Copper-Graphene Composite (CGC) Conductors: Synthesis, Microstructure, and Electrical Performance.

Improving the electrical performance of copper, the most widely used electrical conductor in the world is of vital importance to the progress of key technologies, including electric vehicles, portable devices, renewable energy, and power grids. Copper-graphene composite (CGC) stands out as the most promising candidate for high-performance electrical conductor applications. This can be attributed to the superior properties of graphene fillers embedded in CGC, including excellent electrical and thermal conductivity, corrosion resistance, and high mechanical strength. This review highlights the recent progress of CGC conductors, including their fabrication processes, electrical performances, mechanisms of copper-graphene interplay, and potential applications.

Effect of random fiber networks on bubble growth in gelatin hydrogels.

In hydrodynamics, the event of dynamic bubble growth in a pure liquid under tensile pressure is known as cavitation. The same event can also be observed in soft materials (e.g., elastomers and hydrogels). However, for soft materials, bubble/cavity growth is either defined as cavitation if the bubble growth is elastic and reversible or as fracture if the cavity growth is by material failure and irreversible. In any way, bubble growth can cause damage to soft materials (e.g., tissue) by inducing high strain and strain-rate deformation. Additionally, a high-strength pressure wave is generated upon the collapse of the bubble. Therefore, it is crucial to identify the critical condition of spontaneous bubble growth in soft materials. Experimental and theoretical observations have agreed that the onset of bubble growth in soft materials requires higher tensile pressure than pure water. The extra tensile pressure is required since the cavitating bubble needs to overcome the elastic and surface energy in soft materials. In this manuscript, we developed two models to study and quantify the extra tensile pressure for different gelatin concentrations. Both the models are then compared with the existing cavitation onset criteria of rubber-like materials. Validation is done with the experimental results of threshold tensile pressure for different gelatin concentrations. Both models can moderately predict the extra tensile pressure within the intermediate range of gelatin concentrations (3-7% [w/v]). For low concentration (∼1%), the network's non-affinity plays a significant role and must be incorporated. On the other hand, for higher concentrations (∼10%), the entropic deformation dominates, and the strain energy formulation is not adequate.

Proteomic Analysis of Hippocampus in a Mouse Model of Depression Reveals Neuroprotective Function of Ubiquitin C-terminal Hydrolase L1 (UCH-L1) via Stress-induced Cysteine Oxidative Modifications.

Chronic physical restraint stress increases oxidative stress in the brain, and dysregulation of oxidative stress can be one of the causes of major depressive disorder. To understand the underlying mechanisms, we undertook a systematic proteomic analysis of hippocampus in a chronic restraint stress mouse model of depression. Combining two-dimensional gel electrophoresis (2D-PAGE) for protein separation with nanoUPLC-ESI-q-TOF tandem mass spectrometry, we identified sixty-three protein spots that changed in the hippocampus of mice subjected to chronic restraint stress. We identified and classified the proteins that changed after chronic stress, into three groups respectively functioning in neural plasticity, metabolic processes and protein aggregation. Of these, 5 proteins including ubiquitin C-terminal hydrolase L1 (UCH-L1), dihydropyrimidinase-related protein 2 (DPYL2), haloacid dehalogenase-like hydrolase domain-containing protein 2 (HDHD2), actin-related protein 2/3 complex subunit 5 (ARPC5) and peroxiredoxin-2 (PRDX2), showed pI shifts attributable to post-translational modifications. Further analysis indicated that UCH-L1 underwent differential oxidations of 2 cysteine residues following chronic stress. We investigated whether the oxidized form of UCH-L1 plays a role in stressed hippocampus, by comparing the effects of UCH-L1 and its Cys mutants on hippocampal cell line HT-22 in response to oxidative stress. This study demonstrated that UCH-L1 wild-type and cysteine to aspartic acid mutants, but not its cysteine to serine mutants, afforded neuroprotective effects against oxidative stress; there were no discernible differences between wild-type UCH-L1 and its mutants in the absence of oxidative stress. These findings suggest that cysteine oxidative modifications of UCH-L1 in the hippocampus play key roles in neuroprotection against oxidative stress caused in major depressive disorder.

