Abnormal activation of Wnt/β-catenin-mediated transcription is closely associated with the malignancy of pancreatic cancer. Family with sequence similarity 83 member A (FAM83A) was shown recently to have oncogenic effects in a variety of cancer types, but the biological roles and molecular mechanisms of FAM83A in pancreatic cancer need further investigation. Here, we newly discovered that FAM83A binds directly to β-catenin and inhibits the assembly of the cytoplasmic destruction complex thus inhibiting the subsequent phosphorylation and degradation. FAM83A is mainly phosphorylated by the SRC non-receptor kinase family member BLK (B-lymphoid tyrosine kinase) at tyrosine 138 residue within the DUF1669 domain that mediates the FAM83A-β-catenin interaction. Moreover, FAM83A tyrosine 138 phosphorylation enhances oncogenic Wnt/β-catenin-mediated transcription through promoting β-catenin-TCF4 interaction and showed an elevated nucleus translocation, which inhibits the recruitment of histone deacetylases by TCF4. We also showed that FAM83A is a direct downstream target of Wnt/β-catenin signaling and correlates with the levels of Wnt target genes in human clinical pancreatic cancer tissues. Notably, the inhibitory peptides that target the FAM83A-β-catenin interaction significantly suppressed pancreatic cancer growth and metastasis in vitro and in vivo. Our results revealed that blocking the FAM83A cascade signaling defines a therapeutic target in human pancreatic cancer. Although diagnostic and surgical treatment methods have been developed to aid in lengthening survival and providing symptom relief, few approaches provide a curative effect.2 Moreover, both intrinsic and acquired chemo-resistant behaviors are major factors that significantly decrease the clinical efficacy of chemotherapy for pancreatic cancer.3 Understanding the biology of pancreatic cancer and identifying its putative therapeutic targets in the dysregulated oncogenic pathways are urgently needed for PDAC patients.Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with a 5-year survival rate of 5%.4 β-catenin is the central effector that mediates the transcription activity of numerous oncogenic target genes, such as C-myc, CyclinD1, and AXIN2. Cytoplasmic β-catenin is constitutively phosphorylated for degradation by the destruction complex, which consists of two multidomain scaffolding proteins AXIN1, adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK3β) and casein kinase Iα (CK1α) without Wnt stimulation. Phosphorylated β-catenin is then recognized by the E3 ubiquitin ligase β-transducin repeat-containing protein (β-TrCP) and is thus ubiquitinated and degraded by proteasome.5 Moreover, in the absence of Wnt/β-catenin signaling, Wnt target genes are silenced by co-repressors of Groucho/TLE1 and histone deacetylase (HDAC).6 Upon Wnt stimulation, the destruction complex is dissociated, and the stabilized β-catenin subsequently enters the nucleus and activates the transcription of downstream target genes via the T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) of transcription factors.7Mutations that result in the constitutively activated Wnt/β-catenin signal pathways are thought to be related to pancreatic cancer progression.8 FAM83A is a 434-amino-acid protein and contains DUF1669, serine-rich domains, and proline-rich domains (PRDs).9 A previous study has reported that FAM83A is overexpressed in multiple human tumors, such as lung, cervix, liver, and pancreatic cancer,13 suggesting a tumorigenesis role of FAM83A during cancer development and progression. The UniProt database describes that FAM83A independently functions in activating the PI3K/AKT signaling cascade pathway.11 Moreover, FAM83A has been identified as a candidate cancer-associated gene capable of conferring resistance on EGFR-tyrosine kinase inhibitors (TKIs) through interacting with and causing phosphorylation of c-RAF and PI3K p85, upstream of MAPK and downstream of EGFR.9 Notably, recent studies have indicated that FAM83A promotes the proliferative and invasive abilities of cancer cells via epithelial-mesenchymal transition and the Wnt signaling pathway.15 However, the detailed molecular mechanisms remain to be investigated.Family with sequence similarity 83 member A (FAM83A) was originally identified as BJ-TSA-9, which is highly expressed in lung cancer, without known function(s).In the present study, we figured out the roles of FAM83A and its posttranslational modification in states in canonical Wnt/β-catenin signaling regulation. Mechanically, we demonstrated that FAM83A interacts with β-catenin and acts as a negative component of the cytoplasmic β-catenin destruction complex that inhibits its assembly. We also identified that the FAM83A protein is phosphorylated by the SRC kinase subfamily member BLK at the tyrosine 138 residue, where the phosphorylation level markedly elevates the oncogenic potential of pancreatic cancer cells. Moreover, the peptides that target the FAM83A and β-catenin interaction significantly suppress the activity of canonical Wnt/β-catenin signaling and restrain the proliferation and metastasis of pancreatic cancer cells.To investigate the mechanisms of FAM83A in pancreatic cancer progression, a Flag-tag affinity procedure was performed to purify a FAM83A-containing complex in HEK293T cells, which was then subjected to LS-MS/MS analysis. The results showed that three peptide sequences of β-catenin were found in the complex, which suggests that β-catenin is a potential binding partner of FAM83A Fig. . The int16 we examined whether FAM83A affects the ability of TCF4 to recruit β-catenin. The results revealed that FAM83A depletion decreased the interaction between β-catenin and TCF4, whereas FAM83A overexpression enhanced β-catenin-TCF4 complex formation  and a mutant (FOPflash) reporter assay Fig. . Conversion Fig. . CollectTo investigate the functions of FAM83A in the β-catenin destruction complex and β-catenin stability, we asked whether FAM83A influences the stability of the destruction complex. To this end, the interaction between GSK3β, AXIN1, and β-catenin was evaluated. The results showed that FAM83A depletion increased the binding activity of either GSK3β or AXIN1 to β-catenin, whereas FAM83A overexpression reduced the association between GSK3β or AXIN1 and β-catenin in a dose-dependent manner in HEK293T, PANC-1 and SW1990 cells Fig. . MoreoveFrom our mass spectrometry data shown in Fig. To investigate the roles of FAM83A phosphorylation in Wnt/β-catenin signaling, we first assessed whether FAM83A phosphorylation by BLK affected β-catenin destruction complex assembly. It is shown that BLK overexpression decreased the binding ability of GSK3β and AXIN1 to β-catenin, whereas FAM83A overexpression amplifies its effects. Moreover, BLK knockdown showed opposite effects Fig. . We also17 we assessed whether FAM83A affected the recruitment of HDAC to the TCF4/β-catenin complex. The co-IP assays revealed that FAM83A depletion significantly increased the binding of endogenous HDAC1/2 with β-catenin or TCF4  Fig. . By fusisis Fig. . In vivosis Fig. . Moreovesis Fig. . Consistsis Fig. . These dTo examine the effects of the peptides that target FAM83A-β-catenin interaction on the tumorigenicity of pancreatic cancer cells, in vitro and in vivo assays were performed, and the results showed that CP-FaP2 and CP-FaP3 decreased the ability of DNA synthesis of PANC-1 and AsPC-1 cells compared with that observed in the control cells by using a 5-ethynyl-20-deoxyuridine (EdU) incorporation assay . The phosphorylation states of FAM83A at tyrosine 138 residue are shown to be essential for the activation of Wnt/β-catenin signaling and the development of pancreatic cancer.Recent studies have demonstrated that FAM83A is significantly upregulated in a variety of cancers and is presumed to be a novel oncogene partly through activating the PI3K/AKT and Wnt signaling cascade pathway.4 The DUF1669 domain, which is conservative among the FAM83 members, mediates the signal cascade of the EGFR/RAS/CRAF pathway and EGFR- and RAS-driven oncogenic transformation.20 Consistently, FAM83A is identified using a retroviral cDNA library screen that confers resistance on an EGFR-tyrosine kinase inhibitor in tumorigenic mammary epithelial cells.9 Moreover, recent observations confirm that FAM83 members A-E can independently promote anchorage-independent growth and form complexes with CRAF in mammary epithelial cells.21 Research using mouse models of pancreatic cancer or other cancers has similarly reported nodes of crosstalk between Wnt signaling networks and RAS oncogene. Overexpression of constitutively active β-catenin produces no lung tumor, whereas co-expression of constitutively active forms of both KRAS and β-catenin induces lung tumors, as shown in one model of lung cancer.22 Expression of constitutively active β-catenin also synergizes with the active forms of NRAS to induce melanomas with short latency.23 Inherited inactivating mutations in Wnt signaling negative regulator APC are found in patients with familial adenomatous polyposis (FAP), which can progress to colorectal carcinomas following concomitant activating mutations in KRAS and inactivating mutations in TP53.24 Here, our results revealed positive feedback that the Wnt signaling factor TCF4 binds to the promoter region of FAM83A and initiates its transcription. Our finding also proposes an approach that mediates the interplay between Wnt signaling and RAS cascade activation in the development of pancreatic cancer. Previous reports have shown that the FAM83 protein family has been annotated as containing a putative phospholipase D-like  motif, which lacks conservation at a critical histidine residue that is present in all bona fide PLD enzymes for catalytic activity.25 Both FAM83A and FAM83B were unable to exhibit phospholipase activity.20 However, our data show that the domain aa 150–250, which mediated the interaction between FAM83A and β-catenin, exactly coincided with the PKD-like motif. Hence, future studies are needed to assess the features within the DUF1669 domain that still harbor certain pseudo-PLD roles, such as binding to specific phospholipids, that might affect binding to β-catenin.High-frequency somatic mutations in a subset of genes and constitutively activated signaling pathways, such as the KRAS (KRAS proto-oncogene) and Wnt signaling pathways, are considered to be the main cause of pancreatic cancer development.26 Autophagy is essential for the development of pancreatic cancer.28 It was reported that the Wnt/β-catenin signaling activity directly repressed the transcription of SQSTM1 via TCF4 and suppressed autophagosome formation.29 β-catenin and Disheveled (Disheveled segment polarity proteins) could be selectively degraded and thus showing inhibited Wnt signaling via the formation of an LC3 or SQSTM1 binding complex during nutrient deprivation.31 RACK1 was reported to promote autophagy by enhancing the ATG14-Beclin1-VPS34-VPS15 complex formation32 but suppresses Wnt signaling and gastric tumorigenesis by stabilizing the β-catenin destruction complex.33 However, it is still largely unknown the mechanisms of the existence of high levels of both autophagy and Wnt/β-catenin signaling in pancreatic cancer. In our previous study, we demonstrated that lncRNA PVT1 up-regulates the expression of both Pygo2 and ATG14 and thus regulated Wnt/β-catenin signaling and autophagic activity to overcome gemcitabine resistance through sponging miR-619-5p.34 The positive autophagy initiation factor STYK1 probably enhances Wnt/β-catenin signaling through the AKT/GSK3β cascade.37 Moreover, we also showed that the β-catenin/TCF4 complex elevates the