Alcohol-related facial flushing is a sign of compromised alcohol metabolism and increased risk of certain cancers. This project examined how facial flushing might be used to reduce alcohol use to lower cancer risks. Interviews with Chinese university students identified gender, friendship, and drinking purpose as important variables related to whether someone would encourage a person who flushes when drinking alcohol to stop or reduce their drinking. A questionnaire was developed that incorporated these variables into 24 drinking scenarios in which someone flushed while drinking. Students responded whether they would (a) encourage the flusher to stop or drink less; (b) do nothing while wishing they could; or (c) do nothing because there was no need. Analysis of survey responses from 2912 university students showed a three-way interaction of the variables and implied that the probability students will intervene when a drinker flushes was highest when the flusher was a female, a close friend, and the drinking purpose was for fun and lowest if the flusher was a male, the friendship was general, and the drinking purpose was risky. The results provide important details about the social factors affecting how other people respond to a person who flushes when drinking alcohol. This information is useful for those considering ways to reduce and prevent aerodigestive cancers through education and information programs. Alcohol (ethanol) has been identified as a causative agent for aerodigestive tract cancers ,2. AerodAlcohol (ethanol) metabolism proceeds in two stages. In the first stage, alcohol is metabolized by the enzyme alcohol dehydrogenase (ADH) into acetaldehyde. Acetaldehyde is carcinogenic and toxic, and, in some people, stimulates a histamine reaction visible as a red flush on the face and neck. In the second stage, the aldehyde dehydrogenase (ALDH) isozymes metabolize acetaldehyde into relatively harmless acetic acid that is expelled from the body mostly in the urine. Genetic code for ALDH consists of 19 genes in 11 families . One groOne ALDH gene is inherited from each parent. Individuals who inherit two ALDH2*2 genes (homozygous) are essentially unable to metabolize alcohol, experience extreme reactions from the accumulation of acetaldehyde, and as a consequence do not drink or rarely drink. Heterozygous individuals (ALDH2*2/ALDH2*1) experience a greatly reduced ability to metabolize acetaldehyde—about 18% of normal metabolism . In addiData from alcoholics in Japan  suggest A reduction in alcohol consumption among ALDH-deficient individuals can be achieved if the individuals who flush change their drinking behavior and if those who encourage those individuals to drink more change theirs.The purpose of this study was to identify and examine the interaction of social variables that could affect whether a person was willing to suggest to someone who flushes that he/she stop or reduce drinking.To develop the questionnaire, we initially conducted semi-structured personal interviews with students to identify what students thought were the factors influencing their reaction to a fellow student flushing. In all, 24 Chinese university students participated in the interviews.Data from the interviews identified three factors that were used for questionnaire development. First, students confirmed the importance of gender as a major predictor of how other people would react to someone’s flushing that was identified in prior studies ,22,23. SBecause of the contextual nature of reactions reported by students, the questionnaire was designed with a scenario format. Six drinking scenarios were developed as described in A total of 2912 students from universities in Southwestern, Central, and Northeastern China completed the survey. Data from 2492 (85.6%) of the surveys presented valid results . The description of the sample is provided in A generalized logit mixed model was estimated in Mplus Version 7.0  to evaluFull information maximum likelihood with robust variance estimation was used throughout. Likelihood ratio tests (LRTs) were performed to evaluate the omnibus effect of each predictor on the overall outcome (three response options corresponding to two degrees of freedom), and Wald tests were used to determine the significance for the specific comparisons (two response options corresponding to one degree of freedom). The odds ratios were computed to facilitate interpretation.The survey data upon which the results are based can be accessed at University of Nebraska-Lincoln Data Repository . ResponsThe primary purpose of this study was to determine what factors influenced college student responses to someone’s facial flushing. Specifically, the study objective was to determine how gender, drinking setting/purpose, and friendship influenced how students reacted.suggest,00 = 3.721, p < 0.001, indicating a significant variability in response among students. Similarly, the variance of Level 2 random effects of students on responses for “want to suggest the person stop or reduce drinking, but did not” versus no suggestion was τwant,00 = 2.577, p < 0.001, indicating a significant between-student variance in the outcome.The results from the test of the full model with all interactions is shown in In the follow-up test, holding constant the random effect, the three-way interactive logistic model implied that the probability of suggesting to a flusher to stop or reduce drinking versus not making such a suggestion was the highest when the flushing student was a close female friend in a fun drinking situation and lowest if the flusher was a general male friend in a risky drinking situation.The odds of wanting to suggest that the flusher stop but not actually doing so versus having no such inclination were lower with respect to female flushers and close friends and were also lower with respect to close friends (of either gender) in fun drinking occasions. The three-way interaction among these factors was not significant . HoweverIn the follow-up test, holding constant the type of drinking purpose and the random effect, the two-way interactive logistic model suggested that the probability that a student did not suggest that a flusher stop or reduce but wanted to versus the probability that a student had no such inclination was the highest when the flushing student was a general female friend and the lowest if the flusher was a close male friend.When holding constant gender and the random effect, the probability of wanting to make a suggestion versus having no such inclination was highest when the flushing student was a general friend in a fun drinking situation and lowest when the flusher was a close friend in a risky drinking purpose.Alcohol-induced facial flushing is common in Asian populations and indicates in a given individual a compromised ability to metabolize alcohol and an increased risk for upper aerodigestive cancers. When someone’s heightened risk is visible to other people in a drinking situation, it raises questions about how facial flushing could be used as a cue for people to make a suggestion to the person who flushes to reduce or stop their drinking. This study explored the effects of three social factors—gender, degree of friendship, and drinking purpose—on the likelihood that someone would intervene. The results from this study can benefit the development of educational programs aimed at increasing the number of people who reduce or stop drinking because they flush and who lend support to drinking companions who want to stop drinking or slow down. Chang et al.  have recAlmost all of the students in the sample (99%) reported that they regularly or occasionally observed someone flushing when drinking alcohol, leading us to believe that an educational program for Chinese young adults developed around the flushing phenomenon would be relevant.Our results indicated that the probability that someone would suggest to a flusher that she or he stop or reduce drinking was highest when the flusher was a female and a close friend and when the drinking situation was principally for pleasure. The probability that someone would not intervene even while thinking the flushing person should stop drinking (“bystanders”) was highest either when the flusher was a general female friend or when the flusher was a general friend (regardless of gender) in a fun drinking situation.Finding that the probability that people will suggest to a person who flushes that they reduce or stop drinking is highest when the person flushing is a female and a close friend and when the drinking situation is for pleasure gives rise to the most likely scenario about which to begin education to modify drinking behaviors.An educational approach making use of these findings will have to be sensitive to the Chinese cultural context of gender, friendship, and drinking purposes. Specific implications of using each social factor for educational program development are discussed in the following sections.Earlier studies suggested that being a female flusher provided a degree of protection from pressure to drink ,22,23. SFor bystanders (the students who said they would not intervene but think they should), gender interactions were less pronounced. Again, there was a higher probability that students think they should intervene when the flusher was a female. However, this was not affected by drinking purpose. The probability of a student saying they want to intervene but did not was higher for general rather than close friends for both females and males. This is likely opposite to the probabilities for intervening because students were more likely to actually intervene with a close friend rather than standing by. These results are not necessarily surprising, but given the almost universal acknowledgment by Chinese people that their drinking patterns are deeply culturally ingrained and would be extremely difficult to change, some education on this issue is thus suggested.The students we interviewed said they thought people’s reactions to someone flushing would depend on whether the person who flushed was a haopengyou (好朋友) or a putongpengyou (普通朋友), which we have translated to English as “a close friend” or “a general friend.” Students said that the wish to help a close friend is always balanced against the status roles and drinking etiquette in a drinking situation, so while a person may wish to help out a close friend, they may not be able to take action to intervene. For example, drinking at an official banquet with bosses or dignitaries is a situation where status roles and rules of etiquette prevail over individual wishes. Students believed that, while the degree of friendship could not change the rules of etiquette, it might change the degree to which a person followed the rules.The study results strongly supported the interaction of friendship with the drinking setting, as well as with the gender of the flusher. This was especially true for males more so than females. The probabilities of intervention were much higher when the male was a close friend, whereas they were similar for females regardless of the closeness of the friendship. Similarly, bystanding  was more likely for general friends of either gender. This suggests hesitancy to speak up or take action when the flusher is only a casual acquaintance.Students we interviewed described contrasting types of occasions where drinking together would occur, which we have classified as fun/pleasure occasions and risky occasions. A fun/pleasure drinking occasion was described as a relaxed drinking environment that served as a means to enjoy the pleasures of drinking and friendship. People at fun/pleasure drinking occasions would not necessarily refuse to get drunk, but getting drunk was not the priority. With fun/pleasure as the unspoken goal, anything that might detract from the fun or pleasure of the occasion would be noticed and resisted. For instance, a person’s flushing while drinking could be interpreted as a sign of that person’s discomfort, and by extension a threat to the pleasantness of the occasion, so people in the group might encourage the flusher to stop or drink less.In contrast, at risky drinking occasions, some drinkers come with the purpose of getting drunk or getting others drunk. If the majority of the people in the group shared this objective, an individual’s wishes were easily overridden. Anyone who appeared to get in the way of the group’s objective may become the focus of the group’s attention. If someone in the group flushed and flushing was seen as an obstacle to the group’s intentions, members of the group could either ignore the flusher or informally work together to get the flusher drunk. The group’s dynamics in these risky drinking occasions may mean that flushers will fight to overcome the physical discomfort of their reaction to alcohol and get drunk. In risky situations, someone who flushes might also pressure other people to drink in order to get others to share in the discomfort and to redirect attention away from themselves.The study results thus support the importance of the drinking setting and purpose for taking action. It was always the case that students were more likely to intervene in a fun setting than in a risky setting. This was true across both gender and friendship status. However, the effect of the setting was greater for males than females, as the probability of intervening was much lower for males in risky drinking settings. For bystanders, however, the setting made little difference. Setting did not interact with gender, and the probability of not suggesting but wanting to was only marginally higher for general friends in risky drinking situations. These findings suggest that the effect of drinking setting/purpose on the probability of taking overt action is much stronger than that on the probability of thinking that perhaps they should do something but did not.These results need to be viewed in the context of several limitations. Our results are based on scenarios representing hypothetical situations. Reactions to hypothetical situations may not reflect actual reactions to actual situations. The identification of gender, degree of friendship, and drinking purpose used in this questionnaire was based on interviews from students at only one university. Moreover, while the sample was students from 13 universities in different parts of the country, we cannot claim it is truly representative of all undergraduate university students in China, nor is it representative of China’s general population. Interpreting the results from written questionnaires is always dependent upon the honesty of the respondents. The sample was intentionally homogeneous—undergraduate university students—limiting extrapolation of the results to other populations.New evidence linking alcohol-induced facial flushing to increased aerodigestive cancer risk creates a need to develop prevention and education. This study suggests the effects of social factors on the likelihood that individuals will encourage someone who flushes to stop or reduce drinking. This study identified social factors other than gender, which had been identified in earlier studies, namely the degree of friendship and the drinking purpose, that may increase the likelihood of a person’s suggesting that someone who flushes should stop or reduce drinking.


