SMA is characterized by the degeneration of spinal cord motoneurons (MNs), progressive skeletal muscle atrophy, and weakness. The cellular and molecular mechanisms causing MN loss of function are only partially known. Recent advances in SMA research postulate the role of calpain protease regulating survival motor neuron (SMN) protein and the positive effect on SMA phenotype of treatment with calpain inhibitors. We analyzed the level of calpain pathway members in mice and human cellular SMA models. Results indicate an increase of calpain activity in SMN-reduced MNs. Spinal cord analysis of SMA mice treated with calpeptin, a calpain inhibitor, showed an increase of SMN, calpain, and its endogenous inhibitor calpastatin in MNs. Finally, in vitro calpeptin treatment prevented microtubule-associated protein 1A/1B-light chain 3 (LC3) increase in MNs neurites, indicating that calpain inhibition may reduce autophagosome accumulation in neuron prolongations, but not in soma. Thus, our results show that calpain activity is increased in SMA MNs and its inhibition may have a beneficial effect on SMA phenotype through the increase of SMN in spinal cord MNs.Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by loss of the  SMN complex participates in the assembly of small nuclear ribonuclear proteins (snRNPs), which are involved in the splicing of pre-mRNA2. SMN also accomplishes unique functions in motoneurons (MNs), such as the transport of β-actin mRNA to the growth cones of the axons3. SMN protein deficiency is the basis of the neuromuscular disease spinal muscular atrophy (SMA). SMA is a devastating disorder that leads to progressive muscle weakness and atrophy and represents the most common lethal genetic disease in infants6. SMA is characterized by the degeneration of spinal cord MNs caused by the mutation of the survival motor neuron (SMN1) gene located at the telomeric region of chromosome 5q3, which is responsible for the production of SMN protein7. SMN2 gene, a nearly identical paralog of SMN1 in centromeric region of chromosome 5q3, is present in the human genome as part of an inverted duplication. SMN2 expresses limited amounts of functional full-length SMN protein. In SMA, SMN2 is efficiently transcribed but cannot fully compensate for the loss of SMN1 because a translationally silent, single-nucleotide transition at position 6 in exon 7 causes predominant exon 7 skipping and results in an unstable protein (SMNdelta7)9. The number of SMN2 gene copies inversely correlates with the severity of SMA phenotype and determines disease onset10. Although the genetic causes of SMA are well-known, the mechanisms underlying lower MN degeneration remain unclear. For example, MNs may have a uniquely high demand for efficient messenger RNA splicing or disruption of a specific SMN function in MNs may cause SMA11. Knowledge of the molecular and cellular mechanisms of MN degeneration could be extremely relevant to prevention and/or delay of SMA progression and to optimization of recently developed therapies13.Survival motor neuron (SMN) is a ubiquitously expressed protein located in the cytoplasm and the nucleus of the cells, where it accumulates in Cajal bodies and Gems 15. Calpains are a calcium-dependent family of proteases that participate in calcium-mediated signaling pathways involved in cell processes. Increases of cytosolic calcium in neurons activate calpains and pathological conditions often result in their overactivation18. Autophagy is a regulated process responsible for the degradation of cytoplasmic proteins and organelles by incorporating them into a double-membrane vesicle (autophagosome) that is delivered to the lysosome19. In SMA, autophagy is likely involved in MN death20.The calpain protease system and autophagy are two major regulatory pathways of the cell that have been correlated to neurodegenerative disorders14. In this context, we recently reported that calpain inhibition increases the SMN protein level in cultured spinal cord MNs and that administration of calpeptin  to SMA mice models improves lifespan and motor function21. Based on these results, the aim of the present work was to investigate the calpain pathway in SMN-reduced MNs. We examined calpain activation by monitoring α-fodrin fragments and concluded that calpain activation is increased in cultured human and mice SMA MNs, but not in SMA fibroblasts. In vivo calpeptin treatment increases Smn, calpain, and calpastatin protein levels in spinal cord MNs. Finally, the addition of calpeptin to the culture medium modulated the level of the LC3 autophagy marker in cultured MNs. Our findings suggest that calpain can be overactivated in SMN-reduced MNs and support the hypothesis that calpeptin may be useful in SMA therapies.Evidence exists that calpains exert modulatory effects at multiple levels of autophagy89AhmbSmn1tm1Msd/J (mutSMA) and FVB·Cg-Grm 7Tg(SMN2)89Ahmb Smn 1tm1Msd Tg(SMN2*delta7)4299Ahmb/J (SMNdelta7) mice were kindly provided by Dr Josep E Esquerda and Dr Jordi Caldero (IRBLleida-Universitat de Lleida). MutSMA  and SMNdelta7  were obtained by crossing heterozygous animals. Littermates mutSMA/SMNdelta7 and WT  were used for the experiments.Experiments involved two severe SMA mouse models. FVB·Cg-Tg (SMN2)For MN purification, the heads of 12.5-day embryos (E12.5) were snipped for genotyping; for in vivo treatment, neonatal offspring were tattooed and a fragment of the tail was obtained. The REDExtract-N-Amp Tissue PCR Kit (Sigma) was used for genomic DNA extraction and PCR setup, with the following primers: WT forward 5′-CTCCGGGATATTGGGATTG-3′, SMA reverse 5′-GGTAACGCCAGGGTTTTCC-3′ and WT reverse 5′-TTTCTTCTGGCTGTGCCTTT-3′. All procedures were done in accordance with the Spanish Council on Animal Care guidelines and approved by the Universitat de Lleida Advisory Committee on Animal Services .23. Isolated cells  were pooled in culture medium and plated either in 4-well tissue culture dishes  for western blot analysis  or using 15-mm glass coverslips placed into the 4-well dishes for immunofluorescence experiments . Culture medium was Neurobasal medium  supplemented with B27 , horse serum , l-glutamine  and 2-mercaptoethanol  and a cocktail of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), and hepatocyte growth factor (HGF), as follows: 1 ng/ml BDNF, 10 ng/ml GDNF, 10 ng/ml CNTF, 10 ng/ml CT-1, and 10 ng/ml HGF (Peprotech). At 24 h after plating, 2 μg/ml of aphidicolin (Sigma) was added to the culture medium and was maintained throughout the experiment.MN primary cultures were obtained from the spinal cords of CD1 or WT and mutant SMA mouse embryos at E12.5 essentially as describedMice were individually housed in propylene cages (33 cm × 18 cm × 14 cm) at an ambient temperature of 22 ± 2 °C and relative humidity of 40 ± 10%. Breeder mice were provided with ad libitum water and rodent chow. Mice were maintained on a 12 h:12 h light:dark cycle (light period 07:30–19:30). SMNdelta7 and WT littermates were assigned by simple randomization to receive treatment or vehicle. Males and females were equally included in the study. Calpeptin  was dissolved in DMSO  and injected at a dose of 6 μg per gram of weight in saline solution. Vehicle groups (Sham) received equal volumes of saline solution with the same amount of DMSO. No blinded administration was via subcutaneous (SC) injection in the interscapular region once a day starting from P0 with a polypropylene sterile syringe  and with a 30 G needle (BD Microlance). WT and mutant animals received treatment or vehicle to a maximum of postnatal day 8 (P8). Birth was defined as postnatal day 0 (P0) for the experiments.l-glutamine (Gibco), 1% (v/v) of nonessential amino acids (Gibco), and 20 µg/ml Penicillin–Streptomycin (Gibco). Cells were subcultured every 3–4 days. For western blot analysis, cells were plated at a density of 3000–4000 cells/cm2 in 35 mm tissue culture dishes and maintained in supplemented MEME medium. After 2 days, total cell lysates were collected and submitted to western blot analysis.Human fibroblast cell lines were obtained from the Coriell Institute for Medical Research . The Coriell Cell Repository maintains the consent and privacy of the donor samples. All the cell lines and culture protocols in the present study were carried out in accordance with the guidelines approved by institutional review boards at the Universitat de Lleida and IRBLleida research center. Two human fibroblast cell lines from patients with SMA  and one unaffected control  were purchased and cultured following manufacturer instructions. Cells were maintained in minimum essential medium eagle (MEME) (Sigma) supplemented with non-inactivated fetal bovine serum (Gibco) (15% v/v), 0.5 M of SMN2 2 copies; delta exon7–8 in SMN1). Control and SMA cells were differentiated to MNs following the protocol described previously24 with minor modifications. Briefly, the human iPSCs were cultured on a layer of irradiated mouse embryonic fibroblasts (MEFs) (Gibco). To generate neuroepithelial cells (NEP), iPSCs were dissociated with Accutase (Gibco) following manufacturer indications and plated on Geltrex (Gibco)-coated plates in MEF-conditioned medium. After 24 h, fresh