bluuebunny/crossref_metadata_2025 huggingface dataset:
Associations between Breast Milk Viral Load, Mastitis, Exclusive Breast-Feeding, and Postnatal Transmission of HIV
Authors: Lunney Kevin M. , Iliff Peter , Mutasa Kuda , Ntozini Robert , Magder Laurence S. , Moulton Lawrence H. , Humphrey Jean H.
Published: February 2010

Background . Exclusive breast-feeding is protective against postnatal transmission of human immunodeficiency virus (HIV), compared with mixed breast-feeding. Accordingly, exclusive breast-feeding for 6 months is the World Health Organization's recommendation to HIV-infected mothers for whom exclusive replacement feeding is not acceptable, feasible, affordable, safe, or sustainable. The mechanism of exclusive breast-feeding protection is unknown but is hypothesized to be mediated through reduced mastitis.

Methods . We compared breast milk and plasma specimens of exclusive breast-feeding and mixed breast-feeding HIV- positive mothers archived from the ZVITAMBO trial in which mixed breast-feeding was associated with a 2-fold increased risk of postnatal transmission at 18 months. Plasma HIV load, breast milk HIV load and sodium/potassium ratio were measured as a proxy for subclinical mastitis.

Results . Mixed breast-feeding was not associated with mastitis or breast milk HIV load. Mastitis was associated with breast milk HIV load, and this effect increased with increasing maternal plasma HIV load; mastitis was associated with postnatal transmission only when maternal plasma HIV load was high (>3.7 log 10 copies/mL). Initiation of breast-feeding within an hour of delivery was associated with exclusive breast-feeding (adjusted odds ratio, 1.62; 95% confidence interval, 1.02–2.58).

Conclusions . Exclusive breast-feeding is associated with reduced postnatal transmission of HIV from mother to child, but this protection is not mediated by reduced mastitis or breast milk HIV load. The deleterious effect of mastitis increases as the mother's plasma HIV load increases.
Lifting of characters for nonlinear simply laced groups
Authors: Adams Jeffrey , Herb Rebecca
Published: February 2010

One aspect of the Langlands program for linear groups is the lifting of characters, which relates virtual representations on a group

G
G

with those on an endoscopic group for

G
G

. The goal of this paper is to extend this theory to nonlinear two-fold covers of real groups in the simply laced case. Suppose

G
~

\widetilde G

is a two-fold cover of a real reductive group

G
G

. A representation of

G
~

\widetilde G

is called genuine if it does not factor to

G
G

. The main result is that there is an operation, denoted

Lift
G

G
~

\text {Lift}_G^{\widetilde G}

, taking a stable virtual character of

G
G

to a virtual genuine character of

G
~

\widetilde G

, and

Lift
G

G
~

(

Θ
π

)

\text {Lift}_G^{\widetilde G}(\Theta _\pi )

may be explicitly computed if

π
\pi

is a stable sum of standard modules.
Vascular Endothelial Growth Factor: Much More than an Angiogenesis Factor
Authors: Senger Donald R.
Published: February 2010

Vascular endothelial growth factor (VEGF) is best known as a cytokine essential for embryonic vasculogenesis and for the angiogenesis associated with various pathologies including cancer. However, VEGF also serves other functions that are less widely recognized. An early study ( Berse et al., 1992 ) revealed widespread expression of VEGF transcripts in adult tissues devoid of ongoing neovascularization, thereby predicting additional VEGF functions distinct from angiogenesis. More recent studies have confirmed that VEGF does indeed serve multiple additional functions, including normal maintenance of endothelial and neural cell compartments. These findings have important implications for the use of VEGF antagonists and VEGF receptor antagonists in patients for which inhibition of pathological angiogenesis is the therapeutic goal.
Genome-Wide Identification of a Large Repertoire of Ralstonia solanacearum Type III Effector Proteins by a New Functional Screen
Authors: Mukaihara Takafumi , Tamura Naoyuki , Iwabuchi Masaki
Published: February 2010

The gram-negative plant-pathogenic bacterium Ralstonia solanacearum utilizes the hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS) to cause disease in plants. To determine the entire repertoire of effector proteins possessed by R. solanacearum RS1000, we constructed a transposon carrying a calmodulin-dependent adenylate cyclase reporter that can be used to specifically detect rip (Ralstonia protein injected into plant cells) genes by monitoring the cAMP level in plant leaves inoculated with insertion mutants. From the new functional screen using this transposon, we identified 38 new Rip proteins translocated into plant cells via the Hrp T3SS. In addition, most of the 34 known effectors of RS1000 could be detected by the screen, except for three effectors that appear to be small in size or only weakly expressed. Finally, we identified 72 Rips in RS1000, which include 68 effector proteins classified into over 50 families and four extracellular components of the Hrp T3SS. Interestingly, one-third of the effectors are specific to R. solanacearum. Many effector proteins contain various repeated amino acid sequences or known enzyme motifs. We also show that most of the R. solanacearum effector proteins, but not Hrp extracellular components, require an Hrp-associated protein, HpaB, for their effective translocation into plant cells.
The Intra- and Intercellular Movement of Melon necrotic spot virus (MNSV) Depends on an Active Secretory Pathway
Authors: Genovés Ainhoa , Navarro Jose Antonio , Pallás Vicente
Published: February 2010

