Phytochromes (Phys) encompass a diverse collection of bilin-containing photoreceptors that help plants and microorganisms perceive light through photointerconversion between red light (Pr) and far-red light (Pfr)-absorbing states. In addition, Pfr reverts thermally back to Pr via a highly enthalpic process that enables temperature sensation in plants and possibly other organisms. Through domain analysis of the Arabidopsis PhyB isoform assembled recombinantly, coupled with measurements of solution size, photoconversion, and thermal reversion, we identified both proximal and distal features that influence all three metrics. Included are the downstream C-terminal histidine kinase-related domain known to promote dimerization and a conserved patch just upstream of an N-terminal Period/Arnt/Sim (PAS) domain, which upon removal dramatically accelerates thermal reversion. We also discovered that the nature of the bilin strongly influences Pfr stability. Whereas incorporation of the native bilin phytochromobilin into PhyB confers robust Pfr → Pr thermal reversion, that assembled with the cyanobacterial version phycocyanobilin, often used for optogenetics, has a dramatically stabilized Pfr state. Taken together, we conclude that Pfr acquisition and stability are impacted by a collection of opposing allosteric features that inhibit or promote photoconversion and reversion of Pfr back to Pr, thus allowing Phys to dynamically measure light, temperature, and possibly time. Phys typically exist in two conformers, a dark-adapted, red light-absorbing Pr state that is biologically inactive, and a metastable, far-red light-absorbing Pfr state that is biologically active. Through the reversible photointerconversion of Pr and Pfr and thermal reversion of Pfr back to Pr, Phys act as photoswitches in various signaling pathways that detect light fluence rate, duration, and spectral quality. Moreover, at least some plant Phys also perceive temperature through enthalpic effects on the rate of thermal reversion4, and can possibly measure photoperiod via the nighttime depletion of Pfr5. The cumulative effects of Pr/Pfr interconversion impact a number of physiological processes, including photoprotection, redox sensing, phototaxis, sporulation, pathogen virulence and chromatic adaptation in microorganisms, and seed germination, chloroplast development, shade avoidance, circadian entrainment, flowering time and senescence in plants8.The biological perception of light is mediated by a collection of photoreceptors that connect light absorption to various signal transduction cascades crucial to organismal development and survival. Influential photoreceptors among plants, bacteria, and fungi are the phytochromes (Phys), a superfamily of chromoproteins that enables light detection through a covalently-linked bilin (or linear tetrapyrrole) prosthetic group8. The canonical PSM sequentially includes a variable N-terminal extension (NTE), Per/Arnt/Sim (PAS) and cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domains that form a tight complex through a unique figure-of-eight knot, and a Phy-specific (PHY) domain that is integral to Pfr acquisition and transmission of the light signal to the OPM. Whereas proteobacterial and fungal Phys link the bilin biliverdin IXα (BV) via the C32 position of the A pyrrole ring vinyl sidechain and a conserved cysteine in the NTE9, cyanobacterial and plant Phys link the bilins phycocyanobilin (PCB) and phytochromobilin (PΦB), respectively, via C31 of the A pyrrole ring ethylidine side chain and a conserved cysteine in the GAF domain11. Regardless of the linkage site, the incorporated chromophores assume near equivalent positions within the GAF domain cleft, where an intricate network of non-covalent contacts  enables the unique reversible absorption properties of Phys1. The OPM varies within the superfamily; it is often a histidine kinase domain in microbial Phys, and a histidine kinase-related domain (HKRD) with possibly other activities in the plant versions12.Phys are head-to-head homodimers with each polypeptide harboring an N-terminal photosensory module (PSM) that binds the bilin followed by a C-terminal output module (OPM) that supports dimerization and often promotes signal transmission to downstream effectors16. Following photoexcitation of Pr, the bilin reversibly switches from a 15Z to 15E configuration. The net effect is a ~180° rotation of the D pyrrole ring, followed by sliding the bilin within the GAF domain pocket and reorientation of adjacent amino acids18. Subsequently, a structurally unique hairpin loop , extending from the PHY domain to contact the GAF domain surface near the bilin, transitions from a two-stranded, antiparallel β-sheet as Pr to a helical conformation as Pfr. Accumulating evidence posits that this reconfiguration rearranges the hairpin/GAF domain contact, which then tugs on the PHY domain. The end result is to effectively toggle the position/activity of the sister OPMs rigidly tethered to the PSM through a long helical spine20, but the end conformation remains unclear at present.Through a variety of structural and biophysical studies, a model for Phy photoconversion is emerging4. Within the GAF domain pocket, a number of amino acids are essential for acquisition of Pfr, with substitutions of key bilin/protein contacts either blocking the Pr → Pfr transition or stabilizing/destabilizing Pfr once formed24. In fact, million-fold differences in the rate of thermal reversion are possible by substituting individual residues in this pocket, without significant impact on absorption or photoconversion21.Given how sensitive Phy signaling is to Pr/Pfr interconversion, it is not surprising that multiple features both proximal and distal to the bilin are crucial to facilitate or modify this exchange. This is especially evident for plant Phys, some of which must be delicately balanced to perceive both light and temperature25. For example, manipulating the contact points between the hairpin and GAF domain either substantially promotes or dampens Pfr → Pr thermal reversion27, whereas the NTE in plant Phys is important for stabilizing the Pfr state and subsequent signaling once formed30. Interestingly, the effects of the NTE on thermal reversion can also be reversibly adjusted by phosphorylation in planta, thus suggesting that controls on Pfr stability by this region are physiologically relevant32. Even the OPM impacts Pfr stability. For example, removal of the OPM histidine kinase domain in the Deinococcus radiodurans bacteriophytochrome (BphP) greatly dampens thermal reversion despite being largely separated from the GAF domain in the 3-D dimeric structure of the photoreceptor33.Outside of the GAF domain, additional features also strongly influence thermal reversion, suggesting that the overall conformation of the Phy dimer allosterically impacts Pfr stability even at considerable distances away from the bilinArabidopsis thaliana PhyB, a prominent isoform crucial for both light and thermal perception in plants26, and now widely used for the optogenetic control of cellular events . PhyB-type isoforms harbor the canonical PAS-GAF-PHY modular architecture of the PSM but are uniquely preceded by a long glycine/serine-rich NTE36. Like other plant Phys, the PSM of Arabidopsis PhyB is followed by a PAS-PAS and HKRD region that helps promote dimerization, nuclear import, and possibly signaling through interaction with a family of PHYTOCHROME-INTERACTING FACTOR (PIF) transcriptional repressors37.To further define how various features within the dimeric Phy molecule impact Pr/Pfr interconversion, we analyzed the quaternary and spectroscopic properties of a collection of mutations within Remarkably, we found that several features, both proximal to the bilin and anticipated to be substantially distant within the 3-D structure, have strong effects on Pfr acquisition and stability, and thus critical to proper light and thermal perception. Even subtle substitution of the native chromophore PΦB for PCB strongly dampens thermal reversion. Defining how these modular features evolved to modify photo- and thermosensing of the various Phy isotypes should provide a framework to reengineer these photoreceptors for agronomic benefit and as improved optogenetic reagents.38. Analysis of the GAF domain by itself was not attempted given that its intimate connection to the N-terminal PAS domain through the figure-of-eight knot appears to be required for proper folding21. The truncations were expressed and assembled with PΦB in Escherichia coli, purified via appended tags bearing a hexahistidine (6His) moiety followed by a tobacco etched virus (TEV) protease cleavage site, and separated from the tag by TEV protease digestion. As shown in Figs To provide further insights into how various modular features within Phys influence their dimeric structure and Pr/Pfr interconversion, we first studied a series of C-terminal truncation mutants  of Arabidopsis PhyB that sequentially eliminated the HKRD, the pair of internal PAS domains, and the PHY domain, respectively, from the full-length 1172-residue bili-protein Fig. . The 1–921, while the full-length chromoprotein should be dimeric based on prior analyses of the PhyA isoform40 and PhyB assembled with PCB in yeast41. Here, we confirmed by SEC that both the 1–450 and 1–624 fragments are monomers with observed masses of 73.6 and 97.3 kDa, respectively, which are consistent with the behavior of a non-spherical monomer, and that the full-length polypeptide is indeed a homodimer with an observed mass of 320 kDa  indicated that the 1–624 fragment of PhyB is monomeric in solutionkDa Fig. . Similaries Fig. .D. radiodurans BphP33, full-length Arabidopsis PhyB appears to assemble as a head-to-head dimer in solution with an obvious cavity separating the sister PSMs, which was seen as a quadrate constellation of densities form by the separate PAS/GAF and PHY domains , which resulted in 50% and 90% transformation of Pr to Pfr for the 1–908 and 1–624 fragments, respectively, but only trace conversion to Pfr for full-length PhyB as judged by appearance of the Q-band peak at 730 nm  could we obtain near saturating Pfr occupancy for the full-length chromoprotein , which was not detected by a comparable depletion of the Pfr absorption band , suggesting that NTE features emerge after this dividing line that are important to all Phy lineages. Taken together, it appears that the NTE in PhyB is positioned to stabilize Pfr, and thus might have evolved in plants to counteract the destabilizing influence of the PAS-PAS and HKRD regions, or vice versa.Interestingly, when we generated an alignment of Phy sequences from a variety of PhyB isoforms from seed and seedless plants along with the other four Phy isoforms from Arabidopsis, we discovered that the critical residues are within a patch best conserved within the PhyB subfamily, which also includes its hyD Fig. 36. Notab49,chemical complementation of PΦB synthesis mutants , and the expanding collection of optogenetic strategies based on PhyB38. However, comparison of our data generated using PΦB relative to previous studies using PCB showed non-trivial photochemical differences in the behavior of PhyB. As judged by zinc-induced fluorescence of the bound bilins following SDS-PAGE, recombinant PhyB assembled with PCB with efficiencies equal to that seen for PΦB  was hypsochromically shifted ~14 nm (693 nm) compared to the PΦB- containing form. Despite these shifts, the Pr → Pfr photoconversion rates for the PCB-containing preparations in red light were similar