Posthearing Ca(2+) currents and their roles in shaping the different modes of firing of spiral ganglion neurons.

Whereas prehearing spiral ganglion neurons (SGNs) rely faithfully on outputs from spontaneously active developing hair cells, the electrical phenotypes of posthearing neurons are shaped by distinct rapid and graded receptor potentials from hair cells. To date, technical difficulties in isolation of fragile posthearing neurons from the rigid bony labyrinth of the inner ear have hindered analyses of the electrical phenotype of SGNs. Therefore, we have recently developed new strategies to isolate posthearing mouse SGNs for functional analyses. Here, we describe the coarse and fine properties of Ca(2+) currents, which sculpt the firing properties of posthearing SGNs. Murine SGNs express multiple Ca(2+) channel currents to enable diverse functions. We have demonstrated that suppression of Ca(2+) currents results in significant hyperpolarization of the resting membrane potential (rmp) of basal SGNs, suggesting that Ca(2+) influx primes rmp for excitation. In contrast, removal of external Ca(2+) has modest effects on rmp of apical SGNs. The blockade of Ca(2+) currents with a mixture of specific blockers attenuates spontaneously active SGNs. Paradoxically, different subtypes of Ca(2+) currents, such as R-type currents, may activate resting outward conductances since blockage of the current results in depolarization of rmp. In keeping with whole-cell current data, single-channel records revealed multiple diverse Ca(2+) channels in SGNs. Additionally, there were differential expressions of distinct Ca(2+) current densities in the apicobasal contour of the adult cochlea. This report provides invaluable insights into Ca(2+)-dependent processes in adult SGNs.

Mature mice lacking Rbl2/p130 gene have supernumerary inner ear hair cells and supporting cells.

Adult mammalian auditory hair cells (HCs) and their associated supporting cells (SCs) do not proliferate, and HC death leads to irreversible neurosensory hearing loss and balance impairment. In nonmammalian vertebrates, loss of HCs induces mitotic proliferation of adjacent nonsensory SCs and/or direct SC transdifferentiation to generate replacement cells. This results in the structural and functional recovery of the nonmammalian sensory systems. Potential replacement of mammalian auditory HCs, either by transplanting cells or by transforming existing cells through molecular therapy, has long been proposed. However, HC replacement strategies with clear therapeutic potential remain elusive. The retinoblastoma (pRB) family of cell cycle regulators, Rb1, Rbl1 (p107), and Rbl2 (p130), regulate the G(1)- to S-phase transition in proliferating cells. In the inner ear, the biochemical and molecular pathways involving pRBs, particularly p107 and p130, are relatively unexplored and their therapeutic suitability is yet to be determined. In this study, we analyzed the cochleae of adult p130 knock-out (p130(-/-)) mice and showed that lack of the p130 gene results in extra rows of HCs and SCs in the more apical regions of the cochlea. No evidence of transdifferentiation of these supernumerary SCs into HCs was observed in the p130(-/-) mouse. Nevertheless, unscheduled proliferation of SCs in the adult p130(-/-) cochlea coupled to downregulation of bona fide cell cycle inhibitors provides a mechanistic basis for the role of p130 as a regulator of SC and HC mitotic quiescence in the more apical regions of the cochlea. Interestingly, p130(-/-) mice exhibited nearly normal peripheral auditory sensitivity.

Conditional deletion of the human ortholog gene Dicer1 in Pax2-Cre expression domain impairs orofacial development.

