Immune checkpoint inhibitors (ICIs) are commonly used to treat lung cancer patients, but their use can lead to immune-related adverse events (irAEs), which pose a challenge for treatment strategies. The impact of irAEs on the incidence of COVID-19 pneumonia in lung cancer patients during the ongoing COVID-19 pandemic is unclear. This study aims to investigate the association between irAEs and COVID-19 pneumonia in lung cancer patients receiving ICIs.We conducted a cross-sectional study of lung cancer patients who received ICIs and were infected with COVID-19 due to the Omicron variant between December 2022 and February 2023 in China. We collected data on irAEs and COVID-19 outcomes. Logistic regression analyses were used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between irAEs and the incidence of COVID-19 pneumonia.A total of 193 patients were enrolled, with 72 patients (37.30%) in the irAEs group and 121 patients (62.70%) in the non-irAEs group. Twenty-six patients (13.47%) developed COVID-19 pneumonia and 6 patients (3.11%) progressed to severe cases after COVID-19 infection. Multivariate logistic regression showed that the lung cancer patients who experienced irAEs was significantly associated with a higher incidence rate of COVID-19 pneumonia .Our study suggests that lung cancer patients receiving ICIs and experiencing irAEs may have a higher risk of developing COVID-19 pneumonia due to the Omicron variant. Therefore, close monitoring of these patients during the COVID-19 pandemic is necessary to mitigate this risk. Immune checkpoint inhibitors (ICIs) are commonly used to treat lung cancer, either alone or in combination with chemotherapy and radiotherapy , 2. WhilThe COVID-19 pandemic has created significant disruptions in healthcare and social and economic systems worldwide –13. IrAETherefore, this cross-sectional study seeks to investigate the association between irAEs and COVID-19 pneumonia in lung cancer patients infected with the Omicron variant in China. By understanding the impact of irAEs on COVID-19 pneumonia incidence, we can improve the management and monitoring of these patients during the ongoing pandemic.This cross-sectional study was conducted at the Second Affiliated Hospital of Nanchang University in China between December 8, 2022 and February 1, 2023, during the Omicron variant epidemic. We reviewed lung cancer patients diagnosed with COVID-19, who received at least one cycle of immune checkpoint inhibitors (ICIs), including programmed cell death protein 1 (PD-1)/pro-programmed death-ligand 1 (PD-L1) inhibitors, either as monotherapy or combination therapy at our institution. All enrolled patients had confirmed lung cancer by pathology and COVID-19 diagnosis through reverse transcriptase-polymerase chain reaction (RT-PCR) using nasopharyngeal swabs. Patients diagnosed solely on the basis of rapid antigen testing were excluded. This study adhered to the Declaration of Helsinki principles and followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines . The insWe collected data on patient characteristics, including age, sex, tumor stage, Eastern Cooperative Oncology Group (ECOG) performance status score at the time of COVID-19 infection, previous treatment history, ICI-related data, irAE-related data, and routine blood tests. The Common Terminology Criteria of Adverse Events version 5.0) was utilized to grade irAE at its peak severity .0 was ut.We used R software (version 3.6.3) to analyze all data. Categorical variables were reported as frequency and percentage, while continuous variables were expressed as mean ± standard deviation. Groups were compared using independent sample t-tests or analysis of variance (ANOVA). Univariate and multivariate logistic regression analyses were conducted to evaluate the connection between immune-related adverse events (irAEs) and COVID-19 pneumonia incidence; Odds ratios (ORs) with 95% confidence intervals (CIs) were reported. Statistical significance was defined as P < 0.05.We excluded 46 patients with non-lung cancer, leaving 326 patients diagnosed with both lung cancer and COVID-19 infection for further investigation. Out of these, 133 patients who did not receive immune checkpoint inhibitor (ICI) treatment were excluded, resulting in a total of 193 patients enrolled in the study. Of these, 72 patients (37.30%) experienced immune-related adverse events (irAEs), while 121 patients (62.70%) did not belong to the irAEs group . Prior research has established that reduced CD4+/CD8 + T cells and lymphocyte count are associated with severe COVID-19  and mortWhile our study has limitations - including its retrospective design and small sample size - it provides valuable real-world data from a Chinese institution during a specific timeframe. What’s more, our study specifically focused on lung cancer patients used ICIs. This focus was chosen because distinguishing immunotherapy-related pneumonitis from COVID-19 pneumonia when both occur simultaneously can be challenging, and CT scanning is often necessary for tumor evaluation and monitoring adverse reactions. Our analysis was limited to the Omicron variants (BA.2 and BA.5), and given the emergence of new strains, more investigation is needed to explore the association between irAEs and COVID-19 infection outcomes in cancer patients. Nonetheless, our findings underscore the importance of close monitoring and timely intervention for lung cancer patients receiving ICIs who contract COVID-19.In conclusion, our study firstly evaluated the impact of irAEs on COVID-19 pneumonia incidence in lung cancer patients receiving ICI therapy. We found that patients who developed irAEs were at higher risk of contracting Omicron variant COVID-19 pneumonia. This highlights the importance of increased vigilance for COVID-19 infections in this patient population, along with closer monitoring, including timely chest CT scans, and potentially more aggressive treatment if necessary.


Polyethylene oxide (PEO)-based solid-state electrolytes for lithium-ion batteries have garnered significant interest due to their enhanced potential window, high energy density, and improved safety features. However, the issues such as low ionic conductivity at ambient temperature, substantial ionic conductivity fluctuations with temperature changes, and inadequate electrolyte interfacial compatibility hinder their widespread applications. Electrospinning is a popular approach for fabricating solid-state electrolytes owing to its superior advantages of adjustable component constitution and the unique internal fiber structure of the resultant electrolytes. Thus, this technique has been extensively adopted in related studies. This review provides an overview of recent advancements in optimizing the performance of PEO solid-state electrolytes via electrospinning technology. Initially, the impacts of different lithium salts and their concentrations on the performance of electrospun PEO-based solid-state electrolytes were compared. Subsequently, research pertaining to the effects of various additives on these electrolytes was reviewed. Furthermore, investigations concerning the enhancement of electrospun solid-state electrolytes via modifications of PEO molecular chains are herein detailed, and lastly, the prevalent challenges and future directions of PEO-based solid-state electrolytes for lithium-ion batteries are summarized. The escalating energy crisis and environmental issue necessitate the developments of eco-friendly and sustainable energy alternatives . Among dTo mitigate these issues and promote the development of lithium-ion batteries, researchers have proposed substituting the conventional electrolyte and separator with mechanically robust solid-state electrolytes, as shown in Generally, solid-state electrolytes are primarily classified into two types: inorganic ceramics and organic polymers. Inorganic ceramic-based solid-state electrolytes possess superior electrical conductivity, yet their inadequate mechanical properties and high interface impedance with the electrode limit their use in solid-state electrolytes. In contrast, although the electrochemical properties of organic polymer-based solid-state electrolytes are inferior to those of inorganic ceramic-based solid-state electrolytes, their excellent plasticity and extraordinary mechanical properties make them preferred choices for solid-state electrolytes in lithium-ion batteries.Commonly, the most used materials for organic polymer-based solid-state electrolytes of lithium-ion batteries include polymethyl methacrylate (PMMA) , polyacrCurrent methodologies for preparing solid-state electrolytes include casting, hot pressing, and electrospinning. Among them, the solution casting method is simple and low-cost and easily adjusts the thickness of the film . The hotIn summary, the introduction of electrospinning in the production of PEO-based solid-state electrolytes of lithium-ion batteries leverages the advantages of both PEO as a solid-state electrolyte material and the unique properties of electrospun micro/nanofibers. Accordingly, an increasing number of researchers are devoting their efforts toward studies related to electrospun PEO-based lithium-ion solid-state electrolytes. Presently, these explorations predominantly concentrate on enhancing the electrochemical, mechanical, and thermal performances of electrospun PEO-based lithium-ion solid-state electrolytes. Although noticeable advancements have been accomplished in recent years, the commercialization of electrospun PEO-based lithium-ion solid-state electrolytes demands a further refinement. In this review, a variety of strategies for performance improvement of electrospun PEO-based solid-state electrolytes have been concluded . FirstlyThe efficiency of electrospun PEO-based solid-state electrolytes for lithium-ion batteries is mainly dependent on their electrochemical characteristics, mechanical properties, and thermal stability. Among them, electrochemical performance is paramount, which is determined by several significant factors such as lithium-ion conductivity, lithium-ion transfer number, and electrochemical stability window. Currently, there exists a considerable disparity between the electrochemical performance of solid-state electrolytes and conventional liquid electrolytes, which poses a significant barrier to further advancements of PEO-based solid-state electrolytes.