Secondary metabolites of mulberry leaves exert anti-lung cancer activity through regulating the PD-L1/PD-1 signaling pathway

Lung cancer ranks the top of malignancies that cause cancer-related deaths worldwide.The leaves of Morus alba L are traditional Chinese medicine widely applied in respiratory diseases.Our previous work has demonstrated the anti-lung cancer effect of secondary metabolites of mulberry leaf,but their mechanism of action has still not fully elucidated.We synthesized Moracin N(MAN)-Probe conjugated with alkyne to label lung cancer cells and identified protein targets by chemical proteomic analysis.MAN and its probe exerted similar growth-inhibitory effect on human lung cancer cells.Chemical proteomic results showed that MAN targeted the programmed death ligand 1(PD-L1)checkpoint pathway and T cell receptor(TCR)signaling pathway,indicating its immune-regulatory function.Cell-free surface plasmon resonance(SPR)results showed the direct interaction of MAN with PD-L1 protein.Molecular docking analysis demonstrated that MAN bound to E158 residue of PD-L1 protein.MAN downregulated the expression levels of PD-L1 in a time-and dose-dependent manner and disrupted the PD-L1/programmed death 1(PD-1)binding,including other secondary metabolites of mulberry leaves Guangsangon E(GSE)and Chalcomoracin(CMR).Human peripheral blood mononuclear cells(PBMCs)co-cultured with MAN-treated A549 cells,resulting in the increase of CD8^(+)GZMB^(+)T cells and the decrease of CD8^(+)PD-1^(+)T cells.It suggested that MAN exerts anti-cancer effect through blocking the PD-L1/PD-1 signaling.In vivo,MAN combined with anti-PD-1 antibody significantly inhibited lung cancer development and metastasis,indicating their synergistic effect.Taken together,secondary metabolites of mulberry leaves target the PD-L1/PD-1 signaling,enhance T cell-mediated immunity and inhibit the tumorigenesis of lung cancer.Their modulatory effect on tumor microenvironment makes them able to enhance the therapeutic efficacy of immune checkpoint inhibitors in lung cancer.

Hematopoiesis reconstitution and anti-tumor effectiveness of Pai-Neng-Da capsule in acute leukemia patients with haploidentical hematopoietic stem cell transplantation

BACKGROUND With the rapid development of haploidentical hematopoietic stem cell transplantation(haplo-HSCT),primary poor graft function(PGF)has become a lifethreatening complication.Effective therapies for PGF are inconclusive.New Chinese patent medicine Pai-Neng-Da(PND)Capsule exerts dual effect in promoting hematopoiesis recovery and regulating immunity.Still,the application of PND capsule in hematopoietic stem cell transplantation,especially in the haplo-HSCT setting,has not yet been reported.AIM To evaluate the role of PND capsule in acute leukemia patients with haplo-HSCT.METHODS We retrospectively collected data of acute leukemia patients who underwent haplo-HSCT at the Affiliated People’s Hospital of Ningbo University between April 1,2015 and June 30,2020.Twenty-nine consecutive patients received oral PND capsule from the sixth day to the first month after haplo-HSCT were included in the PND group.In addition,31 patients who did not receive PND capsule during haplo-HSCT were included in the non-PND group.Subsequently,we compared the therapeutic efficacy according to the western medical evaluation indexes and Chinese medical symptom scores,and the survival between the PND group and the non-PND group,using the chi-square test,Fisher’s exact test,and the Kaplan-Meier method.RESULTS The duration of platelet engraftment was shorter in the PND group than in the non-PND group(P=0.039).The PND group received a lower frequency of red blood cells and platelet transfusions than the non-PND group(P=0.033 and P=0.035,respectively).In addition,PND capsule marginally reduced the rate of PGF(P=0.027)and relapse(P=0.043).After 33(range,4-106)months of follow-up,the 3-year relapse-free survival(P=0.046)and progression-free survival(P=0.049)were improved in the PND group than in the non-PND group.Also,the therapeutic efficacy of the PND group according to Chinese medical symptom scores was significantly better than that of the non-PND group(P=0.022).Moreover,the adverse events caused by PND capsule were mild.Nevertheless,there were no significant differences in the duration of neutrophil engraftment,the risk of infection within 100 days after haplo-HSCT,the acute graft-versus-host disease,or the 3-year overall survival between the two groups.CONCLUSION PND capsule could promote hematopoiesis reconstitution,improve the therapeutic efficacy of Chinese medical symptom scores,present anti-tumor effectiveness,and prolong the survival of acute leukemia patients with haplo-HSCT.