Cdt2 and SCFSkp2 couple to degrade p21 prior to the G1/S transition. Mathematical modelling reveals that a bistable switch, created by CRL4Cdt2, promotes irreversible S-phase entry by keeping p21 levels low, preventing premature S-phase exit upon DNA damage. Thus, we characterize how p21 regulates the proliferation-quiescence decision to maintain genomic stability.Following DNA damage caused by exogenous sources, such as ionizing radiation, the tumour suppressor p53 mediates cell cycle arrest via expression of the CDK inhibitor, p21. However, the role of p21 in maintaining genomic stability in the absence of exogenous DNA-damaging agents is unclear. Here, using live single-cell measurements of p21 protein in proliferating cultures, we show that naturally occurring DNA damage incurred over S-phase causes p53-dependent accumulation of p21 during mother G2- and daughter G1-phases. High p21 levels mediate G1 arrest via CDK inhibition, yet lower levels have no impact on G1 progression, and the ubiquitin ligases CRL4 Cell cycle arrest after DNA damage is achieved by the expression of the CDK inhibitor p21. Here the authors show that spontaneous DNA damage incurred in unperturbed cell cycles, leads to cell populations exhibiting a bistable state, with p53 and p21 regulating the proliferation-quiescence decision. Cell cycle regulation balances the requirement of proliferation during tissue growth and homeostasis, with the need to ensure that damaged DNA is not propagated to future generations. Checkpoints have evolved to achieve control of cell cycle progression in response to DNA damage. The importance of DNA damage checkpoints is highlighted by the fact that their dysregulation is the fundamental basis of oncogenesisin vivo is controversial111213The tumour suppressor and transcriptional regulator p53 is stabilized in response to DNA damage and regulates the expression of numerous target genes involved in the DNA damage response2658Live measurements of p53 protein levels have shown that unperturbed cells exhibit transient pulses of p53 expression1921Cdt2 and SCFSkp2, couple to remove p21 prior to the G1/S transition with markedly different rates. By combining single-cell measurements with mathematical modelling, we show that this p21 control system has the hallmarks of bistability, ensuring that the G1/S transition is irreversible. Our data therefore support a model in which p21 mediates a p53-dependent G1pm arrest in response to DNA damage occurring in S-phase.Using a live single-cell imaging approach, we show that DNA damage accrued during S-phase, in non-transformed cycling cells, results in heterogeneity in p21 levels that is p53-dependent. We find that p21 accumulates during mother G2- and daughter G1-phases, where it modulates the proliferation-quiescence decision in daughter cells via CDK2 inhibition. Strikingly, we find that p21 accumulation below a threshold does not influence G1 progression. At these lower levels of p21, two ubiquitin ligases, CRL4CDKN1A gene  and daughter cell G1 , as well as G1D length between sisters . Instead, we find G1D p21-GFP levels correlate better with combined G2M+G1D length (R=0.68**) than with G1D length alone (R=0.62**) suggesting that G2M length is not predictive of G1D because of noise in the timing of mitotic entry (Methods). As such, we find that the maximum p21-GFP level reached in G1D is most reflective of the time between S-phase exit in the mother cell and S-phase entry in daughter cells. Given that much weaker correlations are observed between phase lengths before and after S-phase (Methods), we suggest that stochastic events occurring in S-phase determine p21 levels in, and the length of, the combined G2M+G1D phase.The lack of p21 degradation during mitosis would allow daughter cells to inherit p21 protein from their mother. Indeed, there is a positive correlation between p21-GFP levels in the mother cell G2 . Following both daughter cells after mitosis, in 70% (45/64) of cases both daughters cycle, in 16% (10/64) both daughter cells enter a G1pm arrest (twin arrest), while in 14% (9/64), one daughter arrests and the other cycles (single arrest). If arrest occurred by chance following mitosis, we would expect fewer than 4.4% of mitoses to result in twin arrest . As such, conditions leading to arrest are frequently inherited by both daughter cells. Finally, where either single or twin arrest occurs in daughter cells, mean G2M p21-GFP levels are higher than those in G2M of cycling daughter cells  driving heterogeneity in p21 levels. Multiple transcription factors have been linked to p21 regulationWe further analysed the dependence of p21 expression on p53 by adding the inhibitor Nutlin-3 to stabilize p53 proteinCDKN1A gene using two different gRNAs, p21KO1 and p21KO2  of p21-depleted cells entered a G1pm arrest, compared to 17.2% (11/64) of control siRNA-treated cells , but we d p21KO2 . We obsed p21KO2 , in spitd p21KO2 . As obsed p21KO2 . As suchR=−0.67**). In addition, 13.7% (7/51) of the population enter a CDK2low state . We only observed one p21-depleted cell entering a CDK2low state (1/68), suggesting that the CDK2low G1pm arrest state is due to p21-dependent inhibition of CDK activity, consistent with previous workConsistent with p53 siRNA treatment in p21-GFP hTert-RPE1 cells, p53 depletion in CDK2 activity sensor expressing cells leads to a more homogenous G1 length and cells do not enter G1pm arrest 0/25; . HoweverM and G1D and, at sufficiently high p21 levels in G1, CDK2 activity is inhibited leading to a p21-dependent G1pm arrest. However, at low-intermediate levels, p21 is a poor CDK2 inhibitor and a G1 delay is not p21-dependent.These data further support the notion that p53 signalling induces p21 accumulation over G2Given our observation that p21 heterogeneity in unperturbed hTert-RPE1 cells is p53-mediated, reflective of the time between S/G2 in the mother and G1/S in the daughter, and that p21 starts to increase in G2, we postulated that p21 levels increase in response to endogenous DNA damage occurring during S-phase in the mother cell. To assess this, we immunostained unperturbed hTert-RPE1 cells with the DNA damage marker γH2AX and noted a striking correlation between p21-GFP expression in G1 and G2 cells and the presence of a single nuclear focus of γH2AX . We founR=0.39*; To explore the link between endogenous DNA damage and p21 expression, we generated hTert-RPE1 cells co-expressing p21-GFP and mRuby-53BP1. 53BP1 localizes to DNA Double Strand Breaks (DSBs) during G1 ref. . Live ceThese data suggest that DNA damage is inherited from the mother cell leading to 53BP1 localization to DSBs in daughter cells, that correlates with both a G1 delay and G1pm arrest. Despite p21 levels correlating with the presence of 53BP1 foci and a G1 delay, only G1pm arrest, but not G1 delay, is p21-dependent.Our data suggest that stochastic DNA damage occurring during normal DNA replication could generate the observed heterogeneity in p21 expression. To determine if p21 expression can be induced by impaired DNA replication during S-phase, we treated cells with low dose aphidicolin. Aphidicolin treatment caused an increase in the proportion of cells with a single γH2AX focus, in the number of γH2AX foci per nucleus, and in p53 expression . In timeTo further examine if DNA damage during S-phase can induce p21 accumulation in G2, we treated cells with an ATR kinase inhibitor  to induce destablization of stalled DNA replication forksTo determine if p21 and/or p53 is required for the cellular response to impaired DNA replication, we depleted p21 or p53 by siRNA and treated hTert-RPE1 cells expressing mRuby-PCNA and the CDK2 activity sensor with aphidicolin. p53-depleted cells treated with aphidicolin were unable to arrest in G1 (0/32) , G1 lengSkp2 and CRL4Cdt2 have been implicated in p21 degradation at the G1/S transition34353637Since p21-GFP is absent in all cells during S-phase, we hypothesized that, while high levels of p21 promote cell cycle arrest, its removal is required for efficient DNA replication. Both SCFR=0.76**), in Skp2-depleted cells G1 length was longer and showed weaker correlation with p21-GFP levels . Therefore, while G1pm arrest in the presence of high serum is characterized by high p21 expression, G1pm arrest after serum withdrawal is not.Given that passage through the RP depends on mitogen-signalling and thus serum concentration, we wanted to determine how p21-GFP levels change in hTert-RPE1 cells during G1pm arrest in response to serum starvation. As cells exit mitosis in the absence of serum, the majority  enter G1pm arrest . ImportaSince we did not observe a correlation between p21-GFP levels and G1pm arrest after serum starvation, we wanted to know if cells could arrest in G1 in response to serum withdrawal in the absence of p21 protein. We serum-starved WT, p21KO1 and p21KO2 hTert-RPE1 cells and measured their cell cycle profiles by FACS. We found that p21KO cells arrested in G1 as efficiently as WT cells in response to serum withdrawal . This inThus there exist two molecularly distinct G1pm arrest states—one that occurs in the presence of serum and is p21-dependent, and another that occurs following serum withdrawal that is p21 independent. Furthermore, the p53- and p21-dependent G1pm arrest we observe in cells grown in high serum is principally caused by endogenous DNA damage.M and continues into G1D, where, at sufficiently high levels, p21 inhibits CDK activity and promotes G1 arrest following mitosis.In this study, we sought to characterize and understand the role of p21 in regulating the proliferation-quiescence decision and genome stability in unperturbed cells. In response to DNA damage generated during S-phase, p21 accumulation begins in G2Skp2-mediated p21 degradation being turned off in G1 by increasing APC/CCdh1 activity46Skp2-inactivation and additional DNA damage occurring over mitosis.Our experiments demonstrate that in unperturbed cycling cells, heterogeneity in p21 levels is fully accounted for by p53. This is consistent with a recent report demonstrating that, in cells expressing both p53-YFP and p21-CFP, p21-CFP levels peak following peaks in p53-YFP ref. . The aut44et al.Spencer Skp2 and CRL4Cdt2. Thus we provide experimental evidence for, and extend on, previous work which concluded that the decision to remain quiescent or proliferate is bistable because of feedback regulation between p21 and CDK2 via SCFSkp2 +10% FBS (Sigma) and 1% Penicillin-Streptomycin  at 37 °C and 5% COPCNA gene was tagged at the N-terminus with a cDNA encoding the mRuby fluorophore using AAV-directed homologous recombination. To generate the targeting construct, Left and Right Homology Arms  flanking mRuby cDNA were amplified by PCR from hTert-RPE1 genomic DNA using the following primers: P1 NotI: 5′–cggccgcgaatcttcagttccctcaacaa-3′ and P2: 5′-ctttgatcaggctgtttgccatggtggcggagtggcaacaac-3′ to amplify the LHA including the first 20 bp of mRuby; P3: 5′-gttgttgccactccgccaccatggcaaacagcctgatcaaag-3′ including the first 20 bp of PCNA LHA; and P4 XbaI: 5′-ctcgaatctagatcctcctccccctccgcccaggccggc-3′ to amplify mRuby and create a Gly-Gly-Gly-Ser-Arg linker in front of the first PCNA exon. P5 XbaI: 5′-tctagattcgaggcgcgcctggtc-3′ and P6 NotI: 5′-gcggccgcacggaggaggatcacttgagg-3′ to generate the RHA.One allele of the endogenous LHA and mRuby fragments were fused by PCR using P1 and P4 and cloned by three-way ligation, together with the RHA into the NotI sites of pAAV-MCS (Stratagene). The complete targeting construct was packed into pAAV viruses using an AAV helper-free system (Stratagene), according to the manufacturer's instructions, followed by transduction of hTert-RPE1 cells. Four days after transduction, single mRuby positive integrands were sorted by flow cytometry into 96-well plates and analysed for correct targeting of PCNA by Western blotting.CDKN1A gene was tagged at the C-terminus using CRISPR-mediated gene tagging. The gRNA 5′-TTTGAGGCCCTCGCGCTTCCAGG-3′ (PAM site in bold) downstream of the CDKN1A stop codon was selected using crispr.mit.edu. Forward and reverse oligos for the gRNA were annealed and ligated into the BbsI cut pX330 bicistronic Cas9 and sgRNA expression plasmidThe endogenous CDKN1A homology arms from hTert-RPE1 genomic DNA using the high-fidelity Q5 DNA polymerase (NEB). To PCR amplify the LHA we used the forward primer with a NotI site: 5′-gcggccgccaggccctttgccaagct-3′ and the reverse primer with a SalI site: 5′-gtcgacgggcttcctcttggagaa-3′. To PCR amplify the RHA we used the forward primer with a SpeI site 5′- actagttccgcccacaggaagcctgcagttctgg-3′ (underlined base represents mutated base to change the PAM site in the recombined gene) and the reverse primer with a NotI site: 5′-gcggccgcatacccctaacacagagataaccccact-3′. GFP cDNA was PCR amplified from peGFP-C1 vector (Clontech) with the forward primer with a SalI site and a poly-glycine linker: 5′-gtcgacggaggaggagtgagcaagggcgaggag-3′ and the reverse primer with a SpeI site: 5′-actagtttacttgtacagctcgtc-3′. 