The coronavirus disease 2019 (COVID-19) pandemic has caused recurring and major outbreaks in multiple human populations around the world. The plethora of clinical presentations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been described extensively, of which olfactory dysfunction (OD) was established as an important and common extrapulmonary manifestation of COVID-19 infection. The aim of this protocol is to conduct a systematic review and meta-analysis on peer-reviewed articles which described clinical data of OD in COVID-19 patients.This research protocol has been prospectively registered with the Prospective Register of Systematic Reviews . CINAHL, ClinicalTrials.gov, Cochrane Central, EMBASE, MEDLINE and PubMed, as well as Chinese medical databases China National Knowledge Infrastructure (CNKI), VIP and WANFANG, will be searched using keywords including ‘COVID-19’, ‘coronavirus disease’, ‘2019-nCoV’, ‘SARS-CoV-2’, ‘novel coronavirus’, ‘anosmia’, ‘hyposmia’, ‘loss of smell’, and ‘olfactory dysfunction’. Systematic review and meta-analysis will be conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and the Meta-analyses Of Observational Studies in Epidemiology (MOOSE) guidelines. Articles will be screened according to pre-specified inclusion and exclusion criteria to extract studies that include new clinical data investigating the effect of COVID-19 on olfactory dysfunction. Included articles will be reviewed in full; data including patient demographics, clinical characteristics of COVID-19-related OD, methods of olfactory assessment and relevant clinical outcomes will be extracted. Statistical analyses will be performed using the Comprehensive Meta-Analysis version 3.This systematic review and meta-analysis protocol will aim to collate and synthesise all available clinical evidence regarding COVID-19-related OD as an important neurosensory dysfunction of COVID-19 infection. A comprehensive search strategy and screening process will be conducted to incorporate broad clinical data for robust statistical analyses and representation. The outcome of the systematic review and meta-analysis will aim to improve our understanding of the symptomatology and clinical characteristics of COVID-19-related OD and identify knowledge gaps in its disease process, which will guide future research in this specific neurosensory defect.PROSPERO registration number: CRD42020196202.The online version contains supplementary material available at 10.1186/s13643-021-01624-6. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the aetiological agent of the coronavirus disease 2019 (COVID-19) global pandemic, has infected over 102 million people worldwide, accounting for over 2,200,000 deaths as of 27 September 2020 . ConsistRecent systematic reviews and meta-analyses regarding COVID-19-related OD have found significant discordance between subjective reporting of smell changes and objective quantitation of olfaction –13, suggIn this systematic review and meta-analysis protocol, we aim to investigate the demographic characteristics of COVID-19 patients presenting with OD, and to ascertain whether there is any age, sex, or ethnic predisposition to COVID-19-related OD. In addition, we will investigate the potential associations between olfactory neurosensory impairments and other otolaryngologic or neurologic disorders in COVID-19 infection. Finally, we aim to determine the prevalence of COVID-19-related OD as an isolated symptom, including its onset and duration, and whether OD may be a prognostic indicator for COVID-19 disease severity.This systematic review will include peer-reviewed articles which described clinical data on OD in patients of all ages who were confirmed with SARS-CoV-2 infection by reverse transcription polymerase chain reaction (RT-PCR) tests.R2 index and p-value] [The systematic review protocol has been registered on the Prospective Register of Systematic Reviews . The research progress will be periodically updated on PROSPERO. The systematic review and meta-analysis will be carried out according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)  and Metap-value] .st January 2020 to the date of completion of data extraction. Search keywords include ‘COVID-19’, ‘coronavirus disease’, ‘2019-nCoV’, ‘SARS-CoV-2’, ‘novel coronavirus’, ‘anosmia’, ‘hyposmia’, ‘loss of smell’, and ‘olfactory dysfunction’. Additionally, articles published within this time period will be searched from the following Chinese medical databases: China National Knowledge Infrastructure (CNKI), VIP and WANFANG, to ensure greater scope of representation from different geographical and ethnic populations. The detailed search strings of each database can be found in Supplementary Table For the systematic review, the research group will search CINAHL, ClinicalTrials.gov, Cochrane Central, EMBASE, MEDLINE and PubMed for articles published from 1Subsequently, the search results will be combined and duplicates will be removed by Excel . Eligible articles will be screened by four authors  by the article titles and abstracts, followed by full text examination. Disagreements will be resolved by another author (T.W.H.C.).Potentially eligible articles will be categorised using Microsoft Excel into three groups according to the article titles and abstracts: (A) articles containing clinical data on COVID-19; (B) epidemiological-modelling studies, animal models and experiments, and laboratory investigations which did not contain sufficient clinical data; and (C) guidelines, editorials, commentaries and review articles that did not contain new clinical data. After initial categorisation, full text of the articles containing clinical data [under category (A)] will be examined for their eligibility for inclusion. The design of the study selection strategy is summarised in Fig. The inclusion criteria for systematic review include (1) COVID-19 diagnosis confirmed by SARS-CoV-2 RT-PCR tests; (2) studies which reported clinical data on olfactory disturbances, either qualitatively or quantitatively; and (3) written in English or Chinese. The exclusion criteria include (1) articles which did not report individual clinical data on olfactory disturbances; and (2) articles that did not contain new clinical data. Case reports and case series of insufficient sample size (i.e. < 10 patients) will be included in the systematic review, but not the meta-analysis.The methodological quality of studies will be determined using the Newcastle–Ottawa Scale (NOS) with a maximum of nine points (stars) for observational studies . ‘SelectData will be extracted independently by four authors . Disagreements will be resolved by mutual consensus. For included articles, the following data will be extracted: (1) basic information of the articles ; (2) patient demographics ; (3) disease characteristics ; (4) relevant investigation outcomes ; (5) the method(s) used to assess olfaction ; (6) relevant imaging and endoscopic findings; and (7) any treatment provided.I2 statistics [p value less than 0.05 will be deemed statistically significant. All analyses will be performed using the Comprehensive Meta-Analysis version 3 . Descriptive statistics will be used for outcomes which are not suitable for meta-analyses.The prevalence of OD in COVID-19 patients will be computed for each of the studies. Pooled estimate of the prevalence of COVID-19-related OD will be calculated using the random effects meta-analysis, as the included studies involved different centres, different populations and different tools for olfactory assessment. Analysis of heterogeneity will be performed using the atistics . Publicaatistics . Egger’satistics . The outThis systematic review and meta-analysis will be the most up-to-date and comprehensive study that evaluates COVID-19-related OD. Meticulous search strategy will be applied to identify all relevant peer-reviewed articles from multiple medical databases, thereby increasing the sensitivity and specificity of the search strategy. One potential limitation of this meta-analysis will be the heavy reliance on observational studies, which may be prone to biases and confounding factors. However, quality assessment procedures as mentioned will help in the selection of articles. Strict adherence to the PRISMA and MOOSE guidelines will help to improve the reporting quality of the study. Additionally, this research protocol has been prospectively registered on PROSPERO, which aims to maintain transparency throughout the study process. Any amendments made in the process of the systematic review or meta-analysis will be clearly indicated on PROSPERO. The outcome of this systematic review and meta-analysis will be crucial in quantifying the global prevalence and disease burden of COVID-19-related OD and serve to identify knowledge gaps in understanding its disease course. This article will be instrumental for future research regarding this important neurosensory defect in the COVID-19 pandemic.Additional file 1 : Table S1. Search strings according to medical database platforms. PRISMA-P checklist. MOOSE checklist. PROSPERO registration.


Saccharomyces cerevisiae, Escherichia coli, and Corynebacterium glutamicum have been engineered towards microbial synthesis of such compounds in recent years. However, developed strains and processes often suffer from insufficient productivity. Usually, tightly regulated intracellular malonyl-CoA availability is regarded as the decisive bottleneck limiting overall product formation. Therefore, metabolic engineering towards improved malonyl-CoA availability is essential to design efficient microbial cell factories for the production of polyketides and fatty acid derivatives. This review article summarizes metabolic engineering strategies to improve intracellular malonyl-CoA formation in industrially relevant microorganisms and its impact on productivity and product range, with a focus on polyketides and other malonyl-CoA-dependent products.Malonyl-CoA is an important central metabolite serving as the basic building block for the microbial synthesis of many pharmaceutically interesting polyketides, but also fatty acid–derived compounds including biofuels. Especially Key PointsMalonyl-CoA is the central building block of polyketide synthesis.• Increasing acetyl-CoA supply is pivotal to improve malonyl-CoA availability.• Improved acetyl-CoA carboxylase activity increases availability of malonyl-CoA.• Fatty acid synthesis as an ambivalent target to improve malonyl-CoA supply.•  Polyketides are an outstanding group of secondary metabolites with regard to their structural diversity and the number of their clinical applications Hopwood . Not onlSaccharopolyspora erythraea synthesizing the 6-deoxyerythronolide B scaffold of erythromycin A condenses one propionyl-CoA starter unit with six methylmalonyl-CoA extender units . Based on their overall architectural structure, which defines starter unit selection, chain elongation, degree of reduction, and cyclization patterns, PKS are classified into three types (types I–III)  , a pentaketide from the medical plant E. coli, deletion of acetyl-CoA consuming acetate and ethanol forming pathways resulted in a 15-fold improved malonyl-CoA availability, allowing for the synthesis of phloroglucinol (1280 mg/L)  Fig. . Besides/L) Fig. . This stS. cerevisiae was demonstrated to increase cytosolic acetyl-CoA availability for the biosynthesis of n-butanol  was reduced stepwise to 5.5% compared with the wild-type CS activity by exchanging the native promotor of the CS-encoding gltA gene  cycle or glyoxylate shunt is the main fate of acetyl-CoA. In this context, disruption of the glyoxylate shunt involved in the transport of acetyl-CoA between the cytosol and cellular compartments in E. coli, S. cerevisiae, and C. glutamicum, increasing ACC activity is important to make use of the available acetyl-CoA pool. Whereas bacterial and plant chloroplastic ACCs are organized as complexes of distinct dissociable polypeptides, mammalian, fungal, and plant cytosolic ACCs are single polypeptide chains possessing all catalytically active domains  instead of four subunits for catalytic activity, rendering this enzyme an often exploited alternative to heterotetrameric ACCs for improving intracellular malonyl-CoA availability  are regarded as effectors interacting with FasR , encoded by the FasR-controlled genes fasA and fasB  as well as different biotechnologically interesting flavoring phenylbutanoids  using E. coli , represent powerful high-throughput tools for rapidly isolating mutants from genetically diverse strain libraries (Flachbart et al.


