Zebrafish spontaneously regenerate the retina after injury. Although the gene expression profile has been extensively studied in this species during regeneration, this does not reflect protein function. To further understand the regenerative process in the zebrafish, we compared the proteomic profile of the retina during injury and upon regeneration. Using two-dimensional difference gel electrophoresis (2D-DIGE) and label-free quantitative proteomics (quadrupole time of flight LC-MS/MS), we analysed the retina of adult longfin wildtype zebrafish at 0, 3 and 18 days after Ouabain injection. Gene ontology analysis indicates reduced metabolic processing, and increase in fibrin clot formation, with significant upregulation of fibrinogen gamma polypeptide, apolipoproteins A-Ib and A-II, galectin-1, and vitellogenin-6 during degeneration when compared to normal retina. In addition, cytoskeleton and membrane transport proteins were considerably altered during regeneration, with the highest fold upregulation observed for tubulin beta 2 A, histone H2B and brain type fatty acid binding protein. Key proteins identified in this study may play an important role in the regeneration of the zebrafish retina and investigations on the potential regulation of these proteins may lead to the design of protocols to promote endogenous regeneration of the mammalian retina following retinal degenerative disease. Fish and amphibians have the ability to completely regenerate a functional retina after injury and this capability continues throughout adult life134in vitro1112in vivo. Currently, most of our understanding of the retinal regenerative process in the zebrafish is based upon gene expression of isolated retina914The zebrafish has been increasingly used as a model to study complex tissue regeneration of the retina. Several models of retinal insult in this species including surgical removal, light damage and neurotoxin damage result in complete recovery of the functional retina78The aim of the current study was to identify protein expression changes in the zebrafish retina that may regulate the regenerative processes after injury in this species. We compared the protein expression profile of normal zebrafish retina with that of retina damaged by intravitreal injection of ouabain undergoing degeneration and during the process of regeneration. We conducted 2D Difference Gel Electrophoresis (2D-DIGE) and label free proteomic analysis on these samples. We have shown that metabolic processing is negatively regulated, and fibrin clot formation was upregulated during retinal degeneration in the zebrafish. In addition, apolipoproteins A-Ib and A-II and the carbohydrate-binding protein extracellular matrix factor galectin-1 were highly upregulated in the degenerated zebrafish retina as compared to normal retina. Similarly, in the regenerating retina we observed activation of cytoskeletal and extracellular matrix proteins during regeneration. More specifically, cytoskeletal proteins such as tubulin beta 2A are highly upregulated in the regenerating zebrafish retina as compared to control. The validation of galectin-1 expression was supported by RT-PCR and western blot.Factors identified are likely to be important for the regenerative process in the zebrafish retina and further investigations on the roles of these molecules in the mammalian retina may aid to design regenerative approaches to induce endogenous repair of the human retina.Examination of the zebrafish retina after injection of 200 μM ouabain showed evident morphological changes occurring after toxin-induced damage. Bright field images were taken from the same site of the peripheral retina of treated and control animals at various time intervals after injection . The thiAnalysis of the proteomic profile of zebrafish retina by label free mass spectrometry identified the presence of 2042 proteins in the control retina, 1514 proteins in the degenerated retina and 1445 proteins in the regenerating retina. Further examination of the proteins detected in the zebrafish retinae identified several proteins that were differentially expressed between control, degenerated and regenerating retina. Using the normalised abundancies, proteins considered as differentially expressed were either ≥2-fold upregulated or ≤0.5-fold downregulated between the different time points. In the degenerated retina, 192 proteins were found to be differentially expressed as compared to the control retina, with 21 being ≥2-fold upregulated and 171 being ≤0.5-fold downregulated . Fewer cIndividual protein analysis showed that only one protein, annexin A2A, was ≥2-fold upregulated in both the degenerated and regenerating zebrafish retina as compared to control. In the degenerated retina the glycoprotein vitellogenin 6 exhibited the highest fold upregulation when compared to control retina (32.12-fold increase) . This waApproximately 135 spots were detected in the 2D-DIGE gel containing samples of control, degenerated and regenerating zebrafish retinae . CompariProteins that were differentially expressed between degenerated and control zebrafish retina by 2D-DIGE and label free proteomics were subject to gene ontology (GO) enrichment and pathway analysis to identify key functional areas of interest. Differentially expressed proteins between regenerating retina (18 dpi) and control retina obtained from the label free mass spectrometry were also subject to gene ontology and pathway enrichment. Out of the 55 proteins in this dataset, 16 could not be mapped by the enrichment. It was shown that proteins involved in the structural constituent of the cytoskeleton were the most significantly enriched within this group. These proteins included tubulin alpha 1B chain and an uncharacterised tubulin (Q6PE34), which were ≤0.5-fold downregulated and tubulin beta 2B chain, tubulin beta 5, and vimentin, which were ≥2-fold upregulated in the regenerating retina as compared to control retina. Proteins involved in GTPase activity and transporter activity were also significantly enriched . InteresTo validate our MS proteomic results, we conducted PCR and western blot analyses to examine the mRNA and protein expression. We selected three of the highly upregulated proteins to which antibodies were readily available for the zebrafish, including adenylate kinase isoenzyme1, tubulin beta 2 and galectin 1. Analysis of mRNA for galectin 1 correlated with proteomic results, and showed a significant increase in expression in the degenerated retina (3 dpi) as compared to normal retina (P = 0.0461). In contrast, mRNA levels of tubulin beta 2 did not correlate to the proteomics results and showed a significant decrease in the degenerated retina as compared to control (P = 0.0270) that returned to normal levels in the regenerating retina. A significant reduction in mRNA expression of adenylate kinase isoenzyme was observed in the degenerated retina as compared to control, and regenerating retina (P < 0.0020) . SimilarThis study has applied a proteomics approach to investigate molecular events occurring during degeneration and regeneration in the zebrafish retina. We observed that upon degeneration proteins involved in fibrin deposition and cytoskeletal proteins were highly upregulated, whereas energy metabolism in the retina is mostly negatively regulated. Similarly in the regenerating retina, cytoskeletal proteins such as tubulin beta 2 were highly upregulated and proteins involved in transporter activity were downregulated. Validations of galectin1 by western blot and RT-PCR correlated with results. However mRNA and protein expression of tubulin beta 2 and adenylate kinase largely conflicted with proteomic findings. The differences for beta tubulin 2 expression can be accounted for as fragments of this particular protein was identified across seven fractions and then averaged, as generated by Progenesis software. In comparison, galectin1 and adenylate kinase was identified in only one out of the ten fractions analysed. It is also well documented that mRNA and protein expression levels may differ in the same systemZebrafish retinae are small in size (1 mm × 1 mm), hence samples used in this study were pooled to obtain a single representative sample. Creating a sample pool can also reduce the sample bias and biological variance, leading to more precise data estimatesAnalyses of zebrafish retina by 2D-DIGE and label-free mass spectrometry were comparable, supporting the validity of the results. Data presented by 2D-DIGE analysis showed that apolipoprotein A-1b (ApoA1b), fibrinogen beta polypeptide, and heat shock protein 90-alpha2 were ≥2-fold upregulated in the degenerating retina as compared to the control retina. These results supported the findings from the label free mass spectrometry analysis, which also showed upregulation of apolipoproteins ApoA2, ApoA1b and ApoC in the degenerated retina. Previous observations have also shown increased apolipoprotein expression, including ApoA1 and ApoE, after injury in zebrafish tissues, that are capable of regeneration including the eye and fin20Gene ontology and pathway enrichment analyses of zebrafish proteins can be challenging due to the incomplete proteome database for this species. This may lead to partial or incorrect annotation of the dataset, and results should therefore be carefully analysed. Despite these limitations gene ontology analysis showed that proteins involved in energy metabolism were highly represented. However, in both the degenerated (3 dpi) and regenerating (18 dpi) retina the majority of these proteins were negatively regulated in comparison to control retina. Metabolic regulation in the retina is tightly controlled and is one of the most energy demanding tissues, with photo-transduction, neurotransmitter signalling, oxygen consumption and glucose metabolism all playing a significant role in energy consumption25in vitro investigations of vitellogenins have shown hemagglutination activity, as well as inhibition of bacterial growthVitellogenin 6, a glycoprotein involved in the response to activation of oestrogen receptors, showed the highest fold upregulation in the degenerated retina as compared to control retina. Although vitellogenin has been mainly described in the embryo and early developmental stages, expression has also been observed in adult tissues such as skin, brain and eye, however the function in these tissues is largely unknown29Many changes were observed in the regulation of cytoskeleton proteins between the control, degenerated and regenerating retina. Specifically, striking changes in the expression of several tubulins were observed between the control, degenerated and regenerating retina, with tubulin beta 2A showing the greatest fold increase in the regenerating retina as compared to control. Various tubulins are involved in retina remodelling and previous studies have shown changes in α-1 tubulin levels in Müller glia from the regenerating zebrafish retina37Neural retina remodelling may also occur through extracellular matrix regulation. Galectin 1, a beta galactoside-binding protein, was observed to be 2-fold upregulated in the degenerated zebrafish retina as compared to controls. This protein is expressed in the cytoplasm and can be translocated into the nucleus, or secreted into the extracellular space, where it can bind to cell surface. Galectin-1 