−6 S/cm at 25 °C).The ionic conductivity, a critical indicator in evaluating electrochemical performance, represents the transport rate of charged ions within a material. The low conductivity confines the battery to operate at a relatively small charge/discharge rate, thereby making it challenging to satisfy the operational requirements of standard devices. Plus, the semi-crystalline structure of PEO results in a high degree of crystallinity inside itself at room temperature, characterized by an abundance of internal crystalline regions. Direct usage of PEO as a solid-state electrolyte base for lithium-ion batteries would severely inhibit lithium-ion transportation within and between PEO chain segments owing to these crystalline regions. Consequently, these would lead to inefficient lithium-ion transport, thereby decreasing the ionic conductivity of PEO-based solid-state electrolytes , lithium perchlorate (LiClO4), lithium tetrafluoroborate (LiBF4), lithium chloride (LiCl), and lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) ,8079,80]ofillers . This ca2 and tin dioxide (SnO2) to PEO during electrospinning. The experimental results indicated that the inclusion of nanoparticles improved the tensile strength of the electrospun fibers, suggesting a potential enhancement in the mechanical properties of electrospun PEO-based solid-state electrolytes. Banitaba et al. [2, Al2O3, and clay nanoparticles on the electrochemical properties of electrospun PEO-based solid-state electrolytes. Their findings demonstrated that the doped nanoparticles influence the electrical conductivity of the precursor solution, affecting the electrospinning process. A moderate amount of nanoparticles can effectively raise the fiber stretching rate and diminish the fiber diameter. However, an excessive amount of nanoparticles causes an increase in the solution viscosity, leading to the intertwining of polymer chains and the subsequent gradual increments in fiber diameters [Zaccaria et al.  investiga et al.  comparediameters . Additioiameters .2 as nanoparticle fillers to fabricate electrospun PEO-based solid-state electrolytes, which exhibited superior mechanical properties and cycling stabilities. In some non-electrospun solid-state electrolytes, the doping of graphene improved their usability for practical applications. Based on this study, Abdollahi et al. [Another study by Banitaba et al.  employedi et al.  applied 2 into the core of electrospun PEO micro/nanofibers using a coaxial electrospinning method and subsequently incorporated SiO2 into their shells  and prepared PEO-based solid-state electrolytes via casting method  into electrospun PEO-based solid-state electrolytes has been found to enhance their cycling stability . Comparer shells a. This an et al.  investigg method g. These 0.33La0.557TiO3 (LLTO) nanowire fillers via electrospinning and then cast PEO solutions onto them to create PEO-based solid-state electrolytes. When the content of the LLTO nanowire filler was 5 wt%, the ionic conductivities at room temperature and 60 ℃ increased to 5.53 × 10−5 S/cm and 3.63 × 10−4 S/cm, respectively. Such fillers can also increase the electrochemical stability of the prepared solid-state electrolyte whose electrochemical window achieve a value of 4.75 V. Additionally, Wang et al. [7La3Zr2O12 (LLZO) nanowire fillers. With an LLZO nanowire filler of 10 wt%, the ionic conductivities of the resulting solid electrolyte reached 9.87 × 10−5 S/cm and 8.66 × 10−4 S/cm at 30 °C and 60 °C, respectively. Notably, these electrolytes maintained a relatively stable voltage and exhibited adequate discharge-specific capacity even after several cycles.Nanowire fillers, as compared to nanoparticles, can form a large number of continuous ion transport channels in the solid-state electrolyte, thus increasing the migration rate of lithium ions and achieving a higher ionic conductivity . In contg et al.  also preg et al.  employedThus far, nanomaterials used in electrospun PEO-based solid-state electrolytes can be primarily classified into three categories: nanoparticles, nanotubes, and nanowire frameworks, as shown in The third additive for enhancing the performance of electrospun PEO-based solid-state electrolytes involves blending PEO with other polymers. This approach allows leveraging the advantageous properties of both materials while mitigating their inherent disadvantages. Notably, this method can decrease PEO crystallinity, thereby enhancing the ionic conductivity of electrospun PEO-based solid-state electrolytes. The preparation process for precursor solutions of polymer blends is straightforward, and their composition can be easily adjusted, thus offering a greater convenience relative to other methods .−3 S/cm and an electrochemical window of 4.8 V at 303 K as well as superior specific capacity and coulombic efficiency. Prasanth et al. [PVDF owns an excellent chemical stability and a high dielectric constant , which ih et al.  prepared−4 S/cm while maintaining superior mechanical properties  are considered an alternative polymer matrix for PEO-based solid electrolyte due to their exceptional mechanical properties, thermal stability, and safety . Tang et2 nanoparticles to the PEO and PVDF–HFP–g-AMPSLi solution to obtain two electrospinning precursor solutions, respectively. The two electrospun nanofibers displayed a stable three-dimensional network structure after a hot pressing. These nanofibers demonstrated ionic conductivity and electrochemical windows of 5.4 × 10−5 S/cm and 3.8 V at 60 °C, respectively. Moreover, they exhibited a superior multiplicative performance and a remarkably stable cycling performance in battery tests.Additionally, surface coverage of electrospun PEO fibers with other polymers also presents a viable strategy for improving the performance of PEO-based solid-state electrolytes. Hafner et al.  applied −5 S/cm at 60 °C.Efficient electrospun PEO-based solid-state electrolytes can also be fabricated by applying PEO as an auxiliary material onto other electrospun polymer fibers via casting. For instance, Wang et al.  producedPAN allows the cell to function at elevated temperatures and thus serves as an effective material of solid-state electrolyte due to its outstanding thermal stability. Ma et al.  soaked e−4 S/cm at a temperature of 60 °C while exhibiting adequate stability in electrical cycling tests.Thermoplastic polyurethane (TPU), known for its excellent elastomeric properties, is often selected as a matrix material of the solid-state electrolyte due to its excellent mechanical features . However−4 S/cm at 30 °C.Hydrogen-bonding interactions between the amide bond on the poly (PMIA) benzene ring, PEO molecular chain, and lithium salt anion can inhibit PEO crystallization processes and promote LiTFSI dissociation, thereby enhancing ionic conductivity of the resulting solid-state electrolyte. Moreover, hydrogen bonding between PMIA and PEO can lead to the formation of a three-dimensional network structure in the solid-state electrolyte, improving its mechanical properties and thermal stability . Gao et In brief, this section mainly involves blending PEO with other polymer materials endowing robust mechanical properties, such as PVDF, aiming to enhance the mechanical features of electrospun PEO-based solid-state electrolytes. Furthermore, due to potential interactions between the distinct polymer materials, controllable adjustments in their proportions can effectively decrease the overall crystallinity, thereby boosting the ionic conductivity of the solid electrolyte.An alternative method to enhance the performance of electrospun PEO-based solid-state electrolytes involves the modification of PEO molecular chains. This avenue primarily relates to the formation of copolymers from PEO monomers and one or more additional monomers through polymerization reactions. This includes the production of alternating copolymers, random copolymers, block copolymers , and gra−4 S/cm.Block polymers are macromolecules comprising two or more chemically distinct polymers joined by covalent bonds. Appropriate selections of different polymers with varied molecular weights can yield a multitude of tailor-made materials with specific physicochemical properties. Despite the extensive applications of block copolymers in various fields, their utilization in electrospun PEO-based solid-state electrolytes for lithium-ion batteries remains in the nascent stages. Block copolymers composed of PEO and polystyrene (PS) demonstrate favorable microstructures when they are combined with lithium salts , making Watanabe et al.  fabricatCopolymerizing PEO with other polymers also facilitates the preparation of PEO-based solid-state electrolytes with excellent properties. For instance, random copolymer PEO–co-PPO-based polymer electrolytes exhibit low crystallinity, superior mechanical properties, and adequate thermal stability. However, no scholarly work has yet reported the preparation of such materials into PEO-based solid-state electrolytes via electrospinning for lithium-ion batteries.With the rapid developments of wearable electronic products, flexible, compact, and high-energy-density batteries have recently attracted lots of attention. Lithium-ion batteries prepared with a solid-state electrolyte can maintain a high energy density while exhibiting a desirable safety performance, which could potentially be applied on a large scale for wearable electronic products in future.Compared to other polymer materials, PEO is widely used in solid-state electrolytes due to its excellent lithium salt solubility and ionic conductivity. However, current PEO-based solid-state electrolytes face challenges such as poor mechanical properties and a narrow electrochemical window. Although they show good ionic conductivity, the developments of PEO-based solid-state electrolytes still lag behind that of traditional liquid electrolytes. To further advance the development of high-energy-density and ultra-safe lithium-ion batteries, this review discusses various strategies to enhance the electrochemical and mechanical performance of electrospun PEO-based solid-state electrolytes. In general, selecting suitable lithium salts with appropriate concentrations can improve the electrochemical performance of the obtained PEO-based solid-state electrolytes, although the impact is usually modest. Additionally, incorporating plasticizers into the PEO-based solid-state electrolyte can significantly improve its electrochemical ability but may compromise its mechanical properties and the safety. Therefore, current research efforts are primarily focused on enhancing their electrochemical and mechanical capabilities by incorporating inorganic nanoparticles or composing with other polymer materials. Inorganic nanofillers can enhance mechanical properties and improve the ion conduction by optimizing the transfer channels within electrospun micro/nanofibers. Similarly, blending electrospun PEO with other high-molecular-weight polymers not only reduces the crystallinity within the material but also combines their respective advantages to realize both high ionic conductivities and strong mechanical properties. And this preparation process has low production costs and an easy operation, facilitating a scale-up production. Finally, modification of PEO molecular chains can inhibit the growth of lithium dendrites and optimize the electrochemical performance abilities of the PEO-based solid-state electrolytes. Generally speaking, employing a multi-strategy approach can combine the strengths of different strategies and lead to further improvements in the performance of the resulting material. This allows for a comprehensive enhancement of various aspects of the material’s properties. This review aims to advance the future research on obtaining electrospun PEO-based solid-state electrolytes with superior performance for large-scale applications.Although the aforementioned improvement strategies have contributed to the enhancement of the electrochemical and mechanical performance of electrospun PEO-based solid-state electrolytes, these electrolytes still encounter significant challenges in practical applications. For instance, significant changes of their ionic conductivity still exist as temperature varies, which seriously restricts its applications in lithium-ion batteries. Moreover, the technology of preparing PEO-based solid-state electrolytes by electrospinning is not yet mature, and there is still room for optimization of electrospinning process parameters. In addition, this technique is not yet feasible for large-scale industrialization. Therefore, the future optimization for electrospun PEO-based solid-state electrolytes primarily focuses on the following three aspects: (1) exploration of novel PEO-based materials is needed to simultaneously enhance ion conductivity and mechanical properties and reduce their sensitivity to temperature fluctuations. (2) optimization of the electrospun fiber’s morphology, diameter, arrangement, and other structures is needed to achieve a better ion transport efficiency and electrochemical performance; (3) realization of the industrial-scale production of electrospun PEO-based solid-state electrolyte is also necessary.


