In recent years, progress in nanotechnology provided new tools to treat cancer more effectively. Advances in biomaterials tailored for drug delivery have the potential to overcome the limited selectivity and side effects frequently associated with traditional therapeutic agents. While autophagy is pivotal in determining cell fate and adaptation to different challenges, and despite the fact that it is frequently dysregulated in cancer, antitumor therapeutic strategies leveraging on or targeting this process are scarce. This is due to many reasons, including the very contextual effects of autophagy in cancer, low bioavailability and non-targeted delivery of existing autophagy modulatory compounds. Conjugating the versatile characteristics of nanoparticles with autophagy modulators may render these drugs safer and more effective for cancer treatment. Here, we review current standing questions on the biology of autophagy in tumor progression, and precursory studies and the state-of-the-art in harnessing nanomaterials science to enhance the specificity and therapeutic potential of autophagy modulators. Autophagy encompasses a number of self-catalytic mechanisms which enable the elimination of macromolecules and organelles, protecting from any potential toxicity upon damage and making available their constituent building blocks for anabolism and energy obtention –3. Its pAutophagy plays several roles in tumor biology which depend on different context features. Sustained autophagy at later disease stages can promote cancer cell survival by ensuring the removal of damaged organelles and macromolecules, and the fulfilling of the high metabolic demands of proliferating tumor cells exposed to stressful conditions, such as nutrient deprivation, oxidative stress, hypoxia, or exposure to anti-cancer agents. However, induction of autophagy can also promote cell death, likely through excessive degradation of cellular constituents and organelles , 3. ThusDespite the antitumor therapeutic potential of autophagy modulators, their use in a clinical setting has been limited so far. Currently available autophagy modulators show poor bioavailability because of low solubility in aqueous media and non-targeted delivery, leading to modest therapeutic efficacy and favoring undesired effects. Novel approaches are thus warranted to harness the potential of these modulators.Nanotechnology is revolutionizing biomedicine and pharmaceutical industry; this new field is generally referred to as nanomedicine. Several nanoparticles (NPs) have been proposed as drug carriers and imaging tools . Nanoparintrinsic ability of altering the complex network of signaling pathways and molecules involved in autophagy regulation, and thus represent an exciting therapeutic approach against cancer disease. [Besides acting as nano-carriers, nanoparticles of specific materials may have the disease. .In this review, we aim to provide an updated picture of the different pharmacological approaches designed so far to treat cancer, focusing on existing clinical trials where different autophagy modulators are carried by NPs, including siRNA cellular nanodelivery and modulation of autophagy by magnetic hyperthermia.Macroautophagy—commonly simply referred to as autophagy—is a degradative process by which intracellular macromolecules and organelles are engulfed by autophagic vesicles, delivered to lysosomes, and broken down to their molecular building blocks. Autophagy is a crucial process to ensure cell survival in the face of different sources of stress and damage, such as nutrient starvation . Upon inAutophagy regulation is intimately linked to energy status and nutrient sensing. AMP-activated protein kinase (AMPK) is a central regulator of autophagy. AMPK-dependent phosphorylation inhibits mTORC1 through TSC2 and Raptor in response to cellular energy deficit, and activates ULK1, which is a required step to trigger the autophagic machinery . In enerMechanistic target of rapamycin (mTOR) participates of two distinct functional complexes, mTORC1 and mTORC2 . mTORC1 The observation that mTORC1 pharmacological inhibition is sufficient to induce autophagy even in the presence of nutrients, highlights the role of mTORC1 complex as a powerful repressor of autophagy . GeneticmTORC2 is largely insensitive to rapamycin , and incAutophagy has been involved in physiological processes including normal development, and in a variety of human diseases, including cancer, neurodegenerative diseases and muscular disorders . Growth Constitutive autophagy may have a protective role in tumor cells by removing damaged organelles or recycling misfolded macromolecules . MoreoveHowever, a large body of evidence also suggests that induction of autophagy in tumor cells can promote cell death—cell death-type II—through enhanced degradation of cellular components and organelles. Autophagy has thus been considered tumor-suppressive in several experimental systems. In fact, tumor cells frequently exhibit driving features, such as increased ROS production and induction of genetic mutations, that imply an attenuation of excessive autophagy . ImpaireAutophagy is often associated with tumor survival and chemoresistance , 2, 22. The only compounds intervening autophagy currently approved by FDA for clinical use are the antimalarial drug chloroquine (CQ) and its derivative, hydroxychloroquine (HCQ) . HCQ is Of note, CQ activity is strongly diminished in conditions of acidic pH; this may explain the limited effect exhibited by CQ in vivo, since the tumor microenvironment is frequently acidic due to enhanced glycolytic rates and tissue damage .HCQ enhanced the activity of erlotinib, an EGFR inihibitor, in a phase I study conducted in advanced non-small-cell lung cancer patients . In renaHowever, exacerbated autophagy induction upon cytotoxic drug treatment or by direct autophagy induction, can also lead to tumor cell death. For example, temozolomide, an alkylating agent with a pro-autophagy activity, displays synergistic induction of apoptosis in glioblastoma with dasatinib, a tyrosine kinase inhibitor . HistoneThe relationship between cancer metabolism and autophagy is being evaluated in a clinical trial NCT01206530) combining HCQ with chemotherapy in patients with advanced colorectal cancer . Another0 combiniThe growing number of ongoing trials reflects the relevance that autophagy modulation can have in combinatorial treatments to overcome the existing resistance in some cancer therapies. Recent studies have highlighted that autophagy, besides leading to severe metabolic changes and chemoresistance, also plays an immunomodulatory role and may be exploited to enhance tumor immunotherapy .Several hurdles hamper the use of currently available drugs intervening autophagy in clinical settings. Autophagy modulators often exhibit low bioavailability due to both low solubility in aqueous media and non-targeted delivery, as well as reduced activity in an acidic microenvironment . Novel sRecently, nanotechnology-based therapeutic solutions have been developed, generally referred to as nanomedicine . SeveralNanomedicine may favor the preferential accumulation of systemically administered chemotherapeutics at tumor sites via the enhanced permeability and retention effect (EPR). In this case, the leaky vascular system and reduced lymphatic drainage characteristic of the tumor microenvironment increases the specificity of the therapeutic system in in vivo settings .Extensive research focusing on cancer nanomedicine has generated nanostructures capable of overcoming biological barriers and transport chemotherapeutic agents to targeted sites while minimizing harmful effects on healthy tissues  MoreoverOverall, nanomaterials have been explored as potent modulators of autophagy through several mechanisms, and are being actively studied as new therapeutic tools against cancer . A list Treatment with cisplatin and chloroquine in micelles formed by self-assembling hybrid dendritic-linear-dendritic block copolymers (HDLDBCs) increased cytotoxicity in tumor cells, while maintaining a limited cytotoxic activity in non-transformed cells . Lys-05,n vivo [In addition to their cargo activity, some nanoparticles have the intrinsic ability of regulating various stages of autophagy, thus constituting an additional pharmacological tool in tumor therapy . Metal-bn vivo . D- and n vivo .3O4 NPs (HTCC–MNPs) increase autophagy through the co-localization of LC3 with the lysosomes, inducing cytotoxicity in gastric carcinoma cells rather than not tumoral cell line [Novel chitosan chloride (HTCC)/alginate-encapsulated Feell line .Albumin-based nanoparticles, such as albumin-stabilized gold nanoclusters (ABN), show interesting features in biological environments—including in vivo settings—such as low toxicity and remarkable stability. These nanomedicines have been recently exploited for autophagy induction by delivering AZD8055 (ABN-AZD), a powerful mTOR kinase inhibitor, for the treatment of uveal melanoma . NotablyHence, many nanostructures may acquire exceptional medical and toxicological relevance due to their inherent chemical activity within the cellular environment, or by delivering drugs and bioactive compounds active in autophagosome formation or in the related signaling pathways. Hence, autophagy targeting with innovative tools, as nanostructure-based strategies, is acquiring clinical importance as a synergist approach for cancer therapy.As mentioned above, PI3K/AKT/mTOR pathway inhibitors  are well-known autophagy inductors –79 and t2 weekly, for 4 weeks) did not show any toxicity and strongly inhibited mTOR targets S6K and 4EBP1 with initial proofs of response and disease stabilization in patients with unresectable and metastatic solid malignancies [Nanoparticles of albumin–bound rapamycin  make unnecessary the use of toxic solvents due to their ability to bind hydrophobic drugs to albumin. In preclinical studies these nanocomplexes were