Molecular mimicry: Basis for autoimmunity

Structural similarity between a viral protein and a self-component can trigger an autoimmune response, which is the basis of molecular mimicry. Alternatively an invading virus can induce an inflammatory response which in turn can initiate an attack by hitherto dormant T cells on a specific self-antigen, a phenomenon which is referred to as Bystander Activation. Several viruses share amino acid sequences with target self-proteins. A widely studied viral interaction is the structural mimicry of a small portion of coxsackie virus to a specific region of the enzyme glutamic acid decarboxylase (GAD) which is expressed by the β cells of the islet of Langerhans in the pancreas leading to the destruction of insulin producing cells and the onset of Type I insulin dependent diabetes mellitus (IDDM). Knowledge of specific epitopes in GAD susceptible to autoimmune attack can permit devising therapeutic strategies for the prevention and suppression of IDDM.     

Engineering G-quadruplex aptamer to modulate its binding specificity

The use of aptamers in bioanalytical and biomedical applications exploits their ability to recognize cell surface protein receptors. Targeted therapeutics and theranostics come to mind in this regard. However, protein receptors occur on both cancer and normal cells; as such, aptamers are now taxed with identifying high vs. low levels of protein expression. Inspired by the flexible template mechanism and elegant control of natural nucleic acid-based structures, we report an allosteric regulation strategy for constructing a structure-switching aptamer for enhanced target cell recognition by engineering aptamers with DNA intercalated motifs (i-motifs) responsive to the microenvironment, such as pH. Structure-switching sensitivity can be readily tuned by manipulating i-motif sequences. However, structure-switching sensitivity is difficult to estimate, making it equally difficult to effectively screen modified aptamers with the desired sensitivity. To address this problem, we selected a fluorescent probe capable of detecting G-quadruplex in complicated biological media.     

Assessment of the inhibition of Dengue virus infection by carrageenan via real-time monitoring of cellular oxygen consumption rates within a microfluidic device

A microfluidic device combined with a light modulation system was developed to assess the inhibitory effect of carrageenan on Dengue virus (DENV) infection via real-time monitoring of cellular oxygen consumption rates (OCRs). Measuring cellular OCRs, which can reflect cellular metabolic activity, enabled us to monitor the process of viral infection in real time and to rapidly determine the antiviral activity of potential drugs/chemical compounds. The time variation of the cellular OCR of single cells that were infected in situ by DENV at different multiplicity of infection (m.o.i.) values was first successfully measured within a microfluidic device. The influence of the timing of carrageenan treatment on DENV infection was then examined by real-time monitoring of cellular OCRs in three groups. Cells that were pre-treated with carrageenan and then infected with DENV served as a pre-treatment group, cells to which carrageenan was added simultaneously with DENV served as a virucide group, and cells that were pre-infected with DENV and then treated with carrageenan served as a post-treatment group. By monitoring cellular OCRs, we could rapidly evaluate the inhibitory effect of carrageenan on DENV infection, obtaining a result within 7 h and showing that carrageenan had strong and effective anti-DENV activity in the three groups. In particular, a strong inhibitory effect was observed in the virucide group. Moreover, once the virus enters host cells in the post-treatment group, the immediate treatment with carrageenan for the infected cells has higher efficiency of antiviral activity. Our proposed platform enables to perform time-course or dose-response measurements of changes in cellular metabolic activity caused by diseases, chemical compounds, and drugs via monitoring of the cellular OCR, with rapid and real-time detection. This approach provides the potential to study a wide range of biological applications in cell-based biosensing, toxicology, and drug discovery.     

Examination of the role of transient receptor potential vanilloid type 4 in endothelial responses to shear forces

Shear stress is the major mechanical force applied on vascular endothelial cells by blood flow, and is a crucial factor in normal vascular physiology and in the development of some vascular pathologies. The exact mechanisms of cellular mechano-transduction in mammalian cells and tissues have not yet been elucidated, but it is known that mechanically sensitive receptors and ion channels play a crucial role. This paper describes the use of a novel and efficient microfluidic device to study mechanically-sensitive receptors and ion channels in vitro, which has three independent channels from which recordings can be made and has a small surface area such that fewer cells are required than for conventional flow chambers. The contoured channels of the device enabled examination of a range of shear stresses in one field of view, which is not possible with parallel plate flow chambers and other previously used devices, where one level of flow-induced shear stress is produced per fixed flow-rate. We exposed bovine aortic endothelial cells to different levels of shear stress, and measured the resulting change in intracellular calcium levels ([Ca²⁺]_i) using the fluorescent calcium sensitive dye Fluo-4AM. Shear stress caused an elevation of [Ca²⁺]_i that was proportional to the level of shear experienced. The response was temperature dependant such that at lower temperatures more shear stress was required to elicit a given level of calcium signal and the magnitude of influx was reduced. We demonstrated that shear stress-induced elevations in [Ca²⁺]_i are largely due to calcium influx through the transient receptor potential vanilloid type 4 ion channel.     

