Aberrant activation of Wnt signaling has been implicated in human osteosarcoma, which may provide a genetic vulnerability that can be targeted in osteosarcoma treatment. To test whether Wnt activation is necessary for osteosarcoma growth, colony formation, invasion, and metastasis, we treated human osteosarcoma cells with a small molecule inhibitor of Wnt/β-catenin, PRI-724, which suppresses Wnt/β-catenin-mediated transcription. We found increased protein levels of endogenous active-β-catenin in five human osteosarcoma cell lines. Treatment with PRI-724 was sufficient to inhibit human osteosarcoma 143B and SJSA-1 cell proliferation. Suppressed Wnt signaling was confirmed by decreased protein levels of the Wnt target Cyclin D1. Furthermore, we revealed significant inhibitory effects on cell migration, invasion, and colony formation in the human osteosarcoma cells. Using deposited data from next generation sequencing studies, we analyzed somatic mutations and gene expression of components in the Wnt/β-catenin pathway. We found somatic mutations and upregulated gene expression of many components in the Wnt/ β-catenin pathway, indicating activated Wnt signaling. Taken together, our results illustrate the critical role of Wnt/β-catenin signaling in human osteosarcoma pathogenesis and growth, as well as the therapeutic potential of Wnt inhibitors in the treatment of human osteosarcoma. Osteosarcoma (OS) is the most common type of bone cancer in adolescents and young adults with an incidence rate of 4.4 per million people per year in the age group of 0-24 years for all races and both genders , 2. MoleCCND1 [Recent studies have shown that osteosarcoma cancer stem cells play major roles in chemo-resistance, tumor recurrence, and metastasis , 9. CancCCND1 . ActivatCCND1 , 17, 18.CCND1 .Deregulation of canonical Wnt/β-catenin signaling in human osteosarcoma samples and cell lines has been described in recent studies –23; howeTo gain a better understanding of Wnt activation signaling pathway in human OS cells, we first performed Western blotting to detect active β-catenin, total β-catenin, and β-actin protein levels in human OS cell lines 143B, Saos-2, SJSA-1 , U-2 OS, and MG-63. Breast cancer lines MCF7 and MDA-MB-231 were used as controls for high-level expression of active β-catenin protein whereas hMSC cell line was used as a control for low-level expression of active β-catenin protein. As shown in Figure To test if Wnt activation is necessary for osteosarcoma cell proliferation, migration, invasion, and colony formation, we treated human osteosarcoma 143B cells with a small molecule inhibitor of the Wnt/β-catenin pathway, PRI-724  Among these altered genes, Wnt antagonists FRZB, SFRP1, and WIF1 were decreased in tumor samples while Wnt ligands WNT5A, FZD1, DVL3 and target transcription factors CCND1, MYCB, SNAI2 were increased in tumor samples  . We useds Figure . Moreove1 Figure . Among tc cancer . In addic cancer . We obse protein . Consist protein , implicaMYC, APC, CTNND1, CCNE1, CDK4, NF1, DLG2, and BAP1. Mutation types include copy number alteration (amplified or deleted), in frame deletion, structural variation, frame shift, copy gain/loss, and point mutations (e.g. missense mutation). For example, copy number amplification of the oncogene MYC has been found in most types of cancer [APC may lead to increased nuclear functional β-catenin protein in OS [Apc1638N/+;twist null/+ mutation mimics the human disease [Next generation sequencing (NGS), using either whole-genome sequencing or exome sequencing of human OS samples, has helped us decipher the genetic causes of OS at a much higher resolution and in an unbiased fashion. In the past few years, several groups have performed NGS analysis on a total of 111 human OS samples and their normal tissue pairs , 38–43. f cancer , 46; copin in OS –49. The  disease . Howeverin vivo animal models to determine whether activated Wnt signaling has a causative role in OS development , corresponding to a concentration of 55 μM. Thus, the ICG-001 concentrations used in the present study (25 μM) in vitro may be achievable in patients. Our study using PRI-724 in human OS cell lines may pave the way to additional preclinical studies in mice and clinical trials on efficacy of the drug in patients with OS.A growing body of evidence links Wnt/β-catenin signaling to tumorigenesis of many cancer types and implicates its role in the development of cancer drug resistance . Due to CCNE1, E2, and A2 [The molecular mechanisms of action of PRI-724, which serves as a cytostatic drug, have been explored over the past 14 years among many types of cancer cells , 58, 59., and A2 . In the , and A2 . PRI-724, and A2 , 34.Apc;twist deficit) based on mutations of Wnt pathway [CSCs are responsible for cancer metastasis and reoccurrence, and cannot be easily eliminated by regular anti-proliferative drugs . The clo pathway . Moreove2. Unless stated otherwise, cells in 100 mm tissue culture dishes  were treated with 25 μM PRI-724  for 24 hours. Human mesenchymal stem cell (hMSCs)  were cultured in Minimum Essential Medium , containing 10% FBS  and 100 U/ml penicillin, 100 mg/ml streptomycin sulfate . The corresponding amount of DMSO (vehicle) was used as a control. Western blotting was performed according to the modified procedure described previously [Cell lines MCF7(HTB-22), MDA-MB-231HTB-26), 143B(CRL-8303), Saos-2(HTB-85), SJSA-1 (CRL-2098), U-2 OS (HTB-96), and MG-63 (CRL-1427) were all purchased from ATCC and maintained in growth medium containing 10% Fetal Bovine Serum (FBS)  and 1% Penicillin-streptomycin  at 37°C under a humidified atmosphere containing 5% CO, 143B at a density of 2,000 cells per well and treated with PRI-724 at 0, 1, 10, 25, 50, or 100 μM for 24, 48 or 72 hours. 20 μl of 20 μM Sytox Green  was added into each well. The plate was incubated in the incubator for 15 mins and read by plate reader SpectamaxM5  at ex/em 485/530 with a 515 nm cut off for dead cell reading. 20 μl of 6% Triton X-100  was added to each well and the plate was incubated in the incubator for 45 mins and was read again for total cell reading. The value of total cell reading subtracted by the dead cell reading was considered as viable cell reading . All expCells were seeded in six-well plates and allowed to grow to confluence. Using a sterile 200-μl pipette tip, a straight scratch was drawn on the monolayer of cells. The cells were then washed three times with phosphate buffered saline (PBS). Fresh growth medium containing 25 μM PRI-724 or DMSO was added, and the cells were allowed to close the wound. Photographs were taken later at the same position of the wound. The width of the scratch wound was measured with NIH ImageJ. The relative migration at an indicated time point was calculated by comparing to the wound width at the initial time point.in vitro migration and invasion assays were carried out using the 8-μm pore-sized Transwell chamber system . Prior to loading the cells into the upper chamber, the lower chamber was filled with medium containing 10% FBS with 25 μM PRI-724 or DMSO. Cells were starved overnight and added to serum-free medium containing 25 μM PRI-724 or DMSO in the upper chamber at a concentration of 4X104 cells per well. For invasion assays, membranes were coated with a layer of 0.2 mg/ml Matrigel . After incubation for 24 hours, the cells on the upper surface of the well were removed completely by a Q-tip. The wells were fixed in 10% formalin and stained with Crystal violet . The plate with inserts were then imaged with an upright microscope  and the relative number of cells that migrated through the Transwell pores was quantified using NIH ImageJ  [The MD, USA) .2 atmosphere at 37°C until there was visible clonal colony formation. Images were taken after the colonies were washed gently with PBS , fixed with 10% formalin, and stained with 0.5% Crystal violet solution. Assays were performed at least three times.Cells were seeded in six-well plates at a density of 200 cells per well and cultured in full medium containing 25 μM PRI-724 or DMSO. Cells were grown for 7 to 14 days in a humidified, 5% COWe used published data (GEO accession dataset: GSE99671)  from 18


Mycoplasma hyorhinis infection promotes gastric cancer cell motility. The β‐catenin signaling pathway is critical to determining malignant cancer cell phenotypes; however, the association between M hyorhinis and the β‐catenin signaling pathway is unclear.We previously identified that M hyorhinis.We performed subcellular fractionation and immunofluorescence staining to observe β‐catenin accumulation in the nucleus. The expression of downstream β‐catenin genes was detected by quantitative RT‐PCR. Gastric cancer cell motility was examined by transwell chamber migration and wound healing assays, and a co‐immunoprecipitation assay was used to detect the proteins associated with the membrane protein p37 of M hyorhinis infection promoted nuclear β‐catenin accumulation and enhanced the expression of downstream β‐catenin genes. M hyorhinis‐promoted gastric cancer cell motility was counteracted by treatment with the β‐catenin inhibitor XAV939 or β‐catenin knockdown. We further detected a protein complex containing LRP6, GSK3β, and p37 in M hyorhinis‐infected cells. M hyorhinis also induced LRP6 phosphorylation in a GSK3β‐dependent fashion. Knockdown of LRP6 or GSK3β abolished M hyorhinis‐induced cell motility.We found that M hyorhinis infection, thereby contributing to M hyorhinis‐induced gastric cancer cell motility.Our results reveal that the β‐catenin signaling pathway could be activated by  There were more than 900 000 new cases and 700 000 gastric cancer‐related deaths worldwide in 2012.Mycoplasma hyorhinis major membrane protein p37 revealed varying positive rates of M hyorhinis infection in several cancer types.M hyorhinis‐positive cancer tissues.M hyorhinis infection rate was higher in tissues with high differentiation than in tissues with low differentiation,M hyorhinis is associated with tumor cell malignancy. Namiki et al demonstrated that persistent mycoplasma  infection could promote the chromosomal instability and the malignant transformation of mammalian cells.M hyorhinis‐induced gastric cancer cell migration.M hyorhinis‐induced migration and invasion in vitro and metastasis in vivo.M hyorhinis‐infected tumor cells, MMP‐2 activity was significantly increased, which in turn stimulated EGFR phosphorylation and contributed to metastasis.M hyorhinis could promote IL‐6 expression via the TLR4 receptor, leading to IL‐6‐mediated STAT3 signaling activation.M fermentans reduces the activity of TopoI through PARP1 by MAPK activation, thereby altering cellular gene expression and conferring camptothecin resistance. All these studies suggested mycoplasma infection as a possible promoting factor in cancer initiation and progression. However, the signaling pathways mediating mycoplasma‐induced malignant phenotypes have not been fully identified.In our previous study, immunohistochemical staining of cancer tissues with a PD4 monoclonal antibody against M hyorhinis‐induced motility in gastric cancer cells.The Wnt/β‐catenin