PHD1  belongs to a family of prolyl hydroxylases (PHDs) that are involved in the control of the cellular response to hypoxia. PHD1 is also able to regulate mitotic progression through the regulation of the crucial centrosomal protein Cep192, establishing a link between the oxygen-sensing and the cell cycle machinery. Here, we demonstrate that PHD1 is phosphorylated by CDK2, CDK4 and CDK6 at S130. This phosphorylation fluctuates with the cell cycle and can be induced through oncogenic activation. Functionally, PHD1 phosphorylation leads to increased induction of hypoxia-inducible factor (HIF) protein levels and activity during hypoxia. PHD1 phosphorylation does not alter its intrinsic enzymatic activity, but instead decreases the interaction between PHD1 and HIF1α. Interestingly, although phosphorylation of PHD1 at S130 lowers its activity towards HIF1α, this modification increases the activity of PHD1 towards Cep192. These results establish a mechanism by which cell cycle mediators, such as CDKs, temporally control the activity of PHD1, directly altering the regulation of HIF1α and Cep192. Summary: CDK-mediated phosphorylation of PHD1 at serine 130 controls target specificity and confers cell cycle regulation of PHD1. Decreased oxygen levels, or hypoxia, present a major stress to the cell. Many of the crucial cellular processes, such as ATP production through oxidative phosphorylation, cell division and cell cycle progression are all highly energy-demanding processes that require oxygen . ExposurHypoxia activates a variety of transcription factors , but the2+ as cofactors . PHDs require molecular oxygen as a co-substrate to carry out hydroxylation, but they also have a requirement for α-ketoglutarate (α-KG) and Feofactors . As α-KGofactors . More reofactors . Currentofactors . Geneticofactors , whereasofactors .During normoxia, hydroxylation of HIFα creates a binding site for the von Hippel-Lindau (VHL) tumor suppressor E3 ligase complex. Binding of VHL results in polyubiquitylation and proteosomal degradation of HIFα. During hypoxia, when oxygen levels are decreased, PHD activity is reduced, leading to stabilization of HIFα and dimerization with HIF1β, resulting in a transcriptionally active complex. HIFs have been shown to regulate a large number of genes involved in a variety of cellular processes, such as metabolism, apoptosis, autophagy, angiogenesis and cell proliferation . The resOne of the most energy-consuming processes within the cell is the cell cycle and, hence, cell division. This process must be tightly regulated to ensure there is no hyperproliferation and/or mis-segregation of genetic information. Errors within the cell cycle can ultimately lead to disease states, such as cancer. Cell cycle control is achieved through multiple mechanisms, but amongst the most important regulators are cyclin-dependent kinases (CDKs) . CDKs arSeveral studies have shown that hypoxia also affects the cell cycle. Early work has demonstrated that, upon exposure to hypoxia, cells are reversibly arrested in G1 or S phase . The mecIn more recent years, the roles of the PHDs in processes other than hypoxia have become more apparent. Recently PHD1 has been shown to regulate the transcription factor FOXO3A . PHD3 haHere, we show that PHD1 is regulated by the cell cycle at the post-translational level. We show that PHD1 is phosphorylated at S130 in a CDK-dependent manner. PHD1 phosphorylation reduces its interaction with HIF1α, but increases the association between PHD1 with Cep192. Functionally, this results in increased levels of HIF1α protein and increased transcriptional activity in response to hypoxia, and in reduced levels of Cep192 protein. These results indicate that the behaviour of PHD1 towards different substrates can be altered by specific post-translational modifications.Xenopus and the fruit fly . Immunoprecipitation of GFP from the GFP, PHD1–GFP, PHD1-S130A–GFP or PHD1-S130D–GFP cells, revealed that the phospho-specific antibody only detected a band in the extracts derived from wild-type PHD1, demonstrating its specificity . Collectively, these results show that PHD1 is subject to phosphorylation on S130 in cells.Although, endogenous PHD1 levels are difficult to detect in cell lines other than breast cancer lines , we usedFig. S1C). We could also detect an interaction between PHD1 and CDK2 at the endogenous level . By contrast, gain-of-function experiments revealed that when increased levels of CDK4 were present, there was a concomitant increase in the levels of S130 phosphorylation of PHD1 . CDK2 was immunoprecipitated from cells, and kinase assays were performed using bacterially expressed recombinant PHD1, followed by western blot analysis using the anti-phospho-PHD1 antibody . In this case, mutation of S130 only slightly reduced the phosphorylation signal, suggesting that other sites on PHD1 are being targeted by CDK1 in vitro. Taken together, these results suggest that no single CDK is exclusively responsible for the phosphorylation of PHD1, indicating a redundancy between them. This is to be expected, as genetic studies have shown that only CDK1 is essential, as knockout of CDK1 results in significant developmental defects in mice . We compared hydroxylation of the HIF peptide over time using recombinant purified GST–PHD1, GST–PHD1-S1301A and GST–PHD1-S130D proteins. No significant difference in hydroxylation activity between the wild-type PHD1 and the two mutants could be detected . These data indicate that phosphorylation of PHD1 at S130 has little or no effect on the intrinsic enzymatic activity of PHD1.As PHD1 is a proline hydroxylase, we next determined whether phosphorylation of PHD1 at S130 could impact on its intrinsic enzymatic activity. Previous work performed in bacteria has shown that PHD1 is potentially phosphorylated on S132  and thistrometry A. This aFig. S2B). In addition, endogenous PHD1 was depleted by siRNA and, as expected, when MG132 was added, we saw an accumulation of HIF1α in all cell types. However, there were decreased levels of HIF1α in the PHD1–GFP and PHD1-S130A–GFP cells, whereas HIF1α levels in PHD1-S130D–GFP cells were similar to the cells expressing GFP alone . Hypoxia does not alter PHD1 phosphorylation at earlier times of exposure but does result in a significant reduction after 24 h . This is to be expected, as exposure to hypoxia for this period results in G1 arrest . However, for the PHD1-overexpressing cells, the highest induction of HIF1α was observed in the PHD1-S130D–GFP cells , indicating that PHD1 hydroxylase activity is still present when S130 is phosphorylated.In cells exposed to hypoxia, PHD1 overexpression results in a reduction of HIF1α levels and its targets as expected . These results show that levels of HIF1α targets are always higher in PHD1-S130D–GFP cells when compared with cells expressing wild-type PHD1. In this analysis, PHD1-S130A–GFP activity was comparable to wild-type PHD1, for all the genes analysed .To understand whether the changes in HIF1α levels observed in the PHD1-S130D–GFP cells are altering HIF1α transcriptional activity, we assessed HIF activity by investigating the levels of several HIF target genes. Levels of HIF1α targets were always higher in PHD1-S130D–GFP cells when compared with cells expressing wild-type PHD1. However, additional differences were also observed for PHD1-S130A–GFP cells when compared with cells expressing wild-type PHD1, with higher levels of PHD2, PHD3 and CAIX  observed in these cells D. This cFig. S2H). We thus chose a 3-h DFX treatment because, at this time point, HIF is stabilised but still hydroxylated, and hence PHD activity changes could still be monitored. When we investigated HIF1α levels in the different stages of the cell cycle, we could observe that cells in G1 had lower levels of PHD1 S130 phosphorylation as well as lower HIF1α levels . Cells were incubated under hypoxia for 4 h prior to treatment with cycloheximide for the indicated periods of time. We measured p53 levels as a positive control for the treatment, as it is known to have a high turnover rate . PHD1 levels were stable throughout the timecourse of the experiment in all cell lines . Interestingly, although PHD1–GFP and PHD1-S130A–GFP cells led to a substantial decrease in HIF1α half-life when compared with GFP cells, PHD1-S130D–GFP cells had a slower rate of degradation of HIF1α . These results are in agreement with the increased HIF1α levels observed in PHD1-S130D–GFP cells when exposed to either MG132 or hypoxia.Our analysis so far has revealed that PHD1 phosphorylation does not alter intrinsic enzymatic activity Fig. S3B), again using MG132 treatment to stabilise HIF1α prior to immunoprecipitation. In this system, we could again detect a reduction in the level of PHD1 that interacts with HIF1α in the cells transfected with the PHD1-S130D–GFP construct . By contrast, there was no difference between PHD1–GFP- and PHD1-S130A–GFP-transfected cells in the levels of interaction between PHD1 and HIF1α. These results suggest that phosphorylation of PHD1 at S130 disrupts the interaction between HIF1α and PHD1.To determine the mechanism behind the loss of PHD1 targeting of HIF1α in PHD1-S130D–GFP cells, we next investigated whether this modification alters the ability of PHD1 to interact with HIF1α in cells. To this end, we treated cells with MG132, to stabilise HIF1α, and immunoprecipitated endogenous HIF1α from all the PHD1 cell lines. Normal IgG was used as a negative control for this approach B. As canFig. S3C). This is to be expected, as this construct lacks the nuclear localisation signal. Furthermore, we could detect good expression levels of all of the constructs in cells when these were analysed by western blotting . Interaction assays with the deletion constructs revealed that both the N-terminal and C-terminal regions of PHD1 were able to interact with HIF1α in cells , indicating that phosphorylation is modulating PHD1 activity only in cells, and not affecting PHD1 intrinsic hydroxylase activity.Given the reduced levels of Cep192 in PHD1-S130D cells, we hypothesised that once phosphorylated, PHD1 had enhanced activity towards Cep192. To investigate this, we analysed whether PHD1 could interact differently with Cep192 in wild-type versus PHD1 S130 mutant cells. This analysis revealed that increased levels of PHD1 were immunoprecipitated with Cep192 in phospho-mimicking PHD1 cells C. By conFig. S4B). These results suggest that competition between the HIF and Cep192 substrates can occur.To determine whether S130 phosphorylation correlated with Cep192 levels, we analysed the synchronisation release samples described in Taken together, we conclude that S130 phosphorylation by interphase CDKs is an important determinant of which substrate PHD1 will target in cells F.in vitro, it regulates PHD1 activity in cells, determining target selection between HIF and Cep192. These results provide a mechanistic link between the cell cycle and the regulation of PHD1 activity in cells, allowing for its different functions to be carried out at specific stages of the cell cycle.In this report, we have identified and characterised the functional significance of a CDK-dependent phosphorylation site on PHD1. Our results show that phosphorylation of PHD1 on S130 is dynamic and regulated by CDK activity, cell cycle stage and oncogenic signals. Interestingly, although phosphorylation of PHD1 on S130 does not alter PHD1 hydroxylase activity 2+ and α-KG  are involved in the regulation of PHD1 phosphorylation on S130, but not CDK1. Previous data have indicated that CDKs show a degree of functional redundancy and can compensate for each other, with only CDK1 being essential . Our datll cycle . This stin vitro, when recombinant protein was analysed, but instead altered the interaction between PHD1 and HIF1α in cells. This could be due either to inability to bind a cofactor in cells or to a direct interference of the phosphorylation site with binding to HIF1α. Our analysis revealed that the N-terminal region of PHD1 is able to bind HIF1α without the help of the hydroxylation domain, suggesting that this latter point might be the case. The N-terminus of PHD2 has also been shown