non-toxic and very efficient in different cancer types since they moderated cell viability and hamper mTOR downstream signaling in several xenograft tumor models, including multiple myeloma and breast tumor –84. Furtgnancies , nivolumgnancies , FOLFOX,gnancies , temozolgnancies  in patieAnother phase I/II study NCT02009332) is taking place to determine the efficacy and dose limiting toxicities (DLT) of ABI-009 nanoparticles combined with gemcitabine as innovative therapy for non-muscle invasive bladder cancer (NMIBC) patients . Intrave32 is tak18F) fludeoxyglucose (FDG) activity which was linked to increased treatment response or stable disease [2 did not display any toxicity, and the pharmacokinetics of rapamycin showed a coherent relation between dose and plasma concentration, with no significant molecular interplay between rapamycin and nab-paclitaxel detected [Interestingly, the combination of ABI-007 with rapamycin has been investigated in several phase Ib clinical studies  –98. The  disease . The 40 detected .The discovery and characterization of non-coding RNA species in the last decades have revolutioned our understanding of genome regulation, and several strategies have aim at leveraging on such molecules for cancer therapy . Among tThese molecules display unique proprieties such as good affinity for the target, no immunogenicity and ease of chemical modification, that render them excellent therapeutic systems, particularly to overcome challenges presented by traditional drugs . Their mAutophagy-related miRNAs represent an essential control mechanism on top of all other autophagy-regulatory pathways that were characterized so far. Recently, we have witnessed a drastic increase in the number of studies dissecting miRNA-autophagy relationships . InteresInnovative strategies for cancer therapy based on nucleic acids for autophagy modulation have been evaluated in different in vitro and in vivo models as well as have been proposed in clinical studies –118. How2O3) nanoparticles (SPIONs) have been linked to anti-HER2 antibody and autophagy inhibitor miRNA-376B. These modified nanostructures efficiently delivered the bioactive microRNA into HER2-positive breast tumor cell lines and in a xenograft nude mice model of breast cancer, and lead to autophagy inhibition [Superparamagnetic iron oxide  were designed to carry siRNA targeting survivin, an autophagy regulator gene that has been observed to promote the accumulation and stabilization of IKKα in the nucleus as well as interact with the pro-autophagy functional complex ATG12-ATG5 , 122. ThIn summary, many types of nanomaterials may be modified with specific nucleic acids and drugs to act as nanocarriers to interfere with the complex autophagy process or the related signaling. Therefore, these nanoformulations could be employed for the inhibition of protective autophagy or for the activation of autophagic cell death in combination to chemotherapy injuries and thus leading to a tumor suppressor phenotype which, enhances therapeutic efficacy. Given the potential of gene therapy in modulating autophagy, many clinical studies  have been performed to evaluate the clinical significance of modified nanocarriers in delivering therapeutic nucleic acids able to modulate autophagy process in targeted tissues –130. A lThe high potential of encapsulated miRNAs for clinical use is supported by of MRX34, a lipid nanoparticle loaded with miR-34 mimics, the first microRNA-associated therapeutic molecule tested in a clinical trial . Indeed,Kasinski et al. showed the therapeutic efficacy of delivered miRNA by using the co-encapsulated miR-34a and let-7b in NSCLC mice resistant to conventional anticancer therapy. Results showed that dual treated animals had lower tumor burden and higher survival . These dLiver cirrhosis or fibrosis, as the endpoint of chronic hepatic damage, is a strong indicator of high risk for the development of hepatocellular carcinoma (HCC), the main source of cancer-related deaths . AutophaHeat shock protein 47 (HSP47) is a molecular chaperone required for collagen folding and maturation. Moreover, HSP47 plays an important role in collagen accumulation in fibrotic areas and disorders associated with desmoplasia . IntriguA promising preclinical study has developed vitamin A–coupled liposomes able to deliver siRNA targeting HSP47 in hepatic stellate (HS) cells, which have key roles in liver homeostasis and vitamin A uptake and storage . In thisAmong different available nanocarriers, metallic nanoparticles have high importance due to their inherent reactivity and physicochemical properties, which can be leveraged on for therapeutic purposes. Therapeutic strategies based on hyperthermia  consist ®therapy (trade name), the first MNP based therapy for prostate and brain tumors in the world, has been recently evaluated in a clinical setting (NCT02033447 (prostate) [® Therapy with an open label, randomized and controlled trial aimed to determine its efficacy and safety alone or in combination with radiotherapy versus radiotherapy alone. In 2019, the Phase 1 clinical study on the focal ablation of intermediate-risk prostate cancer was completed by MagForce USA, Inc. Hence, to synergistically combine MNPs-MH with other therapeutic approaches, namely chemotherapy, radiotherapy, immunotherapy or photothermal/PDT, will allow a further enhancement of the efficacy against tumors. Although researchers have made a remarkable progress, several challenges stand, as detailed in [Furthermore, various works highlight magnetic hyperthermia as a promising adjuvant strategy to radiation and chemotherapy against cancer . Alternarostate)  and DRKSrostate)  for MNPsailed in .Interestingly, hyperthermia has been reported to trigger macro-autophagy across many experimental conditions and in most cases, the induction of this pathway increased cell survival and reduced programmed cell death . MechaniWhile nanoparticles have a tremendous potential for antitumoral therapy and extensive research is still been undertaken to assess the viability of their use in the clinic, concerns have raised in this field since despite big efforts along decades, results obtained in terms of technology/knowledge transfer have been so far limited , 147. ToAlthough the EPR effect has been confirmed by many studies in nanoparticles, it is still difficult to evaluate its real advantage with respect to free drugs in vivo. The problem resides in the fact that most approved anticancer nanomedicines were analyzed by comparing standard care with the combination of nanomedicine and standard care, instead of comparison with free drugs. Since the free drug may be ineffective for cancer patients, clinical trials using a free drug as a control may not be possible due to ethical concerns . MoreoveRegarding the second effect considered, surface modification of nanoparticles  limiting uptake by the RES—most prominent in liver and spleen parenchyma—, decreases drug clearance, promotes sustained availability of drug-loaded nanoparticles in the blood stream, and reduces tissue-specific toxicity. This may be relevant to reduce dose requirements and intrinsic toxicity issues, as exemplified by liposome formulation Doxil, with reduced accumulation at the myocardium and cardiotoxicity, as compared to free drug ./in vivo models to the clinic may be due to the fact that established cell lines conventionally used to validate nanoformulations may do not reflect the heterogeneity existing among human individuals. Even during in vivo assays, the use of subcutaneous injection of tumor cells and xenografts may also provide artifactual results with respect to spontaneous tumor model or metastasis studies [A further criticism on the lack of translation from in vitro studies .Overall, it should be considered that different anticancer drugs have distinct physicochemical, pharmacokinetic, and pharmacodynamic properties, which determine their unique clinical efficacy and safety profiles in human cancer patients. Moreover, due to tissue specificity of many tumors, different nanodelivery platforms should be designed for different drugs focusing the application on tumor specificities. Indeed drug, specificities  as well as tumor features , should be taken into account.Cancer is still an unstoppable challenge worldwide, so novel diagnostic and treatment strategies are needed. Among different approaches explored by scientists, nanomedicine emerges as a novel alternative, based on its virtually endless variety of nanomaterials potentially suitable for cancer therapeutics. Thus, different scientific disciplines, such as engineering, chemistry, physics, nanotechnology, materials science or medicine, are integrated to achieve precision systems, which also leverage on existing compounds. However, even though standardization, stability and reproducibility are required for this goal, tailored features are mandatory for the successful application of personalized medicine.Advanced nanoparticles have been revealed as potential smart drug delivery systems to improve the therapeutic effect of current standard drugs and increase patient survival rates. Undoubtedly, there is still a long journey from the nanocarriers design to translation to the pharmaceutical market as viable products. Although thousands of research articles describe great outcomes of drug delivery systems with different nature and properties in multiple in vitro and in vivo cancer models, only a small fraction has successfully reached trials for their use in the clinic. This limited clinical translation of new nanoparticles is mainly due to incomplete therapeutic efficacy and off-target toxicity in vital organs. Nonetheless, results and evidence from previous clinical trials should guide not only the optimization of nanocarrier formulations, but also setting clinical studies considering tumor heterogeneity through the identification of stratified populations, instead of unbiased cancer patient cohorts.