to those containing PΦB, including the substantially slower rate for the full-length chromoprotein Fig. .More striking were the substantial changes in Pfr → Pr thermal reversion for PhyB assembled with PCB. For the full-length chromoproteins, the Pfr reversion rate was substantially slower for PCB- versus PΦB-containing preparations, where the slow rate constant was impacted to a greater extent than the faster rate constant Fig. ; Table 16. How Phys respond to all these features is not completely understood. Certainly, one strategy by plants  is to link individual Phys with specific sensory transduction chains to engender distinct signaling outcomes. Another would be to adjust the Pr/Pfr transitions of Phys mechanically to imbue unique signaling potentials. For the latter strategy, Phy interconversion could be viewed as a delicate balance among several features both proximal and substantially distal to the chromophore that modify exchange between the two endstates. By adjusting these features even for Phy isoforms within the same organism, a rich panoply of signaling potentials could be possible, thus enabling broad environmental perception, including measurement of light, temperature, and possibly time.Phys are unique among photoreceptors by their ability to assess numerous environmental parameters to generate a coherent response based on the amount of Pfr generated, rates of Pr/Pfr interconversion, and the final acquired ratio of the two states. These factors are interconnected through the relative magnitudes of the rate constants for photoconversion and thermal reversion, the temperature dependence of each , and the intensity and color of the impinging light. Through these parameters, Phys can measure: (i) fluence rate (intensity), (ii) fluence (photon counting), (iii) fluence duration (time), (iv) shading by detecting alterations in the Pr/Pfr ratio caused by changes in the ratio of impinging red and far-red light, (v) temperature through enthalpic effects of Pfr → Pr reversion, and possibly (vi) the annual changes in photoperiod by diurnally quantifying the nighttime loss of Pfr (hourglass)24. Here, we extend this influence to the NTE, which had been previously documented to accelerate thermal reversion when removed29. Fine mapping of the NTE pin-pointed the causative motif to a region centered on Gln59-Gln60. This motif not only strongly impacts thermal reversion but also bathochromically shifts the absorption of Pfr, implying an influence on the bilin/protein interface.Numerous studies have revealed a critical role for the bilin/protein contacts within the chromophore-binding pocket of the GAF domain in generating the unusual absorption properties of Phys as compared to those of free bilins, and for impacting photoconversion and thermal reversion21, which might allow residues 50–60 to interact with amino acids in intimate contact with the bilin or with the PHY-domain hairpin in close proximity. In support, recent hydrogen/deuterium exchange mass spectrometry revealed that the conformation of the NTE and/or its association with the GAF domain are altered upon photoconversion46. Notably, Pfr-enhanced deuterium uptake was seen for residues 55–58 and 62–87, which is indicative of increased solvent accessibility. One likely scenario is that Pfr formation releases these NTE residues from a buried state to form new more solvent-exposed contacts that enhance Pfr stability. As implied by our truncation studies, absence of these contacts then destabilizes Pfr. Intriguingly, the NTE of PhyB (and possibly other plant Phys) is modified by phosphorylation. In particular, modifications at Ser86 and Tyr104 not only impacts signaling by the photoreceptor but also substantially accelerates thermal reversion32, thus implicating this NTE/GAF domain contact as an important control point for regulating Pfr signal strength.How the NTE induces these effects on a chromophore almost completely enveloped within the GAF domain remains unclear in the absence of a 3D structure. Structural details for the NTE immediately upstream of the PAS domain (residues 90–100) suggest that it folds back on the PAS/GAF domains33. Whereas removal of the HKRD dramatically accelerates photoconversion, subsequent deletions of the PAS-PAS region and HKRD regions combine to dramatically dampen thermal reversion, implying that the OPM constrains photoconversion to Pfr and then destabilizes Pfr once formed. Presumably, the sum total of these changes would substantially desensitize PhyB to light. The influences of the OPM on these parameters could reflect structural features within these domains that directly impinge on the PSM. Possibilities include altering the helical spine that transverses nearly the entire length of the polypeptide and/or adjusting the splay/twist reorientation of the OPM relative to the PSM as the paired PHY domains attempt to separate due to the photoconversion-induced hairpin contraction1. Alternatively, the effects could reflect the importance of dimerization to Pfr stability, as has been implicated from similar truncation studies of D. radiodurans BphP with its canonical HK OPM19. In support of the latter, Klose et al.44 recently provided evidence that dimerization through the HKRD leads to cooperativity in thermal reversion; i.e., when one subunit as Pfr in the Phy dimer reverts to Pr, the reversion rate of the second becomes faster.Even more dramatic are the allosteric effects of the C-terminal domains on PhyB photochemistry and thermal reversion despite their substantial distance away from the bilin-binding pocket Fig. 15,33. Whin vitro studies detected a much more rapid rate of thermal reversion for full-length PhyB than that measured in planta44, suggesting that the cellular environment also impacts the process. Included are the aforementioned modification of the NTE by phosphorylation, and/or the interaction of PhyB with other signaling components, including PIFs that were recently shown to strongly influence thermal reversion in vivo53. Conceivably, other modifications and binding partners could alter photoconversion and/or thermal reversion rates leading to a highly regulated light-feedback system that can dampen or enhance the photosensitivity of PhyB in response to external conditions.Our et al.54 studying the PSM of potato PhyB expressed recombinantly in yeast, implying that the effects could be universal among plant Phys. Modeling PCB into the 3D structure of the PhyB PSM21 suggests that one consequence is the ethyl group having more conformational freedom to interact with neighboring amino acids. As Pr, the C18 vinyl group of PΦB is tightly flanked by Met274, Tyr276, Val286, Tyr303, and Met365. Fitting PCB into this position predicted that its ethyl group unlikely perturbs this solvent-excluded hydrophobic interaction  as compared to that assembled with PCB used by cyanobacterial Phys . Notabl3.It is commonly appreciated that plant Phys evolved from using PCB, as in cyanobacterial Phys, to PΦB to better align their absorption with those of chlorophylls and thus enhance detection of shade. A second possibility emerging from our data is that the switch to PΦB also enhanced the use of Pfr → Pr thermal reversion as an environmental signal. Whereas the reversion rate of Arabidopsis PhyB assembled with PΦB appears sufficiently fast relative to the Pr → Pfr photoconversion rate to enable thermoperception and the diurnal measurement of darkness, the reversion rate for PhyB assembled with PCB might be too slow to be physiologically relevant. For example, point mutants of Arabidopsis PhyB that significantly dampen thermal reversion lack temperature-dependent responses at physiological temperatures57. While this choice has benefited strategies where a stable Pfr species is needed, the alternative use of PΦB might avail new applications where a decelerating Pfr signal would help. Our finding that the 1–908 truncation of PhyB has an innate tendency to form dimers should also be considered when using this truncation in optogenetic applications that require a monomeric photoreceptor. Thus, the expression of the 1–908 fragment might need to be controlled to avoid dimerization-induced artifacts.Coincidently, PCB is commonly used as the chromophore for optogenetic applications involving PhyB58. Clearly, further spectroscopic characterizations of Phys in the contexts of their photo- and thermosensing and downstream signaling should reveal this evolutionary interplay, which in turn can be exploited to engineer Phys with novel environmental signaling potentials.The accumulating structural data for Phys indicate that they are highly modular dimeric receptors with numerous features allosterically contributing, either positively or negatively, to their signaling potential even at a distance. Presumably by adjusting the balance between these features, Phys could evolve novel isoforms capable of detecting specific environmental signals. For plant Phys, one can imagine that thermal reversion was selectively accelerated in the PhyB isoform to effectively compete with the rates of photoconversion, thus enabling better measurement of photoperiod and temperature, and signaling in the light-rich environments of mature plants, while PhyA could have dampened thermal reversion to better detect the low light levels seen by germinating seedlings. One can even speculate that adjusting the interconversion balance between Pr and Pfr led to the extreme properties found for bacterial bathy-Phys that generate a dark-adapted Pfr state without photoexcitationA. thaliana PhyB gene designed for recombinant expression was appended to codons for an N-terminal 6His tag followed by a TEV protease cleavage site (MGSSHHHHHHSSENLYFQG), and inserted into the modified pBAD Myc-His plasmid (Invitrogen)21. Truncations were generated by the PIPES59 or Gibson assembly methods60, whereas the site-directed mutants were generated by Quickchange mutagenesis (Stratagene) in combination with appropriate oligonucleotide primers. The 6His-TEV-PhyB apoproteins were co-expressed in E. coli BL21(DE3) cells with the pL-PΦB or pL-PCB plasmids harboring genes encoding the Synechocystis PCC6803 heme oxygenase (Ho1), and either the Arabidopsis HY2 or Synechocystis PCC6803 PcyA synthases that direct production of PΦB or PCB, respectively61. Cells were first grown at 37 °C to 1 OD600nm in Terrific Broth containing 1 mM MgSO4 and 250 μM 5-aminolevulinic acid, and incubated for an additional hour at 16 °C. Isopropyl β-D-1-thiogalactopyranoside was then added to 1 mM to induce the expression of the PΦB or PCB biosynthetic genes, which was followed one hour later by addition of 0.2% L-arabinose to induce expression the PhyB polypeptide21. Cultures were grown overnight at 16 °C, harvested by centrifugation, and immediately frozen at liquid nitrogen temperatures. Unless otherwise noted, all culture growths, purification of PhyB as Pr, and subsequent manipulations of samples were performed at 0–4 °C in darkness or under dim green safelight conditions.An intron-less g, and followed by filtration through a G-25 Sephadex column equilbrated in LB. The filtrate was loaded onto a Ni2+-nitrilotriacetic acid (Ni-NTA) column (GE) pre-equilibrated in LB containing 30 mM imidazole, washed with the same buffer, and the bound proteins were eluted with LB containing 300 mM imidazole. Fractions containing PhyB were exchanged by ultrafiltration (Amicon Ultra-15 filters) into anion exchange buffer , and 10 mM 2-mercaptoethanol) also containing 20 mM NaCl, and subjected to anion exchange chromatography using a HiTrap Q