Circadian oscillators are networks of biochemical feedback loops that generate 24-hour rhythms in organisms from bacteria to animals. These periodic rhythms result from a complex interplay among clock components that are specific to the organism, but share molecular mechanisms across kingdoms. A full understanding of these processes requires detailed knowledge, not only of the biochemical properties of clock proteins and their interactions, but also of the three-dimensional structure of clockwork components. Posttranslational modifications and protein–protein interactions have become a recent focus, in particular the complex interactions mediated by the phosphorylation of clock proteins and the formation of multimeric protein complexes that regulate clock genes at transcriptional and translational levels. This review covers the structural aspects of circadian oscillators, and serves as a primer for this exciting realm of structural biology. T. TS. elomer Fig. b. It has PCC7120  and fromTe) BP-1 , 62. TheTe) BP-1 , 63, 64,KaiC is a kinase/phosphatase/ATPase: The principal clock component, KaiC, is the only protein with known enzymatic activity in the cyanobacterial clock. It acts as an autokinase, an autophosphatase, and an ATPase, exhibiting these functions both in vitro and in vivo [ in vivo –67. The  in vivo .KaiC functions as kinase/phosphatase: The key phosphorylation sites identified in the KaiC CII domain are Ser431 and Thr432. Phosphorylation at these sites occurs at the subunit–subunit interface  [n cycle) . There an cycle) –76; and n cycle) . Work bycis-trans isomerization of a peptide (D145–S146) accompanying the ATP hydrolysis  and its period-modulating variants in the pre- and post-hydrolysis states (PDB entries 4TL9 and 4TLA) revealed two structural bases of slow KaiC CI ATPase activity . First, gures in ) resultsgures in .2+  and KaiC mutants   reveal t2+ Fig. e. The glKai protein interactions and the phosphorylation cycle: Both in vitro and in vivo, KaiA is an enhancer of KaiC phosphorylation, while KaiB antagonizes the action of KaiA [ of KaiA , 81, 82. of KaiA , 83. Kai of KaiA . KaiB in of KaiA , 69. The of KaiA , resulti of KaiA . In contflexible → SpTflexible → pSpTrigid → pSTvery-rigid → STflexible. KaiA interaction with exposed A-loops of the flexible KaiC CII ring activates KaiC autokinase activity. KaiC hyperphosphorylation at S431 changes the flexible CII ring to a rigid state that allows a stable complex formation between KaiB and KaiC. The resulting conformational change in KaiB exposes a KaiA binding site that tightens the binding between KaiB and the KaiA linker, thus sequestering KaiA from A-loops in a stable KaiCB(A) complex and activating the autophosphatase activity of KaiC [Structural studies , 85 prov of KaiC . KaiB biKaiB is the only known clock protein that is a member of a rare category of proteins called the metamorphic proteins , 88. Thefs-cryst  and CIcryst (truncation at the N-terminus of the isolated CI domain of the KaiC monomer) complex (PDB 5JWO) shows an interface that primarily consists of the residues from the fold-switched C-terminal half of KaiB and the B loop of the CIcrys [A high-resolution  in pre- and post-hydrolysis states displayed large conformational changes in the KaiC CI domain at the ATP binding site after ATP hydrolysis. Residue F200, near the ATP binding site and the α6 and α7 helices, moves “downward” as a result. Residues Q154 and Y155 of α6 then constitute the KaiBfs-cryst–CIcryst interface. Another 3.87Å resolution crystal structure –phosphomimmic KaiC S431E complex hexamer, crystallized in the presence of ATP, showed densities of ADP between each CI subunit [ATP hydrolysis in the KaiC CI ring is a pre-requisite for KaiC interaction with fsKaiB , 91. A cd to ADP  with the subunit  as oppos subunit . The str subunit .Fig. 6KaDynamic structural analysis of Kai CI ring tryptophan mutants using fluorescence spectroscopy demonstrated a link between slow ATP hydrolysis and the KaiC CI binding to KaiB. The structural change triggered by slow ATP hydrolysis results in a structural rearrangement in the CI ring at the inner hexamer radius side (includes α7) and the D145–S146 peptide, without altering the overall hexameric framework of the KaiC CI ring. A slow KaiC CI ring conformational change (from pre- to post-hydrolysis state) coupled with the phosphorylation of KaiC results in a KaiC conformation that is receptive to the incoming active KaiB. This conformational switch in KaiC, coupled with ATPase activity and KaiC phosphorylation state, signals KaiC–active KaiB complex assembly and provides an explanation for the slowness of the cyanobacterial clock .cryst  in complex with KaiBfs-cryst–CIcryst provides the molecular level understanding of the co-operative assembly of the Kai components and the regulation of output signaling pathways by the Kai oscillator. Ternary complex analysis indicates that the presence of KaiA results in an increase in the affinity of KaiB for KaiC CI domain  at the KaiAcryst dimer interface and blocks it. Thus, KaiB binding to KaiA induces changes in KaiA conformation and, as a result, KaiA inhibits itself from binding to KaiC. Structure-guided mutagenesis of the α5 helix and α7 and α9 helices of KaiA weakened ternary complex formation. Mutations in the β2 strand of fsKaiB disrupted the antiparallel β-sheet formation, eliminating the interaction between KaiAΔN and fsKaiB–KaiC CI complex. The mutation did not affect complex formation between fsKaiB and KaiC CI. The analogous mutations in kaiBSe disrupted the circadian rhythms in vivo [Analysis of the ternary complex also reflects on the auto-inhibitory role of KaiA Fig c. Bound  in vivo .6B1, C6B2 …. C6B6) followed by the formation of higher order KaiCBA complexes (C6B6A2 …. C6B6A6) that peaks in 12 hours, followed by the dephosphorylation phase wherein the KaiCBA complex disassembly is not the reverse of complex assembly. Incubation at 4°C favored autophosphorylation with KaiCBA complex levels increasing even after 24 hours. A protocol devised on these observations is used to obtain Kai complex assemblies "frozen" in various states for structural analysis. KaiCBA complex assembly could be obtained with near complete occupancy of the KaiA binding site by prolonged incubation of KaiC, B, and A in 1:3:3 molar ratio. Structural maps of KaiC6B6A12 and KaiC6B6 complex assemblies obtained at 4.7Å and 7Å resolution using mass spectrometry and single particle cryo-electron microscopy (EM) and fitted with previous crystal structures of the individual Kai proteins reveal that KaiCB assembly consists of three stacked rings of which the bottom two correspond to KaiC, and KaiB forms the top ring  binds to the distal LRE region of the frq promoter to activate frq transcription. frq mRNA levels peak in the early subjective morning and subsequently lead to FRQ accumulation that peaks in the late subjective day [frq transcription [The n system , 94, 95.n system , 47. Togtive day , 15, 96.tive day . The twocription . FRQ is cription , 98.frq and vivid (vvd) are two of them) [Neurospora circadian rhythms [Neurospora clock. VVD is the only LOV domain containing a protein for which the crystal structure has been solved in the light and dark state, by Zoltowski et al. [Among the core-clock components, WC-1 consists of three PAS domains: PAS-A, PAS-B, and PAS-C. Of the three PAS domains, PAS-A belongs to a specialized class of light, oxygen, or voltage (LOV) domain and functions as a blue-light photoreceptor. The function of PAS-B is unclear, and PAS-C is required for the interaction between WC-1 and WC-2 , 100. WCof them) , 105–107 rhythms . VVD andi et al.  (see belperiod (per), in Drosophila and subsequent analysis of its expression led to the first molecular model of an animal circadian oscillator [Drosophila and mammalian clock genes share a high level of sequence similarity and have orthologs. The primary feedback loop of the clock -PAS (Period-Arnt-Single-minded) transcription factor family, and they heterodimerize to activate the transcription of genes containing E-box cis-regulatory elements in their promoter region: Period (dPer) and timeless (dTim) in Drosophila and period genes  and cryptochrome genes (Cry1 and Cry2) in mouse [Drosophila) proteins translocate to the nucleus and repress their own transcription by acting on CLOCK/BMAL1 (dCLK/dCYC) activity [Tim is retained in mammals (mTim); however, unlike a central role for dTim, the function of mTim in the mammalian circadian clock is not clear. An essential role similar to that of dTim is performed by Cry genes in the mammalian circadian clock [Drosophila and mammals, regulate the circadian clock in a light-dependent (photoreceptors) and a light-independent manner [Identification and isolation of the first clock gene, cillator , 109. Thock Fig. c, d consin mouse , 110–114activity , 115–119an clock –122. Intt manner , 123–127Drosophila, dCLK/dCYC activates the transcription of vrille (vri) and Par domain protein 1ɛ (Pdp1ɛ) by binding to the VRI/PDP1-box (V/P) of the clk promoter to form the second loop [The core-clock loop integrates with other regulatory systems that further fine-tune the mammalian clock system, wherein CLOCK/BMAL1 activates transcription of the members of the orphan nuclear receptors family (Rev-ErbA/NR1D (Nuclear receptor family 1 group D). Rev-erbα and β, and Retinoic acid receptor (RAR)-related orphan receptors ) in mammals via recognition of their E-box elements –131. RORond loop , 135.The interactions of PERIOD proteins: Crystal structures of fragments of Drosophila PERIOD (dPER residues 232–599) and mouse PERIOD  proteins  mutant, which has a temperature-dependent 29-hour long period phenotype, existed as a monomer in the solution [2 homodimerization [Structural analysis of dPER has shown the importance of the PAS-A-αF interface in homodimer formation in solution. A dPERsolution . The anasolution . Consistrization . The cryrization , 52.Despite the conserved domain composition of the mPER proteins, the different interacting interfaces of the homodimers could play a role in defining their distinct functions. Of the three mammalian period proteins, mPER1 and mPER2 have been shown to be more important for maintaining the circadian rhythmicity. mPER2 regulates the expression of the clock genes (interaction with REV-ERBs), while mPER1 maintains their stability and subcellular localization via protein–protein interactions –155. Kno2 homodimer [Analytical gel filtration analysis of the mPER homodimers in solution revealed a higher affinity for the mPER1 homodimer than for mPER2 and mPER3. Structural analysis of the PAS-A/αC interface Fig. c, f showomodimer , 52. TheLL coactivator motif was observed in the PAS-A βE strand of mPER2 [LL) and in mPER3 (PXXLT) is buried deep in the hydrophobic pocket formed by a Trp (in PAS-A) and a Leu residue in the N-terminal cap in mPER1 and mPER3, but not in mPER2. In addition, the coactivator motif in mPER2 is preceded by a less ordered βD-βE loop in the motif, suggesting that the motif in mPER2 is more easily available for interaction with nuclear receptors based on the higher flexibility of the adjoining regions [period and Rev-erbs knockdowns showed cell-specific phenotypes [The N-terminal cap was observed to be unstructured in mPER2, whereas it formed a long helix followed by a β-strand in mPER1 and a shorter helix in mPER3. Sequence analysis of the mPER proteins predicts the presence of a HLH motif N-terminal to the PAS-A domain. In the absence of a basic region of the bHLH transcription factors, the mPERs HLH region might be engaged in heterodimeric interactions with other HLH proteins. Analytical gel filtration and mutation studies showed that mPER3 utilizes the HLH motif as a second interface to further stabilize