Research and Development of the Effective Components of Panaxdiol Saponin as New Chinese Patent Medicine for Treating Hemocytopenia

Pancytopenia （hemocytopenia） such as primary immune thrombocytopenia （ITP）, aplastic anemia and chronic neutropenia （agnogenic leukocytopenia） were often treated by glucocorticoids, androgen and immunosuppressive agents at present, but the response to these treatments has not been always satisfactory, and may cause serious adverse events. Our research has identified a biological active component in ginseng extract and the active component, panaxadiol saponins component （PDS-C）, was isolated from total saponins of ginsenosides, and formulated into capsules named as Painengda （派能达）. We successfully obtained approval from State Food and Drug Administration （SFDA） of China in 2010 to conduct clinical trials of PDS-C as class-five new Chinese patent medicine. Phase Ⅰand phase Ⅱ clinical trials of PDS-C and Painengda Capsule were carried out in the treatment of ITP and agnogenic leukocytopenia. Thecomposition and content of PDS-C have been analyzed and defined by high-performance liquid chromatography mass spectrometry （HPLC-MS） and HPLC using specific monomers of ginsenosides as the reference standards. PDS-C is very efficacious for treating mice and rats with ITP and aplastic anemia, and myelosuppression caused by chemotherapy or radiation. Our animal model studies and cell biology and molecular biology experiments demonstrated that PDS-C possessed dual activities, namely that of promoting proliferation and differentiation of hematopoietic progenitor cells, and that of regulating the immune function. PDS-C and Painengda Capsule as a new Chinese patent medicine have been successfully transferred to industry. We believe that PDS-C is effective and safe in the treatment of refractory hemocytopenia. The advantages are that it is effective in small doses, it is convenient to use because of its oral administration, its lack of adverse events, it could be used alone or in combination with pharmacological agents, which improve the efficacy and decrease adverse events.

Ginsenoside Rd Induces Differentiation of Myeloid Leukemia Cells via Regulating ERK/GSK-3βSignaling Pathway

Objective To investigate the role of ginsenoside Rd(GRd)in acute myeloid leukemia(AML)cell differentiation.Methods AML cells were treated with GRd(25,50,100 and 200µg/mL),retinoic acid(RA,0.1g/L)and PD98059(20 mg/mL)for 72 h,cell survival was detected by methylthiazolyldiphenyl-tetrazolium bromide and colony formation assays,and cell cycle was detected by flow cytometry.Cell morphology and differentiation were observed by Wright-Giemsa staining,peroxidase chemical staining and cellular immunochemistry assay,respectively.The protein expression levels of GATA binding protein 1(GATA-1),purine rich Box-1(PU.1),phosphorylated-extracellular signal-related kinase(p-ERK),ERK,phosphorylated-glycogen synthase kinase-3β(p-GSK3β),GSK3βand signal transducer and activator of transcription 1(STAT1)were detected by Western blot.Thirty-six mice were randomly divided into 3 groups using a random number table:model control group(non-treated),GRd group[treated with 200 mg/(kg·d)GRd]and homoharringtonine(HTT)group[treated with 1 mg/(kg·d)HTT].A tumor-bearing nude mouse model was established,and tumor weight and volume were recorded.Changes of subcutaneous tumor tissue were observed after hematoxylin and eosin staining.WT1 and GATA-1 expressions were detected by immunohistochemical staining.Results The cell survival was inhibited by GRd in a dose-dependent manner and GRd caused G0/G1 cell arrest(p<0.05).GRd treatment induced leukemia cell differentiation,showing increased expressions of peroxidase and specific proteins concerning erythrogenic or granulocytic differentiation(p<0.05).GRd treatment elicited upregulation of p-ERK,p-GSK-3βand STAT1 expressions in cells,and reversed the effects of PD98059 on inhibiting the expressions of peroxidase,GATA-1 and PU.1(P<0.05).After GRd treatment,tumor weight and volume of mice were decreased,and tumor cells underwent massive apoptosis and necrosis(P<0.05).WT1 level was decreased,and GATA-1 level was significantly increased in subcutaneous tumor tissues(P<0.05 or P<0.01).Conclusion GRd might induce the differentiation of AML cells via regulating the ERK/GSK-3βsignaling pathway.

