To compare corneal morphological parameters between diabetics and age matched non-diabetic control subjects and to evaluate the correlation of these parameters in relation to duration of diabetes mellitus (DM), glycemic status and severity of diabetic retinopathy.(DR).This cross sectional comparative study was conducted at the Department of Ophthalmology, PNS Shifa Karachi from February 2016 to January 2017. Patients with ages between 10 to 80 years of either gender who were diagnosed to have DM were recruited in the study. Control group comprised of age matched healthy volunteers who did not have DM. Corneal morphological parameters  was evaluated in each subject with non-contact specular microscope and findings were endorsed on a pre devised proforma.2, while mean CED of control group was 2574.46 ± 279.97 cells/mm2 [p = 0.04]. Between group differences in mean average cell size, CV of cell size and hexagonality was statistically not significant. Analysis of corneal endothelial parameters among subgroups of patients with no DR, with NPDR and PDR did not show statistically significant difference. Moreover, patients with diabetes of more than 10 years duration had significantly lower CED (p <0.01) and larger average cell size (p= 0.03). Duration of DM was significantly correlated with type of DR, HbA1c level, CED, polymegethism and hexagonality.Data of 298 eyes  was evaluated. Mean corneal endothelial cell density (CED) of diabetic population was 2494.47 ± 394.10 cells/mmMean corneal endothelial cell density (CED) was found to be significantly lower in diabetic population as compared to healthy controls. Lower CED and hexagonality with higher coefficient of variation (CV) and average cell size had been reported by various authors.After approval of hospital ethical review committee, this cross sectional comparative study was conducted at the Department of Ophthalmology, PNS Shifa Naval hospital Karachi from February 2016 to January 2017. Written informed consent was obtained from each subject before enrolment and study was conducted in accordance with the declaration of Helsinki. Patients with ages between 10 to 80 years of either gender who were diagnosed to have DM were recruited in the study through non probability convenience sampling. The diagnosis of diabetes mellitus was based on criteria of the American Diabetes Association (ADA) and included all the patients who were already under treatment of physician.Statistical analysis of the data was done using SPSS version 13.0. All the data were tested for normality before analysis. Descriptive statistics i.e. means ± standard deviation (SD) for quantitative variables and frequencies and percentages for qualitative variables were used. Independent sample ‘t’ test and One way analysis of variance (ANOVA) was used to compare quantitative data between groups, while chi square test for independence was used to compare qualitative data. Pearson’s correlation coefficient test was performed to find association of different study variables. A p-value < 0.05 was considered statistically significant.2 (range: 1094.7 – 3358.9 cells/mm2), while mean CED of control group was 2574.46 ± 279.97cells/mm2 (range: 1856.4 – 3240.6 cells/mm2) [p = 0.04]. Between group differences in mean average cell size, CV of cell size and hexagonality was statistically not significant  was evaluated. Mean age of diabetic population was 54.13 ± 9.97 years (range: 30-75 years), while mean age of control group was 52.01 ± 12.10 years (range: 32-80 years). Demographic and clinical profile of both groups is given in nificant . Analysinificant . Howevernificant . MoreoveHyperglycemia has profound effect on cornea with approximately 70% of diabetics having corneal complications known as diabetic keratopathy.132 vs 2574.46 cells/mm2; p=0.04). Significantly lower CED values in diabetic population as compared to healthy controls had been reported in various other studies.9Evaluation of corneal morphological parameters i.e. CED, CV and hexagonality has been done worldwide with conflicting reports. Corneal morphological parameters do differ among various races and ethnic groups with age being the major confounding factor affecting the CED, CV and hexagonality. In this study, both groups were age matched to eliminate the age related bias in corneal parameters among groups. In our study, Mean CED of diabetic population was significantly lesser as compared to normal controls  for the first time in Pakistani population. Limitations of the study include lack of multivariate analysis, not performing gold standard test (glucose tolerance test) to exclude diabetes in controls and not taking into account possible confounding factors like smoking, IOP and corneal diameter. Results of this study provide a greater insight into the understanding of corneal morphology in diabetic population especially in the context of pre-operative evaluation. In fact, Shenoy et al. concluded that evaluation of corneal endothelium in diabetic patients should be part of the protocol for eye care of diabetic patients.24Mean CED was found to be significantly lower in diabetic population as compared to healthy controls. Moreover, duration of DM was significantly correlated with type of DR, HbA1c level, CED, polymegethism and hexagonality.QUI: The conception and design of the study, acquisition of data, analysis and interpretation of data, drafting the article and final approval.MAM: Acquisition of data and final approval of manuscript.


Aging is a complex phenomenon involving functional decline in multiple physiological systems. We undertook a comparative analysis of skeletal muscle from four different species, i.e. mice, rats, rhesus monkeys, and humans, at three different representative stages during their lifespan  to identify pathways that modulate function and healthspan. Gene expression profiling and computational analysis revealed that pathway complexity increases from mice to humans, and as mammals age, there is predominantly an upregulation of pathways in all species. Two downregulated pathways, the electron transport chain and oxidative phosphorylation, were common among all four species in response to aging. Quantitative PCR, biochemical analysis, mitochondrial DNA measurements, and electron microscopy revealed a conserved age-dependent decrease in mitochondrial content, and a reduction in oxidative phosphorylation complexes in monkeys and humans. Western blot analysis of key proteins in mitochondrial biogenesis discovered that (i) an imbalance toward mitochondrial fusion occurs in aged skeletal muscle and (ii) mitophagy is not overtly affected, presumably leading to the observed accumulation of abnormally large, damaged mitochondria with age. Select transcript expression analysis uncovered that the skeletal inflammatory profile differentially increases with age, but is most pronounced in humans, while increased oxidative stress  is common among all species. Expression studies also found that there is unique dysregulation of the nutrient sensing pathways among the different species with age. The identification of conserved pathways indicates common molecular mechanisms intrinsic to health and lifespan, whereas the recognition of species-specific pathways emphasizes the importance of human studies for devising optimal therapeutic modalities to slow the aging process. Aging is a complex phenomenon involving functional declines in multiple physiological systems with the passage of time. Focusing on skeletal muscle, a group of international scientists identified pathways involved in healthspan and by determining global gene expression profiles across species they exposed common mechanisms fundamental to the aging process. Their experimental design involved comparative analysis of mice, rats, rhesus monkeys and humans, targeting three key time points during their respective lifespans. Pathways related to oxidative stress, inflammation and nutrient signaling, which function collectively to affect the quality and status of mitochondria, emerged across all species in an age-influenced manner. The identification of conserved pathways reveals molecular mechanisms intrinsic to health and survival, whereas the unveiling of species-specific pathways emphasizes the importance of human studies for devising optimal therapeutic modalities to slow the aging process. Some of the key processes thought to muscle loss include mitochondrial dysfunction, oxidative stress, chronic inflammation, and perturbations to the insulin-like-growth factor (IGF-1) and the mechanistic target of rapamycin (mTOR) signaling pathways.7Aging is a complex and inevitable phenomenon that almost certainly involves the gradual accumulation of damage to the cellular machinery, eventually leading to the progressive deterioration of normal biological functions, and an increased risk of disease and death.8 The former allows mitochondria to transmit among dividing cells, meet increased ATP demands, and maintain mitochondrial quality, primarily through autophagy.10 Fusion is the counterpart biological event, and involves the mixture of mitochondrial compartments to (i) preserve the equilibrium of oxidative phosphorylation (OXPHOS) proteins critical to respiration, (ii) carry out energy exchange, and (iii) complement defects in the mitochondrial genome.12 Additionally, it has been uncovered that mitochondrial electron transport chain (ETC) components can exist as supercomplexes, facilitating the electron flow, and hence making the system more efficient.14 Destabilization of the supramolecular organization may contribute to the development of an aging phenotype, as it has been shown that a lack of supercomplex formation leads to mitochondrial activity impairment, and reduced ATP levels.15Mitochondria are considered the powerhouse of the cell, and use oxygen to generate energy in the form of ATP, creating reactive oxygen species (ROS) as by-products in this process. Importantly, these organelles are highly dynamic, exhibiting the ability to carry out fission and fusion.17 Recent reviews by Nair and others20 have highlighted the specific involvement in human sarcopenia of reduced mitochondrial DNA (mtDNA) and protein content, impaired enzymatic activity and elevated markers of oxidative damage. However, a report by Lustgarten et al.21 found that oxidative stress, introduced by deletion of the radical scavenger MnSOD in mice, resulted in mitochondrial impairment, but not muscle atrophy during aging. Thus, the molecular choreography between mitochondria and aging is multi-factorial and requires further investigation.In many studies, age-dependent mitochondrial dysfunction has been reported to drive life-threatening disorders, such as frailty, and accelerate the aging process.22 Nevertheless, a revised look at this theory has suggested that harmful imbalances in redox homeostasis or signaling may be more central to the aging phenomenon than to the oxidative damage itself.23Often associated with defects in mitochondrial function is oxidative stress. Elevated levels of ROS can lead to increased macromolecular damage, namely to lipids, proteins and nucleic acids, which in turn can promote cellular dysfunction and organismal aging. Although the free radical theory of aging has experienced its setbacks, there is still significant evidence indicating an age-dependent accumulation of oxidative modifications in the main cellular components in skeletal muscle.24 The inflammatory response is also a common denominator of major diseases such as diabetes, obesity and cancer.25 This event has been proposed to arise