Sepharose column (GE) in combination with a 20–500 mM linear gradient of NaCl in AEB. PhyB-containing fractions were pooled, exchanged into LB, and subjected to a second round of Ni-NTA chromatography using a 30–140 mM linear gradient of imidazole in LB.PhyB-containing cell pellets were thawed and disrupted by sonication in a fourfold excess of lysis buffer , 500 mM NaCl, 1 mM 2-mercaptoethanol, 0.05% Tween 20, 1 mM phenylmethanesulfonyl fluoride, one tablet per liter Roche or Pierce complete EDTA-free protease inhibitor) also containing 30 mM imidazole. Cell lysates were clarified by centrifugation at 35,000 x9.Purified PhyB fractions as Pr were incubated for three hours at 4 °C with ~1 mg of 6His-TEV protease. Subtractive nickel affinity chromatography was conducted by exchanging samples back into LB containing 30 mM imidazole and passing them through a Ni-NTA column equilibrated in LB to retain the 6His-TEV protease. PhyB samples were concentrated and flash frozen to liquid nitrogen temperatures as 30 µL drops. Prior to analysis, thawed samples were passed through a Superdex 200 column to provide exchange into the appropriate buffers and to remove contaminants and PhyB breakdown products. Bilin occupancy was assessed by zinc-induced-fluorescence of the chromoproteins following SDS-PAGE62. Glycerol was added to the TEV protease preparations (~1 mg/ml) at a 50% (w/v) concentration, and stored at −20 °C.TEV protease bearing a 6His tag was expressed in BL21(DE3) cells, and enriched via Ni-NTA chromatography as above, exchanged into cation exchange buffer (CEB: 0.3 mM tris(2-carboxyethyl)phosphine), and 10 mM Tris-HCl (pH 7.5)), and subjected to HiTrap SP Sepharose (GE) chromatography (GE) using a 0–600 mM linear gradient of NaCl in CEBSEC (0.3 ml/min flow rate) of full-length PhyB and various truncations was conducted at 10 °C using an ÄKTA FPLC  equipped with the Superdex 200 HR 10/30 column (GE) equilibrated in 150 mM KCl, 50 mM HEPES-NaOH , and 0.3 mM tris(2-carboxyethyl)phosphine. Prior to loading, samples (100 µL) were fully converted to Pr using far-red (730 nm) light. Apparent molecular mass based on elution volume was calculated using Unicorn software  in combination with size standards (BioRad).33. Micrographs were recorded in a low dose mode (15 electrons/Å2) at a 50,000x magnification using a Gatan  UltraScan 4000 CCD camera (4096 × 4096 pixels), which corresponded to 2.12-Å/pixel sampling at the specimen level. Particle selection and image processing were conducted using the EMAN and EMAN2 software packages as described previously15.2D class averages of PhyB as Pr were generated by negative staining electron microscopy. Full-length PhyB or the 1–908 truncation were fixed onto carbon films, stained with phosphotungstic acid, and imaged by a JEOL JEM–2010F transmission electron microscope  operating at 200 kV as described previously4EDTA, 10 mM 2-mercaptoethanol, and 150 mM KCl. Absorption spectra as Pr or following various fluences of red light (630 nm) were collected at 25 °C with a Cary 60 spectrophotometer (Agilent). Unless specified, Pfr spectra were recorded after steady state had been achieved using continuous 630 nm irradiations. Pr → Pfr and Pfr → Pr photoconversions were driven with LED lights having peak emissions of 660 nm or 740 nm, respectively, using a LiCor LI-185B photometer to determine fluence rate. For all photoconversion reactions the sample concentration was adjusted so the maximal absorption of the Q band was 0.4 as Pr. To ensure consistency between measurements the same cuvette was used for all experiments and was loaded with the same volume of sample. Pfr → Pr thermal reversion was measured at 25 °C in the dark. Time-resolved spectra were collected from 630–850 nm in 10-nm intervals, and the scan rate was set at 24,000 nm/min to minimize the red- or far-red light exposure. Examples of the raw kinetic traces can be found in Supplementary Fig. i = ΔAbsi⋅exp(-k⋅t) + Abs(0)i, where i represents the ith wavelength, Abs is the absorption, ΔAbs is the reaction amplitude, k is the rate constant, t is time, and Abs(0) is the absorption at time zero. If two exponentials were required to explain the results, the data were fit to a simple sum of two exponentials using the equation described above.For all spectroscopic experiments, the PhyB samples were exchanged into 50 mM HEPES-NaOH (pH 7.9), 1 mM Na63 and displayed with BoxShade (http://www.ch.embnet.org/software/BOX_form.html). The extreme N-terminal sequences, which are poorly conserved, were manually compressed to ease viewing.Amino-acid-sequence alignment of representative members of the plant Phy family was generated using the Clustal Omega serverSupplementary Information


Kidney transplantation is the optimal treatment for end-stage renal disease in children. However, long-term graft survival has not significantly improved among pediatric patients. To investigate the determinants of long-term graft survival among Iranian pediatric recipients of kidney transplantation.  In a single-center cohort study, we studied 314 pediatric kidney transplantations performed from 1989 to 2013 at Dr. Shariati Hospital, Tehran, Iran. Different variables were collected for each patient and graft survival rates were calculated. After a mean±SD follow-up period of 15.8±4.0 years, the mean±SD graft survival rate was 14.5±0.5 years; the 1-, 5-, 10-, and 20-year mean graft survival rates were 90%, 81%, 62%, and 62%, respectively. The corresponding patient survival rates were 100%, 99.4%, 97.8%, and 96.5%, respectively. Pre-emptive transplantation (p=0.006), and living graft donation (p=0.002) led to higher graft survival, while acute rejection (p=0.002), and primary disease of primary hyperoxaluria (p=0.001) led to lower graft survival. Chronic rejection was the most frequent cause of graft loss. Short-term graft survival still outpaces the long-term outcome. Modifying the mentioned determinants, with more intense immunosuppression for greater prevention of acute and chronic rejection, and increased rate of pre-emptive transplantation and living donor transplantation, long-term graft survival may significantly improve in future. End-stage renal disease (ESRD) is an overwhelming illness especially in children. A recent study estimates the incidence of chronic kidney diseases (CKD) at 16.8 per million children population; the kidney transplantation rate in Iran is 7.2 per million children population .Recent advances in pre- and post-transplantation management, immunosuppressive medications, surgical techniques, and donor selection have contributed to improved patient and graft survival among pediatric kidney recipients -8. NonetThis was a 25-year retrospective cohort study on pediatric patients who underwent kidney transplantation at Dr. Shariati Hospital, Tehran, Iran, between 1989 and 2013. All patients who aged 18 years or younger at the time of transplantation (n=297) were included in the study. A total of 17 patients underwent retransplantation that resulted in 314 pediatric kidney transplantation during the study period. All the graft nephrectomies were performed using the classical open extraperitoneal technique. This study was approved by the Medical Ethics Committee of Tehran University of Medical Sciences.For each patient, data including age; sex; cause of ESRD; history of dialysis before transplantation; donor’s age, sex, and type; immunosuppressive regimen; presence of acute rejection and malignancy; cause of graft loss and mortality; and serum creatinine levels were obtained and analyzed to find independent factors of graft survival. Regarding the age, recipients and donors were categorized into age groups for easier analysis. In case of encountering a missing variable for a patient, the patient was excluded for that variable and the analysis was performed using other patients’ data.The immunosuppressive regimen consisted of a triple combination of corticosteroid plus two other drugs based on the availability and applicability at the time of transplantation. Regarding the induction treatment, those patients who underwent transplantation after 1999 received thymoglobulin; others did not receive such treatment due to unavailability of the drug before 1999.Acute rejection episode was defined as an abrupt increase in baseline serum creatinine level, and was biopsy-proven thereafter. The provided treatment for acute rejection consisted of 3-day high dose corticosteroids pulse therapy. In case of corticosteroid-resistance, thymoglobulin was administered. Graft loss was defined as absence of graft function due to patient death, graft injury using the RIFLE criteria, or the need to chronic dialysis and/or retransplantation. Therefore, graft survival was considered the period between the time of kidney transplantation and either graft loss, last date of follow-up with a functioning graft, or patient death. Meanwhile, patient survival was defined as the period between the time of kidney transplantation and either death, or last date of follow-up.Statistical AnalysisStudent’s t test, Mann-Whitney U test, χ2 test, one-way ANOVA, and Kruskal-Wallis one-way analysis of variance were used when applicable. Cox proportional hazard model was used to investigate the associations between patient characteristics and the graft outcome. A p value <0.05 was considered statistically significant.IBM SPSS Statistics for Windows  was used for data analysis. Results are presented as mean±SD, median, and range for continuous variables, and as numbers and percentages for categorical variables. To estimate the graft and patient survival rates, Kaplan-Meier survival analysis was used and comparison of graft survival rates was made using the log-rank test. Comparing different variables, independent-samples A total of 1904 kidney transplantations was performed at Dr. Shariati Hospital, Tehran, Iran, during 1989–2013. Of these, 314 (16.5%) transplantations were done on pediatric recipients. The mean±SD follow-up period was 15.9±4.0 (range: 0.5–20) years. The mean±SD graft survival was 14.5±0.5  years. The 1-, 3-, 5-, 10-, and 20-year graft survival rates (SEM) were 90% (2%), 82% (2%), 81% (2%), 62% (4%), and 62% (4%), respectively . AlthougThe mean±SD graft survival was significantly higher (p=0.006) among those who underwent pre-emptive transplantation  than those who underwent hemodialysis  or peritoneal dialysis  before the transplantation . MoreoveThe graft survival was higher among recipients of living donor grafts, and notably, as shown in Considering the immunosuppressive regimen, the recipients who received the combination of steroid plus cyclosporine plus azathioprine had a significantly (p=0.001) worse graft survival.The causes of ESRD among the recipients were glomerular diseases (33.5%), congenital anomalies of the kidney and urinary tract (36%), tubulointerstitial diseases (12.5%), and unknown (18%). When looking at these causes, recipients with primary hyperoxaluria (PH) had the worst (p=0.001) and those with neurogenic bladder had the best graft survival rates (p=0.002). Other causes did not make significant differences regarding the graft survival. Seventeen patients underwent retransplantation. Surprisingly, these patients had a 10-year graft survival of 100% that was significantly (p=0.033) higher than others.A total of 28.6% of the recipients experienced at least one episode of acute rejection and had a significant (p=0.002) lower mean±SD graft survival  than those without any acute rejection episodes . However, the mean±SD graft survival was lower among those with