Surface protein profiling and subtyping of extracellular vesicles in body fluids reveals non-CSF biomarkers of Alzheimer's disease.

Noninvasive and effortless diagnosis of Alzheimer's disease (AD) remains challenging. Here we report the multiplexed profiling of extracellular vesicle (EV) surface proteins at the single EV level in five types of easily accessible body fluids using a proximity barcoding assay (PBA). A total of 183 surface proteins were detected on the EVs from body fluids collected from APP/PS1 transgenic mice and patients with AD. The AD-associated differentially expressed EV proteins could discriminate between the control and AD/AD model samples with high accuracy. Based on machine learning predictive models, urinary EV proteins exhibited the highest diagnostic potential compared to those on other biofluid EVs, both in mice and humans. Single EV analysis further revealed AD-associated EV subpopulations in the tested body fluids, and a urinary EV subpopulation with the signature proteins PLAU, ITGAX and ANXA1 could diagnose patients with AD in blinded datasets with 88% accuracy. Our results suggest that EVs and their subpopulations from noninvasive body fluids, particularly urine, are potential diagnostic biomarkers for AD.

Investigating the Potential Shared Molecular Mechanisms between COVID-19 and Alzheimer's Disease via Transcriptomic Analysis.

SARS-CoV-2 caused the COVID-19 pandemic. COVID-19 may elevate the risk of cognitive impairment and even cause dementia in infected individuals; it may accelerate cognitive decline in elderly patients with dementia, possibly in Alzheimer's disease (AD) patients. However, the mechanisms underlying the interplay between AD and COVID-19 are still unclear. To investigate the underlying mechanisms and associations between AD progression and SARS-CoV-2 infection, we conducted a series of bioinformatics research into SARS-CoV-2-infected cells, COVID-19 patients, AD patients, and SARS-CoV-2-infected AD patients. We identified the common differentially expressed genes (DEGs) in COVID-19 patients, AD patients, and SARS-CoV-2-infected cells, and these DEGs are enriched in certain pathways, such as immune responses and cytokine storms. We constructed the gene interaction network with the signaling transduction module in the center and identified IRF7, STAT1, STAT2, and OAS1 as the hub genes. We also checked the correlations between several key transcription factors and the SARS-CoV-2 and COVID-19 pathway-related genes. We observed that ACE2 expression is positively correlated with IRF7 expression in AD and coronavirus infections, and interestingly, IRF7 is significantly upregulated in response to different RNA virus infections. Further snRNA-seq analysis indicates that NRGN neurons or endothelial cells may be responsible for the increase in ACE2 and IRF7 expression after SARS-CoV-2 infection. The positive correlation between ACE2 and IRF7 expressions is confirmed in the hippocampal formation (HF) of SARS-CoV-2-infected AD patients. Our findings could contribute to the investigation of the molecular mechanisms underlying the interplay between AD and COVID-19 and to the development of effective therapeutic strategies for AD patients with COVID-19.

Transcriptome and DNA methylome analysis of peripheral blood samples reveals incomplete restoration and transposable element activation after 3-months recovery of COVID-19.

Comprehensive analyses showed that SARS-CoV-2 infection caused COVID-19 and induced strong immune responses and sometimes severe illnesses. However, cellular features of recovered patients and long-term health consequences remain largely unexplored. In this study, we collected peripheral blood samples from nine recovered COVID-19 patients (median age of 36 years old) from Hubei province, China, 3 months after discharge as well as 5 age- and gender-matched healthy controls; and carried out RNA-seq and whole-genome bisulfite sequencing to identify hallmarks of recovered COVID-19 patients. Our analyses showed significant changes both in transcript abundance and DNA methylation of genes and transposable elements (TEs) in recovered COVID-19 patients. We identified 425 upregulated genes, 214 downregulated genes, and 18,516 differentially methylated regions (DMRs) in total. Aberrantly expressed genes and DMRs were found to be associated with immune responses and other related biological processes, implicating prolonged overreaction of the immune system in response to SARS-CoV-2 infection. Notably, a significant amount of TEs was aberrantly activated and their activation was positively correlated with COVID-19 severity. Moreover, differentially methylated TEs may regulate adjacent gene expression as regulatory elements. Those identified transcriptomic and epigenomic signatures define and drive the features of recovered COVID-19 patients, helping determine the risks of long COVID-19, and guiding clinical intervention.

Single-cell transcriptomic analysis reveals circadian rhythm disruption associated with poor prognosis and drug-resistance in lung adenocarcinoma.

