Jmjd6a is expressed in developing Xenopus laevis eye during optic vesicle formation and retinal layer differentiation stages. Knockdown of Jmjd6a by an antisense morpholino resulted in eye malformation including a deformed retinal layer and no lens formation. We further found down-regulation of gene expression related to eye development such as Rx1, Otx2, and Pax6 in Jmjd6a morpholino injected embryos. Jmjd6 interacts with splicing factor U2AF25 and GSK3β RNA in the anterior region of Xenopus embryos. Knockdown of Jmjd6a led to deletion of GSK3β RNA exon 1 and 2, which resulted in generation of N’-terminal truncated GSK3β protein. This event further caused decreased phosphorylation of β-catenin and subsequently increased β-catenin stability. Therefore, our result may suggest that Jmjd6a plays an important role in Xenopus eye development through regulation of GSK3β RNA splicing and canonical Wnt/β-catenin signaling.It has been suggested that Jmjd6 plays an important role in gene regulation through its demethylation or hydroxylation activity on histone and transcription factors. In addition, Jmjd6 has been shown to regulate RNA splicing by interaction with splicing factors. In this study, we demonstrated that  During vertebrate eye development, spatiotemporal orchestration of eye-specific gene expression occurs between different cell types from different embryonic origins including the neural ectoderm, surface ectoderm, and periocular mesenchyme . Eye devAmong cellular signaling pathways, the canonical Wnt/β-catenin signaling pathway plays an essential role in eye development –6. Wnt/βJmjd6 demonstrates multiple defects in the developing brain, heart, notochord, and somite in zebrafish, frog, and mouse  in the presence of complete protease inhibitors (Roche) and 1mM phenylmethylsulphonylfluoride. Yolk was removed by centrifugation for 10 min at 4°C. The extract was incubated with anti-Jmjd6 antibody  for overnight at 4°C and subsequently incubated with protein A/G agarose beads (Sigma‐Aldrich) for 4 hr at 4°C. The beads were washed extensively and mixed with SDS sample buffer. For negative control, rabbit IgG  was used. Western blotting was performed with anti-Jmjd6 , anti-α/β tubulin , anti-U2AF65 , anti-β-catenin , anti-phospho-β-catenin (Ser33/37/Thr41) , and anti-GSK3β  antibodies.2, 1% Triton X-100] for 20 min with frequent mixing. After centrifugation for 15 min at 2500g, the resulting nuclei pallet was incubated with RIP buffer . Anti-Jmjd6 antibody  or rabbit IgG  coupled to protein G agarose bead  was incubated with the nuclear extracts for overnight at 4°C. After washing the beads with ice-cold RIP buffer, DNase I  was treated. The RNAs were extracted with phenol/chloroform and precipitated by ammonium acetate/ethanol. Oligonucleotides are listed in Supporting Information  containing multiple copies of Tcf-binding site was injected with CMV-Renilla luciferase plasmid (5 pg) into two blastomeres of the animal-dorsal region at the 8-cell stage. The anterior regions of the injected embryos were collected at stage 26 and a luciferase assay was performed using the Dual-Luciferase Assay System (Promega). Renilla luciferase activity served as an internal control for normalizing the firefly luciferase activity.P ≤ 0.05 and **P ≤ 0.01.All quantitative data are presented as mean ± standard deviation (SD). for three independent experiments. For examination of gene expression, more than 30 embryos were analyzed in each experiment. The differences between two groups were evaluated by a paired t-test. Significance values were *Jmjd6a and Jmjd6b, which are expressed maternally and zygotically [Jmjd6a and Jmjd6b were gradually decreased during Xenopus early development  against Xenopus Jmjd6a (Jmjd6a by MO efficiently reduced the endogenous level of Jmjd6 protein (Jmjd6a mRNA without 5’-UTR (Δ5’UTR Jmjd6a), which cannot be paired with Jmjd6a MO, restored Jmjd6 expression . In severe cases, no lens was formed (nd and 3rd row). In addition, an ectopic eye, which developed incompletely, was found in embryos with no eye formation . As expected, co-injection of Jmjd6a mRNA without 5’-UTR (Δ5’UTR Jmjd6a) with Jmjd6a MO resulted in a reduced number of embryos with abnormal eye , Rx1 , and Pax6  at tailbud stages . We found that expression of Otx2, Rx1, and Pax6 decreases in the eye primordia at stage 22 in the Jmjd6a MO injected side compared with the un-injected or control MO-injected side of embryos  analysis in Jmjd6a MO-injected embryos at stage 26  , 21, 22.s U2AF65 . To idenelopment –38. To cSK3β RNA . However embryos . However embryos . We confstage 26 . The abert codon . Becauseproteins . Consist embryos . Moreove embryos . These rGSK3β RNA splicing, we next investigated the effect of Jmjd6a knockdown on canonical Wnt/β-catenin signaling using a TOP-flash luciferase reporter containing multiple TCF binding sites. We injected the TOP-flash reporter with Jmjd6a MO into Xenopus embryos at stage 26. Although basal activity of luciferase was observed in the TOP-flash reporter-injected embryos, Jmjd6a MO co-injection resulted in an increase of luciferase activity (Jmjd6a mRNA without 5’-UTR (Δ5’UTR Jmjd6a) suppressed increased luciferase activity by Jmjd6a knockdown. Consistently, phosphorylation of β-catenin was decreased and stability of β-catenin was increased in Jmjd6a MO-injected embryos . Moreover, ectopic eyes were found in case of anophthalmia [Jmjd6a altered gene expression related to eye development including Otx2, Rx1, and Pax6. Otx2 is expressed in the entire optic vesicle at the initial stage of eye development, and its expression is restricted to the presumptive RPE at later stages [Rax, which is a mouse homolog of Rx1, is intensively expressed in the developing retina in mouse [Xenopus eyes [Pax6 is an important transcription factor for eye development because ectopic expression of Pax6 alone is sufficient to induce ectopic eyes in fly and frog embryos [Pax6 is expressed highly in the early optic vesicles and the surface ectoderm, and its expression remains in all eye components at the optic-cup stage. Its expression becomes restricted to the lens, corneal and conjunctive epithelia, iris, and inner portion of the neuroretina [In this study, we found that knockdown of  and INL . Howeverhthalmia . We furtr stages . Rax, whin mouse  and the pus eyes . Pax6 is embryos , 45. Paxroretina .GSK3β RNA in Xenopus embryos. Knockdown of Jmjd6a resulted in GSK3β RNA lacking exon 1 and 2, thereby generating an N’-terminal truncated form of GSK3β protein with a molecular weight of approximately 35 kDa. A few studies have been described the importance of the N’-terminal region of GSK3β, for instance the N’-terminal of GSK3β may regulate its kinase activity on β-catenin because of the presence of critical lysine resides (K85 and K86) located at the ATP binding site of GSK3β [Jmjd6a may induce decreased β-catenin phosphorylation and consequently increased β-catenin stability.Previous studies have shown that Jmjd6 regulates RNA splicing by interaction with splicing factors , 22, 40.of GSK3β –49. Moreof GSK3β , 51. ThuJmjd6a knockdown, overexpression of constitutively active β-catenin results in disorganization of the retina layers in mouse [It is well known that canonical Wnt/β-catenin signaling pathway plays a critical role in vertebrate eye development. For instance, canonical Wnt/β-catenin signaling is active in the dorsal optic vesicle and presumptive RPE at the optic vesicle stage. It is subsequently restricted to the peripheral RPE , 52, 53.in mouse .GSK3β RNA, resulting in inclusion of exon 1 and 2. Subsequently, GSK3β phosphorylates β-catenin and β-catenin is degraded during Xenopus optic vesicle formation and retinal cell differentiation Click here for additional data file.S2 Figin situ hybridization of Jmjd6b was performed at indicated stages (n = 3). (A) At late neurula stage (stage 20), Jmjd6b is expressed in the eye primordia (white arrow), brain primordia (green arrow), and neural tube (red arrow). Posterior view is shown. (B) At early tailbud stage (stage 25), Jmjd6b expression is detected in the eye (white arrow) and brain region (red arrow). Lateral view is shown. (C) At stage 30, Jmjd6b is expressed in the eye (white arrow). Lateral view is shown. (D) At stage 40, Jmjd6b expression is not detected. Lateral view is shown.Whole-mount (TIF)Click here for additional data file.S3 FigTo confirm proper MO injection, plasmid containing RFP (red fluorescence protein) cDNA was co-injected into one blastomere at the 8-cell stage. (A) Lateral view of un-injected side of embryo. (B) Dorsal view of embryo. RFP-injected side of embryo is shown to the right. (C) Lateral view of RFP-injected side of embryo. Note the red fluorescence in RFP-injected side of embryo.(TIF)Click here for additional data file.S4 FigGSK3β exon (4~11) was amplified by real time-PCR in the anterior region of Jmjd6a MO-injected embryos (stage 26) using exon specific oligonucleotides. Relative expressions were normalized with GSK3β RNA exon 10 because its expression was not changed based on EF1a expression. Data represent mean ± SD .Each (TIF)Click here for additional data file.S5 FigGSK3β RNA without exon1 and 2 was injected into Xenopus embryos and the lysate was analyzed by western blotting using an antibody recognizing the full length of GSK3β. An extra 35 kDa band of GSK3β is detected (*). Endogenous full length of GSK3β is denoted as **.Synthesized (TIF)Click here for additional data file.S1 Table(PPTX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


