Mammalian circadian clocks are driven by a transcription/translation feedback loop composed of positive regulators (CLOCK/BMAL1) and repressors (CRYPTOCHROME 1/2 (CRY1/2) and PER1/2). To understand the structural principles of regulation, we used evolutionary sequence analysis to identify co-evolving residues within the CRY/PHL protein family. Here we report the identification of an ancestral secondary cofactor-binding pocket as an interface in repressive CRYs, mediating regulation through direct interaction with CLOCK and BMAL1. Mutations weakening binding between CLOCK/BMAL1 and CRY1 lead to acceleration of the clock, suggesting that subtle sequence divergences at this site can modulate clock function. Divergence between CRY1 and CRY2 at this site results in distinct periodic output. Weaker interactions between CRY2 and CLOCK/BMAL1 at this pocket are strengthened by co-expression of PER2, suggesting that PER expression limits the length of the repressive phase in CRY2-driven rhythms. Overall, this work provides a model for the mechanism and evolutionary variation of clock regulatory mechanisms. The molecular mechanisms that define the periodicity or rate of the circadian clock are not well understood. Here the authors use a multidisciplinary approach and identify a mechanism for period regulation that depends on the affinity of the core clock proteins for one another. In eukaryotes, these clocks are made up of transcription/translation feedback loops (TTFLs) with regulatory steps at many levels that tune the period and provide environmental input into the system.Cell autonomous molecular clocks with robust 24-h rhythms have evolved to coordinate physiological processes with daily changes in the environment. At the molecular level, clocks appear to have evolved independently a number of times and are comprised of a variety of different components3. This transcription factor regulates the expression of many targets including the genes encoding the repressors: Cry1/2 and Per1/2/34. CRY and PER proteins translocate to the nucleus where they bind and repress CLOCK and BMAL1 activity5. Both CRY and PER are necessary to maintain rhythmicity in both cellular and organismal milieus9, and it is known that degradation of the repressors PER and CRY is a critical period-determining step15.The CRYPTOCHROMES (CRYs) play a critical regulatory role in clock function for both animals and plants. Though the precise molecular implementation of the TTFL varies across species, the mammalian circadian clock provides a clear illustration of how CRY functions. In this system, the TTFL is principally defined by a heterodimeric transcription factor, composed of basic helix–loop–helix (bHLH) PER-ARNT-SIM (PAS) domain-containing proteins CLOCK and BMAL118 and CRY’s secondary pocket has recently been reported to mediate an interaction with CLOCK’s PAS-B domain20. Notably, mutation of the secondary pocket residue R109 to a glutamine has been shown to reduce CRY1’s repressive capacity and abrogate binding between CRY1 and CLOCK without disrupting the global CRY1 fold21. Additionally, it was recently reported that a human mutation that truncates CRY1’s C-terminal tail strengthens the interaction between CRY1 and the CLOCK/BMAL1 heterodimer while also lengthening period in carriers22. Finally, crystallographic data demonstrates that PER2 and FBXL3, part of an E3 ubiquitin ligase complex that targets CRY for degradation, share an overlapping interface on CRY that includes an ancestrally important active site, a flavin-adenine dinucleotide (FAD)-binding pocket24. Despite these reports, it has been difficult to develop clear structure–function relationships for the wealth of functionally relevant mutations in CRY that have been reported28 aside from mutations that have an effect on stability32.Several groups have begun to identify the functional interfaces on CRY involved in specific protein–protein interactions with CLOCK and BMAL1. In particular, BMAL1’s transactivation domain (TAD) has been shown to interact with a C-terminal portion of CRY33. Thus they provide an excellent model system for understanding how subtle variation in sequence and structure can give rise to regulatory diversity and functional differences in clock timing. For example, the animal CRYs are broadly classified into types I and II. Only type II CRYs (such as CRY1 and CRY2 in mice) function as direct repressors of their transcriptional activators34, whereas type I CRYs, such as the single CRY in Drosophila (dCRY), function as blue light-sensitive inputs to a TTFL composed of CLOCK and BMAL1 homologs and the repressors PER and TIMELESS35. Understanding how sequence variation in the type I and II CRYs leads to distinct functional roles thus provides one avenue to identify structural regions involved in differential regulation.CRYs from all organisms share a stereotyped structure of a conserved Photolyase (PHL)-homology region, a C-terminal coiled-coil-like helix (CC helix), and a highly variable C-terminal extension known as the tail. However, they have evolved to perform a diversity of functional roles in the clock36. Second, although deletion of both CRYs results in arrhythmicity, individual null mutations result in opposite phenotypes—short (mCry1−/−) and long (mCry2−/−) periods, respectively9. Moreover, the CRYs are associated with a highly divergent set of DNA-binding complexes within the genome and a substantially different phase of peak binding37. The peak of occupancy for CRY2 is early in the evening in phase with PER1 and PER2, whereas CRY1 occupancy peaks in the late night and early morning, several hours after the peak occupancy of PER1 and PER237. The sequence determinants of the regulatory differences between CRY1 and CRY2 are poorly understood. Overall, greater insight into the mechanisms governing how CRY determines periodicity is necessary for targeted development of therapies for circadian disorders.Within the type II CRYs, individual family members show further functional differences. The mammalian CRYs—here represented by mouse CRY1 and CRY2—have distinct functions despite high sequence identity and similarity. First, CRY1 is a stronger repressor of CLOCK/BMAL1-mediated transcriptional activation than CRY2Thus, major open questions include: (1) how do certain mutations in CRY lead to acceleration or deceleration of the clock; (2) what residues are involved in functional interfaces on CRY that mediate specific protein–protein interactions; (3) how are functional differences between type I and II CRYs manifested at a structural level; and (4) how do the structural features that differentiate CRY1 and CRY2 manifest divergent functional characteristics? To understand the determinants for CRY-mediated regulation of CLOCK/BMAL1 and for functional variation between the CRYs, we studied the pattern of amino acid conservation and co-evolution across the CRY/PHL family (CPF). This analysis reveals a co-evolving network of amino acids spanning the primary and secondary pockets that is conserved across both the CRYs and PHLs, indicating a shared family-wide functional architecture. We show the secondary pocket gates interactions with CLOCK and BMAL1 and likely differentiates type I and type II CRYs in their ability to directly repress CLOCK and BMAL1. Furthermore, we show that subtle changes at this pocket result in substantial differences in periodicity in cycling cells due to changes in affinity between CRY1 and CLOCK/BMAL1. Finally, we observe that a few small differences between CRY1 and CRY2 at this surface underlie CRY2’s periodicity characteristics, again the result of a weakened affinity for CLOCK/BMAL1. Overall, these results define a mode of period regulation of the circadian clock that is orthogonal to the canonical model of period regulation arising from the stability of the PER and CRY repressor proteins.38. The FAD molecule is essential for enzymatic activity, while the secondary cofactor modulates the dynamics of the reaction39. Interestingly, in a previous targeted mutational screen, we identified several positions in CRY1 and CRY2 that weaken repressive capacity and localize to the region on CRY structurally analogous to the PHL secondary pocket ((E103 (E121 in CRY2), G106, and R109), Fig. To better understand the sequence determinants of CRY function, we conducted a statistical study of conservation and co-evolution across the CRYs and the PHLs, an evolutionarily related family of photoactivatable enzymes that catalyze the repair of ultraviolet-induced DNA lesions Fig. . PHLs ha41. These co-evolving groups are called sectors, and typically comprise physically contiguous networks of amino acids that link active sites to distantly positioned surfaces. Sectors correspond to allosteric mechanisms in several model systems, and recent work has shown that sectors create hotspots for allosteric regulation, enabling the evolution of new regulation in proteins through local modifications43. While distinct co-evolution-based approaches have also been developed to identify direct structural contacts in proteins , SCA uniquely focuses on discovering the collective networks of residues that underlie long-range coupling within a protein.To investigate the possibility that functional coupling between the primary and secondary pockets is a conserved feature across both the CRYS and PHLs, we conducted the statistical coupling analysis (SCA), a method for inferring groups of conserved, co-evolving positions associated with complex functions such as allostery in a protein family45. This relationship permits us to test whether an allosteric mechanism inferred by SCA is conserved or is divergent in members of the protein family. For example, the family of Hsp70-like molecular chaperones contains both allosteric and non-allosteric members, and sector positions display the property of (1) cleanly separating these functional subfamilies and (2) displaying lower conservation in the non-allosteric group45. In contrast, the finding that sector positions do not separate subfamilies by function and do not show differences in overall conservation in