2LHA, RHA and eGFP PCR products were ligated into the blunt-end cloning vector pJet1.2 (Fermentas) for restriction digest. LHA, RHA and eGFP digested inserts were ligated into NotI cut pAAV-MCS vector by 4-way ligation at a ratio of vector:inserts of 1:2:2:2, using T4 DNA ligase (NEB). All constructs were checked by sequencing before transfection into cells.For the homology donor plasmid, we PCR amplified To generate endogenously tagged p21-GFP cells, the pX330 p21 gRNA plasmid and the p21 homology donor plasmid were transfected into hTert-RPE1 cells at a ratio of 1:1 using Lipofectamine 2000, according to the manufacturer's instructions (Invitrogen). Cells were allowed to expand for 7 days before FACS sorting to select GFP positive cells. Individual GFP expressing cells were sorted into 96-well plates containing 50:50 conditioned:fresh growth media. Clones were left to expand for 10–14 days and then visualized on the Opera high-throughput microscope (PerkinElmer) to determine which clones had GFP expression in the nucleus. Candidate clones were expanded and further validated, as described in Samples were directly lysed by addition of Laemmli buffer. Whole cell lysates were loaded onto 4–20% Tris-Glycine Novex precast gels (ThermoFisher) followed by transfer to PVDF membranes. After protein transfer, membranes were briefly washed in MeOH and then dried between sheets of filter paper. When completely dry, primary antibody diluted in 5% milk in TBS was added and membranes were incubated overnight at 4 °C with rocking. Membranes were washed three times in TBS+0.05%TritonX-100 (TBS/T) and anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (CST) were diluted 1:2,000 in 5% milk in TBS and incubated with membranes for 1 h at RT, with rocking. Membranes were washed three times in TBS/T and visualized using Pierce ECL Western Blotting Substrate (ThermoScientific). All blots were scanned on the Azure c300 imaging system.Antibodies used in this study for Western blotting are p21 , Skp2 , Cdt2 , p53  and GAPDH .To determine if p21-GFP could bind to CDK2, we performed immunoprecipitation of GFP-tagged p21 using the μMACS GFP Isolation kit (Miltenyi). Immunoprecipitations were performed simultaneously from asynchronous mRuby-PCNA hTert-RPE1 cells and p21-GFP mRuby-PCNA hTert-RPE1 cells, according to the manufacturer's instructions. mRuby-PCNA hTert-RPE1 cells served as a negative control since they do not express GFP. Eluted samples were separated by SDS-PAGE, transferred to PVDF and probed with anti-p21 and anti-CDK2 antibodies.Cells were plated at a density of 50,000 cells per well in 6-well plates. Duplicate cell counts were taken every 24 h using the Countess cell counter (Invitrogen).D10A GFP nickase vectorWe generated two CDKN1A nickase targeting vectors, based on the methods described in ref. For genotyping of p21KO lines, genomic DNA was extracted using the Flexigene DNA kit, according to the manufacturer's instructions. The p21 gene was amplified using nested PCR and primers: CDKN1A_KO_FWD1 5′-ATGTCAGAACCGGCT-3′ and CDKN1A_KO_REV1 5′-TTAGGGCTTCCTCTT-3′, followed by CDKN1A_KO_FWD2 5′-GGATGTCCGTCAGAACCCAT and CDKN1A_KO_REV2 5′-GTGGGAAGGTAGAGCTTGGG-3′. The resultant PCR product was ligated into the pJet1.2/blunt Cloning Vector (Fermentas). Plasmid DNA was purified from individual colonies using the Qiagen Miniprep kit and sent for sequencing using the pJet_forward primer. The results of the genotyping are shown in 2 and 80% humidity. Cells were plated on 384 well CellCarrier (PerkinElmer) plates at a density of 1,000 cells/well 24 h prior to imaging. Cells were imaged using a 20X (N.A. 0.45) objective at 10 min intervals for 24–72 h in Phenol-Red Free DMEM+10% FBS and 1% P/S. Image processing was performed in Matlab using custom-made scripts.Live cell imaging was performed on the Opera HC spinning disk confocal microscope (PerkinElmer), with atmospheric control to maintain cells at 37 °C, 5% COP=0.938 and 20% versus 40% P=0.243; and G1 length with no correction: 20% versus 30% P=0.916 and 20% versus 40% P=0.607.Since laser irradiation can induce DNA damage, and thus p21 expression, we wanted to be sure that our imaging conditions were not inducing p21 expression. Therefore, we imaged hTert-RPE1 mRuby-PCNA p21-GFP expressing cells using two 488 nm laser exposures where the laser power was greater than the 20% used in our other experiments; exposure time remained constant (120 ms). The results are shown in Inhibitors used were ATRi , Aphidicolin (Sigma), Camptothecin (Sigma), Etoposide (Sigma) and Nutlin-3 (Sigma). All drugs were reconstituted in DMSO and stored in aliquots at −20 °C.4 cells ml−1 was added, and cells were incubated at 37 °C. siRNAs used in this study were OnTARGETplus pools (Dharmacon).For live cell imaging, cells were either transfected 24 h prior to imaging, or, in cases where we wanted to capture the first cell cycle after protein loss, 6 h prior to imaging. Cells were transfected with 20 nM final concentration of siRNA using Lipofectamine RNAiMax, according to the manufacturer's protocol (Invitrogen). Briefly, per well of a 384 well plate, 40 nl of Lipofectamine RNAiMAX was mixed with siRNA in 10 μl OPTIMEM. To this, 20 μl cells in media, at a density of 5 × 10GFP-53BP1 plasmid was a generous gift from Jiri Lukas−1 G418 (Sigma), using the BD FACS Aria. After 10–14 days selection, single-cell clones were visualized for appropriate expression of 53BP1 and those clones were expanded.hTert-RPE1 cells were plated in six well plates to ensure a density of 80% confluency on the day of transfection. Cells were transfected with 4 g of plasmid using Lipofectamine 2000, according to the manufacturer's instructions (Invitrogen). After 24 h incubation, cells were transferred to a T-25, and after a further 24 h, cells were sorted to single-cell density in 96-well plates containing 50% fresh media:50% conditioned media with 0.25 mg ml−1 Hoescht 33258 (Sigma) diluted in PBS for 15 min at RT.Cells were fixed in an equal volume of media to warm 8% formaldehyde in PBS for 5 min at 37 °C. Cells were permeabilized in PBS/0.2% TritonX-100 for 5 min at RT and then blocked in 2% BSA in PBS (Blocking buffer) for 1 h at RT. Cells were incubated in primary antibody diluted in Blocking buffer for either 2 h at RT or overnight at 4 °C. Cells were washed three times in PBS and then incubated with Alexa-conjugated secondary antibodies (ThermoFisher) diluted in Blocking Buffer for 1 h at RT. Cells were washed three times in PBS and incubated in 1 μg mlCells were imaged on the Opera HC spinning disk confocal microscope (PerkinElmer). All imaging data was uploaded to the Columbus image analysis database (PerkinElmer) for visualization and analysis.For RPA2 immunostaining, cells were plated onto glass coverslips. Cells were briefly washed in PBS and then pre-extracted using ice-cold PBS/0.2% TritonX-100 for 1 min. Cells were then fixed in warm 4% formaldehyde and processed as usual. Cells on coverslips were imaged on a Zeiss LSM710 confocal microscope using a 60x oil, N.A. 1.4 objective.Primary antibodies used for immunostaining in this study are: p21 , p53 , 53BP1 , γH2AX , P-S1981-ATM , RPA2 .All quantitative image analysis on fixed cells was performed using Columbus software (PerkinElmer). Individual protocols are described below.Measuring nuclear intensities of proteins. Nuclei were segmented based on Hoescht intensity. Nuclei at the edge of the image and nuclei<100 μm2 were excluded. Mitotic nuclei were also excluded by excluding nuclei with Hoescht intensity above a defined threshold . The fluorescence intensity of individual proteins was then calculated in each interphase nucleus.Measuring cytoplasmic intensities of proteins. Nuclei were segmented as above. To measure cytoplasmic intensities of proteins, we defined a cytoplasmic nuclear ring region of four pixels beyond the nucleus. This was to exclude any effect cell shape or area may have on cytoplasmic intensity readings. The cytoplasmic fluorescence intensity of individual proteins was then calculated in each ring region.Counting nuclear foci. Nuclei were defined as above. To define and count γH2AX foci within nuclei, we needed to distinguish between the bright, individual foci observed in G1 and G2 cells, from the dim, multiple γH2AX foci observed in most S-phase cells. We segmented foci using the ‘Find Spots' function in Columbus. Specifically, we used Method A and defined the Relative Spot Intensity as 0.290 and the Splitting Coefficient as 1. We validated our automated focus finding against manual scoring of foci.Excluding S-phase cells. When scoring p21-GFP levels and foci in cells, we wanted to exclude S-phase cells from the analysis since p21 levels are negligible in all S-phase cells. To exclude S-phase cells, we used a Linear Classifier in Columbus. Inputs to the Linear Classifier were a combination of Nuclear Morphology , Hoescht Texture Properties (SER Features), PCNA Intensity Properties, and PCNA Texture Properties (SER Features).Live imaging data was analysed using custom image analysis scripts  is negated from the original image.Images from channels with a nuclear probe are combined to maximize the quality of nuclear segmentation, this principally involves channels containing the p21-GFP and mRuby-PCNA signal.Marker Controlled Watershed Segmentation.Nuclei are subsequently segmented from the filtered, combined, image using Segments with intensity or size below a threshold are filtered out, as these correspond to mis-segmented background noise.Segmented images are saved as equally sized zero matrices with each nuclei region uniquely labelled.Nuclear segmentation:Cell area: area in pixels of the segmented nucleiMajor axis length: length of the ellipse that is equivalent based on second order moments to the segment53Minor axis length: width of the ellipse that is equivalent based on second order moments to the segment53Eccentricity: eccentricity of the ellipse that is equivalent based on second order moments to the segment53Equivalent diameter: diameter of the circle with the same area as the segmentSolidity: percentage of pixels in the convex hull surrounding the segment which are also in the segmentPerimeter: perimeter length of the segment2 (ref. Roundness: defined as 4π × area/perimeter2 ref. )PCNA mean intensitys.d. of PCNA intensityKurtosis of PCNA intensityFoci strength: PCNA intensities below the mean are removed, the s.d. of pixels above the mean is then calculated. This identifies bright spots, and ignores dark spots in the nucleus, for example, nucleoli.p21 mean intensityp21 s.d.K-mean clustering (K = 2) is performed on nuclei segments, and the mean silhouette score of the two clusters calculated. This feature identifies dumbbell shapes and is indicative of double nuclei segments.These