L7G stimulated EpSC proliferation through β-catenin and c-Myc. We further examined the effect of L7G on EpSC proliferation in skin tissues by treatment of human skin explants with L7G and examined the number of EpSCs by immunohistochemical stain of EpSC markers α6 integrin and β1 integrin. We found that treatment of human skin tissue explants with L7G significantly increased the thickness of the epidermis and increased the numbers of α6 integrin-positive and β1 integrin-positive cells at the basal layer of the epidermis. Taken together, these results indicate that L7G promotes EpSC proliferation through upregulating β-catenin, c-Myc, and cyclin expression. L7G can be used to expand EpSCs for generating epidermal autografts and engineered skin equivalents.Skin epidermal stem cells (EpSCs) play critical roles in skin homeostasis and the repair of skin injury. Luteolin-7-glucoside (L7G) has been reported to accelerate skin wound healing through its anti-inflammatory and antioxidative activity. But its effect on EpSCs is not clear. In the present study, we examined the effect of L7G on the proliferation of human EpSCs and explored the mechanisms involved. MTT assay showed that L7G promoted EpSC proliferation in a dose- and time-dependent manner. BrdU incorporation assay and Ki67 immunofluorescence staining confirmed the proproliferative effect of L7G on EpSCs. Cell cycle analysis showed that treatment of EpSCs with L7G decreased the cell number in the G1 phase and increased the cell number in the S phase. In addition, L7G significantly enhanced EpSC migration. Mechanistic studies showed that L7G significantly induced the expression of  Whenin EpSCs , 7. Othein EpSCs , 9.EpSCs are becoming important sources for novel therapeutic approaches in the management of wounds. Transplantation of cultured keratinocytes which contain EpSCs, either in cell suspension or on various delivery systems, has been well established for the treatment of extensive, deep, and chronic wounds . FurtherA large number of plants and plant extracts have been used in traditional medicines worldwide for the treatment of skin injury. Luteolin-7-glucoside (L7G) belongs to flavonoid glycosides. It is reported that L7G promotes skin wound healing by accelerating wound reepithelialization and increasing collagen deposition , 12. Theα6 integrin, β1 integrin, PCNA, c-Myc, β-catenin, cyclin D1, and GAPDH were obtained from Abcam . PI/RNase Staining Buffer, FITC-conjugated rat anti-human α6 integrin antibody, PE-conjugated mouse anti-human CD71 antibody, FITC-labeled rat IgG2a, and PE-labeled mouse IgG2a were from BD Biosciences . FITC-conjugated goat anti-mouse IgG antibody (BA1101) and Cy3-conjugated goat anti-rabbit IgG antibody (BA1032) were obtained from Boster . MaxVision™ HRP-Polymer anti-Mouse IHC Kit (KIT-5001) was provided by Fuzhou Maixin Biotech. Co. Ltd. .Luteolin-7-glucoside was purchased from Nature Standard . XAV-939 and 10058-F4 were from Selleck . Thiazolyl Blue Tetrazolium Bromide (MTT) was from Beyotime Biotechnology . Y-27632 was obtained from STEMCELL Technologies. The keratinocyte growth medium 2 (KGM2) was purchased from PromoCell . Dispase II and collagen IV were from Sigma . The BrdU cell proliferation kit and antibodies against Ki67, CK19, 2pieces and digested with 0.25% dispase II overnight at 4°C. The epidermis was peeled off and digested with 0.25% trypsin for 10 min at 37°C. The keratinocyte cell suspension was filtered and centrifuged. The cell pellet was washed with PBS, resuspended in KGM2 supplemented with 0.125 ng/ml epidermal growth factor, 4 μl/ml bovine pituitary extract, 0.06 mM CaCl2, and 100 IU/ml of streptomycin and penicillin, and seeded in 25 mm culture dishes coated with collagen IV. Cells were incubated for 10 min at 37°C in a humidified chamber with 5% CO2. Nonadherent cells were then immediately rinsed off. The adherent EpSCs were cultured with fresh medium supplemented with 10 μM Y-27632 to promote EpSC proliferation and inhibit its differentiation [Human skin was obtained from the extra fingers removed from male polydactyly patients (under 15 years old) during plastic surgery in Ruihua Affiliated Hospital of Soochow University, Suzhou, China. The protocol was approved by the Ethics Committee of the hospital. EpSCs were isolated from the skin tissue with dispase and trypsin as previously described , 14. Briα6 integrin antibody and PE-conjugated mouse anti-human CD71 antibody at 4°C for 1 h in the dark. FITC-labeled rat IgG2a and PE-labeled mouse IgG2a were used as isotype-matched negative controls. The cells were resuspended in PBS containing 2% FBS. FACS analyses were performed on a FACSCalibur (BD Pharmingen™).Human EpSCs were stained with FITC-conjugated rat anti-human The expression of biomarkers of EpSCs and Ki67 was examined by immunofluorescence staining. Briefly, EpSCs were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100, and incubated with normal serum from the same species as the secondary antibody for 30 min. Then, the cells were washed with PBS and incubated with the primary antibody overnight at 4°C. After washing, EpSCs were incubated with a fluorescence-conjugated secondary antibody for 1 h. The nuclei were stained with DAPI for 5 min. The fluorescence signal was detected with a fluorescence microscope .μM L7G combined with the β-catenin inhibitor (2 μM XAV-939) or the c-Myc inhibitor (2 μM 10058-F4) for 48 h. Twenty microliters of MTT at 5 mg/ml was added to each well, and the plate was incubated for 3 h at 37°C. The culture medium was removed, and 200 μl of DMSO was added to each well to solubilize formazan crystals. The optical density (OD) was measured at 490 nm with the Multiskan™ Spectrum . The experiments were performed in sextuplicate.Human EpSCs seeded on 96-well plates were stimulated with different concentrations of L7G for different periods of time or stimulated with 1 μM L7G for 48 h. Cell proliferation was examined using the BrdU cell proliferation kit according to the manufacturer's instructions. The OD value was measured using the Multiskan™ Spectrum at 450 nm.Human EpSCs seeded on 96-well plates were treated with or without 1 μM L7G for 24 h were trypsinized, washed with PBS, and then fixed in cold 80% ethanol. After the removal of ethanol by centrifugation, cells were stained with PI/RNase Staining Buffer  for 20 min in the dark according to the manufacturer's protocol. The DNA content was analyzed by using a flow cytometer (Beckman Coulter).Cell cycle phase distribution was analyzed by flow cytometry. Briefly, EpSCs treated with or without 1 5/well) were seeded on a 24-well culture plate. After overnight incubation, the confluent monolayer of cells was wounded with a 200 μl pipette tip to create a uniform cell-free zone. Cell debris was removed by washing with PBS. Wounded monolayer was cultured in EGF free KGM2 with or without 1 μM L7G. The cells were photographed under an inverted phase contrast microscope  after 0, 12, and 24 h. The width of the cell-free zone was analyzed using the ImageJ software .The migration of human EpSCs was examined by in vitro wound closure assay. Human EpSCs (2 × 10β-catenin: GCAGTTCGCCTTCACTATGGA (forward), ATCTTGTGGCTTGTCCTCAGAC (reverse); c-Myc: AGGAACTATGACCTCGACTACG (forward), AGTAGCTCGGTCATCATCTCCAG (reverse); cyclin D1: AACTACCTGGACCGCTTCCT (forward), CCACTTGAGCTTGTTCACCA (reverse); cyclin A2: TCCAAGAGGACCAGGAGAATATCA (forward), TCCTCATGGTAGTCTGGTACTTCA (reverse); cyclin E1: GTCCTGGCTGAATGTATACATGC (forward), CCCTATTTTGTTCAGACAACATGGC (reverse); and β-actin: CGTGGACATCCGCAAAGAC (forward), CTGCTGTCACCTTCACCGTTC (reverse).Total RNA was extracted from human EpSCs using the TRIzol reagent following the manufacturer's protocol . cDNA was reverse transcribed from RNA by using the oligo (dT) 18 primer and M-MLV reverse transcriptase . PCR reaction was carried out with 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds. PCR products were separated by 1% agarose gel electrophoresis, visualized by ethidium bromide staining. The expression level of target genes was semiquantified with the ImageJ software. The sequences of PCR primers (5′-3′) are as follows: β-catenin, cyclin D1/E1/A2, and GAPDH were used. Target proteins were detected with SuperSignal West Pico Chemiluminescent Substrate , quantified with the ImageJ software .EpSCs were lysed in RIPA lysis buffer and centrifuged to remove the debris. The protein concentrations were measured using a Bradford protein assay kit . Western blotting was performed following standard protocols. The primary antibodies against c-Myc, μM L7G in a 50/50 (v/v) mix of DMEM (1 g/l glucose) and Ham's F-12 medium (Gibco) supplemented with 10% FBS, 2 mM L-glutamine, and antibiotics. The epidermal side of the skin is kept at the air-liquid interface at 37°C under 5% CO2 in a humidified atmosphere, and the medium was changed every other day. After 2-5 days, the skin tissues were embedded in OCT compound  and frozen for histological and immunohistochemical assays.The normal skin explant culture was performed as described previously , 17. Briμm using a cryostat (Leica) and stained with hematoxylin and eosin (HE). For immunohistochemical assay, the sections were fixed with acetone at 4°C for 10-20 min, incubated with 0.3% H2O2 for 15 minutes, washed with PBS, and blocked with goat serum for 30 min. Then, the sections were incubated with the primary antibody against α6 integrin, β1 integrin, or proliferating cell nuclear antigen (PCNA) at 4°C overnight, washed with PBS, and incubated with the secondary antibody from the MaxVison™ HRP-Polymer anti-Mouse IHC Kit for 15 min. After washing with PBS, the sections were incubated with the DAB-coloring agent  for 10 min in the dark, washed with water, and counterstained with hematoxylin. Sections were examined and photographed under a microscope. The positive signals of immunohistochemical stain were analyzed using the ImageJ software.Frozen human skin tissues were sectioned at 5 t-test. A P value of less than 0.05 was considered significantly different.All results are expressed as mean ± SD. Statistical differences between the test and control groups were analyzed with two-tailed Student's α6 integrin and low levels of CD71, two well-recognized marks of EpSCs [α6high/CD71low is higher than 90% (β1 integrin and CK19, another two markers of EpSCs, by immunofluorescence staining, and found that more than 90% of the cells are β1 integrin and CKl9 positive (Human EpSCs were selected from epidermal keratinocyte suspension according to their rapid attachment to collagen type IV , 19. Aftof EpSCs . The popthan 90% . We alsopositive . These rμM L7G promotes EpSC proliferation in a dose-dependent manner (μM L7G for different periods of time showed that L7G enhanced cell proliferation in a time-dependent manner (To investigate the effect of L7G on EpSC proliferation, EpSCs were incubated with different concentrations of L7G and measured for cell proliferation by MTT assay after 24 and 48 h, respectively. We found that 0.1-1 t manner . Stimulat manner . Treatmet manner  and the t manner . Furthert manner . All togμM L7G for 12 or 24 h significantly accelerated cell migration and reduced the width of the scratched cell-free zone (We further examined the effect of L7G on EpSC migration through the wound closure assay. Compared with untreated EpSCs, treatment of EpSCs with 1 ree zone .