BACKGROUND: Orofacial clefts are common worldwide and result from insufficient growth and/or fusion during the genesis of the derivatives of the first pharyngeal arch and the frontonasal prominence. Recent studies in mice carrying conditional and tissue-specific deletions of the human ortholog Dicer1, an RNAse III family member, have highlighted its importance in cell survival, differentiation, proliferation, and morphogenesis. Nevertheless, information regarding Dicer1 and its dependent microRNAs (miRNAs) in mammalian palatogenesis and orofacial development is limited. AIMS: To describe the craniofacial phenotype, gain insight into potential mechanisms underlying the orofacial defects in the Pax2-Cre/Dicer1 CKO mouse, and shed light on the role of Dicer1 in mammalian palatogenesis. MATERIALS AND METHODS: Histological and molecular assays of wild type (WT) and Pax2-Cre/Dicer1(loxP/loxP) (Dicer1 CKO) mice dissected tissues have been performed to characterize and analyze the orofacial dysmorphism in Pax2-Cre/Dicer1(loxP/loxP) mouse. RESULTS: Dicer1 CKO mice exhibit late embryonic lethality and severe craniofacial dysmorphism, including a secondary palatal cleft. Further analysis suggest that Dicer1 deletion neither impacts primary palatal development nor the initial stages of secondary palatal formation. Instead, Dicer1 is implicated in growth, differentiation, mineralization, and survival of cells in the lateral palatal shelves. Histological and molecular analysis demonstrates that secondary palatal development becomes morphologically arrested prior to mineralization around E13.5 with a significant increase in the expression levels of apoptotic markers (P < 0.01). CONCLUSIONS: Pax2-Cre-mediated Dicer1 deletion disrupts lateral palatal outgrowth and bone mineralization during palatal shelf development, therefore providing a mammalian model for investigating the role of miRNA-mediated signaling pathways during palatogenesis.

A radiographic method for distinguishing noncavitated from cavitated proximal carious lesions: A proof of concept clinical trial.

OBJECTIVES: We propose a new topical radiographic contrast method for distinguishing noncavitated from cavitated radiolucencies. Laboratory tests and a pilot clinical trial were designed to test the feasibility and efficacy of the method. STUDY DESIGN: Twenty-two adults with 27 proximal radiolucencies had conventional bitewing (BW) examinations. After exclusion, 21 surfaces were evaluated. A concentrated solution of sodium iodide was placed in the interdental spaces via a microsyringe and BWs were again exposed. A class II cavity preparation was made in the adjacent tooth and polysiloxane impressions were made of the study surfaces. The impressions were scanned by visible light, creating a high resolution 3D replica. Cavitations, if present, were measured. RESULTS: Nine surfaces were noncavitated and 12 surfaces were cavitated. The microsyringe dispensed a variable volume of liquid, which affected the accuracy of the test. The sensitivity for cavitation was 50%, specificity was 88.9%, and accuracy was 66.7%. This compares to a reported 60% sensitivity, 62% specificity, and 62% accuracy for BW examinations. Intraexaminer reliability for classifying noncavitated or cavitated lesions using the kappa test was 0.649. CONCLUSIONS: This method needs improvement but was an advance over conventional BWs and could result in reduction of restorations in low- and high-risk patients.

PLCγ-activated signalling is essential for TrkB mediated sensory neuron structural plasticity.

BACKGROUND: The vestibular system provides the primary input of our sense of balance and spatial orientation. Dysfunction of the vestibular system can severely affect a person's quality of life. Therefore, understanding the molecular basis of vestibular neuron survival, maintenance, and innervation of the target sensory epithelia is fundamental. RESULTS: Here we report that a point mutation at the phospholipase Cγ (PLCγ) docking site in the mouse neurotrophin tyrosine kinase receptor TrkB (Ntrk2) specifically impairs fiber guidance inside the vestibular sensory epithelia, but has limited effects on the survival of vestibular sensory neurons and growth of afferent processes toward the sensory epithelia. We also show that expression of the TRPC3 cation calcium channel, whose activity is known to be required for nerve-growth cone guidance induced by brain-derived neurotrophic factor (BDNF), is altered in these animals. In addition, we find that absence of the PLCγ mediated TrkB signalling interferes with the transformation of bouton type afferent terminals of vestibular dendrites into calyces (the largest synaptic contact of dendrites known in the mammalian nervous system) on type I vestibular hair cells; the latter are normally distributed in these mutants as revealed by an unaltered expression pattern of the potassium channel KCNQ4 in these cells. CONCLUSIONS: These results demonstrate a crucial involvement of the TrkB/PLCγ-mediated intracellular signalling in structural aspects of sensory neuron plasticity.

Spatiotemporally controlled overexpression of cyclin D1 triggers generation of supernumerary cells in the postnatal mouse inner ear.

