MA) values for left the anterior descending (LAD), left circumflex (LCX), and right coronary artery (RCA) in patients without plaques on coronary CT angiography (cCTA), taking into account tube voltage influence.To determine normal pericoronary adipose tissue mean attenuation  men; median age 49 years ) who underwent cCTA with third-generation dual-source CT for the suspicion of CAD between 2015 and 2017. We selected patients without plaque on cCTA. PCATMA was − 90.3 ± 11.1 HU. PCATMA in men was higher than that in women: − 88.5 ± 10.5 HU versus − 91.5 ± 11.3 HU (p = 0.001). PCATMA of LAD, LCX, and RCA was − 92.4 ± 11.6 HU, − 88.4 ± 9.9 HU, and − 90.2 ± 11.4 HU, respectively. Pairwise comparison of the arteries showed significant difference in PCATMA: LAD and LCX (p < 0.001), LAD and RCA (p = 0.009), LCX and RCA (p = 0.033). PCATMA of the 70 kV, 80 kV, 90 kV, 100 kV, and 120 kV groups was − 95.6 ± 9.6 HU, − 90.2 ± 11.5 HU, − 87.3 ± 9.9 HU, − 82.7 ± 6.2 HU, and − 79.3 ± 6.8 HU, respectively (p < 0.001).The overall mean ± standard deviation (SD) PCATMA varied by tube voltage, with minor differences in PCATMA between coronary arteries . PCATMA values need to be interpreted taking into account tube voltage setting.In patients without plaque on cCTA, PCAT• In patients without plaque on cCTA, PCATMAdiffers slightly by coronary artery .• Tube voltage of cCTA affects PCATMAmeasurement, with mean PCATMAincreasing linearly with increasing kV.• For longitudinal cCTA analysis of PCATMA, the use of equal kV setting is strongly recommended.The online version of this article (10.1007/s00330-020-07069-0) contains supplementary material, which is available to authorized users. MA) , with the following parameters: mean and SD of PCATMA (− 75.1 ± 8.6 HU and − 77.0 ± 8.5 HU); correlation between groups 0.5. The effect size was calculated to be 0.2222108. With α = 0.05, power = 0.8, and two-tailed analysis, the needed sample size was 161. We added 20%, yielding a total sample size of 192, to decrease type I and type II error ratios.The sample size was calculated using paired sample n) and frequencies (%). Associations of age, sex, and BMI with PCATMA were tested using multivariable regression analysis. PCATMA values by sex were compared using unpaired sample t-testing. PCATMA comparisons between combinations of two coronary arteries were made using paired sample t-testing; values between three coronary arteries were compared using repeated ANOVA testing. In order to determine the effects of tube voltage on PCATMA, patients were grouped according to tube voltage as follows: 70, 80, 90, 100, 120 kV. For PCATMA and EAT volume comparison of multiple kV groups, one-way ANOVA testing was used. Post hoc pairwise comparisons of PCATMA were performed between each two kV groups. p values < 0.05 were considered statistically significant. For multi-paired t-testing a Bonferroni correction was applied, adjusting the p value accordingly. SPSS  was used for statistical analysis.Normality testing for continuous variables was performed with the Shapiro-Wilk test. Continuous variables were represented as mean ± SD. Categorical variables were recorded as numbers (n = 6), insufficient image quality (n = 5), incomplete coronary image coverage (n = 1), pacemaker artifact (n = 1), and streak artifact (n = 1) , respectively. In multivariable linear regression analysis, kV, age, and gender were significantly associated with PCATMA (p < 0.05) while BMI was not (p = 0.235). Mean PCATMA of LAD, LCX, and RCA was − 92.4 ± 11.6 HU, − 88.4 ± 9.9 HU, and − 90.2 ± 11.4 HU, respectively (p < 0.001). There were significant differences between all combinations of coronary arteries: PCATMA-LAD and PCATMA-LCX (p < 0.001), PCATMA-LAD and PCATMA-RCA (p = 0.009), PCATMA-LCX and PCATMA-RCA (p = 0.033) . Post hoc pairwise comparisons of the kV groups demonstrated significant differences between each two groups except for the 80 kV and 90 kV (p = 0.222), and 100 kV and 120 kV groups (p = 0.267).Mean PCATage Fig. . Mean .Mean EAT volume showed a positive linear association with tube voltage Fig. . Mean  , with minimal bias but with some variability between readings   were usually scanned with higher kV to get better image quality. In view of the influence of kV on PCATMA analysis, it is recommended that for the longitudinal follow-up and comparison of cCTA-based PCATMA values, the cCTA is performed at equal tube voltage setting.One of the main results of this study is that the use of different kV levels has considerable impact on PCATr 120 kV , 25. Howr 120 kV , 28. OurMA studies about relationship with CAD focused on analysis of the RCA alone, while the LAD and LCX could provide additional information and increase the accuracy of outcome prediction [MA measurements in all three coronary arteries, showing that PCATMA was slightly but significantly different between the LAD, LCX, and RCA. This difference could be caused by differences in anatomy and surrounding tissues, indicating that PCATMA values and corresponding cutoff values based on RCA measurements cannot be directly transferred to the other coronary arteries. Our study results showed that LAD had slightly but significantly lower PCATMA compared with the RCA and LCX. As is known in literature, atherosclerosis development also differs between the coronary arteries. The LAD is subject to atherosclerosis more often and at an earlier stage in comparison with RCA and LCX [MA is an important hypothesis-generating finding. This finding suggests that PCATMA could be related to vessel vulnerability for atherosclerosis. This hypothesis should be further investigated in pathophysiological and prospective clinical studies.The majority of PCATediction , 21. Our and LCX –31. The MA between our 10-mm methods and 40-mm method. PCATMA measurement width around the coronary could potentially affect measurement accuracy. Prior studies measured PCATMA using approximately 3 mm thickness  around the coronary vessels [MA using a more constrictive measurement width, making it more suitable for LAD and LCX measurements, and potentially more sensitive to inflammatory changes. Manual PCAT measurements using the method described here can be performed in a similar time span compared with manual EAT measurements. Fully automated software, as mentioned by some researchers [MA evaluation within 30 s, increasing the time efficiency of PCATMA analysis and enabling use in clinical practice.To analyze all three coronary arteries, an adjusted measurement method was used in our study. Previously, a measurement length of 40 mm was used , 21, 25  vessels , 21, 25. vessels . To takeMA and EAT both are measures of adipose tissue, they represent different processes [MA quantifies fat at the per-vessel level or per-lesion level as an indicator of coronary inflammation, while EAT provides a measure of the volume of the entire epicardial fat system as a marker for paracrine effects of fat. Thus, PCATMA may provide a more specific, focal assessment of coronary risk and vulnerability. PCATMA was shown to have additional diagnostic value with more precision and specificity compared with EAT measurements [MA and FFR were related at the per-vessel level [MA of RCA was able to assist in risk stratification of cardiovascular mortality [MA, total plaque volume, and diameter stenosis has shown high diagnostic accuracy for prediction of hemodynamically significant coronary stenosis [While PCATrocesses . PCATMA urements . Studiesel level  and thatortality . The comstenosis . Interesstenosis  found thMA between men and women that could not be explained by differences in kV distribution. Men are known to get CAD more frequently than women and at an earlier age [MA difference between men and women follows the same trend. This finding deserves further exploration in a study with comprehensive cardiovascular risk factor assessment and more diverse range of coronary atherosclerosis. Sex-related differences in PCATMA could be caused by several factors. Men have a higher amount of EAT than women [MA in men and women [Besides differences between kV levels and coronary arteries, our results show a slight but important difference in PCATlier age . The PCAan women , 35, relan women  and in tan women . Additiond women .MA values while there was no plaque development yet. There could be factors other than tube voltage affecting PCATMA measurements such as obesity or diabetes, inflammatory processes, or specific medication. Further work is needed to explore all potentially influencing factors. With regard to the measurement itself, the anatomical variation of LCX compared with LAD and RCA was relatively large and could have affected the PCATMA measurement. However, with our adjusted measurement protocol, we found excellent correlation between repeated measurements within and between readers with limited bias, indicating the validity of the PCATMA measurement.This was a single-center retrospective study of patients with a clinical indication for cCTA. There was no follow-up, since a normal cCTA result led to discharge from the cardiology outpatient clinic. Some of the patients might have been at higher risk to develop CAD, reflected in already altered PCATMA varied considerably by tube voltage in patients without plaque on cCTA, with minor differences in PCATMA between coronary arteries . cCTA kV setting needs to be taken into account when interpreting PCATMA values.In conclusion, our results showed that PCATESM 1(DOCX 374 kb)


Drosophila Kib levels independently of Yorkie-mediated transcription. We find that Hpo signaling complex formation promotes Kib degradation via SCFSlimb-mediated ubiquitination, that this effect requires Merlin, Salvador, Hpo, and Warts, and that this mechanism functions independently of other upstream Hpo pathway activators. Moreover, Kib degradation appears patterned by differences in mechanical tension across the wing. We propose that Kib degradation mediated by Hpo pathway components and regulated by cytoskeletal tension serves to control Kib-driven Hpo pathway activation and ensure optimally scaled and patterned tissue growth.The Hippo (Hpo) pathway regulates tissue growth in many animals. Multiple upstream components promote Hpo pathway activity, but the organization of these different inputs, the degree of crosstalk between them, and whether they are regulated in a distinct manner is not well understood. Kibra (Kib) activates the Hpo pathway by recruiting the core Hpo kinase cassette to the apical cortex. Here, we show that the Hpo pathway downregulates  Drosophila in somatic mosaic screens for tumor-suppressor genes (Mob as tumor suppressor (Mats). Activation of the Hpo pathway results in a kinase cascade that culminates in the phosphorylation of a transcriptional co-activator Yorkie (Yki) by Wts, which inhibits Yki nuclear accumulation. Conversely, inactivation of the Hpo pathway allows Yki to translocate into the nucleus where, together with its DNA-binding partners such as Scalloped (Sd), it promotes transcription of pro-growth genes. As a result, inactivation of the Hpo pathway is characterized by excessive tissue growth. Mutations that disrupt Hpo pathway activity can lead to various human disorders including benign tumors and carcinomas  signaling pathway is an evolutionarily conserved inhibitor of tissue growth that was first identified in or genes . Centralrcinomas .A distinct feature of the Hpo pathway is the remarkably complex organization of its upstream regulatory modules . The corSlimb (SCFSlimb) E3 ubiquitin ligase complex, which leads to Ex degradation  cells. We found that Kib was ubiquitinated and that depletion of the core pathway kinases Hpo or Wts resulted in dramatically decreased Kib ubiquitination -tagged ubiquitin in cultured tination . Taken tTo better understand how the Hpo pathway controls Kib levels via ubiquitination, we sought to identify the machinery that mediates this process. Protein ubiquitination occurs via an enzymatic cascade that culminates in the covalent attachment of ubiquitin molecules to substrates by E3 ubiquitin ligases . We firs676DSGVFE681 that matches the consensus Slimb degron (S677A) known to be important for proper substrate