Enhancement of Strength-Ductility Synergy of Al-Li Cast Alloy via New Forming Processes and Sc Addition.

In this study, concurrent enhancements in both strength and ductility of the Al-2Li-2Cu-0.5Mg-0.2Zr cast alloy (hereafter referred to as Al-Li) were achieved through an optimized forming process comprising ultrasonic treatment followed by squeeze casting, coupled with the incorporation of Sc. Initially, the variations in the microstructure and mechanical properties of the Sc-free Al-Li cast alloy (i.e., alloy A) during various forming processes were investigated. The results revealed that the grain size in the UT+SC (ultrasonic treatment + squeeze casting) alloy was reduced by 76.3% and 57.7%, respectively, compared to those of the GC (gravity casting) or SC alloys. Additionally, significant improvements were observed in its compositional segregation and porosity reduction. After UT+SC, the ultimate tensile strength (UTS), yield strength (YS), and elongation reached 235 MPa, 135 MPa, and 15%, respectively, which were 113.6%, 28.6%, and 1150% higher than those of the GC alloy. Subsequently, the Al-Li cast alloy containing 0.2 wt.% Sc (referred to as alloy B) exhibited even finer grains under the UT+SC process, resulting in simultaneous enhancements in its UTS, YS, and elongation. Interestingly, the product of ultimate tensile strength and elongation (i.e., UTS × EL) for both alloys reached 36 GPa•% and 42 GPa•%, respectively, which is much higher than that of other Al-Li cast alloys reported in the available literature.

Human red blood cells release microvesicles with distinct sizes and protein composition that alter neutrophil phagocytosis.

Extracellular vesicles (EVs) are membrane-bound structures released by cells and tissues into biofluids, involved in cell-cell communication. In humans, circulating red blood cells (RBCs), represent the most common cell-type in the body, generating daily large numbers of microvesicles. In vitro, RBC vesiculation can be mimicked by stimulating RBCs with calcium ionophores, such as ionomycin and A23187. The fate of microvesicles released during in vivo aging of RBCs and their interactions with circulating cells is hitherto unknown. Using SEC plus DEG isolation methods, we have found that human RBCs generate microvesicles with two distinct sizes, densities, and protein composition, identified by flow cytometry, and MRPS, and further validated by immune TEM. Furthermore, proteomic analysis revealed that RBC-derived microvesicles (RBC-MVs) are enriched in proteins with important functions in ion channel regulation, calcium homeostasis, and vesicular transport, such as of sorcin, stomatin, annexin A7, and RAB proteins. Cryo-electron microscopy identified two separate pathways of RBC-MV-neutrophil interaction, direct fusion with the plasma membrane and internalization, respectively. Functionally, RBC-MVs decrease neutrophil ability to phagocytose E. coli but do not affect their survival at 24 hrs. This work brings new insights regarding the complexity of the RBC-MVs biogenesis, as well as their possible role in circulation.

Effects of Al-Ti-C Refiner and Forming Processes on the Microstructure and Properties of Al-Zn-Mg-Cu Alloys.

In this paper, the refinement effect of Al-5Ti-0.2C refiner on Al-Zn-Mg-Cu alloys was first investigated, and then the effects of three forming processes, i.e., Gravity Casting (GC), Squeeze Casting (SC), and Squeeze Casting after Ultrasonic Treatment (UT-SC), on microstructure and properties of Al-Zn-Mg-Cu alloys were studied. The results show that the refining effect of Al-5Ti-0.2C refiner is obvious; first, the average grain size of the alloy decreases and then increases with the increase in Ti content from 0.15 wt.% to 0.3 wt.%. The optimal amount of added Al-5Ti-0.2C is 0.2 wt.% Ti content. The good refining effect is attributed to the formation of TiC particles and Al3Ti compounds by the refiner, which can all be the nucleus of solidification. The poor refining effect when the Ti content was more than 0.2 wt.% is due to the formation of coarse Al3Ti particles. The results of three forming processes that cast Al-Zn-Mg-Cu alloys under the addition of Al-5Ti-0.2C with 0.2 wt.% Ti content show that the mechanical properties under the UT-SC process are the best; the tensile strength in the as-cast state reaches 367 MPa, and the elongation is 3.84%. The effect of tiny TiC particles in the refiner on the microstructure and properties of Al-Zn-Mg-Cu alloys is also discussed.

SnRNA sequencing defines signaling by RBC-derived extracellular vesicles in the murine heart.