Heterogeneous nuclear ribonucleoprotein K inhibits heat shock-induced transcriptional activity of heat shock factor 1.

When cells are exposed to heat shock and various other stresses, heat shock factor 1 (HSF1) is activated, and the heat shock response (HSR) is elicited. To better understand the molecular regulation of the HSR, we used 2D-PAGE-based proteome analysis to screen for heat shock-induced post-translationally modified cellular proteins. Our analysis revealed that two protein spots typically present on 2D-PAGE gels and containing heterogeneous nuclear ribonucleoprotein K (hnRNP K) with trioxidized Cys132 disappeared after the heat shock treatment and reappeared during recovery, but the total amount of hnRNP K protein remained unchanged. We next tested whether hnRNP K plays a role in HSR by regulating HSF1 and found that hnRNP K inhibits HSF1 activity, resulting in reduced expression of hsp70 and hsp27 mRNAs. hnRNP K also reduced binding affinity of HSF1 to the heat shock element by directly interacting with HSF1 but did not affect HSF1 phosphorylation-dependent activation or nuclear localization. hnRNP K lost its ability to induce these effects when its Cys132 was substituted with Ser, Asp, or Glu. These findings suggest that hnRNP K inhibits transcriptional activity of HSF1 by inhibiting its binding to heat shock element and that the oxidation status of Cys132 in hnRNP K is critical for this inhibition.

Single-cell detection of mRNA expression using nanofountain-probe electroporated molecular beacons.

New techniques for single-cell analysis enable new discoveries in gene expression and systems biology. Time-dependent measurements on individual cells are necessary, yet the common single-cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP-E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP-E is used to transfect a DNA-based beacon that detects glyceraldehyde 3-phosphate dehydrogenase and an RNA-based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time-dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time.

Nanofountain probe electroporation (NFP-E) of single cells.

The ability to precisely deliver molecules into single cells is of great interest to biotechnology researchers for advancing applications in therapeutics, diagnostics, and drug delivery toward the promise of personalized medicine. The use of bulk electroporation techniques for cell transfection has increased significantly in the past decade, but the technique is nonspecific and requires high voltage, resulting in variable efficiency and low cell viability. We have developed a new tool for electroporation using nanofountain probe (NFP) technology, which can deliver molecules into cells in a manner that is highly efficient and gentler to cells than bulk electroporation or microinjection. Here we demonstrate NFP electroporation (NFP-E) of single HeLa cells within a population by transfecting them with fluorescently labeled dextran and imaging the cells to evaluate the transfection efficiency and cell viability. Our theoretical analysis of the mechanism of NFP-E reveals that application of the voltage creates a localized electric field between the NFP cantilever tip and the region of the cell membrane in contact with the tip. Therefore, NFP-E can deliver molecules to a target cell with minimal effect of the electric potential on the cell. Our experiments on HeLa cells confirm that NFP-E offers single cell selectivity, high transfection efficiency (>95%), qualitative dosage control, and very high viability (92%) of transfected cells.

Breaking the Intrinsic Strength-Ductility Tradeoff in Graphene-Metal Composites.

Small carbon materials, such as graphene, offer excellent mechanical strength. Micro/nano carbon materials are often dispersed into a metal matrix to form bulk composites with mechanical enhancement. Despite technical progress, such composites intrinsically suffer from a trade-off condition between strength and ductility because the load transfer path forms between mechanically strong yet chemically inert micro/nano carbon materials or between the carbon-metal interfaces. In other words, conventional carbon and metal composites become stronger with increasing carbon contents, but the weak interfaces also increase, leading to premature failure. In this regard, crucial advances are presented toward breaking the strength-ductility trade-off condition by utilizing Axially bi-Continuous Graphene-Nickel (ACGN) wires. This innovative ACGN achieves excellent combined strength and ductility-the highest among the current Ni-, Al-, and Cu-based carbon-enhanced metal matrix composites. For example, the ultimate strength and failure strain of 25-µm-diameter ACGN wires are improved by 71.76% and 58.24%, compared to their counterparts. The experimental and theoretical analyses indicate that the graphene-nickel interplay via their axially bi-continuous structure is the main underlying mechanism for the superb mechanical behavior. In specific, the continuous graphene, in addition to effective load-sharing, passivates the free surface of fine wire, forming dislocation pileups along the graphene-nickel interface and, therefore, hindering localized necking.