recognition by Slimb . For these experiments, we used the UASp promoter . KibS677A-GFP accumulated to much greater levels than wild-type Kib-GFP when expressed in the wing disc pouch using the nub>Gal4 driver  Slimb could still weakly bind KibS677A-GFP and promote its degradation, albeit with reduced efficiency, and (2) loss of Hpo pathway activity could lead to greater cortical Kib accumulation at the expense of the total cytoplasmic pool. In support of the first possibility, KibS677A-GFP weakly associated with Slimb , and Wts in S2 cells  and can rylation .coiled-coil regions , a C2-like domain, a putative atypical protein kinase C (aPKC)-binding domain, and three 1-857-GFP) but retaining the degron motif was strongly upregulated upon Hpo depletion, similar to wild-type Kib  was insensitive to Hpo depletion even though it retained the Slimb degron motif  or together (KibΔWW1&2-GFP), expressed at markedly higher levels than wild-type Kib  resulted in a protein that localized at the apical cortex but did not form puncta  still increases upon Hpo pathway inactivation. Additionally, we show that Kib is phosphorylated and ubiquitinated in a pathway-dependent manner, and that Kib ubiquitination is mediated via SCFSlimb.In this study, we show that the Hpo pathway negatively regulates Kib levels via a previously unrecognized post-translational feedback loop. Several key results indicate that this feedback is independent of Yki transcriptional activity. First, loss of Mer leads to a dramatic increase in Kib levels without a detectable increase in Yki transcriptional activity. Second, removing Sd, which blocks Yki-mediated transcription, does not suppress the elevated Kib levels upon Hpo pathway inactivation. Third, the abundance of Kib-GFP expressed under a Yki-insensitive promoter  to Kib-organized signaling complexes and thereby regulate Kib-mediated pathway activation. We currently know little about how dynamic Hpo pathway output is in developing tissues, largely because there are no available single-cell resolution reporters for pathway activity. Our findings suggest that pathway output mediated by Kib could be tightly and dynamically regulated in response to mechanical tension or other factors that affect the degradation mechanism described here.It might seem paradoxical that Kib degradation, which is dependent on signaling complex assembly, is greater at the periphery of the wing pouch where net Hpo pathway activity is thought to be lower . HoweverAnother question our study raises is the functional significance of having both transcriptional and post-translational negative feedback mechanisms that regulate Kib levels. Feedback regulation is a common feature in cell signaling, and transcriptional negative feedback can serve to limit the output of a signaling pathway over time . In the hh>Gal4, en>Gal4, ap>Gal4, nub>Gal4.For expression of UAS transgenes, the following drivers were used: To generate mutant clones, the following crosses were performed:e1 40A FRT/CyO, dfdYFP; Kib::GFP/TM6, Tby w hsFlp; Ubi-RFP 40A FRT X ex4 19A FRT/FM7, actGFP; MKRS/TM3, Ser, actGFP X hsFLP, w1118, Ubi-RFP-nls 19AFRT; Kib::GFP/TM3, Ser, actGFPMere1 Yki-YFP ykiB5/CyO, dfdYFPy w hsFlp; Ubi-RFP 40A FRT X ex4 19A FRT/+; Yki-YFP/CyO, dfdYFP X hsFLP, w1118, Ubi-RFP-nls 19AFRT;MKRS/TM3, Ser, actGFPMere1 40A FRT/CyO, dfdYFP; ban3-GFP/TM6, Tby w hsFlp; Ubi-RFP 40A FRT X ex4 19A FRT/FM7, actGFP; MKRS/TM3, Ser, actGFP X hsFLP, w1118, Ubi-RFP-nls 19AFRT; ban3-GFP/TM3, Ser, actGFPMer47 Mer4 19A FRT/FM7, dfdYFP; Sco/CyO, dfdYFP X hsFLP, w1118, Ubi-RFP-nls 19AFRT; Kib::GFP/TM3, Ser, actGFPsd47 19A FRT/FM7, dfdYFP; FRT 42D hpoBF33/CyO, dfdYFP X ey>Flp Ubi-GFP 19A FRT; FRT 42D Ubi-RFP/CyO, dfdYFP; Ubi>Kib-GFP/+sd4 19A FRT/+; nub>Gal4/CyO, dfdYFP X hsFLP, w1118, Ubi-RFP-nls 19AFRT; UASp-Kib-GFP/TM3, Ser, actGFPMer4 19A FRT/+; nub>Gal4/CyO, dfdYFP X hsFLP, w1118, Ubi-RFP-nls 19AFRT; UASp-KibS677A-GFP/TM3, Ser, actGFPMerBF33/CyO, dfdYFP X y w hsFLP; FRT 42D Ubi-RFP/CyO, dfdYFP; UASp-Kib-GFP/+nub>Gal4 FRT 42D hpoBF33/CyO, dfdYFP X y w hsFLP; FRT 42D Ubi-RFP/CyO, dfdYFP; UASp-KibS677A -GFP/+nub>Gal4 FRT 42D hpoTCCGGTACCGGCTCCGGC-3′, and the entire Kib-GFP-FLAG cassette was first cloned into UAStattB backbone to generate UASt-Kib-GFP-FLAG, with unique NotI (immediately 5′ of the Kozak sequence) and KpnI (in the linker region) restriction sites flanking Kib sequence. To make Kib1-857, Kib858-1288, and Kib484-1288, the corresponding regions were amplified  using primers KibS677A  of ~50 μm in diameter were placed under the cover slip. The mounted samples were immediately imaged on Zeiss LSM 880 or LSM 800 confocal microscopes. Throughout the paper, apical tissue views were shown as maximum projections of the most apical optical sections  generated using Image J; for basal views, single sections ~10.5 μm below the apical surface were shown. Widefield fluorescence imaging of live wing imaginal discs was done using a Zeiss Axioplan 2ie microscope with an Orca ER camera and Zeiss AxioVision software.Throughout the paper (except in escribed . BrieflypMT-Kib-GFP-FLAG (this study), ΔWW1&2-GFP-FLAGpMT-Kib (this study), pAc5.1-Slimb-6x-myc , 1-600, pMT-FLAG-Hpo,pAFW-Mer, pAHW-Mer and pAC5.1-V5-Wts (The following constructs were used in co-immunoprecipitation experiments: 1-V5-Wts .6 S2 cells (S2-DGRC) were transfected with total of 500 ng of the indicated DNA constructs using dimethyldioctadecylammonium bromide . For GFP or Myc IPs, guinea pig anti-GFP (1:1250) or mouse anti-Myc (1:1000) antibodies were used. Pierce Protein A (Thermo Scientific) magnetic beads were used to precipitate antibody-bound target proteins. For immunoblotting, the following antibody concentrations were used: rabbit anti-GFP (1:5000), mouse anti-Hpo (1:5000), mouse anti-α-tubulin (1:2500), mouse anti-Myc , mouse M2 anti-Flag , mouse anti-V5 (1:2500), and rabbit anti-hemagglutinin (HA) (1:5000). Immunoblots were scanned using an Odyssey CLx scanner (LI-COR Biosciences).Briefly, 3.5×10e Sigma;  at 250 μethylenediaminetetraacetic acid, 0.5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 0.9 M glycerol, 0.1% Triton X-100, 0.5 mM Dithiothreitol, and Complete protease inhibitor cocktail (Roche) at one tablet/10 ml concentration.Cells were harvested and lysed on ice in buffer containing 25 mM Hepes, 150 mM NaCl, 1 mM nub>Gal4 with Ubi>Kib-GFP alone or together with an indicated RNAi transgene were immediately flash-frozen in a bath of dry ice and 95% ethanol and stored at −80°C. On the day of IP, the discs were briefly thawed on ice and lysed in buffer described above. PhosSTOP (Sigma Aldrich) phosphatase inhibitor cocktail was added to the lysis buffer to inhibit phosphorylation (one tablet/10 ml of buffer). Kib-GFP was immunoprecipitated with guinea pig anti-GFP antibody (1:1250). A control sample was treated with λ-phosphatase. Samples were run on 8% polyacrylamide gel, with 118:1 acrylamide/bisacrylamide  expressing rylamide , to bettFor ubiquitination assays, pMT-HA-Ub  was co-tFor dsRNA-mediated knockdown experiments, T7 primers , annealiImage J was used to quantify mean fluorescence intensity in clones vs. control region in  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The Hippo pathway is a highly conserved pathway controlling tissue growth during development. One of its upstream regulators is Kibra, which activates the pathway by assembling the Hippo signaling complex to regulate the activity of the downstream transcriptional effector, Yorkie. However, as shown here, activating the Hippo signaling complex also results in the phosphorylation of Kibra itself, which promotes its own degradation via the E3 ubiquitin ligase.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by K VijayRaghavan as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Yorkie-independent negative feedback couples Hippo pathway activation with Kibra degradation" 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.eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife, either of which would be linked to the original paper.Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife is supported, provided the authors can clarify the points raised by the reviewers.This is an interesting, well-written manuscript, which addresses the post-transcriptional regulation of Kibra, a regulator of the Hippo pathway, an important pathway controlling growth. The authors convincingly show that Kibra stability is regulated by Slimb, and that Hippo pathway kinases Hpo and Wts promote Kibra phosphorylation and thus Slimb-dependent degradation. They have strong biochemical and imaging-based analyses. They define a function for a Slimb degron, and conduct structure-function to explore Kibra domains in protein turnover. Overall this is an interesting, well-conducted story presenting novel data that will be of interest to the cell signaling and growth control fields. Publication in Summary:Many of the points can be easily addressed by the authors, but they should particularly pay attention to their comments to Figure 7.Reviewer #1:In this manuscript, the authors present data to further unravel the complex regulation of the Hippo pathway, an important pathway controlling growth. Their data point to a negative feedback loop mediated by the core Hippo kinase cassette, which regulates one of its upstream regulators, Kibra, in a Yorkie-independent way. In addition, they provide some evidence that Kibra degradation is patterned across the disc. These are novel data and provide additional information on how such an important pathway is controlled to prevent excess growth of tissues, at least in some regions of the fly wing disc.1) They show that knock-down of Sav, Hippo or Warts results in increased Ubi>Kib-GFP levels, but I do not understand why they also "… regulate its localization at the junctional cortex.". In both control and sav-, hpo- and wts-RNAi Ubi>Kib-GFP can be found at the junction .2) Figure 2I. The authors show dramatic decrease of ubiquitinated Ubi>Kib-GFP upon hpo- and wts-RNAi, as well as less phosphorylated Ubi>Kib-GFP upon single knock-down. No explanation is given why in one experiment they used single knock down (H), but double knock-down in (I). In addition, the statement that Kib levels are regulated "via phosphorylation-dependent ubiquitination" is an assumption, although it may be correct given findings published previously.3) Subsection “Slimb regulates Kibra levels via a consensus degron motif”: If loss of Slmb leads to increased Ex  and loss of either Slmb or Ex leads to increased Ubi>Kib-GFP , why should co-depletion of Ex and Slimb "… suppress the increase in Ubi>Kib-GFP levels”?4) Given their conclusion suggesting that the Kibra pathway is patterned, I wonder whether the authors always have studied the same region of the disc when comparing different genotypes. This is not always obvious from the figures.Reviewer #2:eLife.This interesting, well-written manuscript addresses the post-transcriptional regulation of the Hippo pathway regulator Kibra. The authors convincingly show that Kibra stability is regulated by Slimb, and that Hippo pathway kinases Hpo and Wts promote Kibra phosphorylation and thus Slimb-dependent degradation. They have strong biochemical and imaging based analyses. They define a function for a Slimb degron, and conduct structure function to explore Kibra domains in protein turnover. Overall this is an interesting, well conducted and novel story that is appropriate for publication in Endogenous Kib ( tagged with GFP) abundance is increased in merlin clones, but not in expanded clones, even though ex clones dramatically increase nuclear yorkie and ban3>gfp, and merlin does not. These data indicate loss of ex has a more potent effect on Yki than loss of merlin, and suggest Merlin acts independent of Yki and transcription. Consistent with this model, loss of Sd does not affect Kib levels in the eye. Importantly, ubiquitin driven KibraGFP is increased by loss of Merlin, Sav, Hpo or Wts. The authors note strong junctional accumulation of Kibra upon loss of Hpo or Wts. The authors show Kibra is phosphorylated and ubiquitinated upon Hpo/Wts activation. They show that Kibra has a Slimb degron (DSGVFE), and that mutation of this motif leads to stabilization of Kibra, and makes its levels insensitive to Hpo pathway activation. They also conducted structure function analyses that indicate that the WW domains of Kibra are important for its degradation upon Hpo pathway activation. Because Slimb promotes Ex degradation, loss of Slimb leads to increased Ex, and hence increased Hpo/Wts activity. The authors show overexpressed Ex does not in itself stabilize Kibra. While it is clear that Kibra phosphorylation is promoted by Hpo/Wts, it is still unclear if this is a direct phosphorylation The authors propose a model in which Kibra promotes Hpo/Wts activity, which then locally promotes Kibra degradation. It is not clear how it this local signaling is isolated from other junctional Hpo and Wts.The weakest part of this very strong manuscript is Figure 7. If the intent is to show that Kibra is in a pattern in the wing, it would be good to include the staining and intensity quantification of endogenous Kibra in A and B comparison. In Figure 7C and D, the authors should include pictures of examples of adult wings of the different genotypes that they provide the size analysis. Details of the landmarks on the wings they used to define distal and proximal width would be helpful. The manuscript is very strong without this figure, and given the complex feedback loops, it is hard to know if patterned expression drives growth or is a readout of growth.Reviewer #3:Drosophila wing disc to explore mechanisms regulating of the abundance of the Hippo pathway upstream regulator, Kibra. They demonstrate that the increase in Kibra protein levels observed upon loss of Hippo pathway activity cannot be fully explained by the known Yki-dependent transcriptional feedback mechanism, and uncover a previously unsuspected post-translational layer of regulation of Kibra protein levels. They show that Kibra is phosphorylated and ubiquitylated in S2 cells, but less so upon knockdown of hpo and wts. They show that knockdown of the ubiquitin ligase substrate recognition subunit Slmb stabilises Kib, and identify a putative phospho-degron for Slmb within Kibra. They further show that mutation of this phospho-degron diminishes Kib ubiquitylation and renders Kibra insensitive to loss of Hippo pathway activity. They then use deletion constructs to narrow down the domains of Kibra necessary for degradation by their new mechanism and find that the regions essential for Kibra to activate the Hippo pathway are the ones necessary for degradation via their mechanism. Finally, they describe a non-uniform pattern of degradation of their Kibra stability reporters across the wing pouch.Tokamov et al. have used the eLife, provided the authors can clarify the points below.The paper is well written and easy to read. The data are of high quality and the conclusions drawn are supported by the evidence presented. The findings are interesting, novel and should be of interest to the cell signalling and growth control fields. I support publication in Experimental points:1) The authors make a good case that Kibra can be phosphorylated in a Hpo/Wts-dependent manner (even if it may not be directly by Hpo or Wts themselves), and that Hpo and Wts are required for Kibra stability. However, they don't directly test whether Wts kinase activity itself is required for Kibra degradation by Slimb. As they suggest that the assembly of a complex involving Kibra and several pathway members is necessary for degradation, it would be important to test if Wts kinase activity is required, or rather if it is the formation of this complex itself that is necessary to recruit Slimb. For example, this can be done by re-expressing wild type or kinase dead Wts in the S2 cell setup of Figure 3E (dsRNAs targeting the UTRs of wts would have to be used), and/or by looking at the effect of wild type or kinase dead Wts transgenes on ubi-kib-GFP expression in vivo.2) To directly connect the Kibra degron identified by the authors with Hippo pathway activity, it would be good to look at expression of a Yki target gene such as ex-lacZ upon Kib-wt and Kib-S677A expression.Text/presentation changes:1) "These results suggest that the upstream regulation of the Hippo pathway is highly compartmentalized and that the activity of the core kinases is tightly associated with its distinct upstream regulators in a manner that allows little crosstalk between them".Discussion – "Such tightly compartmentalized activity of the Hippo pathway is also suggested by the punctate appearance of all of these proteins at the cell cortex".The authors should clarify in discussion what they mean with these statements. Is it that different microdomains of upstream regulators define hotspots of core kinase cascade activity that have different functions? What would this mean functionally for Yki inactivation?2) The subsection “The Hippo pathway promotes Kibra degradation in a highly compartmentalized manner and independently of pathway activation by Expanded” refers to Figure 5F-G, but it should read 5D-E.3) In Figure 6A (schematic of Kib truncation constructs), construct 1-857 has been drawn without a GFP tag at the end. I assume this is an omission.4) The discussion about tension in the wing pouch (Discussion) is confusing. Surely if tension at the periphery of the pouch is higher, leading to decreased Hpo/Wts activity, then Kibra degradation should be lower there, not higher as observed? It therefore doesn't follow that "The results from this study may lead to the elucidation of how external cues, such as mechanical tension, modulate Kib-mediated Hippo signaling and pattern tissue growth." The authors should clarify this, or remove this part.5) The authors should include scale bars in their images.6) It would be nice to know which of their images are of live wing discs, and which are fixed (the appearance of reporters such as these can be different in live vs. fixed tissue). Could they list this in the Materials and methods? Reviewer #1:In this manuscript, the authors present data to further unravel the complex regulation of the Hippo pathway, an important pathway controlling growth. Their data point to a negative feedback loop mediated by the core Hippo kinase cassette, which regulates one of its upstream regulators, Kibra, in a Yorkie-independent way. In addition, they provide some evidence that Kibra degradation is patterned across the disc. These are novel data and provide additional information on how such an important pathway is controlled to prevent excess growth of tissues, at least in some regions of the fly wing disc.1) They show that knock-down of Sav, Hippo or Warts results in increased Ubi>Kib-GFP levels, but I do not understand why they also "…… regulate its localization at the junctional cortex.". In both control and sav-, hpo- and wts-RNAi Ubi>Kib-GFP can be found at the junction .We agree with the reviewer that this point is confusing to the reader. Because it is not important for the main focus of this manuscript, we have eliminated this statement from the text.2) Figure 2I. The authors show dramatic decrease of ubiquitinated Ubi>Kib-GFP upon hpo- and wts-RNAi, as well as less phosphorylated Ubi>Kib-GFP upon single knock-down. No explanation is given why in one experiment they used single knock down (H), but double knock-down in (I). In addition, the statement that Kib levels are regulated "via phosphorylation-dependent ubiquitination" is an assumption, although it may be correct given findings published previously.We initially used double knock-downs in this experiment to eliminate as much kinase activity as possible. We have since repeated these experiments using single knock-downs, which similarly affect Kibra ubiquitination. Figure 2I has been replaced with single knock-down experiments to avoid confusion over this point. Additionally, we agree that the notion that ubiquitination is phosphorylation dependent has not been shown directly and so have modified the text accordingly.3) Subsection “Slimb regulates Kibra levels via a consensus degron motif”: If loss of Slmb leads to increased Ex  and loss of either Slmb or Ex leads to increased Ubi>Kib-GFP , why should co-depletion of Ex and Slimb "… suppress the increase in Ubi>Kib-GFP levels”?We thank the reviewer for pointing out that we were not sufficiently clear here. Our point was that because Kibra and Expanded have previously been shown to interact, our concern in this experiment was that the increased Kibra abundance observed upon depletion of Slimb could be due to increased recruitment by Expanded (whose abundance is greatly increased by Slimb depletion). The co-depletion experiment simply shows that increased Kibra is not caused by increased recruitment by Expanded. We have modified the text to make this point clearer to the reader.We should also point out that the reviewer was mistaken in that Figure 1 shows endogenous Kibra (GFP-tagged via Crispr) rather than Ubi>Kib-GFP. In this case, we interpret the relatively modest increase in Kibra abundance to the previously identified feedback loop in which Yorkie promotes Kibra transcription. Ubi>Kib-GFP was used in subsequent experiments to eliminate the possibility of Yorkie-mediated transcriptional feedback.4) Given their conclusion suggesting that the Kibra pathway is patterned, I wonder whether the authors always have studied the same region of the disc when comparing different genotypes. This is not always obvious from the figures.With the exception of Figure 2, which includes images from the eye imaginal disc, all images are from the blade primordium (pouch) of the wing imaginal disc. We have added a statement in the Results indicating this. The main figures that involve wing imaginal discs show images of the entire wing pouch together with higher magnification insets which are indicated by boxes on the lower magnification panels . This allows the reader to see exactly what part of the wing blade is shown. As is apparent from the figures, we do not observe significant differences across the wing pouch in the effects we have described.Reviewer #2:This interesting, well-written manuscript addresses the post-transcriptional regulation of the Hippo pathway regulator Kibra. The authors convincingly show that Kibra stability is regulated by Slimb, and that Hippo pathway kinases Hpo and Wts promote Kibra phosphorylation and thus Slimb-dependent degradation. They have strong biochemical and imaging based analyses. They define a function for a Slimb degron, and conduct structure function to explore Kibra domains in protein turnover. Overall this is an interesting, well conducted and novel story that is appropriate for publication in eLife.Endogenous Kib ( tagged with GFP) abundance is increased in merlin clones, but not in expanded clones, even though ex clones dramatically increase nuclear yorkie and ban3>gfp, and merlin does not. These data indicate loss of ex has a more potent effect on Yki than loss of merlin, and suggest Merlin acts independent of Yki and transcription. Consistent with this model, loss of Sd does not affect Kib levels in the eye. Importantly, ubiquitin driven KibraGFP is increased by loss of Merlin, Sav, Hpo or Wts. The authors note strong junctional accumulation of Kibra upon loss of Hpo or Wts. The authors show Kibra is phosphorylated and ubiquitinated upon Hpo/Wts activation. They show that Kibra has a Slimb degron (DSGVFE), and that mutation of this motif leads to stabilization of Kibra, and makes its levels insensitive to Hpo pathway activation. They also conducted structure function analyses that indicate that the WW domains of Kibra are important for its degradation upon Hpo pathway activation. Because Slimb promotes Ex degradation, loss of Slimb leads to increased Ex, and hence increased Hpo/Wts activity. The authors show overexpressed Ex does not in itself stabilize Kibra. While it is clear that Kibra phosphorylation is promoted by Hpo/Wts, it is still unclear if this is a direct phosphorylation The authors propose a model in which Kibra promotes Hpo/Wts activity, which then locally promotes Kibra degradation. It is not clear how it this local signaling is isolated from other junctional Hpo and