BACKGROUND & AIMS: :Accumulating evidence indicates that Toll-like receptor (TLR) signaling adapter protein interactions with Toll/Interleukin-1 Receptor (TIR) domains present in sensory neurons may modulate neuropathic pain states. Following ligand interaction with TLRs, TIR serves to both initiate intracellular signaling and facilitate recruitment of signaling adapter proteins to the intracytoplasmic domain. Although TLR TIR is central to a number of TLR signaling cascades, its role in sensory neurons is poorly understood. In this study we investigated the degree to which TLR TIR decoy peptide modified to include a TAT sequence (Trans-Activator of Transcription gene in HIV; TAT-4BB) affected LPS-induced intracellular calcium flux and excitation in sensory neurons, and behavioral changes due to TLR4 active metabolite, morphine-3-glucuronide (M3G) exposure in vivo. TAT-4BB inhibited LPS-induced calcium changes in a majority of sensory neurons and decreased LPS-dependent neuronal excitability in small diameter neurons. Acute systemic administration of the TAT-4BB reversed M3G-induced tactile allodynia in a dose-dependent manner but did not affect motor activity, anxiety or responses to noxious thermal stimulus. These data suggest that targeting TLR TIR domains may provide novel pharmacological targets to reduce or reverse TLR4-dependent pain behavior in the rodent.

背景与目标： : 越来越多的证据表明，Toll样受体 (TLR) 信号衔接蛋白与感觉神经元中存在的Toll/Interleukin-1受体 (TIR) 结构域的相互作用可能调节神经性疼痛状态。配体与tlr相互作用后，TIR既可启动细胞内信号传导，又可促进信号衔接蛋白向胞浆内结构域的募集。尽管TLR TIR是许多TLR信号级联的核心，但对其在感觉神经元中的作用知之甚少。在这项研究中，我们调查了修饰为包含TAT序列 (HIV中转录基因的反式激活因子; TAT-4BB) 的TLR TIR诱饵肽对LPS诱导的细胞内钙通量和感觉神经元的兴奋以及由于TLR4活性代谢物引起的行为变化的影响程度，morphine-3-glucuronide (M3G) 体内暴露。TAT-4BB抑制了大多数感觉神经元中LPS诱导的钙变化，并降低了小直径神经元中LPS依赖性神经元的兴奋性。TAT-4BB的急性全身给药以剂量依赖性方式逆转了M3G-induced的触觉异常性疼痛，但不影响运动活动，焦虑或对有害热刺激的反应。这些数据表明，靶向TLR TIR结构域可以提供新的药理学靶标，以减少或逆转啮齿动物的TLR4-dependent疼痛行为。

BACKGROUND & AIMS: :The main objective of this work is to provide an update on synthetic nucleic acid analogues and nanoassemblies as tools in gene therapy. In particular, the synthesis and properties of peptide-oligonucleotide conjugates (POCs), which have high potential in research and as therapeutics, are described in detail. The exploration of POCs has already led to fruitful results in the treatment of neurological diseases, lung disorders, cancer, leukemia, viral, and bacterial infections. However, delivery and in vivo stability are the major barriers to the clinical application of POCs and other analogues that still have to be overcome. This review summarizes recent achievements in the delivery and in vivo administration of synthetic nucleic acid analogues, focusing on POCs, and compares their efficiency.

背景与目标： : 这项工作的主要目的是提供有关合成核酸类似物和纳米组件作为基因治疗工具的更新。特别地，详细描述了肽-寡核苷酸缀合物 (poc) 的合成和性质，它们在研究中具有很高的潜力并作为治疗剂。POCs的探索已经在治疗神经系统疾病，肺部疾病，癌症，白血病，病毒和细菌感染方面取得了丰硕的成果。然而，递送和体内稳定性是POCs和其他类似物临床应用的主要障碍，仍需克服。这篇综述总结了合成核酸类似物的递送和体内给药方面的最新成就，重点是poc，并比较了它们的效率。

