Supplying −800 mV to the microbiome after repeated exposure to acidic pH resulted in up to 2.6 kg/m3/day hydrogen , 0.7 kg/m3/day formate, and 3.1 kg/m3/day acetate ( = 4.7 kg CO2 captured).Production of hydrogen and organic compounds by an electrosynthetic microbiome using electrodes and carbon dioxide as sole electron donor and carbon source, respectively, was examined after exposure to acidic pH (∼5). Hydrogen production by biocathodes poised at −600 mV vs. SHE increased>100-fold and acetate production ceased at acidic pH, but ∼5–15 mM (catholyte volume)/day acetate and>1,000 mM/day hydrogen were attained at pH ∼6.5 following repeated exposure to acidic pH. Cyclic voltammetry revealed a 250 mV decrease in hydrogen overpotential and a maximum current density of 12.2 mA/cm Microbial electrosynthesis is a process by which microbes grow as a biocathode and couple electrical energy to the capture and conversion of CO2 into compounds such as methane or organic acids The ability to capture carbon dioxide  buffered at near neutral pH 2 will maintain the pH close to neutrality and thereby support acetogenesis 2 production by the microbiome, which has implications for H2 and organic acid production. This extends our understanding of how these microbiomes perform electrosynthesis, and suggests a platform for the electrosynthetic production of fuels and chemicals Electrosynthesis of acetate and other short-chain fatty acids from COAn electrosynthetic microbiome enriched from brewery wastewater obtained from Palmetto Brewery  was used in this study 2 CMI-7000 cation exchange membrane . A total of 25 grams of graphite granules (Showa Denko) were used in each chamber for the anode and cathode. A 0.95 cm outer diameter fine extruded graphite rod (Graphite Store) was cut into 3 cm long current collectors wound with 0.81 mm diameter titanium wire (Sigma Aldrich). All carbon electrodes were pretreated by washing in acetone and drying, followed by immersion in 1 M NaOH, and 1 M HCl for 24 hours each with deionized water rinses between each step. Reference electrodes were made with a 1 mm diameter AgCl coated silver wire  immersed in 4 mm glass capillary tube (ChemGlass) containing 3 M KCl saturated with Ag/AgCl and to which a Vycor tip (Koslow) was attached using Teflon heat-shrink tape (BASi). The reference electrodes were immersed in a 7 mm diameter Luggin capillary containing 1 M KCl. The cathode and anode chambers were each filled with 50 mL of media. Reactors were poised at −600 mV vs. the standard hydrogen electrode (SHE) unless indicated otherwise. Chronoamperometry and cyclic voltammetry were recorded using a VMP3 potentiostat and EC-Lab Software (Bio-Logic Science Instruments). Voltammetric sweeps ranged from −800 to 0 mV vs. SHE at 1 mV/sec. Reactors were sparged with 100% carbon dioxide at 15 mL/min, except in graphite rod cathode yield tests. Another larger customized reactor (Adams & Chittenden) was designed with a 20 cm2 cation exchange membrane. The anode and cathode chambers each contained 100 g graphite granules and 100 mL of phosphate buffered media. This reactor was used to test the biocathodes at potentials lower than −600 mV vs. SHE. A total of 10 reactors were examined and the inoculation scheme is depicted in Reactors were customized three-electrode, two-chamber glass cells (ChemGlass) separated by a 2 cmAliquots of media were filtered and analyzed for pH using a pH meter (Mettler-Toledo) and fatty acid content via HPLC (Shimadzu) using the method described in Marshall et al. Samples for SEM were incubated in a 0.1 M sodium cacodylate buffer with 2% gluteraldehyde for 3 hours, then incubated in a 2.5% osmium tetroxide, and finally dehydrated with an ethanol dilution series using 0%, 25%, 50%, 75%, % and 100% at 5 minute intervals. Samples were stored in a desiccator before being sputtered with Au and Pd using a Denton Vacuum sputter coater. Images were mounted on a stage with conductive carbon tape and imaged using a JEOL JSM-5600LV Scanning Electron Microscope.http://www.earthmicrobiome.org/). Sequences are publicly available under the MG-RAST IDs: Reactor 4 electrode 4562455.3, and Reactor 4 supernatant 4562456.3 (http://metagenomics.anl.gov/).Reactor 4 was analyzed for microbial composition at 5 months after inoculation. Electrode attached cells were sampled by collecting graphite granules while planktonic cells were collected by filtering supernatant through a Sterivex filter (Millipore). Both samples were stored in Soil Preservation Solution (MoBio Laboratories) at −80°C. DNA from electrode-attached cells was extracted using a PowerSoil DNA Isolation Kit (MoBio Laboratories). DNA from planktonic cells was extracted using PowerWater Sterivex DNA Isolation Kit (MoBio Laboratories). Primers amplifying the V4 region (F515/R806) of 16S ribosomal sequence was amplified using Golay barcoded primers An acetogenic/methanogenic microbiome sans NaBES can, in certain cases, mitigate the competitive advantage of the favorable thermodynamics and H2 scavenging ability of methanogens due to the commensurate kinetics of H2 utilization between some acetogens and methanogens Acetobacterium spp. have been reported to outcompete certain methanogens when H2 is not limiting 2 was used to lower the electrolyte resistance and maintain the cathode potential at a lower overall applied voltage, and avoid the bicarbonate buffer that is more supportive of methanogenesis. The first test with this medium was done with Reactors 4–6 . This likely contributed to the pH change since without the gradient>100 mM acetic acid was needed to lower the pH significantly compared to the lowering of pH by only 30 mM acetate with the gradient. In order to determine if the response of the microbiome was due to the change in pH only, two reactors with active acetogenic microbiomes (Reactors 4 and 5) received fresh media and 50 mM NaBES added to the anode and cathode chambers.  Acetate was produced early in the experiment (13–19 mM/day) while hydrogen production settled below 25 mM/day and the pH stayed between 6.5 and 7 . Each ca2 surface area) were incubated in the graphite-granule bed cathode of Reactor 4 for 48 days and exposed to 5 medium exchanges and cycles of acidic pH (<5). The rods were then transferred to serve as a defined surface area biocathode without graphite granules in three independent reactors (Reactors 7–9) with 50 ml of phosphate buffered medium plus 50 mM NaCl substituted for NaBES in each chamber.Three graphite rods  suggests that the dead microbiome might have left a redox-active species adsorbed to the cathode. Further examination of the mechanisms of how the electron transfer occurs within the biocathode is beyond the scope of this study, but the results indicate that a living and intact microbiome is necessary for the development of high current and organic acid production by these biocathodes.A CV analysis was performed on all of the biocathodes after their transfer to the new reactors while the catholyte was sparged with 100% COutrality . All thr −800 mV  inset. M −800 mV . Autocla2, sealed, and poised at −600 mV vs. SHE. Productivity for two of the three biocathodes (Reactors 8 & 9) was similar over a 4-day test, but the cathode that generated the least current in the CV analysis (Reactor 7) performed poorly in relation to the others  on the electrodes. Protein densities were comparable at this time (14.3 ± 0.9 µg/cm2) and the average current density to protein for the three biocathodes was of 6.5 ± 1.2 A/g. The coulombic efficiency for each reactor was similar with nearly 90% to hydrogen early on and over 40% to acetate at the end of the experiment  with a larger membrane and working volume (100 mL) was inoculated with granules from Reactor 4. Relieving the membrane limitation allowed the reactor to be operated at −800 mV vs. SHE. Under these conditions, high hydrogen and organic acid production was achieved as the pH decreased . The hydAcetobacterium woodii has been shown to produce a maximum of 123 mM/day acetate when incubated under a partial hydrogen pressure of 1700 mbar A. woodii would produce 421 mM/day in a nutrient enriched medium stirred at 1200 rpm under 40% hydrogen A. woodii to overexpress phosphotransacetylase and acetate kinase or the four THF dependent enzymes of the Wood-Ljungdahl pathway in order to overcome carbon flux bottlenecks. These mutants produced acetate at 480 mM/day in the same medium and conditions. The current densities and product formation by the electrosynthetic microbiota from the current study indicate that electron flux from an electrode to a microbial community is similar to these rates of hydrogenotrophic acetogenesis, albeit without the energy required to pressurize or stir the reactor. For example, the electrosynthetic microbiome has produced>50 mM/day acetate, while an additional 325 mM/day could potentially be generated based on the electron equivalents for maximum hydrogen production rates observed using the system.Acetobacterium sp., particularly on the electrode, where it comprised 90% of the relative abundance  is that these organisms capture electrons directly from an electrode and synthesize acetate Acetobacterium could possibly be contributing to the productivity of the microbiome, but it is difficult to discern DET to acetogenesis when hydrogen is concomitantly produced in such high amounts. Current densities are sufficiently high at cathodic potentials so that CV scans of the biocathodes are dominated by the hydrogen reaction, and hydrogen forming at the electrode surface is readily visualized bubbling vigorously off of the biocathode surface when the pH is low or after the community has grown/adapted to higher hydrogen production following an acidic pH challenge  transfer may also play an important role in the metabolism of the electrosynthetic microbiome. For example, 3/day hydrogen (1 kg H2 ≈ 1 GGE) production by an electrosynthetic microbiome. High acetate production occurred (up to 3.1 kg/m3/day) when the pH was>5, with 1.5 kg of CO2 captured per kg acetate produced. CV analysis indicated a lowering of the overpotential at the cathode, and current densities as high as 12.2 mA/cm2. Oxygen and autoclave treatment ameliorated the cathodic current. Acetobacterium may solely facilitate these reactions but alternatively, interspecies interactions by the microbiome may be required to produce high levels of hydrogen and organic acids during microbial electrosynthesis.Repeated exposure to acidic pH resulted in up to 2.6 kg/mFigure S1Inoculation scheme of the 10 reactors used in this study. Reactors 1-4 were inoculated from a previous electrosynthetic microbiome. NaBES or NaCl at 50 mM was added to the cathode (C) or anode and cathode (A + C) of each reactor. Reactors 1–3 were operated with bicarbonate buffered medium with 50 mM NaBES in the catholyte. They were replenished with fresh media wherein 50 mM NaCl was substituted for the NaBES in the catholyte. Reactor 4 was operated with a phosphate buffered medium with 50 mM NaBES in the catholyte or in both catholyte and anolyte. Reactors 5 and 6 were two replicate reactors inoculated from Reactor 4 and under the same conditions. Yield tests and CV were performed in Reactors 7–9 which contained phosphate buffered media with 50 mM NaCl in the anolyte and catholyte and rods initially incubated in Reactor 4. Reactor 10 was the larger membrane reactor inoculated with granules from Reactor 4 and poised at lower potentials in phosphate buffered media with 50 mM NaBES in the anolyte and catholyte.(PDF)Click here for additional data file.Figure S2Replicates of Reactor 1 and the conditions presented in. Biocathodes of Reactors 2 (A and B) and 3 (C and D) were incubated in bicarbonate buffered media with 50 mM NaBES (A and C) or 50 mM NaCl (B and D) in the catholyte and poised at −600 mV vs. SHE.(PDF)Click here for additional data file.Figure S3Transferability and replication of the electrosynthetic microbiome. Granules were transferred from Reactor 4 (A) into Reactors 5 (B) and 6 (C) and exposed to lowered pH in phosphate buffered media containing 50 mM NaBES in the catholyte −600 mV vs. SHE.(PDF)Click here for additional data file.Figure S4Abiotic controls. Hydrogen production (solid lines) in low and high pH (dashed) sterile and sealed reactors. Graphite granule cathodes were poised at −600 mV vs. SHE in phosphate-buffered medium with 50 mM sodium BES and with (blue) or without (red) 100 mM acetic acid.(PDF)Click here for additional data file.Figure S5Inactivation of an active biocathode. Cyclic voltammogram of an active rod biocathode in phosphate buffered medium at pH = 6.3 with 50 mM NaCl in the anolyte and catholyte, and 100% CO2 sparge (black). The active rod was exposed to sterile flowing air (40 mL/min) for 20 hours and the scan was repeated under 100% CO2 sparge (blue). The O2 inactivated rod was then autoclaved on a gravity cycle for 30 min and the scan was repeated again under 100% CO2 sparge (red). An abiotic sterile control (gray) and the autoclave and O2 inactivation treatments showed far less cathodic current densities than the active biocathode.(PDF)Click here for additional data file.Figure S6Improved production at lower potential. Sequential media replacements (A and B) of phosphate buffer medium with 50 mM BES in the anolyte and catholyte in Reactor 10 poised at −800 mV vs. SHE unless otherwise indicated.(PDF)Click here for additional data file.Figure S7Screen shot of hydrogen gas evolving off of biocathode. Video screenshot of a biocathode in phosphate buffered media poised at −600 mV vs. SHE.(PDF)Click here for additional data file.Table S1Summarized parameters and maximum productivities for the reactors in this study. Maximum productivities are in mM/day, and g/m2/day is in parenthesis for the rods. Presence of NaBES or NaCl in the catholyte or catholyte and anolyte is noted by C or C & A, respectively.(PDF)Click here for additional data file.Movie S1Brief video of the biocathode evolving hydrogen gas at −600 mV vs. SHE.(MOV)Click here for additional data file.


