Traditional injection and extraction devices often appear painful and cumbersome for patients. In recent years, polymer microneedles (MNs) have become a novel tool in the field of clinical medicine and health. However, the cost of building MNs into any shapes still remains a challenge. In this paper, we proposed hydrogel microneedles fabricated by high-precision digital light processing (H-P DLP) 3D printing system. Benefits from the sharp protuberance and micro-porous of the hydrogel microneedle, the microneedle performed multifunctional tasks such as drug delivery and detection with minimally invasion. Critical parameters for the fabrication process were analyzed, and the mechanical properties of MNs were measured to find a balance between precision and stiffness. Results shows that the stiffness and precision were significantly influenced by exposure time of each layer, and optimized printing parameters provided a balance between precision and stiffness. Bio-compatible MNs based on our H-P DLP system was able to execute drug injection and drug detection in our experiments. This work provided a low-cost and fast method to build MNs with 3D building, qualified the mechanical performance, drug injection, drug detection ability of MNs, and may be helpful for the potential clinical application. Traditional injection and extraction devices such as hypodermic needles often appear painful and cumbersome for patients. In recent years, polymer microneedles (MNs) have become a novel tool in the field of clinical medicine and health . With thMost of these polymer microneedles are typically fabricated by template driven . The shaIn this paper, we brought hydrogel MNs based on 3D printing. A self-built high-precision digital light processing (H-P DLP) system based on light curing was utilized to print MNs. The H-P DLP system has high printing accuracy and is capable of meeting precision of building micro-needle . BesidesThe raw material of MNs was liquid at first, the monomer in solution would crosslink under the illumination of blue light. The preparation of precursor of MNs was as followed: First, transfer 10 mL Polyethylene glycol diacrylate  into a brown sealing bottle, then 1% wt Phenylbis  phosphine oxide  was added into the bottle. Stirring for 24 h. All operations were down under dark environment because of the light sensitivity of photo-initiator. Then we use H-P DLP system made in our previous work to build precursor into solid state MNs . The simDue to the significant impact of exposure time on the precision and stiffness, groups of printing experiments with exposure time of ranging from 50 ms to 900 ms were carried out. MNs with ideal precision and stiffness were used in following drug injection and drug detection experiments.Universal material testing machine was utilized to measure the mechanical properties of MNs see . As showAfter mechanical tests of MNs, the balance of between precision and stiffness was found. Then to certify the skin penetration, drug injection, and detection ability of MNs, 5 wt% alginate hydrogel was used as artificial skin in drug injection and drug detection experiments. Since 5 wt% alginate hydrogel has similar mechanical properties to human skin ,25. AlgiExposure time has great influence on the mechanical performance of printed MNs. Long exposure time could enhance the stiffness of MNs, however, MNs were bigger than designed MNs. Short exposure time could avoid over exposure, but the stiffness of MNs would decrease. It is crucial to find a balance between precision and stiffness. In this section, the impact of exposure time on precision was analyzed in detail. MNs with exposure time ranging from 50 ms to 900 ms were printed, and the side face pictures of those MNs are shown in The real height of needles was defined as H1, and the curing height of the interval between needles was defined as H2. H1 and H2 were displayed in The side face pictures of mechanical properties testing process are displayed in The impact of exposure time on the mechanical properties of MNs is shown in 1,D1 were coordinate values of starting point in curves and P2,D2 were coordinated values of changing point next to starting point. It represented the pressure increased after the displacement increased and could indicate the stiffness of MNs. As exposure time increased, the K of MNs ascended at the same time according to K was defined asComparing to other MNs, MNs with 300 ms exposure time have well precision and stiffness. In consequence, 300 ms MNs were used in following drug injection and drug detection experiments. The influence of water content is shown in Penetration test was also performed by the universal material testing machine while the MNs and artificial skin were fixed on the sensor and the base, respectively. The pressure-displacement curve of artificial skin penetration process is shown in MNs were fabricated from precursor with the help of blue light. Monomers crosslinked with each other and formed grid structure. Thus, MNs had many micro pores on its surface as shown in As described in The images of artificial skin area in all recorded pictures were imported into Matlab and transferred into gray pictures. The gray values were calculated as followed: Summing gray values of every pixel up, then the total value was divided by the numbers of pixels. Mean integrated density was obtained by methods above. The concentration is calculated as shown in Similar method in drug injection was used in simulation of drug detection. MNs was soaked in DI water for 24 h while artificial skin was soaked in 0.005 wt% rhodamine B solution for 24 h. Then MNs without rhodamine B was stuck into artificial skin with rhodamine B. Samples were put under green laser at 532 nm and fluorescence picture was taken every 2 min. The initial state and final state pictures are displayed in The images of white dotted area in all pictures recorded were imported into Matlab and transferred into gray pictures. The same method was used to calculated the mean integrated density. The quantitative data of drug detection is displayed in 3D building is a novel and low-cost method in manufacturing MNs comparing to conventional template driven fabrication. We could use H-P DLP system to build MNs into many shapes needed with bio-compatible materials in different printing parameters. The precision and stiffness of MNs could be controlled by the exposure time. When exposure time as over 300 ms, the precision would decline as exposure time ascend while the stiffness would increase at the same time. There was a balance between precision and stiffness. After groups of mechanical tests and measurement, we regard 300 ms as an ideal exposure time for building MNs. There are limitations of existed MNs such as solid MNs, coated MNs, and commercial 3D printed MNs. Solid MNs is relatively rough and could cause irreversible wound and dosage of coated MNs is limited, besides, it is difficult to control and quantify ,11. PhotIn this paper, we have done the experiments and result analysis of printing parameters on the mechanical properties of microneedles, which provides a method to change the mechanical properties of microneedles. In addition, we also achieved the simulation experiments of micro needle puncture of artificial skin and the simulation experiments of drug injection and drug extraction by MNs. What is more, the results of drug injection and drug detection were analyzed quantitatively, which may provide a certain reference for micro needle using in biological injection and drug extraction. Due to the low-cost, painless, short manufacturing time, well biocompatibility, and drug delivery performance properties of 3D built MNs, it is exciting to adapt MNs to the market. We expect to apply 3D printed MNs with properties above to clinical usage in the future.


The mirror neuron system has been argued to be a key brain system responsible for action understanding and imitation. Subsequently, mirror neuron system dysfunction has therefore been proposed to explain the social deficits manifested within autism spectrum condition, an approach referred to as the broken mirror hypothesis. Despite excitement surrounding this hypothesis, extensive research has produced insufficient evidence to support the broken mirror hypothesis in its pure form, and instead two alternative models have been formulated: EP-M model and the social top-down response modulation (STORM) model. All models suggest some dysfunction regarding the mirror neuron system in autism spectrum condition, be that within the mirror neuron system itself or systems that regulate the mirror neuron system. This literature review compares these three models in regard to recent neuroscientific investigations. This review concludes that there is insufficient support for the broken mirror hypothesis, but converging evidence supports an integrated EP-M and STORM model.The mirror neuron system has been argued to be a key brain system responsible for understanding the actions of others and for imitation. It has therefore been proposed that problems within this system could explain the social difficulties experienced by people with autism spectrum condition. This idea is referred to as the broken mirror hypothesis. However, research has produced insufficient evidence to support the broken mirror hypothesis in its original form. Therefore, two other models have been suggested: EP-M model and the social top-down response modulation (STORM) model. All models suggest something is different regarding the mirror neuron system in autism spectrum condition: either within the mirror neuron system itself or within the systems that control the activity of the mirror neuron system. This literature review compares these three models in regard to recent neuroscientific investigations. This review concludes that there is insufficient support for both the broken mirror hypothesis, but converging evidence supports an integrated EP-M and STORM model. The discovery of mirror neurons, in macaque monkeys, was considered by some to be a major breakthrough into unravelling the neural basis of action understanding, imitation and also Autism spectrum condition (hereafter ASC/autism) . The mirHowever, the function and even the very existence of the human MNS remain controversial. There have been methodological issues in MNS-related research, including the use of unsatisfactory controls and confounding variables, such as the effect of attention on specific electroencephalographic responses , 2017, aA main tenet of the EP-M model  is that The STORM model  proposesThese models are not mutually exclusive: Given continued neuroscientific research into the MNS in ASC, a review and comparison of these two models is timely. The present literature review aims to critically assess studies published since Hamilton’s reviews, to capture the extent to which each model is supported by recent neuroscientific findings. Behavioural evidence (i.e. imitation deficits in autism) has been reviewed elsewhere, demonstrating robust deficits in imitation in autistic individuals . InteresHowever, all three models – the BMH, EP-M and STORM – are inherently neuroscientific models that make claims regarding the role of specific brain networks and regions. Therefore, our review shall focus on neuroscientific methodologies (i.e. methods of inquiry that draw upon measures of brain activity or structure), to complement reviews of behavioural work. Within this definition of ‘neuroscientific’ also falls neurofeedback training (NFT) and post-mortem data, neither of which were included in Hamilton’s reviews.Each model yields specific predictions for neuroscientific data. If the BMH is valid, then participants with ASC should show a global deficit in the MNS, leading to a disadvantage in tasks reliant upon this system: as long as the task utilises mirroring mechanisms, there should be a significant difference between ASC participants and NT participants on measures of mirror neuron activity. Whether this difference is hyperactivity or hypoactivity in relation to NT controls is not specified, and both have been argued to support the theory .The EP-M model would predict that ASC individuals are only disadvantaged on tasks in which participants process non-goal-directed actions, as these depend on the