It is reported that the elongated amylosic chain at the nonreducing end in enzymatic polymerization twines around guest polymers with suitable structures and moderate hydrophobicity, which is dispersed in aqueous polymerization media, to form amylosic nanostructured inclusion complexes. As the image of this system is similar to how vines of a plant grow around a support rod, this polymerization has been named ‘vine-twining polymerization’. In particular, the helical inclusion behavior of the enzymatically produced amylose toward hydrophobic polyesters depending on their structures, e.g., chain lengths and substituents, has been systematically investigated in the vine-twining polymerization field. Furthermore, amylosic supramolecular network materials, such as hydrogels, are fabricated through vine-twining polymerization by using copolymers, where hydrophobic polyester guests or maltooligosaccharide primers are covalently modified on hydrophilic main-chain polymers. The vine-twining polymerization using such copolymers in the appropriate systems induces the formation of amylosic nanostructured inclusion complexes among them, which act as cross-linking points, giving rise to supramolecular networks at the nanoscale. The resulting materials form supramolecular hydrogels, films, and microparticles.This review article presents the biomimetic helical inclusion of amylose toward hydrophobic polyesters as guests through a vine-twining polymerization process, which has been performed in the glucan phosphorylase (GP)-catalyzed enzymatic polymerization field to fabricate supramolecules and other nanostructured materials. Amylose, which is a representative abundant glucose polymer (polysaccharide) with left-handed helical conformation, is well known to include a number of hydrophobic guest molecules with suitable geometry and size in its cavity to construct helical inclusion complexes. Pure amylose is prepared through enzymatic polymerization of α- Since t7), is examined in the presence of suitable hydrophobic polymers, dispersed in aqueous buffer media , iBased on the above results, the author concludes that the following characteristics are typically required for the polymeric guests that induce helical inclusion by amylose in the vine-twining polymerization system. Because the vine-twining polymerization is carried out in aqueous buffer as the solvent for the GP-catalyzed enzymatic polymerization, polymeric guests should contain relatively polar groups in their main chains for their dispersibility in such aqueous media. Polymeric guests should also exhibit moderate hydrophobicity for both dispersion in aqueous media and hydrophobic interaction with the amylosic cavity. Because a sufficient cavity size is not provided from amylose to include bulky polymeric gusts, slender polymers without bulky substituents can be conceived to be suitable guests for the vine-twining polymerization.d-alanine) (PDAla), poly (PLAla), and poly (PDLAla), only PDAla was helically included in the amylosic cavity in the field of GP-catalyzed polymerization. The helical directions of amylose and PAlas are key factors in explaining the stereoselective inclusion behavior in the vine-twining polymerization. Both amylose and PDAla construct a left-handed helical conformation, suitably giving rise to helical complexation between the two polymeric chains to obtain the amylosic inclusion complex.According to the above characteristics, poly(tetramethylene carbonate) (PTMC), with relatively polar carbonate groups in the main chain, that is, a hydrophobic polycarbonate, was found to act as a polymeric guest for the vine-twining polymerization to construct an amylosic inclusion complex . PolycarHydrophobic polyesters also provide suitable characteristics as polymeric guests for vine-twining polymerization. The author has extensively investigated the helical inclusion behavior of amylose toward such polyester guests depending on their structures. The following section deals with the significant findings in the vine-twining polymerization using hydrophobic polyester guests.7 primer in aqueous buffer, which dispersed the polyester guests, amylose–polyester inclusion complexes were produced as precipitates. The characterization results of the precipitates fully supported the inclusion complex structures. In the same operation, a hydrophobic polyester with a shorter methylene chain than PBL was used, that is, poly(b-propiolactone) (PPL), and an incomplete inclusion of PPL in the amylosic cavity was observed. Furthermore, poly(glycolic acid) (PGA), which has an additionally shorter methylene chain, did not form the amylosic inclusion complex in the GP-catalyzed enzymatic polymerization in aqueous buffer, where a pure amylose was produced. The results of PPL and PGA were owed to the poor dispersibility of these polymers in aqueous media, as a result of their high crystallinities. When the copolyester, poly(glycolic acid-co-ε-caprolactone) (P(GA-co-CL)), was used as a polymeric guest, the corresponding amylosic inclusion complex was obtained using the vine-twining process in GP-catalyzed enzymatic polymerization. This was because of its higher dispersibility in aqueous media than that of the pure PGA, due to reduction in crystallinity by incorporating the PCL chains . It. It38]. 2CH2C(C=O)OCH2CH2CH2CH2O-) also forms the amylosic inclusion complex through vine-twining polymerization . A . A l-lacR)-3-hydroxybutyrate] (PRHB), as a guest polyester. As a result, only its oligomer with low molecular weight (approximately 500) gave rise to an amylosic inclusion complex under specific conditions in the GP-catalyzed enzymatic polymerization field as follows [R)-3-hydroxybutyrate] (ORHB) is first examined at 70 °C, higher than that for the general vine-twining polymerization (at 45–50 °C), catalyzed by thermostable GP (from Aquifex aeolicus VF5), which shows stability at such a higher temperature. This polymerization system produces a water-soluble amylosic oligomer (short α(1γ4)-glucan) without double-helical assembly, called single amylose, that weakly interacts with ORHB. The polymerization mixture is then cooled to 45 °C over 7 h to carry out the further enzymatic propagation from the amylosic oligomer, catalyzed by GP, that helically includes ORHB to obtain the amylosic inclusion complex , that is, poly[( follows . The enz complex .7-block-PLLA (a primer–guest conjugate)   42]. Th. Th7-blo7–block-PLLA conjugates, which comprise a primer segment, linked to the carboxylate or hydroxy terminus of PLLA in the molecule. The GP-catalyzed enzymatic polymerization in the presence of both conjugates produces the inclusion supramolecular polymers according to the abovementioned process through the vine-twining polymerization process  was also carried out through the GP-catalyzed enzymatic polymerization using a branched Glc7-PLLA (Glc7-PLLA2) conjugate according to the vine-twining polymerization process . Furthermore, a cryogel with porous morphology was produced through lyophilization of the hydrogel.The synthesis of a hyperbranched supramolecular polymer composed of a continuum of amylose-PLLA inclusion complexes (amylose-PLLA process  44]. A . A 2) waThe vine-twining polymerization process for the formation of inclusion complexes with polyester guests has been applied to the hierarchical nanoarchitecture of amylosic network materials, which appropriately form supramolecular materials, such as hydrogels and films . Cross-lgraft-δ-valerolactone) ((PAA-Na)-g-PVL) was used. When GP-catalyzed enzymatic polymerization is examined in the presence of (PAA-Na)-g-PVL in aqueous buffer, the reaction mixture completely turns into the hydrogel form . Be. BegraftA-g-PCL) , a self-graft-poly(ε-caprolactone) (NaCMC-g-PCL)   49]. Th. ThgraftC-g-PCL) . The gel7 primer-modified Pg-GA and NaCMC  at elevated temperatures has been found to produce a water-soluble amylosic product with a single chain  in an equilibrium state between polymerization and phosphorolysis , the enzymatic polymerization from the primer-modified Pg-GA was first conducted with or without PLLA at 80 °C for single-amylose-chain production [The fabrication of macroscopic structures from amylose-grafted Pg-GA networks was attempted . Becauseoduction . Double-The present review comprehensively overviewed the helical inclusion of amylose toward various hydrophobic polyester guests in the biomimetic vine-twining polymerization process. The investigation examined, regarding GP-catalyzed enzymatic polymerization, whether amylosic inclusion complexes form from the hydrophobic polyesters depending on their structures. Polyesters, such as PCL, PVL, and PBL, which exhibit dispersion ability in aqueous polymerization media, are helically included in the amylosic cavity via the vine-twining polymerization process. The highly crystalline polyesters, that is, PPL and PGA, show low dispersibility in aqueous media, leading to difficulty in the progress of vine-twining polymerization. Dispersion in the emulsion system and a reduction in the crystallinity by incorporating the other polyester in the main chain from PPL and PGA, respectively, induce the vine-twining process in the GP-catalyzed enzymatic polymerization. The helical directions of the polyesters with methyl substituents depending on chirality strongly affect the helical inclusion by the amylosic cavity in the vine-twining polymerization process. Amylosic supramolecular networks at the nanoscale are hierarchically fabricated through the construction of cross-linking points from the amylose–polyester inclusion complexes in the vine-twining process in the GP-catalyzed enzymatic polymerization using polyester-grafted and primer-modified hydrophilic polymers. The products formed supramolecular materials, such as hydrogels, films, and microparticles. A variety of polyester structures are available via simple ring-opening polymerization of the corresponding cyclic monomers, compared with other hydrophobic polymers containing polar groups, such as polyethers and polycarbonate. Accordingly, a systematic study on the vine-twining polymerization using the polyester guests has previously been investigated, as mentioned above. In the future, new hydrophobic polyesters will be found to act as guests for vine-twining polymerization, which could also be potentially used for the fabrication of amylosic supramolecular network nanomaterials, like gels, with unique functions. In addition, the recycling and reuse of GP are key techniques for the scale-up of the present amylosic supramolecular network materials in practical applications. Such procedures will be developed by employing enzymological technology in the GP-catalyzed enzymatic polymerization.


Functional arterial blood pressure waveform (ABW) features are incompletely understood in the context of burn injury and resuscitation. In this work, we analyzed the variability of pulse pressure (PPV), systolic pressure (SPV), stroke volume (SVV), and five measures of heart rate variability (HRV) in time and frequency domains as potential indicators of burn resuscitation adequacy in a large total body surface area (TBSA) porcine model with varying degrees of intravenous fluids.Data were collected from 21 anesthetized and mechanically-ventilated pigs , which were instrumented for continuous hemodynamic monitoring for 24 hours. Urinary output (UO), hematocrit (HCT) and cardiac output (CO) were also measured at various time points. The animals were divided into Paradigm 1 (P1): no resuscitation (N=7); Paradigm 2 (P2): fluid titration to UO (N=8); and Paradigm 3 (P3): deliberate over-resuscitation with consistent high fluid rates (N=6). We calculated PPV, SPV, SVV, and five widely used HRV measures: normalized low-frequency power (NLF), high-frequency power (NHF), very-low-frequency power (VLF), low-frequency-high-frequency (LFHF) ratio, and the root-mean-squared of the duration difference in consecutive beats (RR-RMS). All features were averaged on an hourly basis and their potential in tracking reference CO and SV was investigated via linear regression and correlation analysis after calibrating the data to SV and CO on an individual basis.PPV, SPV, SVV, and RR-RMSE showed statistically distinguishable trends for different paradigms, as depicted in Figure 1, along with averaged trends for lab measurement. PPV, SPV, and SVV tracked the CO the best and were comparable to UO, however, SVV was far superior in tracking SV. A regression analysis between these four features and UO showed a very low level of agreement despite comparable tracking of SV and CO. HRV measures did not distinguish resuscitation paradigms and were inferior in tracking CO and similar in tracking SV to aforesaid features.PPV, SPV, SVV, and RR-RMSE may potentially complement UO in the hemodynamic assessment of the burn patients and outperform other HRV measures in general. Future work will optimize the extraction of strategic informative features of ABW analysis in the various fluid status states post-burn injury.Titrating burn resuscitation rates to have protocols including PPV, SPV, SVV, and RR-RMSE alongside other standard metrics may offer precision medicine strategies to fine-tune IV fluid rates.