pathway, also called the canonical Wnt pathway, is crucial to embryonic development and adult tissue homeostasis. Aberrant activation of the Wnt/β‐catenin pathway results in deregulated cell growth and malignant transformation.22.1Anti‐GSK3β #22104‐1‐AP), anti‐Wnt2 (#27214‐1‐AP), and anti‐β‐catenin #51067‐2‐AP) antibodies were purchased from Proteintech . Anti‐pSer1490‐LRP6 (#2568) and anti‐LRP6 (#3395) were from Cell Signaling Technology . Anti‐β‐tubulin (#ZS‐9104) was purchased from Zhongshan Technology . Anti‐Histone 2B (#GR193918‐1) was purchased from Abcam . Anti‐p37 monoclonal antibody PD4 was generated and characterized previously.2104‐1‐AP1067‐2‐AP2.2M hyorhinis was handled as previously described.M hyorhinis ) was cultured for 24 hours at 37°C in modified Hayflick's medium supplemented with 20% heat‐inactivated fetal bovine serum (FBS). Three generations of M hyorhinis were cultured continuously. M hyorhinis was collected after centrifugation at 12 000 g for 20 minutes. After washing once with PBS, M hyorhinis was resuspended in PBS and stored at −80°C. We used color change units (CCUs) to calculate the M hyorhinis titer. Cells were serum‐starved for 24 hours prior to adding 105 CCU/mL of M hyorhinis. M hyorhinis inactivation was performed by autoclaving at 121°C and 15 psi for 15 minutes.2.32 at 37°C. The culture media and FBS were obtained from Invitrogen . siRNAs  were transfected into cells with siRNA‐Mate Reagent (GenePharma). The following siRNAs were used in this study: negative control, 5′‐UUCUCCGAACGUGUCACGUTT‐3′, 5′‐ACGUGACACGUUCGGAGAATT‐3′; GSK3β #1, 5′‐ GCUGGAGUAUACACCAACUTT ‐3′, 5′‐ AGUUGGUGUAUACUCCAGCTT ‐3′; GSK3β #2, 5′‐ GGACAAGAGAUUUAAGAAUTT ‐3′, 5′‐ AUUCUUAAAUCUCUUGUCCTT ‐3′; GSK3β #3, 5′‐ GGACUAUGUUCCGGAAACATT ‐3′, 5′‐ UGUUUCCGGAACAUAGUCCTT ‐3′; LRP6 #1, 5′‐CCACAAAUCCAUGUGGAAUTT‐3′, 5′‐AUUCCACAUGGAUUUGUGGTT‐3′; LRP6 #2, 5′‐GGUUCUGACCGUGUAGUAUTT‐3′, 5′‐AUACUACACGGUCAGAACCTT‐3′; LRP6 #3, 5′‐GCAGAUAUCAGACGAAUUUTT‐3′, 5′‐AAAUUCGUCUGAUAUCUGCTT‐3′; Wnt2 #1, 5′‐CCAGGGUGAUGUGCGAUAATT‐3′, 5′‐UUAUCGCACAUCACCCUGGTT‐3′; Wnt2 #2, 5′‐GCUGGCAGGAAGGCUGUAATT‐3′, 5′‐UUACAGCCUUCCUGCCAGCTT‐3′; Wnt2 #3, 5′‐GCUGACUGGACAACCGCUATT‐3′, 5′‐UAGCGGUUGUCCAGUCAGCTT‐3′; β‐catenin #1, 5′‐CCUUCACUCAAGAACAAGUTT‐3′, 5′‐ACUUGUUCUUGAGUGAAGGTT‐3′; β‐catenin #2, 5′‐GCUCAUCAUACUGGCUAGUTT‐3′, 5′‐ACUAGCCAGUAUGAUGAGCTT‐3′; and β‐catenin #3, 5′‐GUCAACGUCUUGUUCAGAATT‐3′, 5′‐UUCUGAACAAGACGUUGACTT‐3′.The human gastric cancer cell lines MGC803 and BGC823 were maintained by Peking University Cancer Hospital & Institute, and the human gastric cancer cell line AGS was obtained from the ATCC. All cells were cultured in RPMI 1640 medium supplemented with 10% FBS in 5% CO2.46 cells were collected in 800 μL of buffer A  for 20 minutes on ice, followed by the addition of another 200 μL of buffer A with 0.5% NP‐40. After centrifugation at 1500 g and 4°C for 5 minutes, the supernatant was re‐centrifuged at 18 000 g and 4°C for 20 minutes to obtain cytoplasmic proteins. The pellet was washed twice with buffer A and homogenized directly in 1× SDS‐PAGE loading buffer to obtain nuclear proteins.A total of 5 × 102.5g for 10 minutes at 4°C, the protein concentrations of the cleared lysates were quantified with a BCA Protein Assay Kit . Samples (50 μg per lane) were resolved by 10% SDS‐PAGE, transferred to nitrocellulose membranes, and blocked with 5% non‐fat milk/TBST. Proteins were probed with primary antibodies overnight at 4°C. The membranes were washed three times with TBST and incubated with HRP‐conjugated secondary antibodies for 45 minutes at room temperature and then washed with TBST three times. Signals were detected by enhanced chemiluminescence. For quantification, the optical densities of the protein bands were analyzed by ImageJ software.Cells were washed twice with cold PBS and then lysed in buffer containing 25 mmol/L Tris‐HCl pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% NP‐40, 10% glycerol, and 1× protease inhibitor cocktail. After centrifugation at 15,000 2.6M hyorhinis for 24 hours. Next, the cells were fixed with 2% fresh paraformaldehyde/PBS for 15 minutes at room temperature, washed three times with PBS, permeabilized with 0.5% Triton‐X100/PBS for 20 minutes at room temperature, and blocked with 5% bovine serum albumin (BSA) at room temperature for 1 hour. Rabbit anti‐β‐catenin  was then incubated with the cells overnight at 4°C, followed by washing with PBS three times and probing with a FITC‐conjugated anti‐rabbit antibody for 45 minutes at room temperature in the dark. After washing, cells were counterstained with DAPI (1 μg/mL) for 10 minutes and mounted with 90% glycerol/PBS. A Zeiss LSM780 confocal microscope  was used to acquire images with fixed settings  and exposure time at room temperature. No Z‐stacking or deconvolution was applied.Cells were seeded on glass coverslips, grown to 80% confluence, and infected with 2.75 cells/mL, and 150 μL was transferred into the top chamber of each transwell apparatus. The cells were incubated for 24 hours at 37°C. The chambers were then fixed with methanol and stained with hematoxylin. Then, the chambers were washed with water, and the top surface of each membrane was cleared of cells with a cotton swab and counted in five randomly selected microscopic fields per well.Cells were starved overnight (12 hours) before the migration experiment. A tissue culture‐treated 6.5‐mm transwell chamber with 8.0‐μm pore membranes  was used. Each well of 24‐well plates was filled with 800 μL of medium containing 10% FBS as a chemoattractant. Cells were resuspended in serum‐free medium at a density of 5 × 102.8Cells were cultured to full confluence in 6‐well plates. The cells were washed with PBS, and wounds were made with sterile pipette tips. Then, culture medium with 5% FBS was added. Images of wound closure at the indicated time points were captured using a Nikon TiU microscope .2.93 cells/well) and cultured overnight. Then, the corresponding reagents were added. After 24, 48, and 72 hours, cell proliferation was quantified by cell confluence with a CloneSelect Imager .Cells in each group were seeded into 96‐well plates . cDNA was synthesized with a kit from Promega  according to the manufacturer's protocol. qRT‐PCR was performed using the SYBR premix system . The sequences of the specific primers were as follows:β‐catenin, forward primer 5′‐TGCAGTTCGCCTTCACTATG‐3′, reverse primer 5′‐ ACTAGTCGTGGAATGGCACC‐3′; c‐MYC, forward primer 5′‐CCCTCCACTCGGAAGGACTA‐3′, reverse primer 5′‐GCTGGTGCATTTTCGGTTGT‐3′; COX2, forward primer 5′‐GCTCAAACATGATGTTTGCATTC‐3′, reverse primer 5′‐GCTGGCCCTCGCTTATGA‐3′; telomerase reverse transcriptase (TERT): forward primer, 5′‐TCACGGAGACCACGTTTCAAA‐3′, reverse primer, 5′‐TTTCAAGTGCTGTCTGATTCCAAT‐3′; ENAH, forward primer, 5′‐TCTATCACCATACAGGCAACAAC‐3′, reverse primer, 5′‐GCACAGTTTATCACGACCTGA‐3′; GAPDH, forward primer 5′‐TGAAGGTCGGAGTCAACGG‐3′, reverse primer 5′‐CCTGGAAGATGGTGATGGG‐3′. PCR programs were 94°C for 2 minutes, followed by 40 cycles of 94°C for 15 seconds, 56°C for 20 seconds and 72°C for 30 seconds, followed by 72°C for 2 minutes. All gene expression levels were normalized to GAPDH, and the data were analyzed using the 2‐ΔΔCt method.2.11P values were calculated by t test using Graph Pad Prism 5. A P value less than 0.05 was considered statistically significant.The values represent the mean ± SD of at least three independent experiments with triplicate samples. 33.1M hyorhinis infection on the β‐catenin pathway. Total cell lysates, cytoplasmic proteins and nucleoproteins from M hyorhinis‐infected and uninfected gastric cancer cells were analyzed by Western blot. In the total cell lysates, there was no obvious change in β‐catenin levels upon M hyorhinis infection. However, nuclear β‐catenin was increased by M hyorhinis infection . However, XAV939 counteracted M hyorhinis‐induced gastric cancer cell motility in wound healing  mutation was detected in patients with familial adenomatous polyposis (FAP) and in sporadic colon cancer.M hyorhinis‐induced gastric cancer cell motility. Our data demonstrated that the β‐catenin pathway is activated by M hyorhinis infection and contributes to M hyorhinis‐induced cell motility, possibly by transactivation of downstream β‐catenin genes. We further found that GSK3β and LRP6 are essential for M hyorhinis‐induced β‐catenin activation, while the role of Wnt2 is less important. The monoclonal clonal antibody PD4 specifically recognizes lipoprotein p37,M hyorhinis.M hyorhinis infection caused greater LRP6 phosphorylation in AGS cells than in MGC803 cells. It is likely that the responsiveness of different cells to M hyorhinis infection could be different, and the kinetics of LRP6 phosphorylation in different cells could also be distinct.In this study, we revealed that the β‐catenin pathway plays an important role in M hyorhinis‐induced p‐LRP6 was decreased. This result further indicates that M hyorhinis can promote β‐catenin signaling through the GSK3β‐LRP6 pathway. Because the β‐catenin signaling pathway regulates multiple malignant cancer cell phenotypes,M hyorhinis‐induced β‐catenin activation may affect other aspects of tumorigenesis warrants further investigation.In the Wnt/β‐catenin pathway, the serine/threonine protein kinase GSK3β functions as a negative regulator of β‐catenin.Helicobacter pylori‐induced activation of EGFR‐PI3K/Akt signaling resulted in GSK3β suppression and β‐catenin accumulation through VacA or OipA.M hyorhinis infection activated the EGFR‐PI3K/Akt signaling axis in gastric cancer cell lines.M hyorhinis infection, likely through physical association with p37 protein.M hyorhinis infection.Cross‐talk between the β‐catenin pathway and other pathways has been reported. For example, M hyorhinis‐induced nuclear β‐catenin accumulation was impaired by LRP6 or GSK3β knockdown, while the effect of Wnt2 knockdown was minimal (Figure M hyorhinis‐induced motility, but the effect of Wnt2 knockdown was partial (Figure M hyorhinis‐stimulated β‐catenin pathway activation is dependent mainly on LRP6 and GSK3β, whereas Wnt2's contribution is relatively smaller.We next detected p37's associations with GSK3β and LRP6. l Figure G. The wol Figure . We therM hyorhinisM hyorhinis infection,M hyorhinis to fully induce β‐catenin signaling, which will be explored in future studies.The p37 protein is the most abundant membrane protein of M hyorhinis‐induced motility, this study may provide valuable evidence for patient‐directed therapies for M hyorhinis‐positive gastric cancer patients.Nevertheless, by identifying the contributing role of β‐catenin, as well as LRP6 and GSK3β, in 5M hyorhinis infection. Targeting the key factors in the β‐catenin signaling pathway, including β‐catenin, GSK3β, and LRP6, could counteract M hyorhinis‐induced gastric cancer cell motility.We found that the β‐catenin signaling pathway is activated by Not applicable.Not applicable.The authors declare that they have no competing interests.CS conceived the project. CS and XL designed the experiments. XL performed the experiments. CS and XL analysed the data. XL wrote the manuscript. All authors read and approved the final manuscript.


