Head and neck cancer patients are at a high risk to suffer from malnourishment, a risk that increases in postoperative condition and with the use of enteral nutrition (EN). Until now patients who are suffering from indigestion in the intensive care unit (ICU) received treatment in the form of prokinetic drugs, drugs that can lead to serious side effects and only can partially improve digestion functions. Acupuncture was used successfully in several clinical trials to improve postoperative indigestion in cancer patients without any reported adverse events. The study aims are to investigate acupuncture effect in combination with prokinetic drugs in the treatment of indigestion in postoperative oral and hypopharyngeal cancer patients in the ICU.Single-center, double-blind randomized control trial will compare between 2 equal groups. A total of 28 patients that will meet the inclusion criteria: age 30 to 80, postplastic surgery for oral cancer or hypopharyngeal cancer, developed feeding intolerance 2 times in the first postoperative day, Apache score <20, and needed EN. Patients will be randomly divided (1:1) into treatment group or control group for 3 treatments in 3 days along with routine ICU treatment. The main outcome measurement will be the number of days a patient needs to reach his total energy expenditure.The results will shed light on the effectiveness and safety of acupuncture in a double-blind design treating postoperative ICU cancer patients. In addition, the study presents a revolutionary double-blind design that if, will prove as successful might influence the way double-blind acupuncture studies are performed today.The study will be conducted in the surgical ICU department, of China medical university hospital, Taichung 404, Taiwan. The study is conducted on stable ICU patients and is anticipated to have minimum risk for adverse events. Patients enrollment and data collection will start from May 15, 2019. The study expected completion time: June 2021. Postoperative intensive care unit (ICU) patient often required parenteral nutrition or enteral nutrition (EN) to prevent malnourishment. EN is considered as the superior nutrition choice that assists the maintenance of gut mucosal integrity, improved utilization of nutrients, reduced respiratory infections, and hospital total stay.,4 Unfortunately EN carry a number of side effects such as delayed gastric emptying, diarrhea, constipation, reflux, nausea, vomiting, gastrointestinal (GI) bleeding, contamination, and EN feeding tube clogging.–9 To prevent the side effect of EN, prokinetic drugs are often used. A recent systemic review and meta-analysis found that although prokinetic drugs can reduce high gastric residual volume, prokinetic drugs failed to decrease vomiting, diarrhea, mortality risk, and length of stay in the ICU. Metoclopramide, a popular and commonly used prokinetic drug, can lead to serious neurological and cardiac adverse reactions.Up to 80% of head and neck cancer patients will suffer malnourishment during the course of the disease, malnutrition can increase the risk of postoperative complications as well as the rate of mortality.–14 Acupuncture is rarely a part of the complementary treatment that is used in the ICU. A few studies used acupuncture in the ICU without any adverse events.–18 A review investigating acupuncture for postoperative delayed gastric emptying patients including abdominal surgery, esophageal cancer, and laparotomy found promising effectiveness of acupuncture when comparing to prokinetic drugs. Two additional studies suggest that electrical acupoint stimulation might be superior to prokinetic drug usage in the treatment of delayed gastric emptying in EN patients in the ICU resultant from stroke or traumatic brain injury.,20 Another study proves that acupuncture, along with Chinese herbal medicine, can decrease acute GI injury incidence in elderly patients with severe sepsis.Acupuncture is an ancient treatment that was used for thousands of years and is considered to be a safe treatment when applied by a certified acupuncturist.As a control treatment, we will use nonspecific acupuncture, which uses “real” acupuncture but the points selected have no relation to improving digestion function. We believe that the use of nonspecific acupuncture might enable proper blinding to the acupuncturist. As far as we know, there is no double-blind randomized controlled study that investigates the effect of acupuncture on total energy expenditure (TEE) days and other accompanying digestive signs present in postoperative oral and hypopharyngeal cancer patients receiving EN. The goal of our study is to conduct a double-blind randomized controlled trial to investigate the effect of acupuncture on treating indigestion factors of postoperative oral and hypopharyngeal cancer patients receiving EN in the surgical ICU; furthermore, we hypothesize that specific acupuncture (treatment group) will have a greater beneficial effect on digestion indicators when compared with the control.22.1www.clinicaltrial.gov (NCT03934294). The goal of our study is to assess the efficacy of acupuncture in treating indigestion and accompanying indigestion symptoms of surgical ICU patients. This will be achieved through analysis of 2 parallel groups; treatment group (ACU): specific acupuncture treatment alongside routine ICU treatment; control group (CON): nonspecific acupuncture treatment with routine ICU treatment  and registered study protocol on An informed consent form will be filed and signed by the legal representative of the patient before the initiation of our study. Patients will be randomly divided into 2 equal groups to ACU, and CON. Two qualified acupuncture doctors with at least 2 years of experience, from the acupuncture department of China medical university hospital, will perform the ACU and the CON treatments, ICU routine treatment will be carried out by ICU staff in all groups . Prokinetic drug treatment will be prescribed by an ICU medical doctor if needed in all study groups. Researchers, statisticians, doctors, acupuncturists, and patients along with nurses (with the exception of study nurse) will be blinded to the patient allocation. Patients’ assessment will be done on a daily basis by ICU medical doctors and ICU nurses who will be blind to the patients’ allocation.2.2A total of 28 patients that required EN and admitted to the surgical intensive care department in china medical hospital for estimated ICU duration stay of at least 3 days will be enrolled in this study if the patient met the study inclusion criteria and after patient's legal representative will sign an informed consent form.2.3Age 30–80Apache score <20Patients needed ENPatients who developed feeding intolerance 2 times in the first postoperative day.Postplastic surgery, including oral cancer or hypopharyngeal cancer2.4CoagulopathyProlong prothrombin time activated partial thromboplastin time >4 timesThrombocytopenia—low platelet countClinically unstable: receiving 2 inotropic agents or Fraction of Inspired Oxygen >70%Estimated ICU stay <3 days2.5Patients recruiting will be done from May 2019 to July of 2021 or until a total of 28 patients will be recruited. A study personal (KP-Y) that is responsible for communicating with patient's families and legal guardians will inform the patients family of the study along with its potential pros and cons and will ask the patients legal guardian to fill an informed consent form in case of willingness to join the study.2.6The study characteristics, objectives, risks, and benefits along with details on patient's rights according to the declaration of Helsinki will be explained to the patient's legal guardian by a study personal (KP-Y). In the event of willingness to enroll in the study, an informed consent form will be signed by the patient's legal guardian that can decide on withdrawal from the study at any moment. In case of withdrawal from the study, the patient's available data will be reserved for the final analyses (for the full informed consent form see Supplementary Information).2.7Patients will be randomized to 1 of 2 groups by study nurse (EB-A): ACU/CON using a computer-based simple random sampling with 1:1 ratio without stratification using the IBM SPSS statistics version 22 software . A number (1–28) will be affiliated to each patient at the time of ICU admission and after achieving informed consent. The number and patient name will be written down on a nontransparent envelope by the study nurse. The study nurse will provide an envelope containing a sheet depicting the acupoint name, location, and picture (ACU/CON) to the acupuncture doctor 1. Acupuncture doctor 1 (blind) will mark the acupoints on the patients’ body with a green sticker and leave the room, and then acupuncture doctor 2 (blind) will enter the room and perform acupuncture on the marked points. Thirty minutes after needle insertion, acupuncture doctor 2 will withdraw the needles. Acupuncturists, ICU nurses, ICU doctors, researchers (except EB-A) and statisticians will all be blind to group allocation and to the meaning of the patient's numbers until all acupuncture treatments have been completed. The 2 acupuncturists will also be blind to the treatment goals. Both acupuncture doctors will not be allowed to speak with the patients or themselves about the study goals or patients’ allocation ; nonspecific acupuncture with routine ICU treatment (CON). The patients will receive everyday treatment for 3 days for a total of 3 treatments. The treatment will start after 2 failed tube feeding sessions in both groups.2.9In addition to routine ICU treatments, patients in the specific acupuncture group (ACU) will also receive daily bilateral traditional Chinese medicine style acupuncture on the following acupuncture points: ST36 (Zusanli), ST37 (Shangjuxu), ST39 (Xiajuxu), PC6 (Neiguan), and LI4 (Hegu). The acupoints indications in this group are specific to treat indigestion-related conditions  will mark the points on the patients’ body with a green sticker and leave the room. Acupuncture doctor 2 (blind) will disinfect the marked acupoints with an alcohol pad containing 70% alcohol and will perform acupuncture on the marked points with perpendicular needle insertion into the muscle layer, followed by up and down manipulation and 180° rotation of the needles in both directions to generate a “De Qi” sensation. During the session the patients will stay in a supine position. Needle retention time will be 30 minutes. The needles will be withdrawn by acupuncture doctor 2, who will not be allowed to communicate with the patients. At the end of the treatment, acupuncture doctors 1 and 2 will be asked to guess if they treated the specific or nonspecific acupuncture group to assess and maintain the integrity of the blindness in the study.2.10Patients’ in the nonspecific acupuncture group (CON) will receive routine ICU treatment and a total of 3 daily indigestion-related traditional Chinese medicine style acupuncture treatments at the following acupoints: LI 15 (Jianyu), SJ 14 (JianLiao), LU3 (Tianfu), GB35 (Yangjiao), BL 59 (Fuyang). The selected control points are not indicated for the treatment of digestion-related pathologies and are not reported to improve digestive function  will mark the points in green marker, and acupuncture doctor 2 (blind) will disinfect the acupoint with a 70% alcohol pad and insert the needles into the marked points. A total of 10 bilateral sterile “Yu