Extracellular vesicles (EVs) mediate intercellular signaling by transferring their cargo to recipient cells, but the functional consequences of signaling are not fully appreciated. RBC-derived EVs are abundant in circulation and have been implicated in regulating immune responses. Here, we use a transgenic mouse model for fluorescence-based mapping of RBC-EV recipient cells to assess the role of this intercellular signaling mechanism in heart disease. Using fluorescent-based mapping, we detected an increase in RBC-EV-targeted cardiomyocytes in a murine model of ischemic heart failure. Single cell nuclear RNA sequencing of the heart revealed a complex landscape of cardiac cells targeted by RBC-EVs, with enrichment of genes implicated in cell proliferation and stress signaling pathways compared with non-targeted cells. Correspondingly, cardiomyocytes targeted by RBC-EVs more frequently express cellular markers of DNA synthesis, suggesting the functional significance of EV-mediated signaling. In conclusion, our mouse model for mapping of EV-recipient cells reveals a complex cellular network of RBC-EV-mediated intercellular communication in ischemic heart failure and suggests a functional role for this mode of intercellular signaling.

Electrophoretic mobility shift as a molecular beacon-based readout for miRNA detection.

MicroRNAs are short, non-coding RNA sequences involved in gene expression regulation. Quantification of miRNAs in biological fluids involves time consuming and laborious methods such as Northern blotting or PCR-based techniques. Molecular beacons (MB) are an attractive means for rapid detection of miRNAs, although the need for sophisticated readout methods limits their use in research and clinical settings. Here, we introduce a novel method based on delayed electrophoretic mobility, as a quantitative means for detection of miRNAs-MB hybridization. Upon hybridization with the target miRNAs, MB form a fluorescent duplex with reduced electrophoretic mobility, thus bypassing the need for additional staining. In addition to emission of light, the location of the fluorescent band on the gel acts as an orthogonal validation of the target identity, further confirming the specificity of binding. The limit of detection of this approach is approximately 100 pM, depending on the MB sequence. The method is sensitive enough to detect specific red blood cell miRNAs molecules in total RNA, with single nucleotide specificity. Altogether, we describe a rapid and affordable method that offers sensitive detection of single-stranded small DNA and RNA sequences.

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation.

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.

High-Sensitivity Glycan Profiling of Blood-Derived Immunoglobulin G, Plasma, and Extracellular Vesicle Isolates with Capillary Zone Electrophoresis-Mass Spectrometry.

We developed a highly sensitive method for profiling of N-glycans released from proteins based on capillary zone electrophoresis coupled to electrospray ionization mass spectrometry (CZE-ESI-MS) and applied the technique to glycan analysis of plasma and blood-derived isolates. The combination of dopant-enriched nitrogen (DEN)-gas introduced into the nanoelectrospray microenvironment with optimized ionization, desolvation, and CZE-MS conditions improved the detection sensitivity up to ∼100-fold, as directly compared to the conventional mode of instrument operation through peak intensity measurements. Analyses without supplemental pressure increased the resolution ∼7-fold in the separation of closely related and isobaric glycans. The developed method was evaluated for qualitative and quantitative glycan profiling of three types of blood isolates: plasma, total serum immunoglobulin G (IgG), and total plasma extracellular vesicles (EVs). The comparative glycan analysis of IgG and EV isolates and total plasma was conducted for the first time and resulted in detection of >200, >400, and >500 N-glycans for injected sample amounts equivalent to <500 nL of blood. Structural CZE-MS2 analysis resulted in the identification of highly diverse glycans, assignment of α-2,6-linked sialic acids, and differentiation of positional isomers. Unmatched depth of N-glycan profiling was achieved compared to previously reported methods for the analysis of minute amounts of similar complexity blood isolates.

Detection of Extracellular Vesicle RNA Using Molecular Beacons.

Extracellular vesicles (EVs) have recently emerged as intercellular conveyors of biological information and disease biomarkers. Identification and characterization of RNA species in single EVs are currently challenging. Molecular beacons (MBs) represent an attractive means for detecting specific RNA molecules. Coupling the MBs to cell-penetrating peptides (CPPs) provides a fast, effective, and membrane-type agnostic means to deliver MBs across the plasma membrane and into the cytosol. Here, we generated RBCs-derived EVs by complement activation and tested the ability of MBs coupled with CPP to detect miRNAs from RBC-EVs. Our results showed that RBC and RBC-EVs miRNA-451a can be detected using MB-CPP, and the respective fluorescence levels can be measured by nano-flow cytometry. MB-based detection of RNA via nano-flow cytometry creates a powerful new analytical framework in which a simple addition of a reagent allows profiling of specific RNA species present within certain EV subsets.