Chemical alterations in DNA induced by genotoxic factors can have a complex nature such as bulky DNA adducts, interstrand DNA cross-links (ICLs), and clustered DNA lesions . Complex DNA damage (CDD) has a complex character/structure as compared to singular lesions like randomly distributed abasic sites, deaminated, alkylated, and oxidized DNA bases. CDD is thought to be critical since they are more challenging to repair than singular lesions. Although CDD naturally constitutes a relatively minor fraction of the overall DNA damage induced by free radicals, DNA cross-linking agents, and ionizing radiation, if left unrepaired, these lesions cause a number of serious consequences, such as gross chromosomal rearrangements and genome instability. If not tightly controlled, the repair of ICLs and clustered bi-stranded oxidized bases via DNA excision repair will either inhibit initial steps of repair or produce persistent chromosomal breaks and consequently be lethal for the cells. Biochemical and genetic evidences indicate that the removal of CDD requires concurrent involvement of a number of distinct DNA repair pathways including poly(ADP-ribose) polymerase (PARP)-mediated DNA strand break repair, base excision repair (BER), nucleotide incision repair (NIR), global genome and transcription coupled nucleotide excision repair , mismatch repair (MMR), homologous recombination (HR), non-homologous end joining (NHEJ), and translesion DNA synthesis (TLS) pathways. In this review, we describe the role of DNA glycosylase-mediated BER pathway in the removal of complex DNA lesions. Endogenous oxidative stress and environmental factors induce multiple damage in cellular DNA, among them bulky DNA adducts, interstrand DNA cross-links (ICLs), and clustered DNA lesions , which are distinguished from singular lesions by their complex characters and structures - and (5′R)-8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG) are generated by hydroxyl radical attack at C5′ sugar by hydrogen atom abstraction resulting in the formation of a covalent bond between the C5′ and C8 positions of the purine nucleoside   lesion bypass during DNA replication and BER.Reactive oxygen species (ROS) are commonly responsible for the formation of non-bulky DNA base damage in cellular DNA . MajoritAristolochiaceae which are popular supplements in Chinese medicine. AAs are structurally diverse blends corresponding to nitrophenanthrene carboxylic acids composed of two main constituents: principally 8-methoxy-6-nitrophenanthro--1,3-dioxolo-5-carboxylic acid  and its 8-demethoxylated form (AA-II) , as slimming treatment, suffered from rapidly progressive interstitial nephritis  are a group of acids found in the flowering plant family N6-yl)aristolactam (dA-AL) and 7-(deoxyguanosin-N2-yl) aristolactam I (dG-AL) adducts .Bulky DNA adducts generated by environmental carcinogens cause mutations that often drive malignant transformation of affected cells. As we described above, bulky DNA adducts are removed in the NER pathway; however, certain bulky DNA lesions that cause a minimal decrease, or an enhancement in the stabilities of the DNA duplex can be very resistant to the repair machinery. Benzo[a]pyrene (B[a]P) is a well-known representative of PAH carcinogens present in the environment Luch, . Primarya,l]pyrene , another PAH present in about ten-fold lower concentration in the environment, is 100 times more tumorigenic than B[a]P in rodent model systems . ICLs are highly cytotoxic DNA lesions that prevent DNA strand separation during DNA replication, transcription, and recombination. Indeed, DNA cross-linking agents, like mitomycin C, cis-diamminedichloroplatinum(II), and melphalan, are widely used against hyperplastic diseases, such as cancer.cis-DDP) reacts with the N7 atom of the deoxyguanosine residue forming initially one covalent bond with DNA then covalently binds to other neighboring guanine residues resulting in DNA cross-linking amine functional group which reacts with guanine and adenine at the NApiaceae and Fabaceae)  is a product of  Tomasz, . Inert b Tomasz,   is an essential step in promoting aflatoxin carcinogenesis. The FAPY-AFB1 adduct seems to be the most stable of all AFB1-DNA adducts and is quite resistant to DNA repair machinery , a potent carcinogen produced by Aspergillus species, mainly found as food contaminant in animal farms and poultry production through the diet. AFB1 exposure of infant mice induces hepatocellular carcinoma after reaching adulthood  and 1, N2-propano-2'-deoxyguanosine  are major DNA adducts caused by acetaldehydes. Several studies indicated that endogenous aldehydes are a significant source of genotoxicity in the human hematopoietic system and that the presence of the proficient Fanconi anemia (FA) pathway is essential to protect cells from DNA damage induced by these reactive compounds. Hematopoietic stem cells (HSCs) are a primary target for aldehyde-induced DNA damage, and FA-deficient patients suffer from bone marrow failure due to p53/p21-mediated cell death and senescence . Half of the population of Northeast Asian descent and about 5–10% of Northern European descent harbor a dominant mutation in the ALDH2 gene, which greatly reduces the enzyme activity and leads to acetaldehyde accumulation after alcohol consumption. Individuals with a mutant form of ALDH2 have a greater risk of liver damage and susceptibility to many types of cancer -initiated BER and mismatch repair (MMR) pathway are able to eliminate normal DNA bases in mismatched DNA duplexes.E. coli MutY and human MMR can act in an aberrant manner: MutY removes adenine (A) in a template strand opposite to the misincorporated 8-oxoguanine residue , hypoxanthine (Hx), 8-oxoA, and even abasic site in the TpG/CpX sequence context, where X is a modified residue. MBD4 removes T only when it pairs with εA, but not with Hx and other modified adenine residues. In vitro reconstitution demonstrated that TDG can catalyze aberrant removal of T in the specific sequence context that leads to TpG, CpA → CpG mutations  paired with a damaged adenine (A) residue in the DNA duplex  is a human mismatch-specific adenine-DNA glycosylase homologous to the Similar to the alkyl-purine DNA glycosylases (AlkA and ANPG) which remove regular purines ] is employed to treat various types of cancers, including lung, head and neck, ovarian, and other organs. Cisplatin interacts preferentially with guanine residues in DNA and generates mono-adducts and intra- and interstrand cross-links. Exposure of the cells to cisplatin triggers a strong DNA damage response signal, which often leads to the irreversible apoptosis. Studies of the roles of DNA glycosylases and other BER enzymes in the removal of ICL have generated conflicting results, suggesting that the involvement of a given DNA repair pathway strongly depends on cellular context and lesion structure. In cisplatin-ICL, the cytosine residues adjacent to the cross-linked guanines undergo extra-helical flipping which exposes them to water and subsequently stimulates spontaneous deamination and conversion of cytosines to uracils -induced ICLs in living cells by McNeill et al.  demonstrE. coli Fpg, phage T4 endonuclease V, and human NEIL1 can excise the imidazole ring opened form of guanine-C8-N-hydroxy-2-aminofluorene adduct, UV-induced cyclobutane dimer, and psoralen-thymine monoadduct, respectively , a secondary metabolite produced by soil-dwell bacteria E. coli cationic alkylpurine DNA glycosylase homologous to AlkZ, has been identified. YcaQ can excise a broad range of DNA base lesions, including ICLs induced by nitrogen mustard. These studies demonstrate that the DNA glycosylase-initiated BER pathway is an alternative ICL repair pathway in bacteria, which may provide insight for potential mechanisms implicated in drug resistance in cancer cells  in a pre-catalytic complex with DNA duplex containing a 6-4 photoproduct in the absence of divalent metal cations. This 3D structure revealed a novel dual dinucleotide flip mechanism for recognition of bulky cross-linked dipyrimidines. Archaeon SacUVDE flips the two purines opposite to the damaged pyrimidine bases into a dipurine-specific pocket, whereas the damaged pyrimidines are also flipped into another cleft  is a DNA endonuclease that recognizes and incises DNA 5′ next to cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs)  DNA glycosylases are present in mammalian cells; these proteins show structural homology to the Fpg and Nei proteins of Escherichia coli and initiate the BER pathway to remove oxidized bases from DNA  disorder. FA is a recessive cancer-prone syndrome which is characterized with bone marrow failure and hypersensitivity to ICLs and, to a milder extension, to ionizing radiation and oxidative stress . In addition, the mammalian NER pathway is not efficient when acting upon ICLs, for the reason that both strands in duplex DNA are damaged and that the repair proteins cannot discriminate between two strands to excise the lesion  aberrant repair via stimulation of mutations in the specific regulatory regions of genome could play a capacitor role in evolution of warm-blood animals (Talhaoui et al., Mismatch-specific DNA glycosylases such as NEIL3 is a particular DNA glycosylase that possesses an extended C-terminal domain, containing three additional zinc finger-binding domains that specifically interact with ssDNA and ADP-ribose polymers (Chen et al., YB, DK, AAI, DB, and MS wrote the nature of DNA damage part of the review. YB, ST, RG, BM, and MS wrote the DNA repair part of the review. YB, AAI, and MS prepared figures. All authors discussed and contributed to analysis of published literature and to writing the manuscript.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.


