Coronary vasospasm is a known side effect of 5-FU (fluorouracil) therapy. Beyond switching to non-5FU-based chemotherapy, there are no established treatments for 5-FU associated coronary vasospam. Our objective was to assess the safety and efficacy of re-challenge with 5-FU after pre-treatment with calcium channel blockers (CCBs) and long-acting nitrates among patients 5-FU associated coronary vasospasm.We conducted a retrospective study of patients with 5-FU coronary vasospasm at a single academic center. By protocol, those referred to cardio-oncology received pre-treatment with either combination [nitrates and CCBs] or single-agent therapy [nitrates or CCBs]) prior to re-challenge with 5-FU. Our primary outcome was overall survival. Other important outcomes included progression-free survival and safety.Among 6,606 patients who received 5-FU from January 2001 to Dec 2020, 115 (1.74%) developed coronary vasospasm. Of these 115 patients, 81 patients continued 5-FU therapy, while 34 stopped. Of the 81 who continued, 78 were referred to cardio-oncology and prescribed CCBs and/or nitrates prior to subsequent 5-FU, while the remaining 3 continued 5-FU without cardiac pre-treatment. Of the 78, 56.4% (44/78) received both nitrates and CCBs, 19.2% (15/78) received CCBs alone, and 24.4% (19/78) received nitrates alone. When compared to patients who stopped 5-FU, those who continued 5-FU after pre-treatment (single or combination therapy) had a decreased risk of death  and a trend towards decreased cancer progression . No patient in the pre-treatment group had a myocardial infarct after re-challenge; however, chest pain  recurred in 19.2% (15/78) among those who received cardiac pre-treatment vs. 66.7% (2/3) among those who did not (P = 0.048). There was no difference in efficacy or the recurrence of vasospasm among patients who received pre-treatment with a single agent (nitrates or CCBs) or combination therapy (14.7% (5/34) vs. 25.0% (11/44), P = 0.26).Re-challenge after pre-treatment with CCBs and nitrates guided by a cardio-oncology service was safe and allowed continued 5-FU therapy. HoweverThe incidence of recurrent cardiotoxicity with 5-FU rechallenge without any adjustment in treatment is 90% , 7. TherWe conducted a retrospective analysis of all patients who received 5-FU at a single academic center  from January 2001 to December 2020. Individuals were flagged based on keyword search for ‘5-fluorouracil’ in their charts using RPDR (Research Patient Data Registry). 6,606 individual patients were identified from this search. The results were narrowed based on keyword search for ‘5-fluorouracil’ and ‘vasospasm’ among these individuals. Vasospasm was defined as the new occurrence of a typical chest pain syndrome at rest in the presence of recent 5-FU with or without ECG or biomarker changes. We further searched the list of 6,606 patients for the diagnosis of myocardial infarction based on ICD  codes, entered within a year of receiving 5-FU, to ensure completeness of our dataset. With this combined approach, 115 patients with vasospasm were identified. The diagnosis of 5-FU associated coronary vasospasm was independently adjudicated by two cardiologists. Demographics, baseline medical history and last known follow-up or date of death were collected by manual chart review from electronic medical records. Baseline parameters were identified from the oncology consult note prior to 5-FU initiation. Data regarding 5-FU dosing were obtained from pharmacy dispense records. The institutional review board approved the study, and the requirement for written informed consent was waived.Referral to cardio-oncology was based on provider preference . Once paBaseline characteristics are presented as continuous variables summarized as mean ± standard deviation (SD), or categorical variables summarized as counts with percentages (%). Differences between continuous variables were assessed using the t-test, and between categorical variables using the chi-square test. Overall survival (OS) and progression free survival (PFS) between the two groups were analyzed using Kaplan-Meier curves and log-rank test. A multivariable variable Cox-regression model was constructed to adjust for baseline variables that were differentially distributed between the two groups. All statistical tests were 2-sided and 5% was set as the level of significance. Statistical analysis was performed using STATA version 15.1 .Among 6,606 individual patients who received 5-FU, 115 patients (1.74%) developed 5-FU associated coronary vasospasm. Overall 81 patients were re-challenged with 5-FU therapy, while 34 stopped 5-FU . Among tBaseline demographics, cancer and medical histories, and 5-FU regimens of all vasospasm patients are outlined in Baseline characteristics, medical history and medication use of patients who developed vasospasm (N = 115), and comparison of patients who continued 5-FU (Fluorouracil) with cardiac pre-treatment (N = 78) versus those who stopped 5-FU therapy (N = 34) after development of vasospasm. *Not shown are 3 patients from total of 115, who continued 5-FU therapy without cardiac pre-treatment . ContinuThe median overall survival (OS) for patients who continued 5-FU with cardiac pre-medications was 47 months (95% CI 23.4 –NR months) as compared to 18.3 months (95% CI 9.2–35.5 months) for those who stopped 5-FU (P = 0.003) . SimilarChest pain  recurred in 19.2% (15/78) among those who received cardiac pre-treatment vs. 66.7% (2/3) among those who did not (P = 0.048). Among the 63/78 patients who successfully tolerated additional 5-FU therapy with cardiac pre-medications, 9 patients were also transitioned to bolus only regimens. There was no difference in the recurrence of vasospasm symptoms among patients who received pre-treatment with a single agent (long-acting nitrates or CCBs) or combination therapy, 14.7% (5/34) vs. 25.0% (11/44), P = 0.26. Furthermore, among patients who received single agent pre-treatment, there was no difference in the recurrence of vasospasm symptoms among those who received long-acting nitrates or CCBs, 21.1% (4/19) vs. 6.7% (1/15), P = 0.24.In this retrospective study of patients with 5-FU associated coronary vasospasm, we demonstrate the efficacy and safety of re-challenging patients after pre-treatment with cardio-protective medications. The median overall survival (OS) of patients who continued to receive 5-FU with cardiac pre-medications was longer by 28.7 months when compared to those who stopped therapy. Furthermore, patients who continued 5-FU therapy after pre-treatment had a decreased risk of death, when compared to those who stopped. Patients who received cardiac pre-treatment were also less likely to experience recurrent vasospasm compared to those who continued 5-FU without pre-treatment. This approach appeared safe as, of the recurrent vasospasm cases who were pre-treated with CCB and or long-acting nitrates, none had a myocardial infarction. As the reported incidence of recurrent cardiotoxicity with 5-FU rechallenge without changing the treatment approach is 90% , 7, our There are limited data on the successful re-challenge of 5-FU vasospasm patients with or without pre-treatment with anti-anginal therapies. Tsavaris et al. observed that ‘intensive cardiologic monitoring and prophylactic nitrate administration may result in fairly good subsequent tolerance,’ among the 20 patients who developed coronary vasospasm in their study . SimilarOur methodical approach to a cardiac evaluation and pre-treatment therapy  highlighThis was a retrospective study design; however, given the low incidence of coronary vasospasm amongst all comers receiving 5-FU therapy, a prospective design would not have been feasible. The relatively large scale of the study allowed us to show a statistically significant difference in overall survival between patients who were rechallenged and those who stopped 5-FU chemotherapy. Lastly, we acknowledge some potential selection bias when comparing patients who stopped 5-FU and those who continued 5-FU therapy after pre-treatment, since patients who stopped 5-FU therapy were possibly sicker than patients who were able to continue after pre-treatment. We attempted to address this limitation by controlling for confounders that were different between the two groups, namely age, initial stage of cancer, smoking, colorectal and upper gastrointestinal cancers. Furthermore, we were unable to directly compare patients who continued 5-FU therapy with and without pre-treatment due to the very small number of patients who continued 5-FU therapy without pre-treatment .(DOCX)Click here for additional data file.S1 File(PDF)Click here for additional data file.