more than one acute rejection episodes  compared to recipients with one episode . The difference was not significant (p=0.28), however. Graft loss occurred following 27.1% of transplantations; the most common cause was chronic rejection that occurred in 37 (44%) recipients. A total of 13 (15%) patients experienced disease recurrence, mostly those with the primary diseases of focal segmental glomerulosclerosis (FSGS) and PH. The other causes of graft loss were acute rejection in 12.9%, discontinued immunosuppression due to noncompliance in 11.8%, renal vein thrombosis in 3.5%, and deaths with functioning graft in 12.9%.The frequency of post-transplantation malignancy among our patients was 2.9%, including six recipients with lymphoproliferative diseases and three with other malignancies. These malignancies occurred after a median period of 4.5 years post-transplantation. The patient survival rate among these recipients was 100%. The 1-, 3-, 5-, 10-, and 20-year patient survival rates (SEM) among our recipients were 100% (0%), 100% (0%), 99.4% (1%), 97.8% (1%), and 96.5% (2%), respectively. Totally, 11 patients died during the follow-up period. The cause of death for all these patients was systemic infection that caused the death after a median period of 11.5 years post-transplantation. The mean±SD serum creatinine level of patients after 1, 3, 5, 10, and 20 years of transplantation was 1.04±0.20, 1.08±0.18, 1.14±0.30, 1.20±0.23, and 1.24±0.17 mg/dL, respectively.Cox proportional hazard analysis revealed that dialysis history , acute rejection , and donor type  were significant factors to predict the graft outcome.Kidney transplantation is the treatment of choice for pediatric patients with ESRD. However, a recent study in Iran revealed that only less than half of these children have the chance to undergo kidney transplantation . MoreoveHerein, we presented our experience in pediatric kidney transplantation over 25 years. Although a few studies have previously investigated the outcome of kidney transplantation among children in Iran , 13, 14,More than half of our recipients were boys, compatible with previous studies in Iran and other countries , 10-16. About two-thirds of our transplant patients were 7–14 years old. We did not have the experience of kidney transplantation in infants and very young children. It was previously indicated that pediatric kidney transplantation in developing countries is usually limited to older children . HoweverIt is generally accepted that pre-emptive transplantation should be considered in children when possible, and that the major criterion is the availability of a suitable donor , 19. MeaIn different countries, the donor sources are different based on the religious, cultural, and ethnical variations . Some coAll of our patients received steroid as part of their immunosuppressive therapy. The most frequent combination immunosuppressive regimen among our patients was steroid plus cyclosporine plus mycophenolate mofetil. Our patients received the drug combinations based on the availability and applicability at the time of transplantation. Different studies have reported the use of different combinations of immunosuppressive agents , 32. It It is reported previously that the most common causes of ESRD among children are congenital or inherited anomalies, and glomerulonephritis , 8. A reDifferent studies have reported various rates of retransplantation based on their study design and transplantation outcome , 15, 16.Previous studies have demonstrated that episodes of acute rejection are associated with poor long-term survival rates , 14, 36.Similar to previous reports, chronic rejection was the most common cause of graft loss among our patients , 13, 15.Children are at increased risk for developing malignancies following kidney transplantation compared with adults and the general population , 38. A pMany studies report various predicting factors of graft survival including recipient age, sex, and race, donor age and type, dialysis history before the transplantation, previous transplant history, transplant year, acute rejection, delayed graft function, and GFR , 20, 23.It is stated before that kidney transplantation allows more than 90% of pediatric patients to survive for at least 20 years . The patOur study was limited in some aspects. As this was a retrospective study over 25 years, we could not have complete data about some variables such as growth, HLA mismatch, panel reactive antibody test results prior to transplantation, detailed types of graft rejections, and delayed graft function status, so that we had to eliminate these variables in our analysis. Furthermore, although we provided overall 20-year graft and patient survival rates, we could not investigate the associations of different variables with graft survival at 20 years of transplantation, because many of the studied patients have not yet reached the 20-year milestone. We did also not investigate the graft outcome in different eras, as it was frequently performed before , 15, 16.In conclusion, the main findings of the current study were as follows: (1) the 20-year graft and patient survival rates were 62% and 96.5%, respectively; (2) pre-emptive transplantation and living kidney donation were positive factors in graft survival; (3) acute rejection and immunosuppressive regimen of steroid plus cyclosporine plus azathioprine were associated with lower graft survival; (4) acute rejection, dialysis history before the transplantation, and donor type were the significant predictors of long-term graft outcome among children; (5) chronic rejection was the most common cause of graft loss; (6) patient survival yet surpasses the graft survival in pediatric kidney transplantation; (7) short-term graft survival still outpaces the long-term outcome in the pediatric kidney transplantation; (8) still greater improvements in graft survival, particularly long-term, are needed; (9) as the effective factors on graft survival are modifiable, with more intense immunosuppression for greater prevention of acute and chronic rejection, and increased rate of pre-emptive and living donor transplantation, long-term graft survival may significantly improve among children in the near future.


To report a surgical method for treating corneal oedema in a case of late-onset Descemet membrane detachment after penetrating keratoplasty.A 55-year old patient presented with sudden visual loss in his left eye 28 years after penetrating keratoplasty for keratoconus. Slit-lamp biomicroscopy revealed a distortion of the corneal graft anatomy with protrusion of the graft and peripheral thinning and steepening in the residual host tissue, accompanied by corneal graft oedema. Anterior segment optical coherence tomography revealed detachment of Descemet membrane localized to the area of the graft oedema. We proceeded with a full-thickness, partially circumferential incision in the graft-host junction, followed by repositioning and re-suturing of the graft in place, and intracameral air injection in order to achieve reattachment of Descemet membrane.Corneal graft repositioning in combination with re-bubbling may represent an effective therapeutic option in keratoconic patients with peripheral thinning in the residual host corneal tissue and subsequent Descemet membrane detachment. Until recently, repeat-penetrating keratoplasty (PK) has been considered the gold standard for the management of PK graft failure or the recurrence of the primary disease that led to the initial transplantation. Hence, patients had to be re-subjected to various PK-related complications, including delayed wound healing, prolonged visual recovery, high post-operative astigmatism and increased incidence of graft rejection , 2. CurrWe present a case of spontaneous Descemet membrane (DM) detachment 28 years after an uneventful PK for keratoconus and report the outcome of a new, modified surgical approach for the treatment of this entity.A 55-year old male patient presented to our department complaining of sudden visual loss in his left eye, 28 years after an uncomplicated PK for keratoconus. The patient reported no history of trauma or eye rubbing. Upon presentation, best-corrected visual acuity (BCVA) was 20/40 in the right eye and hand movement (HM) in the left eye. Slit-lamp biomicroscopy showed a bulging, full-thickness graft with distorted curvature and marked peripheral thinning and steepening. Diffuse corneal stromal oedema was observed (9–6 o’clock) that spared the inferior nasal area of the graft Fig. . No signo degrees (9 o’clock hours), surrounding the area of the corneal oedema and the underlying detached Descemet membrane. Following this, the graft was repositioned and sutured into place using interrupted 10-0 Nylon sutures. Lastly, air was injected in the anterior chamber in order to achieve reattachment and promote adherence of the previously detached DM.We decided to proceed with corneal graft repositioning and re-bubbling. More specifically, a circumferential, full-thickness incision in the previous graft-host junction was made, using fine corneal scissors. The incision was extended for 270On the first post-operative day, corneal oedema had resolved and DM was found reattached. Patient’s BCVA was 20/40 in the left eye. The postoperative course was uneventful and the graft remained clear after a follow-up of 3 months Fig. . AS-OCT Spontaneous DM detachment represents a recently described clinical entity. To our knowledge, this is the third case of spontaneous, late-onset DM detachment after uncomplicated PK to be reported in literature . PreviouIn our case, similar to previous reports, the corneal graft showed neither scarring nor signs of rejection. The existing endothelial dysfunction and subsequent oedema were attributed to the spontaneous DM detachment. For this reason, re-bubbling or posterior lamellar keratoplasty, such as DSAEK, might have appeared as a more favourable surgical option. Nevertheless, significant thinning and steepening in the peripheral corneal host tissue was observed in our case. This observation was suggestive of possible progression of keratoconus in the residual host tissue. This finding might had complicated any attempt of performing DSAEK, as well as increased the chance of DSAEK failure. Therefore, we decided to attempt restoration of the structural anatomy of the corneal surface at first, by repositioning the graft, and then proceed with re-bubbling. This combination of revisional surgery and re-bubbling may not only have improved the observed peripheral corneal thinning and prevented its further progression, but also increased the possibility of successful DM reattachment. Furthermore, since peripheral thinning and steepening seem to have been managed successfully, as shown by postoperative AS-OCT, and patient’s visual acuity has improved significantly, further improvement in his refractive profile is expected, especially following future removal of sutures. Nevertheless, safe and definite results regarding our patient’s postoperative corneal astigmatism may be reported only after suture removal.Although the exact aetiology for the late changes in keratometric corneal power after PK in patients with keratoconus has not been elucidated, it may include progression of keratoconus in the host rim or recurrence of ectasia in the donor graft –8. SinceIn conclusion, corneal graft repositioning in keratoconic patients with signs of progressive peripheral corneal thinning and subsequent DM detachment, may represent an alternative, effective therapeutic option.