SARS-CoV-2-targeted GapmeR can also be given to frontline healthcare workers as a prophylactic protection. The recombinant ACE2 protein, the efficacy of which is being evaluated in clinical trials, will bind to the spike (S) glycoprotein of extracellular SARS-CoV-2 and potentially block viral infectivity. We propose that combining inhalable SARS-CoV-2-targeted GapmeRs with recombinant ACE2 could provide a viable and rapidly implementable more effective therapeutic approach for eradicating SARS-CoV-2 and save millions of lives.Here we report our perspective on applying GapmeR technology in combination with recombinant angiotensin-converting enzyme 2 (ACE2) in the treatment of COVID-19 patients. GapmeR is a cell-permeating antisense single-stranded DNA molecule that can be designed to specifically target intracellular severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Once internalized into host cells, such as lung alveolar cells, GapmeR molecules can bind to the viral RNA. This RNA/DNA hybrid will then be degraded by the RNase H enzyme abundantly present in the host cells. GapmeRs can be delivered to COVID-19 patients through inhalation or  In an accelerated effort to develop therapeutic modalities and vaccines to manage this devastating pandemic, collaborative teams of regulatory bodies, health professionals, clinicians, and scientists in both academic and industrial settings have been engaged and integrated. Although there has been some success in developing new vaccines, repurposing previous coronavirus antibodies, and other potential therapeutic modalities, there is no effective therapy available for the ongoing COVID-19 pandemic. Through expedited regulatory processes, the US Food and Drug Administration (FDA) has granted Emergency Use Authorization (EUA) for remdesivir  on 1st May 2020 to treat severe COVID-19 cases (Media). Surprisingly, there has been relatively little exploration of directly targeting the SARS-CoV-2 genome.COVID-19 or the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten human health worldwide. As of 15th July 2020, there are over 13.4 million confirmed cases and over 580,000 deaths worldwideWe now know the complete genome of SARS-CoV-2, which is a single-stranded positive-sense RNA of about ∼30 kb and is encapsulated within a membranous envelope. As with other coronavirus, SARS-CoV-2 contains four main structural proteins – spike (S) glycoprotein, membrane (M) protein, envelope (E) protein, and nucleocapsid (N) protein . The infin vitro and in vivo. Studies have validated the use of small interfering RNA (siRNA) to safeguard host from viral infection by inhibiting the replication of viral genome, by blocking the expression of viral antigens and accessory genes and by interfering with the assembly of viral particles (RNA interference (RNAi) is a powerful biological process inherently applied by the host cells to destroy intracellular RNA viruses. For the past two decades, researchers have been widely capitalizing on RNAi mechanisms to silence cellular gene expression articles . Previouarticles . Indeed,in silico antisense design tools , which increase the binding affinity of GapmeR to the target RNA. In addition to specificity and highly efficient gene silencing, we have shown that GapmeRs can easily internalize into target cells  by macropinocytosis or gymnosis ; thus regn tools . Care mugn tools  should bin silico analysis. We believe that once internalized, virus-specific GapmeRs would bind to SARS-CoV-2 RNA within a host cell. RNase H enzymes present in the host cell will then specifically degrade the DNA/RNA hybrid and thus destroy SARS-CoV-2 , S protein and M protein. While designing SARS-CoV-2 targeting GapmeRs, we have considered multiple parameters, including RNA secondary structure, accessibility of target region, and avoidance of off-target effects using robust bioinformatics algorithms and RS-CoV-2 .via inhalation as aerosol or in powder form, either using a customized handheld nebulizer device or through the ventilator. Using either of these simple non-invasive inhalation devices, one can easily achieve high enough concentration of GapmeR molecules in the lung sufficient for type II alveolar cell uptake. GapmeRs could conceivably be administered to frontline healthcare worker dealing with COVID-19 patients to provide them prophylactic protection. Since GapmeR molecules are highly stable in human serum  screen multiple GapmeR designs against highly conserved regions of SARS-CoV-2 genome, (ii) carefully select potent candidates with high stability and no off-target effect, (iii) demonstrate their delivery to the lung using multimodal in vitro, organoids, and animal model systems appropriate for COVID-19 preclinical research (iv) determine dosing regimens for various infection scenarios, including for example, cases where the virus titer is increasing, and (v) achieve a good therapeutic window with acceptable toxicity profiles while suppressing viral loads in well-controlled experiments.It is worth noting that, like any other drugs, GapmeRs may have unforeseen off-target effects, including for example, unwanted interactions with host proteins . While such interactions between SARS-CoV-2-targeted GapmeRs and host proteins may not be strong, they may trigger unintended signaling pathways, cytokine release, and yield confounding physiological effects. In our previous work using gene expression analysis, we did not find any major immunological off-target effects of a panel of GapmeR molecules that we designed against multiple targets . Neverth in 2016 . Single  in 2016 . It woulresearch , (iv) de2. While viral replication is reduced through the use of hrsACE2, SARS-CoV-2 is not eliminated. It should be noted that SARS-CoV-2 can target multiple cell types expressing ACE2 and that, in addition to alveolar epithelial cells, many human tissues and organs express the ACE2 (In a recent study, the ACE2 , which mthe ACE2 . This coIn conclusion, there is an urgent need to radically rethink current management of the devastating COVID-19 pandemic. Although there are a set of rapidly increasing challenges, logistic, and regulatory hurdles ahead to bring a proposed GapmeR technology to clinics, these can be overcome by engaging collaborative academia-industry partnerships, setting-up joint screening, and testing facilities and making research funding available to support new concepts. We believe that targeted GapmeR in combination with recombinant ACE2 protein could provide a viable rapidly implementable therapeutic approach for COVID-19 and similar infections in the future.https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/virus?SeqType_s=Nucleotide&VirusLineage_ss=SARS-CoV-2,%20taxid:2697049&utm_source=gene&utm_medium=referral&utm_campaign=COVID-19.Publicly available datasets were analyzed in this study. This data can be found here: NV and DK conceived the idea. NV, MF, SD, and DK wrote the manuscript. All authors approved the final manuscript 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.


The reduction of the dismally high mortality of current end-stage renal disease patients maintained on conventional standard haemodialysis (HD) remains an unmet medical need. Online haemodiafiltration (HDF) modes with various sites of fluid substitution  are increasingly used worldwide as promising alternatives to conventional HD. Large scale cohort studies, post hoc analyses of randomized trials, and individual participant meta-analyses suggest that post-dilution and pre-dilution, especially with high substitution volumes, improve outcomes compared with conventional standard HD. However, there is no definitive proof of a survival advantage of HDF over standard HD. The different modes of high-volume HDF should be considered a therapeutic platform allowing to personalize and tailor routine HDF treatment. The selection of the HDF mode should be made according to individual patient characteristics. Utilizing high retention onset membranes, expanded haemodialysis (HDx) can achieve the same solute removal performance as HDF. Subgroups of high-volume OL-HDF patients could benefit from HDx. Ongoing and future trials should provide definitive proof for the superiority of high-volume OL-HDF over conventional HD or HDx to give guidance for the most favourable mode of dialytic therapy for clinical use. Patients with end-stage renal disease (ESRD) maintained on dialysis have a shortened life expectancy and impaired quality of life compared to their peers without kidney disease, despite improvements in medical treatment. Death due to cardiovascular diseases—including arrhythmias, cardiac arrest, congestive heart failure, acute myocardial infarction and atherosclerotic heart disease is the largest known category of cause-specific mortality in dialysis patients. There is no convincing evidence of better survival with one type of dialysis  compared to another  .Online haemodiafiltration (OL-HDF) represents the most technologically advanced convective form of blood purification and has the potential to improve outcomes in ESRD patients. OL-HDF is defined as a combination of diffuse and convective solute transport using a high-flux membrane with an effective ultrafiltration rate of at least 20% of the blood flow rate in combination with the use of an online-generated sterile and non-pyrogenic solution for fluid substitution .T, 4.T3, 4This literature review describes the content and quality of knowledge about clinical benefits associated with high-volume OL-HDF modes in ESRD patients and gives a critical appraisal of the clinical relevance of the reported data.Convective therapies coming under the umbrella of high-efficiency HDF involve larger volumes of infusion than other convective therapies. Various modes of OL-HDF, differing in the site of replacement fluid infusion are in use. However, only four modalities of OL-HDF are currently used in different countries: post-dilution HDF, pre-dilution HDF, mixed-dilution HDF and mid-dilution HDF Table  2]. The. The2]. High-volume high-efficiency post-dilution offers—at least in theory—the most effective removal of middle molecule solutes. This mode is widely spread in Europe and other parts of the world. However, high-efficiency post-dilution OL-HDF is often limited by inappropriately high haemoglobin levels due to correction of renal anaemia and increased protein concentration which could result in a critically increased trans-membrane pressure, membrane fouling and consequently to impaired removal of solutes. Haemoconcentration generally limits the rate of ultrafiltration to a filtration fraction of 20–30% of the blood flow rate in post-dilution HDF [Ultrafiltration rate is not limited by the blood flow rate in-pre-dilution HDF. However, pre-dilution HDF reduces the efficiency of both the diffusion and convective components of solute removal. To match the efficiency of post-dilution OL-HDF, the inherent dilution should be compensated for by increasing the substitution volume by a factor of two in the pre-dilution mode –5. In miSuccessful high-volume post-dilution HDF depends on high blood flow rates ≥ 350 mL/min), an excellent vascular excess (arteriovenous fistula blood flow rates ≥ 600 mL/min), an ability to achieve adequate anticoagulation throughout the HDF session, and the absence of any clinical disorder that increases blood viscosity  . The results of these studies suggested, albeit not consistently, that post-dilution OL-HDF is associated with less morbidity and lower mortality than conventional HD.The observed clinical advantages provided by high-volume HDF techniques, particularly post-dilution OL-HDF encompass (a) better intra-treatment haemodynamic stability; (b) less inflammation induced malnutrition, atherosclerosis and erythropoietin resistance; (c) fewer cases of dialysis related amyloidosis and uraemic polyneuropathy; (d) improved derangement in calcium-phosphate homeostasis and less vascular calcification; (e) higher treatment dose by better preservation of residual renal function and higher KT/V; (f) improved quality of life with less insomnia, restless leg syndrome, itching or joint pain , 8, 11.There is some controversy on the potential effects of post-dilution OL-HDF for the reduction of mortality risk. Well-performed, large scale cohort studies, post hoc analysis of three large RCTs and meta-analyses of four RCTs suggest that post-dilution OL-HDF could improve patient mortality. Most randomized controlled trials were not designed to study the effect of effective dose. Nevertheless, reported patient survival was greater for those receiving greater convective volume exchange. Reduced mortality (by 22%) with post-dilution HDF was observed with pooled analyses of patient data of the four trials, due to a reduction of cardiovascular events (by 31%), with authors estimating the prevention of one cardiovascular death for every 75 patient years of treatment , 13. CurRetrospective small observational studies suggest that pre-dilution OL-HDF may ameliorate various uraemic symptoms, such as loss of appetite, resulting in an improved nutritional status and preserved muscle volume, relief of shoulder pain and lower prevalence of dialysis related amyloidosis, itching, insomnia and restless legs syndrome , 16. A sThe association of improved survival with pre-dilution OL-HDF has been recently evaluated by retrospective analyses. A small Greek study noted a survival benefit of pre-dilution OL-HDF over peritoneal dialysis 2–2.5 years after treatment initiation . The stuPreliminary results of its clinical application indicate that trans-membrane pressure (TMP)- modulated mixed-dilution OL-HDF could be one of the most powerful strategies to prevent or delay the occurrence of some long-term dialysis complications and to promote improved survival of ESRD patients . HoweverA recent retrospective multi-centre 1-year study aimed to explore whether mixed-dilution OL-HDF improves anaemia management compared to traditional post-dilution HDF. 174 matched prevalent ESRD patients were included. 87 patients had received mixed-dilution HDF (mean substitution volume 38 ± 5 L/session) and 87 patients were on post-dilution HDF  and opens an avenue to personalized treatment of ESRD Table . High-voHDx has emerged as a potential breakthrough of haemodialysis techniques , 23. MedThe MCO HDx may be a valuable option for ESRD patients who don't reach the large blood flow rates required for high-volume OL-HDF or when HDF is not available. Patients treated with high-volume OL-HDF could also benefit from HDx whenever HDF needs to be suspended.Limited studies have demonstrated potential clinical benefits of MCO membranes in reducing cardiovascular risk by reduction of vascular calcification  and by dThe current uncertainty of the optimal dialysis strategy for ESRD patients results in a heterogenous delivery of care, which is not driven by the best evidence but by empiricism and local centre performance. Definitive proof for a survival benefit of high-volume OL-HDF is still needed. Currently, the mode of OL-HDF should be established according to characteristics and needs of individual patients. It would be worthwhile if further randomized trials prove the superiority of high-volume HDF over conventional or HDx.