due to an overall condition of oxidative stress, which activates signaling pathways, such as the nuclear factor-κB-driven (NF-κB) transcriptional response. The resulting production of pro-inflammatory cytokines and ROS further augment this perturbed oxidative redox state, leading to a vicious cycle.Another major etiological event in aging muscle is low-grade chronic inflammation, or the so-called inflamm-aging phenomenon.26 The kinase Akt plays an important role in this pathway, as Akt stimulates (i) protein synthesis by activating the kinase mTOR and glycogen synthase kinase 3β (GSK-3β) and (ii) protein degradation by repressing the transcription factor FoxO.27 This insulin/IGF-1/mTOR network also plays a complex role in the aging process. In the case of muscle growth, the pathway is activated, whereas reduced signaling has been associated with longevity, both in humans and model organisms.28The nutrient-sensing insulin/IGF-1/mTOR pathway is a highly conserved pathway in evolution that is involved in the regulation of cell growth, survival, and energy metabolism.While the above processes all have supporting evidence for their involvement in the aging phenomenon, there are also reports that have raised questions about their participation in this process. To attempt to consolidate the current picture regarding skeletal muscle aging, we have analyzed four distinct species with vastly different lifespans, i.e., humans, monkeys, rats and mice, to identify genetic pathways that are commonly regulated. Conserved pathways are of specific interest because they may reveal molecular mechanisms intrinsic to aging, which ultimately play a major role in modulating human health and lifespan. A particular strength of this study is the inclusion of multiple age-groups that span the comparative life stages across species, and the involvement of an advanced-age group, which allows for the detection of gradually changing molecular events and those that occur strictly later in life.Aging has been associated with alterations in body composition in various species, including humans, and such changes can promote adverse health outcomes and retard successful aging. We determined the effects of aging on bodyweight and body composition across four different species—C57Bl/6J male mice, Fischer 344 male rats, male rhesus monkeys, and male humans—at three different ages chosen to represent similar stages during their respective lifespans (see below), broadly defined as young, middle-aged and old Table . All spe31 our results show that muscle mass among all species declines with age  fall at similar time points along the lifespan curves of each organism Fig. . Principa priori defined gene sets.32 PAGE analysis revealed that as one moves from mice to humans, pathway complexity increases, and as mammals age, there is predominantly an upregulation of pathways in all species , a computational method that determines differences between genes and pathways using ies Fig. . Notablyies Fig. . To defiies Fig.  from A–DTo gain insights into the molecular events underlying the putative mitochondrial dysfunction, we examined the effects of aging on mitochondrial content in skeletal muscle. The protein complexes of the OXPHOS system are encoded by the mitochondrial and nuclear genomes, and a coordinated control of these two genomes is required to maintain mitochondrial integrity. Quantitative PCR of a number of nuclear-encoded and mitochondrial-encoded transcripts revealed that the former are largely unchanged between young, middle-aged, and old mouse samples Fig. . Convers33 while in monkeys, mitochondrial size increased at middle-age, before decreasing in the old animals  in combination with in-gel activity using digitonin for solubilization, as this retains inner mitochondrial membrane supercomplexes , the two most abundant cardiolipin species in muscle tissue, in subsarcolemmal and intermyofibrillar mitochondria in young and old monkeys. While, the data is not quantitative, the preliminary results suggest that the L4/L3O ratio trends lower in aged skeletal muscle ) and fission (fission 1 protein (Fis1) and dynamin-related protein (Drp1)). Mfn1 levels were significantly upregulated with age in both mice and rhesus monkeys, with the effect being most pronounced in the oldest animals, whereas no differences were detected within the human group  and programmed mitophagy (NIX/BNIP3). The mitochondrially targeted serine/threonine kinase PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin ligase Parkin are known to selectively promote the degradation of damaged mitochondria.ies Fig. . In addiies Fig. . In conties Fig. . NonetheAs mitochondrial oxidative stress is considered a hallmark of cellular aging, we evaluated age-related effects on oxidative stress markers in skeletal muscle of mice, rhesus monkeys and humans. 4-hydroxynonenal adducts, a marker of lipid peroxidation, were increased in skeletal muscle of old mice and humans compared to young, whereas no age-related effect was detected in the muscle of monkeys Fig. . Moreove42 An increased transcript level in the pro-inflammatory cytokine interleukin 6 (IL-6) was observed in skeletal muscle of old mice  and the eukaryotic translation initiation factor 4E–binding protein (4E-BP), which both play important roles in the initiation of mRNA translation, were unchanged with age in all species .50 Thus, research over the last decade has commonly found an association between mitochondrial dysfunction and major phenotypes associated with aging, including muscle decline. Importantly, mitochondrial transcript levels of humans appeared the most preserved, at least until middle age, likely suggesting an evolution-driven fitness more robust than other species. That said, the observed species-specific alterations in gene expression emphasize the importance of studying aging in humans in order to understand the mechanisms that modulate life- and health-span in people.Notably, our array studies identified only two “pathways” that were downregulated in the species studied herein: the mitochondrial ETC and OXPHOS. Previous transcription profiles of aging in the skeletal muscle of worms, flies, mouse, rats, and humans also indicate that genes encoding mitochondrial components decrease with age.52 In general agreement with prior work,51 our analysis revealed discoordination between the regulation of the mitochondrial and nuclear genomes. Specifically, we observed downregulation of the mitochondrial-encoded genes in all species with age, although the timing, middle-age or old, was different among species. Overall, the results regarding the expression patterns of the nuclear and mitochondrial genomes at oldest ages are consistent with impaired compartment coordination and likely reflect the observed mitochondrial dysfunction.Coordination of expression of the nuclear and mitochondrial genomes is crucial for optimal organism function, and studies have found that this coordination is disturbed with age.15, who reported that in mice mitochondrial activity is impaired and ATP content decreased due to a lack of supercomplex formation. Notably, a previous study using primary skin fibroblasts from long-lived individuals (centenarians) found that supercomplexes were increased, possibly as a compensatory mechanism to facilitate efficient respiratory chain activity.53 It is likely that this apparent discrepancy represents adaptation changes that are not seen in the old of the cohort studied herein, but that are unique to centenarians.OXPHOS component analysis uncovered that muscle extracts from old rhesus monkeys and humans contained an aberrant composition of several of the mitochondrial membrane respiratory complexes, including a tendency towards a decrease in the supercomplexes with age . In addition, despite the fact that there was species-specific variability in some of the individual parameters, our studies revealed an age-related reduction in mitochondrial content in all species, as assessed by citrate synthase (CS), mtDNA, and TEM experiments, implying an underlying role for mitochondrial dysfunction in the aging process. These findings agree well with the recent work of Ikeda et al.54 creating giant mitochondria in the process that are unable to be efficiently removed by the autophagosome. Our observation that there are no consistent changes in mitophagy with age in any of the species suggests that while this system may remain intact, it is incapable of dealing with the hyperfusion network, ultimately resulting in mitochondrial dysfunction. We note that prior evidence has revealed a decline in the autophagic process during normal aging in invertebrates and higher organisms.55Mitochondrial morphology is altered during aging in a process governed by the fission and fusion machinery. In our studies, we observed that fusion is increased in all species with age (as evidenced by the higher protein levels of Mfn1/Mfn2 relative to younger controls), whereas fission shows variable and limited differences. An imbalance in mitochondrial dynamics towards fusion is associated with the appearance of abnormal mitochondria, including those that are enlarged or more rounded in shape, as seen by TEM in mouse and monkeys, respectively. This hyperfusion phenotype may stem from an attempt to cope with an increase in mtDNA mutations by dilution,57 Thus, our collective studies support that with age, mitochondrial damage progressively increases, leading to altered mitochondrial composition and morphology, which further exacerbates the mitochondrial dysfunction and promotes increased fusion as a compensatory mechanism for the consequent ATP demands.Additionally, we measured a gradual increase in oxidative stress markers over the lifespan of all species. This finding is by-and-large consistent with prior studies that have indicated an increase in oxidative macromolecular damage with age, presumably resulting from aberrant mitochondrial activity.58 It is tempting to speculate that the distinct inflammatory responses among the different species stems at least in part from the fact that humans exist in an environment accompanied by unique exposures and lifestyles, including a widespread contact with antigenic elements. Our results, therefore, indicate that in addition to oxidative stress and mitochondrial dysfunction, inflammation plays a significant role in the aging process, as previously proposed.60Another notable feature of our analysis was the increase in variable inflammatory markers with age in all species. Inflamm-aging was most pronounced in humans, involving a unique age-related upregulation of the transcription factor NF-κB as previously shown.62 One notable observation of our study was the deregulated AKT response with age. In mice, the AKT response, as measured by phosphorylated AKT, was found to be downregulated, as was mTORC1, indicating a favoring of protein degradation, consistent with the increased GSK3β and FOXO1. Conversely, in humans, the AKT response was generally upregulated, revealing species-specific alterations during aging. Based on our IPA, we surprisingly observed that mTORC1 was upregulated in humans and monkeys, indicating that protein synthesis may be promoted as a consequence of the aging process. It is possible that the age-related muscle atrophy in primates is accompanied by a compensatory response that attempts to retain muscle integrity, but fails due to impaired cellular function, as suggested by a previous study.63 Our data in total paints a picture in which age-dependent alterations in oxidative stress, inflammation, and