Wide Screening of Phage-Displayed Libraries Identifies Immune Targets in Planta

Microbe-Associated Molecular Patterns and virulence effectors are recognized by plants as a first step to mount a defence response against potential pathogens. This recognition involves a large family of extracellular membrane receptors and other immune proteins located in different sub-cellular compartments. We have used phage-display technology to express and select for Arabidopsis proteins able to bind bacterial pathogens. To rapidly identify microbe-bound phage, we developed a monitoring method based on microarrays. This combined strategy allowed for a genome-wide screening of plant proteins involved in pathogen perception. Two phage libraries for high-throughput selection were constructed from cDNA of plants infected with Pseudomonas aeruginosa PA14, or from combined samples of the virulent isolate DC3000 of Pseudomonas syringae pv. tomato and its avirulent variant avrRpt2. These three pathosystems represent different degrees in the specificity of plant-microbe interactions. Libraries cover up to 26107 different plant transcripts that can be displayed as functional proteins on the surface of T7 bacteriophage. A number of these were selected in a bio-panning assay for binding to Pseudomonas cells. Among the selected clones we isolated the ethylene response factor ATERF-1, which was able to bind the three bacterial strains in competition assays. ATERF-1 was rapidly exported from the nucleus upon infiltration of either alive or heat-killed Pseudomonas. Moreover, aterf-1 mutants exhibited enhanced susceptibility to infection. These findings suggest that ATERF-1 contains a microbe-recognition domain with a role in plant defence. To identify other putative pathogen-binding proteins on a genome-wide scale, the copy number of selected-vs.-total clones was compared by hybridizing phage cDNAs with Arabidopsis microarrays. Microarray analysis revealed a set of 472 candidates with significant fold change. Within this set defence-related genes, including well-known targets of bacterial effectors, are over-represented. Other genes non-previously related to defence can be associated through this study with general or strain-specific recognition of Pseudomonas.

Patterns of succession in benthic infaunal communities following dredging and dredged material disposal in Monterey Bay

This report is the final product of a two-year study conducted for the Office, Chief of Engineers, by the Moss Landing Marine Laboratories, Moss Landing, California, under Contract No. DACW39-74-C-OI51 with the Environmental Effects Laboratory (EEL), U. S. Army Engineer Waterways Experiment Station (WES), Yicksburg, Mississippi. (PDF contains 192 pages)

微尺度压痕实验的数值模拟及相关问题研究

目前国际上占主导地位的纳米压痕技术是由Oliver与Pharr提出并发展,目前的纳米压痕可以给出整个加、卸载过程的载荷—位移曲线以及硬度与弹性模量随压痕深度变化的曲线,从而提供了丰富的、比较精确的信息,为利用它探索材料比较完整的力学特性提供了可能.为达到该目的,就必须对压痕实验的加、卸过程进行较为深入的研究.作为主要的研究工具,有限元方法模拟微压痕过程在探讨通过实验数据得到更多、更准确的材料表层力学性能参数以及解释实验现象等方面发挥着重要作用.基于计算机速度与容量的原因,较早进行微压痕过程有限元模拟的BhattacharyaandNix、LaursenandSino都使用圆锥压头模拟维氏显微硬度标准正四棱锥Vicker压头与纳米压痕仪标准正三棱锥Berkovich压头,因为圆锥压头具有旋转对称性,可用二维旋转对称单元(二维实体单元)进行计算从而降低计算规模.即便如此,以当时大型计算机的水平,对规模为400~2000个四节点矩形单元的有限元模型进行一次完整的加、卸载过程也需要1~2天.到目前为止,微尺度压痕实验的数值模拟沿用二维模型.事实上,由于加工工艺的限制,微尺度压痕仪的压头如Berkovich与Vicker压头均不个旋转对称性;就微观尺度而言,实际的表层材料都是非均匀的.这些特征均不能由二维模拟体现,所以该文首先建立三维有限元模型,模拟带滑动接触的微尺度压痕加、卸载过程.在此基础上重点讨论了压头几何效应的问题,如二维模拟与三维模拟的关系、显微硬度与纳米的压痕硬度的关系、不同压头下材料的应力应变场、压痕间距与压痕边界的效应等,最后针对微尺度压痕实验中出现的压痕硬度随压痕深度减小而升高的现象,讨论了影响不同压痕深度硬度值的因素.