Gene names Ompk36 and Ompk35 are swapped incorrectly throughout the manuscript. All instances of Ompk35 should appear as Ompk36, and all instances of Ompk36 should appear as Ompk35. Corrected versions of Tables


More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1–TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1–TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex. In vivo, TRF2 forms a stable complex with Repressor activator protein 1 (Rap1)  . The Rap1 (Rap1) . While T1 (Rap1) , Rap1 de1 (Rap1)  nor telorecently . All thein vitro  domain mediating homodimerization of TRF2, a flexible linker region comprising Rap1 binding motif and a C-terminal DNA Myb domain  Figure . The myein vitro , no signin vitro ,19. The rap1/trf2 was cloned into pHGWA vector (The cDNA sequences of Rap1 and TRF2 were synthesized by Source BioScience and cloned to pDONR/Zeo vector (Life Technologies) using two sets of primers (Supplementary Table S1) and BP clonase enzyme mix from Gateway technology (Life Technologies). Resulting plasmid pDONR/Zeo A vector  using LR6-tags were expressed in bacterial cells as described elsewhere (Escherichia coli BL21(DE3) and Rap1 in E. coli BL21(DE3)RIPL carrying the vector pHGWA -polyacrylamide gel which was subsequently stained using Bio-Safe Coomassie G 250 (Bio-Rad). Mass spectrometry measurements were used to confirm that proteins were expressed in full length and high purity. Supplementary Figure S1 shows SDS-polyacrylamide gel electrophoresis of TRF2 and Rap1 used in the studies.GGTTAGGGTTAGGGTTAGGGTTAG, G-strand sequence; overhang is in bold italic), denoted as Ov  and 35 bp long (GTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAG) denoted as R2 (two telomeric repeats) and R5 (five telomeric repeats), respectively, along with 47 nt long DNA substrate containing 24 nt overhang  buffer was used. Reactions containing the same amount of fluorescently labeled DNA (3 pmol) and increasing amounts of protein TRF2 (3–15 pmol) were prepared. DNA was labeled with fluorophore Alexa Fluor 488. The influence of protein Rap1 on DNA-binding affinity of TRF2 was detected in reaction mixtures composed of a constant amount of labeled DNA (5 pmol), a fixed amount of protein TRF2 (10 pmol) and increasing amounts of protein Rap1 (20–80 pmol). In both cases, the reactions were supplemented with buffer  to a volume of 15 and 3 μl of 6× loading buffer . Reaction mixtures were incubated for 15 min on ice and then loaded on horizontal 5% (w/v) non-denaturing polyacrylamide gels in 0.25×TBE buffer. The electrophoresis proceeded at 1 V/cm for 45 min and for an additional 3 h at 2 V/cm at 4°C. Fluorescently labeled DNA in gels were analyzed with a FLA 7000 imaging system (Fujifilm).Kd) was determined from the curve representing the dependence of FA on the concentration of protein added to the solution in the cuvette. Fitting analyses were carried out using programs SigmaPlot 12 (Systat Software) and DynaFit4  . A dissociation constant (in Ltd.) .Rap1 was labeled with fluorescent dye Alexa Fluor 594 or 488. Subsequently, labeled proteins were separated from free fluorophores by gel filtration using a PD 10 Desalting Column . The experimental instrument setting for fluorescence measurement of Rap1 conjugated with the fluorophore Alexa Fluor 594 was 584 nm and 611 nm with the same width of slits 7 nm for excitation and emission. To measure TRF2 binding to DNA that was conjugated with the fluorophore Alexa Fluor 488, the excitation wavelength was set to 492 nm and emission wavelength to 516 nm with the width of slits 9 nm. The integration time was 3 s. The FA titration experiments were carried out in a 10×4 mm quartz-glass cuvette with chamber for magnetic bar stirrer. FA was measured at 25°C in the buffer containing 50 mM NaCl and 50 mM sodium phosphate (pH 7.0) if not stated otherwise. All fluorescence measurements were performed on a FluoroMax-4 spectrofluorometer (Horiba Jobin Yvon) with an L-format set up equipped with automatically adjustable polarizers for excitation and emission lights under control of an Origin-based FluorEssence software (version 2.1.6).The contribution of electrostatic interactions was determined from the linear dependence of the binding constants on the increasing concentration of NaCl. The electrostatic component of binding originates from the formation of ion pairs between the cationic amino acid residues of the protein and the negatively charged phosphate groups of DNA or amino acid residues of the other protein. The electrostatic component of binding was determined from the binding constant dependence on ionic strength as described in  and in SKa), reaction stoichiometry (n) and binding enthalpy (ΔH) were obtained from the fit. Binding free energy (ΔG) and entropy change (ΔS) were determined from the equation:ITC experiments were carried on a VP-ITC instrument  at 25°C. Proteins were diluted in the same buffer 50 mM NaCl and 50 mM sodium phosphate (pH 7.0) and degassed. The cell (1423 μl) was filled with TRF2 (5 μM). Rap1 (44 μM) was added in 20 injections of 10 μl at 5 min intervals, with a stirring rate 240 rpm. Experimental data were analyzed in Origin 7.0 software using one-site binding model to fit a theoretical titration curve. Binding constant  on GLC and NLC sensor chips (BioRad) in a Phosphate Buffered Saline with Tween 20 (PBST). The detailed conditions of SPR measurements are available in Supplementary Data.6 per M and corresponding dissociation constant 21 nM  were similar in the presence or absence of Rap1 (Supplementary Table S4). Similarly, our SPR data did not provide us with convincing results showing significant effect of Rap1 on TRF2 off-rate constants for fully hybridized duplex DNA nor DNA with 3′ overhang (data not shown). It might be caused by the short length of telomeric DNA that was required for our SPR experiments.To quantify the effect of Rap1 on TRF2 binding affinity to double-stranded DNA, we carried out three FA measurements with different experimental arrangements. In the first arrangement, TRF2 alone was allowed to bind to a fluorescently labeled DNA duplex R2 . In accordance with our EMSA data  in the absence or presence of Rap1 . At first, only TRF2 was allowed to bind DNA. Subsequently, the complex of TRF2 with Rap1 was used in DNA-binding assays. It was revealed that TRF2 binding affinity to telomeric DNA is approximately 5-fold higher in comparison with TRF2 binding affinity to non-telomeric DNA duplex N. In other words, the selectivity, which is defined as the ratio of association constant for protein binding to specific telomeric R2 and non-specific random N DNA, was five . On the other hand, in the presence of Rap1, the binding affinity of TRF2 is more than 8-fold higher to telomeric than to non-telomeric DNA duplexes ; the selectivity was eight. Based on these results, we conclude that TRF2 in complex with Rap1 binds telomeric DNA with nearly 2-fold higher selectivity.Ka) were calculated as reciprocal values of dissociation constants obtained for at least three independent measurements at each salt condition. Logarithms of Ka were plotted against logarithms of salt concentration  interactions, were identified (Supplementary Table S2). The parameter Z revealed that the binding of TRF2 to telomeric DNA resulted in the formation of approximately five ion pairs in average. On the other hand, in the presence of Rap1, TRF2 formed only three ion pairs with DNA. The decreased Z value means that the electrostatic interactions with DNA were less pronounced when the preformed complex of Rap1 and TRF2 bound DNA. The contribution of electrostatic component to overall DNA-binding affinity of TRF2 was calculated from values corresponding to the total binding affinity Ka and non-electrostatic contribution to the binding affinity Kanel. Based on the salt dependence of association constant, the total affinity and corresponding Gibbs free energy of binding was divided into contributions originated from electrostatic and non-electrostatic interactions  than to the full-length protein and therefore induce a more profound displacement of TRF2ΔB from duplex DNA than in case of full-length TRF2. To test this idea, we allowed either full-length TRF2 or TRF2ΔB to bind to DNA R2 until about half saturation range was achieved . When Rap1 was added, we observed that significantly more DNA was released from TRF2ΔB than in case of full-length TRF2. This experiment strongly supports our previous measurements and conclusions about the effect of Rap1 on DNA-binding affinity of TRF2 and the importance of the TRF2 basic domain to non-specific DNA binding.We revealed the molecular mechanism of how Rap1 affects TRF2 binding to DNA by using a combination of quantitative biophysical approaches. The explanation of the origin of selective DNA binding of shelterin protein TRF2 is essential for understanding shelterin functions in genome stability maintenance in humans and mammals. Moreover, our findings could be applied on proteins that bind DNA and participate in gene regulation through selective DNA recognition. The quantitative descriptions include several new observations that provide a more complete understanding of the activity of the critical shelterin subunit TRF2 on DNA, which extents and confirms the previous separate observations of other investigators ,17,18.Kd value for binding of full-length Rap1 and TRF2 is in a very good correlation with the value of Kd for the binding of C-terminal domain of human Rap1 . Additionally, our EMSA experiments have shown that Rap1 does not bind telomeric DNA until Rap1:DNA molar ratio 40:1 . These results are partially contradicting the previous finding that Rap1 binds telomeric DNA directly . The difThe main objective of our study was to quantify how Rap1 affects affinity and selectivity of protein TRF2 to telomeric DNA. We found that, on one hand, the presence of Rap1 increases the selectivity of TRF2 binding to telomeric DNA by 2-fold, but on the other hand, Rap1 decreases TRF2 binding affinity to DNA by approximately 2-fold.Kd which could cause the less pronounced Rap1 effects on total release of DNA in case of EMSA experiments . The observed selectivity increase is in a very good accordance with the results obtained recently from gel retardation assays by Arat and Griffith . Additios Figure . The morA probable explanation of how Rap1 could contribute to the observed reduction in the DNA-binding affinity of TRF2 is a modification of net surface charge after Rap1–TRF2 complex formation. Protein interaction with DNA occurs in two steps. In the first step an electrostatic, non-specific attraction of interacting partners occurs . In the Eventually, we assessed how Rap1 affects TRF2 binding to DNA substrate Ov containing naturally occurring overhang that comprises four telomeric repeats Figure . We obseMoreover, the interaction model takes into consideration dimeric arrangements of TRF2 and Rap1. The sequence selective binding of TRF2 in dimeric form is in agreement with DNA sequence recognition mechanism of other selectively binding proteins that take part in regulatory mechanisms .To test this model we prepared a truncated variant of TRF2 lacking the N-terminal basic domain. In accordance with our hypothesis, when the basic domain is absent, TRF2 should have a significantly lowered DNA affinity. Indeed, when we measured the DNA-binding affinity of TRF2 lacking the basic domain, DNA-binding affinity was decreased to the level of the affinity observed for the binding of TRF2 with Rap1 present . Moreover, we observed that more telomeric double-stranded DNA was released when Rap1 added to TRF2 lacking the basic domain pre-bound to DNA compared to full-length TRF2 . This finding strongly supports our previous measurements and the proposed model of Rap1 effects on DNA-binding affinity of TRF2.et al. have shown by combination of nuclear magnetic resonance, differential scanning calorimetry (DSC) and permanganate probing experiments. It has been suggested by the same laboratory that TRF2 basic domain has evolved to finely regulate TRF2 ability to condense DNA  . The onlin vitro binding studies, one may speculate that Rap1 is needed for efficient directing of TRF2 to its proper binding location at telomeres also in vivo. TRF2 alone could be accumulated in internal chromosomal or peritelomeric regions. After Rap1 binding, Rap1–TRF2 complex might relocate to a single/double-strand junction of telomeric DNA. Although little to no effect on TRF2 telomere binding is observed after Rap1 deletion in both human and mouse cells (Rap1 contribution to the whole shelterin function and signal pathways of the cell is matter of a long discussion ,13,16. Tse cells , it is pse cells . It woulWe conclude that Rap1 serves as a selectivity enhancer of TRF2 with newly found ability to partially remove bound TRF2 from telomeric DNA duplex. The observed Rap1 release activity suggests that Rap1 plays an important role in tuning DNA interactions of TRF2, the central subunit of shelterin protein complex. Our data showing the release of TRF2 from telomeric DNA in the presence of Rap1 suggest that protein Rap1 might prompt the relocation of TRF2 to the preferred single/double-strand junction of telomeric DNA. For the first time here, we used combination of quantitative biophysical approaches to describe and explain molecular origins of Rap1 contribution to selective TRF2 recognition of telomeric DNA. We found that Rap1 neutralizes the electrostatic attraction of TRF2 to DNA. Hence, Rap1 reduces overall DNA-binding affinity of TRF2 in order to improve its binding selectivity toward telomeric DNA. The following studies focused on shelterin protein dynamics using single molecule approaches will be of particular interest as they can reveal relocation dynamics of TRF2 induced by Rap1 and the mechanism of regulation of shelterin binding and distribution on telomeres.Supplementary Data are available at NAR Online.


The following information is missing from the Acknowledgments section:"We thank Drs. Gautam Shirsekar and Jiangbo Fan who generated the data shown in Figure 3C, 3D, and 3E."


Several genetic variants for type 2 diabetes (T2D) have been identified through genome wide association studies (GWAS) from Caucasian population; however replication studies were not consistent across various ethnicities. Objective of the current study is to examine the possible correlation of 9 most significant GWAS single nucleotide polymorphisms (SNPs) for T2D susceptibility as well as the interactive effect of these variants on the risk of T2D in an Indian population.Case-control cohorts of 1156 individuals were genotyped for 9 SNPs from an Indian population. Association analyses were performed using logistic regression after adjusting for covariates. Multifactor dimensionality reduction (MDR) analysis was adopted to determine gene–gene interactions and discriminatory power of combined SNP effect was assessed by grouping individuals based on the number of risk alleles and by calculating area under the receiver-operator characteristic curve (AUC).TCF7L2 (rs7903146) and SLC30A8 (rs13266634) with T2D. MDR analysis showed statistically significant interactions among four SNPs of SLC30A8 (rs13266634), IGF2BP2 (rs4402960), HHEX (rs1111875) and CDKN2A (rs10811661) genes. Cumulative analysis showed an increase in odds ratio against the baseline group of individuals carrying 5 to 6 risk alleles and discriminatory power of genetic test based on 9 variants showed higher AUC value when analyzed along with body mass index (BMI).We confirm the association of TCF7L2 and SLC30A8. MDR analysis demonstrates that independently non-significant variants may interact with one another resulting in increased disease susceptibility in the population tested.These results provide a strong evidence for independent association between T2D and SNPs for in  The risk of an individual towards T2D reflects the environmental influence in the background of genetic predisposition. Owing to the complex etiology of the disease progression, the identification of genetic markers has been slow until 2007. The advent of new technology in the form of microarray chips, has led to the development of high throughput genome wide association study (GWAS). Nevertheless, only a few variants in genes such as KCNJ11, PPARG, SLC30A8, and TCF7L2 were reported to be linked with T2D  were genotyped in all the subjects. Genotyping of KCNJ11 (rs5219), FTO (rs8050136) andTCF7L2 (rs7903146) polymorphisms was carried out using Tetra primer amplification refractory mutation system (TETRA-ARMS) and genotyping of SLC30A8 (rs13266634), IGF2BP2 (rs4402960), HHEX (rs1111875), CDKN2A (rs10811661), KCNQ1 (rs2237892) andCDKAL1 (rs7754840) polymorphisms were carried out by polymerase chain reaction followed by restriction fragment length polymorphisms (PCR-RFLP). Random samples were picked and direct DNA sequencing was performed  to confirm the genotype.Gene variants studied basically corresponds to those identified from GWAS of T2D till the year 2010 and also shown in replicative studies for its association with T2D susceptibility in other population. DNA was extracted from peripheral blood using standard phenol-chloroform procedure. A total of 9 SNPs [http://www.ncbi.nlm.nih.gov/projects/SNP/). The categorical data of SNPs for association analysis with T2D was performed by using Pearson’s chi-squared test to detect differences in allele frequencies between cases and controls. Hardy–Weinberg equilibrium test was performed using a χ2 goodness-of-fit test to assess genotype frequencies. Association analysis was further confirmed by logistic regression after adjusting for age, sex and BMI as covariates and association results of SNPs with T2D was assessed by using odds ratio (OR) and corresponding 95% confidence interval (CI). In our analysis the most frequent homozygous genotype in the control population was considered as the reference category. Further we evaluated the differences in continuous variables  using Students t-test and data are represented as mean ± SD. Bonferroni correction has been used to reduce the chances of obtaining false-positive results (type I errors). A P value of less than 0.005 has been kept as a threshold of significance (after Bonferroni correction). All statistical analysis was performed using SPSS version 16 for windows  and SNPstat Software. Population attributable risk (PAR%) which identify what percentage of total risk for T2D is due to genetic effect of the variant was estimated for those SNPs which showed positive association with T2D susceptibility. For allele dosage analysis to acquire the combined information