the nutrient signaling pathways ultimately affect the quality and status of mitochondria.An additional element that we observed during our analysis is the likely involvement of the nutrient sensing pathways in the aging phenomenon. Associations have been previously introduced between mitochondrial aging and the IGF-1 signaling and mTOR pathway.65Notably, the age-related mitochondria impairment also affect energy metabolism leading to the alteration of beta-oxidation with an increase of intra-fibers lipid accumulation that affects skeletal muscle quality and function, as previously shown both in human and denervation-induced atrophy in mice model.66The current work highlights the molecular denominators of aging process among different mammalian species, including humans, with different lifespan. Mitochondria and their status are at the core of commonalities tightly related to evolution constrains such as natural selection and fitness, but also effects of environments/life style and food intake. Not surprisingly, many mutations that extend lifespan in animal model affect signaling pathways regulating the metabolicrate and human longevity is associated with efficient respiratory chain activity and mitochondrial genetics.An important challenge looking forward is the development of therapeutic strategies that improve mitochondrial function as a means to delay the onset of age-related diseases, keeping in mind the important species-specific and even individual-specific adaptations.Macacamulatta), and humans were used in this study. Study species characterizations can be found in Table vastus lateralis were obtained from the monkeys from ongoing NIA studies. All animal procedures for this study were reviewed and approved by the Animal Care and Use Committee (ACUC) at the Biomedical Research Center (NIA/NIH).Humans who underwent surgery for hip dysplasia were included in this study. During surgery, biopsies from the vastus lateralis muscle were obtained. Age (>20 years) and ability to provide informed consent cooperating with study personnel were inclusion criteria, while exclusion criteria were presence of diseases, i.e., chronic kidney and/or liver diseases, bleeding disorders, severe type 2 diabetes, rheumatic diseases, osteoarthritis, neuromuscular disorders, malignancies and systemic infections, other than chronic steroid use, major psychological problems or history of alcohol or drug abuse, evidence of prior surgery in the involved hip. Written informed consent was obtained from each patient before the kick off study. A thorough medical history, including smoking habits and alcohol consumption, occupation and level of physical activity was taken from each patient and reported in questionnaire forms. Written informed consent was obtained from all participants, and the study was approved by the ethics committees of Istituto Ortopedico Rizzoli and Sant’Orsola-Malpighi University Hospital . With the exception of tissues processed for electron microscopy, muscle samples for all study species were flash frozen in liquid nitrogen and stored at −80 °C until assayed. Gastrocnemius (mice) and vastus lateralis  muscles were used for all biochemical assays and to generate the micro-array data.Young, middle-aged, and old male skeletal muscle samples from C57BL/6 mice, Fisher-344 rats, rhesus monkeys  or Telazol . Total body scans were acquired with the monkey in a supine position using dual-energy X-ray absorptiometry (DEXA) Lunar Prodigy X-Ray Bone Densitometer . Data analysis was done with GE encore Software v.12.3. Humans. Standing height and weight were measured for each participant. Muscle thickness of the vastus lateralis was measured by a portable ultrasound instrument . Thickness measure was obtained as vertical distance between muscle superficial and deep aponeuroses at an equidistant point from right and left borders of the sagittal image.Measurements of lean, fat, and fluid mass in live mice and rats were acquired by nuclear magnetic resonance using the Minispec LF90 . In addition, a different set of mice were euthanized and lower leg muscles  were collected with standardized dissection methods, cleaned of excess fat and tendon/connective tissue, and pair weighed on an analytical balance. Total RNA was isolated from frozen muscle samples by Trizol  and first strand complementary DNA (cDNA) was synthesized using the High Capacity cDNA Reverse Transcription Kit  following the manufacturer’s instructions. Quantitative real-time PCRs were performed with SYBR® Green PCR master mix to observe expression of desired genes on an ABI Prism 7300 sequence detection kit (Applied Biosystems). Quantification was performed using the ΔΔCT method after normalization to the housekeeper gene glyceraldehyde 3-phosphate dehydrogenase (Gapdh) or β-actin. Each analyzed sample was performed in three replicates. Fidelity of the PCR was determined by melting temperature analysis. Total DNA was extracted with DNeasy blood and tissue kit . In mice, mtDNA was amplified using primers specific for the mitochondrial cytochrome c oxidase subunit 2 (COX2) gene and normalized to genomic DNA by amplification of the ribosomal protein s18 (rps18) nuclear gene. In addition, mtDNA was amplified using primers specific for mitochondrial NADH subunit 1 (rhesus monkeys) and ND1 (humans) gene and normalized to genomic DNA by amplification of β-actin nuclear gene. Oligonucleotides used for real-time RT-quantitative PCR are listed in Table S67 The Z ratio (between treatment A and B) is given by z(A)−z(B)/std dev. Individual genes with Z ratio > 1.5, p-value < 0.05, and avg intensity > 0 were considered significantly changed. For PAGE, a list of pathways was obtained from http://www.broad.mit.edu/gsea/msigdb/msigdb_index.html (C2 collection). Our expression data was tested for gene set enrichment using the PAGE method as previously described.32 Briefly, for each pathway a Z score was computed as Z (pathway) = (sm mu)*pow/delta, where mu = mean Z score of all gene symbols on the microarray, delta = standard deviation of Z scores of all gene symbols on the microarray, sm = mean Z score of gene symbols comprising one pathway present on the microarray, and m = no of gene symbols in a pathway present on the microarray. For each Z (pathway) a p-value was also computed in JMP 6.0 to test for the significance of the Z score obtained. These tools are part of DIANE 6.0 and are described at http://www.grc.nia.nih.gov/branches/rrb/dna/diane_software.pdf. In addition to pathway PAGE analysis described above, statistically significant gene results were analyzed using IPA  to identify the top comparative aging network functions. Gene expression data was deposited in NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under the accession numberGEO87109according to MIAME compliance.RNA was isolated from skeletal muscle of mice, rats, rhesus monkeys, and humans at different ages using Trizol Reagent  following the manufacturer’s instructions. RNA was then hybridized to: Mouse, IlluminaMouseRef-8; Rat, Illumina Rat Ref-Tissues were lysed in radio immunoprecipitation buffer supplemented with ethylenediaminetetraacetic acid and ethylene glycol tetraacetic acid  and protease and phosphatase inhibitors . Following centrifugation  protein concentration was quantified using the Bradford assay method . Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and then transferred to nitrocellulose membranes. Western blots were performed according to standard methods. Membranes were blocked in 5% nonfat milk, incubated with the antibody of interest followed by incubation in a secondary antibody conjugated with horseradish peroxidase. Immunoreactive bands were visualized using chemiluminescent (ECL) Plus western blotting detection system , Immobilon Western Chemiluminescent HRP Substrate , or SuperSignal West Femto Maximum Sensitivity Substrate . The quantification was performed by volume densitometry using ImageJ software and normalized to staining with Ponceau S solution or anti-β-tubulin , which served to verify equal protein loading. The primary antibodies used in this study were directed against Mfn1, Mfn2, Drp1, and Fis1 , PINK1 , Parkin, pNF-κB, AKT, pAKT (Ser473), FOXO, pFOXO, GSK-3β, and pGSK-3β , BNIP-3 (Sigma Aldrich), NIX, COX2 , 4HNE  and NF-κB , which were generally used at the manufacturer’s recommended dilution. For detection of protein NF-κB levels in humans, rabbit monoclonal C22B4 anti-NF-κB  was used. Protein carbonylation was measured using an oxyblot protein oxidation kit  according to the manufacturer’s instructions.68 Briefly, skeletal muscle samples from rats, rhesus monkeys and humans were weighed and finely minced with scissors. The minced tissue was homogenized in 250 mM Sucrose, 10 mM Tris-Cl pH 7.5, 2 mM 6-aminohexanoic acid using a pre-cooled motor-driven, tightly fitting glass/Teflon Potter-Elvehjem homogenizer. Aliquots corresponding to 30 mg wet weight were centrifuged 10 min at 10,000×g. Pellets were frozen in liquid nitrogen and stored at −80 °C. In order to solubilize protein complexes, aliquots were resuspended in 50 mM NaCl, 50 mM imidazole pH 7, 2 mM 6-aminohexanoic acid and solubilized by adding 10 µl digitonin (20% stock in water (w/v)). Samples were centrifuged for 20 min at 22,000×g and the protein content of the supernatant was measured. Equal amounts of protein/lane (50 µg/sample for Complex I (CI) in-gel activity stain and native blot; 75 µg for Coomassie stain) were mixed with a 5% Coomassie blue G-250 dye stock suspension to obtain a detergent/dye ratio of 8/1. Blue native electrophoresis was performed as described.68 Due to the limited sample size of the human samples BNE was performed in mini format as previously described.69 Equal amounts of protein/lane  were loaded.Preparation of crude mitochondrial membranes and solubilization of mitochondrial membrane protein complexes were performed as previously described.70Following separation of mitochondrial OXPHOS complexes by BNE, subunit composition was studied using 2D BN/sodium dodecyl sulfate-polyacryl amide gel electrophoresis as described in ref. 2  in 10% acetic acid. Following destaining in 10% acetic acid gels were scanned by an office flatbed scanner (Epson perfection 2400 PHOTO) for documentation.in-gel NADH: nitrotetrazolium blue (NTB) reductase assay followed essentially the protocols described by Zerbetto et al.71 with some modifications according to Wittig et al.72. Briefly, complex I in-gel activity stain was done in 1D BN-gels directly after electrophoresis. 1D BNE gels were incubated in buffer containing 5 mM Tris/HCl, pH 7.4, 0.1 mg/ml NADH and 2.5 mg/ml NTB until the purple colour was developed (approx. 