This study aimed to explore whether melatonin could attenuate VSMC calcification through regulating the Wnt1/β-catenin signaling pathway.  Melatonin has been demonstrated to protect against calcification in cyclosporine nephrotoxicity. The wingless-type MMTV integration site family member 1 (Wnt1)/α-smooth muscle actin (α-SMA), and cleaved caspase-3.  The effects of melatonin on vascular calcification were investigated in vascular smooth muscle cells (VSMCs). Calcium deposits were visualized by Alizarin Red Staining. Calcium content and alkaline phosphatase (ALP) activity were used to evaluate osteogenic differentiation. Western blots were used to measure the expression of runt-related transcription factor 2 (Runx2), α-SMA was found to be increased by melatonin, together with a decrease in apoptosis. Immunofluorescence assay revealed a lower Runx2 protein level in the melatonin group. Melatonin treatment significantly decreased the expression of Wnt1 and β-catenin. Treatment with lithium chloride or transglutaminase 2 abrogated the protective effects of melatonin.  Melatonin markedly ameliorated calcium deposition and ALP activity. Runx2 and cleaved caspase-3 were found to be reduced and β-GP-induced VSMC calcification through the suppression of Wnt1/β-catenin system. Melatonin can attenuate  The caufication . Melatonfication . Severalfication , 7. Melafication . The aimβ-glycerophosphate (β-GP) for 14 days [μM), was added before inducing calcification [β-catenin pathway, VSMCs were treated with lithium chloride   [β-catenin signaling agonist)  [VSMCs were isolated from the aortas of Sprague–Dawley rats (4 weeks) using the explant method described in the previous study . For cal 14 days . For drufication . To inveco, USA)  or transma, USA)  before iAlizarin Red S was performed to visualize the formation of mineralized matrix . The calcium content was determined using a calcium colorimetric assay kit . Alkaline phosphatase (ALP) activity was measured using an ALP kit . Caspase-3 activity was analyzed using a caspase-3 assay kit .α-smooth muscle actin , anti-runt-related transcription factor 2 , anti-cleaved caspase-3 , anti-Wnt1 , anti-β-catenin , and anti-β-actin . After overnight incubation, the membranes were further incubated with the appropriate secondary antibodies at room temperature for 60 minutes. Membranes were detected with an enhanced chemiluminescence reagent.VSMCs were lysed in RIPA buffer  for 30 minutes and then centrifuged at 14,000 ×g for 10 minutes at 4°C. Equal amounts of extracted protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel and electrotransferred onto a polyvinylidene difluoride membrane . Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 for 1 hour at room temperature followed by overnight incubation at 4°C with the following primary antibodies: anti-α-SMA, forward—5′-AAGTATCCGATAGAACAC-3′, reverse—5′-AAACATAATCTGGGTCAT-3′; Runx2, forward—5′-AGGCGCATTTCAGATGATGAC-3′, reverse—5′-ACCTGCCTGGCTCTTCTTAC-3′; β-actin, forward—5′-GATGGTGGGTATGGGTCAGAAGGAC-3′, reverse—5′-GCTCATTGCCGATAGTGATGACT-3′. The ABI PRISM 7500 Sequence Detection System using QuantiTect SYBR Green  was used to quantify differential gene expression. The mRNA expression levels were normalized to β-actin mRNA.The one-step RT-PCR kit  was used to assess the reverse transcription of total RNA. The primers used for polymerase chain reaction were as follows: For immunofluorescence, cells were fixed with 4% paraformaldehyde for 30 minutes, followed by permeation using 0.5% Triton X-100 for 10 minutes. Then, cells were blocked with 5% BSA for 1 hour and incubated with primary antibody against Runx2  and Wnt1  overnight at 4°C. The next day, cells were incubated with secondary antibody  for 1 hour at 37°C. Apoptosis was detected using a TUNEL assay  according to the manufacturer's instructions.P < 0.05.Data were expressed as the mean ± standard deviation (SD) of at least three independent experiments and were analyzed using one-way analysis of variance (ANOVA). The significance level was set at μM of melatonin significantly reduced calcium content in calcifying VSMCs. Therefore, most experiments were performed at the concentration of 5 uM of melatonin. The Alizarin Red S indicated that β-GP promoted the calcification of VSMCs and melatonin significantly inhibited β-GP-induced calcification (P < 0.05) . The mRNA expression of α-SMA was reduced in β-GP group as compared to the control group. The β-GP-induced reduction was mitigated by melatonin, but this effect was blocked by luzindole treatment. Moreover, the effects of melatonin on α-SMA protein expression in β-GP-treated VSMCs were consistent with the mRNA expression data. Runx2 mRNA and protein along with the ratio of cleaved caspase-3 were found to be upregulated in untreated cells, but in VSMCs treated with melatonin prior to β-GP, the levels were downregulated relative to the untreated cells  . We also found that TG2 alleviated the effects of melatonin on β-catenin  and β-catenin, promote Wnt target gene expression, and regulate many physiological processes. β-GP increased the expressions of Wnt, p-GSK-3β, and β-catenin, but they were reduced by pioglitazone treatment in rat vascular smooth muscle cells [β [α/β protein expression and promoted the nuclear localization of β-catenin, while it inhibited GSK-3β phosphorylation in osteoblastic differentiation [Wnt signaling pathways interact with glycogen synthase kinase 3 beta (GSK-3le cells . Yu et acells [β . Park etntiation .β-catenin signaling pathway after spinal cord injury [β-catenin in human mesenchymal stem cells. Melatonin also decreased the levels of cyclic adenosine-3,5-monophosphate and reactive oxygen species [β-catenin. The Wnt1/β-catenin pathway activators, LiCl and TG2, decreased the effects of melatonin on β-catenin and increased VSMC calcification. Taken together, melatonin decreased the expression of Wnt1, which in turn inactivated β-catenin and subsequently suppressed osteogenic differentiation. These effects subsequently attenuated VSMC calcification (A study by Shen et al. showed that melatonin inhibits neural cell apoptosis and promotes locomotor recovery via the activation of the d injury . But the species . Our datfication .β-catenin to further validate our findings.There are a few limitations to our study. First, the findings are only based on in vitro experiments. Second, the siRNAs could be used to knock down Wnt1 or β-GP-induced VSMC calcification through the suppression of Wnt1/β-catenin system.In conclusion, our study indicated that melatonin can inhibit


Klebsiella pneumoniae bacterial infection, we demonstrate that gentamicin dissolving microarray patches, applied to murine ears, could control K. pneumoniae infection. Mice treated with microarray patches had reduced bacterial burden in the nasal-associated lymphoid tissue and lungs compared with their untreated counterparts.Using a murine model of  Klebsiella pneumoniae bacterial infection, we demonstrate that gentamicin dissolving microarray patches, applied to murine ears, could control K. pneumoniae infection. Mice treated with microarray patches had reduced bacterial burden in the nasal-associated lymphoid tissue and lungs compared with their untreated counterparts. This proof of concept study represents the first published data on the in vivo delivery of the antibiotic gentamicin via dissolving microarray patches, resulting in the control of bacterial infection.Using a murine model of  Neonatal infections, including pneumonia and sepsis, remain a significant cause of mortality and morbidity, with an estimated 3 million neonatal deaths occurring every year worldwide . NeonateGEN is a potent aminoglycoside antibiotic with bactericidal activity against Gram-negative bacteria and is widely utilized due to its efficacy and low cost . Similarin vivo model   for transdermal delivery of GEN . MNs arevo model . HoweverIn this study, we aimed to test the therapeutic efficacy of antibiotics delivered via MNs. MNs containing GEN were prepared utilizing simplified manufacturing processes, as previously described  12). MN. MN12). in vivo. We tested the GEN MNs in a Klebsiella pneumoniae murine model of pneumonia. K. pneumoniae is one of the most important Gram-negative pathogens associated with a wide spectrum of infections, including pneumonia, intra-abdominal infections and bloodstream infections  delivered intranasally , and this results in dissemination 24 h postinfection. The inoculum was plated for confirmation of bacterial number/load. Following this, mice (n = 6 to 7/group) were treated as per the schematic in K. pneumoniae infection, GEN MNs were applied to the dorsal surface of each murine ear and held in place using micropore tape for 24 h, after which they were removed and replaced with additional MNs. GEN delivered i.m. to the thigh muscle of the hind limb was included as control. At 48 h postinfection, mice were sacrificed and the organs were harvested for analysis of bacterial burden. Body weights were monitored over the course of infection; however, no significant differences between groups were observed 48 h postinfection (P = 0.0053) and lungs (P = 0.0006) of mice treated with MNs compared with their untreated counterparts, demonstrating the in vivo activity of GEN delivered via MN. Specifically, application of GEN MN resulted almost a two-log reduction in the number of CFUs in the NALT and a 3.4-log reduction in lung CFU. A one-way analysis of variance (ANOVA), followed by correction for false discovery rate, was used for determination of statistical significance. A greater spread of results was evident in mice receiving GEN MNs than that of mice receiving GEN i.m., and this is likely attributable to the variability in the MNs manufactured under small-scale laboratory conditions. All experiments were performed in accordance with the UK Home Office and approved by the Queens University Belfast Ethical Review Committee.We subsequently probed the capacity of GEN MNs to induce therapeutic effects fections , 21. GENpathogen –24. Micenfection . As demoK. pneumoniae infection in mice. While further studies are warranted to demonstrate complete clearance, this is the first reported study utilizing MNs for the treatment of bacterial infection. MNs may be a potentially viable delivery platform for antibiotic delivery, offering the possibility to expand access to lifesaving antibiotic treatment in low-resource settings. As MNs dissolve upon insertion in the skin, they circumvent the generation of sharps waste and associated transmission of blood-borne pathogens. Our ongoing efforts entail the optimization of GEN MNs to increase bioavailability and develop a thorough understanding of the pharmacokinetics and pharmacodynamics of GEN delivered via this route compared with that delivered i.m. In the era of increasing antimicrobial resistance, novel approaches for empirical therapy are necessitated and MNs may offer an ideal solution. Importantly, as MNs bypass the gastrointestinal microbiota, they also offer an alternative delivery option for antibiotics that are currently delivered orally and could potentially prevent dysbiosis of the gut microbiota.In conclusion, the results presented herein collectively demonstrate that MNs containing GEN effectively control