M-route. Tasks in which stimuli have an end goal, and therefore utilise the EP pathway, should produce no significant differences between ASC and NT participants on measures of mirror neuron activity.In contrast to both of the preceding models, the STORM model would predict that differences between NT and ASC groups are not due to deficits within the MNS itself but within its connections to and regulation by frontal regions of the brain. This would suggest that under circumstances that did not require top-down regulation of the MNS, ASC and NT participants should appear the same. However, ASC individuals would be disadvantaged if the task had social relevance or relied upon the executive control of the MNS.A literature search, incorporating Ovid, Web of Science and Google Scholar, was conducted using the following search phases: ‘Autism’ OR ‘ASD’ OR ‘ASC’ OR ‘Autistic’ AND ‘Mirror neuron system’ OR ‘MNS’ OR ‘Broken Mirror Hypothesis’. Initial keyword searches produced 7088 papers . Papers were selected based on the following criteria: (1) they must refer to both ASC and the MNS; (2) they must focus on the MNS rather than the whole action imitation network; (3) they must not have been incorporated in In EEG studies of the MNS, a common index of MNS activity is mu suppression, a characteristic reduction in alpha activity (8–13 Hz) from electrodes over the sensorimotor strip  during action observation or execution . If indeComparing data from six studies, So far, the discussed investigations have focused on the 8–13 Hz rhythm as a single band width and focused dominantly on the central electrodes (positioned above the sensorimotor strip). Finally, Taken together, these EEG studies refute the BMH, as predicted differences in mu suppression were either not reported or are better explained under the alternative models, particularly the EP-M model. More recent studies that have examined mu suppression band differences at other sites and using other sub-bands have also produced findings fitting the STORM model. However, this model struggles to explain some of the findings brought about by studies using mentalising tasks: tasks in which we would expect social cues to modulate MNS activity.TMS approaches to MNS research have typically utilised motor evoked potentials (MEPs). MEPs, movements stimulated by TMS over the motor cortex (e.g. arm flinches), are more pronounced during action observation, compared to a baseline condition. This has been suggested to represent pre-activation in the motor cortex during action observation; therefore, it is argued that differences in MEPs during action observation could provide a measure of the activity of the human MNS . Under tSimilar to No differences in MEPs were reported between ASC and NT participants in either investigation. This is intriguing as Since Cole et al’.s stimuli were object-based and goal-directed (pushing a poker chip through a slot), the EP-M model would predict no MEP differences between ASC and NT participants, since these actions utilise the EP route. The STORM model would predict that MEPs would be unaffected when viewing hand actions, as neither study was presented the task within a social context. However, deciding whether an actor is spiteful or clumsy could be argued to require social cues and therefore, ASC participants in the study by The brain regions typically considered to form the MNS usually include IFG and IPL. Magnetic resonance imaging (MRI) techniques allow for activity in these purported mirror neuron regions to be compared between ASC and NT groups. The BMH would predict global activity differences with the MNS, whereas the EP-M model would predict that differences lie within middle temporal regions, regions that support the M-route, and that activity differences would be specific to stimuli without objects. The STORM model predicts that differences in MNS activity, if any, are due to reduced top-down regulation. Therefore, differences in PFC activity should be present, and during tasks that require social modulation. In addition to differences in the activation of brain regions, diffusion tensor imaging (DTI) techniques allow for differences in the connectivity between regions belonging to the MNS to be investigated. Global connectivity differences would be in line with the BMH, whereas the EP-M model theorises that connectivity differences are specific to the M-pathway. Differences within top-down connections to the MNS would support the STORM account. Recent work using fMRI has reported significant differences in activity from MNS brain areas between the NT and ASC groups . Both stReduced functional connectivity between the IFG and the MTG (i.e. the M-route) has been reported , up-holdGiven potential functional connectivity differences, investigating the white matter connections within the MNS may provide more insight into the abnormalities associated with ASC. Three studies using DTI, or more recent diffusion spectrum imaging (DSI), were identified: In summary, imaging techniques demonstrate that MNS activity and functional connectivity differ between ASC and NT individuals, although the direction of group differences is often contrary to expectations. In particular, the IFG is reported across different studies as having different activity, different functional connectivity and different structural properties, in ASC. This would suggest a dysfunction, as proposed by both the BMH and EP-M. However, DTI studies fail to show structural connectivity differences between participant groups within the MNS itself, and thus atypicalities may be the result of reduced top-down modulation, as argued by STORM.Since near-infrared spectroscopy (NIRS) measures neural activity via oxy-haemoglobin levels, similar to fMRI, predictions from each model are identical to those made about MRI research. At the time of Broadly, these findings support the BMH, in that the IFG, a key MNS area, is underactive, but training targeting behaviours that utilise the MNS can normalise the activity of the IFG. The involvement of the IFG, and its increase in activity after training of facial imitation – a form of imitation argued to rely particularly on the M-route – would also fit with the EP-M model.For the data reviewed thus far, the resolution limitations of neuroimaging prevent discovering whether group differences in MNS activation are due to atypicality in mirror neurons themselves. Here post-mortem studies may be able to shed light on differences at the cellular level. The BMH would predict differences in the IFG and IPL, the EP-M would predict differences in the IFG and MTG and the STORM model would predict differences in frontal brain regions. This study measured neuronal differences within the IFG in eight post-mortem brains from ASC individuals and two NT brains. It was reported that ASC brains had reduced neuron size but equal neuronal frequency and layer volume within the IFG. Since all models would predict some form of neuronal difference within this area, affecting either the top-down pathways to the MNS (STORM model) or the efficiency of the M-pathway or entire MNS (EP-M model and BMH respectfully), such limited investigation cannot differentiate between the models in question. However, this reduction in neuron size could be responsible for the reduced connectivity between the IFG and PFC, shown in DTI studies. Neural tissue from individuals as young as 4 years old were incorporated, suggesting neural differences being present from early life. Therefore, such findings can add to evidence supporting BMH, EP-M and STORM accounts. However, there is no way of confirming whether these neurons had mirror properties.NFT evidence with regard to the BMH argues that, assuming the BMH is valid, normalising MNS activity using NFT techniques should reduce ASC symptoms. In addition to its potential clinical uses, NFT could also provide an important experimental paradigm to acquire causal, rather than correlational, evidence regarding the role of the MNS in autistic symptomatology. NFT studies examining MNS function to date have relied on EEG measures of the MNS, in which children watch a film or play a game which is linked to EEG recordings of mu rhythms. The child must periodically decrease their mu rhythm  to continue the film or game while simultaneously ensuring that theta and beta activity (associated with distraction) remains below a threshold. If a deficit in the MNS is a cause of ASC symptomatology, as the BMH and EP-M models suggest, training based on MNS activity should reduce ASC symptoms (the EP-M model would specify that training M-route-based processes should be what supports a reduction in ASC symptoms). The STORM model would suggest that simply normalising activity will not help reduce autistic symptoms, rather training ought to consider the appropriate modulation of mirroring processes.Studies into the effectiveness of NFT , 2014 clIntriguingly, NFT may have disadvantageous effects on NT participants, including reduced social communication scores  and reduThe NFT literature at present faces several methodological issues. Neither Only To summarise, the NFT literature contains methodological flaws, and the most prominent study in the literature only shows improvements in attention, which could be better explained by the STORM model.The BMH has been a highly influential  theory regarding autism, and it is evident that this theory and the counter-hypotheses that came after it continue to inform neuroscientific research in autism. Consistent with previous reviews, this review found a significant lack of evidence for the BMH. The EP-M model received some support from EEG, TMS and fMRI evidence. Some DTI evidence included in this review cast some doubt over the validity of this model, given that lack of a neuroanatomical connection between the brain region comprising the M-pathway, although such a pathway has been identified in non-MNS related research. The STORM model also received some support, particularly from EEG/NFT data, and (unlike the EP-M model) is not dependent upon the existence of an M-pathway. As noted by Future researchers should consider investigating the nature of top-down modulation in ASC, possibly implemented by the PFC, as opposed to just examining the MNS itself. The PFC has been implicated in allocating the contextual goal behind a task  and has A strength of the STORM account is that it is not dependent upon the existence of the MNS itself. Even though Hamilton incorporated the MNS within the model, the model is mainly discussed in terms of a basic visual-motor mapping system, consisting of the inferior parietal cortex, premotor cortex and primary motor cortex. Therefore, evidence which contradicts the existence of a human MNS e.g.  would noIt is possible that discrepant findings may be explained by factors such as participants’ age and IQ levels. With regard to how age and gender bear upon our analysis of these studies, we see no particular pattern with age: results that refute the BMH occur across studies with different ages of participants and different proportions of males and females. For IQ, at least for the studies that included details regarding the IQ of their samples, it appears that these studies largely only included participants without intellectual disability (i.e. IQ > 70). Thus, it must be borne in mind that the results reported in these studies, and thus the conclusions of our review, are restricted to autistic people without intellectual disabilities.In conclusion, this review extends the current ASC literature by specifically contrasting the BMH, EP-M and STORM model against each other, based on recent neuroscientific evidence. Evidence contradicts predictions made by the BMH and favours an integrated account of the EP-M and STORM models. Future investigations should focus on top-down modulation, rather than simple MNS dysfunctions in ASC. Such investigations should use mentalising or facial imitation tasks, which may be more sensitive to detecting specific processes important to the STORM model  than the often used hand observation tasks. Furthermore, we suggest shifting away from looking for dysfunction in isolated MNS areas, in favour of examining how networks regulate multiple systems, which contribute to social processes in both NT and ASC populations.


