抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Dual colour total internal reflection fluorescence microscopy is a powerful tool for decoding the molecular dynamics of clathrin-mediated endocytosis (CME). Typically, the recruitment of a fluorescent protein–tagged endocytic protein was referenced to the disappearance of spot-like clathrin-coated structure (CCS), but the precision of spot-like CCS disappearance as a marker for canonical CME remained unknown. Here we have used an imaging assay based on total internal reflection fluorescence microscopy to detect scission events with a resolution of ∼2 s. We found that scission events engulfed comparable amounts of transferrin receptor cargo at CCSs of different sizes and CCS did not always disappear following scission. We measured the recruitment dynamics of 34 types of endocytic protein to scission events: Abp1, ACK1, amphiphysin1, APPL1, Arp3, BIN1, CALM, CIP4, clathrin light chain (Clc), cofilin, coronin1B, cortactin, dynamin1/2, endophilin2, Eps15, Eps8, epsin2, FBP17, FCHo1/2, GAK, Hip1R, lifeAct, mu2 subunit of the AP2 complex, myosin1E, myosin6, NECAP, N-WASP, OCRL1, Rab5, SNX9, synaptojanin2β1, and syndapin2. For each protein we aligned ∼1,000 recruitment profiles to their respective scission events and constructed characteristic “recruitment signatures” that were grouped, as for yeast, to reveal the modular organization of mammalian CME. A detailed analysis revealed the unanticipated recruitment dynamics of SNX9, FBP17, and CIP4 and showed that the same set of proteins was recruited, in the same order, to scission events at CCSs of different sizes and lifetimes. Collectively these data reveal the fine-grained temporal structure of CME and suggest a simplified canonical model of mammalian CME in which the same core mechanism of CME, involving actin, operates at CCSs of diverse sizes and lifetimes.

https://pure.mpg.de/pubman/item/item_3071073_2/component/file_3071074/PLoS_Biol_2011_9_1000604.pdf

A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis.

Biological organisms use complex molecular networks to navigate their environment and regulate their internal state. The development of synthetic systems with similar capabilities could lead to applications such as smart therapeutics or fabrication methods based on self-organization. To achieve this, molecular control circuits need to be engineered to perform integrated sensing, computation and actuation. Here we report a DNA-based technology for implementing the computational core of such controllers. We use the formalism of chemical reaction networks as a 'programming language' and our DNA architecture can, in principle, implement any behaviour that can be mathematically expressed as such. Unlike logic circuits, our formulation naturally allows complex signal processing of intrinsically analogue biological and chemical inputs. Controller components can be derived from biologically synthesized (plasmid) DNA, which reduces errors associated with chemically synthesized DNA. We implement several building-block reaction types and then combine them into a network that realizes, at the molecular level, an algorithm used in distributed control systems for achieving consensus between multiple agents.

