963 resultados para SIGNAL AMPLIFICATION


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The globular C1q-domain-containing (C1qDC) proteins are a family of versatile pattern recognition receptors via their globular C1q (gC1q) domain to bind various ligands including several PAMPs on pathogens. In this study, a new gC1q-domain-containing protein (AiC1qDC-1) gene was cloned from Argopecten irradians by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNA of AiC1qDC-1 was composed of 733 bp, encoding a signal peptide of 19 residues and a typical gC1q domain of 137 residues containing all eight invariant amino acids in human C1qDC proteins and seven aromatic residues essential for effective packing of the hydrophobic core of AiC1qDC-1. The gC1q domain of AiC1qDC-1, which possessed the typical 10-stranded beta-sandwich fold with a jelly-roll topology common to all C1q family members, showed high homology not only to those of Cl qDC proteins in mollusk but also to those of C1qDC proteins in human. The AiC1qDC-1 transcripts were mainly detected in the tissue of hepatopancreas and also marginally detectable in adductor, heart, mantle, gill and hemocytes by fluorescent quantitative real-time PCR. In the microbial challenge experiment, there was a significant up-regulation in the relative expression level of AiC1qDC-1 in hepatopancreas and hemocytes of the scallops challenged by fungi Pichia pastoris GS115, Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Listonella anguillarum. The recombinant AiC1qDC-1 (rAiC1qDC-1) protein displayed no obvious agglutination against M. luteus and L. anguillarum, but it aggregated P. pastoris remarkably. This agglutination could be inhibited by D-mannose and PGN but not by LPS, glucan or D-galactose. These results indicated that AiC1qDC-1 functioned as a pattern recognition receptor in the immune defense of scallops against pathogens and provided clues for illuminating the evolution of the complement classical pathway. (C) 2010 Elsevier Ltd. All rights reserved.

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C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles as pattern recognition receptors (PRRs) in the innate immunity. In this study, the full-length cDNA of a C-type lectin was cloned from scallop Chlamys farreri (designated as Cflec-5) by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach The full-length cDNA of Cflec-5 was of 1412 bp. The open reading frame encoded a polypeptide of 153 amino acids, including a signal sequence and a conserved carbohydrate-recognition domain with the EPN motif determining the mannose-binding specificity The deduced amino acid sequence of Cflec-5 showed high similarity to members of C-type lectin superfamily. The quantitative real-time PCR was performed to investigate the tissue distribution of Cflec-5 mRNA and its temporal expression profiles in hemocytes post pathogen-associated molecular patterns (PAMPs) stimulation. In healthy scallops, the Cflec-5 mRNA was mainly detected in gill and mantle, and marginally in other tissues The mRNA expression of Cflec-5 could be significantly induced by lipopolysaccharide (LPS) and glucan stimulation and reached the maximum level at 6 h and 12 h, respectively But its expression level did not change significantly during peptidoglycan (PGN) stimulation The function of Cflec-5 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta Gami (DE3) The recombinant Cflec-5 agglutinated Pichia pastoris in a calcium-independent way The agglutinating activity could be inhibited by D-mannose. LPS and glucan, but not by D-galactose or PGN. These results collectively suggested that Cflec-5 was involved in the innate Immune response of scallops and might contribute to nonself-recognition through its interaction with various PAMPs (C) 2010 Elsevier Ltd All rights reserved