Plant viruses hijack endogenous host transport machinery to aid their intracellular spread. Here, we study the localization of the p7B, the membrane-associated viral movement protein (MP) of the Melon necrotic spot virus (MNSV), and also the potential involvement of the secretory pathway on the p7B targeting and intra- and intercellular virus movements. p7B fused to fluorescent proteins was located throughout the endoplasmic reticulum (ER) at motile Golgi apparatus (GA) stacks that actively tracked the actin microfilaments, and at the plasmodesmata (PD). Hence, the secretory pathway inhibitor, Brefeldin A (BFA), and the overexpression of the GTPase-defective mutant of Sar1p, Sar1[H74L], fully retained the p7B within the ER, revealing that the protein is delivered to PD in a BFA-sensitive and COPII-dependent manner. Disruption of the actin cytoskeleton with latrunculin B led to the accumulation of p7B in the ER, which strongly suggests that p7B is also targeted to the cell periphery in an actin-dependent manner. Remarkably, the local spread of the viral infection was significantly restricted either with the presence of BFA or under the overexpression of Sar1[H74L], thus revealing the involvement of an active secretory pathway in the intracellular movement of MNSV. Overall, these findings support a novel route for the intracellular transport of a plant virus led by the GA.
Viral-Induced Systemic Necrosis in Plants Involves Both Programmed Cell Death and the Inhibition of Viral Multiplication, Which Are Regulated by Independent Pathways
Authors: Komatsu Ken , Hashimoto Masayoshi , Ozeki Johji , Yamaji Yasuyuki , Maejima Kensaku , Senshu Hiroko , Himeno Misako , Okano Yukari , Kagiwada Satoshi , Namba Shigetou
Published: February 2010

Resistant plants respond rapidly to invading avirulent plant viruses by triggering a hypersensitive response (HR). An HR is accompanied by a restraint of virus multiplication and programmed cell death (PCD), both of which have been observed in systemic necrosis triggered by a successful viral infection. Here, we analyzed signaling pathways underlying the HR in resistance genotype plants and those leading to systemic necrosis. We show that systemic necrosis in Nicotiana benthamiana, induced by Plantago asiatica mosaic virus (PlAMV) infection, was associated with PCD, biochemical features, and gene expression patterns that are characteristic of HR. The induction of necrosis caused by PlAMV infection was dependent on SGT1, RAR1, and the downstream mitogen-activated protein kinase (MAPK) cascade involving MAPKKKα and MEK2. However, although SGT1 and RAR1 silencing led to an increased accumulation of PlAMV, silencing of the MAPKKKα-MEK2 cascade did not. This observation indicates that viral multiplication is partly restrained even in systemic necrosis induced by viral infection, and that this restraint requires SGT1 and RAR1 but not the MAPKKKα-MEK2 cascade. Similarly, although both SGT1 and MAPKKKα were essential for the Rx-mediated HR to Potato virus X (PVX), SGT1 but not MAPKKKα was involved in the restraint of PVX multiplication. These results suggest that systemic necrosis and HR consist of PCD and a restraint of virus multiplication, and that the latter is induced through unknown pathways independent from the former.
Cucumber Mosaic Virus 2b Protein Subcellular Targets and Interactions: Their Significance to RNA Silencing Suppressor Activity
Authors: González Inmaculada , Martínez Llucia , Rakitina Daria V. , Lewsey Mathew G. , Atencio Félix A. , Llave César , Kalinina Natalia O. , Carr John P. , Palukaitis Peter , Canto Tomás
Published: February 2010

The RNA silencing suppressor activity of the 2b protein of Cucumber mosaic virus (CMV) has been variously attributed to its nuclear targeting, its interaction with and inhibition of Argonaute 1 (AGO1), or its ability to bind small RNAs in vitro. In addition, the 2b ortholog of Tomato aspermy virus forms aggregates and binds RNAs in vitro. We have further studied the relationships between CMV 2b protein silencing suppressor activity and its subcellular distribution, protein-protein interactions in vivo, and interactions with small interfering RNAs in vitro. To do this, we tagged the protein with fluorescent markers and showed that it retained suppressor activity. We showed that the 2b protein is present in the nucleolus and that it self-interacts and interacts with AGO1 and AGO4 in vivo. Using a battery of mutants, we showed that the putative nuclear localization signals and phosphorylation motif of the 2b protein are not required for self-interaction or for interaction with AGO proteins. The occurrence of neither of these interactions or of nucleolar targeting was sufficient to provide local silencing-suppression activity. In contrast, the ability of the 2b protein to bind small RNAs appears to be indispensable for silencing suppressor function.
MoRic8 Is a Novel Component of G-Protein Signaling During Plant Infection by the Rice Blast FungusMagnaporthe oryzae
Authors: Li Ya , Yan Xia , Wang Hong , Liang Shen , Ma Wei-Bin , Fang Min-Yan , Talbot Nicholas J. , Wang Zheng-Yi
Published: February 2010

