Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and ...

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, ...

Fluorescence Micropipette Aspiration Assay to Investigate Red Blood Cell Mechanosensing

Micropipette aspiration assays have long been a cornerstone for the investigation of live-cell mechanics, offering insights into cellular responses to ...

Wuyi University

Forcyte Biotechnologies

The University of Sydney

Apo (apolipoprotein) CIII mediates the metabolism of triglyceride (TG)-rich lipoproteins. High levels of plasma apoCIII are positively correlated with the plasma TG levels and increase the cardiovascular risk. However, whether apoCIII is directly involved in the development of atherosclerosis has not been fully elucidated. Approach and Results: To examine the possible roles of apoCIII in lipoprotein metabolism and atherosclerosis, we generated apoCIII KO (knockout) rabbits using ZFN (zinc finger nuclease) technique. On a normal standard diet, apoCIII KO rabbits exhibited significantly lower plasma levels of TG than those of WT (wild type) rabbits while total cholesterol and HDL (high-density lipoprotein) cholesterol levels were unchanged. Analysis of lipoproteins isolated by sequential ultracentrifugation revealed that reduced plasma TG levels in KO rabbits were accompanied by prominent reduction of VLDLs (very-low-density lipoproteins) and IDLs (intermediate-density lipoproteins). In addition, KO rabbits showed faster TG clearance rate after intravenous fat load than WT rabbits. On a cholesterol-rich diet, KO rabbits exhibited constantly and significantly lower levels of plasma total cholesterol and TG than WT rabbits, which was caused by a remarkable reduction of β-VLDLs-the major atherogenic lipoproteins. β-VLDLs of KO rabbits showed higher uptake by cultured hepatocytes and were cleared faster from the circulation than β-VLDLs isolated from WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits.

Apolipoprotein CIII Deficiency Protects Against Atherosclerosis in Knockout Rabbits.

ARHGEF16 is a recently identified Rho-family guanine nucleotide exchange factor (GEF) that has been implicated in the activation of Rho-family GTPases such as Rho G, Rac, and Cdc42. However, its functions in colon cancer cell proliferation and migration are not well understood. In this study, we showed that ARHGEF16 was highly expressed in clinical specimens of colon cancer. In colon cancer cells, ARHGEF16-stimulated proliferation and migration in vitro and in vivo. Furthermore, we identified a nonreceptor tyrosine kinase, FYN, as a novel partner of ARHGEF16. Knocking down FYN expression decreased ARHGEF16 protein level in colon cancer cells. We further demonstrated that ARHGEF16-induced colon cancer cell proliferation and migration were dependent on FYN since knockdown FYN abolished the ARHGEF16-induced proliferation and migration of colon cancer cells. The FYN-ARHGEF16 axis mediates colon cancer progression and is a potential therapeutic target for colon cancer treatment.

FYN is required for ARHGEF16 to promote proliferation and migration in colon cancer cells.

High ceftaroline non-susceptibility in Staphylococcus aureus isolated from acute skin infections in 15 tertiary hospitals in China.