Wts.First, we thank the reviewer for these supportive comments. With regards to the final point, how this effect can be localized to just medial, Kibra-dependent signaling, we have added new findings to the revised version of the manuscript that we believe are relevant to this issue. As reviewer 3 noted, the data in the original version of the manuscript did not distinguish between a structural vs. enzymatic role for the pathway kinases Hippo and Warts. While the manuscript was being reviewed, we addressed this question using dsRNAs to deplete endogenously expressed Hippo or Warts in S2 cells and expressing RNAi-resistant, kinase dead mutant forms of either kinase. Interestingly, we found that the kinase dead forms of either kinase promote Kibra ubiquitination in S2 cells, indicating that it is formation of the signaling complex , rather than activation of the kinases, that recruits the Slimb E3 complex that ubiquitinates Kibra. We think this discovery nicely explains how this feedback mechanism is isolated from junctional signaling mediated by Expanded because it indicates that it is formation of the complex itself rather than activation of the kinases that promotes Kibra ubiquitination and degradation.The weakest part of this very strong manuscript is Figure 7. If the intent is to show that Kibra is in a pattern in the wing, it would be good to include the staining and intensity quantification of endogenous Kibra in A and B comparison.We thank the reviewer for this comment because it points out that we were not sufficiently clear in explaining the experiments presented in Figure 7. A key point that we now realize may not have been clear was that in these experiments Kibra, either wild-type or the degron mutant, was expressed ectopically under Nubbin>Gal4, rather than under its endogenous promoter. We did this because we did not want endogenous transcriptional patterning or transcriptional feedback from Yorkie to confound differences in protein degradation. So, although endogenous Kibra displays a similar abundance pattern to ectopically expressed wild-type Kibra , we do not think that the endogenous pattern is directly relevant to the question we are asking here. Rather, we think the relevant comparison is between wild-type and degron mutant Kibra, where both forms are expressed identically at the transcriptional level and in a manner not subject to pathway feedback. As the figure shows, in this system the pattern of wild-type and degron mutant Kibra abundance across the wing blade is quite different, suggesting that Kibra degradation is patterned (non-uniform) across the blade. We note that this pattern is strikingly similar to the pattern of observed junctional tension that has been reported previously, leading us to propose that tension might regulate Kibra degradation.bantam to produce small clones of rapidly growing cells in the wing imaginal disc. Because these clones are bounded by slower growing cells, they become compressed and cytoskeletal tension decreases . We set up this system in the background of either wild-type or ΔWW1 mutant Kibra-GFP expressed under the Ubi promoter. Like the degron mutant, ΔWW1 is not subject to pathway promoted degradation (because it is unable to assemble the complex), but unlike the degron mutant this transgene is not lethal under the Ubi promoter. While we see a robust increase in Kibra-GFP (wild-type) abundance in compressed bantam-expressing clones, we do not see any increase in the ΔWW1 mutant Kibra protein.Additionally, while the original version of the manuscript was under review we used a different experimental approach, designed in the Irvine lab, to further test the hypothesis that tension regulates Kibra abundance. This system uses expression of the microRNA Taken together, we feel these two lines of experimental evidence provide evidence that the feedback regulation of Kibra we have identified here is, at least in part, regulated by mechanical tension. While we acknowledge that a fuller understanding of this will depend on understanding its underlying mechanistic basis, we think these interesting observations provide a developmental context that emphasizes the significance of the feedback we have discovered and therefore improve the impact of the work.In Figure 7C and D, the authors should include pictures of examples of adult wings of the different genotypes that they provide the size analysis. Details of the landmarks on the wings they used to define distal and proximal width would be helpful.Agreed – this has been done.The manuscript is very strong without this figure, and given the complex feedback loops, it is hard to know if patterned expression drives growth or is a readout of growth.Please see the comments just prior, and also those that follow in response to reviewer 3.Reviewer #3:Drosophila wing disc to explore mechanisms regulating of the abundance of the Hippo pathway upstream regulator, Kibra. They demonstrate that the increase in Kibra protein levels observed upon loss of Hippo pathway activity cannot be fully explained by the known Yki-dependent transcriptional feedback mechanism, and uncover a previously unsuspected post-translational layer of regulation of Kibra protein levels. They show that Kibra is phosphorylated and ubiquitylated in S2 cells, but less so upon knockdown of hpo and wts. They show that knockdown of the ubiquitin ligase substrate recognition subunit Slmb stabilises Kib, and identify a putative phospho-degron for Slmb within Kibra. They further show that mutation of this phospho-degron diminishes Kib ubiquitylation and renders Kibra insensitive to loss of Hippo pathway activity. They then use deletion constructs to narrow down the domains of Kibra necessary for degradation by their new mechanism and find that the regions essential for Kibra to activate the Hippo pathway are the ones necessary for degradation via their mechanism. Finally, they describe a non-uniform pattern of degradation of their Kibra stability reporters across the wing pouch.Tokamov et al. have used the The paper is well written and easy to read. The data are of high quality and the conclusions drawn are supported by the evidence presented. The findings are interesting, novel and should be of interest to the cell signalling and growth control fields. I support publication in eLife, provided the authors can clarify the points below.Experimental points:1) The authors make a good case that Kibra can be phosphorylated in a Hpo/Wts-dependent manner (even if it may not be directly by Hpo or Wts themselves), and that Hpo and Wts are required for Kibra stability. However, they don't directly test whether Wts kinase activity itself is required for Kibra degradation by Slimb. As they suggest that the assembly of a complex involving Kibra and several pathway members is necessary for degradation, it would be important to test if Wts kinase activity is required, or rather if it is the formation of this complex itself that is necessary to recruit Slimb. For example, this can be done by re-expressing wild type or kinase dead Wts in the S2 cell setup of Figure 3E (dsRNAs targeting the UTRs of wts would have to be used), and/or by looking at the effect of wild type or kinase dead Wts transgenes on ubi-kib-GFP expression in vivo.We completely agree with the reviewer's points here. In fact, while the original version of this manuscript was under review, we performed exactly the experiment requested. The results convincingly show that the kinase dead forms of Hippo and Warts promote Kibra ubiquitination quite effectively and thus argue that the kinases play a structural rather than enzymatic role in this process. This finding is quite exciting for two reasons. First, it potentially explains the remarkable compartmentalization between Kibra vs. Expanded promoted pathway activation because it argues that the physical presence of the kinases in a complex with Kibra, rather than their enzymatic activity (which might readily spread from one compartment to another), promotes Kibra degradation. Second, this finding suggests that another kinase is required to phosphorylate the degron sequence, a question we hope to solve in the future.2) To directly connect the Kibra degron identified by the authors with Hippo pathway activity, it would be good to look at expression of a Yki target gene such as ex-lacZ upon Kib-wt and Kib-S677A expression.While we agree in principle, in practice the difference in growth is subtle and these reporters are generally less sensitive to gain of upstream pathway function than its loss. Accordingly, we have added a statement in the text indicating that while the decreased growth caused by Kib-S677A is consistent with decrease Yki function, we have not formally shown this.Text/presentation changes:1) "These results suggest that the upstream regulation of the Hippo pathway is highly compartmentalized and that the activity of the core kinases is tightly associated with its distinct upstream regulators in a manner that allows little crosstalk between them".Discussion – "Such tightly compartmentalized activity of the Hippo pathway is also suggested by the punctate appearance of all of these proteins at the cell cortex".The authors should clarify in the Discussion what they mean with these statements. Is it that different microdomains of upstream regulators define hotspots of core kinase cascade activity that have different functions? What would this mean functionally for Yki inactivation?We thank the reviewer for these useful comments. We think the new data described above, showing that the kinases have a structural role in promoting Kibra degradation, substantially address the first point. That is, because it is the physical presence of the kinases in the complex, rather than their activity, the basis of the compartmentalization of this effect becomes clear. We have added text in the Discussion to clarify this point.We agree that the second point is not really clear and since it is not a primary focus of this manuscript we have deleted it from the Discussion.2) The subsection “The Hippo pathway promotes Kibra degradation in a highly compartmentalized manner and independently of pathway activation by Expanded” refers to Figure 5F-G, but it should read 5D-E.Agreed.3) In Figure 6A (schematic of Kib truncation constructs), construct 1-857 has been drawn without a GFP tag at the end. I assume this is an omission.Yes, thank you for catching this mistake.4) The discussion about tension in the wing pouch (Discussion) is confusing. Surely if tension at the periphery of the pouch is higher, leading to decreased Hpo/Wts activity, then Kibra degradation should be lower there, not higher as observed? It therefore doesn't follow that "The results from this study may lead to the elucidation of how external cues, such as mechanical tension, modulate Kib-mediated Hippo signaling and pattern tissue growth." The authors should clarify this, or remove this part.Our new finding (described in detail above) that formation of the Kibra signaling complex, rather than activation of Hippo and Warts, promotes Kibra ubiquitination and degradation provides an elegant answer to this important question. We envision that as tension increases the degradation mechanism we have identified becomes more efficient (through mechanisms that remain to be discovered), resulting in degradation of Kibra and dissolution of the complex it assembles before it can efficiently activate the kinases and inactivate Yorkie. We have modified the Discussion to clarify this point.We note that we also have added additional data, described above, showing that experimentally altering tension in the wing epithelium alters Kibra abundance.5) The authors should include scale bars in their images.We have done this.6) It would be nice to know which of their images are of live wing discs, and which are fixed (the appearance of reporters such as these can be different in live vs. fixed tissue). Could they list this in the Materials and methods?Agreed. This information has been added in the Materials and methods section. In short, all imaginal disc images except those of Expanded, Flag, and Scalloped antibody staining were done using live tissues.


