Diffusion through a membrane: Approach to equilibrium

The transfer of solute through a membrane separating two aqueous solutions is studied with the time-dependent diffusion equation for composite media. By introducing new independent and dependent variables it is shown that the differential equations and boundary conditions can be transformed into a dimensionless form which does not explicitly depend on the diffusivities of the media. Laplace transforms are used to derive explicit solutions for the solute concentration as a function of position and time. It is shown that at large time the concentration approaches the equilibrium distribution exponentially. Explicit results are given for the decay time as a function of the parameters of the system. In addition, an accurate and simplified expression is derived for the decay time for the case of small membrane permeability. The accuracy of the analytic solutions for the concentration profiles is tested by comparing them with numerical results obtained by solving the diffusion equations by the method of finite differences. Excellent agreement is found. Research supported in part by a grant from the National Science Foundation.     

Reverse ChIP:研究DNA-蛋白质相互作用的新方法

反向染色质免疫共沉淀技术(reverse chromatin immunoprecipitation assay,Reverse ChIP)是一种在体内状态下分析DNA-蛋白质相互作用的新方法.它用特异的核酸探针捕获靶DNA片段及与其相结合的蛋白质,蛋白质用质谱仪检测,以达到确定靶DNA位点全部相关蛋白质的目的.其可对靶DNA位点相关蛋白质进行全面、系统地鉴定,特别是寻找已知DNA元件相应的调节蛋白.在发现、鉴定靶DNA位点相关蛋白质和研究DNA-蛋白质相互作用中有重要应用价值.     

Diffusion from a Circular Stoma through a Boundary Layer: A FIELD-THEORETICAL ANALYSIS

The case of diffusion of a gas from a single circular stoma through an unstirred boundary layer of finite thickness into a perfectly stirred atmosphere free of convective effects is examined theoretically, with the gas assumed to be at constant concentration across the stoma. The analysis employs a mathematical solution to an analogous problem in electrostatic physics previously obtained by Kuz'min (1972 Sov Phys Tech Phys 17: 473-476). The diffusion flux is shown to be no more than 1% greater than that into a perfectly unstirred atmosphere if the boundary layer is thicker than 40 times the stomatal radius. Under the conditions assumed, for realistic boundary-layer and stomatal dimensions, taking the diffusion flux through the boundary layer to be linear with the stomatal radius would usually involve no significant error. This result may indicate that the principal effect of wind velocity on mass exchange between leaf and atmosphere may be exerted through influencing convection outside the boundary layer rather than through determining the thickness of that layer.     

一种用于抗原-抗体相互作用研究的实验方法

蛋白抗原-抗体相互作用是生物技术领域的重要研究之一.蛋白抗原难以合成、纯化且易于降解,低质量的蛋白抗原可能会导致该研究复杂、耗时、昂贵,甚至不可靠;抗原-抗体相互作用可能涉及易接触的抗原结合位点也可能给其带来挑战.故本研究开发出高效的实验体系,仅使用相应肽段作为抗原,来研究蛋白抗原与抗体的相互作用.以抗A型肉毒杆菌抗体...     

Multiregional Evaluation of the SimPlate Heterotrophic Plate Count Method Compared to the Standard Plate Count Agar Pour Plate Method in Water

A new SimPlate heterotrophic plate count (HPC) method (IDEXX Laboratories, Westbrook, Maine) was compared with the pour plate method at 35°C for 48 h. Six laboratories tested a total of 632 water samples. The SimPlate HPC method was found to be equivalent to the pour plate method by regression analysis (r = 0.95; y = 0.99X + 0.06).     

Diffusion of synthetic biology: a challenge to biosafety

One of the main aims of synthetic biology is to make biology easier to engineer. Major efforts in synthetic biology are made to develop a toolbox to design biological systems without having to go through a massive research and technology process. With this “de-skilling” agenda, synthetic biology might finally unleash the full potential of biotechnology and spark a wave of innovation, as more and more people have the necessary skills to engineer biology. But this ultimate domestication of biology could easily lead to unprecedented safety challenges that need to be addressed: more and more people outside the traditional biotechnology community will create self-replicating machines (life) for civil and defence applications, “biohackers” will engineer new life forms at their kitchen table; and illicit substances will be produced synthetically and much cheaper. Such a scenario is a messy and dangerous one, and we need to think about appropriate safety standards now.     