BACKGROUND & AIMS: BACKGROUND:A combinatorial phage display approach was previously used to evolve a 12-mer peptide (SVSVGMKPSPRP) with the highest affinity for different semiconductor surfaces. The discovery of the multiple occurrences of the SVSVGMKPSPRP sequence in an all-against-all basic local alignment search tool search of PepBank sequences was unexpected, and a Google search using the peptide sequence recovered 58 results concerning 12 patents and 16 scientific publications. The number of patent and articles indicates that the peptide is perhaps a broad range adhesion peptide. METHODS:To evaluate peptide properties, we conducted a study to investigate peptide adhesion on different inorganic substrates by mass spectrometry and atomic force microscopy for gold, carbon nanotubes, cobalt, chrome alloy, titanium, and titanium alloy substrates. RESULTS:Our results showed that the peptide has a great potential as a linker to functionalize metallic surfaces if specificity is not a key factor. This peptide is not specific to a particular metal surface, but it is a good linker for the functionalization of a wide range of metallic materials. CONCLUSION:The fact that this peptide has the potential to adsorb on a large set of inorganic surfaces suggests novel promising directions for further investigation. Affinity determination of SVSVGMKPSPRP peptide would be an important issue for eventual commercial uses.

背景与目标：

BACKGROUND & AIMS: :Tumor-specific immunotherapy with a Wilms' tumor 1 (WT1) peptide has been on clinical trial for leukemia, myelodysplastic syndrome, breast and lung cancers and is producing promising results. In this study, we treated three patients with renal cell carcinoma with an anchor modified, HLA-A*2402 binding WT1 peptide which was emulsified in Freund's incomplete adjuvant. In two patients tumor growth was suppressed and clinical response was evaluated as stable disease by the RECIST criteria after 3 months of weekly immunizations. Notably, development of new metastases has stopped in these patients for a prolonged period. No deleterious side effects were observed. Peptide-specific T cells were expanded in PBMCs of the patients and a substantial fraction of them bore the surface phenotype consistent with a CD8+ cytotoxic effector population. Although established tumors did not regress further, considering the component of the vaccine, i.e. peptide alone, the stabilization effect suggested the potential of WT1 peptide to develop into a more effective vaccine. To our knowledge, this is the first report of WT1 immunotherapy for renal cell carcinoma. Hopefully, the results will stimulate more extensive clinical studies.

背景与目标： : 使用Wilms' 肿瘤1 (WT1) 肽进行的肿瘤特异性免疫疗法已在白血病，骨髓增生异常综合征，乳腺癌和肺癌的临床试验中，并取得了可喜的结果。在这项研究中，我们用锚定修饰的hla-a * 2402结合WT1肽治疗了3例肾细胞癌患者，该肽在弗氏不完全佐剂中乳化。在每周免疫3个月后，两名患者的肿瘤生长受到抑制，并根据RECIST标准评估临床反应为稳定疾病。值得注意的是，这些患者的新转移已经停止了很长时间。没有观察到有害的副作用。肽特异性T细胞在患者的pbmc中扩增，其中很大一部分具有与CD8细胞毒性效应子群一致的表面表型。尽管确定的肿瘤没有进一步消退，但考虑到疫苗的成分，即单独的肽，稳定作用表明WT1肽有可能发展成为更有效的疫苗。据我们所知，这是WT1免疫疗法治疗肾细胞癌的首次报道。希望这些结果将刺激更广泛的临床研究。

BACKGROUND & AIMS: :The antigenicity of the major outer membrane protein of Chlamydia trachomatis serovar C was assessed by using overlapping hexapeptide homologs of serovar C major outer membrane protein and rabbit antisera in a peptide enzyme-linked immunosorbent assay. Five immunogenic sites were found distributed within variable sequences of the protein: four were immunodominant and three were surface exposed on native elementary bodies of serovar C. None was surface exposed on serovars H, I, and J.

背景与目标： : 通过在肽酶联免疫吸附试验中使用血清C主要外膜蛋白和兔抗血清的重叠六肽同源物评估沙眼衣原体血清型C主要外膜蛋白的抗原性。在蛋白质的可变序列中发现了五个免疫原性位点: 四个是免疫优势，三个表面暴露在血清型C的天然基本体上。没有一个表面暴露在血清型H，I和J上。

BACKGROUND & AIMS: :An undecapeptide which potentiates the beat of the ventricle in the African giant snail, Achatina fulica Ferussac, was purified from the atria of the snail. Its primary structure was determined to be H-Ser-Gly-Gln-Ser-Trp-Arg-Pro-Gln-Gly-Arg-Phe-NH2. This peptide was found to have excitatory actions not only on the ventricle but also on the penis retractor muscle, the buccal muscle and the identified neurons controlling the buccal muscle movement of Achatina.

