ATRA treatment slowed P-selectin-mediated rolling of flowing HL60 cells in a mechano-chemical-dependent manner.

All-trans retinoic acid (ATRA)-induced differentiation of acute promyelocytic leukemia (APL) toward granulocytes may trigger APL differentiation syndrome (DS), but there is less knowledge about the mechano-chemical regulation mechanism of APL DS under the mechano-microenvironment. We found that ATRA-induced changes in proliferation, morphology, and adhesive molecule expression levels were either dose or stimulus time dependent. An optimal ATRA stimulus condition for differentiating HL60 cells toward neutrophils consisted of 1 × 10-6 M dose and 120 h of stimulus time. Under wall shear stresses, catch-slip bond transition governs P-selectin-mediated rolling for neutrophils and untreated or ATRA-treated (1 × 10-6 M, 120 h) HL60 cells. The ATRA stimuli slowed down the rolling of HL60 cells on immobilized P-selectin no matter whether ICAM-1 was engaged. The ß2 integrin near the PSGL-1/P-selectin axis would be activated within sub-seconds for each cell group mentioned above, thus contributing to slow rolling. A faster ß2 integrin activation rate and the higher expression levels of PSGL-1 and LFA-1 were assigned to induce the over-enhancement of ATRA-treated HL60 adhesion in flow, causing APL DS development. These findings provided an insight into the mechanical-chemical regulation for APL DS development via ATRA treatment of leukemia and a novel therapeutic strategy for APL DS through targeting the relevant adhesion molecules.

Spatiotemporal characteristics of P-selectin-induced ß2 integrin activation of human neutrophils under flow.

Activation of integrins is crucial for recruitment of flowing leukocytes to inflammatory or injured vascular sites, but their spatiotemporal characteristics are incompletely understood. We discovered that ß2-integrin activation over the entire surface of neutrophils on immobilized P-selectin occurred via mitogen-activated protein kinase (MAPK) or non-MAPK signaling with a minute-level timescale in a force-dependent manner. In flow, MAPK signaling required intracellular Ca2+ release to activate integrin within 2 min. Integrin activation via non-MAPK signaling occurred first locally in the vicinity of ligated P-selectin glycoprotein ligand-1 (PSGL-1) within sub-seconds, and then over the entire cell surface within 1 min in an extracellular Ca2+ influx-dependent manner. The transition from a local (but rapid) to global (but slow) activation mode was triggered by ligating the freshly activated integrin. Lipid rafts, moesin, actin, and talin were involved in non-MAPK signaling. Fluid loads had a slight effect on local integrin activation with a second-level timescale, but served as enhancers of global integrin activation.

Force-enhanced biophysical connectivity of platelet ß3 integrin signaling through Talin is predicted by steered molecular dynamics simulations.

Platelet ß3-integrin signaling through Talin is crucial in platelet transmembrane signaling, activation, adhesion, spreading and aggregation, and remains unclear in mechano-microenvironments. In order to examine Talin-ß3 integrin biophysical connectivity, a series of "ramp-clamp" steered molecular dynamics (SMD) simulations were performed on complex of F3 domain of Talin and cytoplasmic tail of ß3 integrin to imitate different force-loads in platelet. Pull-induced allostery of the hydrophobic pocket in F3 domain might markedly enhance complex rupture-force (> 150pN) and slow down breakage of the complex; the complex should mechano-stable for its conformational conservation under loads (≤ 80pN); increasing force below 60pN would decrease the complex dissociation probability, and force-induced extension of ß5 strand on Talin and binding site residues, ASP740 and ALA742 as well as Asn744, on ß3-integrin were responsible for the force-enhanced linkage of the Talin-ß3 integrin. Force might enhance biophysical connectivity of ß3-integrin signaling through Talin by a catch bond mechanism, which be mediated by the force-induced allostery of complex at clamped stage. This work provides a novel insight into the force-regulated transmembrane ß3-integrin signaling and its molecular basis for platelet activation, and exhibited a potential power of the present computer strategy in predicting mechanical regulation on ligand-receptor interaction under loads.

MD Simulations on a Well-Built Docking Model Reveal Fine Mechanical Stability and Force-Dependent Dissociation of Mac-1/GPIbα Complex.