Background: A critical unmet medical need in clinical management of colorectal cancer (CRC) pivots around lack of noninvasive and or minimally invasive techniques for early diagnosis and prognostic prediction of clinical outcomes. Because DNA methylation can capture the regulatory landscape of tumors and can be measured in body fluids, it provides unparalleled opportunities for the discovery of early diagnostic and prognostics markers predictive of clinical outcomes. Here we investigated use of DNA methylation for the discovery of potential clinically actionable diagnostic and prognostic markers for predicting survival in CRC. Methods: We analyzed DNA methylation patterns between tumor and control samples to discover signatures of CpG sites and genes associated with CRC and predictive of survival. We conducted functional analysis to identify molecular networks and signaling pathways driving clinical outcomes. Results: We discovered a signature of aberrantly methylated genes associated with CRC and a signature of thirteen (13) CpG sites predictive of survival. We discovered molecular networks and signaling pathways enriched for CpG sites likely to drive clinical outcomes. Conclusions: The investigation revealed that CpG sites can predict survival in CRC and that DNA methylation can capture the regulatory state of tumors through aberrantly methylated molecular networks and signaling pathways. Despite remarkable progress in screening and patient management, colorectal cancer (CRC) remains a major public health problem . AccordiThere is ample evidence from the published reports that lifestyle plays a significant role in the development and progression of CRC ,5. At thA number of screening tests are available for CRC, including colonoscopy; fecal immunochemical test; fecal occult blood test; sigmoidoscopy; multi-target stool DNA (mt-sDNA) test; and computed tomography colonography (CTC) ,5. Each Advances in microarray and sequencing technologies have enabled comprehensive characterization of CRC genomes and transcriptomes ,9. GlobaGenes may play a strong role in the pathogenesis of CRC, but epidemiologic studies suggest that CRC risk is largely determined by gene and environmental interactions mediated by epigenetic factors ,16. ReceHowever, to date, there has been little information about the ability of DNA methylation or its derivatives, the CpG sites, to predict clinical outcomes such as survival in CRC. This investigation was conducted to comprehensively characterize methylation signatures associated with CRC and to determine whether CpG sites associated with CRC can predict survival. A second, but equally important objective was to discover aberrantly methylated molecular networks and signaling pathways associated with clinical outcomes. Our working hypothesis was that CpG sites associated with CRC can predict survival. We further hypothesized that CpG sites associated with CRC and predictive of survival map to functionally related aberrantly methylated genes interacting in gene regulatory networks and signaling pathways associated with clinical outcomes. We addressed these hypotheses using publicly available DNA methylation data from TCGA generated from tumor samples derived from individuals diagnosed with CRC and matched control samples. For the purposes of this investigation, clinical outcome was defined as either deceased (dead) or alive as reported in TCGA.The rationale and scientific premise of this investigation was that novel biomarkers such as DNA methylation that could allow and or aid early detection and prediction of clinical outcomes using noninvasive and or minimally invasive methods are of major importance to reducing the burden of CRC. We reasoned that because aberrant DNA methylation is an early event in CRC development, detectable in circulating cell-free DNA and body fluids, and its stable alterations can be easily and rapidly quantified by methylation-specific PCR methods, it provides unprecedented opportunities for the discovery of molecular markers that could aid with early detection of the disease and stratifying patients to guide therapeutic decision making in clinical management of CRC.We used publicly available DNA methylation data on CRC from TCGA, which has been well catalogued, annotated and linked with clinical information . The metn = 37 tumors and n = 37 matched controls) with long-term follow up, which we used in downstream analysis to identify a signature of differentially methylated CpG sites associated with CRC and a signature of CpG sites predictive of survival. The use of only 37 individuals with both tumor and adjacent non-tumor control tissue samples was necessitated by the understanding that DNA methylation can be population-specific. Thus, drawing comparison samples from the same patient group can effectively eliminate confounding factors due to patient differences.DNA methylation (array-based) data, and clinical information were obtained from TCGA . The datWe processed and checked the data for quality using the R package MINFI ,29. Poorp-value by comparing methylation profiles between tumor and control samples using the MINFI method [p-value for each CpG site in each sample, which represented the quality of the signal. The approach calculates the detection p-value by comparing the total signal (M + U) of each CpG site to the background signal level estimated from the negative control probe thereby adjusting for background noise [p-values indicate reliable signals, while larger p-values indicate poorer quality signals. We computed the differences between each sample type and averaged them across samples to determine whether there were overall significant differences in mean methylation levels for each CpG site. We performed this analysis on a matrix of β-values, obtaining a moderate t-statistic and associated p-value for each CpG site. Because the analysis involved performing a large number of hypothesis tests, we employed the false discovery rate (FDR) procedure [p-values corrected for multiple hypothesis testing. The discovered CpG sites were annotated for gene names, positions, DNA methylated regions (DMRS) and ranked on adjusted p-values. We used a Volcano plot to select CpG sites based on differential methylation value calculated as mean (βtumor) − mean , combined with adjusted p values. We performed unsupervised analysis using hierarchical clustering to group aberrantly methylated genes and samples together based on the similarity of their patterns of methylation profiles and the direction of change defined as hypermethylated if up- or hypomethylated if down-regulated. This approach was used to identify genes that were co-regulated and associated with CRC. Due to the small sample sizes, we did not stratify samples by age or ethnicity. However, a quantitative assessment of CpG sites was conducted and correlated with clinical information, specifically dead or alive and tumor stage.Subsequently, using normalized data, we computed the detection I method ,29,30. Wnd noise ,29. Underocedure  to compuHaving identified the CpG sites associated with CRC, we conducted analysis to discover a prognostic signature and to determine whether the CpG sites associated with CRC could predict survival. To address this need, we compared the patterns of DNA methylation between patients who survived and patients who died from CRC using information on CpG sites associated with the disease. Here, we sought to discover novel prognostic markers correlated with different survival indicators for individualized prognosis prediction for CRC patients. Differential methylation patterns between dead versus alive patient groups was achieved by computing the median of beta values for the CpG sites associated with CRC. We categorized patients into high-methylated (up) or low-methylated (down) groups based on probe level DNA methylation. Survival analysis was conducted based on time to death data obtained from clinical information provided in TCGA. We performed survival analysis by comparing the median beta values of DNA methylation levels between the dead and alive groups, using information on differentially methylated CpG sites. Survival was computed as a probability score indicating whether the patient was dead or still alive at a particular time point. Using information on survival and time to death, for each time point, we calculated the number of deaths observed in each group, and the expected number if there were in fact no differences between the two groups.To correlate the information on differentially methylated CpG site level with the patient’s survival, we used the Kaplan–Meier estimator of the survival curve, a tool for visualizing the results of methylation level that involves a time to event analysis . To detep-values to determine the probability of correctly assigning aberrantly methylated genes to the correct networks, functional categories and disease type. Aberrantly methylated gene networks were ranked on Z-scores and predicted functional categories were ranked on p-values. For pathway prediction, we computed the p-values using Fisher’s exact t-test and the FDR to determine the probability of correctly assigning aberrantly methylated genes and associated CpG sites to signaling pathways. The pathways were ranked based on log p-values. We performed Gene Ontology (GO) analysis [To gain insights about the broader biological context in which the aberrantly methylated CpG sites and genes associated with CRC and predictive of survival operate, we performed network and pathway analysis using the Ingenuity Pathway Analysis (IPA) software . Our woranalysis  implemenDespite remarkable progress in patient management, CRC remains a major public health problem worldwide ,2,3,4,5.We performed dimension reduction of DNA methylation data using PCA analysis to identify aberrantly methylated genes and CpG sites separating CRC tumor samples from controls. Here we sought to investigate whether there is significant variation in patterns of DNA methylation profiles within and between tumor samples and matched controls, and whether employing PCA could capture the variations distinguishing tumor from control samples. The results of this investigation are presented in . As showOne of the primary objectives of this investigation was to discover and characterize a DNA methylation signature and regions associated with CRC. To address this issue, we tested the hypothesis that there are significant differences in patterns of DNA methylation profiles between tumor and control samples, by comparing the pattern of DNA methylation between the two sample groups as described in the methods section. Here we sought to identify a signature of CpG sites and genes associated with CRC, which could be used in downstream analysis to identify CpG sites and genes predictive of survival outcomes.p value < 0.05) methylated CpG sites located in DNA methylated regions (DMRs) distributed widely throughout the methylome and mapped in or near 16,733 known genes. Out of all the aberrantly methylated genes discovered, 3097 genes contained a single significant CpG site, whereas 13,636 genes contained two or more significant CpG sites, indicating that DNA methylation is highly prevalent in tumor genomes. When we increased the threshold to a p-value cutoff of 10−15, we discovered 779 highly significantly aberrantly methylated CpG sites mapped to 464 genes. Among the 779 most highly differentially methylated CPG sites discovered, 271 CpG sites mapped to 202 genes and were hypermethylated, whereas 508 CpG sites mapped to 262 genes and were hypomethylated. This suggests that hypomethylation may be more prevalent in tumors than hypermethylation, which is consistent with results from a previous report [Comparison of patterns of DNA methylation between tumor and matched controls samples produced 117,026 significantly differentially  or hypo (down). The genes were ranked based on the number of CpG sites significantly associated with CRC. The investigation revealed extensive CpG sites mapped to a wide range of DMRs associated with CRC. The results showing the 70 most highly