from multiple SNPs, we used allele count model where we summed the number of exact risk alleles carried by each individual in cases and control group.Quanto version 1.2.4 was employed for sample size calculation using minor allele frequency data from dbSNP database  method was employed. MDR is a nonparametric method and therefore no hypothesis concerning the value of any statistical parameter is made. It is also model free, thus no genetic inheritance model is assumed. All interactions and identification of best models were performed using 10 fold cross validation consistency (CVC) and permutation testing was done considering all possible SNP combination. Testing balance accuracy in the context of 10-fold cross-validation was used to assess model quality. An overall best model was selected that had the maximum accuracy in the testing data (i.e. testing accuracy or TA). We also recorded the cross-validation consistency or CVC. This provides a summary of the number of cross-validation intervals in which a particular model was found. Higher numbers indicate more robust results. Those models/SNP combinations with highest testing balanced accuracy (TBA) and CVC was selected as ‘‘best model’. This procedure generates an empirical estimate of the null distribution of testing accuracies and corrects for multiple testing because the same number of models are evaluated in all permutated and real data. All interactions were visualized by constructing an interaction dendrogram according to the method described by Moore et al., 2004 , 27, 28.TCF7L2 (rs7903146) and SLC30A8 (rs13266634) with an allelic OR = 1.46, 95% CI 1.15–1.85, p = 0.001 and OR = 1.33, 95% CI 1.10–1.60, p = 0.002. No significant association was observed between the remaining 7 loci and T2D in this population (p>0.05). Given that, variants in FTO and HHEX have shown to be associated with BMI, we further examined the association of these polymorphisms with BMI in our NGT control subjects and found that FTO (rs8050136) was significantly associated with BMI  controls matched for age, sex and ethnicity. with BMI .p<0.05 were still included in our allele dosage analysis because these were confirmed T2D risk variants and lack of significance may probably due to lower risk allele frequencies. SLC30A8 (rs13266634) and TCF7L2 (rs7903146) which were found to be associated with T2D in our population. We observed that TCF7L2 (rs7903146) accounts for 18.4% and SLC30A8 (rs13266634) accounts for 30.4% PAR. These two genetic regions contribute to a total of 48.8% population attributable risk (PAR) for T2D (FTO (rs8050136) was removed from analysis.The individual risk variants in our study showed similar effect sizes as compared to other large studies from Caucasian population . Several variants in our study though are not associated with T2D at  for T2D . We asse for T2D . The areSLC30A8 (rs13266634), IGF2BP2 (rs4402960), HHEX (rs1111875) and CDKN2A (rs10811661) SNPs. This model has a significant TBA of 0.618 and a CVC of 10/10. However it’s worth mentioning that 3 SNPs showing genetic interaction in this model were not associated with increased risk of T2D in univariate analysis (IGF2BP2 (rs4402960), HHEX (rs1111875) and CDKN2A (rs10811661)). The entropy based dendrogram obtained by MDR evidently showed an intricate and a hierarchical pattern of interaction among the gene variants constituting the polygenic basis of the disease (CDKN2A (rs10811661) and FTO (rs8050136), IGF2BP2(rs4402960) and HHEX (rs1111875) loci showed maximum degree of synergy in their interactions and on the contrary, different degree of redundancy (antagonism) was observed between TCF7L2 (rs7903146) and SLC30A8 (rs13266634). SLC30A8 (rs13266634), IGF2BP2 (rs4402960) and HHEX (rs1111875), and CDKN2A (rs10811661) as high and low risk groups along with its statistical interactions. As per the four locus model, individuals homozygous for the wild type allele in SLC30A8 (rs13266634), IGF2BP2 (rs4402960), HHEX (rs1111875) and individuals homozygous for the mutant allele in CDKN2A (rs10811661) were placed in low risk group while, individuals heterozygous for all four SNPs were placed in high risk group. However, we also observed that individuals heterozygous for at least three of the four alleles were also in high risk group.Consecutively to expand the findings in our data, MDR analysis was applied to detect higher order gene-gene interaction in cases and controls. MDR analysis of the above mentioned variants revealed statistically significant interactions. Best interaction models along with testing accuracy and cross validation consistency are represented in  disease . In partKCNJ11 rs5219) belongs to well replicated biological candidate gene which has shown a clear evidence of its relationship to T2D susceptibility [TCF7L2 (rs7903146) has been shown to have a strongest association with the highest effect size for T2D association across various ethnicities [TCF7L2 (rs7903146) with T2D susceptibility with an effect size of 1.46, 95% CI 1.15–1.85, p = 0.001. Sladek et al., 2007 was the first to identify SLC30A8 (rs13266634) as a novel gene variant associated with T2D and since then, many studies have been performed across various ethnic population but the results have been contradictory [p = 0.002.The outcome of GWAS has significantly contributed to the discovery of number of genetic loci linked to T2D –19. In ttibility . Until n7L2 rs790146 has bnicities , 29, 30.7L2 rs79046 has beC30A8 rs166634 as SLC30A8 and TCF7L2 as a key gene for T2D susceptibility has been well known, the genetic variants in these genes could account for about ~20% of all T2D cases in the Caucasian population [SLC30A8 and TCF7L2 polymorphisms in Caucasian population was 30.4% and 18.4% respectively which is much higher when compared to our population, and this could be attributed to the higher risk allele frequency in Caucasian population. Further in our allele dosage analysis, we have identified a particular subset of individuals at different risk of disease by weighing against the individuals with smallest number of risk alleles in comparison to those with the majority of risk alleles. This in turn allowed us to identify subgroups of the population with distinctly differing risk for disease. In our population, we were able to distinguish 14.5% of individuals carrying >13–14 risk alleles that had four times increased risk of T2D when compared to 1.3% of individuals with >5–6 risk alleles. The high risk group also had over thrice the OR for T2D than those with the lower number of risk alleles. Thus, variants are not predominantly discriminative but only explain a small percentage of heritability of T2D. However, instead of focusing on individuals with elevated risk alleles at the population level, the efficacy of genetic test perhaps is better utilized by means of ROC curves. The nine T2D variants though had an insufficient discriminatory ability with an AUC of 0.536; a marginal increase was obtained when it was analyzed with BMI with an AUC value of 0.624.While the significance of pulation , 36. TheSLC30A8, IGF2BP2, HHEX, and CDKN2A genes towards T2D susceptibility by MDR analysis. Among the five models for all the loci tested (nine SNPs of nine candidate genes), the best gene-gene interaction model identified was a four locus model which includes SLC30A8 (rs13266634), IGF2BP2 (rs4402960), HHEX (rs1111875) and CDKN2A (rs10811661). Individuals heterozygous for at least three out of four loci were shown to cluster in high risk groups. In this model the combination of HHEX (rs1111875) CC homozygotes, SLC30A8 (rs13266634) CC homozygotes, IGF2BP2 (rs4402960) GG homozygotes and CDKN2A (rs10811661) TT homozygotes were associated with reduced risk to T2D. Our study did not show any independent association of IGF2BP2 (rs4402960), HHEX (rs1111875) and CDKN2A (rs10811661) with T2D. However in MDR analysis, combined effect of these polymorphisms with SLC30A8 (rs13266634) was associated with increased risk of T2D which suggest a cross talk between these genes in the pathogenesis of T2D. Further, a synergistic interaction among these SNPs suggests a significant role of epistasis in the susceptibility of T2D. However, it is also observed that all the SNPs which were included in this study showed a weak synergistic effect with each other and this suggests the interaction of susceptibility alleles with environmental and life style factors. Nevertheless, the risk allele frequencies of these variants in the tested population were entirely different with those from Caucasian population and thus, the absence of positive association towards T2D in remaining loci points towards the contribution of these polymorphisms to T2D susceptibility is not convincing across different ethnicities or to a particular genetic background. To the best of our knowledge, this is the first report from south Indian population trying to investigate the role of these polymorphisms individually and cumulatively towards T2D susceptibility. Nonetheless, further replication studies are warranted to validate these and newer susceptibility loci which are identified from large scale GWAS in independent samples collected from other ethnic groups.Another major finding of our study was the establishment of gene interaction model between S1 Table(XLSX)Click here for additional data file.