neuroepithelial induction medium  was added. Cells were maintained in culture for 6 days, changing the medium every other day, and dissociated with Accutase to generate MN progenitors (MNP). MNPs were expanded with NEPIM containing 0.1 μM retinoic acid (Sigma), 0.5 μM purmorphamine (Cayman), and 0.5 mM valproic acid (Sigma).The human iPSCs used in the present work were purchased from Coriell Institute for Medical Research. The control cell line was GM23411*B iPSC  and the SMA cell line was GM23240*B iPSC from a patient with SMA type II .To induce MN differentiation, MNPs were detached with Accutase and cultured in suspension in MN induction medium . Medium was changed every other day. After 6 days, neurospheres were dissociated with Accumax (Invitrogen) and plated on laminin-coated plates in MN maturation medium . For western blot analysis, cells were plated on laminin-coated four-well dishes at 60,000 cells/cmLumbar region 1 and lumbar region 2 (L1 and L2) segment of the spinal cords of calpeptin- or sham-treated SMNdelta7 and WT mice was dissected and fixed in 4% paraformaldehyde (Sigma) for 24 h. Three animals for each condition were used. Cryopreservation with 30% sucrose buffer was done 48 h before mounting segments in tissue freezing medium , sectioned at 16 μm-thickness in a cryostat (Leica CM3000). Slices were permeabilized with 0.3% Triton X-100 and incubated for 1 h with 5% bovine serum albumin (BSA) in PBS. Cultured human and mice MNs were plated on glass coverslips and at the end of the treatment were fixed with 4% paraformaldehyde (Sigma) for 10 min followed by an additional min with cold methanol (Sigma), then incubated for 2 h with 5% BSA in PBS.Primary antibody  was diluted in 0.01% Triton X-100 and incubated overnight. After washing, the secondary antibody was added: anti-mouse ALEXA555 antibody, 1:400, Cat. No. A21422; anti-rabbit ALEXA488 antibody, 1:400, Cat. No. A11008, all from Invitrogen; anti-sheep ALEXA649, 1:400, Cat. No. 713-496-147; anti-mouse ALEXA488 antibody, 1:400, Cat. No. 715-546-150, both from Jackson ImmunoResearch. Counterstain was performed with NeuroTrace 530/615 Red Fluorescent Nissl Stain . Hoechst  to identify nuclear localization in MNs soma. Samples were mounted using Mowiol  medium. Confocal images were obtained using an FV10i Olympus confocal microscope. Quantification of fluorescence was blinded performed using the NIH ImageJ software.22. Spinal cord tissue samples  were disaggregated using Direct Quant 100ST Buffer (DireCt Quant) and a G50 Tissue Grinder (Coyote Bioscience). Total cell lysates of cultured cells or tissue homogenates were resolved in sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene difluoride Immobilon-P transfer membrane filters (Millipore) using an Amersham Biosciences semidry Trans-Blot. The membranes were blotted with anti-SMN antibody , anti-Calpain-1 antibody , anti-Fodrin (Clone AA6) antibody , and anti-Calpastatin antibody . To control the specific protein content per lane, membranes were reprobed with monoclonal anti-α-tubulin antibody . Blots were developed using Luminata™ ForteWestern HRP Substrate (Millipore).Western blots were performed as previously describedt test or by one-way ANOVA with Bonferroni multiple comparisons post-test. Similar variances between the compared groups were assumed. Values were considered significant when p < 0.05.All experiments were performed at least three independent times. For animal studies we selected the minimum number of mice to have enough subjects to accomplish experimental aims. No samples or animals were excluded from the analysis. Values were expressed as mean ± SEM. Statistical analyses were performed using GraphPad Prism (version 5) (GraphPad Software Inc.). Differences between groups were assessed with two-tailed Students 21. In order to determine whether calpain pathway is altered in Smn-reduced MNs, mutSMA and SMNdelta7 E12.5 embryos were genotyped and the spinal cords of WT and mutant mice were dissected. MNs were isolated and cultured in the presence of a cocktail of neurotrophic factors. After 4 days, in vitro protein extracts were collected and submitted to western blot analysis using anti-SMN antibody, anti-α-fodrin antibody, anti-Calpain-1 antibody, or anti-calpastatin antibody. As expected, Smn protein level was significantly reduced in total cell lysates of cultured MNs from both SMA models , compared to the WT controls    Fig.  and SMNd29) Fig.  cultured29) Fig. .Fig. 1FoTaken together, these results suggested an increase of calpain activity in cultured Smn-reduced MNs without an increase of calpain protein level. To elucidate whether increased calpain activity was caused by a reduction of calpastatin protein level, we evaluated the level of this endogenous calpain inhibitor by western blot analysis. We observed that calpastatin was not significantly modified in mutSMA and SMNdelta7 conditions Fig. , suggestTo further evaluate changes of calpain pathway members in SMN-reduced cells, we next explored protein levels of calpain, calpastatin, and fodrin in two human SMA cellular models: fibroblasts and MNs differentiated from iPSC Figs.  and 3.Fip < 0.0001) compared to the clinically unaffected control. In SMN-reduced fibroblasts we observed that 150/145 kDa α-fodrin degradation product was significantly decreased  compared to control condition, indicating that calpain activity was reduced in these cells. Protein level of calpain and calpastatin were not significantly modified in SMA fibroblasts compared to the control  MNs compared to Control   samples compared to Control  compared to the Control condition; (ii) calpain protein level was significantly reduced in SMA samples  compared to the Control condition; and (iii) calpastatin protein levels were not significantly different between SMA and Control conditions (p = 0.720)  Fig. . Westernrol Fig. . When ca20) Fig. . These r21. To further examine the cellular effect of in vivo calpeptin administration we started a treatment protocol using the FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg (SMN2*delta7)4299Ahmb/J (SMNdelta7) mouse model. This severe SMA mouse model has a life expectancy of 10 days, and calpeptin treatment increases lifespan to 14 days21. SC injection of calpeptin  was administrated once a day starting from postnatal day 0 (P0) to postnatal day 8 (P8). Vehicle (Sham) and calpeptin treated mice were sacrificed at P8. In order to determine the level of Smn protein and calpain activation in spinal cord of treated animals, total protein extracts of lumbar fragments were obtained and submitted to western blot using anti-SMN antibody and anti-α-fodrin antibody. Results showed that Smn level was significantly increased in protein extracts from calpeptin-treated SMNdelta7 mice  compared to the vehicle-treated control extracts (SMNdelta7 Sham) , no significant differences were observed compared to the Sham control condition . We also analyzed the level of 150/145 kDa α-fodrin fragments in protein extracts of calpeptin-treated mice. As shown in Fig. p = 0.021), indicating an increased calpain activity in SMNdelta7 spinal cord protein extracts. On the other hand, calpeptin-treated SMNdelta7 condition showed 150/145 kDa α-fodrin (1.11 ± 0.29) levels, which is not different to WT Sham controls, but it is significantly reduced compared to SMNdelta7 Sham (p = 0.031). Together these results suggest that SMNdelta7 spinal cord protein extracts show increased calpain activity which is prevented by SC injection of calpeptin.Calpeptin is a cell-permeable calpain inhibitor which increases Smn protein level in cultured isolated mice spinal cord MNs. In addition, in vivo calpeptin treatment extends SMA mice survival and improves motor functionam) Fig. . Even itp < 0.0001) compared to the WT Sham control (222.8 ± 30.8). No significant differences of Smn fluorescence were observed in WT Sham- and WT calpeptin-treated cells   compared to sham-treated SMNdelta7, indicating that calpain inhibition increases Smn level in spinal cord MNs.In order to know whether Smn protein level is increased in lumbar spinal cord MNs soma of SMNdelta7 calpeptin-treated mice, we performed an immunofluorescence analysis using an anti-SMN antibody. Mice were subcutaneously treated daily with calpeptin from P0 and sacrificed at P8. Lumbar fragments of spinal cords of Sham- or calpeptin-treated WT and SMNdelta7 mice were dissected and cryostat sections of 16 μm-thickness were obtained. MNs were identified in spinal cord sections using Nissl stain, and Smn corrected total cell fluorescence was measured in Nissl-positive MNs. As predictable, quantification of the relative average of fluorescence units demonstrated that Smn level was significantly reduced in Sham-treated SMNdelta7 cells  Fig. . Howeverp < 0.0001) control condition. Calpeptin treatment significantly increases calpain level in both WT (702.7 ± 74.16) and SMNdelta7 (531.8 ± 71.62) conditions compared to WT (p < 0.05) and SMNdelta7 (p < 0.0001) Sham-treated control samples, respectively. This result indicated that calpeptin treatment regulates calpain protein level in spinal cord MNs soma. On the other hand, the analysis of calpastatin levels in the same experimental