significantly differently methylated genes containing both hypermethylated and hypomethylated regions in the tumor genome compared to the control group are shown in To determine the similarity and dissimilarity in patterns of DNA methylation profiles for the top CpG sites that were most highly significantly associated with CRC, we performed a two-way hierarchical clustering using beta values. Here we sought to discover clusters of CpG sites with similar patterns of DNA methylation in the tumor samples and controls by addressing the hypothesis that genes containing CpG sites associated with CRC are likely co-regulated and have similar patterns of DNA methylation in tumor and control samples. For this analysis, we used the 779 CpG sites and 446 genes that were most highly associated with CRC, as these were the most informative and could eliminate spurious patterns.The results of hierarchical clustering are presented in a heatmap in Another objective of this investigation was to determine whether CpG sites associated with CRC could predict survival. Thus, having discovered and characterized the CpG sites associated with CRC, we performed a survival analysis using the information on the most significant CpG sites. In this approach, patients were regrouped for different CpG sites using the beta value of each probe. Samples with beta values greater than the median value were considered the hypermethylated group, and samples with beta values below the median value were considered the hypomethylated group. Out of a total of 37 patients in the study, 36 (97.3%) patients had died at the last follow-up visit. For each probed CpG site, patients were clustered in different groups and survival was computed as the probability of that individual’s survival up to a particular time point as described in the methods section. We performed Kaplan–Meier analysis and a log-rank test for each probe to evaluate its association with overall survival in the set as described in the methods section.p-values. We discovered 13 CpG sites (p < 0.05) mapped to 13 genes , DCLK1, NKX2-2, KIAA1026, ADARB2, MAGI2, SMAD3, GUCY1B3, DOK6, EFS and PCSK2) that were predictive of survival  are shown in survival . Figure  and EFS , which hThe results showing Kaplan–Meier curves displaying the estimated survival probability for two different groups of patients with different methylated CpG sites are presented in A secondary but equally important objective was to discover aberrantly methylated gene regulatory networks and signaling pathways and their functional connectivity. Here we sought to gain insights about the broader biological context in which the CpG sites and aberrantly methylated genes associated with CRC and predictive of survival operate, and to discover the molecular networks and signaling pathways they control. To achieve this objective, we performed network and pathway analysis using IPA as described in the methods section. For this analysis we used the 464 genes containing the 779 CpG sites that were most highly significantly associated with CRC, which included the 13 genes containing the 13 CpG sites predictive of survival.Network analysis produced 25 aberrantly methylated molecular networks with Z-scores ranging from 11 to 47. The molecular networks were enriched for CpG sites mapped to functionally related aberrantly methylated genes with overlapping functions. The top seven networks with Z-scores ranging from 31 to 47 contained aberrantly methylated genes predicted to be involved in cancer, gastrointestinal disease, organismal injury and abnormalities, organismal development, tissue development, cellular development, cellular growth and proliferation, and metabolic disease. In addition, the analysis produced aberrantly methylated networks containing genes predicted to be involved in nucleic acid metabolism, small molecule biochemistry, cell signaling, cell-to-cell signaling and interaction, molecular transport, infectious diseases, cellular assembly and organization, cell morphology, inflammatory response, endocrine system disorders, amino acid metabolism, energy production, post-translational modification, cell death and survival, digestive system development and function, antimicrobial response, connective tissue development and function, cellular function and maintenance, and inflammatory response. A complete list of molecular networks enriched for CpG sites and functionally related aberrantly methylated genes they map to, as well as information on top diseases and molecular functions they are involved in, is contained in 2-adrenergic, endocannabinoid developing neuron, glutamate receptor, dermatan sulfate biosynthesis and axonal guidance signaling pathways. A complete list of all the significant pathways enriched for CpG sites is presented in Pathway analysis revealed 284 signaling pathways enriched for CpG sites mapped to functionally related aberrantly methylated genes associated with CRC. Among the top signaling pathways discovered include synaptogenesis, circadian rhythm, corticotropin releasing hormone, CREB, GI, synaptic long term depression, G-Protein coupled receptor, cAMP-mediated, endocannabinoid cancer inhibition, dopamine-DARPP32 feedback in cAMP, white adipose tissue browning, netrin signaling, dopamine receptor, endocannabinoid neuronal synapse, ephrin receptor, cardiac ÎOverall, the results from functional analysis revealed functionally related aberrantly methylated genes with overlapping functions, interacting in gene regulatory networks and signaling pathways. Most notably, the 13 genes containing 13 CpG sites associated with CRC and predictive of survival were found to interact with other genes containing CpG sites highly significantly associated with CRC, suggesting that genes containing CpG sites predictive of survival may be regulated by or may be regulating other genes. Thus, the investigation addresses the two objectives by (1) showing that CpG sites associated with CRC can predict survival, and (2) revealing aberrantly methylated molecular networks and signaling pathways associated with clinical outcomes.A critical unmet medical need in clinical management of CRC pivots around lack of noninvasive and or minimally invasive techniques for early detection of the disease and lack of molecular markers for stratification of patients to guide decisions in clinical management of the disease. To address this knowledge gap and critical unmet medical need, we analyzed the patterns of DNA methylation to discover signatures of CpG sites associated with CRC and that could predict survival, and network states and signaling pathways they control. The investigations revealed CpG sites mapped to extensive hypermethylated and hypomethylated DMRs associated with CRC. We discovered 13 CpG sites associated with CRC that accurately predicted survival outcomes. In addition, we discovered aberrantly methylated gene regulatory networks and signaling pathways associated with clinical outcomes. The results associating DNA methylation with CRC discovered in this report are consistent with the results from previous reports ,54. The NPBWR1, CDH12, NR5A2, DCLK1, NKX2-2, KIAA1026, ADARB2, MAGI2, SMAD3, GUCY1B3, DOK6, EFS and PCSK2 implicated in CRC [CDH12) gene has been shown to increase cancer cell migration, invasion and progression via promoting EMT by targeting the transcriptional factor Snail [CDH12 has also been shown to be a predictor of prognosis in CRC patients and as an oncogene in CRC cell proliferation [NR5A2 is linked to worse overall survival of CRC patients [DCLK1 gene is reported to be up-regulated and associated with metastasis and prognosis in CRC [The clinical significance of our investigation is that DNA methylation-based molecular markers have the promise for detection and clinical management of CRC. Although the DNA methylation biomarkers we discovered were not clinically validated, which could preclude their implementation in clinical practice, in silico validation revealed important aberrantly methylated genes including d in CRC ,53,54,55or Snail ,56. CDH1feration ,56. It hpatients . The DCLs in CRC . Cytoplas in CRC . The NKXs in CRC . Taken tNetwork analysis revealed molecular networks enriched for CpG sites mapped to aberrantly methylated functionally related genes predicted to be involved in cancer, gastrointestinal disease, cellular development, cellular growth and proliferation, inflammation, and metabolic disease, all of which are highly relevant to the pathogenesis, development and progression of CRC ,62,63,64The discovery of aberrantly methylated signaling pathways including the circadian rhythm, CREB, GI, G-Protein coupled receptor, cAMP-mediated, endocannabinoid cancer inhibition, white adipose tissue browning, dopamine receptor, ephrin receptor, glutamate receptor and axonal guidance signaling pathways implicated in CRC suggests that DNA methylation has promise for the discovery of potential therapeutic targets. Circadian clock genes discovered here are reported to be involved in cell cycle regulation, oncogenesis, tumor suppression, DNA repair, cell proliferation, cell death and cell growth . The cloOther pathways discovered in this investigation associated with CRC included the glutamate receptor, axonal guidance, p53 and VEGF signaling pathways implicated in CRC ,84,85,86Overall, the clinical significance of these findings is that, because DNA methylation can be measured in body fluids such as urine and blood, if confirmed in such tissues, the CpG sites and genes discovered here could serve as noninvasive or minimally invasive molecular diagnostic and prognostic markers. Although this investigation was based on tumor samples, a number of studies have reported DNA methylation-based biomarkers with high sensitivity and specificity for blood-based and urine-based detection of CRC ,88,89. MLimitations: Our investigation provides insights about the association of DNA methylation with CRC and demonstrates that CpG sites associated with CRC could predict survival outcomes. However, some limitations must be acknowledged. First, our discovery cohort consisted of patients with varying stages of CRC and some of them may have received postoperative radiotherapy with or without concurrent chemotherapy. In the absence of information on patient treatment, addressing that issue was beyond the scope of this investigation. However, although the identified CpG sites that were predictive of survival cannot be used for stratification of patients treated with intensive multimodal therapy, we believe that our findings may guide therapeutic decision making by providing clues to future de-escalation therapeutics. Secondly, our study used a limited sample size as opposed to all available samples in TCGA. This is because DNA methylation can be population-specific. While our discovered CpG sites and DMRs show very high associations of DNA methylation with CRC, these high associations may not be the norm as DNA methylation can be population- and tissue-specific.Our investigation shows that DNA methylation-based molecular markers are associated with CRC, and have the promise to predict survival outcomes and discovery of potential therapeutic targets. New DNA methylation-based biomarkers that could aid in early CRC detection and prognosis detected by noninvasive and or minimally invasive methods are of critical importance to reducing the growing burden of CRC. Aberrant DNA methylation is an early event in CRC development and may be detected in body fluids and therefore constitutes a valuable CRC biomarker. Importantly, because DNA methylation is a stable epigenetic alteration that can be easily and rapidly quantified by methylation-specific PCR methods, it provides unprecedented opportunities for clinical implementation. Further research is recommended using large and diverse population cohorts to ensure that promising DNA methylation biomarkers undergo large-scale validation and their performance is compared with current screening techniques prior to their implementation in clinical practice. Future research will also focus on the tumor immune microenvironment.


