Fluorescence response of human HER2+ cancer- and MCF-12F normal cells to 200MHz ultrasound microbeam stimulation: a preliminary study of membrane permeability variation

Targeted mechanical cell stimulation has been extensively studied for a better understanding of its effect on cellular mechanotransduction signaling pathways and structures by utilizing a variety of mechanical sources. In this work, an ultrasound-driven single cell stimulation method is thus proposed, and a preliminary study is carried out by comparing the fluorescence intensities representing a change in cell membrane permeability between MDA-MB-435 human HER2+ cancer cells (∼40-50 μm in diameter) and MCF-12F normal cells (∼50-60 μm) in the presence of ultrasound. A 200 MHz single element zinc oxide (ZnO) transducer is employed to generate ultrasound microbeam (UM) whose beamwidth and depth of focus are 9.5 and 60 μm, comparable to typical cell size. The cells in tetramethyl rhodamine methyl ester (TMRM) are interrogated with 200 MHz sinusoidal bursts. The number of cycles per burst is 5 and the pulse repetition frequency (PRF) is 1 kHz. The temporal variation of fluorescence intensity in each cell is measured as a function of input voltage to the transducer (16, 32, and 47 V), and its corresponding fluorescence images are obtained via a confocal microscope. A systematic method for visualizing UM’s focus by adding Rhodamine B to the immersion medium is also proposed to enhance the precision in aiming the beam at an individual cell.Both types of cells exhibit a decrease in the intensity upon UM irradiation. In particular, normal cells show more fluorescence reduction (down to 0.7 in normalized intensity) than cancer cells (∼0.9) under the same excitation condition of the transducer. With UM being turned off, the normalized intensity level in normal cells is slowly increased to 1.1. The cell images taken before and after UM exposure indicate that the intensity reduction is more pronounced in those cells after exposure. Hence the results show the potential of UM as a non-invasive in vitro stimulation tool for facilitating targeted drug delivery and gene transfection as well as for studying cellular mechanotransduction.     

The development of acoustic cues to coda contrasts in young children learning American English

Research on children's speech perception and production suggests that consonant voicing and place contrasts may be acquired early in life, at least in word-onset position. However, little is known about the development of the acoustic correlates of later-acquired, word-final coda contrasts. This is of particular interest in languages like English where many grammatical morphemes are realized as codas. This study therefore examined how various non-spectral acoustic cues vary as a function of stop coda voicing (voiced vs. voiceless) and place (alveolar vs. velar) in the spontaneous speech of 6 American-English-speaking mother-child dyads. The results indicate that children as young as 1;6 exhibited many adult-like acoustic cues to voicing and place contrasts, including longer vowels and more frequent use of voice bar with voiced codas, and a greater number of bursts and longer post-release noise for velar codas. However, 1;6-year-olds overall exhibited longer durations and more frequent occurrence of these cues compared to mothers, with decreasing values by 2;6. Thus, English-speaking 1;6-year-olds already exhibit adult-like use of some of the cues to coda voicing and place, though implementation is not yet fully adult-like. Physiological and contextual correlates of these findings are discussed.     

Vibration suppression of a cantilever beam using magnetically tuned-mass-damper

Eddy currents are induced by the movement of a conductor through a stationary magnetic field or a time varying magnetic field through a stationary conductor. These currents circulate in the conductive material and are dissipated, causing a repulsive force between the magnet and the conductor. These electromagnetic forces can be used to suppress the vibrations of a flexible structure. A tuned mass damper is a device mounted in structures to reduce the amplitude of mechanical vibrations and is one of the effective vibration suppression methods. In the present study, an improved concept of this tuned mass damper for the vibration suppression of structures is introduced. This concept consists of the classical tuned mass damper and an eddy current damping. The important advantages of this magnetically tuned mass damper are that it is relatively simple to apply, it does not require any electronic devices and external power, and it is effective on the vibration suppression. The proposed concept is designed for a cantilever beam and the analytical studies on the eddy current damping and its effects on the vibration suppression. To show the effectiveness of the proposed concept and verify the eddy current damping model, experiments on a cantilever beam are performed. It is found that the proposed concept could significantly increase the damping effect of the tuned mass damper even if not adequately tuned.     

