Gene immunization comprises mRNA and DNA vaccines, which stand out due to their simple design, maintenance, and high efficacy. Several studies indicate promising results in preclinical and clinical trials regarding immunization against ebola, human immunodeficiency virus (HIV), influenza, and human papillomavirus (HPV). The efficiency of nucleic acid vaccines has been highlighted in the fight against COVID-19 with unprecedented approval of their use in humans. However, their low intrinsic immunogenicity points to the need to use strategies capable of overcoming this characteristic and increasing the efficiency of vaccine campaigns. These strategies include the improvement of the epitopes’ presentation to the system via MHC, the evaluation of immunodominant epitopes with high coverage against emerging viral subtypes, the use of adjuvants that enhance immunogenicity, and the increase in the efficiency of vaccine transfection. In this review, we provide updates regarding some characteristics, construction, and improvement of such vaccines, especially about the production of synthetic multi-epitope genes, widely employed in the current gene-based vaccines. Throughout history, we have experienced how several disease outbreaks have caused health risks, many of them with pandemic potential, culminating in the deaths of millions of people worldwide. The emergence of new diseases accompanied by population growth and globalization indicates the need to obtain new tools capable of reducing the transmission of infectious agents and the risk of future pandemics ,2. In thIn 2020, during the COVID-19 pandemic scenario caused by SARS-CoV-2, the urgent nature of protective measures prompted rapid action by the scientific community through pre-existing studies . In partAs seen in recent epidemic outbreaks, future pandemics are likely to require the continued development of new models and approaches for designing nucleic acid vaccines. Particularly with viral infections, there is a demand for rapid production and updating of vaccine platforms. These improvements are essential not only for diseases not controlled through vaccination, but also in the context of the appearance of mutations that lead to the emergence of variants or the establishment of new serotypes. In addition, it is also essential to invest in the vaccine targets’ presentation, which can be whole genes or constructs based on epitopes predicted in silico, besides the development of adjuvants and immunomodulators ,7. ThereGenetic vaccines consist of immunizing or immunotherapeutic approaches that employ DNA or RNA plasmids as antigen precursors. The gene sequence (of one or more genes) encoding the antigen of interest is taken up and translated into protein by host cells. This so-called third-generation vaccine technology, considered innovative among vaccine platforms, was widely used in the COVID-19 immunization program and has great potential to become increasingly common. In addition, these vaccines can trigger cellular and humoral responses and can be improved through in silico tools, which allow the selection of antigenic epitopes, called synthetic antigen vaccines.The manufacture of vaccines that have RNA or DNA molecules in their composition dispenses with the large-scale cultivation of pathogenic microorganisms in a laboratory with a high level of biosafety, such as BSL3, to the detriment of conventional vaccine strategies, such as those using attenuated or inactivated vaccines. In addition, the absence of the pathogen prevents the virus reactivation. This aspect favors the vaccination of immunocompromised people .Indeed, nucleic acid vaccines also exhibit significant advantages over traditional vaccines regarding their ability to induce CD4+ and CD8+ T cell responses. The inherent immunostimulatory nature of the mRNA molecule and its functionality as an immunoadjuvant is regarded as a strength that can be exploited in vaccine strategies. Transforming these characteristics into a safe and effective clinical product presents the challenge of balancing immune stimulation with the expression of the encoded antigen. Lately, mRNA vaccines have received special attention and exhibit some advantages over DNA vaccines, such as targeting delivery only to the cell cytoplasm, nullifying the risk of genomic integration, and performing its function independently of cell division. They have a transient and controlled expression of the encoded antigen due to a relatively short half-life, and the absence of additional foreign genes ensures their safety. Besides, cell-free manufacture reduces the chances of contamination with bacterial components and facilitates their production under good manufacturing practices. However, RNA vaccines require additional steps in their production and are susceptible to degradation ex vivo and in vivo, while DNA vaccines are more thermostable, facilitating their storage ,10,11.One of the main advantages of nucleic acid vaccines over predecessor vaccine platforms is the improvement of the immune response targeting. Besides, this approach allows the addition of antigens from two or more variants in the same vaccine, rapid production, and subsequent modifications to include new variants. Despite initial concerns regarding the possibility of integrating the vaccine plasmid integration into the host’s genome, DNA vaccines have shown remarkable safety, and no significant evidence of integration has been demonstrated . DNA vacAfter translation, these intracellular antigens are processed inside the proteasome, generating the vaccine epitopes. Then, peptides are transported to the endoplasmic reticulum by the TAP transporter and are linked to MHC-I molecules to be presented to the T cell surface receptors and activate cytotoxic responses. Another pathway of immune response activation can occur from the production and secretion of vaccine antigens from transfected cells, like myocytes. These secreted products are phagocytized by antigen-presenting cells (APCs) and can activate helper responses when presented via MHC-II. The helper response is important because it allows the cross-activation of other cells in the immune system, such as B and TCD8 lymphocytes . More deThis versatility in activating different immunological response pathways makes nucleic acid vaccines useful for both prophylactic and therapeutic purposes. In this sense, since the main objective of prophylactic vaccines is to promote immunological memory, these strategies demand a strong humoral response through specific CD4+ T cells. A vaccine for therapeutic purposes, on the other hand, requires primarily cytotoxic CD8+ T cell responses to recognize and cause apoptosis of chronically infected or tumoral cells ,15.As an example of a prophylactic study, the effects of the mRNA-1273 vaccine against SARS-CoV-2 demonstrated high neutralization and Th1-shifted CD4+ T cell responses in humans. This response profile involved a reduction in the risk of increased vaccine-associated respiratory disease or increasing antibody-dependent replication. Furthermore, these characteristics of mRNA composition and formulation have been associated with prolonged protein expression, induction of antigen-specific follicular T helper cells, and activation of germinal center B cells ,17.Regarding the therapeutic approach, Rittig et al.  carried n = 53), whereas in placebos, spontaneous regression of lesions occurred in 30.6% (n = 11). Furthermore, in this study, the immunological analysis demonstrated significantly greater specific activation of cytotoxic T lymphocytes and increased humoral response in the vaccinated patients . This molecule consists of a double-stranded RNA analog capable of inducing cell signaling through multiple inflammatory pathways. Poly(I:C) is especially used in formulations that target dendritic cells, promoting their maturation . In a stIn addition to TLR ligands, many other classes of immunomodulators can be used through co-injection or even as part of the vaccine construction in nucleic acid vaccines. Cytokines are a class of immunoregulatory proteins critical to the signaling of immune cells and capable of affecting their behavior. Several studies have included plasmids encoding ligands of growth factors, adhesion molecules, death receptors, and other cytokines . An examIL-12 is another cytokine that provides a link between innate and adaptive immunity in response to infections . FurtherIn addition to establishing the profile of these adjuvants, it is necessary to assess how they will be delivered and presented to immune system cells. Many of them are inserted in DNA plasmids, encoding sequences of inflammatory cytokines, chemokines, interferons, and growth factors, and are co-administered with the nucleic acid vaccine . Others Edwardsiella tarda infection induced.Lapuente et al.  observedThe type of vaccine platform represents an important aspect regarding the intensity of the associated immune response. Vaccines that contain live virus, for example, accurately simulate the natural process of infection and, after administration, quickly disseminate through the circulation, reaching their target tissues. These viral particles promote a broad response and contribute to the transmission of signals associated with pathogens, which mobilizes the innate immune system response. Meanwhile, non-live vaccines, such as nucleic acid vaccines, exhibit limited immune response due to the lack of replicative activity, essentially activating the innate immune response locally at the point of administration. In this case, the site and route of administration are relevant factors of choice .For nucleic acid vaccines, the most common routes are intradermal and intramuscular due to the availability of DCs in these tissues that allows a successful immunization without the need for high antigen doses, especially under limited immunogenicity conditions. Another alternative route for non-live vaccine injections is the vascularized muscle tissue because it has a region with high amounts of DCs. Some routes may be less effective, such as subcutaneous injections, since there is a lower distribution of these cells in adipose tissue .Whereas the intramuscular route (IM), followed by electroporation, is pointed as the best administration route for DNA vaccines , for mRNIn addition to the immunization route, it is essential to ensure that nucleic acid vaccines are transported through the biological barriers and can be delivered inside the cell before they become degraded. Hence, improving delivery systems for nucleic acid vaccines constitutes an important research area for vaccine development. In this review, we focused on the electroporation (EP) for DNA and lipidic nanoparticles (LNPs) for mRNA vaccines, which are the most common systems employed to increase the immunogenicity of acid nucleic-based vaccines.Over the years, electroporation has been applied in several preclinical and clinical trials to improve the delivery of nucleic acids and chemotherapeutic drugs to target tissues. This technique emerged as an introductory method for macromolecules and was associated with an increased expression of these molecules in vivo . The proA study demonstrated that electroporation modulated the production of pro-inflammatory cytokines in the skin, elevating local concentrations of transforming growth factor-α (TGF-α) and IL-1 . MoreoveDiken et al.  showed tMiao et al.  used a cThird-generation vaccines represent a huge advance in the field of vaccinology. The COVID-19 pandemic reinforced the importance of this vaccine platform, providing an excellent example of how the pioneer studies initiated decades ago could be applied in an extremely fast, efficient, safe, and cost-effective way—as never seen before—in the fight against the pandemic. This review has compiled research trends on the engineering of third-generation vaccines and the various aspects that can be addressed to increase their efficiency. As presented here, growing knowledge about the immune system has supported the bioengineering of molecules capable of activating immune responses with higher efficiency, specificity, and safety. The challenge now lies in further improving this technology to overcome its limitations, such as the low immunogenicity of DNA molecules and the inherent instability of the RNA molecule. In this regard, recent studies are developing new generations of biomolecules for vaccine delivery, as well as new adjuvants.In addition to the advances described in this work, it is worth mentioning the increasing number of studies that feed the international databases that, in turn, support bioinformatics tools, allowing future assistance in the development of new tools, in addition to the improvement of existing ones. All of this will contribute to increasingly accurate epitope predictions during the vaccine construction stage. Furthermore, thanks to next-generation sequencing, a massive deposit of genomic sequences has been observed, which in turn will contribute to the characterization of MHC profiles of different populations in the world, allowing the development of multi-epitope vaccines with greater population coverage capacity. All these advances in studies and techniques discussed here lead to a positive expectation for the future of vaccinology, helping to ensure the resolution of infections associated with challenging pathogens or even complex and multifactorial diseases such as cancer.