The retinoblastoma family of pocket proteins (pRBs), composed of Rb1, p107, and p130 are negative regulators of cell-cycle progression. The deletion of any individual pRB in the auditory system triggers hair cells' (HCs) and supporting cells' (SCs) proliferation to different extents. Nevertheless, accessing their combined role in the inner ear through conditional or complete knockout methods is limited by the early mortality of the triple knockout. In quiescent cells, hyperphosphorylation and inactivation of the pRBs are maintained through the activity of the Cyclin-D1-cdk4/6 complex. Cyclin D1 (CycD1) is expressed in the embryonic and neonatal inner ear. In the mature organ of Corti (OC), CycD1 expression is significantly downregulated, paralleling the OC mitotic quiescence. Earlier studies showed that CycD1 overexpression leads to cell-cycle reactivation in cultures of inner ear explants. Here, we characterize a Cre-activated, Doxycycline (Dox)-controlled, conditional CycD1 overexpression model, which when bred to a tetracycline-controlled transcriptional activator and the Atoh1-cre mouse lines, allow for transient CycD1 overexpression and pRBs' downregulation in the inner ear in a reversible fashion. Analyses of postnatal mice's inner ears at various time points revealed the presence of supernumerary cells throughout the length of the cochlea and in the vestibular end-organs. Notably, most supernumerary cells were observed in the inner hair cells' (IHCs) region, expressed myosin VIIa (M7a), and showed no signs of apoptosis at any of the time points analyzed. Auditory and vestibular phenotypes were similar between the different genotypes and treatment groups. The fact that no significant differences were observed in auditory and vestibular function supports the notion that the supernumerary cells detected in the adult mice cochlea and macular end-organs may not impair auditory functions.

In silico Design of a Multivalent Vaccine Against Candida albicans.

Invasive candidiasis (IC) is the most common nosocomial infection and a leading cause of mycoses-related deaths. High-systemic toxicity and emergence of antifungal-resistant species warrant the development of newer preventive approaches against IC. Here, we have adopted an immunotherapeutic peptide vaccine-based approach, to enhance the body's immune response against invasive candida infections. Using computational tools, we screened the entire candida proteome (6030 proteins) and identified the most immunodominant HLA class I, HLA class II and B- cell epitopes. By further immunoinformatic analyses for enhanced vaccine efficacy, we selected the 18- most promising epitopes, which were joined together using molecular linkers to create a multivalent recombinant protein against Candida albicans (mvPC). To increase mvPC's immunogenicity, we added a synthetic adjuvant (RS09) to the mvPC design. The selected mvPC epitopes are homologous against all currently available annotated reference sequences of 22 C. albicans strains, thus offering a higher coverage and greater protective response. A major advantage of the current vaccine approach is mvPC's multivalent nature (recognizing multiple-epitopes), which is likely to provide enhanced protection against complex candida antigens. Here, we describe the computational analyses leading to mvPC design.

Inducible and Reversible Dominant-negative (DN) Protein Inhibition.

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based approaches. For example, although chimeric and Cre-LoxP targeting strategies have been widely used, the intrinsic limitations of these strategies (i.e., leaky promoter activity, mosaic Cre expression, etc.) have significantly restricted their application. Moreover, a complete deletion of many endogenous genes is embryonically lethal, making it impossible to study gene function in postnatal life. To address these challenges, we have made significant changes to an early genetic engineering protocol and combined a short (transgenic) version of the Rb1 gene with a lysosomal protease procathepsin B (CB), to generate a DN mouse model of Rb1 (CBRb). Due to the presence of a lysosomal protease, the entire CB-RB1 fusion protein and its interacting complex are routed for proteasome-mediated degradation. Moreover, the presence of a tetracycline inducer (rtTA) element in the transgenic construct enables an inducible and reversible regulation of the RB1 protein. The presence of a ubiquitous ROSA-CAG promoter in the CBRb mouse model makes it a useful tool to carry out transient and reversible Rb1 gene ablation and provide researchers a resource for understanding its activity in virtually any cell type where RB1 is expressed.

Author Correction: Quinoxaline protects zebrafish lateral line hair cells from cisplatin and aminoglycosides damage.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Pocket proteins and cell cycle regulation in inner ear development.