mRNA and protein levels of TSPAN1, a positive regulatory protein that functions in autophagy maturation, through binding directly to the two conservative TCF/LEF-binding elements in the promoter region of the TSPAN1. Furthermore, the upstream microRNA that targets both TSPAN1 and FAM83A is significantly downregulated.2 Here, our data detailed the mechanisms of FAM83A that promote Wnt/β-catenin signaling and together with our previous report defined the crosstalk between autophagy and Wnt in pancreatic cancer development.Macroautophagy (hereafter referred to as autophagy) is a process in which organelles termed autophagosomes deliver cytoplasmic constituents to lysosomes for degradation.39 Treatment of primary PDAC cultures established from patient-derived xenografts with dasatinib or PP2 reduced the clonogenic, self-renewal, and tumor-initiating capacity of pancreatic cancer stem cells (PaCSCs). Insulin receptor 1, which plays a role in GLUT4 and GLUT1 translocation, was reported to be phosphorylated by LCK/YES-related novel kinase (LYN) directly and appears to be required for oncometabolite changes.40 The inhibition of SRC family kinase YES1, LYN, FYN, FRK, and C-SRC using specific small interfering RNA transfection markedly suppressed cell proliferation in PANC-1 cells.41 The recent identification of BLK as a player in the differential phosphorylation signatures of PDAC cells suggests that BLK may play an important role in solid pancreatic malignancies.42 Increased BLK activity was also reported to lead to increased PDX1 activation and enhanced insulin synthesis, which is being investigated as a potential target for pancreatic cancer therapy.44 Consistently, our data also show that FAM83A could be phosphorylated by the non-receptor SRC kinase subfamily members, in which BLK kinase presents central effects. Moreover, the phosphorylation states of FAM83A revealed an oncogenic function and presented a link between Wnt/β-catenin signaling and BLK kinase in the progress of pancreatic cancer. Although the conserved PxxP motif was predicted to interact with Src homology 3 (SH3) domain-containing proteins in the PRD domain of FAM83A,45 our results reveal that the DUF1669 domain-mediated binding of FAM83A to BLK kinase.Intricate communication between pancreatic cancer cells and their surrounding environment is driven by a dynamic signaling network of cellular and matrix remodeling enzymes, cytokines, chemokines, and growth factors, which collectively promote tumor growth and treatment resistance. The non-receptor SRC kinase family signaling network is increasingly recognized as a key player in pancreatic tumorigenesis.The β-catenin transcriptional complex is a high-priority pharmacologic target because of its pathologic role in a broad range of human cancers. Indeed, the chimeric peptide CP-FaP2 or CP-FaP3 interferes with the formation of the FAM83A-β-catenin and β-TCF4 complex, decreasing the stability of β-catenin, and counteracts the oncogenic capability of pancreatic cancer cells in vitro and in vivo. Our data also show that the peptide dosing interval of twice a week resulted in an ideal treatment outcome. In summary, our results detail the roles of FAM83A in the regulation of Wnt/β-catenin signaling through directly binding to β-catenin and suppressing TCF4-mediated transcriptional activity. In addition, our results provide a proof of concept for a therapeutic strategy against pancreatic cancer development.6) were subcutaneously injected into the left and right axillae, or into the tail vein of five female BALB/c nude mice per group for the xenograft model and lung/colon metastasis model, respectively. One or two weeks later, the animals were treated with Wnt inhibitor XAV939 at 50 mg/kg body weight or commercially synthetic peptides at 2 mg/kg body weight via intraperitoneal injection twice a week. The length and width of the mouse xenograft tumors were measured every 5 days with calipers, and tumor volume (V) was calculated with the following formula: V = [(length × width × width)/2]. Experimental mice were euthanized at the end of the observation period, and then the tumors were excised and weighed. The metastasis of tumor cells to the lung or colon was also quantified after euthanasia. The subcutaneous xenograft and metastasis mouse models were used to assess the tumor formation and metastasis ability of pancreatic cancer PANC-1 cells, which were stably expressed wild-type FAM83A, FAM83A Y138A. and FAM83A Y138D mutants with or without XAV939 or functional peptides treatment.All animal experiments were carried out in compliance with a protocol specifically approved for the use of laboratory animals by the Hubei University of Technology Animal Care and Use Committee. Four-week-old (18–22 g) female BALB/c nude mice were purchased from Hunan SJA laboratory animal . PANC-1 cells (3 × 102. The medium was replaced every 2–3 days and the cell was subcultured and used for an experiment at 80–90% confluence. Wnt agonist Wnt3a was purchased from Abcam (ab153563). Wnt inhibitor XAV939 was purchased from MedChemExpress (HY-15147). BLK kinase inhibitor saracatinib and LCK kinase inhibitor PP2 were both purchased from MedChemExpress (HY-10234 and HY-13805). Cycloheximide (CHX) and MG132 were purchased from Selleck (S7418 and S2619).Human pancreatic cancer cell lines PANC-1 (TCHu 98), AsPC-1 (TCHu 8), SW1990 (TCHu201), and CFPAC-1 (TCHu112) were purchased from the cell center of the Institute of Biochemistry and Cell Biology or National Collection of Authenticated Cell Cultures, Chinese Academy of Sciences . HEK293T and HeLa cells were stored in our lab. HEK293T, HeLa, PANC-1, and AsPC-1 cells were cultured in Dulbecco’s modified Eagle’s medium . All culture mediums were supplemented with 10% fetal bovine serum , 100 U/ml penicillin G, and 100 μg/ml streptomycin  at 37 °C in a humidified incubator containing 5% CO35 GAPDH protein levels were used as loading controls. We used the following primary antibodies: anti-Non-phospho (Active) β-catenin (Ser33/37/Thr41) , anti-phospho-β-catenin (Ser33/37/Thr41) , anti-β-catenin , anti-β-TrCP , anti-FAM83A , anti-c-myc , anti-CyclinD1 , anti-AXIN2 , anti-mouse HA , anti-Rabbit HA , anti-mouse DYKDDDDK , anti-Rabbit DYKDDDDK , anti-GAPDH , anti-GFP , anti-phosphotyrosine , anti-phosphoserine , anti-phospho-threonine , anti-GSK3β , anti-AXIN1 , anti-TCF4 , anti-BLK , anti-HDAC1 , anti-HDAC2 , anti- Acetyl-Histone H3 .Immunoprecipitation and Western blotting were performed as described previously.34 Briefly, FAM83A shRNAs or scramble RNA were subcloned into the lentiviral vector pLKO.1-puro . DNA fragments encoding FAM83A and its Y138A or Y138D mutants were subcloned into lentiviruses vector pCDH-CMV-MCS-EF1-turboRFP-T2A-Neo. 5 μg lentiviral constructs were co-transfected with viral packaging plasmids 3 μg psPAX2 and 3 μg pMD2.G into 293 T cells in 10 cm dishes for the lentiviral particle production. The viral supernatant was harvested at 48 h post-transfection and filtered through a 0.22 µm membrane. After applying the viral supernatant to PANC-1 cells with 10 μg/μl of polybrene , selection for puromycin and/or G418 resistance was initiated 48 hours after transfection. The selection media was changed every 3-4 days for several weeks, and clones of puromycin and/or G418 - resistant cells were isolated and expanded for further characterization. The stable cells were maintained with a complete culture medium with 2 μg/ml puromycin and/or 100 μg/ml G418.Lentiviral production was performed as described previously.The HEK293T cells cultured in 10 cm dishes were transiently transfected with an empty vector or Flag-FAM83A for 48 h. The cells were collected and washed with cold PBS and lysed in lysis buffer supplemented with protease and phosphatase inhibitors. The cell lysates were subjected to IP assay using the Flag antibody, and the immunoprecipitate was sent for LS-MS/MS analysis.The lysates of HEK293T cells were co-incubated with purified GST-tagged FAM83A with or without ATP addition. Then the products were sent to western blotting assays using an anti-phosphotyrosine antibody.The FAM83A promoter region which from 2000 bp upstream to 500 bp downstream of the transcription start site was cloned into the firefly luciferase reporter plasmid pGL3-Basic . The renilla luciferase vector was used as an internal control and was purchased from Promega . The primer sequences used for FAM83A promoter qRT-PCR primer sequences were (forward) CACACAGAGATATGTGTGTGTGTATA and (reverse) GCCAGTGAGCTGGGAACTG. The nucleotide sequences of all constructs were confirmed by DNA sequencing. The site mutations or depletions of the FAM83A promoter region luciferase reporter construction were generated using the overlap extension PCR assay or QuikChange II XL Site-Directed Mutagenesis Kit . The luminescence ratio of the experimental reporter (Firefly) to the control reporter (Renilla) was calculated. All experiments were performed in triplicate.Zebrafish and embryos were raised, staged, and maintained according to standard procedures. All zebrafish experiments were approved by the Hubei University of Technology Animal Care and Use Committee. Dechorionized 2 days post fertilization (dpf) zebrafish embryos were anesthetized with 0.003% tricaine , and then positioned on a 10 cm Petri dish coated with 3% agarose. Pancreatic cancer PANC-1 cell suspensions labeled with GFP were loaded into borosilicate glass capillary needles and the injections were performed using a pneumatic pico-pump and a manipulator . Approximately 500 cells were injected at ~60 μm above the ventral end of the duct of Cuvier (DoC), where the DoC opens into the heart. Then the zebrafish embryos were maintained at 33 °C and photographed under an Olympus FSX100 microscope.p value <0.05 were statistically significant. Differences between two groups were analyzed by independent sample t-tests and differences among multiple groups by one-way ANOVA; *represents P < 0.05, **represents P < 0.01, and ***represents P < 0.001.All experiments were performed independently at least three times. All statistical analyses were performed using GraphPad Prism 6.0 software . All data are presented as the mean ± SD (standard deviation) from triplicates. Differences with a Supplementary Material


