Saccharomyces cerevisiae, which contains a conserved nuclear transport machinery. In contrast to prior work, we demonstrate that β-catenin accumulates in the nucleus in a Ran-dependent manner, suggesting the use of a nuclear transport receptor (NTR). Indeed, a systematic and conditional inhibition of NTRs revealed that only Kap104, the ortholog of Kap-β2/Transportin-1 (TNPO1), was required for β-catenin nuclear import. We further demonstrate direct binding between TNPO1 and β-catenin that is mediated by a conserved PY-NLS. Finally, using Xenopus secondary axis and TCF/LEF (T Cell factor/lymphoid enhancer factor family) reporter assays, we demonstrate that our results in yeast can be directly translated to vertebrates. By elucidating the nuclear localization signal in β-catenin and its cognate NTR, our study suggests new therapeutic targets for a host of human diseases caused by excessive Wnt signaling. Indeed, we demonstrate that a small chimeric peptide designed to target TNPO1 can reduce Wnt signaling as a first step toward therapeutics.Wnt signaling is essential for many aspects of embryonic development including the formation of the primary embryonic axis. In addition, excessive Wnt signaling drives multiple diseases including cancer, highlighting its importance for disease pathogenesis. β-catenin is a key effector in this pathway that translocates into the nucleus and activates Wnt responsive genes. However, due to our lack of understanding of β-catenin nuclear transport, therapeutic modulation of Wnt signaling has been challenging. Here, we took an unconventional approach to address this long-standing question by exploiting a heterologous model system, the budding yeast  Wnt signaling plays multiple roles in embryonic development. For example, Wnt signaling is critical for establishing the dorsal embryonic axis; increased Wnt signaling can lead to a secondary axis (twinning of the embryo), while depletion of a key effector of the Wnt signaling pathway, β-catenin, can lead to a radially ventralized embryo . In addiIn Wnt signaling, β-catenin (CTNNB1) relays the message from a Wnt ligand at the plasma membrane to transcription factors in the nucleus . As suchSaccharomyces cerevisiae. Yeast do not have a Wnt pathway or a β-catenin ortholog, which presumably emerged in metazoans with the onset of multicellularity and cell fate specialization . We further demonstrate that a small peptide based on a chimeric PY-NLS sequence can prevent Wnt signaling, opening the door for therapeutics.A major challenge with understanding the β-catenin nuclear import mechanism is the myriad of binding partners that modulate its steady-state distribution and, hence, complicate the direct interrogation of the nuclear transport step . Here, wlization . Yeast alization . Indeed,lization . Using tXenopus β-catenin-GFP (xβ-catenin-GFP) in a wildtype yeast strain expressing an endogenously tagged nuclear envelope membrane protein, Heh2-mCherry, to help visualize the nuclear boundary. Indeed, xβ-catenin-GFP was enriched in the nucleus compared to GFP alone  xβ-catenin-GFP is imported by an NTR, (2) xβ-catenin-GFP piggybacks on an unknown binding partner that is itself imported by an NTR, or (3) xβ-catenin-GFP has an intrinsic ability to cross the NPC free of NTRs. To rule out the latter possibility, we tested whether xβ-catenin-GFP nuclear accumulation was dependent on a functional Ran gradient, which would specifically impact NTR-mediated transport . We thermtr1-1 strain at the non-permissive temperature  repeats sandwiched between two unstructured domains . We geneperature . Thus, wperature , these dperature .mtr1-1 strain (We confirmed that nuclear accumulation of xβ-catenin-(665-745)-GFP was dependent on the Ran pathway using the 1 strain . To ensu1 strain . Further1 strain .Xenopus (Xenopus embryos. By injecting a moderate dose (200 pg) of xβ-catenin mRNA, secondary axes develop in roughly half of the embryos -GFP shows a reduction in nuclear accumulation, which is rescued by the addition of the cNLS  protein sequence to established TNPO1 NLS  fusions of human β-catenin (GST-hβ-catenin) and human β-catenin containing the PM-AA mutations . We immobilized these GST fusions  on GT Sepharose beads and tested binding to purified TNPO1. We observed specific binding of TNPO1 to the GST-hβ-catenin , which wXenopus secondary axis development. First, once β-catenin enters the nucleus, it binds to the TCF/LEF complex to activate transcription of Wnt responsive genes  line has seven tandem TCF/LEF DNA binding sites upstream of GFP and is an effective reporter of Wnt signaling (tnpo1 gene is duplicated (tnpo1 and tnpo2), and both paralogs have nearly identical sequence and function  to visualize GFP transcripts as an assay for Wnt reporter activation and used sibling embryos without the transgene as a WMISH negative control. When we depleted both tnpo1 and tnpo2 using F0 CRISPR, significantly more embryos had weak expression of the GFP transgene compared to UIC embryos (tnpo1/2 sgRNAs#2). Importantly, we detected deleterious gene modification at the appropriate targeted sites using Inference of CRISPR Edits (ICE) analysis  a TCF/LEF reporter and (2) ve genes . A well-ve genes . In Xenoignaling . We crosfunction . Therefoanalysis .Xenopus, mouse, and human cancer cell lines.We next tested the function of mouse Tnpo1/2 in a stable transgenic mouse fibroblast cell line in which luciferase is expressed under the control of TCF/LEF DNA-binding elements. To activate Wnt signaling, we transfected a full-length human β-catenin-GFP that increased luciferase signal 7.7-fold over transfection of GFP alone . Then weXenopus secondary axis assay. We compared the number of secondary axes induced by the wildtype hβ-catenin mRNA to a PM to AA mutated version . We noted a significant reduction in the number of secondary axes induced by the PM-AA mutant β-catenin  M and A1 . To testAs the M9M peptide inhibits the nuclear import of a multitude of TNPO1/2 cargos , we sougOur lack of understanding of the mechanism of β-catenin nuclear transport has been a major stumbling block for opening up new avenues of Wnt-targeted anti-cancer therapies. Numerous transport models have been proposed including piggybacking on TCF/LEF  or APC , althougXenopus, and human cells, supporting that it can be recognized by TPNO1 orthologs across eukaryotes. It turns out, however, that although the β-catenin NLS can be imported in yeast, Kap104 (the yeast TPNO1 ortholog) does not recognize the same spectrum of NLSs as its human counterpart .Further information and request for reagents may be directed to and will be fulfilled by the lead contact, Mustafa K. Khokha  medium + 10% fetal bovine serum + 1% penicillin and streptomycin in a T-75 flask. The engineered 3T3 mouse fibroblast cell line that stably expresses the TCF/LEF luciferase transgene was maintained with DMEM medium + Cell Growth Medium Concentrate (Enzo life sciences). Further experimental procedures for the 3T3 cell line can be found under TCF/LEF luciferase assay method section. Upon 70–80% cell confluency, cells were transfected with plasmids using jetPRIME (Polyplus-transfection) following the manufacturer’s instructions. Cells were fixed with 4% paraformaldehyde/PBS and further processed for immunofluorescence 24–48 hr post-transfection. Antibodies used in this study are listed in Key resources table. Cells were tested for mycoplasma using MycoAlert Detection Kit (Lonza).Xenopus β-catenin-GFP (Addgene #16839), Xenopus cNLS-β-catenin-GFP (Addgene #16838), GST-human β-catenin (Addgene #24193), and NLS-mCherry (Addgene #49313) plasmids were obtained from Addgene. GST-transportin 1 (TNPO1) and Gal-MBP(x3)-GFP plasmids were generous gifts from Dr. Yuh-min Chook at UTSW and Dr. Liesbeth M. Veenhoff at University of Groningen, respectively. Gibson Assembly (New England Biolabs) was used to generate GFP-tagged Xenopus β-catenin truncation constructs following the manufacturer’s instructions. Both the human and Xenopus β-catenin P687A, M688A variants were generated using Q5 site- directed mutagenesis (New England Biolabs) following the manufacturer’s instructions. Subsequently, the β-catenin constructs were sub-cloned into the pRS406 vector containing an ADH1 promotor for yeast studies or a pCS2+ vector for mammalian/Xenopus studies. mRNAs were generated using the SP6 mMessage machine kit (Thermo Fisher Scientific) and RNA clean & concentrator kit (Zymo Research) following the manufacturer’s instructions. We obtained siRNAs directed against mouse TNPO1 (s108857), mouse TNPO2 (s102754), and a control siRNA (4390843) from Thermo Fisher Scientific. To generate CRISPR sgRNAs, we used the EnGen sgRNA synthesis kit (NEB) following the manufacturer’s instructions with the following targeting sequences tnpo1 sgRNA#1 (5’-GGCATGGGGGCCACCTCTTG-3’), tnpo1 sgRNA#2 (5’- GGGTTACGTTTGTCCTCAAG-3’), tnpo2 sgRNA #1 (5’- GGGCGTTTAGCCGCGTTCTA-3’), and tnpo2 sgRNA #2 (5’- GGCGTCATGGATGAGTCCGA-3’)  as previously described  strain. A yeast colony that incorporated the plasmid sequence was cultured and mounted onto a coverslip for live imaging on a DeltaVision wide-field microscope  with a CoolSnapHQ2 CCD camera. Yeast fluorescent images were deconvolved using the iterative algorithm sofWoRx. 6.5.1 . β-catenin-GFP transfected HeLa or HEK293T cells were mounted on Pro-Long Gold coated coverslip for imaging on a Zeiss Axio Observer microscope. All fluorescent images were analyzed with Fiji software. For quantification, the oval selection tool was used to draw a circle in both the nuclear and cytosolic regions on the same image plane to measure florescence intensity.Expression plasmids containing full length and fragments of the β-catenin-GFP coding sequence under the control of the X. laevis embryos were injected with a mixture of either 200 pg of Xenopus or human β-catenin-GFP mRNA and cNLS-mCherry mRNA in one of four cells . Embryos were assessed for a secondary axis via stereomicroscopy at stage 17–19. For the β-catenin localization experiments by fluorescence, stage 10 embryos were fixed in 4% paraformaldehyde/PBS at 4°C overnight on a nutator. Embryos were washed in 1× PBS + 0.1% TritonX-100, and the dorsal blastopore lips were sectioned with a razor blade and mounted on Pro-Long Gold (Invitrogen) coated coverslip before imaging on a Zeiss 710 confocal microscope.