Developmentally regulated transcription factors in Drosophila melanogaster

During early stages of Drosophila development the heat shock response cannot be induced. It is reasoned that the adverse effects on cell cycle and cell growth brought about by Hsp70 induction must outweigh the beneficial aspects of Hsp70 induction in the early embryo. Although the Drosophila heat shock transcription factor (dHSF) is abundant in the early embryo, it does not enter the nucleus in response to heat shock. In older embryos and in cultured cells the factor is localized within the nucleus in an apparent trimeric structure that binds DNA with high affinity. The domain responsible for nuclear localization upon stress resides between residues 390 and 420 of the dHSF. Using that domain as bait in a yeast two-hybrid system we now report the identification and cloning of a nuclear transport protein Drosophila karyopherin-α3(dKap- α3). Biochemical methods demonstrate that the dKap-α3 protein binds specifically to the dHSF's nuclear localization sequence (NLS). Furthermore, the dKap-α3 protein does not associate with NLSs that contain point mutations which are not transported in vivo. Nuclear docking studies also demonstrate specific nuclear targeting of the NLS substrate by dKap-α3.Consistant with previous studies demonstrating that early Drosophila embryos are refractory to heat shock as a result of dHSF nuclear exclusion, we demonstrate that the early embryo is deficient in dKap-α3 protein through cycle 12. From cycle 13 onward the transport factor is present and the dHSF is localized within the nucleus thus allowing the embryo to respond to heat shock.

The pair-rule gene fushi tarazu (ftz) is a well-studied zygotic segmentation gene that is necessary for the development of the even-numbered parasegments in Drosophila melanogastor. During early embryogenesis, ftz is expressed in a characteristic pattern of seven stripes, one in each of the even-numbered parasegments. With a view to understand how ftz is transcriptionally regulated, cDNAs that encode transcription factors that bind to the zebra element of the ftz promoter have been cloned. Chapter Ill reports the cloning and characterization of the eDNA encoding zeb-1 (zebra element binding protein), a novel steroid receptor-like molecule that specifically binds to a key regulatory element of the ftz promoter. In transient transfection assays employing Drosophila tissue culture cells, it has been shown that zeb-1 as well as a truncated zeb-1 polypeptide (zeb480) that lacks the putative ligand binding domain function as sequencespecific trans-activators of the ftz gene.

The Oct factors are members of the POU family of transcription factors that are shown to play important roles during development in mammals. Chapter IV reports the eDNA cloning and expression of a Drosophila Oct transcription factor. Whole mount in-situ hybridization experiments revealed that the spatial expression patterns of this gene during embryonic development have not yet been observed for any other gene. In early embryogenesis, its transcripts are transiently expressed as a wide uniform band from 20-40% of the egg length, very similar to that of gap genes. This pattern progressively resolves into a series of narrower stripes followed by expression in fourteen stripes. Subsequently, transcripts from this gene are expressed in the central nervous system and the brain. When expressed in the yeast Saccharomyces cerevisiae, this Drosophila factor functions as a strong, octamer-dependent activator of transcription. The data strongly suggest possible functions for the Oct factor in pattern formation in Drosophila that might transcend the boundaries of genetically defined segmentation genes.

Regulation of mitochondrial division by the Drp1 receptors

Mitochondria can remodel their membranes by fusing or dividing. These processes are required for the proper development and viability of multicellular organisms. At the cellular level, fusion is important for mitochondrial Ca2+ homeostasis, mitochondrial DNA maintenance, mitochondrial membrane potential, and respiration. Mitochondrial division, which is better known as fission, is important for apoptosis, mitophagy, and for the proper allocation of mitochondria to daughter cells during cellular division.