plays a role in cell adhesion, transmembrane signalling and cell-cell interactions as well as inflammatory responsesIn conclusion, comparison of protein expression profile between the degenerated and regenerating zebrafish retina has highlighted key differences in protein expression. The data presented suggest that retinal regeneration in this species is likely to involve complex cellular processes involving proliferation, remodelling and metabolic changes, which might influence one another throughout the process. The study has highlighted proteins that may be important to examine for their function in order to create a permissible environment for endogenous regeneration of the mammalian retina.et al., in which an injection of 200 μM was given to yield a final concentration of 10 μM in the vitreousLongfin wildtype Zebrafish, 7–9 months were obtained from UCL biological services Fish facility and maintained according to local and Home Office regulations for the care and use of laboratory animals and the UK (Scientific Procedures) Act at the UCL Institute of Ophthalmology. UCL approved all procedures for experimental protocols, which were conducted in accordance with the approved guidelines at UCL Institute of Ophthalmology. Zebrafish were anaesthetised using 1 mg/ml Tricane in aquarium water for 1 min. Injections were performed by making an incision into the temporal side of the zebrafish eye using a double edge sapphire blade , where the needle was inserted and Ouabain  was injected intravitreally. The needle set up consisted of a 10 μl Gastight Hamilton syringe  with a retinal pigment epithelium (RPE) kit . A total of 0.2–0.4 μl of 200 μM Ouabain in injectable saline solution was injected into each eye to produce a final intravitreal concentration of 10 μM. The protocol used was based upon the original paper by Sherpa Zebrafish eyes were fixed for 24 hrs in 4% paraformaldehyde (PFA) at 4 °C. They were dehydrated through a graded ethanol series and then embedded in JB-4 plastic resin as described previouslyRetinal samples were washed in phosphate buffered saline (PBS) prior to homogenisation in the presence of 50 nM Ammonium Bicarbonate + 2%ASB-14  pH8.2, followed by sonication. Protein content was estimated using the Bicinchoninic Acid protein assay kit . Samples were pooled to obtain three separate conditions, consisting of control, degenerated and regenerating zebrafish retina to a total of 80 μg. Samples were run on a 1D gel  as previously describedhttp://www.pantherdb.org/), and gene ontology classification by WebGestalt .Data was processed using ProteinLynx Global Server (PLGS) 2.4 software  and peptides were matched using downloaded Uniprot reference proteome for Danio rerio. The protein sequence for the internal standard, yeast enolase1 (Accession: P00924) was also added to the database. Identified peptide data was imported into Progensis LC-MS (non-linear dynamics) for comparative analysis. Peptides were normalised to housekeeping protein beta actin (E9QD59). Further bioinformatics analysis of the results were conducted on web-based platforms which included the Panther Classification System , given an n number of 1 for each condition. Therefore the high cut-off values of ≥2-fold upregulated or ≤0.5-fold downregulated were chosen to obtain higher confidence in analysing the data from a pooled cohort.http://proteomecentral.proteomexchange.org) via the PRIDE partner repositoryThe mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium database .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Vitamin E is one of the 13 vitamins that are essential to animals that do not produce them. To date, six natural organic compounds belonging to the chemical family of tocochromanols—four tocopherols and two tocotrienols—have been demonstrated as exhibiting vitamin E activity in animals. Edible plant-derived products, notably seed oils, are the main sources of vitamin E in the human diet. Although this vitamin is readily available, independent nutritional surveys have shown that human populations do not consume enough vitamin E, and suffer from mild to severe deficiency. Tocochromanols are mostly produced by plants, algae, and some cyanobacteria. Tocochromanol metabolism has been mainly studied in higher plants that produce tocopherols, tocotrienols, plastochromanol-8, and tocomonoenols. In contrast to the tocochromanol biosynthetic pathways that are well characterized, our understanding of the physiological and molecular mechanisms regulating tocochromanol biosynthesis is in its infancy. Although it is known that tocochromanol biosynthesis is strongly conditioned by the availability in homogentisate and polyprenyl pyrophosphate, its polar and lipophilic biosynthetic precursors, respectively, the mechanisms regulating their biosyntheses are barely known. This review summarizes our current knowledge of tocochromanol biosynthesis in plants, and highlights future challenges regarding the understanding of its regulation. Natural compounds exhibiting vitamin E activity in animal cells belong to the chemical family of tocochromanols, a group of organic molecules with a polar chromanol ring and lipophilic polyprenyl side chain that varies according to the type of tocochromanol . The polfactor X”, as it was originally named, was identified as α-tocopherol [The first article published on vitamin E relates the finding of an unknown nutritional factor that prevents embryo resorption during rodent gestation . Severalcopherol . In addicopherol , notablycopherol , by delacopherol , and by copherol ,8. Althocopherol ,11,12,13Plasmodium falciparum, a non-photosynthetic parasite that causes malaria, synthesizes both α- and γ-tocopherols during its intraerythrocytic stages to avoid oxidative stress [Synechocystis. The present review summarizes our current knowledge on tocochromanol metabolism in plants, including the core tocochromanol biosynthetic pathway that is now well delineated  as a methyl donor [vte1-1 mutant lack all tocopherols, PC-8, and γ-tocomonoenol, indicating that the cyclase indiscriminately uses mono- and dimethyl-benzoquinols carrying either a phytyl, a solanesyl, or a tetrahydrogeranylgeranyl side chain as substrates [HGGT gene notably produce α-tocotrienol [Tocochromanol biosynthesis is initiated by the condensation of the polar aromatic head HGA with various lipophilic polyprenyl pyrophosphates that determine the type of tocochromanol. PPP is the lipophilic biosynthetic precursor of tocopherols, while GGPP is the one for tocotrienols, as is SPP for PC-8, and THGGPP for tocomonoenols  in wild-type Arabidopsis and maize leaves, methyl PC-8 (α-tocochromanol) has not been detected in any wild-type tissue analyzed to date . In contromanols . This laotrienol . In contotrienol . In addin leaves . CollectTMT genes in Arabidopsis and Brassica napus is sufficient to trigger the quantitative accumulation of both α-tocopherol and methyl PC-8 in transgenic seeds, thus demonstrating that the lack of α-tocochromanols in the seeds of these species results from the very low transcriptional activity of the γ-TMT/VTE4 gene in seeds [TMT/VTE4 expression, notably in seeds, is currently limited to a single study performed in soybean. Tocopherol analysis in seeds of 1109 wild and cultivated soybean varieties showed that while almost all of them produce primarily γ-tocopherol, three varieties accumulate up to seven times more α-tocopherol [TMT3 promoter correlated with the higher expression of the methyl transferase, and thus that α-tocopherol level is transcriptionally regulated in soybean seeds as well [TMT/VTE4 gene. To date, despite many independent research initiatives that led to the identification of numerous α-tocopherol QTLs in Arabidopsis [TMT genes in plants is still unknown.In contrast to leaves, the tocochromanol composition of wild-type seeds is much more variable, and three groups of plant species can be currently distinguished according to this criterion. The seeds of some plant species accumulate mostly γ-tocochromanols and very little or no α-tocochromanols. This is notably the case of wild-type Arabidopsis seeds, which accumulate mostly γ-tocopherol and PC-8, very low levels of α-tocopherol, and no methyl PC-8 . The ovein seeds ,35,38. Iin seeds ,40. Thisin seeds ,42,43. Ocopherol . Quantit as well . Collectbidopsis , soybeanbidopsis ,47,48, mbidopsis ,50, wintbidopsis ,52, and bidopsis , the tral-tyrosine (l-tyr), which is produced by the plastidic shikimate pathway (l-tyr is first converted into 4-hydroxyphenylpyruvate (HPP) by tyrosine aminotransferases (TATs). Among the six to 10 TAT genes identified in the genetic model Arabidopsis, the enzymatic activity has been experimentally confirmed only for TAT1  and TAT2 [TAT1/TAT7 and HPPD genes have shown that both lack the typical sequence encoding for a chloroplast transit peptide, suggesting that TAT1/AtTAT7 and HPPD are localized in the cytoplasm [Early feeding experiments of safflower, sunflower, and soybean cell cultures with HGA have shown that an exogenous HGA supply significantly increased tocochromanol biosynthesis in plant cells ,55,56. T pathway . l-tyr iand TAT2 ,58,59,60and TAT2 . In the ytoplasm .HPPD genes isolated from barley, carrot, and coleus also lack chloroplast transit peptide sequences [l-tyr/HPP into the cytoplasm, and other one(s) that import HGA back into chloroplasts  importing HGA back into chloroplasts, are potentially limiting factors for tocochromanol accumulation. To date, neither the membrane chloroplast transporters exporting l-tyr and HPP in the cytoplasm, nor the one importing HGA back into chloroplasts, have been identified in Arabidopsis. In contrast, a cationic amino acid transporter localized in the chloroplast membrane and capable of exporting phenylalanine, tryptophan, and l-tyr from the stroma into the cytosol has been identified in petunia [Genetic data obtained from soybean further illustrate how the cytoplasmic HGA biosynthesis and the exchanges of tocochromanol biosynthetic precursors between chloroplasts and the cytoplasm participate in the regulation of tocochromanol biosynthesis in plants. In this species, the HPPD activity is encoded by a single gene that exhibits two transcription start sites. It has been shown that the long transcript encodes a polypeptide imported into the chloroplast, while the short one encodes a polypeptide that remains in the cytoplasm . This inoacetate , exhibit petunia .l-tyr into chorismate, prephenate, and arogenate  correlates with a much higher accumulation of free HGA [CM/PDH or PDH in chloroplasts implies that HPP is efficiently exported into the cytosol where the HPPD activity is localized, and that the cytoplasmic HGA is imported back into chloroplasts to sustain tocochromanol biosynthesis  and HPP/HGA trafficking are both not so limiting for leaf tocochromanol metabolism in Arabidopsis. In contrast to this, the situation seems very different in tobacco leaves; while the overexpression of the yeast PDH gene does not significantly alter tocochromanol accumulation in transgenic tobacco leaves, its overexpression in combination with HPPD fused to a chloroplast targeting sequence strongly increases tocochromanol accumulation [Interestingly, the overexpression of bacterial ith HPPD ,36. Compfree HGA . This inynthesis . Since tmulation . The reaCM/PDH in