Osteoarthritis (OA) is one of the most common joint degenerative diseases in the world. At present, the management of OA depends on the lifestyle modification and joint replacement surgery, with the lifespan of prosthesis quite limited yet. Effective drug treatment of OA is essential. However, the current drugs, such as the non-steroidal anti-inflammatory drugs and acetaminophen, as well as glucosamine, chondroitin sulfate, hyaluronic acid, are accompanied by obvious side effects, with the therapeutic efficacy to be enhanced. Recently, novel reagents such as IL-1 antagonists and nerve growth factor inhibitors have entered clinical trials. Moreover, increasing evidence demonstrated that active ingredients of natural plants have great potential for treating OA. Meanwhile, the use of novel drug delivery strategies may overcome the shortcomings of conventional preparations and enhance the bioavailability of drugs, as well as decrease the side effects significantly. This review therefore summarizes the pathological mechanisms, management strategies, and research progress in the drug molecules including the newly identified active ingredient derived from medicinal plants for OA therapy, with the drug delivery technologies also summarized, with the expectation to provide the summary and outlook for developing the next generation of drugs and preparations for OA therapy. There are more than 400 million patients affected by osteoarthritis (OA) worldwide, with a global total prevalence of OA of 15%, causing significant economic burdens . OA is rOA is a whole joint disease that affects the hyaline articular cartilage, subchondral bone, ligaments, capsule, synovium and periarticular muscles . The intvia β2 adrenergic receptors in the central route  Cuss that is extensively used in Traditional Chinese Medicine clinical practice. Recent research has discovered that osthole possesses antioxidant, anticancer, anti-inflammatory, and immunomodulatory properties  in IL-1β-stimulated chondrocytes (Morroniside is a significant iridoid glycoside and one of drocytes . Morronidrocytes .Centella asiatica (L.) Urban] that has antioxidative and anti-inflammatory activities. Madecassoside has been demonstrated to successfully suppress IL-1β-induced overexpression of MMP-3, MMP-13, inducible nitric oxide synthase (iNOS), and COX-2, as well as type II collagen and sox9 degradation (Madecassoside is a triterpenoid component obtained from the Gotu kola plant [radation . MadecasTaxillus sutchuenensis (Lecomte) Danser’ total flavonoids. By stimulating the p110/AKT/mTOR signaling pathway, quercitrin can decrease MMP-13 production and enhance collagen II accumulation, promoting cell proliferation and delaying ECM breakdown . Cell proliferation, migration, and differentiation, as well as cartilage homeostasis and joint remodeling, are all influenced by the Wnt/β-Catenin pathway. Emodin has the potential to cure OA, according to the findings.modin is a natural anthraquinone isolated from the root and rhizome of acterial , anti-caacterial  and antiacterial  activitiThe active chemical found in medicinal plants holds a lot of promise for treating OA. However, further clinical trials are needed to validate their usefulness and safety.As a topical disease, innovative delivery options such as intra-articular injection or transdermal delivery have been extensively researched in addition to the traditional oral and systemic administration routes. Many biomaterials have been investigated for the design of novel drug delivery systems for the treatment of OA to improve the efficacy of the delivery vehicles.Oral delivery is still the most common approach in drug administration for OA . HoweverAs a localized disease, OA may be treated through intra-articular (IA) injections . Intra-ain vivo from numerous causes but also slow down their release. It reduces delivery durations and doses while avoiding the harmful side effects of systemic absorption, providing essential theoretical support for OA research and therapy. Triamcinolone acetonide (TAA), a traditional corticosteroid, relieves synovitis and discomfort, but only temporarily. Local release of TAA based on biomaterials may extend pain relief without the need for numerous injections  exhibited resistance to hyaluronidase digestion via the skin. Compared to the oral route or intra-articular injection, transdermal administration offers a number of benefits (via the transdermal method (OA is a localized disease that often occurs in the knee, hand and hip joints. Therefore, skin penetration-based topical administration may commonly meet the needs of OA treatment. Transdermal drug administration can allow the medication to enter circulation at a steady pace and continuously benefits . Transdebenefits . Howeverl method . To enhaNumerous chemical enhancers are capable of disrupting the highly ordered bilayer structures of intracellular lipids found in the stratum corneum by inserting amphiphilic molecules into these bilayers to disrupt molecular packing or by extracting lipids using solvents and surfactants to create nanometer-scale lipid packing defects . HoweverCavitational ultrasound that uses the bubbles to oscillate and collapse at the skin surface to generate localized shock waves and liquid microjets directed at the stratum corneum induces the disruption in the lipid structure of the stratum corneum and can increase skin permeability for many hours. Additionally, thermal effects from ultrasound have been suggested to impact sonophoretic skin contact favorably by increasing the drug diffusion and the skin permeability coefficients .Articular thermotherapy induces vasodilation and increases blood flow around the joints, thereby reducing pain and joint stiffness . Besidesvia convective flow through hollow microneedles. Skin penetration enhancement strategies are shown in Microneedles are tiny and cheap needles that can carry medications through the skin in the least invasive way . FurtherOA is a chronic joint disease characterized by degeneration of articular cartilage and hyperosteogeny around the joint. At present, the molecular mechanism of OA is still not clear. The process of modern science and medicine will help us to know better about the pathogenesis of OA and thus more therapeutic targets can be excavated in follow-up work . LifestyAnother challenge for pharmaceutical treatment in OA lies in the design of cartilage-targeting drug delivery systems. Oral administration frequently leads to fierce gastrointestinal reactions and low bioavailability because of the first-pass effect. Intra-articular injection is an invasive method to the joints. Therefore, various DDSs such as nanoparticles, liposomes and hydrogels based on transdermal/topical delivery systems have caused increasing attention to reducing the frequency of intra-articular injections, increasing patient compliance, and reducing the risks along with the conventional preparations.Furthermore, as a big challenge to deliver drugs into chondrocytes, we can use physical technologies such as ultrasound and iontophoresis to assist the process of drug delivery and build intelligent local drug delivery systems which help researchers to provide new strategies for the treatment of OA. Besides, new approaches that simultaneously deliver chemical drugs and biological drugs aiming to enhance drug efficacy and reduce the side effects might be also combined to develop the next generation therapeutic system for OA treatment. Because the combination of several different types of drugs may work synergistically, the advanced vehicles that can co-deliver multiple drugs or release them in a responsive manner are highly proposed. However, it presents several new challenges that must be dealt with and a thorough biological assessment is needed for determining the drug combination. Another important aspect is the determination of the best mass ratio of each agent within a co-deliver drugs system which needs a systematic study to investigate the effects of different drug ratios on biological efficacy .


There is urgent demand for biologically compatible vascular grafts for both adult and pediatric patients. The utility of conventional nonbiodegradable materials is limited because of their thrombogenicity and inability to grow, while autologous vascular grafts involve considerable disadvantages, including the invasive procedures required to obtain these healthy vessels from patients and insufficient availability in patients with systemic atherosclerosis. All of these issues could be overcome by tissue-engineered vascular grafts (TEVGs). A large body of evidence has recently emerged in support of TEVG technologies, introducing diverse cell sources  and novel fabrication methods . Before TEVG can be applied in a clinical setting, however, several aspects of the technology must be improved, such as the feasibility of obtaining cells, their biocompatibility and mechanical properties, and the time needed for fabrication, while the safety of supplemented materials, the patency and nonthrombogenicity of TEVGs, their growth potential, and the long-term influence of implanted TEVGs in the body must be assessed. Although recent advances in TEVG fabrication have yielded promising results, more research is needed to achieve the most feasible methods for generating optimal TEVGs. This article reviews multiple aspects of TEVG fabrication, including mechanical requirements, extracellular matrix components, cell sources, and tissue engineering approaches. The potential of periodic hydrostatic pressurization in the production of scaffold-free TEVGs with optimal elasticity and stiffness is also discussed. In the future, the integration of multiple technologies is expected to enable improved TEVG performance. Coronary artery disease, the leading cause of adult death in developed countries , is treaTissue engineering involves the combination of engineering technology and medical science toward generating functional biological constructs . In 1986Recent improvements in TEVG technologies have involved diverse cell sources  and novel fabrication methods . In this review, we provide an overview of recent advances in TEVG fabrication. We also propose a new manufacturing method in which supraphysiological periodic hydrostatic pressure promotes layered elastic fiber formation in TEVGs .One of the important goals of TEVG development is to ensure sufficient mechanical strength for implantation. The required strength depends on the implantation site, but, in general, arterial shunts require more robust structure than venous shunts do. Although there is a lack of consensus regarding target strength, it has been common to use the saphenous vein burst pressure value (approximately 1680 ± 307 mmHg) for arterial shunts because the saphenous vein is a major autologous source for coronary bypass procedures . An altehttps://www.iso.org/obp/ui/#iso:std:iso:7198:ed-2:v1:en). Commercially available vascular grafts have much lower compliance than native blood vessels do . Th. Th18]. We examined the effect of PHP at various magnitudes and frequencies on human umbilical artery SMCs , which cRat neonatal aortic SMCs were seeded on a cell culture disk to create the first cell layer. Twenty-four hours after seeding, cells were exposed to PHP (110-180 kPa (65-590 mmHg), 0.002 Hz) for 24 hours, and then cells for the next layer were seeded on top of the first layer . In addiin vitro [These studies have demonstrated the potential usefulness of supraphysiological hydrostatic pressurization in fabricating layered elastic fiber structures in cell-based TEVGs in vitro . Emerginin vitro , fibroblin vitro , and ECsin vitro ). Our noThe field of TEVG generation continues to develop as clinical trials are conducted and novel technologies  are reported. Several fabrication methods have been explored, yet the ideal method for TEVG production is yet to be achieved. One of the remaining issues is the lack of functional elastic fiber formation in TEVGs. Fortunately, our recent findings may further improve TEVG performance. Further integration of mechanics and biology should lead to novel findings and improved TEVG technologies, resulting in favorable outcomes for patients with cardiovascular diseases.