SARS-CoV-2) is a unique accessory protein, yet little is known about its cellular function. We examined the cellular distribution of ORF8SARS-CoV-2 and its role in the regulation of human lung epithelial cell proliferation and antiviral immunity. Using live imaging and immunofluorescent staining analyses, we found that ectopically expressed ORF8SARS-CoV-2 forms aggregates in the cytosol and nuclear compartments of lung epithelial cells. Using in silico bioinformatic analysis, we found that ORF8SARS-CoV-2 possesses an intrinsic aggregation characteristic at its N-terminal residues 1-18. Cell culture did not reveal any effects of ORF8SARS-CoV-2 expression on lung epithelial cell proliferation and cell cycle progression, suggesting that ORF8SARS-CoV-2 aggregates do not affect these cellular processes. Interestingly, ectopic expression of ORF8SARS-CoV-2 in lung epithelial cells suppressed basal expression of several antiviral molecules, including DHX58, ZBP1, MX1, and MX2. In addition, expression of ORF8SARS-CoV-2 attenuated the induction of antiviral molecules by IFNγ but not by IFNβ in lung epithelial cells. Taken together, ORF8SARS-CoV-2 is a unique viral accessory protein that forms aggregates when expressing in lung epithelial cells. It potently inhibits the expression of lung cellular anti-viral proteins at baseline and in response to IFNγ in lung epithelial cells, which may facilitate SARS-CoV-2 escape from the host antiviral innate immune response during early viral infection. In addition, it seems that formation of ORF8SARS-CoV-2 aggregate is independent from the viral infection. Thus, it would be interesting to examine whether any COVID-19 patients exhibit persistent ORF8 SARS-CoV-2 expression after recovering from SARS-CoV-2 infection. If so, the pathogenic effect of prolonged ORF8SARS-CoV-2 expression and its association with post-COVID symptoms warrant investigation in the future.Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a disease that involves significant lung tissue damage. How SARS-CoV-2 infection leads to lung injury remains elusive. The open reading frame 8 (ORF8) protein of SARS-CoV-2 (ORF8 The global coronavirus disease 2019 (COVID-19) pandemic is caused by a novel beta coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This new pathogenic virus infects epithelial cells and macrophages in the lungs, which leads to severe inflammation in the respiratory system and diffuse alveolar damage . HoweverSARS-CoV-2) has a low sequence similarity to SARS-CoV ORF8a, 8b, or 8ab. ORF8SARS-CoV-2 protein is highly expressed in patients with SARS-CoV-2 infection  and human embryonic kidney epithelial cell line HEK293 were obtained from American Type Culture Collection . A549 and HEK cells were respectively cultured in ATCC-formulated F-12K and DMEM medium supplemented with 10% (v/v) fetal bovine serum, 50 U/ml penicillin, and 50 μg/ml streptomycin at 37°C in a humidified incubator with 5% COSARS-CoV-2 (RefSeq MN908947 27894 to 28256) was synthesized by Integrated DNA Technologies  and cloned into a pEGFP-N1 expression vector with C-terminal eGFP fusion, resulting in a new plasmid construct, pEGFP-N1-ORF8. The identity of pEGFP-N1-ORF8 was confirmed by Sanger sequencing. To generate a plasmid construct of ORF8SARS-CoV-2 with C-terminal Flag tag, pEGFP-N1-ORF8 was used as a template for PCR amplification and subcloned into pcDNA3.1 BamHI and EcoRI sites using In-Fusion HD Cloning kit (Clontech). The identity of pcDNA3.1-ORF8-Flag construct was confirmed by Sanger sequencing. A549 or HEK293 cells were seeded onto 6-well plates at a density of 3.5 × 105 cells/well and cultured at 37°C for 24 hours before transfection. Cells were then transfected with empty vector pEGFP-N1 or pEGFP-N1-ORF8 plasmids (2.5 µg/well), respectively, using Lipofectamine 3000  according to the manufacturer’s protocol. In some experiments, cells were co-transfected with a mixture of plasmids (2.5 µg/well) and Poly(I:C) . Culture medium was refreshed after 4 hours, and cells were processed for analysis 24-48 hours post-transfection. Some cells were treated with IFNβ- or IFNγ-containing medium  after transfection and then processed for analysis.The gBlocks gene fragment of ORF8Total protein was isolated from cells using RIPA lysis buffer (Thermo Fisher Scientific) and Halt protease inhibitor cocktail (Thermo Fisher Scientific). Protein concentration was measured using the Pierce BCA protein assay kit (Thermo Fisher Scientific) and cell lysates containing 20 µg of total protein were loaded and separated in 4-20% TGX precast SDS-PAGE gels  followed by transfer onto PVDF membranes (Bio-Rad). Immunoblot analyses were carried out as previously described . Mouse mSARS-CoV-2, cells were transfected with pcDNA3.1-ORF8-Flag construct and the transfected cells were processed for immunofluorescent staining with mouse anti-Flag M2 antibody .Cells were seeded onto a Lab-Tek chambered coverslip (Sigma-Aldrich) and transfected with empty vector pEGFP-N1 or pEGFP-N1-ORF8 plasmids. GFP-live fluorescent images were captured and merged with differential interference contrast (DIC) bright-field images using a Leica Thunder microscope system . For immunofluorescent staining, cells seeded onto Lab-Tek removable chamber slides were transfected with plasmids and Poly(I:C) as described above, then fixed using 4% paraformaldehyde (Thermo Fisher Scientific), permeabilized with 0.1% Triton X-100, and blocked with PBS buffer containing 2% normal goat serum  and 1% bovine serum albumin (Sigma-Aldrich). The slides were incubated at 4°C overnight with chicken anti-GFP primary antibody . After incubation, slides were washed with PBS and incubated for 1 hour at room temperature in the dark with goat anti-chicken IgY (H+L) antibody labeled with Alexa Fluor 488 . Finally, slides were washed with PBS and mounted with 4’,6-diamidino-2-phenylindole (DAPI)-contained mounting solution (Vector Labs). Slides were reviewed under a Leica Thunder microscope system and further processed by Adobe Photoshop software v21 . For image quantification, 8-14 random images were acquired, and approximately 100-200 GFP-positive cells were counted from each group. To further confirm the subcellular distribution of ORF8Cells were plated in 24-well plates and co-transfected with plasmids with or without Poly(I:C) as described above. Cell proliferation was assessed 24 hours after transfection. In brief, cells were trypsinized and mixed with an equal volume of 0.4% trypan blue stain, then subjected to cell counting using a Countess 3 automated cell counter (Thermo Fisher Scientific). All experiments were conducted in triplicate.To assess transfection efficiency, cells were plated in 6-well plates and transfected with plasmids. Cells were collected 24 hours post-transfection and stained with cell viability dye FVD-eFluor-506 (Thermo Fisher Scientific) at 4°C for 30 min, then subjected to flow cytometry analysis using a BD FACSymphony A5 Cell Analyzer . The GFP-positive live cell population was calculated by FlowJo . For cell cycle analysis, cells were collected and washed twice with PBS, then resuspended in 1 mL PBS and fixed by adding 4 mL ice-cold absolute ethanol at -20°C overnight. After fixation, cells were washed and counter-stained with a PBS-buffered solution containing 0.1% Triton X-100, 3 µM propidium iodide , and 0.5 mg/mL RNase A (Sigma Aldrich) at 4°C for 30 min. Cell cycle distribution was examined using a BD FACSymphony A5 Cell Analyzer, and the proportion of cells in the G0−G1, S, and G2−M phases was determined using FlowJo software. All experiments were performed in triplicate.−ΔΔCT method, with GAPDH as the endogenous reference. PCR reactions were run in duplicate for each sample. Primers used for RT-qPCR were synthesized by Integrated DNA Technologies and are listed in Total RNA was extracted from cells using Trizol (Thermo Fisher Scientific) and purified by Zymo RNA Clean & Concentrator kit . RNA was quantified with the Nanodrop  and single-strand cDNA was generated using the iScript cDNA synthesis kit (Bio-Rad). RT-qPCR was performed using SYBR Green PowerUp PCR Universal Mastermix (Thermo Fisher Scientific) and the QuantStudio™ 6 real-time PCR system (Thermo Fisher Scientific), all according to the manufacturer’ manuals. The fold change in expression levels of target genes was calculated using the 2SARS-CoV-2 sequence was retrieved from NCBI RefSeq MN908947 and submitted to online bioinformatics prediction tool PASTA2 (http://protein.bio.unipd.it/pasta2/). The propensity of ORF8 to form aggregates was assessed based on parallel aggregation probability and free energy.The ORF8t-test or one-way analysis of variance (ANOVA) followed by Fisher’s least significant difference post-hoc test. p<0.05 was considered statistically significant.All experiments were performed at least twice with triplicate samples. Statistical analysis was performed with GraphPad Prism 8 . Data are presented as mean ± s.d. and the statistical significance was assessed with either student’s SARS-CoV-2 is an accessory protein encoded by the SARS-CoV-2 genome. This protein has been detected in sera of COVID-19 patients  of the pEGFP-N1 mammalian expression vector . After transfecting the pEGFP-ORF8SARS-CoV-2 plasmid into A549 cells, expression of eGFP-tagged ORF8SARS-CoV-2 protein (42 kDa) was confirmed by immunoblot with anti-GFP. eGFP (27 kDa) was expressed in the A549 cells transfected with pEGFP-N1 empty vector (EV) . When examined by flow cytometry analysis, approximately 60% of A549 cells were GFP-positive 24 h after transfection with either pEGFP-N1 or pEGFP-ORF8SARS-CoV-2 constructs , suggesting significant uptake of plasmids by A549 cells. This in vitro system was used to study the function of ORF8SARS-CoV-2 in human lung epithelial cells.ORF8patients , 17, sugn vector . After tn vector . After tSARS-CoV-2 plasmid construct. Using live fluorescent imaging, we found that pEGFP-N1-transfected A549 cells exhibited homogeneous GFP fluorescence . In contrast, distinctive fluorescent particles were seen in A549 cells transfected with the pEGFP-ORF8SARS-CoV-2 construct , suggesting that ORF8SARS-CoV-2 forms aggregates in lung epithelial cells. In addition, immunofluorescence staining revealed that ectopically expressed ORF8SARS-CoV-2-GFP protein was either homogenously distributed in A549 cells or formed aggregates in cytoplasmic and nuclear compartments of the cells . Image quantification revealed a higher ratio of intracellular aggregates in pEGFP-ORF8SARS-CoV-2-transfected cells compared to cells transfected with pEGFP-N1 empty vector . To exclude the possibility that eGFP fusion to C-terminal of ORF8SARS-CoV-2 could affect the subcellular distribution of ORF8SARS-CoV-2, cells were transfected with pcDNA3.1-ORF8-Flag construct followed by immunofluorescent staining with anti-Flag antibody. Using this approach, we confirmed a similar subcellular expression pattern of ORF8SARS-CoV-2, suggesting that ectopic expression of ORF8SARS-CoV-2 forms cytoplasmic and nuclear aggregates in the lung epithelial cells without contribution of Tag proteins. Using in silico prediction (http://protein.bio.unipd.it/pasta2/)  , we founrescence . In contrescence . In contSARS-CoV-2 aggregates in lung epithelial cells. A549 cells were stimulated with Poly(I:C) to mimic the activation of cells by viral infection. As expected, treatment with Poly(I:C) profoundly induced the expression of various antiviral genes in A549 cells , indicating activation of the antiviral immune response. Next, cells were co-transfected with a mixture of plasmids and Poly(I:C) to examine the formation of ORF8SARS-CoV-2 aggregates in lung epithelial cells under a Poly(I:C)-mimicked antiviral microenvironment. Immunostaining analysis did not reveal any change in the distribution pattern of ORF8SARS-CoV-2 aggregates in poly(I:C)-treated cells . These data suggest that ORF8SARS-CoV-2 forms aggregates in both the cytosol and nucleus of lung epithelial cells, independent from inflammatory events activated by viral infection.We next examined whether activation of antiviral immune response affects the formation of ORF849 cells , indicatSARS-CoV-2 expression. Cell numbers of pEGFP-N1-transfected and pEGFP-ORF8SARS-CoV-2-transfected A549 cells were compared in the presence or absence of poly(I:C). Cells transfected with pEGFP-ORF8SARS-CoV-2 exhibited a similar growth rate to empty vector-transfected cells when cultured with or without poly(I:C) .Intracellular protein aggregates can be toxic, causing injury or death to cells , 24. ThuSARS-CoV-2 expression on cell cycle progression in A549 cells using flow cytometry analysis. We noted that cell cycle phases  in pEGFP-ORF8SARS-CoV-2-transfected cells were similar to those in pEGFP-N1-transfected cells, regardless of poly(I:C) stimulation  family including ZBP1 , MX1 and MX2 , and DHX58  in A549 cells  grants  and US Department of Veterans Affairs Merit Review Award (I01BX001690). IP is funded by NIH grant R01DK116568. The funders had no role in study design, data collection and analysis, interpretation of data, decision to publish, or preparation 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.