The frontal eye fields (FEFs) and the anterior cingulate cortex (ACC) are commonly coactivated for cognitive saccade tasks, but whether this joined activation indexes coordinated activity underlying successful guidance of sensorimotor mapping is unknown. Here we test whether ACC and FEF circuits coordinate through phase synchronization of local field potential and neural spiking activity in macaque monkeys performing memory-guided and pro- and anti-saccades. We find that FEF and ACC showed prominent synchronization at a 3–9 Hz theta and a 12–30 Hz beta frequency band during the delay and preparation periods with a strong Granger-causal influence from ACC to FEF. The strength of theta- and beta-band coherence between ACC and FEF but not variations in power predict correct task performance. Taken together, the results support a role of ACC in cognitive control of frontoparietal networks and suggest that narrow-band theta and to some extent beta rhythmic activity indexes the coordination of relevant information during periods of enhanced control demands. Frontal eye fields (FEF) and anterior cingulate cortex (ACC) are coactivated during cognitive tasks, but the precise format of their interaction is not known. Here the authors show that phase coupling between ACC -FEF in theta and beta frequency bands better predicts behavioural performance. The frontal eye field (FEF) plays a crucial role in overt135One proposed mechanism for the communication of spatially dispersed neuronal groups is through synchronizing the excitability phases of band-limited activity12121517181920Here we recorded local field potentials (LFPs) and spiking activity from FEF and ACC simultaneously and investigated task-dependent synchronization between the two areas while monkeys performed a memory-guided saccade task and a task with randomly interleaved pro- and anti-saccade trials. The results show that a narrow-band theta and beta rhythmic activity indexes the coordination of relevant information between FEF and ACC during periods of enhanced control demands.n=233 sites; n=87 and n=146 in monkey 1 and 2, respectively) and FEF  while monkeys performed the memory-guided saccade task and the pro-/anti-saccade task  and FEF units . Both ACC and FEF LFPs showed a prominent increase in theta power (3–9 Hz) during the delay period . AdditioP<0.05, Binomial test) but not ACC  channels showed significant spatial tuning of power within the theta frequency band (3–9 Hz) during the delay period in the time window of 400–1,100 ms following target stimulus onset (P<0.05). In addition, a significant number of FEF  but not ACC channels  showed significant tuning in the beta band (12–30 Hz). Thus, we found spatial tuning of LFP power in theta and beta frequencies in FEF but not in ACC. The observed power modulation of the FEF LFPs is in line with previous FEF single-unit recording results, which suggest a high degree of spatial tuning in FEF neuronsWe further examined whether LFP power was tuned to the spatial target location. We found that a significant number of FEF . On the other hand, respectively, 128 (18.99%) and 179 (26.56%) pairs exhibited statistically significant decreased theta and beta synchrony in the delay period relative to the baseline . We observed that, respectively, 234/674 (34.72%), 213/674 (31.6%), and 96/674 (14.83%) of channel pairs exhibited increased phase synchronization across both theta and beta band, theta band only, and beta band only, during the delay period versus baseline. With regard to significant decreased phase synchrony in the delay period versus baseline, the following results were obtained: 39/674 (5.79%) channel pairs exhibited concurrent theta- and beta-band decrease, 89/674 (13.2%) pairs displayed theta- but not beta-band decrease, and 140/674 (20.77%) showed beta- but not theta-band phase synchrony decrease. Furthermore, the population of ACC-FEF channel pairs exhibited increased theta- and beta-band synchrony in the delay period versus baseline of the memory-guided saccade task (We next tested whether ACC and FEF showed task-dependent interactions in the theta (3–9 Hz) and beta (12–30 Hz) band. For a large subset of interareal recording pairs, theta- and beta-band phase synchronization increased from the baseline  to the delay period of the memory-guided saccade task . Of the 674 ACC-FEF LFP-LFP channel pairs, respectively 447 (66.32%) and 330 (48.96%) channel pairs displayed significant increases in theta- and beta-band phase synchronization during the delay period , as shown in We further investigated the directionality of the ACC-FEF interactions by performing Granger-causality analyses. Both during the delay and the baseline period, we observed bidirectional Granger-causal influences between ACC and FEF LFPs, with prominent peaks in the theta and beta range, and a significantly stronger influence from ACC to FEF than vice versa . We validated the relationship of coherence with performance by using an anti-saccade task that triggered more directional error trials than the memory-guided saccade task. Similar to the memory-guided saccade task, the population of the ACC-FEF channel pairs showed significantly decreased theta- and beta-band phase synchronization in the error versus correct anti-saccades during the 400–1,100 ms following the fixation onset during the preparatory period . We confirmed that these statistical differences were not the result of differences in sample size, by performing the same analysis using the same number of trials for correct and error trials.We found that FEF and ACC LFPs exhibit phase synchronization in the theta and beta frequency bands during the delay period. Our next question was whether this synchronization predicted behavioural task performance. To test whether the strength of phase synchronization influenced performance in the memory-guided saccade task, we compared FEF-ACC phase synchronization between correct trials and error trials in which the animal made an eye movement toward the wrong stimulus location. We found that both theta- and beta-band coherence between the ACC and FEF were significantly larger on correct trials than error trials during the delay period  as a measure of spike-field coherenceP<0.001; FEF units: 16/154=10.4%, P<0.001, Binomial test) and ipsiversive trials . In contrast, we found no evidence for significant decreases in phase locking  and ipsiversive trials , whereas we found no evidence for significant decreases   in the delay period versus baseline of both contraversive   right. Mal test) , right. al test) .We also tested for possible influences of firing rate differences between units on the observed spike LFP synchronization. We repeated the spike-field analysis, but only including in the analysis those units with no significant difference of spike rates between the tested conditions. The obtained effects were still statistically significant  was evident in more than half of the LFP-LFP recording pairs, (2) translated to spiking activity in the ACC and FEF with significant interareal spike-LFP synchronization in both anatomical directions, that is, with spikes from ACC coupled to LFPs in FEF and with FEF spikes coupling to LFPs from ACC, (3) indexed correct versus erroneous working memory performance, and (4) exhibited asymmetric directionality, with stronger influence of ACC over FEF than vice versa. These findings provide evidence that functional interactions between brain areas implementing (associated with FEF) and biasing (associated with ACC) working memory and also higher-order cognitive performance proceed through phase-synchronized activation at band-limited theta and beta rhythmic activity1516Neuronal circuits in FEF are well known to encode target locations for overt orienting and covert stimulus selection during working memory, sustained attention, and visual search tasks 14We found that a moderate fraction of ACC and FEF units synchronized to theta LFP activity at distant sites  more strongly during the working memory delay than during the predelay baseline period see  and 6. T153941The role of frontal midline theta in working memory has been previously demonstrated in human studies and the ACC has been suggested as its main sourceAs mentioned in the introduction, beta-band synchronization is suggested to be involved in long-range transmission of information between brain areas171819142027One possible mechanism for ACC-FEF phase synchronization could be based on interactions of a subclass of parvalbumin expressing, fast spiking interneurons, and subsets of pyramidal cells showing resonance to theta rhythmic inhibition14We observed that the theta-band Granger causality increased in the delay period in both ACC→FEF and also FEF→ACC directions. This could indicate that theta-band synchrony is involved in both top-down and bottom-up processes, as has been described previously15In summary, the increased ACC-FEF phase synchrony and also increased influence of the ACC over FEF during the delay period suggests that the ACC-FEF interaction serves a functional role. The increased ACC unit-FEF LFP coupling in the delay period of the contraversive saccades could indicate that the ACC biases FEF to generate a contraversive saccade . Our res1431Macaca mulatta and Macaca fascicularis) weighing 7–9 kg were subjects in this study. Recording chambers were implanted over the right arcuate sulcus and right anterior cingulate sulcus based on previously obtained MRIs. Details for the surgical procedures have been described previouslyTwo male adult macaque monkeys  the colour of which instructed the monkeys to perform a pro- or anti-saccade on stimulus presentation. The fixation point remained illuminated for a period of 1,100–1,400 ms, which we designate as the preparatory period. The monkeys were required to maintain fixation during the preparatory period and then the target stimulus appeared at a distance of 9° either to the right or left of the central fixation point. In pro-saccade trials, the monkeys were required to saccade towards the target stimulus, and in anti-saccade trials, they had to make a saccade towards the mirror location of the target stimulus . To obtaet al.We simultaneously recorded the LFP and unit activity in the ACC and FEF while monkeys performed a memory-guided saccade task and a randomly interleaved