How to make a neuron, a synapse, and a neural circuit? Is there only one ‘design’ for a neural architecture with a universally shared genomic blueprint across species? The brief answer is “No.” Four early divergent lineages from the nerveless common ancestor of all animals independently evolved distinct neuroid-type integrative systems. One of these is a subset of neural nets in comb jellies with unique synapses; the second lineage is the well-known Cnidaria + Bilateria; the two others are non-synaptic neuroid systems in sponges and placozoans. By integrating scRNA-seq and microscopy data, we revise the definition of neurons as synaptically-coupled polarized and highly heterogenous secretory cells at the top of behavioral hierarchies with learning capabilities. This physiological (not phylogenetic) definition separates ‘true’ neurons from non-synaptically and gap junction-coupled integrative systems executing more stereotyped behaviors. Growing evidence supports the hypothesis of multiple origins of neurons and synapses. Thus, many non-bilaterian and bilaterian neuronal classes, circuits or systems are considered functional rather than genetic categories, composed of non-homologous cell types. In summary, little-explored examples of convergent neuronal evolution in representatives of early branching metazoans provide conceptually novel microanatomical and physiological architectures of behavioral controls in animals with prospects of neuro-engineering and synthetic biology. A phylogenetic definition of an organism’s biological system prevents us from considering whether that system has emerged in other organisms outside that definition.”- The origins and rise of neuronal complexity are among the rarest yet globally impactful life transitions, and these events likely occurred over 570–530 million years ago near the Cambrian boundary. Despite more than a century of comparative research, the mechanisms and pathways of nervous system evoluton among 30 + animal phyla are elusive . From brAll studied extant neural systems contain highly heterogeneous neuronal populations with multiple cell types, which is the hallmark of any neural organization. Every neuron in a given nervous system can be unique regarding its connectivity, functions, morphology, and gene expression patterns. But, the rules underlying the neuronal heterogeneity and the entire scope of the neuronal diversity across phyla remain unknown, calling for novel unbiassed NeuroSystematics and/or Periodic System of Neurons with predictive power .How different are neurons, and more importantly, why are they so different? The evolutionary hypothesis can be as follows. Neurons are different not only because they have different functions but also because neurons, as evolutionary units  as taxonomically diverse evolutionary groups with a wide-ranging spectrum of ecological adaptations and novelties in neural architecture . NeuroSystematic would be the foundation to unravel genomic bases controlling neuronal identity and neuronal circuit evolution with the predictive power of novel cell phenotypes—a hypothetical Periodic System of Neurons . PromisiAdmittedly, the cellular and molecular bases of behaviors in sponges, placozoans, and ctenophores are so remarkably distinct compared to the rest of the animals that it would be advantageous to explore the concept of 3 separate ‘designs’ for neuroid architectures  that evoTrichoplax feeding, hundreds of cells and cilia reversible change their behaviors, and some of these cells could also be chemosensory such as gland cells in the rim  larvae eventually leading to settlement and metamorphosis. Sensing environmental cues can be mediated by specialized epithelial secretory flask cells, which possess a cilium and F-actin-rich protrusion, secretory vesicles, and neurite-like processes  and across non-bilaterians is challenging. Researchers frequently use a location by in situ hybridization with various markers in comparative studies. However, most mRNAs are usually not transported to neuronal processes hiding specific cell morphology such as branched neurites, characteristics for many neurons , but not so often in evolutionary neuroscience. Function-based terminology does not always imply homologization and direct phylogenetic relationships among respiratory or circulatory systems, for example. In contrast, it opens to multiple convergent/independent/parallel evolution instances, including the origins and diversifications of nervous systems , Beroe, Dryodora and even unnamed species  epithelial neural net with neurons arranged in specific orthogonal fashion and their branches to tentacles; 2) compact neural-like cells in the aboral organ with the gravity sensor  Neurons are asymmetrical, highly polarized secretory cells, which persistently maintain one or multiple cellular processes (neurites) as well as distinct compartments specialized for directed information processing to other cell types and demonstrate experience-dependent plasticity and (2) Neurons can make polarized and specialized connections (synapses) but do not necessarily do so in all their neurites and nervous circuits, as documented in basal metazoans and various bilaterians. Hormonal-like volume transmission in some cells (or neurites) can support many integrative functions without a specialized synapse and synaptic cleft. This happens if targets are localized within a few micrometers from transmitter release points or if fast chemical transmission is not required .integrating numerous signals and information transmission functions. It was suggested that a neuron could express more genes, gene products, and especially ligand-gated receptors to support its integrative operations than other cell types ((3) Neurons are cells specialized for ll types . Many holl types , which oRegarding the definition of neurons, the combination of the following features of neurons should be considered. Still, they need to be carefully validated in a broad comparative survey that includes representatives of all basal metazoan lineages.with synapses, evolved for generation, integration, and directional propagation of electrochemical signals leading to the release of extracellular messengers—features that enable them to transmit information, primarily chemical in nature, beyond their immediate neighbors (at useful speed) and without affecting all intervening cells en route. Systemic decision-making, short- and long-term neuroplasticity as parts of learning and memory mechanisms are inherent components of any neural organization.Thus, we define neurons as a hierarchical ensemble of polarized heterogenous secretory cells We do not know if all extant neurons have plasticity properties, but the development of phenotypic plasticity in terms of strength of synaptic transmission or (hyper) excitability might be an important trait for the evolution of nervous systems . It woulFirst, we emphasize the definition of neurotransmitters as signaling chemical messengers not involved directly in nutrition and related metabolic pathways  does not ‘know’ what the signal value would be from the information standpoint  and communications to virtually all other systems within an organism. In other words, neurons extensively communicate via multiple receptors to ‘determine’ which signal to send or not to send to other cell types (elaborated integrative activity (thousands of rapid communications between neurons) is the second hallmark of nervous systems, which separates them from other tissues and organs. In contrast to computers, the elaborated integrative activity of nervous systems is primarily based on the elaborated heterogeneity of its units (neurons) and a broad spectrum of chemically different intercellular messengers .ll types . Thus, tprimordial transmitter diversity scenario explains why even simpler extant nervous systems always have multiple neurotransmitters (rather than one or a few for depolarization or hyperpolarization responses in postsynaptic cells), as discussed elsewhere  modification of the genome operation as the fundamentals of long-term memory . As a reThus, we think the presence of chemical synapses is the most crucial feature of any extant neural/neuronal system. This criterion can separate canonical neuronal systems from other integrative systems in animals and beyond. For practical reasons, the criterion of the presence of chemical synapses taxonomically restricts the term neurons as an animal-specific innovation only. It contrasts and prevents confusions with different terminologies, like arguments for the existence of neural systems in plants and other eukaryotes [extensively reviewed in ]. We do Again, on a practical note, the proposed incorporation of synapses in the definition of a neuron can also be a critical criterion that separates ‘true’ neurons/nervous systems from the so-called neuroid cells/neuroid systems in non-bilaterians . Even coAplysia , with about 10% of electrical synapses (mediated by innexins); the rest are chemical synapses.Both canonical gap junction proteins and recently discovered tunneling nanotubes mediate a long-range junctional communication to coordinate metabolic coupling and signaling in a broad diversity of cells and tissues . The elepan—all, throughout and nexus - connection, bond .Non-homologous classes of proteins (connexins and innexins/ = pannexins) make gap junctions  with theIn contrast to pannexins , connexiCaenorhabditis elegans. According to its neuronal connectome reconstructions, 302 neurons can make 8693 synapses, but only 890 (10.2%) are electrical and formed by innexins. The C. elegans genome encodes 25 innexins, and 20 might contribute to neuronal wiring  mediated by gap junctions; electric fields can also mediate inhibitory synaptic action as in the Mauthner cell network . HoweverAdmittedly, electrical and true chemical synapses co-evolved due to increased animal and behavioral complexities , with exBioenergetic studies indicated that the nervous system is very costly. As a result, a preferential selection of some groups of transmitters vs. others might occur . The adindependent innexin radiation correlate with the respective increases in tissue/organ differentiation and needs for physical/metabolic cell coupling unrelated to neuronal functions. The simpler bodyplans in placozoans and sponges are associated with the absence/loss of innexins and ‘needs’ of direct intercellular connections.Innexins/pannexins-based junctions are not found in colonial and unicellular eukaryotes; therefore, they are metazoan but not neuron-specific synapomorphy. Phylogenomics survey pointed out that pannexins evolved in the common ancestor of all metazoans  and inteetc. Thus, gap junctions are much more widespread across cell types and tissues  and broadly used to communicate between other cell types rather than neurons, including the integrative functions during development.Gap junctions, recruited in the evolution of early animals, address the dramatic increase in the number of cell types , by coupling similar cell populations to mediate more stereotyped functions: secretion, contractility, coordinating cilia beating, exchange macromolecules and mRNAs during embryogenesis, regeneration, contribution to mechanistic tissue biology, Pleurobrachia bachei and Mnemiopsis leidyi encode 12 innexins each (8). Analysis of ten ctenophore transcriptomes and two genomes showed that independent diversification of innexins occurred early in ctenophore evolution with several ctenophore-lineage-specific innovations . Still, they are not unique neurons or synapses and are often co-opted for multiple functions. Genes encoding most of these proteins are broadly expressed across other non-neuronal cells and tissues; therefore, they have limited use as specific neuronal or synaptic markers, particularly in the analysis of non-bilaterian systems. This notion suggests: different adhesive molecules  and chaperons could subsequentially or in parallel scaffold individual protein modules to form chemical synapses with a defined synaptic cleft and its components.Generalized extant neurons are cells that make chemical synapses among themselves and other cell types, with few exceptions. The first neural-like integrative systems were non-synaptic with volume transmission . ComparaPleurobrachia, Mnemiopsis, Bolinopsis, and kin  transmitter systems and neural circuits is also a new direction of comparative research across all non-bilaterian lineages. It is an exciting time to unite multiple disciplines in such an endeavor.In summary, little-explored examples of convergent neuronal evolution in early branching metazoans are essential to discover novel molecular and cellular toolkits controlling stereotyped and learned behaviors with prospects of neuro-engineering and synthetic biology. We also predict a greater diversity of neuroid and other systems in prebilaterian metazoans, including sponges and placozoans , than in(1) Neurons (and nervous systems) are functional but not a genetic category.