subfamilies would be consistent with retention of an allosteric mechanism or a conserved functional coupling between sites. For the CPF, we expect SCA to reveal a sector linking the FAD-binding pocket (or primary pocket) with the secondary pocket. If so, we can then use the sector positions to test the functional role of the secondary pocket in the CRY subfamily.A key feature of SCA is the ability to mathematically relate the amino acid motif within sectors (correlated evolution of positions) to phylogenetic and/or functional divergences in the protein family (correlated evolution of sequences)SCA for a multiple sequence alignment of 5385 CPF proteins reveals a single sector of 260 co-evolving amino acid positions that surrounds the primary FAD-binding site and extends to several surface accessible positions, including the secondary cofactor pocket Fig. . These fTo examine this more deeply, we plotted the top eigenvectors of the SCA co-evolution matrix and the corresponding projected sequence divergences. Sector residues emerge along the first eigenmode and are split by the second eigenmode as the first levels of a hierarchy of coevolution Fig. . Residue24. Consistent with repurposing of the PHL active site in the CRYs, we found that PER2 and FBXL3 interact extensively with sector positions near the FAD-binding pocket (where DNA lesions directly interact with PHLs) and around CRY2’s CC helix. A logical hypothesis is that the secondary pocket has also been repurposed for regulatory interactions with CLOCK/BMAL1, with the residues in the yellow subgroup representing candidates for specifically contributing to the allosteric mechanism.We mapped the network of residues identified by SCA onto structures of the CRY2/PER2 CRY-binding domain (CBD) complex  driven by a Per2 promoter) in Cry1−/−/Cry2−/− mouse embryonic fibroblasts  interface (α4) of the secondary pocket cause local disruptions in CRY1’s structure rather than global disruption of its protein fold.In order to test residues identified in the secondary pocket in the SCA for a role in regulation of the clock mechanism, we first focused on the α4 helix between E103 and R109 of CRY1, where many , but not all , amino acids were identified in the SCA Fig. . To teststs Fig. . We teststs Fig. . In addi19. Ultimately, in concert with this report, we conclude that the secondary pocket is a site for interaction with CLOCK specifically. These results also support the predictions from SCA that a subset of residues in the secondary pocket primarily mediate regulation of this protein–protein interaction.In contrast, even severe mutations of the non-sector residues at positions 107 (K107E) and 108 (E108K) of CRY1 did not impair CRY1’s ability to rescue rhythms Fig. , and theDrosophila), some have only a type II CRY , and some have both 47. We hypothesized that an evolving interface for direct interaction between CRY and core clock components might underlie this evolutionary divergence. Our finding that the secondary pocket is an interface for binding between CLOCK and CRY led us to look carefully at other residues in the secondary pocket identified in the SCA. In addition to the “lower” helical boundary of the pocket discussed above (α4), a subset of residues was identified in a flexible loop forming the “upper” boundary of the pocket (located between β2 and α2 in CRY1). An alignment of the α4 helix residues and the “upper” loop residues from various vertebrate and insect sequences reveals divergence at both sites between the two groups  can be found in various combinations in different animals. For example, some insects have only a type I CRY  caused either weakly repressive, short period rescues (P39G), or arrhythmicity  Fig. . Substit1S, G106W48. In order to test whether substitutions at these residues were causing a change in the degradation rate of CRY, we expressed a CRY1::LUC fusion protein in HEK-293A cells and treated with cycloheximide to block new protein synthesis, monitoring the decay in luminescence as a reporter for protein degradation. Introducing a mutation that stabilizes CRY1 (S588D)29 led to deceleration of the rate of luminescence signal decay  affected the rate of decay  and Cry2−/− mice have long endogenous periods (~24.6 h)9. Moreover, CRY1 functions as a stronger repressor of CLOCK/BMAL1-mediated transcriptional activation36. Using the Cry1 promoter to drive Cry2 expression in Cry1−/−/Cry2−/− mouse embryonic fibroblasts, Khan et al.27 reported that they were unable to rescue rhythms with WT Cry2. However, if they swapped residues 313–426 of CRY1 for the homologous residues in CRY2, this chimeric CRY2 was able to rescue rhythms. This experiment suggests that some set of differential residues within this domain is likely to play a critical role in driving repression of CLOCK/BMAL1-mediated transcriptional activation. There are only 12 residues that are different between CRY1 and CRY2 in this 113 amino acid domain. Intriguingly, five of those residues  are located superficially at the “right” boundary of the secondary pocket  and CRY2 were competent to rescue rhythms . The effect of these mutations was again additive  and found a small effect of dose on period length  competition assay  or the tail residues  were swapped Fig. . All fou24. Here we describe how a hotspot of evolutionary changes in the CPF lineage, the secondary pocket, has also been repurposed for a crucial protein–protein interaction with the CLOCK/BMAL1 heterodimer.CRY’s evolution into a role as a direct repressor of CLOCK-mediated and BMAL1-mediated transcriptional activation suggests that specific structural changes have resulted in critical functional advantages. Much focus has been placed on the FAD-binding pocket of this family and significant work has emphasized the role of FAD in the function of plant CRYs and type I animal CRYs. Moreover, structural work from the past few years highlights the extent to which this pocket has been repurposed for protein–protein interactions with FBXL3 and PER251. Moreover, a recent report suggests that a common human mutation, which results in the deletion of 23 residues in CRY1’s tail, results in stronger binding between CRY1 and the CLOCK/BMAL1 heterodimer22. Additionally, mutations on CLOCK’s HI loop cause a complete abrogation of binding between CLOCK and CRY53 and a recent report suggests that CLOCK’s PAS-B domain, which contains the HI loop, interacts directly with the secondary pocket19. Taken together with our results, these data suggest a model in which an interaction between CLOCK’s HI loop and CRY at the secondary pocket brings CRY into proximity with the BMAL1 TAD to create a sustained repressive complex. Tuning the affinity between CRY’s CC helix and the BMAL1 TAD was shown to have a substantial effect on the period of the oscillation18. Similarly, we found that mutations in the secondary pocket that strengthen the interaction with CLOCK and BMAL1 lengthen the period of the oscillation and mutations that weaken the interaction predominantly shorten the period. Critically, the effects on period were independent of CRY’s degradation rate. Historically, spontaneous and induced mutations in fly57 and mammalian61 genomes have helped to identify clock components that affect periodicity primarily by modulating the rate of decay of repressors. Our work demonstrates that the affinity of the core clock proteins for one another plays an important orthogonal role in determining periodicity and is in agreement with two other recent reports with similar findings49. Additionally, it is not yet clear whether modulating the degradation rate of CRY would rescue interactions between mutant CRYs and other core clock binding partners. One might expect that stabilizing CRYs with slightly weakened interactions would rescue periodicity by restoring the equilibrium of activator/repressor complexes. However, this idea has yet to be tested.CRY’s C-terminal α-helix and tail have been shown to form a functionally important complex with BMAL1’s TAD with a ~1–10 μM affinityCry1−/− and Cry2−/− mice has long been mysterious. At a behavioral level, deletion of one or the other leads to a dramatically different output, but molecular analysis suggests a similar functional role. Amidst high sequence identity and similarity between the two proteins62, no obvious structural differences present themselves as possible drivers of functional dissimilitude beyond the divergent tails. The data presented here strongly suggest that the functionality of these proteins stems in large part from the accumulation of subtle structural differences at the secondary pocket, which reduce the strength of the interaction between CRY2 and the CLOCK/BMAL1 heterodimer. Strikingly, co-expression of PER2 markedly improved the strength of the interaction between both CRY2 and the CRY1 7m mutant and the CLOCK/BMAL1 heterodimer. In concert with previous work demonstrating concurrent temporal occupancy for CRY2, PER1, and PER2 in chromatin association37, our data suggest that CRY2 requires either PER1 or PER2 to form a stable repressive complex with CLOCK and BMAL1, restricting its repressive phase to the phase of PER expression. CRY1, due to its stronger physical interaction with CLOCK/BMAL1, is perhaps capable of forming a stable complex without a PER  in an intron of the mCry1 gene body is necessary for the delayed repressive phase of CRY1. A recent report by Edwards et al.68, in contrast, showed that a small fragment of the mCry1 promoter not including the RRE was sufficient to drive rhythmic expression of either CRY1 or CRY2 in brain slices containing the suprachiasmatic nucleus (SCN), a brain region that functions as a master oscillator. Moreover, they found that Cry1 and Cry2 were individually sufficient for rescue in Cry1−/−/Cry2−/− SCN and drove rhythmic output characteristic of Cry2−/− (long periods) or Cry1−/− (short periods) animals, respectively. Our results, in concert with these two reports, suggest that more work is necessary to understand the contributions of transcriptional regulation, protein–protein interaction dynamics, degradation, and intercellular coupling