context showed a comparable profile to the one observed in calpain immunofluorescence measures. Sham-treated SMNdelta7 (30.2 ± 4.9) MNs exhibited decreased calpastatin levels compared to sham WT  controls  and SMNdelta7 (p < 0.05) sham-treated conditions, respectively. These results together suggest that in vivo calpain inhibition using calpeptin administration increases endogenous levels of calpain and calpastatin.To further analyze the calpain pathway in spinal cord MNs of calpeptin-treated mice, we decided to measure by immunofluorescence the level of calpain and calpastatin in cell soma. To this end, lumbar spinal cord sections of 8 days sham- and calpeptin-treated WT or SMNdelta7 mice were processed for immunofluorescence using an anti-calpain antibody or an anti-calpastatin antibody. Results indicate that SMNdelta7 Sham-treated (47.87 ± 4.91) MNs showed significantly reduced levels of calpain compared to WT Sham-treated  compared to the WT controls  or treated with 25 µM calpeptin or 50 nM resveratrol for 12 h; or with 25 µM calpeptin or 100 mM trehalose for 6 h. After treatment, cell lysates were obtained and submitted to western blot using an anti-α-fodrin antibody or an anti-LC3 antibody. Results showed that calpeptin, resveratrol and trehalose treatments significantly increased LC3-II protein level  compared to untreated controls  compared to the untreated controls. Nevertheless, resveratrol and trehalose addition did not significantly modify α-fodrin fragments level  compared to untreated controls  compared to the WT controls  compared to untreated WT controls. However, even LC3-II level was increased in calpeptin-treated SMNdelta7 condition , the differences were not statistically significant compared to untreated SMNdelta7 controls  controls. Calpeptin treatment significantly increases LC3 spots in WT MNs soma  compared to non-treated control cells, but a similar effect was not observed in neurites. However, calpeptin treatment clearly prevents LC3 increase in neurites of SMNdelta7 MNs  compared to the SMNdelta7 control cultures, indicating that calpeptin treatment reduced autophagosomes in neurites of SMA mice. We did not observe the same effect in MN soma. When fluorescent LC3 spots were measured in calpeptin-treated SMNdelta7 cell soma (93.67 ± 6.3), there were no significant differences compared to control non-treated SMNdelta7 cells. This result suggests that calpain inhibition increases LC3 spots in MN soma and prevents autophagosome accumulation in neurites.We next analyzed whether calpeptin treatment regulate LC3 level in Smn-reduced MNs. SMNdelta7 E12.5 embryos were genotyped and the spinal cords of WT and mutant mice were dissected. MNs were isolated and cultured in the presence of neurotrophic factors. After 6 days in vitro, cells were treated with 25 µM calpeptin for 3 h and protein extracts were collected and submitted to western blot analysis using an anti-SMN antibody or an anti-LC3 antibody. LC3-II protein level was significantly increased in total cell lysates of mutant cultured MNs  to the 150/145 kDa-specific fodrin breakdown product has been attributed to the activation of calpain25. We observed an increase of these calpain-specific fodrin products in mice cultured spinal cord MNs and differentiated human SMA MNs, but not in human SMA cultured fibroblasts, suggesting an increase of calpain activity in SMN-reduced MNs. This increase of calpain activity was not the consequence of an increase of calpain protein, because no increase from basal level occurred in cultured mice MNs, and a decrease was observed in human differentiated SMA MNs and in MNs soma of SMNdelta7 mice.The molecular mechanisms leading to MN degeneration in SMA disease are poorly understood. SMN protein reduction causes cellular changes which directly or indirectly provoke the deregulation of several processes involved in cell homeostasis, including autophagy and apoptosis35. Calpain activity can be compartmentalized and calcium changes localized in some neuronal sections in SMA may contribute to the increase of calpain activity in these particular spaces37.Reports of the activation mechanisms of calpains have been controversial. The calcium concentrations necessary for activation of calpains are in the micro- to millimolar range, which is rather beyond the nanomolar calcium levels in cells under normal physiological conditions. Nevertheless, this apparent contradiction can be resolved because the cellular microenvironment may provide the sufficient calcium concentration14. This calpain overactivation can be caused by an increase of intracellular calcium concentration or by a reduction of the natural endogenous inhibitor calpastatin. Calpains form a complex with calpastatin, the only known endogenous protein inhibitor of calpain activity in cells34. Depletion of endogenous calpastatin has been described in mouse models of several neurodegenerative disorders40. Likewise, calpastatin reduction and increased calpain activity have been related to biochemical changes related to the breakdown of cytoskeletal proteins and activation of cdk5 and caspase-3, leading to MN degeneration in hSOD1G93A mice16. Western blot analysis of calpastatin levels in cultured SMA MNs showed no changes in this protein in these cells; however, when immunofluorescence experiments were performed in spinal cords of P8 SMNdelta7 mice, we observed calpastatin reduction in MNs soma. In vivo calpeptin treatment prevents calpastatin reduction, which suggests that calpain activity can also regulate the members of the pathway. In this context, we observed that basal levels of calpain were increased by calpeptin treatment, reinforcing the hypothesis that calpain activity regulates calpastatin and calpain protein levels in spinal cord MNs. Interestingly, in vivo calpeptin treatment also increased SMN protein in spinal cord extracts and in MNs soma. It is well-known that SMN is a proteolytic target of calpain and its activity modulates SMN level42. Therefore, our results strongly suggest that the in vivo beneficial effect observed in calpeptin-treated SMA mice21 can be mediated to some extent by SMN protein increase in MNs.However, damaging calpain overactivation has been detected in other neurodegenerative disorders, such as Alzheimer disease, amyotrophic lateral sclerosis, Parkinson disease, or the group of polyQ disorders14. Several neurodegenerative disorders exhibit an overactivation of calpains and disturbances of the autophagic pathway. In SMA disease, autophagy pathway is deregulated28 and inhibition of autophagy delays MN degeneration and extends lifespan of SMA mice20. From the results presented in these previous reports it can be suggested that LC3-II autophagosome marker is augmented in SMN-reduced MNs. In the present work we show that LC3-II protein level is increased in the SMNdelta7 mutant isolated MNs compared to the WT controls. It has been previously described that calpeptin treatment increases the number of autophagosomes-like structures in COS-7 cells43; accordingly, calpeptin treatment increased LC3-II protein level in WT conditions, but no significant differences were observed in SMNdelta7 cultures. By immunofluorescence, we showed that calpeptin treatment increased LC3 spots in MN somas of WT cells. However, we did not observe the same effect in neurites: LC3 spots in WT neurites did not increase after calpeptin treatment. Interestingly, calpeptin treatment prevented LC3 increase in neurites in SMA cells, but not in cell soma. Accumulation of autophagosomes in axons can perturb microtubule-associated axonal transport, which can be linked to neurodegeneration affecting autophagic flux. Our results suggest that calpeptin treatment may regulate autophagosome accumulation in neurites. This effect would prevent the neurite collapse that can lead to MN degeneration.Calpains are considered as modulator proteases that can regulate protein functions, and therefore various cellular pathways, including autophagy. In many disease environments and models, calpains are known to negatively regulate autophagy. Consequently, enhanced calpain activation can contribute to the compromised activation of this degradation pathway. Various studies have found that the impact of calpains on autophagy occurs on multiple levels, such as reduction of autophagy initiation or switching from autophagy to apoptosisIn conclusion, calpain pathway is an intracellular proteolytic mechanism involved in the development of several neurodegenerative disorders. The present study found an increase of calpain activity in SMN-reduced cells. We suggest that the positive modifier effect of in vivo calpeptin treatment on SMA mice survival and motor phenotype could be the result of SMN protein and LC3 autophagosome protein regulation in SMA MNs and could offer a viable therapeutic approach. Although infants with SMA appear to have a genetic defect in common, the clinical course of this disease shows high variability. The analysis of new SMA modifiers belonging to several intracellular regulatory pathways which contribute differently to disease progression may lead to the discovery of new treatment strategies for this devastating condition.