Research Progress on Chinese Medicine Immunomodulatory Intervention for Chronic Primary Immune Thrombocytopenia: Targeting Cellular Immunity

Chronic primary immune thrombocytopenia(CITP) is the most common acquired autoimmune disease that seriously threaten the physical and mental health of patients. Compared with Western medicine treatment, the intervention and treatment of Chinese medicine(CM) has shown certain therapeutic advantages. This paper reviewed the new pathogenesis progress on T cell immune abnormality in CITP, and CM studies on interferes effects of cellular immune regulation of CITP in recent years. Qi deficiency failing to control blood and internal obstruction of blood stasis are the two common types of CM syndromes in CITP patients, the corresponding treatments include invigorating Pi(Spleen), supplementing qi, activating blood, as well as tonifying qi and activating yang, regulating Gan(Liver) to invigorate Pi. The authors also mentioned the problems in the research field of CM for CTIP treatment, and put forward new ideas for the research in the future.

Panax Notoginseng Saponins Induced Up-Regulation,Phosphorylation and Binding Activity of MEK,ERK,AKT,PI-3K Protein Kinases and GATA Transcription Factors in Hematopoietic Cells

Objective： To investigate the effects of panax notoginseng saponins （PNS） on expression, regulation and phosphorylation of multiple protein kinases in mitogen activated protein kinase （MAPK） intracellular signal pathway and GATA transcription factors in hematopoietic cells, so as to explore its mechanism of proliferation and differentiation activity on hematopoiesis. Methods： The human granulocytic HL-60, erythrocytic K562, megakaryocytic CHRF-288 and Meg-01 cell lines were treated by PNS, the positive control of K562, CHRF-288 ceils treated by recombination human erythropoietin （Epo） and thrombopoietin （Tpo） respectively. The total cell lysate and nuclei protein were extracted after being treated by PNS, subsequently, analyzed by both Western blot and immune-precipitation. Meanwhile, the nuclei extract was performed for electrophoretic mobility shift assay （EMSA） by using 32p radio labeled double-stranded GATA consensus oligonucleotide. Results： The expression levels of kinase MEK-1, MEK-2, ERK-1, ERK-2, AKT-1, AKT-2 and PI- 3K were increased by PNS treatment to different extent in four cell lines, depending on cellular heterogeneity and sensitivity to PNS, also phosphorylation of MEK-1, ERK-1 was differentially promoted by PNS respectively （P〈0.05, 0.01, 0.001）. The expression levels of transcription factors GATA-1 and GATA-2 were increased, moreover, their DNA binding activities were raised dramatically in PNS treated K562, CHRF-288 and Meg-01 cells compared with the controls respectively （P〈0.05, 0.01, 0.001）. The positive control of K562, CHRF-288 cells treated by Epo or Tpo respectively also displayed up-regulation of protein kinases and GATA transcription factors respectively （P〈0.05, 0.01, 0.001）. Conclusion： The results indicated that intracellular signal pathway initiated by PNS was involved in MAPK pathway and transcription factors of GATA family in hematopoietic cells. PNS displayed the role to promote proliferation and differentiation, by means of increasing expression level and phosphorylation status of multiple protein kinases, also inducing synthesis of GATA transcription factors and up- regulation its DNA binding activity.

Effects of Panaxadiol Saponins Component as A New Chinese Patent Medicine on Proliferation,Differentiation and Corresponding Gene Expression Profile of Megakaryocytes