OPRM1, goes through extensive alternative pre-mRNA splicing to generate numerous splice variants that are conserved from rodents to humans. These OPRM1 splice variants can be classified into three structurally distinct types: (1) full-length 7 transmembrane (TM) carboxyl (C)-terminal variants; (2) truncated 6TM variants; and (3) single TM variants. Distinct pharmacological functions of these splice variants have been demonstrated by both in vitro and in vivo studies, particularly by using several unique gene-targeted mouse models. These studies provide new insights into our understanding of the complex actions of mu opioids with regard to OPRM1 alternative splicing. This review provides an overview of the studies that used these gene-targeted mouse models for exploring the functional importance of Oprm1 splice variants.The mu opioid receptor has a distinct place in the opioid receptor family, since it mediates the actions of most opioids used clinically , as well as drugs of abuse . The single-copy mu opioid receptor gene,  OPRM1 , s, s101], Oprm1 splice variants, including all 7TM, 6TM, and 1TM variants, were lost . Th. Th63]. junction ,111. Satce (MRS) ,112 in mDifferent inbred mouse strains, particularly C57BL/6J (B6) and 129, have divergent responses to mu opioids ,37,38. F35S-GTPγS binding assay in WT B6 mice [On the other hand, morphine tolerance was significantly reduced in the exon 7-encoded C-terminal truncation mice with a B6 background (mE7M-B6)  Figure , which w B6 mice , suggest B6 mice , implyinThree C-terminal truncation mouse models also revealed their unique differences in morphine physical dependence, measured mainly by the number of jumps with naloxone-precipitated withdrawal and morphine reward by CPP . Both mEIn morphine-induced locomotor responses , both mE35S-GTPγS binding and β-arrestin 2 recruitment assays in PathHuntet cell lines, stably expressing individual exon 7-associated full-length C-terminal splice variants, demonstrated that the exon 7-associated C-terminal variants, particularly mMOR-1O, had greater β-arrestin 2 bias toward several mu agonists than exon 4-associated mMOR-1 [Comparison of morphine actions between the mE7M-B6 model and a β-arrestin 2-KO model revealed several similarities . Both mod mMOR-1 ,64, provd mMOR-1 , PxPxxE/d mMOR-1 . DisruptNevertheless, several morphine-induced responses, such as analgesia, locomotion, and reward, as well as fentanyl and methadone tolerance in mE7M-B6 mice, were dissimilar  . More importantly, these studies provide potential therapeutic targets for developing novel analgesics or medications for better pain management. However, these gene-targeted mouse models have a limitation in defining the role of individual variants because many exons are shared by different splice variants, which makes it difficult to create a mouse KO model for a specific splice variant. For example, several full-length 7TM C-terminal variants and one 6TM variant containing exon 7-encoded C-terminal sequences were disrupted in the mE7M mutant mice. Although the exon 7-associated 7TM variants were thought to be involved in the altered morphine actions, it remains unknown which specific variant is responsible. Thus, creating a mouse KO model for a specific splice variant with a conditional setting by using new targeting strategies is essential for studying the pharmacological functions and molecular mechanisms of individual variants in either region-specific or cell-specific fashion. We anticipate seeing such gene-targeted mouse models being developed in the near future, which would be tremendous in advancing our understanding of the roles of OPRM1 alternative splicing in the complex actions of mu opioids.Extensive alternative splicing of the single-copy


Oryza sativa) during an incompatible rice–Magnaporthe oryzae interaction. Here, we show that rice MAP kinase (OsMEK2 and OsMPK1) signaling cascades are involved in iron- and ROS-dependent ferroptotic cell death responses of rice to M. oryzae infection using OsMEK2 knock-out mutant and OsMEK2 and OsMPK1 overexpression rice plants. The OsMPK1:GFP and OsWRKY90:GFP transcription factor were localized to the nuclei, suggesting that OsMPK1 in the cytoplasm moves into the nuclei to interact with the WRKY90. M. oryzae infection in ΔOsmek2 knock-out plants did not trigger iron and ROS accumulation and lipid peroxidation, and also downregulated OsMPK1, OsWRKY90, OsRbohB, and OsPR-1b expression. However, 35S:OsMEK2 overexpression induced ROS- and iron-dependent cell death in rice. The downstream MAP kinase (OsMPK1) overexpression induced ROS- and iron-dependent ferroptotic cell death response to virulent M. oryzae infection. The small-molecule ferroptosis inhibitor ferrostatin-1 suppressed iron- and ROS-dependent ferroptotic cell death in 35S:OsMPK1 overexpression plants. However, the small-molecule inducer erastin triggered iron- and lipid ROS-dependent, but OsMEK2-independent, ferroptotic cell death during M. oryzae infection. Disease (susceptibility)-related cell death was lipid ROS-dependent, but iron-independent in the ΔOsmek2 knock-out mutant during the late M. oryzae infection stage. These combined results suggest that OsMEK2 and OsMPK1 expression positively regulates iron- and ROS-dependent ferroptotic cell death, and blast disease (susceptibility)-related cell death was ROS-dependent but iron-independent in rice–M. oryzae interactions.Mitogen-activated protein kinase (MAPK) signaling is required for plant cell death responses to invading microbial pathogens. Iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death occurs in rice ( Plants have evolved effective innate immune system responses to avert the invasion of microbial pathogens in their natural habitat . Plant iMAPK and ERK  kinase (MEK); and MAP kinase (MAPK or MPK) -like cell death and defense responses in tobacco. Many kinases in MAPK cascades, including MAPKKK, MEK, SIPK/WIPK, and MAPK, are involved in N gene-mediated resistance to tobacco mosaic virus in tobacco (N-mediated resistance to TMV (MEK2 (SlMKK2), SlMPK2, and SlMKK4 in tomato disrupted the resistance to infection by Xanthomonas campestris pv. vesicatoria (Xcv) and Botrytis cinerea (Mitogen-activated protein (MAP) kinase (MAPK) signaling pathways have pivotal roles in plant defense, immunity, and hypersensitive cell death responses to pathogen attack . However or MPK) . These k or MPK) . Phospho or MPK) . MAPKs c or MPK) . A previ or MPK) . Arabido or MPK) . Pathogethaliana . Nearly  tobacco . Tobaccoe to TMV . Silenci cinerea .WRKY45 expression and blast (Magnaporthe oryzae) resistance in rice plants (X. oryzae pv. oryzicola (Xoo) resistance and drought tolerance (Xanthomonas oryzae pv. oryzicola (Xoo) infection in rice  accumulation at the cell death sites in rice tissues  and NADPH-oxidase (Rboh) are ROS sources that have been proposed to be involved in iron- and ROS-dependent ferroptotic cell death  suppressed iron- and ROS-dependent ferroptotic cell death, which ultimately induced susceptible responses to avirulent M. oryzae 007 infection. However, OsMEK2 overexpression in rice DJ induced iron- and ROS-dependent ferroptotic cell death against M. oryzae 007 infection. Treatment of the OsMEK2-knockout mutants with erastin induced the ROS burst and iron accumulation, which caused ferroptotic cell death in ΔOsmek2 knock-out plants in response to M. oryzae infection. Disease (susceptibility)-related cell death at the late stage of M. oryzae infection in ΔOsmek2 knock-out plants is ROS-dependent and iron-independent. During M. oryzae infection, OsMEK2 knock-out and overexpression differentially regulated the expression of OsMPK1, OsMPK6, and the OsWRKY90 transcription factor in the rice MAPK signaling pathways. OsMPK1 overexpression in susceptible rice cultivar Nipponbarre (NB) induced iron- and ROS-mediated ferroptotic cell death against M. oryzae PO6-6 infection. Treatment with ferrostatin-1 suppressed iron- and ROS-dependent ferroptotic cell death in 35S:OsMPK1 overexpression leaf sheaths during infection. These combined results indicate that OsMEK2 and OsMPK1 expression via MAPK signaling pathway positively regulates iron- and ROS-dependent ferroptotic cell death and plant immunity to M. oryzae infection.Our previous study identified rice mitogen-activated protein (MAP) kinase kinase 2 (OsMEK2) as a rice MAP interactor . Rice MAnfection . Here, wOsmek2 knock-out, 35S:OsMEK2 and 35S:OsMPK1 overexpression lines were used in this study. ΔOsmek2 T-DNA insertion knock-out mutant seeds were provided by the Rice Functional Genomic Express Database (RiceGE) managed by the Salk Institute Genomic Analysis Laboratory1 . Rice plants were grown in growth chambers at 28°C under white fluorescent light (150 μmol photons m–2 s–1) with a 16 h photoperiod and 60% relative humidity, as described previously (The WT rice cultivars Dongjin (DJ) and Nipponbarre (NB) and the Δoratory1 . DJ and eviously .Magnaporthe oryzae 007 and PO6-6 were provided by the Center for Fungal Genetic Resources, Seoul National University, Seoul, South Korea3. M. oryzae 007 was avirulent (incompatible) and M. oryzae PO6-6 was virulent (compatible) to the rice cultivar DJ. The rice cultivar NB was susceptible to M. oryzae PO6-6 infection. The fungal cultures were stored at –20°C and cultured on rice bran agar media . M. oryzae strains were grown at 25°C in the dark for 2 weeks. M. oryzae sporulation was induced by removing aerial mycelia from the fungal culture plates, followed by their incubation under a continuous fluorescent light (80 μmol photons m–2 s–1) for 2–3 days at 25°C.The rice blast fungal strains M. oryzae strains were inoculated on rice leaves and leaf sheaths as described previously (M. oryzae conidia were harvested from the sporulated culture plates using a 0.025% Tween 20 (Sigma-Aldrich) solution. The conidial concentration was adjusted to 4 × 105 conidia mL–1. The conidial suspension was spray-inoculated over the surface of 2-week-old rice seedlings. The inoculated seedlings were incubated at 25–28°C for 24 h under dark and moist conditions, and then were moved to normal conditions (16 h light/8 h dark). Disease phenotypes were observed at 5 days after inoculation and classified with respect to susceptible  and resistant  reactions.Conidial suspensions of eviously . M. oryzM. oryzae conidial suspensions (4 × 105 conidia mL–1). Inoculated leaf sheaths were incubated in a moistened box with 100% relative humidity at 25°C under dark conditions. After incubation for different times, epidermal layers were excised from the leaf sheaths, cut into 1.5 cm lengths, and fixed on glass microscope slides. The infected epidermal cells from each of three epidermal sheaths were observed under the microscope and divided into two infection phenotypes: cells with viable IH and cells with HR cell death. For the quantification of invasive hyphae (IH) and HR cell death, approximately 500 infected cells in each of the leaf sheaths were counted at least three times from one representative of three independent experiments.Middle-aged leaf sheaths (5–7 cm lengths) of 4- or 5-week-old rice plants were inoculated with Osmek2 T-DNA insertion mutant seeds from RiceGE . The constructed CaMV 35S:OsMEK2 and CaMV 35S:OsMPK1 were introduced in the rice cultivars DJ and NB, respectively, by Agrobacterium tumefaciens-mediated transformation, as described previously