To the Editor—In the absence of effective treatments for coronavirus disease 2019 (COVID-19), many hospitalized COVID-19 patients receive antibiotics.2 Thus far, the literature does not indicate that antibiotics are effective in treating COVID-19, and the incidence of bacterial coinfections appears low.2 One analysis reported that while 8% of COVID-19 patients experienced a bacterial or fungal coinfection, 72% of COVID-19 patients received antibiotics.2Clostridioides difficile infection (CDI) is associated with broad-spectrum antibiotics frequently used for COVID-19; CDI is thus a significant concern for COVID-19 patients.3 Patients at higher risk of severe COVID-19 frequently also have risk factors for CDI such as advanced age and weakened immune systems.3 COVID-19 treatments themselves, which often involve extended hospital stays, can also increase a patient’s risk of developing healthcare-associated CDI.3 CDI has been identified in patients who received antibiotics as part of their COVID-19 treatment.5Given the patient safety risks posed by CDI, effective antibiotic stewardship remains critical throughout the COVID-19 pandemic. However, pandemic-related changes to healthcare delivery  have made antibiotic stewardship interventions even more challenging. We present a systems engineering approach to evaluate and modify antibiotic stewardship programs within the constraints of COVID-19 responses, with the overall goal of reducing CDI.Antibiotic stewardship initiatives can involve persuasive  and restrictive  approaches; these initiatives are used together with appropriate diagnostic and infection prevention measures. These strategies require complex behavioral changes and can involve significant resources such as real-time access to antibiotic stewardship staff for consultation.6 Prescribers and antibiotic stewardship team members are facing higher and more complex patient loads. The wide-ranging symptoms of COVID-19 may mimic other infections, and a worsening of symptoms is frequently seen 1–2 weeks into the disease that can make it difficult to identify potential coinfections. The length of hospitalization for many patients increases risks of developing healthcare-acquired infections such as ventilator-associated pneumonia that may require additional antibiotic treatment.7 These factors, combined with a lack of effective treatment options for severe COVID-19, have resulted in high levels of antibiotic use among COVID-19 inpatients.2The COVID-19 pandemic response has further increased the complexity of antibiotic stewardship.8The structure and effectiveness of an antibiotic stewardship program is dependent on the individual work-system context, including characteristics of the patient population, organizational culture toward antibiotic stewardship, availability of infectious disease and pharmacy personnel, accessibility of clinical decision support tools, and existing policies to support antibiotic stewardship. A systems engineering approach can be used (1) to fully evaluate the roles that work-system elements play in complex antibiotic stewardship interventions and (2) to develop modifications to these elements to support the implementation of interventions. The Systems Engineering Initiative for Patient Safety (SEIPS) provides a framework for this approach. SEIPS defines work-system elements: person(s), technology and tools, environment, tasks, and organization. The interaction of these elements influences care processes and outcomes.9 Teams can then develop strategies to support antibiotic stewardship within the individual work system to optimize patient and organizational outcomes.Given the urgent needs to ensure appropriate antibiotic use and reduce CDI risk in COVID-19 patients, a systems engineering approach such as SEIPS can be used to understand the various work-system factors that are involved in antibiotic stewardship and CDI prevention. The interaction of these elements drives antibiotic stewardship, COVID-19 treatment, and CDI prevention processes in each work system; thus, they influence critical patient and organizational outcomes. This SEIPS-based approach provides flexibility for teams to evaluate their own work-system–specific barriers and facilitators to antibiotic stewardship practices within their COVID-19 response.Table Research is needed to understand whether, when, and how antibiotics should be used to treat COVID-19 patients while minimizing adverse effects such as CDI. CDI in COVID-19 patients should be investigated to identify risk factors such as use of specific antibiotics, previous CDI, and/or presentation of COVID-19 gastrointestinal symptoms. Research findings should be incorporated into comprehensive evidence-based antibiotic stewardship programs. In the meantime, the urgency of preventing CDI in COVID-19 patients requires adjusting antibiotic stewardship interventions to fit within current COVID-19 protocols. The SEIPS-based approach presented here can help local antibiotic stewardship teams and decision makers adjust existing plans and develop new approaches to support antibiotic stewardship and reduce CDI during the COVID-19 pandemic.


Viral dysregulation or suppression of innate immune responses is a key determinant of virus-induced pathogenesis. Important sensors for the detection of virus infection are the RIG-I-like receptors (RLRs), which, in turn, are antagonized by many RNA viruses and DNA viruses. Among the different escape strategies are viral mechanisms to dysregulate the post-translational modifications (PTMs) that play pivotal roles in RLR regulation. In this review, we present the current knowledge of immune evasion by viral pathogens that manipulate ubiquitin- or ISG15-dependent mechanisms of RLR activation. Key viral strategies to evade RLR signaling include direct targeting of ubiquitin E3 ligases, active deubiquitination using viral deubiquitinating enzymes (DUBs), and the upregulation of cellular DUBs that regulate RLR signaling. Additionally, we summarize emerging new evidence that shows that enzymes of certain coronaviruses such as SARS-CoV-2, the causative agent of the current COVID-19 pandemic, actively deISGylate key molecules in the RLR pathway to escape type I interferon (IFN)-mediated antiviral responses. Finally, we discuss the possibility of targeting virally-encoded proteins that manipulate ubiquitin- or ISG15-mediated innate immune responses for the development of new antivirals and vaccines. Infectious diseases caused by viruses affect the global population at an alarming rate, as can be seen by the current COVID-19 pandemic that has caused millions of infections worldwide. Given the large number of emerging and re-emerging viruses that exist, it is important to understand how these pathogens successfully replicate in the human host and how they induce disease. A hallmark of successful viral pathogens is their potent ability to antagonize or dysregulate human innate immune responses, in particular the induction of antiviral and proinflammatory cytokines. The first step in innate immunity is the detection of the virus where innate immune sensors, also known as pattern-recognition receptors (PRRs), recognize virus-derived molecular signatures  that are distinct from the host, such as viral nucleic acids . In addition, mislocalized, unmasked, or misprocessed cellular RNA (mostly noncoding RNAs) and DNA  can also trigger certain types of PRRs, leading to activation of innate and intrinsic defense mechanisms [5S ribosomal RNA  when these are mislocalized in the cytoplasm and ‘unmasked’ due to downregulation of their binding proteins [RIG-I and/or MDA5 gene-edited cells revealed that RIG-I senses a variety of negative-strand RNA viruses  and vesicular stomatitis viruses (VSV)) as well as some herpesviruses , Epstein–Barr virus (EBV), and Kaposi’s sarcoma herpesvirus (KSHV)), while MDA5 detects infection by picornaviruses and coronaviruses [A number of PRRs have been discovered, each recognizing unique and defined PAMPs. This review focuses on the family of retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), which sense cytoplasmic RNA. The two major members of the RLR family are RIG-I and melanoma differentiation-associated protein 5 (MDA5), which trigger an antiviral program following the detection of distinct RNA agonists . Caaviruses ,7. An inBoth RIG-I and MDA5 are DExD/H-box-containing helicases possessing two N-terminal caspase activation and recruitment domains (CARDs), a large helicase domain, and a carboxyl-terminal domain (CTD). These structural components are necessary for RNA binding and interacting with the common adaptor mitochondrial antiviral signaling protein (MAVS). MAVS acts as a scaffold for RLR signaling by coordinating the recruitment and activation of TANK-binding kinase 1 (TBK1) and inhibitor of nuclear factor κB kinase (IKK), which then phosphorylate IRF3 and IRF7  . The IRFInnate immune signaling must be tightly controlled to prevent excessive inflammatory responses and potential tissue damage. This is achieved through post-translational modifications (PTMs) that regulate each step of the antiviral innate immune response in a dynamic and complex way. They regulate the activity, subcellular localization, and/or protein-protein interactions of sensor and signaling proteins as well as key transcription factors in innate immunity. PTMs that are important for regulating IFN-mediated antiviral responses include the enzymatic addition of phosphate groups (phosphorylation), ubiquitin (ubiquitination), ISG15 (ISGylation), and acetylation, along with other less well-studied modifications like glutamylation and deamidation (Reviewed in ). NotablDuring ubiquitination, ubiquitin moieties, either as a single unit or in multimeric chains, are covalently attached to a target substrate via three enzymes: ubiquitin activating E1), ubiquitin conjugating (E2), and ubiquitin ligase (E3) enzymes. E3 ligases are primarily responsible for substrate specificity of this PTM. Seven polyubiquitin-linkage types have been discovered , each with its own distinct function to regulate the activity or abundance of target proteins. K48-linked ubiquitination and K63-linked ubiquitination are the linkage types most relevant to RLR regulation, and their roles in innate immunity have been extensively studied over the past several years. Modification with K48-linked polyubiquitin chains ‘earmarks’ the substrate protein for proteasomal degradation, while K63-linked ubiquitination  facilities the transduction of immune signaling. In many cases, K63-linked ubiquitin chains allow for the multimerization of sensor or signaling proteins, and the formation of larger signaling platforms. Ubiquitination is removed from the substrate by deubiquitinating enzymes (DUBs), which allows for an “on/off” switch to regulate innate immune signaling . Moreov-/-Usp18 mice and cells from these mice displayed restricted virus replication after VSV or Sindbis virus (SINV) challenge [ISGylation is a PTM in which the ubiquitin-like protein ISG15 (IFN-stimulated gene 15) is conjugated to a target protein also via