Drosophila circadian clock, Period (PER) and Timeless (TIM) proteins inhibit Clock-mediated transcription of per and tim genes until PER is degraded by Doubletime/CK1 (DBT)-mediated phosphorylation, establishing a negative feedback loop. Multiple regulatory delays within this feedback loop ensure ~24 hr periodicity. Of these delays, the mechanisms that regulate delayed PER degradation (and Clock reactivation) remain unclear. Here we show that phosphorylation of certain DBT target sites within a central region of PER affect PER inhibition of Clock and the stability of the PER/TIM complex. Our results indicate that phosphorylation of PER residue S589 stabilizes and activates PER inhibitory function in the presence of TIM, but promotes PER degradation in its absence. The role of DBT in regulating PER activity, stabilization and degradation ensures that these events are chronologically and biochemically linked, and contributes to the timing of an essential delay that influences the period of the circadian clock.In the  Many behaviors, such as when we fall asleep or wake up, follow the rhythm of day and night. This is regulated in part by our ‘circadian clock’, which controls biological processes through the timed activation of hundreds of genes over the 24-hour day.In fruit flies, the proteins that form the core of the circadian clock activate and repress each other in such a way that their expression oscillates over a 24-hour cycle. During the late afternoon and early evening, the Clock protein initiates the production of proteins Period and Timeless: these two molecules then accumulate in the cell, and after binding to each other, they are transported into the nucleus. During the late night and early morning, this Period/Timeless complex inhibits the activity of Clock. After a delay, Period and Timeless are degraded. This allows Clock to be reactivated, restarting the cycle for the next day.Period is critical to help maintain the 24-hour oscillation shown by these proteins. A protein called Doubletime is responsible for making a number of chemical modifications on Period. It is unclear how these changes interact with each other, and how they influence the stability and function of Period when it is associated with Timeless.period to study these processes, and develop a new biomolecular technique to monitor the stability and activity of Period protein in insect cells grown in the laboratory.Here, Top et al. generate mutations in the fruit fly gene The experiments reveal new roles for the chemical changes made by Doubletime to Period. First, after Period associates with Timeless, Doubletime triggers certain modifications that lead to Period being able to inactivate Clock. Second, Doubletime makes another change in a nearby region of Period that results in the Period/Timeless complex being stabilized. Both sets of modifications help the complex to stay active and keep inhibiting Clock for long enough such that a 24-hour rhythm can be maintained. Finally, when Timeless is degraded, Period is released from the complex. At this time, the modifications made by Doubletime promote the degradation of Period, resetting the clock.Fruit flies with mutations that block this mechanism perceive the day as shorter. This shows that the smallest change to clock genes can disorganize behavior. Indeed in humans, health problems such as sleep or mental health disorders are associated with irregular circadian clocks. Understanding the biochemical mechanisms that keep the body clocks ticking could help to find new therapeutic targets for these conditions. Drosophila, the activator complex consists of dClock/Cycle (dCLK/CYC), which initiates transcription of period (per) and timeless (tim). PER and TIM proteins assemble to form an inhibitory complex in the cytoplasm that, after a delay, enters the nucleus to inhibit dCLK/CYC activity. After a second delay, the inhibitory complex is degraded, freeing the activator complex to reinitiate transcription, closing the feedback loop. Although the delay in transcriptional activation is critical for maintaining ~24 hr rhythmicity, its regulatory mechanisms are not well understood.The proteins that comprise circadian clocks are largely conserved across the animal kingdom . These cShort Downstream region , which promotes S47 phosphorylation (A Casein Kinase I/Doubletime (DBT) phosphorylation cascade on PER regulates PER protein stability. Current models of this phosphorylation cascade infer that each DBT phosphorylation event gates later phosphorylation events on PER, thus delaying protein degradation and transcriptional activation. The cascade begins with the PER rylation . Phosphorylation . PER-SD rylation . In otherylation . The purMutation of the S589 site in flies leads to a short behavioral period . Given tTIM binding to PER adds another layer of complexity to the model by protecting PER from hyperphosphorylation and degradation . This suIt is generally assumed that inhibition of dCLK is gated by nuclear accumulation of PER, and release of dCLK inhibition occurs passively, through PER degradation. PER phosphorylation, PER degradation and PER inhibitory activity coincide closely in time and space , which hIn this study, we propose a new model for the regulation of PER by DBT, which includes separable features affecting PER stability and activity. Our findings suggest that the DBT/PER/TIM complex is interdependent in the initial stages of dCLK/CYC inhibition. We show that the initial sites of DBT phosphorylation stabilize the PER/TIM inhibitor complex and activate transcriptional inhibition. The classic per short site S589 serves two functions: first to stabilize the activated PER inhibitor in the presence of TIM and then to mediate its degradation after TIM is degraded. We propose a dual role for DBT in regulating the circadian clock. (1) In the nucleus DBT activates function of the PER/TIM complex as a transcriptional inhibitor and stabilizes it, (2) subsequently promoting PER degradation by midday. The dual function of DBT links PER/TIM inhibitory activity and stability, biochemically and chronologically, and underlies a key delay in the circadian clock that helps establish 24 hr behavioral rhythmicity.per0 mutation with per variants containing substitutions of the DBT-targeted perS site S589, using targeted genomic integration to avoid positional effects on protein expression. As previously reported . Similar to phosphonull S589A mutants, we found that phosphomimetic S589D mutants exhibit a modest advance in evening anticipation and no change in morning anticipation relative to controls . ConsistIt was not clear whether these effects on period length by this residue is solely due to phosphorylation by DBT kinase or whether there may be a second kinase involved. We can distinguish between these two possibilities by testing genetic interactions between specific phosphomutations and kinase overexpression or kinase mutant expression. For example, mutation of specific target residues of the TIM protein blocks the effect of CK2 kinase overexpression on rhythmic behavior . DBT oveO-GlcNAc transferase (OGT) has been shown to influence behavioral rhythmicity in mammals and flies, either by competing with CKIδ for modification of mPER2  or blocking BMAL1/CLOCK (orthologues of CYC/dCLK in flies) ubiquitination in mice, or prolonging PER stability in flies (The activity of in flies . Since win flies . The samin flies . We therdbt that reduce DBT kinase activity produce long (dbtL) and short (dbtS) behavioral rhythms  or phosphomimetic (S589D) per variants in a dbtL genetic background leads to longer behavioral rhythms  exhibits kinetics of nuclear entry similar to wild type  that drive behavioral rhythms in constant darkness  in a similar study that included TIM (but not DBT) in cultured cells suggests that it is equivalent in stability to wild type PER  or a dCLK inactive phase (trough). This analysis revealed that the dCLK active phase of S589A mutants is shorter than both wild type and S589D, which are equivalent . On the per mutants that represent transitional isoforms informed by the phosphorylation cascade model  simultaneously with PER inhibitory activity (YFP fluorescence)  . Co-tranescence) . dCLK exescence) . Cells wescence) . Using ttim expression to stabilize high levels of actin-driven exogenous per expression. Differences in inhibitory activity or stability are not due to poor nuclear accumulation  that precede S589 and S47 phosphorylation . When thThe PER-SD region can also regulate PER stability and activity by blocking S589 phosphorylation . To deteA PER variant not predicted by the phosphorylation model, S589D/4A, is an unstable protein with indeterminate inhibitory activity that resembles S589D  and 6C. Delays in the transcriptional negative feedback loop of the circadian clock ensure that it oscillates with ~24 hr periodicity. Delayed transcription (i.e. delayed PER degradation and transcription re-activation) is one such critical delay. In this study, we present a model in which DBT stabilizes and activates PER inhibition to delay transcription in the circadian clock . DBT begdbtL) and short-period (dbtS) behavioral phenotypes carry mutations that reduce the kinase activity of DBT activity in the S589/PER-SD region.DBT is likely to regulate the stability and inhibitory activity of PER through separate mechanisms. Such functional uncoupling has also been suggested for the Neurospora clock . Regionsy of DBT . Loweredckground . These dy others , while dS2 cells , dbtS mahper2 locus (decrease in transcription), rather than a change in protein stability. On the other hand, S589 phosphorylation (S589D) stabilizes PER in complex with TIM and helps activate PER inhibitory activity. Despite these differences, both S589A and S662G advance evening anticipation behavior, suggesting that phospho-regulation of these sites underlie this aspect of rhythmic behavior. Also, a reduction of DBT activity in mutant flies (dbtL) or reduced CKIδ expression in mice (heterozygous expression) both lead to longer period behavioral rhythms that are independent of residue substitution in positions S589/S662. Thus, despite differences in the PER function that sites S662 and S589 regulate, both mammals and flies utilize distinct mechanisms that employ DBT/CKIδ to separately regulate the activity and stability of Period protein, delaying transcription reactivation in the circadian clock.Distinct phospho-programs also regulate human PER2 stability and inhibitor activity . The humIn vivo, it is difficult to determine if a mutation in per results in lower PER protein levels caused by destabilized protein or overactive transcriptional inhibition. To distinguish between the two possibilities, we must simultaneously measure transcription factor expression and activity. We therefore developed a fluorescence-based transcription assay to probe the relationship between S589 and the PER-SD in regulating stability and inhibitor activity. The previous PER phosphorylation model begins with phosphorylation of PER-SD that blocks PER S589 phosphorylation, and promotes PER S47 phosphorylation  alters its binding affinity to Clock, also changing the delay of transcription inhibition in humans to cause a familial form of delayed sleep phase disorder  was subcloned into the HS-Casper  was a gift from Sheyum Syed. A HindIII site 3’ to the YFP-PEST cassette was used to insert a pAc > clk-cfp cassette to generate the reporter plasmid. S2 cells were obtained from ATCC  maintained in Schneider’s medium (Invitrogen) supplemented with 10% FBS  and tested negative for mycoplasma contamination. S2 cells were transfected using Effectene  as per manufacturer’s protocols. Cells were resuspended in fresh medium one day after transfection and seeded onto Lab-Tek II chamber slides  for imaging.DNA encoding S-Casper  or pAc5.per mini-gene . Individual flies were analyzed for locomotor activity in 12 hr: 12 hr light/dark light regiments (LD) or in constant darkness (DD) after at least three days entrainment, using the Drosophila Activity Monitor System IV . Period was determined using ClockLab Software . UAS-DBT transgenic flies were a gift from Jeffrey Price . UAS-OGT transgenic flies were a gift from Louis Ptáček . Flies overexpressing OGT were generated by recombining the second chromosome carrying the tim-UAS-Gal4 and UAS-OGT transgenes.The ini-gene  was mutaini-gene  with a Cper promoter (plo) were crossed to flies expressing the indicated per transgenes in a per0 background to generate a y,w,per0;plo/[per];+genotype for analysis. Two sets of 80 males were monitored in 3 cm plates with standard fly food supplemented with luciferin , as previously described  were heat shock induced to exogenously express TIM (YFP) or PER (mCherry) alongside actin-driven PER (mCherry) or TIM (YFP), respectively, and actin-driven CFP in an air incubator at 37°C for 30 min. Cells were imaged using a DeltaVision system  equipped with an inverted Olympus IX70 microscope , a CFP/YFP/mCherry filter set and dichroic mirror , a CCD camera , and an XYZ piezoelectric stage for locating and revisiting multiple cells. 