Kuang” acupuncture needles 40 mm with 30 G will be inserted perpendicularly into the depth of the muscle level with up and down stimulation and 180° rotation of the needles to both directions to generate a “De Qi” sensation. The needles will be retained for 30 minutes until removal by acupuncture doctor 2 who will not be allowed to communicate with the patients. During the session the patients will stay in a supine position. At the end of the treatment acupuncture doctors, 1 and 2 will be asked to speculate if they treat the specific or nonspecific acupuncture group to assess and maintain the integrity of the blindness in the study.2.11Patients in all groups will receive metoclopramide 10 mg/8 hours in the case of poor digestion, alongside the individualized drug treatment prescribed by the ICU medical doctor as per individual patient needs.2.12Nasogastric (NG) tubes will be inserted for the patients during surgery for the duration of surgery and postoperative time to service the nutritional need as per the patient prognosis/requirement. NG tubing depth and function will be fixed and checked every 24 hours and will be retained as long as the patient needs it. TEE will be calculated by the weight, height, and physical factors of the patient as assessed by the nutritionist in ICU to calculate daily nutritional needs. In postoperation day 1 ICU staff will start with a feeding amount of 30% of the TEE and gradually increase to 100% by day 4. Every 3 days the nutritionist will reassess the patient's condition (and percentage of nutrition intake).2.13The main outcome measurements will be the number of days of intakes for each patient to achieve the TEE.Secondary outcomes measures are the amount of prokinetic drugs prescribed by the ICU doctor in total dosage, the need of nasojejunal tube (5 consistent days of indigestion with conservative therapy), the need of parental nutrition (patients who cannot digest with daily NG tube drainage of >500 mL/day or severe diarrhea of >1000 mL/day), incidents of vomit or diarrhea , incidents of constipation (no stool passage in 3 days will be considered as constipation), reflux, nausea , GI bleeding (positive occult blood test of the NG tube drainage and in the stool), and incidents of fever episodes (body temperature >38°C) will also be recorded when they occur, albumin blood levels, total ICU stay, total hospital stay, total mechanical ventilation in days , and total mortality will also be measured as a secondary outcome.In order to compare the effect of the control treatment, heart rate and blood pressure will be measured 1 hour before and after the acupuncture in both groups.Acupuncturists will be asked to speculate if they treated the specific or nonspecific acupuncture group to assess and maintain the integrity of the blindness in the study .2.14Daily follow up on TEE, NG tube depth, parenteral/enteral feeding, and prokinetic drug amounts will be collected by ICU staff along with incidents of vomiting, diarrhea, constipation, reflux, nausea, and GI bleeding. After 3 days, the data collected will be used for calculating study results. Mechanical ventilation days needed and total ICU stay along with total hospital inpatient duration and mortality will be collected by the time of hospital discharge or until June 2021 Fig. .2.15Before patients’ enrollment, the patient's legal guardian will be informed on possible acupuncture adverse events including minor bleeding, hematoma, infections, alcohol allergy, etc. Any case of acupuncture-related severe adverse events taking place, the event will be reported immediately to primary investigator and to the ethics committee, adverse events details such as treatment site, acupoint selected, patient recovery outcome, and the number of events on 1 patient will be recorded. Acupuncture will be stopped and treatment groups will be unblind. The patient will be provided with suitable ICU treatment. China medical hospital ethics committee will be in charge of deciding whether the study can continue, should be altered, or shut down.2.16The sample size was calculated using G∗power version 3.1.9.2 software, the sample size was based on data of 51 patients that visit the ICU department 9 months before the experiment and are similar to the study target population. A 95% power was calculated with an effect size of 1.68 and a calculation of 20% patient dropout rate required 14 patients in each group for a total of 28 patients.2.17www.clinicaltrial.gov.In order to maintain the integrity of measured data and providing proper data reporting, patients’ data will be recorded manually as a case report form and uploaded to the hospital electronic database by ICU staff, the manual case reports will be stored in a secure location in the ICU and will be destroyed after trial completion. Regular meeting and presentations will ensure that the entire study staff is aware of the study protocol and follow it. At the beginning of the study, the principal investigator will make sure that patients enrolled are meeting inclusion criteria, the informed consent form is properly signed and all study procedures are following the study protocol. Any modification in the study protocol will be done upon agreement with China Medical University Hospital Ethics Committee and with 2.18t test or the Mann-Whitney U test and categorical variables using the Chi-square (χ2) test or the Fisher exact test with a best applicable basis. Repeated measurements will use the generalized linear model generalized estimating equation to conduct the analysis. The analysis will be performed using the IBM SPSS Statistics version 22.0 , the results will be marked as significant at P value <.05.A researcher who is blind to patient allocation will conduct the statistical analysis. Statistical data will be described as a percentage (n %) for categorical data and as mean ± standard deviations for continuous data. Continuous variables will be analyzed using the 3 In order to treat indigestion prokinetic drugs are commonly used, although prokinetic may carry some adverse reactions. From data collected from our ICU department before our study, oral and hypopharyngeal cancer patients suffering from postoperative feeding intolerance will reach their TEE after 9 days with the use of prokinetic drugs, on the contrary, postoperative oral and hypopharyngeal cancer patients that do not suffer from feeding intolerance will reach their TEE in <3 days. There is a need to use a safe and effective method to improve poor digestion in critically ill cancer patients. Acupuncture has been reported to improve indigestion signs in several studies.,23–25In critically ill patients, the incidence of indigestion can be as high as 46%; this may lead to malnutrition, increase ICU stay, and increased mortality. Secondly, sham needles do not allow a full up and down manipulation which may be crucial in achieving “DE QI” sensation (an important penman in successful acupuncture treatment), and thirdly, in some cases, sham needles failed to achieve a good level of acupuncturist blinding. Our study design involves 2 groups; when both groups are using “real” acupuncture needles at “real” acupoints. The differences between the 2 acupuncture groups are the acupoint selected. When in the treatment group (ACU) the selected acupoints are specific for the treatment of indigestion, the CON is here to present the placebo effect of acupuncture by needling acupoints that do not have any indication to treat indigestion. In both groups, the acupuncturist will perform needles stimulation that will activate the “DE QI” sensation. The acupuncturists performing the treatments will be blind to the study goal to treat indigestion, a fact that we believe will make it difficult to speculate which of the group is receiving the “real” treatment and allow us to generate blinding of the acupuncturist and patients. After a treatment session, we decided to ask the acupuncture doctors to speculate which treatment was the “real” treatment to calculate the level of doctor blindness. The patients will not be asked to speculate on the treatment nature since both groups will receive traditional medicine style acupuncture and due to the nature of ICU patients that may not be in a fully conscious state.For study design, we decided to conduct a double-blind design to minimize the bias. A double-blind design is unusual in acupuncture studies. Double-blind acupuncture studies are often consisting of the usage of sham needles, which carry a number of limitations; firstly, the needling depth is limited 10 mm depth which may not be ideal insertion depth in many acupoints.,20 The 3 acupoints, ST36 (Zusanli), ST37 (Shangjuxu), and ST39 (Xiajuxu) are “He-Sea” points of stomach, large, and small intestines, respectively and are used to benefit those organs, the points were also a part of the acupoint combination used in a recent pilot study in improving gastric emptying. ST36 (Zusanli) and ST37 (Shangjuxu) also showed to improve small intestines motility in animal studies; additionally, ST36 (Zusanli) showed to increased gastric motility through a vagal pathway.,28,29 The acupoint LI4 (Hegu) a “Yuan-Source” point of the Large Intestine channel is usually indicated for treating pain-related conditions alongside with the ability to benefit the digestive system, the points showed a beneficial effect on irritable bowel syndrome and on rats with gastric carcinectomy.,31The acupoints selected in the treatment group (specific acupuncture group) are; ST36 (Zusanli), ST37 (Shangjuxu), ST39 (Xiajuxu), PC6 (Neiguan), and LI4 (Hegu). The acupoint, PC6 (Neiguan) main characteristics are regulating the stomach, relieve nausea and vomiting, and the point was used successfully in reducing gastric emptying. LU3 (Tianfu) is used mainly for mental imbalance and for cough; GB35 (Yangjiao) is used for pain in the knee and leg; and BL 59 (Fuyang) is used for low back and thigh pain. None of the points in this group are used to benefit the digestive system and we believe that the points can be suitable to demonstrate the placebo effect of acupuncture treatment. An acupuncturist might speculate that the patients receiving those acupoints are suffering from shoulder pain.The selected acupoint in the CON (nonspecific acupuncture group) are LI 15 (Jianyu), SJ 14 (JianLiao) LU3 (Tianfu), GB35 (Yangjiao), and BL 59 (Fuyang). The acupoints LI 15 (Jianyu) and SJ 14 (JianLiao) are located on the shoulder and used to treat shoulder conditions.The authors would like to thank the Chinese Medicine Research Center, China Medical University for the support to our study. The authors would like to show their gratitude to Wei Tsu-Hsuan, MD and Chang Chiu-Ming, MD for performing acupuncture.Analysis and interpretation of the data: Ho Wen-Chao, Eyal Ben-Arie, Kao Pei-YuConceptualization: Eyal Ben-Arie, Kao Pei-Yu.Data curation: Eyal Ben-Arie, Kao Pei-Yu.Design: Eyal Ben-Arie, Kao Pei-Yu, Lee Yu Chen, Ho Wen-ChaoFormal analysis: Ho Wen-Chao.Funding acquisition: Lee Yu Chen.Investigation: Eyal Ben-Arie, Kao Pei-Yu.Methodology: Eyal Ben-Arie, Kao Pei-Yu, Ho Wen-Chao, Lee Yu Chen.Project administration: Eyal Ben-Arie, Kao Pei-Yu, Lee Yu Chen.Resources: Lee Yu Chen.Software: Eyal Ben-Arie, Kao Pei-Yu, Ho Wen-Chao.Supervision: Lee Yu Chen.Validation: Eyal Ben-Arie, Lee Yu ChenValidation: Kao Pei-Yu.Visualization: Kao Pei-Yu.Writing – original draft: Eyal Ben-Arie, Kao Pei-Yu.Writing – review and editing: Eyal Ben-Arie, Kao Pei-Yu.