30 min). Reaction was stopped with 50% methanol, 10% acetic acid fixing solution for several hours to remove Coomassie. Gels were placed into water and scanned for further densitometric analysis by the Quantity One Software (Bio-Rad).In gel activity stain was used to quantify complex I containing supercomplexes isolated by 1D BNE. The 73 Citrate synthase were determined by spectrophotometric methods. Results were expressed in nmol/mg protein/min.Citrate synthase activity was determined in approximately 20 µg of protein lysates following the method described by Bernier et al.74 The isolated pellets were suspended in 100 ul of Reagent C from the Mitochondria Isolation Kit  and frozen at −80 °C. Isolated mitochondria were extracted using a previously described method.75 Briefly, to 80 μl of the suspension, 320 μl of water was added and mixed. Ten microliter of 1 mg/ml of myristoyl-cardiolipin was added as internal standard. Two milliliter of a chloroform:methanol (2:1) mixture was added and the aqueous layer was removed. Four hundred microliter of water was added twice to the organic phase and removed. The samples were then subjected to silica gel chromatography and the lipid classes were eluted in solvents with increasing polarity: (95:1 (Isooctane: EtOAc); 20:1 (Isooctane:EtOAc); 75:25 Isooctaine:EtOAc; 75:25:2 ; 75:25:2 . The phospholipids were eluted in the final step with 4 ml of methanol, stream dried, and then reconstituted again in 250 ml of methanol. MS/MS analysis was performed using a triple quadrupole mass spectrometer model API 4000 system from Applied Biosystems/MDS Sciex equipped with Turbo Ion Spray® (TIS) . The data was acquired and analyzed using Analyst version 1.4.2 (Applied Biosystems). Negative electrospray ionization data were acquired using multiple reaction monitoring (MRM). The TIS instrumental source settings for temperature, curtain gas, ion source gas 1 (nebulizer), ion source gas 2 (turbo ion spray), and ion spray voltage were 500 °C, 20 psi, 60 psi, 60 psi, and −4500 V, respectively. The TIS compound parameter settings for declustering potential, entrance potential, and collision cell exit potential were −110—10 V, and −6 V, respectively. The collision energy setting was −50 V for cardiolipin molecular species and −75 V for myristoylcardiolipin. The MRM transitions were (C18:2)4-CL (1448/695.0); (C18:2)3(C18:1)-CL(1450/695.0), and myristoyl-CL (1240/227.3). The chromatographic experiments were carried out on a Shimadzu Prominence HPLC system  following a previously described protocol, with slight modifications.75 Briefly, the samples were introduced to the analytical column using Shimadzu SIL-20A autosampler and maintained at 4 °C in the autosampler tray, and injections of 30 μl were made with a column temperature of 35 °C. The separation of (C18:2)4-CL, (C18:2)3(C18:1)-CL and (C18:2)2(C18:1)2-CL was accomplished using a Symmetry C18 column . The mobile phase consisted of 90:10 acetonitrile:water containing 0.5% of trimethylamine and 0.5% of glacial acetic acid as Component A and 90:10 isopropanol:water containing 0.5% of trimethylamine and 0.5% of glacial acetic acid Component B. A linear gradient was run as follows:0 min 0% B; 5 min 0% B; 70 min 100% B; 80 min 100% B; 85 min 50% B at 0.4 ml/min. The total run time was 90 min per sample.SS and IMF mitochondria were isolated as previously described.ATP content was measured with a commercial kit according to the manufacturer’s instructions .For TEM analysis, gastrocnemius, and vastus lateralis samples from mice and rhesus monkey, respectively, were removed and placed directly into a fixative solution consisting of 2.5% glutaraldehyde and 3% paraformaldehyde in 0.1 M sodium cacodylate buffer . The tissues were post fixed in 1% osmium tetroxide for 1 h at 4 °C in the same buffer, dehydrated and then embedded in Embed 812 resin , after which tissue samples were transferred to pure resin for 24 h. Blocks were formed in fresh resin contained in silicon molds, and the resin was allowed to polymerize for 48 h at 65 °C. Ultrathin sections were obtained and stained with uranyl acetate and lead citrate, and then examined on a Philips CM-10 electron microscope  at 25,000x magnification by a blinded investigator. Ten digital images per sample were analyzed using NIH Image J  software for measurement of mitochondrial area and mitochondrial circularity.t-tests are Mann–Whitney U-test when normality assumption was not obtained. Analyses were performed using Excel 2010 . A p-value of ≤0.05 was considered statistically significant.Data are expressed as means ± standard error of the mean (SEM). Between-group comparisons were analyzed using the Student’s Supplementary Figure 1Supplementary Figure 2Supplemental Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6Supplemental Figure 7Supplemental Figure 8Supplemental Table 1Supplemental Table 2Supplemental Table 3Supplemental Table 4Supplemental Table 5Supplementary file


Although retinoic acid (RA) signaling plays a crucial role in the body patterning of chordates, its function in non-chordate invertebrates, other than its mediation of environmental cues triggering metamorphosis in cnidarians, is largely unknown. We investigated the role of RA signaling in the metamorphosis of starfish (Echinodermata).We found that exogenous RA treatment induced metamorphosis in starfish larvae. In contrast, inhibitors of RA synthesis and RA receptors suppressed metamorphosis triggered by attachment to a substrate. Gene expressions of the RA signaling component were detected in competent larvae.This study provides insight into the ancestral function of RA signaling, which is conserved in the metamorphosis of cnidarians and starfish.The online version of this article (10.1186/s13227-018-0098-x) contains supplementary material, which is available to authorized users. Retinoic acid (RA) plays a critical role in the body patterning of chordates, such as the anterior–posterior patterning of the central nervous system and pharyngeal arches –3. RA syHemicentrotus pulcherrimus after RA treatment [Paracentrotus lividus did not induce specific phenotype except for delaying development [Hox genes had an RA response element [Hox gene expression has been reported.Despite these genomic surveys, the function of RA signaling in non-chordate deuterostomes remains largely unknown, other than the observation of pseudopodial cable growth on micromere-delivered cells from reatment . Sciarrielopment . Althoug element , no addiInsight into the ancestral function of RA signaling can be gained from the study of cnidarians, a basal lineage of animals. Fuchs et al. showed that RA signaling is involved in the life cycle transition of cnidarians . MedusozStrongylocentrotus purpuratus, larvae acquired competence to metamorphose 4.5–6 weeks after fertilization [Molecular mechanisms for metamorphosis have been investigated in echinoderms, primarily echinoids –28. In tlization  and commlization . Previoulization , 24–27. lization , 18, anyPatiria pectinifera, as it acquires competence to metamorphose earlier than sea urchin species (about 1 week after fertilization). We show that exogenous RA treatment induced larval body absorption and juvenile rudiment development in competent larvae. Conversely, the inhibition of endogenous RA synthesis or binding of RA to RAR blocked metamorphosis. In addition, genes related to RA signaling were expressed in the juvenile and larval stages. These results suggest that RA signaling acts as a regulator of metamorphosis after settlement.Here, we investigate the role of RA signaling in asteroid metamorphosis. We used the starfish P. pectinifera around the mainland of Japan: Manaduru (Kanagawa Prefecture), Asamushi (Aomori Prefecture), and Hiraiso (Ibaraki Prefecture). Fertilization and embryo rearing were performed from described previously [Chaetoceros calcitrans  by rearing in seawater with 50,000 cells/ml. We changed seawater with fresh algae every other day.We collected adult specimens of eviously . From 2 N,N-diethylaminobenzaldehyde  or the RAR antagonist RO41-5253 . We prepared 100 mM, 1 M, and 50 mM stocks of RA, DEAB and RO, respectively, in dimethyl sulfoxide (DMSO). Larvae were incubated in 2 ml artificial seawater containing 2 µl of reagents or DMSO in 12-well plates at 22 °C. For the treatment without a substrate, 10 larvae were incubated in one well. In the experiments conducted to induce metamorphosis with a substrate, a single larva was cultured in one well to identify individuals. We observed metamorphosis proceed in the same manner in these densities of larvae. Coral sand from tanks of adult P. pectinifera was used as a substrate for P. pectinifera experiments [The RA signaling pathway was activated by exogenous all-trans RA . RA signaling was inhibited by the Raldh inhibitor We considered the ability to induce metamorphosis to have been achieved when larval body absorption and the development of juvenile rudiment with primary podia were observed Fig. . We consThe metamorphosis ratio was calculated by dividing the number of metamorphosis commenced larvae by the number of treated larvae. In the case of reagent treatment with substrate, metamorphosis ratio was calculated by dividing the number of metamorphosed larvae by the number of settled larvae.We used ANOVA to evaluate differences of reagent treatments on the metamorphosis or settlement, assuming above conditions and batches as main factors and “blocks,” respectively, because preliminary tests in some treatments revealed slight, but not essential, differences among batches. If we had two factors, we assessed an interaction of the two factors. Also, multiple comparison was performed by Tukey’s honestly significant difference test. The R statistical software was used for all statistical analyses .As to the assumption of normal distribution, we use Kolmogorov–Smirnov test and confirmed no significant difference from the assumption of normality. Also, Bartlett’s test was used to test homogeneity of variances prior to ANOVA. In a few experiments, we found significant violations of the equal variance assumption and conducted Welch’s one-way ANOVA to test main effects without the assumption of equality. However, there were no differences in statistical conclusions between the main factor of ANOVA and the Welch’s method. We thus used ANOVA with assumption of equal variance in the following analyses because we needed multi-dimensional analyses simultaneously and because ANOVA is robust to deviations from equal variance, especially if numbers of sample are equal , and norP. pectinifera. Each sample was collected from three biological replicates: The samples of blastula and brachiolaria larva were collected from progeny of three distinct pairs of parents, and the adult epidermis was collected from three mother specimens. Total RNA was extracted using TRIzol reagent  and treated with DNase and cleaned up using the RNeasy kit . Paired-end libraries (200 bp) were prepared using a TruSeq RNA Sample Preparation Kit (Illumina). Sequencing on the Hiseq 2000 platform  was performed at the National Institute of Basic Biology (NIBB) The raw reads were deposited in the DDBJ Sequence Reads Archives (DRA006662). The low-quality reads were filtered using the NGS QC Toolkit57 [To gather sequence of RA signaling related genes, we performed transcriptome analysis on samples of blastula (13 h post-fertilization), brachiolaria larva (14 dpf) and adult epidermis in starfish oolkit57 . De novooolkit57 .raldh, rar and rxr from gene models assembled from our de novo transcriptome data of larval stages for P. pectinifera. The orthologies of these genes were confirmed by molecular phylogenetic analysis  with the default parameters [We recovered nucleotide sequences coding for rameters . Amino arameters . A best-rameters . Confideraldha, raldhb, raldhc, rar, and rxr. We used 40-bp reverse primers including a 20-bp T3 promoter sequence to synthesize Dig-labelled RNA probes for in situ hybridization. Dig-labelled RNA probes for raldha, raldhb, raldhc, rar, and rxr were transcribed from PCR products using T3 RNA polymerase . In situ hybridization was performed as described previously [cDNA was obtained by reverse transcription from larval RNA using PrimeScript 1st strand cDNA Synthesis kit . Additional file As in other echinoderm species, the transition from larval form to adult form proceeds via multiple steps in starfish. Development of the adult rudiment commences well before larvae