Vascular calcification (VC), the ectopic deposition of calcium phosphate crystals in the vessel wall, is one of the primary contributors to cardiovascular death. The pathology of VC is determined by vascular topography, pre-existing diseases, and our genetic heritage. VC evolves from inflammation, mediated by macrophages, and from the osteochondrogenic transition of vascular smooth muscle cells (VSMC) in the atherosclerotic plaque. This pathologic transition partly resembles endochondral ossification, involving the chronologically ordered activation of the β-catenin-independent and -dependent Wingless and Int-1 (WNT) pathways and the termination of peroxisome proliferator-activated receptor γ (PPARγ) signal transduction. Several atherosclerotic plaque studies confirmed the differential activity of PPARγ and the WNT signaling pathways in VC. Notably, the actively regulated β-catenin-dependent and -independent WNT signals increase the osteochondrogenic transformation of VSMC through the up-regulation of the osteochondrogenic transcription factors SRY-box transcription factor 9 (SOX9) and runt-related transcription factor 2 (RUNX2). In addition, we have reported studies showing that WNT signaling pathways may be antagonized by PPARγ activation via the expression of different families of WNT inhibitors and through its direct interaction with β-catenin. In this review, we summarize the existing knowledge on WNT and PPARγ signaling and their interplay during the osteochondrogenic differentiation of VSMC in VC. Finally, we discuss knowledge gaps on this interplay and its possible clinical impact. Cardiovascular disease (CVD) affects more than 420 million people worldwide, causing 18 million deaths annually. A recently published study postulated an increase to 23 million deaths by 2030, which will increase the global economic burden of CVD to USD 1.044 billion. About 70% of these deaths are due to coronary heart disease (CHD) or ischemic strokes, outcomes that are mainly caused by the vascular disease atherosclerosis [The atherosclerotic plaque manifests as single or multiple pathological deposits in the aorta’s vascular wall or in its branching arteries. These deposits mainly consist of living and dead cells, as well as lipids that accumulate in the vessel wall. Ultimately, the plaque may narrow the affected artery, which induces ischemia in the supplied organs. This narrowing results in CVDs such as CHD, peripheral artery disease, as well as acute vascular events like myocardial infarction and stroke. Notably, patients with atherosclerosis frequently suffer from vascular calcifications (VC), an ectopic deposition of calcium phosphate crystals in the atherosclerotic plaque . Atherosclerosis of the coronary arteries often leads to acute and chronic complications associated with coronary artery calcification (CAC). The prevalence of CAC alternates between 50 and 70% and depends on ethnicity, which was confirmed by the Multi-Ethnic Study of Atherosclerosis that compared the prevalence of CAC in the Black, White, Chinese and Hispanic population, finding the highest risk for CAC in the White population . MoreoveAtherosclerotic risk factors include metabolic diseases like type 1 and type 2 diabetes (T1D/T2D), chronic kidney disease (CKD) as well as lifestyle factors like Western diet, smoking and obesity . Lately,This review aims to present a brief overview of VC pathology and the participating cells. At first, we will explain the similarities between VC and endochondral ossification, as well as the contributing role of inflammation. Subsequently, the respective roles of Wingless/Int-1 (WNT)- and Peroxisome proliferator-activated receptor γ (PPARγ) signaling in atherosclerosis and VC will be explained before discussing existing knowledge about their interplay as well as its relevance for VC.Historically, VC was assumed to be a passive consequence of vascular injury. However, research demonstrated that VC is an actively regulated process that involves inflammation, oxidative stress and mechanisms similar to endochondral ossification ,11. The The presence and morphology of VC is a significant feature of an atherosclerotic lesion and seems essential to identify unstable plaques, which could help to predict acute vascular events . CalcifiMicrocalcifications, presumably, can coalesce to larger macrocalcifications, and more extensive calcifications can increase vascular stiffness, an important and independent predictor of CVDs ,16. In cThe Tampere vascular study found that the gene expression in atherosclerotic plaques differs by up to 50% between arterial beds, including genes responsible for inflammation, extracellular matrix modulation and lipid uptake . This geVarious, commonly used drugs and their effects on VC were studied in the last decades, including Statins, PCSK-9 inhibitors, calcium channel blockers, bisphosphonates and Renin-Angiotensin System (RAS) inhibitors ,22. Stat18F-sodium fluoride-positron emission tomography can detect microcalcifications with high sensitivity, a method that received more attention for the diagnosis of VC over the past years [While treating VC seems complex, its proper diagnosis is also puzzling. Visualizing VCs by computer tomography is common practice. However, due to the low resolution of this method, it only allows the detection of large calcifications. The visualization of microcalcifications—the culprit behind plaque ruptures and acute vascular events—remains challenging, making it difficult to classify atherosclerotic calcifications. Consequently, the correct diagnosis of disease progression in the clinical setting appears difficult, limiting the application of drugs to prevent or treat VC. Currently, only st years . Overall, VC is not solely responsible for vascular events since macro-calcifications may offer protection at the cost of vascular stiffness. In addition, clinicians lack proper diagnostic tools and drugs to identify and treat VC. This underlines the urgency to investigate further plaque heterogeneity and the cellular fate of the contributing cells.Early atherosclerotic disease results from a lesion known as a fatty streak, a visible collection of lipid-laden macrophages, called foam cells, in the vessel wall. This lesion develops to pathological intimal thickening, characterized by the migration of VSMCs from the vessel’s medial layer to the intima. Subsequently, a fibroatheroma arises, defined by a fibrous cap and a lipid-rich, necrotic core .During pathological intimal thickening, VSMCs change from a contractile to a proliferative synthetic phenotype. This phenotype transition is characterized by the loss of contractile markers like calponin-1, actin alpha 2 , and by adopting a mesenchymal stem cell (MSC)-like plasticity ,25. The The main osteochondrogenic transcriptions factors SRY-box transcription factor 9 (SOX9) and runt-related transcription factor 2  are found in atherosclerotic plaques, being mainly expressed in osteochondrogenic VSMC ,32,33,34Macrophages act as an inflammatory modulator in atherosclerosis and are derived from monocytes migrating to the atherosclerotic plaque. Classically, the macrophage phenotypes were divided into M1 and M2 macrophages. Although this is a significant simplification since multiple macrophage subtypes are known, we will only refer to M1 and M2 macrophages in the following chapters, to ease the mechanistic understanding of this review .As presented in Various biochemical signaling networks, such as the bone morphogenetic protein (BMP)/transforming growth factor-beta (TGFβ) signaling and the receptor activator of nuclear factor-κB ligand (RANKL)/OPG/TNF–related apoptosis-inducing ligand (TRAIL) pathway are involved in the development of VC. Physiologically, the RANKL-OPG-TRIAL axis is essential for limiting bone loss due to osteoclasts, but dysregulation of this axis appears to play a detrimental role in the progression of VC . Similar2+-mediated signaling, also known as the non-canonical pathways. The WNT ligand family consists of 19 lipid-modified, secreted glycoproteins, able to induce autocrine and paracrine signaling. In the cell, WNT/β-catenin signal transduction follows binding of a WNT ligand to a receptor of the frizzled (FZD) family and the lipoprotein receptor-related protein five or six (LRP5/6). Consequently, β-catenin accumulates in the nucleus and binds to transcription factors of the T-cell factor/lymphoid enhancer-binding (TCF/LEF) family, directing the transcription of the target gene [2+ pathway, are transducing their signals in a different receptor context, involving FZD receptors and different co-receptors such as receptor tyrosine kinase-like orphan receptors one or two (ROR1/2) and related to receptor tyrosine kinase, while inhibiting WNT/β-catenin signaling. In addition, several regulatory molecules, namely the LRP5/6 inhibitors sclerostin, the dickkopf (DKK) protein family as well as the WNT ligand scavenging secreted frizzled receptor protein (sFRP) family, are regulating these molecular pathways. Aberrant WNT signal transduction plays an essential role in CVD, making this pathway a versatile subject to study in pathologies like VC as reviewed by our group [Nusse and Varmus described first parts of the WNT signaling network in 1982. Almost 40 years have passed, and WNT signaling has been shown to determine the fate of stem cells, making it essential for embryonal development and tissue homeostasis . This siget gene . Withoutur group .calvaria and clavicle bones. In contrast, endochondral ossification forms the remaining parts of the skeleton. In this process, chondrocytes develop from MSC and differentiate into osteoblasts. Numerous signaling pathways, including Hedgehog, Notch, BMP, as well as many others, control this developmental mechanism [Bone tissue develops either by intramembranous or endochondral ossification. During intramembranous ossification, MSCs directly differentiate towards osteoblasts to form the echanism . Especiaechanism . 