E1, E2, and E3 enzymes ; hallenge ,17, the hallenge ,19,20,21hallenge .A growing number of studies have demonstrated RLR regulation by a diverse set of PTMs. Dynamic acetylation and deamidation regulate dsRNA binding of RLRs ,24,25. TThe innate immune response mediated by RLRs elicits defense against a multitude of viruses including coronaviruses, influenza viruses, flaviviruses, rhabdoviruses, paramyxoviruses, and picornaviruses . HoweverReversible ubiquitination regulates important steps of the RLR-signaling cascade (Reviewed in ). As sucCommon targets of viral antagonism are the cellular E3 ligase enzymes that activate RIG-I by catalyzing K63-linked ubiquitination. One critical positive regulator of RIG-I-mediated innate immune signaling is the E3 ligase TRIM25, which attaches K63-linked ubiquitin chains to the RIG-I CARDs ,34. UponIAV nonstructural protein 1 (NS1) inhibits the RIG-I-mediated IFN response through a direct interaction with TRIM25. NS1 was first shown to interact with the CCD of TRIM25, preventing TRIM25 dimerization and, thereby, its enzymatic activity. This study also revealed that the E96/E97 residues in NS1, which are located in a highly conserved protein-protein-interacting motif, were required for TRIM25 binding and efficient suppression of RIG-I CARD ubiquitination catalyzed by TRIM25 . More reNS1 proteins of other negative-strand RNA viruses also target TRIM25. Respiratory syncytial virus (RSV) NS1 protein directly binds to TRIM25 at its PRY/SPRY domain. This interaction suppresses the K63-linked ubiquitination of the RIG-I CARDs and disrupts their interaction with the MAVS CARD. Ectopic expression of TRIM25 reverses evasion by RSV NS1 and recoups IFN induction . The V pBunyaviridae, interacts with TRIM25 but not Riplet or RIG-I. NSs inhibits the K63-linked ubiquitination of RIG-I by relocalizing and trapping TRIM25 in viral inclusion bodies. Furthermore, a PXXP motif at amino acid residues 66−69 is known to be important for NSs function, and mutations at these residues abrogated the ability of NSs to block the TRIM25-mediated K63-linked ubiquitination of RIG-I [Recently, mass spectrometry analysis showed that the nonstructural protein NSs of severe fever with thrombocytopenia syndrome (SFTSV), a member of the large family of RIG-I .Abrogation of Riplet-mediated K63-linked ubiquitination of the CTD of RIG-I has been reported for hepatitis C virus (HCV) nonstructural protein 3/4A (NS3/4A) , which iFlaviviridae and required for viral pathogenicity [Not only viral proteins but also viral RNAs bind to TRIM25 to antagonize RIG-I signaling. Subgenomic flavivirus RNA (sfRNA) is a noncoding RNA derived from the 3′ untranslated region of the flavivirus genome and the product of incomplete degradation by the cellular ribonuclease XRN1 . It is agenicity ,54. The genicity . Howevergenicity .Another virus that targets the USP15-TRIM25-RIG-I axis is human papilloma virus (HPV), which encodes the oncoprotein E6. HPV E6 binds to both TRIM25 and USP15 when ectopically expressed or in natively HPV-infected cells. Co-Immunoprecipitation studies indicated that the interaction between TRIM25 and E6 was conserved in high-risk , low-risk (HPV6 and HPV11), and cutaneous beta genus type (HPV5 and HPV8) HPVs. This interaction promotes TRIM25 ubiquitination and degradation, suppresses K63-linked ubiquitination of RIG-I, and blocks the RIG-I-MAVS interaction, ultimately dampening RIG-I signaling  Figure . These dNidovirales order, have DUB activity via their nonstructural protein 2 (nsp2), which belongs to the papain-like protease (PLP) family. Nidovirus PLPs are multifunctional in that they serve as proteases, DUBs, and deISGylases [Bunyaviridae also possess DUB activity via their ovarian tumor (OTU)-like cysteine protease domain, which is located within the L protein along with the RNA-dependent RNA polymerase (RdRp), and the Crimean–Congo hemorrhagic fever virus (CCHFV) L protein also reduces RIG-I-mediated K63-linked ubiquitination [Many studies have demonstrated that several virus-encoded proteins have the ability to act as DUBs to remove ubiquitin moieties from RIG-I. Arteriviruses that, along with Coronaviruses and Roniviruses, comprise the SGylases . Viral PSGylases . Nairovitination  , which has DUB activity, targeting both K48- and K63-ubiquitin linkage types. The usage of PLP2 catalytic mutants  provided evidence that PLP2, a proven IFN antagonist, possesses direct DUB activity. In studies where these mutants were overexpressed, PLP2 blocked the ubiquitination not only of RIG-I, but also of STING, a critical adaptor protein in the intracellular DNA sensing pathway [Many coronaviruses encode viral enzymes that also deubiquitinate RIG-I. Porcine epidemic diarrhea virus (PEDV) in the  pathway . Overexp pathway . Middle Picornaviridae, which are positive-sense, single-stranded RNA viruses, can also deubiquitinate RIG-I, indicating that this mechanism is conserved amongst a large range of viruses. Seneca Valley virus (SVV) encodes the protease 3C protease (3Cpro) that possesses DUB activity both when overexpressed and during native infection. 3Cpro acts to remove both K48- and K63-linked polyubiquitin chains from multiple substrates including RIG-I, TBK1, and TNF receptor associated factor 3 (TRAF3), thereby blocking downstream antiviral gene expression. The authors also identified that the amino acid residues 48 and 160 were integral to the DUB activity and viral replication [pro) is another PLP that inhibits both K48- and K63-linked ubiquitination in vitro and in overexpressed HEK293T cell-based assays. Additionally, the Lpro deubiquitination of RIG-I, TANK1, TRAF6, and TRAF3 negatively regulated type I IFN induction, although the exact ubiquitin-linkage types that were removed were not determined. Catalytic activity  of FMDV Lpro is required for efficient deubiquitination [Studies have shown that certain members of the family lication . Foot-antination .Besides HCV, other hepatitis viruses also evade RIG-I-mediated immune signaling, with differing mechanisms of action. Hepatitis B virus (HBV) X protein (HBx) deubiquitinates a number of substrates modified with K63-linked polyubiquitin including RIG-I and TRAF3, thereby negatively regulating type I IFN production . HepatitRta and orf25 mRNA amounts, strengthening that RIG-I-mediated innate immune signaling plays a crucial role in suppressing KHSV lytic reactivation [RIG-I has recently been discovered to detect DNA viruses such as adenoviruses, HSV-1, KSHV, and EBV, in addition to RNA viruses. During DNA virus infection, RIG-I was shown to recognize RNA polymerase III-generated viral transcripts or host-derived RNAs that are unshielded, mislocalized, or misprocessed ,69,70. Gtivation  .pro) downregulates miR-526, which positively regulates antiviral type I IFN responses. In a screen of 168 miRNAs that regulate IFN induction, miR-526 emerged as a top candidate. It was shown that miR-526 targets CYLD, a DUB that removes K63-linked ubiquitin chains from RIG-I to inactivate the immune response, for degradation [pro overexpression, or native infection of EV71, reduces miR-526 expression, which increases the abundance of CYLD and ultimately suppresses RIG-I activation [In the case of enterovirus 71 (EV71), its 3C protease  was inhibited in RGNNV-infected cells while, conversely, viral replication was enhanced both in vitro and in zebrafish (zb). The authors found that the mechanism of viral evasion was that miR-202-5p targeted zbTRIM25 to inhibit zbTRIM25-mediated zbRIG-I CARD K63-ubiquitination [Red spotted grouper nervous necrosis virus (RGNNV), a major pathogen of fish, induces expression of miR-202-5p, one of the top ten most upregulated miRNAs in this study. Antiviral gene expression  infection, and its knockdown increases antiviral signaling following SeV or vesicular stomatitis mutant virus (VSV Δ51). The RIG-I CARDs interacted with USP27X during immunoprecipitation, which was enhanced during SeV infection. Additionally, ubiquitination assays demonstrated that USP27X reduced RIG-I ubiquitination, specifically the K63-ubiquitin linkage type. This was further substantiated through the use of ol cells . In the ol cells ,27,77.Viruses also can escape detection by RLRs by inducing their degradation. Conceptually, there are two ways of how viruses achieve this: either through direct degradation of RLRs, or by enhancing the expression of cellular proteins that then degrade RLRs.Phlebovirus), but SFNV NSs lacks the last 78 amino acids that are important for IFN antagonism and RIG-I degradation [Toscana virus (TOSV) is a sandfly-borne virus whose NSs protein has E3 ubiquitin ligase activity, transferring ubiquitin from the charged E2 enzyme to the RIG-I CARDs and promoting proteasome-dependent proteolysis . In vitrradation  , SARS-CoV-2 PLpro cleaves ISG15 from IRF3 to inhibit antiviral type I IFN responses . This, aCurrently, SARS-CoV-2 PLpro is the only viral enzyme known to regulate ISGylation in the context of RLR-mediated innate immune signaling; however, as mentioned briefly, other coronavirus PLPs as well as OTU domain-containing viral proteins have been characterized to possess deISGylating activity (Reviewed in ). The PLOur current understanding of the RLR pathway and regulatory PTMs has grown appreciably in the past decade, and a better understanding of these processes is critical for the development of drugs to avoid uncontrolled inflammatory responses. On the other hand, boosting RLR responses through the fine-tuning of their PTMs could be an effective measure against a wide range of viruses, especially new viruses that emerge and those for which no vaccines or antivirals exist. A panacea for all viral infections is unlikely, so understanding the precise molecular mechanisms that each single virus utilizes to subvert immune detection may allow us to target and inhibit viral IFN antagonists and restore host antiviral responses. The identification and specific targeting of viral DUBs or deISGylases, and the modulation of the abundance of cellular RLR-regulatory enzymes or miRNAs that control their expression, are all potential opportunities for translating major discoveries into antiviral drugs. Other conceivable applications are to generate attenuated viruses devoid of specific protein functions that inhibit ubiquitin- or ISG15-dependent antiviral responses for their use in vaccines, or to develop inhibitors against viral DUB enzymes.Furthermore, the role of ISGylation in RLR signaling is a new area of research and other viral proteins may be found to alter this PTM to avoid immune detection. As with viral dysregulation of the host ubiquitinome, identification of viral mechanisms that target critical ISGylation events could lead to the development of much needed antiviral treatments. Exciting advances in this and further understanding of the mechanistic roles of ISGylation in antiviral immunity are exciting avenues for future research. These findings may help not only in the fight against SARS-CoV-2, but could also be applied to other viruses for which no effective treatment exists.