Facile fabrication of forest‐like ZnO hierarchical structures on conductive fabric substrate

Forest‐like ZnO hierarchical structures were synthesized on the conductive fabric substrate via a simple one‐step electrochemical deposition process. By applying externally a high cathodic voltage to the samples, ZnO microrods were aligned on the seed coated layer and then ZnO branches were formed on the pillars of ZnO microrods, which was caused by the strong deposition potential between the pillars of ZnO microrods and the Zn²⁺ dissolved in growth solution. At the external cathodic voltage of –3 V, the optimized forest‐like ZnO hierarchical structures exhibited high density, high porosity, and good crystallinity. These fabricated forest‐like ZnO hierarchical structures are potentially useful for electronic and chemical sensing applications. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The M α and C-integrals

In this paper, we define the ${M_\alpha }$ -integral of real-valued functions defined on an interval [a, b] and investigate important properties of the ${M_\alpha }$ -integral. In particular, we show that a function f: [a, b] → R is ${M_\alpha }$ -integrable on [a, b] if and only if there exists an $AC{G_\alpha }$ function F such that F′ = f almost everywhere on [a, b]. It can be seen easily that every McShane integrable function on [a, b] is ${M_\alpha }$ -integrable and every ${M_\alpha }$ -integrable function on [a, b] is Henstock integrable. In addition, we show that the ${M_\alpha }$ -integral is equivalent to the C-integral.     

The role of tonicity responsive enhancer sites in the transcriptional regulation of human hsp70-2 in response to hypertonic stress

The inducible 70 kDa heat shock proteins (Hsp70) in mice are encoded by two almost identical genes, hsp70.1 and hsp70.3. Studies have found that only hsp70.1 is induced by hypertonic stress while both hsp70.1 and hsp70.3 genes are expressed in response to heat shock stress. It is unclear if the human counterparts, hsp70-2 and hsp70-1, are differentially regulated by heat shock and osmotic stress. This study found that only hsp70-2 was induced by hypertonic stress in human embryonic kidney epithelial cells and fibroblasts, while heat shock stress induced both hsp70-1 and hsp70-2. The human hsp70-2 promoter region contains three TonE (tonicity-responsive enhancer) sites, which were reported to play an important role in the response to hypertonicity. When the reporter plasmids containing different parts of the 5' flanking region of hsp70-2 were transfected into human embryonic kidney epithelial cells or fibroblasts, one TonE site at -135 was found to play a key role in the response to hypertonicity. The inactivation of the TonE site using site-directed mutagenesis led to the complete loss of induction by hypertonicity, which demonstrates the essential role of the TonE site. This suggests that the TonE site and the TonEBP (TonE binding protein) are the major regulators for the cellular response against high osmolarity in human kidney tissue.     

Overexpression of nicotinamide N-methyltransferase in gastric cancer tissues and its potential post-translational modification

Gastric cancer is one of the most common cancers worldwide. The purpose of this study was to find out potential markers for gastric cancer. Tumor and normal tissues from 152 gastric cancer cases were analyzed by two-dimensional gel electrophoresis (2-DE). The images of silver stained gels were analyzed and statistical analysis of spot intensities revealed that spot 4262 showed higher expression (5.7-fold increase) in cancer tissues than in normal tissues (P < 0.001). It was identified by peptide mass fingerprinting as nicotinamide N-methyltransferase (NNMT). A monoclonal antibody with a detection limit down to 10 ng was produced against NNMT in mouse. Using the prepared monoclonal antibody, western blot analysis of NNMT was performed for gastric tissues from 15 gastric cancer patients and two gastric ulcer patients. The results corroborated those of 2-DE experiments. A single spot was detected in gastric ulcer tissues while four to five spots were detected in gastric cancer tissues. In cancer tissues, two additional spots of acidic and basic form were mainly detected on 2-DE gels. This suggests that NNMT receives a post-translational modification in cancer- specific manner.     