Extracellular vesicles are small membranous particles secreted by cells. Extracellular vesicles facilitate the transportation of biomolecules, such as protein, RNA, and DNA fragments, to communicate with neighboring and distant cells. Cancer cells use extracellular vesicles to hijack the immune system and induce cancer-promoting signals. Modifying extracellular vesicles using surface engineering tools allows the addition of biomolecules for targeted delivery, thus modulating the hijacked tumor immune microenvironment to improve therapeutic efficacy. This review article discusses extracellular vesicle modification strategies explicitly focusing on the approaches used for surface engineering. We revisit the work carried out on the surface-engineered extracellular vesicle and its application in immunomodulating tumor microenvironments for cancer immunotherapy.Extracellular vesicles (EVs) are lipid bilayer-enclosed bodies secreted by all cell types. EVs carry bioactive materials, such as proteins, lipids, metabolites, and nucleic acids, to communicate and elicit functional alterations and phenotypic changes in the counterpart stromal cells. In cancer, cells secrete EVs to shape a tumor-promoting niche. Tumor-secreted EVs mediate communications with immune cells that determine the fate of anti-tumor therapeutic effectiveness. Surface engineering of EVs has emerged as a promising tool for the modulation of tumor microenvironments for cancer immunotherapy. Modification of EVs’ surface with various molecules, such as antibodies, peptides, and proteins, can enhance their targeting specificity, immunogenicity, biodistribution, and pharmacokinetics. The diverse approaches sought for engineering EV surfaces can be categorized as physical, chemical, and genetic engineering strategies. The choice of method depends on the specific application and desired outcome. Each has its advantages and disadvantages. This review lends a bird’s-eye view of the recent progress in these approaches with respect to their rational implications in the immunomodulation of tumor microenvironments (TME) from pro-tumorigenic to anti-tumorigenic ones. The strategies for modulating TME using targeted EVs, their advantages, current limitations, and future directions are discussed. Extracellular vesicles (EVs) are small membrane-bound particles that mediate cell-to-cell communication. All cells secrete EVs generated via intrinsic cellular biogenic pathways as the cargo of signaling molecules typically involved in cell regeneration, differentiation, and proliferation . As smalThe cellular uptake mechanism of EVs depends on the recipient cells’ membrane receptor components. The surface proteins and receptors facilitate the initial adhesion of exosomes to recipient membranes . Upon adTumors deploy EVs to alter stromal cell behavior and promote metastasis. EVs are increasingly shown to be involved in the invasive–metastatic cascade. Tumors secrete a heterogenous population of EVs to shape the tumor microenvironment (TME) and change its malignant behavior in response to immune surveillance or therapies . EVs couSurface-engineered EVs allow preferential internalization through one mechanism over another when modified to target a specific signaling pathway. The membrane proteins and lipids modifications can achieve cell-specific delivery or interactions with great promises in cancer immunotherapy . The IntEVs have a unique origin and molecular composition that renders them highly stable in the bloodstream; thus, they have potential in cancer immunotherapy . HoweverAs a transport cargo, the intrinsic property that allows EVs to bypass immune surveillance favors EV fabrication. Cell genetic engineering allows the obtention of desired EV functions to target specific cell types . A good Another EV surface modification strategy is attaching monoclonal antibody-derived chimeric antigen receptors (CARs) for specific binding on cancer cells . CARs arThe surface engineering of exosomes by the aptamer-based DNA nano assemblies has significantly benefited the theragnostic applications  36]. TR. TR36]. One of the most widely used exosome surface proteins to display a targeting motif is LAMP-2B, a lysosome-associated membrane protein (LAMP) member. LAMP-2B is predominantly available on the lysosomes and endosomes, and a smaller fraction is expressed on the cell surface. The abundant expression of the LAMP-2B protein was reported on the dendritic cell-derived exosomes . LAMP-2BSimilarly, the N-terminal of LAMP2B fused with interleukin-3 receptor (IL-3Rα) improved exosome-targeting efficiency at treating chronic myeloid leukemia (CML) . The IL-In addition to LAMP2B, the transmembrane protein platelet-derived growth factor receptor (PDGFR) is another commonly used membrane display. In a study, the PDGFR transmembrane region GE11 (YHWYGYTPQNVI) was genetically fused using a phage-display vector transfected in the 293T cells to produce the exosome with GE11 . These eExosomes highly express signature proteins, such as CD9, CD63, and CD81, that can be fused with the targeting molecules . In partSimilarly, functionally customized exosomes were made through genetic modification to accommodate actively integrated membrane proteins or soluble protein cargos (GIFTed-Exos) . The remIn a proof-of-concept study, genetic manipulation of exosomes was achieved via transfection of lung adenocarcinoma cells, SK-LU-1 cells. The isolated exosomes were shown to promote the transmission of TAMs to the M1 active profiles . LikewisSimilarly, the NIH 3T3 cell lines were engineered to express IL-15/IL-15R𝛼 on the cell membrane. Nanovesicles derived through extrusion overexpressing the IL-15/IL-15R𝛼 (IL-15/IL-15R𝛼-NVs) complex boosted the proliferation, activation, and survival of tumor-infiltrated T-cells via the trans presentation of IL-15 to T-cells and the eliminated tumor. These nanovesicles also promote the activation and proliferation of tumor-specific CD8+ T-cells and TRM cells, effectively inhibiting melanoma growth in mice and increasing the survival rate of mice. An IL-15/IL-15R𝛼-nanovesicle complex was used as cargo to carry the PD-1/PD-L1 inhibitor to a T-cell. The PD-1/PD-L1-inhibitor combined with the IL-15/IL-15R𝛼-nanovesicle complex significantly enhanced the anti-tumor responses . SimilarCXCR4 is a chemokine receptor that regulates T-cell migration. In a study, MSCs were infected with the PGMLV-PA6-containing virus expressing CXCR4 protein and GFP . The derIn another study, MSCs were transfected with the pEGFP-C1-GFE1-LAMP2B plasmid. The functional exosome (GExoI) was decorated with pulmonary targeting peptide GFE1 on the membrane surface. These exosomes were loaded with the PI3Kγ inhibitor (IPI549) to suppress melanoma lung metastasis. In a postoperative mouse model, the accumulation of intravenously injected GExoI in the lungs released IPI549 to block G-MDSC recruitment by interfering with CXCLs/CXCR2/PI3Kγ signaling. The increased percentages of CD4+ and CD8+ T-cells in the lungs inhibited metastasis and immunostimulation of the TME . In anotThe lipid bilayer of the EV surface primarily consists of phospholipids and transmembrane proteins that can be functionalized via chemical conjugation . ChemicaThe covalent modification involves the direct conjugation of ligands to the EVssurface. Functional moieties such as those of carboxylic acid, amines, and sulfhydryl on the EV surface enable covalent conjugation reactions . The widEDC–NHS coupling could be used for conjugating peptides, proteins, antibodies, and so on to the EVs surface for tumor therapy. The EDC–NHS coupling reaction involves using 1-ethyl-3-(3-dimethylamino propyl)-carbodiimide, which mediates the reaction between carboxyl groups and primary amines by forming amine-reactive NHS esters . HosseinAmong all the conventional methods, bio-orthogonal click chemistry is highly efficient and biocompatible, and can be effectively employed for the surface engineering of EVs. Click chemistry  is a reaction between an alkyne and an azide, involving the catalysis of a triazole linkage by copper . Smyth eUnlike covalent modifications, non-covalent modifications correspond to EV conjugation via weak interactions, including electrostatic, hydrophobic, or ligand-receptor interactions by nature . Non-covAmong others, the non-covalent modifications applied in EV modification are ligand-receptor interactions. This method involves the conjugation of ligands to increase EV target specificity. Qi et al., 2016, reported a notable illustration of modified reticulocyte (RTC)-derived exosomes with superparamagnetic nanoparticles (SPMN). They conjugated SPMN with transferrin (Tf) and attached Tf-conjugated SPMN to the RTC-derived exosomes with TF receptors on their surface through the Tf and Tf-receptor interaction . These mEV membranes primarily comprise amphiphilic substances such as phospholipids, cholesterol, and glycolipids, making them conducive to the integration of hydrophobic compounds through hydrophobic interactions. Hydrophobic interactions involve the spontaneous integration of hydrophobic moieties on the EV surface. Aminoethyl anisamide-polyethylene glycol (AA-PEG) was conjugated to vectorized exosomes via hydrophobic interaction. AA-PEG-vectorized exosomes showed high tumor targeting, given their interaction with the sigma receptor that is overexpressed in lung cancer cells. These engineered exosomes (EXO) were attached with mannose, a natural sugar with anti-tumor properties . The EV In terms of folate-mediated EV modifications, folic acid (FA) receptor is a glycoprotein receptor that is overexpressed in cancer cells . ExosomeIn terms of liposome-mediated surface engineering, liposomes are small artificial spherical vesicles used for drug delivery owing to their size and amphiphilic nature . LiposomWith regard to aptamer-based surface engineering, aptamers are short artificial oligonucleotides and can be attached to EV surfaces, given their high binding ability and specificity. Both covalent and non-covalent coupling methods are employed for aptamer conjugation. Hosseini et al., 2022, used the AS1411 aptamer to functionalize exosomes for the targeted delivery of doxorubicin . The AS1Physical surface engineering methods include electroporation, sonication, extrusion, and freeze–thaw. These methods temporarily disrupt the lipid constructs of membranes and permeabilize the EV membrane for cargo loading and surface functionalization. Freeze–thawing, electroporation, and extrusion are common physical methods that can be used for EV surface permeabilization and modifications. These techniques create surface pores to allow modifying agents to pass through or bind to exposed membrane lipids or proteins. The freeze–thaw method disrupts the membrane via the formation of ice crystals. Sato et al., 2016, utilized this strategy and developed hybrid exosome—liposome-fused vesicles that combine the advantage of natural targeting and signaling properties of exosomes with the drug-delivery capabilities of liposomes. The study demonstrates that hybrid exosomes efficiently deliver therapeutic molecules by targeting HeLa cells. Likewise, Cheng et al., 2019, used freeze–thaw techniques to incorporate nuclear localization signal peptides on the EV surface by shaking the peptide and EVs together in an ice bath for four hours. The resulting modified EVs exhibited significantly improved therapeutic effects on inhibiting tumor growth . HoweverElectroporation is a technique that uses an electric field to temporarily disrupt the cell membrane, allowing the introduction of exogenous materials into the membrane of EVs to enhance their tumor targeting and therapeutic capabilities. Although it is a method of choice for loading hydrophilic therapeutic cargo  onto EVs, its implications in EV surface engineering