combination with HPPD in Arabidopsis stimulates seed tocochromanol accumulation, whereas the expression of CM/PDH alone does not significantly alter it  is determined by the polyprenyl pyrophosphate substrate condensed to HGA, the biosynthetic pathways producing the lipophilic tocochromanol precursors  are key components in the regulation of tocochromanol biosynthesis. To date, although the tocotrienol biosynthetic pathway is well characterized, and key tocotrienol biosynthetic genes have been identified in plants, there are currently no data describing the mechanisms regulating the biosynthesis of GGPP used for tocotrienol biosynthesis . This manstructs ,36,56,66Nicotinamide Adenine Dinucleotide Phosphate (NADPH) dehydrogenase C1  is a type II NAD(P)H quinone oxidoreductase that reduces PQ-9 into PQH2-9, using ferredoxin as an electron donor [ndc1 mutants, the leaf PQ pool is more oxidized than in wild-type controls, while PC-8 amounts are reduced by ca 66% [2-9 into PC-8—preferentially using reduced prenyl quinones [ABC1K1 (AT4G31390) and K3 (AT1G79600), have been shown to regulate PQ-9/PC-8 metabolism in Arabidopsis [abc1k1 and abc1k3 mutants exhibit altered PQ-9/PC-8 metabolism. Although the function of these atypical kinases in PQ-9/PC-8 metabolism has been clearly demonstrated, their mode(s) of action remains to be determined. It has been shown that both ABC1K1 and K3 phosphorylate the tocopherol cyclase VTE1. Since the biosynthesis of tocopherols, which requires VTE1 activity as well, was not affected in abc1k3 leaves, it is unlikely that the ABC1K3 mode of action is mediated by VTE1 phosphorylation. In contrast, tocopherol amounts were significantly reduced in abc1k1 leaves, suggesting that the ABC1K1 mode of action might involve the regulation of the tocopherol cyclase activity. The role of ABC1K1 and K3 in tocochromanol metabolism might also be mediated by the synthesis of fribrillin, a group of lipid-associated proteins that are localized in chloroplasts and essential for the biosynthesis of PQ-9, and thus PC-8 [fibrillin-5 mutants exhibit lower PQ-9/PC-8 amounts, and it was shown that mutation in abc1k3 strongly reduces the accumulation of fibrillin proteins [The biosynthesis of PC-8 and its solanesyl benzoquinone precursor plastoquinone-9 (PQ-9) is initiated by the homogentisate solanesyltransferase-dependent condensation of HGA with SPP that comes from the preferential concatenation of GGPP and isopentenyl pyrophosphate ,69,70,71on donor . This eny ca 66% . In contquinones . Two ABCbidopsis ,76. Bothhus PC-8 ,78. Indeproteins .vte5 that lacks 80% and 65% of the tocopherols in seeds and leaves, respectively [g4/chlsyn1 mutant, which carries a mutated chlorophyll synthase that esterifies geranylgeranyl pyrophosphate on chlorophyllide a [g4/chlsyn1 seeds lack 75% of the tocopherols, thus indicating that tocopherol biosynthesis is also mostly chlorophyll-dependent in Arabidopsis seeds as well. Collectively, these data demonstrate that the PPP used for tocopherol biosynthesis primarily originates from the recycling of phytol released during chlorophyll catabolism in both leaves and seeds. This biosynthetic model, including details about enzymes such as Mg-dechelatase, chlorophyllases, and pheophytinase, has been very recently reviewed, and will not be developed here [Regarding the regulation of tocopherol biosynthesis, it is known that the availability of phytol is a major parameter governing the final amount of tocopherols in a plant tissue. It has been shown, for instance, that the supplementation of plant cell suspensions with free phytol is sufficient to significantly increase tocopherol accumulation in these cells ,55,56. Mectively . VTE5 isyllide a . The leaped here .a priori do not synthetize chlorophylls? A comprehensive analysis of vitamin E natural variation in maize grain recently provided an unexpected answer to this puzzling question. Among the 52 QTLs identified for maize grain tocochromanols, QTL5 and QTL24 exhibited scores for phenotypic variance explained among the highest of all identified QTLs [a, of chlorophylls a and b, and of the first chlorophyll catabolite that lacks the Mg atom pheophytin a. In addition, endosperm also accumulated detectable traces of chlorophyll a. Although further studies—notably with genetic evidence—are required to definitively link chlorophyll metabolism and tocopherol biosynthesis in leucoembryophyte seeds, these data indicate that leucoembryophyte seeds might produce tocopherols via the same mechanism originally described in chloroembryophyte seeds [Plasmodium falciparum [If one can easily understand the origin of PPP in the seeds of chloroembryophytes such Arabidopsis or rapeseed, which accumulate significant amounts of chlorophylls in developing embryos, how do leucoembryophyte seeds produce tocopherols, since they ied QTLs . Interesied QTLs . This rete seeds ,83. Thislciparum ,15,16.enhanced vitamin e (eve) 1 and 4 mutants, have been isolated by forward genetics [eve1 and eve4 mutants overaccumulate tocopherols, PC-8, and γ-tocomonoenol, a monounsaturated form of tocochromanols. Interestingly, both eve1 and 4 seeds did not accumulate any tocotrienols, such as transgenic Arabidopsis plants overaccumulating HGA, suggesting that although HGA metabolism is likely increased in these mutants to sustain tocochromanol metabolism, HGA overaccumulation is not the primary source of its enhancement. In addition, γ-tocomonoenol accumulation has never been reported in Arabidopsis HGA overaccumulating lines, further suggesting that the misregulation of HGA biosynthesis is likely not the primary source of the enhancement of tocochromanol accumulation in eve1 and eve4 seeds. The fact that all tocochromanol forms are overaccumulated in eve1 and eve4 seeds likely suggests that both mutations affect the chloroplastic isoprenoid metabolism produced by the methyl erythritol phosphate pathway . The aus embryo . This inas NADPH . Interesl-tyr and chlorophylls, involves a yet-unknown transport system between the cytoplasm and the stroma in plant species in which HGA biosynthesis is localized in the cytoplasm, and is interconnected to fatty acid biosynthesis in plastids, render the understanding of its regulation particularly challenging. Indeed, understanding the regulation of vitamin E biosynthesis will imply that we take up the challenges to understand the regulation of each of these numerous events. Beyond understanding the regulation of vitamin E biosynthesis in plants, it seems important that medical scientists assess the sanitary consequences of the significant vitamin E deficiency independently detected in human populations, including in developed countries. The fundamental role of this vitamin in human reproduction and its benefit in current widespread diseases such as high cholesterol and neurodegenerative pathologies makes it a candidate of choice to improve human health.From the first purification of α-tocopherol from wheat germ oil to the latest model of the tocochromanol biosynthetic pathways introducing tocomonoenol biosynthetic genes, our understanding of vitamin E biosynthesis in plants has been considerably enriched ,21. In c


Objective: This study aims to ascertain the health and lifestyle factors associated with the activities of daily living (ADL) disability of centenarians in rural Chongqing, China. Method: 564 centenarians living in rural Chongqing were selected for this cross-sectional study. Demographic characteristics and self-reported lifestyle factors were obtained from face-to-face interviews. ADL disability was measured using the Katz Activities of Daily Living Scale. Result: Among the respondents, 65.7% were considered ADL disability centenarians. Multivariable logistic regression analysis showed that preference for salt, drinking habits, social activities, physical activity, and failure to follow good diet habits were significantly associated with the ADL disability of centenarians. Conclusion: ADL disability of centenarians was associated with certain lifestyle habits. This outcome suggested that target intervention may help maintain ADL independence even among the oldest of the elderly population. A clear disparity exists between its urban and rural areas. Data from the Chongqing Municipal Bureau show that Chongqing became an aging society in 2000, and the proportions of people aged 65 and over from 2013 to 2015 are 11.88%, 12.02%, and 12.17%, respectively [As aging intensifies, it causes numerous social problems, including those related to health care and shelter for the elderly, that situation is also very serious in Chongqing, China. Chongqing, located in Southwest China, is a fast-growing city with a total area of 824 thousand square kilometres and a permanent population of 3371.84 million [Centenarians should be the focus of attention, as their morbidity is higher and their self-care ability is lower relative to the average elderly. Therefore, they are strongly dependent on society and others . Having Activities of daily living (ADLs) are the essential behaviors that prove the capability of a person to live independently . UnderstExtant literature identified several factors of physical function and disability in elders ,22,23,24The design and methods of this research  have been reported previously . This woA total of 877 individuals (173 men and 704 women) were identified as aged 100 or above. Examining the hukou and current residences of all participants led to the choice of 606 centenarians who had rural hukou as the target sample. Hukou is the household registration system in China . People Exclusion criteria of this study include the following: (1) centenarians with rural hukou but who migrated to urban areas, (2) participants with missing data on ADL status, and (3) respondents with missing data on lifestyle items (more than three items). Forty-two participants were excluded, including seven centenarians who were not considered “real” rural centenarians, 12 individuals with missing ADL status, and 23 individuals with more than three lifestyle items whose data were missing. Finally, 564 centenarians  were included in our survey . All participants were informed of the purpose of the study and their cooperation was voluntary. All participants were interviewed and provided oral consent. Consent was also obtained for the examination of the participants’ ID cards and hukou. To ensure anonymity, their names were not required in the questionnaire. Instead, each centenarian was given a unique code that was written on the questionnaire. An ethical clearance was also obtained from the local health authorities in Chongqing.Face-to-face interview questionnaires were administered by well-trained researchers from local health education institutes and CDCs to collect information on demographic characteristics, health conditions, self-reported lifestyle behaviors, and ADL status. In rare cases , relatives of the participants were interviewed.Demographic characteristics included age, gender, and ethnicity. Date of birth and ethnicity were ascertained from national ID cards. Ethnicity was categorized as Han or ethnic minority. Participants were asked whether they had any current chronic conditions including cancer, stroke, heart diseases, dementia, diabetes, hypertension, osteoarthritis, cardiovascular disease, respiratory disease, and