pro-/anti-saccade task . FEF andThe signal from each electrode was preamplified (Plexon Inc.) and band-pass filtered between 0.5–250 Hz for LFP signals and between 250 and 8,000 Hz for the single /multiunits and digitized at the frequency of 40 kHz. Each area's signal was referenced separately against the corresponding recording chamber. The sampling rate for LFP signals was set at 1,000 Hz. ACC and FEF multineuron activity was isolated offline by applying a threshold±3–5 s.d. above or below the average raw signal to separate the multineuron activity from noise. We further visually inspected the waveforms to confirm that this procedure resulted in normal waveforms and the multiunits with abnormal waveform shape were excluded from the analysis. We will refer to multineuron activity as units throughout the paper.http://www.ru.nl/fcdonders/fieldtrip/)z-score normalized using a baseline of 500–1,000 ms prior to the fixation point onset in both memory-guided saccade task and pro-/anti-saccade task.All of the data analyses were performed with custom-written Matlab code  using the fieldtrip toolbox , we considered the difference between groups as significant. We obtained statistically significant results in all the comparisons that t-test yielded statistical significance; thus, we could validate the t-test results with this permutation test.Here The WPLI-debiased method outputs a single value for a given trial set and thus, we cannot perform a traditional statistics to test whether there is a difference in WPLI-debiased coherence between the baseline and delay period of a single channel pair. To test for a statistically significant difference in coherence between baseline (700 ms before fixation onset) and delay  period in a single channel pair, we used a bootstrapping approach. We created 1,000 bootstraps with replacement of the original trial set and obtained a distribution of the WPLI-debiased for the baseline and delay periods. The difference between baseline and delay was determined to be significant if the mean of the WPLI-debiased in the delay period fell above or below the 99.9th percentile of the WPLI-debiased distribution of the baseline period. To further confirm the results of the bootstrapping procedure, we pooled the baseline and delay WPLI-debiased results and randomly assigned them into two groups. Then, we tested whether the mean difference between the original baseline and delay period falls above or below the 99.9th percentile of the mean difference of the randomly generated groups. The results of this procedure yielded nearly identical results to the bootstrapping procedure we have described above.et al.We have subtracted the evoked response from the raw data to investigate whether or not the observed phase synchronization is due to the induced oscillations. To further examine whether the observed synchronization was due to the induced oscillations, we have subtracted the evoked response using a method described by Truccolo To analyse the directionality of interactions, we performed Granger-causality analysis. Granger-causality analysis is based on modelling the two time series as a vector autoregressive model,M=32 and using the armorf function in the BSMART toolboxet al.In this equation, the two signals are predicted by a linear combination of their own past values, and the past values of the other time series. The residual errors (unexplained variance) are captured by the variables x2 to x1 is now defined asThe frequency-domain Granger causality from f over the amount of power that remains after the predictions from the second signal have been factored in.This expression can be understood as the log fraction of intrinsic power in the first signal at frequency x1 to x2 is stronger than the Granger causality from x2 to x1, then we expect that the reverse holds true when we time reverse the signalset al.It is well established that Granger causality can be sensitive to both uncorrelated and correlated noise that is superimposed on the measurements of the signals of interest. This can lead to a substantial amount of false alarms in the identification of Granger-causality relationships see refs , 67, 68.t-test. We further confirmed the results of the t-test by implementing a permutation test. The permutation test procedure is described in detail above.The Granger-causal influence of the ACC over FEF is shown as GACC→FEF and the Granger-causal influence of FEF over ACC is denoted as GFEF→ACC. The overall influence of ACC over FEF was derived by implementing the (GACC→FEF−GFEF→ACC) equation. The results shown in We also shuffled the time-frequency transformed raw data and computed the corresponding Granger values to obtain the chance level Granger. Then we compared the chance level GACC→FEF and GFEF→ACC values to the original Granger values from the non-shuffled data set. We observed that across theta and beta frequencies, both the GACC→FEF and GFEF→ACC values in the baseline and delay periods were above the chance level.We used the PPC as a measure of spike-field synchrony; for a detailed mathematical definition see ref. M is the number of spikes, Ui is the vector describing the x and y component of the spike-LFP phase for the ith. We only included units with at least 50 spiking events in the tested conditions to obtain a reliable and robust results and increase the statistical power of the analysis as has been shown before15t-test and also a permutation test to probe the significant difference of the PPC across experimental conditions. We have described the permutation procedure above. To correct for multiple comparisons, we have implemented the false discovery rate algorithm as described by Benjamini and YekutieliHere To test for significant spike-field coupling across single ACC-unit with FEF-LFP and FEF-unit with ACC-LFP, we performed a permutation test. We first randomly shuffled the spikes across the tested conditions  and obtained a distribution of the PPC values in the randomized experimental conditions. Then, we tested whether the PPC values of the original non-randomized conditions fell above or below the 2.5th percentile of the randomization distribution. If so, we designated that unit-LFP pair as having a significant modulation across the tested conditions.We have not made the raw data available. However, the computer codes used to perform data analysis could be provided on request.How to cite this article: Babapoor-Farrokhran, S. et al. Theta and beta synchrony coordinate frontal eye fields and anterior cingulate cortex during sensorimotor mapping. Nat. Commun.8, 13967 doi: 10.1038/ncomms13967 (2017).Supplementary Figures and Supplementary References


DEAR EDITOR1 This is achieved by fixing the skin graft with tie over dressing using multiple silk sutures. Repeated tie over dressing is needed in the settings where graft needs to be inspected frequently.1Reconstruction of any soft tissue defect is managed as per conventional reconstructive ladder. The options range from healing by secondary intention, primary closure, skin graft and flaps. Skin graft is one of the commonest methods of reconstruction. For proper take of skin graft, the graft needs to be adherent to the wound bed for first few days. The fixation should not allow any hematoma or seroma formation under the graft.Four patients having recurrent and huge keloids on anterior chest wall in the presternal region were referred from radiation oncology department for possible excision and postoperative radiotherapy. All these patients have previously undergone multiple treatments of their keloids in the form injections and local excisions with out any benefit. The patients were accepted by radiation oncology and were found candidates for immediate radiotherapy after excision of keloid.In all patients keloids were excised and the soft tissue defect was covered by full thickness skin grafts harvested from groin. The skin grafts were immobilized by tie over dressing in an innovative way. In this method the normal saline bottle used for irrigation was cut at its neck . The skiOnce non adherent layer of gauze and adequate padding is applied on the raw area, the tie over threads were passed from inside out of the cut part of collar of saline bottle and pulled at the appropriate tension to keep the dressing in place . The capOn postoperative day 1, the dressing is removed by unscrewing the cap and patient is shifted to receive radiotherapy . After f2Skin grafts after applying on wound bed is dressed by applying non-adherent layer of dressing. The dressing can be secured in place by many methods, but can be applied once only. These traditional methods can stabilize the graft till the first dressing post operatively. In some contaminated wounds, the dressing needs to be removed earlier, especially if there is drainage or foul smell.This approach may also be proper for graft, used to cover defects of some anatomical regions with increases risk of contamination, such as perineal, axillary, and genital or it can be used in areas where base of wound is difficult to immobilize like breast/pectoral region. It is true in cases of our study where repeated dressing is needed for giving local radiotherapy. There are multiple methods that can be used repeatedly for the skin graft stabilization.3 When taking a tie over stitch, both the threads can be left long and only one thread is tied at a time, the other thread is left long for next time.4 This dressing can be reapplied only twice. Repeated tie over dressings are also done using bra hooks,5 and silk loops.6Repeated tie over dressings can be done by keeping interrupted sutures long to be used as tie over dressings. These ties over dressing can be made of sutures or rubber bands.These techniques are difficult for small dressings especially the bra hooks; the silk loops method is very cumbersome and takes long time to do. Our innovative method being discussed is very fast. It hardly takes 5 min in the hands of a Plastic surgeon to complete the dressing. This dressing technique maintains the advantage of conventional tie over dressing with rapidity and repeativity. Excellent graft take can be expected with appropriate methods of stabilization. We recommend an innovative low cost, simple and rapid method of graft fixation that can be used repeatedly.The authors declare no conflict of interest.


