Extracellular vesicles (EVs) containing specific subsets of functional biomolecules are released by all cell types and analysis of circulating EVs can provide diagnostic and prognostic information. To date, little is known regarding the role of EVs both as biomarkers and potential key players in human lung cancer.Plasma EVs were isolated from 40 cancer-free heavy-smokers classified according to a validated 24-microRNA signature classifier (MSC) at high (MSCpos-EVs) or low (MSCneg-EVs) risk to develop lung cancer. EVs origin and functional properties were investigated using in vitro 3D cultures and in vivo models. The prognostic value of miRNAs inside EVs was assessed in training and in validation cohorts of 54 and 48 lung cancer patients, respectively.Different membrane composition, biological cargo and pro-tumorigenic activity were observed in MSCpos vs MSCneg-EVs. Mechanistically, in vitro and in vivo results showed that miR-126 and miR-320 from MSCpos-EVs increased pro-angiogenic phenotype of endothelial cells and M2 polarization of macrophage, respectively. MSCpos-EVs prompted 3D proliferation of non-tumorigenic epithelial cells through c-Myc transfer. Moreover, hypoxia was shown to stimulate the secretion of EVs containing c-Myc from fibroblasts, miR-126-EVs from endothelial cells and miR-320-EVs from granulocytes. Lung cancer patients with higher levels of mir-320 into EVs displayed a significantly shorter overall survival in training [HR2.96] and validation sets [HR2.68].Overall our data provide a new perspective on the pro-tumorigenic role of circulating EVs in high risk smokers and highlight the significance of miR-320-EVs as a new prognostic biomarker in lung cancer patients.The online version contains supplementary material available at 10.1186/s13046-021-02040-3. Lung cancer remains the leading cause of cancer-related deaths, despite the substantial advances made over the past 20 years in its prevention, diagnosis and treatment . The 5-yNon invasive biomarkers able to assess cancer risk and improve early diagnosis  may impaEVs are an evolutionarily conserved group of bilayer membrane vesicles , 8. TheyAmong the different EV’s cargo components, miRNAs have the largest impact on oncology thanks to their ability to act as both oncogenes and tumor suppressors. MiRNAs are endogenous small non-coding RNAs (21–25 nucleotides) that play a pivotal role in the regulation of gene expression. Deregulation of miRNA expression and the consequent alterations in related biological processes can lead to cancer development, as demonstrated by several studies . DetectiIn two large prospective studies, we previously assessed the value of circulating miRNAs for early detection of lung cancer in the context of LDCT screening , 18. NotAlthough many studies have elucidated the function of EV-miRNAs released by cancer cells, to date only a few studies have analyzed EV-miRNAs circulating in cancer-free subjects and focused on their interaction with recipient cells to favor a pro-tumorigenic milieu. Our study aimed to characterize plasma EVs isolated from smokers at risk of developing lung cancer and to elucidate their pro-tumorigenic role, focusing on the functional interactions between EV-miRNAs and specific microenvironmental target cells.NPlasma samples were collected from high-risk heavy-smoker volunteers (age 50–75 years), including current or former smokers with a minimum pack/year index of 30 enrolled in a LDCT screening trial performed at our institution  . MiR-126-EV did not show prognostic value also in the validation cohort (Suppl. Figure To test the consistency of our findings we tested miR-320-EV and miR-126-EV in a validation set of 48 lung cancer patients with the same clinical characteristics (Suppl. Table Several studies have revealed that cancer progression is driven by a complex systemic interplay between tumor's genetic alterations and paracrine effectors within the host microenvironment, thus, the knowledge of these cellular mechanisms is fundamental for early detection and treatment of lung cancer. Supporting cells in the lung microenvironment, such as lung fibroblasts, endothelial cells and circulating immune cells can control several epithelial cell processes, such as proliferation, metabolism and apoptosis. Interestingly, these processes are finely regulated by the concentration of oxygen in the microenvironment. Indeed, hypoxic conditions enhance EVs production by stromal cells , improviIn the present study, we focused on the role of circulating plasma EVs and in particular on their miRNA cargo associated with a high-risk to develop lung cancer, to understand their role during lung carcinogenesis.We previously described that reciprocal ratios of 24 circulating miRNAs predicts lung cancer occurrence up to two years before CT-detection of lung nodules and are able to identify tumor aggressiveness . These sSeveral studies indicate the importance of tumor-derived EV-miRNAs in lung cancer development , 42 but Analysis of plasma-derived EVs from heavy smokers unveiled the presence of several surface proteins, such as HLA, CD45, along with specific markers of blood cell subset as CD14, CD11c, CD56 and CD3. Importantly, in our study, CD31 was downregulated in EVs from MSCpos individuals compared to EVs from MSCneg individuals. The loss of CD31 expression on EVs membranes could be considered an early biomarker for hypoxia . MoreoveIn a mouse model of alveolar hypoxia, the release of proangiogenic factors that promote lung tumor growth, such as VEGF-A and an increase of C-Myc in lung tissues was observed . We demoFurthermore, the protumorigenic PMN phenotype has been demonstrated to be induced by TGF-β release under hypoxic conditions . PMNs isSmoke and hypoxia may act synergistically to generate an immunosuppressive and pro-tumorigenic microenvironment that leads to tumor development. Cigarette smoke can enhance the production of inflammatory factors and reactive oxidative species and induce the activation of HIF and, downstream effectors . VEGF-A Our data suggest that the alteration in miRNAs levels observed in EVs from high-risk smokers could reflect changes in lung stromal cells that are able to generate a pro-tumorigenic microenvironment and sustain tumor growth. Chronic cigarette smoke exposure in subjects with a miRNA –based high risk profile induces intracellular alterations in the lung microenvironment and, consequently, in the EVs released by these cells.We are aware that miRNAs are a minor component of EV-cargo , so otheEVs and their miRNAs content gained attention as diagnostic tool for lung cancer detection or for prognosis of patients. An interesting study showed that 27 miRNAs were present in lung cancer-derived compared to EVs isolated from healthy volunteers . InteresIn conclusion, based on in vitro experiments, hypoxia-induced modulation of the miRNA cargo of stromal-derived EVs generates a pro-tumorigenic and immunosuppressive microenvironment that stimulates the proliferation and growth of premalignant epithelial cells to promote lung tumor development. In this context, circulating EVs enriched in miR-320 play a key role by inducing immunosuppressive features and could also be an useful prognostic biomarker in lung cancer patients.Additional file 1:Additional file 2:


Mycobacterium tuberculosis (MTB) infections have not been reported. Deaths associated with a positive ante-mortem SARS-CoV-2 PCR test and/or respiratory disease symptoms at Chris Hani Baragwanath Academic Hospital in Soweto, South Africa from 15th April to 2nd November 2020, during the first wave of the South African COVID-19 epidemic, were investigated. Deceased adult patients had post-mortem minimally-invasive tissue sampling (MITS) performed to investigate for SARS-CoV-2 infection and molecular detection of putative pathogens on blood and lung samples, and histopathology examination of lung, liver and heart tissue. During the study period MITS were done in patients displaying symptoms of respiratory disease including 75 COVID-19-related deaths (COVID+) and 42 non-COVID-19-related deaths (COVID-). The prevalence of HIV-infection was lower in COVID+ (27%) than in the COVID- (64%), MTB detection was also less common among COVID+ (3% vs 13%). Lung histopathology findings showed differences between COVID+ and COVID- in the severity of the morphological appearance of Type-II pneumocytes, alveolar injury and repair initiated by SARS-CoV-2 infection. In the liver necrotising granulomatous inflammation was more common among COVID+. No differences were found in heart analyses. The prevalence of bacterial co-infections was higher in COVID+. Most indicators of respiratory distress syndrome were undifferentiated between COVID+ and COVID- except for Type-II pneumocytes. HIV or MTB infection does not appear in these data to have a meaningful correspondence with COVID-related deaths.Comparisons of histopathological features and microbiological findings between decedents with respiratory symptoms due to SARS-CoV-2 infection or other causes, in settings with high prevalence of HIV and  The coronavirus disease 2019 (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2), has caused more than 5.3 million deaths as of November 2021 . These sPathologic studies of decedents may provide important information related to disease presentation, therapeutic decision making, and inform trials of strategies designed to prevent severe disease . In the th March 2020 and, subsequently, the first known hospitalized case of COVID-19 at CHBAH was diagnosed on 6th April 2020. The number of COVID-19 cases admitted at CHBAH was low until the end of May 2020, after which the numbers rapidly increased with infections peaking from mid-June to early August (week 26 to 32), the number of COVID-19 cases was again low during October and November 2020 [th April to 2nd November 2020.Chris Hani Baragwanath Academic Hospital (CHBAH) is one of two public hospitals serving the population of Soweto, Gauteng, South Africa, providing tertiary-level hospital care to a population that generally (>90%) depends on the public health service. Details of the population of Soweto and the standard of care practices at the CHBAH have been published previously . The firber 2020 . This stFor this study, the families of the patients dying in-hospital with a positive ante-mortem SARS-CoV-2 polymerase chain reaction (PCR) test and/or respiratory disease symptoms during the hospital stay were contacted after the patient’s death to obtain permission for inclusion in the study. Decedents were considered to have displayed respiratory symptoms if they had been diagnosed with bronchopneumonia, tuberculosis, bronchitis, lower respiratory tract infection, asthma/acute exacerbation of asthma, chronic obstructive pulmonary disease, or lung disease, or if a clinical finding of respiratory distress, tachypnoea, stridor, chest wall retraction, wheeze, bronchial breathing, or two or more of cough, shortness of breath, or difficulty breathing. For each decedent for whom consent was obtained, medical record information was collected on demographic characteristics, duration of hospital stay, HIV infection status and other comorbidities.Post-mortem minimally-invasive tissue sampling (MITS) was used in place of full autopsies. MITS has been validated against complete diagnostic autopsy and has shown high concordance for attributing cause of death, particularly for infectious diseases . MITS saFollowing a biopsy, tissues for histopathology were immediately placed in 10% neutral buffered formalin and fixed for a minimum period of 6 hours, but no longer than 24 hours. After fixation, the biopsies were dehydrated in graded ethanol concentrations, cleared in xylene, and infiltrated with wax, using the Tissue-Tek Vacuum Infiltration Processor manufactured by Sakura. Tissues were then embedded in wax blocks from which 4 micron sections were cut and stained with haematoxylin and eosin (H&E). Protocols for the tissue processing and H&E staining were based on widely established protocols and methodology . A histoNucleic acid amplification test using the Emergency use authorization assay developed by the USA Centers for Diseases Control and Prevention (CDC) was used to detect SARS-CoV-2 in respiratory and blood samples. Results were classified as positive for SARS-CoV-2 when both the N1 and N2 targets of the nucleocapsid gene were detected by PCR with cycle threshold (Ct) values were <40.The presence and abundance of specific viruses, bacteria and non-bacterial agents in lung tissue were also evaluated using PCR based methods. Real-time PCR was performed using the Open Array QuantStudio 12k Flex Real-Time PCR System with TaqMan Array Cards (TAC) according to manufacturer’s instructions  and the pathogens investigated are listed in Mycobacterium tuberculosis (MTB) was performed as previously described [GeneXpert testing for detection of escribed . BrieflyThis is a descriptive report on a convenience sampling and no formal statistical significance was evaluated. Among SARS-CoV-2 decedents stratifications by HIV-infection status and duration of hospital stay before death (≤3 days vs >3 days) are presented.The study was approved by the Human Research Ethics Committee at the University of the Witwatersrand . Informed written consent was obtained from relatives of the deceased.th April to 2nd November 2020, there were 600 deaths with a positive SARS-CoV-2 polymerase chain reaction (PCR) test ante-mortem at Chris Hani Baragwanath Academic Hospital (CHBAH); of these, post-mortem minimally-invasive tissue sampling (MITS) were performed on 68. Additionally, MITS were done in 11 decedents who had a negative ante-mortem test but a positive post-mortem test and 2 SARS-CoV-2 positive decedents who were just tested post-mortem, for a total of 81 SARS-CoV-2 infected decedents. Of these 81 decedents, six tested SARS-CoV-2 ante-mortem but did not manifest any respiratory symptoms. Because the focus of this analysis is the comparison between SARS-CoV-2 infected and–uninfected decedents with respiratory symptoms, these 6 were not included in the analysis. The 75 decedents displaying symptoms of respiratory disease along with one or more positive SARS-CoV-2 tests are referred to as the COVID-positive (COVID+) group. Furthermore, 42 decedents who tested negative for SARS-CoV-2 on ante- or post-mortem sampling were also included; all these displayed symptoms of respiratory disease and are for convenience referred to as the COVID-negative (COVID-) group.From 15The COVID+ decedents were slightly older than the COVID- group (60 vs 51 years), and more likely to be female (46% vs 23%). Hypertension (54% vs 40%) and diabetes (37% vs 16%) were the two comorbidities more common among COVID+ decedents compared to those in the COVID- group. There was a lower prevalence of HIV-infection in the COVID+ group (27%) than in the COVID- group (64%) . Median Mycobacterium tuberculosis (MTB) GeneXpert testing. Overall, more bacteria were detected among the COVID+ group compared to the COVID- group, especially for Acinetobacter baumannii (12% vs 5%), Escherichia coli (11% vs 0%) and Klebsiella pneumoniae (12% vs 2%). Pneumocystis jirovecii was however only detected in COVID- decedents . Detection of MTB was more common among the COVID- (13%) than the COVID+ group (3%); all seven patients positive for GeneXpert showed necrotizing granulomatous inflammation on histopathological examination. Stratification of the COVID+ group by HIV-infection status showed a similar frequency of detection for most pathogens with only Cytomegalovirus (15% vs 4%) and MTB  being more frequently detected in HIV-infected decedents (ecedents .Escherichia coli (13% vs 2%) and Klebsiella pneumoniae (12% vs 5%) detection being higher in the COVID+ than the COVID- group. Decedents that stayed in hospital for >3 days were more likely to be positive for at least one bacterium compared with hospitalization ≤3 days (58% vs 31% among COVID+ and 52% vs 33% in the COVID- group).The post-mortem lung culture results in Klebsiella pneumoniae (11% vs 2%) being more frequently detected post-mortem in COVID+ than in COVID- decedents. Similarly, to lung cultures, the rate of bacterial detection by post-mortem blood culture was higher in those decedents that stayed in the hospital for >3 days compared with ≤3 days (49% vs 22% in COVID+ and 62% vs 10% in COVID-).Blood ante-mortem and post-mortem culture results are shown in Intravascular fibrin-platelet thrombi were found in seven (9%) COVID+ decedents, involving the portal vein in five, hepatic arterioles in one and the hepatic vein also in one . There wThis study documents the lung, liver and heart pathologies of decedents with respiratory symptoms at a large hospital in Soweto, South Africa. The findings presented here highlight the severity of the tissue response in the lungs in those patients who are COVID+, including florid Type-II pneumocyte proliferation, with exaggerated cytomegaly and nucleomegaly, and syncytia formation. Exuberant hyaline membrane formation suggests that surfactant production or dysfunction in Type-II pneumocytes may play an important role in the pathogenesis, although there is no current evidence for this . The desDespite important differences in the underlying populations and the methods utilized, this MITS-based study of decedents presenting with symptoms of respiratory disease shows substantial conformity with prior results from other parts of the world concerning lung pathology among persons testing positive for SARS-CoV-2 infection , 34.Comparison of the traditional approach to the classification (Corrin) of the pathological changes seen in acute respiratory distress syndrome with that proposed by Polak et al, highlights a group of decedents, up to 32% of the COVID+ group, that have co-existent vascular pathology in the form of thromboembolic disease , 30. TheThe identification of pre-existing small foci of fibrosis in the alveolar septal walls is unexplained but as Soweto is a densely populated urban environment this could be consequent to environmental pollutants including exposure to tobacco smoke or previous episodes of unknown lung disease resulting in early interstitial lung disease .The identification of fibrin-platelet thrombi in the liver suggests that this is a manifestation of a systemic coagulopathy in COVID+ decedents and has also been described by other authors . AlthougWhilst the findings in the cardiac biopsies show no clear histopathological changes that could be directly attributed to SARS-CoV-2 infection, the changes reflected show evidence of pre-existing chronic cardiac disease which, as a co-morbidity, may impact on cardiac function and final outcome .Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae were more often detected in COVID+ decedents, suggesting a potential viral-bacterial association that could have precipitated the death. Moreover, a longer duration of hospitalization before death led to a higher rate of bacterial culture positivity maybe due to nosocomial infections.Three important bacteria, i.e. Although the number of COVID-19 reported deaths in Africa is low relative to those reported elsewhere, it may be the case that this is an artifact of limitations related to surveillance rather than a lower underlying rate of fatalities in Africa. Other possible reasons could include differences in age-group demographics, the prevalence of underlying comorbidities and factors that could influence the virus inoculum load. Despite these possible differences, the data presented here suggest that the pathological evidence is similar to what has been seen in other parts of the world.Two aspects of the characteristics shown in Limitations of our study include it describing observed differences among decedents presenting with respiratory disease symptoms based on positive/negative test results for SARS-CoV-2 infection. Entry into the study depended not only on meeting the inclusion criteria, but critically on obtaining informed consent from the relatives of the deceased. It is certainly plausible that decedents for whom consent was obtained differ in important ways from those for whom consent was not obtained. Nevertheless, the ability to gather these data further suggests that the use of the MITS procedure is feasible, and indeed could be used more widely in areas where full autopsies are not widely available or are not culturally appropriate. Also, this was a hypothesis generating descriptive study and no formal statistical analysis was done due to the results’ cell sizes being small. The criteria used to define respiratory symptoms are broad, allowing for the possibility of substantial functionally unobserved heterogeneity among the decedents included in the analyses. Owing to the limited sample size, stratification into more homogeneous groups was not a practical option. Also, the fact that MTB could not be detected by molecular assay detection, but only on GeneXpert test, may suggest that other pathogens also failed detection one the molecular array assay.Nonetheless, our results suggest that MTB and HIV infections are not associated with fatal COVID-19 pathology. Our study corroborates the feasibility of MITS in adults, and shows that pathogens could be detected with molecular assays, as well as, with culture techniques. Although most indicators of respiratory distress syndrome were undifferentiated between COVID+ and COVID- except for Type-II pneumocytes, more data is needed to support these suggestions.S1 FigHaematoxylin and eosin stained section of lung tissue showing: 1) typical microscopic features of severe SARS-CoV-2 infection, including florid Type-II pneumocyte proliferation in the alveolar spaces, lymphocytic interstitial pneumonitis and extensive necrosis of alveolar septal walls; 2) severe hyaline membrane formation lining the alveolar septa with accompanying interstitial lymphocytic inflammation and Type-II pneumocyte proliferation; 3) cytomegaly, nucleomegaly and multinucleation of Type-II pneumocytes in a background of exuberant intra-alveolar Type-II proliferation (arrow and inset), accompanying intra-alveolar inflammation and early collapse of alveolar septa is also seen; 4) syncytial metaplasia of Type-II pneumocytes; 5) progressive SARS-2-CoV-2 pulmonary injury with hyaline membranes (A), Type-II pneumocyte proliferation (B) and early intra-alveolar organising pneumonia with alveolar septal necrosis (C). A lymphocytic infiltrate is present throughout; 6) multifocal intra-alveolar organising pneumonia; 7) multifocal fibrosis of alveolar septa; 8) “honeycombing” with collagenous thickening of alveolar septa and obliteration of the functional air-blood interface, residual proliferating Type-II pneumocytes remain highlighting the continuum between the different pathological phases of the tissue response in acute respiratory distress syndrome; 9) partial occlusion of a pulmonary vein by fibrin platelet strands (long arrow) contrasting with an area of patency (short arrow); 10) occlusion of pulmonary veins by fibrin platelet thrombi (*) contrasting with adjacent patent pulmonary arterioles.(PDF)Click here for additional data file.S2 FigHaematoxylin and eosin stained section of liver showing intraluminal thrombus in a portal vein radicle (arrow).(PDF)Click here for additional data file.S3 FigHaematoxylin and eosin stained section of heart tissue showing: 1) myocarditis with interstitial lympho-histiocytic inflammatory infiltrate, interstitial oedema and focal myocardial fibre disruption; 2) lympho-histiocytic myocarditis (short arrow) and myocardial fibre disruption and necrosis (long arrows); 3) focus of myocardial fibre necrosis (arrow) accompanied by lympho-histiocytic inflammatory infiltration; 4) intra-capillary fibrin-platelet thrombi in interstitial vasculature (arrows).(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.


