bluuebunny/crossref_metadata_2025 huggingface dataset:
Suitability of low-work-function titanium nitride coated transfer mold field-emitter arrays for harsh environment applications
Authors: Nakamoto Masayuki , Moon Jonghyun
Published: March 2011

Low-work-function titanium nitride (TiN) coated transfer mold field-emitter arrays (TiN-FEAs) were fabricated by the transfer metal mold method. This method has potential for realizing stable large-area aerospace nanodevices that are resistant to the harsh plasma atmospheres often experienced by electric propulsion engines in space. Another application is for production of vacuum nanoelectronic devices such as field-emission displays that are suitable for highly oxidizing atmospheres. TiN-FEAs have sharp tips with radii of 6.8 nm, and their work function is just 3.2 eV compared to approximately 5.0 eV for conventional emitter materials such as carbon nanotubes. The turn-on fields of TiN-FEAs are as low as 15.4 V/μm, and their anode-to-emitter distance is 10 μm. Field-emission characteristics of TiN-FEAs were evaluated in situ at a pressure as high as 10−3 Pa and by irradiation with oxygen radicals at 1015 cm−2 s−1, a flux several orders of magnitude higher than orbital satellites typically experience. As the treatment time increased, the turn-on fields of TiN-FEAs changed only slightly from 15.4 to 18.8 V/μm, with no observed change in the work function during the in situ oxygen radical treatment. In summary, the TiN-FEAs exhibited stable field-emission characteristics and a high level of resistance to harsh environments.
The Polyomavirus BK Large T-Antigen-Derived Peptide Elicits an HLA-DR Promiscuous and Polyfunctional CD4+T-Cell Response
Authors: Ramaswami Bala , Popescu Iulia , Macedo Camila , Luo Chunqing , Shapiro Ron , Metes Diana , Chalasani Geetha , Randhawa Parmjeet S.
Published: March 2011

BK virus (BKV) nephropathy and hemorrhagic cystitis are increasingly recognized causes of disease in renal and hematopoietic stem cell transplant recipients, respectively. Functional characterization of the immune response to BKV is important for clinical diagnosis, prognosis, and vaccine design. A peptide mix (PepMix) and overlapping (OPP) or random (RPP) peptide pools derived from BKV large T antigen (LTA) were used to restimulate 14-day-expanded peripheral blood mononuclear cells (PBMC) from 27 healthy control subjects in gamma interferon (IFN-γ)-specific enzyme-linked immunospot (ELISPOT) assays. A T-cell response to LTA PepMix was detected in 15/27 subjects. A response was frequently observed with peptides derived from the helicase domain (9/15 subjects), while the DNA binding and host range domains were immunologically inert (0/15 subjects). For all nine subjects who responded to LTA peptide pools, the immune response could be explained largely by a 15-mer peptide designated P313. P313-specific CD4+T-cell clones demonstrated (i) stringent LTA peptide specificity; (ii) promiscuous recognition in the context of HLA-DR alleles; (iii) cross recognition of homologous peptides from the polyomavirus simian virus 40 (SV40); (iv) an effector memory phenotype, CD107a expression, and intracellular production of IFN-γ and tumor necrosis factor alpha (TNF-α); (v) cytotoxic activity in a chromium release assay; and (vi) the ability to directly present cognate antigen to autologous T cells. In conclusion, T-cell-mediated immunity to BKV in healthy subjects is associated with a polyfunctional population of CD4+T cells with dual T-helper and T-cytotoxic properties. HLA class II promiscuity in antigen presentation makes the targeted LTA peptide sequence a suitable candidate for inclusion in immunotherapy protocols.
Microbiological Screening of Irish Patients with Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy Reveals Persistence of Candida albicans Strains, Gradual Reduction in Susceptibility to Azoles, and Incidences of Clinical Signs of Oral Candidiasis without Culture Evidence
Authors: McManus Brenda A. , McGovern Eleanor , Moran Gary P. , Healy Claire M. , Nunn June , Fleming Pádraig , Costigan Colm , Sullivan Derek J. , Coleman David C.
Published: March 2011

Patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) are prone to chronic mucocutaneous candidiasis, which is often treated with azoles. The purpose of this study was to characterize the oral
Candida
populations from 16 Irish APECED patients, who comprise approximately half the total number identified in Ireland, and to examine the effect of intermittent antifungal therapy on the azole susceptibility patterns of
Candida
isolates. Patients attended between one and four clinical evaluations over a 5-year period, providing oral rinses and/or oral swab samples each time.
Candida
was recovered from 14/16 patients, and
Candida albicans
was the only
Candida
species identified. Interestingly, clinical diagnosis of candidiasis did not correlate with microbiological evidence of
Candida
infection at 7/22 (32%) clinical assessments. Multilocus sequence typing analysis of
C. albicans
isolates recovered from the same patients on separate occasions identified the same sequence type each time. Fluconazole resistance was detected in isolates from one patient, and isolates exhibiting a progressive reduction in itraconazole and/or fluconazole susceptibility were identified in a further 3/16 patients, in each case correlating with the upregulation of
CDR
- and
MDR
-encoded efflux pumps. Mutations were also identified in the
ERG11
and the
TAC1
genes of isolates from these four patients; some of these mutations have previously been associated with azole resistance. The findings suggest that alternative
Candida
treatment options, other than azoles such as chlorhexidine, should be considered in APECED patients and that clinical diagnosis of oral candidiasis should be confirmed by culture prior to the commencement of anti-
Candida
therapy.
Characterization of an F1 Deletion Mutant of Yersinia pestis CO92, Pathogenic Role of F1 Antigen in Bubonic and Pneumonic Plague, and Evaluation of Sensitivity and Specificity of F1 Antigen Capture-Based Dipsticks
Authors: Sha Jian , Endsley Janice J. , Kirtley Michelle L. , Foltz Sheri M. , Huante Matthew B. , Erova Tatiana E. , Kozlova Elena V. , Popov Vsevolod L. , Yeager Linsey A. , Zudina Irina V. , Motin Vladimir L. , Peterson Johnny W. , DeBord Kristin L. , Chopra Ashok K.
Published: March 2011

We evaluated two commercial F1 antigen capture-based immunochromatographic dipsticks, Yersinia Pestis (F1) Smart II and Plague BioThreat Alert test strips, in detecting plague bacilli by using whole-blood samples from mice experimentally infected withYersinia pestisCO92. To assess the specificities of these dipsticks, an in-frame F1-deficient mutant of CO92 (Δcaf) was generated by homologous recombination and used as a negative control. Based on genetic, antigenic/immunologic, and electron microscopic analyses, the Δcafmutant was devoid of a capsule. The growth rate of the Δcafmutant generally was similar to that of the wild-type (WT) bacterium at both 26 and 37°C, although the mutant's growth dropped slightly during the late phase at 37°C. The Δcafmutant was as virulent as WT CO92 in the pneumonic plague mouse model; however, it was attenuated in developing bubonic plague. Both dipsticks had similar sensitivities, requiring a minimum of 0.5 μg/ml of purified F1 antigen or 1 × 105to 5 × 105CFU/ml of WT CO92 for positive results, while the blood samples were negative for up to 1 × 108CFU/ml of the Δcafmutant. Our studies demonstrated the diagnostic potential of two plague dipsticks in detecting capsular-positive strains ofY. pestisin bubonic and pneumonic plague.
Validation and Diagnostic Application of NS and HA Gene-Specific Real-Time Reverse Transcription-PCR Assays for Detection of 2009 Pandemic Influenza A (H1N1) Viruses in Clinical Specimens
Authors: Rönkkö Esa , Ikonen Niina , Kontio Mia , Haanpää Minna , Kallio-Kokko Hannimari , Mannonen Laura , Lappalainen Maija , Julkunen Ilkka , Ziegler Thedi
Published: March 2011

Real-time reverse transcription-PCR assays specific for the nonstructural (NS) and hemagglutinin (HA) genes of the 2009 pandemic influenza A (H1N1) virus were developed and evaluated with clinical samples from infected patients. The tests are characterized by high sensitivity and specificity and performed well throughout the first year of the 2009 pandemic.
Rapid Deletion-Based Subtyping System for the Manila Family of Mycobacterium tuberculosis
Authors: Frink Stephen , Qian Lishi , Yu Steven , Cruz Laura , Desmond Ed , Douglas James T.
Published: March 2011