to periodicity in CRY1-driven and CRY2-driven rhythms. However, it is likely that each of these characteristics of the clock function as nodes for regulation of periodicity.Ukai-Tadenuma et al.Our rescue data demonstrate that divergence at the secondary pocket drives a great deal of the repressive strength and periodicity differences between the two CRYs. However, it is also clear from rescues with CRY2 7m that this divergent structural feature cannot fully explain the differences in periodicity. The CRY1 tail and secondary pocket are together necessary and sufficient to fully convert CRY2’s rescue profile to a CRY1-like profile. We have developed a mechanistic understanding of the secondary pocket’s contribution to periodicity, but the mechanism underlying the tail’s contribution is still unknown. It is, however, interesting to view the results of the chimera rescues in an evolutionary context. Exchanging either the tail or secondary pocket between the two CRYs invariably resulted in near 24 h rhythms, suggesting that in organisms with a single repressive CRY, such as the honey bee, one or the other of these features, but not both, is likely to remain intact. Given the low level of conservation in C-terminal tails, it is tempting to speculate that the delay characteristic of the CRY1 tail is in fact a gain-of-function mutation specific to organisms with two repressive CRYs. The nature of this function is a fertile area for future research.Finally, the secondary pocket presents a potent opportunity for drug design. The data presented here suggest that antagonizing the interaction of CLOCK and BMAL1 at the secondary pocket has the potential to speed up the clock. Thus, small molecules designed to dock at this cavity could accelerate the clock, potentially functioning as a jetlag drug for west-to-east travelers. Advances in computational approaches to drug discovery require targeted selection of protein structural features based on a mechanistic understanding of the underlying biology. The data provided here form the basis for such further inquiry.Primers used in this study are detailed in Supplementary Table Cry1−/−/Cry2−/− mouse embryonic fibroblasts were generated by Andrew Liu and Hiroki Ueda46 and were gifted to us. HEK-293A cells were purchased from Thermo Fisher Scientific. Both lines were grown in Dulbecco’s modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1× Pen/Strep antibiotics at 37 °C under 5% CO2. LumiCycle recording medium was prepared from powdered DMEM without phenol red containing 4.5 g/L glucose and supplemented with 10 mM HEPES, pH 7.20, 100 μM luciferin, 1 mM sodium pyruvate, 0.035% sodium bicarbonate, 2% FBS, 1× Pen/Strep antibiotics, and 2 mM l-glutamine. LumiCycle recordings were performed at 37 °C.69 locally for two iterations. Extraneous header information was removed using a custom python script before performing a secondary alignment using Promals3D with six additional structure sequences (cyclobutane pyrimidine dimer (CPD) PHL from Escherichiacoli, CPD PHL from Anacystisnidulans, 6–4 PHL from Arabidopsisthaliana, CRY from Drosophilamelanogaster, and CRY1 and CRY2 from Musmusculus73. Using a custom python script, GI numbers were collected from each header in a separate file for use in the annotation step of the SCA. SCA calculations were performed using the pySCA toolbox as described44. The annotation step was performed through NCBI using the GI numbers on April 17, 2016. The initial alignment of 9719 sequences consisted of 4344 independent positions. The alignment underwent preprocessing during which highly gapped positions were removed. After removing highly gapped positions, remaining highly gapped sequences were removed using a set cutoff. Sequences with too great of a fractional identity to the reference sequence were also removed. Finally, each sequence was weighted based on the number of sequences with an identity above 80% to the given sequence. This final weighting step allows an effective number of sequences (M′) to be computed based on the remaining number of actual sequences (M). After all of the preprocessing steps, the final alignment contained 5385 sequences representing 2447 effective sequences composed of 459 positions. Critically, an alignment should be large and diverse enough to give reasonable estimates of amino acid frequencies. Two thousand four hundred and forty-seven effective sequences is well above the suggested minimum of 100 effective sequences needed to obtain a reasonable estimate44. During these processing steps, default parameters were used and residues were mapped to the CRY2 structure (PDB: 4I6E)24. Following initial calculation steps, the workflow was performed in an ipython notebook.Approximately 10 000 CRY and PHL sequences were collected from NCBI on March 30, 2016 using the full-length mCRY1 protein sequence (GenBank Accession ID: AAH85499.1) as a search sequence for a PSI-BLAST. PSI-BLAST parameters were adjusted from default to include an Expect Threshold of 0.01, a PSI-BLAST Threshold of 0.005, and a minimum sequence identity of 20%. PSI-BLAST was performed iteratively for two rounds before the sequences were downloaded. The sequences were initially filtered by size using a custom python script to remove sequences smaller than 400 or larger than 800 residues. Remaining sequences were subjected to an initial alignment using the alignment tool MUSCLE44. Conservation values for individual amino acid positions were calculated as a Kullback–Leibler relative entropy (Di), a measure that quantifies the divergence of amino acid frequencies at position i from the distribution of amino acid frequencies expected randomly. Values of Di near zero indicate that amino acid frequencies at that site approach random expectation, while values of more than 3 indicate highly conserved positions (>~83% residue identity). Co-evolution between all pairwise combinations of amino acids over all L positions was computed as an L x L x 20 × 20 conservation-weighted co-variance matrix:a at site i . The basic idea is that positions experiencing a joint functional constraint will co-vary in an alignment; weighting the co-variance by the degree of conservation Here we provide a brief overview of the SCA calculations; the complete mathematical formalism and methods as used here are more completely presented in ref In order to identify correlated groups of positions within the protein, we analyzed the SCA matrix mentclass2pt{minimmentclass2pt{minimhttp://nebasechanger.neb.com/). Template DNA was mixed with 2× Q5 polymerase chain reaction (PCR) Master Mix and primers before being subjected to PCR. Following PCR, 1 μL of each PCR product was combined with 2× Quick Ligation buffer and ligase, along with 1 μL of T4 polynucleotide kinase, and 1 μL of DpnI in a total of 10 μL. The ligation reaction was incubated at room temperature for 5 min and 5 μL of product were used to transform DH5α-competent cells. Colonies were selected for culturing and miniprep, and mutations were verified by Sanger sequencing.Mutagenesis was performed using both the QuikChange II XL kit and, with a few modifications, the Q5 Site-Directed Mutagenesis kit. Manufacturer's instructions were followed for QuikChange mutagenesis. For Q5 Site-Directed Mutagenesis, primers were designed using the NEBaseChanger tool  and Cry2 (residues 516–593) were amplified by PCR using primers that would generate overlaps between the amplified tail product and the linearized target vectors. PCR products were purified using the QIAquick PCR Purification kit and combined with inserts and linearized target vectors in a 2:1 molar ratio. These combined products were then treated with NEBuilder HiFi DNA Assembly master mix containing an exonuclease, DNA polymerase, and DNA ligase to induce assembly of the final vector. This solution was incubated at 50 °C for 15 min and the products were used to transform DH5α competent cells. Colonies were selected for culturing and miniprep, and insertions were verified by Sanger sequencing.CRY tail chimera rescue vectors were generated by Gibson assembly cloning using NEBuilder HiFi DNA Assembly kit. Briefly, the rescue vector containing 5Cry1−/−/Cry2−/− mouse embryonic fibroblasts were plated in 35 mm tissue culture dishes and transfected the same day with 4 μg of a luciferase reporter (pGL3-P(Per2)-Luc) and 150 ng of a Cryptochrome rescue vector (pMU2-P(Cry1)-(intron336)-Cry-Myc, modified with a C-terminal MYC tag)46 using FuGENE 6 according to the manufacturer's instructions. At 72 h after transfection, the cells were synchronized by exchanging growth medium for growth medium supplemented with 0.1 μM dexamethasone and returned to the incubator for 2 h. The medium was then replaced by LumiCycle recording medium and the plates were sealed with vacuum grease and cover glass and transferred to the LumiCycle. Bioluminescence monitoring was performed using a LumiCycle to record from each dish continuously for 70 s every 10 min using a photomultiplier tube at 37 °C. Rescue results were processed using the LumiCycle Analysis software package. The first 10 h of recording were discarded and period, amplitude, phase, and damping rate were calculated using a damped sine wave based on a running average fit for each plate of cells. Rescues were considered arrhythmic if the goodness of fit for the damped sine wave was <80%. Subsequent data were corrected for background noise in each photomultiplier tube channel, which was measured by monitoring an untransfected plate of cells for 24 h and averaging the signal. All CRY1 and CRY2 mutants were run with WT CRY1 as an internal control. In some cases, multiple experiments were combined in the data shown if there were positive internal controls for each dataset. Rescues were excluded only in cases where the transfection did not work, which was obvious based on both a very weak or non-existent luminescence signal and the lack of induction of luminescence shortly after synchronization. Appropriate replicate size was determined empirically from extensive use of these assays in previous work20.A total of 4 × 10mCry2 cDNA was cloned into