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Serine protease inhibitors, critical regulators of endogenous proteases, are found in all multicellular organisms and play crucial roles in host physiological and immunological effector mechanisms. The first mollusk serine proteinase inhibitor (designated AISPI) cDNA was obtained from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the scallop serine protease inhibitor was 1020 bp, consisting of a 5'-terminal untranslated region (UTR) of 39 bp, a 3'-terminal UTR of 147 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 834 bp. The AISPI cDNA encoded a polypeptide of 278 amino acids with a putative signal peptide of 22 amino acids and a mature protein of 256 amino acids. The deduced amino-acid sequence of AISPI contained six tandem and homologous domains similar to that of Kazal-type serine protease inhibitors, including the conserved sequence C-X(7)-C-X(6)-Y-X(3)-C-X(2,3)-C and six cysteine residues responsible for the formation of disulfide bridges, indicating that the AISPI protein from bay scallop should be a member of the Kazal-type serine protease inhibitor family. The temporal expression of AISPI was measured by semi-quantitative RT-PCR after injury or bacterial challenge. After the adductor muscle was wounded or injected with Vibrio anguillarum, the expression of AISPI mRNA in hemolymph was up-regulated and reached the maximum level at 8 and 16 h, respectively, and then progressively dropped back to the original level. The results indicated that AISPI could play an important role in injury healing and immune response in mollusks as it could be induced by injury and bacterial challenge. (c) 2005 Elsevier Ltd. All rights reserved.

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中华绒螯蟹是我国重要的水产经济动物,近年来养殖规模不断扩大,产量持续增加。但是,伴随着养殖规模的扩大,养殖环境也日益恶化并导致了大量疾病的发生,严重制约了中华绒螯蟹养殖业的健康发展。因此,疾病预防和控制对中华绒螯蟹养殖业的可持续发展具有举足轻重的作用。与其他无脊椎动物一样,中华绒螯蟹的免疫系统没有免疫球蛋白和淋巴细胞,而是依靠由细胞免疫和体液免疫构成的固有免疫系统来对病原进行识别和清除。中华绒螯蟹的固有免疫机制的研究有助于推动中华绒螯蟹病害防治工作的开展。 本研究采用大规模EST测序方法,结合末端快速扩增(rapid amplification of cDNA ends,RACE)技术从中华绒螯蟹血淋巴中克隆到了过氧化物还原酶(peroxiredoxin,EsPrx6)和硫氧还蛋白(thioredoxin,EsTrx1)基因的cDNA 全长序列;采用实时荧光定量PCR 技术检测了这两个基因在健康个体中表达的组织分布情况以及鳗弧菌刺激后血淋巴细胞中的时序表达规律;同时,将这两个基因的编码区克隆到pET 系列载体,并在大肠杆菌中实现了重组表达,并进行了体外活性检测。 过氧化物还原酶是一个抗氧化蛋白超家族,在保护机体免受活性氧(reactive oxygen species,ROS)的伤害中发挥着重要作用。中华绒螯蟹Prx6(EsPrx6) 基因的cDNA 全长为1076 bp,5` UTR(untranslated region,UTR) 为69 bp,3` UTR 为347 bp,开放阅读框(open reading frame,ORF)为660 bp,编码219 个氨基酸的蛋白。mRNA 3`-端具有多聚腺苷酸加尾信号(polyadenylation signal)AATAAA 和polyA 尾巴。EsPrx6 的预测分子量为 24 kDa,理论等电点为6.21,具有一个保守的Prx 结构域、一个AhpC 结构域和过氧化物酶催化活性中心PVCTTE,表明EsPrx6 属于1-Cys 型Prx。在所检测的组织中均有EsPrx6 的表达,其中以肝胰腺表达量最高,为血淋巴细胞中表达量的17.4 倍。鳗弧菌刺激后,血淋巴细胞中EsPrx6 的表达下降,到12 h 时,实验组显著低于对照组(P<0.05);随时间推移,表达水平逐渐回升,但在整个实验期间,都没有恢复到起始水平。将EsPrx6 进行体外重组并在大肠杆菌E. coli BL21(DE3)中实现表达,重组EsPrx6 具有预期的抗氧化活性和过氧化物酶活性,其中抗氧化活力为14.69 U/mg 蛋白,高于相同条件下GSH 的抗氧化力(P<0.05),过氧化物酶活力为23.46 U/mg 蛋白。结果表明,EsPrx6 作为一种重要的抗氧化剂,在中华绒螯蟹抵御ROS 可能引起的氧化损伤方面具有重要作用。 硫氧还蛋白是广泛存在于生物体内的一种具有硫醇依赖性的具有还原活性的蛋白。中华绒螯蟹Trx1(EsTrx1)基因的cDNA 全长为641 bp,5` UTR 为17 bp,3` UTR 为306 bp,开放阅读框为318 bp,编码105 个氨基酸。EsTrx1 的预测分子量为12.2 kDa,理论等电点为4.8。EsTrx1 不含信号肽,其氨基酸序列与其他动物的Trx1s 具有高度相似性,如与地中海黄蝎的Trx1 相似度达到73%;而与其他物种Trx2 的同源性很低,相似度仅为14.3-22.8%,表明EsTrx1 属于Trx1 亚族。实时荧光定量PCR 检测发现,EsTrx1 在鳃、性腺、肝胰腺、肌肉、心脏和血淋巴细胞中都有表达。血淋巴细胞中EsTrx1 mRNA 的表达量在菌刺激后上升,刺激后6 h,实验组表达量显著高于对照组和空白组(P<0.05),然后逐渐恢复到刺激前水平。为进一步探讨其生物学功能,将EsTrx1 进行体外重组并在大肠杆菌E. coli BL21(DE3)得到表达,重组EsTrx1 具有预期的氧化还原调节活性,抗氧化活力为3.06 U/mg,且抗氧化活力高于GSH(P<0.05)。rEsTrx1 的二硫键还原活力为5.03,低于凡纳滨对虾的二硫键还原活力(10.44),接近于大肠杆菌(4.93),小牛胸腺(6.50)和小牛肝脏(5.09),而高于鲍鱼Trx2(1.83)活力。结果表明,EsTrx1 在生理条件下能够作为一种重要的抗氧化剂,参与对细菌感染的免疫应答反应。