S-palmitoylation, a reversible post-translational protein modification. Genome sequence databases show the evolutionary acquisition and conservation concerning vertebrate-specific palmitoylation of synaptic proteins including glutamate receptors. Moreover, palmitoylation of some glutamate receptor-binding proteins is subsequently acquired only in some mammalian lineages. Recent progress in genome studies has revealed that some palmitoylation-catalyzing enzymes are the causative genes of neuropsychiatric disorders. In this review, I will summarize the evolutionary development of palmitoylation-dependent regulation of glutamatergic synapses and their dysfunctions which are caused by the disruption of palmitoylation mechanism.Glutamate is the major excitatory neurotransmitter in the vertebrate brain and various modifications have been established in the glutamatergic synapses. Generally, many neuronal receptors and ion channels are regulated by  Vertebrates comprise a lot of animal species classified within the subphylum Vertebrata, which consists of a majority of the phylum Chordata. Variety of species in mammals, birds, reptiles, amphibians, lobe-finned fishes, ray-finned fishes, cartilaginous fishes, and jawless fishes belong to vertebrates. They have diverged from the common ancestral chordate. Other subphyla Cephalochordata and Urochordata in the phylum Chordata are composed of lancelets and sea squirts , respectively. The vertebrate brain has been naturally developed from the primitive chordate nervous system. Almost as if a language is deeply rooted in the accumulated history of speakers -type receptors, basically named after their selective agonist drugs  family proteins play central roles in excitatory synaptic transmission and plasticity in the vertebrate brain  to intracellular cysteine residues of target proteins via thioester bonds. Accumulating genome information in animal species increasingly clarifies that low rates of substitution occur at structurally or functionally significant amino acid residues during molecular evolution against continuous mutation pressure.Essential biological functions of proteins are basically determined by the amino acid sequence of proteins. Moreover, post-translational protein modifications (PTPMs) enable precise and dynamic control of protein localization, membrane trafficking, and fine-tuning of protein functions. In many types of PTPMs, protein ns Resh, . PalmitaPrevious studies have shown the vertebrate-specific conservation of palmitoylated cysteine residues in GluRs orthologs . Concrethyperpolarization-activated cyclic nucleotide-gated channel 2) orthologs  orthologs  domain-containing proteins, such as PSD-95 (postsynaptic density protein 95), GRIP1 (GluR-interacting protein 1), GRIP2, and PICK1 (protein interacting with C-kinase 1), which make complex with GluRs, respectively. These vertebrate PDZ domain proteins have orthologs in other phyla , broadly exist in vertebrates and many invertebrates. The fruit fly Drosophila melanogaster protein Discs large (DLG) and a nematode Caenorhabditis elegans protein DLG-1 are orthologs of vertebrate PSD-95. While vertebrate PSD-95 possess N-terminal palmitoylated cysteine residues, invertebrate PSD-95 orthologs lack these motifs at the corresponding site. N-terminal palmitoylation sites are lost in DLG orthologs from primitive chordates  and hemichordates . Therefore, synaptic regulation through PSD-95 palmitoylation appears unique to vertebrates  complex and regulates AMPA receptors ion channel properties as well as their membrane localization in many animal species. Orthologs of PSD-95, also known as SAP-90 (tebrates .AMPA receptor-binding protein-L), are alternatively spliced to generate palmitoylated isoforms, GRIP1b, GRIP2b/pABP-L. PDZ domains of these proteins interact with GluA2 C-terminus. The neuronal trafficking and localization of vertebrate AMPA receptor complexes can be regulated by these binding partners via palmitoylation  and hemichordates  lack this palmitoylation site. Generally, palmitoylation motifs are lost in invertebrate GRIP1 and GRIP2 orthologs including insects. Although the dGRIP, a Drosophila GRIP1 ortholog, exceptionally contains a cysteine residue at the corresponding position to vertebrate GRIP1, amino acids around the dGRIP cysteine are poorly conserved compared with vertebrate GRIP1b. Thus, it remains unclear whether dGRIP can serve similar functions to mammalian GRIP1b even if dGRIP is palmitoylated. Palmitoylation of iGluRs, PSD-95, GRIP1b, GRIP2b, HCN2, AQP4, and 5-HT receptor orthologs seem to be acquired in the ancestor of vertebrates at about the same time around 500 million years ago . The Cys-containing PICK1 palmitoylation motif is found in species belonging to the superorders Afrotheria , Xenarthra , Laurasiatheria  and Euarchontoglires . The PICK1 palmitoylation-dependent refined control of AMPA receptors should originate in the common ancestor of these eutherian  species  and American pika (the order Lagomorpha in the superorder Euarchontoglires: -Gly-Ser-Trp-Ser-Asp-Ser). Furthermore, PICK1 orthologs in all reported species in the family Bovidae  possess different Cys-containing sequences from the standard eutherian motif, which may imply ongoing molecular evolution of PICK1 palmitoylation  and depalmitoylation by palmitoyl-protein thioesterases (PPTs) serve as a cellular regulatory mechanism from yeast to human , fruit fly , Zebrafish , frog , mouse , and humans . The proportion of PDZ-binding PATs goes up from 6.7% in worms to around 36.7% in vertebrates as DHHC PAT numbers increase. All eight PDZ-binding mouse PATs  are detected in 14 neuronal tissue-expressing PATs (57.1%), indicating that PDZ-dependent interaction of PATs is likely to play crucial roles in the vertebrate brain.Comparison of animal genomes suggests that DHHC PATs homologs seem to have expanded during animal evolution. PDZ-binding motifs are present in C-termini of several DHHC PATs, which are recognized by PDZ domain-containing proteins . PredictSo far, there is no direct evidence that implicates mutations at the palmitoylation sites of iGluRs and that their binding proteins cause any neuropsychiatric disorders. However, palmitoylation of these orthologs was established in the very early stage of vertebrate evolution and they have been extremely well conserved in the whole vertebrate species or in specific eutherian lineage as summarized in this review. This essential synaptic modification system may be protected by multiple layers of regulatory mechanisms to prevent disturbance of vertebrate brain functions. Further accumulation of genome sequence data will fill in the blanks of the animal order, family, and genus list concerning vertebrate divergence. Additional information will reveal the timeline of establishment and divergence of these palmitoylation sites-containing motifs in more detail. Especially, sequence information on urochordates (sea squirts or ascidians), cephalochordates (lancelets), and cyclostomes (lampreys and hagfishes) will clarify the initial acquisition of the mechanism of synaptic palmitoylation.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Inactivated coronaviruses, including severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and Middle East respiratory syndrome coronavirus (MERS-CoV), as potential vaccines have been reported to result in enhanced respiratory diseases (ERDs) in murine and nonhuman primate (NHP) pneumonia models after virus challenge, which poses great safety concerns of antibody-dependent enhancement (ADE) for the rapid wide application of inactivated SARS-CoV-2 vaccines in humans, especially when the neutralizing antibody levels induced by vaccination or initial infection quickly wane to nonneutralizing or subneutralizing levels over the time. With passive transfer of diluted postvaccination polyclonal antibodies to mimic the waning antibody responses after vaccination, we found that in the absence of cellular immunity, passive infusion of subneutralizing or nonneutralizing anti-SARS-CoV-2 antibodies could still provide some level of protection against infection upon challenge, and no low-level antibody-enhanced infection was observed. The anti-SARS-CoV-2 IgG-infused group and control group showed similar, mild to moderate pulmonary immunopathology during the acute phase of virus infection, and no evidence of vaccine-related pulmonary immunopathology enhancement was found. Typical immunopathology included elevated MCP-1, IL-8 and IL-33 in bronchoalveolar lavage fluid; alveolar epithelial hyperplasia; and exfoliated cells and mucus in bronchioles. Our results corresponded with the recent observations that no pulmonary immunology was detected in preclinical studies of inactivated SARS-CoV-2 vaccines in either murine or NHP pneumonia models or in large clinical trials and further supported the safety of inactivated SARS-CoV-2 vaccines. Antibody-dependent enhancement (ADE) effect, defined as the antibodies induced by initial infection or vaccination binding to viral surface proteins and promote viral invasion of host cells, is a major concern for all vaccine developers. Inactivated coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), as potential vaccines have been reported to result in enhanced respiratory diseases (ERDs) in murine and nonhuman primate (NHP) pneumonia models after virus challenge, which poses great safety concerns for the rapid wide application of inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines in humans ,2. No ER5 TCID50. Samples, including nasal swabs, pharyngeal swabs and blood samples, were collected daily for viral shedding and neutralizing titre analyses. Four days after SARS-CoV-2 infection, all animals were sacrificed after anesthesia. Bronchoalveolar lavage fluid (BALF) and the main tissues and organs were collected for proinflammatory mediator, viral load and histopathology analyses.Anti-serum with a neutralizing antibody geometric mean titre (GMT) > 1:128 was collected from rhesus macaque intramuscularly injected on days 0 and 28 with a formaldehyde and β-propiolactone double-inactivated SARS-CoV-2 vaccine developed by the Institute of Medical Biology, Chinese Academy of Medical Sciences (IMBCAMS) based on the KMS-1 strain (GenBank accession number MT226610.1) ,12. Six Although neutralization titres were detected only in 2 (No. 19256 and No. 19060) of the 4 passively immunized monkeys before virus challenge b, all ofAmong all of the inflammatory cytokines  and chemokines  tested in BALF, some monkeys in the anti-SARS-CoV-2 IgG-infused group showed relatively higher levels of MCP-1, IL-8 and IL-33 than the control group d, S1, alOne way in which SARS-CoV-2 is different from SARS and MERS is the prevalence of a large number of asymptomatic and mild infections. Studies have shown that asymptomatic and mild patients are also capable of producing neutralizing antibodies, the levels of which are positively correlated with disease severity . Recent Our study demonstrated that the antibodies purified in vitro from anti-serum of inactivated SARS-CoV-2 vaccination at subneutralizing or even nonneutralizing levels could offer protection from SARS-CoV-2 infection in vivo c. This r5 TCID50) administered in rhesus macaque pneumonia models may lead to excessive activation of the immune cascade in lung tissues, which results in pulmonary pathology. Studies of SARS immunization in animal models have yielded widely varying results in terms of protective efficacy and potential ADE, and different vaccine strategies may lead to differences in whether the vaccine enhances the disease. There is a general consensus that inducing higher titres of antibodies as well as antibodies that target the S protein more effectively can reduce the risk of ADE. It is encouraging, however, that the inactivated SARS-CoV-2 vaccine, which targets the full antigen of the virus, provides dose-dependent protection in a variety of animal models without producing evidence of enhanced immunopathological damage [On the other hand, the elimination of SARS-CoV-2 by macrophages is reported to be associated with the production of multiple antiviral and proinflammatory cytokines , which il damage . Our stuIn conclusion, our results showed that in the absence of cellular immunity, passive infusion of subneutralizing or nonneutralizing anti-SARS-CoV-2 antibodies could still provide some level of protection against infection upon challenge, and no low-level antibody-enhanced infection was observed. The anti-SARS-CoV-2 IgG-infused group and control group showed similar, mild to moderate pulmonary immunopathology during the acute phase of virus infection, and no evidence of vaccine-related pulmonary immunopathology enhancement was found. There results corresponded with the recent observations that no pulmonary immunology was detected in the preclinical studies of inactivated SARS-CoV-2 vaccines in both murine and NHP pneumonia models or in large clinical trials. Although more evidence is needed to confirm or deny the existence of ADE in SARS-CoV-2 infection and the immune damage caused by it, the available evidence has further supported the safety of the SARS-CoV-2 vaccine.Click here for additional data file.Click here for additional data file.