Meanwhile, CH2O can be generated through three main ways, although the process still depends on the decomposition of epoxy functional groups. H2O is produced by free radical collision and dehydration. The production of small-molecule gases has the following sequence: CO2, CH2O, CO, and H2O. The produced gases have the following order according to amount: CO2, CH2O, H2O, and CO.High temperatures caused by partial discharge results in the decomposition of insulating epoxy resins in electrical equipment. In this paper, the ReaxFF force field is used to investigate the decomposition process of epoxy resins cured by anhydride and the formation mechanisms of small-molecule gases. Results show that the initiation reaction is the cleavage of an ester bond linked with an epoxy resin. Produced by the decomposition of ester groups, CO Consequently, the epoxy resins are degraded. In particular, the surfaces of the epoxy resins become scabrous and gradually form grooves, thereby increasing the possibility of breakdown caused by the development of an electric tree in the epoxy resins [Anhydride-cured epoxy resins are widely used as solid insulators in large electrical equipment because of their excellent resistance to leakage traces . Despitey resins ,3,4,5. My resins ,7. Thus,2 and H2O and certain amounts of acetylene, acetone, and other small volatile substances. Gao et al. [There have been many researchers investigating the decomposition mechanism of epoxy resin ,9 and tho et al.  confirmeo et al. ,15,16 st2O, and other main small-molecule products include H2O, CO, and H2 [2O and H2 production rates [The ReaxFF reactive force developed by Duin and Dasgupta et al.  defines , and H2 . Zhang eon rates . In this paper, the ReaxFF force field was used to analyze the thermal decomposition process of epoxy resin cured by acid anhydride at different angles. Several representative small-molecule gas products were then studied to produce a theoretical basis for the aging process and theory of solid insulation in electrical equipment.In this paper, three models of non-cross-linked epoxy resin were built. Module 1 contains one pure epoxy resin (PEP) molecule, which is formed by the dehydration condensation of two bisphenol A diglycidyl ether molecules, as shown in 3. Annealing via an iterative procedure was then performed, beginning with a molecular dynamics calculation by NVT ensemble at 600 K and then by NPT ensemble at 600 K and a constant pressure of 1.01 MPa (1 atm) for 100 ps; in the succeeding iterations, the temperature was decremented by 50 °C and the process was repeated until the specified temperature for the dynamics was 300 K (around room temperature). After which, geometry optimization of 5000 iterations was performed to get a more stable structure. The final densities of the three models were 1.13, 1.13, and 1.17 g/cm3 (The first step is to build three-dimensional periodic models, the initial densities of which were all set to 0.5 g/cm17 g/cm3 . SubsequThe structures before and after the optimization of the three models are presented in The carbon-oxygen bonds in epoxy resins tend to break, as shown in 2, and this result is consistent with the conclusion in this paper [2 is mainly derived from acyloxy groups in acid anhydride. The ⑦ carbon-oxygen bond (2O. is paper . CO2 is gen bond b then brgen bond d. This p57, C2, and C3 over the simulation time is shown in 57 is a reactant. The main chain of the epoxy resin molecule starts to break from about 65 ps, and all the main chains of epoxy resin molecules are broken until 640 ps. The C2 products mainly include ethylene radicals and acetaldehyde radicals, and the C3 products contain propylene radicals, acetone radicals, acrolein radicals, propylene alcohol radicals, and so on.2, CH2O, H2O, and CO contents over the simulation time. The first gas product is CO2 at about 70 ps, followed by CH2O and H2O. Thus, this result agrees with those of Model 2. The most abundant gas product is CO2 because of the numerous acyloxy groups. The limitations in this paper include the simulation temperature and time result in a small amount of CH2O and H2O, and the absence of H2 and CH4. The amount of hydroxyl groups in the model is comparable to the number of epoxy functional groups. However, the amount of CH2O is larger than that of H2O because the activation energy to form CH2O is lower than that of H2O.2 formation is p1~p3, as shown in 2O can be generated in three ways because the right-hand side of the ⑦ carbon-oxygen bond in The main mechanism of COThe initiation reaction of the decomposition of anhydride-cured epoxy resin is the cleavage of an ester bond;2. The other products were generated in the following sequence: CH2O, CO, and H2O. With respect to content, the following order was observed: CH2O, H2O, and CO.The first and the most abundant product is CO2 is produced from the carbonyl oxygen bond in anhydride; CH2O, from the decomposition of epoxy functional group; H2O, by free radical collision and dehydration.COIn this paper, the ReaxFF force field was used to simulate a number of epoxy resin models, and the decomposition process of epoxy resin cured by anhydride and the formation path of main gas products were analyzed. The conclusions are as follows:


Long non-coding (lnc) RNAs are a class of functional RNA molecules greater than 200 nucleotides in length, and lncRNAs play important roles in various biological regulatory processes and response to the biotic and abiotic stresses. LncRNAs associated with salt stress in cotton have been identified through RNA sequencing, but the function of lncRNAs has not been reported. We previously identified salt stress-related lncRNAs in cotton (Gossypium spp.), and discovered the salt-related lncRNA-lncRNA973.Arabidopsis and overexpressed. Along with the increased expression compared with wild type under salt stress conditions in transgenic plants, the seed germination rate, fresh weights and root lengths of the transgenic plants increased. We also knocked down the expression of lncRNA973 using virus-induced gene silencing technology. The lncRNA973 knockdown plants wilted, and the leaves became yellowed and dropped under salt-stress conditions, indicating that the tolerance to salt stress had decreased compared with wild type. LncRNA973 may be involved in the regulation of reactive oxygen species-scavenging genes, transcription factors and genes involved in salt stress-related processes in response to cotton salt stress.In this study, we identified the expression level, localization, function, and preliminary mechanism of action of lncRNA973. LncRNA973, which was localized in the nucleus, was expressed at a low level under nonstress conditions but can be significantly increased by salt treatments. Here lncRNA973 was transformed into LncRNA973 was localized in the nucleus and its expression was increased by salt treatment. The lncRNA973-overexpression lines had increased salt tolerance compared with the wild type, while the lncRNA973 knockdown plants had reduced salt tolerance. LncRNA973 regulated cotton responses to salt stress by modulating the expression of a series of salt stress-related genes. The data provides a basis for further studies on the mechanisms of lncRNA973-associated responses to salt stress in cotton. Trans-acting lncRNAs may act as signals, guides or scaffolds for the chromatin to regulate the expression of target genes located in distant chromosomal domains or even in different chromosomes. Cis-acting lncRNA is located upstream and downstream of the encoded protein and interacts with cis-acting elements of the promoter or co-expressed genes, thereby regulating gene expression at the transcriptional or post-transcriptional level. Plant lncRNAs may play key roles in flowering time, gene silencing, root organogenesis, seedling photomorphogenesis, re-production, and responses to the biotic and abiotic stresses [Long non-coding (lnc) RNAs are a class of RNAs with transcripts greater than 200 nucleotides in length that do not have the capacity to encode proteins , 2. On tstresses –12.Arabidopsis thaliana, lncRNAs, such as cold-assisted intronic noncoding RNA (COLDAIR) and cold-induced long antisense intragenic RNA (COOLAIR), have been demonstrated to mediate chromatin-modifying activities in the transcriptional silencing of FLC (FLOWERING LOCUS C) during vernalization [At4, IPS1 (INDUCED BY PHOSPHATE STARVATION1) and Pi-deficiency-induced long-noncoding RNA1 (PILNCR1), act as miRNA decoys through a target mimicry mechanism, thus sequestering the miRNAs with regulatory roles away from their intended target genes [HID1) negatively controls the expression of PHYTOCHROME INTERACTING FACTOR 3 (PIF3) and regulates seedling photomorphogenesis [Arabidopsis, lncRNA ELF18-INDUCED LONG-NONCODING RNA1 (ELENA1) enhances resistance against Pseudomonas syringae, which can dissociate the FIB2/MED19a complex and release FIB2 from the PR1 promoter to enhance PR1 expression [DRIR) enhances drought and salt-stress tolerance [The functional mechanisms of lncRNAs in many plant species are not yet fully understood and only a few lncRNAs have been fully characterized. In lization –17. Addiet genes –20. As aogenesis . In Arabpression . Furtherolerance .Gossypium (G.) hirsutum, accounts for > 90% of the annual cotton production worldwide. However, water limitations and the salinization of cotton cultivation areas are challenges to cotton production. High salinity also causes severe limitations to cotton growth and yield by inhibiting leaf expansion, reducing chlorophyll content and increasing hyperionic and hyperosmotic stresses [SOS1 [SARP1 [NAC transcription factors [G. barbadense and G. hirsutum using RNA-seq [G. arboreum fibers and leaves [Verticillium dahlia-resistant island cotton (G. barbadense cv. ‘7124’) and susceptible upland cotton (G. hirsutum cv. ‘YZ1’) using RNA-seq [G. hirsutum, and lncRNA883 was co-expressed with its downstream gene Gh_D03G0339. Additionally lncRNA973 might be a mimic target of miRNA399 [Cotton (Gossypium spp.) is an economically important fiber crop, which produces a natural renewable fiber for the textile industry, provides edible protein for livestock feed and is a source of oil and biofuel. The upland cotton stresses . A numbees [SOS1 , SARP1 [1 [SARP1 , NAC tra factors . At pres RNA-seq , 27. Zoud leaves . Lu et ad leaves . Zhang e RNA-seq . Deng etmiRNA399 . However+ and K+ transport, and transcription factors, that may collectively contribute to an enhanced salt-stress tolerance.In this study, we identified and characterized the functions of a lncRNA, lncRNA973. The expression of lncRNA973 was increased by salt treatments. An in situ hybridization analysis revealed that lncRNA973 localized mainly to the nucleus. The overexpression of lncRNA973 enhanced tolerance to salt stress, while reducing its expression had the opposite effect. Transcriptome data and a real-time PCR analysis revealed that lncRNA973 modulates the expression levels of genes involved in the stress response, including reactive oxygen species (ROS)-scavenging, NaX1 and lncRNA973 X2). The corresponding genomic region was ~ 3.2 Kb, with lncRNA973 X1 and lncRNA973 X2 being 2134 bp and 2888 bp in length, respectively. In addition, lncRNA973 X2 had an exon longer than lncRNA973 X1; therefore, the primers for detecting the expression of lncRNA973 X2 were designed to this exon, primers are designed at this exon location to distinguish them from lncRNA973 X1. The expression levels of lncRNA973 X1 and X2 in stem, root and cotyledon were analyzed using quantitative real-time PCR (qRT-PCR), and the expression levels of lncRNA973 X1 in roots and cotyledons were relatively higher, while those of lncRNA973 X2 were relatively lower in these tissues  revealed that lncRNA973 had no coding capacity  was used as a positive control. For the lncRNA973 and GhUBQ7 probes, a sequence specificity analysis was performed in the cotton genome, and specific probes were selected for hybridization. The fluorescence signal of lncRNA973 could be seen mainly in nuclei of root cells that were hybridized with the DIG-labeled probe, and was also weak signals in the cytoplasm -mediated transformation of wild type (WT) Arabidopsis (Col-0 ecotype) was performed. Five independent transgenic lines were obtained, and three overexpression (OX-)  were chosen for further study  were placed on solid 1/2 MS medium supplemented with 0 mM, 100 mM and 150 mM NaCl. Then, we determined the germination rates of seeds treated with different salt concentrations from 1 to 7 d and the phenotypes after seed germination. The OX-lncRNA973 lines displayed no abnormal phenotypes under normal (0 mM) conditions. The germination rates of OX-lncRNA973 lines grown under normal (0 mM) conditions resulted in no abnormal phenotypes compared with that of the WT , which is frequently used for the analysis of gene functions. The VIGS sequences of lncRNA973 and CLOROPLASTOS ALTERADOS 1 (CLA1) genes were independently cloned into a TRV2 vector  were used as positive and negative controls, respectively. Approximately 490 bp fragment of lncRNA973 and 465 bp of CLA1 were used in the VIGS system. At approximately 2 weeks post-infiltration, marker gene TRV2:CLA1-containing plants started to display an albino phenotype in their true leaves , peroxidase (POD) and catalase (CAT), play important roles in stress responses to adversity. The activities of ROS-scavenging enzymes decreased in both TRV2 and VIGS plants treated with salt; however, VIGS cotton exhibited more significant decreases compared with the TRV2 plants Fig. g. Excessnditions . There wnts Fig. h. Lower ned Fig. i. The imnts Fig. j. In addnts Fig. : Fig. S4Δ1-PYRROLINE-5-CARBOXYLATE SYNTHETASE (P5CS) under salt-stress conditions could promote the accumulation of Pro [P5CS, increased twofold in salt-treated VIGS plants compared with TRV2 lines  encodes a vacuolar Na+/H+ antiporter involved in salt resistance that acts in the electroneutral exchange of protons for cations, such as Na+, across the plasma membrane [NHX7 was more highly expressed in VIGS plants than TRV2 plants under salt-stress conditions  is activated by the abscisic acid (ABA) signaling associated with salt-stress responses [SOD, POD and CAT. Their expression levels were significantly inhibited in VIGS plants exposed to salt stress but increased in control plants  transcription factors. The co-expression coefficients between the transcription factors and lncRNA973 were generally greater than 0.99, indicating possible regulatory relationships between them. RT-qPCR was used to detect the expression of these transcription factors in salt-treated and untreated plants. The expression levels of MYB5 and WRKY46 were lower in VIGS plants than control plants under