β-catenin and c-Myc play critical roles in EpSC proliferation [μM L7G significantly and time-dependently induced β-catenin and c-Myc expression (β-catenin and c-Myc, L7G also increased the expression of cyclins D1, A2, and E1 at mRNA level (β-catenin, c-Myc, and cyclins by L7G at protein level was confirmed by Western blot (β-catenin inhibitor XAV-939 or the c-Myc inhibitor 10058-F4 significantly inhibited L7G-induced EpSC proliferation (β-catenin, c-Myc, and cyclins.It has been reported that feration , 21. We pression . Cyclin NA level . The uprern blot . We furtferation . These rμM L7G for 2 and 5 days, respectively, then examined the histology by HE staining and the expression of marker genes of EpSCs and cell proliferation by immunohistochemistry. HE staining showed that treatment with L7G for 5 days significantly increased the thickness of the epidermis (α6 integrin and β1 integrin, two markers of EpSC [α6 integrin-positive and β1 integrin-positive cells and the staining intensity were significantly increased (Figures To investigate if L7G could promote EpSC proliferation in human skin, we cultured human skin tissue explants in medium with or without 1 pidermis . Immunoh of EpSC , 23, wer Figures . Treatme Figures . These rα6 integrin, β1 integrin, and PCNA in the basal layer of the skin epidermis, which is associated with the increased number of α6 integrin-, β1 integrin-, and PCNA-positive cells, as well as the thickness of the epidermis. These data strongly support that L7G could promote skin EpSC proliferation.In the present study, we found that L7G promoted the proliferation of EpSCs in a concentration- and time-dependent manner and promoted EpSC migration in vitro. We further examined the effect of L7G on EpSCs in cultured human skin tissue explants. The immunohistochemistry results clearly showed that treatment with L7G significantly increased the staining of β-catenin in EpSCs (β-catenin signaling has been reported to play important roles in the self-renewal and proliferation of skin EpSCs [β catenin could avoid its binding and degradation by GSK-3β. Zhu and Watt reported that the introduction of the N-terminally truncated β-catenin into human EpSCs promoted EpSC proliferation and colony formation [β-catenin are expressed in the basal layer of human fetal skin and EpSCs. EpSCs also expressed c-Myc, cyclin D1, and cyclin A. Wnt3a stimulated the proliferation and inhibited the differentiation of human EpSCs, indicating that the Wnt3a/β-catenin pathway is important for EpSC proliferation. We found that the β-catenin inhibitor could block L7G-induced EpSC proliferation, indicating that L7G promotes EpSC proliferation through β-catenin. We further found that the expression of c-Myc and cyclin D1, two downstream molecules of β-catenin in cell proliferation, was also upregulated by L7G. c-Myc has been reported to be involved in skin EpSC proliferation [1 in the basal layer of mouse skin significantly induced epidermal cell proliferation [β-catenin-mediated c-Myc and cyclin D1 pathways.Our mechanistic study showed that L7G significantly induced the expression of in EpSCs . The Wntin EpSCs . Deletioormation . Jia et ormation  reportedferation . Our stuferation . In supp1 drives G1/S phase transition. Cyclin A is required for G1/S phase transition, progression through the S phase, and also plays a role in G2/M phase transition. Cyclin E is essential for G/S transition [1, A2, and E1. These results indicate that L7G promotes EpSC proliferation by increasing G1/S phase transition. As cyclins A2 and E1 are not target genes of Wnt/β-catenin signaling, L7G may promote EpSC proliferation through upregulating cyclins mediated by Wnt/β-catenin/c-Myc-dependent and Wnt/β-catenin/c-Myc-independent pathways.We found that treatment of EpSCs with L7G decreased the cell number in the G1 phase and increased the cell number in the S phase, which verifies the proproliferative effect of L7G on EpSCs. Cyclin is a family of proteins which play an important role in regulating the cell cycle. Cyclin Dansition , 29. OurL7G promotes skin wound healing through its antioxidative and anti-inflammatory activity , 30, 31.β-catenin, c-Myc, and cyclins D1, A2, and E1. Our results indicate that in addition to the antioxidative and anti-inflammatory activity, the proproliferative activity of L7G may also contribute to the acceleration of skin wound healing by L7G. L7G is a useful reagent for the expansion of EpSCs for the clinical translation of cell therapy and skin tissue engineering.L7G promotes skin EpSC proliferation through upregulating the expression of


Controlling the formation of MnS inclusions during solidification influences the mechanical properties and machinability of the resulfurized steel. A coupled segregation–nucleation–growth model was developed by the finite-difference method involving solute redistribution, heterogeneous nucleation and growth kinetics. Laboratory solidification experiments were performed under various cooling rates in resulphurised 49MnVS steel. In this work, the influence of cooling rate on solute redistribution and growth size of MnS inclusions were simulated using the current coupled model, and the calculated results can provide a valuable reference for MnS formation. Increasing of the cooling rate led to early precipitation and refinement of formed MnS inclusions. Based on the simulation results and experimental data, mathematical relationships between the growing size of MnS with the cooling rate in the low ductility temperature region and in the whole solidification were obtained. Over the past few decades, MnS inclusions have been considered as detrimental species in steel matrix, and many effects have been made for desulfurization in the processes of hot metal pretreatment and converter steelmaking, etc . HoweverThe evolution of MnS inclusions during cooling and solidification can be calculated and predicted using mathematical modeling. MnS is normally precipitated in the enriched remnant liquid during solidification and many models have been proposed to analyze the precipitation of MnS inclusions based on the redistribution of solute elements . Clyne aIn the present research, a new coupled model of MnS inclusions precipitation and growth during solidification was proposed based on previous work ,20. UsinThe experimental material used in the present study was a non-quenched and tempered resulphurised 49MnVS steel, and its main chemical compositions are shown in After the experiment, a steel sample for metallographic observation was collected from the center of each ingot. Image-Pro Plus 6.0 software  was used to measure the average length of diameters at 2-degree intervals and passing through the object’s centroid. The steel sample was polished and cleaned in ethanol for 5 min using a ultrasonic cleaner . Besides, to obtain the tri-dimensional morphologies of MnS, the non-aqueous electrolytic method presented by Janis et al  was adopD is the diffusion coefficient (cm2/s), Δt and Δx are the time step and the space step, Ln is the length of the interface between node n and node n + 1 (m), while An is the area of node n (m2). In addition, MnS preferentially precipitates in the interdendritic area when the product of Mn and S concentrations exceeds the equilibrium value in remnant liquid [t liquid . The disKMnS is the equilibrium constant of the reaction [Mn] + [S] = (MnS), T is the temperature in K. aMnS = 1 is the activity of pure MnS, i] and [%j] are the concentrations (wt.%) of solute elements i and j, and j to i; as listed in The formation reaction of MnS in steel is expressed as [Mn] + [S] = (MnS), and the equilibrium constant of the reaction is shown in Equation (2). MnS begins to precipitate in the remnant liquid when the supersaturation of Mn and S occurs. formula , [%i] anAccording to the equilibrium relationship of the above formula, it can be concluded that the consumption of solute elements in the liquid phase.wMnS is the precipitation amount of MnS, and b is the ratio of MMn (the molar mass of Mn) to MS (the molar mass of S) resulting from the equilibrium consumption of solute Mn and S.Assuming that all the consumption of Mn and S are used to generate MnS, the precipitation amount of MnS can be calculated through the above equations as expressed in the following equation:σB and σ are the surface energy of matrix and precipitates (J/m2), and θ is the contact angle.It has been often reported that the nucleation of MnS preferentially occurs at grain boundaries and dislocations ,26. The r0 is the critical radius of MnS, and ΔGV is the free energy per unit volume. The chemical driving force associated with the unit volume of MnS can be represented by Equation (10) [VMnS is the molar volume of MnS (22.3 L/mol), It is reasonable to neglect the influence of elastic energy because it can be completely released during the solidification process. Assuming that the precipitated MnS nuclei is a sphere of radius, the critical free energy of homogeneous nucleation is given by Equation (9).ion (10) .(10)ΔGVN0 is the number of nodes in the dislocation network, Z is the Zeldovich constant (0.05), β’ is the atomic impingement rate, bk is the Boltzmann constant (1.38065 × 10−23 J/K), τ is the incubation time, the precipitation of MnS on dislocations is assumed to be instantaneous and therefore, the incubation time is considered to be zero, t is growth time, ρ is the dislocation density, which is about 1013 m−2 in low carbon steel, a0 is the lattice parameter. Moreover, the main kinetic parameters in formation of MnS are shown in The nucleation rate is calculated based on a classical nucleation law as expressed in the following equations ,29.(11)kae), as expressed in Equation (14). Thus MnS with radius r1 is considered to grow in a dynamic circular diffusion domain with radius r2.Ae and Aa are the areas of equivalent diffusion domain and actual diffusion domain.A schematic diagram of MnS precipitation in the calculation domain is presented in r, θ, and z are three coordinate variables in the column coordinate system, based on assumptions as described before, the diffusion of solute elements is locally steady on the cylindrical surface and therefore, the diffusion coefficient does not vary with the solute concentration. The diffusion rate of solid–liquid phases can be simplified as shown in Equation (16), while diffusion flux on different cylindrical surface is shown in Equation (17).The rates of diffusion into solid and liquid phases are calculated according to the Fick’s second law, as expressed in Equation (15).C = C1 at r = r1 and C = C2 at r = r2, respectively. Therefore, the distribution of concentration can be calculated using the above boundary conditions, as shown in Equation (18).J is the diffusion flux, and A is the surface area of formed MnS in the cylindrical coordinate system. The mass balance surrounding one MnS nuclei is expressed in Equation (19).As shown in Combining Equations (16–19), the differential form of MnS growth is obtained as expressed in Equation (20).As evident in the above equation, the direct driving force for MnS growth is the local concentration gradient of the determining elements rather than concentration difference.λ2 is secondary dendrite arm spacing (µm), wC is the carbon content (wt.%) and RC is the cooling rate (°C/s).The precipitation and growth of interdendritic MnS were mainly studied in the present coupled model, and the secondary dendrite arm spacing is calculated using Equation (21) which was proposed by Won et al. .(21)λ2=Tstart), cooling rate (RC), and initial composition of the steel. The phase transverse are considered and the advancing of phase interface and the algorithm is shown in A flow chart of the present coupled model for the formation of MnS is displayed in Tar4) is calculated using the solute concentrations in solid phase at the solid /liquid interface. When the liquidus temperature (TL) and the δ/γ transformation temperature (Tar4) of one node become equal to the actual temperature, the solidification and δ/γ transformation in the node are considered to be complete, and the interface is moved to the next node. The TL and Tar4 are calculated using Equations (22) and (23) [TFe and mi and ni are the slopes of liquidus and Ar4 line according to the pseudobinary Fe-phase diagram, CL,i and Cδ,i are the solute concentration of element i in liquid and solid phase (wt.