2+ pathway. This signal is mediated by Ca2+, which activates calcineurin (CaN) and, subsequently, the Ca2+/calmodulin-dependent protein kinase II (CaMKII). Afterwards, CaMKII dephosphorylates the nuclear factor of activated T-cells (NF-AT). This post-translational modification leads to the translocation of NF-AT to the cell nucleus, where it drives the transcription of the chondrogenic determinant SOX9 [The WNT signaling ligand WNT5a can start the chondrogenic program by inducing signal transmission through the WNT/Caant SOX9 . Consequant SOX9 ,54. Concant SOX9 . On the ant SOX9 ,57. TherDuring chondrocyte maturation, the stabilization of β-catenin plays an indispensable role. The stabilized β-catenin, together with TCF1, can bind to the promoter region of the osteogenic determinant RUNX2 to induce its transcription . This meThe WNT signaling pathways are not only involved in endochondral ossification, but they also received considerable attention in connection with bone and cardiovascular diseases . For insAs mentioned in The expression of WNT5a, an activator of the chondrogenic determinant SOX9, is found commonly in calcified atherosclerotic plaques ,63,64. IRecently published data revealed osteochondrogenic VSMC as a potential source for WNT5a, which seems plausible since WNT5a often is detected near calcifications . HoweverNotably, WNT5a expression often locates near macrophage-rich regions, proposing macrophages as a potential source for WNT5a ,63,64. IAs mentioned in A study on VC in rats with end-stage CKD did not show a significant increase in WNT5a but of the canonical WNT/β-catenin ligand WNT3a. Moreover, a correlation was found between the expression of β-catenin—the central signaling transducer of the WNT/β-catenin pathway—and VC . These r−/− mice abolishes the occurrence of AMC and AIC and the expression of OCN and ALP [Calcified plaques display a high expression of RUNX2, which is essential for the full osteochondrogenic transition of VSMC ,33. Corr and ALP . In essence, the aberrant WNT5a/SOX9 signal transmission in atherosclerosis seems to set the stage to initiate atherosclerotic calcification A. Later The role of the DKK protein and secreted frizzled-related protein (sFRP) family in bone development, atherosclerosis and VC has been discussed in detail recently ,77,78. FDKK1 was the first discovered DKK protein and, like DKK2 and DKK4, is known to antagonize WNT/β-catenin signaling by acting together with Kremen (Kre) co-receptors to direct the endocytosis of LRP5/6, inhibiting further signal transduction . SeveralIn the vasculature, platelets and endothelial cells are sources of DKK1 ,80. Pro-The knowledge about other DKK proteins and their function in atherosclerosis and VC remains limited. In osteoblasts, DKK2 may activate the WNT/β-catenin cascade and modulate osteogenic signaling, whereas DKK4 seems to suppress osteogenic lineage determination in bone marrow-derived MSC ,90,91,92In contrast to the other DKK proteins, DKK3 performs its action through binding of Krm1 or Krm2 and not by binding LRP6, however, the signaling pathways influenced by DKK3 are complex and not fully understood ,93. In aThe sFRP family is well-known to antagonize both β-catenin dependent and independent WNT signaling by scavenging WNT ligands and by interacting with FZD receptors to inhibit the binding of other ligands. Nevertheless, the sFRP protein family may also act as WNT ligand transporters, giving them a putative function in WNT signal transduction. Despite the broad expression of these proteins in aortic VSMC, their role in controlling WNT signaling during VC appears unclear . In fibroblasts, sFRP1 controls chondrogenic fate by attenuating SOX9, RUNX2 and COL2A1, which was associated with decreased WNT/β-catenin signaling . Mice wiVarious clinical studies have found lower sFRP5 serum levels in obese, T2D, and CHD patients—all high-risk groups for VC—suggesting a critical function for sFRP5 in the healthy vasculature ,98,99. MOverall, since they are able to inhibit WNT5a/SOX9 as well as WNT/β-catenin signal transduction, sFRPs may turn out as the first line of defense in VC. However, our understanding is still limited, and further studies are required to explain how the distribution of sFRP, FZD and WNT molecules in different cell types influence VC.PPARγ belongs to a family of nuclear receptors that includes the subtypes PPARβ/δ and PPARα. This receptor family coordinates a wide range of metabolic mechanisms like insulin sensitivity, glucose homeostasis and lipogenesis. PPARγ, in particular, is the main determinant that regulates adipogenesis.Naturally occurring polyunsaturated fatty acids like eicosapentaenoic acid (EPA) and synthetic ligands such as thiazolidinedione’s (TZD) can directly activate PPARγ. Subsequently, PPARγ forms a heterodimer with the retinoid X receptor (RXR), and the resulting receptor complex binds to the PPAR-response element (PPRE) in the promoter region of numerous target genes, ultimately inducing their transcription. In contrast, target genes with PPRE binding sites can also be suppressed by the PPARγ–RXR complex as long as no ligand is available. Additionally, ligand-bound PPARγ binds, without RXR, to specific target sites to repress target genes ,103.arteria carotis, RXRα and PPARγ expression is reduced in the VSMC and macrophage cell populations [−/− mice, New Zealand rabbits, as well as in CAD and T2D patients [VSMC in the vasculature and macrophages express PPARγ, indicating a role for PPARγ in vascular function, inflammation, and, consequently in vascular disease ,105. Indulations . The trepatients ,109,110.In calcified radial arteries from CKD patients and calcified aortas from CKD mice, PPARγ expression decreases while osteochondrogenic VSMC cultured in high-phosphate or -glucose conditions present a similar outcome ,112. ConHigh levels of pro-inflammatory cytokines such as TNFα and IL6 are frequently observed in CKD, T2D, and CAD patients, leading to chronic low-grade inflammation of the vasculature while also correlating with cardiovascular events ,115,116.TNFα is essential for the inflammatory response during atherogenesis, and various pro-atherosclerotic factors induce TNFα expression in macrophages, including TGFβ, interferon-γ, as well as autocrine stimulation through TNFα itself . In contThe single transmembrane protein α-Klotho is a phosphaturic protein essential for regulating mineral homeostasis and vitamin D metabolism. It is mainly expressed in the kidney and functions a co-receptor for fibroblast growth factor (FGF) 23 to promote binding to its target receptors. The membrane-bound α-Klotho can be cleaved by secretases, leading to its secretion in blood, where it fulfills regulatory functions. A loss of α-Klotho in mice is known to provoke a premature aging syndrome, which leads to the appearance of atherosclerosis, VC, and other age-related pathologies . The chronically high concentration of pro-inflammatory cytokines like IL6 and TNFα found in atherosclerosis and T2D attenuates renal and vascular Klotho expression by increasing FGF23 concentration through the NF-κB pathway ,129,130.i influx in VSMC—indicating an interdependency of VC and hyperphosphatemia [During CKD progression, secreted α-Klotho concentration decreases, ultimately leading to hyperphosphatemia and VC ,131,132.phatemia ,132,133.phatemia . Similarphatemia . These ophatemia . Consequphatemia ,132,137.The acetylation of PPARγ is essential to regulate the transcription of various target genes. However, histone deacetylases (HDAC) like HDAC3 are upregulated in mice with CKD, leading to the suppression of PPARγ activity through de-acetylation, which may explain the α-Klotho deficiency in CKD . ConsequActivation of PPARγ also suppresses the renin angiotensin aldosterone system (RAAS) ,139. TheThe following free and Mesh search terms were used to identify peer reviewed articles in English through the electronic databases PubMed and PMC: “ppar gamma” AND “vascular calcification” AND “Wnt”. Reviews, Editorials and conference abstracts were excluded. In addition, all studies were excluded that did not show any relations to WNT and PPARγ in the context of VC. Searches were performed on 10 July 2020  and in tThe suppression of PPARγ, is fundamental for the osteogenic lineage determination of MSC ,48. Howe−/− mice increased VC, which was accompanied by an increased SOX9 and RUNX2 expression [A PPARγ and β-catenin independent WNT signaling interplay was first evidenced by Woldt et al. In their study, PPARγ deficiency in VSMC of LDLRpression . In accopression ,64. The pression . This LRpression ,150,151.pression . Furtherpression ,153. This cascade may be inhibited by the activation of PPARγ via the upregulation of the WNT5a scavenger’s sFRP2 and sFRP5 ,154. CurPPARγ may also interfere with the pro-osteogenic effects of WNT/β-catenin signaling . The act−/− mice display a high expression of β-catenin in the aorta [Aforementioned in he aorta . These fhe aorta ,169. In he aorta . Hence, he aorta . ConsequBesides the transcription of α-Klotho, PPARγ may induce activation of the WNT/β-catenin inhibitor GSK3β, and the transcription of DKK1 ,170,171.Beyond these previous observations, ligand-bound PPARγ may sequester β-catenin through its TCF/LEF binding domain and a catenin binding domain, a process that may occur GSK3β-mediated and -independent as well, leading to the proteasomal degradation of this complex via ubiquitination ,174,175.The WNT/β-catenin signaling target Cyclin D1 initiates the recruitment of HDAC3 to PPARγ, leading to the de-acetylation of PPARγ, ultimately suppressing target gene transcription . NotablyIn this review, we described how the sequential activation of WNT5a-signaling, the WNT/β-catenin pathway and the dysregulation of PPARγ during atherogenesis, is involved in VC by guiding the osteochondrogenic transition of VSMCs. Plaque topography, disease etiology, as well as cell heterogeneity and the complexity of the underlying mechanisms make it essential to investigate these fundamental molecular pathways further. The resulting insights may clarify the relationship of macrophages and VSMC during VC, by identifying key factors relevant for the induction of WNT signaling in atherosclerotic plaques. Besides some new, experimental, pharmaceuticals like the RANKL inhibitor Denosumab, no commercial drug affects the main cause of VC, namely the osteochondrogenic differentiation of VSMC. Therefore, already registered PPARγ agonists may deserve more attention as potential VC inhibitors for patients at the onset of atherosclerosis due to their anti-inflammatory and cell fate modulating properties.