with slight modification  according to the manufacturer’s instructions. Relative gene expression levels were determined using rice 18S ribosomal RNA or rice ubiquitin as an internal standard gene. Gene-specific primers used for the real-time RT-PCR analysis are listed in Total RNA was isolated from rice tissue using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The gene expression levels were analyzed by reverse-transcription PCR (RT-PCR) and real-time quantitative PCR (qRT-PCR). First-strand cDNA was synthesized from 2 μg total RNA in 20 μL reaction mixture using a cDNA synthesis kit (Invitrogen) according to the manufacturer’s instructions. Prepared cDNA (1 μL) was used as a template for both RT-PCR and qRT-PCR. The qRT-PCR was performed using TOPrealg for 15 min at 4°C, the supernatants were precipitated using pre-chilled acetone and solubilized in LB-TT buffer.Rice proteins were extracted using trichloroacetic acid (TCA)/acetone extraction buffer  as described previously . Rice leOsmek2 mutant plants were determined by 10% SDS-polyacrylamide gel electrophoresis (PAGE) followed by immunoblot analysis using rabbit polyclonal anti-MEK2 antibody (EnoGene® E580135-A-SE). Immuno-reactive target bands were detected by Odyssey® CLx Imaging System (LI-COR Biosciences). Equal gel loading was checked by Ponceau S staining.The OsMEK2 protein expression levels in DJ and ΔM. oryzae conidia (4 × 105 conidia mL–1) were mixed with 10 μM erastin  and inoculated on leaf sheaths. The erastin-treated and M. oryzae-inoculated leaf sheath tissues were incubated in the dark at 25°C. The ferroptosis inhibitor, ferrostatin-1 , was treated as described previously  localization in rice leaf sheath cells was visualized using 5- (and 6-) chloromethyl-2′,7′-dichlorofluorescin diacetate acetyl ester (CM-H2DCFDA) and 3,3′-diaminobenzidine (DAB) staining methods as described previously  in 1 × phosphate-buffered saline (PBS) buffer in the dark for 30 min on a horizontal shaker. The incubated sheath samples were washed twice with 1 × PBS buffer for 5 min in the dark. ROS localization inside the epidermal sheath cells was observed immediately under a fluorescence microscope.Cellular ROS  solution for 5 min, followed by overnight destaining with ethanol:acetic acid:glycerol . ROS localization in the DAB-stained epidermal cells was observed under a microscope. The DAB-stained cells were categorized into two phenotypes: Type I, infected cells that display no or weak DAB staining; and Type II, infected cells that display strong DAB staining. DAB-stained 500 cells with different phenotypes were counted from each of infected sheaths. The counted cell numbers were then converted into the percentages of DAB-stained cells.For DAB staining, epidermal layers of rice leaf sheaths were vacuum-infiltrated with 1 mg mLM. oryzae-inoculated rice leaf sheaths were cut into small pieces (0.5 cm length) and incubated in 1 mL of sterilized Milli-Q water for 5 min to remove wound-induced ROS. Then, a piece of epidermal layer was added into a mixture of 30 μL luminol , 1 μL horseradish peroxidase , and 69 μL Milli-Q water in each well of a 96-well plate. Chemiluminescence  was detected from the ROS signals after 5 min incubation using a GloMax® 96 Microplate Luminometer .The chemiluminescence assay was used to measure ROS production in rice leaf sheaths as described previously  with ming for 5 min. The MDA concentration (C) in the resulting supernatant was determined by measuring supernatant absorbances  at 450, 532, and 600 nm, and then calculating MDA concentration according to the following equation: C = 6.45 × (OD532–OD600) – (0.56 × OD450).The malondialdehyde (MDA) assay was performed to determine lipid peroxidation in rice leaf sheath tissues as described previously . Lipid p3+) in rice leaf sheaths as described previously  was observed as a bright blue color under a fluorescence microscope. Prussian blue-stained cells were categorized into two phenotypes: Type I, cells that contain IH but are weakly or not Prussian blue-stained; and Type II, strongly Prussian blue-stained cells with only a few poor hyphae. Prussian blue-stained 500 cells with different phenotypes were counted from each of infected sheaths. The counted cell numbers were then converted into the percentages of Prussian blue-stained cells.Prussian blue staining was performed to detect ferric ion  to create entry clones. The entry clones were recombined into the Gateway binary vector pGWB552 tagged with G3 green fluorescent protein (G3GFP) using LR clonase (Invitrogen) (The full length cDNA of itrogen) .OsMEK2, OsMPK1, and OsWRKY90 were transformed into A. tumefaciens GV3101. Recombinant agrobacteria were prepared for infiltration using a protocol as described previously with slight modification . The agrobacterial suspension was incubated for 2 h at room temperature, and infiltrated into the abaxial leaves of 6-week-old Nicotiana benthamiana plants using a blunt tipped plastic syringe. Subcellular localization of 00:GFP, OsMEK2:GFP, OsMPK1:GFP, and OsWRKY90:GFP in N. benthamiana epidermal cells 36 h after agroinfiltration were microscopically observed following 4′,6-diamidino-2-phenylindole  staining for 10 min. Nuclear localization of the proteins was visualized by counterstaining the nuclei of the cells with DAPI.The binary plasmids containing fication . Briefly2DCFDA-specific fluorescence was visualized under the fluorescence microscope using a combination of excitation (450–490 nm) and emission (515–565 nm) green fluorescence (GF) filters. Subcellular images were also taken using a fluorescence microscope  using bright field, GF (Ex/Em: 488/498–548 nm), and DAPI (Ex/Em: 405/421–523 nm) filters.Images were captured using a fluorescence microscope  with 40× oil-immersion objective lens. CM-HOsMEK1 (Os01g32660), OsMEK2 (Os06g05520), OsMEK3 (Os03g12390), OsMEK4 (Os02g46760), OsMEK5 (Os06g09190), OsMEK6 (Os02g54600), OsMEK7 (Os06g09180), OsMEK8 (Os06g27890), OsMPK1 (Os06g06090), OsMPK6 (Os10g38950), OsWNK1 (Os07g38530), OsWRKY90 (Os09g30400), OsNADP-ME (Os01g52500), OsRbohB (Os01g25820), OsPR-1b (Os01g28450), OsPAL1 (Os04g43760), OsAPX1 (Os0.g17690), OsAPX2 (Os07g49400), OsUbiqutin (Os06g46770), 18S rRNA (XR_003238819.1), AtMKK1 (At4g26070), AtMKK2 (At4g29810), AtMEK3 (NP_198860), AtMEK4 (At1g51660), AtMEK5 (At3g21220), AtMKK6 (At5g56580), AtMKK9 (At1g73500), AtWNK9 (At3g04910), NtNPK2 (BAA06731), SlMKK1 (NP_001234744), SlMKK2 (NP_001234588), NbMEK2 (LOC107818847), and NtMEK2 (AF325168).Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: 5-S/T, where X represents any amino acid residue)  that interacted with OsMAPKs using yeast two-hybrid analysis . Amino aresidue) . The ricresidue)  (Supplemresidue) . OsMEK2 o SlMKK1 . OsMEK2 o SlMKK1 . Based oOsMEK2 was knocked out in rice cultivar DJ by T-DNA insertion mutagenesis  progeny were analyzed with primer sets LP + RP and LP + LB to detect transgene and homo/hetero selection, respectively (Osmek2 #2 andΔOsmek2 #4 mutants (M5) were identified as T-DNA insertion homozygous plants that lacked the specific PCR products.agenesis . The OsM introns . The T-Dth exons . The genectively . The ΔOsOsMEK2 expression in ΔOsmek2 knock-out and 35S:OsMEK2 overexpression lines was examined by quantitative real-time RT-PCR and immunoblotting  in the leaf sheath cells of ΔOsmek2 #2 and #4 knock-out plants, but induced hypersensitive cell death in wild-type (WT) rice DJ and 35S:OsMEK2 #4 and #6 overexpression plants . During M. oryzae 007 infection, OsMEK2 expression was up-regulated from 1 to 12 hpi and then back to the background level at 24 hpi . These results suggest that early induction of OsMEK2 expression is involved in rice defense signaling in the incompatible rice–M. oryzae interaction  in response to avirulent M. oryzae infection. However, OsMEK2 overexpression in rice DJ enhanced the cell death and resistance responses to rice blast disease.We investigated whether e plants . M. oryzt 48 hpi . By cont sheaths . Whole-lyzae 007 . Rice DJ lesions . These cOsMPK1, OsMPK6, and OsWRKY90 expression in leaf sheaths of rice DJ, ΔOsmek2 #2 knock-out and 35S:OsMEK2 #4 overexpression plants during avirulent M. oryzae 007 infection  activation by OsMKK10-2 is required for the induction of OsWRKY45 expression and blast resistance in rice  interacted with and phosphorylated OsMAPKs, such as OsMPK1 and OsMPK6 . We analnfection . OsMEK2  in rice . OsMPK1  in rice . Bimolec in rice  and subs in rice  as its d in rice . Aviruleculation . This inOsWRKY89 .M. oryzae 007 infection in rice, such as pathogenesis-related protein 1b (OsPR-1b), phenylalanine ammonia lyase1 (OsPAL1), ascorbate peroxidase1 (OsAPX1), and OsAPX2  accumulation and lipid [malondialdehyde (MDA)] peroxidation in leaf sheath cells of rice DJ, 35S:OsMEK2 #4 and ΔOsmek2 #2 plants during avirulent M. oryzae 007 infection to determine whether OsMEK2 is involved in iron- and ROS-dependent ferroptotic cell death (2DCFDA (green fluorescence) and DAB (dark brown) staining revealed that ROS (H2O2) strongly accumulated inside and around invasive hyphae (IH) in rice DJ and 35S:OsMEK2 #4 epidermal cells at 30–48 hpi  and cellular membranes in rice DJ and 35S:OsMEK2 #4 cells, whereas ROS-localized fluorescence was absent or weakly visible around invasive hyphae (IH) in ΔOsmek2 #2 cells at 30 hpi  accumulation and localization in rice cells was detected by Prussian blue (blue color) staining of rice leaf sheath cells at 48 hpi with avirulent M. oryzae 007  peroxidation in rice leaf sheath cells at 48 hpi with M. oryzae 007  and Fe3+ accumulation and cell death response in ΔOsmek2 #2 leaf sheaths during avirulent M. oryzae 007 infection (2O2) and Fe3+ accumulation in healthy rice DJ leaf sheaths  and Fe3+ did not accumulate in healthy rice DJ leaf sheaths at 72 and 92 h after treatment with 10 μM erastin  under the control of CaMV 35S promoter. OsMPK1 was distinctly overexpressed in leaf sheath cells of 35S:OsMPK1-transformed plants (OsMPK1 overexpression induced a hypersensitive cell death with poorly grown invasive hyphae (IH) in leaf epidermal cells at 48 hpi (2DCFDA staining showed accumulation of ROS around the IH in 35S:OsMPK1 leaf sheath cells at 36–48 hpi during virulent M. oryzae PO6-6 infection (OsMPK1 overexpression cells at 48 hpi (3+ showed strong accumulation of ferric ion in 35S:OsMPK1 cells at 48 hpi (OsMPK1 overexpression cells than in rice NB cells at 48 hpi (OsMPK1 overexpression cells (OsMPK1 leaf sheaths during infection (OsMPK1 is involved in ROS and Fe3+ accumulation and lipid peroxidation leading to the ferroptotic cell death during M. oryzae infection.Rice MAP kinase (OsMPK1) is an interactor of OsMEK2 and actively involved in nfection . Genomicnfection . Amino anfection . In partnfection . OsMPK1 nfection . We overd plants . 