inherited retinal diseases (IRDs) display an enormous genetic heterogeneity. Whole exome sequencing (WES) recently identified genes that were mutated in a small proportion of IRD cases. Consequently, finding a second case or family carrying pathogenic variants in the same candidate gene often is challenging. In this study, we searched for novel candidate IRD gene-associated variants in isolated IRD families, assessed their causality, and searched for novel genotype-phenotype correlations. Whole exome sequencing was performed in 11 probands affected with IRDs. Homozygosity mapping data was available for five cases. Variants with minor allele frequencies ≤ 0.5% in public databases were selected as candidate disease-causing variants. These variants were ranked based on their: (a) presence in a gene that was previously implicated in IRD; (b) minor allele frequency in the Exome Aggregation Consortium database (ExAC); (c) in silico pathogenicity assessment using the combined annotation dependent depletion (CADD) score; and (d) interaction of the corresponding protein with known IRD-associated proteins. Twelve unique variants were found in 11 different genes in 11 IRD probands. Novel autosomal recessive and dominant inheritance patterns were found for variants in Small Nuclear Ribonucleoprotein U5 Subunit 200 (SNRNP200) and Zinc Finger Protein 513 (ZNF513), respectively. Using our pathogenicity assessment, a variant in DEAH-Box Helicase 32 (DHX32) was the top ranked novel candidate gene to be associated with IRDs, followed by eight medium and lower ranked candidate genes. The identification of candidate disease-associated sequence variants in 11 single families underscores the notion that the previously identified IRD-associated genes collectively carry > 90% of the defects implicated in IRDs. To identify multiple patients or families with variants in the same gene and thereby provide extra proof for pathogenicity, worldwide data sharing is needed.

Coordinated Network Changes Across the Catalytic Cycle of Alpha Tryptophan Synthase

Networks of noncovalent interactions play important roles in the structural dynamics of globular proteins. Allosteric signals can propagate from the surface of an enzyme into its active site through these amino acid interaction networks. We used nuclear magnetic resonance chemical shift covariance analyses on a catalytically inactive variant of the alpha subunit of tryptophan synthase to map its amino acid interaction networks across its entire catalytic cycle. Each enzyme intermediate has unique sets of residues that display high chemical shift correlations across a set of alanine-to-glycine loop substitutions. Moreover, these chemical shift correlations systematically increase or decrease as a function of the catalytic cycle. These studies indicate that the enzyme intermediates have different amino acid interaction networks, and these network transitions are likely important for catalytic cycle progression. The different network interactions likely help to position catalytic machinery and coordinate function with the beta subunit.

Identification and expression of a unique neonatal variant of the GABAA receptor α3 subunit.

The GABAA receptor provides the majority of inhibitory neurotransmission in the adult central nervous system but in immature brain is responsible for much of the excitatory drive, a requirement for normal brain development. It is well established that GABAA receptor subunit expression changes across the course of brain development. In the present study, we have identified a splice variant of the GABAA receptor α3 subunit which appears unique to the developing brain, referred to here as the GABAA receptor α3 subunit neonatal variant (GABAA receptor α3N). RT-PCR and sequence analysis revealed splicing of exon 8 of the α3 subunit. Western blot analysis showed expression of GABAA receptor α3N in the cortex of several neonatal species and significantly reduced expression of this splice variant in the corresponding adult brains. Expression was evident in multiple brain regions and decreased across development in the pig. Fractionation revealed differential cellular localisation in the parietal cortex, hippocampus and thalamus of the full-length GABAA receptor α3 and GABAA receptor α3N. Immunoprecipitation showed direct interaction with the GABAA receptor subunits α1 and γ2 but not with gephyrin.

Functional and structural characterization of a novel malignant hyperthermia-susceptible variant of DHPR-β1a subunit (CACNB1).