50–70 cells were selected based on their CFP expression and CFP, YFP, mCherry channels recorded. Imaged cells were analyzed using a locally written algorithm in Matlab , as previously described . To meas3). Equal volumes  of lysate were used in analysis by immunoblot using 6% or 10% SDS-PAGE gels. Blots were probed with 1:1000 anti-TIM antibody  were taken at the indicated time points (once an hour), with cycloheximide (20 μg/ml) added at hour 1 (1 hour post heat shock recovery). Cells were washed once with PBS and lysed using 200 μl modified RIPA  using FACSDiva v.6.1.1 software. Fluorescence signal was filtered to monitor CFP (405 nm), YFP (488 nm) and mCherry (561 nm). CFP positive cells  were binned into groups of increasing mCherry signal and plotted as a function of YFP signal.S2 cells expressing the reporter/per (amino acids 1–700) were sub-cloned into pGEX-6–1 p plasmid using standard cloning techniques. Bacteria were induced to express GST-fused PER fragment at 17°C at OD600 0.6 and grown overnight. Bacteria were lysed chemically in lysis buffer , RNase, and benozonase nuclease) supplemented with protease inhibitors. PER fragments were purified using glutathione beads affinity chromatography, the GST tag was cleaved by prescission protease , followed by size exclusion chromatography purification, and confirmed by mass spectrometry. SEC-MALS was conducted at room temperature on a Wyatt–WTC050N5 SEC column with buffer containing 500 mM NaCl, 50 mM Tris pH 7.5, and 3 mM DTT.Variants of 11 photons/sec). Blank buffer with no protein was exposed and collected as background for subtraction. Lysozyme and glucose isomerase were used as standards for Rg and molecular weight calculations. RAW (q = 4πsin(θ)/λ, where θ is half of the angle between the incident X-ray beam and the scattered beam, and λ is the wavelength of X-rays.SAXS data was collected in a similar fashion as previously described , but witons. RAW  and Primons. RAW  were usePER fragments  were incubated with trypsin (80 μM) for the indicated times, and compared to undigested controls. Digestion reactions were stopped using 2 μl of 160 μM trypsin inhibitor and 10 μl SDS gel loading buffer. Digested products were analyzed by 4–12% NuPAGE BisTris gel, Coomassie stain, and quantified using ImageJ. Protein fragments were confirmed as PER fragments by mass spectrometry. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by a Senior Editor and three reviewers, one of whom is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Louis J Ptáček (Reviewer #1).Thank you for submitting your article "CK1/Doubletime Activity Delays Transcription Activation in the Circadian Clock" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Drosophila, numerous studies have led to key insights into how phosphorylation regulates PER stability. Key insights have been gained from the classic per-short mutation at Ser589 that abrogates phosphorylation at this site. The per-s mutation accelerates PER degradation, and TIMELESS (TIM) stabilizes PER against DBT-mediated degradation. In this paper, Top et al. show that phosphorylation at Ser589 by DBT has a dual purpose in the transcriptional feedback loop by delaying PER degradation and making it a better repressor. The transition from repression to degradation is temporally linked to TIM. The data is largely based on analyzing an ala mutant of Ser589 (non phospho form) and a novel Asp mutant (phosphomimetic?). To better measure PER stability (levels) and transcriptional repression, they devised a tissue culture based flow cytometry assay that can score both simultaneously. By incorporating prior findings, they suggest a model whereby upstream phosphorylation of PER (SD region) precedes phosphorylation at Ser589 by DBT, which in the presence of TIM, generates a strong PER repressor. Phosphorylation of Ser589 after CT2, stabilizes PER and allows it to function as a repressor. Later on in the cycle, when TIM is degraded, PER becomes unstable and degraded.In animals, PER is the main repressor of CLOCK-BMAL1/CYCLE dependent circadian transcription. PER undergoes a complex hierarchical phosphorylation program that regulates its stability in a daily manner. In per-short domain also affects PER repressor activity, making it less stable but a better repressor. Prior work  showed that DBT and CK2 increase PER repressor function, although the mechanism was not clear. The role of Ser589 on PER stability and the phosphorylation cascade, including how DBT and TIM function in the model presented , have been largely worked out before . Novel insights were gained by analyzing a 589 Asp mutant as it also causes shorter rhythms (but not as short as the Ala mutant).In an earlier paper, Young and co-workers  suggested that phosphorylation of PER at Ser589 and the Essential revisions:1) The key finding we all seem to agree on is that 589D/4D is a stable and strong repressor. This is a new variant of PER that has not previously been studied, as all prior studies used A mutants. This is a key finding that shows a role for 589 in both degradation and inhibition, with regards to the SD region. In addition to this finding, based on the 589D allele still having a shorter period then wildtype, they also make the claim that phosphorylation at 589 makes PER unstable once TIM is gone; i.e., the TIM switch. The fact that maintaining phosphorylation at 589 ALSO affects the period is interesting and novel. However, here the interpretation of what this means is not clear. The problem is that the species they have in their model for degradation of PER at the end of the cycle is actually S589D/4A. If that is the "wildtype" species for degradation  then why does the 589D mutant have a shorter period then wildtype? Also, in their model they go from S589D/4D to S589D/4A for degradation of PER after TIM is lost. This would imply that it is not just the status of S589 that is key to degradation after TIM is lost but you also need to dephosphorylate the SD region. According to their mutant data in flies showing that 589D is shorter then wildtype, this should(?) imply that there is a species of wildtype PER at the end of the cycle whereby 589 is not phosphorylated and more stable than the 589D form that is phosphorylated. So, is there a species of PER whereby a 589D is less stable then a 589A that could account for the difference in period between S589D and wildtype? From Figure 6, S589D/4A is less stable then S589A/4A, so it might be possible that the S589D/4D becomes S589A/4A at some point, but a potential problem is that S589A/4A is too(?) stable-although it seems to be less stable then PER4A. Thus, the main problem I have is with the second part of the story; i.e., how does maintaining phosphorylation of 589 at the end of the cycle make it less stable then the wildtype, giving rise to a shorter period? In other words, they imply that de-phosphorylation of 589 late in the cycle is critical for delayed PER degradation. Why is that not in the model? And if 589 has to be dephosphorylated at some point, what is the PER species that is being degraded in the wildtype at the end of the cycle?2) Also, the TIM switch argument is complicated. Basically, based on Figure 3, they say, "These data therefore suggest that S589 phosphorylation stabilizes PER in the presence of TIM from DBT-mediated degradation, either by blocking phosphorylation of other PER destabilizing sites, or in spite of phosphorylation of these other sites. In contrast, blocking S589 phosphorylation redirects DBT to promote the degradation of the PER/TIM complex, likely by phosphorylating other PER sites. Therefore, S589 acts as a switch, directing DBT to degrade PER or to stabilize PER within the PER/TIM complex."- While all of this is true, calling it a TIM switch is a bit confusing. All it means is what we already know, in the presence of TIM the 589D species is more stable than the 589A species when other sites are also phosphorylated. Otherwise stated, both 589A and 589D species are more stable with TIM, it is just that 589A is less stable then 589D. In do not see how 589A degradation is "TIM-independent", when it is clearly shown in Figure 3 that TIM stabilizes 589A .To summarize:a) How does retaining S589 phosphorylation destabilize PER at the end of the cycle to get a shorter period compared to the wildtype situation as implied from the S589D mutant . And if this implies that 589 has to be dephosphorylated at some point late in the cycle (so that it continues being stable and repressing for a few more hours), what is that species and why is it not shown in the model?b) I do not fully understand the implication of calling the role of TIM vis. a vis. S589 a TIM switch when all phospho-isoforms of PER appear to be less stable without TIM in the presence of DBT. Indeed, from the model it appears that losing SD phosphorylation is required for degradation but not a change in the phosphorylation status of S589.c) Chiu et al., 2011, showed that S589 is highly phosphorylated by ZT20, so it is not clear that there is an early species of S589A/4D, which is basically based on the Garbe et al., model. The Garbe model is based on epigenetics, so it is not a given that phosphorylation at SD blocks phosphorylation at S589. This potential discrepancy should be discussed. In any event, it was not discussed if there is a functional significance to the initial repressor being highly unstable, i.e., does it play a different role compared to the more stable 589D/4D?Therefore, while the role of 589D/4D as a novel, stable and strong repressor is well documented and highly interesting, and an added role for 589 phosphorylation status in the degradation of PER after CT2 is also interesting, the latter part of this story and a specific role for TIM are less solid, making the latter part of the model unclear as to how it was derived based on the data.When presenting the model in Figure 7, the authors need to present their arguments for the time course and nature of the phosphorylated species at each step, with alternatives presented if possible.per short site likens to the phosphorylation cascade reported in mammalian Pers . This paper should be cited and discussion regarding similarities and differences of the reported findings with these published data should be addressed.3) The authors largely ignore related work in mammalian systems showing that CKId has different effects on Per2 depending on the site of phosphorylation. The PER phosphorylation cascade downstream of the classic 4) Another interesting observation are that the results with the S589A and S589D alleles don't all agree with what might be predicted regarding expected DBT phosphorylation effects (assuming 589D mimics a constitutively phosphorylated S589). It would be worth also looking at 2 additional papers: . These papers show that the same Serines in the "cascade region" in mammals are also being O-GlcNAC modified. That is, CKId and OGT are competing for occupancy and modification of the same sites. An interesting question is whether O-GlcNAC modified vs. phosphorylated Serines in this region have different downstream effects. Has anyone looked to see whether the phosphorylation cascade region in dper is O-GlcNAC modified? While the fly and mammalian regions are not really conserved at a sequence level, the similarity of some of the effects in fly suggests they may be similar functionally. If