Water-soluble chemicals, involving a wide range of toxic chemicals in aqueous solutions, remain essential in both daily living or industrial uses. However, most toxicants are evaporated with water through their use and thus cause deleterious effects on the domestic environment and health in humans. Unfortunately, most current low-dose chemical vapor detection technologies are restricted by the use of sophisticated instruments and unable to promptly detect the quantity of diverse toxicants in a single analysis. To address these issues, this study reports the development of simple and fast chemical vapor detection using doctor-blade-coated macroporous poly(2-hydroxyethyl methacrylate)/poly(ethoxylated trimethylolpropane triacrylate) photonic crystals, in which the poly(2-hydroxyethyl methacrylate) has strong affinity to insecticide vapor owing to a favorable Gibbs free energy change for their mixing. The condensation of water-soluble chemical vapor therefore results in a significant reflection peak shift and an obvious color change. The visual colorimetric readout can be further improved by increasing the lattice spacing of the macroporous photonic crystals. Furthermore, the dependence of the reflection peak position on vapor pressure under actual conditions and the reproducibility of vapor detecting are also evaluated in this study. Commercial water-soluble chemicals, such as cyphenothrin, pyrethrins, pyrethroids, xylenes, and trichloroethane, are capable of dissolving in water and serving as insecticides, pesticides, additives, adhesives, degreaser, and industrial solvents . NowadayRecently, numerous reliable technologies, including mass spectrometry, high-performance liquid chromatography, gas chromatography, surface-enhanced Raman scattering, and fluorescence technologies, have been extensively utilized for precisely detecting poisonous compounds ,10,11,12Photonic crystals comprise periodically arranged structured materials with different refractive indices, generating a forbidden energy gap for electromagnetic waves, and thence allow a certain wavelength range of light to be reflected . NoteworHerein, we develop a scalable doctor-blade coating technology for self-assembling macroporous poly(2-hydroxyethyl methacrylate)/poly(ethoxylated trimethylolpropane triacrylate) photonic crystals in a roll-to-roll compatible procedure. A commercial water-based insecticide involving cyphenothrin and imiprothrin is applied to demonstrate the detecting capability. As a result of the poly(2-hydroxyethyl methacrylate) having strong affinity to water and water-soluble chemicals, the as-fabricated photonic crystals rapidly change intrinsic colors upon exposure to water vapors containing water-based chemicals. Furthermore, the color of the macroporous photonic crystals can be recovered after applying a simple water rinsing process. Importantly, the as-constructed photonic crystals are fast-responsive, inexpensive, and portable and exhibit a visual readout for detecting water-soluble chemical vapors.Reagents. The chemicals applied to synthesize silica colloids, containing tetraethyl orthosilicate (98.5 vol.%), absolute ethanol (99.9 vol.%), and ammonium hydroxide (28.5 vol.%), were purchased from Sigma-Aldrich. Deionized water (18.6 MΩ cm) was collected from an HT-15L laboratory deionized water system. Ethoxylated trimethylolpropane triacrylate (ETPTA) monomer, 2-hydroxyethyl methacrylate (HEMA) monomer, ethylene glycol (EG) monomer, and methacrylic acid (MMA) monomer were provided by Sartomer. Initiators 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP) and azobisisobutyronitrile (AIBN) were acquired from Ciba-Geigy and Sigma-Aldrich, respectively. Aqueous hydrofluoric acid (48 vol.%) for wet etching silica colloids was purchased from Merck. All reagents were of analytical reagent quality and utilized directly in this research. The commercial water-based insecticide, involving cyphenothrin (0.1 vol.%) and imiprothrin (0.1 vol.%), applied in this research was obtained from SC-Johnson.Doctor Blade Coating Technology. Monodispersed StÖber silica colloids were purified using absolute ethanol to remove any unreacted reagent, followed by dispersing in ETPTA monomers with HMPP (1 vol.%) as a photo-initiator [nitiator . The silFabrication of Macroporous Poly(HEMA)/Poly(ETPTA) Photonic Crystals. The silica colloids embedded in the polymeric composite were wet-etched by immersing in an aqueous hydrofluoric acid solution (2 vol.%) for 5 min to create macroporous poly(ETPTA) photonic crystals. The macroporous film was peeled off from the glass substrate and immersed in an ethanol-based mixture of HEMA monomers (14.5 vol.%), MMA monomers (0.4 vol.%), EG monomers (0.1 vol.%), and AIBN (1 vol.%). The final monomer volume fraction was controlled to be 15 vol.%. In the mixture, MMA monomers and EG monomers can serve as a polymeric toughener and a lubricant, respectively. After eliminating excess mixture by spinning at 1000 rpm for 1 min, the monomers were photo-polymerized by ultraviolet radiation in the curing system for 2 s to engineer a free-standing macroporous poly(HEMA)/poly(ETPTA) photonic crystals.Experimental Procedures for Insecticide Vapor Detection. The as-fabricated macroporous films were placed in a vacuum degassing chamber, which was evacuated and then aerated with demanded insecticide vapor pressure and water vapor pressure. After that, the chamber was inflated with nitrogen to maintain the total pressure at 1 atm. An optical fiber probe sealed in the chamber was utilized to evaluate the normal-incidence optical reflectance from the macroporous films.Characterization. The photographic images and surface morphologies of the macroporous photonic crystals are characterized by a Nikon Z50 digital camera and a JEOL 6335F field-emission scanning electron microscope, respectively. Optical reflection spectra of the macroporous films were assessed using an Ocean Optics HR4000 spectrometer with Ocean Optics DT-MINI-2-GS deuterium tungsten halogen light source and recorded in the wavelength range from 400 nm to 900 nm.The experimental procedures for engineering macroporous photonic crystal-based insecticide vapor detectors are schematically illustrated in d denote the effective refractive index of the medium and the lattice spacing, respectively [0 to 1.0 P0, whereas P0 represents the saturation water vapor pressure at 28 °C. Additionally, the changes in the peak position and amplitude were not obvious when the water vapor pressure was higher than 1.0 P0. Owing to the fact that the water-based insecticide is composed of a considerable proportion of water, the optical response of the insecticide vapor detecting is similar to that of water vapor detecting (0 (For investigating the vapor-detecting capabilities of the macroporous poly(ETPTA) photonic crystals, normal-incidence specular reflection spectra of the macroporous film were evaluated under various water vapor pressures at 28 ± 1 °C a. It wasectively . This reetecting b. It wascting  photonic crystals are coated with a thin layer of poly(HEMA), which is responsive to water, to engineer macroporous poly(HEMA)/poly(ETPTA) photonic crystals. The as-developed macroporous film templated from 355 nm silica colloidal crystals displays a striking red color because of light reflection from the highly ordered air cavities . As notit 680 nm . The polEven though the appearance of macroporous poly(HEMA)/poly(ETPTA) film on water-based insecticide vapor detecting was not significantly distinguishable from that of macroporous poly(ETPTA) film, it is clear that the macroporous poly(HEMA)/poly(ETPTA) film exposed to the insecticide vapor presented larger red-shifts a. Moreov0. To further investigate insecticide vapor detection limit of the macroporous poly(HEMA)/poly(ETPTA) film, diluted commercial water-based insecticides containing 0.01 vol.% chemicals and 0.001 vol.% chemicals were also applied as analytes in this study. As displayed in In order to verify the extent of swelling in poly(HEMA) layer, a gravimetric analysis was applied to determine the mass of condensed liquid in the macroporous photonic crystals under various vapor pressures. The mass of condensed liquid could then be utilized to calculate the liquid layer thickness. It was found that the calculated liquid layer thickness using a gravimetric analysis  matched 0  photonic crystals templated from 250 nm silica colloidal crystals were fabricated by as-mentioned doctor-blade-coating technology . The mac0 vapor pressure, the reflection peak position of the macroporous poly(ETPTA) film was dynamically altered and remained unchanged after 8 s. Compared with that, the poly(HEMA) coating layer was capable of absorbing condensed water and easily led to the formation of uniform condensed liquid film, resulting from a favorable Gibbs free energy change for mixing poly(HEMA) with water. Therefore, the poly(HEMA)-coated macroporous film allowed a larger red-shift in reflection peak and a shorter detection time duration of 3 s. Importantly, the macroporous poly(HEMA)/poly(ETPTA) film enables an even shorter detection time duration under 0.2 P0 vapor pressure, indicating the high efficiency and effectiveness in water-based chemical vapor detection.The requirement of detection time duration plays a critical role in detecting water vapor containing chemicals for practical applications. To investigate the required time duration, the optical responses of the macroporous films templated from 250 nm silica colloidal crystals under fixed vapor pressures were evaluated . In the It is worthy of attention that the water-based insecticide does not chemically react with poly(HEMA), and thus the condensed insecticide can straightforwardly be removed by water rinsing. Taking advantage of that, the optical properties of the as-engineered macroporous poly(HEMA)/poly(ETPTA) photonic