Wuchereria bancrofti endemic area in Odisha, India. Following screening, 104 consenting adults were randomly assigned to treatment with the standard regimen annually for 24 months (S1), or annually with increased dose (H1) or with increased frequency (6 monthly) with either standard (S2) or increased (H2) dose. Pre-treatment microfilaria counts (GM) ranged from 348 to 459 mf/ml. Subjects were followed using microfilaria counts, OG4C3 antigen levels and ultrasound scanning for adult worm nests. Microfilarial counts tended to decrease more rapidly with higher or more frequent dosing at all time points. At 12 months, Mf clearance was marginally greater with the high dose regimens, while by 24 months, there was a trend to higher Mf clearance in the arm with increased frequency and 800mg of albendazole (76.9%) compared to other arms, . Although higher and/or more frequent dosing showed a trend towards a greater decline in antigenemia and clearance of “nests”, all regimens demonstrated the potential macrofilaricidal effect of the combination. The higher doses of albendazole did not result in a greater number or more severe side effects. The alternative regimens could be useful in the later stages of existing elimination programmes or achieving elimination more rapidly in areas where programmes have yet to start.Although current programmes to eliminate lymphatic filariasis have made significant progress it may be necessary to use different approaches to achieve the global goal, especially where compliance has been poor and ‘hot spots’ of continued infection exist. In the absence of alternative drugs, the use of higher or more frequent dosing with the existing drugs needs to be explored. We examined the effect of higher and/or more frequent dosing with albendazole with a fixed 300mg dose of diethylcarbamazine in a  In order to achieve global elimination of lymphatic filariasis, it may be necessary to consider alternative approaches to mass treatment using higher or more frequent dosing with the existing drugs used in the programmes. Outside Africa, the drugs used are albendazole and diethylcarbamazine given annually. The current study is the first to examine the use of higher or more frequent dosing in a bancroftian filariasis endemic area outside Africa. Four groups of infected subjects were followed for two years using standard techniques for monitoring drug efficacy in Odisha, India. We showed that higher doses of albendazole, or 6-monthly treatment, were more effective than the standard regimen in producing clearance of microfilaria and reductions in antigenemia. Additionally we found that these regimens were more effective in killing the adult filarial worms in the lymphatics. Importantly, the higher doses used did not increase the risk of side effects. These alternative approaches could accelerate lymphatic filariasis elimination either at the end of existing programmes or to permit new programmes to achieve their goals faster. The Global Lymphatic Filariasis Elimination Programme (GPELF) had its inception in 1998 following agreements between the World Health Organization and the international pharmaceutical companies GlaxoSmithKline (GSK) and Merck to donate supplies of, respectively, albendazole and ivermectin for the programme . AlthougThe initial drug regimens used at the initiation of the Global Programme were based on existing approved doses and did not require extensive additional investigation or drug approval. However no attempts to investigate the effectiveness of alternative regimens, either or use of higher or more frequent dosing were made . A studyW. bancrofti infection. Since the Indian programme uses a single dose of 300 mg DEC for all individuals > 15 years, the dose of DEC remained unchanged [The current study was conducted in India to investigate the effect of increasing the dose of albendazole from 400 mg to 800 mg and /or increasing the frequency of dosing for nchanged . The stuW. bancrofti microfilaremia , who checked completeness of records, compliance with GCP and accuracy of data entry.W. bancrofti endemic area within thirty kilometres of Bhubaneswar in the Khurda district of Odisha was chosen for selection of subjects. Community meetings were arranged to provide information on the importance of the filariasis elimination programme and of the study prior to enrolling individuals for the study. In the filaria endemic villages, night blood was collected by finger prick between 19.00 and 23.00hrs from all individuals between 18 and 55 years of age during a door to door survey. Thick smears from 40μl blood were examined for microfilaria. Microfilaremic individuals who provided consent were further screened for eligibility. Subsequently, in eligible subjects, an intravenous blood sample (5ml) was collected between 21.00–22.00 hours and divided into two aliquots; 2ml with EDTA and 3ml without anticoagulant. From 1ml EDTA blood, the microfilaria count was determined using the Nuclepore filtration technique. Haemoglobin was estimated by automated cell counter ; serum ALT and creatinine were measured by automatic biochemistry analyser  and pregnancy was excluded using a β-HCG rapid test on early morning urine samples. W. bancrofti (OG4C3) antigen levels were also obtained using an ELISA kit .A Subjects of either sex between 18 to 55 years of age, with microfilaria counts greater than 50/ml. of blood, who accepted the hospitalization and follow up requirements were included in the study. Female subjects who were either pregnant or lactating were excluded as were subjects with serum ALT level of > 30 units/dl, creatinine level > 1.2 mg/dl or haemoglobin level of <10 gm%.Individuals satisfying the inclusion and exclusion criteria and providing a second consent for study inclusion were allocated sequentially to one of four treatment groups according to a pre-determined list generated in blocks of 26 using Prism software (Graphpad Prism 6.0).Ultrasonography of inguinal, scrotal, axillary, thigh and arm lymphatic systems was performed prior to treatment using a Doppler Ultrasonography unit  using a linear high resolution 7–12 Mega Hz probe, to record any “filarial dance” sign.The individuals, in groups of 4–6, were then admitted to the hospital of the Kalinga Institute of Medical Sciences, Bhubaneswar, for study drug administration, observation and management of adverse events if required. The drugs were taken orally by the subjects around 8.30am after breakfast, under supervision of hospital staff. Where the subject vomited within 1hr of ingestion, the dose was repeated. The subjects were discharged from hospital after two days and followed at home for up to 7 days post drug intake.A 2x2 factorial design with albendazole at two levels  and number of doses per year at two levels  was used. The resulting four regimens are coded as S1, S2, H1 and H2. For each of the regimens 26 patients were allocated with a total of 104 for the study. These numbers were based on data comparing single and multi-dose regimens for the treatment of lymphatic filariasis , 14. We The four treatment arms were as follows:S1: Diethylcarbamazine citrate (DEC) 300mg plus albendazole 400mg. oral single dose repeated annually.S2: DEC 300mg plus albendazole 400mg. oral single dose repeated six monthly.H1: DEC 300mg plus albendazole 800mg. oral single dose repeated annually.H2: DEC 300mg plus albendazole 400mg. oral single dose repeated six monthly.The drug formulations used for the study were authorized drugs marketed in India as Banocide (DEC) 100mg tablets  and Zentel  400mg tablets .4C3 antigen titre, urine β HCG test in females, serum ALT and creatinine were evaluated blind at follow up visits before the subjects received the next drug dose. All subjects were admitted to hospital for drug administration and for 2 days post drug to monitor for side effects / adverse events. Side effects/ adverse events were graded based on a previously used scale [The subjects were followed up every 6 months up to 24 months for evaluation with drug administration as appropriate. Ultrasonography was repeated in those subjects with “filarial dance sign” detectable adult worm at base line, within 48–72 h of the first dose, and at 12 months and 24 months by the consultant ultrasonographer (C H Mohanty) who was unaware of the treatment received by the patients. Microfilarial count, OGed scale  and manaThe clinical and laboratory information were recorded in a predesigned format. The data was entered into Excel and analysed using SPSS version 16 with appropriate tests for parametric and non parametric variables. Correctness of data was ensured by double entry and matching of entered data. The results were expressed as percentage changes and mean reductions at 6, 12 (the primary endpoint), 18 and 24 months. The chi-square test was used to compare the proportions, Student’s t-test for comparing mean differences between two regimens and analysis of variance for comparing all the four group means at 12 months, at 18 months and at 24 months. The net effect of the high levels was quantified using the contrast of a factorial design. The significance level was fixed at 5% level.W. bancrofti endemic area of Khurda district in state of Odisha, India. This covered a rural area of around 150 square kilometres between Bhubaneswar  and Khurda . The population is mostly middle and lower socioeconomic status within an agriculture based economy.The study was initiated in October 2008 and final two year follow ups were conducted in September 2012, enrolling study subjects from 8 villages in the In all, 1751 individuals between 18 and 55 years of age were screened to identify 118 (6.74%) microfilaraemic subjects. 104 individuals who satisfied the inclusion/exclusion criteria and were willing to participate in the study were screened for eligibility. The subjects were randomly allocated to one of the 4 arms of the study and followed up with 6 monthly screening. All 104 individuals (26 in each of the four arms) received DEC + albendazole at baseline in the dosage prescribed for the respective arm. A 40-year-old male allocated to S1 arm died 9 months after the initial treatment following an acute abdominal emergency not considered to be related to the drug or study procedures. The remaining 103 study subjects all completed 24 months follow up.p = 0.259 Student T test). There was a preponderance of males in all 4 treatment groups with approximately 17% females in each group, with most being below 35 years of age. Blood chemistry values were in the normal range in all subjects. The microfilarial density and mean OG4C3 units of the study population at enrolment are provided in p = 0.822). The mean OG4C3 antigen units were also comparable (p = 0.057). Ultrasonography prior to first dose detected adult worm nests with filarial dance sign (FDS) in 13 subjects in each of S1, H1 and H2 arms and 15 in S2 arm (p = 0.219).The age and gender distribution of enrolled subjects (n = 104) was comparable among the four arms (p = 0.021) compared to the standard regimen (S1). At 24 months, Mf clearance was higher in H2 arm (76.9%) compared to other arms,  although this was not statistically significant (p = 0.77). The mean microfilarial density showed a sharp reduction by 6 months from baseline  in all four arms with a slower decline thereafter , produced an extra reduction of 0.74 log units and if albendazole was given at the higher dose (800 mg) (H1 and H2) instead of 400mg, there was an extra reduction of 0.64 log units. Biannual treatment with 800mg (H2) albendazole reduced the Mf count by 1.09 log units which was statistically significant (p = 0.021). It can be concluded that an increase in dose and frequency can reduce Mf counts significantly at the end of 12 months and 24 months compared to the standard regimen (S1).p = 12 M: 0.942, 18 M: 0.94 & 24 M: 0.23). Mean percentage reductions in OG4C3 from individual baseline values are shown in 4C3 antigen clearance was seen to start at 12 months in S1 & H2 arms and at 18 months in S2 and H1 arms and increased over time. At 24 months, OG4C3 clearance ranged from 26.9% to 73.1% (p = 0.007). Since the variance in the antigen units was very large due to wide range of values, logarithmic transformation was used to stabilize variance and one way ANOVA was performed on the log transformed values  if albendazole is given biannually (S2 & H2), instead of annually (S1 & H1) there was an extra mean reduction of 0.66 log units in OG4C3 antigenemia (ii) if albendazole is given as 800 mg (H1 & H2), instead of 400mg (S1 & S2), there was an extra mean reduction 0.31 log units of OG4C3 antigenemia and (iii) if both are given at high level (H2) there is an extra reduction of 0.80 log units in OG4C3At 24 months, (i) If albendazole is given biannually (S2 & H2), instead of annually (S1 & H1) there is an extra mean reduction of 0.53 log units in OG4C3 antigenemia (ii) If albendazole is given at 800 mg (H1 & H2), instead of 400mg (S1 & S2), there is an extra mean reduction of 0.86 log units of OG4C3 antigenemia and (iii) If both are given at high level (H2) there is an extra reduction of 1.38 log units in OG4C3 antigenemia. It can be concluded that increase in dose and frequency reduces OG4C3 log counts at the end of 12 months and 24 months.4C3 clearance was observed later at 18 month follow up  and increased over the subsequent 6 months .Among individuals exhibiting adult worm (USG) at baseline, OGp = 0.669) after the first dose (p = 0.124) or 18 months (p = 0.305) dosing. However, at 12 months, S1 (28%) & H1 (15.4%) annual arms had a higher frequency of adverse events compared to S2 (3.8%) & H2 (3.8%) biannual arms. The adverse events noted were light-headedness (31.7%), fever (22.1%), body ache (4.8%), unsteadiness (4.8%), dizziness (2.8%), drowsiness (2.8%), fatigue (1.9%), chills (1.9%), itching (0.9%), and weakness (0.9%). There was no difference in the pattern of side effects noted in different arms (p>0.05). There were no severe drug related adverse events reported following any treatment, and the events that did occur could be managed conservatively.Following drug administration, the frequency of adverse events was comparable in all four arms  , it is only possible to increase the albendazole dose . Thus th4C3 suggest that there is a significant macrofilaricidal effect at this dose.The primary endpoint for the study was taken as Mf clearance at 12 months after initiation of treatment to be consistent with past studies, but follow up and analysis was continued for a further 12 months to establish whether any differences that might be seen at 12 months were sustained. Microfilarial clearance in all the four arms was progressive over time with a higher rate of decline in the mf prevalence in H2 arm. Although the difference in mf clearance between arms was not statistically significant over the 24 months of follow-up, the decline in mf prevalence in H2 arm gives the impression that if followed further, this arm could attain 100% mf clearance earlier than the other arms. This has the potential to shorten the period required for elimination. This was also indicated by the parallel reduction in microfilarial density where at both 12 and 24 months there was a significantly greater reduction in the high dose twice yearly arm (H2) compared to the other arms. A similar study using high dose twice yearly albendazole and ivermectin in Mali also showed enhanced suppression of microfilaria [Increased adverse events/side effects directly due to the higher dose of albendazole or as a result of interaction with DEC could be a limiting factor to deployment , 18, 19.The results of this study have important implications for GPELF in areas where onchocerciasis is not co-endemic. The annual low dose regimen performed well in this study and clearly remains the treatment of choice for the majority of situations. However, the availability of an alternative DEC-based regimen that uses a higher dose of albendazole with increased frequency could assist programmes to meet their elimination targets. In particular, this regimen may find use in areas/countries where MDA programmes are yet to be initiated, in “hot spots” identified during surveillance of ongoing programmes and in arresting transmission within shorter time frames . HoweverS1 Fig(DOCX)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file.