remain limited due to non-uniformity in terms of incorporation and orientation. On the other hand, microfluidic isolation and molecular analysis of EVs have seen significant progress. However, microfluidic engineering of exosomes has just emerged recently, and a few reports have employed microfluidic technology for engineering exosomes. Their full potential and capability have yet to be thoroughly explored. Due to their inherent customizability, automation, scalability, and capacity for highly efficient mass transport, microfluidic systems can overcome numerous limitations of benchtop systems. Microfluidic lab-on-chip technology has demonstrated the potential of these advantages . DendritExtrusion is a common physical modification technique for transferring EV membranes onto different nanoparticles. For instance, EV membrane-cloaked gold nanoparticles were synthesized by Deun et al., 2020, to limit the uptake of gold nanoparticles by macrophages . A similCoincubation is a common method with which to introduce targeting peptides onto extracellular vesicle (EV) surfaces. This process involves incubating EVs with the targeting peptide of interest, which can bind to specific receptors on the target cells. Kooijmans et al., 2018, demonstrated the potential of EVs by decorating EVs with EGa1-C1C2 for tumor targeting . Here, lPre-clinical studies investigating surface-modified EVs for cancer immunotherapy were categorically outlined in the above sections. While surface-engineered EVs remain to be broadly evaluated in clinical trials, we noted two studies using surface-modified EVs for cancer therapy. One evaluated dendritic cell-derived exosomes (dexosomes or Dex) in advanced NSCLC patients NCT01159288). While the primary endpoint of this trial was not reached, the phase-I and phase-II studies confirmed that Dex boosts the NK cells’ antitumor immunity in patients 288. Whil. The surAlthough EVs have been extensively researched, their clinical translation has been deterred by many factors. The lack of standardized large-scale EV production limits clinical applications . ConventThe emergence of nanotechnology in medical applications has introduced a new era of drug delivery. Due to their high biocompatibility, EVs have shown great promise as therapeutic carriers and in clinical applications. However, there are associated risks in using exosomes, such as potential immunosuppression and reversion to tumorigenesis. As a result, there is a constant pursuit toward achieving safe and effective exosome-based formulations, leading to the external modification of exosomes as a viable approach. EV surface modification using biological, chemical, and physical strategies offers potential solutions to these challenges, improving the effectiveness of EV-based cancer immunotherapies . Genetic modifications enable the control of cells’ inherent EV production process to generate EVs with specific immunostimulatory or immunosuppressive properties. The incorporation of tumor-associated antigens or cytokines and the expression of chimeric antigen receptors (CARs) or T-cell receptors (TCRs) on EVs are some of the promising strategies for improving the specificity and effectiveness of EV-based immunotherapies. These modifications can enhance EV targeting, stability, and immunostimulatory properties to improve cancer therapeutic outcomes. Chemical modifications of EVs have also shown strong potential for improving their targeting specificity and immunostimulatory properties via attached ligands, antibodies, or peptides onto the EV surface, thus redirecting EVs toward specific immune cells or increasing their specificity to cancer cells. The immunomodulatory properties of EVs have also been achieved by adding adjuvants, such as Toll-like receptor agonists, enhancing EVs’ ability to activate immune cells and stimulate an anti-tumor response. Alternatively, physical modifications, such as size reduction and surface coating with polymers, can enhance EV stability and circulation time, improving their ability to reach and interact with immune cells. The new developments in recent years represent an exciting frontier in EV biology and cancer immunotherapy research. There is much to be learned about the optimal approaches for EV modification. However, many obstacles remain to overcome; one is the production of exosomes on a large scale for clinical purposes with more handy EV purification approaches. Rapid EV purification approaches will be an encouraging development in the EV research domain. Any new methods that are yet to come will have to pass through, the scrutiny phase before being adapted for wide application in the field. Additionally, the question of which cell type is optimal for exosome derivation remains unanswered. EVs as delivery vehicles are highly attractive and promising and could be the potential solution to the ongoing quest for clinically viable nanoplatforms. Therefore, conducting more systematic in vivo studies will be crucial for establishing the efficacy and toxicity of exosomes and bringing EVs towards exciting advancement and bringing them closer to clinical implementation.


This cohort study examines data from the Collaborative Study on the Genetics of Alcoholism to assess whether specific diagnostic criteria for alcohol use disorder that are indicative of greater severity could help detect a greater likelihood of progression. Does emphasis on specific criteria for alcohol use disorder (AUD) improve the identification of individuals at risk for developing more severe AUD?In this cohort study, cross-sectional and longitudinal multimodal secondary analyses involving a combined 15 928 individuals indicated that endorsement of criteria empirically designated as representing greater severity of AUD was significantly associated with 2-fold increased likelihood of progression from mild-to-moderate AUD to severe AUD, even after accounting for total criterion count.Emphasis on more severe criteria as indicators of vulnerability for severe AUD in current diagnostic approaches may increase detection of individuals with greater likelihood for disorder progression. Diagnostic and Statistical Manual of Mental Disorders (Fifth Edition) (DSM-5) diagnoses of substance use disorders rely on criterion count–based approaches, disregarding severity grading indexed by individual criteria.Current To examine correlates of alcohol use disorder (AUD) across count-based severity groups , identify specific diagnostic criteria indicative of greater severity, and evaluate whether specific criteria within mild-to-moderate AUD differentiate across relevant correlates and manifest in greater hazards of severe AUD development.This cohort study involved 2 cohorts from the family-based Collaborative Study on the Genetics of Alcoholism (COGA) with 7 sites across the United States: cross-sectional (assessed 1991-2005) and longitudinal (assessed 2004-2019). Statistical analyses were conducted from December 2022 to June 2023.DSM-5 AUD levels  and criterion severity–defined mild-to-moderate AUD diagnostic groups .Sociodemographic, alcohol-related, psychiatric comorbidity, brain electroencephalography (EEG), and AUD polygenic score measures as correlates of A total of 13 110 individuals from the cross-sectional COGA cohort  and 2818 individuals from the longitudinal COGA cohort  were included. Associations with alcohol-related, psychiatric, EEG, and AUD polygenic score measures reinforced the role of increasing criterion counts as indexing severity. Yet within mild-to-moderate AUD (2-5 criteria), the presence of specific high-risk criteria  identified a group reporting heavier drinking and greater psychiatric comorbidity even after accounting for criterion count differences. In longitudinal analyses, prior mild-to-moderate AUD characterized by endorsement of at least 1 high-risk criterion was associated with more accelerated progression to severe AUD  compared with prior mild-to-moderate AUD without endorsement of high-risk criteria , independent of criterion count.In this cohort study of a combined 15 928 individuals, findings suggested that simple count-based AUD diagnostic approaches to estimating severe AUD vulnerability, which ignore heterogeneity among criteria, may be improved by emphasizing specific high-risk criteria. Such emphasis may allow better focus on individuals at the greatest risk and improve understanding of the development of AUD. Diagnostic and Statistical Manual of Mental Disorders (Fifth Edition)1 (DSM-5), is conceptualized as a syndrome of sustained problematic alcohol use and clinically significant impairment. A diagnosis is based on endorsement of 2 or more of 11 criteria assessing behavioral and physical manifestations of heavy alcohol use that occur in a 12-month period.1 Recent US estimates indicate that 11% and 30% of adults meet criteria for past-year and lifetime AUD, respectively.3 This level of disordered alcohol use results in significant social, economic, and public health costs.6Alcohol use disorder (AUD), as defined by the DSM-5 AUD is diagnosed on a continuum of severity based on the number of criteria endorsed ,1 and identifying individuals at high risk for severe AUD is a priority. Several studies document increased comorbid burden and reduced likelihood of recovery as a function of increasing criterion count.9 However, count-based indices are limited by their equal weighting of criteria, suggesting all diagnostic criteria are interchangeable.10 Extensive cross-national psychometric evidence shows that certain AUD criteria  are more likely to be endorsed by those with more severe AUD and may represent superior indicators of risk.14 Thus, criteria heterogeneity is an important factor to consider in the advancement of personalized treatment approaches.15 Whether “high-risk” AUD criteria endorsement results in differing associations with a wide range of psychiatric, genetic, and neurobiological correlates could further guide delineation of risk severity.12 here we investigate the impact of criteria heterogeneity within criterion count–based AUD diagnoses. As other research also suggests that severe AUD may be substantively different from mild or moderate AUD in terms of treatment needs,16 functional impairment,17 and other health sequelae,18 we sought to compare mild-to-moderate AUD  vs severe AUD while accounting for individual differences in criterion count among those with mild-to-moderate AUD. The current study aimed to (1) validate criterion count–based severity using DSM-5 categorizations of AUD, including comparing mild-to-moderate vs severe AUD, in a sample of 13 110 individuals; (2) use item response theory (IRT) modeling to identify criteria indicative of greater severity; and (3) evaluate whether the presence of certain high-risk criteria, identified through IRT modeling, indexes greater hazards of developing severe AUD in a related cohort of 2818 adolescents and young adults.In light of the above evidence,19 AUD probands were recruited primarily from treatment facilities across 7 US collection sites. Families of probands that included 3 or more individuals with alcohol dependence were also recruited and included members with and without alcohol dependence. Comparison families  were also selected from a variety of community sources.20 The institutional review boards at all 7 sites approved this study, and written consent was obtained from all participants. We followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines for cohort studies. Data were restricted to alcohol-exposed individuals  from 2 COGA subsamples: (1) a cross-sectional cohort of 13 110 individuals from 2234 families assessed from 1991 to 2005, and (2) a longitudinal cohort comprising 2818 offspring of individuals from the cross-sectional cohort, born after 1981 and assessed from 2004 to 2019.20The