vision problems.Self-reported lifestyle behaviors included living arrangement, smoking, drinking habit, regular diet, avoiding sweet, high-cholesterol, and fatty food, salt preference, social activities, physical activity, reading, and watching TV or listening to radio. Living arrangement was classified as living alone and living with others. Participants were identified as smokers or non-smokers. Note that both current smokers and ex-smokers were defined as smokers. Drinking was classified into “no drinking” and “regular” drinking. The latter denoted alcohol consumption of more than three times per week for at least one year. Good diet habits refer to having various types of food including staple food, vegetable and/or fruits, and protein-rich food. Information on the diet habits of participants was determined through the following three questions: (1) “Do you eat staple food in every meal?”; (2) “Do you eat a vegetable and/or fruit every day?”; and (3) “Do you eat protein-rich food such as meat, fish, egg, dairy and soybean every day?” Those who reported in the affirmative for all items were categorized as having good diet habits. Salt preference was assessed through two questions: (1) “Do you like salty food such as pickles, preserved meat and salted fish?” and (2) “Do you add extra salt or salt seasoning while having meals?" Participants who responded in the affirmative for at least one of the two items were classified as having salt preference. Social activities were described as visiting or communicating with others, visiting community centers, and participating in recreational activities, such as playing cards or mah-jong (Chinese game played by four people with 144 tiles) with others. Frequency of social activities was classified into three levels: never or rarely (less than once per week), sometimes (1–2 times per week), and often (more than twice per week). In our study, we dichotomized responses for social participation into “yes” or “no” categories: “yes” if the answer was “sometimes” or “often” and “no” if the answer was “never or rarely.” Information on physical activity was collected through the question: “Do you often do housework or have regular physical exercise such as taking a walk, jogging, and Qigong ?” Yes or no answers were recorded accordingly.The ADL status of centenarians was obtained from interviews. The Katz Activities of Daily Living Scale  was utilAll survey data were encoded using Epidata 3.0. Statistical software  was utilized for statistical data analysis. A Pearson chi-square test was used to compare all demographic and lifestyle characteristics between the ADL-able group and ADL-disabled groups. Logistic regression analysis was conducted to assess the association between such characteristics and ADL disability. The factors associated with ADL disability were examined through bivariate and multivariable logistic regression models. Statistical significance was set at 0.05 (two sides). p = 0.031). The prevalence of ADL disability was significantly higher in the participants who reported not having good diet habits (p = 0.003), having a salt preference (p = 0.028), drinking (p = 0.000), not participating in social activities (p = 0.011), and lacking physical activities (p = 0.005) (= 0.005) . The results of multivariable logistic regression analysis, which are similar to the results of bivariate analysis, indicated that females and participants who have current disease were more likely to suffer ADL disability, and four lifestyle factors remained significantly associated with better ADL status . ParticiThe findings and their implications should be examined in the broadest context possible. Future research directions may also be highlighted. Results in the present study indicated an association between ADL disability and several lifestyle factors among centenarians in rural Chongqing. Females were more likely to be ADL-disabled. Chronic diseases increased the likelihood of ADL disability. The absence of good diet habits in centenarians was associated with ADL disability. Light drinking was strongly linked with ADL independence among the respondents. Participants who preferred salty food had poorer ADL functionality relative to those without such preference. Lack of socialization with others was also related to ADL dependence among rural centenarians. Centenarians who lacked physical activities also had poorer ADL ability than their active counterparts. Female centenarians in rural Chongqing were more likely to be ADL-disabled. This finding is consistent with previous studies ,29. A reChronic disease was associated with ADL disability among the oldest of the study population. Previous studies have reported a close association between chronic condition and physical decline or disability in elders ,32. The Failing to maintain good diet habits was linked with ADL disability among centenarians. Food is beneficial for the physical performance of the elderly. Existing literature found that dairy products, vegetables, and fruits are beneficial for physical function in elders . Eating Centenarians with salt preference are more likely to be ADL disability. To our knowledge, few studies have investigated the association between salt preference and ADL disability in a long-lived population. Two possible explanations for the association are as follows. First, centenarians who preferred the salty taste may have excessive salt intake. Such high intake is a direct risk factor for many chronic diseases, including cardiovascular diseases and strokes, resulting in disability ,37. SecoLack of physical activity was associated with ADL disability among centenarians in rural Chongqing. It was known that physical activity benefits the physical health and functionality of elders. As a recent article reports, the elevated level of physical activity among Sardinian centenarians was associated with high ADL scores . Our finLack of social activities was also linked to increasing odds of ADL disability. This finding is consistent with a previous centenarian study in Korea . Extant Any study of centenarians must take into account age inflation ,45. We cOur study has several limitations. First, this research employs a cross-sectional approach and cannot identify the causal relationship between lifestyle factors and ADL ability. Further longitudinal studies are necessary to confirm the relationship between lifestyle and ADL ability among centenarians. Second, in our study, impairment was defined as “cannot do it at all without help.” The ADL items were dichotomized into two categories, yes and no, and we did not record the specific options for these six themes. This lack of detail may have excluded some ADL disability, which means that these results might not recur in other age populations. In rare cases , relatives of the participants were interviewed. We did not record this proportion, and such omission may cause information bias. Third, information of diet habits was based only on self-reported frequency intake of food items. The quantity of the food and nutrients was not analyzed. Furthermore, we asked about salt preference, but did not investigate their actual salt intake. Food preference might be an important determinant of food intake, but the two variables are different. Thus, further research is warranted to collect more detailed information on these two items in Chongqing’s centenarians. Fourth, due to design limitations, some confounding factors, such as education level, body mass index, and cognitive status, were not investigated. Previous studies have found that cognitive abilities are associated with physical disability . Body maIn this research, centenarians in rural Chongqing, China, with beneficial lifestyle habits  had better ADL status. This finding indicates that targeted intervention may help maintain ADL independence even among the oldest in the elderly population. Further longitudinal studies should be conducted to elucidate the factors associated with ADL independence in the extremely elderly population.


Chronic inflammatory demyelinating polyneuropathy (CIDP) is presented by a large heterogeneity of clinical phenotypes. Around 50% of patients suffer from typical CIDP and show better therapy response than atypical variants. The goal of our study was to search for cellular immunological differences in typical versus atypical CIDP in comparison to controls.We evaluated 26  patients with mainly active-unstable CIDP using clinical and immunological examinations  in comparison to 28 healthy, age-matched controls (HC). Typical or atypical CIDP measurements were compared with HC using Kruskal-Wallis test.Atypical CIDP patients showed increased frequencies of T cell subsets, especially CD4+ effector memory T cells (TEM) and CD4+ central memory T cells (TCM) as well as a tendency of higher T cell responses against the peripheral myelin antigens of PMP-22, P2, P0 and MBP peptides compared to typical CIDP. Searching for novel auto-antigens, we found that T cell responses against P0 180-199 as well as MBP 82-100 were significantly elevated in atypical CIDP patients vs. HC.Our results indicate differences in underlying T cell responses between atypical and typical CIDP characterized by a higher peripheral myelin antigen-specific T cell responses as well as a specific altered CD4+ memory compartment in atypical CIDP. Larger multi-center studies study are warranted in order to characterize T cell auto-reactivity in atypical CIDP subgroups in order to establish immunological markers as a diagnostic tool. Distal Acquired Demyelinating Polyneuropathy (DADS) in 25-35% of the cases, Multifocal Acquired Demyelinating Sensory And Motor Polyneuropathy (MADSAM) in 15% and rare variants such as pure sensory CIDP (10-13%), pure motor CIDP (<10%) and focal CIDP (2%) [Chronic inflammatory demyelinating polyneuropathy (CIDP) is the most common autoimmune peripheral neuropathy but remains a rare disease with a prevalence of 0.8-8.9 cases per 100.000 . The disDespite recent progress, the underlying immunopathogenetic mechanisms remain poorly understood . Both huPeripheral myelin antigens are promising auto-antigens in CIDP pathogenesis. Recently, we demonstrated higher frequencies of auto-reactive IFN-γ responses directed against the peripheral myelin antigens PMP-22 and P2 in treatment naïve patients who responded subsequently well to intravenous immunoglobulin (IVIG) treatment. Clinical improvement under IVIG-treatment correlated with the reduction of antigen-specific responses against PMP-22 and P2 .knockout and TNF-α knockout mice [Experimental studies in the EAN model of Guillain-Barré-Syndrom (GBS) support a pathogenic role of another compact myelin P0. Immunization with P0 180-199 is capable to induce EAN in wildtype-, IFN-γ out mice . HoweverMyelin basic protein (MBP) is a major constituent of the myelin sheath in the central and peripheral nervous system . WhereasT cells can be differentiated into CD45RA+ CCR7+ naïve, CD45RA- CCR7- effector memory (TEM), CD45RA- CCR7+ central memory (TCM) and CD45RA+ CCR7-terminally differentiated effector memory (TEMRA) T cells . EspeciaHere, we hypothesize that autoreactive myelin-specific T cell responses as well as T cell memory subsets differ between atypical and typical manifestations of CIDP.We evaluated 26 CIDP patients using clinical and immunological  