(2) Any given neural system is not a single character; it includes different cell lineages with different genealogies and origins.(3) Both centralized and distributed nervous systems could be chimeric and composed of highly heterogenous cell populations, perhaps with unrelated origins.(4) There are no pan-neuronal/pan-synaptic genes, and synapses evolved more than once with different neurotransmitter systems and adhesion molecules forming the synaptic cleft.etc.) or components of relevant transmitter synthetic pathways. However, transmitter phenotype could be changed in development ((5) Neurons with different transmitters evolved independently from different secretory neuroid-like, digestive and/or immune-type cells that might already have these (or similar) transmitters  Novel signaling molecules and neurotransmitter systems should exist in non-bilaterians, and their diversity might exceed the diversity of neurotransmitter systems in bilaterians. In other words, neural systems in early-branching metazoans are molecularly/genetically more heterogenous than more derived bilaterian neural ensembles, which could secondarily lose the primordial diversity of molecular players in the LCAA or the urbilaterian.(7) Phylogeny of systemic memory is based on the early evolution of the synaptic organization; by characterizing neuroplasticity mechanisms in representatives of all four non-bilaterian lineages, it would be reconstructed. Some learning and memory mechanisms might be different in non-bilaterians expanding the scope of neuroplasticity.(8) Degrees of emerging properties across nervous systems and neuronal ensembles significantly differ in basal metazoans, especially in ctenophores vs. bilaterians.(9) Multiple alternative neuroid-like integrative systems are present in all early-branched metazoan lineages with distinct hierarchical organizations, contributing to these species’ learning and memory mechanisms.(10) The role of neuroid-like integrative systems in cognition and behavior of bilaterians may be under-appreciated. . .We agree with the recent statement: “to advance our knowledge of the nervous system, we should adopt a physiological definition . From thConsidering minimal information about neural systems in ctenophores and alternative, integrative systems in sponges and placozoans, many fundamental questions about neuronal identity, plasticity and neuronal homology remained controversial. Based on expression data in sponges , their nGiven these proposed generalized features of neurons, it is reasonable to address the question: what is the molecular/genomic foundation that lets a cell be or not be a neuron?The neural system evolved as a primary integrative system in organisms and considering the extreme diversity of microchemical microenvironments, the presence of multiple signal molecules, and numerous external signals; it would be reasonable to predict that most neurons are tuned to process multiple signals. Co-options of numerous (even redundant) signaling pathways might provide a versatile molecular playground for processing interneuronal communications and functions.Evidence from some vertebrate and molluscan neurons, where deep single-cell sequencing were performed indicated the presence of several dozens of receptors within the same cell. However, as correctly indicated by one of the reviewers, “simpler” mechanoreceptive sensory neurons or specialized olfactory cells can be specialized to process a single signal. But such neurons do not exist alone, and they transmit information to interneurons and other cell types, which integrate numerous signals.One of us earlier proposed that a complex and coordinated transcriptome/epigenomic response in a cell with co-expression of multiple genes (or even a majority of genes in a genome) at any given time is the primary requirement to be a neuron in the first place . It migh


Fibroblasts are incredible cells. Scattered throughout the body, filling any gap among organs and tissues, they were initially imagined as a monotonous and passive cell population that, at best, could produce the extracellular matrix necessary to cement the body components. Year after year, scientific research has accumulated a huge amount of evidence that points to fibroblasts as masters of physiological and pathological processes, while the definition of “fibroblast” has become more and more elusive, due to the lack of specific markers and the variety of different subpopulations.When trying to identify the original vocation of fibroblasts, we can affirm that they are the guardians of tissue homeostasis ,2. AlongDuring a famous bank robbery in Stockholm, the hostages developed positive attitudes towards their jailer, spontaneously protecting him with their bodies when he finally surrendered to police. This paradoxical reaction was named Stockholm syndrome, and fits the behavior of cancer-associated fibroblasts (CAFs) well. In 1986, Dvorak correctly defined cancer as “a wound that does not heal” , and fibA particularly upsetting feature of CAFs is their ability to protect cancer cells from therapy. This phenomenon is particularly relevant, but infrequently investigated.12,13,61 mutations (11 patients) were assessed for the differential analysis of data.While most mechanisms mediating CAFs protection of tumor cells have been described as acting locally, the systemic reaction of fibroblast populations to chemotherapy is rarely considered. An interesting exception is the study by Loupakis et al. , which mThe response of patients with wild-type KRAS mCRC was unique and particularly interesting: AREG incremented soon after therapy and never returned to the baseline levels in the following time-points. EGF showed intermittent spikes, with drops soon after therapy and peaks in the breaks between administrations. Overall survival and progression-free survival were negatively linked to an early (1 h) increase of AREG or a 15 d increase of EGF. The involvement of increased EGFR ligands could partly explain the resistance to Cetuximab therapy of some KRAS wild-type mCRC, while the source of these growth factors was not investigated. AREG is a well-known autocrine growth factor produced by tumor cells in CRC, while EGF is not. Moreover, the completely different kinetics of AREG and EGF after therapy indicates independent sources.A possible answer to this puzzle comes from the interesting study by C.M. Garvey et al. , which pBy in vitro testing of the cytostatic and cytotoxic effects of Cetuximab in the presence of various CAF:CRC cell ratios, the authors showed that a 30% CAF population was enough to prevent CRC epithelial cell crisis, with increased shielding effects at higher ratios. CAFs exerted a double effect, both preserving epithelial CRC cell proliferation and reducing cell death. The conditioned media of CAFs, treated or not with Cetuximab, were used on Cetuximab-treated CRC cells to verify the real influence of Cetuximab on the overall CAF protection. Indeed, Cetuximab treatment increased the basal protective activity of CAF supernatants, indicating an active effect of the drug on CAF secretome. The authors performed a cytokine-array analysis to identify the possible soluble mediators of this effect. Surprisingly, despite the different secretomes shown by three independent CAF primary cultures, the only increased cytokine was always EGF. These data were confirmed in ELISA and showed that two CRC cell lines and one normal colon-mucosa-derived fibroblasts did not upregulate EGF in response to Cetuximab in vitro. Curiously, the EGFR kinase inhibitor Erlotinib did not induce EGF in CAFs, suggesting that EGF release was not a direct response to EGFR pathway inactivity. EGFR triggering by Cetuximab could thus induce a specific CAF response. Could EGF alone be sufficient to reduce Cetuximab inhibitory effect on CRC cells? Tests of EGF on CRC epithelial cells and one 3D organoid culture, in the absence of CAF, showed a clear dose-dependent protection exerted by EGF against Cetuximab challenge, and the persistence of downstream Erk1-2 signaling. Thus, EGF can rescue CRC cells from Cetuximab inhibition. In line with these observations, Cetuximab-treated CAF-derived conditioned medium, neutralized by a specific anti-EGF antibody, was no longer able to increase the basic protection exerted by untreated CAF supernatants on CRC cells. This study suggests that CAFs, and possibly other fibroblast populations far from the tumor mass, could be the main mediators of a systemic resistance to Cetuximab by EGF upregulation.+ breast cancers, that a specific subset of CAFs can determine resistance to the anti-HER2 antibody Trastuzumab, lowering IL2 activity and causing immune exclusion [The immunomodulatory activity of CAFs could also act as a supplementary mechanism of Cetuximab resistance. Cetuximab can activate macrophages and natural killer cells, triggering the antibody-dependent cellular cytotoxicity of tumor cells . It has xclusion . A similRadiotherapy (RT), while used to attack the tumor, also inevitably damages its stroma . A typicCAFs are relevant mediators of tumor resistance to radio-chemotherapy. Accordingly, some early attempts at targeting them are being tested in clinical trials.In KRAS wild-type mCRC, under Cetuximab treatment, CAFs can be the most relevant source of increased EGF levels, reducing drug effectiveness. Cetuximab shows some activity against CRC metastases, where the tumor microenvironment has a different cellular composition, while the primary tumor is refractory to this treatment, helped by the powerful shielding of resident CAFs. While CAFs could be the principal therapeutic target to subvert Cetuximab resistance in mCRC with a wild-type EGFR pathway, the observation that the systemic EGF concentration can also be increased suggests a possible involvement of other fibroblast populations not confined to the tumor. This second mechanism could be particularly relevant when the primary tumor has been completely eradicated but CRC cells still persist in distant metastases.