salt-stress conditions  seeds were germinated in a mixture of peat and vermiculite at 28 °C. Cotton seeds were grown under a 16-h/8-h light/dark cycle at 28 °C/22 °C. Transgenic and WT Arabidopsis  seeds were surface-sterilized using 70% alcohol for 5 min and 2% NaClO for 5 min, then washed five times with sterile ddH2O and germinated on 1/2 Murashige-Skoog (MS) medium. Seeds were first vernalized for 3d at 4 °C in the dark and then germinated in a growth chamber at 22 °C, 16-h /8-h light/dark photoperiod.In this study, the cultivar cription . Upland Upland cotton ‘SN91–11’ plants were grown under standard field conditions. For the tissue/organ-specific expression profiling analysis, cotyledons, stems and roots were harvested from 10-d-old seedlings. Cotton seedlings were subjected to different abiotic stress treatments after the expansion of the first true leaf. Uniformly cultured cotton seedlings were cultivated in Hoagland’s solution supplemented with 250 mM NaCl. The roots, stems, and leaves of the cotton seedling were collected at designated time points . All the tissues/organs were frozen in liquid nitrogen and stored at − 80 °C until total RNA was extracted.cis- and trans-acting. Trans regulation is not dependent on positional relationships, and RNAplex [trans-target genes by calculating the binding free energy of lncRNA973 and its co-expressed protein-coding genes  (TaKaRa) and the Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR System . The reaction was performed using a two-step method. The step was 95 °C for 30 s as the initial denaturation, the second step was 40 cycles of 95 °C for 5 s and 60 °C for 30 s for amplification in a final volume of 20 μL, which contained 2 μL of a 1/10 dilution of cDNA in water, 10 μL of the 2 × SYBR Premix Ex Taq, 0.5 μM concentrations of forward and reverse primers, and ddH2O. The kits used for detecting miRNA expression were Mir-X™ miRNA First-Strand Synthesis and TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa). The miRNA specific primers were designed based on mature miRNA sequences as 5′ primers for qRT-PCR . The RNA concentrations were quantified using a NanoDrop ND-2000 spectrophotometer . The RNA was used for first-strand cDNA synthesis with a the PrimeScript RT-qPCR kit , reverse transcription primers are a mixture of random primers and oligo dT, and the cDNA was then used as a template for lncRNA and mRNAs qRT-PCR amplification. qRT-PCR reactions were performed using SYBR t method , 55. TheRNA in situ hybridization probes were designed as previously described by Duncan et al. , and eacAfter fixation, the tissue was dehydrated with gradient alcohol and then soaked in wax, embedded and sectioned. Paraffin sections were baked at 62 °C for 2 h, and then placed in xylene and alcohol of different concentrations for dewaxing. Tissue samples were digested with proteinase K  at 37 °C, and washed with phosphate buffer saline (PBS) (Servicebio) three times for 5 min each. The pre-hybridization solution was added and incubated at 37 °C for 1 h, Then, 100 μL of hybridization solution, containing the probes at final concentrations of 250 nM, was added to the pre-hybridization solution of each slide, and the slides were hybridized at 37 °C overnight in the dark. The hybridization solution containing the unhybridized probe was removed using a pipette in the morning. After hybridization, the slides were first washed with 2 × SSC at 37 °C for 10 min, then washed with 1 × SSC at 37 °C for two times for 5 min each, and finally washed with 0.5 × SSC at room temperature for 10 min. Then bovine serum albumin (BSA) was added and the slides were incubated for 30 min at room temperature. Finally, after the hybridization solution was removed, anti-DIG-488 was added to the slides and incubated at 37 °C for 50 min before washing with PBS four times for 5 min each. 4′,6-diamino-phenylindole (DAPI) dye solution was added to the slides, and then, they were incubated for 8 min in the dark. Anti-fluorescence quenching sealing tablets were added and sealed after washing.https://www.3dhistech.com/caseviewer).An automatic slice scanning system, 3D HISTECH, was used for imaging. The following wavelengths were used for fluorescence detection: for DAPI, an excitation of 330–380 nm and signal detection of 420 nm; for probes labelled with DIG an excitation of 465–495 nm and signal was detection of 515–555 nm. Maximum projections and an analysis of three-dimensional pictures were performed using CaseViewer . RNAprep Pure Plant Kit Polysaccharides & Polyphenolics-rich (TIANGEN) kit was used to extract the respective RNA from nucleus and cytoplasm, respectively. Reverse transcription and RT-PCR were performed by referring to the above qRT-PCR process. Ubi promoter in the recombinant binary vector pCAMBIA1300-GFP using the Kpn I and Pst I restriction loci. The constructs were transferred into A. tumefaciens strain GV3101 and transformed into WT Arabidopsis using the floral dip method. Transgenic lines were selected on 1/2 MS medium supplemented with 50 mg/L hygromycin. Independent transgenic lines were obtained, plant DNA was extracted, and PCR was performed to amplify and authenticate the lncRNA973 sequences transformed into these transgenic Arabidopsis plants. Three T3 homozygous lines were selected and used for subsequent salt-stress tolerance tests.Using the previously obtained transcribed sequence, two specific primers  and cloned into the TRV2 vector using the T4 DNA ligation cloning method. The lengths of the silencing fragments for lncRNA973 and GhCLA1 were 490 and 465 bp, respectively. The TRV-VIGS constructs were also transformed into A. tumefaciens strain GV3101. Then, strain GV3101, containing the different constructs, was infiltrated into 10-day-old cotton seedling’ cotyledons according to the method described by Gao et al. [CLA1 (CLOROPLASTOS ALTERADOS 1) was utilized as the positive control and the empty vector TRV2:00 was the negative control. The silencing of endogenous gene expression was detected using an RT-PCR assay. All the primers used in the VIGS experiments are listed in Additional file The plasmid TRV2 (pYL156) and TRV1 (pYL192), which were purchased from Changsha Yingrun Biotechnology Co., Ltd., were used for VIGS. The VIGS sequence was designed using the SGN VIGS Tool . The plants were observed and photographed during the treatment.Two weeks after infection, the albino leaf phenotype appeared in plants transformed with the positive control gene Arabidopsis leaves in the field was detected using ethanol and acetone extraction. Approximately 0.1 g of the leaves (with the main stem removed) were placed in a glass test tube, and 10–20 mL of a mixture of ethanol and acetone was added and shaken at room temperature for 16 h in a shaker. The chlorophyll was measured for absorbance at 645 and 663 nm in a spectrophotometer. The total chlorophyll content is calculated by the following equation: ((8.05*A663 + 20.29*A645)) *V (extraction volume)/ (weight of sample) *1000.Leaves of stress-treated and normal control plants of similar developmental stages were used to measure the chlorophyll, relative water, Pro and MDA contents. The chlorophyll content of cotton and The relative water content was determined as described by Wang et al. . Leaves t-test.The MDA level was determined using the thiobarbital acid method as described previously . All theSalt-treated and untreated fresh cotton leaves (~ 0.1 g) at similar stages of development were used to detect ROS-related enzyme activities. The POD, CAT and SOD activity levels were measured and performed according to kit instructions .Additional file 1: Figure S1. Analysis of the lncRNA973 X1 open reading frame and molecular localization of lncRNA973. a. All frames (gray boxes) were identified in the three forward frames. The two longest open reading frames encoded 107 and 53 amino acids (aa). b. RT-PCR analysis the fragment of lncRNA973 from total RNA, nucleus RNA and cytoplasm RNA. Nuclear U6 and cytoplasmic tRNA were used as controls. A house-keeping mRNA (UBQ7) was used as a negative control. (TIF 1175 kb)Additional file 2: Figure S2. Expression of lncRNA973 in transgenic Arabidopsis and wild type (WT). a. T-DNA region of the plasmid pCAMBIA1300-GFP for transformation. lncRNA973 was controlled by Ubiquitin protein promoter Ubi. NOS, used as resistance selection. RB, T-DNA right border; LB, T-DNA left border. b. The full length of lncRNA973 transcript was obtained from cotton by PCR amplification. c. PCR analysis the transcript of lncRNA973 was inserted into the genome of Arabidopsis. d. Relative transcript levels of lncRNA973 in the WT and transgenic lines, OX-lncRNA973–2, OX-lncRNA973–3 and OX-lncRNA973–5. **, p < 0.01 by Student’s t-test. Data represent means ±SD. (TIF 740 kb)Additional file 3: Figure S3. Relative expression level of the lncRNA973 neighboring gene Gh_D04G0660 in the control (TRV2:00) and TRV2:lncRNA973 mutant. (TIF 269 kb)Additional file 4: Figure S4. Total chlorophyll, MDA, and Pro contents and activity of CAT in the WT, TRV2:00, and TRV2:lncRNA973 leaves under salt stress and mock. WT: Uninfected cotton. Mock, with water treatment. NaCl: 250 mM NaCl treatment. *, p < 0.05 and **, p < 0.01 by Student’s t-test compared with untreated (Mock). Data represent means ± SD. (TIF 491 kb)Additional file 5: Figure S5. Relative expression levels of selected genes in salt-treated WT and OX-lncRNA973 Arabidopsis plants. RNA was extracted from WT and OX-lncRNA973 plants, and gene expression levels were measured by RT-qPCR normalized against the Atactin gene. Relative expression levels are shown for a. AtRBOHB, b. AtRBOHD, c. AtP5CS1, d. AtNHX7, e. AtSOD, f. AtCAT, g. AtPOD, h. AtRD22, i. AtRD29A, j. AtRD29B, k. AtNAC3, l. AtWRKY46, m. AtERF, n. AtMYB5. Mock, without salt treatment, NaCl: 200 mM NaCl treatment. *, p < 0.05 and **, p < 0.01 by Student’s t-test compared with untreated (Mock). Data represent means ± SD. (TIF 1397 kb)Additional file 6: Table S1. lncRNA973 co-expressed protein-coding genes information. (XLS 3055 kb)Additional file 7: Table S2. Gene-specific primers and probes used in the research. (XLSX 13 kb)


The MAPPIT  method allows high-throughput detection of protein interactions by very simple co-transfection of three plasmids in HEK293T cells, followed by a luciferase readout. MAPPIT detects a large percentage of all protein interactions, including those requiring posttranslational modifications and endogenous or exogenous ligands. Here, we present a straightforward method that allows detailed mapping of interaction interfaces via MAPPIT. The method provides insight into the interaction mechanism and reveals how this is affected by disease-associated mutations. By combining error-prone polymerase chain reaction (PCR) for random mutagenesis, 96-well DNA prepping, Sanger sequencing, and MAPPIT via 384-well transfections, we test the effects of a large number of mutations of a selected protein on its protein interactions. The entire screen takes less than three months and interactions with multiple partners can be studied in parallel. The effect of mutations on the MAPPIT readout is mapped on the protein structure, allowing unbiased identification of all putative interaction sites. We have thus far analysed 6 proteins and mapped their interfaces for 16 different interaction partners. Our method is broadly applicable as the required tools are simple and widely available. The expansion of the protein interactomics field is driven by a wide variety of techniques for the detection of protein-protein interactions (PPIs). Classical techniques, such as yeast two-hybrid  and affiHigh-resolution structure determination of protein complexes via X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy or cryo-electron microscopy is inherently slow and is often very challenging. Even with major advances and speedup in structure determination, these methods cannot keep up with the rapid growth of the interactome . This leAlternatively, many approaches can predict protein complex structures based on the separate models or structures of the interacting proteins in the complex ,15. SeveIn this paper, we describe a straightforward method combining random mutagenesis with MAPPIT that allows extensive mapping of PPI interfaces in intact human cells. We first set up and optimize MAPPIT assays that specifically detect the interaction between the protein of interest and its interaction partners A. Their E. coli DH10B cells. For each PCR condition, 24 colonies are grown overnight in a 2× Yeast Tryptone medium in a 96-deepwell block. DNA is purified via a 96-well miniprep protocol using the Nucleospin™ Robot-96 plasmid kit . Next, the MAPPIT mutants are sequenced on Applied Biosystems 3730XL DNA Analyzers to determine the PCR condition with the highest number of single missense mutations. In our studies, up to one third of the random mutant clones contain single missense mutations [The DNA insert is randomly mutated via error-prone PCR using Mutazyme II DNA polymerase, following