homodimer formation instead of the PAS-A/αC interface in mPER1 and forms a more stable homodimer than mPER2. Also, a LXof mPER2 , 52, whiof mPER2 . The corenotypes .Structural analysis of the PERIOD protein fragments is a step towards understanding PAS domains and the interactions of the PERIOD proteins. Future mutation studies of the key surfaces found from the structural studies and the interacting partners will provide a detailed understanding of their functions and the mechanism involved, which are not yet clear. Also, the newly developed cell-autnonomous clock model approach can be applied to other cell types which can be utilized to study mutants based on structural analysis to understand the tissue-specific functional differences of various clock genes.The CLOCK–BMAL1 complex: The CLOCK–BMAL1 complex is central to the core oscillator in the mammalian clock. In the primary loop, these positive elements activate the transcription of Per and Cry genes. The PER–CRY complex in return represses their transcription by acting on CLOCK and BMAL1 expression. Another regulatory loop is formed by CLOCK–BMAL1 and Rev-erbs and RORs, wherein the complex activates their transcription. Rev-erbs and RORs subsequently regulate the rhythmic expression of BMAL1 [of BMAL1 , 131An important step towards understanding the mammalian circadian clock has been the crystal structure of the mouse transcriptional activator CLOCK–BMAL1 heterodimeric complex that is central to the oscillator . The 2.3The CLOCK and BMAL1 PAS-A domain consists of a typical PAS fold that is made of five β-strands and several helices. External to the PAS fold is the N-terminal A'α helix that is packed between the β-sheet surfaces of the two PAS-A domains and contributes to the heterodimeric interactions between the two domains. The interactions between CLOCK A'α and the BMAL1 β-sheet, and vice versa, are highly hydrophobic, forming a parallel heterodimer. Simultaneous mutation of the interface residues in both CLOCK and BMAL1 greatly reduced both the heterodimer formation and its transactivation potential compared to single mutations involving the individual proteins. The PAS-B domains of the two proteins are connected to the PAS-A domains by the 15-residue linker L2, which is ordered and buried within the CLOCK–BMAL1 interface in CLOCK, whereas in BMAL1, the linker is exposed to the surface and flexible. The crystal structure showed a translation of 26 Å in the PAS-B domains of CLOCK and BMAL1. The two PAS-B domains interact via surface-exposed hydrophobic residues in CLOCK and BMAL1. Trp427 of BMAL1 stacks with the CLOCK Trp284 located in the hydrophobic cleft between the Fα helix and the AB loop of the CLOCK PAS-B domain Fig. . The tanDrosophila dPER share a significant similarity with mammalian PER proteins and bHLH-PAS transcription factors  [2 and is a conserved feature of mPER1 and mPER3, but not of other PERs or the bHLH-PAS-containing transcription factors [Lack of similarity among the clock proteins indicates that while the mechanisms are conserved across the kingdoms and are fundamental to clock machinery, the proteins are not structurally related, and further research is required to understand the structural differences. The crystal structures of the PAS domain homodimers of dPER and mPERs provide an interesting view of the interactions and their nonredundant functions. The PAS domains of d NPAS2) . WC-1, td NPAS2) , suggestd NPAS2) . The sec factors . Thus, tThe crystal structure of the heterodimeric complex between mouse CLOCK and BMAL1 revealed an unusual 3D arrangement of the two PAS domains in the two proteins. The conformation and the spatial arrangement of the PAS domains of BMAL1 were similar to that observed in the crystal structure of the PAS domains of dPER and mPER. Trp362 in CLOCK is involved in an interaction with CRY. The corresponding Trp427 in BMAL1 interacts with CLOCK. In PERIOD proteins, Trp at a similar position is involved in homodimer formation , suggestStructural insight into Rev-erb interactions: The crystal structure of human Rev-erbβ was reported in a dimeric arrangement  , 175. N-cis-acting targets of the two isoforms and comparison with the BMAL1-binding sites [Bmal1-/-, Per1-/-Per2-/-, Cry1-/-Cry2-/-), suggesting that, together, the two Rev-erbs work with BMAL1 and other core-clock components to regulate circadian rhythms and metabolism. [One study has shown that both Rev-erbα and β  are centng sites  showed aabolism. . Additioabolism. .Arabidopsis thaliana as a model. The present clock paradigm consists of at least three interlocking transcriptional–translational feedback loops , functions in a feedback role similar to that of TOC1 , 201 andPRR9 –211. RepPRR9 , 213. TaThe plant clock components and their interactions have primarily been studied using reporter assays, the yeast two-hybrid assay, and co-immunoprecipitation. However, lack of structural knowledge is largely limiting our understanding of the clock components. In silico approaches have been applied to predict the structural features and thereby gain insight into the underlying functional aspects of some components. However, in the absence of experimental validation, a cautious approach is required. Using such an approach, TOC1 was predicted to be a multidomain protein, having an N-terminal signaling domain as well as a C-terminal domain that might be involved in metal binding and transcriptional regulation. A middle linker predicted to lack structure connects two domains . The N-telf4 hypomorphic alleles showed phenotypes at both morning and evening genes, suggesting a dual role for ELF4 linked with both morning and evening loops [Another study utilizing bioinformatics approaches  has predng loops . ELF4 inng loops .Fig. 13PPRR9 transcription [Identification of the evening complex, comprised of ELF4, ELF3, and LUX, which are all crucial for the transcriptional repression of the morning genes, addresses the importance of protein–protein interactions in a functional rhythmic oscillator . ELF4, pcription .Light is one of the major environmental cues influencing the CC. Organisms have evolved sophisticated light-signaling networks that synchronize the clock to day/night cycles in order to regulate their metabolic and physiological processes.S. elongatus CiKA (SyCiKA) consists of a cGMP phosphodiesterase/adenylate cyclase/FhlA-like domain (GAF) similar to that in other bacteriophytochromes, followed by a characteristic histidine protein kinase (HPK) domain. However, the GAF domain lacks the conserved Cys and His needed for the binding of the chromophore in other bacteriophytochromes. Also, binding with a chromophore was not observed in vivo. C-terminal to the kinase motif is the receiver domain homologous to the receiver domain of the response regulators of the bacterial two-component signaling systems. It lacks a conserved Asp present in the receiver domains of the bacterial RRs that is phosphorylated by the HPK domain, hence the name pseudoreceiver domain (PsR) [Cyanobacterial rhythms are shown to be synchronized indirectly by light via the redox state of metabolism in the cell. The type of input that the clock perceives was previously unclear. Further work revealed Circadian input kinase A (CikA), a histidine kinase bacteriochrome , and ligin (PsR) , 225. A in (PsR) .The solution structure of the PsR of CiKA (PDB 2J48)  consistsCikA− strains showed high levels of phosphorylated RpaA, indicating CikA promotes dephosphorylation of RpaA [The phosphatase activity of CikA is enhanced significantly in the presence of KaiC and KaiB. In vivo,  of RpaA . Also, r of RpaA . To unde of RpaA .fs-nmr ; binds to PsR-CikA in the absence of KaiC CI) and PsR-CikA , which inhibits electron transfer from PQ to cytochrome bf, thus reducing the PQ pool, significantly affected the stability of LdpA, CikA, and KaiA. Additionally, lack of LdpA in DBMIB-treated cells further reduced CiKA stability, suggesting that LdpA can affect CiKA sensitivity to the cellular redox state [CiKA and KaiA co-purify with LdpA . LdpA, aox state . Interesox state , 230, suNeurospora are mediated by the blue light photoreceptors WC-1 and VVD [Neurospora and are driven by the two LOV domains containing WCC complex and VVD [Neurospora clock in response to blue light [frq and vvd) [Known light-induced responses in  and VVD , 232. Li and VVD , 234. VVand vvd) , 107. Liand vvd) . As showand vvd) , 236.vvd is under the control of photoactive WCC, and it accumulates rapidly upon irradiation. VVD indirectly regulates the light input to the Neurospora clock by repressing the activity of the WCC. Studies show that VVD plays a role in modulating the photoadaption state by sensing changes in light intensity [The expression of ntensity . Recent ntensity . Direct ntensity , 238. Thntensity , 234.Neurospora, VVD and WC-1 are the two LOV domain-containing photoreceptors, and in Arabidopsis, the LOV-containing families include phototropins (phot 1 and phot 2) and the ZEITLUPE family , and Flavin-binding Kelch F-box1 (FKF1)). They bind the flavin mononucleotide (FMN) chromophore [The LOV domain forms a subclass of the PAS domain superfamily; it mediates blue light-induced responses in bacteria, plants, and fungi . In Neuromophore .The crystal structure of VVD-36  that retains wild-type behavior studied in both dark- and light-adapted states explains the light-induced conformational changes that are important for VVD activity  studies show that the light-adapted VVD forms a rapidly exchanging dimer relative to the dark-state monomer, with an expanding hydrodynamic radius. Dimer formation was observed to be concentration-dependent. The increase in the hydrodynamic radius was observed to be highly dependent on the length of the N-terminus. Studies of the various light-state N-cap variants indicate that residues 39–42 are important for dimerization and contain a Pro-Gly-Gly signature that is highly conserved among the dimer-forming variants. The aa39–42 segment adopts two distinct conformations in the crystallographic C71V VVD dimer (PDB 3D72), with a 180° rotation about the Pro residue between the two subunits , highligArabidopsis contains five phytochromes (PHYA–E) [Arabidopsis is called the CRY3/Arabidopsis CRY-DASH and has sequence similarity to CRY-DASH from Synechocystis. It localizes in chloroplast and mitochondria. CRY3 shows sequence-independent DNA binding, but its role in biological signaling remains unknown [Light input in plants is mediated by multiple photoreceptors: phytochromes (red/far-red light photoreceptors), cryptochromes (UV-A/blue light photoreceptors), UVR8 (UV receptor), and ZEITLUPE (ZTL), FLAVINBINDING, KELCH REPEAT, F-BOX 1 (FKF1), and LOV KELCH PROTEIN 2 (LKP2) (blue light) are a suite of photoreceptors involved in the photoentrainment of plants . Arabido(PHYA–E) , 245 and(PHYA–E) , 247. A  unknown , 249. ZT unknown .CCA1 and LHY [Arabidopsis, which shows light-dependent effects on clock period [Recent studies suggested several light-signaling pathways involved in clock entrainment, but they are not yet well understood. One such pathway is proposed by the PIF hypothesis . PIFs ne and LHY  in vitro and LHY . Also, F and LHY . Some of and LHY , 255. Ank period . Moreovek period , 256.Phytochromes are red/far-red light-sensing photochromic biliprotein photoreceptors that are involved in the regulation of various developmental processes. Of the five phytochromes found in plants, phyA and phyB are the best characterized . PHYs haDeinococcus radiodurans [Rhodopseudomonas palustris [Synechocystis 6803 Cph1 (PDB 2VEA) for the Pr ground state [The crystal structures of the PAS-GAF two-domain construct of the bacteriophytochrome DrBphP from iodurans  and RpBhalustris  lack thend state  and for nd state . Togethend state , 265. ThThe structures