Cell-cell adhesion molecules play an essential role in cell growth and differentiation. β-catenin and CD44s are two adhesion molecules which their expression changes are correlated to loss of differentiation and gain of an invasive epithelial phenotype. Oral squamous cell carcinoma (OSCC) is the most common malignancy of oral cavity. Aim of this study was to compare β-catenin and CD44s expression in different histopathological grades of OSCC. β-catenin and CD44s expression were evaluated in 10 well differentiated OSCC (group A) and 10 moderately/poorly differentiated OSCC (group B) using immunohistochemistry. β-catenin membranous and nuclear/cytoplasmic expression were significantly different between groups A and B. CD44s membranous expression was insignificant amongst the two groups. Expression of β-catenin and CD44s alter in different histopathological grades of OSCC. It seems that more rate of aberrant cytoplasmic and/nuclear expression and less membranous expression of β-catenin can lead to significantly lower degree of cell differentiation in OSCC.  Cell-cell adhesion molecules play a fundamental role in cell growth and differentiation . These molecules mediate the adhesion of epithelial cells to each other and preserve integrity of tissue structure . Oral squamous cell carcinoma (OSCC) is the most common malignancy of oral cavity accounting for over 90% of all malignancies in this area . Invasion and metastasis are among the characteristics of OSCC. Cell escape from tumor is the first step for invasion and metastasis. This event occurs as a result of changes in expression of adhesion molecules and disruption of cell-cell adhesion . β-catenin and CD44 are two adhesion molecules . β-catenin is a multifunctional 92-kDa protein  which involves in two completely different processes namely intercellular adhesion and signal transduction . CD44 is a cell surface glycoprotein and the main receptor for hyaluronan . Standard isoform of CD44 (CD44s) is the smallest and the most abundant isoform of this protein . Evidently, expression of β-catenin and CD44s changes are correlated to loss of differentiation and gain of an invasive epithelial phenotype . This study aimed to compare expression of β-catenin and CD44s in different histopathological grades of OSCC. PatientsOSCC samples belong to a previous study  were used in current research. They were microscopically evaluated and classified according to histopathological differentiations including 10 cases of well differentiated  and 10 cases of moderately/poorly differentiated OSCC . Ethical approval of the current study was obtained from the Ethical Committee for Research of Hamadan University of Medical Sciences.ImmnuohistochemistryImunohistochemical study was performed by Envision detection system, using β-Catenin  and CD44s  antibodies. Briefly, 4-μm tissue sections were cut from paraffin blocks, mounted on poly-L-lysine-coated slides, deparaffinized and rehydrated. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide for 5 minutes. For antigen retrieval slides were boiled in citrate buffer  inside an autoclave for 30 minutes. Then, the slides were incubated with antibodies for 60 minutes washed in phosphate buffered saline (PBS) for 10 minutes whereas an Envision  was used for 30 minutes. Subsequently, the sections were washed with PBS for 10 minutes and DAB was used as chromogen. Finally, the slides were counterstained with Harris hematoxylin, dehydrated, and mounted.For β-catenin, membranous /cytoplasmic /nuclear staining and for CD44, membranous staining was considered as positive immunoreactions. Immunoreactivity for β-catenin and CD44 was assessed using IHC immunoreactivity score. Percentage of positive membranous cells were scored: 0 (<20% stained cells), 1 (21-40% ), 2 (41-60%), 3 (61-80%), and 4 (>80%) and staining intensities were graded: 0=no expression, 1=weak, 2=moderate and 3=strong. Subsequently, IHCmembranous score was calculated by positive membranous stained cell score × staining intensity score. Also, according to this method β-catenin IHCcytoplasmic and/ nuclear score was determined in the studied groups. Statistical AnalysisData were statistically analyzed using SPSS V16.0 and Independent T test while P<0.05 was considered as significant level.There were 5 males and 5 females in each studied groups of A and B whereas mean age were 59 (range 35-78 years) and 60 (38-82 years), respectively. The sites of cases for group A were buccal mucosa (n=3), alveolar ridge (n=5), labial vestibule(n=1) and tongue (n=1) while for group B were tongue (n=6), floor of the mouth (n=2), gingiva (n=1) and retromolar pad (n=1). Kolmogorov-Smirnov test confirmed normal distribution of β-catenin and CD44s IHCmembranous score as well as β-catenin IHCcytoplasmic and/ nuclear score. Except for more superficial epithelial cell layers, other layers  in normal tissue adjacent to some of OSCC expressed positive membranous staining of β-catenin (and CD44s). However, no evidence of expression in cell cytoplasm and nuclei was observed in them. β-catenin membranous, cytoplasmic and nuclear immunostaining  were obsMean of β-catenin IHCmembranous was 3.400±2.366 in group A and 0.600±0.843 in group B while 2.300±0.483 and 3.800±2.043 were mean of β-catenin IHCcytoplasmic and/ nuclear in group A and B, respectively. β-catenin, IHCmembranous score and IHCcytoplasmic and/ nuclear score were significant differences between group A and B . Mean CD44s IHCmembranous score was 4.700±2.359 in group A and 3.000±1.825 in group B. CD44s IHC scoremembranous was insignificant between the groups (P=0.088).Whole of the samples were positive for β-catenin immunostaining and showed the three types of membranous, cytoplasmic and nuclear staining. In previous studies by Zaid (2014) and Rosado et al., (2013) the percentage of stained samples were smaller than of the current study. Similar to the findings by Laxmidevi et al., (2010), Zaid (2014) and Ravindran et al., (2015) a significant difference was noted in β-catenin membranous expression for different grades of OSCC in this study. Oppositely, Liu et al., (2010), Rosado et al., (2013) and Balasundaram et al., (2014) found no significant difference in expression of β-catenin for different grades of OSCC. In present study, β-catenin cytoplasmic and/nuclear immunostaining were significantly different amongst groups A and B in agreement to the findings by Zaid (2014) but opposite to those of Zhang et al., (2014). Lopes et al., (2009) reported a significant association between cytoplasmic expression of β-catenin and histopathological grade but no significant association was noted with its nuclear expression.Controversy in results of studies regarding expression β-catenin  can be due to a number of factors such as heterogeneity of tissue samples studied, difference in sample size, immunohistochemical staining/antigen retrieval techniques and scoring criteria. However, reduction in β-catenin membranous staining and a shift towards cytoplasmic (and/or nuclear) immunostaining by an increase in OSCC grade is a significant finding of the current study and also among the most of the previous studies . β-catenin is normally located and expressed on the cell membrane of normal cells . In this region, β-catenin plays a role in E-cadherin-mediated cell adhesion . It binds to the cytoplasmic domain of E-cadherin which forms E-cadherin/β-catenin complex  and establishing cell-cell contact . The stability and integrity of this complex can be disrupted by the dissociation of β-catenin from E-cadherin. In this case, cell adhesion is lost and the cells become migratory and motile . According to the current findings: (I) absence of β-catenin membranous immunostaining in a number of neoplastic epithelial cells in groups A and B revealed that in OSCC changes such as cell cohesion loss and cellular motility occur irrespective of its degree of differentiation (II) significantly lower membranous immunostaining in MD/PDOSCC compared to WDOSCC may indicate further dissociation of cells from the primary tumor bulk and easier occurrence of invasion and even metastasis in this group of tumors. Dissociation of β-catenin from E-cadherin decreases its membranous expression and leads to accumulation of free β-catenin in cytoplasm and its subsequent move into the cell nucleus . Normally, free β-catenin in cytoplasm is phosphorylated via attachment to a complex namely degradation complex which is composed of Axin, APC and GSK3 proteins and is degraded by the ubiquitin-proteasome rapidly . Mutation in APC and Axin genes or β-catenin gene itself, dysfunction of GSK3 and/or Wnt signaling pathway activation can impede proper functioning of the degradation complex . This can lead to accumulation of β-catenin in cytoplasm and promote its subsequent translocation into the nucleus. β-catenin serves as an oncogene in nucleus . Binding of β-catenin to members of TCF/LEF family leads to activation of a number of proto-oncogenes such as c-Myc and cyclin D1 .In this study, β-catenin cytoplasmic/nuclear expression was noted in both groups of WOSCC and MD/PDOSCC. Apparently, aberrant activity and expression of β-catenin in OSCC is not exclusive to a certain histopathological grade. Also, significantly higher frequency of this form of expression in group B can be attributed to the occurrence of higher changes and abnormalities in complex intracellular mechanisms of MD/PDOSCC compared to WDOSCC. MD/PDOSCC cases probably suffer from higher abnormalities such as mutation of APC, Axin and/or β-catenin genes, dysfunction of GSK3 or Wnt signaling pathway activation compared to WDOSCC. Significantly higher β-catenin cytoplasmic and/nuclear expression in group B compared to group A suggest that OSCCs with lower differentiation grade possess greater accumulation of genetic defects. These genetic defects lead to loss of epithelial phenotype and migratory cell control as an indication of aggressive behavior of tumor . Whole of the samples showed positive staining for CD44s marker. Simionescu et al., (2008) reported one and five cases of moderately and poorly differentiated OSCC with negative CD44s immunostaining, respectively. Similar to Krump and Ehrmann (2013); in the current study the difference in expression of CD44s was not significant between groups A and B. In contrast, Hema et al. (2014) demonstrated a significant difference in this expression between different grades of OSCC. Additionally, the most expression was noted in WDOSCC group in the present study similar to Simionescu et al. (2008) where higher frequency of cells with positive CD44s immunostaining in WDOSCC group was found compared to PDOSCC group. CD44s is a molecule with a diversity of functions which can play a role in both cell adhesion and cell motility/migration while it has extracellular, transmembrane and cytoplasmic domains. Function of CD44s highly depends on a ligand that binds to its extracellular domain. Hyaluronan is the most recognized ligand for CD44s. It is ubiquitous in extracellular and pericellular matrix and is synthesized as a high molecular weight (HMW) mass. CD44-HMW hyaluronan interaction causes the attachment of CD44 tail to Merlin (a tumor suppressor protein) which induces signals that mediate cell-cell and cell-extracellular matrix adhesion/ECM . However, tumoral cells release hyaluronidase which degrades HA and creates low molecular weight (LMW) hyaluronan fragments . Binding of LMW hyaluronan to CD44 leads to proteolytic cleavage of extracellular domain of CD44. In this process, ERM replaces Merlin and activates the signaling molecules that promote cytoskeleton remodeling and cellular migration . According to the previous studies; occurrence of cleavage in CD44s by LMW hyaluronan can down-regulate CD44s expression . CD44s immunostaining in some OSCC cells, not all of them, in groups A and B suggests that the cleavage of CD44s and induction of CD44 dependent cell migration irrespective to degree of differentiation in this malignancy. CD44s expression was not significantly different between MD/PDOSCC and WDOSCC groups. Although lower expression of CD44s in MD/PDOSCC may suggest greater loss of cell adhesion and easier tumoral cells detachment from each other and from ECM compared to WDOSCC. CD44 can activate or inhibit cell invasion and migration via interaction with LMW or HMW hyaluronan, respectively. In other words, CD44 mediated signaling pathways highly depend on extracellular conditions and changes in the microenvironment and can be significantly variable among different cell types or even among similar cells . Irrespective to a small sample size in this study, absence of a significant difference CD44s may indicate relatively similar microenvironment and ECM of the studied samples. In conclusion, expression of β-catenin and CD44s alter in OSCC irrespective of the related histopathological grade. It seems that more occurrence of abnormal changes in β-catenin, more rate of aberrant cytoplasmic and/nuclear expression alongside with less membranous expression can significantly lead to lower degree of cellular differentiation in OSCC.