4-1BB (CD137) signals depend upon CD28 signals in alloimmune responses

Our previous study has demonstrated that there is a significant delay of Balb/c cardiac allograft rejection in the C57BL/6 4-1BB-deficient knockout recipient. In this study, we examined the effect of combined blockade of the 4-1BB and CD28 costimulatory pathways on cardiac allograft rejection in the C57BL/6-->Balb/c model. A long-term cardiac allograft survival was induced in CD28/4-1BB- deficient mice (>100 days survival in 3 of 4 mice), which was comparable with CD28-deficient mice (>100 days survival in 2 of 5 mice; P<0.2026). There was no long-term cardiac allograft survival in either wild-type (WT) or 4-1BB-deficient mice, even though 4-1BB-deficient recipients showed a significant delay of cardiac allograft rejection than WT mice. An in vitro mixed leukocyte reaction (MLR) assay showed that 4-1BB-deficient and WT mouse T cells had a similar responsiveness to allostimulation, whereas CD28- and CD28/4-1BB-deficient mouse T cells had a defective responsiveness to allostimulation. Furthermore, 4-1BB-deficient mice showed a similar CTL but an elevated Ab response against alloantigens as compared to WT mice, and the alloimmune responses of 4-1BB-deficient mice were abrogated in the CD28-deficient background. Overall, these results indicate that the CD28 costimulatory pathway plays a major role in the alloimmune response and that 4-1BB signals are dependent upon CD28 signals.     

Association of anti-obesity activity of N-acetylcysteine with metallothionein-II down-regulation

People with upper body or visceral obesity have a much higher risk of morbidity and mortality from obesity-related metabolic disorders than those with lower body obesity. In an attempt to develop therapeutic strategies targeting visceral obesity, depot- specific differences in the expression of genes in omental and subcutaneous adipose tissues were investigated by DNA array technology, and their roles in adipocyte differentiation were further examined. We found that levels of metallothionein-II (MT-II) mRNA and protein expression were higher in omental than in subcutaneous adipose tissues. The study demonstrates that MT-II may play an important role in adipocyte differentiation of 3T3L1 preadipocytes, and that N-acetylcysteine (NAC) inhibits the adipocyte differentiation of 3T3L1 cells by repressing MT-II in a time- and dose-dependent manner. Furthermore, the intraperitoneal administration of NAC to rats and mice resulted in a reduction of body weights, and a marked reduction in visceral fat tissues. These results suggest that MT-II plays important roles in adipogenesis, and that NAC may be useful as an anti-obesity drug or supplement.     

A new class of SN2 reactions catalyzed by protic solvents: Facile fluorination for isotopic labeling of diagnostic molecules

Aprotic solvents are usually preferred for the SN2 reactions, because nucleophilicity and hence SN2 reactivity are severely retarded by the influence of the partial positive charge of protic solvents. In this work, we introduce a remarkable effect of using tertiary alcohols as a reaction medium for nucleophilic fluorination with alkali metal fluorides. In this novel synthetic method, the nonpolar protic tert-alcohol enhances the nucleophilicity of the fluoride ion dramatically in the absence of any kind of catalyst, greatly increasing the rate of the nucleophilic fluorination and reducing formation of byproducts (such as alkenes, alcohols, or ethers) compared with conventional methods using dipolar aprotic solvents. The great efficacy of this method is a particular advantage in labeling radiopharmaceuticals with [18F]fluorine (t1/2 = 110 min) for positron emission tomographic (PET) imaging, and it is illustrated by the synthesis of four [18F]fluoride-radiolabeled molecular imaging probes-[18F]FDG, [18F]FLT, [18F]FP-CIT, and [18F]FMISO-in high yield and purity and in shorter times compared to conventional syntheses.