Substantial individual differences exist in how acute stress affects large-scale neurocognitive networks, including salience (SN), default mode (DMN), and central executive networks (CEN). Changes in the connectivity strength of these networks upon acute stress may predict vulnerability to long-term stress effects, which can only be tested in prospective longitudinal studies. Using such longitudinal design, we investigated whether the magnitude of acute-stress-induced functional connectivity changes (delta-FC) predicts the development of post-traumatic stress-disorder (PTSD) symptoms in a relatively resilient group of young police students that are known to be at high risk for trauma exposure. Using resting-state fMRI, we measured acute-stress-induced delta-FC in 190 police recruits before (baseline) and after trauma exposure during repeated emergency-aid services (16-month follow-up). Delta-FC was then linked to the changes in perceived stress levels (PSS) and post-traumatic stress symptoms (PCL and CAPS). Weakened connectivity between the SN and DMN core regions upon acute-stress induction at baseline predicted longitudinal increases in perceived-stress level but not of post-traumatic stress symptoms, whereas increased coupling between the overall SN and anterior cerebellum was observed in participants with higher clinician-rated PTSD symptoms, particularly intrusion levels. All the effects remained significant when controlling for trauma-exposure levels and cortisol-stress reactivity. Neither hormonal nor subjective measures exerted similar predictive or acquired effects. The reconfiguration of large-scale neural networks upon acute-stress induction is relevant for assessing and detecting risk and resilience factors for PTSD. This study highlights the SN connectivity-changes as a potential marker for trauma-related symptom development, which is sensitive even in a relatively resilient sample. Exposure to severely stressful events can lead to a wide range of mental health problems, including post-traumatic stress symptoms . As mostAcute stress has been shown to induce a reallocation of resources from three large-scale neural networks: the salience (SN), default mode (DMN), and central executive network (CEN) , 8. ThisAbnormal hyperconnectivity between the core regions of the SN , and hypo-connectivity between the core regions of the DMN , as well as between core regions of these two networks , were observed in PTSD patients in contrast to healthy controls –15. In aSince aberrant functional organizations of DMN, SN, and CEN have been proposed to underlie a wide range of psychopathologies, including stress-related ones such as depression, anxiety, and PTSD , it is cpredict the perceived stress and PTSD symptom levels in police recruits after continuous exposure to police-operation-related trauma in the training period. We further investigated acquired abnormalities in network responses to stress induction after trauma exposure, using neuroimaging data collected at the follow-up assessment .Using a longitudinal design, the current study aimed to elucidate whether the magnitude of acute-stress-induced network reconfiguration could predict stress-related symptom development after trauma exposure. Specifically, we used acute-stress-induced connectivity changes  of the SN, DMN, and CEN at baseline  to Based on the literature, we expected to observe predictive effects of acute -stress-induced delta-FC in SN and DMN on trauma-related symptom development. More specifically, the adaptive responses to acute stress that were indicated by increasing SN and decreasing DMN connectivity at the baseline assessment, as shown in our previous study , were exN = 86) reported that their core trauma, the trauma most central to their symptoms, had occurred already before our baseline assessment (Wave-1). As we cannot disentangle predisposed and acquired factors for these participants due to the lack of data before Wave-1 assessment, data from these participants were excluded. Further exclusion included data from the participant whose baseline PCL-5 scores met PTSD diagnosis cutoff  or who exhibited excessive motion artifacts  [An initial sample of 321 police recruits from Dutch Police Academy participated in the current study in accordance with the principles of the Declaration of Helsinki and with the approval from the Independent Review Board Nijmegen (IRBN), the Netherlands. All participants gave their written informed consent before the study upon their first lab visit (Wave-1). Exclusion criteria included any current psychiatric or neurological disorder, history of, or current endocrine or neurological treatment, current use of psychotropic medication, and current drug or alcohol abuse (full details in protocol article ). A grouSupplemental Materials and Methods) [The baseline assessment  took place in parallel with the early police curriculum of mostly in-class theoretical trainings. In this assessment, participants filled out questionnaires measuring their baseline levels of perceived stress  and stress-related symptoms , prior to the participation in all other experimental tasks. Acute stress induction was conducted in the late afternoon  to ensure stable salivary cortisol levels, which consisted of a SECPT  and MA  task (see details in the Methods) , 30, 31.Methods)  was addiMethods) ).Fig. 2Hfit MRI scanner  with a 32-channel head coil. A multi-band T2*-weighted EPI sequence with acceleration factor 8 (MB8), optimized from the recommended imaging protocols for the Human Connectome Project, was used to acquire a total number of 500 volumes of BOLD–fMRI images . High-resolution structural images (1 × 1 × 1 mm3) were also acquired, using a T1-weighted MP-RAGE sequence .Acquisition of the resting-state fMRI data occurred before and after the stress-induction experiment in both Wave-1 and Wave-2 assessments, where participants were instructed to lie still and watch a small white cross at the screen center. Imaging data were acquired using a 3-T Siemens Magnetom PrismaPerceived stress-level and PTSD-symptom levels were measured at both the baseline (Wave-1) and follow-up (Wave-2), prior to the implementation of the acute-stress-induction experiment (see “Procedure”). Additionally, CAPS interviews were conducted at Wave-2 assessment. While sum scores of CAPs were used to indicate post-traumatic symptom levels, the change scores of PSS  and PCL (delta-PCL) were calculated to indicate the development of post-traumatic stress levels.To index acute endocrine- and subjective-stress responses, salivary samples and self-reported ratings of negative affect were measured throughout the stress-induction experiment in Wave-1 and Wave-2 assessments. Following our previous practice , increasSupplemental Materials and Methods.Preprocessing of rs-fMRI data included motion correction, 5 mm spatial smoothing, ICA-AROMA-based denoising , and higResting-state networks (RSNs) of interest  were identified from the components of a group-level independent-component analysis (ICA) that showed the highest spatial correlation with preselected functional ROIs  from the Stanford FIND atlas . Changespredict stress-related symptom development . In contrast, to assess potential acquired changes in network-connectivity strength consequential to trauma, we calculated changes of these delta-FC coefficients over time (from Wave-1 to Wave-2) and looked at the associations with symptom development.To indicate the magnitude of changes in functional connectivity as a function of acute-stress induction, coefficients of delta-FC were extracted using the predefined local and global networks. Both local and global delta-FC coefficients from Wave-1 assessment were used to p < 0.0167 , and averaged the resulting maps across participants. This essentially created a group-level map of regions showing consistent changes in connectivity with the network of interest after stress induction. Changes in the averaged beta-values across all these regions were taken as a summary measure of stress-related global connectivity changes for that network  [To obtain the local-level delta-FC coefficients, we used the RSN maps derived from group-level ICA with a threshold of Z > 3 to reduce noise while retaining the overall spatial patterns of each network. The changes in the beta-values within the resulting mask were then used to index the network-connectivity changes after stress induction. For the more global-level delta-FC coefficients, we calculated the differential RSNs per network at the individual level  before and after stress induction , thresholded at Methods) . This twAdditionally, as cortisol-stress reactivity was previously found predictive of subsequent PTSD-symptom development in a limited number of studies , 37, we Delta-PSS, delta-PCL, and CAPS sum scores were calculated to indicate each individual’s post-traumatic stress levels. These change scores were used as the outcome measures in our analyses for predictive and acquired effects of symptom development. We further explored the development of specific symptom clusters, using the sum scores of each subcluster in delta-PCL and CAPS measures Table .Spearman rank correlation was used for all correlation analyses to mitigate the influences from extreme values and reduce the chance of false positives. Concerning the results for our a priori hypotheses , FDR corrections were applied to account for the number of analyses involving three outcome measurements . For more exploratory analyses concerning the delta-FC of CEN for which we had no a priori hypotheses, more stringent FDR corrections were considered to account for two levels of network connectivity  and three outcome measurements. Follow-up tests on subcluster symptom scores were carried out only if significant predictive or acquired effects were observed. Concerning these analyses, FDR corrections were conducted to account for the number of analyses involving all four subcluster symptom scores, for the local- and more global-level connectivity, separately. In case of significant results concerning delta-PCL or delta-PSS , semipartial Spearman correlation was further conducted to control for the baseline PCL or PSS level from Wave-1 assessment. Finally, we used a generalized additive model (GAM) to explore whether the predictive effects of hypothesized neural measures remain significant when accounting for influences of stress reactivity at hormonal and behavioral levels from baseline assessment, and the impact of trauma exposure . Similar analyses were conducted for acquired effects to account for changes in cortisol and negative-affect stress responses between two assessments. Unlike multiple linear regression that estimates a single parameter for each predictor, GAM finds unspecified (nonparametric) functions that relate the predicted Y (dependent variable) values to the predictor values, and thus allows nonparametric fit , 39.pcor function from RVAideMemoire package [gam function from mgcv package [Apart from the coefficient extraction that was carried out using FSL , all oth package  and gam  package  specificcortisol(4677.36) = 76.82, p < 0.0001; Faffect(4719.87) = 51.50, p < 0.005). Similar significant effects were observed in Wave-2 (Fcortisol(4644.18) = 123.4, Faffect (4675.06) = 51.03, p’s < 0.0001). In short, our experimental manipulation successfully induced acute stress, indicated by increases in cortisol and negative effect for both waves  and follow-up Wave-2) assessments. Specifically, increases in salivary cortisol and the reported negative affect were observed in Wave-1 following stress induction, as