features are defined in the single-cell track matrices presented online. In these matrices the first column gives cell index, the second time point and third migration distance. Subsequent columns are features as defined above.For CDK2 activity data, p21 intensities are replaced with CDK2L-GFP nuclear intensity, CDK2L-GFP ring region intensity, and the ratio of these two values. The ring region, here defined as a seven pixel wide ring surrounding the nucleus, has been shown to perform effectively in measuring nuclear translocationMean silhouette score: Feature extraction: features describing morphology and fluorescent marker levels and localization for each channel are obtained for each segmented object in each frame. Importantly, here we extract intensity and texture features from unfiltered images. The reasoning behind this is being that across the experiments we perform, average intensity levels can vary over several orders of magnitude for example, under Nutlin-3 treatment; plate-wise or well-wise filtering here can mask true levels of p21 relative to controls. Specifically the features we extract are:Segment probability: segments can be one of five defined objects, either correctly segmented  or incorrectly segmented . Probabilistic tracking maximizes the likelihood that a track corresponds to the movements and divisions of a single nucleus. Thus for tracking, probabilities are assigned to each of the five possibilities mentioned, for each segment, in each frame such that tracking can maximize the likelihood a single nucleus is followed. This is performed by manually selecting examples of each of the defined objects, for example, mitotic nuclei, or multiple nuclei, from all segmented objects within a graphical user interface (GUI). Subsequently, a support vector machine classifier is trained on these manually selected examples. The classifier is used to assign a probability to every segment, in every frame, of it being one of these objects. As such, every segmented object, in every frame, is assigned five posterior probabilities corresponding to the likelihood of it being: one nucleus; mitotic nucleus; nucleus exiting mitosis; no nuclei; more than one nuclei.Probabilistic tracking: The goal of automated cell tracking is to generate a set of tracks that correspond to individual nuclei as they move across the field of view (FOV), and to capture cell division events, such that the lineage of nuclei is recorded. Briefly, in the automated probabilistic tracking algorithm usedManual correction: automated tracking performs the majority of work in tracking the hTERT-RPE1 cells, however these cells are highly motile, and some of the conditions we use increase motility even further. As a result, the error rate in tracking ranges from 1 mistake, per cell, per 300 frames; to 1 mistake, per cell, per 20 frames. In the cell cycle analysis we are performing it is essential we obtain near 100% accuracy. As such, we developed a GUI to efficiently amend errors in automated tracking, as well as compare the feature values of tracked cells against the original confocal microscopy images. This allows us to achieve nearly 100% accuracy in tracking, and more importantly if we identify outliers, or unexpected results in later analysis we can return to the videos and visually inspect whether the result is genuine or the product of a tracking error.For automated tracking of hTert-RPE1 cells expressing fluorescent markers in higher throughput we developed a cell-tracking pipeline in the commercial scripting language Matlab. The pipeline tracks cells and extracts data from sequences of uncompressed TIF images, captured from live cell imaging. Segmentation is based on well-established methods, while tracking is an implementation of a probabilistic tracking algorithm defined in refs We define G1/S transition as the point at which both mRuby-PCNA intensity begins to increase and we see the appearance of textured PCNA in the nucleus. This is characterized by a small jump in the PCNA foci strength feature . As furtWe define the S/G2 transition as the point at which mRuby-PCNA foci are at their maximum strength ; after tMitosis was defined by gross morphology changes, principally this is seen as a spike in cell area lasting for one or two frames, corresponding to nuclear envelope breakdown, followed by the nuclear area becoming significantly smaller . All tran=47/219) that observed for sampling both daughters, where only mitosis in which both daughters are subsequently tracked for greater than 60 frames are used (23% n=29/128). The single lineage-sampling regime is used outside of calculations comparing daughters, as here n is greater. In all conditions at least two technical replicates were analysed, and tracks from each replicate were visually compared to ensure dynamics were consistent, prior to pooling of the data.To determine correlations, between p21 levels and phase length, for both control data and depletion experiments, a single-cell lineage was used. This was selected based on the daughter cell captured within the FOV for the longest period of time. The likelihood of arrest observed using this sampling regime very closely matched . No adjustment for this decrease was made, such that data remains faithful to the original imaging data sets, though baseline levels for arrest thresholds are defined at 15 a.u.For all p21-GFP traces except p21-GFP values, used for population mean, s.d. and correlation measures in the main text are derived from the maximum p21-GFP value reached within a single cell in the cell cycle phase defined. The maximum p21-GFP value was used rather than the mean p21 value, as this generally gave stronger correlations to cell cycle phase length, though in all cases analysed for both mean and maximum the mean value also provided a statistically significant correlation.P=1.8677e-04; G1 length, P=0.0012). Following log transformation, the sample fell under the normal distribution . Therefore, we calculated the Pearson correlations on the log-transformed data as well, since Pearson's correlation does not fully describe the association of data which does not fall under a bivariate normal distribution. While correlation values did change, at no point did a previously significant result become insignificant. As such, for simplicity and to match that data presented in figures, correlations determined on the untransformed data are given in the main text.While both maximum G1 p21 levels and G1 length were not normally distributed as determined by a one sample Kolmogorov–Smirnov test  with phase lengths following S-phase are also quoted. Results following log transformation of the max p21 levels versus length data are also given.M) and daughter G1 (G1D), ii) p21-GFP levels in G2M and G2M length, and iii) p21-GFP levels in G1D and G1D length; we were initially surprised to find no correlation between G2M length and G1D length. However, in this comparison noise in the timing of mitosis has the dual effect of increasing G2M length and decreasing G1D length, and vice versa. Thus, if noise in mitotic timing is indeed masking the relationship between cell cycle phase length and p21 levels, we reasoned that the combined length of G2M and G1D would correlate better with the maximum p21-level reached than either length alone. We found this to be the case with R=0.68** for maximum p21 level in G2M/G1D against length of G2M/G1D, versus against length of G1D (R=0.62**) or G2M (R=0.52**) alone. This result was calculated from the merged experimental repeats (n=4). Within each repeat this result held as well (see Given that we observe strong correlation between i) p21-GFP levels in the mother G2 (G2well see .μ=0.25 a.u. per min) at the end of S-phase arrested in G2, while those displaying a slower rate of p21-GFP accumulation (μ=0.1 a.u. per min) underwent mitosis and arrested in the following G1 (R=0.46**). Thus G2 arrest appears to be dependent on the rate of p21 accumulation following S/G2 transition. Finally, we note that those cells that were earlier in S-phase at the time of Nutlin-3 addition, are more likely to arrest compared to those exiting S-phase sooner, this is evident from correlation observed between S/G2 transition time and whether a cell undergoes G2 arrest or not (R=0.34*). We suggest that this is due to accumulated p53 stabilization over S-phase. This means that the longer a cell is in S-phase in the presence of Nutlin-3, more p53 is stabilized and more p21 mRNA accumulates. Thus when a cell exits S-phase and p21 degradation is switched off, p21 protein accumulates faster, increasing the likelihood of G2 arrest.In cells exiting S-phase following Nutlin-3 treatment we observed a G2 arrest that was dependent on the rate of p21 accumulation, meaning, those cells with the fastest rate of p21-GFP accumulation , cyclins (mRNACy), and p53 (mRNAp53).Protein transcription and translation: Part of the noise in cell cycle progression stems from the low copy numbers of mRNAs that encode for regulatory proteinsp21), cyclins (Cy) and p53 (p53) are produced from their mRNAs according to the following equations.where mRNAs are synthesized and degraded with the constitutive rates Cyt corresponds directly to the total level of CDK2:Cyclin complexes with Cy denoting the level of active CDK2:Cyclin. Protein degradation rates are as follows.with Skp2 denotes the relative level of the SCFSkp2 ubiquitin ligase. Similarly, p21 degradation occurs with rate Cdt2 (Cdt2), which is recruited to chromatin-bound PCNA24RCa). For p53, stabilization has been shown to occur in response to DNA damageDam) and an inhibition constant jp53.The constitutive rates of cyclin and p21 degradation are kp2 refs , 59, butCDK2 inhibition. At high levels, p21 can act as an efficient inhibitor of CDK2 (ref. Cy) into an inactive one (CyP21).DK2 ref. . Hence, where RC) converting them into replication complexes that are primed for PCNA loading (RCp).S-phase entry and DNA synthesis: Licensing of replication origins occurs during late mitosis or early G1-phase resulting in the formation of pre-replication complexesn at a CDK2:Cyclin threshold of jCy. Primed replication complexes can revert back to an unprimed state with a small rate PCNAa) or PCNA with a p21 molecule bound to its PIP-box (PCNAi) forming either active (RCa) or inactive (RCi) replication complexes, respectively.Here, priming occurs by CDK2 phosphorylation with rate RCp) creates inaRCs65DNA) and once DNA replication is finished, replication complexes disassemble with rate where H the Heaviside function.Here, PCNAa) is replenished from a cytoplasmic pool and that p21 can bind to PCNA creating PCNA:p21 complexes (PCNAi).PCNA dynamics: We assumed that free PCNA in the nucleus  can either occur randomly, independent of the cell cycle stage, or specifically during DNA replication.Here, Computation. A deterministic version of the model was prepared using the Systems Biology Toolbox 2 .