Magnaporthe oryzae (Mo) is a model pathogen causing rice blast resulting in yield and economic losses world-wide. CK2 is a constitutively active, serine/threonine kinase in eukaryotes, having a wide array of known substrates, and involved in many cellular processes. We investigated the localization and role of MoCK2 during growth and infection. BLAST search for MoCK2 components and targeted deletion of subunits was combined with protein-GFP fusions to investigate localization. We found one CKa and two CKb subunits of the CK2 holoenzyme. Deletion of the catalytic subunit CKa was not possible and might indicate that such deletions are lethal. The CKb subunits could be deleted but they were both necessary for normal growth and pathogenicity. Localization studies showed that the CK2 holoenzyme needed to be intact for normal localization at septal pores and at appressorium penetration pores. Nuclear localization of CKa was however not dependent on the intact CK2 holoenzyme. In appressoria, CK2 formed a large ring perpendicular to the penetration pore and the ring formation was dependent on the presence of all CK2 subunits. The effects on growth and pathogenicity of deletion of the b subunits combined with the localization indicate that CK2 can have important regulatory functions not only in the nucleus/nucleolus but also at fungal specific structures such as septa and appressorial pores. Saccharomyces cerevisiae, also contains two different alpha- and two different β-subunits (b1 and b2) and deletion of both catalytic subunits is lethal  accumulates in the nucleolus, localizes in structures near septal pores, and assembles to form a large ring structure perpendicular to the appressorium penetration pore and is essential for normal growth and also plant infection.Our results show that M.oryzae Ku80 mutant constructed from the wild type Guy11 strain was used as background strain since it lacks non-homologous end joining which facilitates gene targeting  gene amplified from pCX62  gene, HPH resistance). Then the fusion fragments were transformed into protoplasts of the background strain Ku80. The positive transformant ΔMockb1  in 1 L DD-water for 30 min. After cooling pH was adjusted to 6.5 using NaOH and 20 gl−1 agar (MDL No MFCD00081288) was added before sterilization by autoclaving (121°C for 20 min). Conidia morphology was observed using confocal microscopy (Nikon A1+). For conidial germination and appressoria formation conidia were incubated on hydrophobic microscope cover glasses . First strand cDNA was synthesized with the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa). For quantitative real-time PCR, Alignments, phylogenetic trees, analysis to find orthologs, and homologs to well-described proteins in other fungal species is available at FigShare and linked from stefan@olssonstefan.com) or ZW (wangzh@fafu.edu.cn).Further information and requests for resources and reagents should be directed to and will be fulfilled by SO  and two MoCKb regulatory subunit orthologs  based on BLASTp (NCBI) analysis using protein sequences for the CK2 subunits of Targeted deletions of the two regulatory subunits succeeded . For abbThe most obvious visible phenotype of the CKb mutants (strain b1 and b2) in culture was reduced growth rate  and coniTo assess the localization of the three CK2 subunits, we constructed N-terminal GFP fusions of all three proteins  using qPCR and noted it was downregulated 2- to 3-fold compared to the background control strain Ku80  was observed in b1GfpA and b2GfpA , thus, we used mycelial plugs to test for infection  allowed sufficient conidia production to perform conidial inoculations. Small lesions were observed in both cases, indicating that Ckb subunits are not required for pathogenesis  localizes to nuclei  top row MoCKb mutants and the localization of the GFP-labeled MoCK2 proteins showed that all identified MoCK2 components are needed for normal function and also normal localization. Localization to septa requires all three subunits, presumably as the holoenzyme. Mutation of either CKb subunit blocks nucleolar localization of the other CKb subunit. Surprisingly, nucleolar localization of CKa was observed in both CKb mutants. This indicates that the holoenzyme is not required for CKa localization to the nucleolus. It seems likely that CKb1 and CKb2 must interact with each other in order to interact with CKa (Meggio and Pinna, The analysis of the A. oryzae (Figure 4B in Han et al., The pattern of localization to septa  observedin vitro (Glover, in vivo (Hübner et al., The crystal structure of CK2 suggests that it can form filaments and higher-order interactions between CK2 holoenzyme tetramer units, and based on this it has been predicted that autophosphorylation between the units could occur to down-regulate its activity (Litchfield,  Glover,  and in vThe large ring observed in appressoria , 4 may oM. oryzae. CK2 localizes to the nucleus, in particular to the nucleolus but also to hyphal septal pores and most interestingly to appressorial penetration pores. The pore localizations are dependent on all CK2 commponents of the CK2 holoenzyme while the nuclear localization of CKa is not necessarily dependent on the presence of a specific CKb unit. These localizations of CK2 points to a possible involvement in regulating and facilitating the traffic through pores and in line with this also the distribution of protein inside the nucleus.We conclude that CK2 is necessary for normal growth and plant infection by GL, SO, and ZW conceived and designed the experiments. LZ, DZ, YC, WY, and QL performed the experiments. LZ, DZ, DE, SO, and ZW analyzed the data and wrote the paper.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.


GSTM1) gene has been associated with multiple cancers, metabolic and autoimmune disorders, as well as drug response. It is unusually common, with allele frequency reaching up to 75% in some human populations. Such high allele frequency of a derived allele with apparent impact on an otherwise conserved gene is a rare phenomenon. To investigate the evolutionary history of this locus, we analyzed 310 genomes using population genetics tools. Our analysis revealed a surprising lack of linkage disequilibrium between the deletion and the flanking single nucleotide variants in this locus. Tests that measure extended homozygosity and rapid change in allele frequency revealed signatures of an incomplete sweep in the locus. Using empirical approaches, we identified the Tanuki haplogroup, which carries the GSTM1 deletion and is found in approximately 70% of East Asian chromosomes. This haplogroup has rapidly increased in frequency in East Asian populations, contributing to a high population differentiation among continental human groups. We showed that extended homozygosity and population differentiation for this haplogroup is incompatible with simulated neutral expectations in East Asian populations. In parallel, we found that the Tanuki haplogroup is significantly associated with the expression levels of other GSTM genes. Collectively, our results suggest that standing variation in this locus has likely undergone an incomplete sweep in East Asia with regulatory impact on multiple GSTM genes. Our study provides the necessary framework for further studies to elucidate the evolutionary reasons that maintain disease-susceptibility variants in the GSTM1 locus.The deletion of the metabolizing Glutathione S-transferase Mu 1 ( GSTM1 gene, which has been shown to be an important metabolism gene and associated with bladder cancer, autoimmune disorders and response to different drugs. Our results reveal a genetic type common in East Asian populations that may have important evolutionary and biomedical implications.Here, we describe the evolutionary forces that shape the variation in the i.e., deletions, duplications, inversions and translocations of large segments of DNA) comprise a major part of genetic variation among humans  locus has revealed that a specific SV leads to resistance to malaria in African populations  gene in humans  ((NAT2) (G6PD)  , and GluGSTM1 gene deletion is one of the most common whole gene deletions observed among human populations (GSTM1 deletion is higher than 50% (LCE3BC (Late Cornified Envelope 3B and C) and the UGT2B17 (uridine diphosphoglucuronosyltransferase), both of which have been shown to be evolving under non-neutral conditions  flanking both sides of the gene , CEU (Utah residents with Northern and Western European ancestry), and CHB (Han Chinese in Beijing) available from the 1000 Genome Project , the NeaGSTM1 deletion in 1KGP dataset, we used digital droplet polymerase chain reaction experiments to genotype the GSTM1 deletion in 17 humans from 1KGP  . We followed standard protocol provided by Bio-Rad EIF2C1 probe assay (Table S1).To validate the accuracy of the genotyping of the To investigate the accuracy of the 1KGP genotyping of single nucleotide variants, we investigated 74 CHB individuals and 6 sites in the target regions (chr1:110211681-110223007 and chr1:110246810-110255596) reported both in 1KGP and HapMap . Of all the 444 sites, 9 were “NN” in hapmap and 3 showed different results in the two databases. Overall, we found approximately %99.3 concordance between the two datasets.chr1:110216300-110225620 and chr1:110244324-110246143, the deletion was excluded), we found 14 variants among the 7 haplotypes . The only one that was not polymorphic among the seven haplotype dataset was rs61799140. This SNP has been detected by more than a dozen previous studies based on the dbSNP (https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=61799140). As such, we concluded that the absence of polymorphism in this location among the sequenced dataset is likely because it was not represented among the seven haplotypes, rather than it being a false-positive genotype in the 1000 Genomes dataset. Overall, we are confident that 1000 Genomes adequately captures the genetic variation in this locus and that the variant calling which our further analysis based on was accurate.To validate the accuracy of the phasing in 1KGP project data that we used in this study, we compared haplotypes on 50kb on each side of the deletion in our dataset with phased HapMap genomes. Specifically, we were able to find 322 heterozygous sites in 30 phased HapMap CEU genomes. 