Low-Power, Self-Rectifying, and Forming-Free Memristor with an Asymmetric Programing Voltage for a High-Density Crossbar Application

A Pt/NbO_<i>x</i>/TiO_<i>y</i>/NbO_<i>x</i>/TiN stack integrated on a 30 nm contact via shows a programming current as low as 10 nA and 1 pA for the set and reset switching, respectively, and a self-rectifying ratio as high as ∼10⁵, which are suitable characteristics for low-power memristor applications. It also shows a forming-free characteristic. A charge-trap-associated switching model is proposed to account for this self-rectifying memrisive behavior. In addition, an asymmetric voltage scheme (AVS) to decrease the write power consumption by utilizing this self-rectifying memristor is also described. When the device is used in a 1000 × 1000 crossbar array with the AVS, the programming power can be decreased to 8.0% of the power consumption of a conventional biasing scheme. If the AVS is combined with a nonlinear selector, a power consumption reduction to 0.31% of the reference value is possible

Shear Stress Induced by an Interstitial Level of Slow Flow Increases the Osteogenic Differentiation of Mesenchymal Stem Cells through TAZ Activation

<div><p>Shear stress activates cellular signaling involved in cellular proliferation, differentiation, and migration. However, the mechanisms of mesenchymal stem cell (MSC) differentiation under interstitial flow are not fully understood. Here, we show the increased osteogenic differentiation of MSCs under exposure to constant, extremely low shear stress created by osmotic pressure-induced flow in a microfluidic chip. The interstitial level of shear stress in the proposed microfluidic system stimulated nuclear localization of TAZ (transcriptional coactivator with PDZ-binding motif), a transcriptional modulator of MSCs, activated TAZ target genes such as <i>CTGF</i> and <i>Cyr61</i>, and induced osteogenic differentiation. TAZ-depleted cells showed defects in shear stress-induced osteogenic differentiation. In shear stress induced cellular signaling, Rho signaling pathway was important forthe nuclear localization of TAZ. Taken together, these results suggest that TAZ is an important mediator of interstitial flow-driven shear stress signaling in osteoblast differentiation of MSCs.</p></div

Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation

<div><p>Mesenchymal stem cell (MSC) differentiation is regulated by the extracellular matrix (ECM) through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ) was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.</p></div

Computed shear stress distribution.

<p>(A) The rectangular area (9 mm × 4 mm) of the main microchannel was considered the cell culture zone for experimental observation. Due to the tapered geometry near the inlet and outlet ports, higher shear stresses developed in these areas, whereas almost uniform shear stress developed in the cell culture zone. (B) Quantitative analysis showed that over 70% of the cell culture zone had a good uniformity of shear stress (approximately 7% variation) and over 80% of the area had an acceptable uniformity (approximately 20% variation).</p

Rho GTPase is involved in TAZ localization after shear stress.

<p>(A) MSCs were loaded onto microfluidic chips, and shear stress was applied to the chips in the presence of 50 μM Y27632 (Rock inhibitor) and 100 μM Rac1 inhibitor. After 24 h, the cells were fixed and analyzed by immunocytochemistry. A TAZ-specific antibody was used to observe the location of TAZ. Scale bars indicate 20 μm size. (B) Decreased alkaline phosphatase activity in Y27632-treated cells after shear stress. Control and Y27632-treated cells were differentiated into osteoblasts in the presence of osteogenic differentiation media for 4 days with microfluidic shear stress. Alkaline phosphatase activity in the differentiated cells was determined according to the experimental method. (C) Decreased osteogenic marker gene expression in Y27632-treated cells after shear stress. Total RNA in (B) was harvested at 2 days after differentiation and analyzed by reverse transcription and qRT-PCR. The relative expression levels of <i>TAZ</i>, <i>Runx2</i>, <i>DLX5</i>, and <i>Msx2</i> were determined after normalization to the <i>GAPDH</i> level. * for p < 0.05, ** for p < 0.01, <i>t</i>-test.</p

Experimental system.

<p>(A) The system consisted of an inlet reservoir (1000-μL pipette tip), a microfluidic channel where cells were cultured and exposed to shear flow, a coiled tube, and an osmotic pump dipped into a Petri dish containing 0.1 M PEG solution. (B) The coiled tube had a large capacity that served as an outlet reservoir providing clean water to the osmotic pump. An air bubble moved through the tube to prevent the mixing of medium and water. (C) The osmotic flow was generated at the membrane window by the concentration difference between water and PEG solution. (D) Cells in the main microchannel (200-μm height) were exposed to an extremely slow interstitial level of flow.</p

A stiff ECM activates the ERK and JNK signaling pathway.