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, and Supplementary NoteUnperturbed, asynchronous hTert-RPE1 mRubyPCNA p21-GFP expressing cells. mRuby-PCNA is in red and p21-GFP is in green. p21-GFP levels vary both between individual cells and within individual cells as they cycle.hTert-RPE1 mRuby-PCNA p21-GFP expressing cells treated with DMSO (Movie 2) or 5 ìM Nutlin-3 (Movie 3). mRubyPCNA is in red and p21-GFP is in green. Drugs are added at time zero. DMSO-treated cells continue to cycle and display periodic expression of p21-GFP. In cells treated with Nutlin-3, p21-GFP expression starts to increase immediately in G1 and G2 cells. In S-phase cells, cells complete Sphase and only begin to express p21-GFP after S-phase exit.hTert-RPE1 mRuby-PCNA p21-GFP expressing cells treated with DMSO (Movie 2) or 5 ìM Nutlin-3 (Movie 3). mRubyPCNA is in red and p21-GFP is in green. Drugs are added at time zero. DMSO-treated cells continue to cycle and display periodic expression of p21-GFP. In cells treated with Nutlin-3, p21-GFP expression starts to increase immediately in G1 and G2 cells. In S-phase cells, cells complete Sphase and only begin to express p21-GFP after S-phase exit.Unperturbed hTert-RPE1 p21-GFP mRuby-53BP1 expressing cells. mRuby-53BP1 is in red and p21-GFP is in green. A single G2 cell is shown at the start of the movie that expresses a low level of p21- GFP. The cell divides, giving rise to two daughter cells that display different characteristics. The left hand daughter cell displays 53BP1 foci shortly after cell division and goes on to express a high level of p21-GFP. Stills of this cell are shown in Supplementary Fig. 4d for clarity.hTert-RPE1 mRuby-PCNA p21-GFP expressing cells treated with Cdt2 siRNA. mRuby-PCNA is in red and p21-GFP is in green. At the start of the movie, the cell second from the top on the extreme right of the frame is in G1. At 4h20, this cell enters S-phase and subsequently undergoes alternating cycles of DNA replication (indicated by the presence of red mRuby-PCNA foci) and p21-GFP expression. Stills of this cell are shown in Supplementary Fig. 6d for clarity.Custom Matlab scripts for cell segmentation and tracking, cell cycle phase identification and fluorescence quantification over time.Code for deterministic and stochastic models of the p21 control network. "Barr2017_DynamicsOfP21_SBtoolbox": Deterministic version of the mathematical model for the Systems Biology Toolbox 2 for MatLab. "Barr2017_DynamicsOfP21_XPP": Deterministic version of the mathematical model for XPP-Aut. "Barr2017_DynamicsOfP21_SBML": Deterministic version of the mathematical model in the Systems Biology Markup Language level 3 version 1. "Barr2017_DynamicsOfP21_Copasi_Deterministic": Deterministic version of the mathematical model for Copasi. "Barr2017_DynamicsOfP21_Copasi_Stochastic": Stochastic version of the mathematical model for Copasi.


There are abundant examples of natural, engineering and industrial applications, in which “solute transport” and “mixing” in porous media occur under multiphase flow conditions. Current state-of-the-art understanding and modelling of such processes are established based on flawed and non-representative models. Moreover, there is no direct experimental result to show the true hydrodynamics of transport and mixing under multiphase flow conditions while the saturation topology is being kept constant for a number of flow rates. With the use of a custom-made microscope, and under well-controlled flow boundary conditions, we visualized directly the transport of a tracer in a Reservoir-on-Chip (RoC) micromodel filled with two immiscible fluids. This study provides novel insights into the saturation-dependency of transport and mixing in porous media. To our knowledge, this is the first reported pore-scale experiment in which the saturation topology, relative permeability, and tortuosity were kept constant and transport was studied under different dynamic conditions in a wide range of saturation. The critical role of two-phase hydrodynamic properties on non-Fickian transport and saturation-dependency of dispersion are discussed, which highlight the major flaws in parametrization of existing models. It is clear that in many of these applications more than one fluid phase is present in porous media. This means that in these cases the theories and parameterization of transport under single - fluid phase flow are not valid and applicable. Under single-f-luid phase conditions, hydro-dynamic dispersion (D) is empirically proposed to be a power law function of average pore-scale velocity (u), defined as D = Dm + un 8. In this relation, Dm is the molecular diffusion coefficient, and α is the diffusivity coefficient. Obviously α and n are both empirically determined, where the latter one is usually proposed to be equal to unity. This relation has been simply generalized for transport under two fluid phase flow by assuming the saturation-dependency of the proposed parameters D(S) = Dm + (S)un(S) 9. Obviously this formulation does not provide any new insights into the fundamental two-fluid phase transport processes, and can even lead to an over-estimation of dispersion coefficient for a given saturation13.Transport in porous materials is important for many industrial applications and natural processes such as transport of chemicals in enhanced oil recovery, fertilizers in agricultural soils, salt water intrusion into fresh water coastal aquifers, groundwater contaminations, and deliverance of pharmaceutical products to live tissues20. However, lessons learnt from these studies are based on indirect estimations of hydro-dynamic transport properties and fitting “empirically-proposed models” to experimental data.Despite the fundamental importance and serious applied implications of understanding two fluid phase transport in porous media, it has received far less attention compared to the single - fluid phase transport. From a macro-scale perspective, several researchers investigated transport phenomena under unsaturated (two fluid phase) conditions21, high-resolution real-time  imaging has lately allowed us to quantify directly the pore-scale mixing and transport in porous media24 even under two fluid phase conditions26. From the hydro-dynamic point of view, the existence of two fluid phases in a porous medium will create some hydrodynamically stagnant zones in each fluid with very weak (close to zero) velocity fields, which will make the local transport regime to be diffusive. This is in contrast to the remaining area occupied by the same fluid where transport is advective-dispersive. Based on this dual characteristic of the pore network, some mechanistic models such as dead-end pore model27, and the mobile-immobile model (MIM)28 have been developed. These two models employ similar assumptions as they account for mobile and immobile regions at a given saturation, and a diffusive mass transfer between these regions29.In contrary to the column-scale experimentsKaradimitriou et al.25 who directly quantified the stagnant zones and showed that the diffusive mass exchange rate between the mobile and the immobile region in the MIM model is time and flow-rate dependent. Also, although the MIM model could fit the experimental data very well, there was no consistency between the fitted parameters and parameters estimated directly from the experiments. This study is a follow-up study of Karadimitriou et al.25 in which for the first time using direct image analysis, the impact of flow rate on transport properties under the same saturation topology is studied.Major drawbacks in MIM have been pointed out by ref. Karadimitriou et al.25, we conducted 3 sets of dynamic two-phase flow experiments to establish a saturation topology in an elongated micro-model, which comprised 4 independent representative elementary volumes (REVs). While we kept the saturation topology undisturbed, we managed to perform injection of water-based ink in a two-phase (water-Floruinert) micro-model at 4 different rates (each injection rate producing a different Peclet number). In this way, we produced 48 REV-based data sets.The objective here is to employ a direct experimental method (without inverse modelling) to estimate (a) the impact of the Péclet number on the stagnant saturation, (b) examine the relation between the stagnant saturation and total saturation, (c) to provide physically-based explanation for the huge data scattering in stagnant saturation reported the literature. In this paper we aim to present direct estimation of stagnant saturation versus total saturation to investigate whether under well-defined conditions, a meaningful relation (which is missing in the literature) between them exists. It is important to note that we have established a novel experimental procedure such that for a given saturation without any change in saturation topology, we have managed to examine the transport properties at different ink injection rates (reproducing different Péclet numbers). Then, we directly identified the dispersion coefficients of the flowing saturation versus the total saturation and its relation with saturation. To our knowledge, this unique finding provides the first direct assessment of transport under two fluid phase flow conditions. As the continuation of the work of In this paper, after a short review of the experimental methods and data analysis methodology, novel results focused on hydro-dynamic transport under two fluid phase conditions based on direct analysis are presented and discussed.Karadimitriou et al.25. We used a similar Poly-Di-Methyl-Siloxane (PDMS) micro-model, Fluorinert FC-43 as the wetting phase, water as the non-wetting phase, and water-based ink as the dispersed phase, respectively. The micro-model was 4 REV long and each REV was visualized and recorded by a dedicated mono-chrome 5 Megapixel camera synchronically. For more information on the micro-model and the visualization setup, please refer to the ref. The experimental setup, image processing and data analysis used in this work are almost similar to the ones used in the work of The micro-model was initially fully saturated with Fluorinert. Then, water was introduced into the flow network at a given flow rate (0.2 to 0.5 ml/h) to establish a steady-state saturation. After breakthrough of water in the micromodels, the saturation and its topology would not change unless a very high rate was imposed. To avoid any internal remobilization after reaching the steady-state saturation pattern with non-dyed water, dyed-water was introduced at a low flow rate of 0.1 ml/h. Meanwhile we visualized the saturation field to make sure it did not change. The water-based ink and fluorinert are immiscible and the water-based ink got dispersed only in the area occupied by water. Meanwhile, the transport of the ink into the flow network was visualized and recorded. Then, flow was inversed and water was reintroduced into the flow network at the same flow rate until all the ink had been washed away. This process of introduction and removal of ink was carefully performed such that the saturation topology does not change. This allowed us to maintain exact fluids topology to perform another ink injection experiment at a higher rate. The process was repeated for a number of flow rates for the introduction and removal of ink, namely 0.2, 0.6, and 1 ml/h. In total, 3 two-phase flow experiments were performed (resulting in 12 different saturations per REV) and ink injection and removal were performed at 4 different rates.The images acquired from the four individual cameras, each focusing on a different REV, were used to extract information on phase saturation, resident concertation, and the identification of flowing and stagnant regions.saturation mask” of the steady-state distribution of the fluid phases in the network was prepared. In this way it was possible to isolate the area occupied exclusively from water, as this was the area where ink would be dispersed. By assigning different grayscale intensity values to the pixels belonging to the water-filled area, and Fluorinert-filled area, these two phases were distinguished in the saturation mask.In order to identify saturation of each phase in the flow network, a “Karadimitriou et al.25. A nonlinear calibration between grayscale intensity and local concentration was used to convert the grayscale intensity in the images to real concentration values. All images were processed in order to have a uniform illumination, and they were cross-correlated to the saturation mask. Materials  and the optical setup were identical to the ones employed in Karadimitriou et al.25, where more detailed information on the methodology can be found.Resident concentration was estimated in the same way as reported in C′(t)) would take its maximum value, referred to as tmax. Based on the 1-D Ogata-Bank