Objective： To investigate the effects of panaxadiol saponins component （PDS-C） isolated from total saponins of panax ginseng on proliferation, differentiation and corresponding gone expression profile of megakaryocytes. Methods： Bone marrow culture of colony forming assay of megakaryocytic progenitor cells （CFU-MK） was observed for the promoting proliferation mediated by PDS-C, and differentiation of megakaryocytic blasts caused by PDS-C was analyzed with flow cytometry in CHRF-288 and Meg-01 cells, as well as proliferation, differentiation-related genes expression profile and protein expression levels were detected by human gone expression microarray and western blot. Results： In response to PDS-C 10, 20 and 50 mg/L, CFU-MK from 10 human bone marrow samples was increased by 28.9± 2.7%, 41.0% ± 3.2% and 40.5% ± 2.6% over untreated control, respectively （P〈0.01, each）. Flow cytometry analysis showed that PDS-C treated CHRF-288 cells and Meg-01 cells significantly increased in CD42b, CD41, TSP and CD36 positive ratio, respectively. PDS-C induced 29 genes up-regulated more than two-fold commonly in both cells detected by human expression microarray representing 4000 known genes. The protein expression levels of ZNF91, c-Fos, BTF3a, GATA-1, RGS2, NDRG2 and RUNX1 were increased with western blot in correspond to microarray results. Conclusion： PDS-C as an effective component for hematopoiesis, play the role to enhance proliferation and differentiation of megakaryocytes, also up-regulated expression of proliferation, differentiation-related genes and proteins in vitro. KEYWORDS panaxadiol saponins, megakaryocyte, gone expression profile, proliferation, differentiation

Effects of Danshen Injection(丹参注射液) on Inhibiting Proliferation and Inducing Apoptosis through Down-Regulation of Mutant JAK2 Gene and Its Protein Phosphorylation in Human Erythroid Leukemic Cells

Objective： To explore the effects of Danshen Injection （丹参注射液） on inhibition proliferation, inducing apoptosis and its possible mechanisms on human erythroid leukemic （HEL） cells. Methods： The commercial Chinese patent medicine of Danshen Injection was extracted and isolated from Chinese herb of Salvia miltiorrhiza bung. The inhibition effects of proliferation were assayed by 3-（4,5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （M＇l-r） method in HEL cells treated by Danshen Injection at various concentrations for 48 h. The cellular apoptosis was observed in morphology, analyzed by flow cytometry with annexin V and propidium iodide （PI） staining, and examined by DNA degradation ladder on agarose gel electrophoresis. Meanwhile, the expression levels of mutant Janus kinasez （JAK2） gene and phosphorylation-JAK2 （P-JAK2） protein were detected by allele specific-polymerase chain reaction and Western blot. ]Results： The proliferation of HEL cells was effectively inhibited by Danshen Injection in a dose-dependent manner, with suppression rates from 19.46 ± 2.31% to 50.20 ± 5.21%. Typical apoptosis cells was observed in Danshen Injection treated HEL cells, the rates of annexin V positive cells increased obviously in a dose-dependent manner, as well as the DNA degradation ladder of apoptosis revealed on gel electrophoresis. The expression levels of mutant JAK2 gene and P-JAK2 protein reduced gradually with increasing dosage of Danshen injection. Conclusion： Danshen Injection could not only significantly inhibit the proliferation, but also induce apoptosis in HEL cells; down-regulation of the mutant JAK2 gene and P-JAK2 protein expressions are probably one of its molecular mechanisms.

Ginseng-Derived Panaxadiol Saponins Promote Hematopoiesis Recovery in Cyclophosphamide-Induced Myelosuppressive Mice:Potential Novel Treatment of Chemotherapy-Induced Cytopenias

Objective：To investigate the potential efficacy of panaxadiol saponins component（PDS-C）,a biologically active fraction isolated from total ginsenosides,to reverse chemotherapy-induced myelosuppression and pancytopenia caused by cyclophamide（CTX）.Methods：Mice with myelosuppression induced by CTX were treated with PDS-C at a low-（20 mg/kg）,moderate-（40 mg/kg）,or high-dose（80 mg/kg） for 7 consecutive days.The level of peripheral white blood cell（WBC）,neutrophil（NEU） and platelet（PLT） were measured,the histopathology and colony formation were observed,the protein kinase and transcription factors in hematopoietic cells were determined by immunohistochemical staining and Western blot.Results：In response to PDS-C therapy,the peripheral WBC,NEU and PLT counts of CTX-induced myelosuppressed mice were significantly increased in a dose-dependent manner.Similarly,bone marrow histopathology examination showed reversal of CTX-induced myelosuppression with increase in overall bone marrow cellularity and the number of hematopoietic cells（P〈0.01）.PDS-C also promoted proliferation of granulocytic and megakaryocyte progenitor cells in CTX-treated mice,as evidenced by significantly increase in colony formation units-granulocytes/monocytes and-megakaryocytes（P〈0.01）.The enhancement of hematopoiesis by PDS-C appears to be mediated by an intracellular signaling pathway,this was evidenced by the up-regulation of phosphorylated mitogen-activated protein kinase（p-MEK） and extracellular signal-regulated kinases（p-ERK）,and receptor tyrosine kinase（C-kit） and globin transcription factor 1（GATA-1） in hematopoietic cells of CTX-treated mice（P〈0.05）.Conclusions：PDS-C possesses hematopoietic growth factor-like activities that promote proliferation and also possibly differentiation of hematopoietic progenitor cells in myelosuppressed mice,probably mediated by a mechanism involving MEK and ERK protein kinases,and C-kit and GATA-1 transcription factors.PDS-C may potentially be a novel treatment of myelosuppression and pancytopenia caused by chemotherapy.