crystals can be wholly recuperated, and the recovered macroporous film can be reused for water-soluble chemical vapor detecting. The reproducibility of insecticide vapor detecting was investigated utilizing a macroporous poly(HEMA)/poly(ETPTA) film templated from 250 nm silica colloidal crystals. The reflection peak red-shifted from 485 nm to 566 nm as the macroporous film was exposed to saturated insecticide vapor and then blue-shifted to 485 nm after a raising treatment. Importantly, the utilization of macroporous poly(ETPTA) scaffold limited inhomogeneous deformation of poly(HEMA) coating layer during the cycles. As shown in In summary, a scalable doctor-blade coating technology was developed to engineer macroporous poly(HEMA)/poly(ETPTA) photonic crystals for colorimetric detection of water-soluble chemical vapor. Owing to a high swelling degree of poly(HEMA) in the presence of water and water-soluble chemicals, the macroporous film templated from 250 nm silica colloidal crystals behaves a significant reflection peak shift and a corresponding color change from blue to yellow on exposure to water-based insecticide vapor. The photonic stopband shift can be further improved by increasing lattice spacing of the macroporous photonic crystals. The reflection peak red-shifts with the increase of insecticide vapor pressure even in the presence of water vapor pressure. This suggests that the photonic crystals are capable of evaluating low-dose broad-spectrum water-soluble chemical vapor pressure under actual conditions. Importantly, the use of macrporous poly(ETPTA) scaffold allows a highly reproducible and reversible optical response to water-soluble chemical vapor detection within a very short time. It is believed that the doctor-blade-coated macroporous photonic crystals, coated with various materials with similar solubility parameters to selected chemicals, provide a universal and novel strategy for chemical vapor detections without applying any instrumentation or label.


The current pandemic of coronavirus disease 19 (COVID-19) has affected millions of individuals and caused thousands of deaths worldwide. The pathophysiology of the disease is complex and mostly unknown. Therefore, identifying the molecular mechanisms that promote progression of the disease is critical to overcome this pandemic. To address such issues, recent studies have reported transcriptomic profiles of cells, tissues and fluids from COVID-19 patients that mainly demonstrated activation of humoral immunity, dysregulated type I and III interferon expression, intense innate immune responses and inflammatory signaling. Here, we provide novel perspectives on the pathophysiology of COVID-19 using robust functional approaches to analyze public transcriptome datasets. In addition, we compared the transcriptional signature of COVID-19 patients with individuals infected with SARS-CoV-1 and Influenza A (IAV) viruses. We identified a core transcriptional signature induced by the respiratory viruses in peripheral leukocytes, whereas the absence of significant type I interferon/antiviral responses characterized SARS-CoV-2 infection. We also identified the higher expression of genes involved in metabolic pathways including heme biosynthesis, oxidative phosphorylation and tryptophan metabolism. A BTM-driven meta-analysis of bronchoalveolar lavage fluid (BALF) from COVID-19 patients showed significant enrichment for neutrophils and chemokines, which were also significant in data from lung tissue of one deceased COVID-19 patient. Importantly, our results indicate higher expression of genes related to oxidative phosphorylation both in peripheral mononuclear leukocytes and BALF, suggesting a critical role for mitochondrial activity during SARS-CoV-2 infection. Collectively, these data point for immunopathological features and targets that can be therapeutically exploited to control COVID-19. The outbreak of coronavirus disease 19 (COVID-19), first recognized in Wuhan, China, rapidly became a pandemic of major impact not only on global public health but also on economy and social well-being . UnderstTranscriptomics of peripheral blood cells has been a powerful tool to characterize human immune responses to diverse pathogens, including respiratory viruses –6. Gene GSE147507) and microarray data related to SARS-CoV-1 infection (GSE1739) or Influenza A virus (IAV) infection ; and ArrayExpress for NanoString nCounter data related to SARS-CoV-2 infection (E-MTAB-8871). DESeq2-normalized counts were used for the RNA-seq dataset CRA002390 (2 counts per million with EdgeR package for R (Datasets used in this study included public transcriptomes available at the Genome Sequence Archive (GSA) or human GSA in National Genomics Data Center, Beijing Institute of Genomics (BIG), Chinese Academy of Sciences for RNA-seq data related to SARS-CoV-2 infection (CRA002390 and HRA000143); Gene Expression Omnibus (GEO) for RNA-seq data related to SARS-CoV-2 infection GSE14507 and mge for R . DetaileData were analyzed with the positional framework Gene Set Enrichment Analysis (GSEA) , using pp < 0.001 were then linked by the number of genes shared between two gene modules.To construct the network of BTMs from peripheral blood mononuclear cell (PBMC) transcriptomes, genes were pre-ranked by the Wald test statistics score calculated with DESeq2 package comparing each gene in COVID-19 patients and healthy controls, as described . BTMs de2 fold change of experimental samples over healthy controls. Gene modules significantly associated with at least 50% of the datasets were selected by a nominal p < 0.001 for PBMCs and whole blood. The datasets were not merged at the single-gene-level. Each dataset was composed by a different number of genes and samples, and different types of samples (p < 0.001). To enforce confidence in the enrichments, we also retained only the BTMs that were associated with at least 50% of the datasets, independently of infection, sample type and regulation. Metabolic pathways from KEGG database were selected by a FDR adjusted p < 0.05 for PBMCs from COVID-19 patients.To perform the BTM-driven meta-analysis between respiratory viruses, gene lists from each dataset were pre-ranked by log samples . The out2 fold change of experimental samples over healthy controls and used as input in pre-ranked GSEA. BTMs and KEGG metabolic pathways were selected by relaxed significance  and consistent up- or downregulation in both datasets. For lung biopsies (GSE147507), one sample from COVID-19 patients shows a distinct read count profile and was considered an outlier as described , genes were also pre-ranked by logescribed . The remgplots for R and hierarchical clustering with the package amap for R, using Euclidian distance metric and Ward linkage. The bubble plots were generated with the package ggplot2 for R. GraphPad Prisma v. 8 was used to perform t-tests on NanoString nCounter data and generate bar plots.Networks were visualized and generated with Cytoscape v3.7.2 . Heat map < 0.001) to select significant BTMs , there were nine significant BTMs and three KEGG metabolic pathways that were consistently up or downregulated among both datasets  . Using adatasets . BTMs redatasets  and neutdatasets  in lung datasets . None ofHere, we used a robust modular transcriptomics approach that captured significant changes of cellular patterns in peripheral blood of COVID-19 patients, including T lymphopenia and reduced numbers of NK cells . SeveralWe demonstrated that compared to SARS-CoV-1 or IAV, SARS-CoV-2 infection fails to induce significant type I interferon responses in PBMCs  or wholeWe expect that several factors will contribute to differences in transcriptional profiles of larger cohorts of COVID-19 patients, especially those bearing comorbidities associated with severe disease. Higher expression of angiotensin-converting enzyme 2 (ACE2) has been suggested as a potential mechanism of susceptibility of individuals with comorbidities associated with COVID-19 . HoweverStrikingly, we observed the modulation of several metabolic pathways in PBMCs and BALF, while oxidative phosphorylation was the only significant metabolic pathway overlapping in both compartments. This suggests a critical role for mitochondrial activity during COVID-19. Many metabolites composing the pathways identified in the current study have been quantified via metabolomics of plasma or serum from COVID-19 patients , 50. MasGSE147507) and microarray data related to SARS-CoV-1 infection (GSE1739) or Influenza A virus (IAV) infection ; and ArrayExpress for NanoString nCounter data related to SARS-CoV-2 infection (E-MTAB-8871)/b.Publicly available datasets were analyzed in this study. This data can be found here: Genome Sequence Archive (GSA) or human GSA in National Genomics Data Center, Beijing Institute of Genomics (BIG), Chinese Academy of Sciences for RNA-seq data related to SARS-CoV-2 infection (CRA002390 and HRA000143); Gene Expression Omnibus (GEO) for RNA-seq data related to SARS-CoV-2 infection (LG: selected the data, performed data analysis, interpreted the results, and wrote the manuscript. CS, HS-C, and SF: interpreted the results and critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli, two Klebsiella spp., two Acinetobacter baumannii, two Pseudomonas aeruginosa) with multiple single and two-antibiotic combinations. ATP bioluminescence and viable counts were determined at each timepoint , with and without apyrase treatment. Correlation between ATP bioluminescence and viable counts was determined for apyrase-treated and non-apyrase-treated samples. Receiver operator characteristic curves were plotted to determine the optimal luminescence threshold to discriminate between inhibitory/non-inhibitory and bactericidal/non-bactericidal combinations, compared to viable counts. After treatment of bacteria with 