Loss of neurosensory cells of the ear, caused by genetic and non-genetic factors, is becoming an increasing problem as people age, resulting in deafness and vestibular disorders. Unveiling useful mechanisms of cell cycle regulation may offer the possibility to generate new cells out of remaining ones, thus providing the cellular basis to induce new hair cell differentiation in the mammalian ear. Here, we provide an overview of cell cycle regulating genes in general and of those studied in the ear in particular. We categorize those genes into regulators that act upstream of the pocket proteins and into those that act downstream of the pocket proteins. The three members of the pocket protein family essentially determine, through interaction with the eight members of the E2F family, whether or not the cell cycle will progress to the S-phase and thus cell division. The abundant presence of one or more members of these families in adult hair cells supports the notion that inhibition of cell cycle progression through these proteins is a lifelong process. Indeed, manipulating some of those proteins, unfortunately, leads to abortive entry into the cell cycle. Combined with recent success to induce hair cell differentiation through molecular therapy, these approaches may provide a viable strategy to restore lost hair cells in the inner ear.

Quinoxaline protects zebrafish lateral line hair cells from cisplatin and aminoglycosides damage.

Hair cell (HC) death is the leading cause of hearing and balance disorders in humans. It can be triggered by multiple insults, including noise, aging, and treatment with certain therapeutic drugs. As society becomes more technologically advanced, the source of noise pollution and the use of drugs with ototoxic side effects are rapidly increasing, posing a threat to our hearing health. Although the underlying mechanism by which ototoxins affect auditory function varies, they share common intracellular byproducts, particularly generation of reactive oxygen species. Here, we described the therapeutic effect of the heterocyclic compound quinoxaline (Qx) against ototoxic insults in zebrafish HCs. Animals incubated with Qx were protected against the deleterious effects of cisplatin and gentamicin, and partially against neomycin. In the presence of Qx, there was a reduction in the number of TUNEL-positive HCs. Since Qx did not block the mechanotransduction channels, based on FM1-43 uptake and microphonic potentials, this implies that Qx's otoprotective effect is at the intracellular level. Together, these results unravel a novel therapeutic role for Qx as an otoprotective drug against the deleterious side effects of cisplatin and aminoglycosides, offering an alternative option for patients treated with these compounds.

A mouse model of miR-96, miR-182 and miR-183 misexpression implicates miRNAs in cochlear cell fate and homeostasis.

Germline mutations in Mir96, one of three co-expressed polycistronic miRNA genes (Mir96, Mir182, Mir183), cause hereditary hearing loss in humans and mice. Transgenic FVB/NCrl- Tg(GFAP-Mir183,Mir96,Mir182)MDW1 mice (Tg1MDW), which overexpress this neurosensory-specific miRNA cluster in the inner ear, were developed as a model system to identify, in the aggregate, target genes and biologic processes regulated by the miR-183 cluster. Histological assessments demonstrate Tg1MDW/1MDW homozygotes have a modest increase in cochlear inner hair cells (IHCs). Affymetrix mRNA microarray data analysis revealed that downregulated genes in P5 Tg1MDW/1MDW cochlea are statistically enriched for evolutionarily conserved predicted miR-96, miR-182 or miR-183 target sites. ABR and DPOAE tests from 18 days to 3 months of age revealed that Tg1MDW/1MDW homozygotes develop progressive neurosensory hearing loss that correlates with histologic assessments showing massive losses of both IHCs and outer hair cells (OHCs). This mammalian miRNA misexpression model demonstrates a potency and specificity of cochlear homeostasis for one of the dozens of endogenously co-expressed, evolutionally conserved, small non-protein coding miRNA families. It should be a valuable tool to predict and elucidate miRNA-regulated genes and integrated functional gene expression networks that significantly influence neurosensory cell differentiation, maturation and homeostasis.

Diversity of Ca2+-activated K+ channel transcripts in inner ear hair cells.