Interaction of leukocyte integrin macrophage-1 antigen (Mac-1) to platelet glycoprotein Ibα (GPIbα) is critical for platelet-leukocyte crosstalk in hemostasis and inflammatory responses to vessel injuries under hemodynamic environments. The mechano-regulation and its molecular basis for binding of Mac-1 to GPIbα remain unclear, mainly coming from the lack of crystal structure of the Mac-1/GPIbα complex. We herein built a Mac-1/GPIbα complex model through a novel computer strategy, which included a flexible molecular docking and system equilibrium followed by a "force-ramp + snapback" molecular dynamics (MD) simulation. With this model, a series of "ramp-clamp" steered molecular dynamics (SMD) simulations were performed to examine the GPIbα-Mac-1 interaction under various loads. The results demonstrated that the complex was mechano-stable for both the high rupture force (>250 pN) at a pulling velocity of 3 Å/ns and the conformational conservation under various constant tensile forces (≤75 pN); a catch-slip bond transition was predicted through the dissociation probability, examined with single molecular AFM measurements, reflected by the interaction energy and the interface H-bond number, and related to the force-induced allostery of the complex; besides the mutation-identified residues D222 and R218, the residues were also dominant in the binding of Mac-1 to GPIbα. This study recommended a valid computer strategy for building a likely wild-type docking model of a complex, provided a novel insight into the mechanical regulation mechanism and its molecular basis for the interaction of Mac-1 with GPIbα, and would be helpful for understanding the platelet-leukocyte interaction in hemostasis and inflammatory responses under mechano-microenvironments.

One-Step Exfoliation/Etching Method to Produce Chitosan-Stabilized Holey Graphene Nanosheets for Superior DNA Adsorption.

Holey graphene (HG) features universal applications in adsorption because of the large surface areas and the abundant active sites across the nanopores, but it is difficult to produce HG nanosheets straightforwardly from bulk graphite with current etching methods. Herein, for the first time, we developed a one-step sonication-assisted liquid-phase exfoliation/etching method to produce HG nanosheets from bulk graphite by taking advantage of chitosan for stabilization. With the cavitation bubble collapse stress during the intense sonication, the graphite powders were exfoliated and nanopores of tunable diameters from 40 to 200 nm were generated across the graphene nanosheets. Importantly, with chitosan as the stabilizing agent to reduce the fluid collapse stress transferred onto the graphene nanosheets, the lateral size of HG could be as large as 30 µm. Using this approach, several holey layered crystals (graphite, hexagonal boron nitride, and tungsten disulfide) were fabricated with adequate nanostructures, including lateral size, nanosheet thickness, and nanopore size. Notably, the nanoporous structure endowed the graphene nanosheets with superior high double-stranded DNA adsorption (1253 µg/mg, the highest until now) and excellent DNA protection capacity. Based on this, the HG nanosheets were developed for the surface-mediated reversal gene transfection, displaying appreciable efficiency with the traditional methods.

Force-dependent calcium signaling and its pathway of human neutrophils on P-selectin in flow.

P-selectin engagement of P-selectin glycoprotein ligand-1 (PSGL-1) causes circulating leukocytes to roll on and adhere to the vascular surface, and mediates intracellular calcium flux, a key but unclear event for subsequent arresting firmly at and migrating into the infection or injured tissue. Using a parallel plate flow chamber technique and intracellular calcium ion detector (Fluo-4 AM), the intracellular calcium flux of firmly adhered neutrophils on immobilized P-selectin in the absence of chemokines at various wall shear stresses was investigated here in real time by fluorescence microscopy. The results demonstrated that P-selectin engagement of PSGL-1 induced the intracellular calcium flux of firmly adhered neutrophils in flow, increasing P-selectin concentration enhanced cellular calcium signaling, and, force triggered, enhanced and quickened the cytoplasmic calcium bursting of neutrophils on immobilized P-selectin. This P-selectin-induced calcium signaling should come from intracellular calcium release rather than extracellular calcium influx, and be along the mechano-chemical signal pathway involving the cytoskeleton, moesin and Spleen tyrosine kinase (Syk). These results provide a novel insight into the mechano-chemical regulation mechanism for P-selectin-induced calcium signaling of neutrophils in flow.

[Application of γ imaging in chromatographic separation and protein molecular site density determination].

We in this study measured the site density of E-selectin in order to explore the practical pliability using radionuclide labeling method and γ-imaging of single photon emission computer tomography (SPECT). This method required labeling of antibody with 125I using Indogen method and binding of the labeled antibody to E-selectin. Labeled E-selectin was separated and purified in a Sephadex G25 column. The different fractions of the eluants were imaged, analyzed and quantified with SPECT method. For measuring the saturation curve of E-selectin, 130 µL of E-selectin solution with different concentrations were added in a 48-well plate and incubated overnight at 4℃. After incubation, 130 µL of labeled antibody solution were added and kept incubated for 30 min. The resulted mixture was washed, and the radioactivity in each sample was detected by SPECT. The levels of radioactivity were translated to site densities, and were used to plot a standard curve. The labeled product was quantitatively analyzed with SPECT. The labeling rate of E-selectin was 78%. The saturation curve of different concentration samples showed that when the concentration was in the concentration range of 0-1 mg/mL, the standard curve was y=6 045.7 x-51.166, R2=0.997 9. Based on this finding, it could be concluded that γ-imaging is an important tool for analysis of radiolabeled product and determination of site density.