An insertional mutagenesis screen was used to investigate the biology of plant infection by the devastating rice blast pathogen, Magnaporthe oryzae. Here, we report the identification of a new mutant, LY-130, which is defective in multiple steps during infection-related morphogenesis and pathogenicity. Analysis of the mutation confirmed an insertion into gene MoRIC8, which encodes a 480-amino-acid protein that is a putative homologue of the Ric8 regulator of GTP-binding protein (G-protein) signaling, previously described in animals. Targeted gene deletion mutants of MoRIC8 were nonpathogenic and impaired in cellular differentiation associated with sporulation, sexual development, and plant infection. MoRic8 physically interacts with the Gα subunit MagB in yeast two-hybrid assays and appears to act upstream of the cyclic AMP response pathway that is necessary for appressorium morphogenesis. Taken together, our results indicate that MoRic8 may act as a novel regulator of the G-protein signaling during infection-related development of rice blast fungus M. oryzae.
Physics and complexity
Authors: Sherrington David
Published: February 2010

This paper is concerned with complex macroscopic behaviour arising in many-body systems through the combinations of competitive interactions and disorder, even with simple ingredients at the microscopic level. It attempts to indicate and illustrate the richness that has arisen, in conceptual understanding, in methodology and in application, across a large range of scientific disciplines, together with a hint of some of the further opportunities that remain to be tapped. In doing so, it takes the perspective of physics and tries to show, albeit rather briefly, how physics has contributed and been stimulated.
Synthetic mimetics of protein secondary structure domains
Authors: Ross Nathan T. , Katt William P. , Hamilton Andrew D.
Published: February 2010

Proteins modulate the majority of all biological functions and are primarily composed of highly organized secondary structural elements such as helices, turns and sheets. Many of these functions are affected by a small number of key protein–protein contacts, often involving one or more of these well-defined structural elements. Given the ubiquitous nature of these protein recognition domains, their mimicry by peptidic and non-peptidic scaffolds has become a major focus of contemporary research. This review examines several key advances in secondary structure mimicry over the past several years, particularly focusing upon scaffolds that show not only promising projection of functional groups, but also a proven effect in biological systems.
Energy efficiency, resilience to future climates and long-term sustainability: the role of the built environment
Authors: Kelly M. J.
Published: February 2010

Just under half of all energy consumption in the UK today takes place indoors, and over a quarter within our homes. The challenges associated with energy security, climate change and sustainable consumption will be overcome or lost in our existing buildings. A background analysis, and the scale of the engineering challenge for the next three to four decades, is described in this paper.
A simple approach to polymer mixture miscibility
Authors: Higgins Julia S. , Lipson Jane E. G. , White Ronald P.
Published: February 2010

Polymeric mixtures are important materials, but the control and understanding of mixing behaviour poses problems. The original Flory–Huggins theoretical approach, using a lattice model to compute the statistical thermodynamics, provides the basic understanding of the thermodynamic processes involved but is deficient in describing most real systems, and has little or no predictive capability. We have developed an approach using a lattice integral equation theory, and in this paper we demonstrate that this not only describes well the literature data on polymer mixtures but allows new insights into the behaviour of polymers and their mixtures. The characteristic parameters obtained by fitting the data have been successfully shown to be transferable from one dataset to another, to be able to correctly predict behaviour outside the experimental range of the original data and to allow meaningful comparisons to be made between different polymer mixtures.
The path to fusion power
Authors: Smith Chris Llewellyn , Cowley Steve
Published: February 2010

The promise, status and challenges of developing fusion power are outlined. The key physics and engineering principles are described and recent progress quantified. As the successful demonstration of 16 MW of fusion in 1997 in the Joint European Torus showed, fusion works. The central issue is therefore to make it work reliably and economically on the scale of a power station. We argue that to meet this challenge in 30 years we must follow the aggressive programme known as the ‘Fast Track to Fusion’. This programme is described in some detail.
Electrons in one dimension
Authors: Berggren K.-F. , Pepper M.
Published: February 2010

In this article, we present a summary of the current status of the study of the transport of electrons confined to one dimension in very low disorder GaAs–AlGaAs heterostructures. By means of suitably located gates and application of a voltage to ‘electrostatically squeeze’ the electronic wave functions, it is possible to produce a controllable size quantization and a transition from two-dimensional transport. If the length of the electron channel is sufficiently short, then transport is ballistic and the quantized subbands each have a conductance equal to the fundamental quantum value 2
e
2
/
h
, where the factor of 2 arises from the spin degeneracy. This mode of conduction is discussed, and it is shown that a number of many-body effects can be observed. These effects are discussed as in the spin-incoherent regime, which is entered when the separation of the electrons is increased and the exchange energy is less than
kT
. Finally, results are presented in the regime where the confinement potential is decreased and the electron configuration relaxes to minimize the electron–electron repulsion to move towards a two-dimensional array. It is shown that the ground state is no longer a line determined by the size quantization alone, but becomes two distinct rows arising from minimization of the electrostatic energy and is the precursor of a two-dimensional Wigner lattice.
Biophysics and systems biology
Authors: Noble Denis
Published: February 2010