+-ATPase (PMA1 gene), a Heh2-mCherry fusion to mark the nuclear envelope and a mutated TOR1 gene . In this strain, individual, endogenous NTRs are tagged with the FRB domain at the C-terminus by homologous recombination of a PCR product that contains an FRB sequence, a 3× HA epitope, and a selective marker, HIS3, flanked by a 60 bp homology arm of endogenous NTR coding sequence (ADH1 promoter was transformed into the Anchor-Away line. These lines were treated with 1 mg/ml of rapamycin (5–15 min of incubation) or vehicle alone (DMSO) before imaging.To employ the Anchor-Away approach, we used a yeast strain that harbors a FKBP12 fusion of the endogenous plasma membrane Hsequence . IntegraA 3T3 mouse fibroblast cell line that has a stable integration of the luciferase reporter gene under the Wnt responsive TCF/LEF promoters was used for this assay (Enzo life sciences). Cells were maintained with DMEM medium + Cell Growth Medium Concentrate (Enzo life sciences). Prior to the transfection, cells were seeded on a 24 well plate in media containing DMEM + Cell Assay Medium Concentrate (Enzo life sciences). Subsequently, cells were transfected with siRNA (25 pmol) first, then GFP or human β-catenin-GFP DNA (0.5 µg) for 48 hr and 24 hr, respectively, using JetPRIME per the manufacturer’s instructions. Luciferase was quantified using the Luciferase Assay System (Promega) and the Promega Glomax luminometer according to the manufacturer’s instructions. For the M9M peptide experiment, cells were transfected with an M9M or M9M-A peptide dose ranging from 0.635 µg to 0.5 µg using ProteoJuice Protein transfection following the manufacturer’s instructions for 20 hr, and Wnt signaling was activated either by Wnt3a ligand (50 ng/ml) or human β-catenin-GFP DNA (0.5 µg) for 16–24 hr.X. tropicalis Tg(pbin7Lef-dGFP) were crossed with wildtype X. tropicalis. Fertilized embryos were injected with sgRNAs targeting tnpo1 and tnpo2 and Cas9 protein at one-cell stage and collected at stage 10 for in situ hybridization as previously described (Heterozygous escribed . Digoxig3T3 mouse fibroblast cells were lysed in radioimmunoprecipitation assay (RIPA) buffer to harvest protein samples. Nuclear and cytoplasmic proteins were extracted from colorectal cancer cell lines (HCT-116 and DLD-1) following the manufacturer’s instructions (NE-PER Thermo Fisher). Protein levels are normalized, and immunoblots were carried out in bolt 4–12% Bis-Tris plus gels following standard protocols. Antibodies used in this study are listed in Key resources table.Escherichia coli strain and cultured in Lysogeny broth (LB) with antibiotics to mid-log phase (OD600 0.6–0.8). To induce expression of the recombinant proteins , isopropyl β-d-1-thiogalacto pyranoside was added at a final concentration of 1 mM for 3 hr. All cultures were harvested in 50 ml batches and stored at –80°C until further use. Glutathione Sepharose (GT) beads (Millipore Sigma) were washed and equilibrated in lysis buffer 1 . Bacterial pellets containing GST tagged proteins were resuspended with the ice cold lysis buffer 1, sonicated, and spun down at 4°C at 30,000× g for 20 min. The supernatant was collected into a new 50 ml conical tube and incubated with 200 µl of GT bead slurry for 1 hr at 4°C. Subsequently, the GST-GT bead slurry was collected and washed with lysis buffer 1 (excluding the protease cocktail mix). The GST tag was removed from hTNPO1 using proTEV Plus Protease (Promega), and the protease enzyme was further removed from hTNPO1 protein by Ni-NTA Magnetic Beads (NEB) as per the manufacturer’s instructions. To pulldown His6-RanQ69L, bacterial pellets were resuspended with ice cold lysis buffer 2 , sonicated, and spun down at 4°C at 30,000× g for 20 min. The supernatant was collected into a new 50 ml conical tube and incubated with 200 µl of equilibrated Ni NTA bead slurry for 1 hr at 4°C. The His-Ni bead slurry was collected and washed with lysis buffer 2. His6-RanQ69L was eluted from Ni beads by adding the elution buffer  and rotating at 4°C for 1 hr. The supernatant containing His-RanQ69L was collected by centrifugation at 500× g for 3 min and dialyzed in the buffer  at 4°C overnight. Subsequently, RanQ69L was incubated in GTP buffer (400 uM GTP in 5 mM EDTA) 4°C for 1 hr. hNTPO1 protein was incubated with GT beads preloaded with GST fusion protein  for 1 hr at 4°C. Then, RanQ69L protein  was added to the mix for 1 hr at 4°C. The beads were washed with the lysis buffer and eluted with SDS-PAGE sample buffer. Protein samples were separated by SDS-PAGE and detected with Coomassie (BioRad).pGEX-6P1, pGEX-human β-catenin, pGEX-human transportin 1, or pET28a-His6-RanQ69L were transformed into the BL21 Statistical significance was defined as p<0.05 (*), 0.002 (**), 0.0002 (***), and 0.0001 (****). The double axis assay and in situ data were analyzed by Fisher’s exact tests. Otherwise, unpaired two-tailed Student’s t-tests or a one-way ANOVA test were used to determine significance of mean ratio of nuclear to cytosolic fluorescence intensity in GraphPad Prism 8.4.3. In Wnt/β-catenin signaling, Wnt growth factor binding to cell surface receptors results in the stabilization and nuclear translocation of the transcriptional coactivator β-catenin, but how β-catenin is translocated to the nucleus has been a longstanding problem in the field. The authors show that the yeast Kap104/mammalian TNPO1 mediates nuclear translocation of β-catenin using a conserved TNPO1 nuclear localization sequence in the C-terminal region of β-catenin, and mutation of this sequence or knockdown of TNPO1 diminishes nuclear localization and Wnt sigaling. The data demonstrate that β-catenin nuclear translocation is Ran dependent and that TNPO1 binding is a significant, although not exclusive, contributor to β-catenin translocation, and could represent a new therapeutic target. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kevin Struhl as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Yuh Min Chook (Reviewer #1).Thank you for submitting your article "Kap-β2/Transportin mediates β-catenin nuclear transport in Wnt signaling" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The reviewers agree that the findings here are significant and contribute to our understanding of β-catenin signaling. However, there were several problems that need to be addressed, including the constructs used for assessing nuclear translocation of β-catenin fragments, the pulldown assay, and a general concern about TNPO1 as a therapeutic target.Xenopus double axis assay . Further, a significant fraction of β-catenin Δ 665-745-GFP was in the nucleus . If TNPO1/2 are only partially responsible for β-catenin nuclear translocation, targeting the TNPO1/2-β-catenin interaction might not be a good therapeutic strategy for cancer. This can be provided as a speculative discussion point, but not a conclusion of the paper.1. The fluorescence data show significant nuclear localization of β-catenin even in the absence of TNPO1 activity. Thus, there may be both Ran-dependent and -independent mechanisms for its nuclear localization, and this should be made clear, especially since there are prior findings along these lines. Given this, it is premature to claim that TNPO1 would be a target for inhibiting β-catenin signaling in cancer if it is only 50% effective. Double knockdown of TNPO1/2 only decreased WNT reporter activity by 60%. Β-catenin P687A M688A mutant still retained significant activity in the 2. Related to (1), inhibition of TNPO1/2-dependent nuclear import pathway was not confirmed in a relevant  cancer model system, in which β-catenin levels are inappropriately enhanced and inhibition of β-catenin nuclear entry would needed to support the idea that the current findings hold relevance for the development of future cancer therapies. The importance of TNPO1/2 for nuclear translocation of β-catenin should be tested in more mammalian cell lines, especially since a IPO11-β-catenin connection has already been proposed. TNPO1/2 knockdown and/or β-catenin P687A M688A viral transduction in various cell lines, including cell lines with β-catenin or APC mutations, and nuclear β-catenin levels quantified. Does the MPM9 peptide and/or TNPO1/2 depletion inhibit β-catenin mediated transcription in cancer cell lines carrying either β-catenin or APC mutations?Xenopus embryos . It would be more convincing if authors can extend this finding to common mammalian cell lines such as HEK293, Hela or colon cancer cell lines such as DLD1. This can be done by lentiviral transduction and should be a very easy experiment for them.3. The difference between β-catenin-GFP and β-catenin δ 665-745 is quite clear in 4. Since GFP is small and can enter the nucleus passively, it might not be a good fusion partner for mapping nuclear localization activity of β-catenin truncation fragments . Based on Figure 2B, it appears that many fragments of β-catenin have some level of NLS activity. It would be more convincing if authors can fuse different β-catenin fragments to MBP(3x)GFP, which is excluded from the nucleus . A control with an established TNPO1 cargo like hnRNP A1 or a strong YP-NLS would be helpful as well. It is also not clear whether the authors used MBP(3x)GFP to test the activity of the PY like motif in HeLa cells ; if not they should do so.5. The pull-down binding assay of GST-β-catenin + TNPO1 in Figure 4B could benefit from some straightforward improvement. Lane 4 shows TNPO1 binding, but at very sub-stoichiometric amounts. This may be due to having partially active bacterially expressed GST- TNPO1, which is not easy to produce. While there appears to be a decrease in binding to the PM/AA mutant, the experiment would be more decisive if RanGTP is added to the pulldown in lane 4 to demonstrate specific and Ran-sensitive TNPO1-β-catenin interactions.6. It is possible that TNPO1/2 are required for nuclear translocation of cofactors of β-catenin; the C-terminus of β-catenin is involved in transcription activation, and it is thought to bind to many nuclear factors. Can the authors rule this out?Other changes for the introduction/discussion:a. The authors are correct that NTRs are mostly functionally conserved between the budding yeast and mammalian systems. However, PY-NLS/cargo binding functions of Kap104 are only partially conserved when compared to those of TNPO1. Since the key finding here is on Kap104 and TNPO1, the authors should present this information more carefully and thoroughly in the introduction. Please refer to studies of Kap104 binding to the PY-NLS and conservation of PY-NLS recognition across eukaryotic TNPO1 in Suel et al., PLoS Biology 2008.TNPO1 binds to PY-NLSs that carry either basic (e.g. hnRNP M) or hydrophobic (e.g. hnRNP A1 and FUS) N-terminal motifs/epitopes. Please note that Kap104 binds only PY-NLSs that carry the basic epitope  but not the ones that have hydrophobic N-terminal epitopes . This is also why M9M  works in mammalian cells but not in yeast.b. It is suggested that the authors simply use the term PY-NLS, which is defined very broadly (see the Soniat and Chook 2015 review and other reviews on nuclear transport and NLSs), for the NLS of β-catenin. Sequences of PY-NLSs are incredibly diverse and defined only by very loose sequence motifs, and this class of signals is simply named after the most easily recognized and most conserved PY sequence element. The Suel et al., paper shows clearly how other hydrophobic residues function well in the place of the tyrosine in the PY motif of this signal. For example, the yeast mRNA export factor Nab2 carries a PY-NLS that has a PL motif. Soniat et al., (Structure 2016) also showed how a PY-NLS can sometimes not even have a PY motif, and the 2008 Suel et al., paper explained why this is the case.c. The PY dipeptide motif is simply the most easily recognized and most conserved PY sequence element. It is NOT the most energetically important element of the PY-NLS; it is just one of several binding elements usually present in the signal so the PM/PA mutation should not be expected to completely inactivate the NLS let alone nuclear localization. The PY is in fact weak  and does not contribute much to TNPO1 or Kap104 binding in some PY-NLSs. An example is the M9 sequence of the PY-NLS of hnRNP A1, which has a really weak PY motif .eLife. Your revised article has been evaluated by Kevin Struhl (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Kap-β2/Transportin mediates β-catenin nuclear transport in Wnt signaling" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:1. Given the emphasis on the potential therapeutic importance, the concern remains that the requirement of this NLS for β-catenin localization in mammalian cells is not solidly established. The TNPO knockdown experiment has many caveats . Therefore, it was felt that an experiment to test whether a β-catenin mutant lacking this NLS has decreased nuclear localization in mammalian cells should be performed. Note that even if the result is negative, you can discuss the caveats.2. The knockdown experiments in the HCT116 and DLD1 cells show a weak effect  – is it possible to show longer exposures? 