reflected in the main effects of sampling time  with a range between 0 and 17. The most frequently experienced trauma were encountering suicide , severe (traffic) accidents (23.6%), and physical assault =1.48, p = 0.14) or overall PTSD-symptom levels (PCL: t(189)=0.77, p = 0.44). Nevertheless, closer inspection revealed large variance in individual-symptom trajectories  = 2.22, p < 0.05; other symptom clusters p > 0.05). At Wave-2, the average clinical interview (CAPS) score of overall PTSD symptoms was 1.79 , with three participants having developed full-blown PTSD according to DSM-5 criteria  and when baseline level PSS was controlled for . This predictive effect of SN-DMN coupling remained as the only significant predictor in our follow-up analysis using generalized additive model (GAM) when influences of baseline cortisol and subjective affect, as well as trauma exposure amount were considered . Against our hypotheses, we observed no predictive effects of delta-FC in SN on self-reported PTSD symptoms (delta-PCL) or clinician-rated PTSD levels (CAPS scores), nor with respect to hypothesized DMN-connectivity changes after stress induction .In testing our a prior hypotheses concerning the SN and DMN, a decreased coupling between SN and DMN core regions  following stress induction at baseline was found predictive of larger increases in perceived stress level after trauma exposure  and more global levels . After FDR corrections, only the effect of local CEN delta-FC remained significant (pfdr < 0.019). Follow-up tests on the subcluster symptoms revealed that delta-FC within CEN predicted levels of alteration in mood and cognition , as well as hyper-arousal symptoms . These effects also remained significant after multiple-comparison correction  were predicted by increased delta-FC, both at the local .In comparison to these baseline neural responses to acute-stress induction, we did not find cortisol reactivity, nor negative effect, predictive of stress-related symptomology development  = 3.52, p < 0.001), we did not find any associations between this increased cortisol-stress response and symptom level changes . No difference in negative affect levels upon acute stress induction was found between Wave-1 and Wave-2 assessments, nor did we observe any significant associations between negative affect and symptom changes . Furthermore, we did not observe significant associations between connectivity changes of DMN or CEN and symptom changes . However, we did observe an increased coupling between the overall SN and anterior cerebellum  from Wave-1 to Wave-2 that was associated with higher PTSD symptom levels . Yet, this effect just missed significance when correcting for multiple comparisons (pFDR = 0.057). Follow-up tests examining sub-cluster symptoms suggested that this effect might have been driven by intrusion symptom , with the participants showing higher intrusion-symptom levels also exhibiting larger neural coupling in response to acute stress induction , and after accounting for the influence of changes in cortisol and negative-affect stress responses between two assessments . No significant effects were observed for delta-PCL, nor for delta-PSS scores.Cments  predicted higher perceived stress level after trauma exposure. Acute stress has been shown to immediately prompt SN engagement at the potential cost of neural resources that would otherwise have been allocated to other brain circuits , 8. ThisInterestingly, SN reconfiguration upon acute-stress induction seemed to not only signal a risk factor for later symptom development, but also indicate acquired abnormalities that were associated with increasing symptom levels. Our finding of SN–cerebellum coupling in participants with relatively high PTSD symptoms is in line with a growing number of studies that has linked cerebellum to emotional processing and regulation, particularly to negative emotional memories –47, as wFurthermore, our exploratory analyses for CEN found that acute-stress-induced increases in connectivity of this network prior to trauma exposure could predict the elevated levels of overall post-trauma symptoms, and of specific negative mood/cognition and hyperarousal symptoms Figure . These rContrary to our prediction, however, we did not observe any associations between acute stress-induced delta-FC of DMN at baseline and long-term consequences of trauma exposure. However, the finding of decreased connectivity between SN and posterior DMN suggests that the regions in DMN may function distinctively in response to stressful events over time, hence, the mean coefficient indicating overall connectivity patterns of all regions within the network at baseline could not capture individual variability in longitudinal symptom development. Additionally, we did not find evidence in support of cortisol reactivity predicting PTSD-symptom development, which was reported in a few recent longitudinal studies , 55. DisIn an attempt to investigate the predictive factors for PTSD-symptom development and to disentangle predictive from acquired effects of the neural networks, we focused on a relatively healthy and resilient sample, whose baseline levels of depression, anxiety, and PTSD-symptom scores at Wave-1 assessment were significantly lower in comparison with a group of age- and sex-matched control participants . Notably, these observations were in line with the fact that all police students were prescreened for the enrollment based on their physical and psychological performance, and with the literature suggesting higher resilience in police than in civilians . With liIn conclusion, the current study used connectivity changes of large-scale neural networks in response to an acute-stress challenge to predict subsequent stress-related symptoms after trauma exposure in police recruits. Whereas SN–DMN connectivity prospectively predicted the longitudinal changes in perceived stress level, increased SN–cerebellum connectivity was acquired in participants with higher PTSD-symptom levels. These findings suggest that acute-stress-induced SN-connectivity changes may serve as a potential marker of PTSD vulnerability.SUPPLEMENTAL MATERIAL


Macrophage-derived exosomes have been implicated on the modulation of inflammatory processes. Recent studies have shown that macrophage-derived exosomes contribute to the progression of many diseases such as cancer, atherosclerosis, diabetes and heart failure. This review describes the biogenesis of macrophage-derived exosomes and their biological functions in different diseases. In addition, the challenges facing the use of macrophage-derived exosomes as delivery tools for drugs, genes, and proteins in clinical applications are described. The application of macrophage-derived exosomes in the diagnosis and treatment of diseases is also discussed. Rab GTPin vitro after stimulation by different cytokines  macrophages was reported in the early 1990s. This phenotype was characterized by high clearance of mannosylated ligands, enhanced expression of MHC II antigens, and reduced secretion of pro-inflammatory cytokines compared with the classically activated M1 macrophages induced by IFN-γ . This clytokines . Differeytokines . Mills eytokines . This imytokines . The carytokines . Succiniytokines . These sThree types of macrophage-derived exosomes including unpolarised M0 macrophage-derived exosomes (M0-Exos), polarised M1 and M2 macrophage-derived exosomes (M1-Exos and M2-Exos) have been explored . VariatiTumor microenvironment is a local homeostatic environment comprising tumor cells, macrophages, fibroblasts, and extracellular matrix. It plays an important role in development, recurrence, metastasis, and chemotherapy resistance of cancer . MacrophMacrophage-derived exosome-mediated cell-cell communication plays an important role in atherosclerotic processes. Oxidized low-density lipoprotein (ox-LDL) promotes dysregulation of the metabolism of lipoproteins and deposition of lipoproteins in the arterial wall. In addition, ox-LDL is implicated in initiation and development of atherosclerosis (AS). ox-LDL stimulates macrophage-derived exosomes and mediates endothelial cell growth and tube-forming capacity. Notably, blocking exosome secretion rescues endothelial cell growth and tube-forming capacity . Nguyen in vivo and in vitro studies of ATM-secreted exosomes report that miRNAs in the exogenous genes cause modulate insulin signaling  on inflammatory bowel disease (IBD) induced by dextran sodium sulfate (DSS). The findings showed that although all types of M2 macrophage-derived exosomes reduced severity of IBD, M2b macrophage-derived exosomes were more effective compared with M2a and M2c macrophage-derived exosomes. M2b macrophage-derived exosomes carry Chemokine (C-C Motif) Ligand 1 (CCL1) protein to the colon, which interacts with its ligand C-C chemokine receptor 8 (CCR8), and promotes Th2 cells polarization, thus increases levels of Treg cells and reduces production of pro-inflammatory cytokines in the colon  . DiabetiSpasmolytic polypeptide-expressing metaplasia (SPEM) is the initial step of gastric precancerous lesions, which can progress to heterogeneous hyperplasia or even carcinoma with chronic inflammatory stimulation. Macrophages may be involved in this inflammatory process. Xu et al. reported that Deoxycholic acid-stimulated exosomes secreted by macrophages promote cellular communication between macrophages and gastric epithelial cells, thus facilitating development of SPEM, however, the study did not elucidate the mechanism . Macrophin vitro and in vivo without adverse effects  cells to gemcitabine. In the study by Yoav et al., artificial dsDNA (barcode fragments) was transfected into mouse peritoneal macrophages and injected into mice bearing PDAC tumors. The concentration of barcode fragments was 4-fold higher in primary tumors and liver metastases than in normal tissue. This effect was mediated by the transfer of miR-365 in macrophage-derived exosomes. MiR-365 impaired activation of gemcitabine by upregulation of the triphospho-nucleotide pool in cancer cells and the induction of the enzyme cytidine deaminase; the latter inactivates gemcitabine . Elsewheer cells . The deler cells . Recent er cells .In summary, macrophage-derived exosomes have important role in the treatment of diseases such as tumors, atherosclerosis, and diabetes. When used as delivery vehicles, they bind to receptors on target cells thereby delivering loaded drugs such as proteins and nucleic acids. The function of macrophage-derived exosomes is influenced by macrophage polarization, which regulated by the surrounding inflammatory environment. Inflammation is usually an alternating process, thus the role of macrophage-derived exosomes should be viewed in a dynamic light.Compared to artificially targeted nanocarriers, macrophage-derived exosomes are safer and can be easily modified for application in gene therapy. The following issues also need to be resolved in future studies: 1) Currently, there is no uniform protocol to isolate, purify, and preserve exosomes. Moreover, some of the existing isolation and purification methods are not effective. 2) The low yields of exosomes from specific donor cells limits their application as targeted drug carrier systems. 3) There are few pharmacokinetic studies on the use of macrophage-derived exosomes as drug delivery tools, which limits their development as biopharmaceuticals. It is believed that with the development of biotechnology, macrophage-derived exosomes will play a key role in the diagnosis, prevention, and treatment of diseases in the future.