acquire competence. Adult skeletogenesis was observed in penta-radial manner as early as 6 dpf, when development of brachiolar arms commences. Development of the brachiolar arms is tightly linked with the acquisition of competence. Murabe et al. indicated that adhesive papillae on the brachiolar arms sense environmental cues such as proper substrate for promoting metamorphosis . When coP. pectinifera larvae become competent to metamorphose upon attachment to a substrate.Although development of the brachiolar arms commences at 6 dpf, we do not know when the competence to receive cues for this metamorphosis is acquired. Thus, we first sought to identify the stage at which P. pectinifera. Note that we are not certain the coral sand was the most favorite substrate, and thus, larvae may initiate metamorphosis earlier when they attach to other substrates. We introduced coral sand to the wells of larvae of various ages . ANOVA revealed a significant effect of RO on the ratio of metamorphosed larvae .To examine whether RA functions as an internal mediator of signaling to a metamorphosis cue, we treated larvae with RO, known to be the selective RARα antagonist Fig. j. As shoP = 0.000 and 0.632 in RO and batch, respectively, ANOVA; Fig. P = 0.256 and 0.058 in RO and batch, respectively, ANOVA; Fig. These results suggest that exogenous RA drives metamorphosis through binding to RAR. Therefore, we investigated whether RAR plays any role when larvae respond to a substrate and commence metamorphosis. We treated competent larvae with 1 µM RO at 14 dpf. As shown above, more than 90% of 14-dpf larvae responded to the introduction of coral sand and commenced metamorphosis . However, DEAB significantly inhibited metamorphosis after settlement. Significance of DEAB was found in metamorphosis , although there was no significant difference between the latter two in metamorphosis . These results indicate that endogenous RA synthesis was essential for the commencement of metamorphosis after receipt of the signaling cue by the adhesive papillae, whereas it was not essential for settlement on the substrate.To assess whether endogenous RA synthesis was required for metamorphosis, we treated 14-dpf larvae with DEAB, a known inhibitor of Raldh . Considering RA is also core element of metamorphosis regulation in basal group of metazoan (cnidarian), our results suggest that ancestral function of RA signaling is involved in metamorphosis.Additional file 1: Figure S1. Phylogenic tree of Raldh  and Aldh2 (Aldehyde hydrogenase 2) subfamily. The phylogenic tree was constructed by RAxML. raldhs of starfish made sister grouping with hemichordate aldh1as. Numbers at node show bootstrap value. The selected amino acid substation model was LG + F + G. The process to construct was described in material and method and the set of sequences is provided in Additional file Homo sapiens (Human), Mm; Mus musculus (Mouse), Xt; Xenopus tropicalis (Western clawed frog), Dr; Danio rerio (Zebrafish), Bf; Branchiostoma floridae (Amphioxus), Ci; Ciona intestinalis (Transparent sea squirt), Sk; Saccoglossus kowalevskii (Acorn worm), Sp; Strongylocentrotus purpuratus (Purple sea urchin), Pp; Patiria pectinifera.Additional file 2: Figure S2. Phylogenic tree of RAR, RXR and THR (Thyroid hormone receptor). The phylogenic tree was constructed by RAxML. The selected amino acid substation model was LG + F + G. The set of sequences is provided in Additional file Homo sapiens (Human), Mm; Mus musculus (Mouse), Xt; Xenopus tropicalis (Western clawed frog), Dr; Danio rerio (Zebrafish), Bf; Branchiostoma floridae (Amphioxus), Pm; Polyandrocarpa misakiensis (Tunicate), Bl; Branchiostoma lanceolatum (Amphioxus), Ci; Ciona intestinalis (Transparent sea squirt), Sk; Saccoglossus kowalevskii (Acorn worm), Sp; Strongylocentrotus purpuratus (Purple sea urchin), Pp; Patiria pectinifera, Dm; Drosophila melanogaster (Fruit fly), Rc; Reishia clavigera (Sea snail), Ls; Lymnaea stagnalis (Great pond snail), Tc; Tripedalia cystophora (Jellyfish).Additional file 3: Table S1. Accession numbers of the gene used for construction of phylogenic tree. Amino acid sequences to construct tree were obtained from Uniprot or Echinobase (http://www.echinobase.org/Echinobase/), Genbank.Additional file 4: Table S2. Sequences of primer for amplification of raldha, raldhb, raldhc, rar and rxr. We used 40-bp reverse primers including a 20-bp T3 promoter sequence to synthesize Dig-labelled RNA probes for in situ hybridization. Bold characters mean consensus sequence for T3 promoter.Additional file 5: Table S3. Number of metamorphosed/treated larvae of each batch in RO + RA treatment experiment.Additional file 6: Table S4. Number of metamorphosed/settled larvae of each batch in RO 1 µM or DMSO treatment.Additional file 7: Table S5. Number of settled larvae/treated larvae of each batch in RO 1 µM or DMSO treatment.Additional file 8: Table S6. Number of settled/treated larvae of each batch in DEAB 100, 300 µM or DMSO treatment.Additional file 9: Table S7. Number of metamorphosed/settled larvae of each batch in DEAB 100, 300 µM or DMSO treatment.Additional file 10: Supplementary dataset 1. The dataset for phylogenetic analysis of Raldh and Aldh2.Additional file 11: Supplementary dataset 2. The dataset for phylogenetic analysis of RAR, RXR and THR.


Baló’s concentric sclerosis (BCS) is a rare inflammatory demyelinating disorder of the central nervous system characterised by concentric layers of demyelination. It is unclear whether BCS is a variant of multiple sclerosis (MS) or a disease entity in its own right.To compare the cerebrospinal fluid (CSF) features of BCS to those of MS.Retrospective analysis of the CSF profile of all patients with BCS reported in the medical literature between 1980 and 2017.p < 0.000001). OCB frequency was low both in ‘historic’ patients  and in more recent patients . OCB-positive and OCB-negative patients did not differ significantly with regard to age, sex, disease duration, number of Baló-like lesions on MRI, number of relapses, treatment or final outcome. In accordance with the high rate of OCB negativity, Link’s IgG index was negative in 63% of all tested samples (p < 0.000001 vs. MS). CSF pleocytosis was present in 28%  and elevated CSF total protein levels in 41% (31/76) of samples.In total, the results of 146 lumbar punctures (LP) in 132 patients were analysed. The most striking finding was a lack of CSF-restricted oligoclonal bands (OCB) in 66% (56/85) of all LP in the total BCS group, in 74% (14/19) in the subgroup of patients with both MRI and histological evidence for BCS, and in 82% (18/22) in the subgroup of patients with highest radiological confidence . OCB disappeared in 1/2 initially OCB-positive patients. These findings are in stark contrast to MS, in which OCB are present in ≥ 95% of patients and are thought to remain stably detectable over the entire course of disease (OCB and IgG index frequencies in BCS are much more similar to those reported in neuromyelitis optica or myelin oligodendrocyte glycoprotein antibody-associated encephalomyelitis than to those in MS. Our findings suggest that in most cases, BCS-like lesions denote the presence of a disease entity immunologically distinct from MS. In addition, we provide data on the demographics, clinical course and radiological features of BCS based on the largest cohort analysed to date. Baló’s concentric sclerosis (BCS) is a rare demyelinating disorder of the central nervous system (CNS) characterised by concentric layers of demyelination as detected by MRI or histopathology, and named after the Hungarian pathologist József Baló 1895-1979) 895-1979 . BCS is In a number of studies, we have demonstrated highly significant differences in cerebrospinal fluid (CSF) features between patients with MS and patients with other autoimmune disorders of the CNS. In particular, we have found significantly lower frequencies of CSF-restricted oligoclonal bands (OCB) and other markers of intrathecal IgG synthesis in patients with AQP4-IgG-positive neuromyelitis optica (NMO) spectrum disorders (NMOSD), myelin oligodendrocyte glycoprotein antibody-associated encephalomyelitis (MOG-EM), acute demyelinating encephalomyelitis (ADEM) and paraneoplastic neurological disorders (PND) than in MS, as well as significant differences in intrathecal IgG composition and dynamics and in blood–CSF barrier function –14. ThisTo address the question of whether BCS is a variant of multiple sclerosis (MS) or an immunologically distinct disease entity in its own right, we therefore set out to systematically compare the CSF features of BCS with those in MS.We performed a systematic review of all BCS cases published in English, German, French, Spanish, Japanese, Chinese, Russian or Italian in journals indexed in the PubMed database of the US Library of Science at the US National Institutes of Health between 1980 and 2017. Relevant publications were identified using the following search expression: Balo[title/abstract] OR Balo’s[title/abstract] OR Balos[title/abstract] OR ‘concentric sclerosis’[title/abstract]. Further publications/cases were identified by screening the reference lists of all PubMed-retrieved publications and from the authors’ own files. Overall, 215 publications that made mention of Baló’s concentric sclerosis in the abstract and/or title were identified and information on the CSF findings of 132 patients with BCS was collected. Results from follow-up lumbar punctures (LP) were available from 12/132 (9%) patients . In total, results from 146 LP in 132 patients diagnosed with BCS were available for retrospective analysis.The following CSF parameters were assessed: CSF-restricted oligoclonal IgG bands (OCB); Link’s IgG index ; immunoglobulin G/A/M (IgG/A/M) CSF levels and IgG/A/M CSF/serum ratios (QIgG/A/M); measles/rubella/zoster reaction (MRZR) as defined by a positive reaction to at least two of the three viral agents; CSF albumin concentration and albumin CSF/serum ratio (QAlb); CSF white cell count (WCC); CSF cytology; CSF total protein concentration (TP); frequency of an albuminocytologic dissociation (ACD), i.e. CSF TP elevation in the absence of CSF pleocytosis; CSF myelin basic protein concentration (MBP); and CSF lactate concentration. A CSF white cell count ≥ 5/μl was classified as increased. An age-dependent upper reference range for CSF L-lactate was applied . The upper reference limit for total CSF protein was set at 50 mg/dl [In addition, the following demographic, clinical and paraclinical parameters were assessed: sex, ethnic origin, age at onset, age at LP, time between last attack and LP, attack and long-term outcome, treatment status at the time of LP, number and location of Baló-like lesions, frequency and number of coexisting non-Baló-like lesions, initially suspected diagnosis, level of diagnostic certainty , and outcome at last follow-up .U test and Fisher’s exact test (two-tailed) were used to compare groups. Spearman’s r was used to test for correlations between groups. Due to the exploratory nature of this study, no correction for multiple testing was performed. All analyses were performed retrospectively; accordingly, no CSF or serum samples were obtained for this study.The Mann–Whitney The sex ratio  was 1:1.54. The median age of disease onset was 32 years (range 4–57). In 68 patients, the diagnosis of BCS was based upon typical radiological features; in 35, on typical histopathological features; and in 25, on both typical radiological and typical histopathological features. Histopathological evidence was obtained from biopsy