analytical solution for concentration versus time, 2tmax is the characteristic time when the Fickian transport in the flowing part of the network has saturated the flowing network. Due to the very small characteristic transport time in the flowing part of the network, compared to the very large characteristic transport time in the stagnant zone, the mass transfer from the flowing part of the network to the stagnant one is negligible for this ink injection rate. Then, we refer to the image taken at time equal to 2tmax (with respect to the ink entrance time). In this image, those pixels carrying concentration belong to the flowing part of the network, and those pixels which do not carry ink belong to the stagnant zone.To identify the flowing and stagnant regions in the water-filled part of the flow network, for the fastest ink injection rate (1 ml/h) we identified the time when the temporal gradient of concentration averaged over the REV  with two distinct characteristic transport time scales. Therefore, the extracted data showed the non-Fickian transport in longer time scales controlled by diffusion in the stagnant regions. The early-time data were fitted by the one-dimensional advection-dispersion analytical solution0. Thus, for fitting of the results C0 will be the maximum average resident concentration measured under a given condition. Obviously, in a fully-saturated medium the maximum resident concentration should correspond to the concentration at the boundary of the pore structure. This is a straight-forward consequence of the fact that the pore network is fully percolating by design.Note that the average resident concentration in the flow network under two fluid phase conditions does not reach the inlet concentration CThe data supporting the conclusions can be obtained in the tables provided in the supplementary document.2 indicates the 2-norm of a vector. Xi, In Table mentclass2pt{minimOne can see a clear non-monotonic trend between dispersion coefficient and saturation at a flow rate of 0.1 ml//h, which maximizes at a saturation of 0.6. At higher flow rates this non-monotonicity diminishes.et al. flow rates the saturation and tortuosity cases are identical, the impact of the dynamics of two-phase flow on the variation of the dispersion coefficient is not relevant. However it is visible that at small saturation values (0.27 and 0.34) the range of variation of the dispersion coefficient is much more pronounced compared to the fully-saturated case. This suggests that there is no unique Péclet number-dispersion coefficient relation for a given porous medium, and this relation is saturation dependent.For the lowest injection rate, the advective transport in the flowing part of the water-filled part of the network is comparable to the diffusive flow between the flowing and the stagnant part of it, and the variation of the dispersion coefficient with saturation is small. As the pore velocities increase for increasing ink injection rates, dispersion (rather than diffusion) gradually becomes the dominant transport mechanism. This can be interpreted as the reason for which the variation of the dispersion coefficient increases with respect to saturation with increase of ink injection rate (i.e. Péclet number defined as the ratio of advective to diffusive flux), as shown in Fig. In order to explore the saturation-dependency of dispersion coefficient versus pore-scale velocity relation, we plotted the results obtained by fitting the Equation  to the em + (S)un(S), is questionable. Out fitting results shown in Figure α and n terms are only functions of saturation is not well supported. There is an erratic relation between the parameters and saturation that indicates saturation alone is not definitely enough to relation saturation and dispersion coefficient and backbone saturation tortuosity may need to be included.The applicability of the generalized power law relation between pore scale velocity and dispersion relation, D(S) = DAs discussed earlier the stagnant regions created by the two fluid phase configuration control the non-monotonicity in dispersion coefficient-saturation relation and the saturation-dependency of the dispersion coefficient-pore velocity relation. Thus, we need to directly estimate the stagnant saturation created at different water saturation states. Following the methodology explained earlier, we estimated the stagnant saturation in the two fastest flow rates as shown in Fig. 13. Thus, from a physical point-of-view the obtained results from the existing models could be potentially not representative and even incorrect25. Due to the significant discrepancy of literature data, no conclusion could be drawn about this trend.Figure Again, we notice that at saturation close to 0.35 the higher stagnant fraction is resulted. The saturation at which stagnant saturation fraction maximizes corresponds to the maximum dispersion coefficient obtained in Fig. 33, the resident water(referred to as formation brine) has a very different composition compared to the chemically- designed injected water. Thus, the mixing between the formation brine as the injected water, which is a slow process, will deteriorate the quality of the injected water and the oil recovery performance. Based on the findings of this paper, stagnant saturation is highly variable with saturation which means the mixing time-scale between stagnant and flowing water will be saturation dependent and oil recovery performance will alter by saturation.This adds a new dimension to the complexity of the "low salinity water flooding" process. On top of the previously established concepts related to to the impact of the petrophysical properties at large scale and the critical role of surface force interactions of crude oil-brine-rock at sub micron scale, the stagnant regions will play also an important role in the performance of the low salinity water flooding at an intermediate scale.The results of this study have serious implications for industrial applications among which enhanced oil recovery is a major one. In various enhanced oil recovery methods, such as surfactant flooding or low salinity water flooding, transport of chemicals through a two- or three-phase system is the central process. However, modelling of transport in this multiphase system is still in its infancy. Our results show that in order to improve the predictive modelling capabilities, a new theoretical modeling framework is needed to integrate the non-monotonic saturation-dependency of dispersion coefficient and stagnant saturation integrated into two-phase dynamic simulators. Obviously, the existence of stagnant regions will prevent the transport of chemicals to the target zones (even in rather homogenous reservoirs) with the time scale of water flooding, which will impede the performance of the technology. Moreover, in applications such as low salinity water floodingThis study presents a micro-model experimental research to investigate the transport mechanism under two-phase conditions in porous media. For a given saturation, saturation topology, tortuosity, and effective permeability were kept constant for all Péclet numbers in order to eliminate their effect on the distribution of the experimental data. While keeping tortuosity constant, it focuses on the existence of any relation between stagnant saturation and total saturation and the correlation between the stagnant saturation and dispersion coefficient.The dispersion coefficient shows a clear non-monotonic trend with saturation, especially at smaller Peclet numbers, which maximizes at intermediate saturations. The dispersion coefficient under two-phase conditions can be one order of magnitude larger than that of single-phase flow.At different saturation values, ranging from 0.27 to 1.0, we have been able to establish a steady-state saturation topology and inject ink tracer at four different rates. That allowed us to investigate the impact of Péclet number on dispersion coefficient keeping the tortuosity and effective permeability constant.Keeping the tortuosity and effective permeability constant at a given saturation, flow does not impact the relation between stagnant saturation and total saturation, which is in agreement with theoretical understandings. For laminar linear flow, the stagnant saturation should be independent of flow rate and it should be only a function of saturation topology. That highlights that a theoretical framework needs to be developed such that for a given saturation topology the stagnant and flowing volume fractions (i.e. immobile and mobile saturations) are independent from flow hydro-dynamics.Our results show that the coefficients involved in the estimation of dispersion coefficient as a power law of pore scale velocity are not functions of saturation only. Further studies need to be done in order to properly establish a relation between dispersion coefficients and pore scale velocity.The results imply a relation between the fraction of stagnant saturation  and total dispersion coefficient. The saturation, at which the dispersion coefficient maximizes, coincides with the maximum stagnant saturation fraction.Based on the results of the micro-model experiments, the following key conclusions can be withdrawn:Supplemdentary InformationSupplementary Tables


Arteriovenous malformation (AVM) is the most common form of intracranial vascular malformations in adults. Intracranial pediatric AVMs are rare. AVM located in the vicinity of the brain stem in children are even more rare.This study reports a rare case of acute obstructive hydrocephalus following aqueductal stenosis caused by an unruptured grade IV perimesencephalic arteriovenous malformation. An 11-year-old boy admitted to the hospital with progressive headache, nausea and vomiting throughout a month. A Computerized Tomography (CT) showed an obstructive hydrocephaly. A Magnetic Resonance (MR) imaging revealed a mesencephalic AVM compressing the aqueduct. The patient deteriorated in hours and an emergency ventriculoperitoneal shunting was performed. He did well in the early postoperative period. AVM examined with Digital Subtraction Angiography (DSA) in detail for maintaining the definitive treatment by means of endovascular embolization, microsurgery and stereotactic radiosurgery; but the patient was lost to follow up.A Pubmed search revealed 34 cases of hydrocephalus caused by an unruptured AVM in the literature, and only four cases were less than 18 years old with unruptured AVM locating in brain stem or posterior fossa. Although focal neurologic deficit, seizure and headache are the most common symptoms, acute neurologic deterioration due to hydrocephalus may be the presenting symptom in these cases. The decrease in intracranial pressure by changing the flow of cerebrospinal fluid (CSF) via an emergency ventriculoperitoneal (VP) shunting or Endoscopic Third Ventriculostomy (ETV) can be a lifesaving procedure that gives a chance for further treatment modalities. Intracranial pediatric AVMs are rare , 2. AVM This study illustrates a case of an unruptured pediatric perimesencephalic AVM that presented with acute neurological deterioration due to acute obstructive hydrocephalus in a child.21B). A prompt cranial MR revealed serpiginous signal void structures around the midbrain compatible with an AVM and enlargement of the vein of Galen and internal cerebral veins causing mechanical compression of the aqueduct causing an acute obstructive hydrocephalus caused . Cerebral DSA was planned for diagnosis and treatment planning, however; due to rapid deterioration in patient’s consciousness DSA was postponed and emergency right VP shunting was done in the same day. His conciseness improved, headache and vomiting disappeared immediately in the early postoperative period. Control CT scan showed good ventricular decompression . DSA was performed via right femoral access two weeks after