examinations in comparison to 28 healthy, age-matched controls. CIDP patients who met the diagnostic criteria of European Federation of Neurological Sciences (EFNS) 2010 were divided into “typical” vs. “atypical” according to EFNS 2010 [ELISPOT assay was performed using peptides of seven peripheral myelin antigens and CEF as positive control for T cell responses Table . CEF is 7cells/ml in freezing medium A  at 4 °C. The same volume of freezing medium B  at 4 °C was added before cell suspensions were transferred into cryovials  and set in one at 4 °C prechilled Nalgene Cryogenic Freezing Container  which was placed in −80 °C overnight. After 12-24 h, cryovials were transferred into liquid nitrogen tanks for storage until ELISPOT.To evaluate T cell responses efficiently we preserved PBMC in liquid nitrogen over a maximum of 6 months. Blood was sampled in CPT tubes for ELISPOT and in EDTA tubes for flow cytometry. PBMC were isolated within 2 h after venipuncture by 1500 g centrifugation for 20 min. After washing, we diluted the PBMC at a concentration of 2x10Thawed cell suspensions were transferred into a 15 ml tube containing 10 ml of ice cold PBS. After two washing steps, cells were pipetted in complete RPMI medium  and counted manually using Trypan blue-staining and light microscopy.5cells/well in triplicates for each antigen and positive (CEF) or negative control (medium). CEF, a peptide pool containing viral antigens functioning as a positive control for T cell responses, was added at 9 μg/ml [6 cells.IFN-γ ELISPOT assay in this study was performed on human PBMC as previously described . We plat 9 μg/ml . The perFlow cytometry analyses were performed on lymphocyte- and T cell-subpopulations in EDTA whole blood within 12 h after venipuncture.Flow cytometric analysis was performed as we described recently . Brieflyt-test or Mann-Whitney-test when p < 0.05. For group differences with regard to sex, prior treatment, disease activity and therapy response, Fisher’s exact test was used. For age and INCAT score, unpaired t-test was used. For time since diagnosis, Mann-Whitney-test was used. Level of significance was defined as p < 0.05 for all comparative tests.All statistical tests were performed using GraphPadPrism 6.0 software. The study was assessed as an exploratory analysis. Typical or atypical CIDP measurements were compared with healthy, age-matched controls (HC) using Kruskal-Wallis test followed by post-hoc unpaired We recruited 17 (65.4%) male and nine (35.6%) female patients. Mean age was 59 years (range 32-78). 20/26 (76.9%) patients were included in active-unstable stages of the disease, 1 (3.8%) with active-stable CIDP and five (19.2%) in clinical remission . 12 (46.Therapy-responders were classified as 9/9 (100%) typical and only 8/17 (47%) atypical CIDP patients. As controls we used age-matched, healthy patients (HC). For ELISPOT-analyses 14 HC  and for FACS-analyses 28 HC  were evaluated.T cell responses against the peripheral myelin antigens, P0 180-199 and MBP 82-100 were measured by IFN-γ ELISPOT in a cohort of 26 CIDP patients. Due to spontaneous IFN-γ-production, 6 patients  were excluded for further ELISPOT analysis.p < 0.05), MBP 82-100 (p < 0.001) , PMP-22 51-64 (p = 0.1050), PMP-22 120-130 (p = 0.1451), P0 180-199 (p = 0.1894) and MBP 82-100 (p = 0.1841) , PMP-22 51-64 , PMP-22 120-130 , P2 14-25 , P0 180-199 , MBP 82-100   and CD4+ T cells (p < 0.001) were higher in patients with atypical CIDP variants in comparison to typical CIDP patients  and CD4+ TCM (p < 0.01) in Fig. Investigating CD4+ T cell subpopulations, CD4+ memory T cell subsets were significantly increased in atypical vs. typical CIDP patients, as shown for CD4+ TEM (p = 0.1745) and CD8+ TCM (p = 0.1475) tended to be increased in atypical compared to typical CIDP patients. Further compared to HC, atypical CIDP patients had significantly elevated CD8+ TCM-frequencies (p < 0.05)  Fig. .In the present study, typical CIDP differed from the group of atypical variants. Here, we found a stronger activated immune system in patients suffering from atypical variants of CIDP defined by a trend towards increased peripheral myelin antigen-specific  T cell responses associated with a specific altered CD4+ memory compartment of increased CD4+ TEM and CD4+ TCM counts in the blood. Further we detected elevated T cell responses against antigens P0 180-199 and MBP 82-100 in CIDP patients which have not described before.We confirmed or previous findings that changes of the T memory compartment is a common finding especially in untreated patients , 17, whiRecently, it has been demonstrated that CIDP patients show a diminished pro-regenerative function of Schwann cells leading to the axonal loss and therefore incomplete clinical recovery after treatment which is probably caused by inflammatory mediators . Thus, dSince we included mainly clinically unstable patients who had partly received treatment before, we are not able to answer this question at present. Based on previous results of reduced CD4+ memory subsets in GS-treated patients , it mighEarlier publications detected P0 IgG-antibodies in CIDP patients and P0 180-199 specific T cell responses in spontaneous autoimmune polyneuropathy-mice . AlthougUp to now, only little is known about the role of MBP 82-100 in the pathogenesis of CIDP even though MBP has been detected as part of the myelin sheath of peripheral nerves. Nevertheless, there is long-standing evidence that MBP 82-100 can induce neuroinflammation in autoimmune diseases . GlatiraThere is growing evidence for the autoimmune potential of antigens which are derived from non-compact myelin of the nodal/paranodal region such as neurofascin 155 of 186 leading to antibody response in distinct subgroups of CIDP or multifocal motoric neuropathy (MMN) –33. ThusSeveral limitations may have affected our results. First, our clinically heterogeneous group of atypical patients was too small to distinguish between subgroups of atypical CIDP, which would be necessary to characterize atypical subtypes and to define specific cut offs for our immunological parameters. Second, differences in gender and INCAT score between typical and atypical CIDP patients might have influenced our immunological findings. Third, we aimed to recruit treatment-naïve patients in active-unstable stages of the disease. However, only 46% of patients (12/26) were treatment-naïve at enrollment. Previous immunosuppressive and –modulating therapy might have influenced our immunological findings.Higher myelin-antigen specific T cell responses together with elevated T cell memory subsets were found in atypical compared to typical CIDP patients suggesting different patterns of immune responses in clinically distinctive CIDP subgroups. Myelin as well as nodal/paranodal proteins might serve as candidate autoantigens to establish robust immune markers for CIDP subtype differentiation. Given the clinical diversity of CIDP a larger cohort study is warranted in order to establish those markers with reliable cut-off values.


OR6B3 is one of the most highly expressed ORs. Immunohistochemical stainings of retina sections localized OR2W3 to the photosensitive outer segment membranes of cones, whereas OR6B3 was found in various cell types. OR5P3 and OR10AD1 were detected at the base of the photoreceptor connecting cilium, and OR10AD1 was also localized to the nuclear envelope of all of the nuclei of the retina. The cell type-specific expression of the ORs in the retina suggests that there are unique biological functions for those receptors.Several studies have demonstrated that the expression of odorant receptors (ORs) occurs in various tissues. These findings have served as a basis for functional studies that demonstrate the potential of ORs as drug targets for a clinical application. To the best of our knowledge, this report describes the first evaluation of the mRNA expression of ORs and the localization of OR proteins in the human retina that set a stage for subsequent functional analyses. RNA-Sequencing datasets of three individual neural retinae were generated using Next-generation sequencing and were compared to previously published but reanalyzed datasets of the peripheral and the macular human retina and to reference tissues. The protein localization of several ORs was investigated by immunohistochemistry. The transcriptome analyses detected an average of 14 OR transcripts in the neural retina, of which  The odorant receptors (ORs) provide the basis for the detection of volatile odorant molecules in the environment. OR genes were previously determined to be expressed exclusively in the olfactory epithelium . HoweverIn the current study, we are the first to describe the complete expression profile of the ORs in the human retina by RNA-Sequencing (RNAseq) analysis using next-generation sequencing. We also validated the protein expression and the protein localization of several ORs by immunohistochemistry. The identification of the ORs expressed in the human retina provides the basis for the further functional characterization and potential application as therapeutic targets.The RNAseq analysis of human retinae was conducted with anonymized probes from three different donors. For the western blotting analysis and the immunohistochemistry, human donor eyes  with no history of retinal disease were obtained from the Department of Ophthalmology, University Medical Center Mainz, Germany and Department of Ophthalmology, Ludwig Maximilian University, Munich, Germany. We followed the guidelines of the declaration of Helsinki and the study was approved by the local ethics boards of the clinical and the experimental study contributors (Nr. 331-09).GENterprise Genomics  using the Illumina NextSeq500 sequencing platform as paired-end reads with a 101-nucleotide (dataset Retina2) or with a 151-nucleotide (datasets Retina1 and Retina3) length (Supplementary Table S1). We analyzed the mRNAseq data as described previously (1 (accession number: GSE30611), as previously described .For the transcriptome analysis, the RNAs from the human retinae were isolated using the RNeasy Plus Mini Kit  according to the manufacturer’s protocol, including the DNaseI digestion. The mRNA isolation from total RNA and sequencing analysis was performed by eviously . For theeviously . Bowtie,eviously . The BAMeviously . The FPKeviously . We reaneviously  and fromeviously . Six of escribed . The humescribed . The datescribed . All datTo determine a FPKM cutoff value for OR expression, we estimated the background of detection. For this purpose, we used modified gtf-files where the annotated transcripts of the ORs were shifted to random positions in the intergenic regions basically as described by The total RNA from the retinae was reversely transcribed using the iScript cDNA Synthesis Kit  according to the manufacturer’s instructions. The equivalent of approximately 50 ng of RNA was used for each of the RT-PCR experiments. We designed primers that detect approximately 150–450 bp fragments of the OR open reading frame . The PCR was performed under standard PCR-conditions with the Mastercycler ep Gradient S . All experiments were conducted in triplicate.The polyclonal antibody rabbit anti-OR6B2/3 was purchased from Novus Biologicals . It was generated against a synthesized C-terminal peptide derived from