Fractalkine  is a transmembrane chemokine expressed by neurons in the central nervous system (CNS). CX3CL1 signals through its unique receptor, CX3CR1, that is expressed in microglia. Within the CNS, fractalkine acts as a regulator of microglia activation in response to brain injury or inflammation. During the last decade, there has been a growing interest in the roles that the CX3CL1/CX3CR1 signaling pathway plays in the neuropathology of a diverse array of brain disorders. However, the reported results have proven controversial, indicating that a disruption of the CX3CL1 axis induces a disease-specific microglial response that may have either beneficial or detrimental effects. Therefore, it has become clear that the understanding of neuron-to-glia signals mediated by CX3CL1/CX3CR1 at different stages of diseases could provide new insight into potential therapeutic targets. Hence, the aim of this review is to provide a summary of the literature on the emerging role of CX3CL1 in animal models of some brain disorders. Fractalkine FKN, CX3CL1) is a member of the CX3C chemokine family and is constitutively and abundantly expressed in neurons. Fractalkine expression can also be induced in microglia, astrocytes, and vascular endothelial cells . CX3CL1 CL1 is a The altered functionality of the CX3CL1/CX3CR1 pathway in several different pathological states may, on the other hand, promote the activation of microglia and stimulate the release of inflammatory factors ,17,18,19The loss of the crosstalk that leads to the activation of microglia differently affects the patterns of brain disorders. This loss of crosstalk may either be beneficial for promoting neuronal survival or cause harm to the surrounding cells depending on disease stage and progression . The speIt is now widely recognized that brain ischemia triggers a widespread inflammatory reaction that, in conjunction with excitotoxic and oxidative responses, significantly contributes to ischemia-induced neuronal death. Inflammation driven primarily by activating inflammatory glial cells that reside in the CNS together with infiltrating cells of the peripheral immune system  produces several proinflammatory factors , which lead to a disruption of cellular homeostasis and structural damage of brain tissue. The recognition of inflammation as the key response to brain injury has stimulated the use of new therapies. A large body of data from cerebral ischemia of different severities consistently shows that at least some neuroprotective effects, including reduced brain damage, can be achieved by decreasing the level of microglial activation ,29. A brcx3cl1 or deficiency in cx3cr1 (GFP/GFPcx3cr1), reduced post-ischemic brain injury. The neuroprotective effect was observed based on the decreased neurological deficit, the diminished release of inflammatory markers , and the ameliorated neuronal death CX3CR1 geactivity ,85. HoweFurthermore, fractalkine, the ligand of the CX3CR1 receptor, appears to be protective in some contexts, whereas it contributes to neuronal damage in other contexts. Comparison of the soluble or membrane-bound forms of fractalkine showed that overexpression of soluble fractalkine in knock-out mice has the ability to restore neurogenesis and LTP, whereas membrane-bound CX3CL1 does not have such beneficial effects . This fiAlzheimer’s disease (AD) is one of the most common neurodegenerative disorders and the fifth leading cause of death for individuals aged 65 or older. AD accounts for approximately 80% of all cases of dementia due to progressive cognitive impairment and decreased memory formation associated with neuronal dysfunction . AD is aThe two pathological hallmarks of AD are extracellular senile neuritic plaques, of which amyloid beta is the principal component , and an A body of recently collected evidence indicates that the fractalkine ligand and its microglial receptor (CX3CL1/CX3CR1) affect Aβ and Tau pathologies by regulating microglial movement and recruiting monocytes into the brain. For research purposes, different genetic models  exhibiting specific aspects of the AD disorder were used ,92,93. BAs a result, the total levels of Aβ (particularly Aβ 40 and 42)  and the Conversely, a follow-up study surprisingly revealed that ablation of the fractalkine receptor resulted in enhanced Tau pathology, as demonstrated by increased Tau phosphorylation and aggregation, which correlates with worsened behavioral and cognitive deficits ,99. ReacCX3CR1 gene, namely, V249I and T280M, could represent new factors that regulate the onset and progression of pathology in patients with AD. There is data described association of variant CX3CR1-V249I with neurofibrillary pathology. The analysis provides further evidence of the involvement of the CX3CR1 pathway in the pathogenesis of AD [In addition, two variants of the is of AD .Current evidence has shown that interruption of CX3CL1/CX3CR1 signaling affects the neuron–microglia interaction in AD pathology and it may trigger beneficial or detrimental effects. The beneficial effect is expressed by inhibition of inflammation and amyloid clearance, whereas the negative effect is associated with the enhancement of Tau phosphorylation. The discrepancies in the effects caused by the ligand/receptor axis may result from the use of different models, different stages of disease development, and the utilization of different experimental strategies.Recently, a novel idea that Tau can directly bind to CX3CR1 and then compete with the natural ligand of this receptor was proposed . This coAn important issue regarding AD pathology is the definition the biological functions of the fractalkine isoforms. It is possible that the signals initiated by either the membrane-bound isoform or soluble isoform may play different roles in amyloid beta clearance and Tau phosphorylation. Importantly, studies have confirmed that overexpressing the soluble form reduced the effect of Tau and did not affect amyloid pathology. This remains consistent with the documented increase in Tau pathology in CX3CR1 null mice and the interruption of the fractalkine signaling pathway . FurtherAccording to a recently published report, other chemokines and their receptors may compensate for deficiency in CX3CR1. One of these studies suggested the involvement of the chemokines CCL2 or CXCL16 . It was Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disease that mainly affects lower  and upper  motor neurons (MNs) . The mosG93ASOD1 mice accelerates disease progression and exacerbates neuronal death [Recent findings have confirmed that intercellular communication between motor neurons and microglia plays an important role in the pathogenesis of ALS ,111,112.al death . This obal death . In addial death . HoweverThis finding directly contradicts a study performed on a much smaller population of individuals, where the presence of V249I was associated with shorter survival ,115. ThuParkinson’s disease (PD) is the second most common neurodegenerative disease, affecting 1–2% of the population over the age of 65 ,117. TheAlthough the mechanisms that trigger brain degeneration in PD are unknown, several etiological factors are involved in contributing to the disease, and mitochondrial dysfunction, oxidative stress, proteosomal dysfunction, and neuroinflammation thought to be the key components in the pathogenesis of PD ,122,123.Increasing evidence has indicated that the chemokine CX3CL1 and its receptor, CX3CR1, play important roles in modulating the inflammatory response in PD and in other neurological disorders ,126,127.A series of experiments performed on mice deficient in the microglial receptor CX3CR1 showed that dopaminergic neuronal loss was more pronounced after the neurotoxins MPTP or 6-OHDA had been administered ,76. ConvFurthermore, a detailed study by Morganti et al. about the relative contributions of the soluble and uncleaved membrane-bound fractalkine isoforms allowed these authors to discover, through the use of rAAV gene therapy, that only the soluble fractalkine isoform attenuates the neurotoxic effect of MPTP toxin . ImportaCx3cr1−/− mice. Most likely, the mechanism that governs the microglial response after alpha-synuclein has been administered is different from the mechanism that is specific to toxin-induced PD [Taken together, the current data were obtained in studies of neurotoxin models with CX3CR1 deficiency and include contradictory evidence about axis signaling and its effects on the progression of disease. Thome et al. reported that in an alpha-synuclein overexpression model of PD (AA2SYN), deficiency of the receptor attenuates inflammation and fails to exacerbate neurodegeneration . Moreoveduced PD .Finally, the differences in the fractalkine signaling pathways could be attributed to the natures of the models and the events that trigger the degenerative processes. For example, the absence of the CX3CL1/CX3CR1 axis yields no nigral dopaminergic neuron loss in either intranasal MPTP- or 6-OHDA-treated mice. In contrast, the absence of the CX3CL1/CX3CR1 axis was deleterious after intraperitoneal MPTP infusion .In the field of PD, fractalkine signaling appears to have potent effects on the neuroinflammatory processes in PD models, but these effects are complex and depend on the nature of the initiating events. It is important to better understand the state of fractalkine signaling in human disease in order to develop neuroprotective strategies to delay the onset or progression of the disease.Over the past decade, the understanding of the role the fractalkine pathway plays in brain pathology has greatly advanced. Although there is a growing body of literature, which is supported by extensive studies on the role of the CX3CL1/CX3CR1 signaling pathway in brain diseases, the published results are relatively controversial. While disruption of the fractalkine signaling pathway is beneficial in some pathological states (ischemia), it is detrimental in other neurodegenerative diseases (PD). Furthermore, analysis of AD models suggests that deletion of CX3CR1 may lead to both neuroprotective and detrimental effects. In other neurodegenerative diseases, such as ALS, the reported results are rather rare and do not allow us to precisely determine the role of fractalkine signaling. Furthermore, there is also no complete agreement about the role of the fractalkine isoforms in the development of pathological processes. It only seems logical that future research should be focused on understanding the precise mechanism by which disrupted CX3CL1/CX3CR1 signaling affects microglia and leads to either beneficial or deleterious actions. In summary, emerging evidence suggests that the CX3CL1/CX3CR1 axis is an attractive potential therapeutic target due to its ability to control inflammation in the neurological disorders. Therefore, one of the most urgent issues is to unravel the frame windows where the modulation of neuroinflammatory response suits the different stages of the pathologies.