the operation. Bilateral injection of internal carotid arteries showed enlarged posterior cerebral arteries bilaterally, supplying a diffuse AVM by the perforating arteries of the posterior lateral, medial choroidal and posterior communicating arteries. Venous drainage was into enlarged internal cerebral veins and vein of Galen. There was also some reflux to the deep tentorial and adjacent cortical veins due to high flow. The late capillary and venous phase was prolonged because of venous hypertension . The AVM was assessed as grade IV according to the Spetzler-Martin AVM grading system. A multimodal treatment protocol including endovascular embolization and stereotactic radiosurgery to portions of residual AVM was suggested. Patient was discharged on his family’s request as neurologically intact except for a mild right-sided facial palsy and was lost to follow-up.An 11-year-old male from abroad admitted to outpatient clinic with progressive headache, nausea and vomiting which had been presented for a month. Neurologic examinations showed bilateral papillary edema and right-sided central facial nerve palsy. He had been investigated for right sided facial palsy at home and was diagnosed with an AVM located in the perimesencephalic region two years ago. Head CT showed enlargement of lateral and third ventricles with transependymal cerebrospinal fluid oozing (Fig. 33.1Hydrocephalus caused by an unruptured AVM may be seen in both gender and in every age but rare less than 18 years -8. Only 3.2Hydrocephalus is mostly expected to be happening after the hemorrhage of an AVM into the ventricular system or subarachnoid space . Hydroce3.3et al. [Hydrocephalus can occur as a result of the venous outflow and hemodynamic unbalance , 8, 10; et al.  suggesteet al. . In the 3.4Microsurgical resection , endovasObstructive hydrocephalus due to an AVM can be treated by a VP shunting , 18, 21 Some authors primarily chose to treat AVM in cases with mild symptoms of obstruction that not required emergency , 23.Patient’s family requested discharge although they were fully informed about the disease and treatment options due to financial concerns and patient was lost to follow up.via an emergency VP shunting or ETV can be a lifesaving procedure that gives a chance for further treatment modalities.Presentation of a deeply located intracranial unruptured AVM with acute obstructive hydrocephalus is a rare entity in children. Although focal neurologic deficit, seizure and headache are the most common symptoms, acute neurologic deterioration due to hydrocephalus may be the presenting symptom in these cases. The decrease in intracranial pressure by changing the flow of CSF


In Arabidopsis, the plasma membrane-localized magnesium transporter MGT6 mediates Mg2+ uptake under Mg-limited conditions, and therefore is important for the plant adaptation to low-Mg environment. In this study, we further assessed the physiological function of MGT6 using a knockout T-DNA insertional mutant allele. We found that MGT6 was required for normal plant growth during various developmental stages when the environmental Mg2+ was low. Interestingly, in addition to the hypersensitivity to Mg2+ limitation, mgt6 mutants displayed dramatic growth defects when external Mg2+ was in excess. Compared with wild-type plants, mgt6 mutants generally contained less Mg2+ under both low and high external Mg2+ conditions. Reciprocal grafting experiments further underpinned a role of MGT6 in a shoot-based mechanism for detoxifying excessive Mg2+ in the environment. Moreover, we found that mgt6 mgt7 double mutant showed more severe phenotypes compared with single mutants under both low- and high-Mg2+ stress conditions, suggesting that these two MGT-type transporters play an additive role in controlling plant Mg2+ homeostasis under a wide range of external Mg2+ concentrations.Magnesium (Mg) is one of the essential nutrients for all living organisms. Plants acquire Mg from the environment and distribute within their bodies in the ionic form via Mg Mg2+ also contributes to membrane stabilization and active conformation of macromolecules (2+) and potassium (K+) in the soil (3+) in acidic soils or other heavy metals severely inhibit the uptake of Mg2+, resulting in Mg deficiency in the plants. These problems lead to reduction in crop yield as well as higher susceptibility to some plant diseases. On the other hand, high levels of Mg are found in serpentine soils featuring a low Ca/Mg ratio  is an essential macronutrient for plants. Being the most abundant free divalent cation in living cells, Mgolecules . Both lothe soil . MoreoveMg ratio . Genome ransport . Althougderstood .2+. While the MgtE channel and the P-type ATPases MgtA/B do not seem to have any close homologs in plants, there is a major family of Mg2+ transporters (MGTs) related to bacterial CorA proteins  and SALK_064741 (mgt7). The double mutant mgt6 mgt7 was generated by crossing mgt7 to mgt6 mutant, and progeny of F2 generation was screened for double homozygous mutations in MGT6 and MGT7 using a PCR-based genotyping approach.Wild-type and mutant plants were grown in the soil at 22°C under the 16-h-light/8-h-dark condition in the greenhouse. Hydroponically grown plants were generally kept in the 1/6 strength MS solution under the short-day condition (8-h-light/16-h-dark) in the greenhouse. Fresh liquid solutions were replaced twice a week.Arabidopsis seeds of different genotypes were sterilized with 10% bleach for 5 min and washed in sterilized water for 3 times. Seeds were sown on the solid plates supplemented with different concentrations of Mg2+. The basal medium contained 1/6 strength of MS salt  supplemented with Mg2+ at the indicated concentrations.For the post-germination assay, seeds were first sown on MS medium solidified with 1% phytoagar. After germination, 5-day-old seedlings were transferred onto 1/6 Mg4. After 2-week culture, the plants were treated with solutions containing different concentrations of Mg2+.For phenotypic assay in the hydroponics, 7-day-old seedlings were transferred to liquid solutions containing 1/6 MS salts supplemented with 1.25 mM MgSOmgt6 mutant, a 3.5-kb genomic fragment including the MGT6 coding region as well as 1.5 kb of the 5′ flanking DNA upstream of the starting codon was amplified by PCR from Arabidopsis genomic DNA with forward (5′-ACGGATAAATGTGGGGATGCTTG-3′) and reverse (5′-CCAAATCAAATCAACCCATAAAC-3′) primers. The PCR product was cloned into the SmaI site of the binary vector pCAMBIA1300. After sequencing, the construct was transformed into Agrobacterium tumefaciens strain GV3101 and introduced into mgt6 mutant plants by the floral dip method . After being digested by DNase I (Invitrogen) to decontaminate DNA, cDNA was generated from RNA samples at 42°C using SuperScript II reverse transcriptase (Invitrogen). The resultant cDNA samples were used for PCR amplification with the gene-specific primers. Quantitative real-time PCR was performed on the DNA Engine Opticon System (MJ Research) using the SYBR Green Realtime PCR Master Mix to monitor double-stranded DNA products. Data were calculated based on the comparative threshold cycle method. The relative expression of each Mg-starvation marker gene was double-normalized using the housekeeping gene Arabidopsis seedlings were transversely cut with a sharp blade in the middle position of the hypocotyl so that each individual seedling was divided into two parts. Subsequently, different parts of each material were re-assembled and grafted on half MS medium supplemented with 1.2% agar, 0.5% sucrose, 3 mg/L Benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate], 0.02 mg/L IAA (indole acetic acid) and 0.04 mg/L 6-BA (6-benzylaminopurine). The grafted seedlings were grown vertically in the growth chamber for another 10 days to allow the formation of the graft union. Successfully unified seedlings with the same size and status were then transferred to the hydroponic culture for further experiments.Reciprocal grafting experiments were performed as previously described with minor modifications . Seeds w2O for 10 s. The samples were then thoroughly dried up in the oven at 80°C. The dry matters were collected in the 15 mL centrifuge tubes (ions free) and digested with 1 mL ultrapure HNO3 (Sigma-Aldrich) in the water bath at 95°C for 4 h. Digested samples were diluted to the appropriate concentrations with ddH2O, and the elemental concentrations were determined by inductively coupled plasma optical emission spectroscopy .Plant samples were harvested from root and shoot tissues, respectively, and briefly washed with ddHMGT6 in transgenic plants by RNA interference resulted in growth retardation under low-Mg conditions  upstream of the stop codon . RT-PCR analyses showed that full-length MGT6 transcript was not detectable in the mgt6 mutant, while MGT4 gene located in the same chromosome is normally expressed . Consistent with earlier findings, mutation in MGT6 leads to hypersensitivity to Mg limitation in that the mgt6 mutants experienced growth defects at the germination stage . When germinated on the medium containing no Mg2+ or 0.01 mM Mg2+, the mgt6 mutants showed shorter roots and smaller and pale cotyledons . In the presence of 0.25 mM Mg2+, mgt6 seedlings appeared more normal, albeit still smaller than the wild-type . Early seedling establishment during germination became comparable between wild-type and mutant plants when external Mg2+ reached 1.5 mM . Statistical analysis of root length  and seedling fresh weight  verified the hypersensitivity to Mg deficiency in the mgt6 mutant.In a previous study, we have shown that knock-down of nditions . To furtmgt6 mutant in the absence of other divalent cation nutrients. Whereas mgt6 consistently displayed growth defects in the absence of Mg2+, seedling growth appeared indistinguishable between wild type and mgt6 on the medium lacking other divalent cations including Ca2+, Fe2+, Mn2+, and Zn2+ ; the primary roots were shorter  and the seedling fresh weight was significantly reduced . Because Mg2+ is the central structural cation for chlorophyll, we analyzed the chlorophyll content in the young leaves and found that the mutant had a lower chlorophyll level under extremely low-Mg conditions . When the medium Mg2+ levels reached a moderate range , the growth of mgt6 mutants appeared comparable to that of wild-type , although primary root length or seedling fresh weight was slightly affected . Notably, in the presence of 6 mM Mg2+ that is regarded as high, the mgt6 seedlings exhibited a strong growth defect , with much lower fresh weight  and reduced chlorophyll content  than wild-type plants. These data suggested that the mgt6 mutant is not only compromised under low-Mg levels but also hypersensitive to high-Mg stress.To extend the phenotypic analysis of the mgt6 mutant resulted from MGT6 mutation, we conducted a complementation test. A genomic fragment of MGT6 was introduced into the mgt6 mutant. Several homozygous transgenic lines with a similar MGT6 transcript level to that in wild type were obtained , but the growth difference was much more pronounced between wild-type and mgt6 plants under extremely low (0.01 and 0.05 mM) and high-Mg2+ (10 mM) conditions, as revealed by the root and shoot biomass . These results suggest that MGT6 is essential for plant growth at all developmental stages under a wide range of Mg2+ concentrations in the environment, and particularly plays an important role in plant adaption to low- and high-Mg2+ stresses.To assess the function of MGT6 in mature plants, we grew wild-type and 2+ homeostasis is affected by loss of MGT6 function under various conditions, we measured metal content in the roots and shoots of the wild type and mgt6. We first employed the plant materials cultivated in vitro after 2 weeks’ growth on the plates. As expected, compared with wild-type plants, we observed a