As a result, we can obtain all the MQQs over As the basic cryptographic structure for multivariate quadratic quasigroup (MQQ) scheme, MQQ has been one of the latest tools in designing MQ cryptosystem. There have been several construction methods for MQQs in the literature, however, the algorithm for judging whether quasigroups of any order are MQQs over Galois fields is still lacking. To this end, the objective of this paper is to establish a necessary and sufficient condition for a given quasigroup of order  With the development of quantum computer, post-quantum cryptography (PQC) has gained intensive attention in recent years. Multivariate Public Key Cryptography (MPKC) is one among a few serious candidates to have risen to prominence as post-quantum options. In the last two decades, MPKC was developed rapidly, with many schemes being proposed, attacked and then amended. Based on multivariate quadratic quasigroups (MQQ), Gligoroski et al. recently proposed a novel type of MPKC-MQQ schemes (including both the signature scheme and the encryption scheme) , the objective of this paper is to extend the previous work  of type k of them are of degree 1, where [i et al. ] A quasipkd to be an MQQ over pkd is an MQQ over d Boolean functions of MQQ if it is.In this section, we first establish a necessary and sufficient condition for a given quasigroup of order n; i with their counterparts on row j of j (column i) to their counterparts on row i (column j) of For convenience the following notations are adopted: see Golub and Loan  according to the bijection kd-ary binary sequence into d groups from left to right, where every group is a k-ary sequence, and represent every group by a unique element in Let a quasigroup mentclass2pt{minimmentclass2pt{minimd-ary quadratic Boolean function set d-ary sequences in the order of d-ary sequence over According to Lemma mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimBy now we have proved the following necessary and sufficient condition that a given quasigroup is an MQQ over For a given quasigroupof orderpkd, convert every element ofinto a kd-ary sequence overGF(p) according to the bijection, divide every kd-ary sequence into d groups from left to right, and represent every k-ary sequence by a unique element inaccording to the bijectionThenis an MQQ overof typeif and only if the matrix equation Suppose\Now we begin to consider Case 2: By multiplying Writebˇd,then  has solud quadratic functions of MQQ.Suppose  has soluWe summarize the above deduction for Case 2 as the following theorem.Supposeandthen Suppose\To end this section, we summarize our proposed algorithm as follows:pkd in a vector with the form of  and and\docurding to .Compute no MQQ”.If If Write out of MQQ”.Compute …,fd} by  and outpIf rding to , 23) an an\documrding to .Compute no MQQ” .If If Write out of MQQ”.Compute …,fd} by  and outpAlgorithm for checking whether a given quasigroup of order In this section, we use two examples which are dealing with quasigroups of order A quasigroup entclass1pt{minimaSuppose GF(3) are given in Table A quasigroup Suppose entclass1pt{minimapkd is an MQQ over p, k and d, which is beneficial for applying MQQ-design to various platforms. The validity of the theorems and the effectiveness of the proposed algorithm have been verified by two examples.In this paper, a necessary and sufficient condition, which reveals that a given quasigroup equation  has solu


We also show that for 1 < D < 1.5 the Pohrt-Popov behavior (α = 1/(3 − D)) is valid, however for 1.5 < D < 2, the exponent α is different and equal to 2(D − 1)/D. The diversity between the model developed in the work and the Pohrt-Popov one is explained in detail.In this work we describe a theoretical model that predicts the interfacial contact stiffness of fractal rough surfaces by considering the effects of elastic and plastic deformations of the fractal asperities. We also develop an original test rig that simulates dovetail joints for turbo machinery blades, which can fine tune the normal contact load existing between the contacting surfaces of the blade root. The interfacial contact stiffness is obtained through an inverse identification method in which finite element simulations are fitted to the experimental results. Excellent agreement is observed between the contact stiffness predicted by the theoretical model and by the analogous experimental results. We demonstrate that the contact stiffness is a power law function of the normal contact load with an exponent  This fact is of tremendous practical importance since the size and shape of the real contact area affect a large number of physical properties relevant to mechanical engineering and electrical and heating conduction problems. The interfacial contact stiffness that frequently dominates the overall static and dynamic characteristics of mechanical systems is one of the most significant properties. Examples for which interfacial contact stiffness is prominent are constituted by friction dampers in jet engines3, rotor systems5, gears6, head-disk and brake interface7 and micro electro-mechanical systems (MEMS)8.In the design of mechanical components the surface roughness plays a significant part. Roughness makes the actual contact area noticeably smaller than the nominal contact surface in a considerable number of relevant cases9, the ultrasonic method10, the continuous stiffness measurement11, and the digital image correlation12. Several test rigs have also been designed for specific contact conditions, and some are suitable for high sliding velocities and small displacements14, while others are applicable for high loads, large displacements, and relatively low velocities15. A theoretical model is however still needed to predict the interfacial contact stiffness for the entire range of the design parameters of the mechanical component. The most widely used model is the Greenwood–Williamson (GW) statistical one16. The precision of the GW model is however limited, since the parameters used to describe the roughness strongly depend on the resolution of the roughness-measuring instrument. Works by Majumdar and Bhushan18 show however that a variety of surfaces with roughness are self-affine. Based on the representation of the surface as a fractal geometry, the scale-invariant fractal parameters can indeed characterize in an adequate manner the roughness at all length scales19. Since those seminal works, recent interest in the contact mechanics of areas with roughness has focused on the adoption of models dealing with fractal and self-affine rough surfaces. The most relevant study to this article is the contact stiffness model of plane machined joints based on fractal theory by Jiang20. This model assumes that the asperities are sufficiently widely spaced to be treated as resistances in parallel, however only elastic asperities are considered for the calculation of the contact stiffness.Interfacial contact stiffness is mainly estimated through experimental measurements. A variety of measurement technologies have been developed for the purpose during the last decades, such as the contact resonant frequency methodst resonant frequency of the blade with the normal contact load. To verify our model, the contact stiffness corresponding to the normal load is identified by correlating the experimental results to the simulation results based on the contact resonant frequency method.In our work we present a generalized model of the contact stiffness between areas with roughness described in terms of fractal profiles. The model considers the whole interfaces represented by fractal surfaces, and both plastic and elastic asperities are included. We also describe the design and use of a test rig to simulate the dovetail joint of a turbo machinery blade under operating status. The rig can accurately measure the dependency of the 1Kn is the partial derivative of the normal load P versus the relative displacement ∆ of the rigid plane from the equivalent rough surface:The contact between two surfaces with roughness can be modeled as an equivalent rugged surface contacting a smooth rigid plane, and with a large number of separate asperities of different sizes spreading over the irregular surface contacting the rigid plane. The contact stiffness ∆ of the rigid plane from the equivalent rugged surface is equal to the largest interference of a single asperity:As illustrated in Fig. 21. When the truncated micro-contact area of the largest asperity In the case of fractal rugged surfaces, the theoretical model representing the contact stiffness is obtained by associating equations  and 2 wiWhen E* and H represent the equivalent Young’s modulus and the hardness of the softer material of the mating surfaces. The parameter D constitutes the fractal dimension of the surface topography, G is the fractal roughness parameter while γ is a non-dimensional quantity related to the profile describing the fractal surface.In (4) st frequency  of a blade-dovetail joint system from frequency response functions (FRF) under different normal contact loads. The resonant frequency is determined from the peak point of the FRF, while the equivalent modal damping ratio is determined using the half-power method. The measured damping ratio is To verify the proposed theoretical model, we measure the 1entclass1pt{minimast resonant frequency. More details about the experiment and the identification are provided in the following Sections. The variation of the theoretical contact stiffness and the experimental results on the normal contact load are shown in Fig. The contact stiffness is experimentally measured by using an inverse identification method, which consists in fitting numerical results from a high-fidelity finite element model representing the blade, to the measured dynamic velocities measured for the 1The contact stiffness from Jiang’s model is calculated by integrating the stiffness of a single elastic asperity:22, for which the contact stiffness is dominated by the presence of larger asperities and it is insensitive to the behavior at the microscopic scale. We can therefore conclude that the integration form of the whole contact stiffness tends to overestimate the contribution provided by smaller asperities under the given distribution density, leading to significantly higher stiffness values than the experimental ones.Jiang’s model assumes that each asperity behaves as an elastic spring, and the equivalent surface can be regarded as a series of springs in parallel, with 23 and the boundary element simulations shown in24. Our theoretical model is able to illustrate the relation between contact stiffness and normal load for different fractal dimensions 11) is \dWhen equation  is negatThe power relation in the force-stiffness function described by equations  and 13)13) is \dD and G of the equivalent surface can be derived based on the profile distributions or structure functions of the two rugged surfaces25. The Young’s modulus of the equivalent contact surface material is calculated from:n of the truncated asperity sizes is mathematically similar to the one representing islands which spread over the surface of the Earth26, and can be written as:The contact between two rough surfaces can be modeled as a single equivalent rugged surface in contact with a smooth rigid plane. The parameters 17 of a single asperity is determined by the fractal surface profile, and is equal to the peak-to-valley amplitude:The asperity interference R at the tip of the asperity by:In equation  \documenBy substituting equation  into 1717, the r12, the critical value of the asperity interference at the onset of plastic flow is proportional to Asperities with different truncated areas have different interferences and are subjected therefore to different deformation states. According to Greenwood and Williamsonb is a constant independent of the asperity size, and H is the hardness of the softer material of two contacting surfaces. The deformation force of a single asperity is assumed to change continuously during the increase of the contact area, which results in:The parameter As a consequence of (20) we have quations –18) int int\docuquations :r is the radius of the micro-contact area a \documentBy inserting equation  into 2727 and neIt can be deduced from equation  that theEquations , 28) an an28) an27. The fractal parameters D and G are determined with reference to the equivalent parameter values of the contact pairs machined by milling16, which is Figure −1. The force-displacement curves of the two springs are measured by compressive tests using a WDW 3100 electronic universal testing machine, with a 1 kN force sensor (accuracy of 4‰) and a 25 mm electronic dial gauge (accuracy of ± 0.005 mm). The strain rate is 0.1 mm∙s−1. The results show the two test springs are linear (average stiffness of 6.89 N∙mm−1) and coincident with each other, and are therefore capable to apply the load to the blade in a uniform manner. The range of the applied load to blade is chosen to be within 0 N ~ 1,400 N to ensure a noticeable change of the resonant frequency when the applied load is varied. Given that the interfacial frictional force is actually varying during the vibration, the average frictional force is used and thus the relationship between the applied load to the blade and the normal contact load at one side is: F is the applied load, P is the normal contact load of one side, θ is the dovetail angle with θ = 90° and μ is the nominal friction coefficient with 28A loading device is custom designed to facilitate the fine adjustment of the load to the blade and the normal load on the dovetail joint. The dovetail root of the blade is designed to extend beyond the ends, and two springs restricted by four bushes apply the load by simulating a centrifugal force see Fig. . SpringsWhen the magnitude of the applied load is relatively low the blade has an easy relative motion with the slot. In a traditional full contact arrangement between the pushrod of the shaker and the loosely restricted blade, the additional mass and stiffness effect provided by the rod would have a significant influence on the dynamic characteristics of the system, and this is unacceptable for the type of measurement performed here. We use instead a base excitation method in which the shaker applies the dynamic load to the base Fig. . Based o30 which has been widely used to indirectly measure the interfacial contact stiffness. Typically, the system resonant frequency is constant and independent of the load, whereas the contact resonant frequency will shift with increasing contact load because both interfacial stiffness and damping change. The damping effect can be ignored in the calculation, since the resonant frequency of the FRF with reference to the velocity response is equal to the natural frequency of the system. An undamped modal analysis is therefore sufficient to determine the resonant frequency.The identification of the contact stiffness is based on the contact resonant frequency methodkni and kti, respectively) can be calculated as:N is the number of nodes on one interface, i is the node number.The finite element model used for the inverse identification of the test planar blade is shown in Fig. st natural frequency corresponding to any given contact stiffness can be obtained by performing an eigenvalue modal analysis with block Lanczos method. We can then obtain the relationship between the interfacial contact stiffness Kn and the measured resonant frequency according to the FEA results. Eventually the contact stiffness can be inversely identified by fitting the simulation results to the measured 1st resonant frequency.The above stiffness is defined by spring elements (COMBINE 14) on each marked node in the finite element model. The 1All data generated or analysed during this study are included in this published article.