As COVID-19 continues to spread worldwide, severe disease and mortality have been observed in obese patients. We discuss how obesity and obesity-associated factors such as ‘meta-flammation’, dietary fat intake and paradoxical suppression of the innate immune response within the pulmonary compartment may be crucial determinants in the host response to a novel viral pathogen. Modulation of immune cell bioenergetics and metabolic potential plays a central role in the innate immune response to infection, and as we strive to combat this new global health threat, immunometabolism of the innate immune system warrants attention. Obese patients <60 years old have a 2·2-fold increased rate of hospitalisation with coronavirus disease 2019 (COVID-19), while a 7·36 increased risk of mechanical ventilation has been reported in patients with BMI 35 kg/m2 or greater. The observation of worse outcomes in obese patients was also seen during the 2009 H1N1 influenza pandemic, with 61 % of fatalities occurring in obese adults. The apparent increased severity of viral pneumonia in this population is critically important given the increasing prevalence of obesity globally. Multiple potential explanations for this association have been proposed, including altered pulmonary physiology in the obese patient, predisposition to thromboembolic complications which are observed with increased frequency in COVID-19 patients and increased incidence of co-morbidities that may impact upon outcome and render ICU management more complex and challenging,8,9. However, a growing body of evidence implicates direct obesity-induced immune dysregulation. Increased lethality of H1N1 influenza observed in obese mice has been linked to impaired T-cell responses, while failed influenza vaccine response is correlated with impaired antibody generation. Likewise, human studies have demonstrated failure of influenza vaccine response in obese adults. Similarly, increased susceptibility of obese patients to severe COVID-19 infection is hypothesised to be linked to impaired successful immune responses against the virus, particularly through diet-derived reconfigurations of protective immunometabolic responses.As epidemiological data on the dramatic COVID-19 pandemic are disseminated, obesity has emerged as a major risk factor for severe disease necessitating intensive care unit (ICU) admission. Thus, the impact of obesity upon innate immunity likely plays a crucial role in the severity of COVID-19 disease. Obesity creates a state of chronic inflammation, termed ‘meta-flammation’, characterised by sustained pro-inflammatory innate immune responses mediated through activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome/IL-1 axis–19. Adipose tissue directly secretes pro-inflammatory TNFα, IL-1 and IL-6, with downstream activation of the transcription factor NF-κB, driving a state of oxidative stress. This meta-flammation underpins the pathogenesis of several obesity-related conditions, including type 2 diabetes mellitus, CVD and non-alcoholic fatty liver disease,22. Similarly, the establishment of ‘trained immunity’, a non-specific priming of progenitor cells of the innate immune system which alters their functional responses, is influenced by meta-flammation associated with these conditions,24.While obesity has been linked to dysregulated adaptive immunity and failure of antibody generation following infection or vaccination, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel virus to which no one has an established adaptive immune response upon first exposure. Initial host defence against a novel virus relies entirely upon the rapid, non-specific innate immune response,26. Diet-induced obese mice demonstrate a substantially higher mortality rate than lean mice following influenza infection, with reduced expression of pro-inflammatory cytokines TNFα, IL-6, IL-1β and interferon-1 in the pulmonary compartment early post-infection,28. Interestingly, however, by day 6 post-infection, the lungs of obese mice contained higher expression of TNFα and IL-1β, with similar expression of anti-inflammatory IL-10 to that of lean mice, suggesting a ‘skewing’ towards a pro-inflammatory state that results in lung tissue damage. One possible explanation for this observation is failure of early macrophage responses in the obese mouse resulting in insufficient viral control by the innate immune system, with later T-cell infiltration causing increased inflammation and tissue damage that contributes to pathogenesis. Lipopolysaccharide-induced obese mice demonstrated impaired macrophage production of TNFα, IL-1β and IL-6 when challenged, with more severe lung pathology following infection with H1N1 influenza. This mirrors the pathogenesis observed in severe COVID-19 disease, where early localised host responses fail to control viral replication, with later surges in inflammation – the ‘cytokine storm’, particularly driven by IL-6, which is associated with increased mortality – causing overt tissue damage that ultimately leads to respiratory failure. Elevated leptin levels characteristic of obesity may also contribute, with a murine model of 2009 H1N1 influenza infection demonstrating reduced IL-1 and IL-6 levels following administration of anti-leptin antibody to obese mice, resulting in improved survival. Interestingly, though attenuated levels of pro-inflammatory cytokines were evident in the pulmonary compartment early post-infection in obese mice, systemic levels of TNFα, IL-1 and IL-10 were elevated at early time points similar to that of lean mice.Despite increased basal levels of pro-inflammatory mediators, obesity-associated meta-flammation is paradoxically associated with attenuated innate immune response to both bacterial and viral infections. Upon stimulation, healthy human pulmonary macrophages undergo a dramatic shift in central carbon metabolism, termed glycolytic reprogramming, with increased reliance on glycolytic metabolism rather than oxidative phosphorylation to generate energy, resulting in activation of the NLRP3/IL-1 axis through consequent impact on transcription factor expression, diversion of metabolic intermediates into biosynthetic pathways and perturbations of the electron transport chain,34. Similarly, ‘trained immunity’, the protective effect of which is being examined in the context of COVID-19 due to Bacillus Calmette–Guérin vaccination, requires such glycolytic reprogramming in progenitor innate immune cells.We have previously described a similar state of baseline hyper-inflammation but paradoxical failure to mount an adequate innate immune response to infection in smokers’ pulmonary macrophages compared with those of non-smokers’,37, with elevated IL-1 production, similar to the chronic hyper-inflammation observed in innate immune cells from obese patients. Despite this, however, smokers’ macrophages are unable to ramp up glycolytic activity and consequent pro-inflammatory and anti-microbial functions in response to pathogenic challenge, due to a state of ‘bioenergetic exhaustion’ induced by this chronic baseline activity. Others have also demonstrated increased ‘polarisation’ of macrophages from smokers lungs at baseline, notably describing increased proportions of M1-skewed macrophages, known to characteristically favour glycolytic metabolism. Interestingly, analyses of bronchoalveolar lavage fluid from SARS-CoV-2-infected patients have similarly identified higher proportions of recruited M1-like macrophages and depletion of lung-native tissue resident alveolar macrophages  in those with more severe disease. Though baseline BMI was not reported in this study, strong epidemiological evidence suggests severe disease to be more prevalent in obese patients,2 and existing studies have shown that obesity-associated metabolic signals alter tissue resident macrophage phenotype away from the classical anti-microbial programme.Smokers’ macrophages, in contrast to non-smokers’, are skewed towards increased glycolytic activity at baseline. Thus, the effect of this increased glucose on immunometabolic functions is being examined. Ongoing and unpublished work examining the importance of glycolysis in circulating monocytes after SARS-CoV-2 infection indicates that excessive glycolysis may actually facilitate viral replication and drive excessive disease inflammation once recruited to the lung. At the same time, recent murine studies of resident lung macrophages, however, suggest these cells preferentially use fatty acid oxidation rather than glycolysis to adapt to the lipid-rich and glucose-deprived environment in the lung,44. Strategies which boost glycolysis and pro-inflammatory function specifically in these cells could be used to improve early anti-viral containment. Additionally, obesity has also been shown to alter the expression of the receptor for SARS-CoV-2 entry on respiratory epithelia, with retrospective analysis of angiotensin-converting enzyme-2 (ACE2) mRNA demonstrating increased expression in lung epithelia in obese high-fat diet-fed mice. Thus, obese patients could suffer from a trifecta of immunometabolic defects at level of lung epithelium (primary infected cell), the alveolar macrophage (the primary responding cell) and circulating monocytes (the primary recruited cell), which increase their susceptibility to disease  and within the context of obesity. To this end, we need to be cautious and proceed with sound nutritional evidence. One case in point being the recent controversy wherein some observational studies linked low vitamin D status, which often coexists with obesity, to adverse COVID-19 outcomes. It is probable that vitamin D, alongside other important nutrients, plays an important role in immune cell metabolism and related immune functionality, as most cells can convert the vitamin D precursor into its active form. This does not imply that vitamin D supplementation in high-risk obese subjects can reduce risk. There are too many confounders underlying associations, not least of which vitamin D status is simply a good biomarker for several micronutrient deficiencies, contributing to malnutrition despite obesity.There are significant gaps in knowledge with respect to the impact of different dietary elements on COVID-19-related immunity (as recently reviewedThe innate immune response to SARS-CoV-2 infection is likely crucial in determining the clinical course of COVID-19 disease, and obesity-associated dysregulation of this response is critically important given the high prevalence of this co-morbidity worldwide. While understanding the direct impact of obesity upon COVID-19 pathogenesis is important, from the diet and lifestyle perspective, there may be additional elements we could better understand which impact innate immune function at multiple levels. Based on parallels that can be drawn with our findings in smokers’ pulmonary macrophages, metabolic characteristics of innate immune response in the obese patient potentially represent a key feature of the unique susceptibility of this population to severe disease. As we struggle to develop therapies and vaccines to combat this new global health threat, immunometabolism of the innate immune system warrants attention from the global scientific community.


