Single-cell atlas of the human brain vasculature across development, adulthood and disease.

A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains incompletely understood1. Here we performed single-cell RNA sequencing analysis of 606,380 freshly isolated endothelial cells, perivascular cells and other tissue-derived cells from 117 samples, from 68 human fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We identify extensive molecular heterogeneity of the vasculature of healthy fetal and adult human brains and across five vascular-dependent central nervous system (CNS) pathologies, including brain tumours and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated genes and pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict substantial endothelial-to-perivascular cell ligand-receptor cross-talk, including immune-related and angiogenic pathways, thereby revealing a central role for the endothelium within brain neurovascular unit signalling networks. Our single-cell brain atlas provides insights into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.

Directional Endothelial Communication by Polarized Extracellular Vesicle Release.

BACKGROUND: Extracellular vesicles (EVs) contain bioactive cargo including miRNAs and proteins that are released by cells during cell-cell communication. Endothelial cells (ECs) form the innermost lining of all blood vessels, interfacing with cells in the circulation and vascular wall. It is unknown whether ECs release EVs capable of governing recipient cells within these 2 separate compartments. Given their boundary location, we propose ECs use bidirectional release of distinct EV cargo in quiescent (healthy) and activated (atheroprone) states to communicate with cells within the circulation and blood vessel wall. METHODS: EVs were isolated from primary human aortic ECs (plate and transwell grown; ±IL [interleukin]-1ß activation), quantified, visualized, and analyzed by miRNA transcriptomics and proteomics. Apical and basolateral EC-EV release was determined by miRNA transfer, total internal reflection fluorescence and electron microscopy. Vascular reprogramming (RNA sequencing) and functional assays were performed on primary human monocytes or smooth muscle cells±EC-EVs. RESULTS: Activated ECs increased EV release, with miRNA and protein cargo related to atherosclerosis. EV-treated monocytes and smooth muscle cells revealed activated EC-EV altered pathways that were proinflammatory and atherogenic. ECs released more EVs apically, which increased with activation. Apical and basolateral EV cargo contained distinct transcriptomes and proteomes that were altered by EC activation. Notably, activated basolateral EC-EVs displayed greater changes in the EV secretome, with pathways specific to atherosclerosis. In silico analysis determined compartment-specific cargo released by the apical and basolateral surfaces of ECs can reprogram monocytes and smooth muscle cells, respectively, with functional assays and in vivo imaging supporting this concept. CONCLUSIONS: Demonstrating that ECs are capable of polarized EV cargo loading and directional EV secretion reveals a novel paradigm for endothelial communication, which may ultimately enhance the design of endothelial-based therapeutics for cardiovascular diseases such as atherosclerosis where ECs are persistently activated.

Circulating small extracellular vesicles mediate vascular hyperpermeability in diabetes.

AIMS/HYPOTHESIS: A hallmark chronic complication of type 2 diabetes mellitus is vascular hyperpermeability, which encompasses dysfunction of the cerebrovascular endothelium and the subsequent development of associated cognitive impairment. The present study tested the hypothesis that during type 2 diabetes circulating small extracellular vesicles (sEVs) exhibit phenotypic changes that facilitate pathogenic disruption of the vascular barrier. METHODS: sEVs isolated from the plasma of a mouse model of type 2 diabetes and from diabetic human individuals were characterised for their ability to disrupt the endothelial cell (EC) barrier. The contents of sEVs and their effect on recipient ECs were assessed by proteomics and identified pathways were functionally interrogated with small molecule inhibitors. RESULTS: Using intravital imaging, we found that diabetic mice (Leprdb/db) displayed hyperpermeability of the cerebrovasculature. Enhanced vascular leakiness was recapitulated following i.v. injection of sEVs from diabetic mice into non-diabetic recipient mice. Characterisation of circulating sEV populations from the plasma of diabetic mice and humans demonstrated increased quantity and size of sEVs compared with those isolated from non-diabetic counterparts. Functional experiments revealed that sEVs from diabetic mice or humans induced the rapid and sustained disruption of the EC barrier through enhanced paracellular and transcellular leak but did not induce inflammation. Subsequent sEV proteome and recipient EC phospho-proteome analysis suggested that extracellular vesicles (sEVs) from diabetic mice and humans modulate the MAPK/MAPK kinase (MEK) and Rho-associated protein kinase (ROCK) pathways, cell-cell junctions and actin dynamics. This was confirmed experimentally. Treatment of sEVs with proteinase K or pre-treatment of recipient cells with MEK or ROCK inhibitors reduced the hyperpermeability-inducing effects of circulating sEVs in the diabetic state. CONCLUSIONS/INTERPRETATION: Diabetes is associated with marked increases in the concentration and size of circulating sEVs. The modulation of sEV-associated proteins under diabetic conditions can induce vascular leak through activation of the MEK/ROCK pathway. These data identify a new paradigm by which diabetes can induce hyperpermeability and dysfunction of the cerebrovasculature and may implicate sEVs in the pathogenesis of cognitive decline during type 2 diabetes.