Aspergillus isolates were isolated. Based on morphological characteristics and sequences of Internal Transcribed Spacer, β-tubulin and calmodulin, the isolates were identified as A. tamarii. Pathogenicity tests of the isolates, conducted using mycelial plugs with wounded and unwounded treatments, showed A. tamarii as the pathogen of banana fruit rot. Rot symptoms were highly severe on wounded banana fruits compared to unwounded fruits, and therefore, wounded banana fruits are more susceptible to A. tamarii infection. To the best of our knowledge, this is the first report of A. tamarii as a causal pathogen of banana fruit rot. This study indicated A. tamarii is one of postharvest rot pathogens of banana.Banana fruit rot is a common postharvest disease of the banana fruit. The appearance of rot symptoms on the surface of the fruits reduces the quality and marketability of banana. From rot lesions on banana fruits, three  Aspergillus tamarii (A. tamarii) from banana fruit rot was identified molecularly based on Internal Transcribed Spacer, β-tubulin and calmodulin sequences.A. tamarii is a pathogen of banana fruit rot, causing highly severe infection on wounded banana fruit.A. tamarii has the potential to severely infect bananas, which affects their quality and marketability.Combined effect of fruit rot pathogens including Musa spp.) is one of the most important fruit crops in Malaysia with planted area of 30,455 ha and production at 330,957 tonne metrics in 2018  and A. flavus followed by A. parasiticus, A. ochraceus, A. carbonarius and A. alliaceus. Aspergillus species infected crops during pre-harvest, after harvest, during processing, handling, storage and marketing  were recovered from banana fruits showing symptoms of fruit rot. The banana fruits were purchased from a fruit stall in Pulau Pinang, Malaysia. Thus, the objectives of the present study were to identify the Aspergillus isolates using morphological and molecular characteristics, and to conduct pathogenicity test to determine whether the isolates are the causal pathogen of banana fruit rot.In the present study, three isolates of Aspergillus isolates MF9 and MF24 were isolated from the banana Mas cultivar, and BPMF17, from Berangan cultivar. The isolates were identified based on colony appearance on malt extract agar (MEA), Czapek yeast autolysate agar at 25°C (CYA25), and Czapek yeast autolysate agar at 37°C (CYA37) as well as the characteristics of conidiophore and conidia , 0.5 μL of 10 mM dNTP mix (Promega), 4.0 μL of 5 mM primers and 0.5 μL of genomic DNA. PCR was performed using a thermocycler (Bio-Rad MyCycler) with initial denaturation at 95°C for 5 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 58°C for ITS region and 56°C for both β-tubulin and calmodulin genes, extension at 72°C for 1 min and final extension at 72°C for 5 min. The products from the PCR were sent to a service provider for DNA sequencing.Molecular identification was performed based on the internal transcribed spacer (ITS) regions, β-tubulin and calmodulin genes. For DNA extraction, mycelia were grown in malt extract broth. Mycelia were harvested after two days of incubation, freeze-dried for 48 h, and subsequently ground to a fine powder in the presence of liquid nitrogen. DNA was extracted using an Invisorb Spin Plant Mini Kit , following the manufacturer’s instructions. The primers used were based on A. tamarii reference strains  were included in the analysis. Several Aspergillus species section Flavi were also included in the phylogenetic analysis, namely A. pseudotamarii (CBS 766.97), A. nomius (NRRL 13137), A. oryzae (CBS 817.72), A. flavus (CBS 501.65), A austwickii (CBS 135406), A. aflatoxiformans (CBS 121.62) and A. parasiticus (CBS 100926). Aspergillus fumigatus served as the outgroup (CBS 511.64). The species chosen was based on a list of Aspergillus species section Flavi by The consensus sequences of ITS, β-tubulin, and calmodulin were compared with sequences in GenBank using Basic Local Alignment Search Tool (BLAST) search. Phylogenetic analysis was then performed using Molecular Evolution Genetic Analysis (MEGA7) software , β-tubulin , and calmodulin . Phylogenetic analysis showed that the three isolates  recovered from banana rot were grouped with the three A. tamarii references strains   . The othMas cultivar. The mycelial plug was taken from the edge of an actively growing mycelium from a 4-day-old culture using a cork borer (0.5 cm). The mycelial plug was placed onto the surface of wounded and unwounded healthy banana fruits. Control banana was inoculated with PDA plug.The pathogenicity test was conducted using the mycelial plug with wounded and unwounded treatments on For the wounded treatment, the surfaces of banana fruits were cut to approximately 0.5 cm in diameter using a sterile scalpel. The mycelial plugs (0.5 cm) were inoculated onto healthy fruit cut surfaces. The inoculated fruits were incubated at room temperature for 7–10 days and the development of fruit rot was observed daily. Three replicates were prepared for each treatment on the same banana fruit and the pathogenicity test was repeated three times.The development of fruit rot was determined according to the severity scale based on the area of rotting tissues and severity of the rotting symptom  modifiedReisolation of the fungus was performed to fulfill Koch’s postulates. Reisolation of the fungal isolates from the rot lesion showed the presence of the same fungal isolates inoculated on the banana fruits. Morphological identification showed that the re-isolated fungal isolates had the same characteristics as the original isolates isolated from the rot lesion of infected banana.Aspergillus tamarii is a type of spoilage fungi that commonly contaminates different types of nuts, and is occasionally isolated from other food products, including wheat, coffee, spices, dried meat, and fish products (A. tamarii has the ability to cause rot in banana fruits after harvest. Moreover, A. tamarii has been reported as a postharvest pathogen of guava (A. tamarii, two species of Aspergillus, A. niger and A. flavus have been reported to be associated with banana fruit rot (Aspergillus in rot lesion including A. flavus, A. fumigatus, A. niger and A. terreus accelerate banana fruit rot.products . Thus, aruit rot . AccordiA. tamarii was the causal pathogen of banana rot, with more severe infection on wounded fruits. In general, wounds on the surface of fruit crops and other crops are caused by abrasion or cuts during harvesting and handling operations, damage during storage, and poor hygiene during transportation and marketing (A. tamarii infection. The wound and presence of postharvest pathogens as well as suitable environmental factors may provide conducive conditions for pathogen infection and disease development. On unwounded fruits, the infection was not as severe as that in wounded fruits. These results suggest that A. tamarii may produce extracellular enzymes such as xylanases (The results of the present study indicated that arketing . Therefoylanases  to facilA. tamarii, prevention of wound or injury during harvest and postharvest handling is essential. Wounds are caused by bruises and abrasions during harvesting, insect feeding in the field as well as by chilling and heat injuries during storage. To minimise wounds on banana fruits, several precautions can be taken such as careful harvesting and handling of the produce, controlling pest population in the field, proper packaging and stored at appropriate temperature (As wounded banana fruits are more susceptible to postharvest pathogens including perature .A. tamarii as a causal pathogen of banana fruit rot. Banana fruit rot caused by A. tamarii may not solely cause serious damage, compared to the crown rot and anthracnose pathogens of banana. Crown rot and anthracnose are major postharvest disease of fruit crops including banana. Nevertheless, the combined effect of the fruit rot pathogens has the potential to severely infect bananas, which affects their quality and marketability.To the best of our knowledge, this is the first report of


Mucuna Pruriens (MP). According to clinical research, the MP extract containing L-DOPA for the treatment of Parkinson’s disease could reduce side effects more than the synthetic one. Unfortunately, MP extracts can be easily degraded. Changes in physical and chemical properties such as the appearance  and the reduction of L-DOPA content in the extract were commonly observed. Therefore, it is necessary to develop an extraction procedure to stabilize the extract of L-DOPA. This study attempted to enhance the extraction process by modifying the traditional acidification approach using hydrochloric acid, citric acid, or ascorbic acid. According to the stability test results, using Phyllanthus emblica water (PEW) as a solvent improved the preservative properties more than other solvents. The color of the PEW-MP powder changed slightly after 12 months of accelerated storage, but the amount of L-DOPA remained the highest (73.55%). Moreover, L-DOPA was only detected in MP and PEW-MP, but not PEW alone (the HPTLC chromatogram at Rf 0.48 and the HPLC chromatogram at Rt 6.0 min). The chemical profiles of PEW and L-DOPA observed in the chromatograms indicated that they are independently separated. As a result, they can be applied to a quality control process. Therefore, PEW was proven to be a powerful solvent for L-DOPA herbal extract that could be readily used as a raw material for herbal products.Levodopa (L-DOPA) is an essential drug for the treatment of Parkinson’s disease. Currently, L-DOPA can be produced by chemical synthesis and can also be found naturally in many herbs, especially  Canavalia ensiformis, Cassia hirsute, and Mucuna pruriens (MP) [L-DOPA is a first-line drug that is used to treat a symptom of Parkinson’s disease (PD). It is produced by chemical synthesis . Howeverens (MP) . Synthetens (MP) . It was ens (MP) . Severalens (MP)  and reduens (MP) .The powder of MP seed has been clinically studied for PD treatment and was found to be more effective and had fewer side effects than synthetic drugs . InteresPhyllanthus emblica is of interest. P. emblica or Amla fruit is a medicinal plant, and its fruit has high vitamin C and other acids that make it taste sour (P. emblica (PE) extracts, which may aid in the prevention of oxidative stress in the brain, which is a contributing factor to Parkinson’s disease and reduce the cause of Alzheimer’s disease [Although MP extract seems to have therapeutic benefits, extraction has some limitations. Several studies are being conducted to extract L-DOPA as efficiently as possible. DOPA was extracted from MP seeds, but it was discovered that L-DOPA was easily degraded by various extraction methods such as soaking, autoclaving, roasting, sprouting, and alkaline fermentation . Among aste sour . Further disease ,17. In a disease ,19. More disease .This stuThe extraction of MP seeds was performed to compare the effect of PEW and other conventional acid solvents. L-DOPA content was one of the main parameters used to compare the efficiency of the extraction method in this study. To quantify the amount of L-DOPA, the HPLC technique was validated and performed.w/w) due to its readily degradable solution. This is consistent with the report of Ariahu et al., (2011), who studied the kinetics of ASA loss in hot water. It was found that the percentage remain tended to decrease when heating in the range of 60–90 °C and long-time exposure effect in ascorbic content as well [w/w without heating in the extraction process [The sample extract with PEW has been proven to have extraction efficiency similar to that of hydrochloric acid (HCA). The result showed that at 2% PEW and HCA, a conventional acid solution, gave a similar yield in both extractive value and L-DOPA content. When increasing the concentration of PEW in extraction contributes to increasing the percentage of productivity. On the other hand, the amount of L-DOPA decreased because PEW extract had a very high concentration, while the proportion of dried extract consisted mainly of PEW. In comparison, the use of ascorbic acid (ASA) in the test resulted in the lowest amount of L-DOPA  Ness and Phyllantus niruri L. used andrographolide as a chemical marker for HPLC quantitative analysis and method validation [Another method to ensure the presence of L-DOPA in PEW was HPLC. This method has been known to be a technique used in an assay of pharmacopeia. The development of a system to detect an active substance is necessary for the quality control of drug products. The HPLC system used in this study successfully separated and detected L-DOPA in the PEW-MP extract . In othelidation . In anotlidation . HPLC isM. pruriens var. pruriens; MPT) and Indian MP  varieties, to prove that PEW could be used for extraction regardless of the plant variety. Both varieties differ in their character but contain L-DOPA. MPT has long and stingy hair on its seed pod, causing skin irritation when touching it [w/w, which is comparable to MPI (3–9% w/w) [In the production process, raw materials may