The reconstruction of large bone defects remains a crucial challenge in orthopedic surgery. The current treatments including autologous and allogenic bone grafting and bioactive materials have their respective drawbacks. While mesenchymal stem cell (MSC) therapy may address these limitations, growing researches have demonstrated that the effectiveness of MSC therapy depends on paracrine factors, particularly exosomes. This aroused great focus on the exosome-based cell-free therapy in the treatment of bone defects. Exosomes can transfer various cargoes, and noncoding RNAs are the most widely studied cargo through which exosomes exert their ability of osteoinduction. Here, we review the research status of the exosome-derived noncoding RNAs in bone regeneration, the potential application of exosomes, and the existing challenges. The reconstruction of large bone defects is a key challenge in reconstructive surgery. Currently, the treatment strategies include autologous and allogenic bone grafting, bioactive materials. However, they all have their limitations in various aspects. Autologous bone grafts have the disadvantages of limited sources of bone and extra surgical injuries. Allogenic bone grafts may cause immunological rejection and disease transmission. As for the bioactive materials, the issues of biocompatibility, structural stability, mechanical strength, and degradability remain to be solved.Stem cell-based engineered bone may address the limitations of the abovementioned methods. Nevertheless, the satisfactory results are hindered by MSCs' limited ability to form enough new bone. Therefore, approaches of enhancing MSC's osteogenic differentiation have been investigated such as gene editing , 2, the In recent years, accumulating researches demonstrated that the paracrine role may account for the efficacy of MSC therapy given that limited cells have engrafted into the sites of injury , 10 and Unlike stem cell therapy, the application of exosomes involves fewer safety considerations. In fact, several clinical studies have proved the safety of exosomes in the treatment of cancers . SeveralIn vivo experiments also revealed that exosomes dramatically stimulated osteogenesis in calvarial defects [β1 pathway, Wnt/β-catenin pathway [Exosomes can promote the osteogenic ability of MSCs, and the effects of exosomes often increase with increasing concentrations. Indeed, some studies reported that exosomes even outperformed the currently used osteoinductive cocktail, the conditioned medium, and the extracellular matrix (ECM) . In vivo defects , bone fr defects , and rad defects . Exosome pathway , and MAP pathway  and by u pathway , it is w pathway , 28.Exosomes exert their influence either through interacting with the extracellular matrix or through internalization into cells. Once released, exosomes can anchor to the ECM and act as the initial sites for mineralized nodule along with matrix vesicles . RecentlHigh-throughput technologies have discovered about 90% of the genome is actively transcribed , but themiRNAs affect the expression of mRNAs by two modes including translational repression and mRNA decay, both of which were realized by the RNA-induced silencing complex (RISC) formed by miRNAs and Argonaute protein (Ago) . When thThe osteogenic induction of MSCs alters the expression of exosomal miRNAs. Several well-known suppressors of osteogenesis, such as miR-144, miR-31, and miR-221, were downregulated in exosomes from osteogenic differentiated MSCs, while positive regulators of osteogenesis like miR-21 were upregulated . miR-31 β-catenin, thus, activating the β-catenin pathway. The effects of exosomes largely relied on miR-27a-3p, given that myoblast exosomes whose miR-27a-3p was inactivated lost their osteogenic-inductive capacity [Exosomal miRNA played an indispensable role in the cross-talk between bone and muscle. Myoblast-derived exosomes could deliver miR-27a-3p to the preosteoblasts and decrease the expression of adenomatous polyposis coli (APC), a negative regulator of capacity . miR-27acapacity .Preosteoblast-derived exosomes contain miRNAs that can regulate the osteogenic differentiation. The preosteoblast exosomes contain abundant let-7 miRNA , which iβ-galactosidase and suppress the osteogenic differentiation of BMSCs. Heme oxygenase-1 (Hmox1) is also a target of miR-183-5p, which has been shown previously to stimulate BMSCs osteogenic differentiation [In aging mice, the miRNA profile of exosomes was quite different from those in young mice. Specifically, the miR-183 cluster is enriched in aged exosomes . miR-183ntiation .Apart from transporting microRNAs directly, exosomes can also transport protein to affect miRNA expression indirectly in recipient cells. In systemic lupus erythematosus (SLE) model Fas-deficient-MRL/lpr mice, exosome transplantation rescued the osteoporotic phenotype by the Fas/miR-29b/Dnmt1/Notch cascade. Exosome infusion provided donor-derived Fas to recipient cells, which facilitated the release of miR-29b into the extracellular environment and the decrease of intracellular miR-29b . miR-29bAntisense (AS) lncRNAs have sequences complementary to their sense counterparts. Antisense lncRNAs mainly function through regulating the expression of their sense transcripts. In some cases, they can form an RNA–RNA duplex with their sense counterparts, stabilizing their sense transcripts and thus increasing the gene expression , 57. In In vitro experiments demonstrated myeloma-derived exosomes could decrease MSCs' osteogenic differentiation ability. In vivo treatment of GW4869, an inhibitor of exosome secretion, attenuated bone loss in multiple myeloma models, with the expression of bone resorption marker beta-isomerized C-telopeptide (β-CTX) decreased and that of osteogenesis-related gene procollagen type I N-terminal propeptide (P1NP) increased. Using a lncRNA sequencing, they identified that antisense lncRNA RUNX-AS1 was enriched in MM-MSCs and MM-MSC-derived exosomes. Mechanismly, antisense lncRNA RUNX-AS1 and RUNX2 formed a RNA duplex at overlapping regions through base pairing, interfered with RUNX2 pre-mRNA splicing, and suppressed RUNX2 mRNA expression. Thereby, the MM-MSC-derived exosomes may transmit the antisense lncRNA RUNX-AS1 to MSCs, contributing to the impaired osteogenic differentiation ability in MM-MSCs [Exosome-derived antisense lncRNAs play a role in affecting the osteogenic ability of MSCs. Multiple myeloma (MM) is a plasma cell cancer characterized by multiple osteolytic damage.  MM-MSCs .Mature cytoplasmic tRNAs can be cleavaged into small RNA fragments: the 5′ and 3′ tRNA halves (30–40 nt in size). The 5′ tRNA halves can silence target mRNAs by complementary base pairing to the 3′ UTRs of protein-coding genes in a manner like miRNA/siRNA , 61. WhiWhen it comes to clinical application, the pharmacokinetics of exosomes should be noted. One study showed that the exosomes predominately existed in the bone and lung 24 hours after injection . Howeverα-primed cells had an elevated level of Wnt-3a compared with unprimed cells, contributing to the enhanced osteoinductive effects of exosomes [α can stimulate BMSC osteogenic differentiation and enhance angiogenic cell functions. Li constructed HIF-1α mutant BMSCs in which HIF-1α expresses continuously even under normoxic conditions. They found that exosomes from HIF-1α mutant BMSCs (BMSC-ExosMoU) had stronger osteoinductive capacity than those from the wild-type group [The osteogenic capacities of exosomes can be improved by modifying either the parent cells or the exosomes. The modifications include biochemical factors and mechanical factors. In fact, mechanical stimuli such as low-intensity pulsed ultrasound (LIPUS) could be used to enhance the osteoinductive capability of MSCs . Whetherexosomes . HIF-1α pe group . Anotherpe group . The thepe group .Despite their great potential in bone regeneration, several challenges existed in the application of MSC-derived exosomes. Among them, the low yield remains to be a major challenge. Several strategies have been explored to maximize yield, including serum starvation and modulating calcium concentration. Nevertheless, those operations could potentially alter the contents and function of exosomes. Another strategy is to increase the supply of MSCs by immortalization. Researchers found that transfection of the c-myc gene provided infinite cell sources for the production of exosomes . HoweverNCT01159288). One study used this kind of engineered exosomes to deliver siRNA to the brain. They pretransfected dendritic cells with a plasmid with the neuron-specific RVG peptide clone into the exosomal membrane protein Lamp2b. These exosomes delivered siRNAs specifically to the brain without non-specific delivery and achieved strong silencing of BACE1, a therapeutic target of Alzheimer's disease [The target specificity of exosomes needs to be further studied and utilized. The internalization of exosomes is realized through cell-exosome interactions involving transmembrane proteins and ECM proteins , 84, of  disease .The procedures of isolation and administration should be taken into consideration when making conclusions. Important differences occur in terms of the quality and the RNA profiling when using different isolation protocols, such as centrifugation, chromatography, filtration, and polymer-based precipitation . Up to nThe role of exosomes in bone regeneration has been well recognized, and the noncoding RNAs play an important role in exosomes-regulated osteogenic differentiation. If the abovementioned challenges are met, the MSC-derived exosomes for cell-free therapy may offer an elegant alternative for the treatment of bone defects.