Biophysics at the systems level, as distinct from molecular biophysics, acquired its most famous paradigm in the work of Hodgkin and Huxley, who integrated their equations for the nerve impulse in 1952. Their approach has since been extended to other organs of the body, notably including the heart. The modern field of computational biology has expanded rapidly during the first decade of the twenty-first century and, through its contribution to what is now called systems biology, it is set to revise many of the fundamental principles of biology, including the relations between genotypes and phenotypes. Evolutionary theory, in particular, will require re-assessment. To succeed in this, computational and systems biology will need to develop the theoretical framework required to deal with multilevel interactions. While computational power is necessary, and is forthcoming, it is not sufficient. We will also require mathematical insight, perhaps of a nature we have not yet identified. This article is therefore also a challenge to mathematicians to develop such insights.
Micrographiaof the twenty-first century: from camera obscurato 4D microscopy
Authors: Zewail Ahmed H.
Published: February 2010

In this paper, the evolutionary and revolutionary developments of microscopic imaging are overviewed with a perspective on origins. From Alhazen’scamera obscura, to Hooke and van Leeuwenhoek’s two-dimensional optical micrography, and on to three- and four-dimensional (4D) electron microscopy, these developments over a millennium have transformed humans’ scope of visualization. The changes in the length and time scales involved are unimaginable, beginning with the visible shadows of candles at the centimetre and second scales, and ending with invisible atoms with space and time dimensions of sub-nanometre and femtosecond. With these advances it has become possible to determine the structures of matter and to observe their elementary dynamics as they unfold in real time. Such observations provide the means for visualizing materials behaviour and biological function, with the aim of understanding emergent phenomena in complex systems.
‘… a metal conducts and a non-metal doesn't’
Authors: Edwards P. P. , Lodge M. T. J. , Hensel F. , Redmer R.
Published: February 2010

In a letter to one of the authors, Sir Nevill Mott, then in his tenth decade, highlighted the fact that the statement ‘… a metal conducts, and a non-metal doesn’t’ can be true only at the absolute zero of temperature,
T
=0 K. But, of course, experimental studies of metals, non-metals and, indeed, the electronic and thermodynamic transition between these canonical states of matter must always occur above
T
=0 K, and, in many important cases, for temperatures far above the absolute zero. Here, we review the issues—theoretical and experimental—attendant on studies of the metal to non-metal transition in doped semiconductors at temperatures close to absolute zero (
T
=0.03 K) and fluid chemical elements at temperatures far above absolute zero (
T
>1000 K).

We attempt to illustrate Mott’s insights for delving into such complex phenomena and experimental systems, finding intuitively the dominant features of the science, and developing a coherent picture of the different competing electronic processes. A particular emphasis is placed on the idea of a ‘Mott metal to non-metal transition’ in the nominally metallic chemical elements rubidium, caesium and mercury, and the converse metallization transition in the nominally non-metal elements hydrogen and oxygen. We also review major innovations by D. A. Goldhammer (Goldhammer 1913
Dispersion und absorption des lichtes
) and K. F. Herzfeld (Herzfeld 1927
Phys. Rev.
29
, 701–705. (
doi:10.1103/PhysRev.29.701
)) in a pre-quantum theory description of the metal–non-metal transition, which emphasize the pivotal role of atomic properties in dictating the metallic or non-metallic status of the chemical elements of the periodic table under ambient and extreme conditions; a link with Pauling’s ‘metallic orbital’ is also established here.
Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal
Authors: Niakan Kathy K. , Ji Hongkai , Maehr René , Vokes Steven A. , Rodolfa Kit T. , Sherwood Richard I. , Yamaki Mariko , Dimos John T. , Chen Alice E. , Melton Douglas A. , McMahon Andrew P. , Eggan Kevin
Published: February 2010

In embryonic stem (ES) cells, a well-characterized transcriptional network promotes pluripotency and represses gene expression required for differentiation. In comparison, the transcriptional networks that promote differentiation of ES cells and the blastocyst inner cell mass are poorly understood. Here, we show that Sox17 is a transcriptional regulator of differentiation in these pluripotent cells. ES cells deficient in Sox17 fail to differentiate into extraembryonic cell types and maintain expression of pluripotency-associated transcription factors, including Oct4, Nanog, and Sox2. In contrast, forced expression of Sox17 down-regulates ES cell-associated gene expression and directly activates genes functioning in differentiation toward an extraembryonic endoderm cell fate. We show these effects of Sox17 on ES cell gene expression are mediated at least in part through a competition between Sox17 and Nanog for common DNA-binding sites. By elaborating the function of Sox17, our results provide insight into how the transcriptional network promoting ES cell self-renewal is interrupted, allowing cellular differentiation.
Coordinating cohesion, co-orientation, and congression during meiosis: lessons from holocentric chromosomes
Authors: Schvarzstein Mara , Wignall Sarah M. , Villeneuve Anne M.
Published: February 2010