Topographical depth reveals contact guidance mechanism distinct from focal adhesion confinement.

Cellular response to the topography of their environment, known as contact guidance, is a crucial aspect to many biological processes yet remains poorly understood. A prevailing model to describe cellular contact guidance involves the lateral confinement of focal adhesions (FA) by topography as an underlying mechanism governing how cells can respond to topographical cues. However, it is not clear how this model is consistent with the well-documented depth-dependent contact guidance responses in the literature. To investigate this model, we fabricated a set of contact guidance chips with lateral dimensions capable of confining focal adhesions and relaxing that confinement at various depths. We find at the shallowest depth of 330 nm, the model of focal adhesion confinement is consistent with our observations. However, the cellular response at depths of 725 and 1000 nm is inadequately explained by this model. Instead, we observe a distinct reorganization of F-actin at greater depths in which topographically induced cell membrane deformation alters the structure of the cytoskeleton. These results are consistent with an alternative curvature-hypothesis to explain cellular response to topographical cues. Together, these results indicate a confluence of two molecular mechanisms operating at increased induced membrane curvature that govern how cells sense and respond to topography.

A 3D printed tensile testing system for micro-scale specimens.

Mechanical property characterization of micro-scale material systems, such as free-standing films or small diameter wires (<20 µm), often requires expensive, specialized test systems. Conventional tensile test systems are usually designed for millimeter scale specimens with the force sensing capability of >1N while microdevice-based testers are intended for micro-/nano-scale specimens operating within a much smaller force range of <10 mN. This disparity leaves a technology gap in reliable and cost-effective characterization methods for specimens at the intermediate scale. In this research, we introduce the cost-effective and all-in-one tensile testing system with a built-in force sensor, self-aligning mechanisms, and loading frames. Owing to the advantages of 3D printing technologies, the ranges of force measurement (0.001-1 N) and displacement (up to tens of millimeters) of our 3D printed tensile tester can be readily tailored to suit specific material dimension and types. We have conducted a finite element simulation to identify the potential sources of the measurement error during tensile testing and addressed the dominant errors by simply modifying the dimension/design of the loading frames. As a proof-of-concept demonstration, we have characterized fine copper (Cu) wires with 10-25 µm diameters by the 3D printed tensile tester and confirmed that the measured mechanical properties match with the known values of bulk Cu. Our work shows that the proposed 3D printed tensile testing system offers a cost-efficient and easily accessible testing method for accurate mechanical characterization of specimens with cross-sectional dimensions of the order of tens of micrometers.

Expression and purification recombinant human dentin sialoprotein in Escherichia coli and its effects on human dental pulp cells.

Dentin sialoprotein (DSP) is cleaved from dentin sialophosphoprotein (DSPP) and most abundant dentinal non-collagenous proteins in dentin. DSP is believed to participate in differentiation and mineralization of cells. In this study, we first constructed recombinant human DSP (rhDSP) in Escherichia coli (E. coli) and investigated its odontoblastic differentiation effects on human dental pulp cells (hDPCs). Cell adhesion activity was measured by crystal violet assay and cell proliferation activity was measured by MTT assay. To assess mineralization activity of rhDSP, Alizarin Red S staining was performed. In addition, the mRNA levels of collagen type І (Col І), alkaline phosphatase (ALP), and osteocalcin (OCN) were measured due to their use as mineralization markers for odontoblast-/osteoblast-like differentiation of hDPCs. The obtained rhDSP in E. coli was approximately identified by SDS-PAGE and Western blot. Initially, rhDSP significantly enhanced hDPCs adhesion activity and proliferation (p<0.05). In Alizarin Red S staining, stained hDPCs increased in a time-dependent manner. This odontoblastic differentiation activity was also verified through mRNA levels of odontoblast-related markers. Here, we first demonstrated that rhDSP may be an important regulatory ECM in determining the hDPCs fate including cell adhesion, proliferation, and odontoblastic differentiation activity. These findings indicate that rhDSP can induce growth and differentiation on hDPCs, leading to improve tooth repair and regeneration.