The Manila family of
Mycobacterium tuberculosis
is a group of clonal isolates seen throughout the Pacific Basin. Commonly used rapid molecular typing methods often leave large groups of Manila family isolates clustered together. Here we describe a simple deletion-based PCR method that improves the discrimination for Manila family isolates, with or without the use of mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) classification, and that is both rapid and affordable. Twenty-eight Manila family isolates, classified by spoligotyping, were collected from around the Pacific Basin from 1995 to 2003 and were tested for known genomic deletions. Nine of 15 regions of difference tested were identified as potentially discriminatory, with 18 distinct patterns; of these 9, 5 were selected for optimal discrimination using 61 Manila family isolates collected from California in 2009. For this geographically limited sample, the single large cluster was reduced to 14 distinct patterns. When the isolates were tested by spoligotyping and MIRU-VNTR, the addition of deletion analysis increased the number of distinct patterns from 43 to 56. In summary, the two study groups, which together form a single group of 89 isolates by spoligotyping, were segregated into 17 subgroups by our deletion-based subtyping system.
Risk factors for patellar tendinopathy: a systematic review of the literature
Authors: van der Worp Henk , van Ark Mathijs , Roerink Saskia , Pepping Gert-Jan , van den Akker-Scheek Inge , Zwerver Johannes
Published: March 2011

Patellar tendinopathy (PT) is an injury with a high prevalence in sports. Knowledge of risk factors is essential for developing preventive measures and rehabilitation programmes. However, risk factors associated with PT have not yet been systematically studied. This review was undertaken to identify risk factors associated with PT. The literature was systematically searched to identify articles that investigated risk factors for PT. There was no strong or moderate evidence that any investigated risk factor was associated with PT. For nine risk factors there was some evidence: weight, body mass index, waist-to-hip ratio, leg-length difference, arch height of the foot, quadriceps flexibility, hamstring flexibility, quadriceps strength and vertical jump performance. Based on the present evidence, reducing body weight, increasing upper-leg flexibility and quadriceps strength and the use of orthotics may be beneficial treatment options. However, it should be stressed that the evidence for the nine identified risk factors was only limited. Therefore, there is a clear need for high-quality studies in order to identify the exact risk factors associated with PT.
Is there a right not to know one's sex? The ethics of ‘gender verification’ in women's sports competition
Authors: Wiesemann Claudia
Published: March 2011

The paper discusses the current medical practice of ‘gender verification’ in sports from an ethical point of view. It takes the recent public discussion about 800 m runner Caster Semenya as a starting point. At the World Championships in Athletics 2009 in Berlin, Germany, Semenya was challenged by competitors as being a so called ‘sex impostor’. A medical examination to verify her sex ensued. The author analyses whether athletes like Semenya could claim a right not to know that is generally acknowledged in human genetics and enforced by international and national genetic privacy laws. The relevance of this right for genetic diagnosis in sports is discussed. To this end, the interests of the athlete concerned and of third parties are balanced according to the expected benefits and harms.Harm is documented in a number of cases and includes unjustified disqualification, severe sex and gender identity crisis, demeaning reactions, social isolation, depression and suicide. Benefits are dubious as most cases of intersex are considered irrelevant for sports competition. It has to be concluded that the benefits to be gained from ‘gender verification’ in sports via genetic testing do not outweigh the grave individual disadvantages. The current practice of athletic associations to largely ignore the right of competitors not to know does not comply with prevailing ethical provisions on the protection of sensitive personal data. Therefore, genetic ‘gender verification’ in sports should be abolished.
Difficulties in Learning Inheritance and Polymorphism
Authors: Liberman Neomi , Beeri Catriel , Ben-David Kolikant Yifat
Published: March 2011