Diffusion of Particles in the Extracellular Matrix: The Effect of Repulsive Electrostatic Interactions

Diffusive transport of macromolecules and nanoparticles in charged fibrous media is of interest in many biological applications, including drug delivery and separation processes. Experimental findings have shown that diffusion can be significantly hindered by electrostatic interactions between the diffusing particle and charged components of the extracellular matrix. The implications, however, have not been analyzed rigorously. Here, we present a mathematical framework to study the effect of charge on the diffusive transport of macromolecules and nanoparticles in the extracellular matrix of biological tissues. The model takes into account steric, hydrodynamic, and electrostatic interactions. We show that when the fiber size is comparable to the Debye length, electrostatic forces between the fibers and the particles result in slowed diffusion. However, as the fiber diameter increases the repulsive forces become less important. Our results explain the experimental observations that neutral particles diffuse faster than charged particles. Taken together, we conclude that optimal particles for delivery to tumors should be initially cationic to target the tumor vessels and then change to neutral charge after exiting the blood vessels.     

Simplified Intestinal Microbiota to Study Microbe-Diet-Host Interactions in a Mouse Model

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Diffusion of bacteriophages through artificial biofilm models

The simple two-chamber diffusion method was improved to study the diffusion properties of bacteriophage (phage) T4 through a model biofilm agarose gel membrane (AGM) embedded with dead host Escherichia coli K12 cells. The apparent diffusion coefficient (D(app) ) of phage T4 was calculated to be 2.4 × 10(-12) m(2) /s in 0.5% AGM, which was lower than the coefficient of 4.2 × 10(-12) m(2) /s in 0.5% AGM without host cells. The phage adsorption process by dead host cells slowed the apparent phage diffusion. The Langmuir adsorption equation was used to simulate phage adsorption under different multiplicity of infections (MOIs); the maximum adsorbed phage MOI was calculated to be 417 PFU/CFU, and the Langmuir adsorption constant K(L) was 6.9 × 10(-4) CFU/PFU. To evaluate the effects of phage proliferation on diffusion, a simple syringe-based biofilm model was developed. The phage was added into this homogenous biofilm model when the host cells were in an exponential growth phase, and the apparent diffusion coefficient was greatly enhanced. We concluded that D(app) of phages through biofilms could be distinctly affected by phage adsorption and proliferation, and that the idea of D(app) and these methods can be used to study diffusion properties through real biofilms.     

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside ‘septin cages’ and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.     

Interactions between macrophages and T-lymphocytes: tumor sneaking through intrinsic to helper T cell dynamics

In a mathematical model of the cellular immune response we investigate immune reactions to tumors that are introduced in various doses. The model represents macrophage T-lymphocyte interactions that generate cytotoxic macrophages and cytotoxic T-lymphocytes. In this model antigens (tumors) can induce infinitely large T-lymphocyte effector populations because effector T-lymphocytes are capable of repeated proliferation and we have omitted immunosuppression. In this (proliferative) model small doses of weakly antigenic tumors grow infinitely large (i.e. sneak through) eliciting an immune response of limited magnitude. Intermediate doses of the same tumor induce larger immune responses and are hence rejected. Large doses of the tumor break through, but their progressive growth is accompanied by a strong immune response involving extensive lymphocyte proliferation. Similarly a more antigenic tumor is rejected in intermediate doses and breaks through in large doses. Initially small doses however lead to tumor dormancy. Thus although the model is devoid of explicit regulatory mechanisms that limit the magnitude of its response (immunosuppression is such a mechanism), the immune response to large increasing tumors may either be a stable reaction of limited magnitude (experimentally known as tolerance or unresponsiveness) or a strong and ever increasing reaction. Unresponsiveness can evolve because in this model net T-lymphocyte proliferation requires the presence of a minimum number of helper T cells (i.e. a proliferation threshold). Unresponsiveness is caused by depletion of helper T cell precursors.     