Mechanical regulation of calcium signaling of HL-60 on P-selectin under flow.

BACKGROUND: Binding of P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) makes neutrophils roll on and adhere to inflammatory site. Intracellular calcium bursting of adhered neutrophils is a key event for subsequent arresting firmly at and migrating into the injured tissue. But, it remains unclear how the cytoplasmic calcium signaling of the cells were modulated by the fluid shear stress. Here, we focus on mechanical regulation of P-selectin-induced calcium signaling of neutrophil-like HL-60 cells under flow. METHODS: HL-60 cells were loaded with Fluo-4 AM for fluorescent detection of intracellular calcium ion, and then perfused over P-selectin-coated bottom of parallel-plate flow chamber. The intracellular calcium concentration of firmly adhered cell under flow was observed in real time by fluorescence microscopy. RESULTS: Force triggered, enhanced and quickened cytoplasmic calcium bursting of HL-60 on P-selectin. This force-dependent calcium signaling was induced by the immobilized P-selectin coated on substrates in absence of chemokine. Increasing of both shear stress and P-selectin concentration made the calcium signaling intensive, through quickening the cytosolic calcium release and upregulating both probability and peak level of calcium signaling. CONCLUSIONS: Immobilized P-selectin-induced calcium signaling of HL-60 cells is P-selectin concentration- and mechanical force-dependent. The higher both the P-selectin concentration and the external force on cell, the more intensive the calcium signaling. It might provide a novel insight into the mechano-chemical regulation mechanism for intracellular signaling pathways induced by adhesion molecules.

Ectopic splenic autotransplantation following traumatic injury: A case report.

A 41-year-old male patient was admitted to the General Hospital of Guangzhou Military Command due to upper abdominal pain persisting for 12 h. Computed tomography (CT) and positron emission tomography/CT scans revealed multiple soft-tissue shadows in the abdominal cavity, peritoneum and Glisson's capsule, but the metabolic activity was at normal levels. A small area of low-density shadows near the tail of the pancreas and multiple shadows of enlarged lymph nodes were identified around the porta hepatis and the pancreas, with a mildly increased metabolic activity. On the basis of the CT images the patient was diagnosed with pancreatitis. Radionuclide imaging showed the absence of the spleen from its normal position (following splenectomy), but abnormal phagocytosis of multiple red blood cells was observed in the abdomen, which was diagnosed as ectopic splenic autotransplantation (ESAT). The patient subsequently recovered well following symptomatic treatment. ESAT in trauma patients requires urgent surgery in order to remove the damaged spleen and artificially cultivate partial splenic tissue.

Unfolding the A2 domain of von Willebrand factor with the optical trap.

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein involved in both hemostasis and thrombosis. VWF conformational changes, especially unfolding of the A2 domain, may be required for efficient enzymatic cleavage in vivo. It has been shown that a single A2 domain unfolds at most probable unfolding forces of 7-14 pN at force loading rates of 0.35-350 pN/s and A2 unfolding facilitates A2 cleavage in vitro. However, it remains unknown how much force is required to unfold the A2 domain in the context of a VWF multimer where A2 may be stabilized by other domains like A1 and A3. With the optical trap, we stretched VWF multimers and a poly-protein (A1A2A3)3 that contains three repeats of the triplet A1A2A3 domains at constant speeds of 2000 nm/s and 400 nm/s, respectively, which yielded corresponding average force loading rates of 90 and 22 pN/s. We found that VWF multimers became stiffer when they were stretched and extended by force. After force increased to a certain level, sudden extensional jumps that signify domain unfolding were often observed. Histograms of the unfolding force and the unfolded contour length showed two or three peaks that were integral multiples of approximately 21 pN and approximately 63 nm, respectively. Stretching of (A1A2A3)3 yielded comparable distributions of unfolding force and unfolded contour length, showing that unfolding of the A2 domain accounts for the behavior of VWF multimers under tension. These results show that the A2 domain can be indeed unfolded in the presence of A1, A3, and other domains. Compared with the value in the literature, the larger most probable unfolding force measured in this study suggests that the A2 domain is mechanically stabilized by A1 or A3 although variations in experimental setups and conditions may complicate this interpretation.