As the sentinels of the immune system, dendritic cells (DCs) play a critical role in initiating and regulating antigen-specific immune responses. Cross-priming, a process that DCs activate CD8 T cells by cross-presenting exogenous antigens onto their MHCI (Major Histocompatibility Complex class I), plays a critical role in mediating CD8 T cell immunity as well as tolerance. Current DC vaccines have remained largely unsuccessful despite their ability to potentiate both effector and memory CD8 T cell responses. There are two major hurdles for the success of DC-based vaccines: tumor-mediated immunosuppression and the functional limitation of the commonly used monocyte-derived dendritic cells (MoDCs). Due to their resistance to tumor-mediated suppression as inert vesicles, DC-derived exosomes (DCexos) have garnered much interest as cell-free therapeutic agents. However, current DCexo clinical trials have shown limited clinical benefits and failed to generate antigen-specific T cell responses. Another exciting development is the use of naturally circulating DCs instead of in vitro cultured DCs, as clinical trials with both human blood cDC2s  and plasmacytoid DCs (pDCs) have shown promising results. pDC vaccines were particularly encouraging, especially in light of promising data from a recent clinical trial using a human pDC cell line, despite pDCs being considered tolerogenic and playing a suppressive role in tumors. However, how pDCs generate anti-tumor CD8 T cell immunity remains poorly understood, thus hindering their clinical advance. Using a pDC-targeted vaccine model, we have recently reported that while pDC-targeted vaccines led to strong cross-priming and durable CD8 T cell immunity, cross-presenting pDCs required cDCs to achieve cross-priming in vivo by transferring antigens to cDCs. Antigen transfer from pDCs to bystander cDCs was mediated by pDC-derived exosomes (pDCexos), which similarly required cDCs for cross-priming of antigen-specific CD8 T cells. pDCexos thus represent a new addition in our arsenal of DC-based cancer vaccines that would potentially combine the advantage of pDCs and DCexos. As the professional antigen presenting cells (APCs), dendritic cells (DCs) play a critical role in the initiation and regulation of innate and adaptive immune responses, and have the unique ability to activate (prime) both naïve CD4 and CD8 T cells . Cross-pThere are two major hurdles of the success of DC vaccines: tumor-mediated immunosuppression and the functional limitations of the commonly used in vitro differentiated DCs ,11. As iAnother major contributor to the lack of clinical benefits by DC vaccines is the functional limitation of ex vivo differentiated DCs, which were used in the vast majority of all DC-based clinical trials . These DSeveral clinical trials using DC-targeted antigens have shown that in vivo DC-targeted vaccines are safe with varied effects on immune responses ,42,43. I+ DCs (cDC2s), which are specialized in immune responses against bacterial and fungi, and CD141 (BDCA3)+ DCs (cDC1s), specialized in cross-presentation of tumor antigens to prime CD8 T cells [To overcome the functional limitations of MoDCs commonly used in DC vaccines, another promising approach is to use freshly purified blood-derived primary DC subsets. Although rare in numbers, recent technological development of antibody-coated magnetic beads enables the isolation of sufficient numbers of naturally circulating DC subsets directly from peripheral for cancer vaccines ,27,48. N+ cDC2s loaded with TAAs (tumor-associated antigens) for prostate cancer and melanoma have shown that they are safe and feasible [+ mDCs [+ cDC1s, the critical player for priming anti-tumor CD8 T cells and generating CD8 T cell immunity, have not been used for cancer immunotherapy, likely due to their low numbers in peripheral blood [+ cDC1 subsets and the low yield of high purity DC subsets in peripheral blood remain the major hurdles for using nDCs for cancer immunotherapy cells [Although initially regarded as the less efficient antigen presenting cells (APCs), pDCs can present antigens to activate both CD4 T cells and CD8 T cells through cross-presentation ,57. pDCsNK)cells , suggestAs we have mentioned in the previous section, clinical trials using naturally circulating pDCs induced tumor-specific CD8 T cell immunity correlated with improved progression-free survival (PFS) . In the The most exciting development in pDC-based cancer vaccines comes from a recent phase I clinical trial (GeniusVac-Mel4) on melanoma patients using pDCs from an allogeneic pDC cell line . This pDExosomes are small inert membrane vesicles about 30–150 nm diameter in sizes that form within late multivesicular endosomal compartments containing proteins, lipids and nucleic acids, and play an important role in intercellular communications and material transfer of their cargo ,76,77. DUp to now, two phase I and one phase II clinical trials with DCexos have been concluded in cancer patients ,90,91. IAlthough we have focused on DCexos as cancer vaccines, it should be noted that exosomes from other cells such as macrophages, NK cells, B cells, T cells, mesenchymal stem cells and tumor cells have also been employed as cancer vaccines ,101. IntAlthough pDC-based vaccines have shown promising results in multiple clinical trials ,29,30, hb–SIINFEKL) complexes, coupled with previous study showing that anti-Siglec-H antibodies were not detected in cDCs [As both human and murine pDCs have been shown to be capable of cross-presentation in vitro ,119,120, in cDCs , suggest in cDCs . Our fur in cDCs   exhibited significant reduced cross-priming, in particular effector differentiation of antigen-specific CD8 T cells upon pDC-targeted vaccinations [Among cDCs, Batf3-dependent cDC1s are recognized as the superior APCs in cross-presenting exogenous antigens including tumor antigens to prime CD8 T cells ,124,125;inations . Taken tHow do cross-presenting pDCs transfer antigens to bystander cDCs? Experiments using transwells and pDC supernatants demonstrated that antigen transfer from pDCs to cDCs was mediated by soluble factor(s). Further studies showed that antigen transfer from pDCs to bystander cDCs was mediated by previously unreported pDC-derived exosomes (pDCexos). Importantly, pDCexos primed naïve antigen-specific CD8 T cells only in the presence of bystander cDCs, similar to cross-presenting pDCs, thus identifying pDCexo-mediated antigen transfer to cDCs as a novel mechanism for pDCs to achieve cDC-dependent cross-priming. The pDCexo-mediated antigen transfer to cDCs is not limited to targeting pDCs via Siglec-H. Using pDC-targeted anti-Bst2-antigen , which wThe identification of previously unreported pDCexos offers an exciting new addition to current DCexo collections. As a clinical trial of pDC vaccine using a human pDC cell line has shown promising results, pDCexo vaccines have the potential to combine the advantages of both DCexo and pDC vaccines . As inerOn the other hand, the identification of pDCexos also raises several interesting questions. As DC-targeted antigens such as anti-DEC-205 has been shown to be about 1000 times more efficient in cross-presentation than soluble protein antigens , it willOur identification of pDCexos also raises the interesting question of whether cDCs loaded with cDC-targeted antigens similarly generate DCexos. If so, whether these cDCexos exhibit enhanced cross-priming capacity compared to cDCexos generated with non-targeted soluble protein antigens, and whether these cDCexos differ from pDCexos in their function in cross-priming. Further studies are required to further characterize these pDCexos and/or cDCexos generated with DC-targeted antigens to determine their potential as cancer vaccines.


Due to the continued high incidence and mortality rate worldwide, there is still a need to develop new strategies for the prevention, diagnosis and treatment of cardiovascular diseases (CVDs). Proper cardiovascular function depends on the coordinated interplay and communication between cardiomyocytes and noncardiomyocytes. Extracellular vesicles (EVs) are enclosed in a lipid bilayer and represent a significant mechanism for intracellular communication. By containing and transporting various bioactive molecules, such as micro-ribonucleic acids (miRs) and proteins, to target cells, EVs impart favourable, neutral or detrimental effects on recipient cells, such as modulating gene expression, influencing cell phenotype, affecting molecular pathways and mediating biological behaviours. EVs can be released by cardiovascular system-related cells, such as cardiomyocytes, endotheliocytes, fibroblasts, platelets, smooth muscle cells, leucocytes, monocytes and macrophages. EVs containing miRs and proteins regulate a multitude of diverse functions in target cells, maintaining cardiovascular balance and health or inducing pathological changes in CVDs. On the one hand, miRs and proteins transferred by EVs play biological roles in maintaining normal cardiac structure and function under physiological conditions. On the other hand, EVs change the composition of their miR and protein cargoes under pathological conditions, which gives rise to the development of CVDs. Therefore, EVs hold tremendous potential to prevent, diagnose and treat CVDs. The current article reviews the specific functions of EVs in different CVDs. By containing and transporting various bioactive molecules, extracellular vesicles represent a significant mechanism for intracellular communication and impart favourable, neutral or detrimental effects on recipient cells, such as modulating gene expression, influencing cell phenotype, affecting molecular pathways and mediating biological behaviours.Extracellular vesicles play significant roles in maintaining normal cardiac structure and function under physiological conditions and change their composition under pathological conditions to promote the development of cardiovascular diseases.Cardiovascular diseases are the leading cause of death, accounting for almost a third of deaths worldwide. Extracellular vesicles play fundamental roles in regulating physiological and pathological functions and hold tremendous potential to monitor and treat cardiovascular diseases.There is still a need to develop new strategies for the prevention and treatment of different cardiovascular diseases, given their continued high incidence and mortality rate worldwide.Little is known about extracellular vesicle-mediated regulation of cardiomyocytes and noncardiomyocytes within the healthy and diseased heart.There is a lack of broad and detailed understanding of extracellular vesicle functions and a need for the development of new and personalized extracellular vesicle-based therapies to treat different cardiovascular diseases.1. Although advances in cardiovascular research and care have increased patient survival, there is still a need to develop new strategies for CVD prevention and treatment, given the continued high incidence and mortality rate in many countries2. Extracellular vesicles (EVs) are enclosed in a lipid bilayer and represent a by-product released by cells , micro-ribonucleic acids  and deoxyribonucleic acids (DNAs), to target cells, EVs impart favourable, neutral or detrimental effects on recipient cells, such as modulating gene expression, influencing cell phenotype, affecting molecular pathways and mediating biological behaviours5.Cardiovascular diseases (CVDs) are the leading cause of death, accounting for almost a third of deaths worldwide6. Whereas exosomes are released continuously from cells, microvesicles and apoptotic bodies are predominantly released from activated or