Circadian rhythm disruption (CRD) represents a major contributor to tumor proliferation. Nonetheless, the role of CRD in the clinical prediction of cancer outcomes has not been well studied. In this study, we developed a computational algorithm, which was implemented in an open-source R package CRDscore, to define the intratumoral status of circadian disruption in three representative single-cell RNA-seq data sets of lung adenocarcinoma. We found that the malignant cells with high CRDscore were characterized by activation of glycolysis and epithelial-mesenchymal transition pathways. Furthermore, cell communication analysis indicated that CRD played a pivotal role in T cell exhaustion, which may be responsible for the poor prognosis of the malignancy. We then validated the findings with public bulk transcriptome datasets involving 22 cancer types. Cox regression analysis revealed that the CRDscore was a valuable prognostic biomarker. A model containing 23 circadian-related genes performed well in predicting immunotherapeutic outcomes in 14 independent cohorts. Importantly, decreased CRDscore was detect by RNA sequencing on H1299 cells with melatonin treatment. Meanwhile, the cells downregulated the expression level of SNAIL and TWIST, which contributed to an invasive phenotype. In conclusion, this study provides a novel computational framework for characterizing CRD status using single-cell transcriptomic data and further confirmed the molecular mechanisms underlying metabolic reprogramming and T cell exhaustion under CRD. The better understanding of the mechanisms may provide new possibilities for incorporating "anticancer approaches based on circadian clocks" into the treatment protocols of precision medicine.

G-CSF and IL-6 may be involved in formation of endometriosis lesions by increasing the expression of angiogenic factors in neutrophils.

Evidence accumulated in recent years has revealed that neutrophils are involved in the initial establishment of endometriosis, which is well-known as a chronic inflammatory disease. So far, why and how neutrophils promote the formation of early endometriosis are still unclear. In this study, using a mouse model of endometriosis, we demonstrated that endometriosis mice (EMs mice) had a significantly increased number of neutrophils in peritoneal fluids and lesions, and increased levels of granulocyte colony-stimulating factor (G-CSF) and IL-6 in serum and peritoneal fluids compared to the control group. In the neutrophils and uterine fragments co-injection experiment, neutrophils regulated by G-CSF and IL-6 had a similar effect to neutrophils from EMs mice, increasing the number, area, weight and microvessel density (MVD) of endometriotic lesions. Blocking the effect of G-CSF and IL-6 in EMs mice resulted in a decrease in the number, area and weight of endometriotic lesions. Following the depletion of neutrophils in vivo using a anti-Ly6G antibody, the MVD in the lesions of mice treated with neutrophils from EMs mice and neutrophils from pG/pI6 mice were significantly reduced. Neutrophils from EMs mice and neutrophils from pG/pI6 mice altered the expression levels of Mmp9, Bv8 and Trail genes compared to the neutrophils from PBS-treated mice. IL-6 together with G-CSF induced a higher expression of phospho-STAT3 and STAT3 in neutrophils. These findings suggest that neutrophils modulated by G-CSF and IL-6 through the STAT3 pathway alter the expression levels of the angiogenesis-related genes Mmp9, Bv8 and Trail, and may promote the establishment of early endometriosis.

Single-cell analysis reveals cell communication triggered by macrophages associated with the reduction and exhaustion of CD8+ T cells in COVID-19.

BACKGROUND: The coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has become an ongoing pandemic. Understanding the respiratory immune microenvironment which is composed of multiple cell types, together with cell communication based on ligand-receptor interactions is important for developing vaccines, probing COVID-19 pathogenesis, and improving pandemic control measures. METHODS: A total of 102 consecutive hospitalized patients with confirmed COVID-19 were enrolled in this study. Clinical information, routine laboratory tests, and flow cytometry analysis data with different conditions were collected and assessed for predictive value in COVID-19 patients. Next, we analyzed public single-cell RNA-sequencing (scRNA-seq) data from bronchoalveolar lavage fluid, which offers the closest available view of immune cell heterogeneity as encountered in patients with varying severity of COVID-19. A weighting algorithm was used to calculate ligand-receptor interactions, revealing the communication potentially associated with outcomes across cell types. Finally, serum cytokines including IL6, IL1ß, IL10, CXCL10, TNFα, GALECTIN-1, and IGF1 derived from patients were measured. RESULTS: Of the 102 COVID-19 patients, 42 cases (41.2%) were categorized as severe. Multivariate logistic regression analysis demonstrated that AST, D-dimer, BUN, and WBC were considered as independent risk factors for the severity of COVID-19. T cell numbers including total T cells, CD4+ and CD8+ T cells in the severe disease group were significantly lower than those in the moderate disease group. The risk model containing the above mentioned inflammatory damage parameters, and the counts of T cells, with AUROCs ranged from 0.78 to 0.87. To investigate the molecular mechanism at the cellular level, we analyzed the published scRNA-seq data and found that macrophages displayed specific functional diversity after SARS-Cov-2 infection, and the metabolic pathway activities in the identified macrophage subtypes were influenced by hypoxia status. Importantly, we described ligand-receptor interactions that are related to COVID-19 serverity involving macrophages and T cell subsets by communication analysis. CONCLUSIONS: Our study showed that macrophages driving ligand-receptor crosstalk contributed to the reduction and exhaustion of CD8+ T cells. The identified crucial cytokine panel, including IL6, IL1ß, IL10, CXCL10, IGF1, and GALECTIN-1, may offer the selective targets to improve the efficacy of COVID-19 therapy. TRIAL REGISTRATION: This is a retrospective observational study without a trial registration number. Video Abstract.