320 (∼99.4%) of these are concordant with the 1KGP dataset. We also investigated Sanger sequencing data from seven deleted haplotypes for which we generated long-range PCR amplicons for an earlier study , Utah residents with Northern and Western European ancestry (CEU) and Yoruba in Ibadan, Nigeria (YRI) populations in 1KGP phase 3 datasets (http://asia.ensembl.org/index.html) was used to calculate and visualize linkage disequilibrium (LD) between single nucleotide variations and that (or LD) between the deletion and single nucleotide variations. Based on the R2 values, we used two target regions for the statistical neutrality tests. Two target regions were selected for the following analyses; target1, upstream the deletion (chr1:110211681-110223007 in the hg19) and target2, downstream the deletion (chr1:110246810-110255596 in the hg19), which showed relatively high R2 with the GSTM1 deletion in CHB (a priori input of haplogroups nor the deletion status (Vcftools (n in CHB . We consn status .STF (GSTM1 is located (chromosome 1) and also match our target region by size (∼9kb). Then, we calculated these statistics observed in the downstream regions of the GSTM1 deletion .We first constructed a null distribution by calculating the FST , Tajima’FST , π  and nucleotide diversity (π).For the simulation analyses, we used ms  to conduThe command line for ms simulator was ./ms 422 1000 -t 91.4 -I 2 216 206 -m 1 2 3.2 -n 2 0.077 -en 0.0005 1 0.25 -en 0.001 2 0.077 -ej 0.00875 2 1 -en 0.0425 1 0.125 -r 5 8787.STF values between YRI and CHB populations for each single nucleotide variant that was generated in the simulations. We then plotted the values with the matching allele frequencies  for each of these variants. Next, we considered the distribution of the simulated π values. To simulate this, we first generated 1000 simulated datasets as described above. We then focused on the segregating sites with alternative allele frequencies at 0.69-0.70, the range of standard deviation of the observed allele frequency of the variations in Tanuki haplogroup. Based on this, we calculated π for each of these haplogroups which carry the alternative allele for each single nucleotide variation in the simulated CHB population .We calculated STF values are high  from the empirical observation. We ran more than 7000 simulations per each condition and obtained 70-120 core sites with the frequency of 0.69+-0.031  in East Asia and 0 in Africa under neutrality. We calculated both side of ROH for the 70-120 core sites from the simulation. We also calculated one side (downstream) of ROH of the 238 Tanuki homozygous individuals in East Asian populations for the Tanuki SNP as there was a recombination hotspot in the upstream of Tanuki region  in our dataset, we conducted bootstrap resampling of individual phased chromosomes. There are 238 individuals in East Asian populations  that are homozygous for Tanuki haplotype. To construct a distribution to investigate whether there is a sampling bias, we randomly resampled from 694 phased Tanuki haplotypes to generate 500 datasets each with 238 homozygous constructs. This allowed us to test whether the observed value of ROH deviates from randomly resampled distribution. We observed no such deviation, suggesting no sampling bias in our analysis .To test for potential sampling bias  in  and estiTanuki haplogroup from a non-Tanuki haplotype  to calculate a coalescent date for the divergence of these two haplogroups. Specifically, based on a more recent (5 million years (Tanuki haplogroup (0.001048 distance from the root haplotype) to be between 300 to 720 thousand years before present.Second, we used the average sequence differences of the on years ) and oldTanuki haplogroup based on the run-of-homozygosity in this locus with method detailed by −8/generation .Third, we calculated the starting date of the putative selection on It is important to note some caveats here. First, the coalescence dates depend on the local recombination and mutation rates, and even our best estimates may not be accurate with our current knowledge. Second, given that this locus has likely evolved under adaptive forces, it would be more appropriate to incorporate dating approaches that consider selection. However, we have little insight into the timing and the strength of the selection, and thus we focused on dating approaches that do not depend on selection coefficient as a parameter.GSTM1 copy number by ddPCR of 17 individuals in YRI population in the 1KGP. Supplemental materials are available at Figshare: Saitou, Marie; Gokcumen, Omer; Satta, Yoko (2018): G3-Supp0626.pdf.; https://doi.org/10.25387/g3.6384782.Table S1 contains genotyping of the GSTM1 deletion common in humans, we first attempted to resolve the haplotype structure of the locus. We calculated the R2 value between the GSTM1 deletion and flanking single nucleotide variants in Han Chinese in Bejing, China (CHB), Utah residents with Northern and Western European ancestry (CEU) and Yoruba in Ibadan, Nigeria (YRI) populations in 1KGP phase 3 datasets , and observable, but imperfect linkage disequilibrium in CHB only extending to >2.5kb on each side of the deletion  between variants on each side of the deletion in CHB  different deletion breakpoints, and (ii) LD to be intact between the variants on each side of the deletion as described by Boettger n in CHB . Overalli.e., in the last 1 Million years).To further understand the haplotypic variation in this locus, we constructed a phylogenetic tree using sequence variation data in the “target” region flanking the deletion for 620 phased haplotypes from YRI, CEU, CHB populations, as well as chimpanzee, Denisovan, Neanderthal genomes . As expeGSTM1 deletion and the neighboring variants reduces the power of disease-association studies  that rely on imputation of the GSTM1 deletion genotype using nearby single nucleotide variants. Even considering multiple single nucleotide variants within a single population, we were not able to impute the deletion accurately in any of the study populations   . This isGSTM1 deletion, we conducted statistical neutrality tests using single nucleotide variation data from the sequences flanking the GSTM1 deletion. Specifically, we first constructed a null empirical distribution by calculating the STF (GSTM1 is located (chromosome 1) and also match our target region by size (∼9kb). Then, we calculated these statistics observed in the downstream regions of the GSTM1 deletion and compared the results with those from the random regions . We found that both XP-CLR and XP-EHH tests showed significant differences as compared to neutrality when CHB population is involved . These tests measures the change in allele frequency in one population has occurred more quickly than expected by drift alone and the difference in extended homozygosity between two populations , indicating that they represent a single haplotype group in East Asian populations  in the target2 region , along with a non-Tanuki haplotype and the chimpanzee reference genome haplotype as outgroups. We surmised that Tanuki haplotype diverged from other human haplotypes at least 300 thousand years ago (∼300K to 720K YBP). Even considering the relatively large deviation of this estimate, this date is far earlier than the out-of-Africa migrations. As such, we conclude that this haplogroup has originated in Africa, but has increased in frequency in Eurasia. It is plausible that some Tanuki haplotypes still remain in African populations, but at a low allele frequency.To determine the age of this haplogroup, we constructed a phylogenetic tree  of all Tanuki haplotypes . We found that a vast majority of Tanuki haplotypes in CHB population has identical sequences, suggesting a rapid and recent increase in the allele frequency in the East Asian populations. This observation explains the unusually high XP-CLR and XP-EHH values observed for CHB population and further support the notion that a incomplete sweep has shaped the distribution of Tanuki haplogroup in this population.To further understand the haplotypic background of the significantly high XP-CLR and XP-EHH values in Asian populations as compared to European and African populations, we constructed a network of Tanuki haplogroup in CHB potentially point to the effect of an local sweep. The alternative, null hypothesis would be that the observed difference in allele frequency is due to the effect of drift alone. To further distinguish between these two scenarios, we conducted multiple empirical and simulation based analyses. First, we investigated whether the allele frequency of Tanuki haplogroup in East Asian and European populations can be explained by drift alone. To do this, we first assumed that the putative target for selection is an allele that reached to approximately 70% allele frequency in East Asia, but remain at 25% allele frequency in Europe . Specifically, we used ms . To verify the accuracy of our simulations, we first compared our simulated sequences to empirical distribution of STF between YRI and CHB populations for randomly selected single nucleotide variants across the genome  for each of these variants observed in the simulation. We found that none of 603 frequency-matched simulated STF is higher than the observed STF for the Tanuki haplogroup  in East Asians as compared to the nucleotide diversity observed in haplotypes that do not belong to the Tanuki haplogroup in the same population (π=0.00076).In parallel to the allele frequency based analyses described above, we conducted tests based on haplotype similarity. We first followed the reasoning outlined by Kim and Satta . BrieflyTanuki and nonTanuki haplotypes across a larger genomic region surrounding the GSTM1, including sequences that are not overlapping segmental duplications in multiple East Asian populations  . These results also corroborates our initial finding that Tanuki haplotypes show unusually low π as compared to simulated expectations . Then, we plotted the π values for these simulated haplogroups. The results showed that π observed in the Tanuki haplogroup is lowest among 389 frequency-matched simulated values  to construct an expected, neutral distribution of nucleotide diversity (π) values. Specifically, we wanted to test whether π calculated for the haplotypes that belong to the Tanuki haplogroup is also translated into extended homozygosity, a hallmark of a selective sweep, we simulated expected Runs of Homozygosity (ROH) assuming demographic parameters outlined in . The averaged ROH of the Tanuki haplogroup was 16Kb.To test whether the low nucleotide diversity observed in Tanuki Haplogroup began . The exact underlying evolutionary reason and the functional impact of the increase in frequency of the Tanuki haplogroup remain excellent venues for future studies. One obvious question is whether the GSTM1 deletion is the actual target of positive selection. Indeed, we discovered that Tanuki haplogroup is significantly, albeit imperfectly, linked with the GSTM1 deletion in the CHB population (R2=∼0.47) (Table S2). It is important to note that despite the low linkage disequilibrium, ∼91% of the Tanuki haplotypes harbor the GSTM1 deletion. We reasoned that if the GSTM1 deletion was the target for the selective sweep that we observed, then other haplotypes