1. The fluorescence data show significant nuclear localization of β-catenin even in the absence of TNPO1 activity. Thus, there may be both Ran-dependent and -independent mechanisms for its nuclear localization, and this should be made clear, especially since there are prior findings along these lines.The reviewer raises an important point, and we agree that there may be other elements beyond the TNPO1 NLS that are required for b-catenin nuclear entry; however, our data argue against any Ran-independent mechanism. For example, we now include new data examining the localization of aa 1-664-GFP (containing the N-terminus and ARM repeats but lacking the TNPO1 NLS), in the temperature sensitive Ran-GEF strain (mtr1-1). As shown in Figure 2—figure supplement 3, the residual nuclear accumulation of 1-664-GFP is in fact dependent on a functional Ran pathway. Thus, as is now more fully addressed in the revised discussion, although the TNPO1 NLS is the “strongest” nuclear localization element, it is possible that there are other NLSs in the rest of the protein that await future characterization.Xenopus double axis assay . Further, a significant fraction of β-catenin Δ 665-745-GFP was in the nucleus . If TNPO1/2 are only partially responsible for β-catenin nuclear translocation, targeting the TNPO1/2-β-catenin interaction might not be a good therapeutic strategy for cancer. This can be provided as a speculative discussion point, but not a conclusion of the paper.Given this, it is premature to claim that TNPO1 would be a target for inhibiting β-catenin signaling in cancer if it is only 50% effective. Double knockdown of TNPO1/2 only decreased WNT reporter activity by 60%. Β-catenin P687A M688A mutant still retained significant activity in the Certainly, until it is tested, we will not know if inhibiting the TNPO1-β-catenin interaction is a good therapeutic strategy for cancer. We have been more explicit about this point in the discussion.2. Related to (1), inhibition of TNPO1/2-dependent nuclear import pathway was not confirmed in a relevant  cancer model system, in which β-catenin levels are inappropriately enhanced and inhibition of β-catenin nuclear entry would needed to support the idea that the current findings hold relevance for the development of future cancer therapies. The importance of TNPO1/2 for nuclear translocation of β-catenin should be tested in more mammalian cell lines, especially since a IPO11-β-catenin connection has already been proposed. TNPO1/2 knockdown and/or β-catenin P687A M688A viral transduction in various cell lines, including cell lines with β-catenin or APC mutations, and nuclear β-catenin levels quantified. Does the MPM9 peptide and/or TNPO1/2 depletion inhibit β-catenin mediated transcription in cancer cell lines carrying either β-catenin or APC mutations?As suggested, we depleted TNPO1/2 by siRNA in two different colorectal cell lines (HCT-116 and DLD-1) and showed a significant reduction of β-catenin protein levels in the nucleus compared to the control siRNA condition by western blot. We have added this data to Figure 6—figure supplement 4.Xenopus embryos . It would be more convincing if authors can extend this finding to common mammalian cell lines such as HEK293, Hela or colon cancer cell lines such as DLD1. This can be done by lentiviral transduction and should be a very easy experiment for them.3. The difference between β-catenin-GFP and β-catenin δ 665-745 is quite clear in Xenopus. We felt it was important to show that β-catenin-GFP localization in the Xenopus context also correlates with the incidence of secondary axes. Then in HeLa cells, we show that β-catenin 665-782-GFP enriches in the nucleus, which does not occur when we mutate the PM residues to AA . Then we demonstrate that this point mutant leads to a loss of direct binding between β-catenin and TNPO1 (this interaction is between human proteins) . Therefore, both the in vitro binding experiment and the localization of these constructs in mammalian cells provides strong evidence of the NLS’s functionality in the mammalian system.In Figure 2 figure supplement 1, we test xbeta-catenin-(665-745)-GFP in HEK293T cells which enriches in the nucleus to a much greater degree than GFP alone. Figure S2C is supportive data for the secondary axis experiments in 4. Since GFP is small and can enter the nucleus passively, it might not be a good fusion partner for mapping nuclear localization activity of β-catenin truncation fragments . Based on Figure 2B, it appears that many fragments of β-catenin have some level of NLS activity. It would be more convincing if authors can fuse different β-catenin fragments to MBP(3x)GFP, which is excluded from the nucleus . A control with an established TNPO1 cargo like hnRNP A1 or a strong YP-NLS would be helpful as well. It is also not clear whether the authors used MBP(3x)GFP to test the activity of the PY like motif in HeLa cells ; if not they should do so.We agree that fragments outside the C-terminus of β-catenin are likely to contain an NLS element. We tested a C-terminal deletion and demonstrate that the nuclear enrichment is lost at the non-permissive temperature in the mtr1-1 strain indicating Ran dependence . In this manuscript, we focused on the C-terminus as it was required for the most significant fraction of the nuclear enrichment. Future studies can investigate the necessary NTRs in the ARM and N-terminus.As the reviewer suggests, we tried to localize MBP(3x)-GFP in HeLa cells. However, as show in 5. The pull-down binding assay of GST-β-catenin + TNPO1 in Figure 4B could benefit from some straightforward improvement. Lane 4 shows TNPO1 binding, but at very sub-stoichiometric amounts. This may be due to having partially active bacterially expressed GST- TNPO1, which is not easy to produce. While there appears to be a decrease in binding to the PM/AA mutant, the experiment would be more decisive if RanGTP is added to the pulldown in lane 4 to demonstrate specific and Ran-sensitive TNPO1-β-catenin interactions.As requested, we purified a his-tagged RanQ69L protein (ie GTP hydrolysis deficient Ran mutant), loaded it with GTP and showed that RanQ69L-GTP selectively forms complexes with TNPO1 and disrupts the TNPO1-β-catenin interaction. We have added this data to Figure 5 and Figure 5—figure supplement 1.6. It is possible that TNPO1/2 are required for nuclear translocation of cofactors of β-catenin; the C-terminus of β-catenin is involved in transcription activation, and it is thought to bind to many nuclear factors. Can the authors rule this out?We have met the standard burden for defining whether a given protein is a transport cargo of an NTR: we define, in multiple systems including a minimal budding yeast model that lacks the majority of physiological β-catenin transcriptional activators, that TNPO1 is required for its nuclear import. We map the TNPO1 binding site to a minimal sequence that resembles established TNPO1 NLSs (demonstrate the sequence is necessary and sufficient for nuclear import in multiple model systems) and then show that this sequence is required for direct binding of TNPO1 to β-catenin via in vitro assays with recombinant proteins. Further, to solidify it is a canonical NTR-NLS interaction, we also demonstrate that the TPNO1-β-catenin complex is disrupted by Ran-GTP. Whether TNPO1 also transports co-factors of β-catenin that are required for its role as a transcriptional activator is certainly possible, but the sum of our data indicate that TNPO1 binds directly to β-catenin for nuclear transport.Other changes for the introduction/discussion:a. The authors are correct that NTRs are mostly functionally conserved between the budding yeast and mammalian systems. However, PY-NLS/cargo binding functions of Kap104 are only partially conserved when compared to those of TNPO1. Since the key finding here is on Kap104 and TNPO1, the authors should present this information more carefully and thoroughly in the introduction. Please refer to studies of Kap104 binding to the PY-NLS and conservation of PY-NLS recognition across eukaryotic TNPO1 in Suel et al., PLoS Biology 2008.We appreciate this point and regret our oversimplification. We have addressed this with proper citations in the discussion.TNPO1 binds to PY-NLSs that carry either basic (e.g. hnRNP M) or hydrophobic (e.g. hnRNP A1 and FUS) N-terminal motifs/epitopes. Please note that Kap104 binds only PY-NLSs that carry the basic epitope  but not the ones that have hydrophobic N-terminal epitopes . This is also why M9M  works in mammalian cells but not in yeast.We thank the reviewer for this insightful information. We have incorporated a discussion of this point in the main text.b. It is suggested that the authors simply use the term PY-NLS, which is defined very broadly (see the Soniat and Chook 2015 review and other reviews on nuclear transport and NLSs), for the NLS of β-catenin. Sequences of PY-NLSs are incredibly diverse and defined only by very loose sequence motifs, and this class of signals is simply named after the most easily recognized and most conserved PY sequence element. The Suel et al., paper shows clearly how other hydrophobic residues function well in the place of the tyrosine in the PY motif of this signal. For example, the yeast mRNA export factor Nab2 carries a PY-NLS that has a PL motif. Soniat et al., (Structure 2016) also showed how a PY-NLS can sometimes not even have a PY motif, and the 2008 Suel et al., paper explained why this is the case.As suggested, we will change to PY-NLS.c. The PY dipeptide motif is simply the most easily recognized and most conserved PY sequence element. It is NOT the most energetically important element of the PY-NLS; it is just one of several binding elements usually present in the signal so the PM/PA mutation should not be expected to completely inactivate the NLS let alone nuclear localization. The PY is in fact weak  and does not contribute much to TNPO1 or Kap104 binding in some PY-NLSs. An example is the M9 sequence of the PY-NLS of hnRNP A1, which has a really weak PY motif .Thank you for this information. We were fortunate that the mutation of the PM-AA disrupted the detectable binding of β-catenin to TPNO1 in vitro and in vivo.1. Given the emphasis on the potential therapeutic importance, the concern remains that the requirement of this NLS for β-catenin localization in mammalian cells is not solidly established. The TNPO knockdown experiment has many caveats . Therefore, it was felt that an experiment to test whether a β-catenin mutant lacking this NLS has decreased nuclear localization in mammalian cells should be performed. Note that even if the result is negative, you can discuss the caveats.To address this concern, we have generated a deletion in the NLS in human β-catenin-GFP. We have compared the localization of this protein with the wildtype β-catenin-GFP and demonstrate a significant difference. The wildtype β-catenin-GFP readily localizes to the nucleus of mammalian HEK293T cells while the nuclear localization DNLS is significantly less. These data are now included in a Figure 2 Supplement 2 panel C.2. The knockdown experiments in the HCT116 and DLD1 cells show a weak effect  – is it possible to show longer exposures?We regret that the exposure made the blot difficult to interpret. We have replaced this figure with a blot with a longer exposure which clarifies the results.