Malignant hyperthermia (MH) susceptibility has been recently linked to a novel variant of β1a subunit of the dihydropyridine receptor (DHPR), a channel essential for Ca2+ regulation in skeletal muscle. Here we evaluate the effect of the mutant variant V156A on the structure/function of DHPR β1a subunit and assess its role on Ca2+ metabolism of cultured myotubes. Using differential scanning fluorimetry, we show that mutation V156A causes a significant reduction in thermal stability of the Src homology 3/guanylate kinase core domain of β1a subunit. Expression of the variant subunit in β1-null mouse myotubes resulted in increased sensitivity to caffeine stimulation. Whole cell patch-clamp analysis of β1a-V156A-expressing myotubes revealed a -2 mV shift in voltage dependence of channel activation, but no changes in Ca2+ conductance, current kinetics, or sarcoplasmic reticulum Ca2+ load were observed. Measurement of resting free Ca2+ and Na+ concentrations shows that both cations were significantly elevated in β1a-V156A-expressing myotubes and that these changes were linked to increased rates of plasmalemmal Ca2+ entry through Na+/Ca2+ exchanger and/or transient receptor potential canonical channels. Overall, our data show that mutant variant V156A results in instability of protein subdomains of β1a subunit leading to a phenotype of Ca2+ dysregulation that partly resembles that of other MH-linked mutations of DHPR α1S subunit. These data prove that homozygous expression of variant β1a-V156A has the potential to be a pathological variant, although it may require other gene defects to cause a full MH phenotype.

Identification of rare noncoding sequence variants in gamma-aminobutyric acid A receptor, alpha 4 subunit in autism spectrum disorder.

Alterations of the gamma-aminobutyric acid (GABA) signaling system has been strongly linked to the pathophysiology of autism spectrum disorder (ASD). Genetic associations of common variants in GABA receptor subunits, in particular GABRA4 on chromosome 4p12, with ASD have been replicated by several studies. Moreover, molecular investigations have identified altered transcriptional and translational levels of this gene and protein in brains of ASD individuals. Since the genotyped common variants are likely not the functional variants contributing to the molecular consequences or underlying ASD phenotype, this study aims to examine rare sequence variants in GABRA4, including those outside the protein coding regions of the gene. We comprehensively re-sequenced the entire protein coding and noncoding portions of the gene and putative regulatory sequences in 82 ASD individuals and 55 developmentally typical pediatric controls, all homozygous for the most significant previously associated ASD risk allele (G/G at rs1912960). We identified only a single common, coding variant, and no association of any single marker or set of variants with ASD. Functional annotation of noncoding variants identified several rare variants in putative regulatory sites. Finally, a rare variant unique to ASD cases, in an evolutionary conserved site of the 3'UTR, shows a trend toward decreasing gene expression. Hence, GABRA4 rare variants in noncoding DNA may be variants of modest physiological effects in ASD etiology.

R/G editing in GluA2Rflop modulates the functional difference between GluA1 flip and flop variants in GluA1/2R heteromeric channels

In α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors, RNA editing and alternative splicing generate sequence variants, and those variants, as in GluA2-4 AMPA receptor subunits, generally show different properties. Yet, earlier studies have shown that the alternatively spliced, flip and flop variants of GluA1 AMPA receptor subunit exhibit no functional difference in homomeric channel form. Using a laser-pulse photolysis technique, combined with whole-cell recording, we measured the rate of channel opening, among other kinetic properties, for a series of AMPA channels with different arginine/glycine (R/G) editing and flip/flop status. We find that R/G editing in the GluA2 subunit modulates the channel properties in both homomeric (GluA2Q) and complex (GluA2Q/2R and GluA1/2R) channel forms. However, R/G editing is only effective in flop channels. Specifically, editing at the R/G site on the GluA2R flop isoform accelerates the rate of channel opening and desensitization for GluA1/2R channels more pronouncedly with the GluA1 being in the flop form than in the flip form; yet R/G editing has no effect on either channel-closing rate or EC50. Our results suggest R/G editing via GluA2R serve as a regulatory mechanism to modulate the function of GluA2R-containing, native receptors involved in fast excitatory synaptic transmission.

Expanding the Role of Thyroid-Stimulating Hormone in Skeletal Physiology.

The dogma that thyroid-stimulating hormone (TSH) solely regulates the production of thyroid hormone from the thyroid gland has hampered research on its wider physiological roles. The action of pituitary TSH on the skeleton has now been well described; in particular, its action on osteoblasts and osteoclasts. It has also been recently discovered that the bone marrow microenvironment acts as an endocrine circuit with bone marrow-resident macrophages capable of producing a novel TSH-β subunit variant (TSH-βv), which may modulate skeletal physiology. Interestingly, the production of this TSH-βv is positively regulated by T3 accentuating such modulation in the presence of thyroid overactivity. Furthermore, a number of small molecule ligands acting as TSH agonists, which allosterically modulate the TSH receptor have been identified and may have similar modulatory influences on bone cells suggesting therapeutic potential. This review summarizes our current understanding of the role of TSH, TSH-β, TSH-βv, and small molecule agonists in bone physiology.