Collaborative Study on the Genetics of Alcoholism (COGA) is a family-based study with deep and repeated phenotyping of substance use disorders (SUDs), comorbid psychiatric disorders, and related traits  designed to examine the genetic substrates of AUD and its development across the life span.18 and statistical considerations for examining effects of criteria heterogeneity separately within mild and moderate AUD , we elected to combine mild and moderate AUD into a single referent group for analyses in both cohorts.The cross-sectional cohort was used to (1) examine correlates of mild, moderate, mild-to-moderate, and severe AUD; (2) identify AUD criteria indicative of heightened risk using IRT analysis; and (3) evaluate whether individuals with mild-to-moderate AUD who endorsed high-risk criteria differed from those who did not, and from those with severe AUD, across relevant factors, including alcohol-related, comorbid psychiatric, and electroencephalography (EEG)-derived traits and polygenic indices. Given extant literature demonstrating substantive differences between severe AUD and mild or moderate AUD,22 Sociodemographic variables included sex, race and ethnicity, current income, educational attainment, and relationship status. Psychiatric lifetime Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition) (DSM-IV)23 diagnoses included major depressive disorder (MDD), antisocial personality disorder (ASPD), and other SUD diagnoses . In addition, several alcohol-related measures were included as correlates in cross-sectional analyses: (1) lifetime endorsement of drinking every day for a week or more, (2) largest number of drinks consumed each day during this period, (3) lifetime endorsement of experiencing blackouts, (4) age at first intoxication, (5) age at regular drinking onset , (6) lifetime maximum number of drinks ever consumed in a single 24-hour period, and (7) lifetime endorsement of seeking professional help or engaging in treatment for drinking problems.AUD criteria, diagnoses, and several correlates were derived from the Semi-Structured Interview for the Genetics of Alcoholism (SSAGA).24 Prior studies have found the P300 component during the standard visual oddball paradigm to be associated with family history of AUD.24 Subgroup differences for parietal delta (1-3 Hz) and frontal theta (3-7 Hz) band EROs (300-700 millisecond window) and parietal P300 amplitude were also included as correlates of AUD severity in cross-sectional analyses. Polygenic scores (PGS) for AUD diagnostic status , derived from a meta-analysis of large-scale genome-wide association study summary statistics,28 were calculated for genotyped individuals of European (n = 5396) and African American (n = 1774) ancestry separately using PRS-CS-auto29 and PRS-CSx,30 respectively (eMethods in COGA includes EEG-derived event-related oscillation (ERO) response measures.The longitudinal cohort included adolescent and young adult participants who were followed up approximately every 2 years . The SSAGA was administered at each biennial assessment. Given the longitudinal design, this sample was used to examine whether prior mild-to-moderate AUD diagnoses, with and without endorsement of high-risk criteria, were associated with increased hazards of progression to severe AUD. Other well-studied correlates assessed via the SSAGA, including alcohol involvement milestones , and MDD, ASPD, and other SUD diagnoses were also examined.31 in R32 34 and adjustedCurves (version 0.9.1)35 packages in R. Low-risk (n = 699) and high-risk (n = 317) mild-to-moderate AUD subgroups were defined by a prior mild-to-moderate AUD diagnosis and differentiated by endorsement of 1 or more high-risk criteria identified in the cross-sectional IRT analysis. Additional survival analyses were conducted examining hazards of progression to severe AUD based on alcohol involvement milestones, MDD, ASPD, and other SUD diagnoses. Covariates for all models included sex, race and ethnicity, and criterion count to examine associations independent of individual differences in the number of prior criteria endorsed. A family grouping variable was used to estimate robust standard errors to account for familial clustering.36 Violations of the proportional hazards assumption for predictor variables were tested using Schoenfeld residuals and resolved by including an interaction term with age at onset of severe AUD. Statistical analyses were conducted from December 2022 to June 2023.Hazards of progression to severe AUD (≥6 criteria) were estimated for individuals who, at any previous time point, had endorsed a single criterion or met criteria for mild, moderate, or mild-to-moderate AUD via Cox proportional hazards survival analyses conducted using the survival (version 3.4-0)20Participants included 13 110 individuals from the cross-sectional COGA cohort  and 2818 individuals from the longitudinal COGA cohort .17 supported our combined mild-to-moderate group as a comparison with severe AUD.As expected, increasing criterion count  was associated with increasing levels of heavy alcohol use and greater psychiatric comorbidity . For insDSM-5 criteria in the cross-sectional cohort revealed that failure to fulfill major role obligations because of drinking, drinking despite physical/psychological problems, giving up/reducing important activities, withdrawal, craving, and spending a great deal of time drinking represented more severe criteria, indexing greater risk . Even after accounting for the number of criteria endorsed, individuals with mild-to-moderate AUD who endorsed high-risk criteria were more likely to consume a greater number of drinks during heavy drinking episodes and periods of frequent drinking; endorse seeking help or treatment; meet criteria for other SUDs, MDD, and ASPD; and have lower theta EROs when compared with individuals with low-risk mild-to-moderate AUD . AUD PGSA majority of individuals who met criteria for severe AUD (77.8%) had a history of mild-to-moderate AUD . Consistent with cross-sectional findings, individuals with high-risk mild-to-moderate AUD were more likely (33.4%) to transition to severe AUD than those with low-risk mild-to-moderate AUD (12.9% transitioned to severe AUD) . The hazDSM-5 diagnostic scheme. IRT analyses revealed 6 high-risk criteria that reflect greater severity, and even after accounting for increasing criterion counts, there were significant differences among individuals who endorsed high- vs low-risk criteria. Moreover, individuals with mild-to-moderate AUD who endorsed 1 of these high-risk criteria were statistically indistinguishable from those with severe AUD with respect to theta EROs and P300 amplitude (after accounting for differences in criterion count). Individuals with moderate or high-risk mild-to-moderate AUD had the greatest hazards of progression to severe AUD across late adolescence and early adulthood. Overall, these analyses suggest that the presence of specific criteria are a superior indicator of risk for progression to severe AUD compared with criterion count alone. This difference is especially pronounced for the mild-to-moderate AUD group.We sought to examine whether endorsement of certain diagnostic criteria was associated with higher risk for severe AUD within the 39 However, these high-risk criteria do not currently comprise a coherent DSM-5 AUD subtype.40 For example, although DSM-5 characterizes both withdrawal and tolerance as physiological components of AUD, withdrawal but not tolerance was identified as high risk. Interestingly, several of the high-risk criteria were markers of preoccupation  and impairment in several domains . These criteria were endorsed far more frequently by individuals with severe AUD and map onto 2 key stages  of one of the neurobiological models developed to characterize severe forms of SUDs.41Many of the 6 empirically identified high-risk criteria have previously demonstrated stronger associations with comorbid psychopathology and greater severity of and discriminatory capabilities for AUD than other criteria.28 findings here highlight the need for criterion-focused genome-wide association studies to resolve key genetic mechanisms that might underlie specific clinical presentations.44Unlike heavy drinking or psychiatric comorbidity, which may arise as a consequence of problematic drinking patterns, PGS and EEG measures provide a glimpse into potential and preexisting neurobiological vulnerabilities. EEG and AUD PGS results generally echoed other findings. Specifically, theta EROs distinguished between individuals with low-risk and high-risk mild-to-moderate AUD and demonstrated similarly blunted neurophysiological responses in both high-risk mild-to-moderate and severe AUD groups in the cross-sectional cohort. AUD PGS primarily exhibited associations with criterion count and overall diagnostic status (AUD vs no AUD) across both ancestry subsamples. As these PGS are calculated using genome-wide association studies of broadly conceptualized AUD status often obtained from electronic health record–derived diagnostic codes,45 note that while individuals with mild-to-moderate SUDs are currently not a high-priority population for treatment efforts, they represent “one reasonable starting point” for defining a state similar to prediabetes, indexing accumulating risk for progression to severe SUD diagnosis, and thus merit heightened vigilance and intervention. While the proposed concept has drawn criticism regarding potentially stigmatizing terminology,48 the importance of improving treatment efforts for all presentations of AUD cannot be overstated.6 In the current study, despite heightened risk, only 27.6% of the high-risk mild-to-moderate AUD group endorsed seeking professional help or engaging in treatment, which, although significantly more than rates endorsed by individuals in the low-risk mild-to-moderate AUD group, is far from ideal.In a recent conceptual proposal, McLellan et al52Our study provides mixed support for combining across mild and moderate AUD. In the cross-sectional cohort, differences between severe and moderate AUD were more pronounced than between moderate and mild AUD. However, endorsement of high-risk criteria was notably lower in those with mild vs moderate AUD, and in the longitudinal cohort, hazards of progressing to severe AUD from mild or low-risk mild-to-moderate AUD were much lower than hazards for moderate or high-risk mild-to-moderate AUD, which were largely equivalent. These findings are generally consistent with research suggesting that mild AUD may more accurately reflect temporally limited drinking problems consistent with endorsement of less severe criteria.14 Notwithstanding, findings here may not generalize to samples more representative of the general population. Second, although the cross-sectional cohort has a wide age distribution, longitudinal data are delimited to younger ages. Third, many DSM-5 criteria assessed using the SSAGA are compound criteria comprising multiple items of varying severity . Disaggregation of compound criteria into individual items, though not always practical in clinical settings, may help further elucidate symptoms signaling increased risk for development of severe AUD.53The current study should be interpreted in light of its limitations. First, identification of high-risk criteria may be influenced by the AUD-enriched family-based study design, though comparisons with prior IRT estimates suggest consistent results.In this cohort study involving a combined 15 928 individuals, findings suggested that certain high-risk criteria may improve the identification of individuals at higher risk of progression to severe AUD. Defining severe AUD vulnerability using these criteria is consistent with extant etiological and clinical insight and may improve treatment penetrance.