Fibroblast growth factor 2-functionalized collagen matrices for skeletal muscle tissue engineering.

Fibroblast growth factor 2 (FGF2) protein plays important roles in wound healing and tissue regeneration. Collagen is clinically used for wound care applications. We investigated the potential value of FGF2-functionalized collagen matrices for skeletal muscle tissue engineering. When C2C12 cells were treated with FGF2, cell adhesion increased after 3 and 5 days compared to the control (P < 0.05). Wound healing activity of FGF2 was slightly higher than the control through cell migration. Cell proliferation activity of FGF2-functionalized collagen matrices on C2C12 cells also increased. Taken together, FGF2 stimulated C2C12 myoblast growth by promoting cell adhesion, proliferation and wound healing activity after injury. The potential effect of FGF2-functionalized collagen matrices was also observed. Thus FGF2 stimulates skeletal muscle development and regeneration, thereby leading to potential utility for skeletal muscle tissue engineering.

Cavitation nucleation and its ductile-to-brittle shape transition in soft gels under translational mechanical impact.

Cavitation bubbles in the human body, when subjected to impact, are being increasingly considered as a possible brain injury mechanism. However, the onset of cavitation and its complex dynamics in biological materials remain unclear. Our experimental results using soft gels as a tissue simulant show that the critical acceleration (acr) at cavitation nucleation monotonically increases with increasing stiffness of gelatin A/B, while acr for agarose and agar initially increases but is followed by a plateau or even decrease after stiffness reach to ∼100 kPa. Our image analyses of cavitation bubbles and theoretical work reveal that the observed trends in acr are directly linked to how bubbles grow in each gel. Gelatin A/B, regardless of their stiffness, form a localized damaged zone (tens of nanometers) at the gel-bubble interface during bubble growth. In contrary, the damaged zone in agar/agarose becomes significantly larger (> 100 times) with increasing shear modulus, which triggers the transition from formation of a small, damaged zone to activation of crack propagation. STATEMENT OF SIGNIFICANCE: We have studied cavitation nucleation and bubble growth in four different types of soft gels (i.e., tissue simulants) under translational impact. The critical linear acceleration for cavitation nucleation has been measured in the simulants by utilizing a recently developed method that mimics acceleration profiles of typical head blunt events. Each gel type exhibits significantly different trends in the critical acceleration and bubble shape (e.g., A gel-specific sphere-to-saucer transition) with increasing gel stiffness. Our theoretical framework, based on the concepts of a damaged zone and crack propagation in each gel, explains underlying mechanisms of the experimental observations. Our in-depth studies shed light on potential links between traumatic brain injuries and cavitation bubbles induced by translational acceleration, the overlooked mechanism in the literature.

Engineering of a multi-functional extracellular matrix protein for immobilization to bone mineral hydroxyapatite.

PURPOSE OF WORK: We have developed a strategy of designing multi-functional extracellular matrix proteins for functionalizing bone tissue engineering scaffolds and other biomedical surfaces to achieve improvements in bone grafting, bone repair and bone regeneration. We developed a novel extracellular matrix protein designed to have a cell adhesive RGD sequence derived from fibronectin and active functional unit of osteocalcin (OC) containing Ca(2+)-binding sites for immobilization to mineral component of bone, hydroxyapatite (HA). The fusion protein, designated FN(RGD)/OC, was expressed in Escherichia coli and purified with affinity chromatography using a His-tag. The resultant FN(RGD)/OC fusion protein preferentially bound to HA, promoted cell adhesive activity, and stimulated differentiation of MC3T3-E1 cell.

Mechanically Induced Cavitation in Biological Systems.