A portable spirometer is a promising alternative to a traditional pulmonary function test (PFT) spirometer for respiratory function evaluation.This study aimed to investigate the accuracy of automated interpretation of the PFT measured by a portable Yue Cloud spirometer in Chinese adults.n = 220). A Yue Cloud spirometer and a conventional Jaeger MasterScreen device were applied to each patient with a 20‐min quiescent period between each measurement. Pulmonary function parameters, including forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), peak expiratory flow (PEF), maximal expiratory flow at 25%, 50%, and 75% of the FVC , and maximal mid‐expiratory flow (MMEF), were compared by correlation analyses and Bland–Altman methods. The Yue Cloud spirometer automatically interpreted the PFT results, and a conventional strategy was performed to interpret the PFT results obtained by the Jaeger machine. Concordance of the categorization of pulmonary dysfunction, small airway dysfunction, and severity was analyzed by the kappa (κ) statistic.The PFT was performed to evaluate subjects prospectively enrolled at Ruijin Hospital (p < 0.001). No significant bias was observed in any of the measured spirometer variables. A satisfactory concordance of pulmonary function and severity classification was observed between the automated interpretation results obtained with the Yue Cloud spirometer vs. a conventional spirometer interpretation strategy .Significantly similar correlations of all variables measured with the two spirometers were observed (all The portable Yue Cloud spirometer not only yields reliable measurements of pulmonary function but also can automatically interpret the PFT results. The accuracy of automated interpretation of pulmonary function test by the novel portable Yue Cloud spirometer was investigated in comparison with a conventional Jaeger MasterScreen device in Chinese adults. The results proved that the Yue Cloud spirometer not only yielded reliable measurements but also could automatically interpret the pulmonary function test results. In Western countries, a portable spirometer has been proven to have a similar consistency compared with traditional spirometers.In the current prospective study, we aimed to validate the concordance of pulmonary dysfunction classification interpreted by an automated portable spirometer (Yue Cloud) compared with a traditional spirometer (Jaeger MasterScreen) in a Chinese cohort.We present the following article in accordance with the STARD reporting checklist.22.1This was a prospective, self‐control, single‐center study. We consecutively recruited adult subjects aged 18 and over who underwent a PFT at Shanghai Ruijin Hospital between February and July 2020. Those who failed to finish the PFT or without complete PFT results were excluded.n = 220) received standardized instructions on the use of a spirometer. Two sets of PFTs were performed for each subject: the first by using a Jaeger MasterScreen machine  and the second by using a Yue Cloud spirometer. The Yue Cloud spirometer is a small, handheld device consisting of a pressure sensor and a digital display, which has been proven to be a reliable alternative to the conventional device for the measurement of pulmonary function.All included subjects . Informed consent was obtained from all participants.2.2Spirometry variables, including the forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), peak expiratory flow (PEF), maximal expiratory flow at 25%, 50%, and 75% of the FVC , and maximal mid‐expiratory flow (MMEF), were measured by the same technician on the same instruments at the pulmonary function laboratory. Small airway dysfunction was also measured. Measurement and calibration were strictly in compliance with the 2005 American Thoracic Society/European Respiratory Society guidelines. A quiescent period of 20 min was set before each measurement. The Yue Cloud spirometer automatically displayed the results of the measured parameters and the category of lung function impairment by the accompanying software. The measurements of pulmonary function obtained by the conventional Jaeger machine were evaluated by the technician based on the curve morphology.2.3The pulmonary dysfunction was classified according to the Chinese guidelines of the PFT as follows2.4The PFT results were categorized as Grade A, B, C, D, or F, according to the criteria for quality control based on the Chinese practice guidelines for PFT measurements Table .12, 12.5XY axis, where X axis represents the difference of the two measurements and the Y axis shows the mean of the two measurements. The 95% limits of agreement (LoAs) are calculated to evaluate the differences between measurements by two methods. The concordance in diagnosis of lung function impairment between devices was assessed by Cohen's kappa statistic. The kappa coefficient (κ), indicating the strength of diagnosis agreement, was calculated. The kappa value was qualified on the basis of the magnitude as follows: 0.4–0.6, moderate agreement; 0.6–0.8, substantial agreement; and 0.8–1.0, almost perfect agreement.p value <0.05 was considered statistically significant.Continuous data were expressed as the mean ± standard deviation, whereas categorical data were expressed as a number with the percentage. Lung function parameters, including FVC, FEV1, PEF, MEF75, MEF50, MEF25, and MMEF, and small airway dysfunction were compared between groups. Pearson's correlation was used to analyze the relationships between variables. Meanwhile, the consistency of parameters measured by the Yue Cloud spirometer and the Jaeger device was assessed by Bland–Altman analysis.33.1The average age of all included subjects was 59 years old, with a median age of 63 (range: 18–85) years old. The male subjects accounted for 55.5% of the population. Among all 220 subjects, 159 (72.3%) had abnormal PFT results.3.2p value <0.001  provided by the conventional Jaeger MasterScreen machine and the Yue Cloud spirometer. The correlations were all above 0.8, with a The Bland–Altman analyses showed that there was no significant bias in any of the spirometry measurements . Differences and 95% LoAs were displayed in Figure 3.3The classifications of pulmonary dysfunction, small airway dysfunction, and severity, as measured by the Jaeger and Yue cloud spirometers, are displayed in Tables When measured by the Jaeger spirometer, the numbers of subjects diagnosed as having obstructive, restrictive, mixed pulmonary dysfunction, or normal pulmonary function were 53 24.1%), 50 (22.7%), 50 (22.7%), and 67 (30.5%), respectively. The corresponding numbers of subjects measured by the Yue Cloud spirometer were 60 (27.3%), 44 (20.0%), 49 (22.3%), and 67 (30.5%), respectively  subjects with small airway dysfunction, whereas 104 47.3%) subjects were determined to have small airway dysfunction by the Yue Cloud spirometer. Among all these subjects, 100 (45.5%) were diagnosed with small airway dysfunction by both methods , 23 (10.5%), 17 (7.7%), 27 (12.3%), and 10 4.5%) subjects were categorized as having mild, moderate, moderate to severe, severe, and very severe disease, respectively, when measured by the Jaeger spirometer. The corresponding numbers of subjects were 67 (30.5%), 26 (11.8%), 15 (6.8%), 29 (13.2%), and 11 (5.0%), respectively, when measured by the Yue Cloud spirometer .Concordance verified by the Kappa statistic showed an excellent agreement in pulmonary dysfunction, small airway dysfunction, and severity evaluation between the Jaeger and Yue Cloud spirometers. The kappa values were all greater than 0.80  was observed between both methods.This prospective study determined the accuracy of pulmonary function assessment performed by a portable Yue Cloud spirometer and its diagnostic value of dysfunction categorization. Compared with the conventional Jaeger spirometer, the Yue Cloud spirometer performed well not only in the evaluation of lung function but also in the determination of classification. In terms of small airway dysfunction, severity classification, and routine dysfunction categorization, excellent concordance .Informed consent was obtained from all participants.Conception and design: R. D. and M. Z. Administrative support: P. W. Provision of study materials or patients: P. W. and J. C. Collection and assembly of data: L. G. Data analysis and interpretation: J. Z. and R. D. Manuscript writing: all authors. Final approval of manuscript: all authors.


Epoxy clay nanocomposites have been proven to have improved mechanical, thermal and physical properties over pristine matrix. Thus, the fields of application of epoxy–clay nanocomposites along with their hybrid glass/carbon fibre reinforced composites have grown tremendously during the last few decades. The present review paper covers the research work performed on epoxy clay nanocomposites. It includes the influence of the processing techniques and parameters on the morphology of the nanocomposite, the methods of characterization and the effects of adding nanoclay on the mechanical and physical properties of composite. The improvements in the liquid barrier properties brought about by the addition of nanoclay platelets to epoxy resin are discussed. The variation of physical and mechanical properties with nanoclay type and content are reviewed along with the effects of moisture uptake on these properties. The advances in the development, characterization and applications of hybrid glass fibre reinforced epoxy–clay nanocomposites are discussed. Findings of the research work on the influence of nanoclay addition and exposure to water laden atmospheres on the behaviour of the hybrid glass fibre epoxy–nanoclay composites are presented. Finally, the potential health and environmental issues related to nanomaterials and their hybrid composites are reviewed. The market of nanocomposites and polymer-based nanocomposites has been growing during the last few decades. The global nanocomposite market size was estimated to be $4.32 billion in 2019 with the expectation to grow $14.37 billion by 2027  reportedA large number of review papers on nanocomposites have been published in the last three decades ,21,22,23The present review focuses on epoxy–nanoclay (E-nc) composites: their structures, mechanical, thermal and physical properties. The resistance of the nanocomposite to water uptake and the variation of mechanical and physical properties with moisture diffusion is discussed. The research work on glass fibre epoxy–nanoclay (GFRE-nc) hybrid composites is reviewed in terms of their fabrication methods, structures and mechanical properties. The last section discusses the possible health and environmental impacts of PNC and GFRE-nc composites. Epoxy resin is a class of reactive prepolymers that comprise a group of cross linkable materials. The characteristic group form a three-member ring structure know as epoxy, epoxide, oxirane, glycidyl or ethoxyline group as shown in Due to higher reactivity of epoxy group, it can react with a wide range of curing agents. These curing agents are generally of two types: (i) catalysts or (ii) hardeners. Catalysts are usually from tertiary amines, and they are used in low concentration below 1%. In most cases, catalysts are used with hardeners to form resin. Aromatic and aliphatic amines and carboxylic anhydride are common examples of hardeners. Aliphatic amines are normally cure at room temperature whereas aromatic amines and carboxylic anhydride are used for curing at higher temperature. The properties of cured epoxy resin vary depending on the chemical structure of curing agents and curing condition. Generally, resins cured at higher temperature develop improved properties such as strength, stiffness and high glass transition temperature compared to room temperature cured resin . In termClays are aluminium or hydrous silicates containing several cations such as oxygen, hydroxyl and silicon. These cations are organized in two-dimensional sheet-like structures. Clay minerals are also referred as layered silicates because they comprised of 1 nm thick silicate layers containing alumina and silica sheets joined together in various proportions and stacked on top of each other with interlayer distance. Clay minerals are classified as dimorphic , trimorphic  and tetramorphic  based on condensation ration of silica to alumina sheet . Nanoclays are sheets of nanoparticles made of layered mineral silicates. They are classified in different categories depending on their nanoparticle morphology, and chemical composition. Commonly used nanoclays for epoxy resins are organically modified montmorillonite clays with filling amounts ranging from 1% to 10%. Organically modified montmorillonite clays, which are naturally occurring inorganic materials, belong to the smectite clays. These layered silicate clays maintain their two-dimensional (2D) crystallographic plane even after mixing with polymers. 