背景与目标： : 从蜗牛的心房中纯化了一种增强非洲巨型蜗牛Achatina fulica Ferussac心室搏动的十一肽。它的一级结构被确定为H-Ser-Gly-Gln-Ser-Trp-Arg-Pro-Gln-Gly-Arg-Phe-NH2。发现该肽不仅对心室具有兴奋作用，而且对阴茎牵开器肌肉，颊肌和控制Achatina颊肌运动的已识别神经元具有兴奋作用。

BACKGROUND & AIMS: :The synthesis and characterisation of iridium(III) bis(2-(2,4-difluorophenyl)pyridinato-N, C2')-2(4-carboxylphenyl)imidazo[4,5-f][1,10]phenanthroline perchlorate, [Ir(dfpp)(2)(picCOOH)](+) and its octaarginine conjugate [Ir(dfpp)(2)(picCONH-Arg(8))](9+) are reported. Both complex and conjugate exhibit intense and long-lived luminescence, which is O(2) and pH sensitive. Conjugation to the polyarginine peptide renders the complex very water soluble. The uptake of the parent iridium(III) complex and conjugate are compared in two mammalian cell lines; SP2 myeloma and Chinese hamster ovary (CHO). Both complexes internalise into the cytoplasm, however dye uptake rate and distribution vary with peptide conjugation and with cell identity. Whereas transmembrane transport is thought to have been facilitated by the dimethyl sulfoxide (DMSO) used as co-solvent (0.05% v/v) for the parent complex, the octaarginine, the dye-conjugate (iridium-R(8)) is membrane permeable in water only. Both complexes exhibit high cytotoxicity, evident through blebbing and vacuole formation within living cells, indicative of apoptosis, within 30min of exposure to the probe. The IC(50) recorded for the cells in the dark was independent, in the case of the parent complex, of the identity of the cell, with IC(50) of 84.8μM and 88μM respectively for SP2 and CHO cells. The IC(50) approximately doubled for the polyarginine conjugate and displayed a significant dependence on cell type with IC(50) of 35μM and 54.1μM respectively for SP2 and CHO cells. These IC(50) values were recorded in the dark. However under irradiation cell death is considerably faster. Evidence from imaging suggests that the conjugate penetrates the nucleus whereas the parent does not, indicating that nuclear penetration may play a role in cytotoxicity.

背景与目标： : 铱 (III) 双 (2-(2,4-二氟苯基) 吡啶-N，C2 ')-2(4-羧基苯基) 咪唑并 [4,5-f][1,10] 菲咯啉高氯酸盐的合成和表征，报道了 [Ir(dfpp)(2)(picCOOH)]() 及其八精氨酸缀合物 [Ir(dfpp)(2)(picCONH-Arg(8))](9)。复合物和缀合物都表现出强烈和长寿命的发光，O(2) 和pH敏感。与聚精氨酸肽的结合使复合物非常水溶性。在两种哺乳动物细胞系中比较了母体铱 (III) 复合物和结合物的吸收; SP2骨髓瘤和中国仓鼠卵巢 (CHO)。两种复合物都内化到细胞质中，然而，染料的吸收速率和分布随肽缀合和细胞特性而变化。而跨膜运输被认为是由用作母体复合物 (八精氨酸) 的共溶剂 (0.05% v/v) 的二甲基亚砜 (DMSO) 促进的。染料偶联物 (铱-R(8)) 仅在水中具有膜渗透性。两种复合物均表现出高细胞毒性，通过活细胞内的起泡和液泡形成明显，表明细胞凋亡，在暴露于探针的30分钟内，在黑暗中记录的细胞的IC(50) 是独立的，在母体复合体的情况下，细胞的身份，对于SP2和CHO细胞，IC(50) 分别为84.8 μ m和88 μ m。对于聚精氨酸缀合物，IC(50) 大约增加了一倍，并且对于SP2和CHO细胞，IC(50) 分别为35 μ m和54.1 μ m的细胞类型显示出显着依赖性。这些IC(50) 值被记录在黑暗中。然而，在辐射下，细胞死亡要快得多。来自成像的证据表明，缀合物穿透细胞核，而亲本没有穿透细胞核，表明核渗透可能在细胞毒性中起作用。