IRS1), rs1531343 (HMGA2), rs8042680 (PRC1), rs7578597 (THADA), rs1333051 (CDKN2), rs6723108 (TMEM163), rs163182 and rs2237897 (KCNQ1), rs1387153 (MTNR1B), rs243021 (BCL11A), and rs10229583 (PAX4) in our sample. Further, we showed that risk allele of the strongest T2D associated SNP in our sample, rs757832 (IRS1), is associated with increased level of TG. We observed substantial difference of T2D risk allele frequency between the Mongolian sample and the 1000G Caucasian sample for a few SNPs, including rs6723108 (TMEM163) whose risk allele reaches near fixation in the Mongolian sample. Further study of genetic architecture of these variants in susceptibility of T2D is needed to understand the role of these variants in heterogeneous populations.The large scale genome wide association studies (GWAS) have identified approximately 80 single nucleotide polymorphisms (SNPs) conferring susceptibility to type 2 diabetes (T2D). However, most of these loci have not been replicated in diverse populations and much genetic heterogeneity has been observed across ethnic groups. We tested 28 SNPs previously found to be associated with T2D by GWAS in a Mongolian sample of Northern China (497 diagnosed with T2D and 469 controls) for association with T2D and diabetes related quantitative traits. We replicated T2D association of 11 SNPs, namely, rs7578326 ( However, we observed that association of T2D risk allele  of rs7578326 (IRS1)   is associated with the increased level of TG. An elevated level of TG has been implicated as a risk factor of T2D, which likely resulted from the diminished activity of insulin causing inhibition of microsomal TG transfer protein activity [IRS1), namely, rs2972146, is reported to be associated with an elevated level of TG as well [r2 = 0.3753, 1000 G phase I), indicating a possibility that the T2D associated SNP or its proxy could be playing a role in pathogenesis of T2D or its related TG metabolism through IRS1 activity. Further functional work will help to understand the role of this SNP. Since we did not have information on previous medical history of treatment for either high cholesterol or T2D for the patients, it is possible that such treatments, if administered previously, could have prevented us from seeing the effect of SNP association with the diabetes related quantitative traits, including TG.None of the SNPs we tested for association with T2D here has been previously implicated to be associated with lipids based on the NHGRI GWAS catalog (available at activity . The majTMEM163) and rs8042680 (PRC1) have substantial allelic differences in our sample compared to Caucasians and the risk alleles have reached near fixed level in Mongolians. On the other hand, a widely replicated T2D SNP, rs7903146 (TCF7L2), in European populations has substantially low risk allele frequency in the Mongolian sample  and is not associated in our sample of modest size. It is likely that the frequency difference of some SNPs between our sample and European population also contributed to the lack of reproducibility in T2D association in our study.Mongolians are one of the people who reside on the Mongolian Plateau in Asia and have heavily depended on nomadic life styles with harsh environments characterized by low temperature and scarce availability of food sources . AlthougAlthough we note that rs6723108 has a wide range of OR estimate , the OR of 7.7 in our study is substantially greater than what was reported in the original study in an Indian population [ IRS1 to be associated with an increased level of TG. The observation of the remarkable allele frequency difference of the T2D SNPs in our sample compared to Caucasians is important in further identifying causative variants for T2D and understanding the role of these SNPs in development of T2D in different ethnic populations.In conclusion, our association study has confirmed association of several previously identified T2D susceptibility loci in the Mongolian sample. We also identified rs7578326 nearThe SNPs were chosen with following considerations: (1) GWAS SNPs found to be associated with T2D in an Asian sample were given higher priority; and (2) subsequently, SNPs found to be associated with T2D in multiple studies were included. Genotyping of six SNPs was not successful, thus these SNPs were included from further analysis.


The gut is the largest immune organ and plays a central role in the promotion of systemic inflammatory responses . Perturb7 CFU Bifidobacterium longum, 10 × 106 CFU Lactobacillus bulgaricus, 10 × 106 CFU Lactobacillus acidophilus. Colonic tissue and serum samples were collected 24 hours after CLP for ELISA and protein expression analysis by western blotting.Sepsis was induced by cecal ligation and puncture (CLP) in Wistar male rats (8 weeks old). They were pretreated with probiotics or vehicle once a day during 7 days before CLP. The chosen probiotic mixture contained 10 × 10Our data demonstrate that probiotic pretreatment improved survival of septic rats Figure  and thisOur results show that probiotics pretreatment fulfills a dual function at the intestinal mucosa: in addition to preventing intestinal permeability disruption, it also attenuates proinflammatory cytokine release, diminishing the exacerbate host's reaction to infection and offering a novel prophylactic strategy to sepsis.


Paramecium we have shown that three conserved proteins FOR20, centrin 2 (CEN2) and centrin 3 (CEN3) participates in this process, with FOR20 and CEN2 being also involved in the transition zone assembly. We established a chronology in basal body assembly: CEN2 is required for FOR20 recruitment, the latter being necessary to recruit CEN3. Our goal now is to integrate others molecules in this cascade.Ciliogenesis is conditioned by a correct positioning/anchoring of the basal body at the cell surface. In Paramecium. OFD1 is a well-studied protein which is involved in human development whose mutations in human males can impair basal body docking. In contrast, only studies in Chlamydomonas indicate that VFL3 could be involved in this phenomenon.We used a combination of electron microscopy, immunocytochemistry, GFP protein tagging and RNAi knockdowns to study the function of OFD1 and VFL3/CCDC61 in Paramecium induces defects in basal body docking, these defects being similar to those observed upon inactivation of FOR20, CEN2 and CEN3; 1) like FOR20 and despite its distal location on anchored basal bodies, OFD1 is recruited early during their assembly; 2) while the recruitments of OFD1 and CEN2 proceed independently, the two molecules are required for the recruitment of FOR20. We also present preliminary results indicating that VFL3/CCDC61 is crucial for maintaining both basal body polarity and positioning and for the recruitment of CEN3, but neither for CEN2 or OFD1.As in human, the depletion of OFD1 in


Enterovirus genus within the Picornaviridae family. The SVDV genome is composed of a single-stranded RNA molecule of positive polarity. Here, we report the first complete sequence of the coding region of a Spanish SVDV isolate (SPA/1/'93).Swine vesicular disease virus (SVDV) is a porcine pathogen and a member of the  Enterovirus genus within the Picornaviridae family. SVDV is the etiological agent of a highly contagious disease of pigs (SVD) and is closely related to the human pathogen coxsackievirus B5 (CVB5) (Swine vesicular disease virus (SVDV) is a member of the 5 (CVB5) . The SVD5 (CVB5) , 3. In t5 (CVB5)  and propThe coding sequence of the Spanish SVDV isolate SPA/1/'93 was compared with other complete coding sequences from different isolates that varied geographically and temporally using Clustal 2.1 . This analysis showed that SVDV SPA/1/'93 shared a higher degree of sequence identity (over 98%) with contemporaneous Italian isolates . The sequence data of the SVDV isolate determined in this study will help future research on the epidemiology and molecular biology of this porcine pathogen.KU291213.The genome sequence of the SVDV SPA/1/'93 has been deposited in GenBank under the accession number


Rapid response teams (RRT) are important systems for identifying patients requiring intensive car e. CompliaTo investigate the correlation between the clinical outcomes of septic patients and the elapsed time before transfer to the intensive care unit (ICU).A retrospective descriptive study performed in a large hospital in São Paulo, Brazil, with all patients admitted to the ICU by RRT activation due to suspected sepsis, sepsis, severe sepsis or septic shock from January to December 2011.39 patients were attended by RRT 5 to 20 minutes after activation. Thirty patients (76.9%) were immediately transferred to the ICU. The elapsed time since assessment to transfer to the ICU ranged from 15 to 30 minutes , 30 minutes to 1 hour , 1 to 2 hours  and ≥3 hours . As for the clinical outcome in the ICU, 20 (51.3%) had a clinical improvement, 14 (35.9%) died and 5 (12.8%) had an initial clinical deterioration with subsequent improvement. Clinical improvement or initial deterioration with subsequent recovery occurred mainly among patients transferred to the ICU within 15 minutes to 3 hours. In patients transferred after 3 hours, death was the most frequent outcome.The clinical outcomes of septic patients early transferred to the ICU are better than the outcomes of patients transferred later.