2 U/mL apyrase for 15 min at 37 °C, correlation to viable counts was significantly higher compared to untreated samples (p < 0.01). Predictive accuracies of ATP bioluminescence were also significantly higher for apyrase-treated samples in distinguishing inhibitory (p < 0.01) and bactericidal (p = 0.03) combinations against CR-GNB compared to untreated samples, when all species were collectively analyzed. We found that ATP bioluminescence can potentially replace viable plating in TKS. Our assay also has applications in in vitro and in vivo infection models.Traditional in vitro time-kill studies (TKSs) require viable plating, which is tedious and time-consuming. We used ATP bioluminescence, with the removal of extracellular ATP (EC-ATP), as a surrogate for viable plating in TKSs against carbapenem-resistant Gram-negative bacteria (CR-GNB). Twenty-four-hour TKSs were conducted using eight clinical CR-GNB (two  Infections caused by carbapenem-resistant Gram-negative bacteria (CR-GNB) is a major public health problem worldwide . Amidst Adenosine triphosphate (ATP) bioluminescence is a rapid bacterial enumeration method which uses firefly luciferase to convert chemical energy in the form of ATP into light energy that can be detected by a luminometer within a rapid turn-around time of 15–30 min ,7,8. NotIn light of the existing literature, we hypothesized that the determination of quantitative bacterial counts using ATP bioluminescence is limited by the presence of EC-ATP, and that eliminating EC-ATP will allow ATP bioluminescence to be accurately employed to quantify viable CR-GNB counts in combination TKSs. Hence, we developed a method to remove EC-ATP, and compared the ability of the ATP bioluminescence assay in quantifying CR-GNB in combination testing via TKSs before and after elimination of EC-ATP.Escherichia coli, two CR Klebsiella spp., two CR Acinetobacter baumannii, and two CR Pseudomonas aeruginosa), collected as part of a national surveillance study in Singapore from 2009–2011, and four American Type Culture Collection (ATCC) strains , were used. Genus identities of the clinical isolates were determined by the individual hospitals, using Vitek 2 ID-GN cards  and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Susceptibility to carbapenems was determined by disk diffusion and interpreted using the Clinical and Laboratory Standards Institute (CLSI) guidelines  but were significantly improved after the removal of EC-ATP (AUROC for each species: 0.97–0.99). In contrast, semi-quantitative predictions of bactericidal antibiotic regimens in TKSs using ATP without EC-ATP removal were already good for most CR-GNB species (AUROC for each species except  species . Hence, The results of our study suggest that ATP bioluminescence can potentially replace the process of viable plating in the conduction of in vitro studies. In future studies, we believe that the accuracy of our assay can be further improved. It has been hypothesized that filaments or protoplasts, which are osmotically fragile cells with high ATP content induced by exposure to beta-lactam antibiotics, may contribute to the inconsistencies in the relationship between ATP bioluminescence and viable plate counts ,38. WheaTo date, in vitro TKSs remain the mainstay for determining effective antibiotic combinations against CR-GNB. Unfortunately, the elucidation of viable bacterial counts, which is a fundamental procedure in the conduction of TKSs, is a slow and labor-intensive process. Our ATP bioluminescence assay with EC-ATP removal can potentially replace the process of viable plating with high accuracy. Our assay has applications beyond in vitro TKSs, such as for in vitro hollow fiber infection models, as well as in vivo animal studies. Future studies will be conducted to further improve and expand the utility of our assay.


Zeta-chain-associated protein kinase-70 (ZAP-70) is a tyrosine kinase mainly expressed in T cells, NK cells and a subset of B cells. Primarily it functions in T cell receptor (TCR) activation through its tyrosine kinase activity. Aberrant expression of ZAP-70 has been evidenced in different B cell malignancies, with high expression of ZAP-70 in a subset of patients with Chronic Lymphocytic Leukemia (CLL), associating with unfavorable disease outcomes. Previous studies to understand the mechanisms underlying this correlation have been focused on tumor intrinsic mechanisms, including the activation of B cell receptor (BCR) signaling. Recent evidence also suggests that ZAP-70, intrinsically expressed in tumor cells, can modulate the cross-talk between malignant B cells and the immune environment, implying a more complex role of ZAP-70 in the pathogenesis of B cell malignancies. Meanwhile, the indispensible roles of ZAP-70 in T cell and NK cell activation also demonstrate that the autologous expression of ZAP-70 in the immune environment can be a central target in modulation of tumor immunity. Here we review the evidences of the link between ZAP-70 and tumor immunology in the microenvironment in B cell malignancies. Considering an emerging role of immunotherapies in treating these conditions, understanding the distinct molecular functions of ZAP-70 in a broader cellular context could ultimately benefit patient care. IGHV mutation analyses (Zeta-chain-associated protein kinase-70 (ZAP-70) is a tyrosine kinase mainly expressed in T cells and NK cells , 2. The analyses . Howeveranalyses , 9. AlthIGHV gene and poor clinical outcomes  and C-X-C chemokine receptor type 4 (CXCR4) expression on B cells is essential for cell migration and homing during B cell development through the binding to their putative chemokine ligands CCL19/CCL21 and CXCL12, respectively . High reBesides BCR mediated signals, Toll-like receptor (TLR) signaling, which can bridge innate and adaptive immune responses, has been found to play a role in CLL activation and proliferation , 39. IntMounting evidence indicates that the ZAP-70 expressed in tumor cells has ramifications for the composition of immune cells in the microenvironment, especially for the number of infiltrating T cells. In studies comparing the immune-phenotype of ZAP-70 positive and ZAP-70 negative CLL patients, tumor ZAP-70 expression was associated with increased CD4 central memory T cells and CD3/CD69+ T cells with decreased CD4/CD8 ratio in the peripheral blood –42. HoweImmunotherapy, including checkpoint blockade inhibition and cell-based immunotherapies, is a fast developing area in cancer treatment. Such treatment modalities have been applied to treat B cell malignancies and demonstrated significant improved outcomes in smaller subsets of patients, who previously relapsed from chemotherapies and targeted therapies . NotablyBecause of its indispensible role for TCR activation, deficiency, or aberrantly high expression of ZAP-70 in T cells expectedly result in immune deficiency [ZAP-70-related severe combined immunodeficiency syndromes (SCID)]  and autoStructurally, ZAP-70 has been found to play a central role in immunological synapse formation in cytotoxic T lymphocytes (CTL) . CTLA-4 −/− ZAP-70−/− mice still maintain natural cytotoxicity, which suggests a redundant role of ZAP-70 in this process, despite driving the activation of NK cell receptor signaling  are T cells expressing artificial T cell receptors which contain both tumor specific- as well as T cell activating motifs . Promisiin vitro studies have previously investigated inhibitors which can suppress ZAP-70 kinase activity or disrupt its protein-binding ability to access downstream TCR related activators  and their inhibition may therefore be therapeutically beneficial, blockage of cytotoxic T cells and NK cells may be less so. Whether different immune cells display different susceptibilities to ZAP-70 inhibition, thus allowing for a wide-enough therapeutic window of antagonists to be beneficial, is unknown, but at least seems possible. We believe this is a substantial problem to be considered in the design of ZAP-70 directed therapies.Immunotherapies, including CAR-T cells, immune checkpoint blockade, and adaptive T cell therapies, have been applied in clinical treatment for B cell malignancies. The safe and precise control of over-reactions of anti-tumor immune responses has been a major issue for the toxicity of cell-based immunotherapies . Based oZAP-70 is not only critical for T cell and NK cell activation, but also associated with poor outcomes of B cell malignancies, especially in CLL. Tumor intrinsic expression of ZAP-70 in B cell malignancies has been shown to enhance cellular signals under ligand stimulated BCR activation. However, the underlying mechanisms known so far cannot fully explain the correlation between ZAP-70 expression and dismal outcome. More evidence has pointed to ZAP-70 driven environmental changes, which may play a central role for triggering innate immune responses and immune cell infiltration .in vitro experiments, more in vivo studies are needed to fully characterize whether such therapies can be applied in a clinical setting.Growing evidence also indicates that the modulation of ZAP-70 activity can be applied to control T cell activation, which has translational potential to mitigate the toxicity associated with cell-based immunotherapies . HoweverA thorough review of the published evidences focusing on defining the role of ZAP-70 in health and disease clearly indicates that it remains an attractive target for therapeutic interventions, more than ever. More experimental evidence is needed to fully understand the biology behind ZAP-70 in B cell malignances in a holistic cellular approach. The simultaneous targeting of ZAP-70 in tumor cells, T and NK cells, may be beneficial in some instances, but also bears the risk to promote tumor growth through impairing immune surveillance.JC and IR designed and wrote this review. AM provided critical editing on the manuscript and the graphs. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cardiovascular diseases (CVDs) are the leading cause of mortality and morbidity globally, representing approximately a third of all deaths every year. The greater part of these cases is represented by myocardial infarction (MI), or heart attack as it is better known, which occurs when declining blood flow to the heart causes injury to cardiac tissue. Mesenchymal stem cells (MSCs) are multipotent stem cells that represent a promising vector for cell therapies that aim to treat MI due to their potent regenerative effects. However, it remains unclear the extent to which MSC-based therapies are able to induce regeneration in the heart and even less clear the degree to which clinical outcomes could be improved. Exosomes, which are small extracellular vesicles (EVs) known to have implications in intracellular communication, derived from MSCs (MSC-Exos), have recently emerged as a novel cell-free vector that is capable of conferring cardio-protection and regeneration in target cardiac cells. In this review, we assess the current state of research of MSC-Exos in the context of MI. In particular, we place emphasis on the mechanisms of action by which MSC-Exos accomplish their therapeutic effects, along with commentary on the current difficulties faced with exosome research and the ongoing clinical applications of stem-cell derived exosomes in different medical contexts. Myocardial infarction (MI) is a major cardiovascular disease (CVD) caused by a sudden full or partial stoppage of blood flow to the myocardium, leading to cardiomyocyte (CMC) death and subsequent irreversible heart muscle necrosis and apoptosis. While recent pharmacological and mechanical advances have significantly contributed to the sharp decline in death rates ,2,3, MI The current standard treatments for MI that aim to mitigate heart damage remain limited in restoring heart function as they fail to address the CMC and vasculature loss underlying the condition . Major sSubstantial evidence suggest that the observed cell therapy-related effects are attributed to the paracrine activity of the injected cells, rather than their successful integration/transdifferentiation into the myocardium ,25,26,27This review will examine the current state of MSC-Exo research and discuss its potential translational application as a novel cell-free agent for conferring cardiovascular protection and regeneration following MI.EVs are prokaryote and higher eukaryote cell-secreted miniscule vesicles  that act as shuttles for a heterogeneous and bioactive cargo mainly composed of proteins, lipids, and nucleic acids ,54. WhilDespite the umbrella term used to generally describe secreted phospholipid bilayer-enclosed vesicles, EV populations are known to be highly heterogeneous ,55,56. TApoptotic bodies (ABs) are the largest (800–5000 nm in diameter) subtype of EVs that are released as a product of apoptotic cell disassembly. Specifically, ABs release occurs during the execution phase of the apoptotic process, when the cell undergoes shrinkage, chromosomal condensation, nuclear and chromosomal fragmentation, and membrane blebbing . While bExosomes, which are the principal focus of this review, originate from the endocytic pathway. Like the larger MVs, exosomes play crucial roles in intercellular communications, but are divergently formed by the inward budding of the multivesicular body (MVB) membrane, which subsequently fuses with the plasma membrane to release intraluminal vesicles (ILVs) into the extracellular space . As such2 content [Although exosomal cargo specificity varies according to the parent cell-type and other environmental conditions ,  content . The afo content ,78,79,80 content ,82,83,84Due to their ability to convey a biological message by shuttling a highly specific parental cell-derived cargo, including miRNAs, mRNAs, proteins, and a variety of bioactive molecules, EVs exert a pivotal role in cell-to-cell signalling. Hence, EV research is a growing interest worldwide and has led to a large number of clinical trials, aimed at investigating their efficacy and safety as cell-free therapy agents. Although EVs were discovered more than three decades ago, their isolation does not yet rely on a gold-standard protocol, but it is rather accomplished by different approaches, widely depending on their source (biofluids or cell culture media) and the following downstream applications ,86.g) ultracentrifugation (UC), the most commonly used method of isolation, allows low yield exosomes isolations from large volumes of initial material  with the disadvantage of it being considerably time consuming and unable to separate EVs from other contaminants [High speed , transmission electron microscopy (TEM), Western blot (WB) for exosomal markers , and nanoparticle tracking assays (NTA) can be used for post-isolation characterization of the exosomes . QualitaMSCs are multipotent progenitor cells, which can be extracted from a wide variety of tissues, including bone marrow, adipose tissue, synovium, and Wharton’s jelly. While there exists some biological variability between MSCs isolated from different tissue origins, the International Society for Cellular Therapy has proposed a set of minimum criteria defining MSCs based on their characteristics: (a) being plastic-adherent when maintained in standard culture conditions; (b) expressing CD105, CD73, and CD90; (c) not expressing (negative markers) CD45, CD34, CD14, or CD11b, CD79a, or CD19 and HLA-DR surface molecules; (d) being able to differentiate into osteoblasts, adipocytes, and chondroblasts in vitro upon growth factor stimulation; and (e) being self-renewable, multipotent, easily accessible, and culturally expandable in vitro .In addition, the genomic stability and lack of ethical issues with the application of MSCs make them exemplary vectors for cell therapy, regenerative medicine, and tissue repairment. Preclinical and clinical studies have reported generally positive immunomodulatory and regenerative effects of MSCs in various medical contexts, including cardiac regeneration. in vitro and in vivo studies have shown that MSCs are capable of differentiating into CMCs ,97, endoIGF-1R [Initial models attempting to provide mechanistic explanations of the therapeutic effects of MSCs described them as migratory cells travelling to and engrafting at the injury sites and subsequently interacting with local cells. However, recent studies have demonstrated that their therapeutic activity is mainly exerted in a paracrine manner rather than via direct stem-cell transdifferentiation. Such investigations have shown that MSC-conditioned medium enhances CMC and progenitor survival after hypoxia-induced injury ,105. ThiIGF-1R ), and reIGF-1R ,111). ThIGF-1R , tumorigIGF-1R . FurtherThe above indicates strong potential implications of MSC-Exos in novel therapeutics for cardiovascular diseases. In fact, a large volume of preclinical studies has confirmed that MSC-Exos reduce the infarct size and improve post-MI cardiac function ,115,116.de novo microvessels’ formation represent a promising strategy for the treatment of acute MI. MSCs contribute to cardioprotection and regeneration in an infarcted myocardium through its paracrine stimulation of angiogenesis in affected cells. Studies have shown that this pro-angiogenic potential is promoted by MSC-Exo-mediated secretion of bioactive factors  and receptor tyrosine kinase Flk-1 (Flk1) while simultaneously down-regulating the anti-angiogenesis genes vasohibin-1 (Vash1) and thrombospondin-1 (Tsp1) in ECs [Ang1 and Flk1 expression, and inhibits the expression of Vash1 [Significant blood vessel neo-formations including T cell proliferation and tube formation have been observed in vitro after MSC-Exos administration ,121. In ) in ECs . These eof Vash1 .in situ tissue healing [Although the mechanistic bases need to be further elucidated, it is clear that MSC-Exo-induced angiogenesis is strictly cargo-dependent. MSC-Exos exposed to ischemic conditions have a high representation of factors involved in canonical angiogenesis-associated pathways, such as the cadherin, epidermal growth factor receptor (EGFR), FGF, and platelet-derived growth factor (PDGF) pathways . Further healing .Further proteomic studies reinforced the hypothesis that the aforementioned pro-angiogenic response is mediated by a consistent transfer of bioactive factors, such as the extracellular matrix metalloprotease inducer (EMMPRIN), matrix metalloprotease-9 (MMP-9), and VEGF between donor (MSCs) and recipient (ECs) cells . Of partIn addition to their protein fraction, MSC-Exos are able to convey their pro-angiogenic signals through a direct miRNA transfer. Hypoxia-elicited MSC-Exos are significantly enriched in pro-angiogenic miR-125b-5p compared with naturally occurring MSC-Exos . miR-21-Apoptosis is a form of programmed cell death characterised by membrane blebbing, cell shrinkage, condensation of chromatin, and DNA fragmentation, followed by a rapid engulfment of the corpse by neighbouring cells. It is distinguished from necrosis by the absence of an associated inflammatory response . ApoptosEvidence suggests that MSC-Exos exert anti-apoptotic effects through the modulation of bioenergetics in target cells. Key features of acute MI include loss of ATP and NADH, increased oxidative stress, and cell death —all procc from mitochondria and an enhancement of caspase-3 activity [The PI3K/AKT pathway is a key intracellular signal transduction pathway involved in the regulation of apoptosis and survival. AKT is the primary protein effector downstream of the PI3K signalling pathway and plays an important role in glucose metabolism by regulating the biological functions of insulin ,163. CMCactivity . This paactivity . Among tactivity . Similaractivity . These oactivity ,169,170.MSC-Exos treatment activates AMPK/mTOR and AKT/mTOR signalling to partly reduce in vitro and in vivo apoptosis through autophagy enhancement . It is lHypoxic preconditioning of parent MSC significantly improves the ability of MSC-Exos to inhibit apoptosis by enriching their miR-22 content, which inhibits apoptosis by targeting methyl CpG binding protein 2 (Mecp2) . HorizonGATA-4-DEs) expressing anti-apoptotic miRNAs, reduce apoptosis and preserve mitochondrial membrane potential in targeted hypoxic CMCs [GATA-4 -DEs. Further analysis showed that miR-19a downregulates PTEN and BIM expression resulting in AKT and ERK signalling pathways activation while inhibiting JNK/caspase-3 activation by targeting the transcription factor SRY-box transcription factor-6 (SOX-6) [GATA-4 found that the cardioprotection induced by MSCGATA-4-DEs is partially mediated by miR-221 [Interestingly, induced changes to gene expression in parent MSCs influence the anti-apoptotic properties of MSC-Exos. GATA-binding protein-4 (GATA-4) overexpression leads to increased growth factor release and EC-mediated angiogenesis . Anotherxic CMCs . Additio (SOX-6) . These o miR-221 , which iMIF) enhances myocardial repair by ameliorating the heart function, reducing heart remodelling, mitochondrial fragmentation, and apoptosis in vivo [MIF-DEs confer enhanced anti-apoptotic effects compared to unmodified MSC-Exos in hypoxic CMCs. Here, the exosomal long coding RNA lncRNA-NEAT1 inhibits miR-142-3p [Furthermore, exosomes from MSCs treated with macrophage migration inhibitory factor (MIF)  have also indicated similar results, as co-culture experiments increase CD3+ T cell apoptosis while reducing B cell proliferation, differentiation, and production of IgM, IgG, and IgA under CpG stimulation [A breakthrough study published in 2015 showed that MSC-Exos restrain the inflammatory response during acute MI by inhibiting immune cell invasion and proliferation in the infarcted zone of the rat heart . Specifimulation . Furthermulation .Later studies aimed at identifying the mechanism by which MSC-Exos reduced the inflammatory response found numerous components involved in the process. MSC-Exos express programmed death-ligand 1 (PD-L1), galectin-1, and membrane-bound TGF-β, which are all essential molecules for inducing immune tolerance . MoreoveMSC-Exo-delivered miR-182 attenuates myocardial I/R injury via TLR4/NF-κB/PI3K/AKT pathway interaction, which in turn regulates macrophage polarisation . MSC-ExoMoreover, the M1 to M2 macrophages shift has implications in tissue fibrosis. The post-MI myocardium is highly characterised by extensive cardiac fibrosis due to the fibroblast and myofibroblast-mediated reparative response to ischemic cell death . The iniM2 macrophage secretion of TGF-β1 induces resident fibroblast to myofibroblast transformation , which iDespite the seemingly contradictory functions of the M2 macrophages, several reports show that MSC-Exos ameliorate fibrosis after MI ,210,230.plethora of preclinical studies in cardiovascular disorders, more efforts are needed to establish standard and consistent methods for exosome production (cell lines and culture conditions), isolation, and storage, which are aimed at reducing the variability between cell-free products. Additional investigations focused on dose and administration of exosome preparations in patients may facilitate their use in future clinical trials. Hence, due to the above translational limitations, in a majority of the ongoing clinical trials, exosomes and their protected cargos including RNAs, small RNAs, and proteins [Due to their potential as therapeutic cell-free drugs and biomarkers, 151 clinical trial studies involving exosomes  are beinproteins , are beiThe NCT03857841 phase I trial assesses the safety and tolerability of intravenous administration of BM-MSC-EVs (UNEX-42) in 18 patients at risk of bronchopulmonary dysplasia, a severe neonatal lung injury . PreclinEpidermolysis Bullosa (EB) includes a group of inherited genodermatoses caused by a lack of collagen VII, which in turn results in skin fragility and mucocutaneous blistering. The phase I/IIA trial NCT04173650, aims at studying effectiveness and safety of AGLE-103 topic administration in the treatment of lesions in EB subjects. AGLE-103 is an allogenic derived drug composed of MSC-EVs derived from normal donors. Previous data showed that the intradermal administration of allogenic MSCs improved the wound healing and prevented blistering by promoting Collagen VII replacement in patients .The NCT03384433 phase II trial aims at studying efficacy and safety of allogenic miR-124 enriched MSC-EVs intravenously administered to five patients with acute ischemic stroke. Specifically, five patients aged 40–80 years will receive 200 µg  of miR-124-enriched allogenic MSC exosomes one month after stroke attack. MiR-124 regulates several biological processes in central nervous system  and exerChronic graft-versus-host disease (GVHD) is a life-threatening complication following allogenic hematopoietic stem cell transplantation, resulting in enhanced inflammatory events triggered by the interaction between donor lymphocytes and foreign antigens . Major oParticipants from NCT04213248 Phase II clinical trial will receive umbilical mesenchymal stem cell (UMSC)-derived exosomes 10 µg/drop, four times a day for 14 days with the aim of relieving the dry eye symptoms. Previous results indicate that MSCs infusions can be therapeutically effective in suppressing dry eye symptoms associated in cGVHD subjects by promoting the generation of regulatory T cells exerting immunomodulatory effects . SubsequThe early phase I trial NCT03437759 aims at inducing functional recovery of large and refractory macular holes (MHs). MHs are thickness defects in the eye macula region of various pathogenetic origin (including idiopathic and traumatic) .+ T cells [Forty-four subjects will receive a 10 μL PBS drop containing 50 μg or 20 μg MSC-Exo into vitreous cavity and will be followed up by physical examinations, fundoscopy, best-corrected visual acuity (BCVA) measurement, and spectral-domain optical coherence tomography (OCT). The main patient inclusion criteria are a diagnosis of MHs larger than 400 μm. Literature studies show that local human umbilical cord-derived MSC-EVs injection ameliorates uveitis in a rat by inhibiting inflammatory cell migration, including neutrophils, macrophages cells, and CD4 T cells .KRAS) gene mutations, occurring to > 90% of the patients [Pancreatic ductal adenocarcinoma is a highly metastatic disease associated to KRAS Proto-Oncogene, GTPase  and maximum tolerated dose (MTD). The ability of iExosomes to enter cells via micropinocytosis and reducing RAS pathway activation, resulting in pancreatic cancer suppression, has been extensively shown in multiple in vitro and in vivo models . RAS pat6/kg of purified exosomes followed by a second injection of an equivalent dose of MVs (180–1000 nm) after 7 days.Type I diabetes mellitus (T1DM) is caused by the pancreatic islets β-cell damage, due to an autoimmune mechanism involving T cells  and otheWhile there is still much to be understood about their nature, MSC-Exos have emerged as a highly promising cell-free vector for conferring regeneration in the heart. As outlined in this review, MSC-Exos may have especially strong implications in MI therapeutics, being able to confer potent angiogenesis ,120,121,While naturally occurring MSC-Exos have been demonstrated to induce desirable effects, it is a natural next step to pursue research of engineered exosomes that are capable of producing more vigorous effects that optimise cardioprotection and cardiac regeneration. Future research on MSC-Exos in this context should therefore focus on identifying the specific molecular cargo of MSC-Exos, and subsequently elucidate the mechanism by which such components are able to produce specific effects in target cells. Alongside this, research into the mechanism of exosome-loading process will be highly valuable as greater understanding of the process would allow for modification of the parent cell, with the aim of producing exosomes encapsulating desirable molecular cargo. While still unrefined, several aforementioned studies in this review have attempted to artificially improve MSC-Exo cardioprotection and regeneration, either through modification of the cells’ local environment ,173,176 Besides exosome modification, one interesting possibility is to explore the idea of the enhancement of cardioprotection and cardiac regeneration through selection of MSCs that secrete the “perfect” exosomes for the required clinical applications. To do this, cells could be subjected to undergo a process of directed evolution. This is a method for artificially selecting a biological species  towards a user-defined goal . It consNevertheless, the application of MSC-Exos—and exosomes in general—in a clinical setting remains a challenge due to limitations in exosome isolation and characterisation. While techniques such as ultracentrifugation and size exclusion chromatography-based methods are readily able to capture exosomes from MSC-conditioned media, it remains a hard truth that the resulting sample is never a pure population of exosomes due to the fact that other biological particles, especially that of MVs, can overlap in size. Developments of methods that can better distinguish MSC-Exos based on their biological properties rather than their physical qualities is likely to improve the pharmacological potency of the samples. More importantly, however, is that obtaining pure fractions of MSC-Exos would shed light on the components of the exosomes preparation that may produce unexpected and/or undesirable effects when applied in a clinical setting (such as an adverse immune response or tumour development) .