In addition, the proposed sealing material exhibited an extremely low LC contamination level of 9 µm, which is essential to the successful operation of LC displays. With respect to electro-optical properties of the LC device, it was found that a dihydrazide derivative with a bulky heterocyclic ring afforded a normal voltage-dependent transmittance curve and fast response time due to the prevention of abnormal homogeneous LC alignment. This study developed highly adhesive and robust epoxy-based sealing materials based on the use of dihydrazides as thermal latent curing agents for advanced LC displays.In this study, highly adhesive epoxy-based sealing materials for liquid crystal (LC) displays were fabricated using different types of dihydrazides as thermal latent curing agents. Their curing characteristics, mechanical properties, LC contamination levels, and electro-optical characteristics were investigated depending on the chemical structure of dihydrazides. The epoxy-based sealing material containing a dihydrazide derivative with a bulky heterocyclic ring afforded a high heat curing conversion of 90.4%, high adhesion strength of 54.3 kgf cm Of late, liquid crystal displays (LCDs) are finding wide use in various applications, including in tablets, monitors, and televisions. Owing to recent developments in television manufacturing technologies, the bezel, which is the plastic part around the screen, is getting thinner because of users’ preference for larger screens. For this reason, LCD sealing materials with improved properties such as higher adhesive strength, lower moisture permeability, and greater contact contamination resistance need to be developed. For increasing the adhesive strength, several methods, such as polymer blending, curing with polythiourethanes, and using epoxy-acrylate oligomers, have been proposed ,2,3. TheEpoxy resins show several desirable properties, such as high chemical and solvent resistances and excellent adhesive properties ,5,6,7. HIn order to improve the tensile strength and humidity resistance of the sealing materials used for LCD panels, a few researchers have used ultraviolet (UV)-curable adhesive sealants. These were synthesized using a UV-curable resin based on a commercial epoxy resin through the alkylation method ,19. Usin2 and have either aliphatic or aromatic spacers; thus, they can react with the epoxide ring of epoxy resins. Dihydrazides with different backbone structures have different curing effects on epoxy resins, thereby leading to the difference in adhesion properties. Further, because dihydrazide derivatives show latent curing characteristics under high temperature operation [In this study, we fabricate highly adhesive epoxy-based sealing materials for LCDs using different types of dihydrazides as the thermal latent curing agent. The dihydrazide compounds contain the reactive amine groups of C=ONHNHperation , they arAs stated above, four types of dihydrazide derivatives, including adipic acid dihydrazide , isophthalic dihydrazide , sebacic dihydrazide , and 4-isopropyl-2,5-dioxoimidazolidine-1,3-di(propionohydrazide) , were utilized in this study and demonstrated in The epoxy resin system for LC displays was prepared by physical blending using a rotation and revolution mixer . The YD-128 epoxy resin (40.0 g) and BisGMA acryl resin (40.0 g) were blended with the fumed silica (16.0 g) and a photoinitiator (2.5 g) at 2000/1300 rpm for 15 min. Then, the dihydrazide (1.5 g) was added to this mixture and blended at 2000/1300 rpm for 5 min. After the completion of the mixing process, the epoxy mixture was defoamed in a vacuum oven at 25 °C for 2 h. This yielded a white viscous liquid of the desired adhesive.−1 and subjected to DSC scanning. Thermogravimetric analysis (TGA) was performed using a thermal analyzer  in the temperature range from 20 to 800 °C. Further, we used Fourier-transform infrared spectroscopy  to determine the curing conversion ratio of each epoxy resin system under the curing conditions corresponding to LCD fabrication. Each epoxy sealing material was cured on a glass substrate under UV radiation with a radiant fluence of 3.0 J cm−2 and then heat-cured at 120 °C for 1 h.To analyze the phase transition behaviors of the used dihydrazide compounds, we used differential scanning calorimetry . Each dihydrazide compound of 10 mg was heated from 50 to 300 °C at the rate of 10 °C min−2 for 150 s. After the UV curing process, the assembly was heat-cured in an oven at 120 °C for 1 h. The resulting sealant sample had a circular shape with a thickness of 5 µm. A similar assembly was used to analyze the lap shear strength. In this case, the glass substrates were assembled in a straight manner, while all the other conditions were the same. The adhesive and lap shear strengths were measured with a universal testing machine  at room temperature according to the ASTM C663 and D3165 standards; the crosshead speed used was 1.3 mm min−1 [In order to determine the adhesive strength of the epoxy-based sealing materials, the resins were cured between two indium tin oxide (ITO) glass substrates. The two ITO glass substrates were assembled in a cross-wise manner, and the diameter of the adhesive sample used was 0.2 cm. This assembly was exposed to UV radiation with an intensity of 20 mW cmmm min−1 ,23,24.TNI = 75 °C, Δn = 0.095, Δε = −3.1) were injected between the substrates. The curing conditions for the adhesive were the same as those during the adhesive strength measurements. To confirm whether the LCs had been contaminated, the fabricated sample cell was examined using polarized optical microscopy .To evaluate whether the epoxy sealing materials contaminated LCs on contact, we fabricated test LC cells using ITO glass substrates covered with a layer of a polyimide  using a spin coater. The PI layer on the ITO glass substrate was subjected to primary baking on a hotplate at 80 °C for 10 min. It was then hard-baked in an oven at 230 °C for 1 h. Next, the prepared sealing adhesive was cured between two PI-layer-coated ITO glass substrates arranged in a cross-wise manner, and LCs  of the LC devices containing epoxy resin systems were measured with an electro-optical measurement system. The system consisted of an optical table  equipped with a 632 nm HeNe laser , a photodetector , a function generator , and an oscilloscope .2 group of the dihydrazides would interact with the oxirane rings of the epoxy resin. Thus, we compared the area of the epoxide-ring-related peak between 921 and 968 cm−1 [The heat curing behaviors of the epoxy resin systems, including different types of dihydrazides, were analyzed using FTIR in the attenuated total reflection mode. We compared the conversion ratios of the epoxy resin systems under the industrial curing conditions for LCDs based on the changes in the area of the C-O-C epoxide peak. In the prepared epoxy sealing materials, only the NH−1 are shown in m) were obtained based on the peak temperature of the endothermic transition. The results indicated that the used dihydrazide derivatives exhibit the potential of latent heat curing agent due to the high-temperature melting transitions above 100 °C. As mentioned above, these dihydrazide compounds showed nonreactivity under a normal room temperature condition but exhibited high reactivity under high curing temperatures. While the IDH compound showed the highest melting transition at 201.1 °C due to the rigid aromatic spacer, the VDH compound exhibited the lowest melting point at 120.4 °C due to presence of the bulky heterocyclic ring. Notably, the melting transition of VDH is very close to the thermal curing temperature (120 °C) for LCD fabrication. In addition, it was confirmed that the VDH compound with a relatively low melting point provided a high heat conversion ratio of epoxy resin due to the efficient melting transition during the high-temperature curing process. Therefore, under industrial curing conditions, the VDH compound as a thermal latent curing agent will show effective curing performance for epoxy-based sealing materials.To investigate the difference in heat conversion ratio under the same curing condition, DSC measurement was performed on the dihydrazide compounds. The endothermic heat curves of the various dihydrazide derivatives obtained at a heating rate of 10 °C min−2. Because VDH melts at 120 °C, its diffusion rate is higher, and this resulted in an epoxy resin system with the greatest reactivity among the others tested. As a result, there was an increase in the number of cross-linking sites between the epoxy and the dihydrazide when it was cured under the conditions used in this study. On the other hand, since IDH has the highest melting point at 201 °C, the lowest adhesion strength (25.7 kgf cm−2) was obtained. Moreover, the bulkier the spacer structure of the dihydrazide used, the less brittle the cured epoxy system will be when it is detached. This was evident from a comparison of the results for the ADH and SDH epoxy resin systems. The adhesive strength of the epoxy resin system containing ADH was a low value of 28.3 kgf cm−2. In contrast, the epoxy resin system containing SDH showed a high adhesion strength of 50.4 kgf cm−2. Because the alkyl chain of SDH is much longer than that of ADH, the adhesive force of the cured epoxy resin system based on SDH was higher.The results of the pull-off test for each epoxy resin system are shown in The results of the lap shear strength measurements are shown in During the LCD fabrication process, the sealant and LCs come in direct contact during the LC injection process. During the curing process, any unreacted components of the sealant may leak into the LCs, causing alignment defects in the final device ,31. For th) values; the results are shown in th values of the ADH and IDH systems are lower than those of the SDH and VDH systems. As shown in th value. In contrast, the epoxy resin systems formed using VDH or SDH exhibited stable V-T curves with relatively high Vth values because their contamination areas were smaller.With respect to the realization of high-quality LCDs, the electro-optical characteristics are of great importance. When a voltage is applied to the top and bottom electrodes, the vertically aligned LCs become horizontally aligned, and light can pass through the LCD cells. Thus, to evaluate the electro-optical characteristics of the epoxy resin systems, we determined their voltage–transmittance (V-T) curves and their threshold voltage (Vth values. The size of the contamination area affects the vertical alignment of the LCs, with a large contamination area resulting in the disturbed movement of the LCs when a voltage is applied. Thus, as the extent of LC contamination decreases, the electro-optical switching characteristics of the LC device show improvement. Consequently, it is confirmed that the epoxy-based sealing material formed using a VDH compound as a latent curing agent affords low LC contamination, a stable V-T curve, and a fast response time due to high heat conversion ratio and a bulky spacer structure [tructure .In this study, we prepared epoxy-based sealing materials for LCDs using different types of dihydrazides as the latent curing agent to achieve high mechanical properties and excellent electro-optical performances. The characteristics of the latent curing agents were analyzed through DSC measurements. The results confirmed that the used dihydrazide compounds were suitable for use as latent curing agents for LCD sealing materials. We also confirmed the correlation between the melting point and the thermal curing conversion of the dihydrazide agents. Under industrial heat-curing conditions, VDH exhibited the lowest melting point and the highest heat conversion ratio, as determined by a FTIR analysis. Moreover, it was observed that, the higher the conversion ratio is, the lower the extent of LC contamination will be. This is because the number of unreacted components decrease with an increase in the conversion ratio. This also influences the alignment properties of the LCs. Further, based on the results of the adhesive and lap shear strength measurements, it was concluded that the spacer structure of the dihydrazides determined the mechanical properties of the cured epoxy resin systems. We found that as the bulkiness of the spacer structure increases, the impact sensitivity of the cured epoxy resin decreases and its flexibility increases. This results in desirable elongation properties and high adhesive strength when an external force is applied. Finally, we evaluated the electro-optical characteristics of each epoxy resin system formed under industrial heat-curing conditions. It was observed that as the LC contamination area decreases, the electro-optical properties, including the threshold voltage and response time, improve. This is because the LC contamination area affects the alignment properties of the LCs, causing them to become horizontally aligned. Thus, it can be concluded that dihydrazides, and especially VDH, can be used as effective latent curing agents for LCD sealants, owing to their high conversion ratios, low ability to contaminate LCs, high mechanical properties, and good electro-optical characteristics.