the pMU2-P(Cry1)-(intron336)-Myc rescue vector backbone using megaprimer mutagenesis. Luciferase-tagged constructs were also generated using megaprimer mutagenesis. Briefly, the relevant insert was copied from a source vector by PCR (using PfuUltra II Fusion HS DNA polymerase with primers containing roughly 30 bp of overlap in either direction with the insertion site in the target vector and 30 bp of overlap with the insert). Following primary PCR, PCR products were purified using the QIAquick kit, and 400 ng of purified product used as a megaprimer for a secondary PCR with the target vector serving as a template. PCR products were treated with DpnI for several hours, and then DH5α-competent cells were transformed with 5 μL. Colonies were selected for culturing and miniprep and insertions were verified by Sanger sequencing. Photinuspyralis Luciferase was fused directly to the C terminus of Cry1 and Cry2 in the pCMV-Tag3C-Myc vector. Full-length Cry2 was inserted at the exact location in pMU2-P(Cry1)-(intron336)-Myc where Cry1 was deleted.Full-length 5 cells per well. Cells were transfected the same day with FuGENE 6. The following constructs were used for all transfections: p3X-FLAG-CMV-10-mBmal1, p3X-FLAG-CMV-10-mClock, pCMV-Myc-Cry121, pCMV-Myc-Cry221, and pcDNA3.1-mPer2-V574. For IPs involving all four clock components, cells were transfected with 45 ng of mBmal1, 1.5 μg of mClock, 600 ng of mPer2, and 150 ng of either Cry1, Cry2, or mutants thereof, in a total of 2.295 μg of DNA. Empty vector (pcDNA3.1-A) was used to even out DNA cocktails. For IPs of only BMAL1, CLOCK, and CRY, cells were transfected with 45 ng of mBmal1, 1.5 μg of mClock, and 150 ng of Cry plasmid in a total of 1.695 μg of DNA. Finally, for IPs of PER and CRY alone, cells were transfected with 600 ng of mPer2 or 300 ng of Cry in a total of 900 ng of DNA. After 48 h, cells were lysed in 200 μL of TGED buffer ) for 30 min before a 10 min centrifugation to remove cellular detritus. Ten percent of the supernatant solution was saved for use as input and the remainder was incubated for 3 h with 40 μL of anti-MYC-conjugated beads to immobilize MYC-CRY. Beads were washed with 1 ml of TGED buffer twice. Protein was released from beads by boiling in 50 μL of sodium dodecyl sulfate (SDS) sample buffer  and analyzed by immunoblot using anti-MYC, anti-V5, and anti-FLAG-HRP (horse radish peroxidase) for CRY, PER2, and CLOCK/BMAL1, respectively. Catalog numbers for the antibodies can be found in Supplementary Table 20.HEK-293A cells were seeded in 6-well tissue culture plates at a density of 4 × 105 cells per well. Cells were transfected (FuGENE 6) same day with 500 ng of a Cry-Luc fusion construct (pCMV-Myc-Cry1-Luc or pCMV-Myc-Cry1-Luc) and 200 ng of an EGFP construct (pEGFP) as a transfection control. At 48 h after transfection, the medium was exchanged for LumiCycle recording medium supplemented with cycloheximide (100 μg/ml). Plates were transferred to the LumiCycle and bioluminescence was monitored from each dish continuously for ~70 s every 10 min using a photomultiplier tube at 37 °C. Data were corrected for background noise from the photomultiplier tube as described above. The first 10 h of recording were normalized to the first data point and half-life was determined by nonlinear, one-phase exponential decay analysis.HEK-293A cells were plated in 35 mm tissue culture dishes at a density of 4 × 10CRY alignments for Fig. 75. PCR-amplified Cry1 and Cry2 cDNAs were inserted into the NheI and AgeI sites upstream of the coding region of CerN and VenN. Full-length Clock cDNA was amplified by PCR with an N-terminal SacII site and a C-terminal blunt polymerization end and ligated into SacII and SmaI sites in CerC. Residues 2–88 and 396–855 of Clock were deleted from the construct by site-directed mutagenesis. Truncated Venus (VenN (1–155 amino acids)) and Cerulean (CerN (1–155 amino acids), CerC (156–239 amino acids)) fragments were previously generated in the pEGFP-C1 (Clontech) backbone by site-directed mutagenesis15.Full-length Venus (Ex515/Em528) and Cerulean (Ex433/Em475) have been described previously76 was mixed with 200 ng CerC-Clock (89–395), 10 ng p3X-Flag-Bmal1, 200 ng of Cry1-VenN or Cry1-CerN, and 200 ng of Cry2-CerN or Cry2-VenN in a total of 735 ng of DNA. A total of 8 × 104 HEK-293A cells were plated in each well of a 24-well black Visiplate. On the same day, cells were transfected with FuGENE 6. Plates were washed with phosphate-buffered saline (PBS) once 48–60 h after transfection, fixed with 4% paraformaldehyde in PBS for 15 min, and then immersed in PBS. A control well for background fluorescence was transfected with non-complementary BiFC vectors . Controls for normalization of the competition experiments were transfected with single complementation pairs , 10 ng p3X-Flag-Bmal1, and 200 ng of Cry2-CerN or Cry2-VenN in a total of 735 ng of DNA).For BiFC competition experiments, 25 ng H2B-mRFP1Fluorescence images were acquired on a Deltavision Personal DV Imaging System equipped with an inverted 10× (for quantification) or 20× (for publication images) 0.45NA UPLFL objective and a Microtiter stage on an Olympus IX71 microscope. Four locations in each well were selected and autofocused with red fluorescent protein 1 (RFP1) fluorescence. Single layer images for quantification or 20-layer Z stacks with 1 mm steps for deconvolution and presentation were scanned in the channel sequence of RFP , YFP , and CFP  filter sets with the Z step first. Image stacks were deconvoluted with the Softworx deconvolution module and maximal intensity Z projections of layers were built.For quantification of BiFC competition results, original image files were imported to and organized by ImageJ (NIH), and exported image sequences were pushed through a custom pipeline run by Cellprofiler. Briefly, the nuclei were first recognized based on RFP fluorescence, and then inverse-masked with strong aggregates in the Venus and Cerulean channels. Mean Venus and Cerulean intensity values of each masked nucleus were measured. About 40–120 cells were identified and measured in each image, and the average values of cells in one image were obtained for further normalization and statistical analysis. Background intensity values from Venus and Cerulean channels were subtracted from competition experiment intensity values. Background subtracted competition values were then normalized to background subtracted intensity values from single complementation wells. Four images from each well were averaged and the experiments were replicated three times.t tests with Welch’s correction were performed by GraphPad Prism software. Welch’s t test was chosen to compare the period means and half-life means because we do not know whether the various mutant populations have equal variances to the WT protein. Protein half-lives were determined by normalizing each luminescence value of a given sample to the initial luminescence reading, and then fitting a one-phase decay curve to the resulting data in GraphPad Prism.Statistical parameters are reported in the figure Legends and indicated in the figures where appropriate. Unpaired Custom scripts used in processing the SCA data, the alignment used for the SCA, the annotated SCA notebook, and the SCA database file are all available upon request. The custom data pipeline for CellProfiler is available upon request.Other data are available from the corresponding author upon reasonable request.Supplementary Information(PDF 7767 kb)Peer Review File(PDF 454 kb)


High-grade gliomas possess internal pathological heterogeneity. Selective sampling of different tumor regions can help in the study of this heterogeneity. In this report, we have described the use of a novel navigation and optical system for the selective regional sampling of a high-grade glioma lesion. A 45-year-old gentleman presented to us with complaints of intermittent frontal headaches for past eight months. On examination, he had subtle pyramidal weakness in left upper and lower extremities. Magnetic resonance imaging (MRI) showed a large contrast-enhancing, space-occupying lesion in the right frontal lobe causing perilesional edema and midline shift. We marked four different regions on the preoperative MRI using apparent diffusion coefficient (ADC) mapping and contrast enhancement pattern in four different combinations using presurgical planning software (BrightMatter™ Plan) . These pre-identified areas were exported into BrightMatter™ Servo , an integrated robotic video microscope with a neuronavigation system where these areas were selectively sampled and sent for analysis. The BrightMatter™ Servo not only helped us to the target areas but also helped to identify a safe trajectory, respecting white matter tracts. Histopathology showed a neoplastic lesion composed of mononuclear round cells with the perinuclear halo in a fibrillary stroma with admixed mini-gemistocytes consistent with the diagnosis of a Grade 3 anaplastic astrocytoma. A selective regional sampling of the gliomas can be reliably performed using BrightMatter™ technologies to study the pathological heterogeneity of these lesions. Gliomas are heterogeneous in their oncogenic expression and pathological characteristics -2. The d™ Servo .Diffusion magnetic resonance imaging is a non-invasive tool for mapping the brain based on the diffusion characteristics of water molecules in its various regions . This im™ Servo is a novel integrated system comprising of a video microscope (BrightMatter™ Drive) that can align to surgical instruments and a neuronavigation system (BrightMatter™ Guide) that can display whole brain tractography. The use of BrightMatter™ Servo has been demonstrated in both cranial and spinal applications [™ Servo.BrightMatterications -8. It ca™ Plan. This showed the tumor to be displacing rather than infiltrating the right-sided superior longitudinal fasciculus laterally, forceps minor, and genu of corpus callosum posteriorly. The tumor appeared heterogenous on diffusion-weighted images (DWI) and ADC mapping. We marked four different regions on the preoperative MRI using ADC mapping and