Extracellular vesicles (EVs) have emerged as important mediators of homeostasis, immune modulation and intercellular communication. They are released by every cell of the human body and accordingly detected in a variety of body fluids. Interestingly, their expression can be upregulated under various conditions, such as stress, hypoxia, irradiation, inflammation, etc. Their cargo, which is variable and may include lipids, proteins, RNAs and DNA, reflects that of the parental cell, which offers a significant diagnostic potential to EVs. In line with this, an increasing number of studies have reported the important contribution of cancer-derived EVs in altering the tumour microenvironment and allowing for cancer progression and metastasis. As such, cancer-derived EVs may be used to monitor the development and progression of disease and to evaluate the potential response to treatment, which has generated much excitement in the field of oncology and particularly in haemato-oncology. Finally, EVs are able to transfer their cargo to target cells, modifying the properties of the recipient cell, which offers great therapeutic potential for EVs (either by specific drug delivery or by delivery of siRNAs and other inhibitory proteins). In this manuscript, we review the potential diagnostic use and therapeutic options of EVs in the context of haematological malignancies. Extracellular vesicles (EVs) consist of a lipid bilayer and have recently emerged as important mediators of homeostasis and cell-to-cell communication. Since their initial description, about 40 years ago, EVs have now been identified to be produced by almost all cell types in the human body and accordingly detected in a variety of fluid types, such as blood, breast milk, semen, cerebrospinal fluid, bronchoalveolar lavage and bile . EVs, asEVs are quite a heterogeneous group of vesicles, both functionally and phenotypically. The International Society of EVs (ISEVs) has broadly categorised them into: (i) those derived from the plasma membrane  and (ii) those derived from the endosomal system (exosomes) and specifically from the intraluminal vesicles (ILV) formed in the multivesicular body (MVB)  4]. Wit. Wit4]. The EV cargo is equally variable and may include proteins, lipids, RNAs  and DNA. This cargo differs depending on its cellular source, reflecting that of the parental cell. More interestingly, EVs have the unique ability to transfer their content to target cells, modifying the biological properties of the recipient cell. This is mediated either by: (i) contact, referring to receptor–ligand interaction; (ii) uptake, referring to internalisation of the EVs; (iii) fusion, referring to fusion of EVs with the plasma membrane; or (iv) a combination of the above . Overall, EVs have a number of unique properties that offer an advantage over cell and gene therapies, such as: (i) they lack a nucleus, so they cannot proliferate and/or differentiate in vivo; (ii) they have no metabolic potential, indicating that their functions cannot be altered by the host microenvironment; (iii) they are stable biological products and can be stored at −80 °C; and (iv) their small size allows them to circulate easily and reach distant places in microcirculation and also cross natural barriers (such as the blood–brain barrier). All the above are of great importance, as they offer an uncountable potential for therapeutic use. Indeed, there is a growing number of clinical trials involving the therapeutic use of EVs, as well as a number of companies exploring their diagnostic potential, with very encouraging results.In this manuscript, we focus on the potential role of EVs in cancer. Specifically, we review the potential therapeutic use and diagnostic potential of EVs in the context of haematological malignancies .Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders, characterised by dysplasia, ineffective haematopoiesis and cytopenias and associated with an increased risk of progression to acute myeloid leukaemia (AML) . CurrentCirculating small noncoding RNA (sncRNA) molecules, and particularly microRNAs (miRNAs), have attracted significant interest and various groups have identified either a unique miRNA signature or a combination of miRNA molecules to predict MDS outcomes ,59. HrusAnother study by Cerisoli et al. , compariEnjeti et al.  investigSince the first description of EV involvement in tumourigenesis by Skog et al. , there hA number of studies also describe the role of EV-derived miRNAs in supporting the survival of leukaemic cells in the bone marrow (BM) microenvironment. For example, EV-derived miR-125b has been shown to inhibit the expression of proapoptotic P53, BAK and Bmf and promote cell cycle , whereasBeyond the contribution of EVs in the development and survival of AML blasts, emerging data also support their involvement in conferring drug resistance. Viola et al.  were oneFinally, EVs have been proposed to be a useful diagnostic tool for disease diagnosis, minimal residual disease monitoring and risk stratification . For exaThe involvement of EVs in pathogenesis, disease diagnosis and monitoring of myeloproliferative disorders (MPDs) has been well described.In the context of chronic myeloid leukaemia (CML), Jafarzadeh et al. demonstrated an altered T-cell function upon exposure to K562 cell-derived EVs . SpecifiThe contribution of EVs in the pathogenesis of thrombotic complications in patients with BCR-ABL1 negative MPDs has been suggested by various groups . SignifiSimilarly, significantly increased EV plasma levels, of platelet and endothelial origin, were detected in patients with essential thrombocythaemia (ET) . FurtherIn line with the above, significantly elevated levels of platelet-derived EVs and decreased levels of megakaryocyte-derived EVs were detected in patients with myelofibrosis (MF) . More inA role for plasma EVs in the pathogenesis of acute lymphoblastic leukaemia (ALL) has been proposed by Yan et al. . The autWith regard to the prognostic role of EVs in ALL, Egyed et al.  identifiA very interesting study by Fei et al.  demonstrEVs have been demonstrated to play a crucial role in the pathogenesis of chronic lymphocytic leukaemia (CLL). An elegant study by Comprot et al.  revealedWith regard to the potential prognostic role of EVs in CLL, there are various studies supporting its utility for prognostication and minimal residual disease (MRD) . It is nEVs have also been suggested to serve as important tools for the development of novel therapeutic strategies. Gartner et al.  manipulaAn important contribution of EVs in pathogenesis, prognostication and disease response of lymphomas has been suggested by several studies . An elegDiffuse large B-cell lymphoma (DLBCL)-derived EVs have been shown to differentiate between different subtypes  and to express a unique pattern of proteins involved in B-cell receptor (BCR) and ErbB signalling and Fc-gamma-mediated phagocytosis and NK-mediated cytotoxicity . More inWith regard to the potential therapeutic use of EVs in the context of lymphoma, Chen et al.  demonstrEmerging data support the role of EVs in generating a favourable ME promoting the survival and growth of multiple myeloma (MM) cells . It has EVs have proven to be a powerful tool in predicting disease progression and poor outcomes in various malignancies. In the context of MM, Manier et al. , investiWith regard to treatment of the disease, EVs have been explored both as potential vehicles of therapeutic cargo and as potential targets for a successful adoptive cell therapy. Garg et al.  demonstrWith the emergence of new concepts relating to the involvement of EVs in the development and progression of malignancies, but also in their capacity to deliver their cargo to target cells, much excitement has been generated in the field of haematological malignancies. In this review, we presented and critically reviewed the most important work relating to EVs in the context of pathogenesis, diagnosis and therapeutics of haematological malignancies. More specifically, it is now well established that EVs support the survival of cancer cells, either by altering the microenvironment or by inducing apoptosis resistance and stimulating angiogenesis. Furthermore, EVs contribute to the development and survival of malignant cells by conferring drug resistance via multiple mechanisms. Besides the above, there is increasing evidence that EVs constitute a useful diagnostic tool for disease diagnosis, minimal residual disease monitoring and risk stratification, conferring valuable information on the disease course and outcomes. Finally, EVs have been suggested to serve as important tools for the development of novel therapeutic strategies, owing to their capacity to carry proteins, RNAs and DNA and to deliver their cargo to target cells, altering their function. The unique properties of EVs, such as the lack of nucleus and their very small size, constitute them superior candidates for successful immunotherapy, compared to currently used cell and gene therapies. All the above underline the particularly important contribution of EVs in cancer diagnostics and therapeutics and open a new horizon for: (i) early, minimally invasive diagnostics; (ii) personalised therapies with minimal/no side effects; and (iii) prompt detection of disease relapse or resistance. Furthermore, with the increasing awareness of the EV contribution to cancer biology, the prognostication and risk assessment for most haematological malignancies  may be improved, changing patients’ outcomes for the better.However, and despite the increasing data supporting the important contribution of EVs in diagnostic, prognostic and therapeutic strategies in haematological malignancies, there are still some obstacles to overcome prior to their clinical translation and large-scale production. These include: (i) absence of a standardised protocol for EV isolation ; (ii) absence of a unique marker for EV detection and characterisation; (iii) heterogeneity in EV function, which is further affected by the isolation method employed, type and culture conditions of the cell source; and (iv) the regulatory requirements for clinical-grade production remain unclear . The inc