Genetic variants of the NMDAR subunits influence on receptor properties and associate with synaptic plasticity

Genetic variants of the NMDAR subunits influence on receptor properties and associate with synaptic plasticity

The exocyst subunit Sec3 is regulated by a protein quality control pathway

Exocytosis involves fusion of secretory vesicles with the plasma membrane, thereby delivering membrane proteins to the cell surface and releasing material into the extracellular space. The tethering of the secretory vesicles before membrane fusion is mediated by the exocyst, an essential phylogenetically conserved octameric protein complex. Exocyst biogenesis is regulated by several processes, but the mechanisms by which the exocyst is degraded are unknown. Here, to unravel the components of the exocyst degradation pathway, we screened for extragenic suppressors of a temperature-sensitive fission yeast strain mutated in the exocyst subunit Sec3 (sec3-913). One of the suppressing DNAs encoded a truncated dominant-negative variant of the 26S proteasome subunit, Rpt2, indicating that exocyst degradation is controlled by the ubiquitin-proteasome system. The temperature-dependent growth defect of the sec3-913 strain was gene dosage-dependent and suppressed by blocking the proteasome, Hsp70-type molecular chaperones, the Pib1 E3 ubiquitin-protein ligase, and the deubiquitylating enzyme Ubp3. Moreover, defects in cell septation, exocytosis, and endocytosis in sec3 mutant strains were similarly alleviated by mutation of components in this pathway. We also found that, particularly under stress conditions, wild-type Sec3 degradation is regulated by Pib1 and the 26S proteasome. In conclusion, our results suggest that a cytosolic protein quality control pathway monitors folding and proteasome-dependent turnover of an exocyst subunit and, thereby, controls exocytosis in fission yeast.

Pharmacology of Cumyl-Carboxamide Synthetic Cannabinoid New Psychoactive Substances (NPS) CUMYL-BICA, CUMYL-PICA, CUMYL-5F-PICA, CUMYL-5F-PINACA, and Their Analogues

Synthetic cannabinoids (SC) are the largest class of new psychoactive substances (NPS), and are increasingly associated with serious adverse effects. The majority of SC NPS are 1,3-disubstituted indoles and indazoles featuring a diversity of subunits at the 1- and 3-positions. Most recently, cumyl-derived indole- and indazole-3-carboxamides have been detected by law enforcement agencies and by emergency departments. Herein we describe the synthesis, characterization, and pharmacology of SCs CUMYL-BICA, CUMYL-PICA, CUMYL-5F-PICA, CUMYL-PINACA, CUMYL-5F-PINACA, and related analogues. All cumyl-derived SCs were potent, efficacious agonists at CB1 (EC50 = 0.43-12.3 nM) and CB2 (EC50 = 11.3-122 nM) receptors in a fluorometric assay of membrane potential, with selectivity for CB1 activation (3.1-53 times over CB2). CUMYL-PICA and CUMYL-5F-PICA were evaluated in rats using biotelemetry, and induced hypothermia and bradycardia at doses of 1 mg/kg. Hypothermia was reversed by pretreatment with a CB1, but not CB2, antagonist, confirming that cumyl-derived SCs are cannabimimetic in vivo.

Genetic variation in GABRβ1 and the risk for developing alcohol dependence.

Associations between the γ-aminobutyric acid type-A receptors (GABAA) and alcohol dependence risk have been reported, although the receptor subunit driving the association is unclear. Recent work in mice has highlighted a possible role for variants in the Gabr β1 subunit (Gabrβ1) in alcohol dependence risk, although this gene does not contain any common nonsynonymous variants in humans. However, the GABAA receptor is a heteropentamer so multiple potential variants within the gene complex could generate the alcohol dependence phenotype. The association between GABRβ1 variants and alcohol dependence risk was explored in a British and Irish population of alcohol-dependent cases (n=450) and ancestrally-matched controls screened to exclude current or historical alcohol misuse (n=555). Twelve common single nucleotide polymorphisms (SNPs) and a rare nonsynonymous variant, rs41311286, were directly genotyped; imputation was then performed across the whole gene. No allelic association was observed between alcohol dependence risk and any of the directly genotyped or imputed SNPs. However, post-hoc testing for genotypic association identified five common intronic SNPs that showed modest evidence for association after correction for multiple testing; two, rs76112682 and rs141719901, were in complete linkage disequilibrium [Pcorrected=0.02, odds ratio (95% confidence interval)=5.9 (1.7-2.06)]. These findings provide limited support for an association between GABRβ1 and the risk for developing alcohol dependence; further testing in expanded cohorts may be warranted.