carrying the GSTM1 deletion also show evidence for selection. However, we did not observe any unusual increase in allele frequency of other haplotypes that carry the deletion. As such, it is highly plausible, as drastic an event as a whole gene deletion is, GSTM1 deletion may not be the main target of the selective sweep that we observed.By carefully resolving the different haplotype groups in the GSTM1 gene has no fitness consequences and hence accumulating loss of function mutations, such as the deletion. Under this scenario, the GSTM1 deletion just swept along with the remainder of the Tanuki haplogroup with scant adaptive consequences. Contradicting this possibility, it was reported that the GSTM1 gene sequence is conserved among great apes, as well as between humans and archaic humans. Specifically, we found only 4 nonsynomous variants between human and chimpanzee GSTM1, and found none among human, Neanderthal and Denisovan GSTM1 genes (Table S3). In addition, only two commonly observed nonsynonymous mutations were reported for GSTM1 gene in human population, both of which have little effect on the GSTM1 function . It is important to note that the deletion of the entire gene in approximately half of human genomes is not currently reported in ExAC database. However, our point here is that if GSTM1 has no fitness effect in humans, we expect to find relatively high frequencies of loss of function variants in addition to the deletion. Instead, virtually all the nondeleted GSTM1 haplotypes carry an intact, open-reading frame, further supporting the notion that both deleted and nondeleted haplotypes are maintained in the population.To further follow up this thread, we asked whether the GSTM1 deletion has fitness effects, but it is independent of the selective sweep we observed for the Tanuki haplogroup. To explain these observations, we hypothesize that Tanuki haplogroup affect the function of multiple genes in the GSTM gene family and not just GSTM1. Consequently, the putative adaptive phenotype is a result of combination of these effects. Such a perspective has precedent where other studies described likely adaptive haplotypes that have effects on multiple metabolism gene families, such as IRX  . As such, one potential argument would be that the selected effect is not the functioning of a single gene, but the overall regulatory impact of the Tanuki haplogroup on the GSTM locus.To test whether GSTM1 deletion and neighboring variants. This finding corroborates our previous work at a deeper evolutionary depth, where we found evidence for multiple gene conversion events differentiating human and chimpanzee GSTM locus , statistical power for a single nucleotide variant based genome-wide association study would still be low. For example, based on e.g., the odds ratio of the phenotype 1.3, 1000 cases and 1000 controls, etc.).Our results provide a case study for the evolutionary impact of common haplotypic variation in a complex locus, involving both functional single nucleotide and copy number variants. One particular challenge in this locus that we verified is the general lack of linkage disequilibrium between common, neighboring variants. Specifically, we found a lack of strong linkage disequilibrium between the 2 < 0.6 with neighboring variants  in East Asian populations, putatively affecting the function of multiple GSTM genes  variants were often considered together as the leading candidates for explaining the genetic basis of bladder cancer susceptibility  gene family, have also been shown to be evolving adaptively (UGT2B17 (uridine diphosphoglucuronosyl-transferase) has been shown to have increased in frequency in East Asian populations under adaptive forces (These insights fit well with our observations for the GSTM locus has been the subject of more than 500 studies within the context of multiple diseases (GSTM1-5) that broadly metabolizes multiple carcinogenic substances, for example, 4-nitroquinoline-1-oxide (NQO)  carrying the deletion allele that has likely evolved under non-neutral conditions and reached a high allele frequency in East Asian populations. This haplogroup has a broad regulatory effect on the metabolizing GSTM gene family. Overall, our study adds to the emerging notion that complex loci involving and structural variants may contribute to adaptive and biomedically relevant phenotypic variation (In this study, we report a particular haplogroup (


Il11SMC). Within days of transgene activation, Il11SMC mice developed loose stools and progressive bleeding and rectal prolapse, which was associated with a 65% mortality by two weeks. The bowel of Il11SMC mice was inflamed, fibrotic and had a thickened wall, which was accompanied by activation of ERK and STAT3. In other organs, including the heart, lung, liver, kidney and skin there was a phenotypic spectrum of fibro-inflammation, together with consistent ERK activation. To investigate further the importance of stromal-derived IL11 in the inflammatory bowel phenotype we used a second model with fibroblast-specific expression of IL11, the Il11Fib mouse. This additional model largely phenocopied the Il11SMC bowel phenotype. These data show that IL11 secretion from the stromal niche is sufficient to drive inflammatory bowel disease in mice. Given that IL11 expression in colonic stromal cells predicts anti-TNF therapy failure in patients with ulcerative colitis or Crohn’s disease, we suggest IL11 as a therapeutic target for inflammatory bowel disease.Interleukin 11 (IL11) is a profibrotic cytokine, secreted by myofibroblasts and damaged epithelial cells. Smooth muscle cells (SMCs) also secrete IL11 under pathological conditions and express the IL11 receptor. Here we examined the effects of SMC-specific, conditional expression of murine IL11 in a transgenic mouse ( Non-striated smooth muscle cells (SMCs) line the walls of hollow organs and the vasculature. In adults, SMCs are not terminally differentiated and their cellular phenotype remains plastic. A variety of extracellular cues such as humoral factors, mechanical or oxidative stress and cell-cell interactions can induce a spectrum of cellular states ranging from contractile SMCs to highly synthetic and proliferative SMCs . SynthetIl11 transgenic mouse to overexpress mouse Il11 in myosin heavy chain 11 (Myh11)-positive smooth muscle cells (Il11SMC). Here we characterized key organs that may be affected by SMC pathobiology in Il11SMC mice to better understand the role of SMC-derived IL11.We recently identified IL11 as a critical driver of fibroblast activation in the cardiovascular system, liver and lung downstream of a variety of pro-fibrotic factors including TGFB1 –10. In aGuidelines on the Care and Use of Animals for Scientific Purposes of the National Advisory Committee for Laboratory Animal Research (NACLAR). All experimental procedures were approved (2014/SHS/0925) and conducted in accordance to the SingHealth Institutional Animal Care and Use Committee (IACUC). All mice were from a C57BL/6JN genetic background and were bred and housed in individually vented cages in the same room under ABSL-1 conditions in the SingHealth Experimental Medicine Centre and provided normal chow  and water ad libitum. All research staff involved in animal studies underwent the Responsible Care and Use of Laboratory Animal Course  prior to study commencement. Animals were euthanized at endpoint by ketamine (100 mg/kg) and xylazine (10 mg/kg) given IP, followed by the removal of vital organs and tissues.This study was carried out in compliance with the recommendations in the Mice were scruffed to restrict motion during tamoxifen administration IP and monitored daily for clinical signs of distress and body weights were measured thrice per week upon tamoxifen induction. When rectal inflammation/bleeding was observed, the wound was gently disinfected with 70% methylated spirits and 10% povidone-iodine. Mice that displayed behavioral abnormalities, weight loss, and/or rectal bleeding were therapeutically treated with buprenorphine (0.1 mg/kg SQ) and enrofloxacin (5 mg/kg SQ) where necessary. Animals that did not recover with treatment or presented with deteriorated symptoms including pronounced weight loss (>20% over 1 week or >10% over 24 hours) and the development of rectal prolapse were euthanized following consultation with a veterinarian prior to the study endpoint and were treated as deaths.Rosa26-Il11 (Gt(ROSA)26Sortm1(CAG-Il11)Cook) mouse Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: NoReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: NoReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: In my opinion this manuscript present major multiple concerns. Of note, while the most striking feature of this mouse model is supposed to be the development of intestinal inflammation resembling inflammatory bowel disease (IBD) (as highlighted in the title), the main feature seems to be involving global and multi-organ abnormalities leading to 37% of surviving mice to day 14. The authors try to describe the development of an IBD-like phenotype with just few supporting data that actually reduce my confidence in the overall study.1)A great deal rests on the characterization of intestinal inflammation. Fecal calprotectin should not be used as the only parameter to properly assess intestinal inflammation. A more detailed assessment of intestinal inflammation should be performed by an expert GI pathologist, who should be able to provide details about location of the inflammation , active inflammation, chronic inflammation, transmural inflammation, percentage of ulceration etc.2) Fibrosis is typically a long process. How do the authors explain the high severity of fibrosis in the different organs following just 1 or 2 weeks of tamoxifen injection?3)In figure 2d, why is the color of the of stained section so different? It looks like a different method for staining was used.4)In figure 3 the western blots are not convincing. The author should show in the main figure the whole gel but not just sections of it. P-STAT3 has been described in the results to be unchanged in heart, lung and liver. Looking at the blots, it looks like that also in the skin there are not changes. Quantification of the bands and statistical analysis should have been also reported in main figure.5)How much IL-11 different tissues make? Is there a way to measure IL-11 protein production from tissue explants? Can the author stain (i.e. by IHC) IL-11 and correlate with levels of intestinal inflammation and fibrosis?6)It is said in multiple places that IL11 secretion from smooth muscle cells drive fibrogenic gene expression. How IL-11 secretion was measured?7)Similarly, which cells in the intestine express the receptor for IL-11 and is there a hint about the function?8)Again, a more detailed assessment of intestinal inflammation should be performed by an expert GI pathologist for the second model described.9)The main effect of IL11 expression seems to be induction of fibrosis across organs. This is not recapitulated in the second model of IL11 expression in fibroblasts. Is the level of calprotectin enough to claim that the second model recapitulates the colonic inflammatory phenotype seen in IL11smc mice? This is a major concern.Reviewer #2: With this manuscript Authors examined the effects of SMC-specific, conditional expression of murine IL11 in a transgenic mouse (Il11SMC). They show that IL11 secretion from the stromal niche is sufficient to drive an IBD-like inflammation in mice. Overall, the manuscript is very well written and the data and overall message are convincing and an important advance in the field. The methods are robust, and the results support the conclusions.Authors should however better discuss the histologic and mucosal differences between the model presented and IBD in order to weight the real impact of this model on the knowledge of IBD pathogenesis.