Clinical Study of Pai-Neng-Da Capsule(派能达胶囊) in the Treatment of Chronic Aplastic Anemia

Objective：To evaluate the efficacy and safety of Pai-Neng-Da Capsule（派能达胶囊t panaxadiol saponins component,PND）,a new Chinese patent medicine,on patients with chronic aplastic anemia（CAA）and to explore the optimal therapeutic regimen for CAA.Method：A total of 36 patients with CAA were enrolled and divided into three groups：the AP group（20 cases,andriol 120 mg/day ＋ PND 240 mg/day）,the ACP group（13 cases,andriol 120 mg/day ＋ cyclosporine 3-6 mg·kd（-1）·day（-1） ＋ PND 240 mg/day）,and the PND group（3cases,PND 240 mg/day）.All patients were treated and followed up for 6 months.Peripheral blood counts,renal and hepatic function and Chinese medical（CM） symptoms of patients were assessed and all indices were gathered at the beginning and end of the study.Result：In the AP group,no significant hematologic difference was observed at the end of 6-month treatment comparing with the beginning.In the ACP group,the blood counts were maintained at the same level after the 6-month treatment.In the PND group,trilineage hematologic improvement was displayed at the end of 6-month treatment comparing with the beginning.No significant difference was showed in renal and hepatic function in all patients.All patients＇ clinical symptom improved according to CM symptom score.The effective rates were 95%,73%and 100%,respectively.Conclusion：PND improved the efficacy and decreased side effects by cutting down the dosage of andriol,and it could also improve patients＇ clinical symptom and quality of life.PND were effective and safe in the treatment of CAA,it could be used alone or in combination with pharmacological agents such as andriol and cyclosporine.

Chinese Medicine Regulates DNA Methylation to Treat Haematological Malignancies:A New Paradigm of"State-Target Medicine"

Aberrant regulation of DNA methylation plays a crucial causative role in haematological malignancies(HMs).Targeted therapy,aiming for DNA methylation,is an effective mainstay of modem medicine;however,many issues remain to be addressed.The progress of epigenetic studies and the proposed theory of"state-target medicine"have provided conditions to form a new treatment paradigm that combines the"body state adjustment"of CM with targeted therapy.We discussed the correlation between Chinese medicine(CM)syndromes/states and DNA methylation in this paper.Additionally,the latest research findings on the intervention and regulation of DNA methylation in HMs,including the core targets,therapy status,CM compounds and active components of the Chinese materia medica were concisely summarized to establish a theoretical foundation of"state-target synchronous conditioning"pattern of integrative medicine for HMs,simultaneously leading a new perspective in clinical diagnosis and therapy.

Treatment of Chronic Aplastic Anemia with Chinese Patent Medicine Pai-Neng-Da Capsule for Replacing Androgen Partially:A Clinical Multicenter Study