BACKGROUND & AIMS: :Whereas ribosomes efficiently catalyze peptide bond synthesis by most amino acids, the imino acid proline is a poor substrate for protein synthesis. Previous studies have shown that the translation factor eIF5A and its bacterial ortholog EF-P bind in the E site of the ribosome where they contact the peptidyl-tRNA in the P site and play a critical role in promoting the synthesis of polyproline peptides. Using misacylated Pro-tRNAPhe and Phe-tRNAPro, we show that the imino acid proline and not tRNAPro imposes the primary eIF5A requirement for polyproline synthesis. Though most proline analogs require eIF5A for efficient peptide synthesis, azetidine-2-caboxylic acid, a more flexible four-membered ring derivative of proline, shows relaxed eIF5A dependency, indicating that the structural rigidity of proline might contribute to the requirement for eIF5A. Finally, we examine the interplay between eIF5A and polyamines in promoting translation elongation. We show that eIF5A can obviate the polyamine requirement for general translation elongation, and that this activity is independent of the conserved hypusine modification on eIF5A. Thus, we propose that the body of eIF5A functionally substitutes for polyamines to promote general protein synthesis and that the hypusine modification on eIF5A is critically important for poor substrates like proline.

背景与目标： : 尽管核糖体可以有效地催化大多数氨基酸的肽键合成，但亚氨基酸脯氨酸是蛋白质合成的不良底物。先前的研究表明，翻译因子eIF5A及其细菌直系同源物EF-P在核糖体的E位点结合，它们与P位点的肽基-tRNA接触，并在促进聚脯氨酸肽的合成中起关键作用。使用未酰化的前tRNAPhe和Phe-tRNAPro，我们表明亚氨基酸脯氨酸而不是tRNAPro对聚脯氨酸合成施加了主要的eIF5A要求。尽管大多数脯氨酸类似物需要eIF5A来进行有效的肽合成，但azetidine-2-caboxylic酸 (脯氨酸的更灵活的四元环衍生物) 显示出松弛的eIF5A依赖性，表明脯氨酸的结构刚性可能有助于eIF5A的需求。最后，我们研究了eIF5A和多胺在促进翻译伸长方面的相互作用。我们证明eIF5A可以消除对一般翻译伸长的多胺要求，并且该活性与eIF5A上保守的hypusine修饰无关。因此，我们建议eIF5A的主体在功能上替代多胺以促进一般蛋白质合成，并且eIF5A上的hypusine修饰对于诸如脯氨酸之类的不良底物至关重要。

BACKGROUND & AIMS: BACKGROUND:N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentrations predict cardiovascular outcome in many settings. There are very few data assessing the utility of NT-proBNP concentrations in the prediction of long-term outcome after cardiac surgery. We assessed the ability of NT-proBNP to predict 3 yr mortality compared with validated clinical risk prediction tools. METHODS:A secondary analysis of a prospectively recruited patient cohort of 1010 patients undergoing cardiac surgery. Baseline clinical details were obtained including EuroSCORE. Multi-variable modelling, area under the receiver operating characteristic curves (AUCs), and net reclassification improvement were utilized. RESULTS:NT-proBNP was a univariable predictor of 3 yr mortality but was no longer a significant predictor in a multivariable model (hazard ratio 1.00 per 250 ng litre(-1), 95% confidence interval 0.98-1.02, P=0.80). The relative and additive predictive values of the preoperative EuroSCORE (both additive and logistic versions) and NT-proBNP concentrations were compared. All were predictive of 3 yr mortality (P<0.001) with almost identical AUCs (0.71 for EuroSCORE, 0.70 for NT-proBNP). When either the EuroSCORE or NT-proBNP concentrations are known, the addition of the other does not improve the ability to predict 3 yr mortality. CONCLUSIONS:Preoperative NT-proBNP concentrations and the EuroSCORE have equivalent, and moderate, predictive accuracy for mortality 3 yr after cardiac surgery. EuroSCORE uses clinical data but is not routinely used for individual clinical risk prediction. NT-proBNP measurement would incur additional costs but can be measured quickly and objectively. With such similar predictive accuracy, factors such as the ease of calculation and cost will likely determine their use in clinical practice.