Hair cells express a complement of ion channels, representing shared and distinct channels that confer distinct electrophysiological signatures for each cell. This diversity is generated by the use of alternative splicing in the alpha subunit, formation of heterotetrameric channels, and combinatorial association with beta subunits. These channels are thought to play a role in the tonotopic gradient observed in the mammalian cochlea. Mouse Kcnma1 transcripts, 5' and 3' ESTs, and genomic sequences were examined for the utilization of alternative splicing in the mouse transcriptome. Comparative genomic analyses investigated the conservation of KCNMA1 splice sites. Genomes of mouse, rat, human, opossum, chicken, frog and zebrafish established that the exon-intron structure and mechanism of KCNMA1 alternative splicing were highly conserved with 6-7 splice sites being utilized. The murine Kcnma1 utilized 6 out of 7 potential splice sites. RT-PCR experiments using murine gene-specific oligonucleotide primers analyzed the scope and variety of Kcnma1 and Kcnmb1-4 expression profiles in the cochlea and inner ear hair cells. In the cochlea splice variants were present representing sites 3, 4, 6, and 7, while site 1 was insertionless and site 2 utilized only exon 10. However, site 5 was not present. Detection of KCNMA1 transcripts and protein exhibited a quantitative longitudinal gradient with a reciprocal gradient found between inner and outer hair cells. Differential expression was also observed in the usage of the long form of the carboxy-terminus tail. These results suggest that a diversity of splice variants exist in rodent cochlear hair cells and this diversity is similar to that observed for non-mammalian vertebrate hair cells, such as chicken and turtle.

Developmental expression of Kcnq4 in vestibular neurons and neurosensory epithelia.

Sensory signal transduction of the inner ear afferent neurons and hair cells (HCs) requires numerous ionic conductances. The KCNQ4 voltage-gated M-type potassium channel is thought to set the resting membrane potential in cochlear HCs. Here we describe the spatiotemporal expression patterns of Kcnq4 and the associated alternative splice forms in the HCs of vestibular labyrinth. Whole mount immunodetection, qualitative and quantitative RT-PCR were performed to characterize the expression patterns of Kcnq4 transcripts and proteins. A topographical expression and upregulation of Kcnq4 during development was observed and indicated that Kcnq4 is not restricted to either a specific vestibular structure or cell type, but is present in afferent calyxes, vestibular ganglion neurons, and both type I and type II HCs. Of the four alternative splice variants, Kcnq4_v1 transcripts were the predominant form in the HCs, while Kcnq4_v3 was the major variant in the vestibular neurons. Differential quantitative expression of Kcnq4_v1 and Kcnq4_v3 were respectively detected in the striolar and extra-striolar regions of the utricle and saccule. Analysis of gerbils and rats yielded results similar to those obtained in mice, suggesting that the spatiotemporal expression pattern of Kcnq4 in the vestibular system is conserved among rodents. Analyses of vestibular HCs of Bdnf conditional mutant mice, which are devoid of any innervation, demonstrate that regulation of Kcnq4 expression in vestibular HCs is independent of innervation.

Determination of hair cell metabolic state in isolated cochlear preparations by two-photon microscopy.

Currently there is no accepted method to measure the metabolic status of the organ of Corti. Since metabolism and mitochondrial dysfunction are expected to play a role in many different hearing disorders, here for the first time we employ two-photon metabolic imaging to assess the metabolic status of the cochlea. When excited with ultrashort pulses of 740-nm light, both inner and outer hair cells in isolated murine cochlear preparations exhibited intrinsic fluorescence. This fluorescence is characterized and shown to be consistent with a mixture of oxidized flavoproteins (Fp) and reduced nicotinamide adenine dinucleotide (NADH). The location of the fluorescence within hair cells is also consistent with the different mitochondrial distributions in these cell types. Treatments with cyanide and mitochondrial uncouplers show that hair cells are metabolically active. Both NADH and Fp in inner hair cells gradually become completely oxidized within 50 min from the time of death of the animal. Outer hair cells show similar trends but are found to have greater variability. We show that it is possible to use two-photon metabolic imaging to assess metabolism in the mouse organ of Corti.

Differential expression of KCNQ4 in inner hair cells and sensory neurons is the basis of progressive high-frequency hearing loss.

Human KCNQ4 mutations known as DFNA2 cause non-syndromic, autosomal-dominant, progressive high-frequency hearing loss in which the cellular and molecular basis is unclear. We provide immunofluorescence data showing that Kcnq4 expression in the adult cochlea has both longitudinal (base to apex) and radial (inner to outer hair cells) gradients. The most intense labeling is in outer hair cells at the apex and in inner hair cells as well as spiral ganglion neurons at the base. Spatiotemporal expression studies show increasing intensity of KCNQ4 protein labeling from postnatal day 21 (P21) to P120 mice that is most apparent in inner hair cells of the middle turn. We have identified four alternative splice variants of Kcnq4 in mice. The alternative use of exons 9-11 produces three transcript variants (v1-v3), whereas the fourth variant (v4) skips all three exons; all variants have the same amino acid sequence at the C termini. Both reverse transcription-PCR and quantitative PCR analyses demonstrate that these variants have differential expression patterns along the length of the mouse organ of Corti and spiral ganglion neurons. Our expression data suggest that the primary defect leading to high-frequency loss in DFNA2 patients may be attributable to high levels of the dysfunctional Kcnq4_v3 variant in the spiral ganglion and inner hair cells in the basal hook region. Progressive hearing loss associated with aging may result from an increasing mutational load expansion toward the apex in inner hair cells and spiral ganglion neurons.