%).When the carbon content in steels exceeds 0.53%, the first crystallized phase at the center of dendrite is γ-phase. Otherwise, δ-phase is first generated in the steel liquid during solidification. The δ/γ transformation temperature (and (23) ,31.(22)D0 is diffusion constant (cm2/s), Q is the activation energy (J/mol), R is the gas constant (8.314 J/(kg⋅K)), T is temperature (K). The equilibrium partition coefficients and diffusion coefficient of the solute elements are list in In addition, considering the diffusion of solute elements in the solid phase, the relationship between the diffusion coefficient and temperature can be calculated according to the Arrhenius formula as shown in Equation (24).With the temperature decreasing and solid /liquid interface advancing, the thermodynamics and kinetics of MnS formation were calculated by step. During the solidification, the formation of new phase causes the redistribution of solute in the remnant liquid. MnS precipitates in the remnant liquid when the product of Mn and S concentrations exceeds the equilibrium value. In the meantime, stable MnS nuclei is generated when the nucleation radius of MnS exceeds the critical value, otherwise the unstable MnS dissolves in the liquid. The precipitation of MnS was continuously simulated in combination with the enrichment of solute elements. Accordingly, the subsequent calculation started with the new temperature or time step and terminated at the end-temperature. The material used in this simulation is resulphurised 49MnVS steel, whose chemical composition is shown in fS) of MnS are 0.874, 0.875, 0.876, 0.878 and 0.881 at the cooling rate of 1.0, 5.0, 15.0, 30.0 and 60.0 °C/min, respectively. The concentration of Mn and S in residual liquid under various cooling conditions are shown in The solute element of Mn and S were considered as the determining species in the current calculation, respectively. The effect of cooling rate on the nucleation rate of MnS is presented in The variations of the radius of MnS particles during solidification for the five cooling rates is illustrated in AR) of MnS is shown in AR ≤ 2), followed by ellipsoidal (2 < AR ≤ 3) and rod-like (AR > 3). The percentage of spherical MnS inclusions increases with increasing cooling rate, and the percentage of ellipsoidal and rod-like MnS inclusions decrease with increasing cooling rate. According to the above discussion, it is concluded that for studied 49MnVS steel, increasing cooling rate is beneficial to the formation of ellipsoidal and tiny MnS inclusions.The size distribution of MnS inclusion under various cooling conditions is shown in dMnS) in various cooling samples are shown in dMnS decreases with increasing of the actual cooling rate (υ). The value of dMnS is 2.24, 2.02, 1.82, 1.64 and 1.48 μm at actual cooling rate of 0.47, 0.95, 3.96, 6.30 and 9.03 °C/min, respectively. While the standard deviation of dMnS is 2.07, 1.66, 1.20, 1.16 and 1.08. The actual cooling rate (measured by thermocouple B) of each sample is 0.47, 0.95, 3.96, 6.30 and 9.03 °C/min corresponding to the setting cooling rate (measured by thermocouple A) of 0.5, 1.0, 4.5, 8.0 and 15.0 °C/min, respectively. The mean diameter of MnS inclusion , and the regression coefficients (R2) was 0.954. In addition, the fitting curve was present in To detect the correlations between R2 was 0.999.Based on the coupled model, the simulated diameter of MnS was 7.83, 4.42, 2.99, 2.34 and 1.83 μm at the cooling rate of 1.0, 5.0, 15.0, 30.0 and 60.0 °C/min. Similarly, the relationship between cooling rate and calculated MnS diameter was obtained via linear regression analysis as shown in Equation (26), and The comparison of experimental data and calculated value versus cooling rate in common logarithmic coordinates are shown in fS = 0.8). Hence, it is reasonable to say that the growth of MnS inclusions in the present experimental occurred in the low ductility temperature region of 49MnVS steel. The growth time of MnS inclusions for present experimental was shorter than the previous studies and the current simulated results, resulting in smaller size of formed MnS.It should be pointed out that the present sample was obtained at a quenching temperature of 1390 °C. The solidus temperature of studied 49MnVS steel is 1391 °C according to the equilibrium calculation of MnS precipitation, as shown in dMnS-H) and in the whole solidification (dMnS-L) showed the same trend, and the gap between dMnS-H and dMnS-L was shrinking. It also shows that rapid cooling condition can preserve the in-situ state of the sample at high temperature. The size of formed MnS increases with decreasing cooling rate, causes the increased difference between dMnS-H and dMnS-L due to the difference in growth time.Combining the experimental results and calculated values, it can be concluded that the size of MnS inclusions was strongly affected by the cooling rate and rates higher than 100 °C/min were necessary for realizing inclusions which were less than 1.5 μm. With increasing of cooling rate during solidification, the size of MnS grew in the low ductility temperature region ((1)The MnS begins to precipitate in the remnant liquid when local solubility product of manganese and sulfur exceeds the equilibrium value, and the segregation ratio of solute Mn and S are approximately 1.57 and 6.87 at the beginning of MnS formation.(2)The solute element of S was the determining species in deciding the nucleation and precipitation of MnS, while the solute element Mn affected the precipitated amount of MnS owing to the high partition coefficient in solid phase.(3)dMnS-H and dMnS-L with υ were given by the mathematical expressions as follows: The current coupled model can be used to simulate the growth of MnS inclusions in resulfurized steel, and the calculated size of the formed MnS inclusions fits well with the previous experimental results. The value of the MnS size is evidently affected by cooling rate, and the relationships between A mathematical model has been developed to simulate the solute segregation and growth of manganese sulfide during solidification. Additionally, laboratory experiments were carried out to clarify the effect of cooling rate on the MnS inclusions of resulphurised 49MnVS steel. Based on the simulated results and experimental data, the following conclusions are drawn:


Recent data have shown that right-sided colon cancer carries poorer prognosis compared to left-sided tumors. This study was aimed to evaluate the progression-free survival, overall survival of patients with metastatic colon cancer of right-sided versus left-sided primaries treated with triplet chemotherapy regimen. The medical records of patients with metastatic colorectal cancer treated on phase I-II trial of combination Irinotecan, oxaliplatin, capecitabine, and bevacizumab were reviewed for sidedness of the primary. The analysis was performed for progression-free survival and overall survival according to the sidedness and other known prognostic factors. Out of 53 patients treated with triplet therapy, 11 had right sided and 42 had left-sided primaries. The median age for right-sided primaries was 46 (range 24-55) compared to 53 (range 32-74) in left-sided primaries. Median progression-free survival was 14 months for right vs 18 months for left sided tumors  and median overall survival was 21 months for right vs 29 months for left sided tumors . First-line triplet chemotherapy may overcome the difference in prognosis between right sided and left sided primaries in metastatic colorectal cancer. A larger analysis is warranted. Metastatic colorectal cancer carries a poor prognosis. Cure, for the most part, is only possible in a small subset of patients with resectable metastasis, and rarely with chemotherapy alone. Various clinical, pathological and molecular prognostic factors have been identified to influence the clinical outcome of metastatic colorectal cancer. This include but not limited to age, the organ involved, carcinoembryonic antigen, performance status at the time of metastasis, Neutrophil-Lymphocyte ratio, histological grade, RAS and BRAF mutation, microsatellite status and many more . Tumor location in right vs left sides of the colon has been identified as an independent prognostic factor for survival in metastatic disease . Two recent meta-analyses have clearly shown that right-sided colon cancer carries poorer prognosis compared to left-sided tumors, irrespective of the stage, race, adjuvant chemotherapy, number and quality of studies included . Recent retrospective data have suggested lack of benefit for right-sided metastatic colon cancer from the addition of anti-epidermal growth factor receptors anti-bodies to chemotherapy . There is also smaller evidence from post-hoc analysis which showed that patient with right-sided metastatic disease benefit more from upfront triplet chemotherapy regimens than patients with left-sided tumors . We have previously published our results with the combination of capecitabine, oxaliplatin, irinotecan, and bevacizumab in the first-line setting of metastatic colorectal cancer within a phase I-II trial . In this analysis, we report the outcome of patients with right-sided versus left-sided primary tumors in patients treated on the above study and examine if triplet therapy does overcome the poor prognosis of right-sided metastatic colorectal cancer. The medical records of patients treated with triplet chemotherapy consisting of irinotecan, oxaliplatin, and capecitabine  in combination with bevacizumab on a prospective clinical trial , were retrospectively reviewed for the location of the primary tumor. Right-sided tumors were defined from cecum to hepatic flexure. Left-sided tumors from splenic flexure to and including the rectum. Transverse colon patients were excluded. Patients who had multiple primaries were grouped with right-sided patients if at least one of primaries was right sided. Prognostic factors that might affect survival was recorded for right vs left sided primaries. Those include age, gender, performance status, KRAS status (NRAS was not tested as many of the paraffin blocks were exhausted), adjuvant chemotherapy, and the number of metastatic organs involved. Response rate (RR), progression-free survival (PFS) and overall survival (OS) was calculated for right sided and left sided primaries separately. As previously described, RR was assessed using response criteria in solid tumor (RECIST V1.1) . All patients received the combination of capecitabine, oxaliplatin, Irinotecan, and bevacizumab as per the doses described in the earlier publication of 5-8 cycles then maintained on capecitabine and bevacizumab. Univariate analysis was performed on the prognostic factors listed above.Ethical considerationThe original study was approved by the institutional review board. All patients consented to the study. The retrospective review of sidedness was approved by the hospital ethical committee/Institutional review board. Patient