Structure transition in myosin association with the change of concentration: solubility equilibrium under specified KCl and pH condition

We observed, for the first time, the elementary process for the ordered self-assembly formation of myosin in solution. It was realized exclusively under the specific condition of 200 mM KCl, 5 mM phosphate buffer, pH 7.08, at 15-20 degrees C, which is called the transition-generating condition (TGC). Described more in detail: pure myosin extracted from rabbit skeletal muscle exhibited the structural transition in its association form only when the myosin concentration c was changed under TGC. The myosin solubility was saturated in both edges of the total myosin concentration c > 10.0 mg/mL (solubility region II) and c < or = 0.25 mg/mL (solubility region I). In the intermediate region, the association structure of myosin changed stepwise with decreasing c. The steps were classified into four regions: region I (c < or = 0.25 mg/mL), II (0.25 < or = c < or = 0.50 mg/mL), III (0.50 < or = c < or = 5.0 mg/mL), and IV (c > 5.0 mg/mL). In each region except II, the plot of the relative soluble myosin concentration c(aq)/c against c(-1) gave a straight line of different slopes, certifying that myosin constructs self-assemblies by the closed association mechanism and that the self-assembly takes dual structures in each region. In region II, a drastic transition occurred in the self-assembled dual structures. Here, a highly associated (insoluble) giant assembly would break into soluble assemblies composed of several myosin molecules. The solubility region I originates a driving force for this structural transition. The basic binding unit of the self-assembly would be a parallel myosin-dimer constructed by the intermolecular axial staggers of 14.3 and 43 nm, as is observed by X-ray diffraction for the thick filament assembly or light meromyosin paracrystals. Myosin could take a single rod-like chain form only in an extremely low concentration region of c < or = c(aq,0) (= 0.053 mg/mL). The association behavior revealed in the present study suggests strongly that the complementary charge cluster and its electrostatic interaction between parallel myosin rods play a crucial role for the ordered self-assembly formation and that the specific electrostatic atmosphere of the solution under TGC is essential to the association mechanism in skeletal muscle myosin, or the thick filament formation of the mammals.     

Light‐induced stabilization of ACS contributes to hypocotyl elongation during the dark‐to‐light transition in Arabidopsis seedlings

Hypocotyl growth during seedling emergence is a crucial developmental transition influenced by light and phytohormones such as ethylene. Ethylene and light antagonistically control hypocotyl growth in either continuous light or darkness. However, how ethylene and light regulate hypocotyl growth, including seedling emergence, during the dark‐to‐light transition remains elusive. Here, we show that ethylene and light cooperatively stimulate a transient increase in hypocotyl growth during the dark‐to‐light transition via the light‐mediated stabilization of 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthases (ACSs), the rate‐limiting enzymes in ethylene biosynthesis. We found that, in contrast to the known inhibitory role of light in hypocotyl growth, light treatment transiently increases hypocotyl growth in wild‐type etiolated seedlings. Moreover, ACC, the direct precursor of ethylene, accentuates the effects of light on hypocotyl elongation during the dark‐to‐light transition. We determined that light leads to the transient elongation of hypocotyls by stabilizing the ACS5 protein during the dark‐to‐light transition. Furthermore, biochemical analysis of an ACS5 mutant protein bearing an alteration in the C‐terminus indicated that light stabilizes ACS5 by inhibiting the degradation mechanism that acts through the C‐terminus of ACS5. Our study reveals that plants regulate hypocotyl elongation during seedling establishment by coordinating light‐induced ethylene biosynthesis at the post‐translational level. Moreover, the stimulatory role of light on hypocotyl growth during the dark‐to‐light transition provides additional insights into the known inhibitory role of light in hypocotyl development.     

Alteration of integrin-dependent adhesion and signaling in EMT-like MDCK cells established through overexpression of calreticulin

Calreticulin (CRT) is a multi-functional Ca(2+) -binding molecular chaperone in the endoplasmic reticulum. We previously reported that kidney epithelial cell-derived Madin-Darby Canine Kidney cells were transformed into mesenchymal-like cells by gene transfection of CRT. In this study, we investigated the altered characteristics of cell adhesion in these epithelial-mesenchymal transition (EMT)-like cells. Several extracellular matrix substrata were tested, and cell adhesion to fibronectin was found to be specifically increased in the CRT-overexpressing cells compared to controls. The expression of integrins was significantly up-regulated in subunits α5 and αV, resulting in an increase in the formation of complexes such as α5β1 and αVβ3. These integrins also contributed to the enhanced binding of fibronectin. In the CRT-overexpressing cells, the phosphorylation of Akt, a downstream target of integrin-linked kinase (ILK), was up-regulated on attachment to fibronectin or collagen IV. Integrin-associated signaling through ILK was also promoted on attachment to fibronectin, suggesting some of the correlation between ILK and Akt in the CRT-overexpressing cells. Furthermore, on treatment with 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester, a membrane-permeable Ca(2+) chelator, the enhanced Akt signaling was suppressed with a concomitant decrease in the formation of complexes between integrins and ILK in the CRT-overexpressing cells. In conclusion, these findings demonstrate that CRT regulates cell-substratum adhesion by modulating integrin-associated signaling through altered Ca(2+) homeostasis in the CRT-overexpressing EMT-like cells, suggesting a novel regulatory role for CRT in EMT.     