This article reports on difficulties related to the concepts of inheritance and polymorphism, expressed by a group of 22 in-service CS teachers with an experience with the procedural paradigm, as they coped with a course on OOP. Our findings are based on the analysis of tests, questionnaires that the teachers completed in the course, as well as on observations made during the course. The article suggests that the difficulties are mostly caused by the learners’ ignorance about a programming model for inheritance and polymorphism. Such a model is presented in the appendix. The article offers a classification of the difficulties into the following four clusters: (1) alternative (partial) models, (2) analogies to the use of inheritance and conversion in day-to-day life or in imperative programming, (3) lack of understanding of hierarchies and their role in inheritance, and (4) difficulties due to approaches to teaching, and their impact on the students understanding, as well as, with difficulties that arose in previous stages of learning OOP and have yet not been resolved.
Enhancing Clustering Quality through Landmark-Based Dimensionality Reduction
Authors: Magdalinos Panagis , Doulkeridis Christos , Vazirgiannis Michalis
Published: March 2011

Scaling up data mining algorithms for data of both high dimensionality and cardinality has been lately recognized as one of the most challenging problems in data mining research. The reason is that typical data mining tasks, such as clustering, cannot produce high quality results when applied on high-dimensional and/or large (in terms of cardinality) datasets. Data preprocessing and in particular dimensionality reduction constitute promising tools to deal with this problem. However, most of the existing dimensionality reduction algorithms share also the same disadvantages with data mining algorithms, when applied on large datasets of high dimensionality. In this article, we propose a fast and efficient dimensionality reduction algorithm (FEDRA), which is particularly scalable and therefore suitable for challenging datasets. FEDRA follows the landmark-based paradigm for embedding data objects in a low-dimensional projection space. By means of a theoretical analysis, we prove that FEDRA is efficient, while we demonstrate the achieved quality of results through experiments on datasets of higher cardinality and dimensionality than those employed in the evaluation of competitive algorithms. The obtained results prove that FEDRA manages to retain or ameliorate clustering quality while projecting in less than 10% of the initial dimensionality. Moreover, our algorithm produces embeddings that enable the faster convergence of clustering algorithms. Therefore, FEDRA emerges as a powerful and generic tool for data pre-processing, which can be integrated in other data mining algorithms, thus enhancing their performance.
SEATTLE
Authors: Kim Changhoon , Caesar Matthew , Rexford Jennifer
Published: March 2011

IP networks today require massive effort to configure and manage. Ethernet is vastly simpler to manage, but does not scale beyond small local area networks. This article describes an alternative network architecture called SEATTLE that achieves the best of both worlds: The scalability of IP combined with the simplicity of Ethernet. SEATTLE provides plug-and-play functionality via flat addressing, while ensuring scalability and efficiency through shortest-path routing and hash-based resolution of host information. In contrast to previous work on identity-based routing, SEATTLE ensures path predictability, controllability, and stability, thus simplifying key network-management operations, such as capacity planning, traffic engineering, and troubleshooting. We performed a simulation study driven by real-world traffic traces and network topologies, and used Emulab to evaluate a prototype of our design based on the Click and XORP open-source routing platforms. Our experiments show that SEATTLE efficiently handles network failures and host mobility, while reducing control overhead and state requirements by roughly two orders of magnitude compared with Ethernet bridging.
The role of physicality in tangible and embodied interactions
Authors: Hornecker Eva
Published: March 2011

The boundaries between "the digital" and our everyday physical world are dissolving as we develop more physical ways of interacting with computing. This forum presents some of the topics discussed in the colorful multidisciplinary field of tangible and embodied interaction.

Eva Hornecker, Editor
Anion Channels/Transporters in Plants: From Molecular Bases to Regulatory Networks
Authors: Barbier-Brygoo Hélène , De Angeli Alexis , Filleur Sophie , Frachisse Jean-Marie , Gambale Franco , Thomine Sébastien , Wege Stefanie
Published: March 2011