so, then phosphorylated vs. GlcNAC modified vs. unmodified may underlie the unexpected results of S589 vs. D589.5) There is no need for the SAXS data, etc. as it does not add anything to the Discussion. Essential revisions:1) The key finding we all seem to agree on is that 589D/4D is a stable and strong repressor. This is a new variant of PER that has not previously been studied, as all prior studies used A mutants. This is a key finding that shows a role for 589 in both degradation and inhibition, with regards to the SD region. In addition to this finding, based on the 589D allele still having a shorter period then wildtype, they also make the claim that phosphorylation at 589 makes PER unstable once TIM is gone; i.e., the TIM switch. The fact that maintaining phosphorylation at 589 ALSO affects the period is interesting and novel. However, here the interpretation of what this means is not clear. The problem is that the species they have in their model for degradation of PER at the end of the cycle is actually S589D/4A. If that is the "wildtype" species for degradation  then why does the 589D mutant have a shorter period then wildtype? Also, in their model they go from S589D/4D to S589D/4A for degradation of PER after TIM is lost. This would imply that it is not just the status of S589 that is key to degradation after TIM is lost but you also need to dephosphorylate the SD region. According to their mutant data in flies showing that 589D is shorter then wildtype, this should(?) imply that there is a species of wildtype PER at the end of the cycle whereby 589 is not phosphorylated and more stable than the 589D form that is phosphorylated. So, is there a species of PER whereby a 589D is less stable then a 589A that could account for the difference in period between S589D and wildtype?We thank the reviewer for the constructive and helpful comments.We are encouraged that the reviewers agree that the S589D/4D is a strong and stable repressor, and that S589 (with PER-SD) plays a dual role in helping repress CLK-mediated transcription and PER stability. (A) The key problem the reviewers have in this section is our placement of S589D/4A at the end of the PER life cycle. (B) Another problem the reviewers have is the emphasis on TIM as a regulator of PER stability, even in the context of the different mutant isoforms. (C) Finally, there is some confusion regarding the phospho-state of S589, why both S589D, and S589A can be less stable than wild type and the relationship of the changed stability to behavioral period.The reviewers raise important points, which we clarify through changes in the text. We have also reviewed and revised our model in Figure 7.A) After measuring the stability of the S589D/4A variant, we reasoned that it fit well nearer the end of the PER cycle. However, it is also possible that the S589D/4A isoform represents a transitional state in an alternate “short circuit” pathway to induce PER degradation, or may not be relevant for stability. Our model emphasizes that the S589D/4D isoform of PER is a strong and stable repressor and that DBT phosphorylation events mediate the stability of PER, a novel function for DBT. To avoid confusion, we agree with the reviewers’ concern about placing the S589D/4A PER isoform in our model. We have therefore removed the reference to S589D/4A in the schematic of our model .We have also clarified the text to reflect our revisions: “This transition state [S589D/4A] can occur if S589 phosphorylation blocks PER-SD phosphorylation  and may represent (i) a later stage isoform of PER at the end of transcriptional inhibition, (ii) an isoform that commits PER to degradation prematurely, or (iii) may not be an isoform relevant to the PER degradation pathway.”B) It is likely that, in the absence of TIM, PER is rapidly degraded by DBT regardless of its phospho-state. We have included a western blot for the reviewers to support this claim . We thera) We have added the sentence: “Thus, S589 phosphorylation has a stabilizing effect when TIM is present.” This emphasizes that TIM stabilization of PER cooperates with the S589 phospho-state.b)We have added the sentence: “The changes in the stability of the PER variants correlate well with the differences in rhythmic behavior exhibited by the relevant fly mutants .” This emphasizes the correlation of changes in behavior with the stability of each S589 PER variant.c) We have removed the phrases “but that phosphorylation of S589 causes destabilization of PER in its absence” and “during the early stage of the circadian cycle (after CT2)”. We believe that this distracts from the emphasis we wished to place on S589 phosphorylation cooperating with TIM to stabilize PER.C) With regard to why both S589D and S589A exhibit a short behavioral rhythm, the reviewers raise this issue in their review, section “a”, below. We offer a thorough explanation below. We have also modified the text to state, “The changes in the stability of the PER variants correlate well with the differences in rhythmic behavior exhibited by the relevant fly mutants .” As mentioned above (B) to draw attention to the correlation between protein stability in neurons  and rhythmic behavior .From Figure 6, S589D/4A is less stable then S589A/4A, so it might be possible that the S589D/4D becomes S589A/4A at some point, but a potential problem is that S589A/4A is too(?) stable-although it seems to be less stable then PER4A. Thus, the main problem I have is with the second part of the story; i.e., how does maintaining phosphorylation of 589 at the end of the cycle make it less stable then the wildtype, giving rise to a shorter period? In other words, they imply that de-phosphorylation of 589 late in the cycle is critical for delayed PER degradation. Why is that not in the model? And if 589 has to be dephosphorylated at some point, what is the PER species that is being degraded in the wildtype at the end of the cycle?The reviewers here ask about the importance of S589 and how the phospho-state of S589 relates to the phosphorylation state of PER-SD in terms of PER stability. There is concern regarding the need for dephosphorylating S589 as a part of the mechanism of PER degradation.We do not mean to imply that S589D/4D becomes S589A/4A, but instead suggest that S589D/4D may become S589D/4A or S589A/4D through de-phosphorylation. S589D/4A is a highly unstable isoform of PER . The few S589D/4A variants that escape degradation and are available to revert back to the non-phosphorylated form (S589A/4A) would be fed through the same pathway until the number of PER molecules are reduced to levels insufficient to block CLK activity. S589A/4D on the other hand can easily be re-phosphorylated by DBT to form S589D/4D again. As we state in the manuscript, this detail of the proposed model is more speculative than other parts of the model that are directly supported by our data. Therefore, in order not to detract from the model, we removed S589D/4A from our schematic in Figure 7 . For the discerning reader who may wonder about this possible transition state, we have added a small section in the discussion on S589D/4A and clarified the text as follows:“Phosphatases play a role in maintaining 24 hour behavioral rhythms . […] Thus the ordered phosphorylation of PER-SD and then S589 ensures that the circadian clock ticks forward.”We also do not mean to imply that S589 is dephosphorylated as part of the mechanism of PER repression or PER degradation . We propose, based on data from Garbe et al. (2013) and Chiu et al. (2008), that the S589A/4D form is the predecessor isoform to the S589D/4D form. Indeed, S589 does not appear to be dephosphorylated as PER is being degraded, as shown by western blot by Chiu et al. (2011) from whole head protein extracts.2) Also, the TIM switch argument is complicated. Basically, based on Figure 3, they say, "These data therefore suggest that S589 phosphorylation stabilizes PER in the presence of TIM from DBT-mediated degradation, either by blocking phosphorylation of other PER destabilizing sites, or in spite of phosphorylation of these other sites. In contrast, blocking S589 phosphorylation redirects DBT to promote the degradation of the PER/TIM complex, likely by phosphorylating other PER sites. Therefore, S589 acts as a switch, directing DBT to degrade PER or to stabilize PER within the PER/TIM complex."- While all of this is true, calling it a TIM switch is a bit confusing. All it means is what we already know, in the presence of TIM the 589D species is more stable than the 589A species when other sites are also phosphorylated. Otherwise stated, both 589A and 589D species are more stable with TIM, it is just that 589A is less stable then 589D. In do not see how 589A degradation is "TIM-independent", when it is clearly shown in Figure 3 that TIM stabilizes 589A .2a) The reviewers emphasize the confusion regarding the term “TIM switch”.2b) The reviewers also raise the issue that S589A and S589D stabilization by TIM was previously known, and2c) That it is unclear how S589A degradation could be “TIM-independent”.2a) We recognize that there is some confusion regarding the term “switch”. Our data resolve a longstanding paradox of S589 mutation and its effect on period length by showing that S589 phosphorylation stabilizes and promotes PER inhibitory function in the presence of TIM and helps mediate PER degradation in the absence of TIM. We realize that the term “switch” does not capture this complex mechanism. Although we have not used the term “TIM switch”, we did call S589 a switch. To clarify this issue, we have removed most references to the term “switch” throughout the text and inserted more precise language. The text now reads:“Our results indicate that phosphorylation of PER residue S589 stabilizes and activates PER inhibitory function in the presence of TIM, but promotes PER degradation in its absence.”“The classic per short site S589 serves two functions: first to stabilize the activated PER inhibitor in the presence of TIM and then to mediate its degradation after TIM is degraded”“Therefore S589 acts to direct DBT to degrade PER or to stabilize PER within the PER/TIM complex.”“Thus, PER degradation through S47 can be regulated through two routes of phosphorylation, diverging with S589 phosphorylation.”2b) It was not known before our study that S589D is more stable than the S589A variant, in the presence of TIM. To clarify the text and historical context, we have now included the citation suggested by the reviewers. The article cited by the reviewers  use the original perS mutant, S589N, for their studies, not S589A. Substitution at this position would indeed block the ability of a kinase to phosphorylate the protein, but substitution with asparagine can lead to ambiguity. Asparagine has a dipole on its side chain. In this regard, it may be able to act as a weak phosphomimetic under some circumstances. It is for this reason we avoided including the S589N substitution in our studies, instead opting for the clearer S589A and S589D substitutions in PER.S and TIM, without exogenous DBT expression in cultured cells. Since, in cultured cells, exogenous DBT is required for DBT phosphorylation of S589 (and other sites) , we reasoned that endogenous DBT may not be sufficient to see all the effects of DBT-mediated degradation on PER and TIM that are driven by strong promoters in the Li and Rosbash study.Additionally, the paper by Li and Rosbash use PER/PERThe text has been modified for clarification as follows:S) in a similar study that included TIM (but not DBT) in cultured cells suggests that it is equivalent in stability to wild type PER . However the side chain of asparagine does have a dipole that has the potential to act as a phosphomimetic while simultaneously blocking phosphorylation by DBT.”We added the sentence “Analysis of the S589N variant of PER  We did not mean to suggest that S589A degradation was “TIM-independent”. The source of this misunderstanding may have been: “In other words, these data suggest that while both S589A and S589D mutants exhibit shorter circadian periods because of their effects on PER stability ” – The phrase in brackets has been removed for clarity. We instead meant that the stability of PER was variable with respect to