Metastatic prostate cancer is the most common cancer in males and the fifth cause of cancer mortality worldwide. Despite the major progress in this field, leading to the approval of novel anti-androgens, the prognosis is still poor. A significant number of patients acquire an androgen receptor splice variant 7 (AR-V7), which is constitutively activated and lacks the ligand-binding domain (LBD) while maintaining the nuclear localization signal and DNA-binding domain (DBD). This conformational change, even in the absence of the ligand, allows its retention within the nucleus, where it acts as a transcription factor repressing crucial tumor suppressor genes. AR-V7 is an important oncogenic driver and plays a role as an early diagnostic and prognostic marker, as well as a therapeutic target for antagonists such as niclosamide and TAS3681. Anti-AR-V7 drugs have shown promise in recent clinical investigations on this subset of patients. This mini-review focuses on the relevance of AR-V7 in the clinical manifestations of castration-resistant prostate cancer (CRPC) and summarizes redemptive therapeutic strategies. Prostate cancer is the second most common cancer in men and the fifth cause of mortality worldwide, accounting for 33,330 deaths in the United States in 2020 . AndrogeIn order to overcome castration resistance, the United States Food and Drug Administration (FDA) has approved the use of second-generation ADT agents targeting androgen synthesis (abiraterone acetate) and androgen receptor  following favorable Phase III trial results ,9. CabazData from the Phase III ALSYMPCA trial show that radium-dichloride (radium-223), an alpha emitter selectively targeting bone metastases with alpha particles , increasOver the past decades, preclinical and clinical research on tumor samples from CRPC patients has uncovered diverse resistance mechanisms leading to persistent androgen receptor addiction . Indeed,Several androgen receptor splice variants have been identified in PCa cell lines and xenograft tumors at the mRNA level ,26. In 2This mini-review focuses on how the structure of AR-V7 impacts its own function, leading to prostate cancer cell proliferation and resistance to second-generation hormonal therapies. In the era of precision oncology, better knowledge of AR biology, including AR splice variants, will help select mCRPC patients likely to derive little clinical benefit from potent drugs such as abiraterone and enzalutamide and offer them more effective treatment leading to a paradigm shift in mCRPC management.Various clinical markers  are generally used to predict prognosis in advanced prostate cancer ,31. In aThe Gleason grading system, originally proposed by Donald Gleason in 1966, has undergone several important changes, which have been incorporated into the 2016 World Health Organization (WHO) Classification of Tumors of the Urinary System and Male Genital Organs blue book . AlongsiImaging techniques are commonly employed to assess the extent of prostate cancer spread and play a pivotal role in therapeutic decision-making . Over thFor many years, the cornerstone treatment of advanced prostate cancer has been traditional androgen deprivation therapy (ADT), accomplished with surgical castration or chronic administration of gonadotropin-releasing hormone analogs. This regimen is highly effective in patients with biochemical recurrence following radiation or surgical procedure for localized prostate cancer ; howeverNo consensus has been reached on the best treatment sequencing for mCRPC patients, nor do current clinical data support unambiguous guidance concerning the best second-line treatment. Many physicians routinely prescribe taxanes in this setting, although a substantial subgroup of patients may still benefit from oral, less toxic, and better tolerated ARS inhibitors. In addition, an empiric sequencing approach could result in toxicity, higher treatment costs, and delays in patients administered with potentially more effective regimens ,47.In recent years, AR-V7 genomic alterations have been associated with progression to mCRPC, patients with detectable AR-V7 in liquid biopsies usually manifesting more aggressive disease and shorter survival . EmerginBoth preclinical and clinical studies have demonstrated a correlation between abiraterone or enzalutamide resistance and AR-V7 expression, as well as decreased OS in CRPC patients ,51,52. IThe canonical AR gene, located on the X chromosome (Xq11-12), exceeds 90 kb and contains eight exons encoding the AR protein, a ligand-dependent nuclear transcription factor and member of the steroid hormone receptor family . AR is aAR-V7 mRNA retains the first three canonical exons, followed by variant-specific cryptic exon 3 (CE3) within intron 3 ,64,65. ANumerous molecular alterations are reported to be associated with AR Axis-Targeted Therapy (AATT) resistance . AlthougOf paramount importance is the ability of AR-V7 to repress the transcription of tumor suppressor genes . Unlike p = 0.03) and higher OS than AR-V7 positive patients .The expression of AR-V7 has been extensively investigated in both normal and neoplastic prostate tissue. Using a recombinant rabbit monoclonal antibody validated for immunohistochemistry, Sharp et al. demonstrated that AR-V7 is rarely observed in primary prostate tumor specimens (<1%) but is frequent in metastatic biopsy specimens after ADT (>75%), suggesting that AR-V7 increases under the selective pressure of hormonal treatment . AR-V7 pSeveral studies comparing outcomes of mCRPC patients administered second- and third-line therapies have identified AR-V7 in CTCs by means of qRT-PCR and immunohistochemistry. Antonarakis et al. were the first to show that the presence of AR-V7 transcripts in CTCs from mCRPC patients is correlated with second-line AATT resistance . Of 31 ep < 0.001). During AATT, OS and PFS were significantly shorter in AR-V7-positive than in AR-V7-negative patients. In AR-V7-positive patients, multivariate analysis revealed that HR for death was lower in those treated with taxanes compared with those treated with AATT . This study concluded that: (1) AR-V7 positivity increases with the number of treatment lines; (2) AR-V7 positive CTCs can predict a lack in clinical AATT benefit and (3) AR-V7 positive CTCs in men with mCRPC can predict resistance to ARS inhibitors but not to taxanes , differently from conventional therapies targeting the ligand-binding domain. For this reason, EPI-001 was initially considered useful for mCRPC treatment. The successor to EPI-001, EPI-506, a prodrug of ralaniten (EPI-002), was investigated in a Phase I study on mCRPC, but was discontinued after showing only minor PSA decline. Though well tolerated, it proved ineffective probably on account of its high metabolization rate, indeed, 19 metabolites were detected in patient plasma . AR-V7 antagonists, niclosamide and TAS3681, and possibly PROTAC ARV-110, therefore remain the most promising drugs for mCRPC patients progressing under second-generation androgen-depriving therapeutics. Despite recent therapeutic advances, CRPC still remains a lethal disease. AR-V7 is a constitutively active AR isoform that does not require ligand binding and is difficult to treat with commercial androgen-depriving second-generation drugs. AR-V7-positive CTCs have been associated with poor clinical outcome and AATT resistance, thus making AR-V7 a valid biomarker to estimate patient prognosis. Novel drugs such as niclosamide and TAS3681, capable of targeting this variant, have been developed. Future randomized and large clinical trials testing these molecules in patients with AR-V7 positive CRPC are warranted.


There is an urgent need for highly efficacious antiviral therapies in immunosuppressed hosts who develop coronavirus disease (COVID-19), with special concern for those affected by hematological malignancies.+CD20+ B-lymphocyte populations due to previous treatment with anti-CD20 monoclonal antibodies. The patient presented with severe COVID-19 pneumonia due to prolonged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and was treated with two courses of the antiviral plitidepsin on a compassionate use basis. The patient subsequently achieved an undetectable viral load, and his pneumonia resolved.Here, we report the case of a 75-year-old male with chronic lymphocytic leukemia who was deficient in CD19Treatment with plitidepsin was well-tolerated without any further hematological or cardiovascular toxicities. This case further supports plitidepsin as a potential antiviral drug in SARS-CoV-2 patients affected by immune deficiencies and hematological malignancies.The online version contains supplementary material available at 10.1186/s13045-021-01220-0. To the Editor,Patients affected by hematological malignancies constitute a unique population with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. This population has demonstrated an increased risk of persistent infection with SARS-CoV-2, and severe outcomes and mortality due to coronavirus disease 2019 (COVID-19) [In addition to the malignancy itself, short- and long-term effects on lymphocytic populations can be produced by anticancer treatments. Treatment impact on the immune system is of particular note in the case of anti-CD20 monoclonal antibodies , which, despite variability among patients, generally lead to the reduction (or depletion) of B lymphocyte subpopulations that persists for months.Here, we report the clinical and virological course of a prolonged viral SARS-CoV-2 infection in an adult patient previously treated for CLL with obinutuzumab, who was in complete remission of his hematological malignancy. Treatment with plitidepsin was granted for compassionate use as authorized by the Spanish Agency for Medicinal Products (AEMPS) (AUT334100148189/21) [A 75-year-old male with a previous history of CLL arrived to the emergency department (ED) with 1-week of dry cough and tiredness in January 2021. SARS-CoV-2 infection was confirmed by nasopharyngeal (NP) swab samples, and real-time, reverse transcription-polymerase chain reaction  value was 23 at the time plitidepsin was initiated , a second equivalent course of plitidepsin was started. On day 74, the patient received his first negative RT-PCR test and was subsequently negative for SARS-CoV-2 in three consecutive samples. The patient was discharged from the hospital on day 82 and did not experience any signs of SARS-CoV-2 relapse during outpatient follow-up.Prolonged viral replication of SARS-CoV-2 is increasingly being recognized as an emerging clinical problem among immunocompromised individuals. Subacute or chronic COVID-19 pneumonia can cause persistent lung damage and may lead to viral escape phenomena –7. An unPlitidepsin is a known inhibitor of SARS-CoV-2 replication, whose antiviral mechanism of action has been recently described using a drug-resistant mutant model . In brieThe current report has obvious limitations. Though it only describes one patient, the successful outcome observed here should be evaluated in the context of the poor prognosis for immunocompromised patients with hematologic malignancies. Another limitation of this study is that we were unable to amplify and sequence viral RNA from swab samples collected, so the precise SARS-CoV-2 variant that infected this patient remains unknown. We were also unable to capture changes to the patient’s cytokine profile during hospital admission and during plitidepsin infusions.To our knowledge, this is the first evidence of the successful use of a potential SARS-CoV-2 antiviral in a patient with hematological malignancy and depleted B cells B cells due to previous CLL therapy and with prolonged viral replication. The patient was followed up for six months since being discharged from the hospital with no signs of SARS-CoV-2 relapse or reinfection.Additional file 1. Text summarizes methods used, analytical procedures, complications, therapies prescribed during hospitalization and further considerations; Table 1S–4S show analytical parameters that include: absence of immunoresponse against SARS-CoV-2; an increase in lymphocytic populations over time and serum biochemical results during the clinical event. A summary of ISARIC mortality score is also shown. Figure 1S Timeline of qPCR Ct over time, patient shows a reduction in the viral load after the treatment with the second cycle of plitidepsin. Figure 2S Bilateral pneumonia improvement after plitidepsin treatment determined by thorax imagen.