Anion channels/transporters are key to a wide spectrum of physiological functions in plants, such as osmoregulation, cell signaling, plant nutrition and compartmentalization of metabolites, and metal tolerance. The recent identification of gene families encoding some of these transport systems opened the way for gene expression studies, structure-function analyses of the corresponding proteins, and functional genomics approaches toward further understanding of their integrated roles in planta. This review, based on a few selected examples, illustrates that the members of a given gene family exhibit a diversity of substrate specificity, regulation, and intracellular localization, and are involved in a wide range of physiological functions. It also shows that post-translational modifications of transport proteins play a key role in the regulation of anion transport activity. Key questions arising from the increasing complexity of networks controlling anion transport in plant cells (the existence of redundancy, cross talk, and coordination between various pathways and compartments) are also addressed.
The Cryptochromes: Blue Light Photoreceptors in Plants and Animals
Authors: Chaves Inês , Pokorny Richard , Byrdin Martin , Hoang Nathalie , Ritz Thorsten , Brettel Klaus , Essen Lars-Oliver , van der Horst Gijsbertus T. J. , Batschauer Alfred , Ahmad Margaret
Published: March 2011

Cryptochromes are flavoprotein photoreceptors first identified in Arabidopsis thaliana, where they play key roles in growth and development. Subsequently identified in prokaryotes, archaea, and many eukaryotes, cryptochromes function in the animal circadian clock and are proposed as magnetoreceptors in migratory birds. Cryptochromes are closely structurally related to photolyases, evolutionarily ancient flavoproteins that catalyze light-dependent DNA repair. Here, we review the structural, photochemical, and molecular properties of cry-DASH, plant, and animal cryptochromes in relation to biological signaling mechanisms and uncover common features that may contribute to better understanding the function of cryptochromes in diverse systems including in man.
Evolution and Diversity of Plant Cell Walls: From Algae to Flowering Plants
Authors: Popper Zoë A. , Michel Gurvan , Hervé Cécile , Domozych David S. , Willats William G.T. , Tuohy Maria G. , Kloareg Bernard , Stengel Dagmar B.
Published: March 2011

All photosynthetic multicellular Eukaryotes, including land plants and algae, have cells that are surrounded by a dynamic, complex, carbohydrate-rich cell wall. The cell wall exerts considerable biological and biomechanical control over individual cells and organisms, thus playing a key role in their environmental interactions. This has resulted in compositional variation that is dependent on developmental stage, cell type, and season. Further variation is evident that has a phylogenetic basis. Plants and algae have a complex phylogenetic history, including acquisition of genes responsible for carbohydrate synthesis and modification through a series of primary (leading to red algae, green algae, and land plants) and secondary (generating brown algae, diatoms, and dinoflagellates) endosymbiotic events. Therefore, organisms that have the shared features of photosynthesis and possession of a cell wall do not form a monophyletic group. Yet they contain some common wall components that can be explained increasingly by genetic and biochemical evidence.
Convergent Evolution in Plant Specialized Metabolism
Authors: Pichersky Eran , Lewinsohn Efraim
Published: March 2011

Plants synthesize a multitude of compounds that contribute to adaptation to their ecological niches. Such compounds serve as attractants of other living organisms beneficial to the plants or as defense against other biotic as well as abiotic agents. Selection for increased fitness, a never-ending process, has resulted in each plant lineage synthesizing a distinct set of specialized metabolites appropriate for its environment. The total number of specialized metabolites found in the plant kingdom far exceeds the capacity of any one plant genome to encode the necessary enzymes, and just as a plant lineage acquires the ability to make new specialized compounds during evolution, it also loses the ability to make others. Although the ability of plants to make novel, specialized metabolites continues to evolve, there are also many examples in which different plants have independently evolved the ability to make compounds already present in other plant lineages or to make different compounds that fulfill the same role—both are examples of convergent evolution. Here, we discuss many examples of convergent evolution in specialized metabolism. There are many genetic and biochemical mechanisms that can give rise to convergent evolution, and we conclude that, overall, convergent evolution in plant specialized metabolism is surprisingly common.
Folate Biosynthesis, Turnover, and Transport in Plants
Authors: Hanson Andrew D. , Gregory Jesse F.
Published: March 2011

Folates are essential cofactors for one-carbon transfer reactions and are needed in the diets of humans and animals. Because plants are major sources of dietary folate, plant folate biochemistry has long been of interest but progressed slowly until the genome era. Since then, genome-enabled approaches have brought rapid advances: We now know (a) all the plant folate synthesis genes and some genes of folate turnover and transport, (b) certain mechanisms governing folate synthesis, and (c) the subcellular locations of folate synthesis enzymes and of folates themselves. Some of this knowledge has been applied, simply and successfully, to engineer folate-enriched food crops (i.e., biofortification). Much remains to be discovered about folates, however, particularly in relation to homeostasis, catabolism, membrane transport, and vacuolar storage. Understanding these processes, which will require both biochemical and -omics research, should lead to improved biofortification strategies based on transgenic or conventional approaches.
Signaling Network in Sensing Phosphate Availability in Plants
Authors: Chiou Tzyy-Jen , Lin Shu-I
Published: March 2011