A microfluidic platform for real-time and in situ monitoring of virus infection process

Microfluidic chip is a promising platform for studying virus behaviors at the cell level. However, only a few chip-based studies on virus infection have been reported. Here, a three-layer microfluidic chip with low shear stress was designed to monitor the infection process of a recombinant Pseudorabies virus (GFP-PrV) in real time and in situ, which could express green fluorescent protein during the genome replication. The infection and proliferation characteristics of GFP-PrV were measured by monitoring the fluorescence intensity of GFP and determining the one-step growth curve. It was found that the infection behaviors of GFP-PrV in the host cells could hardly be influenced by the microenvironment in the microfluidic chip. Furthermore, the results of drug inhibition assays on the microfluidic chip with a tree-like concentration gradient generator showed that one of the infection pathways of GFP-PrV in the host cells was microtubule-dependent. This work established a promising microfluidic platform for the research on virus infection.     

生物信息流操纵:作物病虫害导向性防控的新科学

作物病虫害是威胁全世界农业生产的重大自然灾害。目前,病虫害防治的核心思想仍然是简单杀灭,主要依靠以病虫微生物的基础代谢、生理生化系统和神经受体为靶标的化学农药,过程中易导致人畜中毒、农产品污染和生态环境破坏等一系列严重的问题。下一代病虫害防治学术思想的重大突破将是对作物—昆虫—病原微生物生物间信息流及行为进行操纵。在基础研究方面,"生物间信息的识别、解码与操纵"也是现代生命科学的前沿与热点学科——生物间相互作用的分子机理一旦被阐明,往往带动通用生物技术的发展突破。例如RNA干扰(RNAi)现象、植物转化技术和TALEN基因组编辑技术等科学发现和技术进步,已经为整个生命科学领域作出了重大贡献。中科院"作物病虫害的导向性防控——生物间信息流与行为操纵"战略性先导科技专项(B类)集合了各学科的优势力量,系统地分析在作物重大病虫害发生过程中种间信息识别、解码、传递和控制的过程,从中解析关键可操纵节点,发展新一代病虫害田间行为操纵的新策略与新技术。该专项目前已经取得了系列重大研究成果,为竞争国际科学前沿地位、保障我国粮食生产安全作出了基础性、前瞻性的贡献。     

Reconfigurable microfluidic device with integrated antibody arrays for capture,multiplexed stimulation,and cytokine profiling of human monocytes

Monocytes represent a class of immune cells that play a key role in the innate and adaptive immune response against infections. One mechanism employed by monocytes for sensing foreign antigens is via toll-like receptors (TLRs)—transmembrane proteins that distinguish classes of foreign pathogens, for example, bacteria (TLR4, 5, and 9) vs. fungi (TLR2) vs. viruses (TLR3, 7, and 8). Binding of antigens activates a signaling cascade through TLR receptors that culminate in secretion of inflammatory cytokines. Detection of these cytokines can provide valuable clinical data for drug developers and disease investigations, but this usually requires a large sample volume and can be technically inefficient with traditional techniques such as flow cytometry, enzyme-linked immunosorbent assay, or luminex. This paper describes an approach whereby antibody arrays for capturing cells and secreted cytokines are encapsulated within a microfluidic device that can be reconfigured to operate in serial or parallel mode. In serial mode, the device represents one long channel that may be perfused with a small volume of minimally processed blood. Once monocytes are captured onto antibody spots imprinted into the floor of the device, the straight channel is reconfigured to form nine individually perfusable chambers. To prove this concept, the microfluidic platform was used to capture monocytes from minimally processed human blood in serial mode and then to stimulate monocytes with different TLR agonists in parallel mode. Three cytokines, tumor necrosis factor-α, interleukin (IL)-6, and IL-10, were detected using anti-cytokine antibody arrays integrated into each of the six chambers. We foresee further use of this device in applications such as pediatric immunology or drug/vaccine testing where it is important to balance small sample volume with the need for high information content.     