not be available as needed due to various factors. Plants similar in species or other plants can be substituted. The purpose of this experiment was to compare two main sources of L-DOPA available in Thailand, Thai MP  ,30.w/w in MPT and MPI, respectively  and 8.11 (pKa2) [The pH value is another indicator of the stability of the extract because L-DOPA is known to be stable under acidic conditions. In general, the extract must maintain optimal conditions with a specific pH value. For L-DOPA, the optimal pH to maintain the condition is in the range of 2.0–4.0 . This ex1 (pKa2) ,38. More1 (pKa2) . TherefoThe pH of each solution slightly increased after extraction between 4.20 and 4.81. After storage and dissolving in water to test the pH, each sample changed only slightly, even at 6 and 12 months, without significant difference . In thisw/w followed by PEW (7.72% w/w), CTA (6.74% w/w), and ASA (3.88% w/w). However, the least amount of L-DOPA obtained in the ASA extract may be due to the fact that the preparation of ASA in solution was prone to degradation faster and higher than other solvents during the extraction process with MP seed powder [L-DOPA in the extract can be degraded over time. In the accelerated condition, the L-DOPA content in the extract powder determined by HPLC  was found powder . TherefoThe stability of L-DOPA was observed after being kept for 6 and 12 months in the accelerated condition. The accelerated condition at 40 °C ± 2 °C/75% RH ± 5% RH and the minimum time period covered by data at submission 6 months used in this study refers to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) . All samHowever, since accelerated storage for the worst-case study and extreme environmental conditions can lead to a rapid decrease in % L-DOPA, the PEW-containing extracts were compared with different storage conditions: freezer (−4 ± 2 °C), ambient of Bangkok Thailand (28 ± 2 °C) and accelerated (40 ± 2 °C), for information on extending the shelf life of L-DOPA, extracted with PEW . It was Alpinia galanga extracts in a 7: 3 ratio, and showed their protective effect on H2O2-induced oxidative stress in endothelial cells and its inhibitory effect on lipid peroxidation [From the stability test results, PEW showed a good trend and contributed to the preservation of the physicochemical values of the extracts, probably because PEW contains important bioactive compounds that help reduce oxidation reactions. PEW has many benefit case studies. Chansriniyom et al., (2018) combined PEW and xidation . PEW wasxidation . In cosmxidation . PEW wasxidation .With many benefits of PEW, this study added to the evidence that PEW could be a solvent to extract other herbs. It provides good extraction results and maintains the quality of the MP extract. Furthermore, it was found that PEW extract alone contributed to the inhibition of the causative activity of Parkinson’s disease in the experimental rat . Thus, iThe MP seeds were purchased from a local market in Bangkok, Thailand, and collected at room temperature. Then, they were heated in a household microwave oven (TOSHIBA Microwave 700 W series MWP-MM20P(WH)) for 10 min until their seed coat was broken. The seeds were then finely ground and used for testing.Phyllanthus emblica extract was purchased from 100% organic Gourmazia from India. Citric acid 1-hydrate 99.5% was purchased from Elago Enterprises Pty Ltd. . L-DOPA reference standard , >99.9% from USP . All solvents were analytical grade, including ethyl formate 98% and toluene 99.5% , formic acid 98% , and methanol 99.9% .Ascorbic acid (99.5% assay) was purchased from Chemipan Corporation Co., Ltd. . P. emblica water (PEW). The abbreviation is defined in 15 g of seed powder was extracted with 150 mL of different acidified agents (pH = 3) that were hydrochloric acid, ascorbic acid, citric acid, and v/v/v/v), and the running distance was 85 mm. The NP-PEG reagent was then used for derivatization. The plates were documented for the chemical profile under UV 366 nm by the Visualizer II using Vision CATS software .Chemical Profile Fingerprint Identification test following a standard operating protocol of the International Association for the Advancement of High-Performance Thin Layer Chromatography . Then, tv/v) formic acid. The standard L-DOPA was 200–1000 ng/μL. The samples were then analyzed by HPLC  using a column . Mobile phase A was assigned to 0.1% (v/v) formic acid in the water, and phase B was methanol by gradient programmed separation as follows: 1% B at 0 min to 10 min and 1–4% B from 10 to 20 min with a flow rate of 0.5 mL/min. The detection was measured at a wavelength of 282 nm. That method validation to confirm the effectiveness of the method followed based on ICH guidelines [−1, respectively.The method for quantitative L-DOPA by HPLC was modified from the report of Duan et al., (2021) . The samidelines . The calThe dried extract of MP seeds from the optimal extraction process was placed in a zip-lock bag and kept in accelerated condition at 40 °C ± 2 °C and relative humidity of 75% ± 5%. All samples were observed at 0, 6, and 12 months. Physical and chemical properties, including color, water pH dissolution, and L-DOPA amount, were recorded. The color quality of the extracts was determined by comparing the color charts of the color grab application range between light brown to a fine brown powder and was reported in the CIE L * a * b value system. The acidity of the extract was measured as the pH value . Some of the extracts were stored in a −4 °C freezer for analysis of L-DOPA content compared to accelerated.p < 0.05 was significant.Analysis of variance one-way ANOVA was used. The significance of the differences was selected from Duncan’s multiple range test comparison tests. In the data, Mucuna pruriens seed with Phyllanthus emblica water yields good results in both physical and chemical properties. The color of the extract changed slightly, its pH was stable, and PEW did not react with L-DOPA. Moreover, there was a higher percentage of L-DOPA content compared to the control group, which is a hydrochloric acid solution. Therefore, our study proved that the improved extraction method using PEW could be used as a promising method to extract L-DOPA from MP seeds that could improve stability, avoid chemical solvents, and yet would also combine the benefit of PEW for the treatment of Parkinson’s disease. Finally, the extract could serve directly as a raw material in the production of herb products at the industrial level.The extraction of L-DOPA from A petty patent was drafted and submitted to the Department of Intellectual Property, Thailand.


Caenorhabditis elegans (C. elegans) is a small, free-living, non-parasitic nematode. ~38% of the genome of C. elegans has predicted orthologues in the human genome, making it a valuable tool to study the function of human counterparts and human diseases. To date, 8 unconventional myosin genes have been identified in the nematode, from 6 different classes with high homology to human paralogues. The hum-1 and hum-5  genes encode myosin of class I, hum-2 of class V, hum-3 and hum-8 of class VI, hum-6 of class VII and hum-7 of class IX. The hum-4 gene encodes a high molecular mass myosin (307 kDa) that is one of the most highly divergent myosins and is a member of class XII. Mutations in many of the human orthologues are lethal, indicating their essential properties. However, a functional characterisation for many of these genes in C. elegans has not yet been performed. This article reviews the current knowledge of unconventional myosin genes in C. elegans and explores the potential use of the nematode to study the function and regulation of myosin motors to provide valuable insights into their role in diseases.Unconventional myosins are a superfamily of actin-based motor proteins that perform a number of roles in fundamental cellular processes, including (but not limited to) intracellular trafficking, cell motility, endocytosis, exocytosis and cytokinesis. 40 myosins genes have been identified in humans, which belong to different 12 classes based on their domain structure and organisation. These genes are widely expressed in different tissues, and mutations leading to loss of function are associated with a wide variety of pathologies while over-expression often results in cancer.  Myosins are ubiquitously expressed, multifunctional motor proteins that converts the energy generated from the hydrolysis of ATP to facilitate movement. They are involved in a wide range of cellular processes including cargo transport, formation of actin-based projections at the plasma membrane, steady state distribution of membrane compartments, cytokinesis, and muscle contraction. Myosins share a common domain organisation comprised of head, neck and tail regions . The myoSaccharomyces cerevisiae and Schizosaccharomyces pombe both encode genes for myosins from class I, II and V [Drosophila melanogaster and Caenorhabditis elegans express in addition to the basic myosin repertoire of I, II and V also myosins from class VI and VII [The functional diversity of myosin motors is reflected in the large number of genes within this superfamily. At least 35 classes of myosins have been identified in eukaryotes, 12 of which are found in the human genome . Class III and V ,14,15,16 and VII ,18,19. A and VII . Many ofC. elegans is a microscopic soil nematode that is now established as a powerful genetic model organism [C. elegans genome has predicted orthologues in the human genome. To date, eight unconventional myosin genes from six different classes with high homology to human paralogues have been identified in the nematode [hum-1 and hum-5  genes encode members of myosins of class I, hum-2 of class V, hum-3 and hum-8 of class VI, hum-6 of class VII and hum-7 of class IX. The hum-4 gene encodes a high molecular mass myosin (307 kDa) of class XII one of the most highly divergent myosins. Mutations or deletions of most of these myosins in humans are lethal, indicating their essential properties [C. elegans and discuss the potential use of this model organism to study the role of myosin motors in human disease.organism , being sorganism . This isnematode  ) (Caenorhabditis Genetics Center), however, details of associated phenotypes have not yet been described. HUM-5, on the other hand, is expressed in an array of tissues that includes intestinal tissue, body wall muscle, and sensory neurons [hum-5(ok1885)), but no phenotypic analyses have yet been published on the strain.HUM-1 is expressed in various tissues, including within the mechanosensory posterior ventral process and outer labial lateral neurons and the germline ,25. Its germline ,50. MYO1germline ,51,52. A neurons ,27. HUM- neurons . Its mam neurons ,55,56. AThe human orthologues of HUM-1 and HUM-5, MYO1E and MYO1D, respectively, have been implicated in a variety of diseases. A missense mutation in MYO1E causes focal segmental glomerulosclerosis and a homozygous truncation mutation in the motor domain of MYO1E has been linked to nephrotic syndrome, both childhood—onset forms of kidney disease ,58. Incrhum-2 gene is an orthologue of human MYO5A, a class V myosin [Drosophila contain a single MYO5 gene called didum, while the yeasts Saccharomyces cerevesiae and Schizosaccharomyces pombe both have two MYO5 genes, MYO2 and MYO4, and myo51 and myo52, respectively [Drosophila didum, and 30–33% with the yeast myosins of class V. HUM-2 contains a very long neck region with six IQ motifs, which is common to all class V myosins, and allows a large step size of 36 nm, the pitch of the helical actin filament. The tail region contains three coiled-coil domains, important for dimerization, and