Rational Design of a Bifunctional AND-Gate Ligand To Modulate Cell-Cell Interactions.

Protein "AND-gate" systems, in which a ligand acts only on cells with two different receptors, direct signaling activity to a particular cell type and avoid action on other cells. In a bifunctional AND-gate protein, the molecular geometry of the protein domains is crucial. Here we constructed a tissue-targeted erythropoietin (EPO) that stimulates red blood cell (RBC) production without triggering thrombosis. The EPO was directed to RBC precursors and mature RBCs by fusion to an anti-glycophorin A antibody V region. Many such constructs activated EPO receptors in vitro and stimulated RBC and not platelet production in mice but nonetheless enhanced thrombosis in mice and caused adhesion between RBCs and EPO-receptor-bearing cells. On the basis of a protein-structural model of the RBC surface, we rationally designed an anti-glycophorin-EPO fusion that does not induce cell adhesion in vitro or enhance thrombosis in vivo. Thus, mesoscale geometry can inform the design of synthetic-biological systems.

A Novel Implementation of Magnetic Levitation to Quantify Leukocyte Size, Morphology, and Magnetic Properties to Identify Patients With Sepsis.

BACKGROUND: We have developed a novel, easily implementable methodology using magnetic levitation to quantify circulating leukocyte size, morphology, and magnetic properties, which may help in rapid, bedside screening for sepsis. OBJECTIVE: Our objectives were to describe our methodological approach to leukocyte assessment, and to perform a pilot investigation to test the ability of magnetic levitation to identify and quantify changes in leukocyte size, shape, density, and/or paramagnetic properties in healthy controls and septic patients. METHODS: This prospective, observational cohort study was performed in a 56,000/y visit emergency department (ED) and affiliated outpatient phlebotomy laboratory. Inclusion criteria were admittance to the hospital with suspected or confirmed infection for the septic group, and we enrolled the controls from ED/outpatient patients without infection or acute illness. The bench-top experiments were performed using magnetic levitation to visualize the leukocytes. We primary sought to compare septic patients with noninfected controls and secondary to assess the association with sepsis severity. Our covariates were area, length, width, roundness, and standard deviation (SD) of levitation height. We used unpaired t test and area under the curve (AUC) for the assessment of accuracy in distinguishing between septic and control patients. RESULTS: We enrolled 39 noninfected controls and 22 septic patients. Our analyses of septic patients compared with controls showed: mean cell area in pixels (px) 562 ±â€Š111 vs. 410 ±â€Š45, P < 0.0001, AUC = 0.89 (0.80-0.98); length (px), 29 ±â€Š2.5 vs. 25 ±â€Š1.9, P < 0.0001, AUC = 0.90 (0.83-0.98); and width (px), 27 ±â€Š2.4 vs. 23 ±â€Š1.5, P < 0.0001, AUC = 0.92 (0.84-0.99). Cell roundness: 2.1 ±â€Š1.0 vs. 2.2 ±â€Š1.2, P = 0.8, AUC = 0.51. SD of the levitation height (px) was 72 ±â€Š25 vs. 47 ±â€Š16, P < 0.001, AUC = 0.80 (0.67-0.93). CONCLUSIONS: Septic patients had circulating leukocytes with especially increased size parameters, which distinguished sepsis from noninfected patients with promising high accuracy. This portal-device compatible technology shows promise as a potential bedside diagnostic.

Various Rejuvenation Behaviors of Zr-Based Metallic Glass by Cryogenic Cycling Treatment with Different Casting Temperatures.

The rejuvenation behavior of an Zr50Cu40Al10 (at.%) metallic glass upon cryogenic cycling treatment has been investigated. At a high casting temperature, the microstructure of the glass is quite homogenous and thus, internal stress cannot be generated during cycling. Therefore, the glass cannot be rejuvenated by cryogenic cycling treatment. In the contrary, by lowering the casting temperature, nano-sized heterogeneity can be induced and subsequently generates the internal stress and rejuvenates the glass. Once the glass is rejuvenated, the more induced free volume can plasticize the glass with a higher plastic strain. These findings point out that the synthesis conditions can tailor the heterogeneity of the glass and subsequently affect the following rejuvenation behavior upon thermal treatment. It can also help understand the mechanisms of rejuvenation of metallic glass upon cryogenic cycling treatment.