Brain network integration underpins differential susceptibility of adolescent anxiety.

BACKGROUND: Parenting is a common and potent environmental factor influencing adolescent anxiety. Yet, the underlying neurobiological susceptibility signatures remain elusive. Here, we used a longitudinal twin neuroimaging study to investigate the brain network integration and its heritable relation to underpin the neural differential susceptibility of adolescent anxiety to parenting environments. METHODS: 216 twins from the Beijing Twin Study completed the parenting and anxiety assessments and fMRI scanning. We first identified the brain network integration involved in the influences of parenting at age 12 on anxiety symptoms at age 15. We then estimated to what extent heritable sensitive factors are responsible for the susceptibility of brain network integration. RESULTS: Consistent with the differential susceptibility theory, the results showed that hypo-connectivity within the central executive network amplified the impact of maternal hostility on anxiety symptoms. A high anti-correlation between the anterior salience and default mode networks played a similar modulatory role in the susceptibility of adolescent anxiety to paternal hostility. Genetic influences (21.18%) were observed for the connectivity pattern in the central executive network. CONCLUSIONS: Brain network integration served as a promising neurobiological signature of the differential susceptibility to adolescent anxiety. Our findings deepen the understanding of the neural sensitivity in the developing brain and can inform early identification and personalized interventions for adolescents at risk of anxiety disorders.

Study of the Mechanism of Hydrolysis of Hemicellulose from Lignocellulose during Alkali Thermal Pretreatment by Density Functional Theory and Experiment.

The covalent bond fracture of hemicellulose leads to hemicellulose hydrolysis during lignocellulosic alkali thermal pretreatment, which has not previously been reported. Density functional theory was used to study the mechanism of hydrolysis of the hemicellulose model compounds under alkali conditions. There are four reaction paths for xylose formation, among which the reaction path with the lowest energy barrier is that in which the nucleophile captures H30 to generate water. The deprotonated hydroxyl group attacks the carbon on the glycoside bond, resulting in the cleavage of the glycoside bond and the formation of a new carbon-oxygen covalent bond, with an energy barrier of 154.2 kJ/mol. The nucleophile further attacks the glycosidic bond to form a new xylose residue with an energy barrier of 111.9 kJ/mol. When the glycosidic bond breaks, the orbital interaction with the largest proportion causes the transfer of ∼0.511 electron from the glycosidic bond oxygen to the deprotonated hydroxy oxygen. In situ Fourier transform infrared spectroscopy is used for the identification of functional groups during the alkali thermal pretreatment. As the temperature increases, the feasibility of the reaction increases. This study lays a theoretical foundation for the development of the alkali thermal pretreatment of lignocellulose.

The risk-taking behavioural intentions of pilots in adverse weather conditions: an application of the theory of planned behaviour.