一种仿真瑞利信道的有效方法

改进了一种基于正弦和的仿真瑞利信道的方法,消除了它生成的复随机过程的同相和正交分量的概率密度函数及该复随机过程的包络平方的自相关函数是广义非平稳的缺点. 运用改进后的方法生成的复随机过程的自相关函数,及其同相和正交分量的自相关函数、互相关函数也与理论参考值相等. 仿真结果表明,改进后的方法生成的复随机过程的幅度、相位的概率密度函数及平均电平通过率、平均衰落持续时间能更好地逼近理论参考值.     

An in vitro model mimicking the complement system to favor directed phagocytosis of unwanted cells

BackgroundOpsonization, is the molecular mechanism by which target molecules promote interactions with phagocyte cell surface receptors to remove unwanted cells by induced phagocytosis. We designed an in vitro system to demonstrate that this procedure could be driven to eliminate adipocytes, using peptides mimicking regions of the complement protein C3b to promote opsonization and enhance phagocytosis. Two cell lines were used: (1) THP-1 monocytes differentiated to macrophages, expressing the C3b opsonin receptor CR1 in charge of the removal of unwanted coated complexes; (2) 3T3-L1 fibroblasts differentiated to adipocytes, expressing AQP7, to evaluate the potential of peptides to stimulate opsonization. (3) A co-culture of the two cell lines to demonstrate that phagocytosis could be driven to cell withdrawal with high efficiency and specificity.ResultsAn array of peptides were designed and chemically synthesized p3691 and p3931 joined bound to the CR1 receptor activating phagocytosis (p < 0.033) while p3727 joined the AQP7 protein (p < 0.001) suggesting that opsonization of adipocytes could occur. In the co-culture system p3980 and p3981 increased lipid uptake to 91.2% and 89.0%, respectively, as an indicator of potential adipocyte phagocytosis.ConclusionsThis in vitro model could help understand the receptor–ligand interaction in the withdrawal of unwanted macromolecules in vivo. The adipocyte-phagocytosis discussed may help to control obesity, since peptides of C3b stimulated the CR1 receptor, promoting opsonisation and phagocytosis of lipid-containing structures, and recognition of AQP7 in the differentiated adipocytes, favored the phagocytic activity of macrophages, robustly supported by the co-culture strategy.How to cite: Bartsch IM, Perelmuter K, Bollati-Fogolin M, et al. An in vitro model mimicking the complement system to favor directed phagocytosis of unwanted cells. Electron J Biotechnol 2021;49. https://doi.org/10.1016/j.ejbt.2020.09.010.     

海杂波统计特性分析及海面动目标检测

利用IPIX雷达回波数据分析了海杂波的统计特性.并利用LFM信号在分数阶Fourier域良好的能量聚集性,提出基于分数阶Fourier变换的海面动目标检测方法.此方法能较好的聚集动目标回波能量,而对海杂波回波的能量聚集不明显,可以较好的检测出动目标.最后采用实测海杂波数据做了仿真分析,证实了此方法的有效性.     

Stabilizing a class of mixed states for stochastic quantum systems via switching control

The global stabilization of a class of mixed states for finite dimensional stochastic quantum systems with degenerate measurement operator is investigated in this paper. We construct a measurement operator and control Hamiltonian that make the target state one of the system equilibria. Based on the proposed Lyapunov function, a control law is designed following Lyapunov’s method to steer system state to the target mixed state from an initial state in the convergence domain, which is obtained through the analyses of invariant set based on LaSella’s invariance principle. When the initial state isn’t in the convergence domain, a constant control is used to steer the system state so that it enters the convergence domain in finite time. The constant control and the control designed by Lyapunov method compose a switching control strategy, which can steer system state to the target mixed state from any arbitrary state in the state space, i.e., the target mixed state is globally stable under the switching control. The convergence of the switching control is proved based on state sample trajectories. Moreover, the numerical experiments on a three dimensional stochastic quantum system are performed to demonstrate the effectiveness of switching control.     

氧化型低密度脂蛋白诱导LOX-1 在基因和蛋白水平表达

目的研究氧化型低密度脂蛋白(oxidized LDL, ox-LDL)是否能在基因和蛋白两个水平诱导内皮细胞表达凝集素样氧化低密度脂蛋白受体(lectin-like oxidized LDL receptor, LOX-1),以探讨LOX-1在动脉粥样硬化形成和发展中的作用.方法将不同浓度ox-LDL(20,40,60,80 mg/L)与内皮细胞共孵育24 h及浓度40 mg/L的ox-LDL作用内皮细胞不同时间(0、3、6、12、24 h),反转录聚合酶链反应检测LOX-1 mRNA水平表达,细胞酶联免疫法测定LOX-1蛋白水平表达.结果加入Ox-LDL 20 mg/L使LOX-1 mRNA和蛋白表达量增加(P＜0.01),40 mg/L使其表达量达最高峰,随后逐渐下降.而同一浓度下从0 h～24 h的趋势是LOX-1 mRNA和蛋白逐渐增加(P＜0.01),在12 h增加最明显.结论氧化型低密度脂蛋白呈剂量依赖性和时间依赖性地上调LOX-1 mRNA和蛋白表达,LOX-1可能在动脉粥样硬化形成和发展中起重要作用.     