With the advent of omics technologies, tremendous progress has been made in understanding the signaling pathways in normal and disease states across different species. Multi-omics approaches can be categorized into two groups: molecular profiling (MPro) and molecular perturbation (MPer) .WGS approaches have been extensively used to unravel the different types of genomic alterations in cancer and facilitate understanding of the mutational landscape of cancer genomes. Using WGS, Dr. Bandapalli's group identified candidate predisposing genes in families with a reported recurrence of Hodgkin-lymphoma (HL), a lymphoproliferative malignancy of B-cell origin. These variants were prioritized using an in-house pipeline “FCVPPv2.” The authors used this pipeline along with gene/variant panels based on cancer predisposing genes and variants prioritized in the largest familial HL cohort study reported to date, to identify high penetrance germline variants in the HL families. Furthermore, pathway and network analyses of these variants have provided additional molecular cues relevant to the molecular pathogenesis of HL that may aid the development of targeted therapy and the screening of individuals who are at risk of developing HL .MicroRNAs play a key role(s) in regulating gene expression via either degradation of a transcript or the inhibition of its translation. Dr. Skoblov's group presents initial insights into the complexity of human microRNA-mRNA interactions. They performed a comprehensive computational analysis on HEK293 and Huh7.5 datasets and reported interesting features of human mRNA interactome, along with more than 46,000 experimentally confirmed mRNA-miRNA duplex regions. As part of this study, they also developed a web-based tool, publicly available at Zhang et al.).In addition, Dr. Fang's group performed a comprehensive analysis of hsa-let-7i-5p miRNA in normal and pathogenic fibroblasts and studied its role ranging from controlling cellular phenotype to molecular signaling particularly TGF-β signaling .In a transcriptome study, Dr. Evelo's group performed computational analysis on transcriptomic data derived from cattle breeds with different intramuscular fat deposition to identify pathways that define marbling in beef cattle. A total of 17 pathways were significantly dysregulated between well-marbled vs. lean-marbled beef including MAPK and insulin signaling, and immune response associated pathways .Dr. Guan's group carried out a genome-wide analysis in gastric cancer and identified 548 and 2,399 differentially methylated sites and lncRNAs, respectively. The lncRNAs were able to discriminate between normal vs. cancerous samples of gastric origin .Dr. Jolly's group carried out a comprehensive and comparative analysis of methods utilizing different transcriptomics signatures to quantify the status of EMT—a cell biological process involved in cancer metastasis and chemoresistance. They showed that these methods exhibited a concordance among themselves in quantifying the extent of EMT in a given sample and that tumor cells can undergo varying degrees of EMT across tumor types. While any of the three methods can capture the generic trend in the EMT status of a given cell (or population), the multinomial logistic regression EMT scoring method has an additional advantage of being able to predict from the transcriptomic signature of a population, whether it is comprised of “pure” single hybrid E/M cells at the single-cell level, or an ensemble of E and M cell subpopulations .Another study, by Dr. Imhof's group in the context of hemolytic disorders, described a new ontology and knowledge graph “HemeKG,” which is publicly available at Lin et al.).In a study on the brain disorder Schizophrenia, Dr. Lane's group used an ensemble boosting predictive framework along with random undersampling, to assess the status of schizophrenia in the population of Taiwan by examining the levels of D-amino acid oxidase protein and its interaction partner, the D-amino acid oxidase activator, in the N-methyl-D-Aspartate receptor pathway, as well as by using melatonin levels in the tryptophan catabolic pathway. They also evaluated the performance of the ensemble boosting algorithm and compared it with other widely used machine learning algorithms, including support vector machine, and multi-layer feedforward neural networks. Notably, they showed that it performs better in distinguishing schizophrenia patients from healthy controls .Using an integrative approach, Dr. Domingo-Fernandez and his group demonstrated that the choice of pathway database could impact the results of statistical enrichment analysis and predictive modeling. They also developed an integrative pathway resource called “MPath” which showed that using multiple pathway databases or integrated resources could provide more biologically consistent results and improved prediction performances, as opposed to using equivalent pathways from different databases  strongly linked to OC. A large majority of Frizzled receptors including FZD6 and FZD8 were involved in the ß-catenin canonical signaling pathway .Dr. Zayed's group employed an integrative and systematic bioinformatics in treating ovarian cancer, and identified not only the DEGs involved in the cell cycle, but also the hub genes including core genes .In a study on the autoimmune disease SLE, Dr. Zayed's group used a high-throughput transcriptomics platform to identify dysregulated signaling pathways. They found that four genes including M. tuberculosis, Dr. Gupta's group studied Rv1915/ICL2a protein as there has been difficulty in harvesting the soluble protein. They overcome this by expression of C-terminal truncated Rv1915/ICL2a in the heterologous host E. coli BL21 (DE3) .In a study on This collective effort brings together studies covering models from prokaryotic to eukaryotic organisms using different omics approaches to delineate either signaling or molecules directly or indirectly related to signaling in cancer and/or other diseases and aberrant conditions.JS coordinated the Research Topic. MKK coordinated the editorial. JS, LB, SK, and MKK contributed to the development of the Research Topic, suggested and invited the participants, and helped with the peer review process. All authors have approved the final version of the editorial.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.