<p>(A) hMSCs were plated on a 1.37 or 4.47 kPa hydrogel, and after 12 h, cell lysates were prepared and analyzed by immunoblotting. To assess the activity of ERK and JNK, phosphorylated ERK (p-ERK) and phosphorylated JNK (p-JNK) antibodies were used, respectively. As a control, total ERK and JNK protein was analyzed. The levels of phosphorylated ERK and JNK were increased in hMSCs cultured on the stiff matrix. GAPDH was used as a loading control. (B) Immunocytochemistry of p-ERK and p-JNK in hMSCs on a 1.37 or 4.47 kPa hydrogel. Cells in panel (A) were fixed and subjected to immunocytochemical staining with a p-ERK or p-JNK antibody. The signals for p-ERK or p-JNK were green fluorescence. DAPI was used for nuclear staining. (C) Fluorescence signals in panel (B) was quantified by image J software and corrected total cell fluorescence was calculated by fluorescence signal with elimination of background signal. AU is arbitrary unit.</p

Inhibition of ROCK/F-actin represses the transcriptional activity of TAZ in hMSCs.

<p>(A) hMSCs cultured on a 4.47kPa hydrogel were treated with a ROCK inhibitor (Y27632, 50 μM) or an F-actin inhibitor (latrunculin A, 0.5 μM). After 12 h of treatment, total RNA was prepared, and <i>CTGF</i> and <i>CYR61</i> expression was assessed by qRT-PCR. (B) The CTGF-luc reporter gene construct or the pGL3-basic control vector was transfected into hMSCs, and after 16 h, the transfected cells were plated on 4.47 kPa hydrogels. After 24 h, luciferase reporter gene activity was analyzed. To inhibit ROCK or F-actin, cells were pretreated with 50 μM Y27632 or 0.5 μM latrunculin A 12 h before reporter gene analysis. A Renilla luciferase-expressing vector was used as a transfection control. Luciferase activity was normalized to Renilla luciferase activity. (C) hMSCs on 4.47 kPa hydrogels were differentiated into osteoblasts for 6 days in the presence of 50 μM Y27632 or 0.5 μM latrunculin A. DMSO was used as the vehicle control. The expression of osteoblastic marker genes, including <i>DLX5</i>, <i>MSX2</i>, osteocalcin, and <i>RUNX2</i>, were analyzed by qRT-PCR. Target gene expression was normalized to <i>GAPDH</i>. (D) hMSCs were transfected with 6OSE2-luc or pGL3-basic (control) along with a Renilla luciferase-expressing construct. Then, the cells were plated on a 4.47 kPa hydrogel. After 24 h, luciferase reporter gene activity was analyzed. To inhibit ROCK or F-actin, the cells were pretreated with 50 μM Y27632 or 0.5 μM latrunculin A 12 h before the reporter gene assay. Luciferase activity was normalized to Renilla luciferase activity. (***p < 0.005, t-test). (E) Total RNAs of hMSCs in panel (A) were prepared and qRT-PCR was assessed to analyze the transcription of <i>TAZ</i>. Gene expression was normalized to <i>GAPDH</i>.</p

Shear stress stimulates osteoblast differentiation.

<p>(A) Increased alkaline phosphatase activity by shear stress. MSCs were loaded onto the chips and differentiated into osteoblasts in the presence of osteogenic differentiation media for 4 days. Alkaline phosphatase activity in the differentiated cells was stained according to the experimental method. Control cells were incubated in the osteogenic differentiation media without low shear stress. (B) Increased osteogenic marker gene expression by shear stress. Total RNA in (A) was harvested at 2 days after differentiation and analyzed by reverse transcription and qRT-PCR. The relative expression levels of <i>TAZ</i>, <i>Runx2</i>, <i>DLX5</i>, and <i>Msx2</i> were determined after normalization to the <i>GAPDH</i> level. ** for p < 0.01, <i>t</i>-test.</p

Inhibition of the ERK or JNK signaling pathway induces TAZ cytoplasmic localization on stiff hydrogels.

<p>(A) hMSCs on a 4.47 kPa hydrogel were treated with 10 μM U0126 or 10 μM SP600125. After 12 h of treatment, cells were subjected to immunostaining with an anti-TAZ antibody. The red fluorescence signal shows the location of TAZ, and DAPI was used to stain the nuclei. The results show that even in cells on a stiff matrix, TAZ is localized evenly to the cytoplasm and nucleus following ERK or JNK inhibition. (B) Approximately 100 cells in panel (A) were counted, and TAZ localization was analyzed in these cells. The counting procedure was done using the Image J program. The number of cells that showed an even cytoplasmic-nuclear or cytoplasm-dominant TAZ localization was higher in the presence of an ERK or JNK inhibitor than in the absence of either inhibitor. (C) Cell lysates in panel (A) were prepared, and the activity of the Hippo signaling pathway components LATS and MST kinase was analyzed by immunoblotting. The phosphorylation status of LATS and MST kinase was analyzed with p-LATS1 and p-MST1/2 antibodies, respectively. Total LATS1 and MST2 levels were detected as a loading control.</p