of the Pr (2VEA) and Pfr (3C2W) states show that, on excitation, transition from Pr→Pfr leads to Z–E isomerization of the chromophore D ring, consistent with Pr–Pfr photochemistry. Differences were also seen in the position of several tyrosine residues around the D ring in 3C2W. These Tyr residues were shown to be important for photoconversion .Arabidopsis phytochrome mutants studied previously, Nagtani [Based on the structures of the bacterial phytochromes and the  Nagtani  detailed Nagtani .The LOV domain-containing ZTL/FKF1/LKP2 family is involved in the regulation of photoperiodic-dependent flowering and the entrainment of the circadian clock . The strThe LOV domain in the ZTL/FKF1/LKP2 family undergoes photochemical cycles similar to phot-LOV domains in vitro , 268–270Arabidopsis where a CRY mutant showed abnormal growth and development in response to blue light [Drosophila, Arabidopsis, Synechocystis, Homo sapiens) [Cryptochromes (CRYs) are flavoproteins that show overall structural similarity to DNA repair enzymes known as DNA photolyases . They weue light . In respue light , 278–281sapiens)  whose mesapiens) , 249.Cryptochromes have 1) an N-terminal photolyase homology region (PHR) and 2) a variable C-terminal domain that contains the nuclear localization signal (absent in photolyase and CRY-DASH proteins and with no obvious sequence similarity to known protein domains). The PHR region can bind two different chromophores: FAD and pterin , 281.Synechocystis [Arabidopsis [10-helix. The α domain is the FAD binding region and consists of fourteen α-helices and two 310-helices. The two domains are linked by a helical connector comprised of 77 residues. FAD binds to CRY1-PHR in a U-shaped conformation and is buried deep in a cavity formed by the α domain [Arabidopsis mediates a constitutive light response [Arabidopsis CRY1 to be functional and to mediate blue light-dependent inhibition of hypocotyl elongation even in the absence of CCT1 [Arabidopsis [. The study showed HBI1 acts downstream of CRYs and CRY1 interacts directly with HBI1 through its N-terminus in a blue-light dependent manner to regulate its transcriptional activity and hence the hypocotyl elongation [Arabidopsis in a blue light-dependent manner [In the absence of any high-resolution structure for a CRY protein, the functional analysis of this blue-light receptor was not clear earlier. Although the structure of CRY-DASH is known from hocystis , it doeshocystis . The crybidopsis , solved bidopsis –285, ledα domain . In contα domain , the CRYα domain –285. In α domain , explainα domain , 287. CRresponse , 287. Ho of CCT1 . Another of CCT1 . The twobidopsis –292. Theongation . Previout manner . These sDrosophila and mammals. Interestingly, studies have shown that cry genes, both in Drosophila and mammals, regulate the circadian clock in a light-dependent [cryb mutant [Drosophila did not respond to brief light impulses under constant darkness, whereas overexpressing wild-type cry caused hypersensitivity to light-induced phase shifts [Drosophila is mediated through light-dependent degradation of TIM. Light-activated CRY undergoes a conformational change that allows it to migrate to the nucleus where it binds to the dPER–dTIM complex, thus inhibiting its repressive action [Drosophila clock can also occur via compound eyes, as external photoreceptors and Hofbauer-Buchner eyelets behind the compound eyes, where rhodopsin is present as the main photoreceptor [Drosophila [.Identification of the cryptochromes in plants subsequently led to their identification in ependent –125 and ependent , 127. Anb mutant  in Drosoe shifts . Light se action . dCRY ble action . Howeverreceptor –300. CRYreceptor . In the osophila .Arabidopsis and Drosophila cryptochromes showed that the presence of both the PHR domain and C-terminal extension is essential to cryptochrome-mediated functions. However, like a functional N-terminal domain of Arabidopsis CRYs independent of the CCTs, studies on N-terminal domain constructs lacking the C-terminal domain of Drosophila CRY demonstrate it to be functional. A Drosophila cry mutant allele (crym) expressing only the N-terminal CRY domain was observed to be capable of light detection and photoransduction independent of the C-terminus [Drosophila lines overexpressing CRYΔ lacking the C-terminus resulted in a constituively active form that did not degrade [The C-terminal extensions that are characteristic of CRYs in the Cryptochrome/Photolyase family gained considerable attention owing to their crucial role in various cryptochrome functions . Dterminus . Also, t degrade . CRYs unArabidopsis (6-4) photolyase-PHR-CRY1-CCT domains showed that the features of both domains are obligatory for the repressive action of the CRY protein. The C-terminus is not sufficient to mediate the transcriptional repressor function [Drosophila, the C-terminal extension has been shown to be critical to the role of dCRY as a magnetoreceptor [Drosophila [Drosophila clock showed increasingly slow rhythms in response to an applied magnetic field in the presence of blue light. The magnetosensitivity was also affected by the field strength. cry mutants with an impaired FAD or mutants lacking cry were observed to be unresponsive to the applied magnetic field. Drosophila clock neurons overexpressing CRYs showed robust sensitivity to an applied field [Studies report direct interaction between CRY and COP1/phyB/ZTL/LKP1/ADO1 in plants, and mPER in animals, mediated via the C-terminus. Studies of chimeric proteins made by fusion of function , 281. Inreceptor , 306. Mareceptor . Lack ofosophila , 306. Thed field .Drosophila and mammalian) and show overall similarities. There are, however, significant differences and these are implicated in defining their diverse functions [Structural studies on the animal cryptochromes contributed immensely to the understanding of their function. Structures have been solved for both full length and truncated CRYs . Unlike photolyases, where an Asn residue can only interact with the protonated N5 atom, the corresponding Cys416 residue of dCRY readily forms a hydrogen bond with unprotonated N5 and O4 of FAD, thus stabilizing the negative charge and preventing further activation to FADH.-, which is the form required for DNA repair in photolyases [°- formation and decay and regulating the dynamics of the light-induced tail opening and closing. Additionally Phe534, Glu530 (tail helix), and Ser526 (connector loop) stabilize the tail interaction with the PHR in the dark-adapted state [The structure of animal CRY did not reveal any cofactor other than FAD. In CRYs, flavin can exist in two forms: the oxidized FADtolyases . Structued state . These aThe structure of the apo-form of mCRY1 by Czarna et al.  shows anStructural analysis  of the pd ~ 40 μm). FAD adopted a similar U-shaped conformation as observed in other CRYs and photolyases. However, it was reported that the FAD moiety is only partially buried in the binding pocket [The crystal structure of the apo form of mCRY2-PHR  showed ag pocket . In , but not in enucleated mice, suggesting that nonvisual photoreceptors could play a role in photoentrainment of the mammalian circadian clock , 320. StOpn4-/-) showed less sensitivity to brief light perturbations under DD [Opn4-/- mice were not completely absent, indicating the involvement of other photoreceptors in the entrainment process. mCRY1 and mCRY2 are found in the inner layer of the retina [Drosophila cryptochromes upon light irradiation, suggesting a possible role as photoreceptors in mammals [Melanopsin (Opn4), a new opsin molecule that has emerged over the past decade as a potential photoreceptor for photoentrainment, is enriched in the ipRGCs , 322. Miunder DD . Howevere retina . Also, h mammals , 325. Ho mammals , 127. Wo mammals  demonstr mammals . Thus, l mammals , 322. RH mammals , 320. InAn exciting chapter of circadian clock research, which is focused on structural aspects, has brought with it new challenges. Whereas the structural aspects of the circadian clockwork in prokaryotes are relatively well studied, the picture regarding eukaryotic CCs is fragmentary, trivial, and far from complete. Much is to be done. A targeted protein complex, which is a structural feature common to all the clocks, has recently gained center-stage in bench science. Multimeric protein complex formations have been shown to be important for the regulation of several core oscillators. We know that the proteins contain identical conserved domains with their typical folds. However, structural analysis of the CLOCK–BMAL1 complex and the PERIOD homodimers suggests that the dynamics of the assembly and disassembly of hetero-multimeric protein complexes is dependent on the differential spatial arrangement of the domains. Additionally, the CLOCK/BMAL1 proteins show potential for a differential electrostatic surface that endowes the complex with asymmetry, indicating that differential surface potential might be responsible for the disparity in their interaction with PER/CRY and, hence, for distinct functions.Neurospora, FRQ interaction with FRH and kinases results in WCC phosphorylation, thus repressing its activity [Drosophila CLK protein is not only sequential, but is also compartment-specific. Although phosphorylation of FRQ is crucial for its transcriptional repression activity, Cha et al. [Sequential phosphorylation is another feature that influences protein–protein interactions in circadian clocks. The dynamics of the cyanobacterial KaiC phosphorylation cycle have been observed to be driven by regulated cycles of interaction with KaiA and KaiB that trigger the enzymatic switch in KaiC. However, both the precise time point for the switch and an understanding of how the information relayed between the phosphorylation/dephosphorylation event and the physical protein–protein interaction triggers the switch are issues that remain to be elucidated. Sequential phosphorylation has also been observed in the eukaryotic clock. Protein–protein and/or protein–DNA interactions coupled with progressive phosphorylation and dephosphorylation events have been shown to be important for stability, subcellular distribution, and the function of the core-clock components , 165. PEactivity , 104. CCactivity . Howevera et al.  showed tFbxl3-mediated ubiquitination. The crystal structure of the mCRY2 PHR–Kloo1 complex shows that Kloo1 is buried deep in the pocket and mimics the cofactor [The dynamics of phosphorylation and dephosphorylation are also important for the transmission of external environmental cues and for resetting the clock. A light-dependent conformational change of the photoreceptors directs a downstream cascade of phosphorylation and protein–protein interactions that defines the period length and the phase shifts. Another interesting mechanism of clock resetting has been observed in the cyanobacterial clock, where the metabolic state of the cell entrains the clock in a light-dependent manner. Circadian metabolic rhythms are also observed in higher organisms . Feedingcofactor .The cyanobacterial CC is an enzymatic clock wherein KaiC, central to the clock, exhibits all the enzymatic activities. The eukaryotic circadian system is, instead, a complex network of transcription factors, regulatory proteins, kinases, and phosphatases. The common elements in the CC systems in different kingdoms of life are fairly well known. However, notwithstanding the coarse models we have, enough differences have been brought about by the different evolutionary paths and different environmental adaptations to justify detailed studies of CCs in different organisms. From this perspective, the efforts invested by us and others, especially with regard to the structural dissection of the circadian systems, are timely and well placed.