Autoimmune thyroid disorders—An update

Background: Autoimmune thyroid disease (AITD), a common organ specific autoimmune disorder is seen mostly in women between 30–50 yrs of age. Thyroid autoimmunity can cause several forms of thyroiditis ranging from hypothyroidism (Hashimoto’s thyroiditis) to hyperthyroidism (Graves’Disease). Prevalence rate of autoimmune mediated hypothyroidism is about 0.8 per 100 and 95% among them are women. Graves’ disease is about one tenth as common as hypothyroidism and tends to occur more in younger individuals. Both these disorders share many immunologic features and the disease may progress from one state to other as the autoimmune process changes. Genetic, environmental and endogenous factors are responsible for initiation of thyroid autoimmunity. At present the only confirmed genetic factor lies in HLA complex (HLA DR-3) and the T cell regulatory gene (CTLA 4). A number of environmental factors like viral infection, smoking, stress & iodine intake are associated with the disease progression. The development of antibodies to thyroid peroxidase (TPO) thyroglobulin (TG) and Thyroid stimulating hormone receptor (TSH R) is the main hallmark of AITD. Circulating T Lymphocytes are increased in AITD and thyroid gland is infiltrated with CD4+ and CD8+ T Cells. Wide varieties of cytokines are produced by infiltrated immune cells, which mediate cytotoxicity leading to thyroid cell destruction. Circulating antibodies to TPO and TG are measured by immunofluorescense, hemagglutination, ELISA & RIA. TSHR antibodies of Graves’ disease can be measured in bioassays or indirectly in assays that detect antibody binding to the receptor.     

Microfluidic platform for isolating nucleic acid targets using sequence specific hybridization

The separation of target nucleic acid sequences from biological samples has emerged as a significant process in today''s diagnostics and detection strategies. In addition to the possible clinical applications, the fundamental understanding of target and sequence specific hybridization on surface modified magnetic beads is of high value. In this paper, we describe a novel microfluidic platform that utilizes a mobile magnetic field in static microfluidic channels, where single stranded DNA (ssDNA) molecules are isolated via nucleic acid hybridization. We first established efficient isolation of biotinylated capture probe (BP) using streptavidin-coated magnetic beads. Subsequently, we investigated the hybridization of target ssDNA with BP bound to beads and explained these hybridization kinetics using a dual-species kinetic model. The number of hybridized target ssDNA molecules was determined to be about 6.5 times less than that of BP on the bead surface, due to steric hindrance effects. The hybridization of target ssDNA with non-complementary BP bound to bead was also examined, and non-specific hybridization was found to be insignificant. Finally, we demonstrated highly efficient capture and isolation of target ssDNA in the presence of non-target ssDNA, where as low as 1% target ssDNA can be detected from mixture. The microfluidic method described in this paper is significantly relevant and is broadly applicable, especially towards point-of-care biological diagnostic platforms that require binding and separation of known target biomolecules, such as RNA, ssDNA, or protein.     

Endothelins -- clinical perspectives

Endothelins (ET) are a group of endogenous peptides, which have a strong and long-lasting vasoconstrictive effect. Three isoforms of endothelins coded by three different genes have been identified to date. Endothelin-1 (ET-1) is the most potent vasoconstrictive agent currently identified, and it was originally isolated and characterized from the culture media of aortic endothelial cells. Two other isoforms, named endothelin-2 (ET-2) and endothelin-3 (ET-3), were subsequently identified, along with structural homologues isolated from the venom ofActractapis engaddensis known as the sarafotoxins. The biological effects of endothelin production are determined via activation of one or two G-protein coupled receptors, endothelin receptors A (ETRA) and B (ETRB1 and ETRB2). Recently endothelin receptor C (ETRC) was discovered, however, its functions and distribution still remain unclear. The effects mediated by ET-1 via ETRA are vasoconstriction, bronchoconstriction and secretion of aldosterone. Agonists related to the ETRB1 activation cause vasodilatation by stimulating NO, PGI2 and endothelium-derived hyperpolarizing factor (EDHF). In contrast, coupling to ETRB2 causes vasoconstriction. Involvement of ET has been demonstrated in the pathophysiology of certain disorders. In this review, we discuss the physiological and pathophysiological role of endothelium-derived ET-1, the pharmacology of its two receptors, focusing on the role of ET-1 in the development of some pathophysiological conditions.     