Acute kidney injury (AKI) is defined as a sudden event of kidney failure or kidney damage within a short period. Ischemia-reperfusion injury (IRI) is a critical factor associated with severe AKI and end-stage kidney disease (ESKD). However, the biological mechanisms underlying ischemia and reperfusion are incompletely understood, owing to the complexity of these pathophysiological processes. We aimed to investigate the key biological pathways individually affected by ischemia and reperfusion at the transcriptome level.AKT, KRAS, and those related to Rho signaling, were downregulated, suggestive of injury responses during reperfusion. Semaphorin 4D and plexin B1 levels were also downregulated.We analyzed the steady-state gene expression pattern of human kidney tissues from normal (pre-ischemia), ischemia, and reperfusion conditions using RNA-sequencing. Conventional differential expression and self-organizing map (SOM) clustering analyses followed by pathway analysis were performed. Differential expression analysis revealed the metabolic pathways dysregulated in ischemia. Cellular assembly, development and migration, and immune response-related pathways were dysregulated in reperfusion. SOM clustering analysis highlighted the ischemia-mediated significant dysregulation in metabolism, apoptosis, and fibrosis-related pathways, while cell growth, migration, and immune response-related pathways were highly dysregulated by reperfusion after ischemia. The expression of pro-apoptotic genes and death receptors was downregulated during ischemia, indicating the existence of a protective mechanism against ischemic injury. Reperfusion induced alterations in the expression of the genes associated with immune response such as inflammasome and antigen representing genes. Further, the genes related to cell growth and migration, such as We show that specific biological pathways were distinctively involved in ischemia and reperfusion during IRI, indicating that condition-specific therapeutic strategies may be imperative to prevent severe kidney damage after IRI in the clinical setting. In the kidney, ischemia-reperfusion injury (IRI) is characterized with the temporary deficiency of oxygen due to restricted blood flow, followed by the sudden restoration of oxygen supply. The result is acute kidney injury (AKI), which may vary from a subtle kidney dysfunction to the need for renal replacement therapy . PhysiolAKI is closely interconnected and integrated with chronic kidney disease (CKD). AKI is a risk factor of incidence of CKD, which itself is a risk factor of AKI episodes , 7. MoreThe various pathophysiological characteristics of AKI pose difficulties to evaluate the underlying mechanism, thereby contributing to poor patient prognosis . For insIn the present study, we investigated the key genes and biological pathways affected separately after ischemia and reperfusion during IRI in human kidney tissues. We performed RNA sequencing (RNA-seq) to examine changes in the gene expression pattern via conventional differential gene expression and self-organizing map (SOM) clustering analyses . The impWe reveal the specific genes and pathways that are involved separately in ischemia and reperfusion during IRI in the human kidney. We suggest that a condition-specific therapeutic approach may be imperative for the effective prevention of severe kidney damage after IRI in the clinical setting. Further investigations are warranted to understand the functions of the newly discovered biological signatures related to IRI.− 11.73 m− 2, mean hemoglobin: 14.2 g/dL) .To identify the changes induced by ischemia and reperfusion at the transcriptomic level, we analyzed the steady-state gene expression pattern during pre-ischemia , ischemia, and reperfusion using RNA-seq Fig. b. Total http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was conducted to assess the quality of RNA-seq data. The RNA-seq data were quantified using an alignment-free tool, Salmon, developed by Patro et. al in 2017 that estimates the relative abundance of all transcripts [FastQC  was used. Genes in each cluster were further analyzed to identify significantly enriched canonical pathways.To identify the trajectory patterns of gene expression across all three time points, pre-ischemia, ischemia, and reperfusion, SOM clustering  analysiswww.qiagen.com/ingenuity, Spring 2019, QIAGEN, CA, USA) was used to identify enriched biological pathways. A p-value or Benjamini-Hochberg adjusted p-value was calculated using Fisher’s exact test, and a cut-off value of less than 0.05 was used to identify significantly enriched canonical pathways based on the Ingenuity Knowledge Base.The Ingenuity Pathway Analysis (IPA) software , ischemia, and reperfusion conditions in human kidney samples using RNA-seq Fig. b. RNA-se2 fold-change| ≥ 1 and p-value < 0.05 as criteria to identify significant DEGs between the groups  ischemia versus pre-ischemia to identify the genes affected by ischemia; 2) ischemia versus reperfusion to detect the genes affected by reperfusion after ischemia; (3) reperfusion versus pre-ischemia to analyze the genes affected by the complete IRI process. We used |log ischemiaAs a result, 603 DEGs  were significantly dysregulated between ischemia and pre-ischemia samples and 135 DEGs  were found to be significantly dysregulated between reperfusion and ischemia samples. However, 1389 DEGs  were dysregulated between reperfusion and pre-ischemia conditions. The top 20 DEGs in each comparison group are shown in Supplementary Tables SCYP1A2, CYP2C8, CYP2C9, CYP2J2, CYP3A4, CYP3A7). Uridine diphosphate glucuronosyltransferase  is involved in the metabolism of various molecules, including steroids, hormones, and drugs  conditions showed that reperfusion induced changes in various pathways related to lipid and drug metabolism such as nicotine, serotonin, and melatonin degradation, FXR/RXR, and PXR/RXR signaling pathway, consistent with the results between ischemia and pre-ischemia conditions Fig. . Differek-means clustering, SOM has been extensively used for the analysis of large-scale gene expression data [23) for further analyses. As a result, 38,014 transcripts were used for SOM input. The 7 × 7 grid structure was chosen for SOM output to enhance biological interpretability. The results were visualized as a colored grid panel with blue hexagons and a yellow-black similarity color scheme between the hexagons  and reperfusion (genes upregulated or downregulated in reperfusion versus ischemia). Modules of interest among 49 modules were selected based on the criteria rules Table . As a reFAS) and tumor necrosis factor (TNF) related to complex signaling pathways for cell death were down-regulated, suggestive of the presence of a protective mechanism against cell death. In addition, intracellular and secondary messenger signaling pathways such as protein ubiquitination, adipogenesis, and apelin adipocyte signaling pathway were identified. Lipid accumulation and deposition is known to induce lipotoxicity, thereby leading to ischemia-mediated kidney injury [We performed pathway analysis using IPA for the selected clusters to identify significantly enriched pathways. The genes dysregulated by ischemia (Cluster 1 and 2) were mainly enriched in apoptosis-related pathways, including aryl hydrocarbon receptor and death receptor signaling pathway Table . During y injury . FurtherThe genes dysregulated by reperfusion (Cluster 3 and 4) were mainly related to cellular functions of growth, proliferation, and migration  , ischemia and reperfusion (reperfusion versus ischemia), and pre-ischemia and reperfusion (reperfusion versus pre-ischemia) conditions. While the conventional differential expression analysis process only compared two conditions, we applied unbiased clustering algorithm, SOM, to identify specific trajectories of interest across all three conditions. In particular, we focused on specific gene expression patterns to identify the effects of ischemic and reperfusion separately during IRI. (1) The genes dysregulated in ischemia versus pre-ischemia; (2) those dysregulated in reperfusion versus ischemia. We then performed pathway analysis to investigate the affected pathways in ischemia and reperfusion during IRI process using selected genes from differential expression analysis and SOM clustering.Pathway analysis for DEGs in each comparison group revealed the enrichment of metabolism-related pathways in ischemia. On the other hand, cellular assembly, development and migration, and immune response-related pathways were enriched in reperfusion. Pathway analysis for the genes selected from SOM revealed apoptosis, xenobiotic metabolism, and fibrosis-related pathways to be enriched in ischemia and cell growth and migration and immune response-related pathways to be significantly enriched in reperfusion.FAS, CAS2/6/7, PARP6/8/11/12/14, TNF, and TNFRSF1/10/10B/25, fibrosis-related genes, collagen (COL1/3/ 4/5/6/7/12//15/16/18), and adipogenesis-related genes were downregulated, suggesting the existence of a protective process from cell damage against ischemia injury in the kidney tissue.In general, the interruption of the blood supply during ischemia induces changes in specific metabolic pathways. We found that melatonin/serotonin degradation and FXR/RXR pathway related to lipid metabolism as well as bupropion and acetone degradation, estrogen biosynthesis, and PXR/RXR pathway related to drug metabolism were enriched in ischemia. Melatonin and its metabolites have been regarded as scavengers of free radicals or stimulators of antioxidant enzymes, and play protective roles in kidney ischemic injury . Thus, cAKT1/2/3, KRAS, MAPKAP1, EIF2B1/4, CDK11A, RPL8/12/13A/17/24/30/31, ARPC2/3/5/5 L, RAC1, and RHOC/T2/V, crucial for regeneration and repair system after reperfusion injury were downregulated. Thus, it indicates that a dysregulation in cell growth and migration pathways in response to reperfusion occurs.Reperfusion after ischemia triggers a robust inflammatory response within the kidney by blood re-supply. The immune response-related pathways, including inflammasome and antigen presentation pathway, were dysregulated after reperfusion in our study, and this effect was not observed during ischemia. Considering the other mechanisms of reperfusion, cell development, growth and migration-related pathways such as FGF, mammalian target of rapamycin (mTOR), eukaryotic initiation factor 2 (eIF2), semaphorin, integrin, and actin-based motility by Rho signaling were identified. SEMA4D) and plexin B1 (PLXNB1), a receptor of semaphorin related to semaphorin signaling, were found to be downregulated under reperfusion condition. Semaphorin 3A (Sema3A) promotes kidney injury followed by AKI [Semaphorin 4D (d by AKI ; howeverd by AKI –40. In cWe reveal that specific biological pathways were uniquely involved in ischemia and reperfusion during IRI. Metabolism, apoptosis, and fibrosis-related pathways were significantly dysregulated under ischemia conditions, whereas cell development, growth, migration, and immune response-related pathways were affected by reperfusion following ischemia. Therefore, we suggest that a condition-specific therapeutic strategy may be necessary to prevent severe kidney damage after IRI in the clinical setting. Although our study has limitations such as the small number of samples and relatively short duration of ischemia and reperfusion, we believe that it will contribute to the understanding of the mechanisms underlying IRI.Additional file 1: Supplementary Table S1. Clinical characteristics of patients. Supplementary Table S2. Information of RNA sequencing analysis. Supplementary Table S3. Top 20 most significantly DEGs between ischemia vs. pre-ischemia. Supplementary Table S4. Top 20 most significantly DEGs between reperfusion vs. ischemia. Supplementary Table S5. Top 20 most significantly DEGs between reperfusion vs. pre-ischemia. Supplementary Table S6. Pathways for DEGs between ischemia and pre-ischemia using IPA. Supplementary Table S7. Pathways for DEGs between reperfusion and ischemia using IPA. Supplementary Table S8. Pathways for DEGs between reperfusion and pre-ischemia using IPA.