Antimicrobial use (AMU) in livestock is a debated topic, mainly due to the risk of associated development of antimicrobial resistance. There is focus on reducing AMU in the Danish pig production, which accounts for the largest proportion of AMU for animals in Denmark. Due to special restriction on AMU in organic pig production, the AMU in organic pig production is lower than in conventional pig production. There is concern that reduced AMU could jeopardize animal health and welfare, if it reflects insufficient treatment of sick animals, which might be reflected by the prevalence and types of lesions found at meat inspection. However, little is known about the associations between AMU and meat inspection findings in pigs from organic farms. Furthermore, excess amount of antimicrobial product after a treatment cannot be re-prescribed in organic pig herds. The initial prescription is recorded in the national database VetStat, but the unused amount is not deducted leading to uncertainty when reporting AMU. The objectives of this study were to 1) describe AMU patterns based on prescription data for organic pig production and compare with those of the conventional pig production for year 2016, 2) study the associations between herd-level AMU prescription data and meat inspection data for organic pig herds and 3) validate herd-level AMU prescription data in VetStat against treatment records collected on-farm in organic Danish pig herds.Gastrointestinal indications account for the largest proportion of AMU in both organic pig herds  and conventional pig herds . A larger proportion is prescribed for respiratory indications in organic than conventional weaners and arthropathic indications in finishers. No associations between AMU and meat inspection data were found. This needs further investigation as the prevalence of lesions at slaughter was slightly (non-significantly) higher in herds with no registered AMU than with AMU prescriptions. Only 8 out of 31 herds had recorded their AMU sufficiently detailed to compare, and using VetStat as a proxy for AMU led to 9–88% overestimation of the actual use in 7 out of these 8 herds and 120% underestimation in one herd. In Denmark, the veterinary AMU at herd-level has been monitored in the database VetStat since 2000 [Over the last decades there has been increasing concern regarding antimicrobial use (AMU) in food producing animals and development of antimicrobial resistance (AMR), due to the risk that AMR can spread from animals to the humans . A prerence 2000 . Also, tnce 2000 , 11, 14.nce 2000 . So far,nce 2000 , 12. AMUThere have been many initiatives to reduce AMU in Danish pig production in general, and the total AMU measured in kg active compound has declined by 28% over the last 10 years . The DanPrescription data from VetStat has previously been used as a proxy for AMU in conventional pigs. However, it might be challenging to do the same for organic pig production. According to national legislation, any excess antimicrobial product after a treatment course in organic herds cannot be stored on farm and re-prescribed for next treatment as in non-organic herds . TherefoDescribe AMU patterns based on prescription data for organic pig production and compare with those of the conventional pig production for year 2016Study the associations between herd-level AMU prescription data and meat inspection data from organic pig herdsValidate herd-level AMU prescription data in VetStat against treatment records collected on-farm in organic pig herdsBased on the presented issues and concerns related to AMU in organic pig production this study had three objectives:The first objective of the study was to describe AMU patterns based on prescription data for organic pig production and compare these with those of the conventional pig production. In Denmark, there were 122 organic pig herds recorded in the Danish Central Husbandry Register (CHR) in 2016. This corresponds to nearly 2% of the total number of herds recorded in CHR that year. As shown in Table As shown in Table N = 25) and sow + finisher herds (N = 19). The distribution of finisher pen places in the two groups is shown in Fig. The second objective of this study was to study the associations between herd-level AMU prescription data and meat inspection data from organic herds. In total, 44 organic herds were included in this part of the study. The herds were grouped into finisher herds  and without AMU (N = 11) for finishers in 2016. Herds without AMU registered for finishers had a slightly higher median prevalence of lesions at slaughter, specifically arthropathy lesions, than herds with AMU registered. A more similar distribution in the two groups was observed when considering the prevalence of gastro-intestinal and respiratory lesions .In total, 11 herds had no records of antimicrobials prescribed for finishers in 2016. Therefore, herds were divided into two groups; herds with AMU (ns Table . Howeverp = 0.001)  Fig. .Fig. 2ScN = 7), no current production of pigs (N = 7) or due to personal reasons (N = 3). Finally, farmers representing 31 herds were willing to participate. Herd visits were performed in 20 herds and copies of treatment records were collected in each farm. Furthermore, treatment records were sent by e-mail from nine herds and finally two herds were interviewed by phone. However, for only eight herds in total, it was possible to collect sufficiently detailed data. The remaining herds were excluded since the AMU was registered as ml/kg, why the specific amounts used or the weights of the animals were not recorded. Thus, the presented results represent data from the remaining nine herds, for which their treatment records were compared to the prescription data from VetStat covering the same period. This comparison and the number of animals in each herd are shown in Table The third objective of this study was to validate herd-level AMU prescription data in VetStat against treatment records collected on-farm in organic pig herds. Out of a target population of 54 organic pig herds, six herds were non-responders (could not be reached by telephone or e-mail). In total, farmers representing 17 herds were unwilling to participate due to time pressure  and the next frequently used way was to return it to the veterinarian or pharmacies for destruction (33%). Finally, 11% of the farmers said the excess amounts were re-prescribed by their veterinarian.The first part of the study showed that the prescribed AMU in organic pig herds is much lower than in conventional pig herds. Previous studies covering AMU in organic pig production is limited. For the present study, the use in weaners were 14 times lower, whereas for finisher pigs it was 4 times lower.There might be different reasons for the low use. It might be due to the higher weaning age as suggested by Sjölund et al., . In thisFurthermore, the withdrawal period from antimicrobial treatment to slaughter for organic pigs is twice the period for conventional pigs . This me2 are allowed in organic herds and that the animals need to have access to an outdoor area [For finisher herds, organic and conventional herds are more closely related than for sow herds, which might also explain why a smaller difference are seen between AMU for organic and conventional finishers than for weaners. Organic and conventional finisher pigs are often housed in similar stables. The main differences are that a fewer number of finishers per moor area , 5. ThesIn general, organic pig herds have fewer animals, which biologically would give a lower disease pressure, and again result in less infections and possibly also a lower need for AMU. Previous studies have also found a relationship between herd size and AMU in conventional pig herds  are only allowed to be treated once in organic pig herds. If they need more than one treatment, they must be slaughtered as conventional pigs, and the farmer looses the supplement organic payment from the slaughterhouse. According to Alban et al. , this miPig raised free-range are expected to be challenged in different ways than indoor raised pigs, which can explain different disease patterns between organic and conventional pig production. This is also reflected in the prescription patterns presented in the first part of the study. For example, a higher proportion of AMU for respiratory indication in organic finishers, which might be explained by a lower use of vaccines against respiratory diseases in the organic pig production . Alban et al.,  showed tThe second aim of this study was to compare prescription patterns and meat inspection at herd-level. A previous study on meat inspection data from organic and conventional herds, showed that three types of lesions were more often seen in organic pigs and free-range  whereas four other types of lesions were more often seen in conventional pigs . The number of lesions in total were equally distributed between production types. However, there were different disease problems occurring in the different types of production, which is probably a result of the different conditions and challenges indoor and outdoor, respectively .No associations between the prescribed AMU and lesions were found, which might reflect that AMU might not be a good indication for disease status, at least not in herds with a low use. In Denmark, free-range (non-organic) production is similar to organic production, but less restricted on the AMU. The AMU is higher in free-range pig herds  despite similar outdoor herd conditions. At least, these two types of production would be expected to have the same types of disease and therefore the same need for AMU. However, that is not reflected in the prescription data from VetStat.Previous studies on the associations between AMU and lesions found at slaughter are limited, especially when studied at herd-level as in the present study. This might be due to a number of limitations with using meat inspection data. In Denmark, weak associations were found between routine meat inspection findings and results from systematic health monitoring when considering pericarditis, pleuritis and lung lesions . FurtherIn the third part of the study, we found that VetStat data is not a good proxy for AMU at herd-level in organic pig herds, and that this would most likely result in an overestimation. This was the case for 7 out of 8 herds. The farmers had different ways of handling the excess amount of antimicrobial products. Most alarming was that 11% of the farmers said the excess amounts were re-prescribed by the veterinarian. This is conflicting with the current legislation, requiring that the excess amount of antimicrobial product is discarded .For one of the herds, the excess amount of antimicrobial products were negative, which means that the amount recorded in VetStat is actually lower than what is reported in the farm records. This reflects another problem with using VetStat for estimating AMU in organic herds. Organic herds often gets their medicine directly from their veterinarian. When antimicrobials are sold from the pharmacies (still with a prescription), the receiving herd is automatically registered. When the product are handed over directly from the veterinarian, it requires that the veterinarian report for which CHR number the products are prescribed for. If not, this will result in an under-estimation of the actual use for that prescription.Many errors in the treatment records were detected, making it difficult to compare with data from VetStat. A total of 22 herds were excluded from the study due to incorrect treatment records. This is an interesting finding, since it might reflect a general problem with registering the AMU despite a legal obligation to do so. However, problems with AMU registration at herd-level is not only seen in organic pig production, but also in the conventional pig production. A campaign by the Danish Veterinary and Food Administration (DVFA) in a subset of Danish conventional finisher herds revealed that in 19% of the cases, the farmer did not report all antimicrobial treatments or did not follow the instructions given by the veterinarian in relation to dosage or indication [Excluding a large proportion of the herds also resulted in a small sample of herds with a large variation in differences between VetStat and treatment records. This makes it difficult to extrapolate these results. It would be of relevance to consider this as a focus point in future campaigns by the Veterinary Authorities. In addition, when working with AMU data from organic pig herds from VetStat, researchers should make interpretations with these results in mind.Compared with conventional production,the prescribed AMU in organic pig herds is 14 times lower for weaners, and 4 times lower for finishers. Furthermore, a larger proportion of AMU is prescribed for respiratory indications in organic weaners and arthropathy indications in organic finishers. No clear association between the AMU and lesions at slaughter were found. However, prevalence of lesions was slightly (non-significant) higher in herds with no registered AMU than with AMU prescriptions. Discrepancies were seen when comparing AMU registered in VetStat and herd treatment records in organic farms. Many errors in the treatment records in organic farms were detected, making it difficult to compare with data from VetStat.Prescription data from 2016 originating from two Danish databases were used in all three parts of the study. Firstly, herd-level antimicrobial prescription data were extracted from VetStat. These data were used as a proxy for AMU in this study. Each prescription was converted into milligram active compound and animal daily dose (ADD), based on a list for each product in VetStat, provided by the Danish Veterinary and Food Administration (DVFA). In Vetstat, the indication for which each product is prescribed is given. Based on this information the AMU for the three predominant indications used in both conventional and organic pig production were summarised. These were gastro-intestinal, respiratory and arthropathy indications.Finally, information regarding herd type and number of animals in each herd were extracted from CHR.Both the CHR and VetStat databases are owned and maintained by the DVFA.In the first part of the study, all herds registered as either organic or conventional pig herds in 2016 were included. These herds were grouped according to herd type, and the two types of herds were compared descriptively in terms of antimicrobial use and herd size.The AMU measured in ADDs were divided by the number of animals produced in 2016. These numbers were based on slaughterhouse statistics for conventional and organic pig production, respectively.For the second part of the study, all organic herds fulfilling the following enrolment criteria were included: Herds with either; 1) No sows and > 150 finisher (finisher herds) or 2) Sows > 50 and finisher > 150 (sow + finisher herds). By using the number of pen places in CHR, it was possible to calculate the average ADD/100 finishers/day in 2016. Furthermore, herds with meat inspection data available were included.Meat inspection data were obtained from the Danish slaughterhouse database. A criterion of a minimum slaughter weight at 110 k was set to only included finisher pigs sent for slaughter. Based on these data, the number of pigs produced in each herd were found. Based on the codes in the meat inspection data, lesions found at slaughter were divided into three main groups of lesions as previous described . These tFor many of the organic herds, the ADD/100 finishers/day recorded in VetStat were zero. Therefore, it was impossible to consider the ADD/100 finisher/day as a continuous variable in the analysis. Instead, herds were divided into two groups; herds with and without AMU in 2016. Patterns in lesions found at slaughter for the two groups were evaluated descriptively and the differences were analysed with student’s t-tests using the software R, version 3.3.2.The organic herds included in the third part of the study were also herds with either; 1) No sows and > 150 finisher (finisher herds) or 2) Sows > 50 and > 150 (sow + finishers herds). All herd owners were contacted through e-mail and telephone for participation. For farmers willing to participate, a herd visit was scheduled. At the herd visits, primary data consisting of the treatment records and information on handling of excess amount of antimicrobial products were collected. In order for a herds to have sufficiently detailed data, the farmer had registered the actual amount used for a treatment, the duration, and the weight of the animal treated. Herds with sufficiently detailed data were included in the calculations of excess antimicrobial products. This was calculated as the difference between the products prescribed in VetStat and the products used according to the treatment records.


CDK5 activation promotes ischemic neuronal death in stroke, with the recognized activation mechanism being calpain-dependent p35 cleavage to p25. Here we reported that CDK5-Tyr15 phosphorylation by zinc induced CDK5 activation in brain ischemic injury. CDK5 activation and CDK5-Tyr15 phosphorylation were observed in the hippocampus of the rats that had been subjected to middle cerebral artery occlusion, both of which were reversed by pretreatment with zinc chelator; while p35 cleavage and calpain activation in ischemia were not reversed. Zinc incubation resulted in CDK5-Tyr15 phosphorylation and CDK5 activation, without increasing p35 cleavage in cultured cells. Site mutation experiment confirmed that zinc-induced CDK5 activation was dependent on Tyr15 phosphorylation. Further exploration showed that Src kinase contributed to zinc-induced Tyr15 phosphorylation and CDK5 activation. Src kinase inhibition or expression of an unphosphorylable mutant Y15F-CDK5 abolished Tyr15 phosphorylation, prevented CDK5 activation and protected hippocampal neurons from ischemic insult in rats. We conclude that zinc-induced CDK5-Tyr15 phosphorylation underlies CDK5 activation and promotes ischemic neuronal death in stroke. Ischemic brain injury develops as the result of ischemia/reperfusion with multiple mechanisms involved including inflammation, excitotoxicity, oxidative stress and apoptosis2. Zinc is a trace element, which is enriched in the brain, with crucial functions in the physiology and pathophysiology of the central nerves system3. In ischemic brain injury, increased Zn2+ in cytoplasm, which were originated from extracellular influx and intracellular release from metallothioneins (MTs) or organelles play a key role in promoting ischemic neuronal damage, leading to necrotic, apoptotic and autophagic cell death4. Zn2+ accumulation precedes calcium deregulation in ischemia-damaged neurons; uptake of Zn2+ by mitochondria is associated with mitochondrial depolarization and consequent Ca2+ deregulation5. Zn2+ also acts on a selective zinc-sensing receptor ZnR to induce intracellular release of Ca2+6. Thus, excitotoxic [Zn2+]i rise is an early event before Ca2+ deregulation in the promotion of neuronal death. However, the underlying molecular signaling of Zn2+-induced neuronal death has not been fully elucidated.Acute ischemic stroke is the most common type of stroke and occurs as a result of vascular occlusion7. CDK5 shows neuron-specific activity because its activation requires association with the neuron-specific activator p35 or p25, the truncated form of p35 by the cleavage of calpain9. CDK5 activity is also regulated by phosphorylation at Tyr15, which induces CDK5 activation12. Abnormal CDK5 activity has been reported to contribute to pathogenesis of several neurological diseases such as Alzheimer’s disease14, Parkinson’s disease15, amyotrophic lateral sclerosis (ALS)16 and stroke18. In a transient forebrain ischemic rat model, CDK5 was activated specifically in hippocampal CA1 region and induced cell death through phosphorylating N-methyl-D-aspartic acid (NMDA) receptors, with the other regions of the hippocampus uninfluenced19. At the same time, chelatable zinc also accumulated specifically in degenerating neurons in the hippocampal CA1 and other presynaptic zinc-containing brain regions, preceding neurodegeneration20. These facts raise the possibility that zinc may play a role in CDK5 regulation in ischemic brain injury.CDK5 is a serine–threonine kinase, which is structurally similar to the mitotic cyclin-dependent kinases4-incubated cells. Src kinase inhibition or expression of an unphosphorylable mutant Y15F of CDK5 abolished Tyr15 phosphorylation, prevented CDK5 activation and protected hippocampal neurons from ischemic insult. Our data suggest that zinc-induced CDK5-Tyr15 phosphorylation promotes CDK5 activation and the following ischemic neuronal death in stroke.Here we explore the regulatory effect of zinc on CDK5 in cultured cells and in animal models with middle cerebral artery occlusion (MCAO). The results show that CDK5 activation was accompanied with Tyr15 phosphorylation in the hippocampus of the rats that had been subjected to MCAO, both of which were reversed by pretreatment with zinc chelator; whereas p35 cleavage and calpain activation in ischemia were not reversed. Zinc induced CDK5 activation through Src kinase-dependent Tyr15 phosphorylation in ZnSOAdult (3 months old) male Sprague-Dawley (SD) rats weighing 250–300 g and adult (3 months old) male C57Bl/6 mice weighing 18–22 g were housed individually under standard conditions of temperature and humidity, and a 12-h light/dark cycle (lights on at 08:00), with free access to food and water before use. Adequate measures were taken to minimize pain or discomfort during surgeries. All animal experiments were approved by the Animal Care and Use Committee of Huazhong University of Science and Technology, and performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.21. Briefly, after weighing, animals were deeply anesthetized. A 4-mm distal nylon monofilament  segment was coated with 0.21–0.22 mm diameter silicone  for mice, and an 11-mm distal monofilament  segment was coated with 0.28–0.30 mm diameter silicone for rats. MCAO was performed by inserting the monofilament via the common carotid artery into the left internal carotid artery, advanced 9–10 mm (in mice) or 20–21 mm (in rats) past the carotid bifurcation until a slight resistance was felt. Body temperature and respiratory rate were monitored during surgery. Body temperature of the animal was maintained at 37 ± 0.5 °C throughout the procedure using a heat pad. For inducing MCAO, the filament was left in place for 60 min, and then withdrawn for reperfusion. In the sham group, the filament was inserted only 5 mm above the carotid bifurcation. Mice were excluded from further studies if excessive bleeding occurred during surgery, if the operation time exceeded 90 min, or if hemorrhage was found in the brain slices during postmortem examination.Transient acute focal cerebral ischemia was induced by intraluminal MCAO, as described previouslyFor rat clioquinol (CQ) treatment, one dose of CQ (10 mg/kg) or vehicle  was injected into the SD rats intraperitoneally (i.p.) 60 min before surgery. For rat 4-Amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolopyrimidine (PP2) treatment, one dose of PP2  or vehicle (DMSO) was injected into the SD rats intracerebroventricularly (i.c.v.) 60 min before surgery. For treatment on cultured cells, the final concentration of PP2 was 1 μM.After 24 h of MCAO/reperfusion, mice and rats were weighed and sacrificed. The brain was removed rapidly and frozen at –20 °C for 20 min. Coronal slices were made at 2 mm intervals from the frontal poles. Sections were immersed in 1% 2,3,5-tripenyltetrazolium chloride  in phosphate-buffered saline (PBS) at 37 °C for 20 min. The presence or absence of infarction was