wild type in the presence or absence of TIM. Figures 2D, 2E, 3A and B illustrate that the S589A PER variant and TIM are less stable compared to wild type PER/TIM in the presence of DBT. S589D on the other hand exhibits stability equivalent to wild type for both PER and TIM. Thus, we conclude that phosphorylation of S589 stabilizes the PER/TIM inhibitory complex. In the s-LNvs in vivo , the S589A variant of PER and TIM are less stable than wild type conditions after CT02, while the S589D variant demonstrates stability equivalent to wild type until TIM is degraded. When TIM is degraded, PER is rapidly turned over. In this regard, we conclude that S589 phosphorylation is stabilizing To remove any confusion about TIM-dependence with regard to the phospho-state of S589 or PER degradation, we have also removed or modified references to TIM dependence to place emphasis on S589 regulation of PER stability.We removed the phrase in parentheses referring to TIM dependence, mentioned above.We removed the words “TIM-dependent” in the abstract. The sentence now reads:“Our results indicate that phosphorylation of PER residue S589 stabilizes and activates PER inhibitory function in the presence of TIM, but promotes PER degradation in its absence.”To summarize:a) How does retaining S589 phosphorylation destabilize PER at the end of the cycle to get a shorter period compared to the wildtype situation as implied from the S589D mutant . And if this implies that 589 has to be dephosphorylated at some point late in the cycle (so that it continues being stable and repressing for a few more hours), what is that species and why is it not shown in the model?The reviewers ask for clarification on how S589D can be less stable than wild type PER at CT06, if wild type PER is phosphorylated to appear similar to S589D.We do not know the precise phospho-state of PER at CT06 in the s-LNvs. Determining its form would require purification of PER from the s-LNVs and mass spectrometry analysis, which is beyond our current technical ability. We do not think that S589 is de-phosphorylated at the end of the PER life cycle. This also appears to be the case in Chiu et al., 2011, where they demonstrate that an S589 phospho antibody reacts with protein from fly head extracts to reveal S589 is phosphorylated even as PER is degraded.certainty of “phosphorylation”, accelerating the degradation process.However, it is clear from our data that S589D is less stable than wild type PER in the s-LNvs in the absence of TIM (CT06). This difference may lie in the variability of phosphorylation of this site in a biological system. Substitution of S589 with the phosphomimetic aspartate forces the protein to appear phosphorylated 100% of the time. In “normal circumstances”, it is possible for serine phosphorylation to often be incomplete, allowing for variation in any given process. We therefore suggest that the higher average stability of wild type PER at CT06 may be due to partial phosphorylation of sites that lead to PER degradation. The S589D substitution offers b) I do not fully understand the implication of calling the role of TIM vis. a vis. S589 a TIM switch when all phospho-isoforms of PER appear to be less stable without TIM in the presence of DBT. Indeed, from the model it appears that losing SD phosphorylation is required for degradation but not a change in the phosphorylation status of S589.We hope we were able to address the issue raised about the term “switch” above.We also hope that our discussion regarding S589D/4A and the changes we have made to the manuscript sufficiently addresses this question (described above). By removing the reference to S589D/4A isoform in our model, we place more emphasis on the S589D/4D variant of PER that suggests that DBT plays a role in PER function that has not been previously described and would appear contradictory to its known role in PER degradation: stabilizing the PER/TIM complex through phosphorylation and activation of PER inhibitory activity.c) Chiu et al., 2011, showed that S589 is highly phosphorylated by ZT20, so it is not clear that there is an early species of S589A/4D, which is basically based on the Garbe et al., model. The Garbe model is based on epigenetics, so it is not a given that phosphorylation at SD blocks phosphorylation at S589. This potential discrepancy should be discussed. In any event, it was not discussed if there is a functional significance to the initial repressor being highly unstable, i.e., does it play a different role compared to the more stable 589D/4D?We thank the reviewer for this insightful comment. Yes, the evidence in Chiu et al. imply that the S589A/4D transition state may be a short-lived isoform. While we included S589A/4D in the isoforms tested because of its pivotal role in the Garbe model, our data do not support a different role than the more stable 589D/4D, which we did not address explicitly in the initial manuscript. In response to the reviewer, we have amended the discussion to include a brief discussion of this issue. The Discussion has been modified as follows:“In vivo, S589 is phosphorylated at ~ZT20, and appears to remain phosphorylated as PER is degraded . […] There is no apparent need for an unstable PER protein early in transcriptional inhibition and it is more likely that both the S589 and PER-SD sites cooperate to regulate PER stability and function.”Therefore, while the role of 589D/4D as a novel, stable and strong repressor is well documented and highly interesting, and an added role for 589 phosphorylation status in the degradation of PER after CT2 is also interesting, the latter part of this story and a specific role for TIM are less solid, making the latter part of the model unclear as to how it was derived based on the data.When presenting the model in Figure 7, the authors need to present their arguments for the time course and nature of the phosphorylated species at each step, with alternatives presented if possible.We are grateful that the reviewers appreciate our novel contributions on the role of S589 phosphorylation state in regulating PER function and stability and a new role for DBT in stabilizing the PER/TIM inhibitory complex. The reviewers point out that a specific role for TIM in our model is less clear.We have altered our model based on the suggestions made by the reviewers . We have further clarified the text with alternatives presented, particularly for the role of the 589D/4A isoform (see above). We hope that the changes made satisfy the concerns raised.We have altered our model based on the suggestions made by the reviewers and amended the Discussion: “DBT begins phosphorylation of PER in the PER-SD  (Step 1), initiating inhibition of dCLK-mediated transcription by PER. […] Thus, the steps that delay dCLK-mediated transcription activity rely on the activation and stabilization of PER by DBT phosphorylation, before TIM degradation ensues.”We have changed the figure legend to reflect the modified model: “Non-phosphorylated PER is stable, but unable to repress CLK-mediated transcription. […] After CT06, TIM is degraded, releasing PER for degradation, ending the PER life cycle.”3) The authors largely ignore related work in mammalian systems showing that CKId has different effects on Per2 depending on the site of phosphorylation. The PER phosphorylation cascade downstream of the classic per short site likens to the phosphorylation cascade reported in mammalian Pers . This paper should be cited and discussion regarding similarities and differences of the reported findings with these published data should be addressed.The reviewers suggest citation and discussion of the work by Xu et al. This is an excellent point and we apologize for this oversight.Drosophila and mammalian regulation of PER via phosphorylation. In summary, while there appear to be differences in specific effects , both mammals and flies appear to share in common the principle of distinct mechanisms by which DBT/CK1d regulates activity and stability of PER. The text has been modified to include the following paragraph in the Discussion, centered on this very interesting issue:We have now amended the Discussion to highlight this paper and address the similarities and differences between the “Distinct phospho-programs also regulate human PER2 stability and inhibitor activity . […] Thus, despite differences in the PER function that sites S662 and S589 regulate, both mammals and flies utilize distinct mechanisms that employ DBT/CKIδ to separately regulate the activity and stability of Period protein, delaying transcription reactivation in the circadian clock.”4) Another interesting observation are that the results with the S589A and S589D alleles don't all agree with what might be predicted regarding expected DBT phosphorylation effects (assuming 589D mimics a constitutively phosphorylated S589). It would be worth also looking at 2 additional papers: . These papers show that the same Serines in the "cascade region" in mammals are also being O-GlcNAC modified. That is, CKId and OGT are competing for occupancy and modification of the same sites. An interesting question is whether O-GlcNAC modified vs. phosphorylated Serines in this region have different downstream effects. Has anyone looked to see whether the phosphorylation cascade region in dper is O-GlcNAC modified? While the fly and mammalian regions are not really conserved at a sequence level, the similarity of some of the effects in fly suggests they may be similar functionally. If so, then phosphorylated vs. GlcNAC modified vs. unmodified may underlie the unexpected results of S589 vs. D589.The reviewers bring up the possibility that the S589 site is GlcNAc modified, as an explanation of why S589A and S589D do not exhibit opposing changes in behavioral rhythmicity.We agree that it is surprising that the phosphomimetic mutant S589D does not give the inverse phenotype of the S589A mutant, but we hope that we were able to make the case for S589D. S589D, while stabilized in the presence of TIM, is degraded more rapidly (relative to wild type) in the absence of TIM.We considered the possibility of GlcNAc modification of this region. We have successfully used the S589A and S589D PER mutants to block the phenotypic effects of DBT overexpression in flies , demonstrating that there is a genetic interaction between S589 and DBT, and modification of S589 is rate limiting. We conducted a similar experiment in which we overexpressed O-GlcNAc Transferase (OGT) in the different S589 mutant backgrounds. In this case, we were not able to demonstrate any genetic interaction between S589 and OGT. We therefore concluded that O-GlcNAc-ylation was unlikely to be involved in regulating the function of S589.We have included these data as supplemental data in the Results section to address this concern by the reviewers:O-GlcNAc transferase (OGT) has been shown to influence behavioral rhythmicity in mammals and flies, either by competing with CKIδ for modification of mPER2  or blocking BMAL1/CLOCK (orthologues of CYC/dCLK in flies) ubiquitination in mice, or prolonging PER stability in flies . […] The same experiment conducted in the S589A and S589D mutant backgrounds similarly lengthened rhythmic behavior of the mutant flies . We therefore conclude that S589 does not genetically interact with OGT.”“The activity of We have also included additional information in the Materials and methods section: “UAS-OGT transgenic flies were a gift from Louis Ptáček . Flies overexpressing OGT were generated by recombining the second chromosome carrying the tim-UAS-Gal4 and UAS-OGT transgenes.”5) There is no need for the SAXS data, etc. as it does not add anything to the Discussion.While we agree that the SAXS data do not add positive results to our model, these and other biophysical data were included to rule out the possibility that the mutations studied might alter multimerization or cause other global conformational changes, such as gross disruption of protein folding. Our data suggest that they may instead alter local or other subtle conformational changes. If the reviewers agree, we would like to keep this information in supplemental to address such concerns that other readers may have regarding the overall effects of our residue substitutions.