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对虾病害在世界范围内的广泛传播,给水产养殖和沿海农村经济造成了重大损失。深入开展对虾免疫机制研究并在此基础上寻找对虾疾病防治的有效方法已成为当务之急。研究表明,当对虾等甲壳动物受到外界病原刺激时,其体内的吞噬细胞在吞噬活动中会激活磷酸己糖支路的代谢,引起呼吸爆发,产生多种活性氧分子。另外,受到病原侵染的对虾还会产生其他多种免疫反应,这些免疫反应将消耗大量的能量(ATP),产能的呼吸链会加速运转,由此也会引发大量活性氧的产生。这些活性氧分子可以杀灭入侵的病原微生物,但同时由于活性氧分子反应的非特异性,它们也会对宿主的细胞、组织和器官造成严重伤害,进而导致对虾生理机能的损伤和免疫系统的破坏。所以,消除对虾体内因过度免疫反应产生的过量氧自由基将能够增强其抵御病原侵染的能力,提高免疫力。本论文从中国明对虾体内克隆了线粒体型超氧化物歧化酶(mMnSOD)、胞质型超氧化物歧化酶(cMnSOD)、过氧化氢酶(Catalase)和过氧化物还原酶(Peroxiredoxin)等四种与免疫系统相关的抗氧化酶基因,分析了它们的分子结构特征,组织分布及应答不同病原刺激的表达变化模式,并对其中的mMnSOD基因和Peroxiredoxin基因进行了体外重组表达、分离纯化和酶活性分析。 采用RACE技术从中国明对虾血细胞中克隆了两个超氧化物歧化酶(SOD)基因,通过序列比对分析发现,其中一个为mMnSOD基因,另一个为cMnSOD基因。mMnSOD基因的cDNA全长为1185个碱基,其中开放阅读框为660个碱基,编码220个氨基酸,其中推测的信号肽为20个氨基酸。多序列比对结果显示中国明对虾mMnSOD基因的推导氨基酸序列与罗氏沼虾、蓝蟹的推导氨基酸序列同源性分别为88%和82%。Northern blot结果表明,该基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。半定量RT-PCR结果显示,对虾感染病毒3 h时,该基因在血细胞和肝胰脏中的转录水平显著升高。此外,通过构建原核表达载体,本研究对该基因进行了体外重组表达,并对纯化的重组蛋白进行了质谱鉴定和酶活分析。cMnSOD基因的cDNA全长为1284个碱基,其中开放阅读框为861个碱基,编码287个氨基酸。多序列比对结果显示中国明对虾cMnSOD基因的推导氨基酸序列与斑节对虾和凡纳滨对虾的同源性高达98%和94%。组织半定量结果显示,cMnSOD基因在对虾被检测的各个组织中均有表达。 另外,半定量RT-PCR结果表明,对虾感染病毒23h时,该基因在肝胰脏中的转录上升到正常水平的3.5倍;而感染后59 h时,该基因在血细胞中的转录上升到正常水平的2.5倍。 利用根据其他生物过氧化氢酶保守氨基酸序列设计的简并引物,结合RACE技术,从中国明对虾肝胰脏中克隆到了过氧化氢酶基因的部分片段,片段长1725个碱基。多序列比对结果发现目前所得中国明对虾Catalase基因部分片段的推导氨基酸序列与罗氏沼虾和皱纹盘鲍Catalase氨基酸序列的同源性分别达到95%和73%。通过实时荧光定量PCR技术对中国明对虾Catalase基因在各个组织中的分布情况及病毒感染后该基因在血细胞和肝胰脏中的转录变化进行了研究。结果发现,该基因在肝胰脏、鳃、肠和血细胞中表达水平较高,在卵巢、淋巴器官和肌肉中的表达水平相对较弱;感染病毒23 h和37 h时,对虾血细胞和肝胰脏中该基因mRNA的表达量分别出现显著性上升。 依据中国明对虾头胸部cDNA文库提供的部分片段信息,结合SMART-RACE技术,从中国明对虾肝胰脏中克隆到了过氧化物还原酶基因(Peroxiredoxin), 该基因的cDNA全长为942个碱基,其中开放阅读框为594个碱基,编码198个氨基酸。中国明对虾Peroxiredoxin基因的推断氨基酸序列与伊蚊、文昌鱼和果蝇等Peroxiredoxin基因的推断氨基酸序列同源性分别为77%、76%和73%。其蛋白理论分子量为22041.17 Da,pI为5.17。Northern blot结果表明,Peroxiredoxin基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。实时荧光定量PCR结果显示,弧菌感染后,该基因在对虾血细胞和肝胰脏中的转录水平都有明显变化并且表达模式不同。另外,对该基因进行了体外重组表达,并对纯化的重组蛋白进行了质谱鉴定和酶活性分析。酶活性分析表明,复性后的重组蛋白能在DTT存在的条件下还原H2O2。