determined by examining TTC-stained sections for the areas on the side of infarction that did not stain with TTC.21 Corrected infarct volume = contralateral hemisphere volume – .Serial sections were photographed using a digital camera and the area of infarct was quantified with Image J . The area of infarct , the area of ipsilateral hemisphere  and the area of the contralateral hemisphere  were measured for each section by an investigator blinded to the experimental group. The volume was calculated by summing the representative areas in all sections and multiplying by the slice thickness, then correcting for edema, as previously described:The rats and mice were deeply anesthetized and then fixed by trans-cardial perfusion with 0.9% NaCl, followed by 4% paraformaldehyde in 100 mM phosphate buffer. After perfusion, the brains were post-fixed in the same solution for 2 days at 4 ℃, followed with dehydration in 30% saccharose for 9 days at 4 °C. Then, the samples were embedded in optimum cutting temperature compound , frozen and sectioned at 30 μm using freezing microtome . Coronal sections of the brain were immersed in 1% toluidine blue for 3 min, followed by dehydration in 75, 85, 95 and 100% ethanol solutions, transparented using xylene, placed under cover slips and observed under microscope . The neuron numbers per ×40 magnification in the CA1 area were quantified by an investigator blinded to the experimental group, using Imaging-Pro Plus 6.0 .Mutant Y15F-CDK5 was generated from wild-type mouse CDK5 through mutagenesis by company . The sequences of wild-type CDK5 or Y15F-CDK5 were then inserted to a pcDNA3.1-GFP vector to generate pcDNA3.1-GFP-CDK5 and pcDNA3.1-GFP-Y15F-CDK5 plasmids. The plasmids were transfect into N2a cells with Lipofectamine 2000  to induce CDK5 overexpression. Adeno-associated viruses (AAVs) pAAV-CMV-CDK5-EGFP and pAAV-CMV-Y15F-CDK5-EGFP expressing wild-type or Y15F mutated CDK5 were constructed and packaged in OBIO . Virus particles were infused (1.0 μl at 0.1 μl/min) into the CA1 of the right hippocampus.Rats or mice were deeply anesthetized and trans-cardial perfusion with PBS was performed before the brains were removed. Affected hippocampus tissues were homogenized in ice-cold RIPA buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM sodium orthavanadium, 100 μM phenylmethylsulfonyl fluoride (PMSF). Total protein concentration was determined by BCA protein assay . Aliquots of homogenate with equal protein concentration were separated by 10% (v/v) sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) gel, and then transferred to nitrocellulose membrane . After blocking in 5% (w/v) nonfat milk for 30 min at room temperature, the membranes were then incubated with primary antibodies at 4 °C overnight, followed by incubation with anti-goat, anti-rabbit or anti-mouse IgG conjugated to IRDye for 1 h at room temperature and visualized using the Odyssey Infrared Imaging System . The primary antibodies used were as follows: CDK5 , phospo-CDK5 at Tyr15 , p35 , p25 , Src , Fyn , phospo-Src at Tyr 529 , phospo-SFK at Tyr 416  and DM1A .2, 2 mM MnCl2, 1 mM EDTA, 1 mM EDTA, 0.1 mM dithiothreitol, 2 μl ATP  and Histone-H1 . Samples were subjected to 10% (v/v) SDS/PAGE gel for western blotting. The antibodies used for samples were: CDK5 , Histone-H1 , phospo-Histone-H1. Calpain activity was measured by ELISA following the manufacturer's instruction through using a calpain activity assay kit .To determine CDK5 activity, brain tissue or cell samples were homogenized in ice-cold lysis buffer containing 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 2 mM EDTA, 1 mM sodium orthovanadate and proteinase inhibitor cocktail . After clearing debris by centrifuging at 12,000 rpm at 4 °C, protein concentrations in the extracts were determined using the BCA protein assay. The extracts (100 μg protein) were incubated with monoclonal mouse anti-CDK5  or monoclonal mouse anti-GFP  overnight at 4 °C, followed by the addition of 20 μl of Protein A + G agarose  for 3 h at 4 °C. Immunoprecipitates were washed four times with lysis buffer, and beads were then resuspended in kinase buffer, containing 20 mM Tris-HCl (pH 7.6), 20 mM MgClMice brain tissues were fixed and sliced as that in Nissl staining, sections were permeabilized in 0.3% Triton X-100 for 30 min, followed by incubation with 5% normal goat serum for 30 min to block nonspecific sites at room temperature. Primary antibody incubation  was performed for 48 h at 4 °C. Alexa Fluor 546-conjugated secondary antibody (1:200) was used for fluorescence labeling. 4,6-Diamidino-2-phenylindole (DAPI) was used to label the nuclear. The image was observed with the LSM780 confocal microscope .t-test was used. The significance was set at p < 0.05. All results shown correspond to individual representative experiments.Data were expressed as mean ± SEM and analyzed using SPSS 20.0 statistical software . The one-way analysis of variance (ANOVA) procedure followed by Least Significant Difference (LSD) post hoc test was used to determine the differences among groups. For data from two groups, two-tailed Student’s To explore the mechanism underlying CDK5 activation in brain ischemic injury, we established an animal model in which focal ischemia is induced by selective unilateral occlusion of the MCAO in rats. Reperfusion is achieved after 1 h of MCAO by removing the embolism. Unilateral MCAO caused marked CDK5 activation at 6 h after reperfusion Figs. , with a 22 and attenuate the ischemia-induced zinc accumulation in the CA1 pyramidal neurons23, thus we used CQ to reveal the effect of zinc on CDK5 activation in focal cerebral ischemia , administration of Zn2+ chelator reduced the infarct volume significantly in mild focal ischemia, and this protective effect was lost when insult severity was increased25. These interesting results indicate that intracellular Zn2+ accumulation is an early trigger of the ischemic neurodegenerative cascade.Zn29. Correspondingly, pharmacological or genetic inhibition of CDK5 could reduce infarct size, prevent neuronal death and promote functional recovery in animal models33. However, as CDK5 is required in lots of neuronal physiological functions, administration of CDK5 inhibitors are reported to affect physiological functions of CDK5 and lead to serious side effects34. Therefore, targeting the upstream factors inducing aberrant CDK5 activation in ischemic brain injury is a therapeutic strategy in stroke patients.Aberrant CDK5 activation has been identified to be a principal cause of neuronal death during stroke. Increased CDK5 protein levels or activities were detected in stroke animal models or patients9. Increased p25 levels35 and calpain activities37 were observed in ischemic injury. In some animal models and stroke patients, the p35 expression level is also increased38. On the other side, CDK5 activation by Tyr15 phosphorylation was not investigated in stroke, even though it has been identified in numerous studies related to CDK5 activation in neurite and spine retraction, dendrite outgrowth and Abeta-triggered neurodegeneration39. Given the coincident Zn2+ accumulation and CDK5 activation in damaged neurons in stroke, and Zn2+ is capable of inducing tyrosine phosphorylation of multiple proteins42, it is of interest to investigate if Zn2+ may also modulate CDK5 activity through Tyr15 phosphorylation in ischemic brain injury.One recognized mechanism underlying CDK5 activation in ischemic injury is calpain-dependent cleavage of p35 to p25, as the cleavage increases the solubility of the CDK5 active complex p25/CDK5 thus enhances it cytoplasmic activity38. In some stroke models, the increase of p25 is accompanied with decreased but not increased p35 level30. This difference may depend on the different time point for observation. In a time point when the expression of p35 significantly increased, the increased total p35 level may override the decrease of p35 by cleavage. However, p25 accumulation is a common change in all stroke models, suggesting that p25 contributes to CDK5 activation in ischemic injury. Thus, CDK5 is activated in ischemic stroke and this activation may be caused by CDK5-Tyr15 phosphorylation and/or p25 accumulation.In the present study, we first confirmed the CDK5 activation in ischemic stroke by detecting CDK5 activity in the hippocampal tissues from rat models with MCAO, analysis of the protein levels in the same tissue showed significantly increased Tyr15 phosphorylated CDK5 levels, indicating a possible involvement of Tyr15 phosphorylation in activating CDK5 in this stroke model. The p35 and p25 levels were also increased, which was consistent with previous findings2+ in regulating CDK5 in stroke. CQ is a widely used membrane-permeable zinc chelator43, it is reported that CQ can effectively reduce chelatable zinc in brain22 and attenuate the ischemia-induced zinc accumulation in the CA1 pyramidal neurons23, thus we used CQ to reveal the effect of zinc on CDK5 activation in focal cerebral ischemia. Pretreatment of CQ efficiently reversed CDK5 activation, reduced infarct volume and prevented hippocampal CA1 neurons from death induced by MCAO, without affecting the cerebral blood flow. At the same time, Tyr15 phosphorylation of CDK5 was also reversed. However, the p35 cleavage to p25 was not reversed. On the contrary, more p35 proteins were cleaved to p25, which was consistent with the calpain activity assay result: MCAO induced calpain activation, whereas CQ pretreatment further increased calpain activity. There are two explanations for this result: (1) Zinc has an inhibitory effect on calpain; (2) CQ directly activate calpain through other unknown mechanism. The first hypothesis was identified by the following observation in our study that Zn2+ inhibited calpain in ZnSO4-incubated cells. Another zinc chelator TPEN also caused calpain activation in cultured cells44. Thus, in our experimental system, Zn2+ chelation by CQ induced CDK5 inhibition in rats with MCAO, this effect was not mediated by decreased p35 cleavage, but more possibly by reduced Tyr15 phosphorylation. In another word, Zn2+ may cause CDK5 activation through Tyr15 phosphorylation.Next, we explored the effect of chelatable Zn4 to reveal the direct effect of zinc on CDK5. Our data showed that Zn2+ activated CDK5 through a Tyr15 phosphorylation-dependent manner. Mutation of Tyr15 to non-phosphorable Phe blocked the CDK5 activating by Zn2+. Furthermore, Zn2+ induced CDK5-Tyr15 phosphorylation through Src kinases activation, as Src kinases inhibitor PP2 completely reversed Zn2+-induced Tyr15 phosphorylation and CDK5 activation. The activation of Src kinases by zinc was also identified in our previous study24 and by other researchers42. Thus, zinc activates Src kinases, the latter, causes CDK5-Tyr15 phosphorylation and activation. For a validation of this molecular signaling pathway in ischemic injury, we performed rescue experiments by pretreatment with PP2 or expression of Y15F mutant of CDK5 in hippocampal CA1 regions in MCAO mice. Both strategies could reverse CDK5-Tyr15 phosphorylation and activation, and alleviate the neuronal damage. All these findings support that zinc-induced CDK5-Tyr15 phosphorylation underlies CDK5 activation and promotes ischemic neuronal death in stroke 45 as an adjunctive therapy; or in the patients treated with mechanical thrombectomy through intra-arterial delivery to prevent the reperfusion injury. Its application as a neuroprotective intervention in stroke is worthy of further exploration and identification.Our study reveals a new signaling pathway underlying ZnSupplementary Figure 1Supplementary Figure 2supplementary information