Cavitation bubbles form in soft biological systems when subjected to a negative pressure above a critical threshold, and dynamically change their size and shape in a violent manner. The critical threshold and dynamic response of these bubbles are known to be sensitive to the mechanical characteristics of highly compliant biological systems. Several recent studies have demonstrated different biological implications of cavitation events in biological systems, from therapeutic drug delivery and microsurgery to blunt injury mechanisms. Due to the rapidly increasing relevance of cavitation in biological and biomedical communities, it is necessary to review the current state-of-the-art theoretical framework, experimental techniques, and research trends with an emphasis on cavitation behavior in biologically relevant systems (e.g., tissue simulant and organs). In this review, we first introduce several theoretical models that predict bubble response in different types of biological systems and discuss the use of each model with physical interpretations. Then, we review the experimental techniques that allow the characterization of cavitation in biologically relevant systems with in-depth discussions of their unique advantages and disadvantages. Finally, we highlight key biological studies and findings, through the direct use of live cells or organs, for each experimental approach.

An Axially Continuous Graphene-Copper Wire for High-Power Transmission: Thermoelectrical Characterization and Mechanisms.

The demand for high-power electrical transmission continues to increase with technical advances in electric vehicles, unmanned drones, portable devices, and deployable military applications. In this study, significantly enhanced electrical properties (i.e., a 450% increase in the current density breakdown limit) are demonstrated by synthesizing axially continuous graphene layers on microscale-diameter wires. To elucidate the underlying mechanisms of the observed enhancements, the electrical properties of pure copper wires and axially continuous graphene-copper (ACGC) wires with three different diameters are characterized while controlling the experimental conditions, including ambient temperature, gases, and pressure. The study reveals that the main mechanism that allows the application of extremely large current densities (>400 000 A cm-2 ) through the ACGC wires is threefold: the continuous graphene layers considerably improve: 1) surface heat dissipation (224% higher), 2) electrical conductivity (41% higher), and 3) thermal stability (41.2% lower resistivity after thermal cycles up to 450 °C), compared with pure copper wires. In addition, it is observed, through the use of high-speed camera images, that the ACGC wires exhibit very different failure behavior near the current density limit, compared with the pure copper wires.

Construction and expression of a recombinant fibronectinIII10 protein for integrin-mediated cell adhesion.

Specific sequences of cell-adhesive peptide/proteins are often required for the bio-inert synthetic polymers to improve cell adhesion. We have developed a recombinant fibronectin fragment (FN(III)10), the central cell-binding domain containing RGD motif, to endow biomaterial surfaces with abilities to promote an integrin-mediated cell adhesion. Immobilized FN(III)10 stimulated adhesion of MC3TC-E1 cells in an integrin-dependent manner.

Mechanisms of cell damage due to mechanical impact: an in vitro investigation.

The dynamic response of cells when subjected to mechanical impact has become increasingly relevant for accurate assessment of potential blunt injuries and elucidating underlying injury mechanisms. When exposed to mechanical impact, a biological system such as the human skin, brain, or liver is rapidly accelerated, which could result in blunt injuries. For this reason, an acceleration of greater than > 150 g is the most commonly used criteria for head injury. To understand the main mechanism(s) of blunt injury under such extreme dynamic threats, we have developed an innovative experimental method that applies a well-characterized and -controlled mechanical impact to live cells cultured in a custom-built in vitro setup compatible with live cell microscopy. Our studies using fibroblast cells as a model indicate that input acceleration ([Formula: see text]) alone, even when it is much greater than the typical injury criteria, e.g., [Formula: see text] g, does not result in cell damage. On the contrary, we have observed a material-dependent critical pressure value above which a sudden decrease in cell population and cell membrane damage have been observed. We have unambiguously shown that (1) this critical pressure is associated with the onset of cavitation bubbles in a cell culture chamber and (2) the dynamics of cavitation bubbles in the chamber induces localized compressive/tensile pressure cycles, with an amplitude that is considerably greater than the acceleration-induced pressure, to cells. More importantly, the rate of pressure change with time for cavitation-induced pressure is significantly faster (more than ten times) than acceleration-induced pressure. Our in vitro study on the dynamic response of biological systems due to mechanical impact is a crucial step towards understanding potential mechanism(s) of blunt injury and implementing novel therapeutic strategies post-trauma.