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  16 Nov 2019Dear Cristiano Pagnini,Thank you for sending our paper to review, we are grateful for the opportunity to improve our manuscript further and trust that the updated version addresses your concerns.The manuscript now follows PLOS ONE’s style requirements and we report additional details in your Methods section regarding animal care. Original uncropped and unadjusted blot/gel image data were in S2 Fig .We were extremely encouraged by the comments of Reviewer 2 who stated “Overall, the manuscript is very well written and the data and overall message are convincing and an important advance in the field. The methods are robust, and the results support the conclusions”. This is in keeping with some reviews of our recent work at other journals. We were, therefore, most surprised by the comments of Reviewer 1 that are very much at odds with those of Reviewer 2, indeed at the opposite end of the spectrum. We believe Reviewer 1’s comments are misplaced and based on some erroneous interpretation of the data. For example, reviewer 1 ignores RNA-based inflammation markers that we show throughout the manuscript. The reviewer also disregards the clinically important disease endpoints of IBD such as florid diarrhoea, rectal prolapse, and bleeding and asks instead for more detail of intermediate phenotypes, which do not predict disease severity or patient outcomes. Taken together with the well-established marker for intestinal inflammation calprotectin, our data show that the genetic models develop an IBD phenotype. IL11 is highly upregulated in human IBD patients - notably in stromal cells in the bowel wall - and its expression predicts treatment failure (we cite three studies showing this) and thus our data showing that IL11 causes the IBD with clinically relevant disease phenotypes will be of great interest: it establishes IL11 as an IBD disease gene for the first time. Despite these concerns, we have addressed the points raised by the reviewers. In particular, we performed a number of new experiments and provide substantial additional data and analyses to address reviewer 1’s comments. The manuscript has been revised accordingly and is further improved. We also adjusted the title to specifically address a concern of Reviewer 1. We point out reviewer 2 asked only for a change to the discussion, which we provide.We strongly believe that this manuscript will be of broad interest to the readership of the journal and presents novel and clinically relevant results that may influence therapy.Please find below our detailed responses and we look forward to hearing from you soon.Sincerely,Sebastian Schäfer and Stuart Cook Point-by-point response the ReviewersReviewer #1: In my opinion this manuscript present major multiple concerns. Of note, while the most striking feature of this mouse model is supposed to be the development of intestinal inflammation resembling inflammatory bowel disease (IBD) (as highlighted in the title), the main feature seems to be involving global and multi-organ abnormalities leading to 37% of surviving mice to day 14. The authors try to describe the development of an IBD-like phenotype with just few supporting data that actually reduce my confidence in the overall study.While we focused our studies on the most prominent phenotype in transgenic mice - the IBD phenotype - we studied multiple organs and reported data across these organs. As stated in the abstract “The bowel of Il11SMC mice was inflamed, fibrotic and had a thickened wall, …. In other organs, including heart, lung, liver, kidney and skin there was a phenotypic spectrum of fibro-inflammation.” As such, this manuscript is not only about the bowel and IL11 is thought to be important in a range of fibro-inflammatory diseases. However, the bowel phenotype was the most prominent and, see our next answer, the upregulation of IL11 in IBD patients makes this aspect of the animal model particularly exciting. We disagree with the reviewer that we presented “just few” supporting data for the IBD phenotype and now provide a large amount of additional layers of evidence, as requested by this reviewer (see below). To address the reviewers concerns regarding the overall message of the manuscript, we have adjusted the title to include reference to the cross-tissue inflammation while retaining the focus of the manuscript on the overt IBD phenotype. The revised title now reads “Transgenic interleukin 11 expression causes cross-tissue fibro-inflammation and an inflammatory bowel phenotype in mice”. We trust this addresses the reviewer’s concern on this matter. 1) A great deal rests on the characterization of intestinal inflammation. Fecal calprotectin should not be used as the only parameter to properly assess intestinal inflammation. A more detailed assessment of intestinal inflammation should be performed by an expert GI pathologist, who should be able to provide details about location of the inflammation , active inflammation, chronic inflammation, transmural inflammation, percentage of ulceration etc.The manuscript does not rely solely on the characterization of intestinal inflammation. It is an established fact that IL11 is the most upregulated cytokine in non-responding patients suffering from Crohn’s Disease or Ulcerative Colitis . Thus, by definition, IL11 is a gene relevant in a range of IBDs. This manuscript addresses a critical gap in the literature, where no gain-of-function studies exist that examine the effect of species-matched mouse IL11 on the murine gut. We overexpress mouse IL11 in the mouse in two cell-types important for IBD: Fibroblasts and SMCs. Hence, this model should give important insights into the pathogenesis of human disease related to the disease gene IL11. We point out that this is a rapid model that develops in weeks and not a chronic inflammation model. Inflammation is present throughout the bowel in both models, as shown in Fig 2b and new Fig 8b, however we have focused on the colon phenotype due to overt characteristics of rectal prolapse suggesting the lower bowels to be highly inflamed. Additionally, while there is no obvious ulceration in our models, we have however noticed significant leukocyte aggregation structures and inflammation in some sections - see new data Fig 2h and Fig 3- and mice have died with blood in the intestinal cavity that suggests acute ulceration and bleeding that results in death. In addition, we did not rely solely on fecal calprotectin to assess intestinal inflammation but also profiled other well established pro-inflammatory markers such as Il-6, Ccl2, and Ccl5 on the RNA level , which are all significantly elevated in the bowel of Il11SMC mice. Gross histology also clearly shows red and swollen intestines that are clearly indicative of a prominent inflammatory response in this organ. Most importantly, IBD is characterized by a number of pathologies in addition to inflammation. The Il11SMC model presented here also presents with acute large and small intestine disease  along with florid diarrhea, rectal prolapse, and bleeding. This is accompanied by prominent fibrosis. In the revised manuscript, we have followed up on the reviewer’s comments on other markers of inflammation and immunohistochemically stained for IL11, together with CD45 (leukocytes), LAMP-2, and LGALS3, markers that are commonly present in activated epithelial cells and immune cells. These, notably LGALS3, are elevated in the Il11SMC colon and there is widespread activation within Payer’s patches upon IL11 expression in SMCs .2) Fibrosis is typically a long process. How do the authors explain the high severity of fibrosis in the different organs following just 1 or 2 weeks of tamoxifen injection?IL11 is an extremely potent pro-fibrotic factor. We have shown previously that IL11 can induce fibrosis in the cardiovascular system , lung  and liver  within just weeks. Transgenic overexpressing of IL11 in fibroblasts or SMCs was hypothesized to be highly fibrogenic and the results shown in the manuscript are in line with previous observations. The data also show that IL11 expression in the stromal compartment is pro-inflammatory, which is a novel finding. 