Flavobacteriia and Gammaproteobacteria were the predominant bacterial classes across all investigated Arctic habitats. Bacterial community richness and diversity peaked in deep-sea samples . Algal aggregate-associated bacterial communities were mainly recruited from the sea-ice community, and were transported to the seafloor with the sinking ice algae. The algal deposits at the seafloor had a unique community structure, with some shared sequences with both the original sea-ice community (22% OTU overlap), as well as with the deep-sea sediment community (17% OTU overlap). We conclude that ice-algal aggregate export does not only affect carbon export from the surface to the seafloor, but may change microbial community composition in central Arctic habitats with potential effects for benthic ecosystem functioning in the future.In summer 2012, Arctic sea ice declined to a record minimum and, as a consequence of the melting, large amounts of aggregated ice-algae sank to the seafloor at more than 4,000 m depth. In this study, we assessed the composition, turnover and connectivity of bacterial and microbial eukaryotic communities across Arctic habitats from sea ice, algal aggregates and surface waters to the seafloor. Eukaryotic communities were dominated by diatoms, dinoflagellates and other alveolates in all samples, and showed highest richness and diversity in sea-ice habitats (∼400–500 OTUs).  Melosira arctica, which forms long filaments attached to the ice matrix, can build up patchy, but dense accumulations  that sea-ice algae aggregates select for specific members of eukaryotes and bacteria; (ii) that these originate from sea ice rather than the water column; (iii) that these are exported by rapidly sinking aggregates that transport sea-ice life to the deep sea, but (iv) get quickly overgrown by sediment microbes. Furthermore, this study contributes baseline knowledge of microbial diversity in the central Arctic Ocean in times of rapid sea-ice melt.Polarstern expedition ARK-XXVII/3 (PS80) in August and September 2012 at the end of the productive season . Surface seawater temperatures were between -1.8 to -1.5°C and salinity ranged from 30.3 to 33.2 , when compared to the remaining stations where values ranged from 0 to 20 g C m-2 , or with a conductivity–temperature–depth (CTD) rosette sampler  at 0–2 m depth. Between 0.5 and 2 L of melted sea ice or water were filtered through 0.22 μm polyethersulfone membranes  with a multichannel peristaltic pump . Three parallel deep-sea sediment cores were retrieved from each sampling site by a TV-guided multicorer and subsamples of the uppermost centimeter of each core were pooled per site. At station Ice1, we obtained a total of nine sediment cores, allowing us to prepare three replicate sediment samples  following the manufacturer’s instructions for maximum yields, with minor modifications. Instead of using the kit’s enclosed solution S5, DNA extracts were eluted in a final volume of 80 μl TE-buffer . By using one standardized extraction kit across all sample types, we aimed to minimize potential biases toward differential extraction yields for individual microbial taxa and thus increase comparability. DNA concentration and purity were determined by fluorescence-based Qubit® quantitation assays . Amplicon libraries of the bacterial V4-V6 region of the 16S rRNA gene and the eukaryotic V4 region of the 18S rRNA gene were generated according to the protocol recommended by Illumina . For selected sediment, sea-ice algae and deposited algae samples we obtained technical replicates, by preparing several sequencing libraries from the same DNA extract . For merging forward and reverse reads we used pear  before clustering the reads into 613,270 bacterial and 59,853 eukaryotic OTUs by applying a local clustering threshold of d = 1 and the fastidious option in swarm . Only 11–12% of the OTUs associated with ice-algae aggregates met our definition of dispersed, abundant community members, i.e., they were present in >50% of aggregate samples and represented by at least 100 sequences. These OTUs represented the majority of sea-ice algae associated sequences (Table 1). Melt-pond waters showed the lowest eukaryote and bacterial OTU richness of the sampled ice habitats, but had a relatively large fraction of abundant bacterial types (23%). Bacterial communities in sea ice also exhibited relatively low richness, while eukaryotic communities showed highest richness in sea-ice samples .We saw indications for higher eukaryotic and bacterial community richness in sea-ice algal aggregates than in the surrounding melt-pond water, and in the case of bacteria, also than in adjacent sea ice . We derived the relative contribution of these shared OTUs . They shared only 29% with the algae-associated abundant types , and 16% with the abundant sediment eukaryotes. Bacterial richness and diversity was lowest in the digestive tract content of holothurians feeding on the deposited aggregates on the seafloor , and these bacteria were mostly unique to the holothurian digestive tract, with only 4% overlap with the abundant, dispersed types of the sediment community . Deep-sea surface sediments contained a significantly richer and more diverse bacterial community than sea ice, seawater and algae deposits, and showed almost no overlap with water column and sea-ice communities, while eukaryotic richness in sediments was at the lower end of the observed range across all sampled environments .Bacterial communities on sunken ice-algal aggregates deposited at the seafloor  during the summer season were slightly richer than the communities on photosynthetically active aggregates in melt ponds, communities in sea ice and in surface seawater. They still shared 22% of their abundant OTUs with the original ice-algae associated community, but also 17% with the abundant, dispersed sediment bacteria  and eukaryotes . In both datasets, the communities in sea-ice algal aggregates showed high within-group variation. Notably, eukaryotic community structure in deep-sea sediment and exported ice-algal aggregates showed a much larger dissimilarity than bacterial communities . Overall, the dissimilarities of bacterial and eukaryotic community structures were significantly correlated across all habitats   of Illumina OTUs clustered the samples into five groups according to their habitat . Dinophyceae were the predominant group in surface seawater, but the majority of its representatives could not be classified . One dominant genus was Karlodinium, which was also observed in individual deposited algae aggregates at the seafloor, and in very high sequence abundance in Ice7 deep-sea sediment, where we observed high algae deposition. Eukaryote community composition in sea ice comprised green-brown algae and Dinophyceae, mainly the genus Scrippsiella, members of the Gymnodinium clade and unclassified Suessiaceae in the upper part of the cores, and mostly unclassified diatoms at the bottom of the ice . The genera Pseudo-nitzschia and Melosira exhibited a patchy distribution with low relative abundance (<1%) at all stations except for Ice7. All of these groups were also present in melt-pond waters, in addition to other algae from the Pelagophyceae and a larger contribution of Thecofilosea, including the nanoflagellate Cryothecomonas. In deep-sea sediments, Labyrinthulomycetes, dinoflagellates and other flagellates from the Imbricatea dominated eukaryote communities . At Ice7 we observed a relatively high contribution of the diatom Chaetoceros, the only diatom detected in the sediment outside of the algal deposits.At the time of sampling in late summer, contribution of diatom sequences in surface seawater was minor , yet with pronounced differences in their predominant representatives at the genus level . In sea ice and melt ponds, Flavobacteriia were mainly represented by the genus Flavobacterium, with reads of this single genus constituting 9–71% of the total community in the ice surface and 3–45% in the bottom of the ice. Also the genera Polaribacter, Psychroflexus, Nonlabens, and Ulvibacter were sequence- abundant in ice. Among these, Polaribacter was the only genus, which was also a dominant flavobacterial member in surface seawater. Here, the genera Owenweeksia, Formosa and the NS5 marine group were the most sequence-abundant Flavobacteriia . Microbial community composition of the deep-sea floor was quite distinct from the water column, and the contribution of Flavobacteriia was markedly reduced . Most of its representatives in surface sediments could not be classified at genus level and clustered as unclassified Flavobacteriaceae.Gammaproteobacteria in sea ice were dominated by Paraglaciecola, which displayed a similarly large variability in its contribution as Flavobacterium, ranging from 0 to 33% in sea-ice surface communities and 2–40% in bottom communities . Other important Gammaproteobacteria in sea ice belonged to the genera Colwellia and Glaciecola. In melt-pond waters Gammaproteobacteria representation was lower , but Paraglaciecola and Glaciecola were also dominant members . In the water column, the most prominent Gammaproteobacteria taxa, Balneatrix, SAR86 clade and ZD0405 clade  showed only minor representation in sea ice, and were absent from the deep-sea samples. The most dominant Gammaproteobacteria in the sediment were members of the JTB255 marine benthic group, and these were restricted to deep-sea samples. Deep-sea surface sediment additionally showed a strong contribution of Deltaproteobacteria and Acidimicrobiia .Alphaproteobacteria contributed most to the communities in surface seawater, deep-sea sediment and the digestive tract content of holothurians, while their sequence abundance in sea ice and melt ponds was low . While the genus Planktomarina and members of the SAR11 Surface1 clade predominated in the water column, Rhodospirillaceae were the most dominant representatives at the seafloor. The bacterial community in the holothurian digestive tract was very different from any other environment  and almost entirely comprised of the alphaproteobacterial genus Sedimentitalea and the genus Rubritalea from the Verrucomicrobiae .Figure 3). Interestingly, the community overlap between ice-algal aggregates associated with melt ponds and the exported ice algae at the seafloor was comparable to or higher than the overlap between aggregates and surface seawater for both bacteria and eukaryotes , despite the >4000 m descent. At the seafloor, deposited ice-algal aggregate communities exhibited similar proportions of community overlap with the surrounding sediment as with the sea-ice bottom and surface seawater. A large overlap was also observed between eukaryotic communities in the deposited ice algae and the digestive tract content of the deep-sea dwelling holothurians . Sea ice and sediment, as well as surface seawater and sediment communities showed lowest community overlap.Based on OTU presence/absence, the microbial community associated with sea-ice algae aggregates showed highest community overlap with both melt-pond water and sea-ice communities. We found lower similarity in composition with the underlying community in surface seawater  and Lentimonas (Opitutae) (Supplementary Information Discussion). Eukaryotic generalists included members of the genera Cryothecomonas (Thecofilosea), the NIF-3A7 and NW617 clades (Thecofilosea) and unclassified Dinophyceae, but also the photosynthetic Micromonas (Mamiellophyceae) and unclassified Prasinophytae. We identified a larger number of OTUs in the bacterial and eukaryotic dataset that were shared members between sea-ice algae aggregates in melt ponds, sea