the guidelines of the Genemorph™ II Random Mutagenesis kit  B. This putations ,19.E. coli colonies of the optimal PCR condition are plated and single colonies are inoculated in 96-deepwell blocks for DNA miniprep purification, as described above  reporter plasmids, as optimized in step 1 D. On the3)4Mg(OH)2·5H2O, 5.34 mM MgSO4·7H2O, 66.6 mM DTT, 0.2 mM EDTA, 270 µM coenzyme A , 530 µM ATP , 470 µM luciferin . The luminescence is measured with the EnSpire plate reader .On day three, 4 out of 8 wells for each transfection mixture are stimulated with leptin or Epo. The 4 other wells are left unstimulated D. After First, the “MAPPIT signal” is determined by dividing the average of the luciferase counts of the 4 stimulated wells by the average of the luciferase counts of the 4 unstimulated wells E. Then, We also calculate the relative MAPPIT signals of the wildtypes, as described above, and plot the variation. The lowest and highest wildtype relative MAPPIT signal determines lower and upper cut-off value, respectively. A. The MAFirst, we create an “attribute list” in a fixed .txt format in which the relative MAPPIT signal of a mutant is assigned to its corresponding residue number. This attribute list is then loaded in the free modelling program University of California San Francisco (UCSF) Chimera via its “define attribute” tool . The relWe successfully applied random mutagenesis combined with MAPPIT for the interaction interface analysis of six targets in four unrelated protein families: the antiviral host restriction factor apolipoprotein B messenger RNA-editing catalytic polypeptide-like G (Apobec3G) , the rinAll tested mutants are available as individual clones and are easily cloned into other vectors. This permits retesting of a large number of mutations in different assays without the need of extra mutagenesis. The biological importance of new potential interfaces identified via MAPPIT was confirmed via orthogonal assays, such as co-immunoprecipitation and signalling assays ,19. ProtOur MAPPIT studies could nicely reproduce effects that were observed in vivo. As a viral defence against the human host restriction factor Apobec3G, this protein is degraded by HIV-1 Vif via ubiquitination mechanisms. A D128K mutation protects simian Apobec3G against degradation via HIV-1 Vif. Via MAPPIT, we could demonstrate that the Apobec3G D128K mutation specifically breaks the Apobec3G-Vif interaction . Mice coMutations can affect an interaction by an effect on folding or stability. For important interpretations of mutants in diseases, such folding/stability effects should be tested experimentally. Mutations can also affect an interface via local folding effects, which may be harder to detect. For example, the L265P mutation was predicted to destabilize the protein by FoldX predictions ,29. MoleWhile MAPPIT can help to demonstrate defects in protein interactions by disease-associated mutations, proper interpretation of disease-causing mutations requires additional studies, such as orthogonal interaction assays, and determination of expression level and turnover, activity assays and if possible in vivo studies.MAPPIT operates in intact mammalian cells, providing a natural environment with endogenous cofactors and regulatory proteins, and is therefore able to detect protein interactions requiring PTMs such as phosphorylation. Several observations indicate that MAPPIT baits and preys behave like their unmodified proteins, and that they can be the target of normal post-translational modifications like phosphorylation and ubiquitination. The Vif prey is rapidly degraded like untagged Vif, which is a target of proteasomal degradation via ubiquitination. Using Vif as MAPPIT prey required introduction of a mutation in the Vif SOCS box, abrogating its function in an E3 ubiquitin ligase complex, and preventing degradation of the Vif prey . A MyD88The MAPPIT signal is also affected by mutations disrupting the structure or proper folding of the protein. For example, most mutations in the core of the protein affect the MAPPIT signal. As mutations at the protein surface can equally disrupt the protein structure, it is mandatory to carefully inspect possible structural effects. As a tool for estimating the mutational effect on the protein structure, we use the FoldX prediction program . HoweverWe analyse expression levels of prey or bait mutants to exclude an effect on the MAPPIT interaction due to differential expression. Expression levels of MAPPIT preys can be determined via Western blot, as these contain a Flag-tag. Expression of the MAPPIT baits can be determined in a  MAPPIT experiment with a MAPPIT prey specifically recognizing the receptor portion or JAK2 portion of the bait. However, expression levels of bait or prey mutants are not a good measure for their proper folding or structural integrity. Very often, mutations that clearly disrupt the structure or folding of the protein hardly affect the expression level.Our method identifies all regions required for protein interaction and some of these do not necessarily represent the actual interface between bait and prey. This is relevant for proteins binding their target as a homodimer or requiring a third interaction partner. In MAPPIT, both bait and prey proteins are free to homodimerize or even homo-oligomerize. We do find that mutations in the homodimerization interface of the mutated target affect its heterotypical interaction. Similarly, for interactions that require an extra third protein or other ligand, mutations in the binding site of these ligands also affect the MAPPIT readout . In this respect, NMR methods, crystallography and cryo-EM can reveal the details of the actual interface between proteins, while extensive mutagenesis can only indicate the position of the interfaces on the interaction partners, without revealing how these surface areas interact to form the interface. However, our method does reveal how specific mutations affect the interaction, which cannot be directly deduced from most other methods and can be very complementary with structure determination.Our combination of MAPPIT and random mutagenesis allows the extensive and unbiased validation of interfaces in high- or low-resolution structures. Interface residues identified via MAPPIT can also guide the docking process in in silico data-driven protein-protein docking . The met


Moreover, the catalytic activity of GOx could be further improved by the hyperthermia of MNS-GOx upon near-infrared laser irradiation. Most intriguingly, MNS-GOx could achieve “turn-on” MR imaging and “turn-off” PA imaging simultaneously. The theranostic capability of MNS-GOx was evaluated on A375 tumor-bearing mice with all tumor elimination. Our findings integrated molecular imaging and starvation-based synergistic cancer therapy, which provided a new platform for cancer nanotheranostics.Cancer theranostics based on glucose oxidase (GOx)-induced starvation therapy has got more and more attention in cancer management. Herein, GOx armed manganese dioxide nanosheets (denoted as MNS-GOx) were developed as cancer nanotheranostic agent for magnetic resonance (MR)/photoacoustic (PA) dual-modal imaging guided self-oxygenation/hyperthermia dually enhanced starvation cancer therapy. The manganese dioxide nanomaterials with different morphologies  were synthesized by a biomimetic approach using melanin as a biotemplate. Afterwards, the manganese dioxide nanosheets (MNS) with two sides and large surface area were selected as the vehicle to carry and deliver GOx. The as-prepared MNS-GOx can perform the circular reaction of glucose oxidation and H Meanwhile, tumor microenvironments also contain a high content of H2O2. As we know, the decomposition of H2O2 can produce water and O2, thus promising the melioration of tumor hypoxia. Manganese oxide (MnO2) nanomaterials can catalyze decomposition reaction to produce O2 to modulate solid tumor hypoxia for effective starvation therapy2 and GOx will accelerate glucose consumption to improve the starvation therapeutic effect. Recently, MnO2-based theranostic agents have been widely explored2 nanoparticles2 nanoplatform2 nanosheets2 nanomaterials need polyethylene glycol (PEG) modification to improve their biocompatibility and water solubilityThe products of glucose oxidation are including gluconic acid and hydrogen peroxide . The melanin coated MnO2 have good biocompatibility and strong NIR absorption. Afterwards, GOx armed manganese dioxide nanosheets (denoted as MNS-GOx) were explored for cancer nanotheranostics with following characteristics  the enhancement of catalytic activity of GOx by the hyperthermia of MNS-GOx; (iii) activatable magnetic resonance (MR)/photoacoustic (PA) dual-modal imaging. The theranostic capability of MNS-GOx was carefully investigated both in vitro and in vivo. The as-prepared MNS-GOx has great potential in traceable cancer therapy, especially for MR/PA dual-modal imaging guided self-oxygenation/hyperthermia dually enhanced starvation cancer therapy.Herein, we used melanin as the biotemplate to directly synthesize MnOs Scheme : (i) the2 nanosheets (MNS) were synthesized by the biomimetic synthesis method using melanin as the biomimetic template. Typically, 0.4 mL melanin (2 mg/mL) and 3.2 mL ethanol were added into 48 mL deionized (DI) water. The pH value of the mixed solution was adjusted to 7.4~7.8. Then the solution was heated to 90 °C, 0.32 mL potassium permanganate (KMnO4) (0.05 M) was slowly added under vigorous stirring. The system was kept stirring for 30 min at 90 °C. The product was purified and concentrated by an ultrafilter (30 kDa).MnO in vitro and in vivo experiments, respectively.GOx was conjugated on the surface of MNS through the 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)/N-Hydroxysuccinimide (NHS) reaction between carboxyl groups of MNS and amine groups of GOx. In brief, 10 mg EDC and 5 mg NHS were added into 4 mL MNS (1 mg/mL). After 2 h, 40 µL GOx (2 mg/mL) was added and the mixture solution was stirred for another 8 h. The MNS-GOx was washed with DI water for several times and suspended in water for further use. In further experiments, the ratios of 200:1 and 1:1 of MNS: GOx were prepared for2 nanomaterials were observed by high-resolution field emission transmission electron microscope (TEM) . The thickness of MNS was measured by atom force microscope (AFM) . X-ray diffraction (XRD) pattern of MNS was detected by X-ray diffractometer . Dynamic light scattering (DLS) and Zeta potential was measured on Zetasizer Nano-ZS90 . Fourier-transform infrared (FT-IR) spectra were collected on a FT-IR spectrometer . UV/Vis absorption spectra were measured on a Cary 60 UV/Vis spectrophotometer . The Mn element concentration of MNS was quantified by an inductively coupled plasma-atomic emission spectrometry (ICP-AES) .The morphologies of MnO2+ was detected by ICP-AES. Meanwhile, an isometric buffer solution was added to keep the total volume same.The pH-responsive degradations of MNS and MNS-GOx were monitored by UV/Vis spectrophotometer. The optical density (OD) at 400 nm of MNS (100 µg/mL) or MNS-GOx (100 µg/mL) in PBS solutions  was measured over time. Additionally, 4 mL of MNS (100 µg/mL) or MNS-GOx (100 µg/mL) was loaded into a dialysis bag with the molecular weight cut-off of 3500 Da. Then the bag was immersed into 50 mL of buffer solutions with different pH values  under vigorous stirring at room temperature. At different time points, 2 mL of buffer solution was taken out and the released Mn2 for 3 min. The photothermal stability of MNS (200 µg/mL) were irradiated upon different laser power density of 1 W/cm2 for four on/off cycles . Real-time thermal imaging was recorded by a SC300 infrared camera  and quantified by FLIR Examiner software.The aqueous solutions of MNS (0~200 µg/mL) were stored in Eppendorf tubes, and exposed to 808 nm laser at power density of 1 W/cmT1 relaxivity was detected by a 3 T clinical MRI scanner . An fast spin echo (FSE) sequence with 19 different values of T1 (TR (repetition time)=5000 ms; TE (ehco time) =100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1200, 1300, 1500, 2000 ms) for T1 measurements. T1 relaxation times were calculated by fitting the signal intensities with increasing T1 to I(t)=I0[1-k•exp(-t/T1)] by using a nonlinear least-squares fit of the Levenberg-Marquardt algorithm.MNS and MNS-GOx with different Mn element concentration  were dispersed in PBS solutions with different pH values  for 4 h. Then the max) of GOx obtained from the Michaelis-Menten plots at different temperatures2 for 5 min).30 µL of MNS-GOx (1 mg/mL) or GOx (1 mg/mL) was added into 1 mL of glucose solution (30 mM) under oxygen gas flow (5 mL/min). The glucose concentrations were immediately detected by Yuwell 590 glucometer at different time points . Moreover, the glucose solutions were heated up to 30, 40, 50 or 60 ºC. The kinetic parameters  for 4 h and then exposed to an 808 nm laser at 1 W/cm2 for 5 min. Then the old dulbecco's modified eagle medium (DMEM) media