Despite advances in surgery and chemotherapy, ovarian cancer remains one of the most lethal malignancies. Hence, the implementation of novel treatment approaches is required to improve the outcomes of the disease. Immunotherapy has been proven to be effective in many tumors and has already been incorporated into clinical practice. In this review, we describe key strategies in immunotherapy of ovarian cancer and summarize data from clinical studies assessing immunological prospects which could improve ovarian cancer treatment approaches in the future. The most notable current strategies include checkpoint blockade agents, the use of vaccines, adoptive cell transfer, as well as various combinations of these methods. While several of these options are promising, large controlled randomized studies are still needed to implement new immunotherapeutic options into clinical practice. Ovarian cancer is the most lethal malignancy of all gynecological cancers  is clinically used in combination with chemotherapy in adjuvant and recurrence treatment  signaling) may enhance or suppress the immune response. Immune checkpoints act as a negative feedback mechanism, modulating effector cell response to protect the host against autoimmunity and maintain self-tolerance. These pathways play an important role during tumorigenesis, as they are the main mechanism of tumor cell immune resistance. The best-known and widely used checkpoints include T-cell surface molecules, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1) , similarly to dendritic cells, migrate to the lymph nodes and present antigens to T cells, which in turn are responsible for attacking cancer via TCRs  protein on cancer cells causes attenuation of T-cell action. In patients with ovarian cancer, PD-L1 expression is associated with a worse prognosis and is correlated with the suppression of local immune response  or hot (inflamed). Hot tumors show higher T-cell infiltration and activity level than cold tumors, resulting in a better response to immunotherapy. Researchers are investigating whether combined therapies and stimulation of the tumor’s immune system can enhance the response to immunotherapy  adoptive transfer for advanced-stage disease. The results demonstrated a substantial duration of response compared to conventional chemotherapy  in combination with other therapeutic options, including checkpoint blockade  has been shown to be present in numerous cancer cell types, including epithelial ovarian cancer, which indicates it as a potential vaccine target (Odunsi et al. Implementation of dendritic cell-based vaccines is yet another approach to treatment, also being investigated in patients with ovarian cancer. Vaccination with the autologous dendritic cell-based vaccine with whole tumor lysate after systemic chemotherapy resulted in a decrease in progression rate, as well as improved overall survival in ovarian cancer (Tanyi et al. The assessment of the clinical use of vaccines in cancer patients has certain limitations (Friese et al. Therapeutic enhancement of immune response may lead to autoimmune disorders. Previous studies have already reported among the most common hypothyroidism, hyperthyroidism, skin rush, and colitis (Matulonis et al. Even though the immune system has a crucial role in the pathogenesis of ovarian cancer, the clinical application of immunotherapy has been limited to small pilot studies in ovarian cancer. The main directions for the development of therapeutic approaches in patients with ovarian cancer include the checkpoint blockade, vaccination-based approaches, as well as adoptive cell transfer Fig. . However


Printing technology and mounting technology enable the novel field of hybrid printed electronics. To establish a hybrid printed system, one challenge is that the applied mounting process meets the requirements of functional inks and substrates. One of the most common requirements is low process temperature. Many functional inks and substrates cannot withstand the high temperatures required by traditional mounting processes. In this work, a standardized interconnection and an automated bump-less flip-chip mounting process using a room temperature curing conductive adhesive are realised. With the proposed process, the conductive adhesive selected for the standardized interconnection can be dispensed uniformly, despite its increase of viscosity already during pot time. Electrical and mechanical performance of the interconnection are characterized by four terminal resistance measurement and shear test. The herein proposed automated process allows for fabrication of hybrid printed devices in larger batch sizes than manual assembly processes used beforehand and thus, more comprehensive evaluation of device parameters. This is successfully demonstrated in a first application, a novel hybrid printed security device. The room temperature mounting process eliminates any potentially damaging thermal influence on the performance of the printed circuits that might result from other assembly techniques like soldering. Printed electronics are an emerging technology that offers formerly unknown possibilities for product design by using non-standard materials as inks or substrates ,2,3,4. LPrinted electronics are fabricated by printing functional inks, frequently formulated from nanoparticles and solvents onto a substrate. In subsequent postprocessing steps , the solvents are removed and the functional structure formed. To date, conductive traces, numerous sensors, passive components, transistors and batteries can be printed ,16,17,18flexible . As manyflexible ,22,23.For both types of hybrid printed systems, the printed device has to be connected to the silicon-based part of the system at low process temperatures down to room temperature. To meet this requirement, a scalable automated room temperature mounting process is developed in this work. In order to select the appropriate bonding technique, state of the art mounting technologies for unpackaged electronic components are investigated. For silicon bare dies, wire-bonding is well established . PrintinDue to the high requirements on low processing temperature for the intended applications, a room-temperature bump-less flip-chip mounting process based on isotropic conductive adhesive is chosen to realise the electrical and mechanical interconnections.The first application is a novel security device, a physically unclonable function (PUF) . The PUFIn this section, the materials - conductive adhesive, substrates and the processes as well as related equipment for establishing the room temperature mounting process are outlined.Isotropic conductive adhesive is used as interconnection material both with a PCB test vehicle to assess its mechanical and electrical interconnection properties and for mounting an ITO-coated glass containing printed structures onto an adapter PCB.The epoxy based silver-filled two-component room temperature curable adhesive Elecolit 325 from Panacol is selected as isotropic conductive adhesive. The target diameter of a conductive adhesive dot forming an interconnection between ITO-coated glass and adapter PCB is To investigate the electrical and mechanical performance of the conductive adhesive interconnection, a test vehicle is designed and fabricated from PCB material as shown in The test substrate has 14 pairs of Cu-pads for test chip mounting  used in these test series, the manufacturer specifies a dispensing pressure tolerance of In order to identify dispensing parameters, systematic dispensing tests are conducted by repeatedly dispensing matrices of 40 dots on a foil substrate . A PUF describes a hardware-security primitive, which allows for fingerprinting. Here, intrinsic variation in printed electronics is exploited to generate unique identifiers. The intrinsic variation source, which is labeled as PUF core, consists of the inkjet-printed inverter array, which is fabricated in the central area of the ITO-coated glass. The input-/output ports of the inverter array are connected to the standard interconnection interface on the edges of the ITO-coated glass. The inverter array is based on low-voltage electrolyte-gated transistors (EGT), whose channel is realised by inkjet-printing of an indium (III) nitrate hydrate (H2InN3O10)-based precursor. For the subsequent formation of the indium-oxide (In2O3) thin-film semiconductor channel, an annealing step at 400 °C is applied. ITO-coated glass both fulfils the high temperature requirement resulting from the annealing step and allows creation of wiring, resistive meander structures, EGT terminals and input-/output ports by laser structuring. The gate insulator and the top-gate electrode are inkjet-printed using a composite solid-polymer-electrolyte (CSPE) and poly:poly (styrenesulfonate) (PEDOT:PSS), respectively. The deployed circuit architecture and devices can be found in .The developed mounting process enables seamless integration of the printed PUF cores on ITO-coated glass onto a specially designed adapter PCB . The adaIn this paper, we have introduced a novel automated room temperature bumpless flip-chip mounting process for hybrid printed electronics based on isotropic conductive adhesive. Firstly, the dispensing process of the room temperature curing adhesive Panacol Elecolit 325 was optimised in order to compensate for increasing adhesive viscosity that occurs already during pot time. By implementing a function to correct these time dependent viscosity changes, the reproducibility of dispensed adhesive dots and thus of the electrical interconnections is guaranteed over the entire processing time. PCB test vehicles were used to perform four point resistance measurements and shear tests to characterize the electrical and mechanical performance of the interconnection. Further, a standardised interconnection interface has been defined between ITO-coated glasses containing inkjet-printed structures and an adapter PCB with peripheral electronic components. The developed mounting process and interconnection interface were applied to automatically mount novel printed security devices to adapter PCBs. A printed security device consists of four PUF cores in the centre of the ITO-coated glass.The proposed automated mounting process enables reproducible fabrication of hybrid printed devices based on ITO-coated glass in larger batch sizes than the manual assembly process used beforehand. Thus, more comprehensive evaluation of the device parameters is now possible. The standardized interconnection interface allows for straightforward transfer of the process to any other circuit printed on ITO-coated glass. Due to the room temperature processing, any thermal influence of the mounting process on the performance of the complex printed circuits can be excluded. The herein described dispensing time correction methodology can be adapted to different room temperature curing adhesives and/or different dot diameters. The optimised dispensing process and the design of the standard interconnection both hold potential for further miniaturisation and thus, higher integration density. First tests have shown that with the same adhesive, dots down to 200 Thus, the developed automated room temperature bumpless flip-chip mounting process is not limited to the interconnection of devices printed on ITO-coated glass to adapter PCBs. It is a promising approach for future utilization in numerous hybrid printed electronic circuits that are based on novel and often temperature sensitive materials both on the side of substrates and inks. Furthermore, it can be applied to enable novel hybrid systems by combining the functionality of standard electronic components with emerging research and application fields in flexible electronics, e.g., ,36.