Problem of Diminished cRGD Surface Activity and What Can Be Done about It.

RGD peptides play a pivotal role in growing and diverse areas of biological research, ranging from in vitro experiments probing fundamental molecular mechanisms of cell adhesion to more applied in vivo strategies in medical imaging and cancer therapeutics. To better understand the outcomes of RGD-based approaches, we quantified the degree to which cyclic RGD (cRGD) activity is blocked by nonspecific binding of commonly used medium constituents. First, we show that recombinant αVß3 integrins can be used as a highly sensitive cell-free sensor to quantitatively and reliably characterize the activity of cRGD-functionalized surfaces via surface plasmon resonance (SPR). Next, SPR experiments were utilized to measure the extent of blocking of cRGD-functionalized surfaces by the commonly used agents BSA, PLL-g-PEG, and fetal calf serum (FCS)-supplemented media, using recombinant αVß3 integrin as a probe for cRGD binding activity in the presence of blocking agents. All three additives were highly efficient blockers of cRGD activity, as exemplified by cell culture media containing 1% FCS which reduced the cRGD activity by 33-fold. We then developed a strategy to combat these deleterious effects by employing the recombinant integrins as a protective cap. We show that the unblocked cRGD activity can be preserved in the presence of PLL-g-PEG by employing the αVß3 integrin as a removable protective cap, both in cell-free and in vitro experiments. In vitro studies with MDA-MB-231 cells cultured atop cRGD-functionalized surfaces found that cell adhesion and migration prevented by PLL-g-PEG were restored when this protective cap approach was used.

Protein engineering, expression, and activity of a novel fusion protein possessing keratinocyte growth factor 2 and fibronectin.

Growth factor-induced proliferation and differentiation often require adhesion of cells to the extracellular matrix proteins such as fibronectin (FN). In this study, we aimed to investigate the effect of protein engineering of the keratinocyte growth factor 2 (KGF2) fused to the FN on the mitogenic activity of KGF2. The fusion protein (KGF2-FN10), which was expressed in Escherichia coli, showed significantly enhanced mitogenic activity of KGF2 on human keratinocytes. Moreover, KGF2-FN10 fusion protein showed significantly increased activity to differentiate keratinocytes from native KGF2. In conclusion, these results suggest that KGF2-FN10 fusion protein has certain advantages over native KGF2 and may offer a novel strategy to potentiate the therapeutic effect of KGF2.

Prognostic Role of TMED3 in Clear Cell Renal Cell Carcinoma: A Retrospective Multi-Cohort Analysis.

Transmembrane p24 trafficking protein 3 (TMED3) is a metastatic suppressor in colon cancer and hepatocellular carcinoma. However, its function in the progression of clear cell renal cell carcinoma (ccRCC) is unknown. Here, we report that TMED3 could be a new prognostic marker for ccRCC. Patient data were extracted from cohorts in the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC). Differential expression of TMED3 was observed between the low stage (Stage I and II) and high stage (Stage III and IV) patients in the TCGA and ICGC cohorts and between the low grade (Grade I and II) and high grade (Grade III and IV) patients in the TCGA cohort. Further, we evaluated TMED3 expression as a prognostic gene using Kaplan-Meier survival analysis, multivariate analysis, the time-dependent area under the curve (AUC) of Uno's C-index, and the AUC of the receiver operating characteristics at 5 years. The Kaplan-Meier analysis revealed that TMED3 overexpression was associated with poor prognosis for ccRCC patients. Analysis of the C-indices and area under the receiver operating characteristic curve further supported this. Multivariate analysis confirmed the prognostic significance of TMED3 expression levels (P = 0.005 and 0.006 for TCGA and ICGC, respectively). Taken together, these findings demonstrate that TMED3 is a potential prognostic factor for ccRCC.