the contrast enhancement pattern in all four possible combinations. These signal combinations included low ADC and high-contrast (Area 1), low ADC and low-contrast (Area 2), high ADC and high-contrast (Area 3), and high ADC and low-contrast (Area 4). The marked areas are shown in Figures ™ Plan where information from different MRI sequences and tractography can be integrated and evaluated to mark different target areas. The patient underwent an awake craniotomy with the help of the BrightMatter™ Servo. These pre-identified areas were exported from the BrightMatter™ Plan to the BrightMatter™ Servo. Using the ADC mapping and tractography, BrightMatter™ Servo navigated us to the target areas through a safe trajectory. To minimize the effect of brain shift, samples were taken before debulking of the tumor. Approximately 1 cm2 samples were obtained from each of these target areas. The resection was helped by a video microscope. After completion of selective sampling, we continued our resection with the help of the BrightMatter™ Servo to achieve a gross total resection.A 45-year-old male patient presented to us with complaints of intermittent frontal headaches associated with nausea and vomiting for the previous eight months. On examination, he was alert and oriented with normal higher mental functions and intact cranial nerves. He had subtle pyramidal weakness in the left upper and lower extremities. MRI of the brain showed a large contrast-enhancing, space-occupying lesion in right frontal lobe causing peri-lesional edema and midline shift. The lesion was also compressing the lateral ventricles. The tractography was generated using the BrightMatterast Area , low ADCPostoperatively, the patient remained well; there were no new neurological deficits and the recovery was unremarkable. He was discharged after three days. MRI of the brain done 24 hours postoperatively showed a gross total resection.Histopathology showed a neoplastic lesion composed of mononuclear round cells with a perinuclear halo in a fibrillary stroma with admixed mini-gemistocytes, consistent with the diagnosis of a Grade 3 anaplastic oligoastrocytoma. Pathological features of different areas are provided in Table High-grade gliomas are genetically and pathologically heterogeneous lesions . SelectiThe BrightMatter™ Plan can be used in the analysis of tractography, preoperative MRI images, identification of targets, and choosing safe trajectories to each of the targets. The plan thus made can be exported to the BrightMatter™ Servo where the information can help navigate (BrightMatter™ Guide) and perform sampling (BrightMatter™ Drive). The risk of injury to normal cortex and white matter while attempting selective sampling of different areas can be minimized using this integrated system.Use of a video microscope is becoming popular among neurosurgeons -8. Howev™ Servo to study the pathological and genetic heterogeneity of these lesions. The technique can potentially minimize the errors of sampling while performing a tumor biopsy.  The selective regional sampling of the gliomas can be reliably performed using BrightMatter


Supplemental Digital Content is available in the text Recently, it has been reported that the relative dose intensity (RDI) of adjuvant chemotherapy (AC) influences survival in various cancers, but there are very few reports about RDI in pancreatic ductal adenocarcinoma (PDAC). The optimal timing for initiation of AC for PDAC also remains unknown. The aim of this study was to identify the significance of RDI and the time interval between surgery and initiation of AC on survival of patients with PDAC. Clinicopathological factors that affect RDI were also investigated.A total of 311 consecutive PDAC patients who underwent curative resection between May 2005 and January 2015 were enrolled. Patients who underwent neoadjuvant chemoradiation, had UICC stage IV disease, or had early recurrences within 6 months were excluded, and the remaining 168 cases were analyzed.P < 0.001). Patients with no AC (n = 34) showed the worst OS (MST: 20.8 months). Whether the AC was initiated earlier or later than 8 weeks after surgery did not influence survival, either in patients with RDIs ≥80% (P = 0.79) or in those with <80% (P = 0.73). Patients in the S-1 monotherapy group (n = 49) showed significantly better OS than patients in the gemcitabine monotherapy group (n = 51) . Univariate analysis conducted after adjusting for the chemotherapeutic drug used identified several prognostic factors; male gender (P = 0.01), intraoperative blood transfusion (P = 0.005), lymph node metastasis (P = 0.03), and postoperative WBC count (P = 0.03). Multivariate analysis identified intra-plus postoperative blood transfusion (P = 0.002) and high postoperative platelet-to-lymphocyte ratios (PLR) (P = 0.04) as independent predictors of poor RDI.Patients with RDIs ≥80% (n = 79) showed significantly better overall survival (OS) compared to patients with RDIs <80% (n = 55) : 45.6 months, 26.0 months, Efforts to maintain RDI had a greater impact on survival than the struggle to start AC early after surgery. Intra-plus postoperative blood transfusion and a high postoperative PLR could be predictive markers of reduced RDI in AC of PDAC patients. Avoidance of perioperative blood transfusions where possible and nutritional support during the perioperative period could maintain adequate RDI and may lead to improved long-term outcome. The European Study Group for Pancreatic Cancer (ESPAC) demonstrated through several large-scale randomized controlled studies that AC should be the standard of care for resectable PDAC;–7 these studies demonstrated that gemcitabine or fluorouracil plus folic acid significantly improved the prognosis of PDAC compared to surgery alone. In Japan, following the results of the Japan Adjuvant Study Group of Pancreatic Cancer (JASPAC)-01, adjuvant S-1 chemotherapy was established as a standard of care for resectable PDAC.The prognosis of pancreatic ductal adenocarcinoma (PDAC) remains poor despite recent improvements in surgical technique,–13 In PDAC patients, however, there are only two reports concerning RDI of AC and prognosis,15 and the factors affecting RDI. Furthermore, starting AC within less than 8 weeks has been recommended in colon cancer management,,17 although the optimal timing for initiation of AC for PDAC remains unknown.In several cancers treated with AC, a decreased relative dose intensity (RDI) has been associated with a poor prognosis.The aim of this study was to investigate the effect of the RDI and the time interval between surgery and initiation of AC on survival of patients with PDAC. Clinicopathological factors affecting the RDI of AC were also explored.22.1We enrolled 311 consecutive PDAC patients who planned to undergo curative resection at the Department of Gastroenterological Surgery, Nagoya University Hospital, Japan, between May 2005 and January 2015. The histological diagnosis of PDAC was confirmed for all patients. Written informed consent for inclusion in the study, as required by the Institutional Review Board of Nagoya University, was obtained from all patients.Of these 311 patients, 177 patients were excluded for the following reasons: received neoadjuvant chemoradiation (n = 41), diagnosed as having distant metastasis during surgery (n = 21), underwent middle pancreatectomy due to different preoperative diagnosis (n = 1), surgical death (n = 2), followed by another hospital after discharge (n = 59), and curative resection without adjuvant chemotherapy (AC) (n = 34). As a result, 153 patients received AC after surgery at our institution . In these patients, 19 developed early recurrence within 6 months.2.2 The pathological stage of PDAC was assessed according to the TNM classification system of malignant tumors published by the International Union Against Cancer .All PDACs were determined to be surgically resectable before surgery. Operative methods were determined based on tumor location. All patients underwent regional lymph node dissection. If portal vein infiltration was suspected during surgery, combined portal vein resection was performed. Reconstruction after pancreatoduodenectomy (PD) was performed according to the modified Child method.2.3 was disclosed in our country. After the disclosure, essentially all the patients with PDAC were administered S-1. The gemcitabine plus S-1 regimen (n = 34) was only used for patients registered in clinical trials. The S-1 regimen consisted of 80 to 120 mg, depending on body surface area, administered twice a day for 4 weeks and repeated every 6 weeks for 4 courses, or for 2 weeks and repeated every 3 weeks for 8 cycles. The gemcitabine regimen consisted of 1000 mg/m2 given by intravenous infusion over a 30-minute period. Patients receiving gemcitabine were scheduled for 6 cycles every 4 weeks followed by a 1-week pause. In the gemcitabine plus S-1 regimen, patients received gemcitabine  plus S-1  every 2 weeks for 12 cycles.The AC regimen used for each patient was either S-1 monotherapy (n = 49) or gemcitabine monotherapy (n = 51). Gemcitabine had been the drug of choice for AC until the result of JASPAC-01AC was planned to be 6 months in duration and was initiated as soon as possible after the patient was discharged from the hospital. Postoperative blood test data were evaluated at the onset of AC, and both blood tests and clinical symptoms were checked every visit. Computed tomography was performed every 3 months after discharge to monitor relapse.2.4 For each drug within each regimen, the total dosage that the patient received was divided by the total dosage specified by the corresponding standard regimen. Since the median value of the RDI was 80%, we set a cutoff value of 80% and defined an RDI ≥ 80% as complete AC and an RDI <80% as incomplete AC.RDI was calculated for each patient according to the method proposed by Hryniuk and Bush.2.5Postoperative complications were evaluated by means of a modified Clavien grading system. A complication of grade III or higher was considered a clinically significant event. The classification system of the International