This paper examined pilots' risk-taking behavioural intentions based on the theory of planned behaviour, as well as the impact of experience on behavioural intentions in adverse weather conditions. Two hundred and seventy-three airline pilots and flying cadets were divided into two groups and asked to complete a questionnaire based on two decision-making scenarios. This questionnaire measured pilots' intentions to take risks, along with the attitude towards the behaviour, subjective norms, perceived behavioural control (PBC), risk perception, and self-identity. The results showed that attitude, subjective norm, PBC, and risk perception explained 52% of the variance in behavioural intentions. Additionally, pilots' risk-taking decisions can be influenced by experience. Inexperienced pilots had a relatively stronger intention to take risks and a more favourable attitude towards risky behaviour. Moreover, pilots were more likely to rely on their own direct experience in the decision-making process. Practitioner summary: This study examined the pilots' risk-taking intentions under adverse weather conditions using a questionnaire based on the TPB theory. Results demonstrated that the TPB model can be applied to the risk-taking scenario and that experience can influence pilots' decisions. These findings have implications for improving flight safety and lowering accident rates.

MiR-30 promotes fatty acid beta-oxidation and endothelial cell dysfunction and is a circulating biomarker of coronary microvascular dysfunction in pre-clinical models of diabetes.

BACKGROUND: Type 2 diabetes (T2D) is associated with coronary microvascular dysfunction, which is thought to contribute to compromised diastolic function, ultimately culminating in heart failure with preserved ejection fraction (HFpEF). The molecular mechanisms remain incompletely understood, and no early diagnostics are available. We sought to gain insight into biomarkers and potential mechanisms of microvascular dysfunction in obese mouse (db/db) and lean rat (Goto-Kakizaki) pre-clinical models of T2D-associated diastolic dysfunction. METHODS: The microRNA (miRNA) content of circulating extracellular vesicles (EVs) was assessed in T2D models to identify biomarkers of coronary microvascular dysfunction/rarefaction. The potential source of circulating EV-encapsulated miRNAs was determined, and the mechanisms of induction and the function of candidate miRNAs were assessed in endothelial cells (ECs). RESULTS: We found an increase in miR-30d-5p and miR-30e-5p in circulating EVs that coincided with indices of coronary microvascular EC dysfunction (i.e., markers of oxidative stress, DNA damage/senescence) and rarefaction, and preceded echocardiographic evidence of diastolic dysfunction. These miRNAs may serve as biomarkers of coronary microvascular dysfunction as they are upregulated in ECs of the left ventricle of the heart, but not other organs, in db/db mice. Furthermore, the miR-30 family is secreted in EVs from senescent ECs in culture, and ECs with senescent-like characteristics are present in the db/db heart. Assessment of miR-30 target pathways revealed a network of genes involved in fatty acid biosynthesis and metabolism. Over-expression of miR-30e in cultured ECs increased fatty acid ß-oxidation and the production of reactive oxygen species and lipid peroxidation, while inhibiting the miR-30 family decreased fatty acid ß-oxidation. Additionally, miR-30e over-expression synergized with fatty acid exposure to down-regulate the expression of eNOS, a key regulator of microvascular and cardiomyocyte function. Finally, knock-down of the miR-30 family in db/db mice decreased markers of oxidative stress and DNA damage/senescence in the microvascular endothelium. CONCLUSIONS: MiR-30d/e represent early biomarkers and potential therapeutic targets that are indicative of the development of diastolic dysfunction and may reflect altered EC fatty acid metabolism and microvascular dysfunction in the diabetic heart.

Identification of HnRNPC as a novel Tau exon 10 splicing factor using RNA antisense purification mass spectrometry.