confidentiality was maintained at all time. Statistical analysisPatient characteristics were summarized using frequencies for categorical variables and medians with ranges for continuous variables. Further, categorical variables and continuous variables were compared using the Chi-squared test and Wilcoxon rank-sum test, respectively.Survival probabilities were calculated using the Kaplan-Meier estimator with variance estimated using Greenwood’s formula. Survival curves were compared using log-rank test.OS was calculated from the date of registration till the date of last follow up or death. Death of any cause was considered as an event. PFS was calculated from the date of registration till the date of progression or death. Patients who were alive progression-free were censored at the date of last follow up. Disease progression and death were considered as events for this outcome.Hazard ratios were calculated using Cox proportional hazard model after testing for proportionality assumption. In case of assumption, violation variable will be included as a time-dependent covariate.Fifty-three patients were enrolled in the prospective phase I-II trial of the combination of irinotecan, oxaliplatin, capecitabine, and bevacizumab in advanced colorectal cancer. Out of those, 11 had right-sided tumors and 42 had left-sided tumors. The characteristics of each group are outlined in The prognostic significance of sidedness for the primary tumor in metastatic colorectal cancer has been extensively studied in the past. Two large recent meta-analyses have established that metastatic colorectal cancer from right-sided primary has shorter survival than left-sided ones . In the meta-analysis presented by Yahagi and his colleagues, 15 studies were included with a total of 108,474 patients. In this meta-analysis, right-sided colon cancer had significantly worse overall survival than left-sided colon cancer with HR of 1.14, 95 % CI 1.06–1.22, p<0.01). Subgroup analyses demonstrated significant prognostic differences in western countries , a nationwide database , and a stage-adjusted analysis . The second meta-analysis published by Petrelli and colleagues, 66 studies were included with a total of 1,437,846 patients. In their report, left-sided colonic tumors were associated with a significantly reduced risk of death . This risk reduction was independent of stage, race, adjuvant chemotherapy, year of study, number of participants, and quality of included studies. It is important to note that both meta-analyses included patients of all stages. Non-of them looked at the aggressiveness of therapy (triplet vs doublet). Whether increasing intensity of therapy in patients with metastatic disease, change the prognostic significance of sidedness is not clear. It appears that sidedness is prognostic in patients treated with fluoropyrimidine alone with HR for survival in both studies for right-sided vs left-sided tumor 1.5 (adjusted P = 0.0001) and 1.592  respectively . Sidedness has also been of statistical significance in patients treated with modern doublet chemotherapy combined with targeted therapy. This has been clearly demonstrated in the CALGB/SWOG 80405 trial, the FIRE-3 trials, CRYSTAL trial and the meta-analysis published by ESMO . In the CALBG/SWOG 80405 trial, 1137 patients with wild-type K-ras were treated with FOLFOX or FOLFIRI with either Cetuximab or bevacizumab. Median overall survival for right-sided tumors was 19.4 months and for left-sided tumor was 34.2 months with HR for right-sided tumors of 1.56 . The difference was less marked for 252 patients with mutant K-ras treated prior to the trial amendment with median OS of 23.1 vs 30.3 months for right and left-sided tumors  . In the CRYSTAL trial, with RAS wild-type metastatic colorectal cancer, 280 (76%) had left-sided tumors and 84 (23%) had right-sided tumors. Median survival for right-sided tumors treated with FOLFIRI plus cetuximab was 18.5 months vs 28.7 months for left-sided ones with HR of 1.93 , while the median survival for right-sided tumors treated with FOLFIRI was 15 months vs 21.7 months for left-sided ones with HR of 1.35  . In the final RAS wild-type population of FIRE-3 trial (n = 400), 306 patients had left-sided tumors (76.5%), and 88 had right-sided tumors (22%). Median survival for right-sided tumors treated with FOLFIRI plus cetuximab was 18.3 months vs 38.3 months for left-sided ones with HR of 2.84 , while the median survival for right-sided tumors treated with FOLFIRI plus bevacizumab was 23 months vs 28 months for left-sided ones with HR of 1.48  .The only trial addressing the benefit of triplet therapy in right-sided tumors vs left-sided tumors was the TRIBE trial . In this trial, 508 patients with untreated metastatic colorectal cancer were randomized to receive either FOLFIRI plus bevacizumab or FOLFOXIRI plus bevacizumab. In a subgroup analysis, right-sided tumors had more progression-free survival benefit with HR of 0.66 for the FOLFIXIRI arm than left-sided tumors with HR of 0.82, p=0.20.Our study is the first one to our knowledge looking at the prognostic significance of sidedness of primary tumor in patients with metastatic colorectal cancer treated primarily with first line triplets’ therapy. Despite that, the difference in median overall survival between right sided and left sided tumors were higher in left-sided tumors, the hazard ration for both PFS and OS was in favor of left-sided tumors with no statistical significance likely because of small sample size. Of note, it is worth mentioning that triplets therapy in patients with metastatic colon cancer have considerable toxicity and is only feasible in patients with good performance status. The study has its own limitations including a small sample size, single institution and the presence of imbalance in the patients’ characteristics between right and left sided groups.In conclusion, our study demonstrated no significant difference in survival in patients with right versus left primary metastatic CRC treated with triple first-line chemotherapy and bevacizumab. This finding is hypothesis generating and need to be confirmed. A pooled analysis of all data published of triplets’ first-line chemotherapy in metastatic CRC is warranted. Disclosure and acknowlegmentThis study has not received any financial and/or material support that was received for the research or the creation of the work. Also, none of the authors have any relationships  or financial involvement with an organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. No writing assistance has been used in the creation of the manuscript.The original study was approved by the institutional review board. All patients consented to the study. The retrospective review of sidedness was approved by the hospital ethical committee/Institutional review board. Patient confidentiality was maintained at all time.


Melanocortin 2 receptor accessory protein 2 (MRAP2) has a critical role in energy homeostasis. Although MRAP2 has been shown to regulates a number of GPCRs involved in metabolism, the key neurons responsible for the phenotype of gross obesity in MRAP2 deficient animals are unclear. Furthermore, to date, all the murine MRAP2 models involve the prenatal deletion of MRAP2.Mc4r-cre mice. We assessed energy homeostasis, glucose metabolism, core body temperature, and response to MC3R/MC4R agonist MTII.To target Melanocortin 4 receptor (MC4R)-expressing neurons in the hypothalamic paraventricular nucleus (PVN), we performed stereotaxic surgery using AAV to selectively overexpress MRAP2 postnatally in adult Mc4r-crePVN-MRAP2 female mice on a standard chow diet had less age-related weight gain and improved glucose/insulin profile compared to control Mc4r-crePVN-GFP mice. These changes were associated with a reduction in food intake and increased energy expenditure. In contrast, Mc4r-crePVN-MRAP2 male mice showed no improvement on a chow diet, but improvement of energy and glucose metabolism was observed following high fat diet (HFD) feeding. In addition, an increase in core body temperature was found in both females fed on standard chow diet and males fed on HFD. Mc4r-crePVN-MRAP2 female and male mice showed increased neuronal activation in the PVN compared to controls, with further increase in neuronal activation post MTII treatment in females.Our data indicate a site-specific role for MRAP2 in PVN MC4R-expressing neurons in potentiating MC4R neuronal activation at baseline conditions in the regulation of food intake and energy expenditure. •Postnatal overexpression of MRAP2 regulates energy balance, thermogenesis and glucose metabolism.•Overexpression of MRAP2 in MC4R expressing neurons increases PVN neuronal activation.•There is a sex difference in extent of metabolic protection, with a more marked lean phenotype in females. In particular, there is the paradox that while Mrap2−/− mice become obese without detectable changes in food intake or energy balance, Mc4r−/− mice have hyperphagia and reduced energy expenditure (EE) Mrap2−/− mice remain responsive to treatment with MTII, a MC3R/MC4R agonist, while the anorexic response to MTII is abolished in Mc4r−/− mice, suggesting at least some preservation of MCR function centrally Although data point to an MC4R dependent function, Mrap2−/− and Mrap2+/+ mice The phenotype of gross, early onset obesity without detectable change of food intake and energy expenditure, replicated in an independent Mrap2 deficient model, is particularly intriguing With data pointing to the PVN and MC4R as the key to unraveling the obesity phenotype in MRAP2 deficient mice and because all existing mouse models to date involve developmental deletion of MRAP2, we undertook this study to assess the effect of postnatal overexpression of MRAP2 in MC4R neurons of the PVN. Furthermore, using this methodology we are able to exclude the effects of MRAP2 interaction with non-PVN MC4R GPCRs, in particular those GPCRs that have been described to interact with MRAP2 in the arcuate nucleus 22.1ad libitum, unless otherwise stated. Mice were fed on a standard chow diet (SD)  for up to 6 months of age. For high fat diet (HFD) experiments, male mice were exposed to HFD  starting at 2 weeks after PVN injection. All animals were kept in temperature and humidity-controlled rooms, in a 12 h dark and 12 h light cycle, with lights on from 7:00 AM to 7:00 PM.All animal studies on male and female mice were approved by Yale University Institutional Animal Care and Use Committee. Animals had free access to water and food was provided 2.2Mc4r-t2a-cre mice were generated as previously described Mc4r-t2a-cre mice were genotyped by polymerase chain reaction . Amplification of a wild-type (WT) allele generated a 3.6-kb product, and a 4.1-kb product in the case of a mutant allele using the following primers: cre 350 FRT5: 5′-ctgtcacttggtcgtggca-3′, Mc4r-2A-creStop F1 FRT3: 5′-gatcatgtgtaacgccgtc-3, Mc4r-2A-creStop R1 FRT3: 5′-catgtcaattcataacgccc-3.2.3Mc4r-cre mice were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine (IP) and placed in stereotaxic apparatus. A