The functions of proteins involved in fission have been best characterized in the yeast model organism Sarccharomyces cerevisiae. Mitochondrial fission in mammals has some similarities. In both systems, a cytosolic dynamin-like protein, called Dnm1 in yeast and Drp1 in mammals, must be recruited to the mitochondrial surface and polymerized to promote membrane division. Recruitment of yeast Dnm1 requires only one mitochondrial outer membrane protein, named Fis1. Fis1 is conserved in mammals, but its importance for Drp1 recruitment is minor. In mammals, three other receptor proteins—Mff, MiD49, and MiD51—play a major role in recruiting Drp1 to mitochondria. Why mammals require three additional receptors, and whether they function together or separately, are fundamental questions for understanding the mechanism of mitochondrial fission in mammals.

We have determined that Mff, MiD49, or MiD51 can function independently of one another to recruit Drp1 to mitochondria. Fis1 plays a minor role in Drp1 recruitment, suggesting that the emergence of these additional receptors has replaced the system used by yeast. Additionally, we found that Fis1/Mff and the MiDs regulate Drp1 activity differentially. Fis1 and Mff promote constitutive mitochondrial fission, whereas the MiDs activate recruited Drp1 only during loss of respiration.

To better understand the function of the MiDs, we have determined the atomic structure of the cytoplasmic domain of MiD51, and performed a structure-function analysis of MiD49 based on its homology to MiD51. MiD51 adopts a nucleotidyl transferase fold, and binds ADP as a co-factor that is essential for its function. Both MiDs contain a loop segment that is not present in other nucleotidyl transferase proteins, and this loop is used to interact with Drp1 and to recruit it to mitochondria.

Structural Characterizations of the Dimeric Anti-HIV Antibody 2G12 and the HIV-2 Envelope Glycoprotein

More than thirty years after the discovery that Human Immunodeficiency Virus (HIV) was the causative agent of Acquired Immunodeficiency Syndrome (AIDS), the disease remains pandemic as long as no effective universal vaccine is found. Over 34 million individuals in the world are infected with the virus, and the vast majority of them have no access to the antiretroviral therapies that have largely reduced HIV to a chronic disease in the developed world. The first chapter of this thesis introduces the history of the virus. The key to the infectious mechanism of the virus lies in its envelope glycoprotein (Env), a trimeric spike on the viral surface that utilizes host T cell receptors for entry. Though HIV-1 Env is immunogenic, most infected patients do not mount an effective neutralizing antibody response against it. Broadly-neutralizing anti-Env antibodies (bNAbs) present in the serum of a minority of infected individuals are usually sufficient to prevent the progression to full blown AIDS. Thus, the molecular details of these bNAbs as well as the antibody-antigen interface are of prime interest for structural studies, as insight gained would contribute to the design of a more effective immunogen and potential vaccine candidate. The second chapter of this thesis describes the low-resolution crystal structure of one such antibody, 2G12 dimer, which targets a high mannose epitope on the surface of Env. Patients infected with HIV-2, a related virus with ~35% sequence identity in the Env region, can generally mount a robust antibody response sufficient for viral control for reasons still unknown. The final two chapters of this thesis focus on the first reported structural studies of HIV-2 Env, the molecular details of which may inform HIV-1 therapy and immunogen design.