α within the whole range of fractal dimension D(1 < D < 2). For 1 < D < 1.5 the Pohrt-Popov behavior (D < 2, the exponent α is slightly larger and equal to One generalized model of the interfacial contact stiffness between fractal rough surfaces is developed by taking into account the effect of both elastic and plastic deformations of asperities. It can be inferred from the model that typically the contact stiffness is a power law function of the normal contact load with an exponent


Zirconia restorations have been suggested as a more durable and more appealing alternative to metal restorations. However, their mechanical properties may be negatively affected by fatigue due to superficial stresses or low temperature degradation. This study aimed to assess the fatigue fracture strength of three-unit implant-supported full contour zirconia and pre-sintered cobalt-chromium (Co-Cr) alloy posterior fixed partial dentures (FPDs).In this in-vitro experimental study, 28 posterior three-unit implant-supported FPDs were fabricated of full contour zirconia and pre-sintered Co-Cr alloy, and were cemented on implant abutments. To simulate the oral environment, FPDs were subjected to 10,000 thermal cycles between 5–55°C for 30 seconds, and were then transferred to a chewing simulator . Afterwards, fatigue fracture strength was measured using a universal testing machine. Data were analyzed by Mann-Whitney U test.The mean and standard deviation of fracture strength were 2108.6±440.1 N in full contour zirconia, and 3499.9±1106.5 N in pre-sintered Co-Cr alloy. According to Mann-Whitney U test, the difference in this respect was statistically significant between the two groups (P=0.007).Since the fracture strength values obtained in the two groups were significantly higher than the maximum mean masticatory load in the oral environment, both materials can be used for fabrication of posterior three-unit FPDs, depending on the esthetic demands of patients. Porcelain-fused-to-metal (PFM) fixed partial dentures (FPDs) have long been used due to their optimal long-term mechanical properties ,2, and aIn the past, cobalt-chromium-molybdenum (Co-Cr-Mo) alloy could be fabricated by the CAD/CAM process in industrial centers. Two different approaches have been described for restoration fabrication by the CAD/CAM systems using these alloys, namely the additive manufacturing using laser sintering, and the subtractive manufacturing using costly milling machines for milling of high strength materials.Limited CAD/CAM systems have been designed for dental laboratories, which can cut back the materials in the fully sintered form; however, they are complicated to work with. The newly introduced pre-sintered Co-Cr-Mo alloy available in the market with the brand name Sintron can be used in a desktop milling machine, with faster preparation time and lower cost.The soft Co-Cr-Mo alloy is first subjected to dry preparation. It contains adhesive materials such as organic coupling agents, which are burned out following sintering at high temperature under argon atmosphere. This results in about 10% reduction in its volume . The CADAll-ceramic FPDs are esthetically favorable, and zirconia restorations have been suggested as a durable alternative to metal restorations. However, these restorations must meet the biomechanical requirements, and must have a durability similar to that of PFM restorations . MonolitPrevious studies have reported more advantages for monolithic crowns, including requiring less tooth preparation –19, and In this in-vitro experimental study, sample size was calculated to be 14 restorations in each group according to a previous study , assuminTwo implants  with 4mm diameter and 11.5 mm height were used. The abutment had 4.5 mm diameter and 8.5 mm height with 1.5 mm gingival height. First, the first fixture was mounted in a polymethyl methacrylate resin block  using a surveyor. Then, the second fixture was mounted parallel to the first fixture using a surveyor, in a way that the distance between the centers of the two fixtures was 19 mm , in ordeThe CAD/CAM system was used for the fabrication of these bridges. The fabricated resin block was first scanned to determine the implant site and implant position using a 3D scanner , and this scan was processed using 3D Designer software. A bridge with 4×4 mm2 contour dimensions and 12 mm pontic width was fabricated. The minimum thickness was 2 mm. Occlusal anatomy was determined according to the Library 13 of 3Shape software . Arrangement was done in iCAM version 4.6 software, and the file was transferred to Remote Dental 2.0 software. Next, the full contour zirconium oxide blocks  were milled by a milling machine , and zirconia bridges were sintered in a furnace  at 1500°C for four hours, according to the manufacturer’s instructions.The frameworks were fabricated by the CAD/CAM system as explained earlier for the zirconia group using pre-sintered Co-Cr blocks  with the same design as that of the full contour zirconia framework by 1.2 mm cut back for application of porcelain veneering. After completion of preparation of pre-sintered Co-Cr blocks by the milling machine, the frameworks were sintered in a furnace  at 1285°C for six hours under argon atmosphere, according to the manufacturer’s instructions. To ensure that these frameworks have the same design and contour as those of full contour zirconia frameworks, a silicon impression was made of full contour zirconia FPD using putty impression material  to provide an index for veneering of the pre-sintered Co-Cr framework. Using this index, opaque and body porcelain  were applied on the framework, according to the manufacturer’s instructions.Before cementation, the surfaces of FPD and abutments were cleaned with 92% alcohol, and glass ionomer   was usedAfter cementation, all the FPDs were subjected to 10,000 thermal cycles  between 5–55°C (±2°C) for 30 seconds to simulate the oral environment. For dynamic cyclic loading, the FPDs were placed in a chewing simulator , and were subjected to 100,000 cycles with 50N load and 0.5Hz frequency  24]. Th. Th24]. This setting corresponded to two to four months of clinical service of the restoration in the oral cavity ,26. NextData were analyzed using SPSS version 24 . Normality of data was checked using Kolmogorov-Smirnov test. Since data were not normally distributed, non-parametric Mann-Whitney U test was used for statistical analysis. Level of significance was set at 5%.Since data were not normally distributed, Mann-Whitney U test was used for statistical analysis, which showed that the mean fatigue fracture strength of pre-sintered Co-Cr alloy was significantly higher than that of full contour zirconia (P=0.007).Full contour zirconia crowns are becoming increasingly popular due to adequate flexural strength (+1000 MPa), which is higher than the maximum occlusal loading during normal mastication. They have also shown flexural strength higher than 2000 N . OptimalPrefabricated dental ceramic blocks are either semi-sintered or fully sintered zirconia blocks . PreparaClinical data suggest that defects of all-ceramic FPDs are commonly seen around the connector. An in-vitro study on all-ceramic Y-TZP FPDs with 4×4 mm2 connector dimensions showed that they had a 100% success rate after two years . TherefoBeuer et al  demonstrFormation of a strong bond between the opaque layer and alloy is critical for optimal durability and survival of metal ceramic restorations (MCRs). Several studies have demonstrated that increased thickness of the oxide layer covering the alloy is the main factor that decreases the bond strength of MCRs –35. In tThe authors revealed that Sintron CAD/CAM alloy was comparable to conventional alloys in terms of bond strength .Our study showed that Sintron alloy had significantly higher fracture strength than Zolid. In 2009, Bonfante et al evaluated the prevalence of fracture and defects in three-unit MCR and Y-TZP implant-supported bridges using fatigue testing, and concluded that MCR has higher fracture strength, and is the gold standard for posterior dental areas .In 2013, Eroğlu and Gurbulak evaluated the fatigue and non-fatigue behavior of 60 restorations in three groups of zirconia ceramics (ZC), galvano-ceramics (GC), and PFM on metal dies in canine to second premolar region of the maxilla, and found that ZC had the highest fracture strength . Also, ZC group did not show a significant difference between fatigue and non-fatigue behavior. PFM ranked second in this respect , and GC ranked last. Significant differences were noted between fatigue and non-fatigue behavior of the latter two groups [In the present study, the mean fracture strength was 2108.6 N in Zolid, and 3499.9 N in Sintron group. Regarding the fracture strength value obtained in zirconia group, we used monolithic zirconia in our study, while Eroğlu and Gurbulak used veneered zirconia. The values in the two studies were in a close range for zirconia, and the existing difference is due to the fact that the bridge fabricated in our study had a 10mm-pontic width to replace the first molar, and was implant-supported; whereas, they fabricated a bridge with a 5mm-pontic width to replace a first premolar, which was supported by a metal die.Moreover, the zirconia blocks used in the two studies were also of different types. Eroğlu and Gurbulak reported lower strength values than ours for MCRs, which may be attributed to the type of alloy used, and the process of fabrication, which were different in the two studies.They used Ni-Cr alloy for the fabrication of PFM restorations, while we used Co-Cr alloy, since Ni-Cr has lower strength, and may cause allergic reactions in the clinical setting. Also, Co-Cr alloy has higher strength and corrosion resistance than Ni-Cr. Furthermore, Eroğlu and Gurbulak used the routine casting process, which is time-consuming, and has the risk of procedural errors. In the current study, Co-Cr alloy was milled by the CAD/CAM machine. This process is simple and fast, and has lower risk of errors since it is fully automated. In 2014, Johansson et al reported that monolithic Y-TZP ceramics showed significantly higher fracture strength than other groups (2795 and 3038 N). The fracture strength of veneered Y-TZP group (2229 N) was significantly higher than that of monolithic lithium disilicate and veneered highly translucent Y-TZP groups [The fracture strength value reported by Johansson et al, for monolithic zirconia group was higher than that in our study, which may be due to the following reasons: First, they evaluated single crowns, while we evaluated three-unit posterior bridges. Second, Johansson et al. performed fatigue test by applying 10,000 cycles at 10° angle with 1Hz frequency, while we performed fatigue test by using 100,000 cycles applied vertically to the pontic with 0.5Hz frequency. Third, the monolithic zirconia blocks used in the two studies were from different commercial brands. Finally, it appears that higher fracture strength in Sintron group is due to higher fracture strength of its Co-Cr framework.On the other hand, since fractures in Sintron group were in the form of porcelain chipping in our study, it seems that thinner superficial oxide layer and the homogenous and uniform surface of this alloy provide adequately strong bond to porcelain, which leads to higher fracture resistance. In monolithic zirconia group, after thermocycling and cyclic loading, the zirconia surface is exposed to liquids, and undergoes low temperature degradation.Consequently, the energy required for transformation toughening  decreases. Moreover, during cyclic loading, zirconia undergoes macro and micro-cracks due to its brittle nature, which gradually jeopardize its strength, and result in fracture under lower magnitudes of load.In the present study, the fracture strength values found for Zolid and Sintron were higher than the reported maximum mean masticatory load in the oral environment. Thus, both types of restorations can be successfully used for patients requiring three-unit posterior bridges, depending on their esthetic requirements.