35S:OsMt 48 hpi . DAB andnfection . Chemilut 48 hpi . Prussiat 48 hpi . Lipid  and respiratory burst oxidase homolog  are involved in Fe in rice . InteracOS burst . RbohB aOS burst . Here, wDP-ME2-3  and OsRb-tagged 35S:OsMEK2 (OsMEK2:GFP), OsMPK1:GFP, and OsWRKY90:GFP in pression . The nucOryza sativa) during Magnaporthe oryzae infection -related cell death was ROS-dependent but iron-independent in the susceptible ΔOsmek2 mutant plants.Plant mitogen-activated protein kinase (MAPK) cascades are involved in signaling multiple defense responses, the HR, and cell death responses during pathogen invasion and infection . We recenfection . Ferroptnfection . OsMEK2 nfection . Here, wM. oryzae infection in rice plants. In the present study, OsMEK2 knock-out in rice DJ plants induced a susceptible (disease) response to M. oryzae infection in ΔOsmek2 knock-out plants; however, OsMEK2 overexpression in 35S:OsMEK2 plants redeemed hypersensitive cell death response against M. oryzae infection. These results suggest the HR-mediated resistance signaling of OsMEK2 during M. oryzae infection. OsMEK2 knock-out and overexpression differentially regulated OsMPK1, OsMPK6, and OsWRKY90 expression in ΔOsmek2 and 35S:OsMEK2 plants, especially during early stages of M. oryzae infection. These results indicated that OsMEK2 expression distinctly induced the downstream OsMPK1 and OsMPK6 signaling responses to M. oryzae infection. OsMPK1 physically interacts with the OsWRKY80 (OsMEK2 expression distinctly induced pathogenesis-related protein 1b (OsPR-1b) but not ascorbate peroxidase1/2 (OsAPX1/2) during M. oryzae infection. PR-1 proteins are markers of defense responses to pathogen infection in rice  is essential in plants for HR cell death and defense- and disease-related iron homeostasis , which is activated during early M. oryzae infection. MAPKs could phosphorylate WRKY transcription factors to subsequently activate NADPH oxidases (Rbohs), which are essential for potent and prolonged ROS burst (OsMEK2) signaling seems likely to activate OsMPK1, OsWRKY90, and NADPH-oxidase (OsRbohB), ultimately leading to the iron- and ROS-dependent ferroptotic cell death response. In our study, overexpression of 35S:OsMPK1 significantly induced iron and ROS accumulation during infection. However, the ferroptosis inhibitor, ferrostatin-1 (Fer-1), suppressed iron- and ROS-dependent ferroptotic cell death, which ultimately led to the restored normal invasive hyphal growth in 35S:OsMPK1 overexpression plants. MPK6 (OsMPK1 in current study) reduced susceptibility in rice cultivar Zhonghua 11 against Xanthomonas oryzae pv. oryzae infection. Our results suggest that the OsMEK2-OsMPK1-OsWRKY90 cascades positively regulate ferroptotic cell death in rice against M. oryzae infection. Rice MPKK10.2 and MPK6 cascades induced resistance against X. oryzae pv. oryzae infection . X. oryzae infection. Rice OsMPK7 interacts with and phosphorylates OsWRKY30 to mediate resistance against X. oryzae infection ; however, significant iron accumulation did not occur. Iron-independent and ROS-dependent cell death at late infection stages in the compatible rice–M. oryzae interaction is distinct from ferroptotic HR cell death, but similar to necrosis-like cell death. Necrotic cell death caused by compatible plant interactions with necrotrophic pathogens is dependent on ROS accumulation (M. oryzae infection at the late stages are involved in disease (susceptibility)-related cell death in rice, as suggested previously  interactions between plants and pathogens . In thismulation . A toxinmulation . Cell demulation . ROS acceviously . HoweverM. oryzae interactions (M. oryzae 007 into rice cells activates rice MAP kinases (OsMEK2 and OsMPK1) via different hypothesized MAPK signaling pathways. The superoxide (O2–) produced from apoplastic oxygen (O2) can be converted to hydrogen peroxide (H2O2) by superoxide dismutase (SOD) (2O2) produced by plasma membrane-bound Rbohs during ETI migrates across the plasma membrane using aquaporin channels and into the cell (3+) and lipid ROS triggers lipid peroxidation and subsequent ferroptotic cell death (M. oryzae into rice cells activates rice MAP kinases (OsMEK2 and OsMPK1) via different hypothesized MAPK signaling pathways. The perception of PAMPs or pathogen effectors via membrane-bound PRRs or NLRs, respectively, activates OsMAP kinase cascades in rice cells, as proposed previously  (de novo synthesis of OsRbohB and its trafficking to the plasma membrane is involved in iron- and ROS-dependent ferroptotic death in rice cells. Rice MAP kinase 1 (OsMPK1) also may target OsWRKY90 to bind to specific sequences of some defense-related genes such as OsPR-1b. Disease (susceptibility)-related cell death is lipid ROS-dependent, but iron-independent, in the compatible rice–M. oryzae interaction  . Apoplasthe cell . The incll death . The inveviously . MAP kineviously . OsMEK2 eviously . OsMPK1 eviously . OsMEK2  nucleus , which mOsRbohB) . The MAPOsRbohB) . The de eraction . Iron acractions . Iron acThe original contributions presented in the study are included in the article/N-SJ designed the research. SD, NKN, RS, YC, JW, and H-GL carried out all the experiments. SD, NKN, BKH, and N-SJ analyzed the data and wrote the manuscript. BKH and N-SJ reviewed and edited the final manuscript. All authors read and agreed to the published 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Enterobacter cloacae is a Gram-negative rod with chromosomally-induced Amp-C β-lactamase with multidrug-resistant potential. Joint Trauma System guidelines for treating combat wounds include prophylaxis with cefazolin and ertapenem, potent inducers of Amp-C. We evaluated clinical characteristics, antibiotic utilization, and outcomes associated with battlefield-related E. cloacae infections. E. cloacae isolates  were collected from the Trauma Infectious Disease Outcomes Study (6/2009-12/2014). Inclusion required E. cloacae isolation from a clinical infection. Amp-C-inducing β-lactams were classified based on induction potential and lability to the Amp-C β-lactamase as Amp-C induction levels. All initial solitary (those with single isolates) and serial E. cloacae isolates, 253 met inclusion criteria – 64 patients had only initial isolates, 54 patients had serial isolates. Patients were largely male (99%), median age 23 years (IQR 21-27), with injury severity score of 34 (IQR 24-45). Initial isolates were wound (70%), respiratory (22%), blood (7%), urine (1%), and other (1%). Patients commonly had blast injuries (89%), required ICU admission (95%), and had a median hospital stay of 57 days (IQR 39-82). Patients with serial isolates showed a trend towards earlier clinical infection . They were also less likely to receive carbapenems prior to E. cloacae isolation compared to those with only initial isolates (4% vs 38%) and more likely to receive 1st generation cephalosporins . The serial isolate group received more days of 1st generation cephalosporins . Cumulative antimicrobial therapy trended towards significance and was greater with the serial isolates . There was no difference in number of surgical interventions between those with and without serial isolates (P = 0.54). Overall, 6 patients died. Of 653 E. cloacae infections after battlefield trauma were frequently encountered and associated with exposure to 1st generation cephalosporins. Serial infections did not correlate to worse patient outcomes but displayed a trend towards an overall greater duration of antibiotic use.William N. Bennett, V, MD, Abbvie (Shareholder)Amgen (Shareholder)Nabriva (Shareholder)


Caenorhabditis elegans development, we knocked down twenty-four genes encoding KH-domain proteins in several miRNA sensitized genetic backgrounds. Here, we report that a majority of the KH domain-containing genes genetically interact with multiple miRNAs and Argonaute alg-1. Interestingly, two KH domain genes, predicted splicing factors sfa-1 and asd-2, genetically interacted with all of the miRNA mutants tested, whereas other KH domain genes showed genetic interactions only with specific miRNAs. Our domain architecture and phylogenetic relationship analyses of the C. elegans KH domain-containing proteins revealed potential groups that may share both structure and function. Collectively, we show that many C. elegans KH domain RBPs functionally interact with miRNAs, suggesting direct or indirect coordination between these two classes of post-transcriptional gene expression regulators.MicroRNAs (miRNAs) and RNA-binding proteins (RBPs) regulate gene expression at the post-transcriptional level, but the extent to which these key regulators of gene expression coordinate their activities and the precise mechanisms of this coordination are not well understood. RBPs often have recognizable RNA binding domains that correlate with specific protein function. Recently, several RBPs containing K homology (KH) RNA binding domains were shown to work with miRNAs to regulate gene expression, raising the possibility that KH domains may be important for coordinating with miRNA pathways in gene expression regulation. To ascertain whether additional KH domain proteins functionally interact with miRNAs during  C. elegans genome is predicted to encode more than 180 miRNAs  and RNA-binding proteins (RBPs). The human genome is predicted to encode at least 2000 miRNAs  transcripts  domain ; otIs114 [Plim-6-gfp + rol-6(su1006)], OH812 otIs114 [Plim-p-gfp + rol-6(su1006)], PS3662 syIs63 [cog-1::gfp + unc-119(+)], OH7310 otIs193 [Pcog-1::lsy-6 + rol-6(su1006)]; sy1s63, VT1296 mir-48 mir-241(nDf51) col-19::gfp (maIs105), MT7626 let-7(n2853), VT2223 lin-31(n1053); col-19::gfp (maIs105); alg-1(ma202), HW1113 , HW1114 , HW1159 , and BW1932 hbl-1p::gfp::NLS::hbl-1 3'UTR (ctIS39). All strains were grown at 20°C with the exception of MT7626 let-7(n2853) which was grown and maintained at 15°C to prevent excess bursting.Worm culture and maintenance was performed as previously described (Brenner 1974). Bristol N2 was used as the wild-type strain. Strains used in this study are OH3646 bcc-1 and E02D9.1 which were obtained from the Vidal RNAi library (fubl-3 clone was generated by using forward 5′-GCCCACTAGTGGACTAACTGCAACGTTCAA-3′ and reverse 5′-GTGGGTACCATTTGCCGCCTCAGAATTG-3′. The Y6A2AR.32 clone was generated using forward 5′-GCTCAGATCTTGCCACGTTTCATGCGAAAC-3′ and reverse 5′-GTAGGTACCGGAAGCTCTTCCTCTCACAA-3′. The B0280.17 clone was generated using forward 5′-GGCCAGATCTCTTCTAGTTCGTGAAATCAA-3′ and reverse 5′-ATAGGTACCGCAGTCTCGGGAGGAAAG-3′. The amplified genomic fragments containing restriction sites were digested using SpeI and Kpn1 (fubl-3) or BglII and KpnI (Y69A2AR.32 and B0280.17) and were ligated with the digested L4440 vector using NEB (M2200) Quick Ligation protocol. Ligated plasmid was then transformed into E. coli HT115 bacteria. Sequence insertion into the L4440 plasmid was confirmed via Sanger sequencing (using M13 forward sequencing primer). Although the Ahringer clone targeting mex-3 was obtained, RNAi of mex-3 in lsy-6(ot150) and mir-48 mir-241(nDf51) resulted in highly penetrant embryonic lethality preventing scoring of the F1 progeny of the RNAi treated animals.RNAi constructs (pL4440) were obtained from the Ahringer RNAi library . fubl genes, and the asd-2/gld-1 pair which show a very low level of overlap in targeted sequence, allowing for the possibility that some cross-gene RNAi targeting may occur.Despite the overall relatedness of protein architecture among member of phylogenetic clades , BLAST (Plim-6::gfp (otIs114) and lsy-6(ot150); Plim-6::gfp (otIs114) worms were placed on RNAi as embryos and F1 progeny were scored as L4 or young adults to increase the ease of detecting fluorescent signal in ASEL neurons. Each group of genes was scored alongside the negative control (empty L4440 vector) and our positive control (dcr-1 RNAi). Worms were scored as cell fate defective when lim-6::gfp was undetectable in the ASEL neuron soma.cog-1::gfp (syIs63) and