Thermosetting polymers have been widely used in many industrial applications as adhesives, coatings and laminated materials, among others. Recently, bisphenol A (BPA) has been banned as raw material for polymeric products, due to its harmful impact on human health. On the other hand, the use of aromatic amines as curing agents confers excellent thermal, mechanical and flame retardant properties to the final product, although they are toxic and subject to governmental restrictions. In this context, sugar-derived diepoxy monomers and anhydrides represent a sustainable greener alternative to BPA and aromatic amines. Herein, we report an “in-situ” sol–gel synthesis, using as precursors tetraethylorthosilicate (TEOS) and aminopropyl triethoxysilane (APTS) to obtain bio-based epoxy/silica composites; in a first step, the APTS was left to react with 2,5-bis[(oxyran-2-ylmethoxy)methyl]furan (BOMF) or diglycidyl ether of bisphenol A (DGEBA)monomers, and silica particles were generated in the epoxy in a second step; both systems were cured with methyl nadic anhydride (MNA). Morphological investigation of the composites through transmission electron microscopy (TEM) demonstrated that the hybrid strategy allows a very fine distribution of silica nanoparticles (at nanometric level) to be achieved within a hybrid network structure for both the diepoxy monomers. Concerning the fire behavior, as assessed in vertical flame spread tests, the use of anhydride curing agent prevented melt dripping phenomena and provided high char-forming character to the bio-based epoxy systems and their phenyl analog. In addition, forced combustion tests showed that the use of anhydride hardener instead of aliphatic polyamine results in a remarkable decrease of heat release rate. An overall decrease of the smoke parameters, which is highly desirable in a context of greater fire safety was observed in the case of BOMF/MNA system. The experimental results suggest that the effect of silica nanoparticles on fire behavior appears to be related to their dispersion degree. Thermosetting polymers have been widely used in many industrial applications as adhesives, coatings and laminated materials because of their peculiar physical, chemical, electrical and adhesive properties ,2. At prThe chemistry of sol-gel may represent a way to design high performing epoxy/silica hybrid nanocomposites. It is well known that it allows silica nanoparticles as small as a few nanometers in diameter be obtained ,10,11 anRecently, very few environmentally-friendly and additive-free bio-epoxy resins have been developed ,18,19; t2,5-bis[(oxyran-2-ylmethoxy)methyl]furan (BOMF) is one of the most promising furan-based diepoxy monomers for producing sugar-based furan polymers ,21. In aIn this context, using a sol-gel hybrid strategy for the synthesis of silica/BOMF nanocomposites cured with anhydrides may allow green materials to be obtained with satisfactory mechanical and flame retardant properties. In contrast to the phenyl analogs ,13,15, og).Herein, we exploit an in-situ sol–gel silica synthesis procedure for preparing bio-based epoxy/silica nanocomposites, using 2,5-Bis[(oxiran-2-ylmethoxy)methyl]furan (BOMF) diepoxy monomer, cured with methyl nadic anhydride (MNA), in the presence of 2-methyl imidazole (2-MI) as initiator. For comparison, a second set of nanocomposites was prepared, replacing BOMF with a commercial diglycidyl ether of bisphenol A (DGEBA). The morphology, as well as the thermal and flame retardant properties of the hybrid phenyl-based and furan-based silica/epoxy systems were thoroughly investigated by means of infrared spectroscopy (FTIR), transmission electron microscopy (TEM), thermo-gravimetric analysis (TGA) and differential scanning calorimetry (DSC). In addition, UL94 vertical flame spread and forced-combustion  tests were exploited to study the flame retardant behavior of the synthesized systems. The experimental results were compared with those collected from analysis of a traditional phenyl analogue  hybrid resin system cured with aliphatic polyamine (PA). The in-situ approach allowed a very fine distribution of silica nanoparticles (at nanometric level) to be obtained within the epoxy networks, independently from the type of epoxy monomer employed. The hybrid furan-based silica/epoxy systems showed chemical, physical and fire properties comparable to hybrid phenyl-based counterparts, even though these latter showed a remarkably higher glass transition temperature , (3-aminopropyl)-triethoxysilane  and ethanol  were purchased from Sigma-Aldrich . A commercial epoxy monomer (component A of the resin system SX10) supplied by MATES S.r.l. , consisting of a modified bisphenol A diglycidyl ether and a bio-derived furanic diglycidyl ether, namely 2,5-bis[(oxyran-2-ylmethoxy)methyl]furan , were uEither a fully petroleum-derived or a partially bio-based epoxy system were obtained to respectively cure the commercial epoxy monomer (DGEBA) and the bio-derived one (BOMF), withmethyl nadic anhydride (MNA). For both the epoxides the same protocol was followed: the epoxy monomer was mixed with MNA in order to obtain a homogeneous mixture, and then, the proper amount of initiator was added . Once the initiator was well dispersed, the epoxy mixture was poured into a silicon mold, cured for 1 h at 130 °C and post-cured for 1 h at 180 °C in an oven.A different procedure was adopted for the preparation of epoxy/silica hybrid nanocomposites. More specifically, the coupling agent, 3-Aminopropyl)triethoxysilane (APTS), and the silica precursor, tetraethyl orthosilicate (TEOS), were added to BOMF or DGEBA bis-epoxy monomers, hence promoting an “in situ” sol–gel synthesis prior to the addition of 2-MI and MNA. TEOS loading was kept constant for samples prepared in different batches. The chemical structures of the products used for the production of the epoxy systems and the corresponding hybrids are reported in TEOS/epoxy and TEOS/APTS weight ratios were set at 0.086 and 1.88, respectively. The in-situ formation of the silicate phase required a temperature of 80 °C and reflux conditions. The synthesis procedure was modified and inspired by methodologies reported in the literature ,22.The synthesis was performed in one pot involving the following three steps.1. Mixtures of epoxy (DGEBA) or bio-based epoxy (BOMF) and APTS with epoxy/APTS weight ratio of 23.3 were stirred vigorously at 80 °C for 2 h to get silanized resins.2. Tetraethoxysilane (TEOS), distilled water and ethanol (EtOH) were added to the silanized resins and stirred vigorously at 80 °C under reflux for 90 min. Subsequently, the reaction vessels were opened and kept at 80 °C for 30 min in order to remove ethanol and water.T = 130 °C for t = 1 h; post-cure at T = 180 °C for t = 1 h.3. The amounts of 2-MI and MNA needed for the curing were then added to the mixtures at room temperature. The resulting mixtures were degassed under vacuum and poured into a silicon mold. For each formulation the thermoset networks were obtained by applying the following thermal treatment: cure at The silica content estimated from the stoichiometry was 2 wt. %. The typical reaction batches are presented in −1 with a resolution of 4 cm−1 and 32 scans. A PerkinElmer Spectrum One FTIR spectrophotometer  equipped with a universal ATR sampling accessory and Spectrum Software  was used to perform FT-IR analysis in ATR mode. Spectra were acquired in the range of 4000–600 cmThermogravimetric analysis (TGA) was performed by means of a TA Instrument TGA 500  and Universal Analysis 2000 Software , heating each sample from 25 to 750 °C at 10 °C/min, under nitrogen and air with a flow of 60 mL/min.−1 nitrogen flux. The glass transition temperature (Tg) was calculated as the midpoint between onset and endpoint of the specific heat variation.Differential scanning calorimetry (DSC) was carried out by means of a TA Instrument DSC Q2000  and Universal Analysis 2000 Software , applying the following protocol: the sample was equilibrated at −60 °C, than heated up to 200 °C at 10 °C/min, cooled down to −60 °C at the same rate, kept at that temperature for 1 min and heated up again to 250 °C. All the analyses were performed under a 30 mL∙minTransmission electron microscopy (TEM) was performed on a FEI TECNAI G12 Spirit-Twin operating at 120 kV  and LaB6 source. The microscope was equipped with a FEI Eagle 4K CCD camera and NanoImaging Services Software .3. UL94 flame spread tests were performed according to the D3801−10 standard; the size of the specimens was 120 × 10 × 2 mm3), with a heat flux of 35 kW/m2, in horizontal configuration. Time to ignition , total heat release , peak of the heat release rate  and heat release rate  were measured. Total smoke release , carbon monoxide yield , carbon dioxide yield  and specific extinction area  were evaluated as well. For each sample, the experiments were repeated at least three times in order to ensure reproducible and significant data.Cone calorimetry tests  were performed according to the ISO 5660 standard, using squared samples  are also reported.In nitrogen, any epoxy system degrades according to a two-step mechanism ,27. The T5 for BOMF/MNA_2Si with respect to the other systems. However, the degradation of both BOMF/MNA and BOMF/MNA_2Si samples occurs with higher pyrolysis rates with respect to DGEBA-based systems, as confirmed by the TG and DTG curves and T10 and T50 values. It is also worth noting the very high residues at 700 °C (about 24%), obtained for all the systems but DGEBA/MNA .It is worthy of note that the presence of silica nanoparticles does not significantly modify the degradation mechanism of DGEBA/MNA and BOMF/MNA, which substantially follows the typical decomposition path described above, notwithstanding a slight decrease of Tg) values. The presence of the in situ-formed silica increases the Tg of the DGEBA/MNA system. Conversely, the hybrid strategy seems to negatively influence the BOMF-based system, for which a decrease of Tg was recorded in the corresponding hybrid. These results suggest that strong interactions occur at the silica/matrix interface in the case of the DGEBA/MNA system, for which silica clustering, as assessed using TEM analyses, is negligible. At variance, the formation of aggregates in the BOMF/MNA system lowers the interactions between the two phases in the hybrid system, hence lowering the Tg.The investigation of the overall fire behavior of a flame retarded system is usually carried out by performing either flame spread or forced combustion  tests.The flammability of all the studied systems was tested through UL94 vertical spread test. All the samples  resulted as being not classifiable according to the UL94 vertical spread test, because it was not possible to prevent the flame propagation along the sample, as already observed for DGEBA cured with polyamines (PAs) . HoweverIt is worth pointing out that, in vertical flame spread tests, the DGEBA/MNA and BOMF/MNA systems do not exhibit dripping phenomena when exposed to a Bunsen flame, even in the absence of silica . ConversAs far as forced-combustion tests are considered, Interestingly, a further strong reduction (−40%) of HRR was recorded as the effect of the addition of silica to the DGEBA/MNA system; this value is very close to that recorded (38%) for the DGEBA/PA system containing silica. When comparing HRR of DGEBA/MNA containing 2 wt. % of silica with the corresponding system cured with PA, HRR is lowered by 55% as the result of effects provided by MNA and silica. Conversely, no remarkable decrease of HRR was observed upon addition of silica to BOMF/MNA. This finding is in agreement with results  obtainedAs reported in the literature ,15,37,382 ratio values  appear unchanged with respect to the phenyl analog. However, MNA and silica nanoparticles mainly act in the condensed phase activity, as revealed by CO/COo values , which ao values  39]. . 