Tapirus terrestris), which expands the understanding of the adaptation of this species to habitats. The histological and ultrastructural analysis of the lingual papillae and lingual glands revealed the presence of two types of mechanical papillae, namely the filiform and conical papillae, while papillae with taste buds  formed the second, less numerous group. The filiform papillae differed from those of Equidae or Rhinocerotidae. The presence of nine vallate papillae, localized in groups of two surrounded by a ring, or individually, was unique for the examined female tapir. In addition, the vallate papillae contained irregular pseudopapillae on their surface. The foliate papillae contained several sulci between each folia. The presence of sparse taste buds in the side wall of the vallate papillae and foliate papillae is unique for the tapir. Compared to other Perissodactyla, the number of taste buds in the tapir is limited, although the features of its tongue surface make it possible to distinguish this species from representatives of Equidae or Rhinocerotidae.This is a detailed study of the surface morphology of the tongue and the lingual glands of the lowland tapir (Tapirus terrestris), the exact anatomy of its tongue has not been studied. Samples of the lingual papillae and lingual glands were collected from the tongue of an adult female lowland tapir. The microscopic analysis of the structure of the lingual papillae and the histochemical analysis of the secretion of the lingual glands were analyzed. The tongue of the tapir is divided into the apex, body with a distinct lingual prominence, and the root. Its ventral surface is smooth. The most numerous of the mechanical papillae were the filiform papillae, while numerous conical papillae with a sharp apex or more rounded papillae were present in the root of the tongue. There were also nine vallate papillae and pair of foliate papillae. The foliate papillae contained several folds parted by 12–14 grooves. The mucous secretion produced by the lingual glands was more obvious than the serous secretion. The features of the dorsal surface of the tongue as well as the shape and number of the lingual papillae on the surface of the tongue of the examined female tapir differ compared to Equidae or Rhinocerotidae, the other two representatives of Perissodactyla. However, further study is necessary for the synapomorpy of the tapir’s tongue.Although the anatomy of the gastrointestinal tract has been characterized in the lowland tapir ( Tapirus terrestris) is the most widespread member of the Tapiridae (Perissodactyla) family and inhabits the subtropical and tropical zones of South America  [The lowland tapir (olombia) ,2. The lolombia) . Populatolombia) .The lowland tapir is a herbivore, known to consume a wide variety of plants, but its diet is not fully understood . Tapirs Melastomataceae, Sapotaceae, Rubiaceae, Araceae, Moraceae, and Fabaceae were reported as the most common consumed plants [Spondias mombin, Helicostylis tomentosa, Ficus spp., and Bagassa guianensis [The tapir diet is highly diverse, including seasonal and locally available plants . The divd plants ,10,11. Ad plants , lowlandianensis . Fruit cianensis  and O’Faianensis .proboscis pushes food towards the mouth. The narial structures indirectly grasp food [The tapir possesses a true proboscis similarly to elephants, showing an evolutionary convergence between the order Perissodactyla and the Proboscidea order ,15. Tapiasp food . The jawasp food .Equidae and Rhinocerotidae. The lingual morphology has already been described in representatives of these families. Filiform papillae seem to be similar in their appearance (hair-like or needle-like) in all described odd-toed ungulates [Equidae and Rhinocerotidae families but their number varied from two in the zebra [Diceros bicornis), 23–29 in the white rhinoceros (Ceratotherium simum) [Rhinoceros unicornis) [Equus asinus) [Equus caballus) [Equus quagga chapmani) [Although the anatomy of the tapir’s gastrointestinal tract was precisely described , the detngulates ,21,22,23ngulates  and the ngulates . Vallatehe zebra  and in the zebra ,23, 2–5 he zebra ,22, apprhe zebra  in the bm simum) , and 21–icornis) . Foliate asinus) ,22 and iaballus) ,23. No fhapmani) , in the hapmani) , or in thapmani) . Round fhapmani) .Tapiridae—includes only one genus—Tapirus.The last family belonging to the Perissodactyla—Tapirus bairdii, Tapirus indicus, Tapirus pinchaque, and Tapirus terrestris are known [The four extant species of tapirs as re known . So far,re known . The aimTapirus terrestris) at the Wroclaw Zoo . The animal was 25 years, 3 months, and 27 days old. The District Veterinary Officer permitted sample collection . Pursuant to the law in the European Union, no consent of the local Ethics Committee for Animal Experiments is required for post-mortem sampling.The tongue was collected in 2019 from an adult female lowland tapir  and a macroscopic analysis of the surface of the tongue was performed ).Samples of the lingual papillae and lingual glands were collected from the apex, body, lingual prominence, and root of the tongue for histological and histochemical studies. All samples were placed in 10% buffered neutral formalin . After at least 72 h the specimens were washed in running water (24 h) and dehydrated in a series of alcohol dilutions , then cleared in xylen. In the next step the samples were impregnated with paraffin and cut with using of microtome Slide 2003 ) into 4 µm sections. The samples then underwent routine staining procedures using Masson-Goldner trichrome and Azan trichrome staining. Alcian blue pH 1.0 (AB pH 1.0), alcian blue pH 2.5 (AB pH 2.5), PAS-AB pH 2.5, periodic-acid Schiff (PAS), and Hale’s dialysed iron (HDI) staining methods were used to determine the type of lingual gland secretions ,27. The The samples of the lingual papillae were collected for ultrastructural analysis from the apex, body, lingual prominence, and root of the tongue and placed in 2% glutaraldehyde diluted in 0.1 M PBS at a pH 7.4. The samples were dehydrated in a graded ethanol series , transferred to absolute acetone  and thenThe obtained results were described using the Nomina Anatomica Veterinaria  and NomiThe tongue of the lowland tapir consisted of three parts, namely the apex, body, and root. The lingual prominence was well marked within the body of the tongue. The tongue was 35 cm long. It was almost 8 cm wide at the apex, 6 cm wide at the body, and 11 cm wide at the root. The thickness of the apex was 2.5 cm, 6 cm at the body, and 8.5 cm at the root, respectively. There was no dark pigmentation on the dorsal surface of the tongue within the apex, there was scarce pigmentation on the lingual prominence and most pigmentation on the surface of the root of the tongue and on some lingual papillae A. MoreovThe gustatory papillae consisted of vallate papillae, fungiform papillae, and foliate papillae A,C,D,F. stratum granulosum  were clearly visible at the base of the foliate papillae. The SEM analysis found that the surface of individual folia was smooth .The lingual glands comprised accessory serous glands of the gustatory papillae  A and domAll three families, the Tapiridae, Equidae, and Rhinocerotidae, belong to the Perissodactyla order. However, their diet and living environment differ greatly. Although their functional anatomy is similar, the structural features of their individual organs varies. A detailed analysis of the morphometric features allows better understanding of the functional anatomy of the digestive tract. A properly adapted stomach and intestine enable absorption of nutrients by a particular species, although the preprocessing of food by the tongue and teeth, palate, and cheeks also play an important role in this process.Equus asinus [Equus asinus [Equus quagga chapmani [Equus asinus [Equus quagga chapmani [The tongue of the captive lowland tapir differed macroscopically compared to the Equidae ,21,22,23s asinus . This tos asinus , while is asinus ,20,22,32s asinus ,22, Equuchapmani . The linchapmani  while its asinus  and EquuThe number and distribution pattern of individual lingual papillae differed between species of the three families in the Perissodactyla order. The main difference was in the number and distribution of the gustatory papillae on the surface of the tongue of the lowland tapir. The filiform papillae were the most numerous mechanical papillae in the lowland tapir, similarly to Equidae ,20 or RhEquus asinus [Equus quagga chapmani [The filiform papillae present on the apex of the tongue in the lowland tapir contained fewer secondary processes. In contrast to the filiform papillae in the lowland tapir, there were two types of filiform papillae in the s asinus  and no cchapmani . The shachapmani . The filchapmani . Hence, chapmani . Similarchapmani  which ischapmani , wherebychapmani ,19,20.Equus asinus [Equus quagga chapmani [Equus asinus [Equus asinus lacked taste buds in the apical part of the fungiform papillae [Interestingly, two types of fungiform papillae were identified, which can be compared to the presence of the second type of “lobulated” fungiform papillae in the s asinus . Moreoves asinus , the whis asinus  and blacs asinus ,24, or ichapmani  and Equus asinus . Howeverpapillae , while spapillae  without papillae  and Pfeipapillae  obtainedpapillae ,33. The Equus asinus, contained from 2–5 vallate papillae [Equus asinus [Diceros bicornis had, in contrast, 60 [The arrangement and shape of the eight vallate papillae in the lowland tapir differed from other representatives of the Perissodactyla order. Contrary to the lowland tapir, representatives of Equidae, such as the papillae  of variopapillae . There ws asinus ,22 compas asinus , while trast, 60  or 43 [2rast, 60  vallate rast, 60 , more thrast, 60 ,24 in thrast, 60 ,24.Equus asinus contained fewer mucosal folds than the tapir, namely 7–8 [A pair of foliate papillae was present in the lowland tapir similarly to horse ,23,32,33mely 7–8  or 8–10 mely 7–8 , which amely 7–8 . Unlike mely 7–8 . In the mely 7–8 . In the mely 7–8  which wamely 7–8 ,22.The glands of the tongue in the lowland tapir comprised serous von Ebner’s glands, accessory vallate papillae, and foliate papillae, while units of mixed glands of the root of the tongue were better developed. The secretion of these glands opened on the surface of the root of the tongue between numerous conical papillae. Histochemically, sulfated mucins and carboxylated sialomucins predominated in the mixed glands. Due to no histochemical studies of the tongue in the Rhinocerotidae, the histochemical analysis cannot be compared between animals from this family. On the other hand, the content of lectins in the secretory cells and duct cells of von Ebner’s glands has been studied in horses .The diet of the lowland tapir focuses on certain plants but occasionally also includes fruits ,4,5. TheMany representatives of Artiodactyla ,46,47,48In Marsupialia with a folivorous diet, i.e., the bear cuscus  and koalAdaptation to a fruit diet is seen in typically frugivorous animals, such as some members of the Chiroptera order. Interestingly, the filiform papillae are most varied and have been found to exist in 5–6 types, depending on the fruit bat species ,54,55. TTapirrus terrestris were not detected. The subsequent question is if there are some differences in the lingual surface microstructure of the tongue in accordance with the diet of the captive and wild tapirs? Thus, the further study are necessary for the synapomorpy of the tapir’s tongue.Compared to other Perissodactyla, the number of taste buds in the tapir’s tongue was limited, although the features of its lingual surface make it possible to distinguish this species from representatives of Equidae or Rhinocerotidae. On the other hand, the number of examined samples was reduced thus the similarities and/or differences between female and male of