guide cannula with dummy cannula  was inserted into the PVN according to the atlas of Franklin and Paxinos (Franklin KBJ) , virus was infused at a rate of 40 nl/min  for 15 min and the injector (Plastics one) remained in place for an additional 5 min before. The injector was connected with a Hamilton syringe and infusion was administered at a rate of 33.3 nL/min.The AAV2-CMV-DIO-GFP [AAV-DIO-GFP] and AAV2-CMV-DIO-MRAP2-FLAG [AAV-DIO-MRAP2] virus (VECTOR BIOLABS) were injected bilaterally into the PVN of Mc4r-cre animals. Moreover AAV2-CMV-GFP (VECTOR BIOLABS cat No. 7004) virus was co-injected to determine successful PVN targeting. Eight weeks old 2.4Fed mice were perfused and brains were processed for immunofluorescence staining to confirm the injection site in the PVN, using anti-GFP antibody . Mice in which viral injections were located outside the PVN were studied separately. The sections were incubated overnight in anti-GFP antibody (diluted 1:5000 in 0.1 mol/L sodium phosphate buffer) and then incubated in secondary antibody  for 2 h. Sections were then analyzed with fluorescent microscope.2.5Immunofluorescence staining was performed using anti-FLAG antibody . Brains were sectioned with a vibratome, and sections were incubated for 24 h at room temperature in anti-FLAG antibody (diluted 1:2000). After several washes with phosphate buffer (PB), sections were incubated in secondary antibody  for 2 h at room temperature, then rinsed in PB three times 10 min each time, and incubated for 2 h at room temperature with Alexa Fluor 594 streptavidin . Sections were mounted on glass slide with vectashield (Vector lab) and analyzed with fluorescent microscope.2.6in vivo by MRI . Twelve weeks after PVN injection male and female mice were acclimated in metabolic chambers  for 4 days before the start of the recordings. Animals were continuously recorded for 3 days with the following measurements being taken every 30 min. Measurements include food intake, locomotor activity (in X and Y axes), and gas exchange (O2 and CO2) every 30 min using the TSE LabMaster System. Respiratory exchange ratio (RER) was calculated as a ratio of CO2 production and O2 consumption. Energy expenditure (EE) was calculated according to the manufacturer's guidelines  and analyzed relative to body weight using ANCOVA analysis Body weight was measured weekly after stereotaxic surgery. Body composition was measured 2.7ad libitum. Blood glucose was measured before IP injection and at 15, 30, 60, and 120 min after insulin injection.Glucose tolerance test (GTT) was performed using 2 mg/kg glucose in saline (DeltaSelect) given intraperitoneally (IP) to 16 h fasted animals as previously described 2.8Ad libitum fed mice were anesthetized and transcardially perfused with 0.9% saline with heparin followed by 4% paraformaldehyde. In another set of experiments, mice were injected with either MTII  or equal volume saline. Animals were perfused 1 h post injection. Brains were collected and post-fixed overnight before several sections of the entire hypothalamus were taken at every 50 μm. Sections were washed and incubated with the goat anti-cfos antibody , and rabbit anti-POMC antibody  in PB containing 4% normal donkey serum, and 1% Triton X-100 for 24 h at room temperature. After several washes with phosphate buffer (PB), sections were incubated in the secondary antibodies  for 2 h at room temperature, then rinsed in PB five times, 10 min each time. Sections were then mounted on glass slide with VectaShield antifade (Vector Laboratories). Fluorescent images of five to seven brain sections were captured with fluorescent microscope and analyzed by imaging Software (Image J).2.9Mc4r-crePVN-MRAP2 and control mice using Trizol solution (Invitrogen). Uncoupling protein 1 (UCP1) and Deiodinase 2 (DIO2) mRNA levels in the brown adipose tissue, were measured by real-time PCR. A High Capacity cDNA Reverse transcription Kit (Applied Biosystems) was used for the reverse transcription. Real-time PCR  was performed with diluted cDNAs in a 20-μl reaction volume in triplicates. Primers used for this study are as follows: cat. No. Mm 01244861_m1 for UCP1, cat. No Mm 00515664_m1 for DIO2, and Mm 02619580_g1 for β-actin (Applied Biosystems). The calculations of average Cp values, SDs, and resulting expression ratios for each target gene were based on the Roche LightCycler 480 software.Total RNA from brown adipose tissue was extracted from 2.10All statistical analysis was performed using GraphPad Prism. Data is plotted as mean ± S.E.M. Student's t test was used to compare two groups; for more than two groups one-way ANOVA was performed followed by Bonferroni multiple comparison test. In all analyses, a two-tailed probability of <5%  was considered statistically significant.33.1AAV-DIO-MRAP2 and its control, AAV-DIO-GFP, in the PVN of female  evidenced after 4 weeks post viral injections. No differences in lean mass were observed between the 2 experimental groups  and controls  and in respiratory quotient were observed  compared to Mc4r-crePVN-GFP female mice , male Mc4r-crePVN-MRAP2 mice showed lower body weight gain compared to control male Mc4r-crePVN-GFP mice  showed a significant increase in cfos immunostaining in the PVN compared to vehicle-injected HFD-fed Mc4r-crePVN-GFP male mice (46.88 ± 4.34 cfos positive cells in controls vs 63.77 ± 3.22 cfos positive cells in Mc4r-crePVN-MRAP2 n = 3 per group; Mc4r-crePVN-GFP and Mc4r-crePVN-MRAP2 male mice, no significant difference was observed (107.37 ± 10.00 cfos positive cells in controls vs 85.97 ± 13.21 cfos positive cells in Mc4r-crePVN-MRAP2 n = 6 per group; Mc4r-crePVN-MRAP2 male mice injected with saline compared to Mc4r-crePVN-GFP male mice injected with saline, whilst no difference in food intake was noted between Mc4r-crePVN-MRAP2 and Mc4r-crePVN-GFP male mice injected with MTII (2.97 ± 0.16 g in Mc4r-crePVN-MRAP2 + saline n = 9, 3.55 ± 0.17 g in Mc4r-crePVN-GFP + saline n = 8, 2.70 ± 0.33 g in Mc4r-crePVN-MRAP2 + MTII n = 6, 2.85 ± 0.29 g in Mc4r-crePVN-GFP + MTII n = 5, Mc4r-crePVN-MRAP2 male mice treated with saline compared to Mc4r-crePVN-MRAP2 mice treated with MTII. Together these data suggest a role for MRAP2 in PVN MC4R-expressing neurons in potentiating neuronal activation within the PVN at baseline, and in males without further increase after MTII administration.To assess the response of MTII in 4Mrap2−/− animals pointing to these neurons, located also in the PVN, as key targets of MRAP2 action in metabolism regulation Mrap2−/− and Mc4r−/− mice suggest possible non-MCR modes of action MRAP2 has been shown to have a critical role in mammalian metabolism Mrap2−/− animals Mrap2−/− mice in which MRAP2 is deleted during development. By selectively overexpressing MRAP2 in MC4R neurons of the PVN, we reveal a reduction in food intake and energy expenditure that supports the observed lean phenotype, which is unlike that of Mrap2−/− in which no change in either measures were identified As Sim1-Cre mice express cre activity in sites outside the PVN Mrap2-deficient mice that showed no change in BAT activation or body temperature phenotype Mc4r-crePVN-MRAP2 mice showed a significant increase in BAT activation with increased UCP1 and Dio2 mRNA levels that were associated with increased body temperature compared to control mice in both genders.In addition, unlike data on the global Mc4r-crePVN-MRAP2 mice compared with Mc4r-crePVN-GFP matched control. Whilst female mice overexpressing MRAP2 in PVN MC4R neurons demonstrate overt protection against obesity on a chow diet, this phenotype was only observed in male mice after challenge with high fat diet. Similarly, sex differences were also observed in locomotor activity and glucose handling. We have previously described a sex-specific increase in daytime locomotor activity and exploratory activity in global Mrap2-deficient mice Mc4r-crePVN-MRAP2 mice compared to controls. A difference in glucose clearance and hyperinsulinemia was demonstrated in Mrap2−/− mice on a C57/BL6N background, while on a 129/Sv genetic background no changes in insulin and glucose handling were found Mrap2-deficient mice on a C57/BL6N background Mc4r-crePVN-MRAP2 mice. Indeed, no significant differences were observed in male mice on a chow diet.We also found a sex-specific metabolic difference in the Mc4r-crePVN-MRAP2 and controls. Furthermore, no differences were found in POMC fiber staining in the PVN of controls and Mc4r-crePVN-MRAP2 mice. This suggests that the metabolic phenotype observed might be independent from changes in POMC neuronal activation, thus pointing to the increased neuronal activation in the PVN as the principal cause of the lean phenotype in the Mc4r-crePVN-MRAP2 model as we observe a significant increase in cfos staining in the PVN of Mc4r-crePVN-MRAP2 mice compared to controls irrespective of sex.To determine a possible role for POMC neurons in the regulation of PVN MC4R neurons, we then assessed the activation levels of POMC neurons by cfos immunostaining. No difference in POMC neuronal activation was observed between Mc4r-crePVN-MRAP2 and control mice. MTII induced a significant increase in PVN neuronal activation and a reduction in feeding in male and female Mc4r-crePVN-GFP mice. In female Mc4r-crePVN-MRAP2 mice MTII treatment results in further increase in PVN neuronal activation and decrease in food intake. At baseline, MRAP2 overexpression drives neuronal activation in the PVN in both males and females. However, response to MTII, differed between genders; whilst female Mc4r-crePVN-MRAP2 mice maintained responsiveness to MTII this was lacking in male Mc4r-crePVN-MRAP2 mice.To assess the hypothesis that MRAP2 overexpression in MC4R neurons affect MC4R signaling, thus affecting food intake, we then administrated MTII, a MC3R/MC4R agonist, in Mc4r-crePVN-MRAP2 mice correlates to models of MC4R activation in which reduced food intake, increased energy expenditure and thermogenesis have been described Altogether these results indicate that MRAP2 in PVN MC4R neurons can be manipulated postnatally to result in a lean phenotype in which feeding is reduced and energy expenditure increased along with core body temperature. Importantly, this leads to an increase in neuronal activation with in the PVN. As the number of PVN cfos positive cells are in excess of the number of FLAG positive Mc4r-cre MRAP2 overexpressing cells, the increased cfos would suggest a more global effect on PVN neuronal activation beyond MC4R expressing neurons. The likelihood is that the action of MRAP2 on MC4R PVN neurons is due to the action on enhancing MC4R function as much of the phenotype in 5In conclusion, our data provides the first evidence that MRAP2 acts postnatally, in a sex-specific manner, to play a role in the regulation of food intake and energy expenditure through the enhancement of MC4R neuronal signaling in the paraventricular nucleus of hypothalamus.GB, JDK, and LFC conducted experiments, acquired and analyzed data. LFC and SD designed the research studies and analyzed data. GB, LFC, and SD wrote the manuscript.