Study Group on Pancreatic Fistula (ISGPF) was used to predict postoperative pancreatic fistula.2.6P < 0.05.Continuous variables are expressed as the mean ± standard deviation and the range. Overall survival (OS) was calculated using the Kaplan–Meier method, and the differences in survival curves were analyzed using the log-rank test. Univariate and multivariate logistic regression analyses were performed to analyze factors associated with complete AC. In addition, multivariate Cox proportional hazard model was used to determine independent risk factors associated with OS. Data were analyzed using JMP v10 software . The level of statistical significance was defined as 33.1P < 0.001). Patients with RDIs <80% also had significantly more combined portal vein resections (P = 0.02), intra-plus postoperative blood transfusions (P = 0.007), lymph node metastases (P = 0.001), dissected peripancreatic tissue margins (pDPM) (+) (P = 0.004), lower postoperative serum albumin levels (P < 0.001), lower white blood cell (WBC) counts (P = 0.03), and higher platelet-to-lymphocyte ratios (PLR) (P = 0.02). The postoperative blood transfusion was defined as transfusion during hospitalization. A total of 18 patients received intra-plus postoperative blood transfusion, and the breakdowns were as follows; both intra and postoperative (n = 2), intraoperative (n = 13), and postoperative (n = 3). In addition, there were 34 patients who did not receive AC. These patients were significantly older than the patients with RDIs<80%  and included 3 patients who were under dialysis, but there were no significant differences regarding other clinicopathological factors.Patient demographics and clinical characteristics are listed in Table 3.2P < 0.001]. Patients with no AC showed the worst OS (MST: 20.8 months). Patients were then classified into 4 groups according to both the RDI and the time from surgery to AC as follows: Group 1, RDI ≥80%/ <8 weeks (n = 53); Group 2, RDI ≥80%/ ≥8 weeks (n = 26); Group 3, RDI <80%/ <8 weeks (n = 37); and Group 4, RDI <80%/ ≥8 weeks (n = 18) , and the MST was 45.8 months in Group 1 and 43.8 months in Group 2. Similarly, no effect was observed in the group with an RDI of less than 80% (P = 0.73); the MST was 25.8 months in Group 3 and 29.7 months in Group 4. In the group of patients who started AC within 8 weeks, the RDI significantly affected the OS . Similarly, a trend was observed for RDI to correlate with OS in the group starting AC after the first 8 weeks .The OS of patients stratified by the RDI is shown in Fig. P = 0.02). In the gemcitabine monotherapy group, there was also a significant difference in OS between patients with RDIs ≥80% (n = 20) and patients with RDIs <80% (n = 31)  . The patients in the S-1 monotherapy group showed significantly better OS than patients in the gemcitabine monotherapy group . There were significantly more patients with RDIs ≥80% in the S-1 monotherapy group than in the gemcitabine group  , and there were significantly more patients with advanced pathological UICC stages in the gemcitabine monotherapy group than in the S-1 monotherapy group (P = 0.002) . For these reasons, the following analyses were conducted to identify predictive factors for poor RDI after adjusting for the anticancer drug used during AC.We also analyzed OS according to the chemotherapy regimen. In the S-1 monotherapy group, patients with RDIs ≥80% (n = 32) showed significantly better OS than patients with RDIs <80% (n = 17)  , lymph node metastasis , and postoperative WBC count . We performed a multivariate analysis using 9 factors that showed a meaningful tendency in the univariate analysis (Table P = 0.01), intra-plus postoperative blood transfusion , and postoperative PLR  identified as independent predictors for completion of AC. We also performed univariate and multivariate analyses of clinicopathological factors for OS . Univariate analysis of OS using the Cox proportional hazards model identified the poor prognostic factors as follows: lymph node metastasis, incomplete AC, and low postoperative serum albumin level. The results of multivariate analysis revealed that complete AC was independently associated with good prognosis .Univariate analysis was performed using various preoperative, operative, and postoperative factors and including the drug used for AC (either S-1 (n = 49) or gemcitabine (n = 51)) as covariates Table . This an4,15 In these reports, high RDIs and successful execution of the planned treatment were associated with an improved prognosis. The results of the present study were consistent with these reports, as a decrease in the RDI was strongly associated with a poor prognosis. In addition, the postoperative PLR, one of the nutritional indexes that predict survival, also affected the RDI. This was also consistent with the earlier report that the postoperative serum albumin level was an independent predictor for maintaining the RDI. However, unlike the case of colorectal cancer, a time interval of longer than 8 weeks before initiation of AC did not adversely influence survival.Use of a sufficient dosage and drug administration according to the schedule determined by clinical trials are the basic principles of chemotherapy for cancer. Thus, the RDI is often considered an important prognostic factor among patients who undergo postoperative adjuvant chemotherapy. To our knowledge, however, only two reports have been published concerning the RDI of AC for patients with PDAC.Postoperative nutritional status in terms of the loss in body weight and lean body mass is considered to affect adherence to postoperative chemotherapy in patients with gastric cancer.,22 Because the PNI and GPS greatly depend on the serum albumin level, we substituted the albumin level for these measures. In the current study, the PLR was shown to be a significant predictor of the RDI. Platelets and lymphocytes are considered to be significant parameters related to the immune condition, and the lymphocyte count in particular reflects ability of the individual to eliminate tumor cells. Actually, there are some reports to demonstrate that high lymphocytic infiltration to the tumor site could be associated with superior response to systemic chemotherapy.,25 In PDAC, the PLR was reported to correlate with prognosis after surgery. To the best of our knowledge, there have been no reports implicating correlation between toxicity of anticancer agent and PLR. However, high lymphocytes might be associated with reduced toxicity through enhanced immune status and increased sensitivity to the chemotherapy.Surgery for PDAC is well-recognized as one of the most invasive procedures among abdominal surgeries and is associated with high morbidity, a decrease in food intake and deficiency in the absorption of nutrients. Therefore, the nutritional status of patients with PDAC is also aggravated after surgery, possibly resulting in poor adherence to AC, especially when attempts are made to initiate it early after surgery. Moreover, several scoring systems that reflect nutritional status, such as the prognostic nutritional index (PNI), neutrophil lymphocyte ratio (NLR), and Glasgow prognostic score (GPS), have been associated with a poor prognosis in PDAC patients. Further clinical trials may be warranted to confirm that such nutritional support actually translates into improvement in survival.The fact that early enteral feeding (EEN) after a pancreaticoduodenectomy (PD) was shown to be more useful in maintaining body weight and recovering digestive function than total parenteral nutrition (TPN) may offer clues for counteracting this situation.P < 0.001, data not shown). The RDI of patients who received a PD tended to be lower than those of patients who underwent other methods . Although the operative method was not a meaningful factor in the multivariate analysis in this study, more rigorous perioperative nutritional support might be necessary, specifically for patients who receive a PD.In contrast, the postoperative serum albumin levels of patients who received a PD were significantly lower than those of patients who underwent other operative methods (–30 In addition, intraoperative blood transfusions were shown to be an independent prognostic factor in PDAC. Although the underlying mechanism remains unclear, blood transfusions have an immunosuppressive effect via transfusion-related immune modulation. It was also proposed that following blood transfusion, lipid mediators, pro-inflammatory cytokines, and immunosuppressive proteins in the transferred blood might suppress natural killer cell activity and IL-2 production. This immunosuppression at the cellular level might influence tolerability of the chemotherapy. As shown in Table Additionally, patients who received an intra-plus postoperative blood transfusion tended to have lower RDIs. Previously, some studies have shown that patients with PDAC who receive a perioperative blood transfusion show a poorer prognosis. Although analyses to determine predictive factors for the RDI were performed after adjusting for the drugs used, our data might still suffer from bias due to drug choice. Third, no standard protocol for blood transfusions was used; the decision to transfuse was based on the judgment of the anesthesiologist.There were several limitations of our study. First, this was a retrospective single-institution study with a limited number of patients. Second, the anticancer agent used in the adjuvant therapy was varied and was selected based on the physician's preferences. The outcomes of patients receiving S-1 monotherapy were actually significantly better than those of patients receiving gemcitabine, as has been shown by the JASPAC-01 trial.In conclusion, the maintenance of RDI is important to improve prognosis. Intra-plus postoperative blood transfusion and a high postoperative PLR could be predictive markers of reduced RDI in AC of PDAC patients. In addition, maintaining RDI is possibly more important to patient outcome than starting AC early after surgery. The role of rigorous perioperative nutritional support may be worth exploring in future studies.