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HSP22 is a member of a small HSP subfamily contributing to the growth, transformation and apoptosis of the cell as well as acting as a molecular chaperone. In the present study, CfHSP22 cDNA was cloned from Chlamys farreri by the rapid amplification of cDNA ends technique. The full-length cDNA of CfHSP22 was of 1279 bp, consisting of a 5'-terminal untranslated region (5'UTR) of 122 bp, a 3'UTR of 581 bp with a canonical polyadenylation signal sequence AATAAA and a poly( A) tail, and an open reading frame of 576 bp encoding a polypeptide with a molecular mass of 22.21 kDa and a predicted isoelectric point of 9.69. There was an alpha-crystallin domain, a hallmark of the sHSP subfamily, in the C-terminus, and the deduced amino acid sequence of CfHSP22 showed high similarity to previously identified HSP22s. CfHSP22 was constitutively expressed in the haemocyte, muscle, kidney, gonad, gill, heart and hepatopancreas, and the expression level in the hepatopancreas was higher than that in the other tissues. CfHSP22 transcription was up-regulated and reached a maximal level at 12 h after the bacterial challenge, and then declined progressively to the original level at 48 h. These results suggested that CfHSP22 perhaps play a critical role in response to the bacterial challenge in haemocytes of scallop C. farreri.