Organisms that reproduce sexually must reduce their chromosome number by half during meiosis to generate haploid gametes. To achieve this reduction in ploidy, organisms must devise strategies to couple sister chromatids so that they stay together during the first meiotic division (when homologous chromosomes separate) and then segregate away from one another during the second division. Here we review recent findings that shed light on how Caenorhabditis elegans, an organism with holocentric chromosomes, deals with these challenges of meiosis by differentiating distinct chromosomal subdomains and remodeling chromosome structure during prophase. Furthermore, we discuss how features of chromosome organization established during prophase affect later chromosome behavior during the meiotic divisions. Finally, we illustrate how analysis of holocentric meiosis can inform our thinking about mechanisms that operate on monocentric chromosomes.
Structural basis of YAP recognition by TEAD4 in the Hippo pathway
Authors: Chen Liming , Chan Siew Wee , Zhang XiaoQian , Walsh Martin , Lim Chun Jye , Hong Wanjin , Song Haiwei
Published: February 2010

The Hippo signaling pathway controls cell growth, proliferation, and apoptosis by regulating the expression of target genes that execute these processes. Acting downstream from this pathway is the YAP transcriptional coactivator, whose biological function is mediated by the conserved TEAD family transcription factors. The interaction of YAP with TEADs is critical to regulate Hippo pathway-responsive genes. Here, we describe the crystal structure of the YAP-interacting C-terminal domain of TEAD4 in complex with the TEAD-interacting N-terminal domain of YAP. The structure reveals that the N-terminal region of YAP is folded into two short helices with an extended loop containing the PXXΦP motif in between, while the C-terminal domain of TEAD4 has an immunoglobulin-like fold. YAP interacts with TEAD4 mainly through the two short helices. Point mutations of TEAD4 indicate that the residues important for YAP interaction are required for its transforming activity. Mutagenesis reveals that the PXXΦP motif of YAP, although making few contacts with TEAD4, is important for TEAD4 interaction as well as for the transforming activity.
Visualizing Fetal Mouse Vasculature by India Ink Injection: Figure 1.
Authors: Nagy Andras
Published: February 2010

INTRODUCTION

The technique described in this protocol was first applied in chicken embryos to visualize the circulatory system. The following method is an adaptation of this “classic” method to the mouse. The basic procedure is to dissect 12.5-dpc (days post-coitus) or older embryos with an intact yolk sac and placenta while the circulation is still active. India ink is then injected slowly into a branch of the vitelline vein of the yolk sac. The carbon particles in the ink are distributed into the arterial system by the beating heart. After fixation and clearing, the three-dimensional structure of the fetal arterial system becomes visible.
Calcium Phosphate Transfection of 3T3 Fibroblasts
Authors: Smale Stephen T.
Published: February 2010

INTRODUCTION

Mixing DNA with calcium chloride in a phosphate buffer results in the formation of a DNA/calcium phosphate precipitate which, when layered onto cultured cells, is taken up by endocytosis. The method is used widely for transfecting fibroblasts and other adherent cell types and can be performed easily with reagent-grade chemicals. This protocol describes the calcium phosphate transfection of murine 3T3 fibroblasts. Briefly, cells are grown on 60- or 100-mm tissue culture dishes and passaged 1 d prior to transfection, such that they are ~50% confluent at the time of transfection. This allows the cells to be transfected when most are not in direct contact with other cells. It also allows the cells to reach 100% confluence ~48 h post-transfection, when most transiently transfected cells are harvested. A precipitate containing DNA and calcium phosphate is formed by gradually mixing a phosphate-containing buffer with a solution of DNA and calcium chloride. The mixture is then layered onto a plate containing the adherent cultured cells and growth medium. After the cells have been exposed to the precipitate for a defined period of time, the medium containing the precipitate is removed and fresh medium is added to prevent toxicity. The procedure is suitable for performing both transient and stable transfection experiments.
Transfection by Electroporation of RAW 264.7 Macrophages
Authors: Smale Stephen T.
Published: February 2010

INTRODUCTION

In electroporation-based transfection methods, the DNA and cells are mixed in an empirically defined buffer, which establishes the resistance during the electric shock. The solution is placed in a cuvette that is compatible with the electroporation device being used. The mixture is then subjected to a brief high-voltage shock, which induces or stabilizes pores in the plasma membrane through which the DNA enters the cell. The cells are allowed to recover by a brief incubation at room temperature and then transferred to a tissue culture plate or T-flask along with growth medium and serum. Electroporation is an attractive method for transfection because it is more rapid and involves fewer manipulations than other methods and can be used for many types of adherent and nonadherent cells. This protocol describes transfection by electroporation of RAW 264.7 macrophages.
Preparation of the Gel and Column for Chromatofocusing: Figure 1.
Authors: Mohammad Jamil
Published: February 2010