Investigation of a dengue case in a laboratory worker in North Carolina, USA, revealed that the case-patient prepared high-titer dengue virus stocks soon before illness onset. Improper doffing of gloves with an open finger wound likely resulted in cutaneous exposure. This case reinforces recommendations for enhanced precautions when working with high-titer dengue virus. Aedes mosquitoes and has a median incubation period of 6 days  cause dengue, an acute febrile illness common throughout the tropics  was notified of a dengue case in a laboratory worker. NCDHHS and CDC conducted an investigation to identify the most likely route of exposure.6 cells/mm3).We interviewed the case-patient and reviewed medical records to collect travel history, potential exposures, clinical course, and diagnostic test results. The case-patient reported no recent travel to an area with ongoing DENV transmission. No travel-associated dengue cases were reported in 2018 from the county where the case-patient worked and resided. The case-patient reported illness onset on July 18, 2018, with retroorbital eye pain, fever, myalgia, arthralgia, lethargy, chills, and lymphadenopathy . By JulyEhrlichia IgG. At CDC, reverse transcription PCR .In the 2 weeks before illness onset, the case-patient reported working with a protocol to grow, purify, and concentrate DENV-4. The case-patient reported wearing a single pair of nitrile gloves, eye protection, a lab coat, and closed-toed shoes while working with infectious virus in a certified biosafety cabinet (BSC).6 plaque-forming units (PFU) of DENV-4  training upon joining the laboratory, receiving hands-on training for BSL-2 work, and annually reviewing laboratory safety plans and procedures.The case-patient reported having sustained a compression wound on the ring finger of the left hand on July 9 or 10; the wound later appeared infected and oozing. The case-patient reported not bandaging or covering this wound before donning a single pair of gloves while working on the protocol for virus production and purification. The case-patient demonstrated their technique for doffing gloves : the basThe presence of an open finger wound during work with high-titer DENV coupled with improper glove doffing suggests that laboratory-acquired infection by cutaneous exposure was the most likely route of DENV infection in this case. However, other routes of exposure, including mucosal, could not be ruled out. Three previous cases of laboratory-associated DENV infection have been reported. In Nigeria, a laboratorian responsible for cleaning cages and disposing of mice infected with DENV-1 became infected, although mosquito-borne transmission could not be ruled out (>1 flavivirus complicated interpretation of neutralizing antibody titers and precluded identification of the infecting DENV. Moreover, we could not rule out infection with DENV between collection of the baseline and acute specimens. The difficulty interpreting flavivirus neutralizing antibody patterns during secondary infections is well described (In this case, detection of NS1 antigen independently confirmed acute DENV infection, supported by detection of DENV IgM and >4-fold rise in DENV IgG and DENV neutralizing antibody. However, historic exposure to A study in Belgium conducted during 2007–2012 found that only 40% of laboratory-associated infections occurred following a known exposure event; a definitive cause of exposure could not be identified in nearly one third of cases associated with bloodborne pathogens (3–107 PFU/mL (9–1010 PFU/mL, the concentration of DENV the case-patient handled would have been 100 to 10 million times higher than the concentration found in the average blood specimen of a patient with DENV infection. Although BSL-2 containment is recommended for laboratory work with DENV, enhanced safety precautions including double-gloving are recommended when handling large-scale or high-titer virus (Titers of infectious DENV in human blood resulting from mosquito-borne transmission are typically 10


Immune checkpoint inhibitor (ICI)-induced pneumonitis has been considered a rare immune-related adverse event (irAE). Incidence is estimated at less than 5% of clinical trial patients, with high-grade pneumonitis occurring in less than 1% of patients who receive ICIs.2 4ICI-induced pneumonitis generally develops within the first 3 months of treatment.8et alet alBalaji In both studies, most patients who developed ICI-induced pneumonitis had lung cancer, which is consistent with prior studies.et al describe the clinical management and disease course of 12 patients with primary steroid-refractory ICI-induced pneumonitis . The identified patients were referred to the multidisciplinary immune-related toxicity team or pulmonary outpatient clinic with ICI-induced pneumonitis. In this study, primary steroid refractoriness occurred after a median of 5 doses of ICI, resulting in grade 2 (n=3) and grade 3 (n=9) symptomatic pneumonitis. The most commonly identified radiographic pattern was diffuse alveolar damage. The mean dose of steroids administered was prednisone equivalent 75 mg/day prior to receiving additional immunosuppressive therapy. Clinical management included treatment with IVIG , infliximab , and combination IVIG + infliximab . Almost all patients with primary steroid-refractory ICI-induced pneumonitis required management in the intensive care unit; around half required mechanical ventilation. The authors report that 75% of patients in this study died of steroid-refractory ICI-induced pneumonitis or infectious complications, including all five patients treated with infliximab .Balaji et al describe the clinical course of 26 patients who had either primary steroid-refractory ICI-induced pneumonitis  or steroid-resistant ICI-induced pneumonitis . The authors searched for administration of immunomodulatory agents and evaluated the clinical management and course for patients who clinically and/or pathologically had ICI-induced pneumonitis. All patients with primary steroid-refractory ICI-induced pneumonitis were treated with at least 60 mg of prednisone equivalent. Median time to addition of an immunomodulator for primary steroid-refractory ICI-induced pneumonitis was 7 days, and median time to additional treatment for steroid-resistant pneumonitis during steroid taper was 2.9 months. The study reported more severe disease in patients with primary steroid-refractory ICI-induced pneumonitis  than in patients with resistant pneumonitis . Most of the retrospective study included patients treated with infliximab monotherapy , mycophenolate mofetil monotherapy  or infliximab comination therapy . However, further investigation into the subgroups shows that all patients with primary steroid-refractory pneumonitis (n=12) received infliximab as monotherapy or in combination with mycophenolate, and most patients with recurrent pneumonitis received infliximab, and/or mycophenolate. Of the 26 patients identified, more than half (62%) were hospitalized and, despite aggressive treatment, only 10 had a durable improvement following additional immunosuppression plus steroids. However, when focusing on the primary steroid-refractory patients, only 3/12 (25%) had durable desponses to combination immunosuppression and all 12 patients died with cause of death attritbued to pneumonitis or infectious complications in half of these patients .Beattie Steroid-refractory ICI-induced pnemonitis is a rare irAE with a poor prognosis. Patients with primary steroid-refractory ICI-induced pneumonitis have a high all-cause mortality rate (pneumonitis or infectious complications) and a worse prognosis than patients with recurrent ICI-induced pneumonitis. Identifying ICI-induced pneumonitis may be challenging due to varying clinical presentations, but the most common radiographic pattern is diffuse aveolar damage. It is unclear if rapid escalation from steroid monotherapy to combination therapy with other immune modulators can fully reverse the disease course or improve clinical outcomes in patients with high-grade primary steroid-refractory ICI-induced pneumonitis. However, therapy escalation may lead to durable improvement  in steroid-refractory ICI-induced pneumonitis in up to 30%–40% of patients. Prospective studies on the efficacy of immune modulators or combinations are greatly needed for primary steroid-refractory ICI-induced pneumonitis. Furthermore, in addition to standard endpoints like 90-day morbidity and duration of immunosuppression, these trials should also incorporate clinically meaningful endpoints like FEV1, required level of clinical care, and need for supplemental oxygen. Prospective studies should also evaluate the role of patient-specific factors like current or prior tobacco use and prior radiation on the development of irAEs. Finally prospective studies should aim to incorporate exploratory analyses of peripheral blood, bronchoscopy specimens and tumor samples to investigate potential predictive biomarkers that may indicate an increased risk for the development of pneumonitis. Several prospectitve studies evaluating alternative treatments for steroid-refractory irAEs have recently opened . We eagerly await the results.et al=0/5; Beattie et al=3/12), only 3 (18%) showed durable clinical responses and all had died. This low treatment response rate with infliximab in primary steroid-refractory ICI-induced pneumonitis demonstrates the need for academically driven, prospective research on treatment algorithms for irAEs. Given that Balaji et al demonstrated that patients who received IVIG monotherapy had improved oxygen requirements, a reduced level of care, and reduced ICI-induced pneumonitis-related fatalies, IVIG should be considered as a reasonable alternative to infliximab until prospective irAE data are generated.Infliximab is a commonly used immune modulator in patients with primary steroid-refracory ICI-induced irAEs, including ICI-induced pneumonitis. These retrospective studies, the largest published to date, demonstrated a high mortality rate (due either to progressive pneumonitis or infectious complications from infliximab) in patients with primary steroid-refractory ICI-induced pneumonitis who received infliximab. While patients with primary steroid-refractory ICI-induced pneumonitis have more severe disease than patients with recurrent ICI-induced pneumonitis, these studies raise serious questions about the efficacy of infliximab in treating steroid-refractory, ICI-induced pneumonitis. Of the 17 patients with primary steroid-refractory ICI-induced pneumonitis who received infliximab with high-dose steroids and/or another immunomodulatory agent (Balaji