cog-1::gfp (syIs63); otIs193 worms were placed on RNAi as embryos and F1 progeny were scored at mid-late L4s in order to ensure a strong GFP signal in both vulval and uterine cells. Each group of genes was scored alongside the negative control (empty L4440 vector) and our positive control (dcr-1 RNAi). Worms were considered to have abnormal uterine cog-1::gfp if either the anterior or posterior or both uterine cells were lacking GFP. cog-1 expression was scored as normal when GFP expression was observed in both uterine cells and in vulval cells.col-19::gfp (maIs105) and mir-48 mir-241(nDf51) col-19::gfp (maIs105) animals were placed on RNAi as young L4s and their F1 progeny were scored as young adults. Each group of genes was scored alongside the negative control (empty L4440 vector) and our positive control (dcr-1 RNAi). Worms were scored first for the presence of col-19::gfp in the hypodermal cells. Normal expression was defined as all hypodermal cells expressing col-19::gfp, whereas abnormal expression was defined as GFP signal absent in many or all of hypodermal cells. Worms were also scored for the number of seam cells present between the pharynx and rectal cells; seam cells were identified using the col-19::gfp transgene. lin-31(n1053), col-19::gfp (maIs105); alg-1(ma202) worms were scored in an identical manner when assaying hypodermal col-19::gfp expression.hbl-1p::gfp::NLS::hbl-1 3ʹUTR (ctIS39) animals were synchronized by bleaching and plated on RNAi plates with the RNAi bacteria supplemented with fluorescent beads for accurate staging  worms were grown and maintained at 15°C. Embryos were synchronized by hypochloride/NaOH solution and embryos plated directly onto RNAi plates as previously described (dcr-1 RNAi).Pdpy-30::GFP(PEST)-H2B::lin-41 3ʹUTR (xeSi78); Pdpy-30::mCherry::H2B::artificial 3ʹUTR (xeSi36)] (HW1113),  (HW1114),  (HW1159)  worms were placed on RNAi as L4s and allowed to lay embryos. When the F1 progeny reached the L4 stage, individual hermaphrodites were moved to their own RNAi plates and allowed to lay embryos for 24 h. After 24 h, each animal was moved to a fresh RNAi plate each day for three additional days. Live larvae were counted on each plate (by picking) 24 and 48 h after the parent has been moved to ensure all larvae were counted. Dead embryos on each plate were counted 48 h after removal of the parent. The total number of live larvae and dead embryos for each hermaphrodite was tallied and together encompass brood size. Embryonic lethality was calculated as  × 100%. Larval arrest was rarely seen, but when it did occur these worms were counted as “live larvae” because they had successfully hatched and developed beyond the embryonic stage.C. elegans genome: mask-1 was excluded due to extensive length and sequence/domain variability from the rest of the protein family.Full proteins sequences of the longest isoforms for each protein were collected from Wormbase and entered to the Mega X program. A MUSCLE protein alignment was carried out to provide input for further phylogenetic analysis. In order to construct the tree, we selected the Maximum Likelihood method and bootstrapped the tree-building (1000 iterations) to increase the stringency of the method. A simple LG model was selected for the substitution model, using a Nearest-Neighbor-Interchange (NNI) method. The phylogenic tree shown represents 27 of the 28 KH domain proteins in the To generate the protein domain graphics, we first determined the longest isoform of each individual protein. The amino acid sequence of the proteins were obtained from Wormbase.org and entered into Simple Modular Architecture Research Tool (SMART)  under thTo generate the coverage of each RNAi clone used in this study, primer pairs were obtained from the Ahringer library database, aligned to the appropriate transcript. Each RNAi target was then translated in the appropriate frame and aligned to the complete protein sequence. Predicted NLS sites were determined using cNLS Mapper using a threshold of 5.0  reduction-of-function mutation causes a low penetrance phenotype, with ∼15% of lsy-6(ot150) animals displaying an ASEL cell fate defective phenotype. This cell fate defect can be observed by the loss of the Plim-6::gfp expression within the ASEL neuron  mutant background  cell fate defective phenotype (lsy-6(ot150) allele, with the exception of F54D1.1 and mxt-1, whose depletion caused an occasional loss of Plim-6::gfp expression in ASEL  phenotype (To identify whether KH-domain genes play a role in ckground  and assahenotype . RNAi-me in ASEL . Furtherhenotype .lsy-6(ot150) were also able to regulate a lsy-6 target, cog-1  mutant background and assayed both col-19::gfp expression and seam cell number in young adult animals (mir-48 mir-241(nDf51) young adults fail to properly undergo the adult-specific developmental program, thereby showing a delay in col-19::gfp expression consistent with a delay in normal developmental timing. RNAi of thirteen KH domain genes significantly enhanced the abnormal col-19::gfp expression phenotype observed in mir-48 mir-241(nDf51) animals  mutants compared to the empty vector control (col-19::gfp expression and increased the seam cell number of mir-48 mir-241(nDf51) mutants , a temperature sensitive reduction-of-function mutation, causes a mildly penetrant vulval rupture phenotype at 15°C  assay, using three reporter strains: Pdpy-30::gfp::lin-41 3ʹUTR alone, Pdpy-30::gfp::lin-41 3ʹUTR with let-7(n2853), and Pdpy-30::gfp::lin-41 3ʹUTR delta LCS (two major let-7 binding sites removed from the lin-41 3ʹUTR) . RNAi of each B0280.17 and mxt-1 de-repressed GFP::lin-41 3ʹUTR  and lin-41::gfp delta LCS backgrounds  that primarily associate with miRNAs and are central for miRNA biogenesis and activity , result in more pronounced defects in miRNA activity, likely due to sequestration of miRNA pathway components away from ALG-2  animals display severe heterochronic defects  young adult animals failing to appropriately express adult cell marker col-19::gfp in hyp7 hypodermal cells  is variable, ranging from a complete lack of expression in the seam cells to full seam cell expression  background and assessed col-19::gfp expression in hypodermal cells of young adult animals. We used this background to screen for genes that may normally negatively interact with the miRNA pathways and therefore suppress the alg-1(ma202) phenotype when knocked down. Interestingly, RNAi of E02D9.1 significantly suppressed the abnormal hypodermal col-19::gfp expression in alg-1(ma202) young adults, with ∼60% of alg-1(ma202) animals showing wild type hypodermal col-19::gfp expression in young adults  restored wild type col-19::gfp expression in alg-1(ma202) young adults, something that is never observed in alg-1(ma202) mutants alone  suppressors, these KH domain genes may act in a manner that opposes normal miRNA activity, with their depletion perhaps resulting in decreased miRNA target gene expression.To determine whether any of the KH domain genes might normally have a negative genetic relationship with miRNA pathway components, we performed RNAi knockdown of KH domain genes in the g adults . Althougts alone . As alg-C. elegans development, we assayed the brood size and embryonic lethality of animals with reduced KH domain gene function. Knockdown of seven genes  resulted in significant reduction in brood size   , which i (SMART) . We foun (SMART) . Unsurpr (SMART)  and 9. TC. elegans KH domain-containing RBPs may function with miRNAs to regulate gene expression, we asked whether RNAi knockdown of KH domain genes could modify the phenotypes observed in reduction-of-function miRNA or family mutants. Surprisingly, eighteen of the twenty-four tested genes genetically interacted with at least one miRNA mutant background, suggesting widespread functional interaction between KH RBPs and miRNAs. Interestingly, the KH domain genes fell into two groups: those that modified phenotypes of all miRNA sensitized backgrounds tested and those that genetically interacted with specific miRNA reduction-of-function mutants , participates in the spliceosome assembly by binding 3ʹ branch sites of pre-mRNAs, whereas its partner, U2AF, cooperatively binds the 5ʹ branch site  phenotype enhancement are neuronally expressed, although it remains unknown whether these KH domain genes are specifically expressed in ASEL/R neurons. Future work is needed to characterize precise tissue and cellular expression to fully understand the spatial and temporal overlap among the molecules in question.To better understand the biological context in which miRNAs and KH domain proteins may interact, we compiled spatial KH domain gene expression patterns using existing promoterome  (mir-48 mir-241(nDf51)  (let-7(n2853)  and let-7(n2853), but not lsy-6(ot150)  was previously identified as a functional interactor of RNAi   and mir-1(nDf51)  mutants,1(nDf51) , but not7(n2853) , suggest6(ot150) . The letC. elegans we performed a phylogenetic analysis  phenotype  and B0280.17 (HOW) can enhance the stability of mRNAs   and coulC. elegans functionally interact with miRNA-mediated regulation of gene expression. Further work is essential to characterize the mechanisms through which individual KH domain proteins may affect gene expression and how they might functionally intersect with miRNA pathways. This study highlights a number of candidates for future genetic, molecular, and biochemical characterization and shows the extent to which miRNAs and KH domain RBPs may directly or indirectly coordinate to ultimately regulate gene expression.Overall, our screen showed that many of the KH domain-containing RBPs in


Background: Increased IL-6 level, M2 macrophages and PD-1+CD8+ T cells in tumor microenvironments (TME) have been identified to correlate with resistance to checkpoint blockade immunotherapy, yet the mechanism remains poorly understood. Rab small GTPase-mediated trafficking of cytokines is critical in immuno-modulation. We have previously reported dysregulation of Rab37 in lung cancer cells, whereas the roles of Rab37 in tumor-infiltrating immune cells and cancer immunotherapy are unclear.Methods: The tumor growth of the syngeneic mouse allograft in wild type or Rab37 knockout mice was analyzed. Imaging analyses and vesicle isolation were conducted to determine Rab37-mediated IL-6 secretion. STAT3 binding sites at PD-1 promoter in T cells were identified by chromatin immunoprecipitation assay. Multiplex fluorescence immunohistochemistry was performed to detect the protein level of Rab37, IL-6 and PD-1 and localization of the tumor-infiltrating immune cells in allografts from mice or tumor specimens from lung cancer patients.Results: We revealed that Rab37 regulates the secretion of IL-6 in a GTPase-dependent manner in macrophages to trigger M2 polarization. Macrophage-derived IL-6 promotes STAT3-dependent PD-1 mRNA expression in CD8+ T cells. Clinically, tumors with high stromal Rab37 and IL-6 expression coincide with tumor infiltrating M2-macrophages and PD1+CD8+ T cells that predicts poor prognosis in lung cancer patients. In addition, lung cancer patients with an increase in plasma IL-6 level are found to be associated with immunotherapeutic resistance. Importantly, combined blockade of IL-6 and CTLA-4 improves survival of tumor-bearing mice by reducing infiltration of PD1+CD8+ T cells and M2 macrophages in