human OR6B2. The polyclonal rabbit antibodies against OR2W3, OR5P3, and OR10AD1 were obtained from Sigma-Aldrich . The specificity of OR antibodies was verified using recombinantly expressed rho-tagged ORs in Hana3A cells , or CFTM-640 , respectively. Some of the samples were counterstained with the fluorescein-labeled lectin peanut agglutinin , which specifically labels the extracellular matrix sheath of the cone photoreceptor cells (The two donor eyes were dissected and fixed in melting isopentane (at -168°C) and then cryosectioned as described in or cells  and withTBP) is detected at 7–9 FPKM in the retina samples, whereas the strongly expressed glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene reveals an expression value of approximately 2700 FPKM . To test whether the retina was dissected free of contamination with RPE, we evaluated the expression of typical RPE associated gene transcripts, which were relatively weakly or not at all expressed in Retina1–3 . For example, the strongly expressed rhodopsin gene (RHO) had an expression value of ∼3,300–4,200 FPKM . As shown in Figure 1A, the common neural retina marker genes are exclusively expressed in our generated retina samples. In comparison, the typical RPE-associated gene transcripts are relatively weakly or not expressed in Retina1–3 . We further tested the variability of the human retinae transcriptome datasets using the Pearson product moment correlation and identified high correlations (ρ = 0.950–0.982) within our generated retina data  and also between our data and the published data of the peripheral . The quantitative expression values were calculated for each sample based on the number of FPKM. In total, we detected the expression of approximately 17,100 genes (FPKM ≥ 0.1) of the 22,711 genes represented in the gene model. On a rough scale, a FPKM ≥ 0.1 corresponds to a weak expression level, a FPKM ≥ 10 represents a moderate expression level and a FPKM ≥ 100 indicates a high expression level. For example, the weakly to moderately expressed TATA box binding protein . Initially, we identified 11 to 19 different potential OR transcripts  of 374 annotated OR genes in the three human retina datasets . The comparison of the OR expression profiles of the retina with the reference tissues revealed that the majority of potential OR transcripts are absent in reference tissues, excluding the TG and the DRG. Approximately 46% of the identified putative OR transcripts in the retina were also present in the analyzed TG and 30% in the DRG (mFPKM ≥ 0.3).Next, we analyzed the expression of the OR transcripts in the human retinae compared to the reference tissue samples . In addition to the retina, the OR6B3 could also be detected in the TG and the DRG. Using the IGV, we confirmed the predicted expression of the OR6B3 and also validated the expression of both of the 5′UTRs of OR6B3 by RT-PCR with a forward primer located in the identified exon and a reverse primer located in the ORF of OR6B3 . For OR6B2, reads are mapped only at the positions where the sequence is identical to OR6B3 and virtually never at positions that were specific for OR6B2. Therefore, the sequenced reads originate from the OR6B3 transcript and the expression of the OR6B2 transcripts remains unclear. In addition, a detailed analysis of unique mapping reads indicates that OR6B3 but not OR6B2 is expressed in the human retina, which is also supported by RT-PCR analysis  34, pseudogene; OR1Q1 revealed splicing events within their ORF, which are a typical feature of many of the OR-transcripts  and is in the reference tissues only detectable in TG and DRG.Additionally, we also identified that the whole genome region around the ORF of in situ by using cryosections taken through the retinae of two mature donors . The well-defined layering of the human neural retina makes it relatively simple to determine the spatial distribution of proteins in a retinal section even by light microscopy . The indirect immunofluorescence staining of cryosections throughout the two human retinae from both donors was consistent and revealed the expression of all four ORs  in the human retina . To determine the subcellular localization, we counterstained the sections with the nuclear/DNA marker DAPI and with antibodies against diverse molecular markers of cell compartments . For example we used the antibody detecting centrin 3 as a well characterized marker for the connecting cilium, the basal body and the adjacent centriole of photoreceptor cell cilia at the junction of the inner and outer segment . Faint staining was detected in the inner plexiform layer (IPL). Counterstaining with the antibody against calnexin, an ER marker, showed co-labeling in the photoreceptor IS, particularly in the wider cone IS . Additionally, Western blotting analysis confirmed the presence of OR6B2/3 protein expression in the human retina . Counter-staining with the anti-centrin 3 demonstrated the OS localization of OR2W3 in these photoreceptor cells . The low number, the relative proximal position, and the morphology of the according cells  led us to the assumption that these cells were cone photoreceptors . Counterstaining with a FITC-labeled peanut agglutinin (FITC-PNA), a classical marker for the cone photoreceptors specifically staining the extracellular sheath .Indirect immunofluorescence staining using anti-OR2W3 resulted in intense immunofluorescence in a subset of photoreceptor cells . In the photoreceptor layer, OR5P3 was present in the ciliary region (CR) and in the IS of photoreceptor cells . Higher magnification and co-staining with the ciliary marker anti-Cen3 demonstrated the association of OR5P3 with the ciliary base of photoreceptor cells .We found punctate staining of the anti-OR5P3 in all of the layers of the human retina with lower concentrations at the OLM and the OPL . At higher magnification, co-staining with anti-centrin 3 demonstrated that OR10AD1 associates with the base of the photoreceptor cilia . An overlay of the indirect immunofluorescence of anti-OR10AD1 with the fluorescence of the nuclear DNA marker DAPI revealed that the OR10AD1 localizes in the nuclear envelope of all of the nuclei in the human retina . Interestingly, we observed that the most intense indirect immunofluorescence of anti-OR10AD1 associated with the nuclei of the IPL where the pericarya of the bipolar, horizontal and amacrine cells are located. An additional association with nuclei of photoreceptor cells in the outer nuclear layer was occasionally observed (data not shown). Z-scans of stained nuclei revealed that OR10AD1 is predominantly localized in the nuclear envelope and to a less extend in spots within the nucleus .We detected OR10AD1 in the CR and in the nuclei of the outer and the inner nuclear layer, as well as in the GC of the human retina  is predominantly localized in the satellite glia cells (RTP1 and RTP2) were not detectable in human retina, only REEP1, which enhances the cell surface expression of ORs was expressed in retina according RNAseq data  were found to be ectopically expressed exclusively in the human retina, suggesting a specialized function. One of them, OR5P3 is localized to the base of the photoreceptor connecting cilium, which corresponds to the transition zone of prototypic cilia  canonically coupled to the ORs is also expressed in the photoreceptor cells  is comparable to that of ORs (Supplementary Figure Most of the ‘classical’ downstream molecules that are associated with GPCR signaling at the plasma membrane, such as the G protein, adenylyl cyclase and Ca nucleus . Interesor cells . Beside e cortex . Thus, Oe cortex  such as e cortex . InteresFor the first time, we have described the expression of ORs in the human retina. We identified OR6B3 as one of the highest ectopically expressed ORs in terms of its FPKM/TPM value and by its detection at the protein level. We observed that some ORs are expressed in diverse and other in specific cell types or regions of the retina. The specific localizations within the neural retina in general and on the subcellular level of the photoreceptor cells implicate the distinct functions of ORs outside of the olfactory epithelium (i.e., similar to the spermatozoa Wrote the paper: NJ, KW, LG, UW, and CH. Analyzed the data: NJ, KW, and GG. Designed the experiments: NJ, KW, GG, HH, LG, UW, and AK. Conducted the experiments: NJ, KW, LT, MW, CF, DM, and LW.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Keratitis-ichthyosis-deafness (KID) syndrome is a syndrome which presents with hearing loss and visual and keratinization disorders. In such patients, hearing aids cannot be effectively used in the rehabilitation of hearing loss because of the frequent blockage of the external ear canal with epithelial debris and due to dry and tense skin of the external ear canal. Moreover, severe or profound hearing loss also limits the benefits gained from the conventional hearing aids. On the other hand, cochlear implantation is a method that has been used in limited cases in the literature. This study presents the results of cochlear implantation applied in our clinic to two children who had been diagnosed with KID. Audiological assessments before and after the cochlear implant operation were performed using pure-tone audiometry, immittance audiometry, and auditory brainstem response (ABR), and the postoperative follow-up was conducted using pure-tone audiometry. Skin problems, visual disturbances, and other additional problems complicate the short-term and long-term rehabilitation after implantation in individuals with KID syndrome. Close monitoring should be exercised due to possible skin complications that may develop during the postoperative period. The families and rehabilitation teams should be warned about the possible visual disturbances and skin complications. Keratitis-ichthyosis-deafness (KID) syndrome is a rare congenital disorder characterized by keratitis, congenital ichthyosis, and deafness. KID is inherited by an autosomal dominant transmission, with a mutation present in the Connexin-26 gene, which is a structural protein –4. VisuaThis study presents the results of cochlear implantation applied to two children with KID syndrome. Before the operation, a proclamation form was obtained from these patients' families.Case 1. The patient was born as a term male infant in the year of 2006. The diagnosis of hearing loss was made at the age of 4 since routine newborn hearing screening was not widely practiced across the country during the period he was born in. Moreover, rehabilitation of his other problems had had priority. After the diagnosis, he started to use a bilateral conventional hearing aid. However, his family stated that he could not use the hearing aid regularly because of debris due to the syndrome and extreme dryness of the skin in the external ear canal. After starting to use the hearing aid, auditory training had been added to the physical rehabilitation he had been using due to his gait problem. His language development had been limited despite the training, and he was referred to our clinic for evaluation with respect to cochlear implantation when he was 7 years old. When the patient came to our clinic, he underwent tests showing severe sensorineural hearing loss bilaterally. The auditory brainstem response (ABR) test was performed