were removed and 5 mg/mL 3--2,5-diphenyltetrazolium bromide (MTT) in 100 μL DMEM media was added into each well. After co-incubation for another 4 h, the old DMEM media were replaced with 150 μL dimethylsulfoxide (DMSO) per well, and the absorbance at 490 nm was monitored by a microplate reader. The cytotoxicity was finally expressed as the viabilities of different-treated cells in contrast to the untreated control cells.For 4/well and then cultured at 37 °C for 24 h. MNS or MNS-GOx  was added into each well and co-incubated for 4 h. Then the cells were exposed to an 808 nm laser at 1W/cm2 for 5 min. After co-incubation for another 24 h, the cells were co-stained by Calcein acetoxymethyl ester  and propidium iodide (PI), and then imaged using an Olympus FV1000 fluorescent microscope.A375 cells were seeded into a 12-well plate at 7*101-weighted images were acquired by FSE sequence at 0, 0.5, 1, 4, 8, 24 h and the parameters were as follows: TR=700 ms; TE=14.3 ms; Flip Angle=145 º; matrix size, 160 × 160; slice thickness, 1.5 mm. Signal intensities were measured in defined regions of interest (ROI) with software named Image J. The PA images were recorded by a Vevo LAZR2100 system  equipped with a 40 MHz, 256-element linear array transducer on tumors.The tumor-bearing nude mice (∼20 g) were intratumoral injected with MNS or MNS-GOx (5 mg/kg). Magnetic resonance imaging (MRI) was performed by a UMR 790 3.0T . TIn vivo treatment was performed when the tumor reached 6 mm in average diameter (10 days after implant). The mice were divided into six groups: PBS as the control group; PBS with laser irradiation (PBS + laser); MNS; MNS-GOx; MNS with laser irradiation (MNS + laser); MNS-GOx with laser irradiation (MNS-GOx + laser). For laser irradiation groups, 808 nm laser with the power of 0.6 W/cm2 was used to irradiate tumor tissues for 5 min after intratumoral injection (dose: 5 mg/kg). For each group, the tumor volumes and body weight were measured every two days, and volume of tumors was calculated as (tumor length) × (tumor width/2)0 (V0 is the tumor volume when the treatment was initiated).A375 cells were implanted subcutaneously into nude mice ∼20 g). 0 g. In vThe red blood cells (RBCs) were isolated from serum by centrifugation of the mixture containing 0.5 mL blood sample and 1 mL PBS solution at 4500 rpm for 3.5 min, then washed the RBCs over five times and diluted the purified cells to 5 mL. Then 0.3 mL volume diluted RBCs suspension was added to quadruple volume of PBS solution with different concentrations of MNS (12.5 to 400 µg/mL). The mixtures were vortexed and kept to stand for 4 h at room temperature. Samples were then centrifuged to measure the absorbance of the supernatants at 541 nm by an UV-vis spectroscopy. RBCs treated with deionized water and PBS were set as positive and negative controls.Tumor tissues were collected from tumor-bearing mice in different groups at day 16 and sectioned into slices for hematoxylin and eosin (H&E) staining. For the toxicity evaluation of MNS or MNS-GOx, mice with intratumoral injection of MNS or MNS-GOx (dose: 5 mg/kg) were sacrificed at day 30 (3 mice per group). Then tumor tissues and major organs  were collected, fixed in 10% neutral buffered formalin, embedded in paraffin, cut into 4 µm sections and stained with H&E. Finally, the images of these histological tissue sections were obtained by a BX41 bright field microscopy (Olympus).P < 0.05 was considered as statistically significant.Data were presented as mean ± standard deviation (SD). Statistical differences among experimental groups were analyzed using one-way analysis of variance (ANOVA) followed by two-tailed Student's t test. 4 using melanin as a template under neutral conditions, and then modified with GOx by a cross-linker, as shown in Figure . Due to an abundant of catechol groups of melanin, it can anchor on the surface of metals oxides2 were strongly depended on the solubility of melanin under different reaction conditions. The as-prepared MnO2 nanomaterials were nanoflowers Figure B at acidic condition (pH < 7), ultrathin nanosheets Figure C at neutral condition (pH 7) and nanowires Figure D at alkaline condition (pH 10). The nanoflowers can be formed when the decomposition rate of KMnO4 was faster in acidic condition and the nanowires were probably formed when the solubility of melanin increased in alkaline solutionFigure S1, the hydrodynamic diameter of MNS was about ~70 nm measured by DLS. High resolution TEM image of MNS indicated that the lattice fringe spacing of MNS were 0.232 and 0.288 nm , which can be attributed to (311) and (411) according to the XRD pattern . EDS spectrum confirmed the high concentration of manganese and oxygen elements in MNS, which is further supported by element mapping images in Figure S4. As shown in Figure S5, the thickness of MNS was ~ 2 nm, which indicated the MNS with ultrathin sheet structure.MNS were prepared by one-step reduction of KMnO2 nanomaterials during the synthesis process. It can be adsorbed on the surface of MNS through the abundant catechol groups of melaninFigure , UV-vis-NIR absorption spectrum of MNS contained the broad absorbance of melanin2 (~400 nm)2 concentration, and the typical equation was Y = 0.003X + 0.0102 . Because the released Mn2+ had five unpaired electrons, Mn-based contrast agents have been widely explored as T1 contrast agents. Based on the profile of 1/T1 vs Mn element concentration of MNS Figure D and MNS-GOx Figure E, the r1 MNS increased from 0.084 to 7.0 mM-1s-1 from pH 7.4 to 5.0, while r1 MNS-GOx increased from 3.3 to 22.8 mM-1s-1Figure F. The r1 MNS enhancement is due to the pH-responsive degradation of MNS, while the r1 MNS-GOx enhancement is due to pH/glucose dual-responsive degradation of MNS, which accelerated the release of Mn2+. In order to observe the degradation process of MNS-GOx, their morphology change was recorded by TEM imaging. As shown in Figure , when MNS-GOx was incubated with 5 mM glucose, most of MNS was decomposed at 17 min, and no MNS can be found at 30 min. These results suggested MNS-GOx exhibited pH/glucose dual-responsive performance.MnO2 nanomaterials can catalyze the decomposition reaction of H2O2 to produce O2, which can promote the reaction of glucose oxidationFigure , the glucose oxidation rate of GOx and MNS-GOx were 2.69 and 3.43 mM/min, respectively. The catalytic activity enhancement of GOx by MNS can be attributed to the self-oxygenation of MNS-GOx during the glucose oxidation reaction that is an O2-depandent reaction. Afterwards, we investigated the catalytic activity of GOx at different temperature. The glucose concentrations were recorded by a glucometer, and extra O2 was supplied into glucose solution for sufficient O2 during the reaction. The results shown that Vmax of MNS-GOx increased from 2.69✕103 M/min at 30 °C to 3.43✕103 M/min at 50 °C Figure B. It was consistent with previous reports that the maximum activity of GOx at 50 °C in vitro experiments were conducted on A375 cells for hyperthermia-enhanced synergistic therapy. MNS or MNS-GOx was incubated with A375 cells for 24 h, and then irradiation with or without 808 nm laser . As shown in Figure , cells incubated with MNS kept their viability over 81%. Cell viabilities of MNS + laser, MNS-GOx and MNS-GOx + Laser groups showed concentration-dependent therapeutic effect. Importantly, MNS-GOx + laser group exhibited better therapeutic effect than that of MNS-GOx only. The synergistic therapeutic effect was further evidenced by live/dead cell staining Figure D-F. No dead cells were found in control group Figure D. For MNS + laser and MNS-GOx + laser groups, most of cells were dead in laser spots Figure E-F. These results indicated the catalytic activity of GOx could be further improved by the hyperthermia of MNS-GOx upon 808 nm laser irradiation. Additionally, we assessed the catalytic activity of GOx before and after laser irradiation. As shown in Figure S9, the glucose reaction rates of MNS-GOx kept similar, which suggested the laser irradiation  can preserve the catalytic activity of GOx.MnOIn vivo MR/PA dual-modal imaging was performed on the A375 tumor-bearing mice. MNS or MNS-GOx was intratumoral injected into A375 tumor-bearing mice at 5 mg/kg dose. Then the treated mice were scanned on 3T clinic MRI scanner. Both MNS and MNS-GOx displayed an obvious T1-weighted enhancement in tumor tissues Figure A. Compared with MNS treated mice, mice in MNS-GOx group exhibited stronger contrast effect at each scan time point. Because MNS-GOx are pH/glucose dual-responsive, it can release more Mn2+ in tumor microenvironment than MNS, and the concentration of released Mn2+ will directly affect the longitudinal relaxivity and MRI contrast effect. So MNS-GOx group exhibited stronger contrast effect compared with MNS group. On the contrary to the increase of MR signal, PA signal of gradually decreased after injection over time Figure . As shown in Figure , the quantified MR signal/noise ratio (SNR) of tumors treated with MNS-GOx gradually increased to the peak value of 2.67 at 8 h post-injection, while the SNR of tumors treated with MNS was only increased to 2.0 at 24 h post-injection. As shown in Figure , the quantified PA signals of tumors treated with MNS or MNS-GOx gradually decreased over time. These results suggested MNS-GOx could achieve “turn-on” MR imaging and “turn-off” PA imaging simultaneously. MRI can offer high resolution contrast for soft tissue without tissue depth limits, but restricted by its poor sensitivity and time-consuming. PAI is convenient and time-saving possesses high optical imaging contrast, but limited imaging depth. So the dual-model imaging can integrate different information to provide accurate diagnosis. A “turn on” MRI can light the deep tissues for precision cancer diagnosis, and the “turn off” PAI can monitor the treatment progress for individual treatment.MNS-GOx was composed of Mn element and melanin, which can be used as contrast agents for MRI and PAI, respectively. in vitro synergistic PTT/starvation therapy effect, the in vivo synergistic therapy was conducted on A375 tumor-bearing mice. Mice were randomly divided into six groups: PBS, laser only, MNS, MNS+laser, MNS-GOx and MNS-GOx+laser. For all groups, the intratumoral injection dose was 5 mg/kg. For laser groups, 808 nm laser  was immediately irradiated on A375 tumor tissues after injection. The tumor volumes were measured every 2 days during 16 days. As shown in Figure , the tumors of PBS and PBS + laser groups grew very fast, while MNS-GOx treated group shown great suppression effect compared with MNS group. Interestingly, the tumors of MNS-GOx + laser group were completely eliminated; while MNS + laser group began to regrow at 10 days after treatment.Based on Figure S11), on the other hand, MNS-GOx can perform the circular reaction of glucose oxidation and H2O2 decomposition for enhanced starvation therapy. The self-oxygenation/hyperthermia dually enhanced starvation cancer therapy shown higher tumor suppression effect than any single treatment. Furthermore, the survival rate of mice in MNS-GOx group was greatly prolonged Figure B. All treated mice had no obvious body weight change during different treatments Figure C. The photographs of A375 tumor-bearing mice further evidenced the synergistic PTT/starvation effect Figure D. Hematoxylin and eosin (H&E) staining images of tumor sections after treatments also indicated an obviously membrane fragmentation or shrinkage of nuclei in MNS + laser group and MNS-GOx + laser group Figure E. But no significant damage or inflammation from H&E stained images of main organs , negligible change of blood biochemistry results, and the hemolysis rates of virous concentrations (15.5-400 µg/mL) of MNS were lower than 5.5% -14. These findings indicated that MNS-GOx exhibited a great potential in hyperthermia enhanced starvation synergistic therapy.On the one hand, the increase of tumor local temperature upon laser irradiation can improve the catalytic activity of GOx, thus consuming more intratumoral glucose was explored as cancer nanotheranostics, especially for self-oxygenation/hyperthermia dually enhanced starvation therapy. The as-prepared MNS can decompose H2O2 to supply O2 for the GOx catalyzed glucose oxidation reaction, thus promising the circular reaction of H2O2 decomposition and glucose oxidation. Moreover, the catalytic activity of GOx could be further improved by the hyperthermia of MNS-GOx upon NIR laser irradiation. Most intriguingly, MNS-GOx could achieve “turn-on” MR imaging and “turn-off” PA imaging simultaneously. The theranostic capability of MNS-GOx was evaluated both in vitro and in vivo. Finally, the as-prepared MNS-GOx exhibited pH/glucose dual-responsive performance, activatable MR/PA dual-modal imaging, and hyperthermia enhanced starvation synergistic cancer therapy, which provided a new nanoplatform for cancer nanotheranostics.In summary, three different morphologies of MnOSupplementary figures.Click here for additional data file.