背景与目标：

BACKGROUND & AIMS: :Alzheimer's disease (AD) is a progressive neurodegenerative disorder that shows cognitive deficits and memory impairment. Extract from the leaves of Gotu Kola (Centella Asiatica) have been used as an alternative medicine for memory improvement in Indian Ayurvedic system of medicine for a long time. Although several studies have revealed its effect in ameliorating the cognitive impairment in rat models of AD and stimulating property on neuronal dendrites of hippocampal region, the molecular mechanism of Gotu Kola on neuroprotection still remains to be elucidated. In this study, we report that phosphorylation of cyclic AMP response element binding protein (CREB) is enhanced in both a neuroblastoma cell line expressing amyloid beta 1-42 (Abeta) and in rat embryonic cortical primary cell culture. In addition, the contribution of two major single components to the enhanced CREB phosphorylatioin was examined. Furthermore, inhibitors were applied in this study revealing that ERK/RSK signaling pathway might mediate this effect of Gotu Kola extract. Taken together, we provide a possible molecular mechanism for memory enhancing property of Gotu Kola extract for the first time.

背景与目标： 阿尔茨海默病 (AD) 是一种进行性神经退行性疾病，表现为认知缺陷和记忆障碍。长期以来，从Gotu Kola (积雪草) 的叶子中提取的提取物已被用作印度阿育吠陀医学系统中改善记忆的替代药物。尽管有几项研究表明其在改善AD大鼠模型的认知障碍以及对海马区神经元树突的刺激作用，但Gotu Kola对神经保护的分子机制仍有待阐明。在这项研究中，我们报告了在表达淀粉样蛋白beta 1-42 (Abeta) 的神经母细胞瘤细胞系和大鼠胚胎皮质原代细胞培养物中，环状AMP反应元件结合蛋白 (CREB) 的磷酸化均增强。此外，还检查了两个主要单一成分对增强的CREB磷酸化蛋白的贡献。此外，本研究还应用了抑制剂，表明ERK/RSK信号通路可能介导了Gotu Kola提取物的这种作用。合在一起，我们首次提供了一种可能的分子机制来增强Gotu Kola提取物的记忆。

BACKGROUND & AIMS: UNLABELLED:Heart failure (HF) is, after cirrhosis, the second-most common cause of ascites. Serum B-type natriuretic peptide (BNP) plays an important role in the diagnosis of HF. Therefore, we hypothesized that BNP would be useful in the differential diagnosis of ascites. Consecutive patients with new onset ascites were prospectively enrolled in this cross-sectional study. All patients had measurements of serum-ascites albumin gradient (SAAG), total protein concentration in ascitic fluid, serum, and ascites BNP. We enrolled 218 consecutive patients with ascites resulting from HF (n = 44), cirrhosis (n = 162), peritoneal disease (n = 10), and constrictive pericarditis (n = 2). Compared to SAAG and/or total protein concentration in ascites, the test that best discriminated HF-related ascites from other causes of ascites was serum BNP. A cutoff of >364 pg/mL (sensitivity 98%, specificity 99%, and diagnostic accuracy 99%) had the highest positive likelihood ratio (168.1); that is, it was the best to rule in HF-related ascites. Conversely, a cutoff ≤ 182 pg/mL had the lowest negative likelihood ratio (0.0) and was the best to rule out HF-related ascites. These findings were confirmed in a 60-patient validation cohort. CONCLUSIONS:Serum BNP is more accurate than ascites analyses in the diagnosis of HF-related ascites. The workup of patients with new onset ascites could be streamlined by obtaining serum BNP as an initial test and could forego the need for diagnostic paracentesis, particularly in cases where the cause of ascites is uncertain and/or could be the result of HF.