TME.Conclusions: Rab37/IL-6 trafficking pathway links with IL-6/STAT3/PD-1 transcription regulation to foster an immunosuppressive TME and combined IL-6/CTLA-4 blockade therapy exerts potent anti-tumor efficacy. In particular, how TME regulates PD-1 upregulation on CD8+ T cells is still a puzzle.Immune cells are known to accumulate in tumor microenvironment (TME) and crucial for regulating tumor progression Elevated IL-6 level is linked to chronic inflammatory conditions and observed in many types of cancers Rabs are a large group of small GTPases and function as regulators of vesicle trafficking and protein transport. The activity of Rab proteins is controlled by active GTP-bound and inactive GDP-bound forms. Dysregulation of Rab GTPases has been reported to associate with tumorigenesis. We have previously reported that Rab37 mediates exocytosis of several secretion factors in non-small cell lung (NSCLC) cells  via the Rab37/IL-6/STAT3 transcription axis. Combined blockade of IL-6 and CTLA-4 significantly suppresses lung tumor growth and metastasis in mice. Clinically, NSCLC patients with increased intratumoral Rab37+IL-6+ immune cells where enriched with tumor infiltrated CD163+ M2-TAMs and exhausted PD-1+CD8+ T cells show poor prognosis. Our findings highlight the crucial role of Rab37/IL-6/PD-1 axis in supporting immunosuppressive TME.Here, we report that Rab37-mediated exocytosis of IL-6 in NSCLC-associated macrophages promotes their M2 polarization and further PD-1 upregulation in T cellsRab37 KO target vector is shown in Rab37 gene was deleted before injecting into C57BL/6J (B6) mice using Cre-loxP system to establish the whole body depleted Rab37 KO mice. Embryonic stem cells were selected by neomycin and genotyped by Southern blotting. The selected cells were injected into blastocysts to generate chimeric mice later used for producing Rab37 KO mice. Mouse studies were approved by the Institutional Animal Care and Use Committee of National Cheng Kung University (NCKU) (Permit Numbers: #106255).The specific mouse Rab37 KO mice were detected with mouse cytokines antibody array-membrane (96 targets) . Media derived from BMDMs of WT mice and Rab37 KO mice treated with the conditioned media (CM) from mouse Lewis lung carcinoma (LLC) cells were collected at 0 h (MOCK) or 24 h (LCM). The array was performed according to the manufacturer's instructions.Rab37 positive-related cargo proteins in supernatants of BMDMs derived from wild-type (WT) and 6) were sonicated and supernatants were obtained by centrifugation  and vesicles were enriched from supernatants by high speed centrifugation  using a 40-Ti rotor . The vesicles-containing solution (500 μg) was incubated with anti-V5 antibody to isolate Rab37-specific vesicles and the IL-6 cargos in vesicles were analyzed by Western blot (6) were cross-linked to prepare nuclear lysates using Magna ChIPTM protein G Kit  followed by immunoprecipitation with 5 μg anti-p-STAT3(Y705) antibody . Q-PCR was carried out using ChIP products. The primer sequences are listed in RAW264.7 cells /2 during observation. For orthotopic model, mice were sacrificed at day 21 and the primary injected lung and contralateral lungs were removed and resected for RNA/protein extraction, flow cytometry (FLOW) analysis and preserved in paraffin for immunofluorescence immunohistochemistry (IF-IHC) staining. Blood samples were collected for serum biochemistry examinations for toxicity.All animal experiments were performed in compliance with NCKU institutional guidelines for use and care of animals (Permit Numbers: #106255). For α-IL-6  and α-CTLA-4  treatment, LLC subcutaneous injection and orthotopic injection models were used. For subcutaneous model, 5×10A total of 62 surgically resected lung cancer patients were recruited from NCKU Hospital, after obtaining appropriate institutional review board permission (#B-ER-106-102) and informed consent from the patients. Overall survival was calculated from the day of surgery to the date of death or the last follow-up. Tumor typing and disease staging were performed according to the World Health Organization classification and the TNM classification system, respectively. A total of 16 lung cancer patients receiving clinical α-PD-1 Ab treatment were also recruited (#B-ER-106-102). All patients received regular follow-up at 3-month intervals to evaluate the treatment responses. Partial responders showed no enlargement or newly developed distant metastatic foci on CT scan, while poor non-responders were patients with enlargement or newly developed distant metastatic foci on CT scan. Blood samples were collected at pre-treatment and 3-month on-treatment of α-PD-1 therapy for ELISA analysis. The detailed clinicopathological characteristics of patients are listed in 2 test was used to compare the correlation of Rab37, IL-6, CD45, CD163, CD8 or PD-1 protein expression in lung cancer patients. Overall survival curves were calculated according to the Kaplan-Meier method, and comparison was performed using the log-rank test. Cox regression comparison was performed to analyze the relative risk for patient poor outcome. The level of statistical significance was taken as p value, *, p < 0.05; **, p < 0.01; ***, p < 0.001.Cell studies were conducted in three independent experiments unless indicated otherwise. The number of mouse per group for animal studies was described for each experiment. Two-tailed Student's t test was used. Data represented mean ± SD. Pearson χDetailed methods are enclosed in the online Rab37 KO mice in C57BL/6 background  was significantly lower in T cells from Rab37 KO mice compared with WT mice . LLC-CM treatment increased the PD-1 expression on WT CD8+ T cells compared to those from Rab37 KO mice  as compared to WT cells. Conversely, Rab37 KO BMDMs showed enhanced expression of M1 marker IL-12 gene even upon IL-13 stimulation , constitutively active Rab37-GTP mutant (Rab37Q89L) and dominant negative Rab37-GDP mutant (Rab37T43N) in RAW264.7 murine macrophage cell line. ELISA data showed that IL-6 level was elevated in CM from Rab37WT and Rab37Q89L, but reduced in Rab37T43N RAW264.7 cells  was progressively taken up into Rab37 (green) punctate vesicular structures to form colocalization vesicles (yellow) and then rapidly released for another run of Rab37-mediated trafficking . The immunoblotting results showed that treatment of α-IL-6 attenuated Rab37WT-induced p-STAT3 signals , which was reduced by α-IL-6 treatment  and induced the M1 genes (IL-12 and NOS2) in BMDMs  therapy were abrogated in Rab37 KO mice  on LLC tumor growth in subcutaneous model  was observed especially in late stage patients (+Rab37+IL-6+ cells per region of interest (ROI) were significantly associated with advanced tumor stage   patients showed CD45+Rab37+IL-6+ high expression (Table +Rab37+IL-6+ expression were significantly associated with advanced tumor stage (P < 0.001), lymph node metastasis (P = 0.025) and distal metastasis (P = 0.003)  and exhausted T cells (PD-1+CD8+) were enriched in the area of CD45+Rab37+IL-6+ cells. Additional multiplex IF-IHC of CD163, CD8, PD-1 and panCK was performed on 20 tumor specimens, which were stained for Rab37, IL-6 and CD45. Two representative images are shown in Figure +Rab37+IL-6+, stromal Rab37 was accumulated around the CD163+ M2-TAMs (ROI 1)     (Figure +CD8+ exhausted T cells to elicit an immunosuppressive TME, leading to poor prognosis of NSCLC patients.To verify if CD45+ signals in TME were stronger in tumor derived from advanced stage patients than those from early ones (Figure Our ELISA results in animal model showed a positive correlation of plasma IL-6 level with tumor size Figure J. In add+ T cells, ultimately establishing an immunosuppressive TME (Figure Rab37 gene expression is downregulated by promoter hypermethylation in cancer cells + T cells, Rab37 has been found in recycling endosomes + T helper cells, Rab37 is suggested to be associated with secretion of IL-2, IL-4 and TNF-α Here, we reveal novel findings that Rab37, a regulator of vesicle transport and protein trafficking, acts as a tumor promoter in stromal cells. Rab37 mediates IL-6 secretion and STAT3 transcription activation leading to M2 macrophage polarization and upregulation of PD-1 on CD8+ T cells in TME via stabilization of IL-6 mRNA in macrophages IL-6 is a pleotropic pro-inflammatory cytokine, thereby contributing to the dysfunction of innate and adaptive immune responses against tumors PD-1 mRNA expression in T cells through STAT3 transcriptional control. Indeed, immunoreactivity of p-STAT3 in TME correlates with expression of PD-1+ stromal cell PD-1 mRNA expression in T cells Pdcd1 gene in T cells can be positively regulated by STAT3, NFATc1, FoxO1, and NF-κB, or negatively controlled by Blimp-1 and T-bet Pdcd1 promoter at -3568 ~ -3419, -3506 ~ -3311 and -1709 ~ -1506, which are located close to the two previously reported DNase I hypersensitive sites at -3.7 kb and -1.1 kb Pdcd1 gene locus was dynamically changed during T cell stimulation and cytokine exposure We discovered that macrophage-derived IL-6 served as a paracrine signal to up-regulate . Several studies have shown that inhibition of IL-6 enhances the therapeutic efficacy of α-PD-L1 therapy in melanoma, colorectal cancer, pancreatic cancer and hepatocellular carcinoma Development of effective therapeutics to ameliorate the immunosuppressive TME in cancer patients is eagerly anticipated in vitro and in vivo experiments that infiltration of intratumoral Rab37+IL-6+ immune cells enriched with CD163+ M2 macrophages is a feature of high density of PD-1+CD8+ exhausted T cells. IL-6 is reported to induce the expression of M2-like macrophages and granulocytic myeloid-derived suppressor cells, and protumor T-regulatory/Th17 cell responses, whereas reducing anti-tumor ability of Th1 and CD8+ T cells +IL-6+CD163+CD8+PD1+ defined by IF-IHC or high IL-6 of plasma ELISA as biomarkers would identify patients in dire need of intensive surveillance and further combination therapy with α-IL-6 and α-CTLA-4 antibodies would improve their clinical outcome.Our clinical data corroborated with via the Rab37/IL-6/STAT3 transcription axis to support lung cancer progression. Combined blockade of IL-6 and CTLA-4 suppressed lung tumor growth in mice by increasing tumor-infiltrating CD8+ T cells and M1 macrophages. Clinically, NSCLC patients with high intratumoral CD45+Rab37+IL-6+ or IL-6+CD163+CD8+PD-1+ immunoreactivity or an increase in plasma IL-6 level are found to be significantly associated with poor prognosis or therapeutic resistance. This study uncovers a novel function of Rab37 in suppressing tumor immunity and adds a new insight into the complexity of PD-1 regulation. Our findings provide a rationale and feasible combined therapeutic approach targeting IL-6 and CTLA-4 in lung cancer patients.In conclusion, we found that Rab37-mediated exocytosis of IL-6 from macrophages promotes PD-1 upregulation in T cells Supplementary figures and tables.Click here for additional data file.Supplementary movie S1.Click here for additional data file.Supplementary movie S2.Click here for additional data file.Supplementary movie S3.Click here for additional data file.Supplementary movie S4.Click here for additional data file.Supplementary movie S5.Click here for additional data file.Supplementary movie S6.Click here for additional data file.