From molecules to organs : Microscopy and multi-scale nature of development

Morphogenesis is a phenomenon of intricate balance and dynamic interplay between processes occurring at a wide range of scales (spatial, temporal and energetic). During development, a variety of physical mechanisms are employed by tissues to simultaneously pattern, move, and differentiate based on information exchange between constituent cells, perhaps more than at any other time during an organism's life. To fully understand such events, a combined theoretical and experimental framework is required to assist in deciphering the correlations at both structural and functional levels at scales that include the intracellular and tissue levels as well as organs and organ systems. Microscopy, especially diffraction-limited light microscopy, has emerged as a central tool to capture the spatio-temporal context of life processes. Imaging has the unique advantage of watching biological events as they unfold over time at single-cell resolution in the intact animal. In this work I present a range of problems in morphogenesis, each unique in its requirements for novel quantitative imaging both in terms of the technique and analysis. Understanding the molecular basis for a developmental process involves investigating how genes and their products- mRNA and proteins-function in the context of a cell. Structural information holds the key to insights into mechanisms and imaging fixed specimens paves the first step towards deciphering gene function. The work presented in this thesis starts with the demonstration that the fluorescent signal from the challenging environment of whole-mount imaging, obtained by in situ hybridization chain reaction (HCR), scales linearly with the number of copies of target mRNA to provide quantitative sub-cellular mapping of mRNA expression within intact vertebrate embryos. The work then progresses to address aspects of imaging live embryonic development in a number of species. While processes such as avian cartilage growth require high spatial resolution and lower time resolution, dynamic events during zebrafish somitogenesis require higher time resolution to capture the protein localization as the somites mature. The requirements on imaging are even more stringent in case of the embryonic zebrafish heart that beats with a frequency of ~ 2-2.5 Hz, thereby requiring very fast imaging techniques based on two-photon light sheet microscope to capture its dynamics. In each of the hitherto-mentioned cases, ranging from the level of molecules to organs, an imaging framework is developed, both in terms of technique and analysis to allow quantitative assessment of the process in vivo. Overall the work presented in this thesis combines new quantitative tools with novel microscopy for the precise understanding of processes in embryonic development.

Eigenvalue problems associated with Korn's inequalities in the theory of elasticity

Interest in the possible applications of a priori inequalities in linear elasticity theory motivated the present investigation. Korn's inequality under various side conditions is considered, with emphasis on the Korn's constant. In the "second case" of Korn's inequality, a variational approach leads to an eigenvalue problem; it is shown that, for simply-connected two-dimensional regions, the problem of determining the spectrum of this eigenvalue problem is equivalent to finding the values of Poisson's ratio for which the displacement boundary-value problem of linear homogeneous isotropic elastostatics has a non-unique solution.

Previous work on the uniqueness and non-uniqueness issue for the latter problem is examined and the results applied to the spectrum of the Korn eigenvalue problem. In this way, further information on the Korn constant for general regions is obtained.

A generalization of the "main case" of Korn's inequality is introduced and the associated eigenvalue problem is a gain related to the displacement boundary-value problem of linear elastostatics in two dimensions.

Effects of magnetostriction and superlattice formation in ferromagnetic thin films

The contribution to the magnetic uniaxial perpendicular anisotropy which arises from substrate constraint through magnetostrictive effects has been measured in Ni-Fe and Ni-Co thin films evaporated on substrates at room temperature. This was accomplished by measuring the perpendicular anisotropy before and after removal of the film from the substrate. Data are given for the fcc crystal structure regions of both alloy systems, but data for Ni-Co include compositions with less than 60% Ni which have a small percentage of the hcp phase mixed with the fcc phase. The constraint contribution to the perpendicular anisotropy correlates well with the value of the bulk magnetostriction constant using the equation ∆K_˔=3/2λ_sσ. Measured values of isotropic stress for films thicker than 600 Å were 1.6 x 10¹⁰ dyn/cm². In films less than 600 Å thick the isotropic stress decreased with decreasing thickness. After removal of the films from the substrates, the measured perpendicular anisotropy deviated from the expected geometrical shape anisotropy near pure Ni in both alloys. This indicates that additional significant sources of anisotropy exist at these compositions.

The effect of substrate constraint on the crystalline anisotropy K₁ of Ni-Fe epitaxial films has been studied by use of a film removal technique, which involves the evaporation of an epitaxial layer of LiF on MgO, the epitaxial growth of the metallic film on the LiF, and the stripping of the film with water soluble tape. Films ranging in composition from 50% to 100% Ni have been studied. For compositions below 90% Ni the experimental values agree reasonably well with the first order theoretical prediction, ∆K₁=[-9/4(C₁₁-C₁₂)λ² 100+9/2C₄₄λ²111].