The alignment is a key factor to fully exploit the potential of graphene in reinforcement of polymer composites. However, it is still a challenge to orientate graphene in thermosets because of the insoluble and infusible features of the later. In this paper, we report a facile and scalable hot press method to fabricate aligned graphene nanoplate (GnP)/epoxy composites by utilizing the dynamic character of epoxy vitrimer. The bond exchange and topological rearrangement associated viscous flow of epoxy vitrimer during hot press allows the spontaneous orientation of GnP in matrix because the 2D structure and volume exclusion effect. SEM images demonstrate the orientation of GnP, while tensile test reveals the significantly increased reinforcement effect of GnP on matrix after hot press. Moreover, the dynamic reaction of epoxy vitrimer confers good healability and recyclability to the aligned composites as confirmed by the nearly fully recovered mechanical properties of the healed sample after cutting, and the recycled sample after grinding. This work is expected to provide new opportunity for fabrication of aligned thermosetting composites. Graphene has been recognized as ideal nanofiller for polymer composites due to its excellent mechanical property, high thermal, and electrical conductivity, and large aspect ratio  was purchased from Ao Yu Co. Ltd in Shanghai. Bisphenol A diglycidyl ether (DGEBA) was purchased from TCI Co. Ltd in Shanghai. Fatty acid (Pripol 1040) was supplied by He Da chemical Co. Ltd (Shanghai). Zinc acetate dihydrate, acetone, ethanol, concentrated nitric acid, and concentrated sulfuric acid were obtained from Sinopharm Chemical Reagent Co. Ltd. All chemicals were used as received without further treatment.Expanded graphite (EG) was first obtained by thermal expansion of GIC at 700°C for 1 min. Then 1 g EG was refluxed in 100 mL mixture of concentrated nitric acid and concentrated sulfuric acid (volume ratio of 1:3) at 100°C for 4.5 h to afford acidic EG (EG-COOH). At last, 1 g EG-COOH was suspended in 100 mL ethanol and sonicated in a low power sonic bath for 24 h followed by centrifugation at 1,000 rpm for 30 min, the supernatant containing exfoliated GnP was collected, and the concentration was calculated by drying a certain amount of the dispersion of GnP.The procedure for the fabrication of epoxy and GnP/epoxy composites were similar to previously reported methods . After maintained at 200°C for another 5 min, the specimens were cooled to room temperature. The hot pressed composites were then denoted as hGnP/epoxy.Fourier transform infrared (FT-IR) spectra were obtained from a Nicolet 6,700 spectrometer. Transmission electron microscopy (TEM) images were obtained using JEM-2100. Atomic force microscopy (AFM) images were recorded using a digital Nanoscope IIIa Atomic Force Microscope in tapping mode. Rheological test was performed on ARES-RFS rheometer in a strain-controlled mode, the sample was in disk-shape with diameter of 8 mm and thickness of 2 mm. From the strain sweep experiments at a frequency of 1 rad/s for determination of the region of linear response, the strain of 0.4% was applied for stress relaxation experiments and the relaxation modulus was recorded as a function of time at different temperatures. Differential scanning calorimetry (DSC) test was performed on a Netzsch 204 F1 thermal analyzer from −20 to 80°C at a heating rate of 10°C/min under nitrogen flow. Thermogravimetric analysis (TGA) was performed with a Netzsch TGA 209 F1 instrument at a heating rate of 20°C/min in nitrogen. Tensile tests were measured using MTS materials testing machine at room temperature and humidity of about 50%, the gauge length was 30 mm and strain rate was 50 mm/min, at least five specimens of each sample were measure for accuracy.−1 in EG-COOH, while TGA curves  of epoxy at 200°C is 346 s. The addition of 0.5 and 1 wt% GnP decreases the τ* to 288 and 206 s, suggesting the accelerated stress relaxation, which is attributed to the decreased cross-linking density because of the introduced excess amount of carboxyl groups in the cross-linked network, and the surface exchangeable bonds on GnP after curing that could participate in the relaxation process of the network  of epoxy and composites are summarized in Tg decreases with the addition of GnP due to the reduced cross-linking density of the whole network. The variation tendency of Tg of hGnP/epoxy composites is similar to that of aGnP/epoxy composites. However, in comparison with their corresponding aGnP/epoxy samples, the Tg of hGnP/epoxy composites are all increased. The slightly increased Tg in hot-pressed epoxy (ΔTg = 0.3°C) is ascribed to the more compacted molecular chains after hot press  were then investigated. E) and tensile strength (σ) of epoxy. The aGnP/epoxy with 1 wt% GnP shows the highest modulus and tensile strength of 2.0 and 3.7 MPa, corresponding to increments of 11.1 and 23.3% as compared to epoxy, respectively. The elongation at break (ε) of composites are also increased as a result of the decreased cross-linking density. Further addition of GnP to higher than 2 wt% has lowered the E and σ of composites, which is probably due to the negative effect of lowered cross-linking density has overwhelmed the positive effect of mechanical reinforcement of GnP. As expected, the hot press has significantly enhanced the reinforcement effect of GnP on epoxy. For example, the E and σ of hGnP/epoxy with 1 wt% GnP is promoted to 2.8 and 6.9 MPa, which are 40.0 and 86.5% higher than its corresponding aGnP/epoxy composite, and 55.6 and 130.0% higher than epoxy. The hGnP/epoxy with 2 wt% GnP exhibits more enhancement in E and σ (with increments of 194 and 141% as compared to its aGnP/epoxy composite) after hot press. Note that the E and σ of pure epoxy are also improved by hot press due to the more compacted molecular chains  are much lower than those in composites. In addition, it is also noted that there might exist additional esterification reaction between excess carboxyl groups and hydroxyl groups in matrix during hot press, which could also contribute to the improved mechanical properties of hGnP/epoxy composites, but a comparison made on non-dynamic composites  composites were then employed to directly demonstrate the orientation of GnP in epoxy matrix by hot press . It can Tg, and the deformed shape could be fixed at lower temperature, the internal stress generated by the external force could not be fully released because the restricted chain mobility by covalent cross-linking points. Such stored stress is harmful to the dimensional stability and mechanical properties of network. Moreover, once these deformed samples were used in some high-temperature applications, the high temperature would activate the frozen polymer chains, thus the deformed sample are tend to recover its original shape, such phenomenon is well-known as “shape memory effect” (Rousseau and Xie, We have also carried out control experiments on non-dynamic GnP/epoxy composites to highlight the important of transesterification reaction in the hot press process. The non-dynamic composites were prepared from the same raw materials and procedure but without the addition of zinc ions as catalyst. The tensile test  reveals The dynamic transesterification reaction not only allows the fabrication of aligned hGnP/epoxy composites by simple hot press process, but also confers healability and recyclability to the composites because of the capability of bond exchange. To demonstrate these points, the specimens of hGnP/epoxy were first cut in half, then the fracture surfaces were put together under slight pressure and heated at 200°C for 20 min. As shown in To verify the recyclability of hGnP/epoxy composites, the composite with 1 wt% GnP was first grinded into fine powder, then the powder was hot pressed at 200°C for 30 min, the thickness of the recycled composite was controlled the same as original one. It can be seen that the grinded powder formed integrated composite again after hot press . The tenThis work presents a facile and scalable hot press method for the fabrication of aligned GnP/epoxy composites by using a epoxy vitrimer as matrix. The GnP could be simultaneously orientated in matrix during hot press as confirmed by SEM images due to the ability of epoxy vitrimer to viscous flow at high temperature. The tensile test demonstrated that the mechanical properties of composites were greatly improved after hot press, and the improvement was closely related to the compression ratio of hGnP/epoxy composites, larger compression ratio resulted in greater improvement in mechanical properties. It is showed that with the addition of only 1 wt% GnP, the strength of hGnP/epoxy with compression ratio of 83% was 173.3% higher than pristine epoxy, while the increment was just 23.3% in aGnP/epoxy. The comparison with hGnP/epoxy composites with no catalyst revealed the crucial role of vitrimer character in fabrication of aligned composites. In addition, the tensile test showed that the healing efficiency of hGnP/epoxy composites were generally higher than 95% after cutting in half, and the strength of recycled hGnP/epoxy after grinding into powder was nearly constant with that of original value, proving the good healability and recyclability of hGnP/epoxy composites. We anticipate that the approach of fabricating aligned GnP/epoxy composites assisted by vitrimer chemistry reported here could be applied in design of other aligned thermosetting composites.All datasets generated for this study are included in the manuscript/JC, HH, JF, and YW contributed to the design and synthesis of polymer and composites. JC, JF, YW, JY, and JZ contributed to the characterizations of properties of composites and data analysis. JC, JF, and YW contributed to the writing of the first draft. HH, JF, YW, and ZH contributed to the final revision of the draft.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.