0.33Al1.6−Mg0.33Si0.33Al1.6−Mg0.33SiO10(OH)2 ,31,32,33When organically modified nanoclays are mixed with polymers, they generally result into one or a combination of two of the three main composites categories : (1) conIf d-spacing for clay layers is larger than 5 nm and transmission electron microscopy (TEM) can detect the disordered arrangement, then the polymer-clay nanostructure is called exfoliated nanocomposite ,29,30. Ag) of DGEBA epoxy from 152 °C to 135 °C. This was because of less crosslinking density between clay layers within clay tactoids. The same phenomenon is reported by other researchers [g was given as increasing concentration of long-chain modifier in the matrix [g [g [g [In a study by Al-Qadhi et al. , it was earchers ,40. One e matrix . Anotheratrix [g ,42, on tix [g [g . The typ[g [g [g . Without proper dispersion and distribution of the nanoclay in a polymer matrix, the advantage from the high aspect ratio of the clay platelets is compromised. In fact, clustering of the clay particles can act as defects resulting in adverse effects . That isIn general, there are three methods to mix and disperse nanoclays in polymers: (1) direct mixing of polymer and nanoclay, (2) in situ polymerization in presence of nanoclays and (3) in situ formation of the nanoparticles and in situ polymerization. The first technique is used for thermoplastics. In a second technique, nanoclays are dispersed in the monomer or monomer solution, then polymerized by standard polymerization methods. This is the preferred method for processing epoxy–clay nanocomposites as it has the potential to graft the polymer onto the particle surface. The third method is employed to fabricate hybrid nanocomposites because it allows the mixing of the two phases. Hussain et al.  and PavlOnce the mixing is completed curing agents have to be added and here the process has to be optimized. The selection of the right curing agent and the right quantity is a critical task as it determines the structure and properties of epoxy–clay nanocomposite . Along wAs a general practice, amine and anhydride-based curing agents are used in epoxy clay systems . AnhydriSome efforts  also stuThe right combination of all of the mixing and curing factors is hard to come about. The degree of exfoliation depends on all of these and more. Therefore, optimum values for these parameters should be determined in order to produce the desired structure of epoxy–clay nanocomposite. As discussed earlier, the degree of dispersion of nanoclays in polymer resin determines enhancement in physical, thermal and mechanical properties of the PNC. Though huge advances are made in the fields of nanotechnology and nanoscience, researchers in the area of nanomaterials are still facing challenges in terms of the availability of proper instruments for observing, measuring and manipulating these materials at the nanometre level. Therefore, proper characterization techniques are crucial for understanding how the resulting polymer-clay morphologies affect the nanocomposites physical, thermal and mechanical properties.Several characterization techniques ,28,65,66X-ray diffraction (XRD) is one of the commonly used techniques to find the type of morphology structure and measure the interplanar spacing (d-spacing) between clays using Braggs law. Several researchers ,73,74,75SEM has been used to study the microscopic view of clay dispersion. Several researchers ,68 used Transmission electron microscope is an imaging technique used to obtain a direct view of the dispersion state in materials. This technique has been widely employed to study the morphology of epoxy–clay nanocomposite at the sub-microscale . Figure The water uptake strongly affects the properties of epoxy resins; in fact, water laden atmospheres at high temperatures are detrimental to polymers. Due to the plasticizing effect of water on cured epoxy polymer, the glass transition temperature, of the systems is, in general, reduced by 20 °C for each 1% moisture uptake ,77. The −7 to 1.37 × 10−7 mm2/s. As shown in As mentioned in the introduction, the epoxy-based nanoclay composites with high moisture and gas barrier properties make them attractive for applications such as vehicle parts and matrices for fibre-reinforced plastic structures used in wet environments. Moisture uptake in epoxy–clay nanocomposites has attracted a lot of attention. A number of researchers ,79,80,81It is, however, observed  that nanNanocomposites consisting of epoxy and organically modified nanoclays often show better mechanical properties than those of pristine epoxies. A number of studies reported improvements in mechanical, physical and thermal properties of epoxy because of adding clay ,41,83,84Zabihi et al. , in theiAl-Qadhi and Merah  found thIn addition to the size of the organic modifier and its compatibility with the monomer, the clay content and the mixing techniques are known to affect the tensile properties of the nanocomposites. g) by about 20 °C for each 1% moisture uptake [As reported above, exposure of epoxy to moisture is seen to degrade its glass transition temperature  is increasing to manufacture pipes for potable water, seawater, sewage and other corrosive fluids. One of such application is the use of GFRE pipes in water production and transportation networks. Although GFRE offers excellent mechanical strength under both static and cyclic loading conditions, one major obstacle to a wider use of GFRE in piping systems is the above shown affinity of epoxy resin to moisture absorption ,29,77. TThus, any material modification strategy, which can lead to lower moisture absorption in GFRE composites can be extremely helpful in expanding the applications of GFRE composites to moisture laden service conditions. One such strategy is to generate tortuosities in the path of absorbed liquid molecules to limit their diffusion into the matrix material and along the fibre/matrix interfacial pathways, which can be achieved by complete exfoliation of a filler such as nanoclay into the matrix. As demonstrated above, the complete exfoliation of fillers in polymer matrices remains quite a challenge for researchers. Another dimension is added to this challenge when a nanocomposite is used as matrix for GFRE. As explained above, adding nanoclay to the neat epoxy has, in general, helped reduce its moisture uptake as well as enhance some of its physical, thermal and mechanical properties. The combination of the nanoclay together with glass fibres as reinforcements in a hybrid nanocomposite was the next step that researchers took during the past decade ,108,109.Besides the improvement in static properties of the GFRP material, the addition of nanoclay is found to considerably enhance quasi-static and dynamic properties  as well Zainuddin et al.  observedUnderstanding potential health and environmental issues related to nanomaterials is an important field of scientific inquiry and it is essential to develop complete picture of nanomaterials and their interaction with environment. Manufacturing of nanomaterials usually requires processes such as chemical and physical vapor deposition, which use large amounts of energy and material inputs and these processes release emissions to air, water and soil . SeveralDespite the growing use of nanomaterials in various applications, concerns about their negative impact on human body remain ,115,116.In addition to reporting about toxic effect of nanomaterials, several researchers have used Life Cycle Assessment (LCA) to measure environmental impacts of nanomaterials. LCA is a standard methodology that is used to evaluate environmental impacts of a product or a process throughout its entire life cycle, i.e., from extraction of materials to the end-of-life treatment ,121,122.2 is released for production of one kg of epoxy. Similarly, another study reported that 6.6 kg of CO2 is release while producing one kg of epoxy [2 emissions are primarily generated by energy used during production process for epoxy. Data about CO2 emissions while producing glass fibres are also easily available. It is reported that about 1.5 kg of CO2 is release while producing 1 kg of glass fibres [2 for one kg of glass fibre production [2 numbers related to nanoclay production are not as widely reported. One study estimates that 1522 kg of CO2 is released while producing 1 kg of montmorillonite clay [2 emission per kg production of epoxy nanoclay composites. However, in order to fully understand the environmental profile of these materials environmental impacts related to use, transportation and recycling of these materials in various applications should be analysed. Several studies have used LCA to quantify greenhouse gas emissions for various materials used in nano composites. The values for global warming potential for epoxy and glass fibres are widely reported in the literature. For example, one study  has repoof epoxy . These Cs fibres . Anotheroduction . Majoritite clay . Using tLloyd et al.  have repIn addition to LCA of nanomaterials, several LCA studies related to glass fibre composites are also reported in literature. These studies have analysed environmental impacts of fibres and matrices used in the various applications of glass fibre composites. Results of these studies can help to understand the environmental impacts of nanoclay composites. Rosa et al.  reportedOverall, it is reported that using plant-based fibres in composites improves the sustainability of composites  and seveDue to impending threat of extreme climate change, there is a growing concern about environmental impacts of various novel materials such as nanocomposites. As the population is becoming aware of adverse effects of various technologies, demand for sustainable and green solutions is growing. Therefore, while introducing new materials it important to consider the environmental impacts of these materials. Sustainable materials should balance technical, economic and environmental constraints. By considering environmental impacts of novel materials in early stages of their design, negative environmental and human health effects can be avoided. Given that the demand for nanomaterials including nano composites is rapidly growing and that their environmental impacts are not fully understood, future studies should develop in depth methods to analyse various environmental impacts and human health issues caused by nanocomposites.The addition of inorganic nanoclays to epoxy resin improves its physical, mechanical and barrier properties. The most common nano fillers used with epoxy resins are the organically modified montmorillonite clays with filling amounts ranging from 1% to 10%. Low clay contents are shown to result in better E-nc structures. It is proven by several researchers that E-nc with exfoliated morphology results in noticeable improvements in most of the properties of the nanocomposite as compared with the pristine epoxy. This E-nc morphology has been shown to maximize the interactions between the resin and the nanoclay platelets, resulting in appreciable improvements in the mechanical, thermal and liquid barrier properties of the epoxy. Improvements of more than 68% in tensile strength and 80% in resistance to water absorption were reported. The complete exfoliation of nano fillers in epoxy matrices remains quite a challenge because the degree of exfoliation depends on the type of epoxy, the type of organically modified clays, the clay loading and the mixing and curing methods. Because of its excellent mechanical strength under both static and cyclic loading conditions GFRE has found a large number of applications. One major obstacle to a wider use of GFRE in structures, such as piping systems, is the affinity of epoxy resin to moisture absorption, especially at high temperatures. The combination of the nanoclay together with glass fibres as reinforcements has been shown to lower the affinity to moisture uptake and improve the mechanical properties of GFRE. Addition of the right amount of clay improves the tensile, flexural and impact properties of GFRE. The maximum water uptake decreases with increasing clay loading and moisture diffusion at higher temperatures was higher than that at room temperature. Though exposure to moisture of GFRE-nc resulted in the degradation of its mechanical properties, they always remained comparably higher than the pristine epoxy.There is almost consensus that properly dispersed and distributed nanoclays improve the physical, mechanical and thermal properties of epoxy. However, there are still contradictions on how much improvements and how to arrive at the complete exfoliation of nanofillers in polymer matrices. This requires an effort to establish detailed procedures for each type of organically modified clay and epoxy. The problem of nanoclay filtration during the production of GFRE-nc needs more investigation. Finally, like every novel material, epoxy nanoclay composites have environmental impacts that need to be fully understood. Further research is needed to develop environmental profiles for these materials. Special focus is required to incorporate end-of-life phases into LCA studies. Similarly, analysis of exposure pathways in various applications of these materials should be investigated to fully understand their effect on human health.