Mutations involving CTNNB1, the gene encoding beta-catenin, and other molecular alterations that affect the Wnt/beta-catenin signaling pathway are exceptionally common in hepatocellular carcinoma. Several of these alterations have also been associated with scarcity of immune cells in the tumor microenvironment and poor clinical response to immune checkpoint inhibitor therapy. In light of these associations, tumor biomarkers of beta-catenin status could have the potential to serve as clinical predictors of immunotherapy outcome. This editorial review article summarizes recent pre-clinical and clinical research pertaining to associations between beta-catenin activation and diminished anti-tumor immunity. Potential non-invasive biomarkers that may provide a window into this oncogenic mechanism of immune evasion are also presented and discussed. Patients with clinically-advanced HCC have poor survival, with estimated one-year survival rates of 21% for those with regionally advanced disease and 6% for those with metastatic disease. After a decade of meager advances in systemic therapy, the treatment of unresectable advanced-stage HCC has taken a leap forward with the advent of immune checkpoint inhibitor (ICI) antibodies. In the United States, the first ICI agent to gain regulatory approval for HCC was the anti-programmed death 1 (anti-PD1) monoclonal antibody nivolumab. Accelerated approval for this ICI as a second-line systemic agent for HCC came after a 20% objective response rate was reported in the dose expansion phase of its phase I/II trial (CheckMate-040) that involved patients who were refractory to, or intolerant of, sorafenib. Pembrolizumab, another anti-PD1 antibody, also received accelerated approval as a second-line therapeutic after similar response rates were reported in a non-randomized open-label phase II clinical trial (KEYNOTE-224). Most recently, a regimen combining bevacizumab with atezolizumab, an anti-programmed death ligand 1 (PD-L1) antibody, received clinical approval as a first-line systemic therapy for unresectable HCC, based on its phase III trial (IMbrave150) reporting an objective response rate of 36%, along with better overall survival compared with sorafenib, the prior standard of care.Hepatocellular carcinoma (HCC) is frequently diagnosed at an advanced clinical stage or precluded from surgery by poor underlying liver function. These results mirror closely what was reported in the phase II trial of pembrolizumab . Similarly, in the CheckMate-040 trial of nivolumab, 1%, 18%, and 32% of patients experienced CR, PR, and PD, respectively. This degree of heterogeneity in tumor response has since been recapitulated in observational cohorts of HCC patients receiving IC therapy,8. The observational data also suggest that heterogeneity in tumor response translates into heterogeneity in survival outcome. Clinical response has also been heterogeneous in the first-line treatment setting, with the IMbrave150 trial reporting 5.5% CR, 21.8% PR, and 19.8% PD in Child-Pugh class A patients not previously treated with sorafenib. Durations of clinical response are also highly variable, ranging 6-24 (median 17) months in the CheckMate-040 trial and 1.5-23.6 (median 13.8) months in the KEYNOTE-240 trial,9. Since immune-related adverse events can be serious, a predictive biomarker that can adequately explain this heterogeneity across patients would be of great benefit in the clinical setting for optimizing patient selection.Notwithstanding the hope that immunotherapy brings to patients with advanced HCC, all trials to date have shown substantial heterogeneity in the degree to which tumors respond to these immune-oncology agents. In the recently completed double-blind placebo-controlled randomized phase III trial of pembrolizumab (KEYNOTE-240), only 2% of treated patients experienced a complete response (CR), while 16% and 32% experienced partial response (PR) and disease progression (PD), respectively,4. Furthermore, while high microsatellite instability (MSI-H) and tumor mutation burden (TMB) have been shown to be predictive of clinical immunotherapy response in multiple other cancers, they have not proven very predictive of ICI response in HCC. It is therefore time to start looking beyond these immediate immunotherapy biomarkers. Examining what is known about the underlying mechanisms of tumor immune evasion in HCC and other cancers may help facilitate a broader search for immunotherapy biomarkers that are useful for HCC.Unfortunately, no clinical trial to date has identified a reliable tissue or serum biomarker to predict immunotherapy response in HCC. Although tumor PD-L1 expression has been associated with anti-PD1/PD-L1 ICI response in multiple cancers , this immunohistochemical marker has not yet proven to be a good predictor of response for HCC-18. Of these, the Wnt/β-catenin signaling pathway was among the earliest to be associated with tumor immune suppression,19. In a genetically-engineered mouse model of melanoma that exhibits stabilized β-catenin expression, it was first discovered that aberrant activation of this pathway could lead to the disappearance of immune cells from the tumor microenvironment (TME) and the development of tumor resistance to anti-PD-L1/anti-CTLA-4 monoclonal antibody therapy,20. β-catenin expression in this model was also associated with decreases in the expression of immune-oncology relevant chemokines such as CCL4 that are important for recruiting dendritic cells and consequently T-cells into the TME. The mechanism by which β-catenin reduces CCL4 expression was tied to the induction of ATF3, a transcriptional repressor, and its binding to the CCL4 promoter,20,21. This immune evasion mechanism has since been recapitulated in a similarly engineered model of β-catenin activated HCC, where it was observed that aberrant β-catenin activation led to tumor resistance to anti-PD1 therapy and that reinstating the expression of CCL5, a chemokine that was downregulated in the β-catenin driven tumors, could restore tumor immune surveillance.Multiple lines of mechanistic and clinical research have identified potential associations between tumor immunotherapy resistance and well-known oncogenic pathways and mechanisms,22. Across numerous cancer types, analysis of TCGA data has revealed a significant difference in the percentage of Wnt/β-catenin activating mutations between immune inflamed and non-inflamed tumors, with the largest differences observed in HCC and esophageal cancer. Histologically, the TME of β-catenin activated HCC also displays a paucity of immune cells. Hence, one may speculate that it may be possible to infer the status of anti-tumor immunity in HCC by analyzing tumor biomarkers that capture the status of β-catenin activation.TME infiltration by antigen presenting dendritic cells and T-cells, leading to a so-called inflamed TME, is now recognized as a critical factor that allows anti-PD1 and anti-PD-L1 antibodies to exert their therapeutic effects. This pathway has been shown to play multiple roles in tumorigenesis and tumor immune-evasion,25-29. In the canonical Wnt/β-catenin pathway, the nuclear transcription co-activator protein β-catenin undergoes continual degradation in the cytoplasm by a destruction complex comprised of the scaffolding protein Axin (AXIN1) along with casein kinase 1 (CK1), glycogen synthase kinase 3 (GSK3), and the adenomatous polyposis coli (APC) gene product . Wntpression [32].CTNNB1), the gene that encodes for β-catenin, are among the most common causes in HCC, as they are found in approximately one-third of HCC tumors-36.Aberrant activation of this pathway in cancer can be caused by a number of different molecular mechanisms, including gene mutations, epigenetic alterations, and abnormal regulation of other pathways. Mutations involving catenin-beta-1 ,12,41Mutations involving other Wnt/β-catenin pathway-related genes can also lead to aberrant β-catenin signalingignaling , includi,35,36,42. In particular, mutations involving CTNNB1 occur in an estimated 25%-40% of HCC tumors,16,23,36 The specific CTNNB1 mutations that have been associated with an immune-barren tumor landscape are almost exclusive to areas within exon 3. These areas correspond to coding regions for phosphorylation sites near the N-terminus of β-catenin that are involved in mediating its breakdown. Mutations involving these regions allow β-catenin, a transcription co-activator, to escape destruction and accumulate in the nucleus, where binding to T-cell factor/lymphoid enhancing factor allows it to aberrantly promote transcriptional programs that include those that may influence tumor immune phenotype-45.Several gene mutations consistently overrepresented in HCC are associated with the β-catenin activated state,16,17,46.Consistent with this apparent mechanism of β-catenin mediated immune cell exclusion, clinical associations between markers of Wnt/β-catenin activation and various molecular signs of immunotherapy resistance have been revealed through numerous and exhaustive molecular profiling and genotyping studies of human tumor specimensCTNNB1 and other mutated genes known to cause Wnt/β-catenin activation have been associated with poor clinical response to treatment with ICI antibodies and other targeted agents in HCC,16,33,34. In one study that included 27 ICI-treated HCC patients who underwent pre-treatment tumor mutation profiling, 10/10 of HCC patients with tumors bearing CTNNB1 or AXIN1 mutations experienced progressive disease compared to 5/17 patients with tumors that did not have these β-catenin activating mutations . The gene AXIN1 codes for a protein involved in β-catenin destruction,37,42,47. The specific AXIN1 mutations involved cause loss-of-function, and they are usually mutually exclusive to CTNNB1 mutations,48. In addition to experiencing higher rates of progressive disease, ICI-treated HCC patients with CTNNB1 or AXIN1 mutated tumors also experience significantly shorter progression