to be important for the regulation of HIF1α by PHD2, although in this case through an indirect mechanism involving a chaperone protein  supplemented with 10% fetal bovine serum (Gibco), 50 units/ml penicillin (Lonza) and 50 µg/ml streptomycin (Lonza) for no more than 30 passages at 37°C and 5% COGFP-N1 was obtained from Clonetech. GFP–PHD1 was a kind gift from Eric Metzen  and was used as a template to create the GFP-tagged PHD1-S130A and PHD1-S130D mutations by site directed mutagenesis. For the truncation mutants of PHD1, GFP–PHD1 and GFP–PHD1-S130D plasmids were used as templates. Primer sequences are available upon request.CMV-HIF1α and CMV-Src expression constructs were obtained from Origene. E2F1 and wild-type Myc and mutant expression constructs were a kind gift from Victoria Cowling . HA–CDK1 , HA–CDK4  and HA–CDK2  were as previously described .2 in an in vivo 300 hypoxia workstation . Cells were lysed for protein extracts and RNA extraction in the work station to avoid re-oxygenation. Whole-cell lysates were obtained using Triton lysis buffer . RNA was extracted using Peqlab Total RNA Kit (Peqlab) as per the manufacturer's instructions.Cells were incubated at 1% OMG132 was obtained from Merck/Millipore and used at the final concentration of 20 µM for 3 h. Desferroxamine mesylate (DFX) was obtained from Sigma and used at the final concentration of 200 µM.Hydroxylation assays were performed as in Kinases assays were performed as described in Antibodies were against the following proteins: phospho-serine-CDK substrate , 1:1000; phospho-threonine , 1:1000; phospho-serine , 1:1000; HIF1α , 1:1000; HO-HIF1α , 1:1000; HIF-2α , 1:1000; HIF-1β , 1:1000; β-actin , 1:5000; CAIX , 1:1000; HK2 , 1:1000; BNIP3 , 1:2000; GLUT3 , 1:1000; PHD3 , 1:1000; PHD1 , 1:1000; PHD2 (Bethyl A300-322A), 1:1000; Cep192 , 1:500; HDAC1 , 1:2000; CDK1 , 1:1000; CDK2 , 1:1000; CDK4 , 1:1000; CDK6 , 1:1000; phospho-ERK1/2, 1:1000; cyclin A , 1:1000, cyclin E , 1:1000; E2F1 , 1:1000; Src , 1:1000; cMyc (gift from Victoria Cowling), 1:500; p53 , 1:2000; pericentrin , 1:100; phospho-S130-PHD1 [produced in rabbits by immunisation with phospho-peptide for S130 of PHD1 (CEDGGDAPSphPSKR) and purified by Dundee Cell products], 1:200.® magnetic beads (ChromoTek).PHD1–GFP cells were lysed as described previously  and PHD1Immunoprecipitation eluates were separated on 1D SDS PAGE gels and stained . The protein bands of interest were excised, chopped into ∼1-mm×1-mm pieces and destained at room temperature . After 15 min dehydration in 100% ACN, proteins in the gel pieces were reduced by incubation in 25 mM tris(2-carboxyethyl)phosphine (TCEP) in 100 mM TEAB for 15 min at 37°C and alkylated by adding iodoacetamide to a final concentration of 50 mM and incubating in the dark at room temperature for 30 min.−1 in TEAB). Digestion was performed overnight at 37°C in 50 µl TEAB.After reduction and alkylation, the gel pieces were washed with 50:50 acetonitrile and TEAB to remove excess iodoacetamide, dehydrated in acetonitrile then dried in a vacuum to remove residual organic solvent prior to digestion. For tryptic digestion, the dried gel pieces were rehydrated using sequencing grade modified trypsin (Promega) solution  to the gel pieces and incubating for 20 min at room temperature. The supernatant, now containing tryptic peptides, was transferred to a clean tube. The gel pieces were extracted further with two washes with 100 µl water:acetonitrile (50:50) incorporating 1% formic acid and once wash with 100% ACN. All extracts were combined, dried down and redissolved in 5% aqueous formic acid for liquid chromatography mass spectrometry (LC-MS) analysis.The digests were analysed using a nano-LC (RSLC-Thermo Scientific) coupled to a Q-exactive orbitrap (Thermo Scientific). The peptides were loaded in 5% formic acid and resolved on a 50-cm RP-C18 EASY-Spray temperature-controlled integrated column-emitter (Thermo Scientific) using a 2-h multistep gradient of acetonitrile (5% acetonitrile to 60% acetonitrile). The chromatography was performed at a constant temperature of 40°C. The peptides eluted directly into the sampling region of the mass spectrometer, and the spray was initiated by applying 1.9 kV to the EASY-Spray (Thermo Scientific). The data were acquired under the control of Xcalibur software in a data-dependent mode, selecting the 15 most-intense ions for sequencing by tandem mass spectrometry using higher-energy collisional dissociation (HCD) fragmentation. The raw data were processed using the MaxQuant software package (version 1.3.0.5)  to identsiRNA transfection, quantitative real-time PCR (qPCR), immunofluorescence, synchronisation release and cell cycle analysis experiments were performed as previously described . Primer P-values were calculated with Student's t-tests (unless otherwise indicated) in all the data comparing control to treatment. *P<0.05, **P<0.01 and ***P<0.001.


Background: Control of the medium osmolarity and temperature during long or short time sperm manipulation is essential.Objective: The objectives of the present study were to find the effects of different osmolarities of modified Tyrode's solution and milk on the bull sperm during incubation at above zero temperatures.Materials and Methods: Semen samples were collected twice from five Najdi bull. Centrifuged and most parts of seminal plasma were removed. First experiment: The concentrated semen were splited into nine aliquots to incubate in three different osmolarities  at three temperatures  for 15 and 60 min of incubation. Second experiment: The semen samples were splited, mixed with the same volume of whole cow milk (5 and 25°C) and milk with 7% glycerol (5°C) and incubated for 15 and 60 min. Results: Sperm motility severely affected (p<0.05) by incubation at low ionic tension (200 mOsm/l) especially at low temperature (5°C). The impact of low osmolarity on sperm viability can reduce by increasing the incubation temperature to 39°C. The decreased sperm motility, which was induced by lowering osmolarity, was not improved (p>0.05) by increasing temperature during 1 h of incubation. Milk can protect the sperm viability and motility at cool conditions and there is no beneficial effect of glycerol in combination of milk on sperm incubation at above zero temperatures (p<0.05).Conclusion: Iso- and hyper-osmotic solutions protect bull sperm motility and viability at 25 and 39°C, while milk can be used for protecting sperm at 5°C. Sperm is exposed to different medium osmolarities and temperatures during freezing or manipulation. Cryoprotectants are high molecular weight substances like glycerol, ethylene glycol or di methyl sulfoxide which are cytotoxic at high temperatures. After exposing to the cryoprotectants at low temperatures, the intracellular water is excluded from the sperm and the osmolarity of the intracellular environment increased, this effect is also very dangerous for the sperm, which is called "solution effects" . et al, (2001) reported the beneficial effects of modified Tyrode’s solution for preserving the stallion sperm only when seminal plasma was to be removed  and toxic effects of the cryoprotectants. Cooling rate, equilibration time and temperature are three important factors in sperm freezing programs. Impact of cooling rate on sperm motility may be mediated by changes in osmolarity . On the  removed . Increas removed , species removed , 10 and  removed . For manThe beneficial effect of egg yolk for the preservation of cooled sperm has also been documented . There aBull semenSemen was provided from bulls (n=5) at the research center for Agriculture and Natural Resources of the Khuzestan province, Iran. Semen was collected using an artificial vagina (39°C) and transported to the laboratory in a light protected 35°C flask for 1 hour.Media preparation2PO4, 2.18 NaHCO3, 0.08 MgCl2 6H20, 0.31 CaCl2 2H2O, 0.94 glucose , and 2.48 HEPES 300 mOsm/l ] was the basic medium in experiment 1. The HEPES buffer was used to prevent the rapid changes in the medium pH. The modified Tyrode’s solution with 100 mOsm/l was prepared by diluting 1 part of Tyrode's with 2 parts of deionized water. To provide 500 mOsm/l solution of Tyrode’s, all values pf chemical components (without HEPES) of Tyrode's were multiplied by 1.66 and added to 1 liter of deionized water . Whole cow milk  has been heated (to 95°C in boiling water over 30 to 45 min) to inactivate lactenin in the protein fraction because of its toxicity . The heaExtension of semen9/ml. The concentrated semen was mixed 1:1 (v/v) with various media by gentle stirring, giving a final sperm concentration of approximately 3 × 109/ml. As diluents and semen were combined in equal volumes, the final osmolarities of the suspensions of sperm were the means of the osmolarities of different media with that of semen (approximately 300 mOsm/l). All the procedures of semen extension were performed at the room temperature.Fresh semen samples were centrifuged at 500 g for 25 min to remove most of the seminal plasma to provide the sperm concentration of approximately 6 × 10Sperm analysisAn expert person in this field of study undertook the sperm analysis procedures. The percentage of motile and viable sperm was estimated 15 to 60 min after initial exposure to the media. Before evaluation, samples were subjected to iso-osmotic modified Tyrode’s solution. A part of sperm was mixed with a part of Eosin B/Nigrosin stain  on a slide for 1 min and dried by placing it on a hotplate. A total of 100 sperm per sample was counted with the light microscopy at a magnification of 100. Unstained sperms were considered to be viable . Sperm pExperiment 1: Sperm in Tyrode’s solution varying in osmolarity. The volume of sperm (n=5) in Tyrode’s solution was measured in a factorial experiment with three osmolarities of the Tyrode’s: semen combination , and examined after two different incubation times (15 and 60 min) at 5, 25 and 39°C and replicated with five semen samples from different bulls. The sperm suspension aliquots were placed in a water-jacketed microtube. Then the water-jacketed microtubes were placed in the refrigerator (5°C), room temperature (25°C) or a warm water bath (39°C).Experiments 2: Sperm in milk with and without 7% glycerol Equal volumes of semen samples (n=5) were mixed with milk without glycerol at two temperatures (5 and 25°C) and with milk with 7% glycerol at 5°C. Progressive motility and viability of sperms were evaluated after 15 and 60 min of incubation. The experiment was replicated with five semen samples from different bulls.Statistical analysisIn the first experiment, effects of osmolarity, temperature and duration of incubation and their interaction on the percentages of viable and progressive motile sperms, in a split plot design, were analyzed by ANOVA using General Linear Model (GLM) procedure of SAS (the main plot was the medium osmolarities and two subplots were the temperatures and the durations of incubation). In the second split plot design, the effects of diluents (milk with and without glycerol) through two times of incubation at different temperatures on the percentages of viable and progressive motile sperms were analyzed using GLM procedure of SAS. Least square means (LSmeans) were compared with pdiff test in SAS . Data arExperiment 1vs. 48.1±6.05; p=0.25, respectively) significantly (p=0.0003) reduced the impact low osmolarity (200mOsm/l) on the percentage of viable sperms (23.7±6.05). The highest percentage of viable sperms, at 25°C, was observed (p=0.03) in 300 mOsm/l medium (83.8±6.5) compare to 200 and 400 mOsm/l media . Although, incubation at 39°C (44.4±6.5%) reduced (p=0.01) the impact of 200 mOsm/l medium at 25 and 5°C on sperm viability, higher osmolarities (300 and 400 mOsm/l) are required (p=0.03) to optimize the percentage of viable sperm  at 39°C. The patterns of changes were similar at two different (15 and 30 min) durations of incubation . The highest (p<0.03) sperm motility at 25°C was observed in 300 mOsm/l solution (71.1±7.6) compare to 200 and 400 mOsm/l  solutions. The highest sperm motility (p=0.0001) at 39°C was in 400 mOsm/l (71.5±7.6) compare to 300 (56.7±7.6) and 200 (21.25±7.6) mOsm/l solutions. The