a globular tail domain, which has been shown to enable cargo binding in other members of class V myosins ) have been generated (Caenorhabditis Genetics Center), however, no detailed analysis has been performed so far. Mutations in human MYO5A give rise to the rare, autosomal recessive Griscelli Type 1 disease, characterised by neurological impairment [C. elegans to explore basic mechanistic pathways that might be underpin complex human neurological diseases.Myosins of class V form a dimer and display processive movement in a hand-over-hand fashion. They are kinetically adapted to move processively, because of their large working stroke/step size and high duty ratio, which is the fraction of the time the motor is attached to an actin filament during one complete cycle ,63. MYO5pairment . Future C. elegans possesses two genes for myosins of class VI, hum-3 (spe-15) and hum-8, which are orthologues of the human MYO6 gene. In contrast, humans express a single MYO6 gene that can undergo alternative splicing to produce four different isoforms [jaguar, which shares 44% and 39% sequence identities with HUM-3 and HUM-8, respectively. The sequence differences are scattered throughout the protein, but many motifs and regulatory elements are conserved, such as the GESGAGKT sequence of the ATP-binding P-loop, and the phosphorylatable TEDS-rule site (Thr405 in human MYO6) in the motor domain. In the tail, the RRL motif is conserved, whilst the WWY motif is altered in both Hum-3 (MWY) and Hum-8 (MWF). Comparing the sequences of HUM-3 and HUM-8 yields a 63% sequence identity between them, which is enough divergence to suggest functional variation. Yeasts do not possess orthologues to MYO6 in their genome.Myosins of class VI are unique amongst myosin motors in that they move towards the minus end of actin filaments . C. elegisoforms . Human MC. elegans MYO6s also have sequences corresponding to the two unique inserts characteristic of myosins of this class: a 53-residue insertion (reverse gear or insert-2) between its converter and light chain-binding domain that is solely responsible for the reverse directionality of MYO6, and a second shorter insert (insert-1) near the nucleotide-binding pocket which has been shown to modulate nucleotide binding [HUM-3 and HUM-8 follow a domain organisation typical of class VI myosins, consisting of a motor domain followed by a neck region containing a single IQ motif, and a tail that terminates in a cargo-binding domain (CBD) . Both C. binding ,69. InteMammalian MYO6 is monomeric, has a large working stroke (~18 nm) and a high duty ratio, and is weakly processive as a dimer ,71,72,73C. elegans germline [Human MYO6 is widely expressed, including in neuronal tissues, the inner ear hair cells of the cochlea, and the intestine. MYO6 undergoes alternative splicing of two inserts in its CBD: the small insert  and the large insert . Human MYO6 has four splice isoforms , which have differential interaction networks and binding partners and are expressed in different cell types and tissues . The pregermline ,31,32. Hgermline ,25. InteC. elegans, as HUM-3 knockout worms are almost completely self-sterile and display gross cytological defects in the morphology of budding spermatids and the residual body. The spermatids typically fail to activate to form ameboid spermatozoa and are characterised by the improper partitioning of Golgi-derived fibrous body membrane organelles (FB-Mos) and mitochondria [Very little is known about the functions, biochemical and biophysical properties of either HUM-3 and HUM-8. The disruption of HUM-8 using RNA interference causes cytokinesis defects within the germline, which coincides with the role of human MYO6 in membrane delivery during mammalian cytokinesis ,83. HUM-chondria ,85. Detachondria . Intereschondria ,88,89.Mutations in human MYO6 have been linked to several diseases such as hearing loss and familial hypertrophic cardiomyopathy ,90. Furthum-4 gene encodes a myosin with a very large (~300 kDa) heavy chain that is unique to C.elegans, and is the founding member of class XII [The lass XII . The motlass XII . Whilst HUM-4 contains a unique 200 amino acid N-terminal extension, the function of which is unknown and with no shared homology to known domains. Another unique feature of this class of myosin is the position of the putative coiled-coil region at the carboxyl-terminal region of the tail. It has yet to be elucidated whether HUM-4 does form a dimer, but recent data suggests that human MYO15A is kinetically adapted for processive motility when oligomerised .C. elegans, HUM-4 is expressed in sensory neurons [In humans, myosins of class XV were first discovered associated with congenital, recessive nonsyndromic deafness  and near neurons . These n neurons ,98.The 266 kDa HUM-6 protein is a myosin of class VII, and is an orthologue of human MYO7a, sharing a 53% sequence identity. In Drosophila a myosin of class VII called crinkled is expressed, which is 62% identical to human MYO7a . The HUMC. elegans the orthologue, HUM-6 is expressed in a number of different cell types, including the intestine which contains polarised cellular projections, suggesting an analogous function of HUM-6 to myo7a and its function in maintaining cellular projections [In humans, MYO7a is widely expressed in the cochlea, retina, testis, lung and kidney ,101,102,jections . HUM-6 ijections ,34.C. elegans to model human diseases.Mutations in human MYO7a cause congenital deafness and blindness, clinically known as human Usher syndrome 1B  and non-hum-7 gene encodes a myosin of class IX [myo9a and myo9b while no MYO9 orthologue has been identified in Drosophila. HUM-7 is 34% identical to MYO9A and 30% to MYO9B. The domain organisation of myosins of class IX has several distinguishing features, including a Ras-association domain at the N-terminus of the protein , the ability of individual motor “heads” to operate independently of each other, and their rate-limiting phosphate release. Myosin II is not just found within muscle cells—non-muscle myosin II is ubiquitously expressed in various mammalian cell types. They have been implicated in a wide range of biological processes such as cell adhesion, cell migration, cell division and phagocytosis ,121 to nopathies , the scoC. elegans have four muscle myosin genes, termed mhc-a, mhc-b, mhc-c and mhc-d. They also have three non-muscle myosin genes , and two further genes myo-5 and myo-6 which have yet to be characterised [C. elegans have both striated and non-striated muscles. Non-striated muscles include 20 pharyngeal muscle cells, two stomatointestinal muscles, one anal depressor muscle, one anal sphincter muscle, eight vulval muscles, eight uterine muscles, and 10 contractile gonadal sheath cells [Humans have 14 myosin class II genes, three of which are non-muscle myosin II and one which is a smooth-muscle specific isoform. cterised . C. elegth cells . They alth cells ,123,124.C. elegans muscle components and their human counterparts has been utilised in numerous studies of myosin folding, particularly of the chaperone, UNC-45. UNC-45 (uncoordinated-45) was originally identified as a result of mutations causing structural disruption of thick filaments in body wall muscle in C. elegans [C. elegans, whilst missense mutations result in disorganized and reduced numbers of myosin-containing thick filaments giving rise to a slow-moving, or uncoordinated, phenotype of adult worms [C. elegans UNC-45 protein [C. elegans as model organism to garner insights into a long-standing question in myosin research, namely how myosin is incorporated into thick filaments, highlights some of the landmark discoveries the nematode can provide in the field.The high homology between  elegans ,125,126.lt worms ,128. UNClt worms ,131,132. protein , a key sC. elegans have proven a useful model for studying muscle folding, they have also been proposed to be suitable as a model system for a number of myopathies. MHC-B shares a 54% sequence homology to human beta-cardiac myosin, a protein which counts for ~30% of known missense mutations that give rise to dilated and hypertrophic cardiomyopathy [C. elegans does limit a full-systemic model of cardiac disease. Despite this, it has been proposed that the nematode can be used to model protein–protein interactions in human cardiomyocytes because of the functional interactions in C. elegans body wall muscle [C. elegans myosins have also been used to study myosin storage myopathy [Whilst myopathy . The lacl muscle . The neel muscle . Mutatiomyopathy  and congmyopathy , whilst myopathy .C. elegans myosin, as has been discussed in this review. Moreover, C. elegans is a well-established organism for the study of aging and longevity. The role of unconventional myosin motors in aging has yet to be explored. The conservation of endogenous locations and functions highlights the benefit of studying these unconventional myosin motors in such a well-established model organism.Myosins are fundamentally important for directed movement on actin filaments, a process which is required for a wide range of cellular activities. The human genome has a repertoire of 12 myosin classes, all adapted to perform specific functions within the cell types they are expressed. Many of these myosins have orthologues to C. elegans is a well-established in vivo model system that has emerged as an extremely valuable resource in pharmacological drug discovery. To make potential use of C. elegans in a pharmacological screen for modulators of myosin activity, requires a more complete understanding of the cellular, biochemical, and biophysical characteristics of the C. elegans myosin homologues, which can form the basis of future work.The role of myosin motors in human disease makes it an attractive clinical drug target, fuelling the need to initiate further studies on this family of motor proteins.


Intestinal fibrosis and stricture formation is an aggressive complication of Crohns disease (CD), linked to increased morbidity and costs. The present study investigates the contribution of Wingless-Int-1 (Wnt) signalling to intestinal fibrogenesis, considers potential cross-talk between Wnt and transforming growth factor β1 (TGFβ) signalling pathways, and assesses the therapeutic potential of small-molecule Wnt inhibitors.n=6 pairs). Functional interactions between Wnt activation, TGFβ signalling, and type I collagen (Collagen-I) expression were explored in CCD-18Co cells and primary CD myofibroblast cultures established from surgical resection specimens (n=16) using small-molecule Wnt inhibitors and molecular techniques, including siRNA-mediated gene knockdown, immunofluorescence (IF), Wnt gene expression arrays, and western blotting. Fibrotic SCD tissue was marked by an increase in β-catenin-positive cells. In vitro, activation of Wnt-β-catenin signalling increased Collagen-I expression in CCD-18Co cells. Conversely, ICG-001, an inhibitor of β-catenin signalling, reduced Collagen-I expression in cell lines and primary CD myofibroblasts. TGFβ increased β-catenin protein levels but did not activate canonical Wnt signalling. Rather, TGFβ up-regulated WNT5B, a noncanonical Wnt ligand, and the Wnt receptor FZD8, which contributed directly to the up-regulation of Collagen-I through a β-catenin-independent mechanism. Treatment of CCD-18Co fibroblasts and patient-derived myofibroblasts with the FZD8 inhibitor 3235-0367 reduced extracellular matrix (ECM) expression. Our data highlight small-molecule Wnt inhibitors of both canonical and noncanonical Wnt signalling, as potential antifibrotic drugs to treat SCD intestinal fibrosis. They also highlight the importance of the cross-talk between Wnt and TGFβ signalling pathways in CD intestinal fibrosis.