背景与目标：

BACKGROUND & AIMS: :Amyloid deposits derived from the amylin peptide accumulate within pancreatic islet beta-cells in most cases of type-2 diabetes mellitus (T2Dm). Human amylin 'oligomers' are toxic to these cells. Using two different experimental techniques, we found that H(2)O(2) was generated during the aggregation of human amylin into amyloid fibrils. This process was greatly stimulated by Cu(II) ions, and human amylin was retained on a copper affinity column. In contrast, rodent amylin, which is not toxic, failed to generate any H(2)O(2) and did not interact with copper. We conclude that the formation of H(2)O(2) from amylin could contribute to the progressive degeneration of islet cells in T2Dm.

背景与目标： : 在大多数2型糖尿病 (T2Dm) 病例中，源自胰淀素肽的淀粉样蛋白沉积物积聚在胰岛 β 细胞内。人胰淀素 “寡聚体” 对这些细胞有毒。使用两种不同的实验技术，我们发现H(2)O(2) 是在人淀粉样蛋白聚集成淀粉样原纤维的过程中产生的。Cu(II) 离子极大地刺激了该过程，并且人胰淀素保留在铜亲和柱上。相反，无毒的啮齿动物amylin无法产生任何H(2)O(2)，并且不与铜相互作用。我们得出的结论是，胰淀素形成的H(2)O(2) 可能有助于T2Dm中胰岛细胞的进行性变性。

BACKGROUND & AIMS: :Peptide growth factors play an important role in several intracellular processes, such as cellular growth and differentiation, angiogenesis and apoptosis, as well as in carcinogenesis, since they contribute significantly to the malignant transformation. The prostate gland is abundant in growth factors. The two most known prostatic diseases, prostate cancer (PCa) and benign prostatic hyperplasia (BPH), are among the most common diseases that affect elderly men. This study was conducted using a quantitative real-time RT-PCR method in order to determine mRNA expression levels of peptide growth factors VEGF, FGF2, TGFB1, EGF, and IGF1 in tissue specimens from 42 patients with PCa, 42 with BPH, and 10 normal prostate samples obtained post-mortem from young individuals, in order to examine their association with prostatic hyperplasia and neoplasia. Our results show that in PCa, growth factors VEGF, EGF and FGF2 are overexpressed, while TGFB1 and IGF1 have reduced mRNA levels. In BPH, transcript levels of FGF2 and EGF are normal, while VEGF, TGFB1 and IGF1 exhibit downregulation. Further statistical analysis revealed that PCa patients with high levels of PSA blood levels have decreased FGF2 expression (p=0.016). Additionally, cancer patients with low Gleason score (<7) have increased EGF (p=0.035) and IGF1 (p=0.031) mRNA levels. IGF1 levels are also elevated in tumors with TNM stages T1-T2 (p=0.030). In BPH, older patients have reduced EGF expression (p=0.018), while IGF1 is overexpressed in younger patients (p=0.041). Additionally, the co-expression pattern of the five studied growth factors differs significantly among normal, benign and malignant prostate. These results implicate VEGF, FGF2, TGFB1, EGF and IGF1 in the development of both PCa and BPH, rendering them potential targets for disease detection, monitoring and therapy.

背景与目标： : 肽生长因子在多种细胞内过程中起着重要作用，例如细胞生长和分化，血管生成和凋亡以及致癌作用，因为它们对恶性转化有重要作用。前列腺富含生长因子。前列腺癌 (PCa) 和良性前列腺增生 (BPH) 这两种最著名的前列腺疾病是影响老年男性的最常见疾病。本研究采用实时定量rt-pcr方法，以测定42例PCa患者组织标本中肽生长因子VEGF，FGF2，TGFB1，EGF和IGF1的mRNA表达水平，为了检查他们与前列腺增生和肿瘤的关系，从年轻人的死后获得了10个正常的前列腺样本。我们的结果表明，在PCa中，生长因子VEGF，EGF和FGF2过表达，而TGFB1和IGF1的mRNA水平降低。在BPH中，FGF2和EGF的转录水平正常，而VEGF，TGFB1和IGF1表现出下调。进一步的统计分析显示，PSA血高水平的PCa患者FGF2表达降低 (p = 0.016)。此外，低Gleason评分 (<7) 的癌症患者具有增加的EGF (p = 0.035) 和IGF1 (p = 0.031) mRNA水平。在TNM分期T1-T2的肿瘤中，IGF1水平也升高 (p = 0.030)。在BPH中，老年患者的EGF表达降低 (p = 0.018)，而IGF1在年轻患者中过表达 (p = 0.041)。此外，在正常，良性和恶性前列腺中，研究的五种生长因子的共表达模式显着不同。这些结果表明VEGF，FGF2，TGFB1，EGF和IGF1参与了PCa和BPH的发展，使它们成为疾病检测，监测和治疗的潜在目标。