Objective:To evaluate the efficacy and safety of Pai-Neng-Da Capsule(panaxadiol saponins component,PNDC)in combination with the cyclosporine and androgen for patients with chronic aplastic anemia(CAA).Methods:A total of 79 CAA patients was randomly divided into 2 groups by a random number table,including PCA group[43 cases,orally PNDC 320 mg/d plus cyclosporine 5 mg/(kg·d)plus andriol 80 mg/d]and CA group[36 cases,orally cyclosporine 5 mg/(kg·d)plus andriol 160 mg/d].All patients were treated and followed-up for 6 treatment courses over 24 weeks.The complete blood counts,score of Chinese medical(CM)symptoms were assessed and urine routine,electrocardiogram,hepatic and renal function were observed for safety evaluation.Female masculinization rating scale was established according to the actual clinical manifestations to evaluate the accurate degree of masculinization in female CAA patients treated by andriol.Results:The effective rates were 88.1%(37/42)in the PCA group and 77.8%(28/36)in the CA group based on the standard for the therapeutic efficacy evaluation of hematopathy.There was no significant difference in the white blood cell(WBC)counts,platelet counts and hemoglobin concentration of peripheral blood between two groups after 6 months treatment.The masculinization score of female patient in the PCA group was significantly lower than the CA group(P<0.05).The mild abdominal distention was observed in 1 case in the PCA group.In CA group,the abnormalities in the hepatic function developed in 2 cases and the renal disfunction was found in 1 case.Conclusion:The PNDC possesses certain curative effects in the treatment of CAA without obvious side-effects and can partially replace andriol thereby to reduce the degree of masculinization[Registried at Chinese Clinical Trial Registry(ChicTR1900028153)].

Panaxdiol Saponins Component Promotes Hematopoiesis and Modulates T Lymphocyte Dysregulation in Aplastic Anemia Model Mice

Objective: To investigate the potential efficacy of panaxadiol saponins component(PDS-C) in the treatment of aplastic anemia(AA) model mice. Methods: Totally 70 mice were divided into 7 groups as follows: normal, model, low-, medium-, high-dose PDS-C(20, 40, 80 mg/kg, namely L-, M-, H-PDS-C), cyclosporine(40 mg/kg), and andriol(25 mg/kg) groups, respectively. An immune-mediated AA mouse model was established in BALB/c mice by exposing to 5.0 Gy total body irradiation at 1.0 Gy/min, and injecting with lymphocytes from DBA mice. On day 4 after establishment of AA model, all drugs were intragastrically administered daily for 15 days, respectively, while the mice in the normal and model groups were administered with saline solution. After treatment, the peripheral blood counts, bone marrow pathological examination, colony forming assay of bone marrow culture, T lymphocyte subpopulation analysis, as well as T-bet, GATA-3 and Fox P3 proteins were detected by flow cytometry and Western blot. Results: The peripheral blood of white blood cell(WBC), platelet, neutrophil counts and hemoglobin(Hb) concentration were significantly decreased in the model group compared with the normal group(all P<0.01). In response to 3 dose PDS-C treatment, the WBC, platelet, neutrophil counts were significantly increased at a dose-dependent manner compared with the model group(all P<0.01). The myelosuppression status of AA was significantly reduced in M-, H-PDS-C groups, and hematopoietic cell quantity of bone marrow was more abundant than the model group. The colony numbers of myeloid, erythroid and megakaryocytic progenitor cells in the model group were less than those of the normal mice in bone marrow culture, while, PDS-C therapy enhanced proliferation of hematopoietic progenitor cells by significantly increasing colony numbers(all P<0.01). Furthermore,PDS-C therapy increased peripheral blood CD3^+ and CD3^+CD4^+ cells and reduced CD3^+CD8^+ cells(P<0.05 or P<0.01). Meanwhile, PDS-C treatment at medium-and high doses groups also increased CD4^+CD25^+Fox P3^+ cells, downregulated T-bet protein expression, and upregulated GATA-3 and Fox P3 protein expressions in spleen cells(P<0.05). Conclusion: PDS-C possesses dual activities, promoting proliferation hematopoietic progenitor cells and modulating T lymphocyte immune functions in the treatment of AA model mice.

Chinese Medicine Treatment on Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Graft-versus-host disease(GVHD)is the most common complication after allogeneic hematopoietic stem cell transplantation,and also an important factor affecting the survival and quality of life in patients after transplantation.Currently,immunosuppressive therapy is commonly used for GVHD,but the curative effect is not ideal.How to effectively prevent and treat GVHD is one of the difficulties to be solved urgently in the field of transplantation.In this paper,we summarize the latest progress in pathogenesis,prevention and treatment of GVHD with Chinese medicine(CM).We hope it will provide ideas and methods for exploring the mechanism and establishing a new comprehensive therapy for GVHD with CM.