A new compact silicon grating coupler enabling fibre-to-chip light coupling at a minimized taper length is proposed. The proposed coupler, which incorporates a hollow tapered waveguide, converts the spot-size of optical modes from micro- to nano-scales by reducing the lateral dimension from 15 µm to 300 nm at a length equals to 60 µm. The incorporation of such a coupler in photonic integrated circuit causes a physical footprint as small as 81 µm × 15 µm with coupling efficiency and 3-dB coupling bandwidth as high as 72% and 69 nm respectively. Such issue arises due to dimensional mismatch between nano-scale waveguide devices and micro-scale SMF, typically in the order of 10−3, which results in excessive modal loss while coupling directly. Therefore, the requirement of efficient coupling becomes indispensable to minimize the coupling loss. Among various coupling methods, grating coupler (GC), while compared with widely investigated facet coupler5 involving post-CMOS fabrication complexities of dicing and polishing, has attracted a lot of attention due to its high compatibility with standard CMOS fabrication processes. GC also offers the flexibility to place it on any position of the photonic chip leveraging wafer level testing in mass manufacturing processes7. However, GC’s inherent characteristics in supporting propagation in multiple directions is causing a major hindrance to attain sufficient directionality for desired coupling efficiency (CE) and/or coupling bandwidth (CBW). To achieve higher directionality in GC, bottom mirrors based on either metal10 or distributed Bragg reflector (DBR)11 were incorporated, which usually require customized (non-standard) wafers or CMOS processes. An alternative CMOS compatible silicon nitride (Si3N4) GC with bottom Si grating reflector was proposed12 which, however, still requires multiple etching and lithographic processes. Apart from the approaches with bottom mirrors, directionality were also improved by adding interleaved trenches13 and anti-phase reflection coatings14 with limited success in improving CE and CBW.Although high refractive index (RI) contrast in silicon-on-insulator (SOI) platforms enables multifunctional submicronic integration of photonic components in a single chip, it leads to the challenges of interfacing them with standard single mode fiber (SMF)17 or fully etched photonic crystals18 to produce Gaussian like profile, controlling back reflection requires further engineered structures such as Si overlay19 or shallow-etched grating structure20 at the expense of additional complexity in lithography and/or etching processes. Another noteworthy drawback of GC is the polarization dependency, which is caused by uneven diffraction in transverse electric (TE) and transverse magnetic (TM) modes. Polarization diversity 1D and 2D grating structures, with their relative merits and demerits23, were proposed to attain similar CE/CBW in both TE and TM modes25. Perfectly vertical GC is another area of active investigation since the recent past to realize cost-effective photonic packaging29.Mode-shape difference between fiber and waveguide modes, and back reflection at the point of incidence also curtail CE and CBW of GC severely. Whilst mode-shape can be improved by using apodized grating structureAlthough, in general, issues regarding GC have been investigated widely, the challenge of interfacing nano-scale waveguide devices with SMF still remains, as most of the GC structures convert only the vertical dimensions of the waveguides to nano-scales, leaving the lateral dimensions unaltered. Therefore, to enable complete interfacing, GC structures must be adiabatically tapered down to nano-scales, which demand further analysis of CE and CBW. Moreover, as the efficiency of a tapered waveguide largely relies on the length of adiabatic transition, an undesirably large footprint of the GC structures in PIC is inevitable.32. Smaller footprint GCs with partially overlay dual taper33 and micrometric SOI rib waveguide34 were also proposed. Among them although focusing grating structure offers relatively smaller footprints, it usually happens at the cost of compromised CE and CBW that restrict its application in many modern technologies including optical interconnects, dense-wavelength-division-multiplexing, frequency-comb generation and so forth. In this report, we propose a new compact GC structure by replacing the conventional tapered waveguide with a hollow tapered waveguide (HTW). The proposed HTW reduces the lateral width of the grating waveguide from 15 micrometer (µm) to 300 nanometer (nm) at a length of 60 µm, keeping the height of the waveguide all through 220 nm.To realize smaller footprint GC for a PIC, various techniques have been proposed in the recent past. Focusing GC structure is one of such initiatives, where geometry of the gratings is cylindrical with a common focal point instead of straight linesThe work reported in this article is organized as follows: Section II describes the modelling environment and approaches used to extract the background parameters for the proposed GC. Section III describes the proposed HTW optimized for lateral matching with nano-scale waveguide devices. Section IV presents the results obtained from simulation and performance characterization of the HTW under discussion. Section V performs a comparison of the proposed HTW with conventional and inverse tapered waveguides. Possible manufacturing process and the associated tolerances are discussed in section VI. Finally, the report is ended with concluding remarks in Section VII.35. Theoretically, FDTD method performs its best for grid spacing  approaching towards zeros. But, in practice, Δ is also perceived as a compromise between cost and complexity, and therefore, chosen to find the right balance among computational cost and accuracy. For the analysis under discussion, the modelling environment was created by using Lumerical’s FDTD solution with grid spacing as small as Δx = Δy = 10 nm, which is sufficient to maintain higher accuracy. Reflections at the boundary of the structure was eliminated by using built-in perfectly matched layer (PML)36. The modelling also involved parameter sweeping module where parameters for best performance can be obtained and used to update other parameters by employing Particle Swarm Optimization (PSO)38 for optimum performance. The simulation environment created for the proposed GC is shown in Fig. The design exercise performed in this investigation rely on widely used Finite Difference Time Domain (FDTD) method, which is a numerical analysis technique used for modelling computational electromagnetics by solving Maxwell’s equations. Although, in real, GC is a computation-intensive 3D problem requiring huge computer memory for modelling, a 2D approximation of the structure can also mimic the same functionality with sufficient accuracy, as typical width of a GC e.g. 15 µm) is much larger in compare to its height (e.g. 220 nm) and operating wavelength (e.g. 1550 nm)5 µm is m2 layer as BOX with 220 nm Si on top to ensure single mode operation. The BOX is incorporated with an Aluminum (Al) layer of thickness of 100 nm as reflector which recaptures the light wasted through the BOX due to downward radiation9. The thickness of the SiO2 between top Si layer and Al layer is optimized as tBOX = 1.6 µm that causes constructive interference among guided and reflected waves39. The gratings are written on top Si layer for which design parameters were are calculated by using the following grating equation:neff is the effective refractive index (RI) experienced by the optical modes, ntop is the RI of the cladding on top of Si layer, and θin is the angle ofTo extract the background parameters for the proposed GC, a grating structure enabling typical vertical matching between SMF and nano-scale waveguide was designed, the schematic of which is shown in Fig. neff, which was calculated by using Full-Vectorial Mode Solver, was found to be 2.23. The targeted wavelength was set at λ = 1550 nm from which grating period was calculated as Λ = 636 nm for ntop = 1 (air) and θin = −120. Shown in Fig. tetch) and filling factor (ff) were set to 50% of the height of top Si waveguide and grating period respectively, which later was optimized as 95 nm and 350 nm respectively for maximum efficiency by using PSO. As ff = (wg/Λ) = 0.55, groove width (wgroove) was left to be 286 nm. Also, as the total height of top Si layer is 220 nm, a 125 nm Si base remains as a waveguide after etching 95 nm-depth gratings.incidence with normal to the direction of incidence causing a slightly tilted positioning of SMF to minimize second order reflections. For the fundamental mode, With all these necessary parameters optimized, the E-field distribution and propagation of light along the structure are shown in Fig. A HTW enabling lateral matching between grating waveguide and nano-scale waveguide devices with a smaller footprint is proposed and modelled by using Lumerical’s 2D FDTD solution, the related simulation environment and background parameters of which were discussed in Section II. In HTW, light is guided through a tapered hollow core where the optical modes are converted from loosely confined mode to highly confined mode. HTW is designed by inserting two Si strips one ends of which are connected with grating waveguide and other ends merge together to form a hollow core. For effective recapturing of the mode to couple to the nano-scale waveguide device, the widths of the Si strips are adiabatically increased until it supports a mode to confine within the merged Si strips. The schematic diagram of the compact GC incorporating the proposed HTW is shown in Fig. nwall = 3.5) and air core (ncore = 1), and (ii) the narrow width of the Si strips (wt-ini), which is thin enough to reject any mode inside the strip. Typical cross-section of a Si strip that fulfils the conditions for a single mode operation at 1300 to 1600 nm band is approximately 500 nm (width) ×200 nm (height)41. The height of the strips is matched with grating waveguide to 220 nm, which also satisfies the conditions for single mode operation. To find the optimum width that rejects any part of the optical mode to reside within it, HTW structure is simulated by using Finite Difference Eigenmode (FDE) solver of the Lumerical’s Mode Solutions with strip-width (wt-ini) setting at 500 nm, 300 nm and 100 nm, the results of which are shown in Fig. Optical mode from the grating waveguide is confined within the hollow air core mainly due to two physical occurrences: (i) contrast of RI between Si wall  in the HTW can be calculated as:The