2 ratio2 yields, compared to DGEBA/PA, DGEBA/PA_2Si, DGEBA/MNA and DGEBA/MNA_2Si. It is well known that soot is one of the major decomposition products during the degradation of an epoxy resin, because of its tendency to produce char and smoke. It is reported in the literature that soot includes species with high molecular weight formed in the gas-phase combustion process and residual pyrolyzed fuel particles furan (BOMF) and the obtained epoxy/silica hybrid nanocomposite was thoroughly investigated and compared to the hybrid system prepared using diglycidyl ether of bisphenol A (DGEBA). Methyl nadic anhydride (MNA) was used as curing agent, in the presence of 2-methyl imidazole (2-MI), in order to use a green approach as much as possible for the synthesis processes. Both systems were compared with the hybrid one prepared using diglycidyl ether of bisphenol A (DGEBA) cured with apolyamine.Morphological study of the furan-based epoxy/silica nanocomposite showed that promoting the formation of silica through the sol-gel route (step 2) in the presence of a pre-formed hybrid epoxy/APTS adduct , in different systems, allowed nanocomposites to be obtained with a very fine distribution of silica nanoparticles (size of a few nanometers). Some tendency of the particles to aggregate into clusters or bigger particles was observed in the case of the BOMF/MNA system only.The thermal stability of both epoxy systems was not significantly affected by the presence of silica nanoparticles, although the BOMF-based samples (both unfilled and hybrid) showed higher pyrolysis rates and residues with respect to DGEBA-based systems. In agreement with the morphological study, an increase of the glass transition temperature was observed for DGEBA-based epoxy/silica nanocomposites, for which a homogenous and very fine distribution of silica was observed. As concerns the fire behavior, BOMF and DGEBA monomers cured with anhydride did not suffer melt dripping phenomena and showed high char-forming character and thus a remarkable decrease  of the heat release rate compared to the DGEBA-based systems cured with an aliphatic polyamine. In addition, the bio-epoxy resin and its hybrid system showed an overall decrease of the smoke parameters compared to their DGEBA-based counterparts.These results demonstrate that BOMF-based resins cured with an anhydride may represent a suitable green alternative to DGEBA-based systems in the synthesis of epoxy/silica hybrid nanocomposites, as the furan-based epoxy/silica nanocomposite showed morphological characteristics and thermal properties similar to the “bisphenol A”-based counterpart. In addition, the combined use of BOMF monomer with anhydride as curing agent was able to lower smoke production during forced-combustion tests. Finally, the experimental results suggest that the dispersion degree of silica within the designed epoxy networks plays a key role in determining their overall final properties.


Biological pathways play an important role in the occurrence, development and recovery of complex diseases, such as cancers, which are multifactorial complex diseases that are generally caused by mutation of multiple genes or dysregulation of pathways.We propose a path-specific effect statistic (PSE) to detect the differential specific paths under two conditions . In observational studies, the path-specific effect can be obtained by separately calculating the average causal effect of each directed edge through adjusting for the parent nodes of nodes in the specific path and multiplying them under each condition. Theoretical proofs and a series of simulations are conducted to validate the path-specific effect statistic. Applications are also performed to evaluate its practical performances. A series of simulation studies show that the Type I error rates of PSE with Permutation tests are more stable at the nominal level 0.05 and can accurately detect the differential specific paths when comparing with other methods. Specifically, the power reveals an increasing trends with the enlargement of path-specific effects and its effect differences under two conditions. Besides, the power of PSE is robust to the variation of parent or child node of the nodes on specific paths. Application to real data of Glioblastoma Multiforme (GBM), we successfully identified 14 positive specific pathways in mTOR pathway contributing to survival time of patients with GBM. All codes for automatic searching specific paths linking two continuous variables and adjusting set as well as PSE statistic can be found in supplementary materials. The proposed PSE statistic can accurately detect the differential specific pathways contributing to complex disease and thus potentially provides new insights and ways to unlock the black box of disease mechanisms. Biological pathways play a key role in the occurrence, development and recovery of complex diseases, such as cancers, which are multifactorial complex diseases that are generally caused by mutation of multiple genes or dysregulation of pathways . BesidesRecently, some pathway analytical methods have been proposed to study human physiology, systems biology and modern drug development that provided the computational framework for data pathway analysis and biomarker selection –17. ThesIn order to reduce the computational burden, we proposed a series of simplification process for the topology structure of complex networks. Of note, the nodes on specific path are only influenced by their parent nodes according to Markov Independence property. After simplification, the path-specific effect statistic PSE is estimated under two conditions to detect the differential specific paths. Therefore, the statistic PSE combined the causal effect calculation under causal inference framework with the network comparison in systems epidemiology designs. To assess the performances of the statistic PSE, theoretical proofs and statistical simulations are conducted to evaluate the stability of type I error and power, and a real gene expression data in Mammalian Target Of Rapamycin (mTOR) pathway on survival time of glioblastoma multiforme (GBM) patients are further analyzed to validate the practicability of PSE statistic.Gliomas are the most common type of primary brain tumor, and are histologically differentiated as astrocytomas, oligodendrogliomas, and ependymomas. The World Health Organization (WHO) classifies central nervous system tumors into four different grades: I, II, III and IV. Grade IV glioblastoma multiforme (GBM) is the most frequent, devastating, and malignant astrocytic glioma. It is characterized by a high degree of cellularity, vascular proliferation, tumor cell chemoresistance, and necrosis. Even after neurosurgical resection, followed by aggressive chemotherapy and radiotherapy, GBM is still considered an incurable malignancy. No effective treatment agent against GBM has been identified –24.The proposed PSE statistic was applied to analyze gene expression data in Mammalian target of rapamycin (mTOR) signal pathway Fig.  of 461 wα = 0.05) when sample size reached 1000 for total causal effects. While Table Tables δ = 1 (Case group vs. Control group). Table Table δ of path-specific effects in case and control group, the powers of total causal effect and path-specific effect obviously elevated. Furthermore, we performed sensitivity analysis to observe whether the PSE statistic would be influenced by the parent nodes or child nodes of nodes on specific path. Tables Besides, Tables 1 → X2 → Y linking X1 and Y under two conditions for the fixed effect difference. The PEM with Bootstrap tests detected some false positive specific pathways , a key mediator of phosphatidyl-inositol-3-kinase (PI3K) signaling, has emerged as a compelling molecular target in glioblastoma patients, although clinical efforts to target mTOR have not been successful  ⊥ C. We haveFrom Eq. , for binX1 → X2 → ⋯ → XK → Y with continuous confounders C, for any In specific path C, we haveFor a discrete variable X1 → X2 → Y via adjusting for the binary parent nodes C1 and C2 isIn observational studies, according to back-door criteria and do-calculus , for theX1 → Y the standardized path-specific effect in the exposure or case group was defined asThe proposed path-specific effect statistic (PSE) was based on product of average causal effect (ACE) of each directed edge, and took difference under two conditions . In order to identify the specific path While for non-exposure or control group, the standardized path-specific effect wasand the test statistic PSE wasThe proposed PSE statistic was developed to test the difference of path-specific effects under two conditions.H0 is true; 3) obtain the value of statistic actually observed in study and locate the value in permutation distribution to get the P value; 4) reject the null hypothesis (H0 : PSE1 = PSE0) if the P value is less than 0.05 [H0 : PSE1 = PSE0) if the CI does not include zero [The Standard Norm Bootstrap Confidence Interval:To test whether the specific path contributed to the disease end point, we conducted a series of hypothesis tests. The permutation-based hypothesis tests were conducted as follows: 1) draw a large number of data on disease status  without replacement and estimate PSE in each group, and make difference between two groups and then forms our statistic PSE; 2) Repeat this process to form a permutation distribution under the condition han 0.05 . While bude zero . Three b2)The Basic Bootstrap Confidence Interval:3)The Percentile Bootstrap Confidence Interval:4)Bias correct confidence interval:PSE1 and PSE0  and effects of parent nodes or child nodes on nodes on specific path as well as average causal effect value of every edge on specific path. We simulated a complex network on Myocardial Infarction and selected the target specific path: Calorific excess → Visceral adiposit → Inflammatory z ` milieu →  Atherosclerosis → Myocardial infarction  depends on corresponding parent nodes X1  and X2, logit) = α0 + β1X1 + β2X2. Furthermore, we set different effects under two conditions on some specific paths and then identify the specific paths with different effects using PSE statistic.Simulations were conducted to evaluate the performances of PSE statistic in the situation of varying across path-specific effect difference of ion Fig.  to test PSE1 = PSE0), given the varied sample sizes , 1000 simulations were conducted to assess the type I error of the PSE by Permutation test and the non-parametric bootstrap tests with confidence interval (CI) estimated by Basic bootstrap, the percentile bootstrap and bias-corrected bootstrap methods and asymptotic normal distribution statistic CI. Under H1 (PSE1 ≠ PSE0). Given the sample sizes 1000, 1000 simulations were repeated to assess the power under varied path-specific effect difference (Case group vs. Control group) of specific path itself and their parent nodes or child nodes as well as average causal effect value of every edge on specific path, respectively.Under the null hypothesis 1 → X2 → X3 → Y by separately adjusting for the parent nodes X1 and X2:The specific path effect X(3)1 → X3 → Y by separately adjusting for the parent nodes X2 and X3:The specific path effect XBased on the pathway effects in two groups, we can develop PSE statistic via differenting the pathway effects in two groups.http://cran.r-project.org/).All simulation codes were generated by R software available from CRAN (Additional file 1. Codes for automatic calculating PSE statistic of all specific paths linking any two continuous variables.