apoptotic cells. Exosomes are released from cells via the endolysosomal pathway, and microvesicles and apoptotic bodies are formed by budding from the plasma membrane. Apoptotic bodies (>1000 nm) form by apoptotic cell membrane blebbing to recycle contents and contain cell debris, genomic DNA (gDNA) and proteins. Microvesicles (150–1000 nm) form through outward pinching off of the plasma membrane and contain RNAs, proteins and lipids. Exosomes, the smallest type of EV (30–150 nm), originate within multivesicular bodies and carry RNAs, proteins and lipids. The plasma membrane buds inward, fills with cytoplasmic contents and retains plasma membrane proteins specific to the cell of origin. Early endosomes are formed and then mature into late endosomes. This plasma membrane further buds inward and forms intraluminal vesicles with the aid of endosomal sorting complex required for transport. Once filled with these exosomes, late endosomes become multivesicular bodies, which might either fuse with the plasma membrane and secrete exosomes or deliver their contents to the lysosome for degradation. Molecular mechanisms controlling exosome production are far from fully understood, and some of these mechanisms appear to vary between cell types.EVs encompass three subtypes, exosomes, microvesicles and apoptotic bodies, which have different routes of intracellular formation, sizes and contents7. On the one hand, EVs play biological roles in maintaining normal cardiac structure and function under physiological conditions. On the other hand, EVs change their composition under pathological conditions and contribute to the development of CVDs8. Therefore, EVs hold tremendous potential to monitor and treat CVDs. Increasing evidence suggests that the effects of EVs on target cells are mainly dependent on miRs and proteins transferred by EVs9. Depending on the condition of the source cells, EVs containing miRs and proteins have been shown to regulate a multitude of diverse functions in target cells, maintaining cardiovascular balance and health or inducing pathological changes in CVDs , leucocytes, monocytes and macrophages Fig. 7. On theDs Table . The cur11. Endotheliocytes and platelets have been shown to be the major sources of EVs in patients with stable coronary artery disease (CAD). Atherosclerotic conditions promote the packaging of miR-92a-3p into endothelial EVs, which regulates angiogenesis in recipient endotheliocytes by a thrombospondin 1-dependent mechanism12. Coronary serum EVs isolated from patients with myocardial ischaemia promote angiogenesis via the miR-939-mediated nitric oxide signalling pathway13. EVs derived from activated endotheliocytes and inflammatory cells, such as monocytes, and especially those expressing cell adhesion and procoagulant molecules, indicate early vascular dysfunction14. Low mitochondrial encoded cytochrome c oxidase I (MT-COI) in monocyte-specific EVs from stable CAD patients is used to identify a population that is at risk for new cardiovascular events15. EVs from macrophage-derived foam cells can promote SMC migration and adhesion, which might be mediated by the integration of EVs into the SMCs and the subsequent activation of extracellular regulated protein kinase (ERK) and protein kinase B (PKB)16. Endothelial EV-mediated transfer of miR-126 promotes the repair of endotheliocyte damage caused by hyperglycaemic conditions. Intravascular injection of endothelial EVs containing miR-126 accelerated reendothelialization after electric denudation of the endothelium in vivo17. Moreover, endotheliocytes shed EVs containing miR-126 during atherosclerosis and induce the production of stromal cell-derived factor-1 (SDF-1) in recipient vascular cells. SDF-1 counteracts endotheliocyte apoptosis, recruits progenitor cells and improves plaque stability18. Increased expression of miR-126 and miR-199a in EVs but not plasma has been associated with reduced major adverse cardiovascular events in patients with stable CAD19.EVs and their miRs are key players in mediating inflammatory and coagulative reactions involving endotheliocytes, platelets, SMCs and inflammatory cells and thus contribute to the development of atherosclerosis and angiogenesis20. Hyperglycaemia-activated miR-503 is packaged into endothelial EVs and delivered to vascular pericytes, resulting in reduced expression of Ephrin-B2 (EFNB2) and vascular endothelial growth factor (VEGF) in these cells, followed by impaired migration and proliferation21. Shear stress and Kruppel-like factor 2 (KLF2), a shear-responsive transcription factor, cause miR-143 upregulation in endotheliocytes and control the phenotypes of SMCs. EVs derived from KLF2-expressing endotheliocytes reduce atherosclerotic lesion formation, improve atherosclerotic plaque stability and promote progenitor cell incorporation in the aorta in ApoE−/− mice22. Mesenchymal stromal cell (MSC)-derived EVs attenuate myocardial ischaemia–reperfusion injury in mice by shuttling miR-182, which modifies the polarisation status of macrophages23. Hypoxia-induced MSC-derived EVs facilitate ischaemic cardiac repair through miR-125b-mediated amelioration of cardiomyocyte apoptosis and prevention of cardiomyocyte death in myocardial infarction (MI)24.Endotheliocytes with increased expression of intercellular adhesion molecule 1 (ICAM-1), an adhesion molecule, in the cell membrane show increased endothelial–monocyte adhesion in cell culture, and endothelial EVs play anti-inflammatory roles by reducing endothelial ICAM-1 expression through transferring functional miR-222 into recipient cells25. Due to increased release from activated or apoptotic cells, EVs are increased in myocardial injury and represent biomarkers for MI26. In MI, cardiomyocytes secrete EVs enriched in miRs related to endothelial proliferation and differentiation, including miR-143 and miR-222, as a response to hypoxic conditions and inflammation27. A circulating EV protein network that is also involved in the inflammatory response has been found to be associated with MI28. Moreover, EVs play a fundamental role in the reorganisation of heart muscle after MI, suggesting an enormous potential for interventions by increasing or decreasing miR-containing EVs.Cardiovascular risk factors change the content of plasma EVs, and EVs play a crucial role in intercellular signalling, influencing the development of myocardial injury29.An enlarged left atrium is closely related to atrial fibrillation, and patients with atrial fibrillation develop cardiac tissue thickening or fibrosis. In patients with atrial fibrillation, miR-21-3p has been found in EVs and plasma and associated with abnormal enlargement of cardiac tissue. MiR-199a-3p in extracellular matrix-derived EVs has been shown to promote cardiac tissue growth and affect atrial electrical function by repressing homeodomain-only protein X (HOPX) expression and increasing Gata-binding 4 (Gata4) acetylation30. Cardiac fibroblast-derived EVs enriched in miR-21-3p serve as paracrine signalling-mediators of cardiomyocyte hypertrophy. MiR-21-3p induces cardiomyocyte hypertrophy by targeting sorbin and SH3 domain containing protein 2 (SORBS2). Furthermore, pharmacological inhibition of miR-21-3p reduces the development of cardiac hypertrophy in an Ang II-induced mouse model31. EVs secreted from hypertrophied cardiomyocytes contain heat shock protein 90 (HSP90) and interleukin (IL-6) to maintain the signal transducer and activator of transcription 3 (STAT3) pathway signalling32. EVs released from diabetic cardiomyocytes inhibit the proliferation, migration, and tube formation of endotheliocytes through a mechanism involving the transfer of EVs containing miR-32033. MiR-146a-loaded EVs shed by endotheliocytes are messengers in the development of peripartum cardiomyopathy and biomarkers for the diagnosis of peripartum cardiomyopathy34. A 16 kDa N-terminal prolactin fragment (16 K PRL) induces increased miR-146a expression in endotheliocytes and promotes the release of miR-146a-loaded EVs. MiR-146a suppresses small ubiquitin-like protein modifier 1 (SUMO1) expression and induces cardiac dysfunction in maladaptive hypertrophy35.EVs have been shown to mediate cross-talk between cardiomyocytes and fibroblasts in cardiac hypertrophy and have become disease biomarkers and diagnostic tools for cardiac hypertrophy and heart failure (HF). Previous studies have shown a significant role of miRs and proteins transferred by EVs in the development of HF and protection against undesired modifications to heart muscle. MiR-21-5p contributes to EV-mediated heart repair by enhancing angiogenesis and cardiomyocyte survival through the phosphatase and tensin homologue (PTEN)/PKB pathway. However, the pathological conditions of HF patients impair their regenerative activities by dysregulating miR-21-5p in cardiac-derived EVs36. EVs released from hypertrophic adipocytes impair endotheliocyte function, suggesting a possible role for EVs in obesity-related atherosclerosis37. Adipocyte-derived EVs have been shown to promote macrophage activation by delivering adipocyte-dominant transcripts (mRNAs and miRs) to macrophages38. Adipocyte-derived EVs might inhibit the regulation of macrophage cholesterol efflux and promote the development of atherosclerosis39. Adipocyte-derived EVs promote plaque burden and exacerbate vulnerable atherosclerosis by inducing angiogenesis of the vasa vasorum in diabetic ApoE−/−mice40. MiR-93-5p-enhanced adipose-derived stromal cell-derived EVs prevent cardiac injury by inhibiting autophagy and inflammation and attenuate acute MI-induced myocardial damage41. Visceral fat-derived EVs, regardless of obesity, facilitate macrophage-derived foam cell formation through the prevention of adenosine triphosphate (ATP)-binding cassette transporter 1 (ABCA1)- and ATP-binding cassette subfamily G member 1 (ABCG1)-mediated cholesterol efflux42. Only obese visceral fat-derived EVs are capable of inducing macrophage polarisation. EVs from the abdominal adipose tissue of obese mice but not lean mice activate monocyte differentiation into macrophages and induce insulin resistance43. The lipid modifying enzyme phospholipase D2 (PLD2) has been demonstrated to be involved in EV production and acts downstream of the small guanosine triphosphatase (GTPase) adenosine diphosphate (ADP)-ribosylation factor 6 (ARF6). PLD2 might integrate a significant number of signalling pathways that participate in EV release. Long-term exercise-derived circulating EVs protect the heart from myocardial ischaemia/reperfusion injury via exosomal miR-342-5p44. Long-term exercise training potentially enhances silent information regulator 1 (SIRT1) signalling, attenuates cardiac inflammation, and provides cardioprotection in d-galactose-induced ageing rats45. Cigarette smoke-induced EVs promote thrombin generation in normal human plasma and contribute to increased cardiovascular risk in smokers46.MiR and protein contents have been shown to differ in the EVs from lean and fatty rats47. Human embryonic kidney 293 cells containing SV40 large T antigen (HEK293T cells) overexpressing angiotensin II type 1 receptor (AT1R) secreted the EVs enriched in AT1Rs when subjected to osmotic stretch in vitro48. Likewise, in mice subjected to transverse aortic constriction to induce pressure overload, EVs were released into the serum that had a 100-fold increase in AT1R density. These AT1R-enriched EVs transferred functional AT1R to recipient wild-type HEK293T cells in vitro and to heart muscle in AT1R-knockout mice in vivo. There have been obvious increases in EVs in the plasma of diabetic individuals, and diabetic conditions render EVs ineffective in activating survival