Midkine Prevents Calcification of Aortic Valve Interstitial Cells via Intercellular Crosstalk.

Calcified aortic valve disease (CAVD), the most common valvular heart disease, lacks pharmaceutical treatment options because its pathogenesis remains unclear. This disease with a complex macroenvironment characterizes notable cellular heterogeneity. Therefore, a comprehensive understanding of cellular diversity and cell-to-cell communication are essential for elucidating the mechanisms driving CAVD progression and developing therapeutic targets. In this study, we used single-cell RNA sequencing (scRNA-seq) analysis to describe the comprehensive transcriptomic landscape and cell-to-cell interactions. The transitional valvular endothelial cells (tVECs), an intermediate state during the endothelial-to-mesenchymal transition (EndMT), could be a target to interfere with EndMT progression. Moreover, matrix valvular interstitial cells (mVICs) with high expression of midkine (MDK) interact with activated valvular interstitial cells (aVICs) and compliment-activated valvular interstitial cells (cVICs) through the MK pathway. Then, MDK inhibited calcification of VICs that calcification was validated by Alizarin Red S staining, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blotting assays in vitro. Therefore, we speculated that mVICs secreted MDK to prevent VICs' calcification. Together, these findings delineate the aortic valve cells' heterogeneity, underlining the importance of intercellular cross talk and MDK, which may offer a potential therapeutic strategy as a novel inhibitor of CAVD.

Interleukin-37b inhibits the growth of murine endometriosis-like lesions by regulating proliferation, invasion, angiogenesis and inflammation.

Endometriosis is a gynecological disease with abnormal expression of interleukin (IL)-37 which can suppress inflammation and the immune system. Here we investigated the role of the IL-37b splice variant in endometriosis in vivo and in vitro. In a murine model of endometriosis, in vivo administration of IL-37b significantly inhibited the development of lesions judged by the number (P = 0.0213), size (P = 0.0130) and weight (P = 0.0152) of lesions. IL-37b had no effect on the early stage of lesion formation, however administration in the growth stage of lesions decreased the number (P = 0.0158), size (P = 0.0158) and weight (P = 0.0258) of lesions compared with PBS control, an effect that was not reversed by macrophage depletion. Expressions of inflammatory factors, matrix metalloproteinases and vascular endothelial growth factor-A mRNA/protein were significantly inhibited in ectopic lesions following IL-37b administration, and in uterine segments treated in vitro. In vitro treatment of uterine segments with IL-37b inhibited phosphorylation of Akt and Erk1/2 in uterine segments. Isolated mouse endometrial stromal treated with IL-37b and transfected with pIL-37b plasmid got suppressed cell proliferation, invasion, angiogenesis and the expression of inflammatory factors. In addition, transfection with pIL-37b significantly decreased the phosphorylation of Akt and Erk1/2. IL-37b also inhibited proliferation and the expression of inflammatory and angiogenesis factors in epithelial cell line RL95-2. These findings suggest that IL-37b may inhibit the growth of lesions by regulating proliferation, invasion, angiogenesis and inflammation through Akt and Erk1/2 signaling pathway.

Transcriptome Sequencing Unravels Potential Biomarkers at Different Stages of Cerebral Ischemic Stroke.

Ischemic stroke, which accounts for 87% of all strokes, constitutes the leading cause of morbidity and mortality in China. Although the genetics and epigenetics of stroke have been extensively investigated, few studies have examined their relationships at different stages of stroke. This study assessed the characteristics of transcriptome changes at different stages of ischemic stroke using a mouse model of transient middle cerebral artery occlusion (tMCAO) and bioinformatics analyses. Cerebral cortex tissues from tMCAO mice at days 1, 3, 7, 14, and 28 were removed for RNA-Seq and small RNA-Seq library construction, sequencing, and bioinformatics analysis. We identified differentially expressed (DE) genes and miRNAs and revealed an association of the up-regulated or down-regulated DEmiRNAs with the correspondingly altered DEgene targets at each time point. In addition, different biological pathways were activated at different time points; thus, three groups of miRNAs were verified that may represent potential clinical biomarkers corresponding to days 1, 3, and 7 after ischemic stroke. Notably, this represents the first functional association of some of these miRNAs with stroke, e.g., miR-2137, miR-874-5p, and miR-5099. Together, our findings lay the foundation for the transition from a single-point, single-drug stroke treatment approach to multiple-time-point multi-drug combination therapies.