Mesenchymal stem cells (MSCs) are located in various tissues of the body. These cells exhibit regenerative and reparative properties, which makes them highly valuable for cell-based therapy. Despite this, majority of MSC-related studies remain to be translated for regular clinical use. This is partly because there are methodical challenges in pre-administration MSC labelling, post-administration detection and tracking of cells, and in retention of maximal therapeutic potential in-vivo. This calls for exploration of alternative or adjunctive approaches that would enable better detection of transplanted MSCs via non-invasive methods and enhance MSC therapeutic potential in-vivo. Interestingly, these attributes have been demonstrated by some iron-related genes and proteins.Accordingly, this unique forward-looking article integrates the apparently distinct fields of iron metabolism and MSC biology, and reviews the utility of iron-related genes and iron-related proteins in facilitating MSC detection and therapy, respectively. Effects of genetic overexpression of the iron-related proteins ferritin, transferrin receptor-1 and MagA in MSCs and their utilisation as reporter genes for improving MSC detection in-vivo are critically evaluated. In addition, the beneficial effects of the iron chelator deferoxamine and the iron-related proteins haem oxygenase-1, lipocalin-2, lactoferrin, bone morphogenetic protein-2 and hepcidin in enhancing MSC therapeutics are highlighted with the consequent intracellular alterations in MSCs. This review aims to inform both regenerative and translational medicine. It can aid in formulating better methodical approaches that will improve, complement, or provide alternatives to the current pre-transplantation MSC labelling procedures, and enhance MSC detection or augment the post-transplantation MSC therapeutic potential. Iron in essential for various intracellular activities, and stem cells would be no exception. For example, ribonucleotide reductase is an enzyme that facilitates DNA synthesis and repair, and iron is a cofactor for this enzyme. Also, iron is essential for mitochondrial respiration. It is used in the synthesis of haem and [Fe-S] clusters; specifically, cytochrome c that not only participates in the electron transport chain, but also has a role in apoptosis . CytochrIn a pathological context, specifically pertaining to stem cells, iron has been found to maintain cancer stem cells  and ironMSCs are the most widely researched stem cell types because of their ability to support several physiological processes in the body and their exuberant reparative and regenerative properties. Located in various body tissues, these cells not only show multilineage differentiation but also secrete immune and trophic factors that stimulate endogenous repair mechanisms at the target site. Furthermore, MSCs show tropism towards tumour and inflammation/injury . ExpecteDespite their therapeutic potential, MSCs are not frequently used in clinical settings for amelioration of pathological conditions. Amongst the many reasons for this are the challenges encountered in pre-transplantation MSC labelling and post-transplantation MSC detection via non-invasive methods like the Magnetic Resonance Imaging (MRI). Nanoparticles including iron oxide nanoparticles have been used to enhance MSC detection, but their usage is confounded by various challenges . EvidentTherefore, it is extremely important to search for effective and non-toxic approaches that not only preserve MSC functionality during extraction and in-vitro cultivation stages but also permit the detection of transplanted MSCs non-invasively and help retain and/or enhance their reparative and regenerative potential in-vivo.Iron-related genes and proteins have shown the potential to support many of these pre-requisites for a successful MSC therapy. Thus, this review compiles and critically evaluates the usage of the iron-related genes  as reporter genes because their encoded proteins allow cellular iron accumulation that eases in-vivo MSC detection and tracking via MRI. The review also addresses the roles of deferoxamine (iron chelator) and the iron-related proteins haem oxygenase-1, lipocalin-2, lactoferrin, bone morphogenetic protein-2 (BMP-2) and hepcidin in preserving MSC characteristics in-vitro and in-vivo, and in enhancing MSC therapeutics.It is essential to be able to detect the transplanted cells and track their destination in-vivo to check the efficacy of MSC therapy. Hence, prior to transplantation, MSCs are labelled so that post-transplantation detection and in-vivo cell tracking via non-invasive methods like MRI become feasible. Cell labelling is essential as it distinguishes between the host and transplanted cells. Thus, MRI contrast agents such as iron oxide nanoparticles (IONPs) are used for cell labelling. Upon exposure to a magnetic field, IONP-labelled cells appear as darker regions in the field, allowing easy identification of areas of interest . DespiteThese constraints can be overcome by introducing and overexpressing an iron-accruing reporter gene in the MSCs prior to the MRI assessment. The term ‘reporter genes’ normally denotes reporting of a biological activity such as a signalling pathway or transcription factor binding. Here, in the context of enhancing MRI assessment following post-MSC transplantation, the reporter gene overexpressed in the MSCs imparts iron accumulation ability to the MSCs, thereby increasing the MRI signals given-off by the cell and making long-term tracing possible Fig. . In thisβ-galactosidase, genes of iron-related proteins such as ferritin and transferrin receptor-1 (TfR-1) have been explored as reporter genes for monitoring the location and viability of transplanted MSCs via MRI. Ferritin is an iron storage protein found intracellularly and in the circulation, whereas TfR-1 is the cell surface iron importer protein, which allows regulated entry of transferrin-bound iron into the cells. Thus, the iron accrual ability of both these proteins promotes their utilisation in MSC detection via MRI.In addition to reporter-genes like tyrosinase and In context of using these as reporter genes, ferritin has relatively more applications. This is partly because it can be integrated into the cell genome and its expression remains unaffected by cell division and proliferation, although integration is more dependent on the method used rather than the gene itself . Also, ferritin has two subunits; light chain and heavy chain, where the heavy chain shows stronger ferroxidase activity and can promote cellular iron uptake from the vicinity . When feHowever, another set of studies by Pereira et al. reported slightly different results. At physiological concentration of extracellular iron, while the overexpression of TfR-1 was well tolerated by mouse MSCs, overexpression of ferritin heavy chain-1 affected cellular iron homeostasis, reduced cell proliferation, altered cell phenotype and upregulated the endogenous TfR-1. Unexpectedly, sole overexpression of neither reporter genes (TfR-1 or ferritin heavy chain-1) led to substantial increment in intracellular iron content. Instead, supplementation of the culture medium with iron sources proved to be better at obtaining the required MRI contrast than using these reporter genes . Therefo. It was thereby concluded that for short-term tracking of cells, loading the cells with ferric citrate was more effective than TfR-1 overexpression [Since the inability of TfR-1 as a reporter gene was attributed to its insufficient expression levels, Pereira et al. re-evaluated the potential of TfR-1 to function as a reporter gene by inducing mouse TfR-1 at high levels in Chinese hamster ovary cells that have the ability to express high levels of recombinant proteins. Major increments were observed in TfR-1  and ferritin heavy chain‐1, and the intracellular iron content increased significantly, even in the absence of major iron supplementation to the culture medium. Following this, injecting the reporter-gene-labelled cells in chick embryos in-ovo showed that the MR contrast obtained by supplementing the culture medium of the control cells (without reporter gene) with ferric citrate was almost the same as that in cells with the reporter genepression .On the other hand, co-expression of these iron-related reporter genes enhanced MRI detection and retained biological properties of the transplanted MSCs. When human MSCs expressing ferritin, transferrin receptor and Deltex-1 were transplanted into rabbits with closed penile fracture, the co-expression of ferritin and transferrin receptor increased the iron accumulation capacity and provided sufficient MRI contrast for detecting the distribution and migration of MSCs. Additionally, as Deltex-1 promoted MSC differentiation into smooth muscle cells, the fracture showed healing . This apCollectively, data suggest that although for short-term tracing, loading cells with a suitable iron source may be an option, for long-term longitudinal tracing, co-expression of these reporter genes may give better clinical outcomes. But this may depend on the iron content of the tissue under investigation.Magnetospirillum magnetotacticum is a magnetotactic bacterium, which produces single-magnetic domain crystals and incorporates these into magnetosomes [Magnetospirillum magnetotacticum), usually containing a lipid bilayer around magnetite (Fe3O4). A previous study showed that MagA is an iron transport protein involved in the synthesis of magnetic particles, and also that MagA expressing Escherichia coli cells accumulated iron in vesicles [2) accumulation in magnetosome-like particles, and redistribution and aggregation of existing cellular iron, which increased the size of the magnetic particles and improved MRI sensitivity [etosomes . MagA isvesicles . This wavesicles . Regardlsitivity , 33. ResMagA induced toxic effects in murine MSCs [MagA gene had a huge potential as a magnetic reporter gene for MSC tracking with MRI and for improving MRI detection in-vivo [MagA-expressing MSCs accrued iron in vesicles and increased MRI sensitivity. Likewise, the liver of iron-loaded MagA-expressing transgenic mice showed high iron concentration and increased MRI sensitivity. Excess iron increases ROS and this can induce p38-MAPK signalling [MagA-expressing MSCs showed lower increments in ferritin and p-p38 MAPK expression compared to control MSCs, and decreased the excess-iron-induced inhibition of osteogenic differentiation [MagA-expression in MSCs. This opens new therapeutic avenues for treating the myriad of pathological conditions that show excessive iron loading [Although earlier, Pereira et al. showed that the expression of ine MSCs , Shen et in-vivo . In theignalling . MagA-exntiation . Thus, t loading .MagA-expressing MSCs reduced these effects. Thus, MagA