Triticum aestivum L.). In total, 11 KASP assays developed in this study and five already reported assays were used for their application in wheat breeding. We investigated alleles at 16 loci associated with drought tolerance among 153 Pakistani hexaploid wheat cultivars released during 1953–2016; 28 diploid wheat accessions (16 for AA and 12 for BB) and 19 tetraploid wheat (AABB) were used to study the evolutionary history of the studied genes. Superior allelic variations of the studied genes were significantly associated with higher grain yield. Favored haplotypes of TaSnRK2.3-1A, TaSnRK2.3-1B, TaSnRK2.9-5A, TaSAP-7B, and TaLTPs-1A predominated in Pakistani wheat germplasm indicating unconscious pyramiding and selection pressure on favorable haplotypes during selection breeding. TaSnRK2.8-5A, TaDreb-B1, 1-feh w3, TaPPH-7A, TaMOC-7A, and TaPARG-2A had moderate to low frequencies of favorable haplotype among Pakistani wheat germplasm pointing toward introgression of favorable haplotypes by deploying functional markers in marker-assisted breeding. The KASP assays were compared with gel-based markers for reliability and phenotypically validated among 62 Pakistani wheat cultivars. Association analyses showed that the favorable allelic variations were significantly associated with grain yield-contributing traits. The developed molecular marker toolkit of the genes can be instrumental for the wheat breeding in Pakistan.High-throughput genotyping for functional markers offers an excellent opportunity to effectively practice marker-assisted selection (MAS) while breeding cultivars. We developed kompetitive allele-specific PCR (KASP) assays for genes conferring drought tolerance in common wheat ( Crop improvement strategies have always circumambulated yield-enhancing genes. Therefore, exploitation of gene diversity in breeding germplasm and identification of superior genetic variations are prioritized activities in crop genetic improvement. Such exploitations enable breeders to identify desirable germplasm for breeding and to devise strategies for pyramiding superior genetic variations for targeted traits. Wheat is one of the most important cereal crops, and there is demand for a yield increase of up to 50% by 2050 . Being aTaSnRK2.3-1A/1B  of the aforementioned genes were successfully applied in Chinese wheat cultivars and provided the concept of screening of genotypes for wheat breeding in Pakistan. Various single-marker methods have been developed for single-nucleotide polymorphism (SNP) genotyping, such as cleaved amplified polymorphic sequences (CAPS), and derived cleaved amplified polymorphic sequences (dCAPS). The CAPS and dCAPS markers are relatively low throughput, laborious, and cost ineffective, as they rely upon site-specific cleavage of PCR products with restriction enzymes and require gel electrophoresis to separate products. At present, more than 150 FMs are available for important genes, giving plant breeders a molecular toolkit for the selection of favorable traits . AlthougKompetitive allele-specific PCR (KASP) is a uniplex and flexible genotyping platform which achieves high throughput in a time- and cost-effective way . ConversTriticum aestivum germplasm, nine genotypes of T. urartu (AA), four genotypes of T. boeoticum Bioss (AA), three genotypes of T. monococcum (AA), 12 genotypes Aegilops speltoides (BB), five genotypes of T. dicoccum L. (AABB), three genotypes of T. persicum Vav (AABB), five genotypes of T. dicoccoides Koern (AABB), four genotypes of T. polonicum L., and two genotypes of T. turgidum (AABB) (One hundred and fifty-three wheat cultivars from Pakistan released during 1953–2016 were used to identify favorable haplotype frequencies of drought tolerance-conferring genes . The whem (AABB)  were alsTaSnRK2.9-5A , and 12 μl of each tailed primer (100 μM). Assays were tested in 96-well formats and set up as 5 μl reaction mixture . PCR cycling was performed using the following protocol: hot start at 95°C for 15 min, followed by 10 touchdown cycles , followed by 32–35 additional cycles of annealing . Fluorescence levels were detected and analyzed by using CFX Connect Real-Time PCR detection system (Bio-Rad® laboratories Inc. United States) and QuantStudio 7 Flex Real-Time PCR systems.For the KASP assays developed in this study, the nucleotide sequences of drought tolerance-causing genes were retrieved from the published literature. The diagnostic polymorphic sites were identified, and KASP primers were developed following standard KASP guidelines. The allele-specific primers were designed carrying the standard FAM and HEX tails and with the targeted SNP at the 3′ end. A common primer was designed so that the total amplicon length was less than 120 bp. The primer mixture comprised 46 μl ddHt-test at P < 0.05 was used to check the effect of SNP/haplotype on the studied agronomic traits. Allele/haplotype frequencies were calculated for all loci. PIC and gene diversity (He) were calculated for each locus using https://www.gene-calc.pl/pic.Student’s TaPARG-2A-KASP-10 (C/T), the concentration of each tailed primer increased up to 15 μM to obtain satisfactory results.The results from KASP markers were compared to contrasting gel-based markers for all the genes. All studied KASP assays produced consistent results when compared to conventional PCR markers in 23 diverse wheat genotypes , but forHap-1 (CA) of TaSnRK2.3-1A and Hap-1 (CG) of TaSnRK2.3-1B which are favored for higher GPS and TKW  of TaSnRK2.8-5A showed a non-significant association with higher GPS and TKW. Hap-1 (TA) and Hap-4 (CA) of TaSnRK2.9-5A are also associated with higher GPS and TKW  and higher GPS and TKW. Accessions carrying Hap-H of TaMOC1-7A possess higher GPS and TKW than Hap-L. Hap-3 (GC) of TaLTPs associated with ideal plant height (100 cm) and higher GPS and TKW. TaDreb-B1 and 1-feh w3 accessions carrying the Hap-1 “A-allele” and Hap-1 “C-allele,” respectively, are associated with higher grain yield contributing parameters (Hap-1 (CC) of TaPARG-2A showed an association with higher GPS. Favorable allelic variation of TaPPH-7A-1  also showed an association with higher GPS among Pakistani wheat cultivars  Pakistani wheat cultivars had the desirable haplotype (Hap-1). The favorable haplotype (Hap-1) of TaSnRK2.3-1B was also present in 127 (83.01%) Pakistani wheat cultivars  in given wheat cultivars. The frequency of favored haplotypes for TaSnRK2.9-5A (Hap-1 and Hap-4) was 75.82% and 2.61%, respectively. The favored allele for TaSAP-7B was predominant in 129 (84.31%) wheat cultivars; at TaMOC1-7A, 33 (21.57%) desirable haplotypes (Hap-H). The favorable haplotype (Hap-3) of TaLTPs was present in 146 (95.42%) wheat cultivars. Superior alleles for TaDreb-B1, 1-feh w3, and TaPPH-7A were present in 48 (31.37%), 44 (28.75%), and 67 (43.79%) wheat cultivars. The favorable haplotypes of TaPARG-2A (Hap-2 (1.31%) and Hap-3 absent) were present in very low frequencies among given wheat germplasm  wheat cultivars.TaSnRK2.3-1A/1B, 83.01% wheat accessions contained favored haplotypes. For TaSnRK2.9-5A, TaLTPs-1A, and TaSAP-7B, 78.41%, 95.42, and 84.31% Pakistani wheat accessions possessed favorable allelic variations, respectively. Based on released time, Pakistani wheat cultivars were divided into four groups. From 1953 to 2016, the frequency of favored haplotypes for TaSnRK2.3-1A, TaSnRK2.3-1B, TaSnRK2.9-5A, TaSAP-7B, and TaLTPs-1A increased remarkably. The combined frequencies of favored alleles/haplotypes of aforementioned genes increased from 35.78% in the pre-1965s to 93% in the post-2005s, showing a progressive selection of favored alleles/haplotypes over the years. The frequencies of favored alleles/haplotypes of other drought-conferring genes remain low (<22%) since 1953, suggesting the potential of favored allele/haplotype introgression through FMs developed in this study.Since 1953, the frequency distribution of favored haplotypes of given drought tolerance-responsible genes varied among Pakistani wheat germplasm . For TaSe) in the investigated genes. The studied tetraploid (AABB) accessions showed an average 0.361 (PIC) and 0.418 (He) in the given genes. Hexaploid Pakistani wheat showed an average 0.270 (PIC) and 0.317 (He) in the given genes  among Pakistani wheat accession reduced.To survey the evolutionary history of given drought tolerance-responsible genes, we analyzed the given genes in wheat progenitor accessions. The results showed that during polyploidization events, diversity decreased in a given set of genes. Diploid wheat accessions showed an average of 0.265 (PIC) and 0.0.331  showed inconsistent outcomes in the form of heterozygous conditions for the given alleles. Overall, the conversion rate for newly developed KASP assays was > 98% and were able to convert gel-based PCR markers into breeder friendly gel-free KASP markers.Identification and validation of SNPs is a significant challenge in wheat due to the large genome size, polyploidy, and high percentage of repetitive sequences . Hence, TaSnRK2.3-1A/1B, TaSnRK2.9-5A, TaSAP-7B, and TaLTPs-1A among Pakistani wheat accessions. A moderate frequency of favored haplotypes was observed at TaSnRK2.8-5A, TaDreb-B1, TaPPH-7A, and 1-feh w3, indicating that exploitation of these alleles may be continued to gain a yield increase in Pakistan. This unconscious selection of favorable haplotypes is likely due to the high linkage disequilibrium of important genes selected during selection breeding. The given Pakistani wheat germplasm had high frequencies of unfavorable allelic variations for TaMOC-7A and TaPARG-2A, suggesting the potential of favorable haplotype introgression through functional markers. Previously, selection pressure was observed on TaSnRK2.3-1A/B  can be found in the article/SU was responsible for conceptualization. SU, MA, MSd, MK, MN, RI, and AI performed the experiments and analyzed the data. SU wrote the manuscript. ZA, XM, MSj, and RJ reviewed the manuscript and assisted in the completion of the experiments. All authors contributed to the article and approved the submitted version.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.