The absence of pharmacological treatments to reduce or retard the progression of cardiac valve diseases makes replacement with artificial prostheses  essential. Given the increasing incidence of cardiac valve pathologies, there is always a more stringent need for valve replacements that offer enhanced performance and durability. Unfortunately, surgical valve replacement with mechanical or biological substitutes still leads to disadvantages over time. In fact, mechanical valves require a lifetime anticoagulation therapy that leads to a rise in thromboembolic complications, while biological valves are still manufactured with non-living tissue, consisting of aldehyde-treated xenograft material  whose integration into the host fails in the mid- to long-term due to unresolved issues regarding immune-compatibility. While various solutions to these shortcomings are currently under scrutiny, the possibility to implant fully biologically compatible valve replacements remains elusive, at least for large-scale deployment. In this regard, the failure in translation of most of the designed tissue engineered heart valves (TEHVs) to a viable clinical solution has played a major role. In this review, we present a comprehensive overview of the TEHVs developed until now, and critically analyze their strengths and limitations emerging from basic research and clinical trials. Starting from these aspects, we will also discuss strategies currently under investigation to produce valve replacements endowed with a true ability to self-repair, remodel and regenerate. We will discuss these new developments not only considering the scientific/technical framework inherent to the design of novel valve prostheses, but also economical and regulatory aspects, which may be crucial for the success of these novel designs. Heart valve pathologies have been described as a cause of disability and death since the seventeenth century. They still remain today a relevant contributor of loss of physical comfort and reduction of longevity and result in a considerable socio-economic burden , 2. Disefibrosa, the spongiosa and the ventricularis, each with different structural and mechanical characteristics. The fibrosa, the layer associated with the outflow, or aortic, side of the leaflet, is predominantly composed of collagen fibers arranged circumferentially in parallel bundles and crossing with a typical “χ” geometry at the leaflet “belly” portion, surrounded by a matrix rich in elastin  has been considered the elective option for surgical treatment of heart valve pathologies. Although highly effective, this is an invasive procedure requiring temporary cardiac arrest and extracorporeal circulation, which exposes patients to complications and side effects . For theA recent novel possibility to restore the functionality of a diseased aortic valve with reduced peri-procedural side effects involves trans-catheter aortic valve replacement (TAVR). This technology, exploiting the pliability of pericardial tissue, allows deployment of a completely functional prosthesis using a minimally invasive procedure. These valves are currently approved for patients with severe, symptomatic aortic stenosis in all surgical risk categories given the favorable outcomes in the postoperative period , 27. DesGiven the shortcomings of contemporary valve replacements, over the years, innovative designs have been sought using different approaches and manufacturing philosophies with the aim, in certain cases, to maximize the ease and minimize the cost of the approach , and in other cases to ensure the maximal biocompatibility . We will discuss these two approaches separately, highlighting advantages and drawbacks.ex vivo and short-term in vivo feasibility tests were done, showing a good biocompatibility in the absence of mechanical failure and regurgitation. The lack of long-term tests in vivo still raises the question as to whether these valves offer an advantage over the most advanced mechanical replacements, in particular concerning the need for anti-coagulation therapies to prevent formation of thrombi on the surface of the leaflets.Polymeric valves are manufactured with elastomeric polymers by a simple fabrication procedure using molds. Typically, the process involves “injection molding” whereby synthetic  , 30 or ni) to seed autologous or allogenic-compatible cells inside scaffolds pre-fabricated with biocompatible/biodegradable materials, (ii) to enable tissue formation in bioreactors by exposing the tissue constructs to controlled mechanical loading and, (iii) to promote final tissue maturation, exploiting the ability of the pre-seeded cells to interact with circulating cells to complete the final evolution of the tissue constructs toward native leaflets . For example, in 1995, Shinoka et al.  manufactt et al.  tested ain vivo implantation  fibers. The valve was assembled commencing with non-woven sheets containing PGA and PLLA fibers in a 1:1 ratio bonded by manual and machine needle punching. The scaffold was subsequently seeded with ovine bone marrow cells and cultured for 4 weeks prior to antation . Valve iantation  of a blein vivo implantation, with general deterioration of the geometry and an overall thickening of the leaflets . While cell-mediated degradation can be reduced by optimizing the mechanical and surface characteristics of the polymers  and/or by performing functionalization of the electrospun fibers with natural or synthetic materials, an important factor is also the influence of mechanical load transmission from the scaffold to the cells. In fact, the unequal distribution of the strain forces on the curvilinear structure of the aortic valve leaflets, i.e., from the belly of the leaflet to the commissures , createsin situ strategy,” which exploits the ability of the human body to promote new tissue formation starting from an acellular implant due to the recruitment of circulating cells. In this setting, the postoperative adhesion of autologous cells to the scaffolds is a crucial event expected to provide a structure with performances as similar as possible to that of the native valves  in ovine model. Both phases of the study produced satisfactory results. In fact, the valves exhibited good functionality in terms of leaflets mobility, and did not show major signs of stenosis and thrombus and maintained a stable geometry and good cellular colonization in vivo.The second approach for generating TEHVs is the so-called “e valves –59. An ee valves  using a in situ TEHV is that recently developed by Coyan et al. , and additionally necessitates huge monetary investments inappropriate for the increasing demand. In contrast, in situ TEHVs, which lack a living component, could be produced with an off-the shelf strategy at enormously lower costs and could be easily implemented into the market. The shortcoming of this approach is that the efficiency of the in situ recellularization and tissue maturation is less controllable, given the anticipated patient-to-patient variability due to the effects of age, risk conditions and pathological settings, which could lead to a variable degree of inflammation and failure , this type of scaffold gave good results when mechanical performance and maintenance of cellular viability was considered  can be potentially modulated by mechanical tuning of the hydrogels characteristics, thereby crucially contributing to maintain them in a quiescent/self-renewing phenotype.In view of the growing awareness of the maladaptive interactions between cells and scaffolds used to produce TEHVs, more complex manufacturing concepts are now emerging based on more systematic views of the cells/scaffolds interactions  and the nsidered . Unfortui et al.  employedfibrosa layer. Preliminary bench testing of the resulting valve after 21 days dynamic conditioning, proved that mechanical stimulation enhanced matrix deposition (in particular collagen) by the cells, thus showing the versatility of a “mixed” fabrication approach to elaborate a design that more closely resembled the natural valve architecture. The utility of a tailored deposition of valve scaffolds fibers is, however, still under question, especially from the perspective of long-term scaffold remodeling after in vivo implantation. In fact, for example, Uiterwijk et al.  printing of valve scaffolds or direct bioprinting of valve leaflets using cell-laden polymers . The advic acids , and PEGic acids , furtherde novo into decellularized animal-derived materials, such as entire valves or pericardium. Prompted by remarkable examples such as the re-engineering of decellularized whole hearts , both from Ministero della Salute, Italy.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


An improved understanding of how policies can promote the diffusion of electric vehicles (EVs) is critical to achieving sustainable development. Previous studies of EV diffusion dynamics have paid insufficient attention to consumer preferences. In this paper, a network-based evolutionary game model considering dynamic consumer preference is constructed to study EV diffusion. Through numerical experiments, the evolutionary processes and results of various promotion policies, including carbon taxes, production subsidies, purchase subsidies, and information policy on EV diffusion, are simulated. In particular, this paper explores the differentiated effects of supply-side policies and demand-side policies. The simulation results indicate that: (1) The effectiveness of promotion policies is sensitive to the size of the manufacturer network, and large networks can dampen periodical fluctuations in diffusion rates. (2) Supply-side carbon taxes and subsidies facilitate a steady diffusion of EVs. However, compared with the sustained effectiveness of subsidies, carbon taxes may inhibit the rapid penetration of EVs. (3) Implementing purchase subsidies in the early stages of diffusion is more effective than production subsidies, but the potential uncertainty of demand-side subsidies should be noted. (4) The impact of information policy on the evolutionary trend of EV diffusion is pronounced but is a longer-term impact, requiring a long enough implementation horizon. The transportation sector, an important pillar of socioeconomic development, is energy-intensive and accounts for approximately 30% of total global energy consumption . With glThe diffusion of EV faces many economic and technical barriers, including high upfront costs, technological externalities, and lower consumer preference, making the EV diffusion relatively slow and limited to niche markets. As governments have implemented various EV-related policies to address market failures and promote EV diffusion, understanding and comparing the effects of different policy levers are critical to instituting improved policies to achieve the target of large-scale EV penetration.The effect of promotion policy on the diffusion of EV technology among manufacturers is the focus of a growing body of literature. Many recent studies have investigated the dynamic diffusion of EVs among manufacturers and the effects of various policy interventions. For example, Li et al.  used a nPrevious studies have provided valuable insights into the dynamic process of EV diffusion and the effects of policy interventions on technology diffusion among manufacturers. However, most of them have focused only on the complex interactions between manufacturers in the market and have treated consumers as an exogenous factor in the technology diffusion system. In other words, existing EV diffusion studies tend to model technology diffusion only among manufacturers on the supply side, without considering consumers on the market demand side and their dynamic characteristics. From the perspective of market supply and demand, the static consumer preferences imply that a fixed percentage of consumers repeatedly buy a certain product each year, which not only omits the reality of evolving consumer preferences, but also neglects relevant feedback mechanisms, such as the periodical fluctuations of the market. Both manufacturers and consumers are an integral part of the automotive market, and many theoretical and empirical studies have proven that market demand is an essential driver of products diffusion ,8. PromoRegarding the policy effect, many studies have examined EV-related policies using various methodologies, but most of them discuss policies on one side of the market in isolation and do not explore the differentiated impacts of supply-side policies with demand-side policies, leaving a key knowledge gap. Additionally, considering the reality that the policy mix of China’s EV industry has started to shift from being producer-oriented to being consumer-oriented , it is wIn light of the illustrated realities, there is a clear need to