pathways in cardiomyocytes49. Insulin resistance increases EV secretion, and EVs might modulate insulin signalling because EVs are preferentially internalised by leucocytes and alter leucocyte function50. In the absence of diabetes mellitus, cardioprotective EVs have been shown to activate the toll-like receptor 4 (TLR-4)-extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway. Exosomal mammalian sterile 20-like kinase 1 (Mst1)-enriched EVs released from cardiac microvascular endothelial cells to cardiomyocytes worsen diabetic cardiomyopathy by inhibiting cardiomyocyte autophagy, promoting cardiomyocyte apoptosis and suppressing glucose metabolism51.MiR and protein profiles have been shown to differ in EVs from hypertensive rats compared with normotensive rats54. An engineered hydrogel patch capable of slowly releasing and providing sustained delivery of EVs secreted from induced pluripotent stem cell (iPSC)-derived cardiomyocytes has been developed to promote ejection fraction recovery, reduce arrhythmic burden, prevent cardiomyocyte apoptosis, reduce infarct size and inhibit cell hypertrophy when implanted onto rat hearts after MI55. In a preclinical MI mouse model, localised injection of miR-21-loaded EVs effectively delivered miR-21 into cardiomyocytes and endotheliocytes, drastically inhibited cell apoptosis and significantly restored cardiac function56. EVs engineered by ischaemic myocardium-targeting peptides can specifically target the ischaemic myocardium, and MSC-derived ischaemic myocardium-targeting peptide-containing EVs exert enhanced therapeutic effects on MI57.Additional investigations have explored the use of EVs as a delivery vehicle for various therapies because of their ability to easily cross biological membranes Table 52–54. An60. These EVs modulate cardiomyocytes and endotheliocytes by stimulating cell proliferation, decreasing cell apoptosis, inhibiting cell autophagy, promoting angiogenesis, reducing tissue fibrosis, preventing cardiovascular injury, treating MI and improving cardiac survival. Several studies examining the roles of MSC-secreted EVs have found that these EVs are involved in cardioprotective paracrine effects61. MSCs originate in the bone marrow, and MSC-derived EVs influence both endotheliocytes and cardiomyocytes. MSC-derived EVs affect endotheliocytes and cardiomyocytes by stimulating angiogenesis and decreasing apoptosis by preserving mitochondrial membrane potential through miR-19a enrichment62. MSC-derived EVs can protect cardiac tissue from ischaemic injury by promoting blood vessel formation, reducing infarct size and preserving cardiac function63. MSCs have been shown to mediate cardioprotective paracrine effects by secreting EVs64. MSC–EVs significantly improve cardiac survival, enhance capillary density, reduce cardiac fibrosis and restore long-term function, suggesting that EVs released from MSCs, can act as shuttles and stimulate the proliferation and migration of cardiomyocytes65. Whereas EVs harvested from stem cells in vitro might protect against cardiac injury and promote cardiac repair, EVs secreted from endogenous cardiac cells might play a more deleterious role in the progression of CVDs. Circulating myocardial miRs from infarcted hearts, such as miR-1, miR-208 and miR-499, are carried in EVs and mobilise bone marrow progenitor cells66. Injured cardiomyocyte-derived EVs accelerate transplanted MSC injury in infarcted hearts67.EVs mimic the cardioprotective properties of various stem cells Fig. , includi68. EVs derived from injured cardiomyocytes might impede the survival and promote the apoptosis of bone marrow-derived MSCs in the infarcted heart. Transplanted MSCs reduce autophagic flux in infarcted hearts via exosomal transfer of miR-125b69. cTnI-targeted EVs carrying miR-590-3p from MSCs were endocytosed by cardiomyocytes and thus promoted cardiomyocyte proliferation in the peri-MI area and eventually restored cardiac function as a treatment for MI70. Cell-free delivery of EVs secreted from iPSC-derived cardiomyocytes into the heart promotes heart recovery and treats myocardial injury71. EVs produced by cells under adverse conditions not only contribute to disease pathology but also might be detrimental towards cardiac regenerative therapies.MiR-132, delivered by MSC-derived EVs, promotes angiogenesis in MIScientific studies have sharply increased in the past 10 years, analysing multiple functions of EVs in numerous physiological pathways, from ageing, obesity, CVDs to other diseases. EV research has now clearly gained widespread interest and enthusiasm. However, the promotion of rigorous EV research is an ongoing process due to a lack of well-accepted standards for performing EV studies, which hinders progress in studying the role of EVs as diagnostic biomarkers and therapeutic options for different diseases. The conclusions of several EV studies are not sufficiently supported by the experiments performed or the information reported. The International Society for Extracellular Vesicles (ISEV) proposed the minimal information for studies of extracellular vesicles guideline in 2018. The major goal of this guideline was to guide experimental requirements specific to the EV field and promote standardisation in the EV field. By improving the reliability and reproducibility of EV studies, EVs will have stronger potential as diagnostic biomarkers and therapeutic options for CVDs and other diseases.Proper cardiovascular function depends on the coordinated interplay and communication between cardiomyocytes and noncardiomyocytes. Despite EVs representing a significant mechanism for intracellular communication, little is known about EV-mediated regulation of cardiomyocytes and noncardiomyocytes within the healthy and diseased heart. Moreover, how neighbouring or distant cells can secrete EVs to affect cardiac structure and function is largely unknown. On the one hand, miRs and proteins are transferred by EVs to maintain cardiovascular homeostasis, but on the other hand, the dysregulation of miRs and proteins transferred by EVs gives rise to the development of CVDs. Considering the fundamental roles of EVs in regulating physiological and pathological functions, future studies should be aimed at the broad and detailed understanding of EV functions and the development of new and personalised EV-based therapies.


Nearly 500,000 older Americans die a cancer-related death each year . Following a diagnosis of a serious illness like cancer, maintaining a sense of dignity is central to a patient’s wellbeing. Dignity Therapy (DT) was recently introduced as an intervention to enhance dignity for terminally ill patients . This therapy provides patients opportunities to foster a sense of dignity though making meaning of their lives . To date, whether meaning-making actually occurs as a central mechanism of effective DT has not been tested. The current study investigates (i) how often and in what forms meaning-making occurs during DT, and (ii) how patients’ baseline feelings of dignity relate to meaning-making during DT. Participants were 25 male and female cancer outpatients . They completed the Patient Dignity Inventory  and then participated in Dignity Therapy with a trained provider. Sessions were audio recorded, transcribed, and reliably content-analyzed for meaning-making using an established coding scheme . Content-analysis revealed that all patients made meaning of past life events at least once (range: 1-12 occurrences). Multiple forms of meaning-making emerged, with Finding Benefit and Personal Growth most common. Patients reporting more dignity-related distress prior to DT showed greater meaning-making during the DT session . This study provides foundational evidence that meaning-making is a key mechanism of Dignity Therapy, helping older adults with cancer enhance dignity at end-of-life.


Platelets are small anucleated blood components primarily described as playing a fundamental role in hemostasis and thrombosis. Over the last decades, increasing evidence has demonstrated the role of platelets in modulating inflammatory reactions and immune responses. Platelets harbor several specialized organelles: granules, endosomes, lysosomes, and mitochondria that can synthesize proteins with pre-stored mRNAs when needed. While the functions of platelets in the immune response are well-recognized, little is known about the potential role of platelets in immune tolerance. Recent studies demonstrate that platelet-specific FVIII gene therapy can restore hemostasis and induce immune tolerance in hemophilia A mice, even mice with preexisting anti-FVIII immunity. Here, we review the potential mechanisms by which platelet-targeted FVIII gene therapy restores hemostasis in the presence of anti-FVIII inhibitory antibodies and induces immune tolerance in hemophilia A. It has been shown that when FVIII expression is introduced by HSC transduction with 2bF8 lentivirus followed by transplantation, FVIII expression is detected only in platelets, but not in plasma of hemophilia A mice , 22, 23./6 mice) , 22, 25.The effectiveness of platelet-targeted gene therapy has been further confirmed in hemophilia A rats  and hemoIn contrast to platelet-specific FVIII expression, it has been shown that targeting FVIII expression to hematopoietic cells under a constitutively active promoter may trigger anti-FVIII immune responses. Wang et al.  utilized+ or c-kit+ cells transduced with porcine FVIII driven by the mouse stem cell virus (MSCV) promoter (MSCV-porcine fVIII) using a retrovirus-mediated gene transfer system. Further studies by Ide et al.  for preconditioning, sustained plasma FVIII expression was achieved in mice after receiving MSCV- porcine fVIII-transduced Sca-1+ cells  mice. Further studies using a FVIII-specific memory B cell differentiation assay showed that CD4 T cells from FVIII-immunized 2bF8 lentivirus-transduced recipients could not promote memory B cell maturation into antibody-secreting cells, but memory B cells from FVIII-immunized 2bF8-transduced recipients could differentiate into antibody-secreting cells when co-cultured with CD4 T cells isolated from FVIII-primed untransduced FVIIInull mice. Further studies showed that immune tolerance is transferable when splenocytes from 2bF8-transduced recipients were infused into naive FVIIInull mice (Multiple lines of evidence suggest that both primary and secondary anti-FVIII immune responses are CD4 T cell-dependent –52. Studull mice . TogetheTo further investigate how immune tolerance is established after platelet-targeted gene transfer, Luo et al.  used thePlatelets play fundamental roles not only in hemostasis and thrombosis but also in innate and adaptive immunity. Data from preclinical trials using animal models have demonstrated that platelet-targeted FVIII gene therapy is effective in treating hemophilia A mice even with inhibitors. Platelet-targeted gene therapy can promote antigen-specific immune tolerance through peripheral tolerance mechanisms. The effectiveness of platelet gene therapy in hemophilia A with inhibitors could be attributed to many pivotal factors, including the shielding of neoprotein by platelets from being recognized by the immune system, the presence of a protective protein VWF in the platelets, and proper preconditioning before gene transfer. In conclusion, platelet-targeted gene therapy is a unique approach for gene therapy of hemophilia A even with inhibitors as it can provide not only therapeutic protein but also induce antigen-specific immune tolerance.YC contributed to manuscript writing. QS conceived the structure of the review and wrote the manuscript.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.