samples in 15, from autopsy samples in 42 and from both biopsy and autopsy samples in 3 patients. In 43 cases, a single Baló-like lesion was present at the time of LP as detected by MRI or, if MRI was not available, histology, and multiple  Baló-like lesions were noted in 53 cases. Baló-like lesions were located in the supratentorial brain at the time of LP in 93 cases, in the infratentorial brain in 3, in both the supra- and infratentorial brain in 5 and in the supra- and/or infratentorial brain and the spinal cord in 4. Additional non-Baló-like CNS lesions were present in 58/94 (61.7%) patients at the time of LP. Before the diagnosis of BCS was made, MS was initially suspected in at least 8 patients, stroke in 5 and other diagnoses such as ‘tumefactive demyelinating disease’, ‘brain tumour’ or ‘brain abscess’ in 8 (no data in the remainder). Antibodies to aquaporin-4 (AQP4-IgG) or myelin oligodendrocyte glycoprotein (MOG-IgG) had been tested in 20 and 3 patients, respectively, and were negative in all. The median age at first LP was 32 years (range 4–60). The exact time between onset of the most recent attack and LP was given for 54 LP and was ≤ 30 days in 42 cases; the median interval was 15 days (range 1–190 days). In 58 additional cases, the exact time interval was not specified but LP was performed during an acute attack and thus probably within 30 days of attack onset. At last follow-up , more than one attack had occurred in 27/102 (26.5%) patients. Fifty-eight LP were performed prior to treatment with steroids or immunosuppressants, 4 LP in patients who had been treated with steroids shortly beforehand, 1 LP in a patient treated with immunosuppressants and 1 LP in a patient who had been treated with PEX and steroids 6 months previously ; in the p < 0.000001 when compared to a reference work on OCB in MS [The most striking finding was a lack of CSF-restricted OCB in the majority of cases: 65% (52/80) of all patients were negative for OCB N=51) or only transiently positive (N=1) (Table  or only CB in MS ). Of notp < 0.00001). Moreover, so far, an effect of IS on OCB positivity in MS has been shown only for natalizumab [Immunosuppressive treatment may theoretically affect OCB frequency. However, the difference between BCS patients and MS in terms of OCB positivity was still highly significant after exclusion of all patients treated with immunosuppressants (IS) at the time of LP .N = 25); all of these lesions showed three or more layers of demyelination. In this subgroup of cases with particularly high radiological confidence, the lack of OCB was even more pronounced than in the total cohort .Due to the retrospective nature of the study, MRI quality varied considerably among cases. To exclude the possibility that the low rate of OCB was due to unintentional inclusion of patients with diagnoses other than BCS, we defined (in a fashion blinded to OCB results) a subgroup of cases with available high-quality MRI depicting textbook-like BCS lesions .N = 64), the differences did not reach statistical significance. Moreover, as a potentially important bias, most fatal cases were reported in Chinese, Philippine and Japanese patients in the 1980s and early 1990s, when OCB were not yet generally determined in those countries. However, if only cases published during the past 20 years (1998 to 2017) were considered, again, more OCB-negative than OCB-positive patients had an unfavourable outcome .We were then interested in whether OCB may define distinct subgroups of BCS patients. However, no significant differences were found between OCB-positive and OCB-negative patients with regard to sex , median age at onset (32 vs. 30 years), median age at LP (32 vs. 32 years), disease course at last follow-up (relapsing in 36 vs. 34%), number of Baló-like lesions (more than one in 48.1 vs. 55.6%), location of Baló-like lesions  and co-existing non-Baló-like lesion (present in 48.1 vs. 55.6%). While more patients in the OCB-negative group had an unfavourable outcome .Conversely, no statistically significant differences in OCB frequency were found between male and female patients (31.3 vs. 37.5%), between children/juveniles and adults (50 vs. 32.9%), between patients with a relapsing and patients with a non-relapsing disease course at last follow-up (36 vs 34%), between LP in patients with a single Baló-like lesion and in those with multiple Baló-like lesions (36.8 vs 32.4%) or between LP in patients with additional, non-Baló-like lesions and in those without such lesions (36.4 vs 27.6%). A non-significant trend towards a lower frequency of OCB in patients with no/almost no recovery or fatal outcome compared with patients who had partial to full recovery at last follow-up was noted  [In accordance with the widespread lack of IgG OCB, Link’s IgG index was positive only in 22.6% (7/31) of all samples tested for that parameter. This is in contrast to the reference study in MS, which reported an elevated IgG index in 86.4%  .N = 267; p < 0.000001) [p = 0.013).The CSF WCC was elevated in 27/96 (28.1%) samples. This compares to a CSF pleocytosis rate in MS of 58.4% reported in a reference work on CSF in MS (.000001) . Among pp < 0.0005 vs. BCS). The majority of patients with ACD and available data (90.9% or 10/11) were OCB negative. CSF MBP concentration was determined in seven samples and was elevated in three (42.9%).TP was elevated in 40.8%  of all CSF samples. Most patients with elevated TP and available data were OCB-negative (77.8% or 14/18). A so-called ACD, i.e. an increase in the CSF total protein concentration without an accompanying increase in the number of CSF white cells, was found in 17/72 (23.6%) samples. ACD is typically found in Guillain-Barre syndrome but not usually in MS  were reported only in single patients or not at all, preventing systematic analysis of these CSF parameters.43.2% (35/81) of all CSF examinations showed neither pleocytosis nor elevated TP, and 15.1% (11/73) showed neither CSF pleocytosis or TP elevation nor intrathecal IgG synthesis. Of note, 12 further LP were classified as ‘normal’ by the authors, but no more detailed information was provided. While these LP could not be included in the statistical analysis, they suggest that the low rate of WCC pleocytosis, OCB positivity and IgG index elevation observed in our study may even underestimate the real lack in CSF abnormalities in BCS and, in consequence, the real degree of difference between BCS and MS in terms of CSF pathology.p < 0.000001 compared with MS) and that Link’s IgG index, a quantitative marker of intrathecal IgG synthesis, is negative in more than 75% of cases (p < 0.000001 compared with MS). Given that intrathecal IgG synthesis is considered a diagnostic mainstay of MS [Traditionally, BCS is considered a variant of MS. Here, we demonstrate highly significant differences in CSF profiles between patients with BCS and patients with MS. In particular, we found that CSF-restricted OCB, a qualitative marker of intrathecal IgG synthesis, are missing in almost two-thirds of patients with BCS , further corroborating the notion that BCS and MS are, in most cases, distinct disorders.In addition, we found a significantly lower frequency of CSF pleocytosis in BCS than in MS reference cohorts  and CSF lactose levels, were reported in only a few patients, preventing statistical assessment.In the effort to better understand the significance of OCB in BCS, we looked into possible differences between OCB-positive and OCB-negative patients with BCS with regard to demographics, disease course, MRI findings and disease outcome. However, no marked differences between the two groups were found in terms of age at onset, age at the time of LP, sex ratio, disease course, lesion location or the number of Baló-like lesions on MRI. Conversely, the rate of OCB-positive samples did not differ significantly between male and female patients, relapsing and non-relapsing patients, children and adults, or patients with a single Baló-like lesion and patients with multiple Baló-like lesions. However, a non-significant trend towards an association of OCB negativity with more severe disease outcome was found.We first hypothesised that the presence or absence of intrathecal IgG synthesis might depend on disease activity, as has been demonstrated in AQP4-IgG-positive NMOSD . InteresAlternatively, BCS lesions may not denote a single disease entity but may be a  final pathway of various demyelinating diseases, e.g. reflecting the presence of intralesional hypoxia as recently proposed. If so, BCS lesions could occur in the context of both conventional (OCB-positive) MS and other (mostly or exclusively OCB-negative) inflammatory demyelinating conditions such as MOG encephalomyelitis, ADEM, NMOSD or other as yet unidentified disorders. In potential keeping with that hypothesis, there was substantial heterogeneity among BCS cases in terms of disease course (relapsing vs. monophasic), MRI presentation  and outcome . Moreover, some patients with BCS had initially been diagnosed with MS, ADEM or NMOSD. Finally, at least some patients with BCS may suffer from what has been called ‘pattern III’ MS. ‘Pattern III’ MS is diagnosed on the basis of histopathological features, some of which suggest ischemia-mediated tissue damage, and has only recently been proposed to denote a disease entity distinct from classical MS based on our observation that patients with ‘pattern III’ lesions are typically negative for OCB . While pInterestingly, a recent study reported a loss of aquaporin-4 (AQP4) and other astrocytic proteins in BCS, which in some lesions preceded the loss of myelin, suggesting that BCS might, at least in a subset of cases, be primarily an astrocytopathy, just as AQP4-IgG-positive NMO . HoweverOur study has both strengths and limitations. Among its strengths, we count the very high number of LP examined  and the fact that our results were both highly significant and robust when retested in subgroups with particularly high diagnostic certainty (see the ‘On the other hand, one may count the retrospective nature of our study and the fact that the patients included in the analysis were seen at many different centres among the potential limitations. However, it is virtually impossible to perform a large prospective single-centre study on BCS given the condition’s very low prevalence. Moreover, the study’s retrospective ‘multicentre’ setting reduced potential risks resulting from centre-specific selection bias, a problem inherent to single-centre studies. Second, no CSF results were given in numerous reports on BCS in the previous literature. This might have introduced a bias towards cases with pathological CSF findings. However, this would imply that our results actually underestimate the observed lack of OCB, IgG index elevation and WCC pleocytosis in BCS and would thus make our findings even more relevant.The present study focused on immunological parameters routinely tested in patients with MS and BCS. Further immunological studies aiming at investigating the differences between BCS and MS in terms of immunopathogenesis in greater detail are now required.It is reassuring that our results are in agreement with a previous much smaller study performed on behalf of the MAGNIMS network . FollowiIn summary, our study demonstrates significant differences in CSF findings between patients with BCS and patients with MS, especially in terms of intrathecal IgG synthesis. This suggests differences in immunopathogenesis between BCS and MS and supports the notion of Baló-like lesions denoting the presence of a disorder immunologically distinct from MS in most cases. This could have clinically important diagnostic, nosological, prognostic and therapeutic implications. Of note, the OCB frequency in BCS is more similar to that reported in NMOSD and MOG-EM than in classic MS Fig. . Further