delve into the gaps above in the literature. We seek to answer the following questions. First, regarding consumers as stakeholders, how do dynamic consumer preferences affect the diffusion of EVs among manufacturers? Second, how do various policies implemented for different stakeholders affect the trends and results of EV diffusion? Third, what are the differential effects of supply-side and demand-side policies on EV diffusion?In this paper, we first choose two types of policies, supply-side and demand-side policies, through literature review, and identify the main stakeholders in EV diffusion. Then, we construct a network-based evolutionary game model to study the EV diffusion in the manufacturer network. Subsequently, consumer dynamics within the coordination game framework are introduced on the demand side to extend the EV diffusion model from the perspective of dynamic consumer preferences. Through controlled numerical experiments, we simulate the process and results of EV diffusion under various policies, thus providing further insights into the effects of various policies and their differences and informing the effective implementation of government policies.Academically, this study contributes to the existing literature in three ways: (1) An EV diffusion model that includes dynamic consumer preferences in the manufacturer network is constructed to fill the research gap in the previous literature that treats only manufacturers and governments as stakeholders, ignoring consumer behavior. (2) By analyzing the dynamic effects of supply-side and demand-side policies on EV diffusion and comparing the differences in the effects of differently oriented policies, this paper theoretically enriches the insights on different types of interventions. (3) This paper provides a theoretical tool for governments to develop effective policy intervention mechanisms and design an improved policy package.This paper proceeds as follows: The research related to this paper flows into three major streams: the effect of policy on EV diffusion, consumer preferences, and the network-based evolutionary game. In the following subsections, the studies of these three streams are reviewed, and then knowledge gaps are presented.Policy intervention has always been regarded as a powerful means to facilitate the diffusion of EVs ,11. The Comparatively, demand-side policies are mainly a market demand tools designed to create more demand for EVs by stimulating consumers to purchase EVs and thus indirectly promote the diffusion of EVs in the manufacturer network . Many stConsumer preferences are a crucial element in predicting and analyzing EV diffusion. In previous studies of EV diffusion, the role of consumer preferences is reflected in providing preferences required to support market demand forecasts for new vehicle technologies . An emerTraditionally, consumer preferences are regarded as static limiting analysis of the consequences of a given sequence of parameters in the economics field . With thMany researchers have also explored the impact of consumer preferences on the adoption or diffusion of vehicle technologies. Oliveira et al.  comparedEvolutionary game theory is a classical approach based on game theory and evolutionary biology theory to study how players adjust their strategic choices in the process of constant interaction and mutual adaptation. It has become an important method for understanding the formation process and interpreting the evolution mechanism of group behavior and social system formation processes . For manThe evolutionary game framework assumes that populations are homogeneous. However, realistic socioeconomic systems may have complex topological and statistical features that may lead to statistically inequivalent interaction probabilities of agents. Therefore, scholars have combined complex network theory, which studies the topology of complex systems, with evolutionary game research and proposes the network-based evolutionary game model . Shi et Reviewing the literature on the effects of policies on EV diffusion reveals the following two knowledge gaps.First, few studies have considered consumers as stakeholders in constructing manufacturer evolutionary game models and considered dynamic consumer preferences on this basis. Although consumers are often modeled as necessary influences on firms’ revenue, most existing evolutionary game models only use the form of the Hotelling demand to calculate the constant product demand corresponding to a certain set of parameters. This implicitly assumes that consumers have constant preferences and purchase the product once a year, which is quite unrealistic for some products, especially EVs. The evolution of EV diffusion and consumer preferences is a process of mutual adaptation and selection, thus simplifying the analysis of consumer preferences would ignore the relevant feedback mechanisms underlying the ultimate diffusion dynamics.Second, supply-side and demand-side policies are often discussed in isolation, lacking adequate comparison of effects and mechanisms. For policymakers, choosing among various policies entails information about their mechanisms and probable outcomes, particularly in complex economic systems and in situations with far-reaching ramifications. Both supply-side and demand-side policies are vital to EV diffusion, and more comprehensive perception of their effectiveness and mechanisms is needed.To advance the research streams, this paper proposes a network-based evolutionary game framework for EV diffusion considering dynamic consumer preferences and investigates the effects of different oriented policies on EV diffusion.The main stakeholders in the EV diffusion process include governments, manufacturers, and consumers. The government implements a series of policies, including supply-side and demand-side policies, to directly or indirectly facilitate manufacturers’ shift towards electric vehicle production. Here, supply-side policies aim to reduce the barriers to technology introduction and output and thus directly facilitate manufacturers’ decisions to produce EVs. In contrast, demand-side policies indirectly promote the diffusion of EVs by influencing consumers’ preferences and purchase decisions for EVs. According to the review of EV diffusion promotion policies in Manufacturers are the central players in enabling the diffusion of EVs, and each manufacturer’s decision to produce EVs forms the diffusion of EVs at the market-wide level. This strategic decision comes from the evolutionary game between different agents in the manufacturer network. At the same time, market demand is the foundation for manufacturers to gain revenue. Consumers on the demand side of the market are the demanders of automotive products, and their preferences and purchasing behaviors determine the market demand for EVs as well as ICEVs, thus affecting manufacturers’ payoffs. The evolution of manufacturers’ and consumers’ strategies, shaped by their respective social dynamics, underlies market dynamics at the microlevel. There are The different strategic decisions adopted by manufacturers will have different carbon-reduction rates, The manufacturer agents are embedded in a complex social network, with nodes representing manufacturers and edges representing linkages between manufacturers. Decision and payoff information is shared among connected nodes in the manufacturer’s network.Manufacturers of the same strategic decision produce homogeneous automotive products. The market demand for EVs and ICEVs is supplied evenly by manufacturers producing EVs and ICEVs, respectively.Automotive products have a lifespan, The size of consumers in the market is Given the above problem description and related literature on EV diffusion, we made the following modeling assumptions for the basis of the network-based evolutionary game model:As an important method of combining evolutionary game theory with complex network theory, the construction of a network-based evolutionary game model consists of three main parts: network structure, game model, and evolutionary mechanism. In this paper, a network-based evolutionary game model of EV diffusion is constructed from these three aspects based on the modeling assumptions.In terms of network structure, manufacturers in the real world are considered to be well-connected, with complex competitive and cooperative relationships and “small world” characteristics. In addition, for the automotive industry, previous research has generally concluded that there is no significant heterogeneity among manufacturers ,5. ThereAccording to the problem description and modeling assumptions, the sales volume of each EV manufacturer and the ICEV manufacturer selling products at time Furthermore, considering the government’s influence on manufacturers, the government imposes a carbon tax related to carbon emissions on ICEV manufacturers and EV manufacturers, denoted as:All manufacturers seek to maximize profits, but they cannot directly determine which strategy to adopt using market demand information. They need to observe and compare the strategies adopted by neighboring manufacturers in the network and the corresponding payoffs after each round of the game to conduct social learning and strategy updating. At each step, all manufacturers obtain the payoffs of adopting the corresponding strategies according to the game matrix, and then randomly select a neighbor to compare the profits. When the manufacturer has the same strategy as the chosen neighbor, no strategy change occurs, while the manufacturer with different strategies learns the neighbor’s strategy with the Fermi rule:Inspired by Shi et al.  study onConsumer preferences reflect individuals’ love for different products. Preference theory suggests that when people are confronted with different choices, they will evaluate various information to form preferences and make decisions based on the order of preferences. Studies also highlight the deterministic role of utility functions and social interactions on preferences . Since tIn large groups of consumers, everyone’s opinions and decisions are constantly changing as people communicate and influence each other. The evolution of group preferences for EVs is a dynamic process involving a large number of interacting and adaptive consumers. We consider an automotive market with o et al.  and Ye eo et al. , the utiIn each round of the manufacturer’s decision evolution, consumers also perform a round of decision evolution. After gaining payoffs, each consumer randomly selects a neighbor in the social network for social learning. Without loss of generality, we also use the Fermi rule as the evolutionary mechanism for the consumer. That is, the larger the payoff difference in social comparison, the more likely consumers are to learn from each other’s strategies. The change in consumer preferences at each moment can be presented as:g et al. , the adoWe calibrate our model using data from relevant studies as well as empirical data from China’s automobile industry. In the manufacturer network, the number of manufacturers ) is 0.1 . Regardi) is 0.1 . Referri) is 0.1 , the car k is 10 , the ini) is 0.2 , and all) is 0.2 . In term) is 0.1 .Based on the above parameter settings, we conduct controlled numerical experiments, and for each policy, the simulation changes only the parameters related to that policy. The simulation of each set of parameters lasts for 100 steps (which can be regarded as 100 years), and the final