dramatic decrease in Mg content in both roots and shoots of mgt6 mutants grown under low (0.01 mM) Mg conditions . In the presence of normal (1.5 mM) and high (6 mM) external Mg2+ levels, mgt6 mutants also contained less Mg in both roots and shoots than wild-type plants, when the seedlings were grown on the plates . Because Ca is usually associated with Mg homeostasis, we also measured Ca content in the plants. While Ca content in the root of mgt6 mutant was slightly higher, we surprisingly found that Mg deficiency resulted in a drastic reduction in shoot Ca compared with wild-type . The Ca content, like other parameters of plant growth, was comparable between the wild-type and mutant plants grown under 1.5 mM Mg2+ . The mgt6 mutant retained significantly less Ca in the root and slightly decreased Ca content in the shoot tissue when plants were cultured in 6 mM Mg2+ .In order to investigate how plant Mg2+ levels increased, wild-type plants accumulated elevated amount of Mg in both root and shoot tissues. The mgt6 plants generally showed a significant reduction in root Mg content compared with wild type, except under 10 mM Mg2+ . However, the shoot Mg content between wild type and mgt6 is most strikingly different under 10 mM Mg2+, although under some other concentrations of Mg2+, such as 0.05, 0.25, and 1.5 mM, mgt6 mutant also contained lower Mg content in the shoot compared with wild type . Plant Ca contents are negatively correlated with external Mg2+ levels. Under low-Mg conditions , an obvious elevation in root Ca was observed in the mgt6 mutant , presumably due to the antagonistic interaction between Mg and Ca. These results suggest MGT6 regulates plant Mg2+ homeostasis in both roots and shoots, and functions in a wide range of external Mg2+ concentrations at all developmental stages.We further measured the Mg and Ca content in the hydroponically grown mature plants. As the external Mgmgt6 can be explained by impaired Mg2+ uptake by root under Mg-limited conditions in the mutant, the high-Mg susceptibility of mgt6 remains obscure. Since MGT6 controls both root and shoot Mg2+ homeostasis, we attempted to further investigate the mechanism by which MGT6 contributes to plant Mg2+ tolerance. Because MGT6 is widely expressed in plants, we decided to examine the relative contribution of MGT6 in roots versus in shoots through reciprocal grafting experiments between mgt6 mutants and wild-type plants . When grown under low-Mg2+ conditions (0.01 mM), the shoots with wild-type scions and mgt6 rootstocks appeared to be smaller than that of self-grafted wild-type plants, although the root looked similar. The grafted plants with mgt6 scions and wild-type rootstocks were significantly smaller than wild-type self-grafted plants, but generally larger than mgt6 self-grafted plants. Under the moderate level of Mg2+ (1.5 mM), both groups of the reciprocal grafted plants grew smaller than wild-type self-grafted plants. However, in the hydroponic culture containing 10 mM Mg2+, which is considered to be a toxic concentration, the grafted plants with mgt6 scions and wild-type rootstocks phenocopied the defects seen in the mgt6 self-grafts, whereas the grafted plants with wild-type scions and mgt6 rootstocks resembled the phenotype of wild-type self-grafted plants . We measured root and shoot fresh weight quantitatively, which verified the growth phenotypes . These observations suggested that MGT6 is important in both root and shoot tissues when external Mg2+ is low and moderate. Presumably, MGT6-mediated absorption of external Mg2+ represents the dominant role under these conditions. When the external Mg2+ is extremely high, MGT6 function in the shoot becomes critical to detoxify excessive Mg2+ at the whole plant level. Consistent with this notion, wild-type scions grafted on mgt6 rootstocks lead to significantly lower root Mg2+ content under 0.01 and 1.5 mM Mg2+ conditions . In the presence of 10 mM external Mg2+, shoots from mgt6 grafted onto wild-type rootstocks retained much less Mg2+ in the shoot, similar to that observed in self-grafted mgt6 plants . This further supported the idea that MGT6 fulfills a shoot-based mechanism to detoxify excessive Mg2+, which could involve vacuolar Mg2+ storage based on the observation of lower Mg content in the mutant shoots.While the low-Mg sensitive phenotype of Arabidopsis MGT7 encodes a low-affinity Mg2+ transporter . However, the mgt6 mgt7 double mutant was significantly more sensitive to external Mg2+ than the mgt6 single mutant . The primary root of mgt6 mgt7 was shorter than that of mgt6 under low-Mg conditions , although seedling fresh weight was comparable . The leaf chlorophyll content in mgt6 mgt7was lower compared with mgt6 when high Mg2+ is present in the medium .We examined the growth phenotype of mgt6 or mgt7 mutant background than in the wild type, suggesting that the mgt6 and mgt7 mutants are impaired in low-Mg2+ adaptation. Consistent with the more severe phenotype, the mgt6 mgt7 double mutant displayed enhanced expression of Mg-starvation marker genes compared with the single mutants (Supplementary Figure 2+ transport under a wide range of external Mg2+ concentrations.Gene expression analysis indicated that a handful of gene markers  were morArabidopsis, rice, and maize, encode homologs of the bacterial CorA-type proteins referred to as MGTs/MRS2s (Arabidopsis, we previously showed that MGT6 is capable of facilitating high-affinity Mg2+ uptake from the soil when external Mg2+ concentration is in the sub-millimolar range (MGT6 gene is highly inducible in the root tissues in response to low Mg (2+ homeostasis within a wide range of external Mg2+ levels. The mgt6 knock-out mutant displayed obvious phenotype under high-Mg conditions, suggesting MGT6 exerts physiological functions in plants other than Mg2+ absorption. In higher plants, after absorption from the soil solution by roots, Mg2+ is believed to be transported to the aerial parts via transpiration stream moving through the xylem vessels. However, little is known about the molecular identity of the transporters involved in this long-distance transport. MGT6 might fulfill such a role in the xylem transport of Mg2+. Considering the negative membrane potential, Mg2+ is expected to be loaded passively into the pericycle cells. MGT6 may be responsible for Mg2+ import into pericycle and xylem parenchyma cells. On the other hand, the possibility that MGT6 serves as an “exporter” in this process cannot be excluded. It is generally believed that ion secretion occurs across plasmalemma of the parenchyma cells surrounding the xylem vessels (Arabidopsis, was shown to be a bidirectional Mg2+ carrier that operates in a concentration-dependent manner (2+ efflux from xylem parenchyma cells, pushing Mg2+ influx into the xylem vessel. Future electrophysiological analysis of MGT6 conductance is required to test this hypothesis.In addition to air and water, plant growth and development rely on mineral nutrients taken up by roots and translocated into the shoot tissues through apoplast and symplast pathways, which entail not only transpiration-driven mass flow but also active membrane transport processes facilitated by various ion channels and transporters. Mg is an essential macronutrient in plants with diverse biological functions. However, the molecular mechanisms for Mg transport and homeostasis in plant cells remain largely unknown. Genomes of many plants such as Ts/MRS2s . Some mear range . Consisto low Mg . In the  vessels . Interest manner . Therefo2+ is an essential mineral, high levels of Mg2+, such as those in the serpentine soils, could be toxic to plants. Recently we established that vacuolar sequestering of Mg2+, regulated by the tonoplast CBL-CIPK signaling network, is crucial for plants to survive under high-Mg conditions (2+ from root to shoot, whereas the serpentine-sensitive counterparts are less capable of driving Mg2+ entry into the transpiration stream, resulting in a lower Mg2+ concentration in the shoot (mgt6 mutant under high-Mg conditions showed that mgt6 retained considerably less Mg2+ in the shoot tissue compared to wild-type, accompanied by the growth retardation upon high-Mg stress. These data support the notion that long-distance Mg2+ transport mediated by MGT6 may play a critical role in protecting plants from Mg2+ toxicity at the whole plant level. More importantly, reciprocal grafting test indicated that MGT6 function in the shoot tissue is responsible for the high-Mg2+ tolerance. Considering the plasma membrane localization of MGT6, it is reasonable to speculate that MGT6 probably facilitates Mg2+ entry into the cytosol of leaf cells after the Mg2+ ions in the xylem unload into the apoplastic space. The excessive Mg2+ in the cytosol is subsequently sequestered into the central vacuole via tonoplast-localized Mg2+ transporters. This transport cascade is critical for detoxification of excessive Mg2+, which is reminiscent of a recent model proposed for Ca2+ detoxification in plants (Although Mgnditions . In the he shoot . Consistn plants .mgt6 mgt7 double mutant is more sensitive to low-Mg stress than the mgt6 single mutant, which is also supported by the enhanced activation of Mg-starvation gene expression in the double mutant. Interestingly, under high-Mg conditions, mutation of MGT7 also significantly enhanced the sensitivity of mgt6, although single mutant of mgt7 only exhibited a subtle phenotype under the same condition. These results suggest that MGT7 synergistically works together with MGT6 in the context of Mg2+ homeostasis at the whole plant level. Further investigations will sort out the mode of action for each of them to explain this functional synergy.Another notable finding in this study is that another MGT-type transporter MGT7 partially overlaps with the function of MGT6. With a preferential expression in the root, MGT7 was shown to be important for plant adaptation to low-Mg conditions . In our in situ. It will also be interesting to examine if the targeting of MGT6 or MGT7 would be dynamically altered in different subcellular compartments in response to various Mg2+ concentrations.Subcellular localization of the MGT proteins may prove to be difficult to study. For instance, recent studies reported discrepant cellular localizations for MGT6 in the plasma membrane  and endo2+ in nature. How plants maintain a balanced level of Mg2+ is not well understood. The present study as well as our previous work provides a working model in which MGT6 plays a dual role in controlling Mg2+ homeostasis. When external Mg2+ is limited, expression of MGT6 is induced in root epidermal cells and root hairs, making this transporter primarily responsible for Mg2+ uptake from the soil. When external Mg2+ is sufficient or becomes excessive, MGT6 mediates Mg2+ loading into the shoot tissues, where leaf mesophyll cells can subsequently sequester extra amount of Mg2+ into large vacuoles via yet-unknown transporters. Future efforts should be made in identifying uncharacterized Mg2+ transport proteins in plants. Furthermore, establishing the regulators and signaling pathways that fine-tune the expression and function of these transport systems will be a challenging but urgent task, which will ultimately lead to genetic manipulation of plants for precise adaption to the changing Mg2+ concentrations in the environment.As sessile organisms, plants have to cope with fluctuating concentrations of MgY-WY and R-JT designed and conducted most of the experiments, interpreted the results, and wrote the draft of the manuscript. D-DM, X-XZ, Q-LT, and Y-PL assisted in some experiments and helped analyze the data. LY and J-LQ provided tools and reagents and made helpful discussions. SL supervised and conceptualized the study and finalized the paper. All the authors approved the final version of the manuscript.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.