We pragmatically delineate the trait repertoire that enables organismal niche specialization. We based our approach on the tenet, derived from evolutionary and complex-system considerations, that genomic units that can significantly contribute to fitness in a certain habitat will be comparatively more complex in organisms specialized to that habitat than their genomic homologs found in organisms from other habitats. We tested this in cyanobacteria forming harmful water blooms, for which decades-long efforts in ecological physiology and genomics exist. Our results essentially confirm that genomics and ecology can be linked through comparative complexity analyses, providing a tool that should be of general applicability for any group of organisms and any habitat, and enabling the posing of grounded hypotheses regarding the ecogenomic basis for diversification. Water bloom development due to eutrophication constitutes a case of niche specialization among planktonic cyanobacteria, but the genomic repertoire allowing bloom formation in only some species has not been fully characterized. We posited that the habitat relevance of a trait begets its underlying genomic complexity, so that traits within the repertoire would be differentially more complex in species successfully thriving in that habitat than in close species that cannot. To test this for the case of bloom-forming cyanobacteria, we curated 17 potentially relevant query metabolic pathways and five core pathways selected according to existing ecophysiological literature. The available 113 genomes were split into those of blooming (45) or nonblooming (68) strains, and an index of genomic complexity for each strain’s version of each pathway was derived. We show that strain versions of all query pathways were significantly more complex in bloomers, with complexity in fact correlating positively with strain blooming incidence in 14 of those pathways. Five core pathways, relevant everywhere, showed no differential complexity or correlations. Gas vesicle, toxin and fatty acid synthesis, amino acid uptake, and C, N, and S acquisition systems were most strikingly relevant in the blooming repertoire. Further, we validated our findings using metagenomic gene expression analyses of blooming and nonblooming cyanobacteria in natural settings, where pathways in the repertoire were differentially overexpressed according to their relative complexity in bloomers, but not in nonbloomers. We expect that this approach may find applications to other habitats and organismal groups. Functional niche specialization is a ubiquitous trait in microorganisms, a fact that is reflected in their trait and genomic repertoires . The preClostridium thermocellum), a multienzyme complex with as many as 160 enzymes , and there were no apparent correlations between their ability to form blooms  and general genomic features, although bloomers had a narrower range  and nonbloomers (68 genomes) . Besideser range . All of 10.1128/mBio.01155-20.1FIG S1FIG S1, 1.0 MB.Phylogeny of the 43 cyanobacterial strains used based on 16S rRNA sequences. Download Copyright © 2020 Cao et al.2020Cao et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01155-20.2FIG S2FIG S2, PDF file, 0.2 MB.The relationship between bloom incidence index (BII) and cell shape and genomic features in 113 cyanobacterial strains. Download Copyright © 2020 Cao et al.2020Cao et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01155-20.7TABLE S1Table S1, DOC file, 0.4 MB.The 113 strains used in this study. Download Copyright © 2020 Cao et al.2020Cao et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2-concentrating mechanism)—were involved in utilization of external resources (light and CO2), and thus were formally included as query pathways in the comparison between bloomers and nonbloomers, even though one could also argue they are core pathways for phototrophs. The other query pathways selected have been shown to promote cyanobacterial growth and bloom formation (see references in Aphanizomenon flos-aquae strain NIES-81 (A. flos-aquae NIES-81 has pathways for the assimilation of carbon (CCM), nitrogen (N), phosphorus (P), sulfur (S), free amino acids (AAT), and sugars, as well as trace elements and vitamins (TEVit). It has capabilities against stressors like heavy metals , antibiotics (DrugR), oxidative stress (OSR), UV radiation (MAA), osmotic pressure, and possibly predation (through toxin production). Additionally, buoyancy can be regulated through the biosynthesis of gas vesicles. These pathways had different composition  and complexity (components consisting of multiple subunits) among different strains.We reconstructed 5 core metabolic pathways and 17 query pathways derived from existing ecological tests in the literature . Core pa NIES-81 . More de10.1128/mBio.01155-20.9TEXT S1Text S1, DOCX file, 0.03 MB.Supplemental methods and references. Download Copyright © 2020 Cao et al.2020Cao et al.Creative Commons Attribution 4.0 International license.This is an open-access article distributed under the terms of the 10.1128/mBio.01155-20.3FIG S3FIG S3, PDF file, 1.7 MB.Core and query pathway in this study. Download Copyright © 2020 Cao et al.2020Cao et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01155-20.8TABLE S2Table S2, DOC file, 0.3 MB.The classification of central and query pathways and their protein reference sources. Download Copyright © 2020 Cao et al.2020Cao et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the https://github.com/hshcao/CHABpathways.On average, bloomers had a total of 461 (±57) proteins in the 19 query pathways, accounting for about 14.5% of their entire genomes, whereas nonbloomers encoded only 274 (±42) proteins, accounting for 10.3% of the genes, suggesting there is indeed a tendency for more complex query pathways among bloomers. As a control, core pathways had on average similar number of proteins between bloomers and nonbloomers, 169 versus 160, accounting for 5.3% and 6% of the total genes, respectively. The specific components of the core and query pathways for each strain in our analyses can be found as Tables S3 to S16 at P < 0.05) more complex among bloomers regardless of metric. However, the magnitude of the difference varied considerably with pathway, from almost 7-fold in some (vesicle) to marginal values in others (ORS). The ratios were quite consistent among metrics. As expected, the core metabolic pathways did not differ significantly between bloomers and nonbloomers for any complexity metric. PSII and PSI were only marginally different, however, perhaps because they should be considered core pathways among phototrophs. More specifically, we present the ratio between bloomers and nonbloomers of each of our eight genomic complexity metrics in Microcystis aeruginosa, Planktothrix agardhii, Cyanothece sp., Synechococcus sp., Crocosphaera watsonii, Prochlorococcus marinus and Prochlorococcus sp., we carried out additionally analyses with data sets that used only one random genome for each species. A similar pattern in complexity differences was still observed  even when determined among closely related phylogenetic strains. We conclude that any effects of evolutionary/phylogenetic legacies were insignificant.The pattern shown in P > 0.05) for any of the core pathways or PSI and PSII (R2 >0.4), and only 2 additional pathways had R2 >0.25 (NF and N). Interestingly, besides the significance of the correlations, the slopes of the correlation also showed a similar pattern among the query pathways . Expectedly, we found no significant correlation . For the OSR genes, only the gene gpx was significantly higher in nonbloomers than bloomers , while others were not different .To illustrate in detail how the query pathways identified above were indeed enriched with more components, we visualized the complete set of components in the significant vesicle pathway as opposed to the nonsignificant OSR, as shown in M. aeruginosa strain NIES-843 for the bloom, Oscillatoria nigro-viridis strain PCC7112 for the oligotrophic freshwater plankton, and Synechococcus sp. strain WH8102 for the oligotrophic marine plankton). Significant correlations between the expression levels of pathways and the pathway GCIs were only found in blooming species in two eutrophic lakes  . To that end, ecologically relevant metatranscriptomes of our own from eutrophic freshwater blooms in Lake Erie and Harsha Lake were studied in contrast to those of oligotrophic freshwater and oligotrophic ocean plankton which were publicly available. RNAseq reads were mapped to the genomes of the species closest to those dominating each habitat ( < 0.01)  to D, bu < 0.01)  to H. PaA key understudied question in CyanoHAB research is whether there exists a common ecophysiology underlying the broad array of blooming species. We attempted to address this question from the comparative functional genomics perspective, testing the hypothesis that functional niche specialization will result in preferential sophistication of genomic systems that are differentially important for bloom formers. Such differential importance must be manifest in both encoding and expressing these functions. An initial comparison of pathway complexity among bloomers and nonbloomers found that pathway complexity  was statistically higher among bloomers for most potentially relevant pathways queried (and not for core pathways or core photosynthetic pathways), but also that the effect was not commensurate among pathways. The fact that none of the core metabolism was significantly different agrees with the general pattern of cyanobacterial genome evolution, i.e., the core set of genes is well conserved while accessory pathways are variable . The potWe identified vesicle formation for buoyancy as the most enriched and highly expressed pathway, consistent with the ecological literature. Vesicle formation has been well established as part of the common ecophysiological repertoire among various types of bloomers, conferring buoyancy to adjust their position in the water column for optimal resource utilization , 23, 31.Two pathways that saw significant enrichment among bloomers and a high correlation with BII, were the amino acid uptake systems, and the synthesis of polyunsaturated fatty acids. Cyanobacteria have been known to utilize organic nutrients such as glucose, acetyl-glucosamine, and various amino acids , 35, but36–2 can easily result in C limitation. In agreement with our results, rising CO2 level has been found to intensify phytoplankton blooms in eutrophic waters (2 levels during a dense CyanoHAB (Prochlorococcus sp. and Prochlorothrix). But an ad hoc comparison excluding the latter did not support this notion, in that the significance remained below 0.01. In any event, this is not to say that P is not important. The even distribution of genes and particularly the fact that some blooming species have two sets of phosphate ABC transporters (pstABCD) suggests that P may simply not be more significant in blooms that in other habitats. In agreement with this view, external P additions do not necessarily lead to higher Microcystis abundance in Lake Erie where phosphorus level is already high  acquisition for bloom formation also found confirmation in our study. Particularly for C, N, and S, all correlated rather well with BII and were expressed at levels commensurate with their GCIs. For C, a diffusion limitation effect associated with high population density may enhance the value of sustaining complex carbon concentrating mechanisms (CCM) among bloomers, because heavy drawdown of COc waters , and traCyanoHAB . InteresCyanoHAB . One posCyanoHAB  than ourady high , and theDrug and metal resistance pathways had low correlations with BII and only minor increases in complexity among bloomers, and also showed lowest expression among the query pathways.With respect to the water-soluble mycosporine pathway, which is common among cyanobacteria of many origins , we woulMore generally, the data presented here provide support for the “relevance begets complexity” in comparative genomics. It delineates a tool to bring together comparative genomics and ecology. While this approach was here applied to the case of bloom-forming cyanobacteria, there is in principle no reason why this approach may not find application in a variety of other microbial habitats and organismal groups. On a broader scale, our approach can be combined with metagenome-assembled genomes to infer the link between genomic complexity and ecological niche, where together they could provide a comprehensive conceptual framework for predictive ecology from genomic data .Whole-genome sequences of 113 cyanobacteria  were dowA more precise ranking followed, in which we examined the blooming incidence index (BII) of all strains in the Web of Science. We used as query either the strain denomination (first), species epithet (if the strain denomination yielded insufficient hits), or, in a few cases, the genus name. The records returned were then further separated into genomic/physiological or environmental studies using appropriate key words. Finally, the records were classified into those that reported a bloom and those that did not. The BII for each strain was simply the proportion of environmental citations that mentioned blooming capacity, and it varied between 0 and 1 . We disc2-concentrating mechanism. The reference protein sequences for the central metabolism were extracted from the SEED subsystems  and query pathways , since proteins in central pathways are more conserved than those in the query pathways . Next, the mean expression levels of each pathway were calculated as the average level of all the genes in the pathways. For each water body, the correlations between mean pathway (log transformed) expression levels and (angular transformed) BIIs were carried out for all (query and core) or only query pathways, using R.We tested the correlation with metatranscrioptome of cyanobacteria collected from three types of water: (i) our own metagenomes from a CyanoHAB in eutrophic Lake Erie; (ii) oligotrophic freshwater Sparkling Lake; and (iii) the oligotrophic Arctic Ocean. The reads from each water body were mapped to reference genomes closest to their own dominant cyanobacterial species,