preoperatively and the tests did not reveal wave V and cochlear microphonic even in the bilateral maximal stimulation. In the Acoustic Immittance, bilateral Type A tympanogram was obtained but bilateral, ipsilateral, and contralateral reflexes could not be obtained. No bilateral emission response was observed. Based on the electrophysiological results, the bilateral severe sensorineural hearing loss was diagnosed. Free-field pure-tone audiometry was performed to test the benefits of the hearing aids. At 90 dB SPL, no response could be obtained with the hearing aid at any frequency. At the same magnitude level, no response with the hearing aid to speech stimulus was observed.Based on the assessment by using AGTE (Ankara Development Screening Inventory) by the Department of Pediatric Psychiatry, his development with respect to social skills and self-care was found at the level of 20 months, and his general development was found to be well behind his age. The ophthalmological assessment did not reveal any keratitis findings associated with the syndrome. The preoperative auditory integration assessment using MUSS, MAIS, CAPS, SIR, and Auditory Questionnaire revealed that he used gesture and gabbled in his daily communication. . No complications were observed related to the cochlear implantation during the 21-month postoperative follow-up period.Cochlear implant surgery was performed in May 2014. A wide postauricular incision was performed and skin was elevated posteriorly. The periost was elevated anteriorly. A place was prepared in the bone for the body of the cochlear implant, and the body was fixed in the hole properly in order to prevent bump formation. The cochlear implantation was performed using the trans-mastoid-facial recess approach. A Cochlear® CI422 Slim Straight Electrode was inserted into the right ear. The intraoperative measurements showed that the impedances of all electrodes were within normal limits. Intraoperative Neural Response Telemetry (NRT) was obtained from the five electrodes  electrodes have been used for the basal programming. After that, it was controlled whether the patient had any discomfort with live sound. No problem was observed and parents were informed about the details of the cochlear implant. Regular auditory training was continued in his town. He returned to our clinic for the first 3 of 4 routine scheduled appointments during the first postoperative year but could not come for the fourth due to his travel problem. Thereafter, annual appointments were scheduled. No test could be performed for the first auditory check after the fitting of the cochlear implant because of the poor cooperation of the child. On the third postoperative month, thresholds with the cochlear implant were obtained at the frequencies shown in After waiting for completion of the wound healing process for 1 month after the surgery taking into consideration the skin structure associated with the syndrome, the external part of the cochlear implant was placed. Fitting of cochlear implantation was made by using objective and subjective methods. For this purpose, NRT thresholds obtained from the nineThe patient had 8 intelligible words at the end of the first postoperative year and had very good adoption of the device. His family stated that they are very satisfied with the cochlear implantation.Case 2. The patient was born as a term female infant in the year of 2008. Hearing loss was diagnosed during the routine newborn hearing screening at the age of 10 months, and thereafter she had started to use a bilateral conventional hearing aid. Her medical history obtained from the family revealed that she could not use the hearing aid regularly due to skin problems and could not receive regular auditory training since she could not go out of the house due to her seasonal and sun sensitivity. This condition had been accompanied by motor growth retardation, and she was admitted to our clinic for cochlear implantation when she was 5 years old. When the patient came to our clinic, she underwent tests showing severe sensorineural hearing loss bilaterally.Preoperative ABR test did not reveal wave V and cochlear microphonic in both ears with the maximal stimulation. In the Acoustic Immittance, bilateral Type A tympanogram was obtained but bilateral, ipsilateral, and contralateral reflexes could not be detected. No bilateral otoacoustic emission response was observed. Based on the electrophysiological testing results, bilateral severe sensorineural hearing loss was diagnosed. The hearing aid threshold is at 90 dB SPL, and she had no response to the warble tone stimulus at any frequency. No response to speech stimulus was observed with the hearing aid. These results demonstrated that the patient could not get benefit from the hearing aid.Based on the AGTE performed by the Department of Pediatric Psychiatry, the general development and the other development areas were well behind her age. All development areas  were found to be well below 30%. The preoperative auditory assessment revealed that her auditory integration-development and language skills were limited. It was observed that she was using gesture and gabbling during the communication. The ophthalmological assessment did not reveal any visual problem. . No complications were observed related to the cochlear implant surgery during the 20 months of the postoperative period.Cochlear implant surgery was performed with the same surgical steps like in the previous patient in May 2014. A wide postauricular incision was performed and skin was elevated posteriorly. The periost was elevated anteriorly. A place was prepared in the bone for the body of the cochlear implant, and the body was fixed in the space properly in order to prevent bump formation. The cochlear implantation was performed using the trans-mastoid-facial recess approach. A Cochlear CI422 Slim Straight Electrode was inserted into the right ear. The intraoperative measurements showed that the impedances of all electrodes were within normal limits. Intraoperative NRT response was obtained from the five electrodesTaking into consideration the skin structure associated with the syndrome, the external part of the cochlear implant was placed one month later and the first programming was performed. The second fitting was performed one month later. Thereafter, she could not return for the third fitting because of the pregnancy of her mother for the second child and hospitalization of the patient due to frequent epistaxis attacks. She could return for follow-up in the sixth postoperative month, and her family stated that she could not use the cochlear implant regularly because of her hospitalization history. She could return for the last annual follow-up and after the placement of the external part of the cochlear implant. Unfortunately, the patient could not attend the auditory training due to her seasonal sensitivity. . Hearing thresholds were attempted to be obtained after each fitting session but could not be obtained, because the patient does not come to regular follow-ups. The hearing thresholds obtained with the cochlear implant on the 1st year of cochlear implant use are shown in During the fitting of the cochlear implant, all the impedances of the electrodes were checked at all settings and were found to be within normal limits. All programming of the device was made based on the NRT response obtained from the nine electrodesHer family stated that they are very satisfied with the cochlear implantation despite the mentioned downsides.Hearing loss is a congenital, often severe or profound condition and this sensorineural deficiency is an important marker in the diagnosis of KID . Our patThese patients were referred to us with a KID diagnosis from another hospital. The diagnosis of the disease was made histologically. We wanted to perform a genetic analysis in these patients. However, we did not get these results, because these patients' families did not provide consent to carry out genetic analyses.In patients with KID syndrome, conduction pathologies may also present due to external ear problems associated with the skin structure in addition to the sensorineural hearing loss , 2, 6. TThere are several studies in the literature emphasizing that severe or profound hearing loss requires the use of cochlear implants in order to enhance the speech perception and speech intelligibility , 8.For individuals with hearing loss, lip-reading is important in daily communication even if a hearing aid is used. In KID syndrome, visual problems may be congenital but may also develop with advanced age, thereby complicating lip-reading in this patient population. Cochlear implants should be considered early in the rehabilitation of hearing loss, taking into account factors such as the facts that the use of hearing aids is limited due to skin problems, that hearing aids cannot help the patients in the development of speech due to the severity of hearing loss, and that visual problems that exist or may occur in the future will complicate lip-reading. Also, bilateral implants can be considered in these patients, but in Turkey, the bilateral implant device is not paid for by the state.There are only a limited number of studies about the cochlear implant application in patients with KID syndrome. Barker and Briggs reported that they applied cochlear implantation to three children with KID syndrome, one unilateral and two bilateral. They found that all three benefited from cochlear implants based on the speech tests during the follow-up periods of 42 months, 26 months, and 21 months, respectively  reportedThe scores of the questionnaires and scales to determine the development of auditory integration following cochlear implantation were found to generally increase or remain the same during the follow-up periods. MUSS is a questionnaire which is designed to assess the child's use of speech in different natural situations . It was Another important issue is the surgical incision to be used in these patients. The minimal incision may be preferred in these patients because of skin and flap problems. However, it may be difficult to place the receiver after a minimal incision. For this reason, we preferred a slightly longer incision and we easily placed the receiver into the bed.In conclusion, there are difficulties in the use of hearing aids for auditory rehabilitation of individuals with KID syndrome. Taking into account that such patients may have visual problems, the cochlear implant should be considered as an appropriate method for auditory rehabilitation. However, unlike the routine cases, special attention should be paid to certain issues in cochlear implantation. The complication rate in surgery is higher. Therefore, in surgery preparation of flaps, selection of incisions and wound site monitoring are important. Like in our cases, patients may not be able to receive regular rehabilitation due to the syndrome. Therefore, an organization of home training programs and provision of training to the families come into prominence for such patients. Moreover, delayed diagnosis and rehabilitation of hearing loss due to additional problems associated with the syndrome may change the success expected to be achieved after cochlear implantation. It is important that families are informed at each stage and have realistic expectations.