patterns of changes were similar at two different (15 and 30 min) durations of incubation (There was no difference between 15 (37.5±3.6%) and 60 (37.8±3.6%) min of incubation on sperm progressive motility (p=0.95). Total sperm progressive motility (%) decreased (p=0.004) at 5°C (22.1±4.3) compare to 25 and 39°C . Overall sperm progressive motilities were 12.9±4.4, 53.01±4.4 and 47.1±4.4% at 200, 300 and 400 mOsm media, respectively (p=0.0001). While there was not (p=0.94) any interaction between duration of incubation, temperature and medium osmolarity , a signicubation .Experiment 2The results of this experiment are presented in the Total sperm progressive motilities were 34.6±7.4% and 18.8±7.4% after 15 and 60 min of incubation, respectively (p=0.14). The percentage of progressive motile sperms was not significantly different (p=0.55) between milk at 25°C (32±9.1%), milk at 5°C (24.2±9.1%) and milk with glycerol at 5°C (23.7±9.1%). The patterns of sperm progressive motility changes in different media were similar at two different incubation times ; p=0.77.Incubation of sperm in an ionic base solution at low temperature decreased motility and viability of sperm especially under condition of lower ionic tension (200mOsm/l). Higher osmolarities increased sperm viability and motility in linear fashion thorough the temperature changes at least up to 1 hour of incubation. Data have shown that sperm motility increased significantly at 39°C, similar to the procedure for sperm capacitation during IVF . Using tet al (2001) showed that while some milk fractions  decreased spermatozoal survival, others (beta-lactoglobulin and native phosphocaseinate) were protective  found that solution containing egg yolk (300 mOsm/l) protected the sperm motility during 1 hour of cool storage  found th, 18. Batet al, (2008) showed that the sperm motility was better preserved during the incubation from 30 to120 minutes for the semen diluted in yolk egg-citrate and skim milk-based extenders than from the Ringer extender  reported changes in sperm motility in a temperature-time-medium dependent relation. They found that glycin betaein can improve the sperm motility at lower temperatures, with no effect at 37°C  concluded that Poly Vinyl Alcohol (PVA) can be used to substitute for Bovine Serum Albumin (BSA) and 20°C is more suitable than 15°C for boar semen storage, and in vitro fertilizing capacity of spermatozoa is maintained for at least 8 days in Zorlesco+PVA at 20°C  showed that prefreezing treatment of bovine sperm with cholesterol-loaded cyclodextrin (CLC) increased the percentages of motile and viable sperms  can use for protection of sperm at 5°C incubation.


Previous influenza surveillance at paediatric intensive care units (PICUs) in Germany indicated increased incidence of PICU admissions for the pandemic influenza subtype A(H1N1)pdm09. We investigated incidence and clinical characteristics of influenza in children admitted to PICUs during the first three post-pandemic influenza seasons, using active screening.We conducted a prospective surveillance study in 24 PICUs in Bavaria (Germany) from October 2010 to September 2013. Influenza cases among children between 1 month and 16 years of age admitted to these PICUs with acute respiratory infection were confirmed by PCR analysis of respiratory secretions.p = 0.020), were diagnosed more often with bronchitis/bronchiolitis (p = 0.004), and were admitted to a PICU later after the onset of influenza symptoms (p = 0.041).A total of 24/7/20 influenza-associated PICU admissions were recorded in the post-pandemic seasons 1/2/3; incidence estimates per 100,000 children were 1.72/0.76/1.80, respectively. Of all 51 patients, 80 % had influenza A, including 65 % with A(H1N1)pdm09. Influenza A(H1N1)pdm09 was almost absent in season 2 (incidence 0.11), but dominated PICU admissions in seasons 1 (incidence 1.35) and 3 (incidence 1.17). Clinical data was available for 47 influenza patients; median age was 4.8 years (IQR 1.6–11.0). The most frequent diagnoses were influenza-associated pneumonia (62 %), bronchitis/bronchiolitis (32 %), secondary bacterial pneumonia (26 %), and ARDS (21 %). Thirty-six patients (77 %) had underlying medical conditions. Median duration of PICU stay was 3 days (IQR 1–11). Forty-seven per cent of patients received mechanical ventilation, and one patient (2 %) extracorporeal membrane oxygenation; 19 % were treated with oseltamivir. Five children (11 %) had pulmonary sequelae. Five children (11 %) died; all had underlying chronic conditions and were infected with A(H1N1)pdm09. In season 3, patients with A(H1N1)pdm09 were younger than in season 1 (Active screening showed a continued high incidence of A(H1N1)pdm09-associated PICU admissions in the post-pandemic seasons 1 and 3, and indicated possible underestimation of incidence in previous German studies. The age shift of severe A(H1N1)pdm09 towards younger children may be explained by increasing immunity in the older paediatric population. The high proportion of patients with underlying chronic conditions indicates the importance of consistent implementation of the current influenza vaccination recommendations for risk groups in Germany. Influenza is one of the most common vaccine-preventable viral diseases, with the highest morbidity reported for children and elderly patients , 2. InflBefore the influenza A(H1N1)pdm09 pandemic in 2009/2010, comparisons of clinical characteristics between patients infected with different influenza types (A vs. B) and between patients infected with different influenza A subtypes showed only small differences when controlling for age , 11–13, In Germany, influenza sentinel surveillance on outpatients of all ages  confirmeInformation on the incidence and clinical characteristics of severe paediatric influenza resulting in intensive care treatment and/or fatal outcome is still limited in Germany, and post-pandemic data is thus far available only for the season 2010/11 –20. BaseIn the study presented here, we used active screening to estimate the incidence of laboratory-confirmed influenza-associated PICU admissions in one of Germany’s largest federal states during the first three post-pandemic seasons. Furthermore, we described the clinical characteristics of influenza PICU patients and compared patients with severe A(H1N1)pdm09 disease between the post-pandemic seasons.st, 2010 roughly 2,001,700 children <17 years of age were registered in Bavaria [Prospective, active surveillance was conducted in PICUs of paediatric hospitals in Bavaria, Germany. On December 31 Bavaria , represe Bavaria . The annAll 30 paediatric hospitals in Bavaria equipped for paediatric intensive care treatment of children older than 1 month of age were invited to participate. These PICUs reported a total of 432 intensive care beds , including 207 beds  equipped with ventilation facilities.i) admission to a participating PICU with suspected acute respiratory infection (ARI) of the upper or lower respiratory tract, with ARI-related symptoms ; ii) age at PICU admission due to ARI at least 1 month and below 17 years of age; iii) parental written informed consent. Enrolled children with PCR-confirmed influenza were classed as influenza-associated ARI.From October 2010 to September 2013 , all patients who fulfilled the following inclusion criteria were enrolled: The PICU physician documented demographic characteristics, underlying chronic medical conditions, influenza vaccination status, diagnostic findings, ARI-associated diagnoses and complications, treatment, duration of hospital and PICU stay, and outcome in a structured questionnaire. A respiratory sample, usually a flocked nasopharyngeal or pharyngeal swab, was collected on the day of PICU admission for PCR-confirmation of influenza. Microbiological testing for bacteria or fungi was at the discretion of the PICU physician; pathogens detected at usually sterile sites or in tracheal aspirates were classified as bacterial or fungal co-infection.Mykoplasma pneumoniae. Samples positive for influenza A and B virus RNA in the multiplex PCR were further analysed to determine the subtype and lineage, respectively. Primers and probes specific for influenza A(H1N1)pdm09 were included in the ‘FTD Respiratory pathogens 21’ kit. All samples positive for influenza A virus RNA but negative for influenza A virus (H1N1)pdm09 RNA were tested by a PCR specific for influenza A virus H3.PCR confirmation of influenza was performed either at the local laboratories of the participating PICUs using influenza-specific PCR, or (in the majority of cases) at the central laboratory at the Institute of Virology and Immunobiology of the University of Würzburg using multiplex PCR for respiratory viruses. For the latter, respiratory samples were placed in a viral transport medium . At the central laboratory, they were tested using the commercial multiplex PCR ‘FTD® Respiratory pathogens 21’  to screen for respiratory viruses . Pathogens detected by the test kit included influenza A and B viruses, respiratory syncytial virus (RSV), parainfluenza virus (PIV) 1–4, coronavirus (CoV) NL63, OC43, HKU1, and 229E, human metapneumovirus (hMPV), human bocavirus (hBoV), adenovirus (AdV), rhinovirus (RhV), enterovirus (EV), parechovirus (PV), and additionally p < 0.05, two-sided) using Pearson’s Chi2-test or Fisher’s Exact test for categorical data, and the Mann–Whitney U-test for continuous data.All data was entered into a Microsoft Access database and transferred to IBM SPSS 21.0 for statistical analysis. Data was analysed descriptively . Comparisons between groups were assessed for significance , with 20/14/17 PICUs participating in seasons 1/2/3 . Twelve of the 24 hospitals participated in all three seasons. Reasons stated for non-participation in all or single seasons were time limitations or staff shortages at the PICUs. Participating and non-participating PICUs did not differ significantly in the number of intensive care beds. The 24 participating PICUs enrolled a total of 572 (233/151/188) ARI patients .Among the 572 enrolled PICU ARI patients, a total of 51 PCR-confirmed influenza patients (24/7/20 in seasons 1/2/3) were reported  were confirmed by multiplex PCR, and 10 (19.6 %) by local PCR. Forty-one patients (80.4 %) were diagnosed with influenza A, and 10 (19.6 %) with influenza B pdm09, 0.0/0.11/0.27 for A(H3N2), and 0.07/0.54/0.36 for B.For 47 92.2 %) of the 51 PCR-confirmed influenza PICU patients, questionnaires with clinical data were available . Eighty-five per cent of these were diagnosed with influenza A, including 68 % with A(H1N1)pdm09, and 15 % with influenza B. Sixty per cent were male; median age was 4.8 years, with 51.1 % of children <5 years and 23.4 % <1 year of age   were too young (<6 months of age) to have been immunized against influenza, and for two patients (5.6 %), data on their influenza vaccination status was unavailable. Twenty-nine (80.6 %) patients from this risk group had not been vaccinated against influenza although they would have been eligible. One immunocompromised child (2.8 %) had been vaccinated in October 2012, but was diagnosed with A(H3N2) in January 2013.One or more specific influenza-associated diagnoses/complications were reported for 42 (89.4 %) of the 47 children (Table Escherichia coli (1), Candida sp. (1)) and from tracheal aspirates of seven intubated patients (Candida sp. (2), Pseudomonas aeruginosa (2), Streptococcus sp. (2), Enterococcus faecium (1), Escherichia coli (1) and Moraxella catarrhalis (1)).In addition to influenza, laboratory-confirmed co-infections were reported for 16 children (34.0 % out of 47). Of these, seven (14.9 %) had viral, five (10.6 %) bacterial, one (2.1 %) viral/bacterial, two (4.3 %) viral/fungal, and one (2.1 %) bacterial/fungal co-infections. The most frequent co-infecting virus was RSV (seven children). Microbiological testing was documented for 33 patients (70.2 %), with pathogens detected in usually sterile material from two patients (p = 0.045), and with a longer stay at the PICU . Thirteen (27.7 %) children each were treated with catecholamines and with inhalation therapy, mainly with bronchodilators. A total of 22 children (46.8 %) required mechanical ventilation; of these, 11 (23.4 %) received only intratracheal ventilation, seven (14.9 %) only CPAP, and four (8.5 %) both. One child (2.1 %) with an immunosuppressed condition, infected with influenza A(H3N2), RSV, Epstein-Barr virus and Candida sp., was treated with extracorporeal membrane oxygenation (ECMO).The majority of the 47 PICU patients were treated intravenously with antibiotics (72.3 %), and with antipyretics (70.2 %) Table . ThirteeFive children 10.6 %), infected with A(H1N1)pdm09, died at an age of 4 to 11 years; four were male patients : state after surgery due to pleural effusion/empyema in two children; increased oxygen requirements in two children who had previously already received oxygen therapy at home; damage of the lung after high-pressure ventilation in one child.n = 18) were compared to patients from season 3 (n = 13) pdm09 from season 1  Tables , 3, 4, 5) Tables . Figure N1pdm09 f) Tables . In seasIn the pre-pandemic period, median duration of PICU stay was longer (19 days), and children were more often diagnosed with encephalitis/encephalopathy (25 %) and co-infections (65 %) than in later periods Table . The proDuring the first three post-pandemic seasons 2010/11, 2011/12 and 2012/13, active screening of children with acute respiratory infection admitted to 24 paediatric intensive care units in Bavaria identified a total of 51 PCR-confirmed influenza cases, resulting in annual incidence estimates of 1.7, 0.7, and 1.8 influenza-associated PICU admissions per 100,000 children, respectively. These figures would, by extrapolation, correspond to a total number of 559 children with influenza-associated PICU admission in Germany within the 3-year post-pandemic period, with an annual average of 186 children. This is almost 28 times as high as the annual average of six to seven influenza-associated PICU admissions detected by nation-wide PICU surveillance in Germany during three pre-pandemic years without active screening , 20. ThuStreptococcus pneumoniae, the bacterial pathogen most frequently associated with community-acquired influenza [In our study, the proportions of children with bacteria-associated complications  were similar to the proportions observed during the pre-pandemic period, but appeared higher than those observed during the pandemic 2009/10 . The lownfluenza .Antiviral treatment patterns changed considerably during the post-pandemic period, with a decrease in the proportion of paediatric influenza cases receiving oseltamivir from previously 50 %  and 61 %In our study, about two-thirds of influenza cases and all fatalities were A(H1N1)pdm09-associated. During the post-pandemic seasons 1/2/3, the proportion of A(H1N1)pdm09 cases among the PICU patients was 79 %/14 %/65 % and, thus, considerably higher than the proportion of this subtype reported among outpatients by national influenza surveillance (65 %/1 %/34 %) . This obComparison of PICU patients with A(H1N1)pdm09 between the post-pandemic seasons showed that median age was 1.7 years in the third season and, thus, significantly lower than in the first season. A significant age shift towards younger children, from a median age of 5 to 3 years, had already been observed in a comparison of the pandemic and the first post-pandemic season in Germany . The conIn Germany, paediatric influenza vaccination for pandemic influenza A(H1N1)pdm09 was recommended and funded for all children as monovalent vaccination from October 2009 to July 2010 . For seaTo our knowledge, our study is the first in Europe to investigate paediatric influenza in PICU patients during the first three post-pandemic seasons after the 2009/10 pandemic. The strengths of our study include the multi-centre design covering the majority of PICUs in Bavaria, the active screening for influenza in patients admitted to PICUs, and PCR-confirmation of all influenza cases. A limitation is that the corrected incidence estimates were based on the assumption that participating and non-participating PICUs treated a similar number of severe paediatric influenza patients. Although PICUs of both groups were of similar size, some of the non-participating PICUs, where paediatricians indicated lack of time as reason for non-participation, may have treated a higher number of patients, or patients with higher acuity. Further limitations include potential over- and underreporting in participating PICUs. On the one hand, due to different hospitalization rules some children may have been admitted to PICUs mainly for the purpose of monitoring their course of influenza disease more closely, thus resulting in an overestimate of severe cases. On the other hand, some parents of children with severe influenza may have refused study participation, or children with a fulminant course of influenza disease may have died before they were admitted to a PICU . Thus, tThe incidence estimates of influenza A(H1N1)pdm09-associated PICU admissions, derived from active screening of PICU patients with acute respiratory infections, reached similarly high levels in the first and third post-pandemic seasons. Both incidence estimates were higher than those previously reported by nation-wide PICU surveillance for the pandemic and the first post-pandemic season, suggesting possible underreporting in previous studies without active screening. Comparison of the first and third post-pandemic seasons indicated an age shift of severe A(H1N1)pdm09 towards younger children, which might be explained by increasing immunity in the older paediatric population. The large proportion of children with underlying chronic conditions indicates the need for a more consistent implementation of the current recommendations for influenza vaccination of specific risk groups in Germany. These children could also profit from herd protection, if universal influenza vaccination was successfully introduced in Germany.


Increases in mucosal intestinal permeability may cause pathological leakage of bacterial products, inducing inflammation. Low-grade systemic inflammation has been reported in overweight humans. Probes used for permeability evaluation in humans have not yet been evaluated in dogs.To determine suitability of human permeability probes  for canine obesity models.per os using human doses corrected for weight and reported to be harmless for dogs. Urine samples were collected 2, 4 and 6 h after ingesting probes. Urinary excretion was quantified.Fourteen healthy dogs were examined as baseline group. They were given all four probes The least squares means of mannitol proportion (excreted/ingested ± SE) was 15.6 % (± 2.0), 11.0 % (± 2.1) and 5.5 % (± 2.1) for urine sampled 2, 4 and 6 h after ingestion (p<0.0001). Hence, the majority of mannitol absorption and secretion representing upper gastrointestinal leakage was completed by 4 h. Riboflavin followed a similar temporal profile, defining 4 h as a cutoff for upper vs lower gut permeability. Probes were well tolerated.Data obtained in the study indicates that probes used for humans can be used for intestinal permeability evaluation in dogs.


Xenopus oocyte expression, we found that C1, but not CC2, is critical for PKD2L1 channel function. Our co-immunoprecipitation and dynamic light scattering experiments further supported involvement of C1 in trimerization. Further, C1 acted as a blocking peptide that inhibits PKD2L1 trimerization as well as PKD2L1 and PKD2L1/PKD1L3 channel function. Thus, our study identified C1 as the first PKD2L1 domain essential for both PKD2L1 trimerization and channel function, and suggest that PKD2L1 and PKD2L1/PKD1L3 channels share the PKD2L1 trimerization process.As a transient receptor potential (TRP) superfamily member, polycystic kidney disease 2-like-1 (PKD2L1) is also called TRPP3 and has similar membrane topology as voltage-gated cation channels. PKD2L1 is involved in hedgehog signaling, intestinal development, and sour tasting. PKD2L1 and PKD1L3 form heterotetramers with 3:1 stoichiometry. C-terminal coiled-coil-2 (CC2) domain (G699-W743) of PKD2L1 was reported to be important for its trimerization but independent studies showed that CC2 does not affect PKD2L1 channel function. It thus remains unclear how PKD2L1 proteins oligomerize into a functional channel. By SDS-PAGE, blue native PAGE and mutagenesis we here identified a novel C-terminal domain called C1 (K575-T622) involved in stronger homotrimerization than the non-overlapping CC2, and found that the PKD2L1 N-terminus is critical for dimerization. By electrophysiology and  Xenopus oocytes alone, PKD2L1 channel was found to traffic to the plasma membrane and be activated by Ca2+ and permeable to Ca2+82+ channel on the mammalian cell surface membrane and primary cilia where it regulates Ca2+ concentration and Ca2+-dependent hedgehog signaling10Eight members in the polycystic kidney disease (PKD) family of proteins have so far been identified: PKD1,-1L1, -1L2, -1L3, -REJ; PKD2, -2L1 and -2L2. Among them, only PKD1 and PKD2 are mutated in autosomal dominant PKD (ADPKD)1356in vitro and in vivo data suggest that PKD2L1 and PKD1L3 are synergistically involved in acid sensing pathways. However, unlike PKD2L1 KO mice, PKD1L3 KO mice were not found to have defects in sour tasting2+ channel that regulates the ciliary Ca2+ concentration and Ca2+-dependent hedgehog signaling pathway, which seems to be developmentally important10There are theoretically 15 possible ways of complexing between a PKD1 and PKD2 homologue, which may nicely respond to various tissue-specific needs of sensory functions and regulations. Eg, the PKD2/PKD1 and PKD2L1/PKD1L3 channel complexes were reported to sense fluid flow and acid, in primary cilia and surface membrane, respectively151515Xenopus oocyte expression showed that both the PKD2/PKD1 and PKD2L1/PKD1L3 complexes form heterotetramers with 3:1 stoichiometry, ie, one PKD2 (or PKD2L1) trimer paired with one PKD1 (or PKD1L3) monomer20in vitro and be important for PKD2L1 homomeric assembly and PKD2L1/PKD1L3 surface expression in Xenopus oocytes21222+-activated channel function as wild-type (WT) PKD2L1 and that PKD2L1 and PKD1L3 interact with each other through their TMs, which is required for PKD2L1/PKD1L3 surface expression in HEK cellsHow does a PKD2 homologue complex with a PKD1 homologue? Studies using mammalian cell and Based on the tetrameric assembly of PKD22656in vitro conditions, including trimerization of its purified C-terminus or of the full-length protein over-expressed in Xenopus oocytes2122in vivo and physiological conditions, we performed Western Blot (WB) experiments using mouse tissues under the non-reducing and reducing conditions (see EXPERIMENTAL PROCEDURES). We found that in the kidney, testis and brain under the non-reducing condition, endogenous PKD2L1 displays similar oligomeric patterns from which the band sizes suggest the presence of either PKD2L1 homodimers, -trimers and –tetramers, or its heteromerization with endogenous interacting partner proteins  in cell lysis buffer to inhibit disulfide bond formation by modifying free cysteine residues. We found that the amount of oligomers decreases with the NEM treatment in HEK cells but does not further decrease with additional reducing agents , indicatin vivo data using mouse tissues strongly indicated that PKD2L1 forms homodimers, -trimers and -tetramers.We also employed BN-PAGE to check PKD2L1 oligomeric state under the non-denaturing condition. No disulfide bonds would be formed during sample preparation and/or electrophoresis with BN-PAGE. Our data indicated that PKD2L1 is mainly present as homotrimers, and possibly also as homotetramers or hetero-oligomers with its partner proteins, while monomers and dimers are still detectable under this condition . Only mo28292+-activated non-selective cation channel when over-expressed in Xenopus oocytes in which PKD2L1 traffics to the plasma membrane8in vitro22Xenopus oocytes, and measured Ca2+-induced channel activation currents with the two-microelectrode voltage clamp. We found that both the ΔCT and ΔNT mutants are functionally dead although they continue to traffic to the plasma membrane  still forms trimers, the mutant with the entire CT deleted