Human mesenchymal stem cells (MSCs) remain one of the best cell sources for cartilage, a tissue without regenerative capacity. However, MSC chondrogenesis is commonly induced through TGFβ, a pleomorphic growth factor without specificity for this lineage. Using tissue- and induced pluripotent stem cell-derived MSCs, we demonstrate an efficient and precise approach to induce chondrogenesis through Wnt/β-catenin antagonism alone without TGFβ. Compared to TGFβ, Wnt/β-catenin antagonism more rapidly induced MSC chondrogenesis without eliciting off-target lineage specification toward smooth muscle or hypertrophy; this was mediated through increasing N-cadherin levels and β-catenin interactions—key components of the adherens junctions (AJ)—and increasing cytoskeleton-mediated condensation. Validation with transcriptomic analysis of human chondrocytes compared to MSCs and osteoblasts showed significant downregulation of Wnt/β-catenin and TGFβ signaling along with upregulation of α-catenin as an upstream regulator. Our findings underscore the importance of understanding developmental pathways and structural modifications in achieving efficient MSC chondrogenesis for translational application.  •Classical TGFβ protocol for MSC chondrogenesis results in off-target differentiation•More rapid chondrogenesis is seen using Wnt antagonism only without TGFβ•Wnt antagonism only without TGFβ increase N-cadherin/β-catenin AJ interactions•Wnt & TGFβ pathways decreased & AJ upregulated in human chondrocyte transcriptome Bioengineering; Biological sciences; Molecular medicine; Tissue engineering Current treatments for arthritic diseases are largely limited to symptom palliation using analgesics for mild cases, and immunosuppressants and/or surgery for more serious cases.,,,Human multipotent mesenchymal stem cells (MSCs) are versatile somatic stem cells with immunomodulatory properties. First isolated in adult bone marrow (BM), MSCs have subsequently been found in numerous post-natal organ/tissues,β-catenin/Wnt signaling, a major developmental and oncogenic pathway, is also important in skeletal related-tissue/organ development, especially for bone and cartilage tissue. Clinical studies have found that the low-density lipoprotein receptor-related protein 5 (LRP5), a key component of canonical Wnt/β-catenin pathway, is critical to osteogenesis, with a loss-of-function mutation impeding bone development to cause osteoporosis-pseudoglioma syndromeCOL2A1) and aggrecan (ACAN) but not SOX9; Wnt/β-catenin agonism, on the other hand, resulted in significant downregulation of all 3 genes  containing TGFβ3 along with a small molecule Wnt/β-catenin agonist CHIR which inhibit GSK3β to disrupt the β-catenin destruction complex, or antagonist XAV, a tankyrase inhibitor which stabilizes the β-catenin destruction complex. TGFβ3 was utilized in ChM because it has been found to possess higher chondrogenic potential than TGFβ1. 3 genes C–1E. Of e effect F. These GSE109859 for human primary smooth muscle cells) with Ranked Gene Set Enrichment Analysis (GSEA), gene set of Signaling by TGFβ Family Members was positively enriched in human BM-MSCs compared to smooth muscle cells with normalized enrichment score (NES) equals to 2.17 and the nominal p value less than 0.001  as well as αSMA (ACTA2), a marker of smooth muscle differentiation and also fibrosis,αSMA and/or RUNX2, the master osteogenic transcription factor, during MSC chondrogenesis. Immunofluorescent staining of αSMA on Day 3 of MSC chondrogenic induction showed strong expression in both TGFβ-treated groups compared to chondrogenic basal medium (ChBM) which does not contain TGFβ (αSMA expression (up to 3-fold) in TGFβ-treated groups compared to ChBM  every three days for 20 days  and found that with N-cadherin (CDH2), β-catenin (CTNNB1) is a core participant within the signaling pathway, with numerous components of the AJ: α-catenin (CTNNA1), p120/δ-catenin (CTNND1), and plakoglobin (JUP). Moreover, the three major GTPases responsible for actin cytoskeleton organization—RhoA, CDC42, and Rac1—are all found as downstream effectors in the signaling pathway E, demonsGSE108186 for human primary BM-MSCs; GSE68038 for human primary chondrocytes; and GSE121892 for human primary osteoblasts). Initial analyses using principal component analysis (PCA) demonstrated that chondrocytes, osteoblasts, and MSCs are three highly distinct populations  which can provide a more causal relationship between transcription factors/master regulators and downstream pathways,,,,,in vitro stem cell differentiation studies did not consistently find Wnt inhibition to promote chondrogenesis, these reports utilized biologically derived Wnt inhibitory ligands which are known to have off-target effects as well as potency issues.,in vitro chondrogenesis in human MSCs from multiple sources, and in vivo using murine BM-MSCs. Moreover, in comparison with TGFβ3, the most potent member of the family for induction of chondrogenesis, Wnt/β-catenin antagonism more rapidly induced chondrogenesis without inducing other non-chondrogenic, off-target, lineages. Our findings are further supported by transcriptome analysis of primary human MSCs, chondrocytes, and osteoblasts, which demonstrated that the Wnt/β-catenin and TGFβ family signaling pathways were downregulated in chondrocytes relative to the other 2 cell types .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, B. Linju Yen  consisting of low-glucose DMEM supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin , 1% Insulin-transferrin-sodium selenite media supplement , 2 mM L-glutamine (Gibco-Thermo Fisher Scientific), 10 μM L-Ascorbic acid 2-phosphate (Sigma-Aldrich), 100 nM dexamethasone (Sigma-Aldrich). Addition of TGFβ1 , TGFβ3 , XAV939  or CHIR99021  to ChBM was performed as indicated. Cell pellets were harvested at the indicated day for analysis. Micromass culture-based chondrogenic differentiation was performed by applying 2 × 107 cells/mL of MSCs suspended in expansion medium and 20-μL drops spotted in the center of each well of a 24-well culture plate.,2, the medium was changed to ChBM with indicated regulators for chondrogenic induction.Samples were washed with PBS and fixed with 4% paraformaldehyde, followed by pH 1.0 1% Alcian blue 8GX (Sigma-Aldrich) staining at room temperature overnight to detect sulfated proteoglycan matrix.GAPDH) level were used as housekeeping control. The results are normalized to ChBM to represent effects of indicated modulators. The sequences of primers for each gene are listed in Total RNA was isolated using TRI® reagent (Sigma-Aldrich), and quantified using NanoDrop spectrophotometer .αSMA) (Sigma-Aldrich), β-catenin  or N-cadherin  were stained with 1:100 dilution for 24 hours, followed by species specific secondary antibody incubation overnight. Fluorescent signals were acquired by confocal microscopy  and analyzed by ImageJ .Immunofluorescent staining was performed as previously reported.Micromass cultured iPSC-MSCs were used for PLA and performed according to the manufacturer’s protocol (Sigma-Aldrich). Antibodies against N-cadherin (Abcam) and β-catenin (BD Biosciences) were used for recognition. After ligation and amplification, the PLA signals from each pair of probes in close proximity (<40 nm) were visualized by confocal microscope (Leica TCS SP5 II) and signal counts were analyzed by ImageJ.GSE109859), MSCs (GSE128949 and GSE108186),GSE68038),GSE121892)https://www.qiagenbioinformatics.com).Transcriptome data of human primary smooth muscle cells .All data represent three replicates or more from separate experiments. Analysis of variance (ANOVA) followed by Tukey’s post-hoc test was performed to evaluate significance for comparisons for multiple groups, with


This guest editorial provides an introduction to the Special Section on Computational Approaches for Neuroimaging. Neurophotonics Volume 9 Issue 4special section of  consists of five review articles and five research papers. These contributions are representative of the broad range of optical technologies employed in neuroscience, such as one-photon or multi-photon imaging of functional indicators, e.g., calcium, voltage, and neurotransmitter, functional near-infrared spectroscopies to image hemodynamics, and optical intrinsic signal imaging. Beyond imaging, this special section also covers the rising field of photostimulation.New advances in computational methods are revolutionizing our ability to collect, reconstruct, analyze, and interpret neuroimaging data. In turn, they provide unique new approaches to understand brain activities and functions at subcellular, cellular, circuit and network levels. This special section collates ten contributions that cover many recent computational technologies underlying these developments. These computational techniques address several challenges, including recording of neural activities, reconstruction of neural activities, inference of functional connectivity, and interpretation of the brain function at cellular level and circuit level. This in vivo, which is timely since mesoscale imaging at high resolution has become one of the main frontier in neuroimaging. Carrillo-Reid and CalderonA major focus of emerging computational approaches is towards fast and accurate data processing for functional neuroimaging data. Benisty et al.issue cover) provide a focused review on advances in computer-generated holography (CGH) for targeted neuronal modulation, which highlights novel algorithms and hardware implementations of advanced CGH techniques to shape the light intensity distribution in 3D for optically interrogating neuronal populations in conjugation with optogenetics. Howe et al.Another emerging area is computational imaging, which seeks to synergistically combine novel optical designs and advanced computational algorithms to enable novel capabilities. XueIn summary, computational approaches will continue to play a central role in advancing neuroimaging. Driven by the need to handle massive spatiotemporal data, we expect fast and accurate data processing pipelines that can leverage advanced deep learning frameworks will be a trend in the coming years. In order to overcome physical limitations to achieve large field-of-view, high spatial resolution, ultrafast acquisition speed, deep penetration, high signal-to-noise, and system simplicity, we expect computational optics will continue to advance and push the boundaries of neural imaging technologies and subsequently, our understanding of the brain at work.