free survival . These clinical associations imply that the mutation statuses of these genes may have some value as predictive or prognostic biomarkers for HCC immunotherapy.Mutated ,28,33,35. For example, mutations that result in the loss or inactivation of the adenomatous polyposis coli (APC) gene product are a frequent cause of aberrant β-catenin activation in colon and rectal cancer. However, while it is estimated that over 80% of colorectal cancers harbor APC mutations, they are relatively uncommon in HCC with an estimated frequency of 5%-7%,49. With the recent clinical approval of two cell-free DNA (cfDNA) sequencing panels for mutation profiling with relatively broad (pan-cancer) coverage, it may now be practical to detect these mutations along with the more prevalent ones through non-invasive liquid biopsy. In addition to profiling a large set of cancer-associated genes, these panels also generate blood-based estimates of TMB (bTMB) and MSI-H status, making it conceivable that an integrative genomic profile of relevance to the HCC immunotherapy space may be discovered. Cohort studies as well as carefully designed prospective biomarker trials will be necessary to learn, test, and validate such mutational profiles as predictors of ICI response. Since HCC is a cancer for which tumor tissue is difficult to obtain clinically, these pan-cancer liquid biopsy panels may eventually prove to be a boon to the development of more precise targeted therapy for this disease. However, regarding immunotherapy, mutation status alone may not be able to fully explain the estimated 60%-80% of HCC patients who fail to exhibit an objective response to ICI therapy.Several other gene mutations that can drive Wnt/β-catenin signaling are relatively uncommon in HCC, as well as alterations involving extracellular pathways-40,48, have also been associated with aberrant Wnt/β-catenin signaling in HCC. There are also data suggesting that upregulated lipid metabolism in HCC is associated with β-catenin activation,29,50. This association raises the possibility of inferring tumor β-catenin activation status based on phenotypic assessments of tumor metabolism. Such assessments are clinically feasible and can be performed non-invasively using several molecular imaging methods including positron emission tomography (PET). As a proof of concept with regard to phospholipid metabolism, a recent series of experiments performed in an adenomatous polyposis coli (APC) knock-out mouse model of β-catenin activated HCC suggests that tumor uptake of fluorocholine (FCH), a fluorine-18 labeled PET radiopharmaceutical analog of choline used for in-vivo tracing of tissue phosphatidylcholine synthesis,52, is strongly influenced by β-catenin activation. This novel animal model of de-novo HCC can develop well, moderately, and poorly differentiated tumors that are capable of recapitulating the salient histological and molecular features of β-catenin-driven human HCC. Experimentally, increased tumor FCH uptake in this system was shown to correlate with increased β-catenin protein expression as well as metabolomic and transcriptional fingerprints of canonical Wnt/β-catenin activity. Conversely, control animal models with non-β-catenin-activated tumors were found to demonstrate low tumor FCH uptake. Providing a simple mechanistic explanation for how this pathway promotes increased tumor FCH uptake and choline phospholipid metabolism, it was confirmed that β-catenin activation was able to drive choline membrane transporter expression. Following a translational route, the team that conducted these experiments also obtained and sequenced tumor DNA from 13 patients that had underwent clinical FCH PET/CT and found mutated CTNNB1 in 6/7 patients with FCH-positive tumors and wild-type CTNNB1 in 6/6 patients with FCH-negative tumors.Other molecular mechanisms, including epigenetic alterations. To explore whether β-catenin activating mutations found in tumor DNA could also be detected in cfDNA, we analyzed the pre-treatment plasma samples collected from these patients by performing targeted mutation profiling of cfDNA using an oncology-specific next generation sequencing panel . The results of this liquid biopsy mutation analysis along with the PET imaging and clinical tumor DNA profiling results are shown in In an attempt to corroborate their results, we examined the tumor genomic profiling results from six HCC patients who underwent preoperative liver FCH PET/CT imaging prior to liver resection. These patients were among the participants of a recently completed diagnostic clinical trial of FCH PET/CT in liver cancerCTNNB1 mutations and one being a mutation involving the guanine nucleotide binding protein-alpha stimulating sub-unit (GNAS) gene. These same genes were also found mutated in the corresponding cfDNA of these patients. Interestingly, mutations involving GNAS have been reported to be a cause of upregulated Wnt/β-catenin activity and lipid metabolism in several digestive tract cancers but not yet in HCC,60. Notably, the tumors of all four of these patients showed high uptake of FCH on PET/CT  . FurH uptake , suggest. However, durations of clinical response in the trial ranged from 1.5 months to 23.6 months, and the risk of death overall was reduced by 22% . The implication of these results is that some patients will benefit substantially from these agents, but the benefits will be thinly spread across too many patients in the absence of a robust predictive biomarker that can be used to refine patient selection. Because anti-PD1 agents can lead to prolonged disease control in those who do respond, a predictive biomarker of treatment resistance/response could have substantial value in both the clinical and research domains. For clinical trials, a reliable predictive biomarker may substantially reduce the study sample size required and increase the statistical power for an a-priori treatment effect size. Because immune-related adverse events to ICI therapy are non-trivial, bringing such a biomarker to the clinical practice space would help guide patients with vulnerable tumors to appropriate therapy while protecting those who are unlikely to respond from the hazards of futile treatment and its side effects. From a healthcare economics standpoint, a predictive biomarker would help to enhance the value proposition of ICI treatment by reducing costs associated with wasted treatments and unhalted disease progression.In 2019, the KEYNOTE-240 phase III trial was reported to have failed in achieving its pre-determined statistical endpoints for survival,65,68-70. An understanding of how different non-convergent molecular pathways and phenotypes can shape tumor immunity in HCC may support multimarker integration as an approach to predicting immunotherapeutic response,46,65,70. There is also a growing number of statistical learning and machine learning based approaches to building, integrating, and evaluating multi-biomarker classifiers,71,72, although the optimal method for assigning significance to any incremental gains in biomarker classification performance has been a topic of debate,74. The tools for integrative biomarker design and analysis have also become research tools to elucidate the biologic origin and functional significance of different biomarkers with the potential of shedding more light on their clinical and biological importance.However, robust biomarkers, detectors, predictors, and other classifiers that are singular in nature are rare in the field of cancer. There are multiple biological and statistical reasons for why an integrative biomarker would perform better than a single or narrowly targeted set of biomarkers, allow for the diagnosis of HCC to be predicated on the results of radiographic testing in appropriately selected patients. In those patients with cirrhosis or chronic liver disease, satisfaction of radiographic criteria based on contrast-enhanced CT or MRI (LIRADS-5) confers a positive predictive value of > 98% for the diagnosis of HCC, effectively alleviating the need for liver biopsies to secure a tissue diagnosis. Unfortunately, this non-histopathologic approach to diagnosis can impede clinical and research efforts to advance precision medicine for HCC, as it makes tumor tissue unavailable for genetic testing or molecular profiling. Non-invasive diagnostic tools such as liquid biopsy and molecular imaging have the potential to address this lack of molecular information. Clinical development of such non-invasive methods for HCC for molecular-subclassification, risk-stratification, and treatment selection share the promise of preserving and extending the non-invasive clinical diagnosis and management approach pioneered in HCC.The contemporary clinical approach to the diagnosis of HCC has evolved into something rather unique among solid tumors. Diagnostic algorithms for HCC, such as those based on the National Comprehensive Cancer Networks (NCCN) guidelinesPresently, there is a critical yet unmet need for biomarkers to predict immunotherapeutic response in HCC, since objective clinical responses to the existing approved ICI agents occur in only a fraction of patients. There is now a large body of evidence that associates Wnt/β-catenin activation with tumor immune evasion and immunotherapeutic resistance. Recent work has also shown that it is feasible to assess the Wnt/β-catenin activation status of malignant tumors non-invasively through liquid biopsy and possibly molecular imaging. Further efforts along these lines to develop non-invasive assays of tumor Wnt/β-catenin activation may have the potential to be fruitful in producing much-needed biomarkers for predicting immunotherapeutic response in HCC. However, as with therapeutic agents, these biomarker tools will require rigorous and thorough clinical testing along a well-planned series of clinical trial phases that begins with the assessment of biomarker classification performance and ends with the measurement of clinical impact.