This is a cross-section observational study that investigated the cardiodynamic response to a 6-minute walk test (6MWT) in patients after stroke using impedance cardiography (ICG). Patients diagnosed with stroke were invited to participate in a 6MWT on consecutive days. Heart rate (HR), cardiac output (CO), stroke volume (SV) and cardiac index (CI) were measured by ICG using the PhysioFlow® PF07 EnduroTM at 1-second intervals for 10 minutes prior to, during and for 10 minutes after each 6MWT. Oxygen saturation, perceived exertion score (modified Borg scale) and the distance covered at the end of each 6MWT were recorded. Twenty-nine patients (mean age 55.6±10.9 years) completed the study. The mean duration of stroke after diagnosis was 14.4±19.1 months. There were no differences in the measured data between the first and second 6MWT (mean intraclass correlation coefficient (ICC) range: 0.87-0.95). The 6 minute walk distance (6WMD) covered in the two 6MWTs was 246±126 and 255±130m respectively (p>0.05). Mean measured data for each subject at rest, and at the end of the better performed 6MWT were, respectively: HR 78±11 and 100±18 bpm; CO 5.5±1.2 and 8.9±2.6 l/min, SV 71.3±16 and 89.3±18.6 ml/beat and CI 3.0±0.6 and 4.9±1.3 l/min/m2. After commencement of the 6MWT, the increase in SV took 30 sec before the rise approaching a plateau, whereas HR, CO and CI continued to rise steeply for 90 sec before leveling off to a steady rise. After completion of the 6MWT, all parameters had returned to baseline by a mean of 3.5 min. Sub-group analysis showed that the increase in cardiac output was predominantly contributed by an increase in heart rate in participants diagnosed with stroke for less than 1 year, whereas both stroke volume and heart rate contributed similarly to the increase in cardiac output in participants with diagnosis of stroke for longer than 1 year. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) both returned to baseline within 2 minutes post 6MWT. HR recorded at the end of the 6MWT was 60.8±10.6% of the predicted maximal heart rate and perceived exertion score was 5±2. Correlations between 6MWD and HR, and between 6MWD and SV were weak, with correlation coefficients Spearman's rho (rs) =0.46, and 0.42, respectively (p<0.05). Correlation between 6MWD and CO and CI were higher (rs= 0.66 and 0.63, respectively (p<0.01)). This is the first study to report cardiac responses during a 6MWT in stroke patients. ICG is a reliable, non-invasive, repeatable method of measuring cardiodynamic data in stroke patients.

Noninvasive investigation of the cardiodynamic response to 6MWT in people after stroke using impedance cardiography.

Invasive aspergillosis (IA) due to  is associated with high mortality. In this study, we investigated the clinical and microbiological characteristics of 6 fatal cases of proven or probable IA caused by  in China. Underlying immunosuppression, prior antifungal exposure, and intensive care unit (ICU) hospitalization were important risk factors for invasive  infection. Phenotypic differences were observed for  isolates including slower growth, reduced sporulation, and inability to grow at 48°C, compared with . ITS sequencing was unable to distinguish  from , but sequencing of the , , and  loci enabled reliable distinction of these closely related species. Phylogenetic analysis further confirmed that the ITS region had little variation within the  section Fumigati while the  gene offered the highest intraspecific discrimination. Microsatellite typing results revealed that only loci on chromosomes 1, 3, 5, and 6b generated detectable amplicons for identification. All  isolates showed  resistance to multiple antifungal drugs including amphotericin B (MIC range 4 to 8 μg/ml), itraconazole (MIC 2 μg/ml), voriconazole (MIC of 4-16 μg/ml), and posaconazole (MIC of 0.5-1 μg/ml). However, MECs for the echinocandin drugs ranged from 0.03-0.25, ≤0.008-0.015, and ≤0.015-0.03 μg/ml for caspofungin, micafungin, and anidulafungin, respectively.  is an emerging fungal pathogen in China, causing fatal disease, and clinicians as well as laboratories should be alert to their increasing presence.

Clinical and Microbiological Characterization of Invasive Pulmonary Aspergillosis Caused by  in China.