β-catenin expression was explored by immunohistochemistry (IHC) in formalin-fixed paraffin embedded (FFPE) tissue from patient-matched nonstrictured (NSCD) and strictured (SCD) intestine ( Intestinal fibrosis with stricture formation and associated bowel obstruction is a common and serious complication of Crohns disease (CD) . StrictuIntestinal fibrosis is part of a complex process that involves all layers of the intestine, multiple cell-types and signalling pathways, as well as interactions between genetic susceptibility loci and environmental triggers. Inflammation is an important initial driver of fibrosis, but there is growing evidence that overtime fibrosis can progress independently from inflammation. A key feature of intestinal fibrosis is an increase in activated myofibroblasts, which are the main source of increased production of extracellular matrix (ECM) proteins, e.g. type I collagen (Collagen-I). In CD patients, fibroblast activation has been linked to increased expression of transforming growth factor β1 (TGFβ) and activation of SMAD signalling ,7. HowevCross-talk between TGFβ and Wingless-Int-1 (Wnt) signalling has been reported widely . Both paThe mechanisms by which Wnt signalling promotes fibrosis are complex, cell-type, and context-dependent; and both canonical and noncanonical pathways have been implicated. For example, in many organs, fibrosis is often associated with increased nuclear β-catenin , and accIn the intestine, Wnt signalling is required to maintain epithelial homeostasis, promote Paneth cell differentiation, and maintain α-defensin production . StromalIn the present study, we confirm reports of increased β-catenin in SCD and demonstrate a role of β-catenin in the regulation of Collagen-I in cell lines and primary CD myofibroblasts. We further explore cross-talk between the TGFβ and Wnt signalling in intestinal fibroblasts and highlight a novel role for FZD8 in SCD. Moreover, we evaluate the ability of the small-molecule Wnt signalling inhibitors to attenuate TGFβ-mediated intestinal fibrosis in models of intestinal fibrosis and primary CD fibroblast cultures. Our data provide important preclinical evidence for the potential use of small-molecule inhibitors of Wnt signalling as a potential novel therapy for SCD.The study was conducted in accordance with the latest version of the declaration of Helsinki. Appropriate local Ethics Committee approval was obtained from the London—City Road & Hampstead Research Ethics Committee (15/LO/2127) and Informed consent were obtained prior to patient recruitment.n=6, pairs, Supplementary Table S1) were identified from pathology reports and the relative levels of inflammation and fibrosis assessed in detail by a pathologist (R.F.), as described previously [muscularis propria (MP) calculated. For IHC image analysis, six paired of SCD and NSCD tissues (n=6) were analysed for β-catenin. For quantification of the percentage of β-catenin cells, six fields of view per slide, three from the mucosa and three from MP, were selected. β-catenin-positive cells were then manually scored in a blinded fashion using the ImageJ point picker plugin. The numbers of β-catenin-positive cells were expressed as a percentage of the total number of cells in that field. In the mucosa, only stromal cells were scored, and morphologically identifiable epithelial and endothelial cells or nerve ganglions were excluded. Similarly, endothelial and nerve cells were also excluded from the scoring the MP. One NSCD block did not contain tissue from the MP; therefore, the paired analysis for the MP related to five SCD and NSCD paired tissues (n=5). Typically, 100–300 cells were scored per field of view.Formalin-fixed paraffin embedded (FFPE) surgical resection tissue from the ileum of CD patients with a documented stricture was obtained from the Barts Health NHS Trust histopathology archive. From each CD patient, SCD and nonstrictured CD (NSCD) FFPE blocks  were cultured according to guidelines. For all cell treatments, cells were serum starved overnight prior to 48 h treatment with 10 µM ICG-001 dissolved in DMSO, or 100 nM porcupine inhibitor Wnt-C59 (Selleckchem S7037), or 10 μM FZD8 inhibitor 3235-0367 , alone or in combination with 10 ng/mL-recombinant human TGFβ1 (R&D Systems). For FZD8 siRNA-mediated gene knockdowns in 96-well plates, cells were transfected with either a siRNA-targeted against FZD8  or a nontargeted control (SC-37007) using DharmaFECT 2 reagent (T-2002-02). Transfections were performed according to the Dharmacon™ DharmaFECT™ 1–4 transfection protocol, using 0.1 μL of DharmaFECT 2 per reaction. For protein analysis, siRNA protocols were scaled-up proportionally.5 cells/mL) were plated on top, and 1.5 mL complete medium was added to the well. Medium was changed the following day and parallel gels were stained with Alexa Fluor anti-F-actin dye  and Hoechst 33342  and imaged using the IN Cell 2200 microscope  to ensure viability and homogenous cell distribution in the gel. Drug treatments were administered on day 3 for 48 h before media were collected for ELISA assays and gel sizes were calculated from light microscopy images taken at 0 and 48 h post-treatment using ImageJ software as a proxy for fibroblast remodelling capacity. Results are from four independent experiments (n=4).3D organotypic models were performed in 12-well plates with Transwell® polycarbonate-coated inserts . Semisolid Collagen gels containing CCD-18Co cells were produced by mixing 70% (v:v) rat tail Collagen-I , and 10% (v:v) 10× DMEM  on ice. pH was neutralised using 0.5 M NaOH before being mixed with 10% (v:v) FBS and 10% (v:v) CCD-18Co cells (15 000 cells per gel). A total volume of 0.4 mL was pipetted directly onto the inserts and left to polymerise for 1 h at 37°C, before 0.5 mL of a Caco-2 cell suspension . Results in SCD cultures were compared and contrasted with seven NSCD cultures .Tissue was collected in DMEM media from strictured small intestine following surgical resection. Resected tissue specimens were washed with Hanks’ Balanced Salt solution (HBSS) supplemented with 0.01% dithiothreitol (DTT) and then HBSS-EDTA (1 mM) for 10 min per wash under agitation at 37°C. For primary myofibroblasts cell cultures, the mucosa was mechanically isolated and only the mucosa was incubated with the collagenase solution. The solution was then filtered through a 100-μM cell strainer and the cell pellet washed twice in PBS. Cells were resuspended in 10 mL DMEM supplemented with 10% heat-inactivated FBS, 1% Penicillin–Streptomycin, 1% L-glutamine, 50 μg/mL gentamycin, and 1 μg/mL amphotericin and transferred to T25 flask. Cells in culture were split once they reached 80% confluency (passage 1) and subcultured once per week thereafter. All experiments were performed using cells between passages 1 and 3. Cell morphology and expression of Collagen-I were used to confirm that cells in culture were fibroblasts. In total, nine human SCD-derived primary CD myofibroblasts cultures were analysed for the present study (Cells were cultured in 96-well plates (2500 cells per well). Following treatment with ICG-001 and TGF-β1, cells were fixed , permeabilised , and blocked  prior to incubation overnight at 4°C with the primary antibody; Collagen-I  and/or β-catenin . Cells were subsequently washed (0.1% PBS-Tween) three times for 10 min before addition of a secondary antibody mix comprised of CellMask Deep Red  and DAPI (1 ng/mL), Alexa Fluor® 488-conjugated antirabbit antibody , and/or Alexa Fluor® 555-conjugated antimouse antibody  diluted in blocking reagent for 1.5 h. Cells were washed (0.1% PBS-Tween) three times for 10 min and stored in PBS (200 μL per well) prior to imaging on an IN Cell 2200 Analyser . Per well, nine images were taken using a 10× magnification lens. Using the IN Cell Developer software (V1.9), the images were then analysed and density levels of proteins of interest calculated. Mean protein density levels were calculated from all cells (defined using DAPI and cell mask) in all nine images; typically, several hundred cells are analysed per well. Mean density levels were background-corrected by subtracting the values from the secondary antibody-only control.n=3).Immunofluorescence (IF) analysis of CCD-18Co cells treated with 3235-0367 (C1) and Wnt-C59 (C59) was performed independently using a modified protocol. CCD18-Co cells were plated on coverslips at 20 000 cells per well in a 24-well plate. Following treatment, cells were fixed in 4% paraformaldehyde in PBS (Santa Cruz) for 10 min at RT. Permeabilisation was performed using 0.1% Triton X-100 in PBS for 10 min at RT, followed by blocking using 2% BSA, 50 mM glycine, 0.01% NaN3 in PBS for 1 h. Primary antibodies were incubated in blocking buffer overnight at 4°C , followed by incubation with secondary antibodies at 1:500 . Coverslips were mounted using Vectashield mounting medium with DAPI (Vector Labs). Stained cells were visualised using a Fluorescence microscope (Axioimager D1) with a 20× objective. Intensity analysis was performed using Fiji/ImageJ. For Collagen staining, 8–18 cells per condition and per experiment were selected for intensity measure. For nuclear β-Catenin, 27-120 nuclei were selected for measurement of β-catenin intensity in the nucleus, and for total β-catenin, whole images were thresholded for selection and measurement of the signal in total cell area. Corresponding background intensities were subtracted from all measurements and t-tests were performed on the normalised values for the different experiments (Protein was extracted from cells with RIPA buffer R0278) and protein quantification was determined by DC™ Protein Assay (5000111). Whole cell protein lysates (10–30 μg) were then separated on Bolt 4–12% Bis-Tris plus gels (NW04120BOX) by electrophoresis using the associated manufacturer’s buffers and transferred into polyvinylidene fluoride membrane (IPVH00010). Protein extracts from cells treated with 3235-0367 (C1) and Wnt-C59 (C59) were analysed independently and separated on SDS polyacrylamide gels using a Mini Protean System (BioRad) and transferred to nitrocellulose membranes for 10 min using Trans-Blot Turbo Transfer System . Immunoblotting conditions for each antibody are given below (Supplementary Table S3); the following conditions were optimised for each of the proteins studied: antibody concentration and incubation time, number of washes and time, type of block buffer and concentration, amount of protein, and ECL type. All primary antibodies were incubated overnight at 4°C. Membranes were then washed six times (5 min each) with TBS-T in a shaker (70 rpm). After washing, blots were incubated for 1 h in blocking buffer with HRP-conjugated secondary antibodies diluted and then membranes were developed using chemiluminescence. Image acquisition of the membranes in the chemidoc was performed using the signal accumulation mode. Blots were analysed using computer-assisted scanning densitometry software  or Fiji/ImageJ [78 and prP-values were assigned.CCD-18Co fibroblasts for the Wnt qPCR array were seeded in 6-well plates overnight in complete media prior to treatment with TGFβ1, as described above. Cells were collected for RNA extraction using the miRNeasy Kit (Qiagen). RNA Samples were reverse transcribed using a Reverse Transcriptase Kit , cDNA was diluted 1:1 with TaqMan Mastermix and 20 µL added to a 96-well plate containing dried TaqMan probes of 92 Wnt signalling and four endogenous control genes . Fold-changes were calculated by the 2⁁-ΔΔCT method. Data were corrected for multiple testing before RPLPO and/or ACTB) using the 2-ΔCt method. Analysis of gene expression in RNA isolated from