Plants acquire phosphorus in the form of phosphate (Pi), the concentration of which is often limited for plant uptake. Plants have developed diverse responses to conserve and remobilize internal Pi and to enhance Pi acquisition to secure them against Pi deficiency. These responses are achieved by the coordination of an elaborate signaling network comprising local and systemic machineries. Recent advances have revealed several important components involved in this network. Pi functions as a signal to report its own availability. miR399 and sugars act as systemic signals to regulate responses occurring in roots. Hormones also play crucial roles in modulating gene expression and in altering root system architecture. Transcription factors function as a hub to perceive the signals and to elicit steady outputs. In this review, we outline the current knowledge on this subject and present hypotheses pertaining to other potential signals and to the organization and coordination of signaling.
It Is a Long Way to GM Agriculture
Authors: Van Montagu Marc
Published: March 2011

When we discovered that crown gall induction on plants by Agrobacterium tumefaciens is a natural event of genetic engineering, we were convinced that this was the dawn of a new era for plant science. Now, more than 30 years later, I remain overawed by how far and how rapidly we progressed with our knowledge of the molecular basis of plant growth, development, stress resistance, flowering, and ecological adaptation, thanks to the gene engineering technology. I am impressed, but also frustrated by the difficulties of applying this knowledge to improve crops and globally develop a sustainable and improved high-yielding agriculture. Now that gene engineering has become so efficient, I had hoped that thousands of teams, all over the world, would work on improving our major food crops, help domesticate new ones, and succeed in doubling or tripling biomass yields in industrial crops. We live in a world where more than a billion people are hungry or starving, while the last areas of tropical forest and wild nature are disappearing. We urgently need a better supply of raw material for our chemical industry because petroleum-based products pollute the environment and are limited in supply. Why could this new technology not bring the solutions to these challenges? Why has this not happened yet; what did we do wrong?
Synthetic Chromosome Platforms in Plants
Authors: Gaeta Robert T. , Masonbrink Rick E. , Krishnaswamy Lakshminarasimhan , Zhao Changzeng , Birchler James A.
Published: March 2011

Synthetic chromosomes provide the means to stack transgenes independently of the remainder of the genome. Combining them with haploid breeding could provide the means to transfer many transgenes more easily among varieties of the same species. The epigenetic nature of centromere formation complicates the production of synthetic chromosomes. However, telomere-mediated truncation coupled with the introduction of site-specific recombination cassettes has been used to produce minichromosomes consisting of little more than a centromere. Methods that have been developed to modify genes in vivo could be applied to minichromosomes to improve their utility and to continue to increase their length and genic content. Synthetic chromosomes establish the means to add or subtract multiple transgenes, multigene complexes, or whole biochemical pathways to plants to change their properties for agricultural applications or to use plants as factories for the production of foreign proteins or metabolites.
The Cullin-RING Ubiquitin-Protein Ligases
Authors: Hua Zhihua , Vierstra Richard D.
Published: March 2011

The posttranslational addition of ubiquitin (Ub) helps control the half-life, localization, and action of many intracellular plant proteins. A primary function is the degradation of ubiquitylated proteins by the 26S proteasome, which in turn plays important housekeeping and regulatory roles by removing aberrant polypeptides and various normal short-lived regulators. Strikingly, both genetic and genomic studies reveal that Ub conjugation is extraordinarily complex in plants, with more than 1500 Ub-protein ligases (or E3s) possible that could direct the final transfer of the Ub moiety to an equally large number of targets. The cullin-RING ligases (CRLs) are a highly polymorphic E3 collection composed of a cullin backbone onto which binds carriers of activated Ub and a diverse assortment of adaptors that recruit appropriate substrates for ubiquitylation. Here, we review our current understanding of the organization and structure of CRLs in plants and their dynamics, substrates, potential functions, and evolution. The importance of CRLs is exemplified by their ability to serve as sensors of hormones and light; their essential participation in various signaling pathways; their control of the cell cycle, transcription, the stress response, self-incompatibility, and pathogen defense; and their dramatically divergent evolutionary histories in many plant lineages. Given both their organizational complexities and their critical influences, CRLs likely impact most, if not all, aspects of plant biology.
Genetic Regulation of Sporopollenin Synthesis and Pollen Exine Development
Authors: Ariizumi Tohru , Toriyama Kinya
Published: March 2011