Mechanical stimuli such as tension, compression, and shear stress play critical roles in the physiological functions of red blood cells (RBCs) and their homeostasis, ATP release, and rheological properties. Intracellular calcium (Ca) mobilization reflects RBC mechanosensing as they transverse the complex vasculature. Emerging studies have demonstrated the presence of mechanosensitive Ca permeable ion channels and their function has been implicated in the regulation of RBC volume and deformability. However, how these mechanoreceptors trigger Ca influx and subsequent cellular responses are still unclear. Here, we introduce a fluorescence-coupled micropipette aspiration assay to examine RBC mechanosensing at the single-cell level. To achieve a wide range of cell aspirations, we implemented and compared two negative pressure adjusting apparatuses: a homemade water manometer (- 2.94 to 0 mmHO) and a pneumatic high-speed pressure clamp (- 25 to 0 mmHg). To visualize Ca influx, RBCs were pre-loaded with an intensiometric probe Cal-520 AM, then imaged under a confocal microscope with concurrent bright-field and fluorescent imaging at acquisition rates of 10 frames per second. Remarkably, we observed the related changes in intracellular Ca levels immediately after aspirating individual RBCs in a pressure-dependent manner. The RBC aspirated by the water manometer only displayed 1.1-fold increase in fluorescence intensity, whereas the RBC aspirated by the pneumatic clamp showed up to threefold increase. These results demonstrated the water manometer as a gentle tool for cell manipulation with minimal pre-activation, while the high-speed pneumatic clamp as a much stronger pressure actuator to examine cell mechanosensing directly. Together, this multimodal platform enables us to precisely control aspiration and membrane tension, and subsequently correlate this with intracellular calcium concentration dynamics in a robust and reproducible manner.

Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing.

The von Willebrand factor (VWF), by interacting with the circulatory system and platelets, harnesses hemodynamic forces to form hemostatic plugs or occlusive thrombi. The autoinhibitory modules (AIMs) flanking the VWF-A1 domain were found to contribute to its biomechanical activation. However, how AIM sequences regulate the VWF-A1 binding behavior is controversial and incompletely understood as their structures are currently unsolvable by crystallography. To address this, we first performed molecular dynamics simulations to predict the N-terminal AIM (N-AIM; residues Q1238-E1260) structure. Excitingly, we found that N-AIM could cooperate with C-AIM to form a joint Rotini-like structure, thereby partially autoinhibiting the VWF-A1-GPIbα interaction. Furthermore, we used biomembrane force probe (BFP) assays to experimentally demonstrate that the VWF-A1 containing long N-AIM sequence (1238-A1) exhibited catch-bond behavior as the force first decelerated (catch) and then accelerated (slip) the dissociation. Conversely, VWF-A1 with short N-AIM (1261-A1) displayed bi-variable behaviors with either catch (1261-A1) or slip bonds (1261-A1). Notably, such bi-variable transition happened at low temperatures or high pH levels, whereas Q1238-E1260 stabilized the 1238-A1 catch bond regardless of the environmental factors. The physiological study was complemented by platelet perfusion assays using microfluidics. Taken together, these studies provide new mechanobiology on how N-AIM serves as a mechano-regulator of VWF activity, which inspires future VWF-A1 dependent antithrombotic approaches.

The N-terminal autoinhibitory module of the A1 domain in von Willebrand factor stabilizes the mechanosensor catch bond.

Cancer cells interacting with the extracellular matrix (ECM) in the tumor microenvironment is pivotal for tumorigenesis, invasion, and metastasis. Cell-ECM adhesion has been intensively studied in cancer biology in the past decades to understand the molecular mechanisms underlying the adhesion events and extracellular mechanosensing, as well as develop therapeutic strategies targeting the cell adhesion molecules. Many methods have been established to measure the cell-ECM adhesion strength and correlate it with the metastatic potential of certain cancer types. However, those approaches are either low throughput, not quantitative, or with poor sensitivity and reproducibility. Herein, we developed a novel acoustic force spectroscopy based method to quantify the cell-ECM adhesion strength during adhesion maturation process using the emerging z-Movi technology. This can be served as a fast, simple, and high-throughput platform for functional assessment of cell adhesion molecules in a highly predictive and reproducible manner.

Yao Wang

School of Biotechnology and Health Sciences

Wuyi University

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Fluorescence Micropipette Aspiration Assay to Investigate Red Blood Cell Mechanosensing

Yao Wang

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Wuyi University

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Fluorescence Micropipette Aspiration Assay to Investigate Red Blood Cell Mechanosensing

School of Biotechnology and Health Sciences