results are obtained by averaging the results of 100 independent runs to ensure stability. Since this paper focuses on EV diffusion at the system level, the EV diffusion rate, representing the proportion of EV manufacturers to all manufacturers, is of most interest for the simulation experiment.The carbon tax is charging fees for burning-carbon-based fuels , widely implemented in European countries. In order to study the effect of carbon tax policy on the diffusion of EVs, we conducted simulations at carbon prices igh 1000 , respectAs can be observed from the evolutionary trend, the EV diffusion rate first rises rapidly and then gradually reach equilibrium. The greater the number of manufacturers, the longer it takes to reach equilibrium. For example, it takes about 20 years to reach equilibrium when Additionally, it is worth mentioning that the impact of carbon taxes varies considerably at different evolutionary periods, as shown in Another interesting finding is that the magnitude of the periodical fluctuation of the EV diffusion rate decreases significantly with the network size increase. When the network size increases from 100 to 300, the maximum diffusion rate during the evolutionary process remains almost unchanged. In contrast, the fluctuation of the diffusion rate in equilibrium decreases by about 2/3. This result indicates that the effectiveness of promotion policies is sensitive to changes in network size. Government policy implementation needs to consider the market environment as well as the number of manufacturers, which is especially important when there are market frictions.The simulation results support previous studies on the effects of carbon taxes, such as ,35, whicSubsidies are one of the most common fiscal interventions governments take, and production subsidies are an important source of revenue for EV manufacturers, facilitating the transition to producing EVs. In this section, we illustrate how different production subsidies affect EV diffusion. We run the model with production subsidy and 0.09 . The resIt can be found that production subsidy policies have a more stable impact on EV diffusion trends and results compared with carbon tax policy. As the production subsidy rises, the diffusion rate increases in any period, which indicates the effectiveness of the supply-side subsidy policy. At the same time, it can be observed that the basic evolutionary trend of EV diffusion is to rise rapidly and then fluctuate at a certain level of diffusion rate, which is in line with the stylized fact of the “S” curve in the traditional innovation diffusion model.In addition, the model proposed in this paper can capture periodical market fluctuations due to dynamic consumer preferences. Since we set up repeat purchases with product lifespan, this mechanism is reflected at the system level in the diffusion rate fluctuations with a ten-year cycle in We now elucidate the impact of demand-side policies on the evolutionary trend of EV diffusion and compare the differences in the effects of demand-side and supply-side policies. The effect of consumer subsidies on EV diffusion is given in Purchase subsidies can be found to play a significant role in the diffusion of EVs. Lower levels of consumer subsidy factors adopters ,47.By comparing the differential effects of supply-side production subsidies and demand-side purchase subsidies, we can find that production subsidies are more influential on long-run diffusion equilibrium, while purchase subsidies have a significant impact on both the diffusion process and equilibrium results. Our modeling exercises to extend consumer dynamics in the manufacturer evolutionary game suggests that the effectiveness of policy interventions on EV diffusion may be sensitive to demand-side dynamics, notably the assumptions about interactions between consumers. For policymakers, this further indicates that demand-side policies may be indispensable and that supply-side efforts alone cannot get rid of consumer dependence on the conventional technology, i.e., the lock-in effect.In addition, there is greater uncertainty about the effects of purchase subsidies compared to production subsidies. Lower or higher production subsidies would lead to almost only a ten percent difference in effects in equilibrium, while the effects of different purchase subsidy intensities could vary significantly. A possible reason for this phenomenon is the complexity of the economic system. On the one hand, the effects of purchase subsidies are transmitted from the demand side to the supply side through the supply-demand interaction in the market. On the other hand, the demand promotion by purchase subsidies involves complex and highly nonlinear social processes. Together, these factors cause the purchase subsidy to exhibit similar tipping-point dynamics . TherefoDemand-side information policies that intervene with consumers are gaining attention. Information policies are interventions that reduce negative consumer perceptions of emerging technologies through the Internet or media. We use the information speed factor The evolutionary trend shows that the impact of information policy on EV diffusion has been realized gradually over time. There is almost no pronounced effect in the early stages of evolution, while the evolutionary trend becomes markedly different between 10 and 30 years and then fluctuates in equilibrium. This trend is due to the time-lag effect of information policy. Information policy differs from the substantive incentives of purchase subsidies by facilitating information on emerging technologies to be perceived by consumers, thereby promoting repeat purchases and changes in consumer preferences. This process of changing consumer preferences and manufacturer evolution are in a circular cause-and-effect relationship. Improvements in manufacturer technology and the development of the EV industry promote consumer preferences toward EVs through information policy, which in turn contribute to the diffusion of EVs among manufacturers. Furthermore, the comparison with supply-side policies also reveals the longer-term impact of demand-side information policies. Even if information policy does not achieve the same stable promotion effect as production subsidy policy in the short term, the impact on the long-term effect and evolutionary equilibrium is more pronounced.The longer-term and time-lagged effects of information policy can be found in our results, consistent with a real-world phenomenon in EV diffusion: “hot policies and cold markets” . DespiteThe large-scale diffusion of EVs is crucial to address environmental pollution and energy shortages. Current research focuses on the evolutionary game of EV production among auto manufacturers while treating consumers as an exogenous factor to the diffusion system. Given the interdependence of market supply and demand and the significant role of consumer preferences, we introduce dynamic consumer preferences into the demand-side modeling of EV diffusion. We propose a network-based evolutionary game framework of EV diffusion considering dynamic consumer preferences, and analyze the differential effects of supply-side and demand-side policies through simulation experiments. The following conclusions can be drawn from the detailed analysis above.The effect of EV promotion policies is sensitive to the size of the network. The increase in the number of manufacturers in the network reduces the magnitude of periodic fluctuations in EV diffusion rates.Both carbon tax policy and production subsidy policy acting on manufacturers can steadily contribute to maintaining a higher level of EV diffusion in equilibrium. However, unlike the effectiveness of production subsidies in the whole diffusion process, carbon tax policy has some inhibiting effects in the phase of rapid diffusion.Demand-side purchase subsidy policy is critical to EV diffusion. Especially in the initial stage of diffusion, implementing the purchase subsidy policy to create market demand is a more appropriate promotion policy. Attention should also be paid to the greater uncertainty of purchase subsidy policies due to the complexity of the economic system. The government’s promotion policy package needs to be adapted and changed according to the expected diffusion targets and the current diffusion stage.The impact of information policy on EV diffusion is pronounced, but its policy effects are longer-term and time-lagged. A long-enough horizon of implementation is needed to ensure the effectiveness of the information policy.This paper considers dynamic consumer preferences in a model of EV diffusion among manufacturers and conducts simulation experiments for typical supply-side and demand-side policies. Through the in-depth analysis of the mechanisms, evolutionary trends, and equilibrium results of different policies on EV diffusion, this paper not only enriches the modeling practice of EV diffusion, but also provides insights for adjusting the policy orientation and balancing the policy packages. Due to the complexity of the EV diffusion problem, some limitations still exist in this paper. In terms of consumer behavior, the reasons that consumers purchase EVs is quite complex, while this paper only considers social utility under social norms and economic utility under subsidy policies. Future research can introduce more influential factors, such as environmental awareness  and cons


Trypanosoma cruzi, high genetic heterogeneity of the parasite, the need to use different detection methods associated to the specific stages of the disease and patterns of transmission, Schijman and colleagues, with great propriety and clarity, revisit the accuracy of CD diagnosis in different existing scenarios in endemic and non-endemic areas. A broad panel of diagnostic strategies and their application is presented, ranging from conventional parasitological techniques to more complex serological and molecular assays. Due to the particularities of each test, the authors present specific diagnostic algorithms to be applied for early T. cruzi detection in the acute phase of CD, to assess congenital transmission through the screening of pregnant women and their offspring, epidemiological surveys, surveillance for vector-borne transmission, blood bank and organ donors screening and for the diagnosis of chronic CD. The tests used for the diagnosis of CD should be universal, i.e., capable of detecting all human-infecting parasite lineages. In addition to laboratory tests, the epidemiology and clinical context are pertinent for the interpretation of results.Chagas disease (CD) affects millions of people, mostly poor and neglected populations residing in endemic countries in Latin America. Diagnosing CD is not simple and results in limited access to treatment. Considering the complexity of the disease attributed to factors as, different routes of infection by Methods to prevent the increase in the incidence of the disease are vector control, control of blood banks, and carrying out tests on pregnant women to prevent congenital transmission.In the initial phase of infection, circulating parasites can be detected by direct microscopic observation. Direct testing with fresh blood is more sensitive than stained smear. If trypomastigotes are not observed in the blood of patients suspected of having acute CD, concentration tests (microhematocrit or Strout) should be carried out to increase sensitivity. The sensitivity of microscopic observation is highly dependent on the