from ΔNT (named ΔNT/ΔCT) has no trimer . Since nare dead  and funcFor WT or a mutant PKD2L1 channel to be functional it is necessary that they are in a correct oligomeric state. In an effort to identify a PKD2L1 domain that is essential for both trimerization and channel function we first made two deletion mutants with C1 or CC2 deleted from truncation mutant Flag-ΔNT see . Consist22in vitro data were in support of the involvement of C1 in trimerization.To provide further documentations on the involvement of C1 in trimerization we first employed co-immunoprecipitation (co-IP) assays to determine the effect of C1 on the interaction between two differently tagged WT or mutant PKD2L1 proteins. To enhance the co-expression efficiency, we transfected HeLa cells with GFP-WT first and 12 hr later with Flag-T622X or -K575X. We collected cell lysates 30 hr after the second transfection. We found that the strength of the interaction of WT with K575X (that lacks C1), assessed by the immunoblotting band after normalization by input bands, is 36.5 ± 6.8%  less than the interaction with T622X . We beliH) as being the size of a hypothetical hard sphere that diffuses in the same fashion as the hydrated protein being measured. We expressed and purified CT and its deletion mutants CT-ΔC1 (with C1 deletion) and CT-Δdouble (with both C1 and CC2 deletions) from E. coli (H of CT measured by DLS is 8.88 ± 0.26 nm (N = 6), which is significantly reduced to 5.80 ± 0.17 nm  and 5.49 ± 0.05 nm  for C1 and double deletions, respectively (H)b, where parameters a and b were found to be 0.41 ± 0.05 and 2.48 ± 0.04, respectively, for proteins in the range of 17–440 kDH values are 93 ± 6 kD, 32 ± 2 kD and 28 ± 1 kD, for CT, CT-ΔC1 and CT-Δdouble, respectively, indicating a predominant trimeric structure for CT and monomeric structures for the two deletion mutants under this experimental condition. These data indicated that C1 deletion significantly breaks down trimerization of CT, in support of the importance of C1 for oligomerization.Further, we employed dynamic light scattering (DLS) to examine the role of C1 in the trimerization of purified CT in solution. DLS assesses a protein's apparent hydrodynamic diameter  fragment M1-T50 (GFP-Ctrl) of a similar size. We then carried out BN-PAGE experiments which indeed revealed the presence of a trimer band for GFP-C1 but not for GFP-Ctrl  important for a functional PKD2L1 trimeric state. Indeed, our current studies using a combination of in vitro expression in mammalian cells and Xenopus oocytes, electrophysiology, mutagenesis, protein-protein interaction, and dynamic light scattering identified the C-terminal C1 domain that is the most conserved in CT and critical for both PKD2L1 trimerization and channel function.Because the function of an ion channel presumably relies on its intact oligomeric state deletion of or alterations in an oligomerization domain would substantially affect the channel architecture and consequently channel activity. Up to now it has remained unknown as to which domains of PKD2L1 that are important for both oligomerization and function. Previous studies identified CC2 as a trimeric domain in CT that is important for the surface membrane expression of PKD2L1 and complex PKD2L1/PKD1L32122Our non-reducing SDS-PAGE with mouse tissues showed the same oligomers pattern in kidney, testis and brain and these oligomers were reduced to monomer with reducing agents . This woTRPC, TRPM and TRPN channels were found to contain a conserved intracellular TRP domain downstream of TM6, which is similarly located as C1. This TRP domain was proposed to participate in subunit assembly and/or allosteric modulation of channel gating2+ followed by channel inactivation, presumably through an increase in the intracellular Ca2+ concentration2+ regulates PKD2L1 channel activity through direct binding. CT contains a Ca2+-binding EF hand motif (E637-L665) that negatively regulates the ability of PKD2L1 channel to be activated by Ca2+, suggesting that Ca2+ binds to this domain to prevent the channel from over-activation2+ to CT was indeed reported2+ binding is not affected by mutations of 6 residues to alanine within CC2, suggesting that CC2 is not involved in Ca2+ binding. More importantly, our previous and current studies both demonstrated that CC2 is not important for PKD2L1 channel function. Although arguably, CC2 was found to be important for surface expression of PKD2L1 and complex PKD2L1/PKD1L3 in Xenopus oocytesWe wondered why PKD2L1 needs two distinct trimerization domains in CT of PKD2L1. PKD2L1 channel is activated by CaMembers of the TRP superfamily including PKD2 and PKD2L1 are assumed to assemble into homo- and/or hetero-tetramers with either a PKD1 homologue or another TRP protein, but the molecular mechanisms underlying these assemblies are still unclear. Like PKD2L1, the PKD2 C-terminus was found to form trimers20202+-activated K+ channel (SKCa) forms trimers, as shown by crystallographic analysesCa channel forms homotetramers+ channel Kv7.1 was shown to trimerize but the full-length channel forms tetramers3940It is noted that such a 3 + 1 assembly protocol for forming homo- and heterotetramers has been reported for other channels. Eg, the cytoplasmic C-terminal leucine zipper domain (G488-Y526) of small conductance voltage-gated CaIn summary, our study has found that endogenous PKD2L1 in mouse tissues exhibits oligomeric states including dimers, trimers and tetramers. We identified a novel PKD2L1 C-terminal C1 domain that is associated with stronger trimerization than the previously reported CC2 domain. Importantly, C1 represents the first oligomerization domain critical for PKD2L1 channel function. We thus propose that functional PKD2L1 channel assembly involves both trimerization and dimerization, a mechanism possibly shared by some non-TRP types of ion channel.Human full-length PKD2L1 cDNA was amplified from pCHGFHeLa and HEK cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin . Cells of less than 25 passages were cultured to full confluence before collection. Transient transfection was performed on cells cultured to 70%–90% confluence using Lipofectamine 2000  according to the manufacturer's instruction.Protein samples were separated on 8% SDS-PAGE gels. For the non-reducing SDS-PAGE, samples were mixed with SDS loading buffer without reducing reagents and kept on ice for 5 minutes (min) prior to electrophoresis. For the reducing SDS-PAGE, samples were mixed with loading buffer supplemented with 0.5 M DTT or 5% 2-mercaptoethanol (2-ME), and subject to heating at 65°C for 5 min. 30 μg of total protein was loaded per lane. Flag and GFP (B-2) antibodies were purchased from Santa Cruz Biotechnology . PKD2L1 antibody (H00009033) was purchased from Abnova  for tissue detection. Rabbit antibody against PKD2L1 (PR71) was custom made and used previouslyHeLa cell lysates for BN-PAGE were prepared with NativePAGE Sample Prep Kit (Invitrogen) according to the manufacturer's protocol. Coomassie blue G-250 was added to supernatants at 8:1 detergent:G-250 ratio. Protein complexes were separated at 150 V for 90 min using NativePAGE Novex 3–12% BisTris gels (Invitrogen). For immunoblotting, gel was incubated in 20 mM Tris-HCl (pH 8.3), 0.15 M glycine, and 0.02% SDS for 5 min at room temperature. Proteins were then transferred to polyvinylidene difluoride membranes (at 150 mA for 90 min and 4°C). Membranes were blocked with 3% skimmed milk in PBS buffer with 0.1% tween-20 for 40 min at room temperature and then incubated with antibodies.Xenopus oocytes were spotted onto a nitrocellulose membrane and then let the membrane dry. Glutaraldehyde fixation was applied as previously describedLysates of Xenopus oocytes were washed in PBS, fixed in 3% paraformaldehyde for 15 min, washed 3 times in PBS plus 50 mM NH4Cl, and then permeabilized with 0.1% Triton X-100 for 4 min. Oocytes were then washed 3 times in PBS for 5 min each time, blocked in 3% skim milk in PBS for 30 min, and then incubated overnight with the rabbit anti-PKD2L1 polyclonal antibody . This was followed by 3 times 10-min washes in PBS. Oocytes were then incubated with a secondary AlexaFluor 488-conjugated donkey anti-rabbit antibody  for 30 min, followed by 3-time washes in PBS and mounting in Vectashield . The slides were examined on an AIVI spinning disc confocal microscopy . Plasma membrane intensity of WT or mutant PKD2L1 was assessed by quantifying the plasma membrane immunofluorescence using Volocity 6.2 . Background fluorescence was subtracted and data were normalized to the average PKD2L1 WT intensity.g for 15 min. Equal amounts of total proteins from postnuclear supernatants were pre-cleared for 1 hour (hr) with protein G-Sepharose , and then incubated for 4 hr at 4°C with the antibody against GFP. After the addition of 100 μl of 50% protein G-Sepharose, the mixture was incubated overnight with gentle shaking at 4°C. The immune complexes absorbed to protein G-Sepharose were washed five times with Nonidet P-40 lysis buffer  with proteinase inhibitor and eluted by SDS loading buffer. Precipitated proteins were analyzed by WB using the antibodies against Flag or GFP.Co-IP was performed using lysates of HeLa cells over-expressing GFP-PKD2L1 and Flag-tagged mutant PKD2L1 constructs. HeLa cell monolayer in 100-mm dishes was washed twice with PBS and solubilized in ice-cold CellLytic-M lysis buffer supplemented with proteinase inhibitor mixture (Sigma). Supernatants were collected following centrifugation at 16,000 × DNA fragment encoding C1 was inserted into pET28a(+) that contains upstream GFP gene. BL21 (DE3) strain  was used for expression. Following growth at 37°C to an optical density (OD) of 0.6, cultures were cooled to 30°C, induced with 1 mM isopropyl β-D-thiogalactoside and incubated for 6 hr. Cell lysates from 50 mL culture were prepared with CellLytic-B lysis buffer (Sigma) according to the manufacturer's instruction. Proteins were pulled down with Ni-NTA resin  and eluted from beads with 250 mM imidazole by following the manufacturer's manual.in vitro transcription from a linearized template in the pCHGF vector using the mMESSAGE mMACHINE1 kit . Stage V–VI oocytes were isolated from Xenopus laevis. Defolliculation of oocytes was performed through incubation in Ca2+-free Barth's solutionCapped mRNAs of WT or mutant PKD2L1 were synthesized by 2 and 10 mM HEPES was used. The solution containing extracellular Ca2+ was prepared from the standard solution with the addition of CaCl2 to a final concentration of 5 mM. Duration of application of Ca2+ medium was indicated in time course recordings. Oocyte whole-cell currents were recorded using a Geneclamp 500B amplifier and Digidata 1322A AD/DA converter . The pClamp 9 software  was applied for data acquisition and analysis. Currents and voltages were digitally recorded at 200 μs/sample and filtered at 2 kHz through a Bessel filter. SigmaPlot 12  was used for data fitting and plotting.Two-microelectrode voltage clamp experiments were performed as described beforeE. coli in a solution containing 50 mM NaH2PO4, 300 mM NaCl and 250 mM imidazole, pH 8.0 were passed through a 0.22 μm filter to remove large particles or aggregates and diluted to 0.4 mg/ml before measurements. Correlation data obtained with DynaLS software  were fitted using SigmaPlot 12 (Systat Software) to derive the average apparent hydrodynamic diameter.DLS experiments were performed using a Malvern Zetasizer Nano ZS instrument  at 25°C, as described previouslyData were analyzed and plotted using SigmaPlot 12 (Systat Software), and expressed as mean ± SEM (N), where SEM stands for the standard error of the mean and N indicates the number of experimental repeats. Paired or unpaired Student t-test was used to compare two sets of data. A probability value (P) of less than 0.05, 0.01 and 0.001 was considered statistically significant and indicated by “*”, “**” and “***”, respectively.Conceived and designed the experiment: W.Z., C.F.P., Y.C., H.B.Z., J.F.T. and X.Z.C. Performed the experiments: W.Z., S.H., J.W.Y., J.H., F.Z. and S.H.A. Analyzed the data: W.Z., C.F.P., Y.C., H.B.Z., J.F.T. and X.Z.C. Wrote the paper: W.Z. and X.Z.C.


Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs . The change in spectral binding for nelfinavir was observed at both δAmax (0.004±0.0003 vs. 0.0068±0.0001) and KD (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δAmax (0.0038±0.0003 vs. 0.0055±0.0003) and KD (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced KD in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir-boosted antiretroviral therapy, in HIV-1-infected individuals who abuse methamphetamine.Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δA Cytochrome P450 (CYP) belongs to the family of heme proteins that are involved in the biotransformation of xenobiotics. Among the various CYP isozymes, CYP3A4 metabolizes approximately 50% of the currently marketed drugs including several antiretroviral drugs . Most anin vitro and in vivo studies have shown the role of methamphetamine in altering HIV-1 pathogenesis by various mechanisms, including suppression of innate restriction factors in macrophages and increased viral loads in the brain avir d5. , Table 1® MS C18 column  using a Shimadzu LC-20AD HPLC system . A binary gradient elution method comprising of mobile phases (acetonitrile with 0.1% formic acid and water with 0.1% formic acid) with a flow rate of 0.5 mL/min was employed. For separation of ritonavir, hydroxy ritonavir, lopinavir, and lopinavir M1, a gradient program starting with 35% acetonitrile with 0.1% formic acid for the first minute, followed by a linear gradient to 100% acetonitrile with 0.1% formic acid in 5 min, maintained for 1 min and then to 35% acetonitrile with 0.1% formic in 4 min with a total run time of 11 min was developed. Under the above conditions, ritonavir, hydroxy ritonavir, lopinavir, lopinavir M1, and IS exhibited retention times of 6.41, 5.26, 6.64, 6.36, and 4.33 min, respectively. For separation of nelfinavir and nelfinavir M8, the same gradient profile was used except for starting with 35% of 70% acetonitrile with 0.1% formic acid. Upon using the above method, nelfinavir, nelfinavir M8, and IS were eluted in 5.42, 4.49, and 6.33 min, respectively. Data acquisition and analyses were performed using the Analyst 1.4.2 software package .All the analytes were separated on reverse phase XTerra www.pdb.org). Currently, there are 17 crystal structures of human CYP3A4 available in PDB. On the basis of crystal resolution, residues completeness, and ligand size, 3NXU (at 2.00 Å resolution) was chosen. There are 2 asymmetric molecules in 3NXU. Chain A was used for docking simulations. The missing residues were reconstructed using 1TQN and 3V0M as the templates. Docking of PIs into the CYP3A4 active site in the absence and presence of methamphetamine was accomplished by Gold 5.2 software [The study was performed as described previously with modifications . The inisoftware . The cenD and δAmax were determined by fitting the curve (absorbance vs. concentration) with hyperbolic equation using non-regression analysis in sigma plot 11 . The p-values were determined using One way ANOVA with Dunnet’s post-hoc test and values <0.05 were considered statistically significant.The KD of methamphetamine as 204 ± 18 μM and δAmax at 0.016 ± 0.001. The result from δAmax suggests that methamphetamine causes approximately 20% spectral transition (low to high spin) with CYP3A4 at saturating concentration of methamphetamine. This spectral transition with CYP3A4 is similar to spectral transition obtained from other type I ligands such as ethanol [The current study was undertaken to examine the role of methamphetamine on the spectral binding of PIs with CYP3A4. In order to determine this, first we examined the spectral binding of methamphetamine with CYP3A4. The results showed an increase in absorbance at 390 nm and a decrease at 418 nm, suggesting that methamphetamine shows a type I spectral change with CYP3A4. The result is consistent with its physicochemical properties as it does not contain any reactive group with a loan pair, e.g. imidazole, which usually shows type II spectral change through covalent binding. The titration of methamphetamine with CYP3A4 showed a hyperbolic spectral binding . Therefo ethanol . To deteD obtained from spectral binding is higher than the plasma concentration of methamphetamine (2–10 μM) [The involvement of various CYP enzymes including CYP3A4 in the metabolism of methamphetamine has been shown in previous studies, including ours –22. Our 2–10 μM) , it is l2–10 μM) . CYP3A4 2–10 μM) , and in 2–10 μM) . A leakySince CYP3A4 is involved in the metabolism of both PIs and NNRTIs, it is important to study drug-drug interactions in the presence of methamphetamine . We and max of nelfinavir (0.004 ± 0.0003 vs. 0.0068 ± 0.0001) and a slight increase in δAmax with tipranavir (0.0061 ± 0.0002 vs. 0.0050 ± 0.0005). However, it did not show any change in δAmax with atazanavir and lopinavir   with CYP3A4. Our results showed that methamphetamine causes a significant decrease in the δAopinavir , Table 2In general, methamphetamine did not significantly alter the binding of Type I PIs with CYP3A4, except for nelfinavir. In consistence with the spectral findings, our inhibition studies in microsomal system did not show any significant effect of methamphetamine on type I PIs metabolism.D values. Since the crystal structure of CYP3A4 complexed with MA is not available, we first predicted the possible binding modes of MA in the active site of CYP3A4. Our docking studies suggested two possible binding modes for both R-methamphetamine and S-methamphetamine , inhibition of CYP3A4, and substrate docking of type I PIs with CYP3A4s. Together these results suggest that there is no major conformational change in the active site of CYP3A4 when type I PIs bind with CYP34 in the presence of methamphetamine. Overall, the findings from type I PIs suggest no significant drug-drug interaction between methamphetamine and type I PIs. This is in contrast with our previous study with ethanol-CYP3A4 interaction, in which, ethanol showed altered metabolism of nelfinavir [ex vivo study is also needed to test whether the metabolism, bioavailability, and efficacy of type I PIs is altered in methamphetamine users.Overall, our data suggests that methamphetamine does not alter the spectral change (δAlfinavir . Therefomax of ritonavir (0.0038 ± 0.0003 vs. 0.0055 ± 0.0003) but didn’t affect indinavir (0.0039 ± 0.0002 vs. 0.0044 ± 0.0002)   with CYP3A4. Methamphetamine significantly decreased the δA 0.0002) , Table 2Further, we performed ritonavir docking to CYP3A4 to verify whether methamphetamine alters binding mode of ritonavir. The docking study showed that ritonavir has three sites that can interact with the heme of CYP3A4 . HoweverD among all the drugs studied, which is consistent with the previous finding [max and a decrease in ritonavir metabolism. These findings suggest that methamphetamine acquires selective binding with certain pool of CYP3A4, which can’t be replaced by ritonavir, a strongest CYP3A4 inhibitor known. Furthermore, docking studies suggest that thiazole group of ritonavir moves away from the heme site of CYP3A4 in the presence of methamphetamine suggesting a decrease in the magnitude of binding. Thus, the docking study is consistent with the finding that the δAmax decreases in the presence of methamphetamine and also with decreased metabolite formation. Similarly, an increased binding affinity with ritonavir (relative to lopinavir) in the presence of methamphetamine can be explained by a higher magnitude of decreased binding energy and increased clusters with ritonavir compared to lopinavir Click here for additional data file.