Two case reports showed that the combination of CRT and LVAD benefits the end-stage heart failure patients with prolonged QRS interval significantly. In one of the reports, the patient had the LVAD removed due to the recovery of the heart function. However, the quantification of the combined devices has yet to be conducted. This study aimed at computationally predicting the effects of CRT-only or combined with LVAD on electromechanical behaviour in the failing ventricle with left bundle branch blocked (LBBB) and right bundle branch blocked (RBBB) conditions. The subjects are normal sinus rhythm, LBBB, RBBB, LBBB with CRT-only, RBBB with CRT-only, LBBB with CRT + LVAD, and RBBB with CRT + LVAD. The results showed that the CRT-only shortened the total electrical activation time (EAT) in the LBBB and RBBB conditions by 20.2% and 17.1%, respectively. The CRT-only reduced the total mechanical activation time (MAT) and electromechanical delay (EMD) of the ventricle under LBBB by 21.3% and 10.1%, respectively. Furthermore, the CRT-only reduced the contractile adenosine triphosphate (ATP) consumption by 5%, increased left ventricular (LV) pressure by 6%, and enhanced cardiac output (CO) by 0.2 L/min under LBBB condition. However, CRT-only barely affects the ventricle under RBBB condition. Under the LBBB condition, CRT + LVAD increased LV pressure and CO by 10.5% and by 0.9 L/min, respectively. CRT + LVAD reduced ATP consumption by 15%, shortened the MAT by 23.4%, and shortened the EMD by 15.2%. In conclusion, we computationally predicted and quantified that the CRT + LVAD implementation is superior to CRT-only implementation particularly in HF with LBBB condition. Heart failure (HF) plays a major role in the number of death worldwide . Thus, tLVAD supports the ventricular pumping via mechanical unloading for weakened heart. LVAD was initially used as a bridge to transplantation for patients with end-stage disease , 15. HowIn 2011, a case report article stated that the CRT supported the restoration of end-stage HF in a 15-year-old patient who underwent LVAD implantation . The CRTTo the best of our knowledge, a computational study that quantifies the effect of combined CRT and LVAD has yet to be conducted. This study aims at predicting, computationally with an electromechanical failing ventricles model, the effects of CRT alone and the combination of CRT and LVAD treatment in patients with LBBB or right bundle branch block (RBBB). We used a well-developed electromechanical ventricular model which had been used to observe different heart conditions, including the prolonged EMD in the dyssynchrony HF condition , mechano2+ transient. The LVAD function included the circulatory systems, which connected to the 3D ventricular mechanics. We followed an existing well-developed electromechanical failing ventricle model with the fibers and laminar sheet structures based on diffusion tensor magnetic resonance imaging –37. In tThe electrical component was a failing ventricular mesh with 241,725 nodes and 1,298,751 tetrahedron elements. The electrical mesh has the characteristics of realistic heart compartment, which consists of endocardial, midmyocardial, and epicardial cells following the ten Tusscher et al. human ventricular tissue model . The linThe Purkinje networks induced the electrical activation sequences of sinus rhythm , LBBB, and RBBB . The CRTV represents the voltage difference of intracellular and extracellular, t represents time, Iion represents the sum of all transmembrane ionic currents, Istim represents the current if an external stimulus is applied, and Cm represents the cell capacitance per unit of surface area. Iion consists of major ionic current as follows:INa is fast inward Na+ current, IK1 is inward rectifier K1 current, Ito is transient outward K+ current, IKr is rapid delayed rectifier K+ current, IKs is slow delayed rectifier K+ current, ICaL is L-type inward Ca2+ current, INaCa is Na+/Ca2+ exchanger current, INaK is the Na+/K+ pump current, IpCa and IpK are sarcoplasmic plateau Ca2+ and K+ currents, IbCa is background Ca2+ current, and INa,b is background Na+ current.The electrical propagation signal represents an ion exchange across the myocyte as described by ten Tusscher et al. . The eleρ) and surface-to-volume ratio (S) in x, y, and z directions, respectively. This phenomenon can be described by the following partial differential equation:To represent the electrical propagation through the conduction in 3D spatial, the cardiac tissue in this case could be described by the combination of the Equation  with theIleak represents the calcium released from the sarcoplasmic reticulum (SR) into the cell. Iup represents the calcium pumping to restore the calcium again back to the SR. Irel represents the calcium-induced calcium release current, d is the activation gate of Irel, which was the same with ICaL. Caitotal is the total calcium inside the cell, which consists of Caibufc, the calcium buffer inside the cell, and Cai, the free calcium inside the cell. Accordingly, the Casrtotal is the sum of calcium in the SR, which includes Casrbufsr, the calcium buffer, and Casr, the free calcium inside the SR.The mathematical equation for calcium dynamics for coupling the electromechanical simulation was also described by ten Tusscher et al. :(4)Ileak2+ transient serves as an input to the cell myofilament model representing the generation of active tension within each myocyte in which an ODE set and multiple algebraic equations describe Ca2+ binding to troponin C, cooperatively between regulatory proteins, and cross-bridge cycling. Ventricular contraction results from the active tension generation represented by the myofilament dynamics model described by Rice et al. [dSL/dt = 0. To measure the isotonic contraction, the SL is solved using the following ordinary differential equation (ODE):Force is the sum of the normalized force integrated toward time:Factive(x) is the active force and the Fpassive(x) is the passive force. Fpreload is the constant force at the resting length of the initial sarcomere length, and Fafterload is the force during the isotonic or isometric contraction. Thus, Fafterload is expressed as follows:A Cae et al. . VentricE) is the outcome of cross-bridge detachment rate (gxbT) and the single overlap fraction of thick filaments (SOVFThick):We calculated the ATP consumption by integrating the myofilament model in one cycle proposed by Rice et al. from each node . In the To construct an integrated model of an LVAD-implanted cardiovascular system, we added a compartment of LVAD function between LV and systemic arteries in the circulatory system based on Kerckhoffs et al.  as descr2+ from the electrical simulation as the input to the mechanical simulation. To model the HF condition, we multiplied the constant of passive scaling in the strain-energy function by 5 times to stiffen the myocardium. The mechanical model was simulated for 20 seconds to reach the steady state. We compared the ATP consumption rates and tension activation during end-systolic volume (ESV) and the strain during end-diastolic volume (EDV) by integrating the information of them from each node. EMD was defined as the time interval between mechanical activation time (MAT) and electrical activation time (EAT). MAT was identified when the local strain was shortened to 10% before its maximum, while EAT was identified when the myocyte started to depolarize, which in our case was −50 mV [For the simulation protocol, first we simulated the electrical model with the Purkinje delivering the signal representing sinus rhythm, LBBB, or RBBB until the steady state was achieved. We set the conduction velocity by 60 cm/s and the basic cycle length by 600 ms. Here, we incorporated the dyssynchrony HF conditions (LBBB and RBBB) using CRT-alone or combined CRT and LVAD. We then used Gaussian point for the interpolation of transient Cas −50 mV .2+ deactivation occurred at 250 ms. CRT fastened the Ca2+ activation throughout the ventricle in the LBBB condition but insignificantly in the RBBB condition due to the CRT pacing site.−1. The ATP consumption rate in LBBB condition was the highest at 100 s−1. CRT reduced the ATP consumption rate in the LBBB condition by 5%, while CRT and LVAD reduced the ATP consumption rate by 15%. In the RBBB and RBBB + CRT conditions, the ATP consumption rates were the same as that in the normal condition, 93 s−1. However, with CRT and LVAD support, the ATP consumption rate in the RBBB condition was decreased by 16% to 84 s−1.CRT shortened the longest EAT by 20.2% in the LBBB condition and 17.1% in the RBBB condition. CRT shortened EMD by 10.1% in the LBBB condition but did not shorten EMD in the RBBB condition.Combination of CRT and LVAD treatment shortened EMD more than CRT alone in the LBBB condition (15.2%) and shortened EMD in the RBBB condition (1.3%).CRT reduced the ATP consumption by 5% as well as the tension and strain in the LBBB condition. CRT also slightly increased the LV peak pressure by 6% and increased the CO by 0.2 L/min in the LBBB condition. However, CRT-alone did not affect these mechanical responses in the RBBB condition.Combination of CRT and LVAD reduced the ATP consumption by 15% in the LBBB condition and by 16% in the RBBB condition. It also increased the LV pressure by 10.5% in the LBBB condition and 5.7% in the RBBB condition as well as the CO up to 4 L/min for both conditions, a degree greater than that in the control condition.In general, we performed a simulation and analyzed seven cardiac diseases and therapy conditions by using an electromechanical ventricular model incorporated with a circulatory systems, CRT, and LVAD models. The models including normal sinus rhythm, LBBB, LBBB + CRT, LBBB + CRT + LVAD, RBBB, RBBB + CRT, and RBBB + CRT + LVAD. The major findings of this study are as follows:The pacing site at the tissue greatly affected synchronization. Placement at the LV free-wall showed faster activation throughout the chamber since the signal propagated evenly from the midway between the base and apex throughout the LV tissue. However, the pacing site at the RV endocardial apex showed a longer activation time for the electrical signal to propagate to the base.In the RBBB condition, the electrical activation of the LV chamber was the same as normal sinus despite the electrical activation alteration in the RV. This is the major factor why the mechanical responses of the LV  were also the same as that in normal sinus rhythm. Even the CRT implementation in the RBBB condition did not affect the mechanical responses of the LV chamber. However, we observed significant improvement in CO in the RBBB + CRT + LVAD condition. CRT and LVAD also reduced the ATP consumption and tension and increased the LV and aortic pressure in the RBBB condition.CRT-alone did not fully restore the mechanical responses under the LBBB condition to normal despite the resynchronization of the EAT, which shortened the MAT and EMD to back normal. On the contrary, the use of combined CRT and LVAD reduced the energy consumption (indicated by the ATP consumption rate), tension, increased the LV pressure, and produced CO by 18% more than that in the HF with sinus rhythm condition. In addition, the LVAD did not fully assist the blood distribution in the LBBB or RBBB condition.The computational method allowed us to predict the electromechanical phenomenon in the dyssynchrony heart which underwent CRT and LVAD treatment. It is hardly possible to observe some parameters including ATP consumption, tension, MAT, and EMD from the patient's heart of that condition in experimental procedure. This study quantified the electrical activation and hemodynamic responses in several dyssynchrony HF combined with CRT-only, and CRT and LVAD, which has never been conducted previously. As for the clinical impact, the results of this study can be used as reference to generally predict the effect of the combination of CRT and LVAD devices to the HF patients with LBBB. Though, there are some parameters need to be consider deeply by the cardiologist expert.This computational study has several limitations that need to be addressed. The study did not follow the standard biventricular pacing method . InsteadIn conclusion, although CRT-alone shortened the MAT and EMD to more than normal in the LBBB condition, the mechanical responses in the LBBB condition were not restored to normal. The combined CRT and LVAD shortened the MAT and EMD more than CRT-alone, restored the hemodynamic, and produced a greater CO than normal in the LBBB and RBBB conditions. Using the combined system, LVAD contributed to the MAT reduction by mechanical unloading, shortened the EMD, reduced ATP consumption, and reduced tension, which contributes to the recovery of the heart shape and function. In short, we computationally predicted and quantified that the CRT + LVAD implementation is superior to CRT-only implementation particularly in HF with the LBBB condition.