Generation of a Retinoblastoma (Rb)1-inducible dominant-negative (DN) mouse model.

Retinoblastoma 1 (Rb1) is an essential gene regulating cellular proliferation, differentiation, and homeostasis. To exert these functions, Rb1 is recruited and physically interacts with a growing variety of signaling pathways. While Rb1 does not appear to be ubiquitously expressed, its expression has been confirmed in a variety of hematopoietic and neuronal-derived cells, including the inner ear hair cells (HCs). Studies in transgenic mice demonstrate that complete germline or conditional Rb1 deletion leads to abnormal cell proliferation, followed by massive apoptosis; making it difficult to fully address Rb1's biochemical activities. To overcome these limitations, we developed a tetracycline-inducible TetO-CB-myc6-Rb1 (CBRb) mouse model to achieve transient and inducible dominant-negative (DN) inhibition of the endogenous RB1 protein. Our strategy involved fusing the Rb1 gene to the lysosomal protease pre-procathepsin B (CB), thus allowing for further routing of the DN-CBRb fusion protein and its interacting complexes for proteolytic degradation. Moreover, reversibility of the system is achieved upon suppression of doxycycline (Dox) administration. Preliminary characterization of DN-CBRb mice bred to a ubiquitous rtTA mouse line demonstrated a significant inhibition of the endogenous RB1 protein in the inner ear and in a number of other organs where RB1 is expressed. Examination of the postnatal (P) DN-CBRb mice inner ear at P10 and P28 showed the presence of supernumerary inner HCs (IHCs) in the lower turns of the cochleae, which corresponds to the described expression domain of the endogenous Rb1 gene. Selective and reversible suppression of gene expression is both an experimental tool for defining function and a potential means to medical therapy. Given the limitations associated with Rb1-null mice lethality, this model provides a valuable resource for understanding RB1 activity, relative contribution to HC regeneration and its potential therapeutic application.

Tests of linkage and/or association of the LEPR gene polymorphisms with obesity phenotypes in Caucasian nuclear families.

Genetic variations in the leptin receptor (LEPR) gene have been conceived to affect body weight in general populations. In this study, using the tests implemented in the statistical package QTDT, we evaluated association and/or linkage of the LEPR gene with obesity phenotypes in a large sample comprising 1,873 subjects from 405 Caucasian nuclear families. Obesity phenotypes tested include body mass index (BMI), fat mass, percentage fat mass (PFM), and lean mass, with the latter three measured by dual-energy X-ray absorptiometry (DXA). Three single nucleotide polymorphisms (SNPs), namely Lys109Arg (A/G), Lys656Asn (G/C), Pro1019Pro (G/A), in the LEPR gene were analyzed. Significant linkage disequilibrium (0.394 < or = |D'| < or = 0.688, P < 0.001) was observed between pairs of the three SNPs. No significant population stratification was found for any SNP/phenotype. In single-locus analyses, evidence of association was observed for Lys656Asn with lean mass (P = 0.002) and fat mass (P = 0.015). The contribution of this polymorphism to the phenotypic variation of lean mass and fat mass was 2.63% and 1.15%, respectively. Subjects carrying allele G at the Lys656Asn site had, on average, 3.16% higher lean mass and 2.71% higher fat mass than those without it. In the analyses for haplotypes defined by the three SNPs, significant associations were detected between haplotype GCA (P = 0.005) and lean mass. In addition, marginally significant evidence of association was observed for this haplotype with fat mass (P = 0.012). No statistically significant linkage was found, largely due to the limited power of the linkage approach to detect small genetic effects in our data sets. Our results suggest that the LEPR gene polymorphisms contribute to variation in obesity phenotypes.