Human group 2 innate lymphoid cells do not express the IL-5 receptor

This work was part funded by (for CEB) Airway Disease Predicting Outcomes through Patient Specific Computational Modelling (AirPROM) project (funded through FP7 EU grant), Wellcome Senior Fellowship, (for DJC) Medical Research Council (MRC) and Asthma UK (Centre Grant: G1000758), from the NIHR Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust and King's College London, an equipment grant from the Midlands Asthma and Allergy Research Association (MAARA) and (for CEB and DJC) the National Institute for Health Research (NIHR) Leicester Respiratory Biomedical Unit. BMJR was funded by an MRC and Asthma UK PhD studentship. This paper presents independent research funded by the NIHR.

Fast and Slow Inhibition in the Visual Thalamus Is Influenced by Allocating GABAA Receptors with Different γ Subunits.

Cell-type specific differences in the kinetics of inhibitory postsynaptic conductance changes (IPSCs) are believed to impact upon network dynamics throughout the brain. Much attention has focused on how GABAA receptor (GABAAR) α and β subunit diversity will influence IPSC kinetics, but less is known about the influence of the γ subunit. We have examined whether GABAAR γ subunit heterogeneity influences IPSC properties in the thalamus. The γ2 subunit gene was deleted from GABAARs selectively in the dorsal lateral geniculate nucleus (dLGN). The removal of the γ2 subunit from the dLGN reduced the overall spontaneous IPSC (sIPSC) frequency across all relay cells and produced an absence of IPSCs in a subset of relay neurons. The remaining slower IPSCs were both insensitive to diazepam and zinc indicating the absence of the γ2 subunit. Because these slower IPSCs were potentiated by methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), we propose these IPSCs involve γ1 subunit-containing GABAAR activation. Therefore, γ subunit heterogeneity appears to influence the kinetics of GABAAR-mediated synaptic transmission in the visual thalamus in a cell-selective manner. We suggest that activation of γ1 subunit-containing GABAARs give rise to slower IPSCs in general, while faster IPSCs tend to be mediated by γ2 subunit-containing GABAARs.

Analysis of Germination Capacity and Germinant Receptor (Sub)clusters of Genome-Sequenced Bacillus cereus Environmental Isolates and Model Strains.

Germination of bacterial spores is a critical step before vegetative growth can resume. Food products may contain nutrient germinants that trigger germination and outgrowth of Bacillus species spores, possibly leading to food spoilage or foodborne illness. Prediction of spore germination behavior is, however, very challenging, especially for spores of natural isolates that tend to show more diverse germination responses than laboratory strains. The approach used has provided information on the genetic diversity in GRs and corresponding subclusters encoded by B. cereus strains, as well as their germination behavior and possible associations with GRs, and it provides a basis for further extension of knowledge on the role of GRs in B. cereus (group member) ecology and transmission to the host.

Evolution of specificity in cartilaginous fish glycoprotein hormones and receptors

Glycoprotein hormones (GpH) interact very specifically with their receptors to mediate hypothalamic-pituitary-peripheral gland endocrine signaling. Vertebrates typically have three functionally distinct GpH endocrine signaling complexes: follicle-stimulating hormone, luteinizing hormone, and thyroid-stimulating hormone, and their receptors. Each hormone consists of a common α subunit bound to one of three different β subunits. Individual hormone subunits and receptors are present in genomes of early metazoans, and a subset of hormone subunits and receptors has been recently characterized in sea lamprey. However, it remains unclear when the full complement of hormone and receptor protein families first appeared, and when specificity of interactions between GpH hormones and receptors first evolved. Here we present phylogenetic analyses showing that the elephant shark (Callorhinchus milii) genome contains sequences representing the current diversity of all hormone subunits and receptors in these co-evolving protein families. We examined specificity of hormone and receptor interactions using functional assays testing reporter gene activation by elephant shark follicle-stimulating hormone, luteinizing hormone, and thyroid-stimulating hormone receptors. We show highly specific, dose-responsive hormone interactions for all three complexes. Our results suggest that co-evolution of specificity between proteins in these endocrine signaling complexes occurred prior to the divergence of Chondrichthyes from the chordate lineage.