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Using the LAMP method, a highly specific and sensitive detection system for genetically modified soybean (Roundup Ready) was designed. In this detection system, a set of four primers was designed by targeting the exogenous 35S epsps gene. Target DNA was amplified and visualized on agarose gel within 45 min under isothermal conditions at 65 degrees C. Without gel electrophoresis, the LAMP amplicon was visualized directly in the reaction tube by the addition of SYBR Green I for naked-eye inspection. The detection sensitivity of LAMP was 10-fold higher than the nested PCR established in our laboratory. Moreover, the LAMP method was much quicker, taking only 70 min, as compared with 300 min for nested PCR to complete the analysis of the GM soybean. Compared with traditional PCR approaches, the LAMP procedure is faster and more sensitive, and there is no need for a special PCR machine or electrophoresis equipment. Hence, this method can be a very useful tool for GMO detection and is particularly convenient for fast screening.

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A novel manganese superoxide dismutase (MnSOD) was cloned from bay scallop Argopecten irradians by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of MnSOD was of 1207 bp with a 678 bp open reading frame encoding 226 amino acids. The deduced amino acid sequence contained a putative signal peptide of 26 amino acids. Sequence comparison showed that the MnSOD of A. irradians shared high identity with MnSOD in invertebrates and vertebrates, such as MnSOD from abalone Haliotis discus discus (ABG88843) and frog Xenopus laevis (AAQ63483). Furthermore, the 3D structure of bay scallop MnSOD was predicted by SWISS-MODEL Protein Modelling Server and compared with those of other MnSODs. The overall structure of bay scallop MnSOD was similar to those of zebrafish Danio rerio, fruit fly Drosophila melanogaster, Chinese shrimp Fenneropenaeus chinensis, human Homo sapiens, and had the highest similarity to scallop Mizuhopecten yessoensis and abalone H. discus discus. A quantitative real-time PCR (qRT-PCR) assay was developed to detect the mRNA expression of MnSOD in different tissues and the temporal expression in haemocytes following challenge with the bacterium Vibrio anguillarum. A higher-level of mRNA expression of MnSOD was detected in gill and mantle. The expression of MnSOD reached the highest level at 3 h post-injection with V. anguillarum and then slightly recovered from 6 to 48 h. The results indicated that bay scallop MnSOD was a constitutive and inducible protein and thus could play an important role in the immune responses against V anguillarum infection. (c) 2008 Elsevier Ltd. All rights reserved.

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Heat shock protein 70 (HSP70), the primary member of HSPs that are responsive of thermal stress, is found in all multicellular organisms and functions mostly as molecular chaperon. The inducible HSP70 cDNA cloned from Pacific abalone (Haliotis discus hannai) using rapid amplification of cDNA ends (RACE), was highly homologous to other HSP70 genes. The full-length cDNA of the Pacific abalone HSP70 was 2631 bp, consisting of a 5'-terminal untranslated region (UTR) of 90 bp, a 3'-terminal UTR of 573 by with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1968 bp. The HSP70 cDNA encoded a polypeptide of 655 amino acids with an ATPase domain of 382 amino acids, the substrate peptide binding domain of 161 amino acids and a C-terminus domain of 112 amino acids. The temporal expression of HSP70 was measured by semi-quantitative RT-PCR after heat shock and bacterial challenge. Challenge of Pacific abalone with heat shock or the pathogenic bacteria Vibrio anguillarum resulted in a dramatic increase in the expression of HSP70 mRNA level in muscle, followed by a recovery to normal level after 96 h. Unlike the muscle, the levels of HSP70 expression in gills reached the top at 12 h and maintained a relatively high level compared with the control after thermal and bacterial challenge. The upregulated mRNA expression of HSP70 in the abalone following heat shock and infection response indicates that the HSP70 gene is inducible and involved in immune response. (c) 2006 Elsevier Ltd. All rights reserved.