A 78-year-old man presented to the emergency department with a seven-day history of headache, fever, diffuse myalgias, dry cough, and dyspnea. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was diagnosed by SARS-CoV-2 RNA detection in nasopharyngeal samples. Chest computed tomography (CT) demonstrated predominantly peripheral ground-glass opacities in both lungs A, sugges-Pneumorrhachis (PR) is an uncommon condition defined as the presence of air in the spinal canal, most often resulting from spinal cord injuries or instrumentation; but also occasionally reported in association with spontaneous pneumomediastinum, as in our case. Spontaneous pneumomediastinum and PR can occur when intra-alveolar pressure increases, as occurs with forceful coughing, leads to the rupture of the central pulmonary alveolus. Air can move into the perivascular interstitium and dissect through the fascial planes from the posterior mediastinum or retropharyngeal space through the neural foramina into the epidural space


Rapid and precise diagnostic tests can prevent the spread of diseases, including worldwide pandemics. Current commonly used diagnostic methods include nucleic-acid-amplification-based detection methods and immunoassays. These techniques, however, have several drawbacks in diagnosis time, accuracy, and cost. Nucleic acid amplification methods are sensitive but time-consuming, whereas immunoassays are more rapid but relatively insensitive. Recently developed CRISPR-based nucleic acid detection methods have been found to compensate for these limitations. In particular, the unique collateral enzymatic activities of Cas12 and Cas13 have dramatically reduced the diagnosis times and costs, while improving diagnostic accuracy and sensitivity. This review provides a comprehensive description of the distinct enzymatic features of Cas12 and Cas13 and their applications in the development of molecular diagnostic platforms for pathogen detection. Moreover, it describes the current utilization of CRISPR-Cas-based diagnostic techniques to identify SARS-CoV-2 infection, as well as recent progress in the development of CRISPR-Cas-based detection strategies for various infectious diseases. These findings provide insights into designing effective molecular diagnostic platforms for potential pandemics. Recent global pandemics have highlighted the importance of accurate diagnostic tests for infectious and pathogenic diseases. Immunoassays for infectious diseases are widely used to detect pathogen-associated antigens or antibodies against pathogens in patient serum. Although these assays are relatively simple to perform and require a short turnaround time, they are relatively insensitive, making it difficult to evaluate clinical samples containing low concentrations of target molecules ,2,3. MorAmong the new diagnostic approaches that have been developed recently are those involving clustered regularly interspaced short palindromic repeats (CRISPR) ,9,10. CROf the various types of Cas nucleases identified to date, Cas9 has been the most well studied and is commonly used for genome editing. Bacterial CRISPR-Cas9 was re-engineered into a simple two-component system by combining crRNA and tracrRNA into a single-guide RNA (sgRNA) . This reFollowing the initial use of Cas9 in CRISPR-based diagnostic platforms, different Cas enzymes have been used to improve CRISPR-Dx. For example, Cas12 and Cas13 were each shown to have a unique enzymatic activity that cleaves the target nucleotide sequence followed by the cleavage of neighboring non-target sequences ,25. ThisThis review describes the unique features of Cas12 and Cas13 that play pivotal roles in CRISPR-Cas-based diagnostics. Recent CRISPR-Cas-based diagnostic platforms for molecular diagnosis of various infectious diseases, including COVID-19, are comprehensively described. This review will assist researchers in understanding the advantages and limitations of currently developed CRISPR-Cas-based diagnostic methods and may aid in the development of advanced diagnostic strategies for future epidemics.The Cas12 enzymes are type V RNA-guided DNA-targeting enzymes, consisting of several subtypes . Among tIn contrast to Cas9, the more versatile features of Cas12, especially its trans-cleavage activity, make it more suitable for molecular diagnoses ,26. Thes−18 M) sensitivity. DETECTR clearly offered an advanced molecular diagnostic strategy that is rapid, specific, and sensitive, although it could not distinguish between targets differing by a single nucleotide.The first Cas12-based diagnostic method was developed in 2018 by coupling Cas12a with a fluorophore quencher (FQ)-labeled reporter molecule , was developed in 2019 . CompareMycobacterium tuberculosis complex (CRISPR-MTB), which was developed as a diagnostic method to detect M. tuberculosis [M. tuberculosis in various clinical samples, including sputum, bronchoalveolar lavage fluid (BALF), and cerebrospinal fluid (CSF).CRISPR-rculosis , showed Cas12f (formerly known as Cas14), which is half the size of the other Cas12 enzymes, has collateral activity at non-specific nucleotides while recognizing its target, enabling its use in CRISPR-based diagnoses. Based on this collateral activity, the combination of CRISPR-Cas14a and the fluorescent reporter (Cas14a-DETECTR) was found to successfully target the specific single-nucleotide polymorphism (SNP) responsible for eye color, an SNP not detected using Cas12a . AlthougPseudomonas aeruginosa (P. aeruginosa) using lateral flow readout [P. aeruginosa is an opportunistic human pathogen with multidrug resistance [Although fluorescence-based diagnostic methods are quick and extremely sensitive, they are not suitable for on-site detection, due to the requirement for expensive fluorescence detection equipment. This issue was solved by the development of a lateral flow visual readout platform using a FAM-biotin reporter . Lateral readout . P. aerusistance . CIA detAn electrochemistry-based platform, involving an affordable transduction element and a disposable sensor, has been widely used for pathogen detection . IntegraAlthough a paper-based visual readout is easy and deployable, it is unsuitable for quantification at the point of care ,43. ThisCas13 is a type VI RNA-guided RNA targeting enzyme, a member of the enzyme family that includes Cas13a, Cas13b, Cas13c, and Cas13d ,50. Cas1Leptotrichia wadei (LwCas13a). Similar to Cas12a-mediated DETECTR, SHERLOCK uses a fluorescence reporter system with RPA-based target amplification. However, the biggest difference between DETECTR and SHERLOCK is that SHERLOCK requires in vitro transcription using T7 polymerase to convert amplified DNA to RNA. Despite this additional step, SHERLOCK showed attomolar sensitivity in the detection of Zika virus (ZIKV), Dengue virus (DENV), and cancer-related mutations at single-nucleotide resolution. In addition, SHERLOCK reagents are suitable for long-term storage due to lyophilization and are readily applied to paper strips for field application. Moreover, SHERLOCK only costs $0.61 per test, which is much cheaper than the usual cost of PCR or RT-PCR ($10–$15 per test) [The first Cas13a-based diagnostic platform, called the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) platform, was developed in 2017 . RNA-guier test) .Despite its success in targeting RNA for diagnostic purposes, SHERLOCK has some limitations in the aspect of point of care. Indeed, SHERLOCK is not suitable for quantitation, and it relies on expensive fluorescence detection equipment. To overcome these limitations, more advanced versions of SHERLOCK were developed in 2018 ,44. SHERA method called heating unextracted diagnostic samples to obliterate nucleases (HUDSON) was integrated into SHERLOCK to enable direct detection of nucleic acids in body fluid and to protect viral DNA from degradation . Using hFollowing the development of SHERLOCKv2, the multiplexed CRISPR-Cas13-based nucleic acid detection method was further improved by integrating a high-throughput system . This reIn December 2019, a cluster of patients with atypical pneumonia caused by infection with a novel coronavirus was reported in Wuhan, China . This prCRISPR-Cas-based SARS-CoV-2 detection assays typically target the viral envelope (E), nucleoprotein (N), RNA-dependent RNA polymerase (RdRp), or spike (S) protein a. To inhAlthough fluorescence detection with the naked eye is a user-friendly POC diagnostic tool, it is still not amenable to automation to diagnose large numbers of samples. A prerequisite RNA extraction process, which typically takes up to 1 h, also slows the test speed. These limitations may be overcome by a combination of microfluidics and an electric chip . An elec2+) was found to accelerate the activity of Cas12a [2+), was found to increase the sensitivity of SARS-CoV-2 detection [Because divalent metal ions have been shown to enhance pre-crRNA processing , severalf Cas12a . When taetection . This diSince their original development, CRISPR-Cas systems have been mainly used for genome editing. The ongoing COVID-19 pandemic accelerated advances in CRISPR-Cas-based detection systems for infectious diseases. Compared with the standard RT-PCR method, CRISPR-Cas-based molecular diagnostic methods showed greater sensitivity and target specificity. In addition, CRISPR-Cas-based systems are more time-efficient, less expensive, and easier to perform in the field. Notably, coupling of the unique collateral cleavage activities of Cas12 and Cas13 with various reporter systems has been essential in establishing a wide variety of CRISPR-Cas-based molecular diagnostic platforms. Despite these advantages, CRISPR-Cas-mediated diagnoses of pathogens have several limitations. The nuclease activity of Cas proteins is highly dependent on the specific target sequence, such as G-rich PAM, T-rich PAM, or non-G PFS. The introduction of random mutations into target motifs in virus genomes will prevent pathogen detection. These limitations may be overcome by the simultaneous use of several gRNAs targeting multiple pathogenic regions. In addition, CRISPR-Cas systems have several off-target effects. Careful design of gRNAs using off-target prediction software is required to avoid the recognition of non-specific sequences, as the latter may lead to unexpected false-positive results.Because the genomes of most infectious viruses undergo rapid mutation, enhancing their ability to infect a broader range of hosts and to survive, future diagnostic tools must be sufficiently flexible to select target regions and capable of simultaneously targeting multiple regions . In addi