Delayed auditory feedback (DAF) leads to nonfluent speech where the voice of a speaker is heard after a delay. Previous studies suggested the involvement of attention to auditory feedback in speech disfluency. To date, there are no studies that have revealed the relationship between attention and nonfluent speech by controlling the attention allocated to the delayed own voice. This study examined these issues under three conditions: a single task where the subject was asked to read aloud under DAF (single DAF task), a dual task where the subject was asked to read aloud while reacting to a pure tone (auditory DAF task), and a dual task where the subject was asked to read aloud while reacting to the vibration of their finger (tactile DAF task). The subjects also performed the single and dual tasks (auditory/tactile) under nonaltered auditory feedback where no delayed voices were involved. Results showed that the nonfluency rate under the auditory DAF task was significantly greater than that under the single DAF task. In contrast, the nonfluency rate under the tactile DAF task was significantly lower compared with that of the single DAF task. Speech became nonfluent when attention was captured by the same modality stimulus, i.e., auditory tone. In contrast, speech became fluent when attention was allocated to the stimulus that is irreverent to auditory modality, i.e., tactile vibration. This indicates that nonfluent speech under DAF is involved in attention capture owing to the delayed own voice. It is difficult to adjust speech in environments where such auditory feedback is disrupted. In a loud environment, the sound pressure and pitch of the speech increase to compensate for the disruption of auditory feedback , where the voice of speaker is heard with a delay on the order of a few tens of milliseconds to a few hundred milliseconds, nonfluent speech could occur in the form of word revisions , syllable repetitions , or prolongations . This disfluency effect has been used in previous studies to investigate the relationship between speech and auditory feedback . We set a dual task paradigm in which subjects were asked to read aloud under DAF (this is a single task for control) or read aloud under DAF while reacting to auditory stimulation  or tactile stimulation . We hypothesized that speech would become nonfluent when attention was allocated to the own voice under DAF , while nonfluent speech would become attenuated when attention was allocated to sensory feedback rather than to the own voice .There are also individual differences regarding the DAF effect . We calculated the sample size required for the present experiment . All subjects obtained written informed consent in accordance with the Declaration of Helsinki. We excerpted 18 sentences from a workbook for the Japanese Language Proficiency Test [NPO Research Institute for Japanese Language Education ]. The avIn the experiment, the subjects were asked to read aloud the sentences that appeared on the monitor placed 60 cm in front of their eyes. The subject’s voice was captured during the task using a microphone (SHURE SM58) placed within 15 cm of his/her mouth, and this was provided as feedback through closed headphones (SONY MDR-7506) worn by the speaker over both ears through a mixer (MACIE 802VLZ4) and effector (LEXICON MX300). The headphones were used to prevent the subject’s voice from coming outside the headphones and overlap with the subject’s voice recorded by the microphone and played on the headphones. In addition, the subject’s voice was recorded on an integrated circuit (IC) recorder (OLYMPUS Voice Trek V-863) with a microphone also placed within 15 cm of his/her mouth.During the single task , the auditory feedback of the subject’s voice was given under NAF or DAF . Under NThe delay time was set to 200 ms in the present study because the frequency of nonfluent speech was high in this delay time compared to other delay times  ⇒ tactile DAF condition ⇒ tactile NAF condition ⇒ tactile DAF condition ⇒ tactile NAF condition ⇒ tactile DAF condition ⇒ single NAF condition ⇒ single DAF condition …]. The sentences, the order of stimulus modality (single/tactile/auditory), and the order of feedback condition (NAF → DAF or DAF → NAF) were counterbalanced among the subjects. A fixation point “+” was presented every 5 s before and after each trial, and the subjects were asked to gaze at the center of the point when presented.We calculated the reaction time that elapsed between the presentation of stimulation and button press (within 600 ms) and the omission error rate for each subject. For the reaction time and omission error rate, we performed a 2 (NAF and DAF)-by-2 (tactile and auditory) intrasubject two-way analysis of variance (ANOVA) with Bonferonni correction. As we have analyzed the data twice (when the sample size was 22 and 36), an alpha level of 0.025 was set for the analysis in order to maintain an experiment-wise error rate of 5%.In addition, a professional teacher calculated the nonfluency rate of each subject under each condition by calculating the proportion of nonfluent speech . Phrase is a unit that contains one independent word. Based on the work by Sakai et al. , we diviThe reaction time and omission error rate under the tactile and auditory conditions are listed in F = 42.2, p < 0.005, F = 43.12, p < 0.005, F = 0.55, p = 0.82, The mean of the reaction time in the tactile condition was 260.9 ± 58.7 ms under the NAF condition and 311.6 ± 85.3 ms under the DAF condition. The mean reaction time in the auditory condition was 312.0 ± 69.5 ms under the NAF condition and 365.6 ± 92.0 ms under the DAF condition. There was a significant main effect of the modality  = 30.3, p < 0.005, F = 2.5, p = 0.13, F = 0.3, p = 0.61, The mean of the omission error rate in the tactile condition was 6.3 ± 5.7% under the NAF condition and 15.1 ± 12.2% under the DAF condition. The mean of the omission error rate in the auditory condition was 8.7 ± 7.5% under the NAF condition and 16.7 ± 12.1% under the DAF condition. There were significant main effects of the feedback  = 83.56, p < 0.005, F = 38.84, p < 0.005, F = 44.56, p < 0.005, F = 0.63, p = 0.53, t(35) = 3.56, p < 0.005, d = 0.54). However, it was significantly higher under the auditory condition than under the single condition (t(35) = 7.46, p < 0.005, d = 0.75). The nonfluency rate was significantly higher under the auditory condition than under the tactile condition (t(35) = 9.24, p < 0.005, d = 1.30). In addition, there was a significant difference between NAF and DAF conditions for the single task and dual tasks with tactile and auditory stimulation  = 35.48, p < 0.005, F = 98.02, p < 0.005, F = 53.07, p < 0.005, The mean nonfluency rate in the single task was 2.1 ± 2.9% under the NAF condition and 9.6 ± 6.7% under the DAF condition. The mean nonfluency rate in the tactile condition was 2.3 ± 2.2% under the NAF condition and 6.5 ± 4.7% under the DAF condition. The mean nonfluency rate in the auditory condition was 2.6 ± 3.4% under the NAF condition and 15.4 ± 8.4% under the DAF condition. There were significant main effects of the feedback . In contrast, there was no correlation between the DAF effect and the increased/decreased fluency rate (auditory DAF condition–single DAF condition) under the auditory condition (r = 0.05).There was a significant positive correlation between the DAF effect and the increased/decreased fluency rate (tactile DAF condition–single DAF condition) under the tactile condition . DAF (task-irrelevant stimulus) deviates the subjects from their internal prediction signal generated in parallel with the movements of the subject’s speech, and it is considered to be a highly salient stimulus. In particular, auditory stimuli are reported to be more likely to capture attention than visual stimuli  and the tactile-modality target . Attention allocation to auditory stimuli decreased when attention was directed to sequences of tactile stimulus  tasks.The present study showed that attention capture by delayed own voice could result in nonfluent speech. However, it remains unclear if attention capture by delayed own voice is involved in matching or the subsequent movement correction process. A study that used event-related potential (ERP) showed that this matching and movement correction are different processes (Rietdijk et al., The datasets generated and/or analyzed for this study are not publicly available due to specifications on data availability within the ethics approval. Data are, however, available from the corresponding author upon reasonable request and with the permission of the ethics committee.The studies involving human participants were reviewed and approved by Tsukuba faculty of human sciences, University of Tsukuba, Japan. The patients/participants provided their written informed consent to participate in this study.All authors contributed to the conception or design of the work or the acquisition, analysis, or interpretation of data for the work. OI and DI contributed to the writing of the manuscript. SM contributed to manuscript revision and read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As