INTRODUCTION

The appropriate stationary phase for chromatofocusing depends very much on the intended application. In general, in order to form a linear pH gradient (which is critical for good separation), the stationary phase must provide uniform buffering capacity over the pH separation range of interest. Thus, the functional groups on the matrix must be able to provide high buffering capacity over a broad pH range. Examples of such functional groups are polyethyleneimine (PEI) and a mixture of tertiary and quaternary amines. The most commonly used stationary phases for chromatofocusing are polymer-based stationary phases such as Mono P or polybuffer exchanger (PBE). In this protocol, a column is readied and packed with PBE matrix. The matrix is equilibrated with the buffer that will be used at the start of the separation, and the quality of the packing is assessed using cytochrome c.
Preparation of the Sample for Chromatofocusing: Buffer Exchange and Desalting
Authors: Mohammad Jamil
Published: February 2010

INTRODUCTION

Chromatofocusing separates proteins on the basis of differences in their isoelectric point (pI). Successful chromatofocusing depends on careful preparation of the protein sample, as described in this protocol. The protein must be transferred to the same buffer that will be used to equilibrate the chromatofocusing column. The buffer pH should be higher than the pI of the protein to keep the proteins negatively charged, which facilitates their binding to the column. A protein sample containing particulate matter will decrease the resolution of the separation and shorten the column lifetime. Therefore, it is necessary to remove all insoluble and immiscible material from the sample prior to loading it on the column. The sample is first clarified by filtration or centrifugation and is then transferred into the start buffer using a high-capacity, fast-flowing, size-exclusion column packed with Sephadex G-25.
Removal of Elution Buffer from Protein Sample Fractions after Chromatofocusing
Authors: Mohammad Jamil
Published: February 2010

INTRODUCTION

Chromatofocusing is a technique that separates proteins on the basis of differences in their isoelectric point (pI). Proteins are often eluted from a chromatofocusing column in Polybuffer or Pharmalyte. These buffers typically do not interfere with subsequent amino acid analysis or with the Coomassie blue protein assay. If buffer exchange and/or desalting is required, a number of methods are available to the researcher. Centrifugal filter units work well. As is described in this article, proteins can be concentrated and transferred to another buffer using ammonium sulfate precipitation which, in some cases, is performed in conjunction with hydrophobic interaction chromatography.
Cleaning, Regeneration, and Storage of Chromatofocusing Columns
Authors: Mohammad Jamil
Published: February 2010

INTRODUCTION

Chromatofocusing separates proteins on the basis of differences in their isoelectric point (pI). A chromatofocusing column should be cleaned and regenerated after use, as described in this protocol, to remove any strongly bound substances. The following observations indicate that column cleaning may be necessary: increased back pressure in the column, a color change at the top of the column, loss of resolution, or reduced sample recovery. To avoid these problems, clean the column routinely (e.g., after every fifth or tenth run). More frequent and extended cleaning procedures may be necessary if complex samples are applied. The steps in this article are guidelines. Personal experience with possible contamination should be used to evaluate which cleaning method is suitable.
Four-Dimensional Imaging of Plant Cells During the Cell Cycle
Authors: Fang Yuda , Spector David L.
Published: February 2010

INTRODUCTION

Live cell imaging is an essential approach for studying the structure, dynamics, and functions of cells in a living plant under normal or stressed growth conditions. The tiny flowering plant, Arabidopsis thaliana, provides an ideal system to apply various live microscopy techniques. Its small size allows fluorescent light to penetrate the tissues, and its plantlets contain different cell types with different ploidy levels and differentiation stages. Its 2C nucleus contains only five pairs of chromosomes in which heterochromatin domains are organized as chromocenters, and these domains are easily resolved under the microscope. In addition, the availability of powerful genetic tools facilitates the investigation of the molecular mechanisms underlying various cellular phenomena. In designing live imaging experiments, one must keep in mind that plants sense light, temperature, osmolarity, humidity, gravity, and nutrition. In addition, plants also have strong circadian rhythms of physiological behavior and gene expression. Moreover, plant tissues are normally thick (having multiple cell layers), and can have strong autofluorescence, especially in green leaves. Therefore, optimized culturing and imaging conditions are essential for successful live cell studies in plants. In this protocol, specific chromatin loci (centromeres) in Arabidopsis are tagged with centromere-specific histone 3 variant (HTR12)-fluorescent protein (FP) fusions, and transgenic plants expressing these fusions are generated. Three- and four-dimensional imaging techniques are used to visualize the organization and dynamics of these specific chromatin loci in interphase and through mitosis. The procedure can be modified easily to accommodate other proteins or structures of interest.
Analysis of Gene Expression by In Situ Hybridization on Adult Zebrafish Brain Sections
Authors: Kühn Enrico , Köster Reinhard W.
Published: February 2010

INTRODUCTION

Whole-mount in situ hybridization is a powerful method for the analysis of gene expression in zebrafish. However, the thickness of adult brain tissue often hampers penetration of reagents, making the direct application of protocols normally used for embryonic stages on adult tissues difficult. This protocol presents a detailed procedure for the dissection, preparation, and processing of the adult zebrafish brain for detection of gene expression in situ using digoxigenin (DIG)-labeled RNA probes on vibratome sections: The entire procedure is presented, including synthesis of labeled antisense RNA, sectioning of the adult zebrafish brain, and in situ hybridization. Using this method, all parts of the tissue are easily labeled and expression patterns are reproduced faithfully between trials. Unlike whole-mount preparations, gene expression examined on vibratome sections can easily be compared to a detailed zebrafish brain atlas and assigned to specific tissues.
Purification of Genomic DNA Extracted from Environmental Sources for Use in a Polymerase Chain Reaction: Figure 1.
Authors: Newman Molli M. , Feminella Jack W. , Liles Mark R.
Published: February 2010