spacing between adjacent Si strips  and inverse taper (IT) with 60 µm taper lengths, equal to the optimum length of the proposed HTW structure. The tapers are used to couple light from 15 µm wide grating waveguide to 300 nm wide nano-scale waveguide. Height of the waveguide throughout the structure is kept 220 nm, which makes the cross-section of the nano-scale waveguide as 220 nm (height) ×300 nm (width). CE and CBW predicted for various taper structures are shown in Table Finally an estimation of CBWs of the designed GC without any taper and the proposed GC structure comprising of HTW with/without 33 Si strips are shown in Fig. 2 BOX and 220 nm top Si layer. There could be two-step lithography process necessary to realize two different etch depths for gratings and HTW. In first step the gratings can be defined using electron beam lithography (EBL) technique and the pattern transfers can be realized by means of dry etching until etch depth of 95 nm. In second lithography step, the HTW with 33 Si strips need to be defined which could be achieved by using hard mask, e.g. SiO2 mask on top Si layer and then the top Si layer is etched until the BOX (etch depth of 220 nm) to form the proposed HTW.The fabrication of the structure can be started with a SOI wafer consisting 1.6 µm SiOwgroove) by +/− 10 nm from the designed width of 286 nm show that the peak of spectrum is shifted towards longer wavelength for smaller groove width while the change in opposite direction happens for larger groove width. Shown in Fig. wgroove drops more for positive variation (larger grooves) than that of negative variation . However, irrespective of larger or smaller groove, these variations are quite insignificant (less than 1%) for a groove width variation of +/− 10 nm. The effects of etch-depth variation is also studied and encapsulated in Fig. tetch) range of 90–100 nm. ΔCE will be around 4% only for the change of tetch from 85 nm to 110 nm.Generally, slight variations of the designed parameters are expected in actually fabricated device. The effects of such variations on the performance of the coupler have been studied. The influence of the grating groove is shown in Fig. wt-ini) of the two outer Si trips is investigated. In section III we have found that wt-ini should be thinner than 200 nm to deny any mode to reside within it. So the HTW structure is simulated for wt-ini from 50 nm to 190 nm as shown in Fig. wt-ini range of 80–120 nm, CE drops only 2%, which however changes sharply for wt-ini around 140 nm. This can be attributed to the fact that at such thickness mode starts escaping the hollow core and gradually penetrating into Si strips. The effects of hollow space (wH) was also studied  ×15 µm (width) to enable smaller footprint in PICs, while ensuring least possible coupling loss between optical fibre and nano-scale waveguide devices. Light is transported from 15 µm wide grating waveguide to 300 nm wide Si nano-scale waveguide with a proposed 60 µm long HTW. The 2D FDTD analysis shows that, with the basic HTW structure with 2 Si strips, only 47% of incident light could be coupled to the nano-scale waveguide. To increase CE, HTW structure was improved by implanting additional Si strips into the hollow core that minimize the mode mismatch between HTW and nano-scale waveguide modes. Such HTW structure with optimum number of Si strips exhibits CE of 72% (−1.42 dB) with 1-dB and 3-dB CBW of 43 nm and 69 nm respectively. Using the proposed HTW light can be coupled from grating waveguide to nano-scale waveguide with least possible taper loss, only 6% estimated in this case. The performance of the proposed HTW structure is compared with conventional and inverse tapers for similar taper lengths as of HTW and found that the proposed HTW structure shows superior performance over conventional and inverse tapers. The proposed structure is fully CMOS compatible and can be fabricated based on standard lithography and etching process. The fabrication tolerance also investigated and found that the device possess remarkable high tolerant to the fabrication errors. The results are comparable with most compact designs in literature taking the size, coupling efficiency and bandwidth into account. The coupling efficiency can further be improved by reducing back reflection and implementing apodized gratings to achieve higher mode matching between fibre and waveguide modes.


This result presents a significant step in the realization of GaN:Er epilayers as an optical gain medium at 1.54 μm.We report the quantum efficiency of photoluminescence processes of Er optical centers as well as the thermal quenching mechanism in GaN epilayers prepared by metal-organic chemical vapor deposition. High resolution infrared spectroscopy and temperature dependence measurements of photoluminescence intensity from Er ions in GaN under resonant excitation excitations were performed. Data provide a picture of the thermal quenching processes and activation energy levels. By comparing the photoluminescence from Er ions in the epilayer with a reference sample of Er-doped SiO Previous work has revealed that a number of Er optical centers as well as a variety of energy transfer routes take place in GaN1214182:Er. The SiO2:Er system has been used as an optical gain medium for solid-state lasers at 1.54 μm42 reference sample, has an internal quantum efficiency (IQE) over 98%. Since basically all the Er ions in the SiO2 matrix participate in photon emission, the reference sample can be employed as a benchmark to determine the optical activity of Er in other matrices. A low thermal quenching of 20% from 10 K to room temperature from the isolated Er optical centers in GaN epilayer at the 1.54 μm emission has been demonstrated. Employing a high resolution spectroscopy and temperature dependent measurements of PL intensity under resonant excitation provides an estimate of the quantum efficiency of PL process of Er optical centers as well as the thermal quenching mechanisms in GaN epilayers grown by MOCVD technique.We present in this work a careful analysis of Er luminescence in GaN epilayers by comparing the PL intensity from our GaN:Er epilayers to a well-characterized standard reference sample of SiOnEr) of ~1021 cm−3 were grown on a thin un-doped GaN template of 1.2 μm on top of (1000) c-plane sapphire substrate. The growth temperature of Er-doped GaN layer was 1040 °C. The X-ray diffraction spectra indicated that GaN:Er epilayers have high crystallinity and no second phase formation. The band gap energy of GaN:Er epilayers is about 3.4 eV at room temperature. A detail description of the growth process and epilayer structure has been reported previously10111624The Er doped GaN epilayer samples were prepared by MOCVD method in a horizontal reactor10164I15/2 → 4I9/2 of Er3+ in GaN were obtained using a tunable wavelength Ti:Sapphire laser around 809 nm (1.533 eV) with a repetition rate of 80 MHzThe high resolution PL spectra were conducted using a Horiba iHR550 spectrometer equipped with a 900 grooves/mm grating blazed at 1500 nm and detected by a high sensitivity liquid nitrogen InGaAs DSS-IGA detector. The resolution of PL spectrum is 0.05 nm. The PL experiments were carried out in a variable temperature closed-cycle optical cryostat (Janis) providing a temperature range from 10 K to 300 K. Both resonant excitation and the non-resonant excitation were employed to investigate the optical properties of Er in GaN epilayers3+ site on the PL emission can be determined from optical excitation mechanisms. We have reported direct evidence of two mechanisms responsible for the excitation of optically active Er3+ ions in GaN epilayers grown by MOCVD in our previous workf shell transitions and non-resonant (band-to-band) excitation of the GaN host, the high resolution PL spectra at 10 K reveal an existence of two types of Er optical centers including the isolated and the defect-related Er optical centers in GaN epilayers4I15/2 → 4I9/2) excitation and the band-to-band excitation. Er ions in substitutional sites are considered as an isoelectronic impurity centerThe influence of Er4I13/2) of the isolated Er optical centers, the high resolution spectra as well as the temperature dependence of PL spectra, have been investigated under resonant excitation  is the integrated PL intensity at the temperature of T, I0 is the integrated PL intensity at 10 K, EA1 and EA2 are the activation energies of the thermal quenching processes, C1 and C2 are fitting constants related to the density of non-radiative recombination centers in the sample, and kB is Boltzmann’s constant. As shown in the EA1 = 13 and EA2 = 118 meV.where 4I13/2) excited state to the sublevels of (4I15/2) ground state. When temperature increases, electrons from the lowest level of the first excited state gain energy and populate at the second-lying crystal field split level. Thus, the intensity of the main PL lines decreases and the intensity of hotlines increases with temperature. These hot lines are displaced by 12.6 meV, which is nearly equal to the activation energy of 13 meV. An energy level diagram for the luminescence of isolated Er optical centers is illustrated in the Obviously, the replications of PL lines at higher temperature originate from the transition of the second-lying crystal field split level of the first  over 98%. Thus, we obtain Under the resonant excitation, the decay dynamics of all PL lines of the isolated Er optical centers appear as a single exponential with a decay time constant of 3.3 ± 0.3 ms12352:Er sample can be used to attribute the measured PL intensity to a particular density of excited Er3+ ions. The number of excited Er3+ ions in SiO2 reference sample can be calculated through excitation photon flux when we assume that all Er3+ ions are equivalent and they all contribute to the PL process. As has been discussed previously624The well-characterized SiO2 is the absorption cross-section of Er3+ for the 4I15/2 → 4I9/2 transition4I13/2−2 is the concentration of Er ions in the SiO2 reference sample. As shown in 2:Er sample shows a linear behavior for low excitation density. Under this condition, i.e.2 and SiO2-air) also might play a role, we have employed Fresnel’s equations for calculations of the photon flux from the Ti:Sapphire laser entering the active layer. By substituting this into 3+ ions in GaN sample  indicate a low thermal quenching of 20% from 10 K to room temperature from ErHow to cite this article: Ho, V. X. et al. Photoluminescence quantum efficiency of Er optical centers in GaN epilayers. Sci. Rep.7, 39997; doi: 10.1038/srep39997 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.