BACKGROUND & AIMS: :Brevinin-2R, a member of a new family of antimicrobial peptides isolated from the skin of Rana ridibunda, displays antimicrobial activity against bacteria and fungi. In this study, we have used an assembly PCR method for the fast and extremely accurate synthesis of the brevinin-2R gene. A total of six primers were assembled in a single step PCR, and the assembly was then amplified by PCR to produce the final gene. The synthetic gene was cloned into the pET32a (+) vector to allow the expression of brevinin-2R as a Trx fusion protein in Escherichia coli. The results indicated that the expression level of the fusion protein could reach up to 25% of the total cell proteins. The expression products could be easily purified by Ni-NTA chromatography and released from the fusion protein by factor Xa protease. The peptide displayed antimicrobial activity similar to that of the purified brevinin that was reported earlier. This method allows the fast synthesis of a gene that optimized the overexpression in the E. coli system and production of sufficiently large amounts of peptide for functional and structural characterizations.

背景与目标： : Brevinin-2R是从Rana ridibunda皮肤中分离出的抗菌肽新家族的成员，对细菌和真菌具有抗菌活性。在这项研究中，我们使用了组装PCR方法来快速，极其准确地合成brevinin-2R基因。在单步PCR中总共组装了六个引物，然后通过PCR扩增组装以产生最终基因。将合成基因克隆到pET32a (+) 载体中，以使brevinin-2R作为Trx融合蛋白在大肠杆菌中表达。结果表明，融合蛋白的表达水平最高可达细胞总蛋白的25%。表达产物可以很容易地通过Ni-NTA色谱法纯化，并通过因子Xa蛋白酶从融合蛋白中释放出来。该肽具有与先前报道的纯化的brevinin相似的抗菌活性。这种方法可以快速合成优化大肠杆菌系统中过表达的基因，并产生足够大量的肽以进行功能和结构表征。

BACKGROUND & AIMS: :As a homing peptide, A54 is the most effective peptide specific to the human hepatocellular carcinoma cell. Homing peptide labeled with green fluorescent protein (A54-GFP) was successfully immobilized on the surfaces of magnetic nanoparticles and characterized by Fourier transform infrared spectroscopy as well as fluorescence microscopy. The binding efficiency was analyzed by performing adsorption equilibrium and SDS-PAGE electrophoresis. Specific binding of the nanoparticles functionalized with A54-GFP to human hepatocellular carcinoma cells in vitro was visualized using fluorescence microscopy. The results demonstrated the specificity of A54-GFP-coated magnetic nanoparticle to tumor cell, pointing to its great potential in magnetic cell separation and purification, magnetic resonance imaging (MRI), magnetic hyperthermia, and drug targeting.

背景与目标： : 作为归巢肽，A54是对人肝癌细胞特异性最有效的肽。成功地将绿色荧光蛋白 (A54-GFP) 标记的归巢肽固定在磁性纳米颗粒的表面上，并通过傅里叶变换红外光谱和荧光显微镜对其进行了表征。通过进行吸附平衡和sds-page电泳分析结合效率。使用荧光显微镜观察用A54-GFP官能化的纳米颗粒在体外对人肝癌细胞的特异性结合。结果表明，A54-GFP-coated磁性纳米颗粒对肿瘤细胞具有特异性，表明其在磁性细胞分离和纯化，磁共振成像 (MRI)，磁热疗和药物靶向方面具有巨大潜力。