INTRODUCTION

The ability to amplify genomic DNA in a polymerase chain reaction (PCR) is dependent upon the purity of the DNA template. Environmental genomic DNA often contains contaminants (e.g., polyphenols, humic acids, polysaccharides) that reduce template purity and can be difficult to remove, thereby inhibiting PCR amplification. There is thus a need for a method to purify extracted genomic DNA without reducing DNA concentration. In this protocol, extracted genomic DNA is embedded in agarose plugs and incubated in a formamide and salt (NaCl) solution to remove contaminants. The NaCl works to deproteinize and stabilize the DNA. The formamide serves to denature the DNA (which will subsequently be renatured within the agarose plug) and any contaminants that may be bound to the DNA. The purified DNA is extracted from the agarose plug using a standard commercial agarose extraction method, and the DNA may then be used as a template for PCR. Genomic DNA purified using this method has been shown to serve as an efficient template for PCR, without significant loss of DNA yield. An additional advantage of the method is that it allows the simultaneous processing of large numbers of samples at once.
DNase-seq: A High-Resolution Technique for Mapping Active Gene Regulatory Elements across the Genome from Mammalian Cells
Authors: Song Lingyun , Crawford Gregory E.
Published: February 2010

INTRODUCTION

Identification of active gene regulatory elements is a key to understanding transcriptional control governing biological processes such as cell-type specificity, differentiation, development, proliferation, and response to the environment. Mapping DNase I hypersensitive (HS) sites has historically been a valuable tool for identifying all different types of regulatory elements, including promoters, enhancers, silencers, insulators, and locus control regions. This method utilizes DNase I to selectively digest nucleosome-depleted DNA (presumably by transcription factors), whereas DNA regions tightly wrapped in nucleosome and higher-order structures are more resistant. The traditional low-throughput method for identifying DNase I HS sites uses Southern blots. Here, we describe the complete and improved protocol for DNase-seq, a high-throughput method that identifies DNase I HS sites across the whole genome by capturing DNase-digested fragments and sequencing them by high-throughput, next-generation sequencing. In a single experiment, DNase-seq can identify most active regulatory regions from potentially any cell type, from any species with a sequenced genome.
Live Cell Imaging of Plants
Authors: Fang Yuda , Spector David L.
Published: February 2010

INTRODUCTION

Live cell imaging is an essential approach for studying the structure, dynamics, and functions of cells in a living plant under normal or stressed growth conditions. The tiny flowering plant, Arabidopsis thaliana, provides an ideal system to apply various live microscopy techniques. Its small size allows fluorescent light to penetrate the tissues, and its plantlets contain different cell types with different ploidy levels and differentiation stages. Its 2C nucleus contains only five pairs of chromosomes in which heterochromatin domains are organized as chromocenters, and these domains are easily resolved under the microscope. In addition, the availability of powerful genetic tools facilitates the investigation of the molecular mechanisms underlying various cellular phenomena. In designing live imaging experiments, one must keep in mind that plants sense light, temperature, osmolarity, humidity, gravity, and nutrition. In addition, plants also have strong circadian rhythms of physiological behavior and gene expression. Moreover, plant tissues are normally thick (having multiple cell layers), and can have strong autofluorescence, especially in green leaves. Therefore, optimized culturing and imaging conditions are essential for successful live cell studies in plants. This article discusses the general experimental considerations and design for live plant imaging studies.
Linkage Analysis of Discrete Traits
Authors: Borecki Ingrid B. , Rice John P.
Published: February 2010

INTRODUCTION

Linkage analysis is an important tool for the investigation of the genetic basis of disease traits and has led to the discovery of the single genes underlying such diseases as cystic fibrosis, Duchenne muscular dystrophy, Huntington’s disease, and a wide variety of metabolic disorders. For certain kinds of traits, linkage analysis allows the localization of trait loci on a known genetic map, which can be followed up with fine mapping based on linkage disequilibrium (LD) and ultimate identification of the gene. Successes using this approach have been largely confined to Mendelian monogenic disorders or oligogenic disorders (e.g., breast cancer, Alzheimer’s disease). Complex diseases (e.g., asthma, schizophrenia) involving several to many loci, as well as environmental factors, may require additional strategies to locate risk loci.
Adipocytokines as new promising markers of colorectal tumors: Adiponectin for colorectal adenoma, and resistin and visfatin for colorectal cancer
Authors: Nakajima Takako Eguchi , Yamada Yasuhide , Hamano Tetsutaro , Furuta Koh , Matsuda Takahisa , Fujita Shin , Kato Ken , Hamaguchi Tetsuya , Shimada Yasuhiro
Published: February 2010