operator. In suspected cases of congenital transmission, it is important to test for the mother’s infection. Around 60% of congenitally infected newborns are asymptomatic at birth. In this review, the authors highlight the importance of preventing vertical transmission and the need to early diagnose and treat congenitally infected newborns, in view of the high rate of cure when treatment is administered close to birth.T. cruzi infection becomes more difficult during the chronic phase, in which, the low and intermittent parasitaemia leads to the need for diagnostic tools with higher sensitivity. In this phase, characterised by high levels of anti-T. cruzi IgG, the diagnosis is essentially serological but its results can turn difficult to interpret, and the parallel use of at least two different tests is strongly advised. Given this scenario, the discovery of new diagnostic biomarkers has been a challenge to improve accuracy and, thus, reduce the number of inconclusive serological results. Nowadays, most commercial tests use recombinant antigens, synthetic peptides or antigenic matrices based on chimeric T. cruzi proteins. To avoid interference on the diagnostic test performance across the Americas, these antigens should be conserved and covering the high diversity of parasite lineages. It is recognised that the antigenic diversity of T. cruzi is one important factor leading to a reduced concordance among different commercial tests applied in distinct environments. Here, Schijman and colleagues also discuss the use of rapid diagnostic tests (RDTs), as effective alternatives for large-scale screening and point-of-care (POC) diagnosis of chronically infected individuals residing in resource limited areas in different CD epidemiological scenarios. Fast results, easy handling, high sensitivity/specificity of new developed RDTs are key parameters for their use in healthcare facilities or in remote areas.The diagnosis of T. cruzi in the chronic phase gives positive results in 40-70% of the seropositive patients; so, these methods have a limited diagnosis value in chronic CD patients. The variable sensitivity depends on the level of parasitaemia, sample volume, DNA extraction method, the selected PCR target, the characteristics of the study populations and the genetic diversity of parasite’s lineages. A negative PCR result does not exclude infection and a positive result confirms the presence of the parasite and can be associated to therapeutic failure of anti-parasitic treatments and parasite reactivation in immunosuppressed patients.,T. cruzi genotypes directly from the blood of infected individuals.T. cruzi detection/quantification that were previously validated for use in the in-house TaqMan qPCR protocols. Up to date there are three commercial qPCR kits targeting the T. cruzi satellite DNA and one solely developed for the minicircles DNA that make up the parasite kDNA network.PCR-based assays have proven useful during acute-phase or chronic-phase reactivations due to higher sensitivity compared to parasitological methods. However, molecular detection of ,Trypanosoma cruzi-LAMP technology has been assayed in distinct scenarios of acute CD, as presented by the authors in Table I, showing high agreement with the real-time PCR results. If one considers the urgent need for improving the access to diagnosis (and treatment), mainly in CD endemic regions with scarce and resource-limited laboratories, the use of POC diagnostics would expand access to a larger number of patients. In this context, as stated by Schijman and colleagues, the LAMP technology, due to its own peculiarities could help fill these diagnostic gaps along with easier, faster and less costly platforms for DNA extraction.Loop-mediated isothermal amplification (LAMP) methodology has been developed as an alternative approach for molecular diagnosis, easy to use in field conditions in areas with scarce resources. The inherent characteristics of LAMP , have attracted experts working in CD for the development of commercial kits with great potential application in endemic areas.As general conclusion of this important and attractive review, the authors highlight the urgent need for an increased access to and demand for effective diagnostics by improving the performance of diagnostic algorithms and methods, making them more available to poor and vulnerable populations in endemic countries in Latin America, in order to an effective control of CD.Comments on the article: Schijman AG, Alonso-Padilla J, Longhi SA, Picado A. Parasitological, serological and molecular diagnosis of acute and chronic Chagas disease: from field to laboratory. Mem Inst Oswaldo Cruz. 2022; 117: e200444.


This review has summarized the methods currently available for cardiac sympathetic assessment in clinical or under research, with emphasis on the principles behind these methodologies. Heart rate variability (HRV) and other methods based on heart rate pattern analysis can reflect the dominance of sympathetic nerve to sinoatrial node function and indirectly show the average activity level of cardiac sympathetic nerve in a period of time. Sympathetic neurotransmitters play a key role of signal transduction after sympathetic nerve discharges. Plasma or local sympathetic neurotransmitter detection can mediately display sympathetic nerve activity. Given cardiac sympathetic nerve innervation, i.e., the distribution of stellate ganglion and its nerve fibers, stellate ganglion activity can be recorded either directly or subcutaneously, or through the surface of the skin using a neurophysiological approach. Stellate ganglion nerve activity (SGNA), subcutaneous nerve activity (SCNA), and skin sympathetic nerve activity (SKNA) can reflect immediate stellate ganglion discharge activity, i.e., cardiac sympathetic nerve activity. These cardiac sympathetic activity assessment methods are all based on the anatomy and physiology of the heart, especially the sympathetic innervation and the sympathetic regulation of the heart. Technological advances, discipline overlapping, and more understanding of the sympathetic innervation and sympathetic regulation of the heart will promote the development of cardiac sympathetic activity assessment methods. The cardiac autonomic nervous system (ANS) is one of the most significant structures of the neurohumoral system that regulates cardiac function. ANS can be divided into the sympathetic nerves and the parasympathetic nerves depending on the composition of the nerves , 2. A vaThe ANS is divided into the sympathetic and parasympathetic subsystems, controlled by regulatory centers in the midbrain, hypothalamus, pons, and medulla. The cardiac sympathetic nerve center is located on the medial lateral column of the first to fifth thoracic segment of the spinal cord. The cardiac sympathetic innervation consists of extrinsic and intrinsic components according to anatomical position , 9. The Cardiac sympathetic nerves play a vital role in regulating sinoatrial node, atrioventricular node, and activity of the segmental myocardium, which depend on neurotransmitters  released by synapses at sympathetic nerve terminals and NE receptors on the cell membrane –16.Norepinephrine is mainly synthesized by tyrosine hydroxylase at the sympathetic nerve terminals and stored in the vesicles . Once thNowadays, the methods of cardiac sympathetic activity assessment applied in the clinical practice or under research are based on the anatomy and physiology of the heart, especially the sympathetic innervation and the sympathetic regulation of the heart.Heart rate variability originates from the study on the heart rate patterns and cardiac rhythms, which could date back to 1965. In the 30 years since more and more clinicians had recognized the physiological and pathological significance of HRV. Until 1996, the European Society of Cardiology and the North American Society of Pacing and Electrophysiology published the standard of measurement, physiological interpretation, and clinical use of HRV , 21.Heart rate variability relies on the analysis of every heartbeat, which is directly controlled by sympathetic and parasympathetic activities. Consequently, analyzing the patterns of heart rate, more precisely, analyzing beat-to-beat changes in the R-wave to R-wave intervals can indirectly reflect and evaluate the overall balance state of the cardiac autonomic nerve , 22. HRVTime domain analysis is used to measure and analyze the variability of the R-R interval of sinus rhythm by statistical and geometric methods . Among tFrequency-domain analysis is to analyze the spectrum curve formed by the R-R interval time series signal of sinus rhythm . The speTime- and frequency-domain analyses of HRV are fairly simple and stable methods for sympathetic nerve activity assessment. However, these methods could not extract key information from complex interactions of hemodynamic, electrophysiological, and humoral variables, as well as by autonomic and central nervous regulations, called non-linear phenomena , 21. HenThe application of HRV analysis is not only limited to CVDs, but also can be applied to obesity, tumor, and other diseases with autonomic nervous disorders , 34. WhiBesides HRV, blood pressure variability, resting heart rate, and so on are also closely related to the state of ANS, which, therefore, are available to evaluate cardiac sympathetic activity , 37.Sympathetic neurotransmitters, released by synapses at sympathetic nerve terminals, are the transmitter between the sympathetic and the heart, which could be the target of cardiac sympathetic activity detection , 39.Most of the sympathetic neurotransmitters, such as NE, dopamine, and epinephrine, will be reabsorbed by sympathetic nerve terminals after signal transduction, but there will also be a little NE getting into circulation and can be detected by a peripheral blood test , 41. Pla123I-mIBG) or other radiolabeled neurotransmitter analogs, cardiac neurotransmission imaging with single photon emission computed tomography (SPECT) and positron emission tomography (PET) allows in vivo assessment of presynaptic reuptake and neurotransmitter storage and of regional distribution and activity of postsynaptic receptors ., 44.123Ieceptors . Yasushieceptors .Poor imaging quality and difficulty in distinguishing different cardiac structures were the main problems that limited the application of this technique for a long time in the past. However, the recent development of solid-state gamma camera technology with significantly improved sensitivity, spatial resolution, and energy resolution has enabled high-quality SPECT imaging with a spatial resolution of ≤ 5 mm , 47. In A stellate ganglion can directly regulate the activity of the cardiac sympathetic nerve and then regulate cardiac activity. Enhanced discharge activity of stellate ganglion can accelerate heart rate and raise blood pressure . SympathLavian et al.  directlyStellate ganglion nerve activity recording can be recorded continuously for more than 40 days for 24 h in living animals. It can record the immediate discharge activity of stellate ganglion without affecting the survival and daily activities of animals. It is an important tool to study the cardiac sympathetic nerve activity , 58. HowIn addition to innervating the heart, some postganglionic fibers of the stellate ganglion are widely distributed in the skin and subcutaneous tissue of the neck and chest, and there is extensive cross-linking in the whole neural network , 59. RobRobinson