Alternative splicing in Tau exon 10 generates 3 R- and 4 R-Tau proteoforms, which have equal abundance in healthy adult human brain. Aberrant alternative splicing in Tau exon 10 leads to distortion of the balanced 3 R- and 4 R-Tau expression levels, which is a causal factor to trigger toxic Tau aggregation, neuron dysfunction and patient death in a group of neurodegenerative diseases known as tauopathies. Hence, identification of regulators upstream of the Tau exon 10 splicing events are crucial to understanding pathogenic mechanisms driving tauopathies. In this study, we used RNA Antisense Purification with Mass Spectrometry (RAP-MS) analysis to identify RNA-binding proteins (RBPs) that interact with the Tau pre-mRNA near exon 10. Among the newly identified RBP candidates, we show that knockdown of hnRNPC induces Tau exon 10 skipping whereas overexpression of hnRNPC promotes Tau exon 10 inclusion. In addition, we show that hnRNPC interacts with the poly-uridine (U-tract) sequences in introns 9 and 10 of Tau pre-mRNA. Mutation of these U-tract motifs abolished binding of hnRNPC with Tau pre-mRNA fragment and blocked its impact on Tau exon 10 inclusion. These findings indicate that hnRNPC binds and utilizes these U-tract motifs located in introns 9 and 10 of Tau pre-mRNA to promote Tau exon 10 inclusion. Intriguingly, high hnRNPC expression level is associated with progressive supranuclear palsy (PSP), a sporadic tauopathy with pathological accumulation of Tau species that contain exon 10, which suggests a putative therapeutic role of hnRNPC for PSP treatment. [Figure: see text].

NHS-Ester Tandem Labeling in One Pot Enables 48-Plex Quantitative Proteomics.

Stable-isotope labeling strategies are extensively used for multiplex quantitative proteomics. Hybrid-isotope labeling strategies that combine the use of isotopic mass difference labeling and isobaric tags can greatly increase sample multiplexity. In this work, we present a novel hybrid-isotope labeling approach that we termed NHS-ester tandem labeling in one pot (NETLOP). We first optimized 16-plex isobaric TMTpro labeling of lysine residues followed by 2-plex or 3-plex isotopic mTRAQ labeling of peptide N-termini, both of which with commercially available NHS-ester reactive reagents. We then demonstrated the utility of the NETLOP approach by labeling HeLa cell samples and performing proof-of-principle quantitative 32-plex and 48-plex proteomic analyses, each in a single LC-MS/MS experiment. Compared to current hybrid-isotope labeling methods, our NETLOP approach requires no sample cleanup between different labeling steps to minimize sample loss, induces no retention time shifts that compromise quantification accuracy, can be adapted to other NHS-ester isotopic labeling reagents to further increase multiplexity, and is compatible with samples from any origin in a wide array of biological and clinical proteomics applications.

Overcoming Barriers: The Endothelium As a Linchpin of Coronavirus Disease 2019 Pathogenesis?

OBJECTIVE: Coronavirus disease 2019 (COVID-19) is a global pandemic involving >5 500 000 cases worldwide as of May 26, 2020. The culprit is the severe acute respiratory syndrome coronavirus-2, which invades cells by binding to ACE2 (angiotensin-converting enzyme 2). While the majority of patients mount an appropriate antiviral response and recover at home, others progress to respiratory distress requiring hospital admission for supplemental oxygen. In severe cases, deterioration to acute respiratory distress syndrome necessitating mechanical ventilation, development of severe thrombotic events, or cardiac injury and dysfunction occurs. In this review, we highlight what is known to date about COVID-19 and cardiovascular risk, focusing in on the putative role of the endothelium in disease susceptibility and pathogenesis. Approach and Results: Cytokine-driven vascular leak in the lung alveolar-endothelial interface facilitates acute lung injury in the setting of viral infection. Given that the virus affects multiple organs, including the heart, it likely gains access into systemic circulation by infecting or passing from the respiratory epithelium to the endothelium for viral dissemination. Indeed, cardiovascular complications of COVID-19 are highly prevalent and include acute cardiac injury, myocarditis, and a hypercoagulable state, all of which may be influenced by altered endothelial function. Notably, the disease course is worse in individuals with preexisting comorbidities that involve endothelial dysfunction and may be linked to elevated ACE2 expression, such as diabetes mellitus, hypertension, and cardiovascular disease. CONCLUSIONS: Rapidly emerging data on COVID-19, together with results from studies on severe acute respiratory syndrome coronavirus-1, are providing insight into how endothelial dysfunction may contribute to the pandemic that is paralyzing the globe. This may, in turn, inform the design of biomarkers predictive of disease course, as well as therapeutics targeting pathogenic endothelial responses.

Prevalence of Azole-Resistant Aspergillus fumigatus is Highly Associated with Azole Fungicide Residues in the Fields.