Carthamus tinctorius L. (CTLP-1 and CTLP-2) were purified, and their structures were analyzed by physical and chemical testing. CTLP-1 had a mass of 5900 Da that was composed of arabinose, glucose, and galactose with a mass molar ratio of 6.7:4.2:1. The backbone of CTLP-1 was →1)-α-GalAp-(1→4)-α-Arap-(1→2)-α-Glup--α-Galp--α-Arap--α-Glup-(3→. Both of them exhibited a high reducing power, hydroxyl radical scavenging activity, DPPH radical scavenging activity and ABTS radical scavenging activity, moderate Fe2+ chelating activity and superoxide anion scavenging activity, implying that they might be potential antioxidants.Two polysaccharides from  Carthamus tinctorius L.) is an annual herb native to the sandy lands of Russia, Japan, Korea, and China  curve was then drawn as the standard curve as the abscissa, where M is the standard molecular weight of glucan. The molecular weight of CTLP was determined in the same way.The 2PO4 in 100 mL of acetonitrile, adding 0.5 mL of TEA and 900 mL of ultra-pure water, shaking well, and passing it through the membrane. The column was balanced with phase A for 3 min, and the elution procedure was as follows: 94% mobile phase B (0–4 min); 94–88% mobile phase B (4–9 min); 88% mobile phase B (9–30 min); and 88–94% mobile phase B (30–35 min).CTLP solution was loaded onto a reverse-phase C18 column (4.6 × 25 cm) to achieve chromatographic separation. The injection volume was 20 μL, and the column temperature was 37 °C. Pure acetonitrile (mobile phase A) was passed through the organic membrane (0.22 μm). Mobile phase B was formed by dissolving 0.45 g KH−1; 0, 0.5, 1.0, 1.5, 2.0, and 4.0 mL) were diluted 250 times with purified water, and their absorbance values at 223 nm were determined [4 solution (50 nmol∙L−1) and stored in a brown volumetric bottle to keep light out. The absorbance at 223 nm was repeatedly measured until the final absorbance value remained unchanged. The mixture with constant absorbance (2 mL) was titrated with 0.01 mol∙L−1 NaOH solution, and phenolphthalein was used as an indicator to determine the production of formic acid. Using purified water as the control, the consumption of periodate was calculated with the standard curve.NaIO4 solutions  following the KBr pressed-disk method [The purified CTLP was ground with KBr powder. The crude CTLP was verified in the range of 4000–400 cmk method ,18. Each2O at a concentration of 3% for 3 h and then freeze-dried. The 1H spectra of CTLP was detected with an NMR spectrometer .The CTLP was dissolved in DTo determine the triple-helical structures of the CTLP, refer to the method of our previous research . The abs2) overnight at 23 °C, then stained for 1 h at 23 °C in 1% osmium tetroxide and 1% potassium ferricyanide in cacodylate buffer, washed with ddH2O and then dehydrated by 10 min incubations in a graded acetone series: two incubations each in 30%, 50% and 70% acetone, and four incubations in 90% and 100% acetone. Then the cover slips were critical-point dried and sputter-coated with 4 nm of gold particles. Images were collected in an LEO1550 scanning electron microscope at 2.5 kV using an in-lens detector.For SEM analysis  of CTLP,g. The supernatant was transferred to a test tube with 0.5 mL 0.1% FeCl3 solution and 2.5 mL water. The absorbance of the mixture was determined at 700 nm, and vitamin C (Vit. C) was the positive control.The reducing power of the CTLP was determined by the following method : The sol2+ chelating activity of the CTLP was determined as follows [−1 ferrous chloride solution, 0.2 mL 5 mmol∙L−1 Ferrozine solution and 2.75 mL water. After fully mixing, the absorbance was determined as 562 nm. EDTA was the positive control, and the water was the blank control.The Fe follows : Five po−1 pyrogallol; and 2 mL, 50 mmol∙L−1 Tris-HCl were mixed. Then, the mixture was incubated at 25 °C for 5 min with the addition of hydrochloric acid, and the reaction was stopped. The absorbance was recorded at 560 nm with Vit. C as the positive control.The superoxide anion radical scavenging activity was determined by a previous method . Differe−1 CTLP was mixed 1 mL, 9 mmol∙L−1 salicylic acid-ethanol and 1 mL, 9 mmol∙L−1 FeSO4. The mixture was incubated at 37 °C for 30 min. The absorbance was detected at 510 nm with Vit. C as the positive control.The hydroxyl radical scavenging activity was determined as previously reported . The 1 mVit. C was the positive control.The DPPH radical scavenging activity of the CTLP was detected as follows . 2 mL diVit. C) was again used as the control group.The ABTS radical scavenging activity was detected, as in the previous description . DiffereA0 is the absorbance of doubly distilled water, A1 is the absorbance of the solution with CTLP in the reactive system, and 2A is the absorbance of the solution with CTLP.The scavenging activities above were determined as Equation (2),−1 NaCl solution and CTLP-2 was eluted with 0.2 mol∙L−1 NaCl solution. The absorbance was determined by the DNS method. The CTLP-1 and CTLP-2 solutions were dialyzed in double-steaming water by dialysis bags with an interception molecular weight of 4000 Da at 4 °C for 24 h, and the polysaccharides were collected by freeze-drying the dialysate.The elution curve of safflower polysaccharides adsorbed on the cellulose column was obtained by gradient elution with water and NaCl solutions with gradient concentrations. Two uniform elution peaks are seen in The collected CTLP-1 and CTLP-2 were eluted using a G-100 agarose gel column, and the absorbance of each eluent was determined. As shown in Y = 5.9207-5.9096X (R2 = 0.9984). The relative molecular weights of CTLP-1 and CTLP-2 were 5900 and 8200 Da, respectively. The molecular weights of plant polysaccharides have been reported to affect their biological activities to a certain extent. In general, an overly small molecular weight corresponds to a weak biological activity, whereas a high molecular weight leads to strong activity. Compared with the leaf polysaccharide, the root polysaccharide of Panax ginseng marc was more complex and had a larger molecular weight. The immune activity of the root polysaccharide was higher than that of the leaf polysaccharide [The standard curve of glucan molecular weight was obtained and the regression equation was ccharide . Similarccharide . HoweverThe common monosaccharides in plant polysaccharides are galactose, glucose, arabinose, fructose, xylose, rhamnose, and various other monosaccharides. Y = 0.0735X + 0.0569 (R2 = 0.9986). According to the standard curve, CTLP-1 and CTLP-2 were gradually oxidized by periodate, and the absorbance decreased continuously until becoming stable. This process consumed periodate  and produced formic acid . For both CTLP-1 and CTLP-2, the amount of periodate consumed was more than twice that of formic acid produced. Thus, the CTLP had a bond type that consumed periodate but did not produce formic acid . The oxidized residue of CTLP-1 was approximately 70%, and the non-oxidizable residue was approximately 30%; thus, CTLP-1 had 1→6 glycosidic bonds and a small amount of 1→3 glycosidic bonds. The oxidized residues of CTLP-2 were approximately 50%. The oxidation residues of 1→2 and 1→4 were approximately 10%, and the non-oxidizable residues were approximately 40%. Thus, CTLP-2 had 1→6 glycosidic bonds and a small amount of 1→2 and 1→4 glycosidic bonds.Periodate oxidation primarily occurs on the sugar chains of polysaccharides and selectively acts on the trihydroxyl and hydroxyl groups of the sugar chains. The periodate consumption and formic acid production differ based on the reaction conditions . The sta−1. The peak at 3383 cm−1 is attributed to the stretching vibrations of the –OH and N–H functional groups. This peak is not sharp, indicating that these functional groups were in a non-free state [−1 is mainly caused by the stretching vibration of the –CH2– functional group. The absorption peak at 1664 cm−1 is caused by the stretching vibration of C=O. The peak at 1379 cm−1 is caused by the angular vibration of the N–H functional group. The peak at 1043 cm−1 is mainly caused by the angular vibration of C–H. Finally, the peak at 623 cm−1 is due to the C–O stretching vibration in the C–O–C functional group of the ether bond of the pyran ring [−1, indicating a similar functional group composition to CTLP-1. The absorption peaks at 1043 and 1046 cm−1 indicate that both CTLP-1 and CTLP-2 contained pyran rings. For CTLP-1, the absorption peak at 623 cm−1 indicates the existence of glycosidic bonds with α configuration.The FT-IR spectrum of CTLP-1 A had obvee state . The pearan ring . The spe1H NMR chemical shifts of CTLP-1 are mainly observed in the region of 0.3–2.7 ppm, while those of CTLP-2 mainly occur in the range of 0.3–4.5 ppm. This indicates that the glycosidic bonds in the CTLP were mainly α-glycosidic bonds. CTLP-1 exhibited fewer NMR peaks than CTLP-2, indicating fewer residues in CTLP-1 [As shown in n CTLP-1 . The higFor the mixture of CTLP-1 and CTLP-2 with Congo red, the wavelength of maximum absorption continuously decreased with the increase in sodium hydroxide concentration, due to the safflower polysaccharides reacting with Congo red to form new complexes ,28. As tThe SEM images of CTLP-1 A and CTL−1. This difference may be related to their respective molecular weights and glycosidic bonds [CTLP-1 and CTLP-2 exhibited a certain degree of reducing power in ic bonds . Compare−1, the Fe2+ chelating activities of CTLP-2 and CTLP-1 reached 29.5% and 25.2%, respectively. CTLP-1 and CTLP-2 had weaker Fe2+ chelating activities than EDTA which might be related to the fact that Fe2+ is easier to chelate in alkaline environments [As shown in ronments , while t−1. This may be related to the monosaccharide composition of safflower polysaccharides. The monosaccharide content has a certain effect on the antioxidant activity of polysaccharides, and the specific monosaccharide composition can show different antioxidant activity in different antioxidant indexes [CTLP-1 and CTLP-2 showed concentration-dependent superoxide anion radical scavenging activities C, but bo indexes .Vit. C as shown in −1.We can see that CTLP-1 and CTLP-2 exhibited strong hydroxyl radical scavenging activity compared with −1 (65.2% for CTLP-1 and 60.5% for CTLP-2).In Vit. C, the ATBS radical scavenging activities of CTLP-1 and CTLP-2 were strong enough.We also noted that the ATBS radical scavenging activities of CTLP-1 and CTLP-2 increased with increasing polysaccharide concentration F. The acCTLP1 and CTLP2 exhibited different antioxidant activities in different experiments, and there were some differences between them. The difference between their respective molecular weights and glycosidic bonds may lead to the differentiation. Therefore, the effect of polysaccharide structure on antioxidant activities needs to be further studied.Carthamus tinctorius L. The relative molecular weights of CTLP-1 and CTLP-2 were 5900 and 8200 Da, respectively. CTLP-1 was a heteropolysaccharide composed of arabinose, glucose, and galactose with a molar ratio of 6.7:4.2:1; CTLP-2 was a heteropolysaccharide containing arabinose, glucose, and galactose with a molar ratio of 16.76:4.28:1. The results show that both CTLP-1 and CTLP-2 exhibited relatively high antioxidant activities in vitro and could be further applied as a novel natural antioxidant drug. In the future, we would look to measure the activity of CTLPs in vivo and to research the relationship between its activity and structure.The two CTLP components were obtained and marked as CTLP-1 and CTLP-2 by using a G-100 agarose gel column from