Vitamin D deficiency is a major global public health problem. Recent epidemiological studies have assessed the relationship between vitamin D and multiple outcomes, including cardiovascular disease. However, this evidence is limited and inconclusive. Our purpose in this study was to evaluate the association between dietary vitamin D intake and cardiovascular disease risk in adult Mexican population.We conducted a cross-sectional analysis with the baseline data from 6294 men and women aged 20–80 years participating in the Health Workers Cohort Study. Data on sociodemographic, lifestyle, and medical history factors were collected with a self-administered questionnaire. Dietary intake was evaluated by using a semi-quantitative food-frequency questionnaire. Cardiovascular disease risk was calculated using a recalibration of the Framingham heart disease prediction score. To evaluate the association between vitamin D intake and 10-year cardiovascular disease risk, odds ratios (OR) and 95% confidence intervals (95% CI) were calculated using multiple logistic regression analysis.P for trend = 0.001); 2.0 cm less in waist circumference (P for trend = 0.001) and 0.8 points less in the Framingham cardiovascular disease risk score (P for trend = 0.002) compared with the subjects in the lower quintile of vitamin D intake. Additionally, participants in the highest quintile of vitamin D consumption were less likely to develop elevated 10-year cardiovascular disease risk, compared with those in the lowest quintile .A total of 6294 subjects (1820 men and 4474 women) with a mean age of 42 years, were included. Of these, subjects in the highest quintile of vitamin D intake presented lower levels of triglycerides 14.6 mg/dL (Our data suggest that higher consumption of vitamin D is associated with a reduced risk of cardiovascular disease in Mexican population. Vitamin D (VD) is a prohormone whose main function is to regulate calcium and phosphorus metabolism related to preserve bone mass . Due to In humans, VD is primarily obtained through sun exposure, because very few foods naturally contain vitamin D, diet and dietary supplements are other sources of VD; consequently, the assessment of dietary intake of VD is valuable for nutritional epidemiological studies . VitaminCardiovascular disease (CVD) is the leading cause of death and a major cause of disability worldwide , and theThere are some plausible mechanisms that explain how vitamin D might modify cardiovascular outcomes, vitamin D regulates the renin-angiotensin system , suppresThere are still some questions on this issue, we do not know if optimal vitamin D consumption and concentrations are a cause or a consequence of good health. Therefore, we examined the cross-sectional relation between dietary vitamin D intake and risk of cardiovascular disease as well as cardiometabolic risk factors in subjects participating in the Health Workers Cohort Study (HWCS), in Mexico. We hypothesized that high intake of vitamin D would be negatively associated with cardiovascular disease risk, independently of demographic and lifestyle factors.We conducted a cross-sectional analysis of data from adults participating in the baseline assessment of the Health Workers Cohort Study (HWCS). The participants in this cohort study are middle- to low-income status, residing in urban central Mexico. The study design, methodology, and participants’ characteristics have been detailed elsewhere –30. ConcFor the present analysis we included 6294 subjects aged 20–80 who provided fasting blood samples , and were not previously diagnosed with type 2 diabetes (assessed by self-report or with levels of glucose ≥ 126 mg/dL) (n = 491) or taking lipid lowering medication (n = 567). We also excluded participants who left 10 percent or more food items blank on the questionnaire (n = 635), and who did not consume between 600 kcal and 7000 kcal daily (n = 320).This study was planned and conducted according to the guidelines laid down in the Declaration of Helsinki. All participating institutions’ research ethics committees: Comité de Ética en Investigación, Instituto Mexicano del Seguro Social (No. 12CEI 09 006 14); Comité de ética en Investigación, Instituto Nacional de Salud Pública (No. 13CEI 17 007 36); Comité de Ética, Centro de Investigación en Ciencias Médicas (No. 1233008X0236), revised and approved the study protocol and informed consent forms. Written informed consent was obtained from each participant.Dietary intake data and VD consumption were collected using a 116-item semi-quantitative food frequency questionnaire (FFQ), previously validated in an adult Mexican population .Vitamin D and other nutrient consumption were computed by multiplying the frequency of consumption of each unit of food from the FFQ by the VD and nutrient content of the specified portion size. Composition values for dietary VD and other nutrients were estimated by means of a comprehensive Mexican database of food composition . Informa2) were used to calculate body mass index (BMI kg/m2). Waist circumference was measured at the high point of the iliac crest at the end of normal expiration, to the nearest 0.1 cm, with a steel measuring tape, which was placed below any clothing, directly touching the participants’ skin. In the present study, being overweight or obese was defined as BMI ≥ 25 kg/m2 [As we reported previously –30, part25 kg/m2 . Abdomin25 kg/m2 .A fasting venous blood sample (fasting time was 12 hours) was collected from each participant. Plasma triglycerides were measured with a colorimetric method following enzymatic hydrolysis performed with the lipase technique. High-density lipoprotein cholesterol (HDL-c) was measured by clearance method; in this method non HDL-c lipoprotein is removed in the first step of the reaction (clearance step). Low-density lipoprotein cholesterol (LDL-c) was measured by clearance method; finally, total cholesterol was measured by colorimetric method following enzymatic assay. All biomedical assays were performed using a Selectra XL instrument (Randox).High lipid profile was defined according to the criteria put forth in the report of the National Cholesterol Education Program ATP-III, that defines a high lipid profile as: high serum triglycerides ≥150 mg/dL, high serum total cholesterol ≥200 mg/dL, high LDL-c ≥100 mg/dL, and low HDL-c ≤40 mg/dL in men and ≤50 mg/dL in women .Cardiovascular disease (CVD) risk was calculated using a recalibration of the Framingham coronary heart disease prediction scores , 37. We The main outcome of the current analysis was the development of more than 10 percent risk of CVD in ten years. We defined participants as at low CVD risk when they had less than 10 percent risk in ten years. Subjects who had more than 10 percent risk in ten years were defining has having a CVD risk (moderate/elevated) by the American Heart Association standards .Demographic characteristics , medical history, and lifestyle, including alcohol and tobacco consumption, were collected by means of self-administered questionnaire. Physical activity was assessed using International Physical Activity Questionnaire (IPAQ). Participants were asked about their daily recreational activity, leisure activity, daily activity and any physical labor associated with employment. Participants reported the time they spent each week on activities such as running and walking during a typical week in the previous year. Each activity was given a value in metabolic equivalent tasks (METs) and total METs per week was computed –30.Descriptive analyses of the main variables of interest  across quintiles of total vitamin D intake were performed. Analysis of variance (ANOVA) was used to evaluate mean differences across quintiles of total dietary VD consumption for continuous variables. The chi-square test was used to determine differences in the distribution of categorical variables across VD quintiles.The influence of the total dietary VD intake on lipid profile, BMI, abdominal obesity, and scores of CVD risk was evaluated using multivariate lineal regression models in which these variables were analyzed as a continuous.Finally, to estimate the magnitude of the association between specific categories of total dietary VD intake and abnormal lipids profile, obesity, abdominal obesity, as well as elevated CVD risk, we computed adjusted odds ratios (OR) and 95% confidence intervals (95% CI) using multiple logistic regression models.2 vs ≥ 25 kg/m2). We tested the significance of the interaction with a likelihood ratio test by comparing a model with the main effects of each intake and the stratifying variable and the interaction terms with a reduced model with only the main effects.To assess possible effect modification, we explored analyses stratified by body mass index .All 3  respectively. A higher VD intake was observed in subjects with lower BMI, fewer prevalence of overweight and obesity and abdominal obesity, increased physical activity levels, decreased use of tobacco, and increased use of multivitamins (P for trend <0.001) were observed across increasing VD quintiles. In addition, subjects in the highest quintile of VD intake had a lower prevalence of diabetes and lipid abnormalities compared with subjects in the lowest quintile of VD consumption. Finally, data shows that subjects in the highest quintile of VD intake have a lower likelihood of > 10% risk of 10-year cardiovascular disease than those in the lowest quintile.Participants’ baseline characteristics  are shown in P for trend = 0.01); 0.9 kg/m2 in BMI (P for trend <0.001); 2.0 cm in waist circumference (P for trend = 0.001) and 0.8 points in the Framingham cardiovascular risk score (P for trend = 0.002) compared with the subjects in the lower VD category.The effect of VD on cardiovascular risk markers is shown on P for trend = 0.007).P for interaction <0.001) when subjects with low VD consumption and overweight/obesity were compared with subjects with a low consumption of VD and body mass index < 25 kg/m2  of BMI, and 2.0 cm in waist circumference. In accordance with this result, other studies [In literature, no studies have investigated the association between dietary VD and cardiovascular disease risk factors. Despite the fact, in a set of cross-sectional studies –47, the  studies –45 have  studies .P for trend = 0.006), 35% lower odds of low HDL-c, and 26% lower odds of being obese or abdominal obese than subjects in the lowest quintile of dietary VD intake. However, serum total cholesterol and LDL-c concentrations were not related to VD intakes. Compared to our results, Martins et al. [In our study, logistic regression analysis shows that subjects in the higher dietary VD group have 28% lower odds of hypertriglyceridemia . This result is consistent with a study that found that subjects in the higher group of total VD intake was associated with a decreased risk of CVD; the relative risks (95% CIs) for a comparison of participants who met the Dietary Reference Intake of VD (≥ 600 IU/day) with participants whose VD intake was <100 IU/day was 0.84  for men and 1.02  for women [Our data also suggests an inverse relationship between VD intake and 10-year CVD risk. We observed that subjects in the highest quintile had 49% lower odds of 10-year CVD risk Click here for additional data file.