3)In figure 2d, why is the color of the of stained section so different? It looks like a different method for staining was used.We thank the reviewer for raising this point. Identical methods for staining were used, but sections were done in batches which resulted in slight color differences. Images have been replaced with representative images from identical batches.4)In figure 3 the western blots are not convincing. The author should show in the main figure the whole gel but not just sections of it. P-STAT3 has been described in the results to be unchanged in heart, lung and liver. Looking at the blots, it looks like that also in the skin there are not changes. Quantification of the bands and statistical analysis should have been also reported in main figure.We thank the reviewer for raising this point and agree with the suggestion that will improve the manuscript. We have now moved the uncropped blots from Fig S2 to Fig 4 along with the densitometric analyses. Quantification and statistical analysis are now also shown in the main part of the manuscript. Dermal stat activation is weak, but densitometry and statistical analyses indicate that P-STAT3 is slightly elevated in the skin. Thus we are hesitant to state there are no changes.5)How much IL-11 different tissues make? Is there a way to measure IL-11 protein production from tissue explants? Can the author stain (i.e. by IHC) IL-11 and correlate with levels of intestinal inflammation and fibrosis?We showed IL11 protein upregulation across tissues with quantification and statistics . However, the actual level of IL11 production and its effects on the tissue level is highly dependent on the cellular composition of both the IL11 source (SMCs/fibroblasts) and the target cells (SMCs/fibroblasts/epithelium etc). As there are many possible cellular targets and the exact tissue composition is unknown, we do not believe that a simple correlation will provide any useful insights and point to the clinically relevant endpoints as the most important and defining phenotypes. It is the case that following this first definitive report of IL11 as an IBD gene that further and more detailed studies of cell-type effects will need to be studied using conditional gene knockouts and reporter strains to dissect inflammation-fibrosis cross-talk, which we are currently planning. We hope the reviewer can appreciate this fact.6)It is said in multiple places that IL11 secretion from smooth muscle cells drive fibrogenic gene expression. How IL-11 secretion was measured?IL11 is a secreted protein by its very nature as a cytokine via a protein motif that targets it to the extracellular space. The genetic models we utilize specifically induce IL11 protein expression in the target cells (either SMCs or fibroblasts). Western blots clearly showed upregulation of IL11 protein.7)Similarly, which cells in the intestine express the receptor for IL-11 and is there a hint about the function?https://doi.org/10.1101/801852The reviewer raises an interesting question and there is more than one answer. Smillie et al. Cell 2019 performed a single-cell study of human UC colon and found IL11RA expressed predominantly on fibroblasts and smooth muscle cells. The effect of IL11 on fibroblasts has been well documented by our group in a number of studies. More recently, we have found that IL-11 on fibroblasts is a potent driver of stromal inflammation: Ng B et al. Fibroblast-specific IL11 signaling is required for lung fibrosis and inflammation bioRxiv doi: We suspect that this also plays a role in IBD, as Smillie et al. have also found an “Inflammatory Fibroblast” to be underlying anti-TNF resistance in IBD. However, this requires further study and is beyond the scope of this manuscript. We point out that IL11RA is also expressed on epithelial cells at high levels and it could well be that IL11 secreted from the stroma then impinges on the IECs but investigating this will again require further study.8)Again, a more detailed assessment of intestinal inflammation should be performed by an expert GI pathologist for the second model described.As with the first Il11SMC model, we show inflammation in the second Il11Fib model via calprotectin as well as elevated RNA levels of IL6. We now also show gross histology of this model, which clearly indicates a swollen and inflamed bowel that recapitulates the first model . A significant reduction in colon length was also indicative of murine colitis, which was also shown. Again we would like to point out that the clinically meaningful endpoint of disease is ultimately more important than intermediate phenotypes that may or may not be present in animal models that variably recapitulates all the salient features of human disease. 9)The main effect of IL11 expression seems to be induction of fibrosis across organs. This is not recapitulated in the second model of IL11 expression in fibroblasts. We cited a number of publications where we have previously shown that the second model (Il11Fib) develops severe fibrosis: Cardiovascular , pulmonary  and liver  fibrosis. Perhaps the reviewer missed our citation of these papers that explain this model in some more detail. We point out that it is not expected that transgenic models with Il11 expression in SMC or fibroblasts will behave identically as these are - by definition - different cell types with different functions. Indeed SMC may be regarded as a more ‘inflammatory’ cell type as compared to fibroblasts, by some .Is the level of calprotectin enough to claim that the second model recapitulates the colonic inflammatory phenotype seen in IL11smc mice? This is a major concern.As we discuss in response to questions above, we do not base our observations solely on the elevation of calprotectin. We also do not want to argue, that the Il11Fib or the Il11SMC model are exact phenocopies. Indeed, they are different models and should be investigated as such. We have now amended the text to make this clearer and would like to thank the reviewer for pointing this out. We now present additional data on the second model .Reviewer #2: With this manuscript Authors examined the effects of SMC-specific, conditional expression of murine IL11 in a transgenic mouse (Il11SMC). They show that IL11 secretion from the stromal niche is sufficient to drive an IBD-like inflammation in mice. Overall, the manuscript is very well written and the data and overall message are convincing and an important advance in the field. The methods are robust, and the results support the conclusions.We thank the reviewer for his supportive comments.Authors should however better discuss the histologic and mucosal differences between the model presented and IBD in order to weight the real impact of this model on the knowledge of IBD pathogenesis.We now discuss the histologic differences between the Il11SMC and Il11Fib models and human IBD in greater detail in the manuscript. However, we would like to point out that IL11 upregulation in the colon has been repeatedly detected in IBD patients . Thus, the mechanisms present in the IL11 transgenic animals are highly clinically relevant, even if they do not completely phenocopy human disease (not uncommon in rodent models).AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 20 Dec 2019Transgenic interleukin 11 expression causes cross-tissue fibro-inflammation and an inflammatory bowel phenotype in micePONE-D-19-24461R1Dear Dr. Lim,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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The addition of water on or below the earth’s surface generates changes in stress that can trigger both stable and unstable sliding of landslides and faults. While these sliding behaviours are well-described by commonly used mechanical models developed from laboratory testing , less is known about the field-scale environmental conditions or kinematic behaviours that occur during the transition from stable to unstable sliding. Here we use radar interferometry (InSAR) and a simple 1D hydrological model to characterize 8 years of stable sliding of the Mud Creek landslide, California, USA, prior to its rapid acceleration and catastrophic failure on May 20, 2017. Our results suggest a large increase in pore-fluid pressure occurred during a shift from historic drought to record rainfall that triggered a large increase in velocity and drove slip localization, overcoming the stabilizing mechanisms that had previously inhibited landslide acceleration. Given the predicted increase in precipitation extremes with a warming climate, we expect it to become more common for landslides to transition from stable to unstable motion, and therefore a better assessment of this destabilization process is required to prevent loss of life and infrastructure. These various sliding behaviours are described by the laboratory-based critical-state soil mechanics5 and rate-and-state friction15 models. In general, stable sliding should occur if the frictional resistance increases during sliding and unstable sliding should occur if the frictional resistance decreases during sliding. These changes in the frictional resistance are controlled by material properties16 and/or from shear-induced changes in pore-fluid pressure17. Although these mechanical models can be used to describe various sliding behaviours, less is known about the field-scale environmental conditions or kinematic behaviours that occur during the transition from stable to unstable sliding. In this manuscript, we present new remote sensing observations that show the behaviour of a landslide that transitioned from stable to unstable sliding.Stress and fluid-pressure perturbations from changes in infiltrating precipitation and snowmelt18. Recent studies have investigated the role of climate change on slope stability using historical records of temperature, precipitation, and landslide events with future predictions from downscaled Global Circulation Models20. These studies have found that the occurrence or activity of landslides is expected to increase in areas where the frequency and magnitude of precipitation is predicted to increase and decrease in areas where the frequency and magnitude of precipitation is predicted to decrease. However, interactions between ongoing climate shifts and landslide behaviour are difficult to assess due to uncertainties in both climate and landslide models. Documenting the behaviour of landslides in response to ongoing climate shifts is essential for improving our understanding of the mechanisms that control their velocity and our ability to forecast catastrophic failure.Ongoing natural and man-made climate shifts  in response to increasing concentrations of greenhouse gases are predicted to increase the frequency and magnitude of geohazards such as landslides27. Slow-moving landslides mainly occur within areas of the Coast Ranges that are comprised of the Jurassic-Cretaceous Franciscan mélange, an uplifted accretionary prism complex consisting of a clayey granular matrix with highly sheared sandstone, siltstone, meta-sandstone, shale, serpentinite, and blueschist28. More specifically, the landslides occur in weak, clay-rich regolith29. The California Coast Ranges have a Mediterranean Climate with average precipitation values of ~1.0 m/yr in the Central Coast Ranges and ~1.5 m/yr in the Northern Coast Ranges, 80% of which falls between October and May. However, precipitation can vary significantly from year to year due to changes in the frequency and strength of atmospheric rivers31, El Niño-Southern Oscillation events32, and over longer time scales with the Pacific Decadal Oscillation33. For instance, between 2015 and 2017 there was a transition from historic drought to one of the wettest years on record. Predictions from the Community Earth System Model Large Ensemble (CESM) indicate that over the next century annual mean precipitation could increase up to 8 cm in Central California and 14 cm in Northern California34. CESM simulations also predict a 100 to 200% increase in cumulative seasonal precipitation and a 25 to 100% increase in the frequency of dry-to-wet year extremes that may be similar to the recent precipitation shifts in California35.The California Coast Ranges are an ideal natural laboratory to investigate how stress and fluid pressure changes govern the stable and unstable sliding of landslides. Landslides are pervasive