Triazole resistance in Aspergillus fumigatus is a growing public health concern. In addition to its emergence in the therapy of invasive aspergillosis by triazole medicines, it has been frequently detected in agricultural fields all over the world. Here, we explore the potential link between residues of azole fungicides with similar chemical structure to triazole medicines in soil and the emergence of resistant A. fumigatus (RAF) through 855 500 km2 monitoring survey in Eastern China covering 6 provinces. In total, 67.3%, 15.2%, 12.3%, 2.9%, 1.5%, 0.4%, and 0.3% of the soil samples contained these five fungicides (tebuconazole, difenoconazole, propiconazole, hexaconazole, and prochloraz) of 0-100, 100-200, 200-400, 400-600, 600-800, 800-1000, and >1000 ng/g, respectively. The fractions of samples containing RAF isolates were 2.4%, 5.2%, 6.4%, 7.7%, 7.4%, 14.3%, and 20.0% of the samples with total azole fungicide residues of 0-100, 100-200, 200-400, 400-600, 600-800, 800-1000, and >1000 ng/g, respectively. We find that the prevalence of RAFs is positively (P < 0.0001) correlated with residual levels of azole fungicides in soils. Our results suggest that the use of azole fungicides in agriculture should be minimized and the intervals between treatments expanded to reduce the selective pressure toward the development of resistance in A. fumigatus in agricultural fields.

Ten days of complete fasting affected subjective sensations but not cognitive abilities in healthy adults.

PURPOSE: People may be unable to obtain anything edible for days under some circumstances, but they must maintain their calmness and cognition to navigate solutions. Our aim was to study changes in subjective sensations and cognition in healthy adults during a 10-day complete fasting experiment. METHODS: Thirteen healthy male volunteers voluntarily participated in the 22-day experiment comprising 4 phases: 3 days of baseline consumption, 10 days of complete fasting (only water ad libitum), 4 days of calorie restriction, and a 5-day recovery period. The volunteers' subjective sensations, cognitive performance, and serum energy substances were measured at 6 time points. RESULTS: Across the 6 time points, the trajectories of subjective sensations in response to fasting were "U"- or " ∩ "-shaped curves instead of progressive discomfort or mood enhancement. A significant fasting time effect was found on depression-dejection (baseline: 16.85 ± 2.88; highest score on the third day of completing fasting: 17.69 ± 3.97, P = 0.04) and self-rated anxiety (baseline: 26.23 ± 4.75; highest score on the sixth day of completing fasting: 30.85 ± 5.58, P = 0.01), and the change curves were consistent with the inflection point of the energy substrates shifting from serum glucose to ketone. In addition, basic cognitive functions appeared to be unaffected during the 10-day fast. CONCLUSIONS: Our study showed strong influences on the sensations from the third to sixth days of the prolonged fasting period but no significant effects on basic cognitive abilities associated with the energy substance switch. These findings could contribute to the development and understanding of survival strategies in food-shortage emergencies or of intermittent fasting programmes.

Five-Year Survey (2014 to 2018) of Azole Resistance in Environmental Aspergillus fumigatus Isolates from China.

A total of 191 soil samples from Hangzhou, China, were submitted to detect non-wild-type (non-WT) Aspergillus fumigatus and its associated mechanisms. There were 2 (4.7%), 13 (12.4%), and 31 (23.1%) isolates identified as non-WT in 2014, 2016, and 2018, respectively. The resistant mutations of TR34/L98H, TR46/Y121F/T289A, and TR34/L98H/S297T/F495I were found in 3, 5, and 5 non-WT isolates. The G448S mutation, previously only found in clinical settings, was detected in A. fumigatus from soil samples.

NanoTPOT: Enhanced Sample Preparation for Quantitative Nanoproteomic Analysis.