Hearing loss resulted from multiple intrinsic and extrinsic factors. Secondhand smoke (SHS) and obesity had been reported to be related to hearing loss. This study explored the possible associations of SHS and obesity with the hearing threshold. The relations between SHS and the hearing threshold in subjects from three different body mass index classes were analyzed. Our study included data from 1,961 subjects aged 20–69 years that were obtained from the National Health and Nutrition Examination Survey for the years 1999–2004. After adjusting for potential confounding factors, the subjects with the higher tertiles of serum cotinine levels tended to have higher hearing thresholds than those with the lowest tertile of serum cotinine levels . Notably, the obese subjects with the higher tertiles of serum cotinine levels had significantly higher hearing thresholds for high frequencies and low frequencies than those with the lowest tertile of serum cotinine levels . Our study showed a significant positive association between SHS exposure and hearing thresholds in the adult population, especially in obese individuals. Based on our findings, avoiding exposure to SHS, especially in obese adults, may decrease the risk of hearing loss. Sensorineural hearing loss is an important public health problem with an estimated prevalence of 16.1% in the United States in adults aged 20 to 69 years2456et al. examined a nationally representative cross-sectional dataset of 3307 individuals and found that SHS was significantly related with hearing loss in nonsmokersSHS is an important public health concern. SHS exposure increases the risk of cardiovascular disease to approximately 30% in nonsmoking individuals459Obesity is associated with increased risks of disease, disability, and death611As these previous studies have shown, there is evidence indicating that SHS and obesity are related to hearing impairment. Moreover, these two distinct factors seem to share some possible mechanisms causing hearing impairment, such as atherosclerosis214151616Our study subjects were 1,961 participants from the NHANES 1999–2004 sample. The demographic distribution and clinical characteristics of our study group are presented according to the tertiles of the serum cotinine level . Among tThe results of multiple linear regression analysis based on serum cotinine tertiles are shown in The analyses of the relations between the serum cotinine level, BMI and hearing thresholds are presented in We studied a large and nationally representative group of nonsmoking adults in the United States who were exposed to SHS. On the basis of serum cotinine levels and self-reported information and consistent with a dose-dependent effect, we found that study subjects with the higher tertiles of serum cotinine levels had significantly higher high-frequency and low-frequency hearing thresholds compared with those with the lowest tertile of serum cotinine levels. Notably, this association was only observed in the obese subjects and was not found in normal weight subjects or overweight subjects. Among the obese subjects, those with higher tertiles of serum cotinine levels had significantly higher hearing thresholds than those with the lowest tertile of serum cotinine levels; this relation was not observed in the normal weight subjects or the overweight subjects. In other words, SHS did not seem to have a significant association with a hearing threshold shift in normal weight subjects or overweight subjects, but did have a significant positive association with a hearing threshold shift in obese subjects.et al. reported an association between smoking and hearing loss and also described a significant correlation between SHS and hearing loss. Nonsmokers who lived with a household member who smoked had a higher risk of hearing loss than those who did not live with a smoker et al. found that SHS exposure was associated with increased odds of hearing loss and a dose-response effect was observed1020Previous studies have shown a correlation between SHS and hearing loss or cochlear auditory dysfunction45910202122510The exact mechanism of ototoxicity due to smoking is not clear. It has been proposed that smoking may be deleterious to the inner ear due to a direct ototoxic effect of nicotine or another toxin or to vascular ischemia or hypoxemia in the cochlea from vasospasm, vasoconstriction, increased blood viscosity, production of carboxyhemoglobin, or blood vessel arteriosclerosis132425262142729Although obesity-related co-morbidities can impair hearing, obesity itself may have some specific associations with hearing impairment. In an animal study, obesity could exacerbate the hearing impairment with elevated hearing thresholds32As far as we know, mechanisms of age-related hearing loss include hypoxia, atherosclerosis, inflammation, oxidative stress and production of reactive oxygen species, mitochondrial dysfunction, and subsequent apoptosis of the cochlear cells2141516163435The tonotopic organization of the cochlea results that hair cells at the base of the cochlea respond to high frequencies and those at the apex to the low frequencies4Our study had several limitations. First, it is a cross-sectional study in which the serum cotinine levels and the hearing thresholds were measured at one timepoint, rather than a longitudinal study. Thus, causal inferences cannot be made. Second, no information regarding congenital or genetic hearing loss was available in the NHANES dataset. Third, a recall bias in the medical history data cannot be excluded. Fourth, our analysis dates were obtained from the 1999–2004 NHANES, so our present study focused on the participants aged 20–69. The interpretation of our results should be cautiously. Finally, misclassification of SHS was possible because the exposure estimate was based on the serum cotinine level that has an average half-life of 17 hours.Many epidemiologic studies about evaluating the possible risk factors of hearing impairment were conducted to find the preventable factors. They can give us inspiration of further laboratory research and an improved understanding of the etiologic risk factors related to hearing impairment which results in significant public health benefits. Noise-induced hearing loss is by far the most important preventable cause of hearing loss23235The NHANES is a large cross-sectional study that is designed to represent the non-institutionalized civilian population in the United States. This survey is annually conducted by the National Center for Health Statistics of the CDC. The survey includes an initial household interview and subsequent physical examinations and laboratory tests that are performed at a mobile examination center (MEC) to collect pertinent information.We limited our study subjects to include NHANES participants who were nonsmokers exposed to SHS, as defined by both the personal questionnaire during the household interview and the self-reported questionnaire regarding smoking status that was administered at the MEC, as well as the nicotine level measured by a laboratory test. Our study examined data from 1,961 subjects aged 20–69 years that were obtained from the NHANES for the years 1999–2004 and included the following: demographic data, results of physical examinations, laboratory tests, questionnaire contents, and audiometric testing. Our study excluded individuals with current or ever occupational noise exposure or with incomplete data regarding either SHS exposure or other variables assessed in this study.http://www.cdc.gov/nchs/data/nhanes/au.pdf.According to the NHANES protocol for 1999–2004, after the initial household interview and medical examination, half of the subjects were randomly scheduled for audiometric testing. Subjects who were eligible for testing were excluded if they could not take off their hearing aids or could not tolerate the auditory headphones. Audiometric testing was performed in the MEC sound-isolated room by technicians who were trained by a certified audiologist from the National Institute for Occupational Safety and Health. The audiometric instrumentation contained an interacoustics model AD226 audiometer equipped with standard TDH-39P headphones and EARTone 3A insert earphones. Both ears were tested across the frequencies of 500, 1,000, 2,000, 3,000, 4,000, 6,000, and 8000 Hz with an intensity range from −10 to 120 dB to measure the air-conduction pure tone hearing thresholds. The high-frequency pure-tone average (high-PTA) was calculated as the average of the pure-tone hearing level at 3,000, 4,000, 6,000, and 8,000 Hz. The low-frequency pure-tone average (low-PTA) was calculated as the average of pure tone hearing level at 500, 1,000, and 2,000 Hz. High-PTA and low-PTA were used for evaluation the high-frequency and low-frequency hearing impairment, respectively4Cotinine, a major proximal metabolite of nicotine, is extensively used as a biomarker of exposure to cigarette smoke for either active smoking or SHS2.We classified subjects based on BMI according to the WHO definition, as follows: a BMI ≧ 30 is obese, a BMI of 25 to <30 is overweight, a BMI of 18.5 to <25 is normal weight, and a BMI < 18.5 is underweight. The BMI is defined as the body mass (in kilograms) divided by the square of the body height (in meters) and is expressed in units of kg/mDemographic data that included age, sex, race/ethnicity, status of smoking, and personal history were collected. Race was categorized as non-Hispanic black, non-Hispanic white, or other. Both the personal questionnaire and the self-reported questionnaire were used to determine smoking status. Participants were defined as having diabetes mellitus (DM) if they self-reported a diagnosis by a physician, had a fasting glucose level of ≥126 mg/dL, had a random glucose level of ≥200 mg/dL, or used diabetic medications. Heart disease was considered to be present if participants had experienced or had been diagnosed with angina, myocardial infarction, coronary artery disease, or congestive heart failure. Stroke was determined from self-reports. The current use of ototoxic medications including aminoglycosides, loop diuretics, non-steroidal anti-inflammatory drugs, or anticancer drugs, was ascertained from self-reports. Abnormal otoscopy was determined if there were abnormal results of an initial otoscopic screening examination of the eardrum and external acoustic canal before the audiometric testing. An audiometric impedance tympanometer was used for the tympanometry. Abnormal tympanometry was determined if the compliance was ≦0.3 ml or the middle-ear peak pressure was ≦−150 daPa. Body measurements, including height, weight, and WC  were recorded from participants at the MEC. Uric acid data were determined using the Hitachi 737 analyzer. Levels of high-density lipoprotein cholesterol (HDL-C) were enzymatically measured using the Hitachi 704 analyzer. All measurements used standardized methods with documented accuracy that followed the CDC guidelines. All detailed information regarding biospecimen collection and processing protocols are available at the NHANES website.SPSS  was used to conduct the statistical analyses. Two-sided p-values < 0.05 were considered to indicate significant differences. Some covariates including age, WC, BMI, uric acid, and HDL-C were treated as continuous variables and were presented as the mean value ± standard deviation (SD). Some covariates such as sex and race were regarded as categorical variables and were presented as numbers with percentages. A log transformation was performed to normalize the distribution of the PTA hearing thresholds. Our study used tertile-based analysis that divided the serum cotinine level into tertiles with the individuals in the lowest tertile considered as the reference group. The three tertiles of the serum cotinine level were as follows: T1 < 0.0226 ng/mL, 0.0226 = T2 < 0.0719 ng/mL, and 0.0719 = T3 < 10 ng/mL. Linear regression analysis was used to assess the association of the tertiles of serum cotinine level with hearing threshold. A multiple-model approach was constructed for adjustments of covariates. Model 1 was adjusted for age, gender and race. Model 2 was model 1 after further adjustment for WC, BMI, HDL-C, and uric acid. Model 3 was model 2 after further adjustment for abnormal otoscopy and tympanometry, current use of ototoxic medications, history of DM, heart disease or stroke. The p-values for the trend tests were determined by treating the tertile of the serum cotinine level as a continuous variable (ranging from 1 to 3) to assess the associations across the increase in the tertile of the serum cotinine level with the PTA hearing threshold.The National Center for Health Statistics Institutional Review Board approved the NHANES study, and written informed consent was received from all participants prior to the study. However, our study was exempt from IRB review because we used a publicly available, unidentifiable dataset.How to cite this article: Lin, Y.-Y. et al. Secondhand Smoke is Associated with Hearing Threshold Shifts in Obese Adults. Sci. Rep.6, 33071; doi: 10.1038/srep33071 (2016).