Computational image analysis is one means for evaluating digitized histopathology specimens that can increase the reproducibility and reliability with which cancer diagnoses are rendered while simultaneously providing insight as to the underlying mechanisms of disease onset and progression. A major challenge that is confronted when analyzing samples that have been prepared at disparate laboratories and institutions is that the algorithms used to assess the digitized specimens often exhibit heterogeneous staining characteristics because of slight differences in incubation times and the protocols used to prepare the samples. Unfortunately, such variations can render a prediction model learned from one batch of specimens ineffective for characterizing an ensemble originating from another site. In this work, we propose to adopt unsupervised domain adaptation to effectively transfer the discriminative knowledge obtained from any given source domain to the target domain without requiring any additional labeling or annotation of images at the target site. In this paper, our team investigates the use of two approaches for performing the adaptation: (1) color normalization and (2) adversarial training. The adversarial training strategy is implemented through the use of convolutional neural networks to find an invariant feature space and Siamese architecture within the target domain to add a regularization that is appropriate for the entire set of whole-slide images. The adversarial adaptation results in significant classification improvement compared with the baseline models under a wide range of experimental settings. Advances in whole-slide scanner technology have increased the speed and reliability with which histopathology slides and other microscopic specimens are digitized. As a result of these improvements, there has been a sharp increase in the number of investigators and health-care providers adopting the use of these devices in routine research and clinical workflows. The sheer volume of digitized specimens now being generated at both small and large institutions has grown accordingly. Once digitized, these specimens are well suited for the application of sophisticated pattern recognition and machine-learning algorithms and strategies that can facilitate automated decision-support and computer-assisted diagnosis. Over the course of hundreds of years, scientists and pathologists have gone to great length to develop and optimize staining methods that augment and enhance the contrast of biological components of interest within these samples at the tissue, cell and sub-cellular levels. Hematoxylin & Eosin (H&E) is a popular stain that is applied to specimens, routinely, that results in nuclei exhibiting a bluish color with cytoplasmic regions rendered in pink  in a completely unsupervised manner. We use two approaches to achieve knowledge transfer from the source domain to the target domain. In the first approach, we adopt two off-the-shelf color normalization  dataset  → UP and TCGA → RCINJ. We firstly use the images in source domain to train a binary classification network. The data from source domain is randomly divided into the training and the testing sets at a ratio of 80%  / 20%. The patients with more than one WSI can only contribute the images to the training set or the testing set. During the training process, the images are resized as 256 × 256 and randomly cropped to 224 × 224 to feed into the network. During the testing process, all the patches are resized to 256 × 256, we do the single center-crop for all testing patches. The network is trained from scratch. For the adaptation using color normalization, we utilize the source domain CNN as the network for target domain to determine the prediction from the testing set. For the adversarial adaptation, we optimize the Siamese network at target domain by fixing the parameters of source domain CNN and training the target network and the discriminator network at the same time. The prostate images at the target domain are randomly divided into the training and the testing sets at a ratio of 80 and 20%.Our implementation is based on Tensorflow  → UP and TCGA → RCINJ. The quantitative results for the adaptation are shown in p < 0.05. In addition, we show the result of the ablation study in We further calculate the statistically significance of the accuracy improvement between the adapted network and the baseline network using McNemar Test


Arabidopsis thaliana Fasciclin like arabinogalactan protein 4 (FLA4) locus is required for normal root growth in a linear genetic pathway with the FEI1 and FEI2 loci coding for receptor-like kinases. The two Fas1 domains of FLA4 are conserved among angiosperms but only the C-terminal Fas1 domain is required for genetic function. We show that at low salt deletion of the N-terminal Fas1 domain of transgenic FLA4 leads to enhanced root elongation compared to the tandem Fas1 wild type version. Modeling the hypothetical interaction between FLA4 and FEI1 we show that the predicted interaction is predominantly involving the C-terminal Fas1 domain. Relative conformational mobility between the two FLA4 Fas1 domains might regulate the interaction with the FEI receptor kinases. We therefore speculate that the FLA4 FEI complex might be a sensor for environmental conditions in the apoplast.The  However, the biological role of Fas1 tandems is elusive. The FLA4 protein encoded by the Arabidopsis thaliana Salt Overly Sensitive 5 (SOS5) locus is required for normal root growth and salt tolerance and acts in a linear genetic pathway with two leucine rich repeat receptor kinases encoding loci named FEI1 and FEI2.8 Sequence searches suggest that every angiosperm genome contains at least one putative orthologue of FLA4 containing both, the N-proximal Fas1-1 and the C-proximal Fas1–2 domain in tandem. Therefore, it was surprising that, in a study previously published in The Plant Journal,9FLA4 exerted its role for root growth depending exclusively on Fas1-2 and did not require Fas1-1 for its function. Both full-length FLA4-citrin (F4C) constructs as well as a F4C construct lacking the Fas1-1 (F4C∆Fas1-1) restored normal root growth to sos5 mutants. In fact, in most experiments, when root growth on NaCl-free full-strength Murashige and Skoog medium (MS) was measured, independent UBQ:F4C∆Fas1-1 (sos5-1) lines not only complemented the growth defect of the sos5-1 mutant background, but slightly exceeded the values of UBQ:F4C(sos5-1) lines or the Col gl wildtype. The difference between UBQ:F4C(sos5-1) and UBQ:F4C∆Fas1-1 (sos5-1) lines, however, was significant (P < 0.01) after prolonged growth periods on half strength MS medium containing no or only low concentrations of NaCl (UBQ:F4C(sos5-1) and the truncated UBQ:F4C∆Fas1-1 (sos5-1) lines behaved identically which indicates that under these conditions, the N-terminal Fas1-1 domain is neutral for the role of FLA4 in root growth.The fasciclin 1 (Fas1) domain is a structural feature of many extracellular proteins in all clades of life and has been implicated with adhesion to and signalling influenced by the extracellular matrix. of NaCl . This su10) and another one shows the Fas1-3 and Fas1-4 domains of Drosophila fasciclin I . We used SwissModel to make a homology model of FLA4 using these two crystal structures as a template.14 The FLA4 sequence has a higher identity with TGFBIp than Drosophila fasciclin I with 21.6% and 17.4%, respectively (the input sequence for the Swissprot query and a list of available structures including sequence identity and GMQE and is presented in the online supplement). Furthermore, although both models do not have a high global model quality estimation score (GMQE: 0.5), the model built with TGFBIp has a better agreement between the model structure and experimental structures. This ‘degree of nativeness’ is represented by the QMEAN score which is higher in the model based on TGFBIp with a score of −4.6 compared to model with Drosophila fasciclin I of −5.3. For these reasons, we continued with the model that was based on the TGFBIp structure. Next, a 3D molecular model of FLA4 with glycosylation at eight N-glycosylation sites was made. Representative glycan structures were added from a library created through enhanced sampling to get a large variety of conformers, as explained in reference.15 As a putative interaction partner, a homology model of the extracellular domain of FEI1 was modelled based on the structure of the extracellular domain of Brassinosteroid Insensitive 1- Associated Kinase 1 .Is there a structural-mechanistic explanation for this observation? Because no crystal structures of FLA4 or FEI RLKs are available, we approached this question using molecular modelling. One of the crystal structures of Fas1 domains in the Protein Data Bank belongs to human transforming growth factor-beta-induced protein (TGFBIp) having four Fas1 domains  reported in iG = −9.8 kcal/mol, indicating a hydrophobic interface.The relatively weak support for an interaction with the Fas1-1 domain concurs with the recent observation that Fas1-1 was not essential for complementation.20 others such as the Spätzle morphogen21 or the follicle-stimulating hormone22 interact with the concave LRR surfaces of their respective receptors. In our model the convex surface of the FEI1 ectodomain binds to the surface of the Fas1-2 domain  surface of the LRR-domain,2 domain  that is 2 domain . Intrigu24 Under some conditions such as low salt concentration, this interaction might be negatively modulated in an auto-inhibitory fashion by the Fas1-1 domain.We suggest that the Fas1-1 domain might negatively regulate FLA4 Fas1-2 binding to FEI1. This arrangement allows for regulatory flexibility. The relative conformation between Fas1-1 and Fas1-2 might be affected by post-translational modification  were used. These models take the structure of a protein as a network taking atoms as nodes connected by springs. To predict the collective motion of FLA4, we used the Elastic Network Model server.2 might be the basis for the evolution of Fas1 tandems in eukaryotes and for the conservation of the tandem Fas1 architecture of FLA4 orthologues in angiosperms. Besides being fully consistent with available evidence, our speculative model provides testable predictions on the hypothetical physical interaction between these intriguing regulatory proteins.Alternatively, Fas1-1 might prevent FLA4 from ectopically interacting with other receptor kinases. An auto-regulatory role, previously suggested for human TGFBIp,