With the ever-growing need for protein-level understanding in pathological research, proteomics researchers thrive to examine detailed proteome dynamics using crucial, yet often limited, primary and clinical samples. Aside from mass spectrometer instrumentation advancement, a single-tube-based high-throughput sample processing workflow is imperative to ensure sensitive, quantitative, and reproducible protein analysis for these increasingly sophisticated studies. Leveraging the benefits of an acid-cleavable detergent, RapiGest SF Surfactant (Waters Corporation), we developed and optimized a nanoproteomic workflow that we termed Nanogram TMT Processing in One Tube (NanoTPOT). Through the assessment of proteolytic digestion, tandem mass tag (TMT) labeling, online and offline fractionation strategies, our optimized workflow effectively eliminated the need for sample desalting and enabled compatible sample processing for mass spectrometry analysis. We further applied the NanoTPOT workflow to examine cellular response to stress caused by dithiothreitol in HeLa cells, where we identified and quantified 6935 and 5474 proteins in TMT 10-plex experiments with one microgram of lysate protein and 2000 sorted HeLa cells (roughly half microgram lysate protein) in each channel, respectively. Our workflow has been proven to be an effective alternative for current nanoproteomic sample processing to minimize sample loss in biological and clinical applications.

Step-Wise Assessment and Optimization of Sample Handling Recovery Yield for Nanoproteomic Analysis of 1000 Mammalian Cells.

Protein and peptide adhesion is a major factor contributing to sample loss during proteomic sample preparation workflows. Sample loss often has detrimental effects on the quality of proteomic analysis by compromising protein identification and data reproducibility. When starting with a low sample amount, only the most abundant proteins can be identified, which often offers little insights for biological research. Although the general idea about severe sample loss from low amount of starting material is widely presumed in the proteomics field, quantitative assessment on the impact of sample loss has been poorly investigated. In the present study, we have quantitatively assessed sample loss during each step of a conventional in-solution sample preparation workflow using bicinchoninic acid (BCA) and targeted LC/MS/MS protein and peptide assays. According to our assessment, for starting materials of ∼1000 mammalian cells, surface adhesion, along with desalting and speed-vacuum drying steps, all contribute heavily to sample loss, in particular for low-abundance proteins. With this knowledge, we have adapted slippery liquid infused porous surface (SLIPS) treatment, commercial LoBind tubes, and in-line desalting during sample processing. With these improvements, we were able to use a conventional in-solution sample handling method to identify on average 829 proteins with 1000 U2OS osteosarcoma cells (∼100 ng) with 75-min LC/MS/MS runs, an 11-fold increase in protein identification. Our optimized in-solution workflow is straightforward and also much less equipment- and technique-demanding than other advanced sample preparation protocols in the field.

Multi-omic Landscape of Extracellular Vesicles in Human Carotid Atherosclerotic Plaque Reveals Endothelial Communication Networks.

Background: Carotid atherosclerosis is a multifaceted disease orchestrated by a myriad of cell-cell communication that drives progression along a clinical continuum (asymptomatic to symptomatic). Extracellular vesicles (EVs) are lipid bilayer membrane-enclosed cell-derived nanoparticles that represent a new paradigm in cellular communication. Little is known about their biological cargo, cellular origin/destination, and functional roles in human atherosclerotic plaque. Methods: EVs were enriched via size exclusion chromatography from human carotid endarterectomy samples dissected into plaque and marginal zones (n= 29 patients, paired plaque and marginal zone; symptomatic n=16, asymptomatic n=13), with further density gradient ultracentrifugation for proteomic analysis. EV cargoes were assessed via whole transcriptome miRNA sequencing and mass spectrometry-based proteomics. EV multi-omics were integrated with publicly available bulk and single cell RNA-sequencing (scRNA-seq) datasets to predict EV cellular origin and ligand-receptor interactions and multi-modal biological network integration of EV-cargo was completed. EV functional impact was assessed with endothelial angiogenesis assays. Results: Human carotid plaques contained greater quantities of EVs than adjacent marginal zones. EV-miRNA and protein content was different in diseased plaque versus adjacent marginal zones, with differential functions in key atherogenic pathways. EV cellular origin analysis suggested that tissue EV signatures originated from endothelial cells (EC), smooth muscle cells (SMC), and immune cells. Furthermore, EV signatures from SMCs and immune cells were most enriched in the marginal and plaque zones, respectively. Integrated tissue vesiculomics and scRNA-seq indicated complex EV-vascular cell communication strategies that changed with disease progression and plaque vulnerability (i.e., symptomatic disease). Plaques from symptomatic patients, but not asymptomatic patients, were characterized by increased involvement of endothelial pathways and more complex ligand-receptor interactions, relative to their marginal zones. Plaque-EVs were predicted to mediate communication with ECs. Pathway enrichment analysis delineated a strong endothelial signature with potential roles in angiogenesis and neovascularization - well-known indices of plaque instability. This was corroborated functionally, wherein human carotid symptomatic plaque EVs induced sprouting angiogenesis in comparison to their matched marginal zones. Conclusion: Our findings indicate that EVs may drive dynamic changes in plaques through EV-vascular cell communication and effector functions that typify vulnerability to rupture, precipitating symptomatic disease. The discovery of endothelial-directed processes mediated by EVs creates new avenues for novel therapeutics in atherosclerosis.