MicroRNAs (miRNA) are functional RNAs that have emerged as pivotal gene expressionregulators in cardiac disease. Although several cardiomyocyte miRNAs have been reported toplay roles in heart failure progression among patients with idiopathic dilated cardiomyopathy(DCM), the role of circulating miRNAs has not yet been well-examined.n=20; controls, n=5). Finally, thecorrelations between specific miRNA expression levels and hemodynamic parameters wereanalyzed.After total RNA extraction from the peripheral blood samples of three controlparticipants and six patients with DCM, miRNA profiling was performed using miRNA arrays.Based on the results of this initial screening, real-time polymerase chain reaction (RT-PCR)was used to perform a quantitative analysis of blood samples from a larger number of matchedpatients (DCM, A primary screening of 2,565 miRNAs resulted in the identification of nine miRNAcandidates. Quantitative RT-PCR results revealed significantly increased miR-489 expressionlevels in the DCM group. Moreover, there was a significant positive correlation betweenmiR-489 expression level and left ventricular ejection fraction.Our results suggest that circulating miR-489 could be a potential noninvasivediagnostic biomarker for DCM. Additionally, the quantification of circulating miR-489 may havevalue as a potential prognostic marker for patients with DCM. Several studies involving congestive HF have evaluated therelationship between miRNA expression profiles and underlying pathological conditions, includingcardiac fibrosis, cardiac hypertrophy, ventricular remodeling, and cardiac failure.11 Our grouphas previously revealed associations between reduced catecholamine sensitivity and severalmiRNAs extracted from right ventricular myocardium biopsy specimens in patients with idiopathicdilated cardiomyopathy (DCM).3 Furthermore,other studies have reported upregulated miR-423-5p and miR-361-5p expression in the myocardiumof patients with DCM.12 However, little isknown about the relationship between circulating miRNAs and DCM. The present study aimed toprofile the expression levels of circulating cardiac-associated miRNAs in patients with DCM anddetermine their utility as biomarkers for DCM.MicroRNAs (miRNAs), which are noncoding RNAs that are 21–25 nucleotides in length,are generally considered to act as intracellular endogenous RNAs that regulatepost-translational gene expression.13 After consideringboth the risks and values for patient prognosis, magnetic resonance imaging and endomyocardialbiopsies were not used in this study. Patients with DCM who had a New York Heart Associationfunctional class of I or II with normal sinus rhythm were enrolled in the present study.DCM was diagnosed on the basis of <50% left ventricular ejection fraction(LVEF), as determined by contrast ventriculography, in the absence of the following: >50%coronary artery stenosis, as determined by coronary angiography; arterial hypertension;valvular heart disease; sustained atrial fibrillation; implantation of any mechanical cardiacsupport devices; complications that influence cardiac function, such as diabetes mellitus;chronic kidney disease and peripheral artery disease; and cardiac muscle disease secondary toany systemic disease.p-value for further detailed analysis using QuantitativeReal-time Polymerase Chain Reaction (qRT-PCR).A two-step process was used to investigate miRNA profiles. In six patients with DCMand three age- and sex-matched healthy controls with normal LVEF and coronary perfusion, wefirst performed a screening study using the microarray analysis system. Serum miRNA sampleswere hybridized to 3D-Gene Human miRNA chips (ver. 21) containing 2,565 miRNAs . In this screening study, we selected candidate miRNAs indescending order of n=20; control group,n=5) using a PRISM-7900HT thermocycler . Caenorhabditis elegans miR-39 (cel-miR-39) was spiked to each sample asa control for the extraction and amplification steps, because it has been reported that thereare few differences between individuals in expression levels in RT-PCR. The relativeexpressions were calculated using the comparative ΔCT method with spiked cel-miR-39 levels.DCM-specific miRNA was detected when the expression level was significantly higher than eithermiR-16 or miR-423-3p, which were used as the internal controls.14The qRT-PCR analysis of the candidate miRNAs was performed in a larger number ofage- and sex-matched patients , left ventricularend-systolic dimension (LVDs), left ventricular end-diastolic dimension (LVDd), left atriumdiameter, fractional shortening (%), interventricular septum thickness, left ventricularposterior wall thickness, E/A , and E/E' . Blood tests included serum creatinine, hemoglobin, and B type natriuretic peptide (BNP)measurements.The study protocol was approved by the Ethics Review Committee of Fujita HealthUniversity, and written informed consent was provided by each patient at the time ofregistration.t-test and Fisher’s exact test, and the results were analyzed using Student’st-test. Correlations were tested using Pearson’s correlation coefficient. Tocompare differences in serum miRNA expressions between the DCM and control groups, Mann–WhitneyU tests were used. All statistical analyses were performed using SPSS version11.0 . Covariates that were found to be significant(p<0.05) during univariate analyses were incorporated into themultivariate analyses. All reported p-values are two-tailed, and statisticalsignificance was set at p<0.01 for the microarray analyses andp<0.05 for the qRT-PCR analyses.Variables are presented as the mean±standard deviation (SD), and qualitativedata are presented as percentages. Patient characteristics were assessed using Student’sThe miRNA microarray analysis was performed to identify DCM-associated differencesin circulating miRNA profiles between six patients with DCM and three controls. The baselineclinical characteristics of the DCM and control groups are shown in 17 whose relationships with cardiomyocyte degradation are well-established. Thelevels of five miRNAs  were below thelimit of detection for qRT-PCR. However, the expression level of miR-489 was significantlyhigher in the DCM group than in the control group when using miR-16 as the internal control(p=0.03), and it also had a higher tendency in the DCM group when miR-423-3pwas used as the internal control (p=0.09). In contrast, the levels of theremaining miRNAs and the previously reported three miRNAs were comparable between the twogroups ogroups .p=0.04, r=0.46; When we analyzed the association between serum miR-489 levels and cardiacfunctional markers, there was a significant positive correlation between LVEF and miR-489expression levels (18 a large number of patients with DCM have had positive responses topharmacological and mechanical therapies for left ventricular reverse remodeling, which confersa more favorable long-term prognosis. However, it remains challenging to identify patients withincreased likelihoods of improvement after therapeutic optimization. Several miRNAs in humanheart tissue have been suggested as valuable diagnostic and prognostic markers for HF.19 However, although numerous studies have beenpublished, the reported impact of miRNAs in HF management remains variable.DCM, which is transmitted by autosomal dominant inheritance and often results frommutations in multiple genes, is one of the most common causes of HF. Although DCM has beenpreviously reported to have a poor prognosis,The present study revealed that circulating miR-489 may be a potential noninvasivediagnostic biomarker for DCM, and that expression levels of circulating miR-489 may correlatewith cardiomyocyte viability in patients with this disease. The expression levels of circulatingmiR-489 were significantly higher in patients with DCM than in controls in our study. Moreover,there was a positive correlation between circulating miR-489 levels and LVEF in patients withDCM. Given that DCM is a degenerative myocardial disease, a reduction in LVEF is mainlydependent on the degree of myocardial fibrogenesis. These results therefore indicate thatmyocardial degeneration upregulates circulating miR-489, whereas the progression of myocardialfibrogenesis decreases circulating miR-489. Thus, circulating miR-489 levels may serve as avaluable marker for detecting myocardial degeneration and identifying disease stage andprognosis in DCM.21 miR-489 dysregulation has also been reported in muscle-related disorders; itis downregulated in cardiac cachexia22 andupregulated in muscular dystrophy.23 Thesefindings support the pivotal role of miR-489 in muscular turnover and degeneration, thedepletion of fetal muscle cells, and fibrosis. Moreover, these observations with regard toskeletal muscle abnormalities are consistent with our present findings in cardiomyocytes.Several investigations of the role of miR-489 have recently been published. Althoughrecent reports have mainly focused on the role of miR-489 in suppressing tumor proliferation andmetastasis,We therefore suggest the utility of measuring miR-489 as a noninvasive diagnosticand prognostic biomarker for patients with DCM. Studies elucidating the importance of themiR-489 signaling axis in regulating cell proliferation and metastasis are ongoing. As such, weexpect to further unravel the mechanisms behind the relationship between myocardial degenerationand miR-489 regulation, which would further benefit patients with DCM through prevalent miRNAscreenings.14 However, this methodology isnot standard, and reliable reference genes for quantifying miRNAs in serum samples have not yetbeen well-established.Our study has several limitations. First, regarding the qRT-PCR analysis,multiplicity was not adequately considered. In this study, we conducted the microRNA arrayusing miR-16 and miR-423-3p as the internal controls, based on a report by Sedighehet al.Second, we revealed a significant positive correlation between miR-489 expressionlevels and LVEF. However, it is possible that the number of cases in our study was too small toconclude this association. Moreover, miR-489 expression levels were only significantly higherin the analysis using miR-16 as the internal control. More extensive studies are thereforeneeded to demonstrate the utility of miR-489 measurement as both a diagnostic and prognosticmarker for DCM in the future.