Effects of Si Content and Ca Addition on Thermal Conductivity of As-Cast Mg⁻Si Alloys.

The Mg⁻Si alloys have low CTEs (coefficients of thermal expansion) and other merits, which contribute to their application potential in the electronic industry. However, the details of their thermal properties are still unclarified, and need more research. In this study, the thermal conductivities (TC) of Mg⁻xSi (x = 1 wt.%, 1.38 wt.%, 2 wt.%, 3 wt.%, and 4 wt.%) binary alloys and Mg⁻4Si⁻yCa (y = 0.2 wt.%, 0.4 wt.%, 0.6 wt.%, 0.8 wt.%, and 1.0 wt.%) alloys over the temperature range of 25⁻300 °C were systematically studied. The results show that the TC of Mg⁻xSi binary alloys decreases with the increase of Si content, while it increases slightly near the eutectic composition. The addition of a Ca element to Mg⁻4Si alloys has an obvious modification effect on the Mg2Si phase. When the Ca content increases constantly, the TC of the alloys decreases at first; then, there is a significant increase at the content of 0.8 wt.% Ca, and after that, it continues to decline. The mechanism is mainly related to the precipitation of the CaMgSi phase at 0.8 wt.% Ca content.

Porous NiTi Particle Dispersed Mg-Zn-Ca Bulk Metallic Glass Matrix Composites.

Even though the Mg-based bulk metallic glasses (BMGs) have shown superior anti-corrosion properties compared with their crystalline counterparts, the brittleness of them limits the widespread application of these materials. In the present study, we have firstly introduced porous NiTi shape memory alloy particles into an Mg-Zn-Ca BMG by the direct adding method. This composite showed both improved compressive strength and corrosion resistance in Hank's solutions than its monolithic glassy counterpart. The NiTi dispersoids among the matrix were likely to hinder the main shear band propagation, and also acted as the corrosion barriers. Furthermore, the porous nature of present added particle could further increase the interface areas, which should enhance the reinforcing effects compared with solid ones. This low-cost, high-anticorrosive composite was a good candidate as an engineering material.

Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling.

RNA-seq is increasingly used for quantitative profiling of small RNAs (for example, microRNAs, piRNAs and snoRNAs) in diverse sample types, including isolated cells, tissues and cell-free biofluids. The accuracy and reproducibility of the currently used small RNA-seq library preparation methods have not been systematically tested. Here we report results obtained by a consortium of nine labs that independently sequenced reference, 'ground truth' samples of synthetic small RNAs and human plasma-derived RNA. We assessed three commercially available library preparation methods that use adapters of defined sequence and six methods using adapters with degenerate bases. Both protocol- and sequence-specific biases were identified, including biases that reduced the ability of small RNA-seq to accurately measure adenosine-to-inosine editing in microRNAs. We found that these biases were mitigated by library preparation methods that incorporate adapters with degenerate bases. MicroRNA relative quantification between samples using small RNA-seq was accurate and reproducible across laboratories and methods.

Effects of ultrasonic vibration on microstructure and mechanical properties of nano-sized SiC particles reinforced Al-5Cu composites.

Ultrasonic vibration (UV) treatment has been successfully applied to improve the particles distribution of nano-sized SiC particles (SiCp) reinforced Al-5Cu alloy matrix composites which were prepared by combined processes of dry high energy ball milling and squeeze casting. When UV treatment is applied, the distribution of nano-sized SiCp has been greatly improved. After UV for 1 min, large particles aggregates are broken up into small aggregates due to effects of cavitation and the acoustic streaming. After UV for 5 min, all the particles aggregates are dispersed and the particles are uniformly distributed in the composites. Compared with the Al-5Cu matrix alloy, the ultimate tensile strength, yield strength and elongation of the 1 wt% nano-sized SiCp/Al-5Cu composites treated by UV for 5 min are 270 MPa, 173 MPa and 13.3%, which are increased by 7.6%, 6.8% and 29%, respectively. The improvements of mechanical properties after UV are attributed to the uniform distribution of nano particles, grain refinement of aluminum matrix alloy and reduction of porosity in the composites.

Tie2 protects the vasculature against thrombus formation in systemic inflammation.