Gel formation and low-temperature intramolecular conformation transition of a triple-helical polysaccharide lentinan in water

The gelation behavior of the triple-helical polysaccharide lentinan fractions having different molecular weights in water at 25 degrees C were studied by using a rheometer. The analysis of concentration and molecular weight dependence of shear stress and shear viscosity showed that aqueous lentinan is a typical shear-thinning fluid, possessing potential as a viscosity control agent, and that a weak gel with entangled network structure formed. The dynamic oscillatory behavior of lentinan in the temperature range of 1-15 degrees C was also investigated by rheologic method. The storage modulus G' and complex viscosity eta* increased first with decreasing temperature, and underwent a maximum centered at 7-9 degrees C, and then decreased with further decreasing temperature. This abnormal phenomenon was ascribed to formation of rigid structure in the gel state, which was confirmed by the experimental results from micro-DSC. The micro-DSC curves showed that an endothermic peak appeared at 7-9 degrees C for lentinan in water upon heating, which was attributable to the intramolecular order-disorder structure transition similar to triple-helical polysaccharide schizophyllan. Namely, at lower temperature, the side glucose residues of lentinan (triplix II) formed a well-organized triple-helical structure (triplix I) through hydrogen-bonding with the surrounding water molecules. Moreover, this conformation transition was proved to be thermally reversible. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 852-861, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Recent Developments and Understanding of Novel Mixed Transition‐Metal Oxides as Anodes in Lithium Ion Batteries

Mixed transition‐metal oxides (MTMOs), including stannates, ferrites, cobaltates, and nickelates, have attracted increased attention in the application of high performance lithium‐ion batteries. Compared with traditional metal oxides, MTMOs exhibit enormous potential as electrode materials in lithium‐ion batteries originating from higher reversible capacity, better structural stability, and high electronic conductivity. Recent advancements in the rational design of novel MTMO micro/nanostructures for lithium‐ion battery anodes are summarized and their energy storage mechanism is compared to transition‐metal oxide anodes. In particular, the significant effects of the MTMO morphology, micro/nanostructure, and crystallinity on battery performance are highlighted. Furthermore, the future trends and prospects, as well as potential problems, are presented to further develop advanced MTMO anodes for more promising and large‐scale commercial applications of lithium‐ion batteries.     

LncRNA HOTAIR/miR‐613/c‐met axis modulated epithelial‐mesenchymal transition of retinoblastoma cells

Since lncRNAs could modulate neoplastic development by modulating downstream miRNAs and genes, this study was carried out to figure out the synthetic contribution of HOTAIR, miR‐613 and c‐met to viability, apoptosis and proliferation of retinoblastoma cells. Totally 276 retinoblastoma tissues and tumour‐adjacent tissues were collected, and human retinoblastoma cell lines (ie, Y79, HXO‐Rb44, SO‐Rb50 and WERI‐RB1) were also gathered. Moreover, transfections of pcDNA3.1‐HOTAIR, si‐HOTAIR, miR‐613 mimic, miR‐613 inhibitor, pcDNA3.1/c‐met were performed to evaluate the influence of HOTAIR, miR‐613 and c‐met on viability, apoptosis and epithelial‐mesenchymal transition (EMT) of retinoblastoma cells. Dual‐luciferase reporter gene assay was also arranged to confirm the targeted relationship between HOTAIR and miR‐613, as well as between miR‐613 and c‐met. Consequently, up‐regulated HOTAIR and down‐regulated miR‐613 expressions displayed associations with poor survival status of retinoblastoma patients (P < 0.05). Besides, inhibited HOTAIR and promoted miR‐613 elevated E‐cadherin expression, yet decreased Snail and Vimentin expressions (P < 0.05). Simultaneously, cell proliferation and cell viability were also less‐motivated (P < 0.05). Nonetheless, c‐met prohibited the functioning of miR‐613, resulting in promoted cell proliferation and viability, along with inhibited cell apoptosis (P < 0.05). Finally, HOTAIR was verified to directly target miR‐613, and c‐met was the direct target gene of miR‐613 (P < 0.05). In conclusion, the role of lncRNA HOTAIR/miR‐613/c‐met signalling axis in modulating retinoblastoma cells’ viability, apoptosis and expressions of EMT‐specific proteins might provide evidences for developing appropriate diagnostic and treatment strategies for retinoblastoma.     