/pdf/programmable-chemical-controllers-made-from-dna-2u5cq3iiqs.pdf

Programmable chemical controllers made from DNA

The π-Calculus: A theory of mobile processes by Davide Sangiorgi and David Walker Formal methods have formed the foundation of Computer Science since its inception. Although, initially these formal methods dealt with processes and systems on an individual basis, the paradigm has shifted with the dawn of the age of computer networks. When dealing with systems with interconnected, communicating, dependent, cooperative, and competitive components, the older outlook of analyzing and developing singular systems—and the tools that went with it—were hardly suitable. This led to the development of theories and tools that would support the new paradigm. On the tools end, the development has been widespread and satisfactory: programming languages, development frameworks, databases, and even end-user software products such as word processors, have gained network-awareness. However on the theoretical end, the development has been far less satisfactory. The major work was done by Robin Milner, Joachim Parrow, and David Walker who developed the formalism known as π-calculus in 1989. π-calculus is a process calculus that treats communication between its components as the basic form of computation. It has been quite successful as a foundation of several other calculi in the field and as Milner puts it, it has become common to express ideas about interactions and mobility in variants of the calculus. Introduction The current book serves as a comprehensive reference to π-calculus. Besides Milner's own book on the subject, this is the only other book-length publication on the topic. In many ways, it is much more comprehensive than Milner's: a much wider area of topics are dealt with and in more detail as well. Contents The book is split into seven part. The first part presents the basic theory of π-calculus. However, basic does not mean concise: every topic is discussed in great detail. The section on bisimulation is particularly intensive and provides several insights about the motivation for the theory. Part two discusses several variants of the original calculus. By varying several characteristics of the calculus, such as whether a process can communicate with more than processes at a time, we can obtain these variants. A number of interesting properties of the language are studied by the other when discussing these variants. As can be understood from the title, the calculus' contribution to analyzing mobile processes is a major topic, and it is dealt with extensively starting from part three. The basics are introduced by the way of a sophisticated typing system whose application in speciying complex processes is presented in part four. Part five looks at higher-order π-calculus in which composed systems are considered as first-class citizens. Part six is one of the best in the book and discusses the relation between π-calculus and lambda-calculus, which is an older and more basic calculus. Finally part seven shows how π-calculus can be employed in studying practical, modern software engineering concepts such as object-oriented programming. Impressions One of my disappointments with this book is in how often the reader is left perplexed with some of the theoretical developments, specially in part three. π-calculus is a complicated topic, even for the graduate student audience to which this book is directed, and the author would have done much better by reducing the number of topics and instead focusing on more lucid and detailed explanations. There are several experimental digressions throughout the book, which although interesting, take away from some of the momentum of sequential study. For example, topics such as comparison and encoding of one language to another could be easily moved to a separate section in order to make the content more suitable for self-study. Another issue is the little effort towards making the connection from the theoretical to the practical. The main reason why formal methods have not been adopted in mainstream software development pracitces is that often it is unclear to developers how formalisms can contribute towards the software engineering process. The book would have served its purpose much better if it had spent part of eah chapter discussing the practical application of that chapter's content. For example, congruence checking and bisimulation can be incredbily exciting topics for programmers to learn if they can see practical applications of such powerful techniques. Beyond the above criticism, the book is absolutely indispensible to students and researchers in the field of formal methods. Currently it serves as the primary reference for anyone who wishes to learn the various aspects of π-calculus in detail. Raheel Ahmad

The Ï-Calculus: A theory of mobile processes

Endocytosis includes a number of processes by which cells internalize segments of their plasma membrane, enclosing a wide variety of material from outside the cell. Endocytosis can contribute to uptake of nutrients, regulation of signaling molecules, control of osmotic pressure, and function of synapses. The actin cytoskeleton plays an essential role in several of these processes. Actin assembly can create protrusions that encompass extracellular materials. Actin can also support the processes of invagination of a membrane segment into the cytoplasm, elongation of the invagination, scission of the new vesicle from the plasma membrane, and movement of the vesicle away from the membrane. We briefly discuss various types of endocytosis, including phagocytosis, macropinocytosis, and clathrin-independent endocytosis. We focus mainly on new findings on the relative importance of actin in clathrin-mediated endocytosis (CME) in yeast versus mammalian cells.

Roles for Actin Assembly in Endocytosis

Summary: The Visual DSD (DNA Strand Displacement) tool allows rapid prototyping and analysis of computational devices implemented using DNA strand displacement, in a convenient web-based graphical interface. It is an implementation of the DSD programming language and compiler described by Lakin et al. (2011) with additional features such as support for polymers of unbounded length. It also supports stochastic and deterministic simulation, construction of continuous-time Markov chains and various export formats which allow models to be analysed using third-party tools.

Availability: Visual DSD is available as a web-based Silverlight application for most major browsers on Windows and Mac OS X at http://research.microsoft.com/dna. It can be installed locally for offline use. Command-line versions for Windows, Mac OS X and Linux are also available from the web page.

Contact: aphillip@microsoft.com 

Supplementary Information:Supplementary data are available at Bioinformatics online.

Visual DSD

DNA strand displacement techniques have been used to implement a broad range of information processing devices, from logic gates, to chemical reaction networks, to architectures for universal computation. Strand displacement techniques enable computational devices to be implemented in DNA without the need for additional components, allowing computation to be programmed solely in terms of nucleotide sequences. A major challenge in the design of strand displacement devices has been to enable rapid analysis of high-level designs while also supporting detailed simulations that include known forms of interference. Another challenge has been to design devices capable of sustaining precise reaction kinetics over long periods, without relying on complex experimental equipment to continually replenish depleted species over time. In this paper, we present a programming language for designing DNA strand displacement devices, which supports progressively increasing levels of molecular detail. The language allows device designs to be programmed using a common syntax and then analysed at varying levels of detail, with or without interference, without needing to modify the program. This allows a trade-off to be made between the level of molecular detail and the computational cost of analysis. We use the language to design a buffered architecture for DNA devices, capable of maintaining precise reaction kinetics for a potentially unbounded period. We test the effectiveness of buffered gates to support long-running computation by designing a DNA strand displacement system capable of sustained oscillations.