Field evidences of fluorescent dissolved organic matter (FDOM) as potential fingerprints for agricultural and urban sources in river environment.

Field evidences of the fluorescence differences between agricultural and urban river reaches are still lack. In this study, the middle reaches of Danhe River (DH) and Mihe River (MH) in Shouguang, China, were designed as agricultural and urban river reaches, respectively, to compare the the fluorescence differences in disparate river reaches using excitation-emission matrix coupled with parallel factor analysis (EEM-PARAFAC). Three fluorescence components were identified. C1 (Ex/Em=230,255,295 nm/420 nm) was categorized as humic-like fluorophores, C2 (Ex/Em=230,275 nm/330 nm) was recognized as tryptophan-like substances, and C3 (Ex/Em=215 nm/290 nm) was noted as tyrosine-like and phenylalanine-like compounds. The results showed that the FDOM posed significant differences between agricultural and urban river reaches (P < 0.001). The monitoring sites in DH were rich in C2 (1.90 ± 0.62 Raman Unit (RU), mean ± standard deviation), and the monitoring sites in MH were rich in C3 (1.32 ± 0.51 RU). Redundancy analysis revealed that C2 could be regarded as a fluorescence indicator of agricultural sewage in river environment, while C3 was recognized as a fluorescence indicator of domestic sewage in river environment. In conclusion, this study provided field evidences of FDOM as potential fingerprints for agricultural and urban sources in river environment.

Synergistic antitumor activity of regorafenib and rosuvastatin in colorectal cancer.

Introduction: Colorectal cancer is one of the most prevalent life-threatening malignant tumors with high incidence and mortality. However, the efficacy of current therapeutic regimens is very limited. Regorafenib has been approved for second- or third-line treatment of patients who are refractory to standard chemotherapy diagnosed with metastatic colorectal cancer, but its clinical efficacy needs to be further improved. Accumulating evidence demonstrates that statins also possess potent anticancer activities. However, whether regorafenib and statins pose synergistic anticancer effects in colorectal cancer is still unclear. Methods: Sulforhodamine B (SRB) assays were applied to evaluate the anti-proliferative activity of regorafenib or/and rosuvastatin in vitro, and immunoblotting analysis were applied to detect the effects of regorafenib/rosuvastatin combined treatment on mitogen-activated protein kinase (MAPK) signaling and apoptosis-related proteins. MC38 tumors were applied to investigate the synergistic anticancer effects of regorafenib in combination with rosuvastatin in vivo. Results: We found that regorafenib in combination with rosuvastatin exerted significant synergistic inhibition against colorectal cancer growth in vitro and in vivo. Mechanistically, regorafenib and rosuvastatin combination synergistically suppressed MAPK signaling, a crucial signaling pathway promoting cell survival, as indicated by the reduction of phosphorylated MEK/ERK. In addition, regorafenib in combination with rosuvastatin synergistically induced the apoptosis of colorectal cancer in vitro and in vivo. Discussion: Our study demonstrated the synergistic anti-proliferative and pro-apoptotic effects of regorafenib/rosuvastatin combined treatment in colorectal cancer in vitro/vivo and might potentially be evaluated as a novel combination regimen for clinical treatment of colorectal cancer.