patient-matched NSCD and SCD  intestine was performed using RNA isolated from patients as described previously [For all other gene expression studies, RNA from CCD-18Co cells were reverse transcribed using the ‘High Capacity RNA-to-cDNA Kit’ (Applied Biosystems). The cDNAs were then diluted 1:10 and incubated with Taqman gene expression probes and Universal Mastermix (Applied Biosystems) on a 7500 System RealTime PCR cycler (ABI). The resultant cycle threshold (Ct) values were normalised to the control genes (e.g. eviously .t-test.CCD-18Co passage number 34 (p34) were amplified and transduced with a lentivirus vector containing β-catenin-activated reporter (TOP), and its accompanying control reporter system (FOP) provided by Randall Moon , reportin=4), over four independent cell passages, unless stated otherwise in the figure legend. Differences between treatments were determined by a paired t-test to account for different cell passages. In general, data are presented as fold-changes and significant results relative to control are indicated by * symbol . Solid bars highlight comparisons between specific sets of treatments, and in these instances, * symbol is given above the bar. All qPCR data were log transformed prior to statistical analysis to standardise variance. The effects of treatments in primary myofibroblasts cultures were determined by paired t-test to account for difference between cultures.Experiments using CCD-18Co cells  were typically performed in quadruplicate . Endothelial cells associated with BV in the mucosa and MP also stained strongly for β-catenin  siRNA . Cells -catenin B,C, and -catenin D. KnockdC) siRNA E.TGFβ treatment also increased levels of β-catenin protein (cytoplasm and nucleus) and Collagen-I in intestinal fibroblasts A–E. HoweAPC gene silencing can promote Collagen-I expression, but that TGFβ treatment does not directly activate this pathway, despite increasing β-catenin protein levels. To investigate whether TGFβ may instead regulate noncanonical Wnt signalling, changes in the expression of Wnt pathway-related genes were investigated using a qPCR gene expression array . TGFβ also increased WNT5B expression, which encodes a noncanonical ligand that binds to the FZD8 receptor and suppressed expression of the canonical ligand WNT2B  were selected for analysis; WNT2B, which was the most down-regulated gene in TGFβ-treated CCD-18Co fibroblasts was also analysed. Relative to NSCD control tissue, SCD segments of intestine expressed increased levels of COL1A1, COL1A2, COL3A1, and COL5A2 mRNA , a small-molecule inhibitor of FZD8 ; Wnt-C59Blocking Wnt ligand production by treatment with C59 had similar effects to treatment with C1 on intracellular Collagen-I protein levels and fibronectin A,C,F,H. COL3A1 and ACTA2 expression and limited their up-regulation by TGFβ in CCD-18Co fibroblasts , which inhibits canonical Wnt signalling by disrupting the interaction of β-catenin with CBP . Like C1roblasts J. In comroblasts , ICG-001roblasts A,C,J conroblasts I. The ICnchanged J. Fibronnchanged B,E.ICG-001 did not affect β-catenin protein levels, the nuclear:cytoplasmic β-catenin ratio or CTNNB1 mRNA expression A,B,F,G,JOf the three inhibitors tested, C1 and ICG-001 were selected for further investigation in primary intestinal myofibroblast cultures isolated directly from surgically resected SCD tissue (Supplementary Table S2). Based on the studies of CCD-18Co cells and the pathologically important increase in Collagen-I deposition in strictured intestine, the primary endpoints for C1 and ICG-001 in primary CD cells were changes in Collagen-I protein levels determined by IF and ELISA. The impact of both drugs on β-catenin levels and cell number were also determined.For assessment of ICG-001, 16 cultures were screened from seven CD patients; seven cultures were established from NSCD tissue and nine cultures were established from SCD tissue. In some instances, multiple cultures were derived from a patient from phenotypically distinct segments of intestine, e.g. SCD and NSCD areas. Each culture was treated as an independent replicate. Protein analysis (IF and ELISA) confirmed the ability of ICG-001 to suppress baseline and TGFβ-induced Collagen-I expression within the cells and production of pro-Collagen Iα1 from cells A–C. IF aFor testing of C1, primary cells from five CD cell cultures: two cultures from NSCD tissue and three cultures from SCD tissue were used. For each culture, experiments were repeated five times over independent passages. The results demonstrate that unlike ICG-001, C1 selectively blocks TGFβ-induced Collagen-I expression and production of pro-Collagen-Iα1 A,E,F butIn the present study, we have demonstrated increased β-catenin expression in SCD tissue by IHC. Wnt signalling pathway components, including FZD8 and WNT5B, were also up-regulated in SCD tissue. Furthermore, we identify small-molecule inhibitors of FZD8 (3235-0367 (C1)), Porcupine (Wnt-C59), and β-catenin (ICG-001) as potential novel therapies for intestinal fibrosis in CD.Our data are supported by the previous reports of increased nuclear β-catenin staining in FFPE CD fibrotic lesions relative to healthy controls . Howeverin vitro using the CCD-18Co human intestinal fibroblast cell line, an established model of intestinal fibrosis [e.g. APC gene was sufficient to induce Collagen-I production. Moreover, treatment with ICG-001, which inhibits β-catenin/CBP-dependent transcription [A direct link between β-catenin-dependent Wnt signalling and Collagen-I was demonstrated sis e.g. . Here, wcription , reducedAPC gene, with no overt toxicity [Our data are consistent with the previous reports demonstrating ICG-001 inhibits fibrosis in liver ,48, kidntoxicity . These fin vivo. While in vitro models of intestinal fibrosis cannot model the complex intestinal architecture, in vivo rodent models do not fully recapitulate human disease and have given little insight into mechanisms with translational potential [in vitro models represents a realistic alternative route to identifying new therapeutic drugs.Moreover, in models of kidney fibrosis, blocking β-catenin/TCF-dependent signalling with ICG-001 not only prevented fibrosis but ICG-001 redirected β-catenin and promoted β-catenin/FOXO1 signalling and thus inhibited inflammation, suggesting ICG-001 may offer a dual benefit for CD patients . A seconotential . DevelopAXIN2 in intestinal fibroblasts. This suggests that the TGFβ-induced increases in β-catenin are not sufficient to activate canonical Wnt signalling. Instead, TGFβ treatment markedly increased the expression of components of noncanonical Wnt signalling pathways, e.g. FZD8 and its ligand WNT5B, which were both up-regulated in SCD tissue relative to paired NSCD tissue. As far as we are aware, this is the first report of altered FZD8/WNT5B expression in SCD tissue and provides important patient-based validation of a role for FZD8/WNT5B in SCD. Although we cannot directly infer that these changes are TGFβ-driven, this hypothesis is supported by the mechanistic in vitro data presented here and the wide-spread reports of increased TGFβ in SCD tissue. Notably, TGFβ has also been shown to up-regulate FZD8 in mesenchymal cells derived from human intestinal organoids [In the present study, we also describe cross-talk between the TGFβ and Wnt signalling pathways. TGFβ has previously been reported to induce fibrosis both via regulating β‐catenin‐dependent , and -inrganoids .The FZD8 receptor has been shown previously to associate with the TGFβ receptor complex, highlighting it as potential mediator of cross-talk between the TGFβ and Wnt signalling pathways . The obs50 7 μM, Kd 2.5 μM) [Inhibitors of the WNT and FZD8 signalling were also shown to limit Collagen-I expression in CCD-18Co cells and CD-derived myofibroblasts, highlighting the therapeutic potential of targeting this signalling axis. FZD8 is a particularly attractive therapeutic target given it selectively targets TGFβ-induced Collagen-I and does not influence basal expression, highlighting potential selectivity for pathological activated myofibroblasts. This is likely associated with its low basal expression in intestinal fibroblasts and its marked induction in TGFβ-activated cells. Moreover, targeting FZD8 at the concentration used here (10 μM) does not appear to disrupt canonical SMAD-driven TGFβ-signalling, which, given its important role in suppressing inflammation in CD, is a positive finding. The dose of FZD8 inhibitor used is also consistent with other studies using cell and NMR-based methods that demonstrate micromolar effects for C1 (IC 2.5 μM) .FZD8 in CCD-18Co fibroblasts reported here, and studies showing that FZD8‐deficient mice are resistant to bleomycin‐induced lung fibrosis [The FZD8 inhibitor C1 used in the present study blocks FZD8-dependent Wnt signalling by binding to the conserved cysteine-rich domain of FZD8 ,27. Thisfibrosis .C59, a porcupine inhibitor that blocks Wnt ligand production, was used to target Wnt5B expression in TGFβ-treated fibroblasts. In accord with the previous reports, C59 inhibited ECM gene expression, but less potently than C1. These effects on ECM protein levels were independent of changes in β‐catenin and support a noncanonical action. In this context, this would be consistent with the antifibrotic effects of blocking WNT5B/FZD8 signalling. However, as Wnt-C59 inhibits secretion of all Wnt family members, we cannot directly link its actions to a specific Wnt ligand. Therefore, targeting the FZD8 receptor selectively may offer greater specificity. To our knowledge the FZD8 inhibitor, C1 has not yet been tested as part of a clinical trial. This will be important in determining its potential as a therapy, given the safety concerns surrounding bone fragility that led to termination of a dose-escalation phase 1b trial of Vantictumab, a monoclonal antibody that binds FZD1, FZD2, FZD5, FZD7, and FZD8, in metastatic pancreatic cancer patients .in vivo, where Wnt ligands in the local microenvironment can initiate β‐catenin-dependent canonical Wnt signalling.In summary, our data highlight complex interactions between canonical Wnt and TGFβ-mediated noncanonical FZD8-Wnt5B-mediated signalling pathways in the context of intestinal fibrosis in CD. Both can induce fibroblasts to increase production of ECM proteins, e.g. Collagen-I, and small-molecule inhibitors of both can attenuate this response, and therefore offer opportunities for therapeutic intervention. TGFβ promotes noncanonical Wnt signalling, but does not directly induce canonical Wnt signalling in intestinal fibroblasts despite increasing the protein levels of β‐catenin. However, it is possible that the higher levels of β‐catenin protein observed in TGFβ-stimulated fibroblasts primes those cells to be more Wnt responsive AXIN2, a Wnt-inducible component of the β‐catenin destruction complex. This mechanism could contribute to the stabilisation of β‐catenin in TGFβ-treated intestinal fibroblasts.A proposed model for Wnt-mediated fibrosis in CD is presented . A similCurrently, there are no specific antifibrotic therapies for stricturing disease in CD; therefore, surgery remains the cornerstone of treatment despite its costs, risks, and the subsequent problems associated with disease recurrence.Hence, there is an urgent need for new therapies to transform the way patients with strictures are treated and to improve their quality of life, based on a better understanding of the underlying molecular mechanism. Our data highlight small-molecule Wnt inhibitors, in particular ICG-001 and C1, for further investigation as potential antifibrotic drugs to treat intestinal fibrosis in CD.Click here for additional data file.