Tyrosine kinase 2 (Tyk2) gene has been shown to determine susceptibility to murine virus-induced diabetes. In addition, a previous human genome-wide study suggested the type 1 diabetes (T1D) susceptibility region to be 19p13, where the human TYK2 gene is located (19p13.2).Recently, natural mutation of TYK2 gene at the promoter region and exons were studied among 331 healthy controls, and 302 patients with T1D and 314 with type 2 diabetes (T2D) in the Japanese.Polymorphisms of TYK2 promoter haplotype with multiple genetic polymorphisms, which are in complete linkage disequilibrium, named TYK2 promoter variant, presenting decreased promoter activity, is associated with an increased risk of not only T1D (odds ratio (OR), 2.4; 95% confidence interval (CI), 1.2 to 4.6; P = 0.01), but also T2D . The risk is high in patients with T1D associated with flu-like syndrome at diabetes onset and also those without anti-glutamic acid decarboxylase autoantibody.A TYK2 promoter variant is associated with an overall risk for diabetes, serving a good candidate as a virus-induced diabetes susceptibility gene in humans.The Ministry of Education, Culture, Sports, Science and Technology and of Health, Labor and Welfare of Japan. •TYK2 promoter variant with multiple polymorphisms was identified in the Japanese.•TYK2 promoter variant is associated with an increased risk of both type 1 and type 2 diabetes.•TYK2 promoter variant serves a good candidate as a virus-induced diabetes susceptibility gene in humans. AccumulVirus infection has long been considered to be a possible cause of type 1 diabetes (T1D), as suggested by many clinical and experimental observations . It was RIG-I) and interferon induced with the helicase C domain I (IFIH1) or melanocyte differentiation antigen (MDA)-5 (MDA-5/IFIH1)  . Interfefections   virus-induced diabetes susceptibility in mice   virus (a picornavirus)-induced diabetes , it is s in mice . Highly -D virus . Interes-D virus , where t19p13.2) . HoweverTYK2 gene may be associated with the risk for T1D and also confer a possible link with virus-induced diabetes susceptibility in humans. We thus examined the association of TYK2 gene polymorphisms with T1D and type 2 diabetes (T2D), focusing on association with flu-like syndrome at diabetes onset.These observations suggest that the human 22.1We studied 947 Japanese patients and controls. Those include 302 patients with T1D, 314 patients with T2D and 331 healthy controls. Among the 302 patients with T1D, 73 patients were associated with flu-like syndrome at the onset. Clinical profiles of the Japanese patients with T1D or T2D, and the healthy controls are presented in Patients were designated as T1D if fasting C-peptide was < 0.5 ng/ml and they were in an insulin-dependent condition (IDDM), or as T2D if fasting blood glucose levels were higher than 126 mg/dl and HbA1c levels exceeded 6.5% with non-insulin-dependent status (NIDDM). Patients with T1D were also grouped according to their age at onset, as 0 to 19 (0–19), 20 to 39 (20–39), 40 to 59 (50–59), and 60 to 79 (60–79) years old. The study was conducted according to the guidelines for human study and was approved by the ethical committee of the Kyushu University, Graduate School of Medical Sciences (No. 433-00). Written informed consent was obtained from all subjects including T1D, T2D and healthy controls involved in this study.2.2TYK2 gene. TYK2 sequence reference was NCBI Reference Sequence: NG_007872.1. PCR amplification of the target genes, followed by the direct sequencing of the amplified gene, was conducted. A list of primers used to detect the polymorphisms of the TYK2 gene is presented in Supplementary Table 1.Genotyping had been performed to detect 25 exons and the putative promoter region, 1.3 kb upstream of start codon, of the 2.3TYK2 promoter variant, we used PCR analysis followed by direct sequencing to identify − 930G > A and − 929 T > A at the promoter region, using the following primer sets: F:5′-GAA TCG CTT GAA TCC GGG AG-3′, and R:5′-ACC CTT CTT CTG TGC CAC AC-3′. Thus, we present TYK2 promoter genotypes of wild type and variant type as GT and AA, respectively.To identify the 2.4χ2 test. Odds ratio (OR) and 95% confidence interval (CI) were estimated by Woolf's method. Statistical analysis was done using Stata version 10 .The genotype distribution between the cases and controls was statistically assessed by 33.1TYK2 gene polymorphisms in 22 patients with T1D associated with flu-like syndrome, suggestive of possible viral infection, by PCR amplification followed by direct sequencing. We found seven polymorphisms: − 930G > A, − 929T > A and − 104A > C at the promoter region from transcription start point at exon 1; 1A > G, 62G > A and 63G > A at exon 1, which is an untranslated region; and 15597G/T at exon 8 with an amino acid substitution from valine to phenylalanine (V326F) (We first screened human  (V326F) .TYK2 promoter variant.Among them, 1A > G (rs17000728), 62G > A (rs17000728) and 63G > A (rs2304258) at exon 1, and 15597G/T (V326F) at exon 8 (rs2304256) have been already identified by the 1000 Genomes Project that included the Japanese population . Because3.2We first studied the association of the missense change at exon 8 with diabetes. We compared 244 patients with T1D, 255 patients with T2D and 254 healthy controls, and found no measurable difference in the genotype frequency between diabetic patients and healthy controls (Supplementary Table 2).TYK2 promoter variant; GT/AA and AA combined, and compared with wild type GT in 302 patients with T1D, 314 patients with T2D and 331 healthy controls (TYK2 promoter variant was significantly higher in patients with T1D (odds ratio (OR), 2.4; 95% confidence interval (CI), 1.2 to 4.6; P = 0.01), and also in patients with T2D , compared with healthy controls   (TYK2 promoter variant was significantly more frequent among T1D patients without anti-glutamic acid decarboxylase autoantibody (GAD) , but not among anti-GAD autoantibody-positive patients   . The TYK= 0.005) . In addi = 0.21) .TYK2 promoter variant is distinct from autoimmunity against pancreatic β-cells. We grouped TID associated with flu-like syndrome and analyzed the age at onset and anti-GAD antibody positivity, in association with TYK2 promoter variant. There was no statistical significance in the age at onset (P = 0.16), but has significantly increased frequency in anti-GAD antibody negative T1D  (Supplementary Table 3), consistent with the observation of all T1D patients. There was no gender difference in the frequency of TYK2 promoter variant, among patients with T1D , and also T2D . In the age-specific analysis on subjects with T1D, we found that the TYK2 promoter variant haplotype was associated with a higher risk for diabetes in younger people aged 0 to 19 years  and 20 to 39 years , but not in older patients aged 40–59 years  or 60–79 years  (It is thus suggested that the risk for diabetes conferred by the  = 0.68) .TYK2 promoter variant is associated with an increased risk of T1D with a younger age of onset.Thus, the P = 0.17), however, those with variant type are more associated with anti-GAD negative people , and also with flu-like syndrome at the onset  (Supplementary Table 3), consistent with the observation in all T1D. Since obesity is an important risk for endocrinological disorders (TYK2 promoter variant in T2D. There was no difference in BMI between T2D with TYK2 promoter wild type gene and variant type (P = 0.12) (Supplementary Table 4). In addition, there was increased TYK2 promoter variant rate at statistical significance only in non-obese T2D with less than 26 BMI , but not obese T2D with more than 26 BMI  (Supplementary Table 4), suggesting that obesity is not likely involved in the increased risk associated with TYK2 promoter variant in T2D.In the age group of 20–39, we found no difference in the age at onset  . Similarly, there was a slight decrease in the interferon-induced expressions of TYK2 gene  in peripheral blood mononuclear cells derived from diabetic patients with TYK2 promoter variant (n = 14), compared with those (0.71 ± 0.28) of patients with the wild type TYK2 gene (n = 17), while there was a mild increase in the interferon-induced expressions of JAK1 gene  in peripheral blood mononuclear cells derived from diabetic patients with TYK2 promoter variant (n = 14), compared with those  of patients with the wild type TYK2 gene (n = 17) . The expression levels of ISGs, including PKR, OAS and MxA, induced by interferon stimulation in patients with TYK2 promoter variant were also lower than those of patients with wild type ISGs , which did not reach statistical significance . These results suggest that the increased risk of developing diabetes conferred by the TYK2 promoter variant may be due to reduced TYK2 promoter activity accompanied by the decreased expression of the TYK2 gene and ISGs, while increased expression level of JAK1 gene on IFN stimulation in patients with TYK2 promoter variant may play a complementary role for the deteriorated TYK2 gene expression to maintain ISGs responses. Further investigation is required to clarify the influence of TYK2 promoter variant on cytokine responses in humans.To determine the function of the 4Tyk2 in mice . All thediabetes . Since Tnd IL-23 , suggest obesity . TherefoTYK2 promoter variant was associated with a significantly higher susceptibility to diabetes in anti-GAD antibody-negative patients , of which observation is consistent with Tyk2 gene mutation dependent murine virus-induced diabetes that simulates non-autoimmune rapid onset and fulminant T1D without autoantibody production (Tyk2 gene played an important role not only in the interferon signaling pathway but also in the Th1 type immune response-associated IL-12-dependent signaling pathway (TYK2 gene promoter variant may reduce the risk for the development of Th1 type-dependent autoimmune reactivity to the pancreatic β-cells. In addition, if TYK2 promoter variant confer risk for the development of diabetes due to increased susceptibility to viral-infection, possible induction of autoimmunity against pancreatic β-cells triggered by viral infections, which is a well-documented hypothesis (TYK2 promoter variant-associated susceptibility to type 1 diabetes. Since these data have been obtained in the Japanese population, less prone to T1D than other ethnic groups, the possible role of TYK2 promoter variant needs to be verified in different populations.Interestingly, oduction . It was  pathway . It was  pathway . These opothesis , was notIFIH1 gene, which is an intracellular pathogen recognition receptor for picornavirus including enteroviruses, operating as an inducer of interferon production (IFIH1 and TYK2 genes is both possible and feasible. Unfortunately, at present, there is no appropriate assay system to prove the diabetogenicity of the virus infective for humans, fulfilling ‘Koch’s postulate (In human cases the situation is highly different from that in experimental animals where mice have been infected with a virus and it is possible to prove that infection is causing diabetes, however, accumulation of circumstantial evidence to identify the putative virus-induced susceptibility gene in humans is important. It was reported that polymorphisms of the oduction , is assooduction . Since tostulate . Mouse mS.Na. designed the study, interpreted the data and wrote the manuscript. Y.K-H., K.Hi., K.Ka., M.T., A.M., E.Y., H.Ku, and H.Ka. performed the genetic analyses. N.T., M.M., A.O., Y.M., Y.U., Y.Y., K.K., K.I., K.An., R.I., K.Ha., N.S., S.No., K.Ak., T.Y., J.O., T.O., R.F., K.I., T.H., M.H., Y.K., M.I., T.K., F.K., H.Ka., S.Na., and S.Kondo collected the human samples of the patients and healthy controls, and analyzed the clinical data of the patients. S.Kono performed the statistical analysis.There is no conflict of interest regarding this research.