ice, water column and deposited algae at the seafloor, but that could not be identified as abundant and dispersed members of the sediment community  and of the above diatoms, as well as golden-brown algae (Chrysophyceae) was also revealed by sequencing .Visual inspection by light microscopy showed a predominance of  sea ice . Other dFlavobacteriia and Gammaproteobacteria . The green aggregate from Ice7 included Glaciecola and Paraglaciecola (Gammaproteobacteria), and Winogradskyella (Flavobacteriia) . The genus Flavobacterium (Flavobacteriia) was the most abundant genus in the highly degraded aggregates from Ice1, where it contributed up to 89% to the total bacterial sequences. Flavobacterium was barely detectable (<0.05%) in the much fresher Melosira aggregate from Ice7 . The eukaryotic community in the holothurian digestive tract resembled the sequence composition of deposited algal aggregates, with a high contribution of the diatom Melosira and unclassified alveolates, but also a high contribution of Cryothecomonas and Scrippsiella observed in sea ice , and the overlap with the sea-ice aggregates was 22% at the OTU level. Yet, deposit-associated Alphaproteobacteria were dominated by the genera Octadecabacter, Sedimentitalea and Sulfitobacter, which were also detected in sea-ice algae aggregates, but in much lower relative abundances . Epsilonproteobacteria were entirely represented by the genus Arcobacter, which was not detected in any other environment, except for the surrounding surface sediments. Verrucomicrobiae were represented by the genus Rubritalea, which was also present in sea-ice algae aggregates. The two types of algae deposits that were differentiated by their eukaryotic community composition showed only minor differences in bacterial community composition. Melosira-dominated samples showed higher contributions of the genera Octadecabacter (on average by a factor of 30), Sulfitobacter (on average by a factor of 45), Ulvibacter (on average by a factor of 18) and Winogradskyella (on average by a factor of 21). Labyrinthulomycetes-dominated samples contained a higher proportion of Neptunomonas .2. In autumn 2012, at the time of this study, Arctic sea-ice extent reached a record minimum, a loss that has been unprecedented since the beginning of satellite records. The bottom of the relatively thin ice floes had been populated by diatoms, foremost the colonial Melosira arctica forming large filamentous aggregates. Strong melting resulted in a wide-spread export and rapid deposition of sea-ice algal aggregates at the deep-sea floor at around 4,000 m water depth , and the dominant members were also predominant members of the sea-ice and melt-water communities . The algal aggregates selected specifically for Flavobacteriia, particularly the genus Flavobacterium, as well as the gammaproteobacterial Glaciecola and Paraglaciecola . Both are known to be tightly coupled to phytoplankton bloom dynamics . Betaproteobacteria are known as particle colonizers and were shown to play key roles in the decomposition of aggregates and nutrient cycling, especially in freshwater ecosystems . Our results, therefore, underpin a recruitment of bacterial groups mainly from sea ice, best adapted to the utilization of ice algal material in the ponds, which differed from the phytoplankton material in the surface ocean. We observed first indications for specific associations between bacterial groups and ice-algae aggregates, with 12 dispersed and abundant bacterial OTUs as exclusive members of ice-algae aggregates and algae deposits , a group of heterotrophic protists that play important roles as saprobes of dead algal material and marine snow .Comparative analyses of microbial communities from upper ocean and deep sea showed that these communities barely overlap . Recent ing rate , with aging rate . We obseine snow . Labyrin enzymes . InteresFigure 3a and Supplementary Table Paraglaciecola, Glaciecola (both Gammaproteobacteria), Octadecabacter (Alphaproteobacteria), Psychroserpens and Polaribacter (Flavobacteriia), which have been previously described as psychrophiles, and were observed in sea ice and surface seawater  belonging to both the abundant types present on sea-ice algae aggregates and on deposited aggregates at the seafloor . Accordingly, the bacterial genera of highest relative sequence abundance  in the deposited ice-algae aggregates consisted of benthic groups that were absent from ice-associated environments and only of minor relative abundance in the surrounding sediment surface  and Neptunomonas (Gammaproteobacteria) . The Roseobacter clade is a physiologically versatile group, known for its capability to utilize a wide range of organic and inorganic compounds and its tight interactions with phytoplankton, important for organic matter decomposition . It remains to be further investigated whether this is a transient feature or whether such events, if reoccurring, will result in a gradual shift of deep-sea sediment communities.A considerable amount of bacteria can be exported to the deep sea attached to particles , but thebacteria , 2013. Tic shelf , the Wadic shelf  and the ic shelf . Its capic shelf , denitriic shelf , fix nitic shelf , as wellic shelf  and recyic shelf  indicateediments , potentiediments , but werediments  or in clediments . This geediments , indicatediments . OverallMelosira deposits . Consequently, ice dwelling organisms, and especially multi-year ice communities, which are distinct from those of seasonal ice , which was observed to correlate negatively with salinity (Hence, this study confirms that sea-ice bacterial and eukaryotic communities are distinct from other Arctic habitats such as seawater and sediment (onal ice , may be onal ice . We saw nditions , and memsalinity . Other tsalinity . This masalinity , but maysalinity , likely salinity . Future JR and MF-M designed the study, with contributions by CB and AB. MF-M, CB, and AB collected the samples. JR performed the laboratory molecular and bioinformatic analysis. JR and CB performed the statistical analysis. JR interpreted the data and all authors contributed to the discussion of the results. JR wrote the manuscript, with support and input from all co-authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The prosthetic component of dental implant is attached on the abutment which is connected to the fixture with an abutment screw. The abutment screw fracture is not frequent; however, the retrieval of the fractured screw is not easy, and it poses complications. A retrieval kit was developed which utilizes screw removal drills to make a hole on the fractured screw that provides an engaging drill to unscrew it. To minimize this process, the abutment screw is modified with a prefabricated access hole for easy retrieval. This study aimed to introduce this modified design of the abutment screw, the concept of easy retrieval, and to compare the mechanical strengths of the conventional and hollow abutment screws by finite element analysis (FEA) and mechanical test. In the FEA results, both types of abutment screws showed similar stress distribution in the single artificial tooth system. A maximum load difference of about 2% occurred in the vertical load by a mechanical test. This study showed that the hollow abutment screw may be an alternative to the conventional abutment screws because this is designed for easy retrieval and that both abutment screws showed no significant difference in the mechanical tests and in the FEA. Placement of dental implants has become the main treatment option for oral function recovery in partially or completely edentulous patients. The components of a dental implant consist of fixture, abutment, and abutment screws.Despite the high success rate of implants, it is not free of complications and dental implants occasionally fail due to biological factors or technical complications , 2. The Majority of implant failures nowadays are caused by mechanical factors rather than the implant itself, and so, there are alternative abutment systems that were developed. Only a few studies on the removal of fractured screw in the implant , 11 wereConventional abutment screw measures 2 mm in diameter, 7.8 mm in length, and 0.4 mm in screw thread pitch , and theFor easy retrieval of the fractured screw of the hollow type screw (modified with prefabricated access hole), the hole was fabricated with a reverse screw drill. The reversed screw was tightened with the screw driver allowing stabilization of the fractured portion, and the external thread would be unscrewed from the fixture.3D implant system models were constructed using Solidworks 2016 (Dassault Systèmes) for this study. Mechanical test was performed on the conventional abutment screw and the hollow abutment screw by fabricating 3D models, and finite element analysis (FEA) was implemented to the single artificial tooth system model.N = 3, independent experiments).MTS Bionix 370.02  was used for the vertical load test to compare the mechanical strength of the conventional abutment screw and the hollow abutment screw. Before the vertical load test, the abutment screw was fixed on the jig vertically by applying 30 N·cm of insertion torque  12]. Th. Th12]. IS II Active Implant System  was used in fabricating the 3D model of the single implant system design . The bonHypermesh version 14  was used to construct the model for FEA , and FEAThe values assumed for the modulus of elasticity and Poisson's ratio are given in n = 3, resp.).Sample sizes were estimated for comparison of two groups based on previous study . The ext-test . P < 0.05 was considered significant.All data are presented as means ± SD of independent recordings. Statistical analyses were performed by unpaired two-tailed Hollow abutment screw eliminates the usage of a commercially available screw retrieval kit which was developed to utilize screw removal drills to make a hole on the fractured screw which provides an engaging drill to unscrew it. Hence, the fractured screw could be easily removed using the H-file .In the vertical load mechanical test, maximum compressive load did not show significant difference between the conventional and the hollow abutment screws .  Table As this experiment does not provide a standard for strength evaluation of the abutment screw, it was performed according to the ASTM standard  on metallic bone screw, and the experiment was performed with only the abutment screw. Thereafter, strength evaluation of the conventional and the hollow abutment screws was performed with a mechanical test, and the maximum load difference of about 2% occurred in the vertical load.In the mechanical test of the three specimens, there were differences depending on the specimens' processing conditions. However, all three specimens showed similar load form, and it was considered that not much difference was observed in the conventional and hollow abutment screws.In this study, the compressive strength test and FEA were performed to compare the physical performance of the conventional type and the hollow type abutment screws. However, since the physical performance on fatigue loading is important, it is necessary to test and verify the fatigue of the screw in future studies in the actual implant system.Management of fractured abutment screw is clinically challenging and timely but it is necessary to provide an adequate rehabilitation plan. This study showed that the hollow abutment screw may be an alternative to the conventional abutment screws because this is designed for easy retrieval and that both abutment screws showed no significant difference in the mechanical tests and in FEA.