Due to its unique properties, including strong adhesion force, high heat resistivity, high insulation properties, and strong mechanical properties, epoxy resin is the most commonly used material for a variety of applications, including adhesives, electronic devices for coatings, and somewhere as a matrix for reinforcement of composites as a fiber network. To boost their properties, different other materials are also inserted in their structure and made its composites; silicon is one of them. Corrosion is serious for marine equipment and causes economic loss. To overcome such issues, different types of coating materials are developed. In this review, current methods for coatings of different materials using a silicon dioxide epoxy nanocomposite are discussed in diversity with the currently followed synthetic routes for the preparation of nanosilica epoxy composites and enhanced properties. R1, R2, R3, and R4 represent aryl and alkyl groups  to high performance, diversified functionalized with aromatic groups resins like triglycidyl ether aminophenol and methyl aniline (TGMDA) . BisphenBeing a thermosetting resin, these are cured using a different variety of chemicals in its curing process. The specific combination of epoxy resins and curing chemicals used determines their quality. Due to the versatile properties of these resins used for a variety of applications including fiber reinforcement products, some general adhesives, and coating applications, these applications are due to their strong mechanical properties and good resistivity to heat and chemical changes –13.μm liners, and fillers cannot be used in transparent applications.Fillers are required in several epoxy resin formulations. Fillers increase mechanical properties like rigidity, modulus, and hardness. But sometimes it has a negative impact on the viscosity of resins, making them unsuitable for a variety of applications. Furthermore, the filler is filtered out by the fabric when the resin product containing the filler is exposed to injection manufacturing techniques for fiber-reinforced composites. As a result, many activities that might be advantageous from the use of a filler are incompatible with standard A composite is a material that has been blended from two or more components to produce something new with better qualities. Composites have attractive properties such as thermal stability, dynamic property, and tensile strength, making them very applicable in different applications. So these variations in properties make it a very useful material among all others. The components are macroscopically mixed and are not soluble in one another. The matrix phase is one of the constituents, whereas the reinforcing phase is the other .The introduction of surface-modified nanosphere into the market was first time done in 2002 and 2003. They were created in situ immediately in the epoxy resin to use a modified sol-gel process, with a mean particle size of 20 nm and an exceedingly narrow particle size dispersion  in theirThey offer several benefits, including the fact that because they are 20 nm in size and completely monodisperse, they only have a little effect on resin viscosity at higher concentrations. Unlike fumed silica, they have no highly viscous properties and act like a Newtonian liquid. Because of their small size, they are transparent and can easily enter even close-meshed materials in composite fabrication .2 epoxy resin composites for coatings propyl-trimethoxysilane (TAPS), 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane (KH-550), and aminoethyl)—aminopropyltrimethoxy. Relative to an undoped nanoscale model, the surface-mounted KH792 form of nano-SiO2 showed the most noticeable improvement in thermal characteristics. The Tg was enhanced by 61 K while the storage modulus increased by 276 MPa. The mechanical characteristics of a nano-SiO2 surface-mounted KH-792 model were about three times greater than those of the undoped nanostructured model, and the Tg increased by 36.5 K and the heat transfer efficiency by 24.5%, according to simulation results  furan (BOMF) or DGEBA monomer, to produce silica nanoparticles in epoxy; in the end, both systems were cured with methyl and anhydride (MNA) .2 and the size of SiO2 depicted in Kejing et al. described the synthesis of silicon dioxide at the exterior of graphene nanoplates to improve the reinforcement and hardness of epoxy resins. Experimental results reveal that the prepared material has high reinforcing and mechanical e5ffect as compared to that of containing fillers. The exceptional structure and restrengthening studies of graphene and silicon dioxide decreased the force concentration, which makes the propagation way more difficult and affording more load which was attributed to the chemical bond of cross-linked epoxy resins and graphene/SiO2 particles (MWCNTs). The effects of nano-SiO2 and MWCNTs on the tensile modulus of epoxy composites as well as their strengthening processes were studied. The outcomes show that the mechanical properties of the epoxy composites have significantly improved. Composites made of nano-SiO2/MWCNTs/EP exhibit the finest mechanical properties. As a result of the synergistic achieving and sustaining competitive advantage of nano-SiO2 and MWCNTs on the EP, the degree of stress concentration can be reduced, and more energy can be absorbed. These mechanisms include the microplastic displacement effect, micropores, their divarication influence, and the knock effect of MWCNTs in the EP matrix [Chufan et al. produced adhesives (EP) reinforced with multiwall carbon nanotubes and nano-SiOP matrix .2 particles which were filled with a glass fiber/polyamide composite in a ground-based atomic oxygen effect simulation facility as part of Xin's research to enhance the AO resistance feature of a spacecraft resin matrix composite. The AO resistance of this novel composite was significantly enhanced, while mass loss and erosion yield were significantly reduced. It shows that injecting these nanoparticles with resin is a good way to improve the composite's AO resistance. After a 40-hour exposure experiment, the erosion yield of glass fiber/SiO2/polyimide dropped to 16.4% [An atomic oxygen (AO) exposure experiment was studied for nano-SiOto 16.4% .2 nanoparticle composites that were surface diversified with the bonding agent 3-glycidyloxypropyltrimethoxysilane. The unmodified and modified nanoparticles were mixed in an epoxy resin and cured at 0.5–5 phr using a poly (oxypropylene) diamine (parts per hundred parts of resin). The nanofiller had no influence on the epoxy-amine matrix's curing process, network shape, or disintegration mechanism, but it did increase the mechanical strength of hybrid materials, particularly Young's modulus and hardness. Only 0.38% of unmodified particles remained after thermal deterioration, resulting in significantly more residue, a 30% increase in Young's modulus, and a 40% increase in the intensity, as well as improved toughness and reduced compaction creep, often without negatively impacting the reinforced material's brittleness. Increasing the fiber content resulted in a loss of several characteristics due to increased agglomeration formation [Jelena et al. developed hardener, bisphenol A DGE, and SiOormation .2 nanoparticles, which successfully produced a transformed BF epoxy coating. According to experimental findings, the BF epoxy resin coating modified with three nano-SiO2 nanoparticles had good mechanical performance, limited water infiltration (water absorption of 0.72% after 480 hours), and superior chemical toughness  (tensile strength of approximately 33.4 MPa). The attainability of employing nano-SiO2 microspheres to improve the chemical and mechanical characteristics of BF epoxy resin coatings is demonstrated in this work [Basalt flakes (BFs) have recently become popular in the maritime sector as a one-of-a-kind anticorrosion substance for preventing metal substrate corrosion in this study, BFs were changed by using 1–7 nano-SiOhis work .2 nanosheets were created to alter SiO2 nanoparticles to fabricate SiO2-MoS2 basic nanomaterials as well-dispersed nanofillers, which improved the corrosion protection and mechanical properties of an oil-based epoxy matrix. The findings revealed that MoS2 nanosheets coated SiO2 nanoparticles. Furthermore, MoS2 nanosheets might be used as a SiO2 reinforcing agent to strengthen the contact area between epoxy and SiO2 nanoparticles, thereby improving the epoxy resin's anticorrosive and mechanical qualities simultaneously, depicted in Youqing et al.'s MoS2-epoxy and SiO2-glass-fiber epoxy nanocomposites, using KH-560 as a coupling agent for the synthesis of nanometer-size SiO2 and heated with epoxy CYD-128 at 120°C to reduce the viscosity of CYD-128 epoxy, which were then homogenized and hardener agent added. The results showed that as-prepared material possesses high tensile strength, tensile modulus, and impact strength [Yapang et al. reported the preparation of SiOstrength .3O4 and SiO2 nanoparticles in the epoxy-modified silicone resin an inorganic-organic hybrid of three different types of combinations such as SiO2 dispersion, ferromagnetic Fe2O4 dispersion, and simultaneous dispersion of both, where nanoparticles were synthesized by using the coprecipitation method. It was reported that silica has homogenously dispersed but ferromagnetic-caused agglomeration, where simultaneous addition has good dispersion of both type particles without much loss [Dahao et al. studied the incorporation of the Feuch loss .2 epoxy nanocomposite in a sustainable and ecologically friendly approach. Jieyuan et al. used a eugenol-based epoxy silane-coupling agent to make nano-SiOHarikrishnan et al. detailed how halloysite nanotubes (HNTs) were saturated using biobased epoxy resin by vacuum infiltration, and silica nanoparticles were modified with a UV activator through simple mixing to create a self-healing covering consisting of these three materials. Both were blended with pure epoxy glue made from biobased materials and then hardened .2) nanocrystals, novel superhydrophobic coatings with significant interfacial adhesion were created. Superhydrophobic F-SiO2/epoxy coatings adhered to the surface of PC materials with a high degree of dependability as a result of the exfoliation of PC substrates and the crystallization process caused by acetone. [Jiayao et al. found that by coating polycarbonate (PC) substrates with an acetone solution containing epoxy resin and fluorinated silica , constrained water infiltration (water absorption of 0.72% after 480 hours), and excellent mechanical effectiveness (tensile strength of approximately 33.4 MPa) [Mingdong et al. studied siloxane-based epoxy resin coating for variable temperatures, which yielded good resistance to temperatures as low as -30°C and high as 25°C for 10 days without any change, showing good resistance to mechanical changes and corrosion . Similar3.4 MPa) .2@BN composite coatings on thermal conductivity. The thermal conduction can be reduced by coating thickness and filler amount if increased. Using the fitting model for the thermal conduction suggested that the filler content increment causes an increase in thermal conductivities of boron nitride-silicon dioxide composites, and the thermal conduction increasing elements decreases [Yunmin et al. studied the effect of epoxy/SiOecreases .2 particles in the polypyrrole matrix [Ruben et al. studied silica/siloxane epoxy resin for anticorrosion purposes. Epoxy-siloxane-silica hybrid nanomaterials with good corrosion protection and thermal and mechanical characteristics were created to act as protective coverings for metallic surfaces . The repe matrix .2O3@SiO2 nanocomposite to fabricate on epoxy coatings to enhance its anticorrosion performance. By verifying that the nanocomposite is homogenously dispersed and causes the increase in the anticorrosion activity of epoxy resin, Zhang et al. reported Fe3O4 a5nd SiO2 nanoparticles have a soft covering for soft magnetic composites. Because ferromagnetic Fe3O4 is used, the magnetic dilution effect is reduced, resulting in improved magnetization and permeability. Inclusion of SiO2 in the SMCs, on the other hand, reduces Fe3O4 nanoparticle aggregation and results in higher electrical resistivity, resulting in decreased core loss and enhanced mechanical strength [ESRs modified with ferromagnetic Festrength .In literature , a tremeLiterature always led to diversification toward efficacy in productivity and perfections; one of the recent research studies was carried out on superhydrophobic epoxies, after variable studies on mechanical distortions and in various corrosive surroundings like acidic-, alkaline-, and salinity-based. The experimental results were full of outstanding ability against photooxidative and hygrothermal aging. In response, it was admired that coatings prepared for hydrophobic application have satisfaction in harsh conditions based on PC material . SimilarEpoxy resins being versatile raw material are utilized in a range of industrial applications which include windmill blades, particularly in complex aeronautical parts like wings and fuselages, and used as building coatings and adhesives. In the current study, we have discussed the synthesis of silicon dioxide containing epoxy resins as well as silicon dioxide epoxy composites and their application for coating as adhesives to protect precious material from being ruptured by environmental conditions, using different environmentally friendly and green approaches. Moreover, the study of its enhanced properties such as high thermal stability, with high tensile strength, high productivity, and icephobicity to self-healing coatings, was carried out.