suppressed the exogenous iron-induced oxidative stress and showed the potential to attenuate iron-overload-induced injury to the bone-marrow haematopoietic microenvironment [MagA could be developed to tackle transfusion-induced iron loading. Along the same line, aiming to enhance detection by MRI and as an alternative to IONPs, kerans et al. described the transfection of MSCs with magnetosome-associated genes (mms6 or mmsF) derived from Magnetospirillum magneticum AMB-1. These genes helped the assimilation of intracytoplasmic magnetic nanoparticles that not only facilitated MRI detection but also retained inherent MSC proliferation, differentiation and migration [Also, while iron-treatment inhibited MSC proliferation and osteogenic differentiation, ironment . Based oigration .While MSCs are known to execute regenerative and reparative functions, specific iron-related proteins and compounds confer additional beneficial properties upon the MSCs and therefore, their combination with MSCs can greatly augment MSC therapeutics, as summarised in Fig. HO-1 is inducible by oxidants and inflammatory cytokines, and it is highly expressed in the liver, spleen, and kidneys. It is a rate-limiting enzyme involved in haem metabolism. It degrades cellular haem to produce biliverdin, iron and carbon monoxide. In pathological conditions, the activity of HO-1 can restore homeostasis, and offer cytoprotective effects against oxidative stress through the antioxidant activities of haem-breakdown products biliverdin and bilirubin, and the anti-inflammatory effects of carbon monoxide , 41.Elevation in HO-1 reduced the effects of lipopolysaccharide-induced lung injury in mice  and atteSimilar results were observed when BM-MSCs transfected with HO-1 showed protection against the effects of iron-loading (and the consequently generated ROS); again attributed to IL-10 secretion . Also, iMSCs show immunomodulatory properties, and their ability to induce immunosuppression and consequently demonstrate therapeutic characteristics in treating inflammatory conditions is known, for e.g., in case of graft-versus-host disease. Here, apoptosis of MSCs has been proposed to be an effector , 49. How. Thus, pre-treatment of MSCs with lipocalin-2 helped maintain their regenerative properties for subsequent applications and the approach demonstrated successful cell-based tissue regeneration in-vivo.Acute kidney injury does not have an effective treatment till date. MSCs pre-engineered to overexpress lipocalin-2 and then transfused in a rat model of cisplatin-induced acute kidney injury led to enhanced renal function. These cells upregulated several growth factors and markers of proximal tubular epithelium (AQP-1 and CK18), while reducing the markers of kidney injury (KIM-1 and Cystatin C). This indicated new lipocalin-related modalities for acute kidney injury . MoreoveCancer treatment poses several challenges, one of which is the absence of tumour-specific treatment. While some studies showed that lipocalin-2 promoted carcinogenesis –64, otheLactoferrin is an antimicrobial and anti-inflammatory iron-binding glycoprotein found in various body secretions . In humaIn bone regeneration applications, the elasticity of the matrix on which the cells are grown in-vitro determine the cellular fate i.e., lineage specification. Bioactive compounds and physical features of the biointerfaces determine MSC osteogenic differentiation. For example, soft gels promote adipogenesis whereas stiff 2-D substrates favour osteogenesis . This isDeferoxamine is an iron chelator that is often used for scavenging excess iron in iron-overload conditions. It is a hypoxia mimicking agent that can enhance MSC therapeutics Fig. . Pre-traConditioning human adipose tissue-derived MSCs with deferoxamine increased HIF1-α, and elevated their paracrine potential by increasing neuroprotective factor (nerve growth factor), anti-inflammatory cytokines (IL-4 and IL-5) and pro-angiogenic factor (VEGFα) in the MSC secretome ; the latThe differentiation ability, genomic stability, and therapeutic effects of MSCs dependent on culture conditions. Pre-conditioning of MSCs with deferoxamine creates hypoxia-like environment, which promotes MSC proliferation and survival post transplantation, while maintaining cells in an undifferentiated state, and improving therapeutic potential. Mechanistically, deferoxamine inhibits iron-dependent prolyl-4 hydroxylase activity and thereby, HIF1-α hydroxylation and degradation. Accordingly, deferoxamine treatment to BM-MSCs upregulated HIF1-α target genes including VEGF and increased the expression of nuclear protein-1 Fig. . The latBMPs are a family of proteins that play an important role during embryogenic development and in adult homeostasis. These regulate important processes such as cell lineage commitment, proliferation, differentiation and apoptosis . FurtherHepcidin is an iron hormone produced predominantly by the liver hepatocytes. It is the master regulator of systemic iron homeostasis. Hepcidin is also an antimicrobial peptide  and is sThis review collates and examines the roles of iron-related genes and proteins in enhancing MSC detection and therapeutics. Genes of the iron-related proteins ferritin, TfR1 and MagA can be overexpressed in MSCs and used as reporter genes to increase cellular iron accrual and thereby aid in-vivo MSC detection by MRI. Iron-related proteins such as HO-1, lipocalin-2, lactoferrin, BMP-2 and hepcidin, and the iron-related drug deferoxamine exert antioxidant, antiinflammatory, antimicrobial and/or other beneficial effects, thereby potentiating better clinical outcomes when used to enhance MSC therapeutics.


Dengue hemorrhagic fever (DHF) is a severe form of dengue virus (DENV) infection that can lead to abnormal immune responses, endothelial vascular dysfunction, and hemorrhage pathogenesis. The virion-associated envelope protein domain III (EIII) is thought to play a role in the virulence of DENV by damaging endothelial cells. However, it is unclear whether EIII-coated nanoparticles simulating DENV virus particles could cause a more severe pathogenesis than soluble EIII alone. This study aimed to investigate whether EIII-coated silica nanoparticles (EIII-SNPs) could elicit greater cytotoxicity in endothelial cells and hemorrhage pathogenesis in mice compared to EIII or silica nanoparticles alone. The main methods included in vitro assays to assess cytotoxicity and in vivo experiments to examine hemorrhage pathogenesis in mice. EIII-SNPs induced greater endothelial cytotoxicity in vitro than EIII or silica nanoparticles alone. Two-hit combined treatment with EIII-SNPs and antiplatelet antibodies to simulate DHF hemorrhage pathogenesis during secondary DENV infections resulted in higher endothelial cytotoxicity than either treatment alone. In mouse experiments, two-hit combined treatment with EIII-SNPs and antiplatelet antibodies resulted in more severe hemorrhage pathogenesis compared to single treatments of EIII, EIII-SNPs, or antiplatelet antibodies alone. These findings suggest that EIII-coated nanoparticles are more cytotoxic than soluble EIII and could be used to develop a tentative dengue two-hit hemorrhage pathogenesis model in mice. Additionally, our results indicated that EIII-containing DENV particles could potentially exacerbate hemorrhage pathogenesis in DHF patients who have antiplatelet antibodies, highlighting the need for further research on the potential role of EIII in DHF pathogenesis. Dengue virus (DENV) infection can lead to self-limited dengue fever and live-threatening dengue hemorrhagic fever (DHF) ,2,3. DesWhen a person is infected with dengue virus for the first time, the immune system produces antibodies that help fight the virus. However, if the same person is infected with a different strain of the virus during a secondary infection, the antibodies produced during the first infection may make the second infection worse ,2,3. DurNanotechnology has various applications in biomedical science research, including antimicrobial substances, drug delivery, imaging, tissue engineering, and biosensors ,14,15,16To assess whether EIII-coated nanoparticles exhibit increased cytotoxicity against human HMEC-1 endothelial cells, the cells were treated with recombinant GST (a control protein), EIII, nanoscale silica beads , GST-coated SNPs (GST-SNPs), and EIII-coated SNPs (EIII-SNPs). The cell viability data showed that EIII displayed higher cytotoxicity than GST treatments. Additionally, EIII-SNPs exhibited higher cytotoxicity than the GST-SNPs and treatments of EIII alone C–E. ThisFollowing previously described methods ,27, a twHemorrhage is a hallmark symptom in DHF  and coulCytokine storms are critical for the development of DHF ,29,30. MHypercoagulation may induce disseminated intravascular coagulation (DIC) and then lead to secondary hemorrhage in DHF . HypercoThis study demonstrates that EIII-SNPs (EIII-coated nanoparticles) have advantages over the soluble EIII to perform two-hit dengue hemorrhage mouse model because they induce enhanced cytotoxicity against endothelial cells and display higher cytotoxicity when compared to the GST-SNPs and respective treatments of EIII and SNPs alone. Additionally, EIII-SNPs induce exacerbated thrombocytopenia, worsen hemorrhage pathogenesis, and the induction of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and anti-inflammatory cytokine IL-10 in mice. Moreover, EIII-SNPs induce an exacerbated suppression of anti-coagulant activated protein C and antithrombin III levels in mice, indicating that they may be a more potent and virus particle-like material for the development of dengue two-hit hemorrhage pathogenesis mouse models than soluble EIII.Leishmania infantum protein, which has been shown to induce a stronger and more sustained immune response when coated on poly(lactic-co-glycolic acid) nanoparticles  were indicated  were then analyzed using Photoshop software  to obtain red and green signals without any brightness or contrast adjustments. To further measure the red and green intensities in a particular image of the hemorrhagic lesion, we utilized Image J software . An approximate severity value for the hemorrhage was calculated by subtracting the image intensity of the red images from that of the green images, as previously described [While the grading of hemorrhage in the local Shwartzman reaction can be assessed using a subjective arbitrary scale of 0 to 4 ,54, we aoftware v, Adobe tt-test. A threshold of statistical significance was established with a probability of type 1 error α = 0.05.The quantifiable data were analyzed by calculating the means, standard deviations, and statistics using Microsoft Office Excel 2003, SigmaPlot 10, and SPSS 17. To test the data’s significance, a 1-way ANOVA was conducted, followed by the post hoc Bonferroni-corrected