Study of Hormone Receptors and Epidermal Growth Factor Expression in Invasive Breast Cancers in a Cohort of Western India

The objective of study was to evaluate and correlate the pathological characteristics of breast cancer patients with estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2/neu) detected by immunohistochemistry and/or fluorescent in situ hybridization method. We have conducted 2 year study of 204 cases of breast cancer at HCG-Medisurge Hospitals, Ahmedabad from 2009 to 2011. Significant correlation was found in ER and PR expression whereas no correlation was found in hormonal receptors and Her2/neu expression. ER and PR positivity increased with advancing age in breast carcinoma patients while not affecting Her2/neu expression. The expression of hormone receptors were higher in infiltrating lobular carcinoma and infiltrating duct carcinoma subtypes of breast carcinoma as compared to other subtypes such medullary and in situ carcinoma. High-grade carcinoma patients were predominantly ER/PR negative and Her2/neu positive as compared to lower grade breast carcinoma whereas high-stage carcinoma patients were ER/PR positive and Her2/neu positive as compared to lower stage breast carcinoma.     

三叶半夏组培苗的病毒检测

组织培养阶段植物细胞中的病毒含量较低,难以用常规方法进行可靠检测。本研究采用DAS-ELISA、RT-PCR和核酸杂交三种方法对不同来源的12个三叶半夏（Pinellia ternata （Thunb．）Breit．）组培苗样品中的黄瓜花叶病毒（Cucumber mosaic virus,CMV）和大豆花叶病毒（Soybean mosaic virus,SMV）进行比较检测。结果显示：经DAS-ELISA检测,4个样品携带CMV,4个样品携带SMV,1个样品同时携带2种病毒;经RT-PCR检测。5个样品携带CMV,6个样品携带SMV,1个样品同时携带2种病毒;经核酸杂交检测验证,其结果与RT-PCR的检测结果一致。可见,对于三叶半夏组培苗,DAS-ELISA检测灵敏度不够高,需用灵敏度更高和特异性更强的核酸杂交方法来对其进行病毒检测,确保检测结果准确可靠。     

Flexible sample selection strategies for transfer learning in ranking

Ranking is a central component in information retrieval systems; as such, many machine learning methods for building rankers have been developed in recent years. An open problem is transfer learning, i.e. how labeled training data from one domain/market can be used to build rankers for another. We propose a flexible transfer learning strategy based on sample selection. Source domain training samples are selected if the functional relationship between features and labels do not deviate much from that of the target domain. This is achieved through a novel application of recent advances from density ratio estimation. The approach is flexible, scalable, and modular. It allows many existing supervised rankers to be adapted to the transfer learning setting. Results on two datasets (Yahoo’s Learning to Rank Challenge and Microsoft’s LETOR data) show that the proposed method gives robust improvements.     

Retrovectors packaged in CHO cells to generate GLP-1-Fc stable expression CHO cell lines

BackgroundChinese hamster ovary (CHO) cells are the most dependable mammalian cells for the production of recombinant proteins. Replication-incompetent retroviral vector (retrovector) is an efficient tool to generate stable cell lines. Multiple copies of integrated genes by retrovector transduction results in improved recombinant protein yield. HEK-293 and their genetic derivatives are principal cells for retrovector production. Retrovectors packaged in HEK-293 cells pose a risk of infectious agent transmission, such as viruses and mycoplasmas, from serum and packaging cells.ResultsIn this report, retrovectors were packaged in CHO cells cultured in chemically defined (CD) media. The retrovectors were then used to transduce CHO cells. This method can block potential transmission of infectious agents from serum and packaging cells. With this method, we generated glucagon-like protein-1 Fc fusion protein (GLP-1-Fc) stable expression CHO cell lines. Productivity of GLP-1-Fc can reach 3.15 g/L. The GLP-1-Fc protein produced by this method has comparable bioactivity to that of dulaglutide (Trulicity). These stable cell lines retain 95–100% of productivity after 40 days of continuous culture (~ 48–56 generations).ConclusionsSuspension CHO cells are clean, safe, and reliable cells for retrovector packaging. Retrovectors packaged from this system could be used to generate CHO stable cell lines for recombinant protein expression.How to cite: Li J, Wei S, Cao C, et al. Retrovectors packaged in CHO cells to generate GLP-1-Fc stable expression CHO cell lines. Electron J Biotechnol 2019;41. https://doi.org/10.1016/j.ejbt.2019.07.002.