Pratylenchus coffeae is among the plant parasitic nematodes contributing to yield losses of banana. To determine the status of P. coffeae, a survey was conducted in banana-growing regions of Tanzania and samples collected. The results indicated that in 2015 there was an increase in total counts of P. coffeae extracted from roots compared to that reported in 1999 in Unguja West, North and South. Moreover, we noted its presence for the first time in mainland Tanzania. Generally, the densities of P. coffeae were high on banana roots collected at 500–1000 m above sea level. This information on the status of P. coffeae is important in planning management of nematodes in Tanzania. •Pratylenchus coffeae occurred in about 80% of ten regions where banana was surveyed.•P. coffeae were above 50% for Unguja North, Mbeya and Ruvuma.The incidences of •P. coffeae increased from 74 to 2996/100 g of fresh roots in Unguja island.The population densities of •P. coffeae was reported for the first time.In mainland Tanzania, •P. coffeae occurred on banana roots grown at 500–1000 m a.s.l.High population densities of  In Japan, P. coffeae can cause serious losses of sweetpotato [P. coffeae and is the leading cash crop in Kagera, Kilimanjaro and part of Ruvuma in mainland Tanzania, areas where they also grow banana for staple food and sometimes intercropped with coffee [P. coffeae [P. coffeae could directly affect other important crops in the country. lesions . It is a lesions . Howeverb et al.  in the Zsh roots . Apart fetpotato , the croetpotato . Coffee h coffee . The spi coffeae  and playThe presence and distribution of different nematodes in the area varies with time and is likely due to movement of plant material through the common practice of exchange between farmers or introduction from one country to another . Banana P. coffeae in the banana production systems of Zanzibar and mainland Tanzania. Information on status of P. coffeae will be useful for development of strategic nematode management and improving banana production in Tanzania.This study was conducted to assess the status of 22.1The study was conducted in 10 major banana-growing areas across three agro-ecological zones of mainland Tanzania and one from Zanzibar . The zonIn this survey of the 10 regions, we randomly collected 314 composite samples of each of soil and roots to make a total of 628. The samples were collected following a previously described procedure . A 20 × 2.2P. coffeae . The upper aqueous layer (50 μL) was transferred into a new tube, then 5 μL of cold 3 M NaAc (pH 5.2) and 2× volume of 98% cold ethanol was added, and then samples were precipitated at −20 °C for 1 h. The mixture was centrifuged at 4 °C for 15 min at 20,000 × g RCF. The DNA pellets were washed with cold 75% ethanol and re-suspended in 10 μL of sterilised double distilled water, then stored at −20 °C for further analysis.The lysate was then extracted with an equal volume of phenol/chloroform/isoamyl at the ratio 25:24:1. The solution was mixed well and centrifuged at 4 °C for 5 min at 20,000 × 2.5Pratylenchus spp. The primers were designed from the internal transcribed spacer (ITS)1 and 2 and the 28 subunit (28S) of the ribosomal DNA (rDNA) region of nematodes using the primer design tool of the National Centre for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) as described by Ye et al. [trans-illuminator and cleaned by ExoSap-IT . The cleaned PCR products were added with their respective sequencing primer pairs and sequenced directly by Bioneer Corp, South Korea.The extracted nematode DNA was used as a template for PCR amplification. The PCR was performed using two primer pairs which were designed by authors at Mikocheni Agricultural Research Institute: PC7ITSF (GAGCAGTCGTATTCGTCCGT) and PC7ITSR (AAGTTCAGCGGGTATTCACGTC) that amplifies the ITS1, 5.8S and ITS2 regions; and PC11LSUF (ACAAGTACCGTGAGGGAAAGTTG) and PC11LSUR (TCGGAAGGAACCAGCTACTA) that amplifies the 28S of rDNA. These primers are specific to e et al. . The PCR2.5.1The obtained molecular sequences were compared with other nematode species sequences available in GenBank through NCBI BLASTN homology search. The DNA sequences were analysed by molecular evolutionary genetics analysis (MEGA7) version 7.0 software . The seq2.6P < 0.05.All data on nematode counts were subjected to analysis of variance using the GenStat statistical package . If required, the density of nematodes was square-root transformed. The means were compared using least significant difference at 3P. coffeae found in samples was confirmed using the tail shape of female P. coffeae, which was truncated  and vulva (%) (67.7–80.4 ± 22.4 compared to standard of 76–83); and for males, length (353.8–407.1 ± 64.0.0 μm compared to standard of 450–700 μm) and testis length (113.9–162.1 ± 79.5 μm compared to 278.9 μm). The PCR amplification using primer pairs PC7ITSF/PC7ITSR and PC11LSUF/PC11LSUR showed the correct fragment sizes for all nematode samples. Molecular characterisation confirmed the morphological and morphometric characterisation of P. coffeae nematodes through PCR  fewer roots infected with nematodes at elevations below 500 and above 1000 m a.s.l. Abundance of P. coffeae was highest in soil of elevations 0–500 m a.s.l. and lowest above 500 m a.s.l.  ; in compm a.s.l. .Table 4P4P. coffeae in most regions of mainland Tanzania. The morphology and molecular identification confirmed its presence. The results on sequence and phylogenetic analysis demonstrated high relationship of these nematodes to other Pratylenchus coffeae from the database. Moreover, the data show that P. coffeae was spreading in most banana-growing areas in the Zanzibar islands of Unguja and Pemba. The nematodes survive in the warm areas of these islands, where air temperatures can reach 38 °C [P. goodeyi [P. coffeae collected from different altitudes and temperature showed no significant effect of temperature on abundance of P. coffeae. This is supported by results by Radewald et al. [P. coffeae at 29 °C but infection occurring at a range of 4–32 °C. In addition to temperature, previous data indicate that soil pH, rainfall and humidity affect the nematode population [P. coffeae in Tanzania. The effect of altitude on nematode populations was considered due to its relationship to temperature, because altitude is usually negatively correlated with temperature [P. coffeae only in Zanzibar [P. coffeae was in either roots or soils of the banana samples from surveyed regions with the exception of two regions (Arusha and Pemba North), implying that one source of its invasion of the mainland may be the Zanzibar islands through movement of plant material. For the past two decades, P. coffeae has been newly reported across banana-growing areas in Africa. For example, the presence of P. coffeae was first reported on banana in Uganda after a nematode survey conducted in 1993 [P. coffeae was reported to cause 24% losses in bunch weight and the toppling of banana plants [P. coffeae caused severe symptoms of dead roots, root necrosis and sucker corm lesions in bananas [The survey results indicated the presence of ch 38 °C , and in ch 38 °C . Informa goodeyi ,42,43. Hd et al. , who foupulation ,46, whicperature . AlthougZanzibar . The cur in 1993 . Moreovea plants . Similar bananas . TogetheP. coffeae in about 80% of the samples collected, but there are no research data on the effect of this nematode on banana production. The results indicate a high chance of this nematode species surviving in a wide range of conditions where banana is grown if no effort is made to control movement of plant materials and increase farmer awareness. Traditionally, farmers source new vegetative materials from existing fields and neighbours [P. coffeae, strategic management measures should be considered that must include cultural practices such as thinning of banana mats, sanitation and hot water treatment of planting material. When assessing the banana fields and collecting samples in Zanzibar, we noted that most banana mats contained 4–20 plants and were left to increase after planting the first crop – this high plant density might be one reason for high nematode incidences. The number of other crops intercropped was high, we found mixtures of taro, sweetpotato, cassava, eggplant, pumpkin, coconut, and orange cardamom and weed control was poor. Weeds such as Bermuda grass, nut grass and black nightshade are also host to different nematodes and can support them even when the crop is not in the field [P. coffeae to banana production has been reported by a number of researchers [P. coffeae has been reviewed in detail [P. coffeae.The current study revealed the presence of ighbours . In areahe field . The danearchers  and the n detail , in our P. coffeae in this soil and hence limited its availability. Soil properties and their relationship to incidence and density of P. coffeae imply that agronomic practices necessary for improvement of soil properties may significantly affect management of this nematode. The effects of clay texture and high soil nitrogen in hindering and reducing nematodes have been reported in different studies [P. coffeae to aid small-scale banana growers in Tanzania.This study indicates that incidence and density of nematodes were high in North and South Unguja and no nematodes were found in Arusha. The variation in nematode abundance can be caused by the nature of varieties grown in the two areas. Traditionally, Arusha has extensively grown East African highland banana which is an unique cooking diploid variety called mchare , whereas studies ,57,58. T5P. coffeae was widespread in banana-growing areas of Tanzania, from the mainland to the Zanzibar islands, regardless of climatic conditions. The results showed that important areas were infested; and further research should be concentrated in these regions to minimise the problem. Based on the survey results, we suggest pathogenicity should be studied in Tanzania to improve understanding of the relationship between P. coffeae and banana and the level of effects caused by this nematode. A knowledge and awareness programme is needed to help farmers apply available nematode management practices for improved banana production.This study indicated that