Current rSO2 technologies assume that the oxygen saturation is a fixed ratio of arterial and venous blood. Cerebral arteries have an oxygenation-related vasoactivity that may change the arterial/venous ratio during hypoxia. Thus, absolute rSO2 accuracy may be less important compared with sensitivity to changes in cerebral rSO2.The purpose of this study was to determine the rate and magnitude of response to hypoxia for three different regional oxygen saturation . Resaturation was induced by rapid change in FiO2 to 1.0. After 5 minutes of SpO2 100%, the process was repeated by desaturation to SpO2 70% and rapid return to SpO2 100%. Cerebral and pulse oximetry data were recorded during the study and the time of each FiO2 change and plateau was recorded. rSO2 levels at 10, 20, 40, 60 and 80% of the total SpO2 response were calculated for each device to assess the rate of rSO2 change. The rate of rSO2 change in seconds and total rSO2 change were compared.Ten subjects completed the study and are included in the analysis. One INVOS sensor (SAFB-SM) was placed on the left side and one Equanox (8000CA) or Foresight (1 July 2007 or 1 July 2005) sensor  was placed on the right side of the forehead for bilateral monitoring. Desaturation was induced by adjusting the inspiratory gas mixture of O2 change during desaturation was similar for all devices with an average slope factor of 0.17 for Foresight, 0.16 for Equanox and 0.21 for INVOS. The rate of rSO2 change in seconds during resaturation from SaO2 70% to SaO2 100% was significantly faster for INVOS (42 ± 16) compared with Foresight (57 ± 20) (P < 0.05). There was significant difference in total rSO2 change between INVOS (23 ± 4%) and Equanox (15 ± 4%) during desaturation and resaturation (P < 0.005) and between INVOS compared with Foresight (20 ± 5%) (P < 0.05).The rate of rSO2 devices followed the SpO2 slope during desaturation as expected. The differences between the devices in terms of total rSO2 change reached statistical significance. There were also significant differences in the rate of rSO2 change in seconds between INVOS and Foresight during resaturation. The rate of absolute change in seconds and the magnitude of absolute change may result in better resolution to detect physiological changes. Clinical studies are required to elucidate the clinical relevance of the differences.All rSO


BTB and KCTD11BTB bind Cul3 with high affinity forming stable complexes with 4:4 stoichiometries. Conversely, KCTD12BTB and KCTD15BTB do not interact with Cul3, despite the high level of sequence identity with the BTB domains of cullin binding KCTDs. Intriguingly, comparative sequence analyses indicate that the capability of KCTD proteins to recognize Cul3 has been lost more than once in distinct events along the evolution. Present findings also provide interesting clues on the structural determinants of Cul3-KCTD recognition. Indeed, the characterization of a chimeric variant of KCTD11 demonstrates that the swapping of α2β3 loop between KCTD11BTB and KCTD12BTB is sufficient to abolish the ability of KCTD11BTB to bind Cul3. Finally, present findings, along with previous literature data, provide a virtually complete coverage of Cul3 binding ability of the members of the entire KCTD family.Cullin 3 (Cul3) recognition by BTB domains is a key process in protein ubiquitination. Among Cul3 binders, a great attention is currently devoted to KCTD proteins, which are implicated in fundamental biological processes. On the basis of the high similarity of BTB domains of these proteins, it has been suggested that the ability to bind Cul3 could be a general property among all KCTDs. In order to gain new insights into KCTD functionality, we here evaluated and/or quantified the binding of Cul3 to the BTB of KCTD proteins, which are known to be involved either in cullin-independent (KCTD12 and KCTD15) or in cullin-mediated (KCTD6 and KCTD11) activities. Our data indicate that KCTD6 The BTB  domain is a widespread structural module that is frequently involved in both self and hetero association . ProteinThe analysis of the molecular organization of these proteins indicates that KCTDs are characterized by a modular organization that couples BTB domains, always located at the N-terminus, to C-terminal regions that are frequently unrelated. Comparative analyses of KCTD sequences have highlighted the possibility to cluster KCTD human paralogs in seven clades (denoted with A to G letters), each composed of proteins involved in similar biological processes . NotablyIn this framework, we have here checked the interaction of Cul3 with two proteins (KCTD12 and KCTD15) which play crucial roles in non-cullin dependent functions. In particular, KCTD12, and the other members of the Clade F (KCTD8 and KCTD16), are integral components and modulators of the GABA(B) receptor . KCTD12 BTB), KCTD11 (KCTD11BTB), and KCTD12 (KCTD12BTB) were expressed in E. coli BL21(DE3) and purified by following previously reported protocols. In particular, KCTD11BTB, which comprises residues 15–116 of the long form of the protein (isoform 2 of the UniProt Code Q693B1-2), was obtained by following the procedure reported in Correale et al. . On the other hand, the elution profiles of the mixtures formed by KCTD12BTB and KCTD15BTB with Cul3NTD do not show the presence of high Mw species (BTB and Cul3NTD (insert of BTB and Cul3NTD (insert of NTD (~ 42 kDa), it can be confidently assumed that the BTB domains of these two KCTD proteins do not form stable complexes with Cul3.An initial analysis of the binding of these BTB domains to Cul3 was assessed by gel filtration, which provides estimates of the molecular masses of the proteins/complexes under investigation through the analyses of the elution volumes. The mixing of the proteins KCTD6 appears . This ob species . Indeed,BTB, KCTD11BTB, KCTD12BTB, and KCTD15BTB with Cul3NTD are shown in BTB and KCTD15BTB with this cullin suggest that no binding occurs  and 25 ± 2 nM  for KCTD6BTB/Cul3NTD and KCTD11BTB/ Cul3NTD, respectively. In both cases, the value of the number of sites of KCTDs tetramers per Cul3NTD is ~ 0.25. This analysis suggests that the two proteins form with Cul3NTD the following complexes (KCTD6BTB–Cul3NTD)4 and (KCTD11BTB–Cul3NTD)4.In order to confirm and to quantify the data obtained from the gel filtration analyses, ITC experiments, which quantitative evaluate protein-protein binding through the analyses of the heat exchanges associated with their mixing. The heat exchanges upon titration of KCTD6g occurs . This obg occurs . The fitBTB/KCTD12BTB (CHIM11/12BTB), in which the α2β3 loop of KCTD11 (residues 42–63) was replaced with the residues 61–80 of KCTD12, which are predicted to correspond to the α2β3 of this latter protein  is in close to the value expected for the tetramer (62 kDa)  the α2β3 loop and (b) the α4α5 helical hairpin , 19. In  protein . This vaCTD11BTB . Moreove(62 kDa) . These fith Cul3 . The lacanalysis .BTB and CHIM11/12BTB in Cul3 recognition, we performed molecular dynamics simulations on these two proteins focusing the attention on the dynamic properties of the α2β3 loop. The starting models used in the simulations were generated as described in the Materials and Methods section. The analysis of the indicators commonly adopted to check system stability in MD analyses indicates that both structures are stable in the simulation timescale (100 ns). This is clearly indicated by the time evolution of (a) the root mean square deviations (RMSD) of the trajectory structures from the starting model, (b) the gyration radius, (c) the secondary structure, and (d) the total number of hydrogen bonds , whose side chains are involved in conserved electrostatic interactions  (BTB exhibits a higher flexibility. The somewhat reduced mobility of this loop in KCTD11BTB may be ascribed to the presence of a proline-rich fragment (residues 50–53 PMPP) in the α2β3 central region of the protein sequence.a)Insights into the local mobility of different protein regions have been obtained by the analysis of the root mean square fluctuation (RMSF) values calculated on C–100 ns) . As a geBTB and CHIM11/12BTB is also confirmed by an essential dynamics analysis carried out by using the procedure developed by Amadei et al. . Interestingly, both proteins recognize Cul3 forming stable complexes, which can be easily purified by gel filtration chromatography, with a 4:4 stoichiometry. The affinity of the BTB domains of these tetrameric KCTDs for Cul3 is comparable to that reported for the pentameric KCTD5BTB [BTB with Cul3 has suggested that the cullin binds at the intersubunit interface of the protein tetramer [BTB recognizes this cullin at the tetramer interface. Collectively, these observations suggest that the inter-subunit interface of both KCTD tetramers and pentamers provide large surfaces for tight Cul3 binding. Current data suggest that in monomeric or dimeric BTB-containing proteins a similar tight Cul3 binding is achieved with the contribution of other structural motifs such as the 3-box [The quantification of the binding of Cul3 to KCTD6KCTD5BTB  and muchin KD of  nM. InteKCTD5BTB . Since ain KD of  nM. InteBTB and KCTD15BTB are unable to bind Cul3. Although our data were derived using recombinant proteins expressed in a bacterial system, to the best of our knowledge  than to KCTD12BTB (42% sequence identity) that is also unable to recognize the cullin. In other words, the sequences of cullin binding and cullin independent KCTDs are not segregated in the evolutionary tree. These considerations indicate that the capability of KCTD proteins to recognize Cul3 has been lost more than once in distinct events along the evolution. In particular, the loss of ability in Cul3 recognition may have occurred in the divergence of KCTD12 from KCTD6 and KCTD15 and then in the separation of KCTD15 and KCTD6.ITC and Gel filtration analyses indicate that both KCTD12also Ref . These omparison  and of tBTB to bind Cul3 indicate that the α2β3 loop is crucial for cullin recognition. It is worth mentioning that present MD studies indicate that the inability of CHIM11/12BTB to bind Cul3 cannot be ascribed to the obstruction of the binding site by the α2β3 KCTD12 sequence. Therefore, in KCTD11 and other Cul3-binding KCTDs the α2β3 loop contributes to Cul3 recognition by forming interactions that directly stabilize the complex. The high variability of this region Click here for additional data file.S2 FigBTB are highlighted in blue and red, respectively. The hotspots for cullin recognition are also highlighted.The sequence number of the first residue of the BTB domain of each protein is reported. KCTD19a and KCTD19b refer to the two BTB domains of this protein. Helices and strands of KCTD5(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 FigThe differentiated motions along the first eigenvector are represented in a film-like fashion. Large movements are displayed by the α2-β3 loop region. An arbitrary color scale (from violet to red) is used to represent the movement. For clarity, the motion of a single chain within the tetramers is shown.(TIF)Click here for additional data file.