et al.  implanteSubsequently, Chan et al.  conducteSubcutaneous nerve activity recording avoids the huge trauma caused by SGNA recording that requires thoracotomy. By embedding the recording electrode in the subcutaneous tissue of the chest, SCNA recording can also complete the recording of nerve activity for more than 40 days for 24 h, reflecting the immediate SGNA and even cardiac sympathetic nerve activity , 61. HowThe histological evidence of human skin biopsy shows that there are abundant sympathetic nerves in the arteriovenous anastomosis, arrector pili muscle, and arterioles. Given the feasibility of SCNA record, Jiang et al.  further Jiang et al.  directlySkin sympathetic nerve activity can record cardiac sympathetic nerve non-invasively and continuously for 24 h. There are also several limitations to its usage, patients’ daily activities and even body movements have a great impact on the quality of recorded signals . Of noteSympathetic nerve dysfunction engages in the pathophysiological process of coronary heart disease, hypertension, heart failure, arrhythmia, and other CVDs. It is necessary to evaluate cardiac sympathetic activity. Generally speaking, these cardiac sympathetic activity assessment methods can be divided into three levels. Firstly, to evaluate the global sympathetic activities by plasma catecholamine levels, blood pressure variability, and so on. The evaluation results obtained by these methods may not be consistent with the true cardiac sympathetic activity state, which needs to be combined with other clinical evidence. Secondly, using HRV, cardiac neurotransmission imaging with SPECT and PET or SKNA evaluates the average state of sympathetic activity during a selected period of time. Thirdly, to monitor immediate sympathetic nerve activity by SKNA, which is impossible for other assessment methods.What is noteworthy is that these cardiac sympathetic activity assessment methods are all based on the anatomy and physiology of the heart, especially the sympathetic innervation and the sympathetic regulation of the heart. Therefore, the depth of our understanding of the sympathetic innervation and sympathetic regulation of the heart determines the approaches we can take to evaluate cardiac sympathetic activity. Moreover, the development of cardiac neurotransmission imaging with SPECT, PET, and SKNA gives us a hint that technological advances and discipline overlapping are the important driving forces for improving cardiac sympathetic activity assessment methods.JL wrote the manuscript. LZ modified and polished the manuscript. Both authors contributed to the article 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Tumor-cell-derived extracellular vesicles (EVs) are known to carry biologically active molecules of parental cells, which can actively modulate the tumor microenvironment. EVs produced by tumor cells play significant roles in the development and maintenance of tumor growth, metastasis, immune escape, and other important processes. However, the ability of EVs to induce the transformation of normal cells has hardly been investigated. This review discusses studies that describe the ability of tumor-cell-derived EVs to alter the metabolism and morphology of normal cells, causing changes associated with malignant transformation. Additionally, the horizontal transfer of oncogenes through EVs of tumor cells and the induction of epigenetic changes in normal cells, which leads to genomic instability and subsequent oncogenic transformation of normal cells, are also discussed. It is known that tumor transformation of cells requires a gradual accumulation of multiple genetic changes . Such chHowever, the appearance of newly transformed cells in the tumor population can also be induced by cancer cells themselves. A number of studies have shown that the cultivation of normal cells with cancer cells leads to the appearance of malignant characteristics in healthy cells, including enhanced proliferation and migration, shift to a mesenchymal phenotype, as well as the development of resistance to γ-irradiation . FurtherHowever, the ability of EVs to induce the transformation of normal cells has been little studied and is likely another mechanism for the formation of tumor heterogeneity. In this review, we discuss the currently available data on the ability of tumor-cell-derived EVs to induce carcinogenesis in normal cells, as well as the molecular mechanisms that cause metabolic and structural changes in normal cells.The mechanisms of modulation of the genotype and functions of normal cells directly depend on the content of the EVs. It has already been shown that proteins entering the recipient cell remain functionally active . TransfeVIM) and N-cadherin (N-cad) genes and suppression of the E-cadherin (E-cad) gene after EV treatment, which indicates a shift in the phenotype of normal cells toward mesenchymal [Tumor cell vesicles entering normal cells can also induce a number of changes in cell properties that are associated with malignant transformation. One of these properties is increased proliferation rate, which indicates healthy cell transformation into cancer cells. It has been shown that EVs isolated from primary prostate cancer, as well as from the plasma of patients with prostate cancer, induce an increase in the proliferation of normal prostate cells by 40% and 60%, respectively, compared with EVs isolated from benign prostatic hyperplasia and plasma of healthy people. The authors of the study attribute this to an increase in the expression of the vimentin  cells to normal L-02 cells, can inhibit the G1/S transition in the cell cycle, thereby stimulating the proliferation of normal cells . The abiion rate . Interesion rate . In addiEGF), RRAS, and MAPK3 [It is known that tumor cells resistant to chemotherapeutic drugs mediate the formation of chemoresistance in sensitive cells due to the transfer of miRNAs and other molecules through exosomes . For exand MAPK3 . AlthougReleased by tumor cells EVs were also able to change the homeostasis of the endoplasmic reticulum (ER) of normal cells. Incubation of non-malignant human SV-HUC urothelial cells with tumor-cell-derived EVs led to the induction of ER stress and triggered an unfolded protein response (UPR). In normal cells, in case of prolonged ER stress activation of UPR may initiate apoptotic cell death via the upregulation of the C/EBP homologous protein (CHOP) . Indeed,SNAI1, KLF, and CD133 factors that trigger and maintain EMT [It is also worth noting that many studies noted that tumor-cell-derived EVs trigger phenotypic and functional changes in normal cells that lead to a transition from epithelial to mesenchymal phenotype (EMT), the process which allows the solid tumors to become more malignant, increasing their invasiveness and metastatic activity . For exatain EMT . Migratitain EMT . Such chtain EMT .Tumor formation involves the accumulation of a number of mutations in a cell genome essential for malignant transformation. Often, mutations occur in certain genes associated with carcinogenesis–oncogenes. The phenomenon of horizontal transfer of oncogenes by EVs and other ways has already been shown for tumor cells. For example, glioma cells expressing an oncogenic form of the EGF receptor known as EGFRvIII can transfer it to other tumor cells with normal EGFR via vesicular transport . KRAS mu12 and C-MYC genes induced loss of contact inhibition in mouse embryonic fibroblasts. Moreover, injection of SCID mice with the fibroblasts transformed after interaction with ABs led to the formation of tumors, which indicates the development of the full tumorigenic potential [12 has also been shown for smaller EVs. Thus, for example, mouse fibroblasts acquired increased viability after H-RASV12 transfer from immortalized epithelial cells transfected with a V12 mutant. However, treatment of normal epithelial cells with EVs from mutant cells did not increase the incidence of tumor formation in SCID mice [eber and ebna1 can be transferred and efficiently expressed in normal human fibroblasts [Normal cells have also been shown to be able to uptake ABs carrying the oncogenes of donor cells. For example, the internalization of fragmented DNA containing human H-RASVotential . The abiCID mice . This alCID mice . At the CID mice . Using troblasts .P53 and RIZ1 was observed, which are often regulated by methylation [Another important discovery is that EVs, besides modulating the metabolism of normal recipient cells, also induce genomic instability, the most important hallmark of cancer, that leads to an increase in genetic alterations . It has hylation .To date, the fact that tumor-cell-derived EVs can significantly modulate tumor development is beyond doubt. Numerous studies confirm the ability of EVs to modulate angiogenesis, invasion, and tumor localization by changing the behavior of surrounding non-malignant cells. At the same time, there are not so many studies dedicated to the possibility of EVs inducing carcinogenesis in normal cells. However, the wide variety of biological molecules that contribute to the transformation of normal cells suggests that the contribution of such a factor as vesicular transport in tumorigenesis cannot be ignored. Current studies show that EVs released by tumor cells can significantly change the metabolism of normal cells, enhancing their proliferation, and anchorage-independent growth, and also stimulate the development of chemoresistance. The significance of these changes is confirmed by the fact that normal cells with increased carcinogenic potential become capable of forming tumors in immunodeficient mice. However, it is known that, for malignant transformation, a normal cell must accumulate a number of oncogenic mutations. EV-transferred molecules are capable of altering normal cell metabolism but are unable to induce stable mutations, lack the potential to self-replicate, and their effects on cells are inherently self-limiting due to their inevitable degradation and dilution. Indeed, several of the studies described above have shown that when cancer EV exposure is terminated, normal cell metabolism can be restored after some time.However, the existence of the phenomenon of field cancerization , in whichttp://clinicaltrials.gov, accessed on 17 April 2017) and recurrent glioblastoma (NCT04863950). However, the results have not yet been reported.Since the effect of field cancerization has been described for a large number of tumors, such as bladder cancer and prostate cancer, the modulation of EV secretion in these tumors may be an important part of therapy that will allow preventing further malignancy of the nearby field and reduce the chance of disease recurrence. At the same time, whereas EVs carry out hundreds of different biologically active molecules into normal cells, it is possible that modulation of EV secretion could be a more advantageous approach to control supporting TME as well as preventing the spread of tumor drug resistance. Suppression of EV released by tumor cells can be achieved clinically with drugs such as amiloride, which blocks both biogenesis and macropinocytosis of EVs, as well as imipramine, calpeptin, manumycin A, etc. ,50. MoreThus, the contribution of EVs to the malignant transformation of normal cells requires the attention of researchers, and in order to successfully investigate this area, future studies should be aimed at suppressing EV biogenesis and subsequent analysis of changes in the phenotype of normal cells.