https://royalsocietypublishing.org/doi/pdf/10.1098/rsif.2011.0343

Abstractions for DNA circuit design

Directed cell migration toward spatio-temporally varying chemotactic stimuli requires rapid cytoskeletal reorganization. Numerous studies provide evidence that actin reorganization is controlled by intracellular redistribution of signaling molecules, such as the PI4,5P2/PI3,4,5P3 gradient. However, exploring underlying mechanisms is difficult and requires careful spatio-temporal control of external chemotactic stimuli. We designed a microfluidic setup to generate alternating chemotactic gradient fields for simultaneous multicell exposure, greatly facilitating statistical analysis. For a quantitative description of intracellular response dynamics, we apply alternating time sequences of spatially homogeneous concentration gradients across 300 μm, reorienting on timescales down to a few seconds. Dictyostelium discoideum amoebae respond to gradient switching rates below 0.02 Hz by readapting their migration direction. For faster switching, cellular repolarization ceases and is completely stalled at 0.1 Hz. In this “chemotactically trapped” cell state, external stimuli alternate faster than intracellular feedback is capable to respond by onset of directed migration. To investigate intracellular actin cortex rearrangement during gradient switching, we correlate migratory cell response with actin repolymerization dynamics, quantified by a fluorescence distribution moment of the GFP fusion protein LimEΔcc. We find two fundamentally different cell polarization types and we could reveal the role of PI3-Kinase for cellular repolarization. In the early aggregation phase, PI3-Kinase enhances the capability of D. discoideum cells to readjust their polarity in response to spatially alternating gradient fields, whereas in aggregation competent cells the effect of PI3-Kinase perturbation becomes less relevant.

/pdf/chemotactic-cell-trapping-in-controlled-alternating-gradient-hmxilng7tc.pdf

Chemotactic cell trapping in controlled alternating gradient fields

In non-viral gene delivery, the variance of transgenic expression stems from the low number of plasmids successfully transferred. Here, we experimentally determine Lipofectamine- and PEI-mediated exogenous gene expression distributions from single cell time-lapse analysis. Broad Poisson-like distributions of steady state expression are observed for both transfection agents, when used with synchronized cell lines. At the same time, co-transfection analysis with YFP- and CFP-coding plasmids shows that multiple plasmids are simultaneously expressed, suggesting that plasmids are delivered in correlated units (complexes). We present a mathematical model of transfection, where a stochastic, two-step process is assumed, with the first being the low-probability entry step of complexes into the nucleus, followed by the subsequent release and activation of a small number of plasmids from a delivered complex. This conceptually simple model consistently predicts the observed fraction of transfected cells, the cotransfection ratio and the expression level distribution. It yields the number of efficient plasmids per complex and elucidates the origin of the associated noise, consequently providing a platform for evaluating and improving non-viral vectors.

Predictive modeling of non-viral gene transfer.

The spreading of motile cells on a substrate surface is accompanied by reorganization of their actin network. We show that spreading in the highly motile cells of Dictyostelium is non-monotonic, and thus differs from the passage of spreading cells through a regular series of stages. Quantification of the gain and loss of contact area revealed fluctuating forces of protrusion and retraction that dominate the interaction of Dictyostelium cells with a substrate. The molecular basis of these fluctuations is elucidated by dual-fluorescence labeling of filamentous actin together with proteins that highlight specific activities in the actin system. Front-to-tail polarity is established by the sorting out of myosin-II from regions where dense actin assemblies are accumulating. Myosin-IB identifies protruding front regions, and the Arp2/3 complex localizes to lamellipodia protruded from these regions. Coronin is used as a sensitive indicator of actin disassembly to visualize the delicate balance of polymerization ...

https://www.tandfonline.com/doi/pdf/10.4161/cam.2.2.6190

Simon Youssef

Papers

Visual DSD

Abstractions for DNA circuit design

Chemotactic cell trapping in controlled alternating gradient fields

Predictive modeling of non-viral gene transfer.

Actin-cytoskeleton dynamics in non-monotonic cell spreading.