In unadjusted Cox proportional regression analyses OSA was not associated with higher all-cause mortality risk (Hazard Ratio(HR) = 1.20; 95% Confidence Interval(CI): 0.50–2.85). No association was found between the presence of OSA and the rate of progression of CKD or all-cause mortality in prevalent kidney transplant recipients.Obstructive sleep apnea(OSA) is one of the most common sleep disorders in kidney transplant recipients, however its long-term consequences have only rarely been investigated. Here, we hypothesized that the presence of OSA would be associated with higher risk of mortality and faster decline of graft function in kidney transplant recipients. In a prospective cohort study 100 prevalent kidney transplant recipients who underwent one-night polysomnography at baseline and were followed for a median 75 months. Generalized linear mixed-effects models and Cox regression models were used to assess the association between OSA and the rate of progression of chronic kidney disease(CKD) and mortality. The estimated slopes of estimated glomerular filtration rate(eGFR) in patients with and without OSA were compared using a two-stage model of eGFR change including only OSA as a variable. In this model patients with OSA (eGFR versus time was −0.93 ml/min/1.73 m Obstructive sleep apnea (OSA) is one of the most clinically important forms of sleep-related breathing disorders. The prevalence of moderate and severe obstructive OSA (AHI ≥ 15 and the presence of daytime symptoms of OSA) is 2–4% in the general population2456Previous studies have shown high prevalence of OSA (16–54%) in patients with chronic kidney disease (CKD)8Despite the potential clinical relevance, the long-term consequences of OSA have only rarely been investigated among kidney transplant recipients. The association between OSA and hypertension, accelerated atherosclerosis and vascular damage was confirmed in patients with CKDWe designed this prospective cohort study to determine the association between presence of OSA and long-term outcome, such as the decline of graft function and all-cause mortality in a randomly selected sample of stable, prevalent kidney transplant recipients. OSA was assessed using polysomnography at baseline. Based on the previous findings we hypothesized that the presence of OSA would be associated with faster decline of graft function and higher risk of mortality.The SLeep disorders Evaluation in Patients after kidney Transplantation (SLEPT) - study cohort was described previouslyThe percentage of male patients was significantly higher among patients with versus without apnea-hypopnea index (AHI) ≥ 15/h. The prevalence of mild (5/h ≤ apnea-hypopnea index (AHI) < 15/h), moderate (15/h ≤ AHI < 30/h) and severe OSA (AHI ≥ 30/h) was 18%, 11% and 14% in our kidney transplant recipients2/yr (95% CI: −1.55 to −0.78) (2/yr (95% CI: −1.75 to −0.11) had a similar slope than patients without OSA (eGFR versus time was −1.24 ml/min/1.73 m2/yr (95% CI: −1.67 to −0.81)  . Figure o −0.81) . Similaro −0.81) . Howevero −0.81) . Additioo −0.81) . Similaro −0.81) .2/year as a cut-off level (2/year (2/year (not shown) or >8 ml/min/1.73 m2/year as a cut-off level for rapid decline (not shown). A similar result was found when we adjusted for baseline eGFR (not shown). Moreover, neither the high desaturation index  nor the desaturation index (as a continuous variable) was associated with the rapid decline of eGFR using >4 ml/min/1.73 m2/year  nor the AHI index (as a continuous variable) was associated with the rapid decline of eGFR using >4 ml/min/1.73 mff level . Similar  was 47.8/1000 patient-years . The unadjusted mortality rate was similar among patients with and without OSA ; AHI < 15/h group: 45.6/1000 patient-years (95%CI: 29.1–71.5); p = 0.68). Time to death was also similar in patients with versus without OSA, as shown on the Kaplan-Meier plot . We foun1 point increase = 1.28; 95% CI: 1.04–1.58) (04–1.58) .Moreover, neither the high desaturation index  nor the desaturation index (as a continuous variable) was associated with the all-cause mortality . SimilarIn this prospective cohort study, which is one of the largest studies using polysomnography in kidney transplant recipients, there was no association between the presence of OSA and the rate of decline of graft function in prevalent kidney transplant recipients. In addition, we could not find any association between presence of OSA and all-cause mortality in this population.In this dataset the prevalence of OSA was higher than published by Mallamaci et al. recentlyThe rate of decline of graft function was similar in transplant recipients with and without OSA. This is surprising as previous data showed strong correlation between severity of CKD and prevalence of OSA in CKD population1617We previously showed that high risk of OSA is an independent predictor of graft loss among female kidney transplant recipientsThere was no association between OSA and all-cause mortality or combined outcome in our patients. OSA is an independent predictor of mortality in the general population22829Post hoc power calculations suggest that this study was powered to detect 0.25 or lower hazard ratio of the mortality between the non OSA versus OSA groups with a power of 80%, but the power is insufficient to detect a smaller difference. Based on this data, we can conclude that our study was underpowered and further, larger studies are needed to confirm or reject our results. Patients from a single center were enrolled; therefore, our results are not to be generalized without further considerations. Finally, a substantial proportion of transplant recipients refused to participate  Study”) potentially eligible patients were selected from all prevalent adult transplant recipients  who were regularly followed at a single outpatient transplant center on December 31, 2006. After applying exclusion criteria  on dialysis or transplant, active and acute respiratory disorder, acute infection, hospitalization within 1 month, surgery within 3 months) 1198 patients remained . From this “base population” we randomly selected and approached 150 patients (“Tx study sample”) using the simple random sampling strategy offered by SPSS 15.0 . From thStandard, attended overnight polysomnography was performed in our sleep laboratory . Recordings were manually scored by two somnologists. Sleep stages were determined in 30 s epochs according to Rechtschaffen and Kales34Laboratory, demographic, anthropometric, medication data and single pool (sp) Kt/V were extracted from the medical records. Estimated glomerular filtration rate (eGFR) was calculated using the abbreviated Modification of Diet in Renal Disease (MDRD) study formulaComorbidity was assessed by the modified Charlson Comorbidity Index (CCI)37Transplantation related information collected included current medications, transplant and dialysis “vintage” , time spent on dialysis prior to transplantation, type of transplantation (deceased donor or living donor), history of cumulative acute rejection, HLA mismatch, titer of pre-transplant panel reactive antibodies (PRA), cold ischemic time (CIT), age and gender of donor and history of delayed graft function. Time elapsed since the initiation of the first treatment for ESRD (cumulative ESRD time) was also calculated. Standard maintenance immunosuppressive therapy generally consisted of prednisolone, either CsA or tacrolimus, combined with MMF or azathioprine, everolimus or sirolimus.2 the last value was the last available eGFR or the last value below 15 ml/min/1.73 m2 and no further data was collected. The “rapid decline of graft function” status was defined if the deterioration of eGFR was higher than 4 ml/min/1.73 m2/year. The secondary outcome variable was all-cause mortality, which included all deaths with a functioning graft and deaths occurring after the graft failure . Deaths and re-initiations of maintenance dialysis were ascertained from the hospital database. Deaths were validated by cross-referencing with data from the Hungarian Central Office of Administrative and Electronic Public Service, which is the government agency maintaining official vital status records. In addition, we also performed a sensitivity analysis using combined outcome, defined as death of any cause or return to dialysis or rapid decline of graft function (deterioration of eGFR was higher than 4 ml/min/1.73 m2/year) of graft function. None of our patients were treated for OSA at baseline or during the follow-up period, however CPAP treatment was offered all of them.Patients were followed for an average 75 months . The primary outcome variable was deterioration of graft function. We collected each patient's eGFR in every 6 months for the entire follow-up period. If a patient was started on dialysis or re-transplanted or eGFR was less than 15 ml/min/1.73 mThe study was approved by the Ethics Committee of the Semmelweis University (4/2007). Before enrolment, patients received detailed verbal and written information about the aims and protocol of the study and signed an informed consent. All experiments were performed in accordance with relevant guidelines and regulations.Statistical analyses were carried out using the STATA 12.1 software. Results are presented as percentage, mean  or medians . Continuous variables were compared using Student's t-test or the Mann-Whitney U test and categorical variables were analyzed with chi-square test. In all statistics, two-sided test were used and the results were considered statistically significant if p was less than 0.05.For multivariate analysis, logistic- and Cox regressions were applied. Independent variables were included in the multivariate models based on theoretical considerations. Variance influence factors (VIF) were used to indicate collinearity between independent variables. The association between baseline AHI level and all-cause mortality was assessed using Cox proportional regression analysis and Kaplan-Meier plots with log rank test. Proportional hazards assumptions were tested using scaled Schoenfeld residuals.The association between the presence of OSA (using different AHI cut-off levels) and the slopes of eGFR versus time were examined in generalized linear mixed-effects models allowing for a random intercept and slope using the “XTMIXED” command in STATA. The change in eGFR from baseline until death, start of dialysis, or re-transplantation (whichever occurred first) was studied in all transplant recipients who had at least three serum creatinine measurements  by using a two-stage model formulation. In such a model, the level 1 change describes intra-individual changes in eGFR, and the level 2 model describes how the change coefficients differ across participants. The covariate of interest (AHI as independent variable) is thus included in the level 2 models to explain interindividual differences in intra-individual change (slope).Supplemental tables and figure