ABCC6) gene (OMIM 603234) are known to cause the rare autosomal recessive disease pseudoxanthoma elasticum (PXE). Recently, we have found that copy number variations (CNVs) in pseudogenes of the ABCC6 gene are quite common. The aim of this study was to investigate the frequency and possible contribution of CNV in ABCC6 and its pseudogenes in PXE. Genomic DNA from 212 PXE individuals were examined for copy number by pyrosequencing and quantitative polymerase chain reaction (PCR) and compared with healthy individuals. The frequency of PXE individuals with any CNV was higher than in healthy individuals. The majority of variation comprised known and possibly new deletions in the ABCC6 gene and duplications of the ABCC6P1 and ABCC6P2 genes. ABCC6 deletions and ABCC6P2 duplications were not observed in 142 healthy individuals. In conclusion, by pyrosequencing and quantitative PCR, we were able to detect known and possibly new deletions in the ABCC6 gene that may have caused the PXE phenotype. Pyrosequencing may be used in PXE patients who have obtained incomplete genotype from conventional techniques. The frequency of ABCC6P2 pseudogene duplication was more common in PXE patients than healthy individuals and may affect the PXE phenotype.Single mutations in the ATP-binding cassette transporter ( ABCC6 belongs to a large family of membrane proteins (ABC transporters) that are highly conserved and present in all organisms from bacteria to man  pseudogenes, ABCC6P1 and ABCC6P2 , autosomal recessive disease pseudoxanthoma elasticum , a metabolic disorder characterized by ectopic mineralization of soft connective tissues  . Written informed consent was obtained from all subjects before blood samples were taken. The study was approved by the Norwegian Regional Ethics Committees.DNA from patients diagnosed with PXE was obtained from the PXE International Registry and BioBank. The patients included 212 individuals and were mainly of European descent . Of these, 148 (70%) were female and 64 (30%) were male. PXE phenotypes were categorized according to the five organ systems  and severity  , Chinese (n = 24), Middle East (n = 20), Mexicans (n = 24), and Africans (n = 24).Genomic DNA from healthy individuals from the National Institute of General Medical Science (NIGMS) was purchased from the Coriell Cell Repositories . The populations were Caucasians . For absolute copy number determination of ABCC6, a TaqMan® Copy Number Assay targeting ABCC6 specifically in intron 11 was used . Two pyrosequencing assays were used to determine the relative copy number of ABCC6P1  versus ABCC6 (targeting intron 7 of both genes) and the relative copy number of ABCC6P2  versus ABCC6 and ABCC6P1 . The analyses were performed as described previously  was finally deduced from the TaqMan® Copy Number Assay and the two pyrosequencing assays. Calculation of the absolute copy numbers of ABCC6 pseudogenes was based on relative quantities (ratios) of the ABCC6 gene compared to the ABCC6 pseudogene(s). When one or more assay(s) deviated with respect to absolute copy numbers , the absolute copy numbers of the three genes were interpreted as the most reasonable.The CNV was analyzed in short specific regions involving exon 2, intron 7, and intron 11 of ABCC6 and ABCC6 pseudogenes, and deletions/insertions in other parts of the genes, or other chromosomal reorganization events, may not be identified.The limitation of this method is that CNV is analyzed in short specific regions of Ps < 0.05 were taken as statistical significance.The Fisher's two-tailed exact test was used for testing categorical variables between patients and controls. ABCC6 and ABCC6 pseudogenes were obtained from 207 of the 212 PXE individuals . Five patients were excluded from further analysis because of inconclusive copy number results. Both deletions and duplications were found for ABCC6, ABCC6P1, and ABCC6P2 in the PXE patients. The frequency of individuals with any CNV was higher than in a healthy population of Caucasian (controls)   , we identified a deletion of ABCC6 in 10/169 (∼6%) of the uncharacterized alleles (Table S1). For 10 of the 11 analyzed PXE patients with ABCC6 deletions, none or one mutant allele only had previously been identified. TableABCC6 and ABCC6P2 (#1) and three patients had deletions in ABCC6 only (#2). Five patients had smaller deletions (#3 and #4) in ABCC6 that were observed in either one or two of the analyzed regions (Exon 2 and/or intron 7)   in ABCC6 was accompanied by a duplication of ABCC6P1. This duplicated segment of ABCC6P1 may in fact have contributed to the ABCC6 deletion by gene conversion. Mendelian transmission of ABCC6 pseudogene CNV could be demonstrated for a few families. In one family (three siblings) with three copies of ABCC6P2, the PXE manifestation was present in individuals with and without this duplication, however, eye, skin, and gastrointestinal symptoms were more severe for the two individuals with three copies of ABCC6P2 (data not shown).In this study, we demonstrated that pyrosequencing is a fast and convenient method for the detection of CNV involving deletions of the entire or part of the ABCC6, mutational information was limited (Table S1). The functional consequence of having three copies of ABCC6 is unknown. Previously, by in vitro studies, we found that reduced mRNA expression of ABCC6P1 could influence the mRNA expression of ABCC6 , we observed a higher frequency of patients with gastrointestinal bleeding (G1 or G2 according to Phenodex™) in patients with more than two copies of ABCC6P2 (3/6) compared to patients with two or less copies of ABCC6P2 (11/178) . It would be interesting to investigate this association further as the pathophysiological cause of gastrointestinal bleeding in PXE is unknown. No significant correlation was observed for other clinical phenotypes, cholesterol diagnosis, and CNV of ABCC6 and/or ABCC6 pseudogenes.When correlating CNV of ABCC6 gene that may have caused the PXE phenotype. This method may be used in combination with quantitative PCR in PXE patients who have obtained incomplete genotype from conventional techniques. The frequency of ABCC6P2 pseudogene duplication was more common in PXE patients than healthy individuals and therefore may affect the PXE phenotype.In summary, by pyrosequencing, we were able to detect known and possibly new deletions in the


Partial or complete remission from type 2 diabetes was recently observed after bariatric surgeries. Limited data is available about the possibility of inducing diabetes remission through intensive weight reduction. We retrospectively evaluated diabetes remissions after one year of the Weight Achievement and Intensive Treatment (Why WAIT) program, a 12-week intensive program for diabetes weight management in real-world clinical practice. Among 120 obese patients with type 2 diabetes who completed the program, 88 patients returned for follow-up at one year. Nineteen patients (21.6%) had major improvement in their glycemic control, defined as achieving an A1C <6.5% after one year. Four patients (4.5%) achieved either partial or complete diabetes remission defined as A1C <6.5% and <5.7%, respectively, on no antihyperglycemic medications for one year; 2 achieved partial remission (2.3%) and 2 achieved complete remission (2.3%). At the time of intervention, patients who achieved diabetes remission had shorter diabetes duration (<5 years) and lower A1C (<8%) and were treated with fewer than 2 oral medications. They achieved a weight reduction of >7% after 12 weeks. These results indicate that a subset of obese patients with type 2 diabetes is appropriate for intensive lifestyle intervention with the aim of inducing diabetes remission. Type 2 diabetes has been considered as an incurable chronic disease. Traditionally, the focus of clinical management has been directed toward controlling hyperglycemia and its consequent complications . HoweverSince weight gain plays a vital role in the pathophysiology of type 2 diabetes, especially among genetically susceptible individuals , there iAnother meta-analysis of long-term effects of weight loss on diabetes prevention among obese people showed that intentional weight loss reduces risk of developing diabetes by 25%, while larger weight loss achieved through surgical interventions showed a dramatic risk reduction of about 63% .Bariatric surgery has been reported to improve type 2 diabetes and was shown to be superior to medical intervention .The aim of this study is to evaluate diabetes remission after one year of intensive lifestyle intervention in real-world clinical practice. It also aimed at characterizing those patients who are likely to achieve remission after weight loss.2) with type 2 diabetes enrolled in the Weight Achievement and Intensive Treatment (Why-WAIT) program between September 2005 and June 2008. Why WAIT program is a 12-week multidisciplinary intensive weight management program customized for obese patients with diabetes in real-world clinical practice at the Joslin Diabetes Center in Boston [We retrospectively evaluated obese patients  years at the start of the Why WAIT program. They were treated with a mean of 1.5 antihyperglycemic medications and they lost on average 26.4 lbs (±8.5) (11.1%) by the end of the program and maintained an average weight loss of 26.6 lbs (±20.7) (11.2%) at one year. The mean change in A1C, body weight, BMI, blood pressure, lipid profile, and renal function were shown in Participants who achieved major glycemic improvement at one year had a mean age of 53.3 (±8.5) years, a mean body weight of 237.7 (±29.5) pounds, a BMI of 38.2 (±4) kg/m2, and a mean duration of diabetes of 1.5 (±0.9) years at the start of the program. They were treated with a mean of 1.25 antihyperglycemic medications and lost on average 25.2 lbs (±7.3) (10.9%) by the end of the program and maintained a mean weight loss of 15.9 lbs (±10.7) (6.9%) at one year.Those who achieved partial or complete remission had a mean baseline body weight of 230.9 (±23.2) pounds, a BMI of 35.6 (±1.7) kg/mIn this study we demonstrated that major improvement in glycemic control can be achieved and maintained for one year after nonsurgical intensive lifestyle intervention in real-world clinical practice. Partial or complete diabetes remission at one year was achieved in small percentage of patients. Remission was more likely to occur in patients with shorter duration of diabetes, on few oral antihyperglycemic medications and with A1C < 8% at start of intervention. These results may indicate that a subset of obese patients with type 2 diabetes on antihyperglycemic medications is appropriate for intensive lifestyle intervention that aims at inducing diabetes remission and stopping the use of antihyperglycemic medications.Look AHEAD  study also showed that complete remission of type 2 diabetes after intensive lifestyle intervention is possible but rare , 14. ForThe concept of diabetes remission in established patients who are already treated with antihyperglycemic medications is intriguing as it contradicts the traditional concept that type 2 diabetes is a chronic progressive disease characterized by significant loss of beta-cell function (up to 80%) at the time of diagnosis , 17. BerThe other explanation for our findings is that patients with short diabetes duration may have significant beta-cell reserve more than what was originally thought. It is unlikely that diabetes remission occurs if 50% or more of beta-cell function is lost at the time of diagnosis. It is worth mentioning that the United Kingdom Prospective Diabetes Study (UKPDS), which created that concept, was not originally designed to evaluate beta-cell function . The stuThis study also demonstrated several indicators of possible diabetes remission in response to intensive lifestyle intervention. They include short duration of diabetes of <5 years, A1C < 8% at the time of intervention, and treatment with fewer than 2 oral medications with higher weight loss of >7% as predictor of improvement. Surgical weight loss studies also showed that patients with short diabetes duration are more likely to achieve diabetes remission after surgery [The current widely accepted concept is that oral antihyperglycemic medications are needed after failure of lifestyle intervention in controlling hyperglycemia . HoweverThis study has several limitations. It is a retrospective observational study. The study also included heterogeneous group of patients with variable diabetes duration and on variable number of antihyperglycemic medications including insulin. These limitations impacted significantly the rate of remission since it is unlikely that patients with long diabetes duration achieve diabetes remission after significant weight reduction. The Look AHEAD study, despite being a randomized controlled study, was not designed or powered to evaluate diabetes remission . However