Mucopolysaccharidosis (MPS) is a group of disorders caused by the reduced or absent activity of enzymes involved in the glycosaminoglycans (GAGs) degradation; the consequence is the progressive accumulation of the substrate  in the lysosomes of cells belonging to several tissues. The rarity, the broad spectrum of manifestations, the lack of strict genotype-phenotype association, and the progressive nature of MPS make diagnosing this group of conditions challenging. Musculoskeletal involvement represents a common and prominent feature of MPS. Joint and bone abnormalities might be the main clue for diagnosing MPS, especially in attenuated phenotypes; therefore, it is essential to increase the awareness of these conditions among the pediatric rheumatology and orthopedic communities since early diagnosis and treatment are crucial to reduce the disease burden of these patients. Nowadays, enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are available for some MPS types. We describe the musculoskeletal characteristics of MPS patients through a literature review of MPS cases misdiagnosed as having rheumatologic or orthopedic conditions. Mucopolysaccharidosis (MPS) are inherited lysosomal storage diseases caused by the deficiency of enzymes necessary for the degradation of glycosaminoglycans (GAGs) such as dermatan, heparan, keratan or chondroitin sulphate . The incThe aim of this review is to elucidate the characteristics of MPS and offer an overview of the possible differential diagnosis from a rheumatological and orthopedic point of view.GAGs are linear sulfated chains, typically associated with proteins to form proteoglycans and represent a crucial component of the extracellular matrix (ECM). GAGs not only have a structural function, but they are also involved in many cellular processes, including cells adhesion, signal transduction and activation of specific inflammatory pathways ,10. DysfERT is the only approved medical treatment for MPS that aims to reduce GAGs synthesis, however, it has no effect on inflammation. In MPS VI rats, treatment with infliximab prevented the elevation of TNF-α and NF-kB signaling not only in the blood but also in articular chondrocytes and fibroblast-like synoviocytes . In anotEven with a broad phenotypic spectrum, the involvement of bone, cartilage, ligaments, tendons, joint capsules, and all the soft tissues near the joints represents a common feature among all MPS types ,37. The The impaired skeletal remodeling and the GAG infiltration of tissues around and within the joints are the causes of joint stiffness and contracture in all but one type of MPS (hypermobility is characteristic of MPS IV) ,6,37. JoMPS IV (Morquio syndrome) is the only MPS type characterized by the presence of joint hypermobility due to several abnormalities of periarticular connective tissues and bones  . ProximaThe GAG deposition in the flexor tendons and capsular tissues is responsible for the trigger finger. Triggering is usually a clinical diagnosis made upon the usual locking and catching symptoms along with ultrasound findings that help understand the underlying cause ,50. IndeThe skeletal alterations seen in MPS and the related radiographic changes are regrouped under the term result from impairment in endochondral and membranous bone growth . All bonKyphosis might occur in all MPS types but is almost always present in severe forms of MPS I ,60. The Hip dysplasia in MPS comes from the variable combination of the flattened acetabulum, hypoplasia of the proximal epiphysis in its medial portion, and coxa valga. Therefore, children may develop several dysfunctions and limitations due to progressive instability and a higher risk of dislocation. Hip dysplasia affects several types of MPS , occurring in almost all subjects with Hurler syndrome (severe MPS I) . Hip paiKnee deformity is usually severe in MPS I, MPS IV, and MPS VI requiring surgery ,66,67. THypoplasia of the odontoid process is frequently observed in MPS, especially in Hurler syndrome and Morquio syndrome. Odontoid hypoplasia is the leading cause of atlanto-axial instability predisposing to subluxation that may result in spinal cord compression in combination with GAG infiltration of the dura ,70,71. TThe treatment approach for skeletal abnormalities in MPS is mainly based on individual cases. However, severe and progressive deformities that may have serious consequences  often require invasive procedures ,74. PatiGrowth impairment is a frequent manifestation of all MPS types and is caused by the alteration of the scheduled chondrocyte maturation in the growth plate . The resNowadays, there are not validated biomarkers able to predict the progression of the disease or the response to therapy. TNF-α levels, regardless of ERT therapy, are higher in patients suffering from MPS when compared with healthy controls, and they are associated with MSK symptoms and limitations. However, elevated levels of TNF-α appear not to correlate with general health score . In MPS,On the other hand, elevated levels of IL-6 seem to predict progression in joint contracture, short stature, and hip dysplasia . SimonarPatients with MPS are theoretically at risk of low bone mass density (BMD) due to limited mobility secondary to bone dysplasia and contractures. The development of bone mass in children is a dynamic process of adapting to mechanical stimuli. Physical exercise is necessary to stimulate bone cells in order to reinforce bone structure and promote bone remodeling. Fractures after low energy trauma have been described in MPS patients but their incidence is low ,91,92,93The correlation between underlying mutations and residual enzymatic activity causes the broad phenotypic spectrum, impacting the timing of the manifestations’ development ,3. The iThe most common ocular finding in MPS is corneal clouding which may severely impact visual acuity. It is due to the deposition of GAG granules with a yellowish-grey color in all corneal layers. MPS I and MPS VI have a more severe manifestation than MPS IV and MPS VII; on the other hand, patients with MPS II and III might present very mild corneal involvement . CornealHeart involvement is a common feature across all MPS types representing a significant cause of morbidity and mortality ,102. PatExcept for MPS III, cognitive impairment is uncommonly seen in the early stage of attenuated forms. Nevertheless, MPS I and II patients may develop variable degrees of intellectual disabilities during the disease course ,107.Among the other possible manifestations, the respiratory system is frequently involved  due to the GAG submucosal deposition causing airway obstruction, potentially, in every part of the respiratory tract . TracheoThe accumulation of undegraded GAGs increases these substrates in urine, blood ,113, andPrenatal diagnosis is based on measuring the enzyme activity in cultivated chorionic villi or amniocytes in case of a family history of MPS or hydrops fetalis. Testing for MPS I has been included in the United States newborn screening panel since 2016, and it is currently underway in other countries ,122.The suspicion of a possible diagnosis of MPS should rise based on the concomitant presence of some of the abovementioned manifestations. However, physicians may focus their attention to symptoms related to each own expertise and overlook other possible hints. In this regard the anamnesis plays a crucial role; a careful medical history of the patient and the family may reveal unexpected clues. A semi-structured medical history form has been proposed to help with medical interviews of suspected MPS patients . Further