The aim of this study was to provide information on the molecular characteristic and the phylogenic relationship of infectious bronchitis viruses (IBV) strains in Bushehr province in comparison to other strains reported in the Middle East. Samples were collected from broiler flocks in Bushehr province during 2014 - 2015. These flocks had respiratory problems such as gasping, sneezing and bronchial rales. A number of 135 tracheal swabs were taken from fifteen flocks (nine swabs per flock). Each three swabs collected from each flock were pooled in one tube . The samples were subjected to reverse transcription polymerase chain reaction (RT-PCR). The PCR products of positive samples were analyzed by sequencing of a (392 bp) segment of the spike gene and the related results were compared with the other IBV sequences in GenBank database. Samples from twelve farms (80.0%) were found to be positive. The viruses from seven farms (46.6%) were identified as field viruses closely related to variant 2. The viruses from three farms (20.0%) were characterized as Mass type and were related to vaccine strains. Two different IB viruses (variant 2 and Mass) were detected in samples from two farms (13.3%). The variant 2 genotype detected in Bushehr had high similarity to variant 2 reported from the Middle East. These variants displayed homologies ranging from 72.9% to 76.5%, and 78.8% to 80.0% with H120 and 4/91, respectively. It is necessary to design vaccination program of poultry farms using IBV strains circulating in the region. The IBV and other avian coronaviruses of turkeys and pheasants are classified as group 3 coronaviruses.4 Its genome consists of about 27 kb and codes for four structural proteins: the spike (S) glycoprotein, the membrane (M) glycoprotein, the nucleocapsid (N) phosphoprotein, and the envelope (E) protein.5,6 The spike glycoprotein (S) is anchored in the viral envelope and is post-translationally cleaved into two proteins S1 and S2.7 The S protein is very diverse in terms of both nucleotide sequence and deduced primary protein structure, especially in the upstream part of S1.8 Three hypervariable regions (HVRs) have been identified in the S1 subunit.9-11 The S1 subunit induces neutralizing, serotype-specific, and haemagglutination-inhibiting antibodies.12-17 Amino acid changes in the spike (S) glycoprotein lead to the generation of genetic variants.18,19 The high frequency of new IBV variants is a distinguished characteristic of this virus among other coronaviruses.20 Many IBV serotypes have been described probably due to the frequent point mutations that occur in RNA viruses and also recombination events. Therefore, the characterization of virus isolates which exists in the field is very important.21 More than 50 serotypes of IBV have been identified and new variants continued to emerge despite the use of live attenuated and killed IBV vaccines.22-24Infectious bronchitis (IB) is an acute and highly contagious respiratory disease of chickens characterized by respiratory signs, and in young chickens by severe respiratory distress and a decrease in egg production in layers.18,19 The IBV Massachusetts (Mass) type was first detected in Iran by Aghakhan et al.25 In 1998, a virus similar to the European 793/B type was isolated in Iran (Iran/793B/19/08).26 In recent years, new variants of IBV have been reported from different part of the country.27-29 The aim of this study was to provide information on the molecular characteristic and the phylogenetic relationship of prevalent IBV genotypes circulating in chicken flocks in Bushehr province, Iran.The usage of live attenuated vaccines is the most important preventive measure of the disease, but anti-genically different serotypes and newly emerged variants from field chicken flocks sometimes cause vaccine breaks. Sampling. Samples were collected from broiler flocks in different regions of Bushehr province as mentioned in RNA extraction Viral RNA was extracted from the directly pooled tracheal swabs in RLT buffer  and 10 μL 2-mercaptoethanol  per 1 mL buffer using RNeasy Mini Kit (Qiagen), according to the manufacturer’s protocol.Reverse transcription The reverse transcriptation (RT) reaction was performed using ReverAid™ first strand cDNA synthesis kit , according to the product manual. The resultant cDNA was immediately used in a PCR or stored at –20 ˚C for later use.Amplification of the spike gene Nested reverse transcription polymerase chain reaction (RT-PCR) was performed using spike gene primers as described previously to amplify 392 bp fragment of the spike gene.30 The first round of amplification (495 bp) was performed using SX1 (5ʹ-CACCTAGAGGTTTGT/CTA/TGCAT-´3) and SX2 (5ʹ-TCCACCTCTATAAACACCC/TTT-´3) primers. The PCR reaction was performed in 25 μL reaction mixture containing 1 μL dNTP (10 mM), 0.50 μL of each primer (25 pmol μL-1), 1 μL MgCl2 (50 mM), 2.50 μL 10X PCR buffer, 0.20 μL Taq DNA Polymerase, 2.50 μL cDNA and 16.80 μL dH2O . The amplification was performed using 35 thermal cycles including 94 ˚C for 30 sec, 58 ˚C for 30 sec, and 72 ˚C for 30 sec. The PCR product was used as template for the second round of amplification in which SX3 (5´-TAATACTGGC/TAATTT TTCAGA-´3), and SX4 (5ʹ´AATACAGATTGCT TACAACCACC-´3) primers were used. The PCR reaction was carried out under the above condition.Agarose gel electrophoresis. The PCR products were electrophoresed on 2% agarose gel and visualized by staining with 0.50 μg mL-1 ethidium bromide by UV transilluminator .PCR product purification. The PCR products were purified using PCR purification Kit  according to kit’s manufacture instructions.Nucleotide sequencing, deduced amino acid analysis and phylogenetic tree. Purified RT-PCR products were sequenced by ABI Prism BigDye terminator cycle sequencing ready reaction kit  in a forward direction using primer SX3 and in a reverse direction using primer RX4. Nucleotide sequence of the PCR product (392 bp), which was submitted to NCBI, were compared with the IBV sequences in GenBank database and sequence similarities were analyzed by BLAST. Multiple sequence alignments were carried out with Clustal W and phylogenetic tree was constructed with MEGA software  using the Neighbor-joining tree method with 1000 bootstrap.31GenBank accession number of IBV sequence. The partial S1 gene sequences of IBVs were submitted to the GenBank database under accession numbers KX578825-KX578834.The RNA was extracted and cDNA was synthesized, and further a (392bp) segment of the S1 gene was amplified by nested RT-PCR . SamplesPhylogenetic Analysis. To evaluate sequences variation among the positive samples, the sequences were compared with each other and with the reference sequences . Nucleotide sequence identity between second subgroup isolates ranged from 92.90% to 100% .Comparing mentioned isolates with vaccinal strains used in Iran , the results showed 72.90% to 76.50%, 71.80% to 75.30% and 78.80% to 80% similarity , respectThe second groups include IR/Bu/Mass/SH1229.2/15, IR/Bu/Mass/SH1229.13/14, IR/Bu/Mass/SH1450.12/15.32-34 The implementation of nationwide genotyping of IBV strains is necessary to determine the distribution of virus genotypes and to develop and adopt suitable vaccination strategies. Antigenic characterization of IBV isolates is important for selecting new and appropriate vaccines for the corresponding geographical regions.35 New serotypes or variant strains may emerge due to only a few changes in the amino acid sequence of the S1protein.3 Therefore, the S1 gene of the isolates should be determined to differentiate field and vaccine isolates.36 Regarding the results obtained, 12 IBV isolates were identified. Phylogenetic analysis based on S1gene nucleotide sequences showed that most of the Iranian isolates belonged to two distinct groups.It is imperative to recognize the prevalent strain(s) of infectious bronchitis virus in a region or country, and to select the best vaccine strain and the vaccination program for controlling the disease. The major problem in the immunization against IBV is the presence of various IBV serotypes in the field against available vaccines which cannot induce proper immunity. The aim of the present study was to detect and identify the type of prevailing IBV strains in Bushehr province. Previous studies comparing conventional and nested RT-PCR methods indicated that nested RT-PCR was more sensitive for detection of IBV.Based on nucleotide sequencing of the S1 gene, a number of field isolates in the present study showed maximum similarity to Variant 2 (IS/1494/06 like strain). This is the first report of IBV variant 2 in the broiler flocks of Bushehr province. These variants displayed homologies ranging from 72.90% to 76.50%, and 78.80% to 80% with H120 and 4/91, respectively .et al. who reported a genotype variant 2 (IS/1494/06 like) had been circulating in Iran between 2010 to 2014.27The second group included three strains which were closely related to Massachusetts (Mass) type strains. In the present study, IR/variant2 viruses (IS/1494/06 like) were recognized as major dominant genotypes and the most important IBV type in Bushehr province chicken flocks. They shared the highest identity of 91.80 to 97.60% with Kurdistan-Sulaymania/12, Egypt/Beni-Suef/01, Turkey/10RS-3161/2010, IS/VAR2-06, variant 2, Eg/CLEVB-2/IBV/012 and Libya-06-2012. These findings are in agreement with those of Hosseini et al. reported IR/7/2011, R/8/2011, and IR/9/2011 isolates which appeared different from the mentioned IBV types and formed separate branches in the phylogenetic tree (variant 2).29 Mahmood et al. reported a new IBV isolate in the Kurdistan region from 2008 to 2010 that caused kidney lesions. Their results indicated the circulation of 793/B with variant 2 in poultry flocks in Iraq, which were similar to the findings obtained in the present study.37Homayounimehr et al. reported Variant 2-like viruses (IS/1494/06 like) that were the most predominant IBV type in Iranian chicken flocks. They shared the highest identity of 99.22% with IS/1494/06, Turkey/TR8, and Eg/ CLEVB-2/IBV/012. These findings had high similarity with our results.28Najafi 38 The IB viruses in Egypt, Jordan, Turkey and Libya showed a close relationship to Israeli variants.28,39-43 Following the first report by Meir et al.,44 the variant 2 has been reported from some Middle East countries such as Iran.27-29,42,43 Since these countries have close connections , so these connections can play an important role in spreading of this variant.Our findings are also in agreement with several other studies carried out in the Middle East countries between 2004 and 2015. Some Iraqi researchers studied circulating viruses in Broiler farm and showed that these strains belong to variant 2 (IS/1494-lik) that had high nucleotide sequence identity with IBV isolates from Iran, Israel, Egypt, Turkey, and Kurdistan.25 The present study is the first report on var2 IS/1494/06 in Bushehr province, in Iran, confirming the presence of the Var2 genotype. Ma5, H120, and attenuated 4/91 IBV-based vaccination strategies have been applied to IB control on poultry farms in Iran recently,45,46 and despite their use, diagnosis of IB in the vaccinated chickens is common. The results of this study may partially explain the failure of Massachusetts-type vaccines and therefore necessitates revising the Iranian vaccination strategy against infectious bronchitis. The low identity between most of Iranian isolates with Mass-type vaccine strain, the presence of variant 2, and other new genotypes 4/91 can be regarded as the causes of vaccination failure. The first isolation of IBV in Iranian chicken flocks was reported in 1994.28 Genotypes found in Sulaymania-Kurdistan, Iraq, included group A (very similar to Iranian isolates), and group C (similar to IS/1494 and Egypt/Beni-Seuf/01 isolates).47 These findings are in agreement with the present study.Moreover, secondary infections and immuno-suppressive agents like infectious bursal disease virus and Chicken anemia virus may also lead to vaccination failure and consequently IBV outbreaks among poultry flocks. These substantial reasons can result in immune failure, poor cross-protection between the field virus and vaccine strain, and the continual emergence of new variants.Cross-protection between IBV strains depends on the amino acid similarity of S1. Based on S1 glycoprotein amino acid sequence, Iranian IBV’s homology with H120 vaccine, Massachusetts vaccine and 793/B vaccine ranges from 72.90% to 76.50%, from 71.80% to 75.30% and from 78.80% to 80.00%, respectively. These findings explain the poor vaccine performance in the field and show that the disease outbreaks were associated with IBV variants, which circumvent vaccination immunity. Further, the findings emphasize the need for new control strategies of IBV in Iran.In summary, the present study is the first report of IBV in Bushehr, Iran, illuminating the circulation of a variant of IBV genotype in chicken farms. Heterogeneity with vaccine strains can explain a poor vaccination performance and disease outbreak in this area. The results emphasize the need for new control strategies and re-arrangement of preventative measure of IBV in Bushehr, Iran.