in these mountains due to active uplift, mechanically weak lithologies, and high seasonal precipitation25. Yet, the Mud Creek landslide, which is located within the Franciscan mélange  that resulted in closing of the road between May 2017 and July 2018, however the landslide resulted in a cost of $54 million in repairs38. Since its construction in the 1920’s, CA1 has been closed more than 50 times due to landslides39, and based on the predictions of future climate change35, landslide frequency and activity is likely to increase over the next century.More than 650 slow-moving landslides have been identified and mapped in the California Coast Ranges in recent years, many of which have been active for decades or longer, and almost none of which have failed catastrophicallynge Fig. , suddenlIn this study, we investigate how a shift from drought to extreme rainfall can affect the behaviour of landslides. We measure the time-dependent deformation of three landslides from satellite and airborne InSAR and model precipitation-induced changes in pore-fluid pressure using a simple homogenous 1D diffusion model. We use our remote sensing observations and hydrologic models to explore interactions between precipitation and landslide kinematics. We then discuss our results in the context of the critical-state soil mechanics and rate and state friction fault mechanics frameworks.2 prior to catastrophic failure, but the exposed failure scar has a minimum area of 0.079 km2. This threefold difference in area indicates that slip localized along a different lateral  sliding surface.We use satellite and airborne InSAR data to measure the deformation history of the Mud Creek landslide between 2009 and 2017 and two other landslides (Paul’s Slide and Boulder Creek landslide) in the California Coast Ranges between 2015 and 2017 (Methods). This time period is particularly well-suited for studying the effects of rainfall on landslide behaviour because it spanned the final year (2015) of a historic drought and one of the wettest years on record (2017). The Mud Creek landslide displayed a minimum of 8 years of stable sliding prior to its catastrophic failure , and the velocities within the headscarp region were higher than they had been during the previous two years  pore-fluid pressure increase that causes a reduction in frictional strength and drives accelerated slip varied drastically between WY2015 and WY2017. We used a simple, homogenous 1D diffusion model to simulate the changes in subsurface pore-fluid pressure induced by precipitation events at Mud Creek (Methods). Because we have no direct measurement of pore-fluid pressure, diffusivity, or thickness from these landslides, we explored relative changes in the normalized pore-fluid pressure, defined as the pore-fluid pressure divided by the maximum value over the study period. Our results show that the minimum pore-fluid pressures occurred during the drought of WY2015 and approximately tripled by WY2017 Fig. . Assumin42. These landslides occur within similar lithology and in similar climates to Mud Creek  intrinsic slip- or rate-strengthening material properties15. Additional stabilization may result from an increase in a critical nucleation size relative to the slip patch size. This is an important length scale because rate-and-state friction theory predicts unstable sliding should only occur if (i) the frictional strength decreases during slip events and (ii) the slip patch exceeds the critical nucleation size defined by h* = Gdc/σ’(b-a), where G is the shear modulus, σ′ is the effective normal stress , b and a are friction parameters, and dc is the characteristic slip distance for the evolution of frictional contacts15. Importantly, this length scale suggests that that the increased pore-fluid pressure that drives acceleration also acts to increase the critical nucleation size, and therefore predicts that an increase in pore-fluid pressure should drive increased slip but should not trigger runaway instability. Yet, landslides and faults can both display dynamic sliding due to increased pore-fluid pressures11.During stable sliding, a balance must exist between transient weakening from pore-fluid pressure increase due to precipitation that drives increased slip rates and (i) transient strengthening from a decrease in pore-fluid pressure due to dilation and drainage15. However, we observed that the Mud Creek landslide decreased in size during catastrophic failure , which currently do not describe how large increases in pore-fluid pressure can trigger dynamic instability on stable sliding surfaces and suggests that modifications that include descriptions of critical-state mechanics may be required to better characterize these sliding behaviours.Our findings agree with recent laboratory investigations that found that large pore-fluid pressure increases from wastewater and fluid injection can trigger earthquakes along otherwise stable faults and provide additional field-scale evidence that large stress changes can lead to a transition in stability regimes35, we hypothesize that there may be an increase in the occurrence and activity of landslides in California. And furthermore, there may be an increase in the transition from stable to unstable landslide motion if these precipitation extremes generate sufficiently high pore-fluid pressures. Our results highlight how state-of-the-art InSAR data can be used to quantify the deformation history of landslides prior to catastrophic failure, especially in areas where timely installation of on-the-ground instrumentation is not feasible. We suggest that changes in the long-term characteristic deformation patterns may provide a way to identify changes in landslide stability regimes and to forecast imminent failure, especially on landslide-prone transport corridors such as California Highway 1.Because precipitation and precipitation extremes (i.e. dry to wet years) are predicted to rise in California over the next several decades51. We use a 12 m pixel spacing digital elevation model from the DLR TanDEM-X to remove topographic contributions to the phase and to geocode the interferograms. We apply a standard power spectral filter with a value of 0.5 to reduce phase noise52. The Sentinel-1A/B satellites acquire C-band (5.6 cm radar wavelength) images with a minimum repeat acquisition interval of 6 days  and descending  data collected in the Interferometric Wide swath mode, which has a 250 km swath with 14 m azimuth and 5 m range pixel resolution. Data is processed with 2 looks in range and 1 look in azimuth. We process interferograms with time spans ranging from 6 to 48 days. Prior to our data analysis we removed all poor quality interferograms that have low coherence. Poor quality interferograms were identified by manual inspection of each individual interferogram. The interferograms used in our analysis are listed in Supplementary Tables We perform interferometric processing of synthetic aperture radar (InSAR) images acquired by the Copernicus Sentinel-1 A/B satellites and the airborne NASA Uninhabited Aerial Vehicle Synthetic Aperture Radar (UAVSAR). The InSAR data are processed with the InSAR Scientific Computing Environment (ISCE) software package developed at JPL/Caltech and Stanford25. To overcome issues associated with these deformation thresholds, we used a scalable model to correct for large deformation at the Mud Creek , Iwrap is the filtered original interferograms, j is the basic imaginary unit, and Imodel is the unwrapped deformation model. (4) We unwrap the residual interferogram, Ir using the Statistical-Cost, Network-Flow Algorithm for Phase Unwrapping (SNAPHU)53. (5) Finally, we add the deformation model back to the unwrapped residual interferogram. Each interferogram is inspected to ensure no additional errors are introduced. Refs25 successfully implemented this technique to process interferograms that were previously limited by deformation thresholds at slow-moving landslides in the California Coast Ranges.We process the InSAR data using conventional 2-pass techniques. However, given the large deformation of these landslides, conventional InSAR techniques sometimes fail because InSAR requires the displacement between adjacent pixels to be less than half the radar wavelength for accurate phase unwrapping54. We use the Small Baseline Subset (SBAS) method56 and apply a coherence threshold of 0.2. This coherence filter removes low quality pixels from the time series analysis 36, which is well beyond the deformation threshold of InSAR25, we were not able to reliably measure the last interval. We compared the velocity time series from the ascending and descending tracks and find good agreement of the temporal pattern of the velocity  motion onto the downslope direction57We combine data with 3 LOS measurements (i.e. UAVSAR and Sentinel-1 A/B ascending and descending) to invert for the full 3D velocity field at the Mud Creek landslide  is the LOS measurement (N = 3), A (N x 3) is the model matrix with LOS vectors projected onto the basis vectors such thatQ is the estimated covariance matrix. We calculate Q from the pixel correlation using the Cramer-Rao bounds58γ is the pixel coherence and λ is the radar wavelength.The LOS measurements  to define the surface boundary conditionP is the ground surface pore-fluid pressure, and r is an empirically calibrated infiltration factor that must account for hydraulic properties  and scales the measured precipitation rate to a pressure value. Alternatively, the downward water flux can be specified at the ground surface, and that boundary condition provides essentially the same result59. A solution for equation , even small data noise can lead to a high variation in the strain rate tensor. For example, 1 mm of measurement error in 30 m distance contributes to ~33 micro-strain rate. We therefore calculate the strain rate tensor of one grid point by considering a range of InSAR pixels. We use 30 m grid points and estimate the strain rate based on the 2D horizontal mean velocities of Mud Creek between 2009 and 2017, neglecting the vertical component. Since we assume no discontinuity in space and no vertical strain, we can use a plane-strain mass continuity equationa1, a2, and a3 are fit parameters. Figure For this, we assume zero vertical strain. The principal strain rate values and orientations are essentially the eigenvalues and eigenvectors of the strain tensor, and the dilatational strain is the sum of these eigenvalues. A 2D strain rate tensor can be calculated with at least 3 measurements in space. Because our InSAR measurements provide high spatial coverage (~150 mAll computer codes used in this work is available from the authors upon reasonable request.Supplemenary Movie 1Extended DataSupplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4Supplementary Table 5