Breast Cancer‐Derived Exosomes Reflect the Cell‐of‐Origin Phenotype

A manner in which cells can communicate with each other is via secreted nanoparticles termed exosomes. These vesicles contain lipids, nucleic acids, and proteins, and are said to reflect the cell‐of‐origin. However, for the exosomal protein content, there is limited evidence in the literature to verify this statement. Here, proteomic assessment combined with pathway‐enrichment analysis is used to demonstrate that the protein cargo of exosomes reflects the epithelial/mesenchymal phenotype of secreting breast cancer cells. Given that epithelial‐mesenchymal plasticity is known to implicate various stages of cancer progression, the results suggest that breast cancer subtypes with distinct epithelial and mesenchymal phenotypes may be distinguished by directly assessing the protein content of exosomes. Additionally, the work is a substantial step toward verifying the statement that cell‐derived exosomes reflect the phenotype of the cells‐of‐origin.     

Mechanical stability of low-humidity single DNA molecules

DNA electrostatic character is mostly determined by both water and counterions activities in the phosphate backbone, which together with base sequence, further confer its higher order structure. The authors overstretch individual double-stranded DNA molecules in water-ethanol solutions to investigate the modulation of its mechanical stability by hydration and polycations. The authors found that DNA denatures as ethanol concentration is increased and spermine concentration decreased. This is manifested by an increase in melting hysteresis between the stretch and release curves, with sharp transition at 10% ethanol and reentrant behavior at 60%, by a loss of cooperativity in the overstretching transition and by a dramatic decrease of both the persistence length and the flexural rigidity. Changes in base-stacking stability which are characteristic of the B-A transition between 70 and 80% ethanol concentration do not manifest in the mechanical properties of the double-helical molecule at low or high force or in the behavior of the overstretching and melting transitions within this ethanol concentration range. This is consistent with a mechanism in which A-type base-stacking is unstable in the presence of tension. Binding of motor proteins to DNA locally reduces the number of water molecules and therefore, our results may shed light on analogous reduced-water activity of DNA conditions caused by other molecules, which interact with DNA in vivo.     

SPOCK1 promotes the invasion and metastasis of gastric cancer through Slug‐induced epithelial‐mesenchymal transition

Metastasis is a crucial impediment to the successful treatment for gastric cancer. SPOCK1 has been demonstrated to facilitate cancer metastasis in certain types of cancers; however, the role of SPOCK1 in the invasion and metastasis of gastric cancer remains elusive. SPOCK1 and epithelial‐mesenchymal transition (EMT)‐related biomarkers were detected by immunohistochemistry and Western blot in gastric cancer specimens. Other methods including stably transfected against SPOCK1 into gastric cancer cells, Western blot, migration and invasion assays in vitro and metastasis assay in vivo were also performed. The elevated expression of SPOCK1 correlates with EMT‐related markers in human gastric cancer tissue, clinical metastasis and a poor prognosis in patients with gastric cancer. In addition, knockdown of SPOCK1 expression significantly inhibits the invasion and metastasis of gastric cancer cells in vitro and in vivo, inversely, SPOCK1 overexpression results in the opposite effect. Interestingly, SPOCK1 expression has no effect on cell proliferation in vitro and in vivo. Regarding the mechanism(s) of SPOCK1‐induced cells invasion and metastasis, we prove that Slug‐induced EMT is involved in SPOCK1‐facilitating gastric cancer cells invasion and metastasis. The elevated SPOCK1 expression is closely correlated with cancer metastasis and patient survival, and SPOCK1 promotes the invasion and metastasis of gastric cancer through Slug‐mediated EMT, thereby possibly providing a novel therapeutic target for gastric cancer.     