一种从培养皿上挑取阳性噬菌斑的改进方法

从密集噬菌斑的培养皿上挑选少数几个阳性噬菌斑是一件繁琐而又容易出错的工作。以往的做法是在阳性噬菌斑周围多挑选几个 ,然后通过重复杂交筛选 ,直到准确筛选出阳性噬菌斑为止 ,但该方法对于大规模筛选显得非常笨拙。这里介绍一种改进方法 ,只需一次杂交筛选即可准确挑出阳性噬斑 ,做到省时、省力、省材 ,而且准确性非常高。     

Fluorescent Bone Viewed through Toenails of Living Animals: a Method to Observe Bone Regrowth

We have developed a new method to observe bone and to document growth in living animals. The technique involves injecting calcein, a fluorescent calcium deposition marker, waiting approximately 4 hr for it to clear the vascular system, and observing bone directly through the toenails of lightly anesthetized living animals. Bone regrowth can be monitored in situ by amputating the digit through the nail plate, waiting the desired number of days, and injecting a second fluorescent label, alizarin red. Bone that has regrown since the amputation appears as a red area distal to the green calcein label on toes of lightly anesthetized animals when viewed under FITC fluorescence. This method has been used to demonstrate blocked bone synthesis and to quantitate significant differences in bone growth in control and experimental toes of individual animals. Advantages of this method include its simplicity, the use of fewer animals to collect sequential data, and increased reliability of repeated microscopic measurements using the same animal.     

Fluorescent Bone Viewed through Toenails of Living Animals: a Method to Observe Bone Regrowth

We have developed a new method to observe bone and to document growth in living animals. The technique involves injecting calcein, a fluorescent calcium deposition marker, waiting approximately 4 hr for it to clear the vascular system, and observing bone directly through the toenails of lightly anesthetized living animals. Bone regrowth can be monitored in situ by amputating the digit through the nail plate, waiting the desired number of days, and injecting a second fluorescent label, alizarin red. Bone that has regrown since the amputation appears as a red area distal to the green calcein label on toes of lightly anesthetized animals when viewed under FITC fluorescence. This method has been used to demonstrate blocked bone synthesis and to quantitate significant differences in bone growth in control and experimental toes of individual animals. Advantages of this method include its simplicity, the use of fewer animals to collect sequential data, and increased reliability of repeated microscopic measurements using the same animal.     

A Robust and Adaptable High Throughput Screening Method to Study Host-Microbiota Interactions in the Human Intestine

The intestinal microbiota has many beneficial roles for its host. However, the precise mechanisms developed by the microbiota to influence the host intestinal cell responses are only partially known. The complexity of the ecosystem and our inability to culture most of these micro-organisms have led to the development of molecular approaches such as functional metagenomics, i.e. the heterologous expression of a metagenome in order to identify functions. This elegant strategy coupled to high throughput screening allowed to identify novel enzymes from different ecosystems where culture methods have not yet been adapted to isolate the candidate microorganisms. We have proposed to use this functional metagenomic approach in order to model the microbiota’s interaction with the host by combining this heterologous expression with intestinal reporter cell lines. The addition of the cellular component to this functional metagenomic approach introduced a second important source of variability resulting in a novel challenge for high throughput screening. First attempts of high throughput screening with various reporter cell-lines showed a high distribution of the response and consequent difficulties to reproduce the response, impairing an easy and clear identification of confirmed hits. In this study, we developed a robust and reproducible methodology to combine these two biological systems for high throughput application. We optimized experimental setups and completed them by appropriate statistical analysis tools allowing the use this innovative approach in a high throughput manner and on a broad range of reporter assays. We herewith present a methodology allowing a high throughput screening combining two biological systems. Therefore ideal conditions for homogeneity, sensitivity and reproducibility of both metagenomic clones as well as reporter cell lines have been identified and validated. We believe that this innovative method will allow the identification of new bioactive microbial molecules and, subsequently, will promote understanding of host-microbiota interactions.