Adipocytokines are adipocyte‐secreted hormones associated with some malignancies such as colorectal, breast, and prostate cancer. We hypothesized that changes in the levels of adipocytokines may indicate the carcinogenesis and progression of colorectal cancer and adenoma, and investigated the association of the blood levels of several adipocytokines through a case‐control study. Blood levels of adiponectin, leptin, resistin, visfatin, and C‐peptide at diagnosis were measured in 115 colorectal cancer patients and 115 age‐, sex‐, and body mass index‐matched controls. The same analysis was performed in 72 colorectal adenoma patients and 72 controls. Logistic regression models were used for estimating odds ratios and 95% confidence intervals, and one‐way anova was performed to determine the prevalence of each variable between two or more groups. Resistin and visfatin levels in cancer patients were significantly higher than those of controls on multivariate analysis (P = 0.03 and P < 0.01, respectively). Stage progression significantly correlated with resistin and visfatin levels (P < 0.01 for both). The adiponectin level in adenoma patients was significantly lower than that of controls on multivariate analysis (P = 0.04). Its level was inversely correlated with the number of adenoma (P = 0.02), but not correlated with the size of adenoma. Resistin and visfatin may be good biomarkers of colorectal malignant potential and stage progression. Adiponectin level may be a good biomarker of colorectal adenoma.

(Cancer Sci 2010; 101: 1286–1291)
Secular trends in breast cancer mortality in five East Asian populations: Hong Kong, Japan, Korea, Singapore and Taiwan
Authors: Shin Hai‐Rim , Boniol Mathieu , Joubert Clementine , Hery Clarisse , Haukka Jari , Autier Philippe , Nishino Yoshikazu , Sobue Tomotaka , Chen Chien‐Jen , You San‐Lin , Ahn Sei Hyun , Jung Kyu Won , Law Stephen Chun‐Key , Mang Oscar , Chia Kee‐Seng
Published: February 2010

Breast cancer risk is increasing in most Asian female populations, but little is known about the long‐term mortality trend of the disease among these populations. We extracted data for Hong Kong (1979–2005), Japan (1963–2006), Korea (1985–2006), and Singapore (1963–2006) from the World Health Organization (WHO) mortality database and for Taiwan (1964–2007) from the Taiwan cancer registry. The annual age‐standardized, truncated (to ≥20 years) breast cancer death rates for 11 age groups were estimated and joinpoint regression was applied to detect significant changes in breast cancer mortality. We also compared age‐specific mortality rates for three calendar periods (1975–1984, 1985–1994, and 1995–2006). After 1990, breast cancer mortality tended to decrease slightly in Hong Kong and Singapore except for women aged 70+. In Taiwan and Japan, in contrast, breast cancer death rates increased throughout the entire study period. Before the 1990s, breast cancer death rates were almost the same in Taiwan and Japan; thereafter, up to 1996, they rose more steeply in Taiwan and then they began rising more rapidly in Japan than in Taiwan after 1996. The most rapid increases in breast cancer mortality, and for all age groups, were in Korea. Breast cancer mortality trends are expected to maintain the secular trend for the next decade mainly as the prevalence of risk factors changes and population ages in Japan, Korea, and Taiwan. Early detection and treatment improvement will continue to reduce the mortality rates in Hong Kong and Singapore as observed in Western countries.

(Cancer Sci2010; 101: 1141–1246)
Molecular characterization of swine leucocyte antigen class II genes in outbred pig populations
Authors: Ho C.‐S. , Lunney J. K. , Lee J.‐H. , Franzo‐Romain M. H. , Martens G. W. , Rowland R. R. R. , Smith D. M.
Published: February 2010

The highly polymorphic swine leucocyte antigen (SLA) genes are among the most important determinants of swine immune responses to disease and vaccines. Accurate and effective SLA genotyping methods are required to understand how SLA gene polymorphisms affect immunity, especially in outbred pigs with diverse genetic backgrounds. In this study, we present a simple and rapid molecular‐based typing system for characterizing SLA class II alleles of the DRB1, DQB1 and DQA loci. This system utilizes a set of 47 sequence‐specific PCR primers developed to differentiate alleles by groups that share similar sequence motifs. We applied this typing method to investigate the SLA class II diversity in four populations of outbred pigs (n = 206) and characterized a total of 19 SLA class II haplotypes, six of which were shared by at least three of the sampled pig populations. We found that Lr‐0.1 (DRB1*01XX–DQB1*01XX–DQA*01XX) was the most prevalent haplotype with a combined frequency of 16.0%, followed by Lr‐0.2 (DRB1*02XX–DQB1*02XX–DQA*02XX) with 14.6% and Lr‐0.15b (DRB1*04XX–DQB1*0202–DQA*02XX) with 14.1%. Over 70% of the pigs (n = 147) had at least one copy of one of these three haplotypes. The PCR‐based typing system described in this study demonstrates a reliable and unambiguous detection method for SLA class II alleles. It will be a valuable tool for studying the influence of SLA diversity on various immunological, pathological and physiological traits in outbred pigs.