Benefiting from the unique nanocapsule structure, especially inner CoS/C shell and outer pure C shell, the MnS@CoS/C@C composite as anode material presents excellent cycling performance  and superior rate capability (1133.3 and 650.6 mAh g−1 at 0.1 and 4 A g−1), compared to the control materials (MnS and MnS@CoS/C) and other MnS composites. Kinetics measurements further reveal a high proportion of the capacitive effect and low reaction impedance of MnS@CoS/C@C. SEM and TEM observation on the cycled electrode confirms superior structural stability of MnS@CoS/C@C during long-term cycles. Excellent lithium storage performance and the convenient synthesis strategy demonstrates that the MnS@CoS/C@C nanocapsule is a promising high-performance anode material.MnS has been explored as an anode material for lithium-ion batteries due to its high theoretical capacity, but low electronic conductivity and severe volume change induce low reversible capacity and poor cycling performance. In this work, the nanocapsule consisting of MnS nanopolyhedrons confined in independent, closed and conductive hollow polyhedral nanospheres is prepared by embedding MnCO Especially, its redox potential is lower than those of other TMSs [+ mobility also gives rise to an unsatisfactory rate capability [With the increasing demands of consumable electronics and pure/hybrid electric vehicles and other energy applications, the requirement for advanced energy storage devices, such as lithium-ion batteries (LIBs), becomes higher and higher. It is well-known that anode materials play a significant role in LIBs ,2,3,4. Hher TMSs ,15,16,17her TMSs ,19. In apability ,22,23,24+ in MnS. On the other hand, hybridizing MnS with carbonous materials can effectively improve the electrical conductivity of MnS, and even suppress volumetric changes of MnS to a certain extent [−1 after 400 cycles at 1 A g−1 [−1 was delivered after 1000 cycles at 1 A g−1 [9S8/carbon heterostructures through the chemical vapor deposition treatment of flower-like Mn/Co-glycerate and achieved a high reversible capacity of 608 mAh g−1 after 300 cycles at 500 mA g−1 [Many strategies have been proposed to overcome the above shortcomings of MnS. One of the most effective strategies is to combine a wide variety of MnS nanostructures with conductive materials, such as various carbonous materials ,29,30,31n extent ,33. The  1 A g−1 . S. Gao  1 A g−1 . L. Zhu 0 mA g−1 . These r0 mA g−1 ,36. It hInspired by the above considerations, herein, we design the independent, closed and conductive hollow polyhedral nanospheres composed of two layers of heterogeneous shells, a CoS/C inner shell and a pure C outer shell. Several MnS nanopolyhedrons are in-situ grown inside the hollow polyhedral nanospheres, constructing a unique nanocapsule structure. The composite is denoted as MnS@CoS/C@C. Compared with other composite strategies, the hollow polyhedral nanospheres can not only improve electronic conductivity of MnS, but also avoid MnS from aggregating or falling off from the composite. More importantly, it can also effectively buffer volume change of MnS. Herein, the detailed structural features and the electrochemical properties of MnS@CoS/C@C nanocapsules have been systematically explored. As expected, MnS@CoS/C@C exhibits superior Li storage performance in terms of reversible capacity, cycling performance and rate capability. The unique nanocapsule structure is also expected to be applied in other fields.3 nanopolyhedrons are prepared via a hydrothermal co-precipitation reaction. Under the hydrothermal conditions, citric acid acts as a reducing agent, and reduces Mn7+ in KMnO4 to Mn2+. On the other hand, citric acid is decomposed, and carbonate ions are released. Mn2+ combines with CO32−. Accordingly, MnCO3 is formed. The relevant reactions are shown as follows:The synthetic procedure of MnS@CoS/C@C nanocapsule is schematically illustrated in 3 and ZIF-67, several MnCO3 nanopolyhedrons are assembled into ZIF-67. Furthermore, RF resin is coated on the surface of MnCO3@ZIF-67 through a polymerization reaction between resorcinol and formaldehyde. Finally, a gaseous sulfurization and carbonization procedure are performed. MnCO3 nanopolyhedrons are sulfurized into MnS nanopolyhedrons. The solid ZIF-67 is sulfurized into the hollow inner shell composed of CoS nanoparticles and a small amount of amorphous carbon. RF resin is decomposed into the outer shell composed of pure amorphous carbon. The above structural changes are accomplished by one-step calcination.Afterword, via the unique bridge effect of PVP between MnCO3 nanopolyhedrons. MnCO3 nanopolyhedrons present solid structure, uniform size and sharp edges. Although the size is only about 30 nm, they do not aggregate, which ensures the successful embedding of MnCO3 nanopolyhedrons into ZIF-67. Through controlling the reaction conditions, MnCO3 is restricted to an inadequate growth state, that is, an intermediate morphology close to the cube. 3@ZIF-67. The product presents the typical rhombic dodecahedron, suggesting good crystallinity of ZIF-67. The size of ZIF-67 is also uniform, about 250–450 nm. It can be seen that there is very little scattered MnCO3 nanopolyhedrons outside ZIF-67. The TEM image  and G band (the stretching modes of sp2-type carbon). This demonstrates that RF resin is successfully decomposed to carbon. The intensity ratio of D band and G band (ID/IG) is 0.89, confirming that RF resin-derived carbon is amorphous. Additionally, the two peaks located at 642 and 347 cm−1 can be indexed to MnS [−1 are related to Eg and F2g peaks of CoS [The phases of MnS@CoS/C@C are examined through XRD a. All th@CoS/C@C b. At 135d to MnS . The oths of CoS . XRD and4 and CoSO4. The weight loss in 390–540 °C is attributed to the combustion of carbon in air. The second weight loss in 615–860 °C is more significant. It is related to the decomposition of MnSO4 and CoSO4 into Mn2O3 and Co3O4. Based on the atomic ratio of Mn and Co provided by EDS, the contents of MnS, CoS and carbon are calculated to be 60%, 14.61% and 25.39%, respectively.−1 in the first three cycles. CV curves present a lot of reduction peaks and oxidation peaks from MnS and CoS, indicating that both MnS and CoS participate in electrochemical reactions, and the reactions of MnS and CoS are multi-step. The first cathodic scanning curve exhibits five reduction peaks located at 1.63, 1.20, 0.58, 0.25 and 0.01 V, corresponding to the intercalation of Li+ into MnS, conversion of CoS to metallic Co, conversion of Li2MnS into metallic Mn and Li2S, the formation of the SEI film and intercalation of Li+ into carbon, respectively. The subsequent anodic scanning curves are relatively stable, and show seven oxidation peaks located at 0.25, 1.12, 1.28, 1.90, 2.06, 2.27 and 2.35 V, related to the deintercalation of Li+ from carbon (0.25 V), re-conversion of metallic Mn to Li2MnS (1.12 V), re-conversion of metallic Co to LixCoS (1.28 V), deintercalation of Li+ from Li2MnS (1.9 and 2.35 V) and deintercalation of Li+ from Li2CoS (2.06 and 2.27 V), respectively [The electrochemical properties of MnS@CoS/C@C as anode material for LIBs are studied in coin-type half-cell. ectively ,38,39. T−1 at the 1st, 2nd and 5th cycle are investigated. The control material of MnS is prepared by the sulfurization of MnCO3 nanopolyhedrons. The control material of CoS/C is prepared by sulfurization of ZIF-67. The charge/discharge curves  and scan rate (v), according to the equation (i = bav). Herein, a and b are the variable parameters. It is known that the value of b can be used to estimate the type of the reaction kinetics. In general, a b-value of 0.5 means that diffusion completely controls the electrochemical reaction, whereas a b-value of 1.0 represents that the capacitive effect dominates the electrochemical reaction. a and b can be determined by the plot of log i vs. log v, as shown in b value of the cathodic peak (Peak 1) is fitted as 0.79. The b values of the two anodic peaks (Peak 2 and 3) are fitted as 0.74 and 0.80. The values suggest that the pseudocapacitive effect controls electrochemical reaction of MnS@CoS/C@C. To quantitatively elucidate the contribution ratios of the pseudocapacitive effect and the diffusive effect in the electrochemical reaction, another equation (i = k1v + k2v1/2) is used to separate the current response (i) of CV curves. In the equation, k1v indicates the pseudocapacitive current, and k2v1/2 represents the diffusive current. k1 and k2 are two parameters related to the potential and can be determined by plotting i/v1/2 vs. v1/2 and then performing linearly fitting. k1 and k2 are the slope and the intercept of the fitting line. In this way, the CV curve at scan rate of 1.0 mV s−1 can be decomposed to the pseudocapacitive current and the diffusive current, as shown in −1, the pseudocapacitive contributions are 63.06, 67.56, 74.53 and 83.31%, respectively, as illustrated in The reaction kinetics of MnS@CoS/C@C is further investigated by CV curves at various scan rates in order to find out the reason for the high specific capacity and excellent rate capability. In w). In the high and medium frequency regions, the compressed semicircles are associated with the interfacial SEI film resistance (Ri) and charge transfer resistance (Rct). RΩ represents internal resistance. CPE is a constant phase element. At the 5th cycle, the semicircle becomes smaller, which means that Rct decreases. The fitted results indicate that Rct decreases from 64.6 Ω before cycling to 38.2 Ω at the 5th cycle. The low Rct suggests rapid charge transfer and a rapid interface reaction. RΩ is only 5.7 Ω, which indicates that the outer pure carbon shell and the inner CoS/C shell improve the electronic conductivity of the composite. EIS results demonstrate that the reaction kinetics of MnS@CoS/C@C are fast, which is beneficial to obtain high capacity and good rate performance.The reaction kinetics of MnS@CoS/C@C are further studied by EIS. −1 is observed. SEM image -67: 0.55 g MnCO3 nanopolyhedrons were ultrasonically dispersed in 50 mL methanol. 1.1 g polyvinylpyrrolidone  was added under stirring to prepare solution A. Solution B was prepared by dissolving 2.96 g 2-methylimidazole in 50 mL methanol. Solution C was prepared by dissolving 0.20 g Co(NO3)2·6H2O in 25 mL methanol. The solution B and solution C were poured into solution A under stirring. After reaction for 45 min, the mixed solution was aged at room temperature for 12 h. The dark purple precipitate was separated by centrifugation, washed repeatedly with methanol, and dried overnight at 50 °C in vacuum.Synthesis of MnCO3@ZIF-67@resorcinol-formaldehyde (RF) resin: Solution D was prepared by ultrasonically dispersing 0.35 g MnCO3@ZIF-67 in 12.5 mL ethanol. Solution E was prepared by dissolving 0.23 g cetyltrimenthylammonium bromide (CTAB) in 30 mL deionized water. Solution E was poured into solution D under stirring. After 15 min, 0.12 g resorcinol and 0.2 mL ammonium hydroxide were added to the mixed solution. After 20 min, 0.017 mL formaldehyde solution was added. After reaction for 12 h at room temperature, the product was separated, washed with ethanol 3 times, and finally dried.Synthesis of MnCO3@ZIF-67@RF resin and sulfur powder were placed in a horizontal quartz tube furnace, with a weight ratio of 1:6. The sulfur powder was at upstream. The mixture was then annealed at 600 °C for 2 h with a heating rate of 2 °C min−1 under flowing argon with a flowing rate of 150 sccm.Synthesis of MnS@CoS/C@C: MnCO−1.Scanning electron microscopy  and transmission electron microscope  were utilized to observe morphology and microstructure. Information about the crystal structure were obtained by Rigaku D/max2550VL/PC system X-ray diffractometer (XRD) with Cu-Kα radiation. The chemical composition was determined by INCA energy dispersive X-Ray spectroscopy (EDS) equipped in SEM. Raman spectra were collected from DXR Raman microspectrometer with a 633 nm laser. Thermogravimetric analysis (TGA) curve was collected with a thermogravimetric analyzer (NETZSCH TG209F3) under air flow at a heating ramp of 10 °C min6. Li foil was used as counter electrode. The galvanostatic charge–discharge cycling measurements were operated in the voltage range of 0.01–3.0 V at room temperature on NEWARE CT-3008 battery test systems. The electrochemical impedance spectroscopy (EIS) with a frequency range of 0.01 HZ—100 KHz and the cyclic voltammetry (CV) curves over the potential range from 0.01 to 3.0 V (vs. Li+/Li) were tested on VersaSTAT 3 electrochemical measurement system. After a certain cycling numbers, the MnS@CoS/C@C electrode was taken out from the cell, rinsed with dimethyl carbonate, and then dried in vacuum for SEM and TEM observation.The working electrodes were made by mixing active material (70 wt%), carbon black (Super-P) (20 wt%) and polyvinylidene fluoride (PVDF) (10 wt%) to form a homogeneous slurry. N-methylpyrrolidone was used as solvent. The slurry was then spread onto copper foils with a diameter of 1.2 cm, followed with drying in vacuum at 70 °C overnight to remove NMP. The CR2025 coin-type half-cells were assembled in an Ar-filled glove-box under the contents of moisture and oxygen less than 0.5 ppm. The polypropylene membrane (Celgard 2400) was used as separator. The electrolyte was the mixture of dimethyl carbonate and ethylene carbonate (1:1 in volume) dissolved with 1 M LiPF−1 at 0.1 A g−1), improved cycling stability (481 mAh g−1 at 5 A g−1 after 300 cycles) and excellent rate capability, as well as rapid reaction kinetics, including a high proportion of pseudocapacitive effect and low reaction impedance. The improved electrochemical performance is associated with the unique nanocapsule structure. Two layers of shells, especially outer pure C shell, not only increase electronic conductivity, but also improve structural stability. Abundant void space in the nanocapsule also facilitates electrolyte storage. The synthesis strategy and structure features proposed in this work can motivate innovative exploration of novel hybrid composites for high-performance energy storage applications.In summary, we developed a facile in-situ assembling and gaseous sulfurization/carbonization strategy to construct MnS@CoS/C@C, in which several MnS nanopolyhedrons as cores are perfectly confined inside hollow polyhedral nanospheres composed of an inner CoS/C shell and outer pure C shell. As an anode material for LIBs, the MnS@CoS/C@C electrode exhibits high specific capacity (1133.3 mAh g