Lactic acid fermentation could be used as a potential modification tool for faba bean flour to enable its incorporation in boosting the nutritional profile of gluten-free breads. Gluten-free breads made with fermented or unfermented faba bean flours were compared with commercial soy flour. The amounts of faba- and soy-bean flours were adjusted to obtain the same protein content in bread (16%). Both fermented and unfermented faba bean flour resulted in larger bread volume  compared to bread made with soybean flour (1.5 mL/g). Breads made with unfermented and fermented faba flour had higher porosity  than bread with soy flour (61%). The faba breads also were softer than the soy bread. Fermentation of faba flour prior to bread making significantly increased crumb hardness (584 vs. 817 g). Fermentation increased in vitro protein digestibility (72.3% vs. 64.8%). Essential Amino Acid and Biological Value indexes were significantly higher for breads containing fermented faba flour compared to breads made with unfermented faba and soy flour. The Protein Efficiency Ratio and Nutritional Index increased by fermentation from 33 to 36 and 1.6 to 2.7, respectively. Pre-fermentation of faba bean flour improved the nutritional properties of high-protein, gluten-free faba bread. A sensory panel indicated that fermentation did not affect the crumbliness, evenness of pore size and springiness of breadcrumb. Vicia faba) is an ancient crop, and together with pea, is one of the most important legume crops in Europe. Faba bean seeds, used in both human and animal nutrition, are rich in essential amino acids: isoleucine, leucine, lysine, phenylalanine, threonine and valine [l-3,4-dihydroxyphenylalanine (l-DOPA), contained at high levels in seeds, has been reported as effective in the treatment of Parkinson’s disease [Faba bean  and thus thermal treatments would remove the negative effect during consumption. Others  are heat stable . ProcessLike other legume crops, faba bean is gluten-free and could thus be utilized for gluten-sensitive and celiac people. The prevalence of celiac disease has become more and more common in recent years, therefore, the need for tasty and nutritious gluten-free foods is increasing. The structure and texture of gluten-free foods pose challenges for baking technology. Pulses such as chickpea, pea or soybeans have been used in gluten-free bread baking either in the form of flour or protein concentrate supplemented to a starch base ,20. ChicLactobacillus plantarum decreased ANFs, increased the amount of free essential amino acids, improved in vitro protein digestibility and lowered the starch hydrolysis index [We have previously shown that fermentation of faba bean flour by is index . After fis index . Trypsinis index . The objVicia faba cv Kontu), which were used for the production of faba bean flour, were provided by the University of Helsinki, Finland. The beans were dehulled by using an ultra-fine friction grinder  operated with a 0.5 mm gap, and the hulls were aspirated in a spray drier . Dehulled beans were milled as described in Reference [Faba beans  was routinely propagated at 30 °C in MRS broth . When used for fermentation, lactic acid bacterial cells were cultivated until the late exponential phase of growth was reached (ca. 10 h), washed twice in 50 mM phosphate buffer, pH 7.0, and suspended in the tap water used for fermentation.7 CFU/g of dough) was carried out at 30 °C for 48 h as described in Reference [Faba bean flour was mixed with water to prepare dough in a ratio of 50:50. Fermentation with the lactic acid bacteria  suspending and aliquot of 10 g of samples in 100 mL distilled water.Total titratable acidity (TTA) was determined with the TitroLine Alpha autotitrator on 10 g dough homogenized with 90 mL of distilled water and expressed as the amount (ml) of NaOH 0.1 M to reach pH of 8.5.The Rapid ViscoTM Analyser (RVA)  was used to determine the pasting properties of the faba bean flours. Pasting properties were determined following the standard Newport Scientific Method 1 (STD1). The heating cycle was 50–95 °C in 282 s, holding at 95 °C for 150 s, and then cooling down to 50 °C in 228 s and holding at 50 °C for 120 s. Each cycle was initiated by a 10 s mixing at 960 rpm paddle speed and 160 rpm paddle speed was used for the rest of the test. The RVA studies were carried out using 3.5 g of dried sample and 25 mL deionized water in an aluminium canister. The parameters recorded were peak, final, breakdown and setback viscosities together with pasting temperature. Flour samples were run in triplicate.Faba bean flour and fermented faba bean flour (freeze-dried) were applied in gluten-free bread baking and commercial soy flour was used for control breads. The amounts of bean flours were adjusted to obtain the same final protein content (16%) which would enable the “protein-rich” nutritional claim. Bread S37 was soy bread with 37% soy flour and 63% corn starch, bread F50 was faba bread with 50% faba flour and 50% corn starch, and bread FF50 was faba bread with 50% fermented faba flour and 50% corn starch. The proportion of the rest of the ingredients were fixed for each bread recipe and were as follows: sugar 5.7%, baking powder 2.4%, shortening 4.9%, baker’s compressed fresh yeast 4.9%, salt 1.7%, xanthan gum 2%, emulsifier 0.6%, water 102.7%. All the percentages were based on baker’s percentages. The recipe, mixing, proofing time and baking conditions were optimized in a preliminary work which is not shown here. The dry ingredients were first mixed together and then shortening, yeast and water were added. The temperature of the water was adjusted so that the final dough temperature after mixing was 26 ± 1 °C. Kneading was done for 4 minutes at high speed (level 2/3) with a planetary mixer . Small tins (9 × 5 × 6 cm) were filled with 160 g of batter and then proofed for 45 min at 28 °C and 85% relative humidity The breads were baked in a rack oven  at 180 °C for 17 min. Baking was conducted twice for each bread recipe.The texture profile analysis of the crumb was performed after 1 h and 24 h of baking. Two slices were taken from the centre of each loaf. Uniform slices of 2.5 cm thickness were obtained with a cutter and the analysis was carried out with a TA-XT Plus texture analyser , equipped with a 25 kg load cell and a 25 mm diameter aluminium cylindrical probe. The pre-test and test speeds were identical with a value of 1.7 mm/sand post-test speed was 10 mm/s. A strain value of 40% strain with 5 s pause time between compression cycles was applied. The results are an average of ten replicates.The specific volume of the bread loaves was measured based on the infrared light scanning method . Six loaves of each bread type were measured, and the average value was calculated.n = 5) evaluated the characteristics of the breads based on a previously published protocol [A trained descriptive sensory panel (protocol . The panSamples for X-ray microtomography (XMT) were made by cutting 1 cubic cm bread samples at 3 different locations from the centre of each bread recipe. Samples were scanned using a desktop XMT system  consisting of an X-ray tube, an X-ray detector and a charge-coupled devices (CCD) camera. The X-ray tube was operated at a voltage of 40 kV/250 μA to obtain optimum contrast between void (air cells) and matter  according to a modified method . A 12-biw/v) agar and fixed in 1% (v/v) glutaraldehyde in 0.1 M Na-K phosphate buffer (pH 7.0), dehydrated in a graded ethanol series, and embedded in hydroxyethyl methylacrylate resin . Polymerized samples were sectioned (2 µm sections) in a rotary microtome HM 355S  using a tungsten carbon knife and stained after transferring onto glass slides. Staining of protein and starch in 2 mm thick sections were done with Light Green and Lugol’s iodine respectively, as described in Reference [Bread samples were first embedded in 2% (eference . The staIn vitro starch digestibility was measured based on Reference  and reprw/v), and the undigested protein was precipitated. The volume was made up to 100 mL with distilled water and the mixture centrifuged at 5000 rpm for 20 min. The precipitate was subjected to protein extraction according to Weiss et al. [In vitro protein digestibility was measured based on Akeson and Stahman . One gras et al. . The cons et al. . The in The supernatant, which contained the digested protein, was freeze-dried and used for further analyses. The method of AOAC 982.30a , modifieChemical Score (CS) estimates the amount of protein required to provide the minimal essential amino acid (EAA) pattern, which is present in the reference protein (hen’s egg). It was calculated using the equation of Block and Mitchel . The seqEssential Amino Acids Index (EAAI) estimates the quality of the test protein, using its EAA content as the criterion. EAAI was calculated according to the procedure of Oser , accordiEAAI) − 11.70). The Protein Efficiency Ratio (PER) estimates the protein nutritional quality based on the amino acid profile after hydrolysis. PER was determined using the model developed by Ihekoronye [The Biological Value (BV) indicates the utilizable fraction of the test protein. BV was calculated using the equation of Oser : BV =  between individual means were identified by Tukey’s test.Data were subjected to analysis of variance using IBM SPSS Statistics 21 , and significant differences (Fermentation did not have a significant impact on the protein 35% dm) and soluble starch (42% dm) contents of the faba flour, whereas total dietary fibre (DF) (7% versus 6.7% dm) and fat contents (1.5% versus 0.9% dm) were slightly but significantly reduced. Reduction in total DF and fat content by fermentation of various pulses such as pigeon pea, gram, kidney bean and cowpea has been observed earlier ,35. DF s% dm and Fermentation of faba bean flour reduced setback  and final viscosities  in the RVA analysis of flour slurry, as reported earlier for various starch matrices including, for example, mung bean . Setbacka and 2.1 ± 0.1 b (mL/g) for unfermented and fermented faba bread respectively, and 1.5 ± 0.05 c mL/g for soy bread. GF bread dough generally resembles a cake batter and added proteins act as emulsifiers by forming a film or skin around oil droplets, which prevents structural failures due to coalescence. The good emulsifying capacity of faba bean proteins was thus reduced by fermentation [The impact of flour pre-fermentation on the structure of breads was evaluated both at macro- and micro-scales. Faba breads had higher volume than soy bread, whereas fermentation reduced the volume yield of bread. The volume yields were significantly different among the breads: 2.4 ± 0.1 entation . Fermententation , which aentation . In addientation . Faba flFaba breads had better loaf shape, smooth crust and better crumb colour and structure compared to soy bread a. The miL. plantarum) did not improve the loaf volume, aroma or microbiological shelf life of the gluten-free oat bread [It has earlier been shown that fermentation improves the structure, texture and shelf life of wheat bread . The litat bread . Lactic at bread . The micat bread . This miHardness, chewiness and cohesiveness of the breadcrumb as a function of fermentation-induced changes and storage time are presented in Earlier, freeze drying of faba bean protein isolates was shown to reduce the solubility and emulsification properties in protein/water suspension with a maximum 4% protein content compared to spray-dried counter parts . The effDegradation of the protein matrix by acidification and weakening of the structure reduced the carbon dioxide holding capacity of dough, lowering both the bread volume and porosity that further increased hardness. For all breads, hardness value significantly increased the next day. Chewiness of all faba breads was lower than soy bread . ChewineThe general appearance of faba breads was better than soy bread in terms of evenness, shape and colour intensity of bread . No signc) compared to S37 soy bread (93 ± 4 b). However, there was no significant difference of HI values between FF50 and F50 (83 ± 2 bc). Processing can induce macromolecular interactions between starch and components of the food matrix reducing the availability of starch for digestive enzymes. The chemical changes and interactions between starch-protein that occur in the presence of lactic acid have been suggested to lower the rate of starch hydrolysis [Bread with fermented faba bean flour (FF50) had a significantly lower hydrolysis index  of gluten-free faba breads. EAA and BV indexes as well as PER were significantly increased for breads made with fermented faba bean flour. Furthermore, fermentation increased the NI of faba bread (2.47) to levels even higher than the soy bread (2.1). No significant difference was caused by fermentation on the general appearance of faba breads . A sensory panel indicated that crumbliness, evenness of pore size and springiness of crumb were similar for all breads.Generally, gluten-free products are inferior in their nutritional properties as they are mainly high in carbohydrates and low in protein and dietary fibre. The incorporation of fermented faba bean flour will not only improve nutritional quality, as stated above, but also enable a source of protein  or high-protein  claims in gluten-free products if used as a protein ingredient. The gluten-free faba bean bread of this study would be qualified for “source of protein” and “source of fibre (at least 3 g of fibre per 100 g of product)” claims.