We will then evaluate how negative feedback modulates the spatiotemporal signalling dynamics of the ERK1/2 pathway regarding signalling amplitude and duration as well as subcellular localisation. Aberrant ERK1/2 activation results in deregulated proliferation and malignant transformation in model systems and is commonly observed in human tumours. Inhibition of the ERK1/2 pathway thus represents an attractive target for the treatment of malignant tumours with increased ERK1/2 activity. We will, therefore, discuss the effect of ERK1/2 MAPK feedback regulation on cancer treatment and how it contributes to reduced clinical efficacy of therapeutic agents and the development of drug resistance.The extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signalling pathway regulates many cellular functions, including proliferation, differentiation, and transformation. To reliably convert external stimuli into specific cellular responses and to adapt to environmental circumstances, the pathway must be integrated into the overall signalling activity of the cell. Multiple mechanisms have evolved to perform this role. In this review, we will focus on negative feedback mechanisms and examine how they shape ERK1/2 MAPK signalling. We will first discuss the extensive number of negative feedback loops targeting the different components of the ERK1/2 MAPK cascade, specifically the direct posttranslational modification of pathway components by downstream protein kinases and the induction of  Eukaryotic cells respond to changes in their environment through complex and interconnected signal transduction networks that convert external stimuli into a range of cellular responses. A common motif by which extracellular modulation of cell-surface receptor activity is transduced into a specific cellular response is the three-tiered mitogen-activated protein kinase (MAPK) cascade, with the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway being the most extensively investigated. The principal mechanisms involved in the activation of the ERK1/2 pathway have been well characterised , 4). Oncde novo gene synthesis of specific pathway inhibitors. The principal difference between these two mechanisms is the time required to take effect. While direct posttranslational modification is nearly instantaneous, de novo gene expression and protein synthesis is somewhat delayed following the initial pathway activation. In this review, we will discuss in detail how negative feedback determines ERK1/2 spatiotemporal signalling dynamics and the role of negative feedback regulation in the development and treatment of cancer.To adapt to particular environmental circumstances, the ERK1/2 MAPK pathway must be integrated into the overall signalling activity of the cell. Importantly, the magnitude, duration, and location of MAPK signalling must be strictly controlled to produce the correct biological response. Multiple mechanisms have evolved to perform this role, including positive and negative feedback loops as well as extensive cross talk between parallel pathways. In this review, we will focus on negative feedback and the mechanisms by which it shapes ERK1/2 MAPK signalling. Negative feedback loops targeting the ERK1/2 MAPK cascade fall into two main categories: direct posttranslational modification of pathway components and the induction of Nearly all components of the ERK1/2 MAPK cascade are regulated through negative feedback phosphorylation by downstream kinases. The negative feedback phosphorylation events that have been studied in reasonable detail are summarised in Table ERK1/2 proteins have been reported to directly phosphorylate several receptor tyrosine kinases. For example, ERK1/2 MAPKs can phosphorylate the epidermal growth factor (EGF) receptor (EGFR) at T669 , 8, the The fibroblast growth factor (FGF) receptor has also been shown to be subject to ERK1/2 feedback phosphorylation. FGF signalling leads to FGF receptor 1 (FGFR1) phosphorylation on a conserved serine residue (S777) in its C-terminal tail in an ERK1/2-dependent manner . FurtherAdaptor proteins connect activated RTKs to Ras and the downstream MAPK cascade. Regardless of continuous growth factor stimulation, Ras activation is usually transient. A negative feedback loop from ERK1/2 to adaptor proteins and exchange factors contributes to this transiency. For example, it was found that MAPK pathway activation results in hyperphosphorylation of SOS1, which was enhanced by ERK overexpression  and prevThe FGF receptor substrate 2 proteins (FRS2α and FRS2β) are adaptor proteins that are recruited to activated RTKs via N-terminal PTB domains. They also contain multiple tyrosine phosphorylation sites in their C-termini that allow the binding of the adaptor protein Grb2 . Lax and2+ mobilisation) and that abolishing ERK1/2 feedback through a MEK1/2 inhibitor similarly enhanced TCR signalling. Through co-immunoprecipitation experiments, the authors showed that the mechanism behind the inhibitory role of LAT T155 phosphorylation may involve the disruption of the interaction of LAT with its downstream signalling partner, phospholipase C γ1 (PLC γ1), but not Grb2.Another adaptor protein regulated by ERK1/2 feedback phosphorylation is the linker for activation of T cells (LAT), a single-pass transmembrane protein that is expressed in various immune cells . Upon enA recent paper by Hennig et al.  also impO-tetradecanoylphorbol 13-acetate). Raf-1 hyperphosphorylation was delayed compared to the activation of Raf-1, MEK1/2 and ERK1/2, and it was further enhanced by overexpression of ERK1, suggesting a negative feedback loop. Further studies showed that Raf-1 hyperphosphorylation is dependent upon MEK1/2 activity [The first suggestion that Raf is subject to feedback phosphorylation was provided by Ueki and colleagues , who shoactivity  and thatactivity , 30. Hypactivity .A major advancement towards understanding the role of Raf-1 in the negative feedback regulation of ERK1/2 signalling was made by the identification of six Raf-1 residues that are phosphorylated following mitogenic stimulation . Five ofFeedback phosphorylation at ERK1/2 phosphorylation motifs was also identified within the B-Raf C-terminus (S750 and T753) upon engagement of the B cell antigen receptor in chicken DT40B cells . MutatioA number of inhibitory, proline-directed phosphorylation sites have been reported for MEK1, including T286, T292, and T386 . The T29Drosophila and C. elegans [A number of scaffold proteins have been shown to regulate MAPK signalling in mammalian cells by organising the signalling components into macromolecular complexes and enhancing the efficiency and specificity of the MAPK signalling cascade –46. KSR  elegans –49. Subs elegans , 51. KSR elegans , 53, thuA number of KSR1 residues  corresponding to the minimum ERK1/2 phosphorylation consensus motif (SP or TP) have been shown to be phosphorylated in cycling cells  as well Phosphorylation of KSR1 at the above sites has been shown to have multiple effects on its function. Mutation of these sites, inhibition of MEK1/2, and blocking ERK1/2 binding to KSR1 all resulted in increased and sustained binding of KSR1 to B-Raf, suggesting that KSR1 feedback phosphorylation interrupts the ternary complex of B-Raf, KSR1, and MEK1/2 . SubsequThe molecular mechanisms by which feedback phosphorylation of KSR1 regulate signalling were further analysed in detail in neurons , where KAlmost every step in the ERK1/2 MAPK pathway is targeted by negative feedback phosphorylation  proteins , originaous RTKs –70. In mous RTKs , 72. In ous RTKs –76. In sous RTKs –76. Inteous RTKs . Neverthous RTKs . Phosphoous RTKs , 80. Othous RTKs  for a dede novo expression of proteins, which in turn target the ERK1/2 pathway at multiple levels. These mechanisms are likely to be slower, affecting the later phases of ERK1/2 signalling. This multitude of mechanisms ensures that ERK1/2 signalling dynamics can be controlled in a well-defined manner and that it can be adapted to the particular circumstances of individual tissues and environmental conditions.A multitude of mechanisms has developed to inhibit ERK1/2 MAPK signalling by negative feedback regulation  and positive and negative regulatory events, such as cross-talk and feedback mechanisms. These events determine ERK1/2 signalling strength and duration and critically influence the physiological outcomes. In a classic study, Traverse and colleagues reported that in rat PC-12 cells, stimulation with nerve growth factor (NGF) or EGF resulted in neuronal differentiation or proliferation, respectively . These sSubsequent modelling approaches have further illuminated the role of negative feedback in determining ERK1/2 signalling outputs. Elegant combinatorial work by Sturm and colleagues  showed tMany cellular processes occur in specialised subcellular compartments, and the localisation of ERK1/2 activity to those specific areas is critical to cellular function –18, 31. Aberrant ERK1/2 activation results in deregulated proliferation and malignant transformation in model systems and is commonly observed in human tumours. Inhibition of the ERK1/2 pathway, therefore, represents an attractive target for the treatment of malignant tumours with increased ERK1/2 activity. However, therapeutic agents targeting the ERK1/2 pathway would be expected to also inhibit the substantial negative feedback loops  and expressing wild-type Raf proteins, negative feedback mechanisms significantly reduce the activity of several upstream pathway components, leading to relatively low (but still elevated) levels of MEK1/2 and ERK1/2 activity , as those cells have lost their robustness to perturbations conferred by the negative feedback loops. This has been shown to be the case in vitro [In contrast to tumours with upstream activation (RTKs or Ras), those harbouring mutant B-Raf are generally sensitive to MEK1/2 or Raf inhibition. One reason for this sensitivity is that mutant B-Raf is constitutively active and insensitive to negative feedback . Nin vitro , 107, anin vitro , 109. Itin vitro , 111. HoInhibitors of B-Raf and MEK1/2 are showing substantial promise in the clinic, particularly for those tumours harbouring B-Raf mutations (see above). However, even patients who respond well to these inhibitors show signs of relapse after several months, indicating that resistance to these inhibitors has developed (adaptive resistance) , 113. ThV600E tumours, Ras activity is suppressed by negative feedback  and improve responses in patients with B-Rafmelanoma . Supportmelanoma . Promisimelanoma , 126. Inmelanoma , 127 repV600E melanoma cell lines results in the activation of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway. This may occur as a result of increased activity of different RTKs, due to the release of negative feedback acting at this level. For example, Villanueva and colleagues [V600E–positive colon cancer [Unfortunately, the approach to specifically target ERK1/2 MAPK signalling—even with combination therapy—is not completely effective, since the relief of ERK1/2 feedback by Raf or MEK1/2 inhibition also reduces the inhibition of parallel pathways that can promote survival and reduce the dependency of the tumour on ERK1/2 signalling Fig. . Severallleagues  showed tlleagues  demonstrlleagues . These olleagues , 130. Colleagues . In addin cancer  and cotrn cancer . Anothern cancer . Under tV600E tumours. They also highlight the fact that multiple RTKs and signalling pathways can become activated to rescue anti-apoptotic signalling. One proposed approach to circumvent this problem is the genomic and proteomic testing of tumour samples early in and during the treatment phase [Overall, these studies demonstrate the importance of understanding the mechanisms that are activated upon relief of ERK1/2-dependent negative feedback, as those efficiently promote drug resistance, particularly in B-Rafnt phase , which wThe regulation of the ERK1/2 MAPK pathway is complex and includes numerous negative feedback loops. This negative feedback has likely developed to confer robustness and effective control over this evolutionally conserved signalling pathway. While essential for the normal functioning of the cell, its ability to “rewire” signalling pathways represents a major problem for clinical intervention. Substantial progress has been made during the last few decades in understanding the complex regulation of ERK1/2 MAPK signalling. While there are still a large number of open questions, we are starting to see the benefits of applying this knowledge to targeted cancer treatment in the clinic.