This dose also enhanced cell motility mediated by 100 µM-H2O2, while higher RS-doses augmented the H2O2-induced impaired myoblast regeneration and mitochondrial dehydrogenase activity. In conclusion, low resveratrol doses promoted in vitro muscle regeneration and attenuated the impact of ROS, while high doses augmented the reduced plasticity and metabolism induced by oxidative stress. Thus, the effects of resveratrol depend on its dose and degree of oxidative stress.While reactive oxygen species (ROS) play a role in muscle repair, excessive amounts of ROS for extended periods may lead to oxidative stress. Antioxidants, as resveratrol (RS), may reduce oxidative stress, restore mitochondrial function and promote myogenesis and hypertrophy. However, RS dose-effectiveness for muscle plasticity is unclear. Therefore, we investigated RS dose-response on C2C12 myoblast and myotube plasticity 1. in the presence and 2. absence of different degrees of oxidative stress. Low RS concentration (10 μM) stimulated myoblast cell cycle arrest, migration and sprouting, which were inhibited by higher doses (40–60 μM). RS did not increase oxidative capacity. In contrast, RS induced mitochondria loss, reduced cell viability and ROS production, and activated stress response pathways [Hsp70 and pSer36-p66(ShcA) proteins]. However, the deleterious effects of H In response to damage, satellite cells rapidly undergo several cycles of cell division prior to withdrawal from the cell cycle to terminally differentiate and fuse with the damaged skeletal muscle fibres1Effective regeneration and training adaptations critically depend on the level of generated reactive oxygen species (ROS)3346ROS can activate a number of signalling pathways that have an impact on cell migration, cell cycle transition, cell survival, apoptosis and differentiation5910117To combat oxidative stress and its consequences, dietary supplementation with antioxidants has often been applied. Antioxidant supplementation has been shown to improve expression of anti-oxidant enzymes, muscle function and muscle repair91314in vitro myogenesis and hypertrophy, at least partly via regulation of expression of myogenic regulatory factors and cell cycle progression factorsIn this context, the anti-oxidant and anti-inflammatory polyphenol resveratrol (RS), commonly found in the skin of grapes and in other red fruits, has received extensive attention in the last years, showing cell-protection from oxidative stress-induced damage and inflammation with benefits in a variety of human diseases, including cancer, cardiovascular diseases and aging14151617Despite these reported benefits, many studies show weak beneficial effects and there is even an increasing awareness of detrimental side effects of RS2425262829303132332O2), as a model of oxidative stress.The aim of the present study was to assess the effects of resveratrol on myoblast and myotube plasticity and to what extent these effects differ in the presence or absence of oxidative stress. Thereto, we explored in mouse skeletal muscle-derived C2C12 myoblasts and myotubes the dose response relationship of resveratrol on key phases of skeletal muscle remodelling and oxidative metabolism, in the presence or absence of hydrogen peroxide  on cell proliferation, cell cycle progression , cell miWe found that the effects of RS were dose-dependent. A low dose of RS (10 µM) enhanced cell motility, as seen by the increased number of migrated cells in scratched monolayers, while it was increasingly inhibited by increasing doses . The degHigh concentrations of RS (40–60 µM) induced thinner and elongated myoblasts with an increased appearance of intracellular vesicles (data not shown) and reduced cell number . Compare23in vitro studies have shown that RS can exert a cytotoxic effect, including induction of mitochondrial apoptosis via mitochondrial membrane depolarization2930Several Mitochondrial depolarization may lead to a reduction in the number of mitochondria. Succinate dehydrogenase (SDH) activity has a fundamental function in oxidative energy metabolismTo gain more information about the effect of RS on the properties of myofibrils, we tested the effect of different concentrations of RS on myosin type 1- and total myosin ATPase activities in C2C12 myotubes . The effTen and 20 µM RS treatment had no significant impact after both 24 h or 48 h of incubation . Higher 2O2 on cell migration and cell fusion (2O2 on cell migration were dose-dependent (2O2 (500 and 1000 µM) blocked cell motility almost completely, while it was increased by 100 µM H2O2, (2O2 (500 and 1000 µM) also blocked cell fusion, as indicated by a reduced number of myotubes  with different concentrations of RS and then treated for 24 h with 1000 µM H2O2. Pre-incubation with 10 and 20 µM RS did counteract the inhibitory effect of H2O2 on total ATPase activity  and its implication in the activation of the mitochondrial apoptosis cascade are well recognized2O2. Therefore, we analysed whether RS was able to counteract H2O2-induced mitochondria depolarisation. RS pre-conditioning did not prevent, but even enhanced the depolarization induced by 24 h exposure to 1000 µM H2O2 (372O2 induced p66Shc(A)-Ser36 phosphorylation  phosphorylation.The effects of H µM H2O2 . Notably µM H2O2 , and ele µM H2O2 , two keyrylation  and bothrylation . Althoug2O2 (day2). Only the highest concentration of H2O2 caused a reduction in both metabolism in myoblasts  did show only a modest effect in attenuating the detrimental impact of high doses of H2O2.Our main observations were that the effects of resveratrol on The literature is equivocal when it comes to the effectiveness of RS to improve muscle contractile function and metabolism. It has been reported for instance that RS has positive effects on muscle mass and function1419263939242527414231Several authors indicated that RS promotes the early stage of C2C12 myoblast differentiation23It has recently been demonstrated that high RS concentrations >30 µM) impair cell viability µM impai313242434237The depolarisation of mitochondria may well lead to loss of mitochondria in the cell. In myotubes, the reduced succinate dehydrogenase (SDH) activity, which plays a role in both the citric acid cycle and the respiratory chain, and total mitochondrial dehydrogenase activity after exposure to high doses of RS, indicates that this is indeed the case . A lowerThe reduced ROS generation in myoblasts incubated with RS may at first glance fit the notion that RS is an effective anti-oxidant. Given the observations discussed above, a more likely explanation is that the RS-induced loss of mitochondria underlies the reduction in ROS production.in vivo would cause a reduced fatigue resistance of the muscle.Finally, high doses of RS reduced myosin type1-ATPase activity  to enhanin vivo studies report the potential of RS to diminish mitochondrial oxidative injury and cell death of skeletal muscle cellsin vivo work7Thus, although several 2O2 stimulated in vitro cell motility (2O2) in vitro cell motility was inhibited. Only low doses (10 µM) of pre-conditioning with RS for 24 h attenuated the deleterious impact of high ROS exposure on cell migration , 2 mM L-glutamine and 1% penicillin-streptomycin solution . At 70% confluence, the cells were divided by trypsinisation . DMEM supplemented with 2% FBS, was used as differentiation mediumPoly-gonumcuspidatum extract; 21st Century Alternative, UK), and/or 10, 50, 100, 500 and 1000 µM hydrogen peroxide . The chosen ranges of concentration of RS and H2O2 were based on studies demonstrating the effectiveness of similar concentrations102848Murine C2C12 myoblasts  were cultured on 0.2% gelatin  coated T75 flasks and/or plates in Dulbecco's modified Eagle's medium  in a 37°C humidified chamber at 5% CO5 cells·mL−1 (2 mL per well) in 0.2% gelatin-coated 6-well plates. After attachment (4 h) cells were washed twice with sterile phosphate buffered saline  and the medium replaced with complete DMEM containing 10, 20, 40 or 60 µM RS and/or DMSO. Cells were counted after 24, 48 or 72 h incubation with an automated Coulter counter . Each experiment was performed in triplicate.Myoblasts were seeded in complete DMEM medium at a concentration of 2 × 10g, 10 min, 4°C) and washed in PBS prior to fixation at −20°C in 70% ethanol. Twenty four hours later the fixed cells were recovered by centrifugation, washed in PBS and suspended with gentle vortexing in propidium iodide labelling buffer , at approximately 1 × 106 cells·mL−1TM flow cytometer . The fluorescence data (FL-H channel) on 10,000 event counts were analysed using Cell Quest  and Modfit LT Software . A coefficient of variation of the G1 peak <6 represented acceptable quality dataCell cycle analysis was performed as described in482O2 or vehicle. The effect of the compounds on cell fusion was determined after 72 h by counting the total number of 1% methylene blue stained myotubes (being defined as having at least three nuclei within one cytoplasmatic continuity) present in three random picture fields captured from each well with an inverted microscope. All experiments were performed in triplicate.To induce differentiation, myoblasts were grown to about 50% confluence5 cells·mL−1 were suspended in DMEM plus 2% FBS and 0.25% (w/v) carboxymethylcellulose . After their formation (24 h), single independent spheroids where sub-cultured for 24 h at 37°C, 5% CO2 with/without the presence of scalar amounts of resveratrol and/or vehicle in a matrix of type I collagen The C2C12 spheroids assay was performed to test the effect of scalar amounts of resveratrol on cell sprouting4·mL−1) were added to a 0.2% gelatin-coated 24-well plate in complete DMEM medium. After attachment (4 h) cells were washed twice with PBS and the medium replaced with DMEM (0.1% FBS) and maintained at 37°C and 5% CO2 for 24 h to minimize cell proliferation. Migration assay492O2 or vehicle (DMEM 0.1%) and followed for 24 h. In the experiment of RS pre-conditioning, cells were pre-treated (24 h) with (10 or 20 µM) RS and migration assays (24 h) performed with scalar amount of H2O2 (DMEM 0.1%). All experiments were done in triplicate.C2C12 myoblasts TM, Becton Dickinson, Oxford, UK) and CellQuest software (BD Biosciences). For the RS pre-conditioning, cells were pre-treated (24 h) with 10 or 20 µM RS, followed by 24 h treatment with 1000 µM H2O2. Before each treatment, cells were washed twice with sterile PBS. All experiments were performed in triplicate.After each treatment, 1 × 102O2 and/or vehicle. After treatment, ~1 × 106 cells·mL−1 were incubated for 30 min at 37°C with 5 µg·mL−1 final concentration of the cationic dye 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolyl-carbocyanine iodide , with conditions as described by the manufacture. Analyses were then performed by flow cytometryTM flow cytometer, Becton Dickinson, Oxford, UK) and CellQuest software (BD Biosciences). Cells  were analysed by FL-1 (green fluorescence) and FL-2 (red fluorescence) channels. Gates  were established with the untreated cells (controls)Myoblasts were cultured (24 h) in the presence or absence of scalar amounts of RS, H2O2 (day2). Treatment with H2O2 alone was also performed at day2. Twenty-four hours after incubations, 5 × 103 cells·mL−1 were seeded in complete DMEM medium on 0.2% gelatin-coated 96-well plates. After attachment (4 h) cells were washed twice with PBS and the medium replaced with complete DMEM containing the tetrazolium salt and incubated at 37°C for 4 h. The absorbance of the formazan was read at 490 nm. Data from H2O2 alone and RS preconditioning were compared to control conditions at day2. All experiments were performed in quadruplicate.The activity of mitochondrial dehydrogenases was measured by a colorimetric assay , based on the redox conversion of a tetrazolium salt into a formazan productth day, suggesting functional maturity2O2 or vehicle. In the RS-preconditioning experiments, cells were pre-treated (24 h) with resveratrol and/or vehicle. After the treatments, the media was removed and cells were stained for myosin ATPase as describedC2C12 myoblasts were cultured in reduced DMEM media (2% FBS) for 8 days. Occasionally, a spontaneous contractile activity was observed at the 8th day, they were treated 24 or 48 h with RS  and/or 24 h with scalar amount of H2O2  or vehicle. In the RS-preconditioning experiments, cells were pre-treated  with resveratrol or vehicle. After two washes with PBS, the media was removed and myotubes were cultured for another 24 h (day2 corresponding to the 10th day of myoblasts cultured in differentiation media) in the presence of 1000 µM-H2O2. Staining for SDH activity was performed as described previouslyC2C12 myoblasts were cultured in DMEM (2% FBS) for 8 days. At the 8Protein extraction was performed in ice-cold RIPA buffer with a protein inhibitor cocktail. Thirty μg protein was loaded and separated on 10% SDS-PAGE gels. Protein samples were transferred onto nitrocellulose membranes , stained with amido-black and photographed to verify equal loading and quality of the transfer. The membranes were subsequently incubated with blocking solution and incubated overnight at 4°C with primary antibodies against mouse p66Shc-pSer36 , heat shock protein 70  or mouse actin . The membranes were then incubated with appropriate horseradish-peroxidase-conjugated secondary antibodies  at room temperature. Protein bands were visualised by chemiluminescence detection kit  and signals normalized to the corresponding actin optical density .t-test or ANOVA (SPSS version 12) and considered significant at P<0.05. Experiments were performed in triplicate or quadruplicate as described in Methods.All data were expressed as mean ± s.e.m. Effects of the treatments were assessed using two-tailed Student's A.B. and H.D. conceived the study. A.B. designed and performed the experiments. A.B. and H.D. analysed the data and wrote the manuscript.Supplementary Figures and supplementary figure legends