In order to compare the magnetic properties of epitaxial thin films more completely with the properties of bulk single crystals, Ni-Fe films ranging in composition from 60% to 90% Ni, which were evaporated epitaxially on (100) MgO substrates, have been subsequently annealed at 400°C in a vacuum of less than 10^-7 Torr to form the ordered Ni₃Fe structure near the 75% composition. This ordered structure has been confirmed by electron diffraction.

The saturation magnetization at Ni₃Fe increased about 6% with ordering which is in good agreement with previous bulk data. Measurements of the magnetocrystalline anisotropy energy K₁ for the epitaxial films show the same large changes with ordering as observed in bulk single crystal samples. In the (001) plane the magnetostriction constants ^λ100, ^λ111 are directly related to the induced anisotropy due to a uniform uniaxial strain in the [100] and [110] directions respectively. Assuming that the elastic constants of a film are the same as in bulk material and are unchanged by ordering, the changes in strain sensitivity with ordering for the epitaxial films are found to be in good agreement with values predicted from bulk data. The exchange constant A as measured by ferromagnetic resonance has been measured at the Ni₃Fe composition and found to increase 25% with ordering. This seems to indicate a significant increase in the Curie temperature which has only been inferred indirectly for bulk material.

一种ICF用新型可阵列反射镜架结构

运用并联机构原理设计了一种新型惯性约束聚变（Inetial Confinement Fusion, ICF）用可阵列反射镜架结构,并用坐标变换方法分析了机构的调整正交性与旋转轴正交性之间的关系.装置的实际测试结果表明调整正交性与分析结果一致,并且反射镜架的稳定性以及调节准确度均能符合ICF装置的使用要求.

A global protocol for monitoring of coral bleaching

Coral bleaching and subsequent mortality represent a major threat to the future health and productivity of coral reefs. However a lack of reliable data on occurrence, severity and other characteristics of bleaching events hampers research on the causes and consequences of this important phenomenon. This article describes a global protocol for monitoring coral bleaching events, which addresses this problem and can be used by people with different levels of expertise and resources.

Coral spawning information

Information on reproduction in reef corals is presented. An understanding of its reproductive behaviour is an important factor in helping to preserve the coral reef ecosystems.

Feeding habits of European hake (Merluccius merluccius) in the central Mediterranean Sea

European hake (Merluccius merluccius) is an important predator of deeper shelf-upper slope Mediterranean communities. It is a nectobenthic species distributed over a wide depth range (20−1000 m) throughout the Mediterranean Sea and the north east Atlantic region (Fisher et al., 1987). Notwithstanding the ecological and economic importance (Oliver and Massutí, 1995) of hake in the Mediterranean, many aspects of its biology (e.g., recruitment and reproduction), due to multiple spawning (Sarano, 1986) and the current state of exploitation, are poorly understood (Arneri and Morales-Nin, 2000).

An Economic Analysis of Texas Shrimp Season Closures

Management of the Texas penaeid shrimp fishery is aimed at increasing revenue from brown shrimp, Penaeus aztecus, landings and decreasing the level of discards. Since 1960 Texas has closed its territorial sea for 45-60 days during peak migration of brown shrimp to the Gulf of Mexico. In 1981 the closure was extended to 200 miles to include the U.S. Exclusive Economic Zone. Simulation modeling is used in this paper to estimate the changes in landings, revenue, costs, and economic rent attributable to the Texas closure. Four additional analyses were conducted to estimate the effects of closing the Gulf 1- to 4-fathom zone for 45 and 60 days, with and without effort redirected to inshore waters. Distributional impacts are analyzed in terms of costs, revenues, and rents, by vessel class, shrimp species, vessel owner, and crew.