oral squamous cell carcinoma (OSCC) can develop from potentially malignant disorders (PMDs), it is critical to develop methods for early detection to improve the prognosis of patients. Epithelial–mesenchymal transition (EMT) plays an important role during tumor progression and metastasis. The Wnt signaling pathway is an intercellular pathway in animals that also plays a fundamental role in cell proliferation and regeneration, and in the function of many cell or tissue types. Specific components of master regulators such as epithelial cadherin (E-cadherin), Vimentin, adenomatous polyposis coli (APC), Snail, and neural cadherin (N-cadherin), which are known to control the EMT process, have also been implicated in the Wnt cascade. Here, we review recent findings on the Wnt signaling pathway and the expression mechanism. These regulators are known to play roles in EMT and tumor progression, especially in OSCC. Characterizing the mechanisms through which both EMT and the Wnt pathway play a role in these cellular pathways could increase our understanding of the tumor genesis process and may allow for the development of improved therapeutics for OSCC. Oral cancer is the sixth most common cancer worldwide, and oral squamous cell carcinoma (OSCC) is the predominant type . DespiteThe majority of OSCC lesions develop from PMDs ,8. The WAlthough OED and CIS are defined based on the presence of dysplastic cells in the epithelium, obtaining accurate clinical and histopathological diagnoses remains challenging ,13. TherMolecular or histological markers that allow for a stricter differentiation between the diagnosis of healthy tissue and PMDs are required because the histopathological diagnosis of OSCC has been established in routine paraffin-embedded specimens ,15.2+-dependent transmembrane glycoprotein E-cadherin, which is involved in cell–cell adhesion and rearrangement of epithelial cells [In recent studies, various biomarkers have been used to identify PMDs, including the kinase inhibitor of cyclin-dependence p16INK4a protein (p16) , and theal cells , is decral cells . Recent al cells .Specific carcinogenesis-related biomarkers are simultaneously involved both in the EMT and Wnt signal pathway, their synergistic action leading to PMDs, and finally OSCC . TherefoWnt signaling is known to control diverse biological processes and functions. This signaling can be subdivided into two types: β-catenin-dependent signaling or canonical Wnt signaling. Another type of signaling is β-catenin-independent signaling, known as non-canonical Wnt signaling. Taking cancer stem cells (CSCs) as an example, the canonical Wnt signaling cascade was shown to be involved in the self-renewal of stem cells as well as the proliferation and differentiation of progenitor cells ,26. HoweIn the canonical pathway, the Wnt protein subtype binds to receptors on the cell membrane and triggers the β-catenin cluster in the cell nucleus, subsequently activating the T-cell/lymphoid-enhancing-factor transcription factors (TCF/LEF). Through this pathway, cell proliferation, apoptosis, and transformation are regulated . In the non-canonical pathway, various receptors or co-receptors on the cell surface and extracellular matrix are involved in the pathway . For nonIn total, 19 Wnt genes have been identified that encode secreted cysteine-rich proteins and play a role in initiating Wnt signaling . Wnt1, WThe canonical/non-canonical Wnt pathway is also regulated by several endogenous secreted Wnt inhibitors, including two types: one type binds with Wnt ligands to inhibit both canonical and non-canonical Wnt signaling, including secreted Frizzled-related protein (SFRP) family and Wnt inhibitory factor 1 (WIF1); the other type is a Wnt inhibitor that interacts with LRP5/LRP6 corepressors to block canonical Wnt signaling, e.g., the Dickkopf (Dkk) family . In addiFor physical interactions between cells, a family of adhesion molecules, the cadherins, are particularly important for dynamic regulation of adhesive contacts that are associated with diverse morphogenetic processes . During cis) cadherin dimer formation [Classic cadherin forms a core protein complex known as the cadherin–catenin protein complex. In the extracellular domain, the cadherin consists of five cadherin-type repeats bound together by calcium ions to form a parallel  protein family, which includes three top non-muscle cell cytoskeletal proteins. Vimentin is ubiquitously expressed in normal mesenchymal cells and is known to maintain cellular integrity and provide resistance against stress. Increased Vimentin expression has been reported in various epithelial cancers . Using ccis-elements and associated factors have been identified within the human Vimentin promoter, suggesting that the Vimentin gene is subjected to complex control [Vimentin is encoded by a single-copy gene located on chromosome 10p13. Recently, several  control . One of  control . In vitr control  supportsAs discussed above, β-catenin plays an important role in the canonical Wnt pathway. Upon activation of the Wnt signaling pathway, β-catenin is disassociated from the E-cadherin complex and translocated to the cytoplasm and nucleus. This suggests that the Wnt pathway triggers the transition of β-catenin from juxta-membrane to the core, subsequently activating the TCF/LEF transcription factors that upregulate the Vimentin promoter. Qiao B et al.  used miRSome recent studies have reported that SFRP1 either promotes or suppresses Wnt/β-catenin signaling based on the cellular context, concentration, and the expression pattern of Fzd receptors ,72. HoweAdenomatous polyposis coli (APC) is a tumor suppressor gene; it was cloned from 5q21-22 as a causal gene in familial adenomatous polyposis . This geSnail was first identified in Drosophila, and its homologs have subsequently been found in many other species, including humans . It is aA recent study found that a wide range of signaling pathways induce Snail expression, including TGF-β ,99,100, Yang Y et al.  found thZhou BP et al.  found thGSK-3β is considered an essential component of Wnt cascade regulation. In an unstimulated cell, cytoplasmic β-catenin is maintained at a low level via degradation after binding to the destruction complex, which is composed of scaffolding proteins Axin, conductin, GSK-3β, and APC. In normal cytoplasm, β-catenin is targeted for ubiquitination and degradation in the 26 S proteasome by paired phosphorylation through the serine/threonine kinases CK1 and GSK-3β bound to a scaffolding complex of Axin and APC. A multiprotein complex containing the scaffold protein plays a role in the phosphorylation of β-catenin, leading to formation of a β-catenin homodimer or heterodimer with the related protein. During this process, β-catenin is phosphorylated by CK1 at Ser 45; this in turn primes GSK-3β to phosphorylate serine/threonine residues Ser 33, Ser 37, and Thr 41. Phosphorylation of the last two residues triggers ubiquitylation of β-catenin by β-TrCP and degradation in proteasomes . HoweverTherefore, the upregulation of Snail protein expression is accompanied by the accumulation of β-catenin in the nucleus, reduction of E-cadherin, and increased expression of Vimentin, which is suggestive of EMT . In OSCCNeural cadherin (N-cadherin) is typically expressed in tissues derived from the mesoderm and neuroectoderm, which differs from E-cadherin that originates from normal squamous epithelium. This cadherin is widely expressed in the nervous system and is associated with small adherens-type junctions at synapses ,115,116.In head and neck SCC, some studies have found that N-cadherin expression is limited in OSCC and is not correlated with the downregulation of E-cadherin expression . DI DomeSome studies have observed that the cytoplasm of β-catenin is positively associated with N-cadherin ,125. MorAccording to Hulin-Curtis S et al. , cultureAt the stage of PMDs, tumor cells invade the surrounding stromal tissues and disseminate from the solid tumor mass. As epithelial tumor cells are typically tightly associated with their neighboring cells via E-cadherin-containing adhesion junctions, tumor cells must break these extracellular junctions before they can move out as a group or single cell and invade stromal tissues . To gainEMT may be induced by the secretion of specific factors including vascular endothelial growth factor (VEGF), interleukin (IL), matrix metalloprotease (MMP), tumor necrosis factor α (TNFα), and TWIST ,136,137.At this time, EMT has been well-studied using in vitro cell line models; however, direct observations of EMT in vivo or in clinical specimens are limited. Previous reports have demonstrated an association of EMT with invasiveness in different cancer types ,141 but OSCC is the sixth most common cancer worldwide. Advanced OSCC, with a high lymph node metastasis rate, leads to an unfavorable prognosis and high relapse rate. Although numerous studies have suggested that certain biomarkers can be used for predicting the prognosis of cancer. Almangush A et al. has reported that most studies have focused on only a few well-studied biomarkers, such as p53, Ki67, and p16 . There h