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针对远距离声源发射的水声信号微弱、水声接收设备电源能量有限的特点,提出一种功耗小、对无源元件误差灵敏度低、高增益放大的微弱水声信号通用放大电路。系统采用场效应管共源单调谐放大器为前置放大级,由四级级联低功耗运放构成带通滤波放大电路,省去传统的R、C低通网络,实现了对微弱水声信号的高增益放大和海洋背景噪声的归一化处理。通过计算电路网络传递函数极点证明了电路系统的稳定性。海上使用表明系统具有精度高、适应性强、电路稳定性好、功耗小等优点。

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Web threats are becoming a major issue for both governments and companies. Generally, web threats increased as much as 600% during last year (WebSense, 2013). This appears to be a significant issue, since many major businesses seem to provide these services. Denial of Service (DoS) attacks are one of the most significant web threats and generally their aim is to waste the resources of the target machine (Mirkovic & Reiher, 2004). Dis-tributed Denial of Service (DDoS) attacks are typically executed from many sources and can result in large traf-fic flows. During last year 11% of DDoS attacks were over 60 Gbps (Prolexic, 2013a). The DDoS attacks are usually performed from the large botnets, which are networks of remotely controlled computers. There is an increasing effort by governments and companies to shut down the botnets (Dittrich, 2012), which has lead the attackers to look for alternative DDoS attack methods. One of the techniques to which attackers are returning to is DDoS amplification attacks. Amplification attacks use intermediate devices called amplifiers in order to amplify the attacker's traffic. This work outlines an evaluation tool and evaluates an amplification attack based on the Trivial File Transfer Proto-col (TFTP). This attack could have amplification factor of approximately 60, which rates highly alongside other researched amplification attacks. This could be a substantial issue globally, due to the fact this protocol is used in approximately 599,600 publicly open TFTP servers. Mitigation methods to this threat have also been consid-ered and a variety of countermeasures are proposed. Effects of this attack on both amplifier and target were analysed based on the proposed metrics. While it has been reported that the breaching of TFTP would be possible (Schultz, 2013), this paper provides a complete methodology for the setup of the attack, and its verification.

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Draper, J., Darby, R.M., Beckmann, M., Maddison, A.L., Mondhe, M., Sheldrick, C., Taylor, J., Goodacre, R., and Kell, D.B. (2002) Metabolic Engineering, metabolite profiling and machine learning to investigate the phloem-mobile signal in systemic acquired resistance in tobacco. First International Congress on Plant Metabolomics, Wageningen, The Netherlands

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This dissertation describes a model for acoustic propagation in inhomogeneous flu- ids, and explores the focusing by arrays onto targets under various conditions. The work explores the use of arrays, in particular the time reversal array, for underwater and biomedical applications. Aspects of propagation and phasing which can lead to reduced focusing effectiveness are described. An acoustic wave equation was derived for the propagation of finite-amplitude waves in lossy time-varying inhomogeneous fluid media. The equation was solved numerically in both Cartesian and cylindrical geometries using the finite-difference time-domain (FDTD) method. It was found that time reversal arrays are sensitive to several debilitating factors. Focusing ability was determined to be adequate in the presence of temporal jitter in the time reversed signal only up to about one-sixth of a period. Thermoviscous absorption also had a debilitating effect on focal pressure for both linear and nonlinear propagation. It was also found that nonlinearity leads to degradation of focal pressure through amplification of the received signal at the array, and enhanced absorption in the shocked waveforms. This dissertation also examined the heating effects of focused ultrasound in a tissue-like medium. The application considered is therapeutic heating for hyperther- mia. The acoustic model and a thermal model for tissue were coupled to solve for transient and steady temperature profiles in tissue-like media. The Pennes bioheat equation was solved using the FDTD method to calculate the temperature fields in tissue-like media from focused acoustic sources. It was found that the temperature-dependence of the medium's background prop- erties can play an important role in the temperature predictions. Finite-amplitude effects contributed excess heat when source conditions were provided for nonlinear ef- fects to manifest themselves. The effect of medium heterogeneity was also found to be important in redistributing the acoustic and temperature fields, creating regions with hotter and colder temperatures than the mean by local scattering and lensing action. These temperature excursions from the mean were found to increase monotonically with increasing contrast in the medium's properties.

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We describe a 42.6 Gbit/s all-optical pattern recognition system which uses semiconductor optical amplifiers (SOAs). A circuit with three SOA-based logic gates is used to identify the presence of specific port numbers in an optical packet header.