Single fluorescence probes along the reactive center loop reveal site‐specific changes during the latency transition of PAI‐1

The serine protease inhibitor (serpin), plasminogen activator inhibitor‐1 (PAI‐1), is an important biomarker for cardiovascular disease and many cancers. It is therefore a desirable target for pharmaceutical intervention. However, to date, no PAI‐1 inhibitor has successfully reached clinical trial, indicating the necessity to learn more about the mechanics of the serpin. Although its kinetics of inhibition have been extensively studied, less is known about the latency transition of PAI‐1, in which the solvent‐exposed reactive center loop (RCL) inserts into its central β‐sheet, rendering the inhibitor inactive. This spontaneous transition is concomitant with a large translocation of the RCL, but no change in covalent structure. Here, we conjugated the fluorescent probe, NBD, to single positions along the RCL (P13‐P5′) to detect changes in solvent exposure that occur during the latency transition. The results support a mousetrap‐like RCL‐insertion that occurs with a half‐life of 1–2 h in accordance with previous reports. Importantly, this study exposes unique transitions during latency that occur with a half‐life of ～5 and 25 min at the P5′ and P8 RCL positions, respectively. We hypothesize that the process detected at P5′ represents s1C detachment, while that at P8 results from a steric barrier to RCL insertion. Together, these findings provide new insights by characterizing multiple steps in the latency transition.     

A‐kinase‐interacting protein 1 facilitates growth and metastasis of gastric cancer cells via Slug‐induced epithelial‐mesenchymal transition

A‐kinase‐interacting protein 1 (AKIP1) has previously been reported to act as a potential oncogenic protein in various cancers. The clinical significance and biological role of AKIP1 in gastric cancer (GC) is, however, still elusive. Herein, this study aimed to investigate the functional and molecular mechanism by which AKIP1 influences GC. AKIP1 mRNA and protein expressions in GC tissues were examined by quantitative real‐time PCR (qRT‐PCR), Western blot and immunohistochemistry. Other methods including stably transfected against AKIP1 into gastric cancer cells, wound healing, transwell assays, CCK‐8, colony formation, qRT‐PCR and Western blot in vitro and tumorigenesis in vivo were also performed. The up‐regulated expression of AKIP1 in GC specimens significantly correlated with clinical metastasis and poor prognosis in patients with GC. AKIP1 knockdown markedly suppressed GC cells proliferation, invasion and metastasis both in vitro and in vivo. In contrast, AKIP1 overexpression resulted in the opposite effects. Moreover, mechanistic analyses indicated that Slug‐induced epithelial‐mesenchymal transition (EMT) might be responsible for AKIP1‐influenced GC cells behaviour. Our findings demonstrated that high AKIP1 expression significantly correlated with clinical metastasis and unfavourable prognosis in patients with GC. Additionally, AKIP1 promoted GC cells proliferation, migration and invasion by activating Slug‐induced EMT.     

miR‐655 suppresses epithelial‐to‐mesenchymal transition by targeting Prrx1 in triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. The epithelial‐to‐mesenchymal transition (EMT) is a key contributor in the metastatic process. In this study, we found that miR‐655 was down‐regulated in TNBC, and its expression levels were associated with molecular‐based classification and lymph node metastasis in breast cancer. These findings led us to hypothesize that miR‐655 overexpression may inhibit EMT and its associated traits of TNBC. Ectopic expression of miR‐655 not only induced the up‐regulation of cytokeratin and decreased vimentin expression but also suppressed migration and invasion of mesenchymal‐like cancer cells accompanied by a morphological shift towards the epithelial phenotype. In addition, we found that miR‐655 was negatively correlated with Prrx1 in cell lines and clinical samples. Overexpression of miR‐655 significantly suppressed Prrx1, as demonstrated by Prrx1 3′‐untranslated region luciferase report assay. Our study demonstrated that miR‐655 inhibits the acquisition of the EMT phenotype in TNBC by down‐regulating Prrx1, thereby inhibiting cell migration and invasion during cancer progression.     

Enhancement of Thermoelectric Performance of n‐Type PbSe by Cr Doping with Optimized Carrier Concentration

Ti, V, Cr, Nb, and Mo are found to be effective at increasing the Seebeck coefficient and power factor of n‐type PbSe at temperatures below 600 K. It is found that the higher Seebeck coefficients and power factors are due to higher Hall mobility ≈1000 cm² V^?1s^?1 at lower carrier concentration. A larger average ZT value (relevant for applications) can be obtained by an optimization of carrier concentration to ≈10¹⁸–10¹⁹ cm^?3. Even though the highest room temperature power factor ≈3.3 × 10^?3 W m^?1 K^?2 is found in 1 at% Mo‐doped PbSe, the highest ZT is achieved in Cr‐doped PbSe. Combined with the lower thermal conductivity, ZT is improved to ≈0.4 at room temperature and peak ZTs of ≈1.0 are observed at ≈573 K for Pb_0.9925Cr_0.0075Se and ≈673 K for Pb_0.995Cr_0.005Se. The calculated device efficiency of Pb_0.995Cr_0.005Se is as high as ≈12.5% with cold side 300 K and hot side 873 K, higher than those of all the n‐type PbSe materials reported in the literature.