Disordered coagulation contributes to death in sepsis and lacks effective treatments. Existing markers of disseminated intravascular coagulation (DIC) reflect its sequelae rather than its causes, delaying diagnosis and treatment. Here we show that disruption of the endothelial Tie2 axis is a sentinel event in septic DIC. Proteomics in septic DIC patients revealed a network involving inflammation and coagulation with the Tie2 antagonist, angiopoietin-2 (Angpt-2), occupying a central node. Angpt-2 was strongly associated with traditional DIC markers including platelet counts, yet more accurately predicted mortality in 2 large independent cohorts (combined N = 1,077). In endotoxemic mice, reduced Tie2 signaling preceded signs of overt DIC. During this early phase, intravital imaging of microvascular injury revealed excessive fibrin accumulation, a pattern remarkably mimicked by Tie2 deficiency even without inflammation. Conversely, Tie2 activation normalized prothrombotic responses by inhibiting endothelial tissue factor and phosphatidylserine exposure. Critically, Tie2 activation had no adverse effects on bleeding. These results mechanistically implicate Tie2 signaling as a central regulator of microvascular thrombus formation in septic DIC and indicate that circulating markers of the Tie2 axis could facilitate earlier diagnosis. Finally, interventions targeting Tie2 may normalize coagulation in inflammatory states while averting the bleeding risks of current DIC therapies.

Refinement of LPSO structure in Mg-Ni-Y alloys by ultrasonic treatment.

Effect of ultrasonic treatment (UT) on the microstructures and mechanical properties of Mg99.0-xNixY1.0 (x=0.5, 1.0, 1.5, at.%) alloys was investigated. With the increase of Ni content, the amount of eutectic structure, consisting of Mg, Mg2Ni phase and LPSO structure, increased while the mechanical properties decreased in as-cast alloys. After introducing UT to the melt, the secondary phases in these alloys were refined significantly and distributed uniformly, especially for long period stacking ordered (LPSO) structure. In Mg98.5Ni0.5Y1.0 alloy, the formation of MgNi4Y phase, which was distributed adjacent to the LPSO structure at the grain boundaries, was stimulated by UT. In Mg97.5Ni1.5Y1.0 alloy with UT, not only the width of grain boundaries were reduced, but also both the width of LPSO structure and that of eutectic structure were reduced. The optimal mechanical properties were obtained in Mg98.5Ni0.5Y1.0 alloy, which exhibited 206MPa in ultimate tensile stress and 7.96% in elongation, respectively. After UT, these properties were enhanced to 231MPa and 14.56%, respectively. The other alloys' mechanical properties were also enhanced significantly by UT.

Detection of membrane-bound and soluble antigens by magnetic levitation.

Magnetic levitation is a technique for measuring the density and the magnetic properties of objects suspended in a paramagnetic field. We describe a novel magnetic levitation-based method that can specifically detect cell membrane-bound and soluble antigens by measurable changes in levitation height that result from the formation of antibody-coated bead and antigen complex. We demonstrate our method's ability to sensitively detect an array of membrane-bound and soluble antigens found in blood, including T-cell antigen CD3, eosinophil antigen Siglec-8, red blood cell antigens CD35 and RhD, red blood cell-bound Epstein-Barr viral particles, and soluble IL-6, and validate the results by flow cytometry and immunofluorescence microscopy performed in parallel. Additionally, employing an inexpensive, single lens, manual focus, wifi-enabled camera, we extend the portability of our method for its potential use as a point-of-care diagnostic assay.

Detection and quantification of subtle changes in red blood cell density using a cell phone.

Magnetic levitation has emerged as a technique that offers the ability to differentiate between cells with different densities. We have developed a magnetic levitation system for this purpose that distinguishes not only different cell types but also density differences in cells of the same type. This small-scale system suspends cells in a paramagnetic medium in a capillary placed between two rare earth magnets, and cells levitate to an equilibrium position determined solely by their density. Uniform reference beads of known density are used in conjunction with the cells as a means to quantify their levitation positions. In one implementation images of the levitating cells are acquired with a microscope, but here we also introduce a cell phone-based device that integrates the magnets, capillary, and a lens into a compact and portable unit that acquires images with the phone's camera. To demonstrate the effectiveness of magnetic levitation in cell density analysis we carried out levitation experiments using red blood cells with artificially altered densities, and also levitated those from donors. We observed that we can distinguish red blood cells of an anemic donor from those that are healthy. Since a plethora of disease states are characterized by changes in cell density magnetic cell levitation promises to be an effective tool in identifying and analyzing pathologic states. Furthermore, the low cost, portability, and ease of use of the cell phone-based system may potentially lead to its deployment in low-resource environments.