Pollen acts as a biological protector of male sperm and is covered by an outer cell wall polymer called the exine, which consists of durable sporopollenin. Despite the astonishingly divergent structure of the exine across taxa, the developmental processes of its formation surprisingly do not vary, which suggests the preservation of a common molecular mechanism. The precise molecular mechanisms underlying pollen exine patterning remain highly elusive, but they appear to be dependent on at least three major developmental processes: primexine formation, callose wall formation, and sporopollenin synthesis. Several lines of evidence suggest that the sporopollenin is built up via catalytic enzyme reactions in the tapetum, and both the primexine and callose wall provide an efficient substructure for sporopollenin deposition. Herein, we review the currently accepted understanding of the molecular regulation of sporopollenin biosynthesis and examine unanswered questions regarding the requirements underpinning proper exine pattern formation, as based on genetic evidence.
Mechanisms of Membrane Curvature Sensing
Authors: Antonny Bruno
Published: March 2011

Bacteria and eukaryotic cells contain geometry-sensing tools in their cytosol: protein motifs or domains that recognize the curvature, concave or convex, deep or shallow, of lipid membranes. These sensors contrast with classical lipid-binding domains by their extended structure and, sometimes, counterintuitive chemistry. Among the sensors are long amphipathic helices, such as the ALPS motif and the N-terminal region of α-synuclein, whose apparent “design defects” translate into a remarkable ability to specifically adsorb to the surface of small vesicles. Fundamental differences in the lipid composition of membranes of the early and late secretory pathways probably explain why some sensors use mostly electrostatics whereas others take advantage of the hydrophobic effect. Membrane curvature sensors help to organize very diverse reactions, such as lipid transfer between membranes, the tethering of vesicles at the Golgi apparatus, and the assembly-disassembly cycle of protein coats.
The Structure of the Nuclear Pore Complex
Authors: Hoelz André , Debler Erik W. , Blobel Günter
Published: March 2011

In eukaryotic cells, the spatial segregation of replication and transcription in the nucleus and translation in the cytoplasm imposes the requirement of transporting thousands of macromolecules between these two compartments. Nuclear pore complexes (NPCs) are the sole gateways that facilitate this macromolecular exchange across the nuclear envelope with the help of soluble transport receptors. Whereas the mobile transport machinery is reasonably well understood at the atomic level, a commensurate structural characterization of the NPC has only begun in the past few years. Here, we describe the recent progress toward the elucidation of the atomic structure of the NPC, highlight emerging concepts of its underlying architecture, and discuss key outstanding questions and challenges. The applied structure determination as well as the described design principles of the NPC may serve as paradigms for other macromolecular assemblies.
AAA+ Proteases: ATP-Fueled Machines of Protein Destruction
Authors: Sauer Robert T. , Baker Tania A.
Published: March 2011

AAA+ family proteolytic machines (ClpXP, ClpAP, ClpCP, HslUV, Lon, FtsH, PAN/20S, and the 26S proteasome) perform protein quality control and are used in regulatory circuits in all cells. These machines contain a compartmental protease, with active sites sequestered in an interior chamber, and a hexameric ring of AAA+ ATPases. Substrate proteins are tethered to the ring, either directly or via adaptor proteins. An unstructured region of the substrate is engaged in the axial pore of the AAA+ ring, and cycles of ATP binding/hydrolysis drive conformational changes that create pulses of pulling that denature the substrate and translocate the unfolded polypeptide through the pore and into the degradation chamber. Here, we review our current understanding of the molecular mechanisms of substrate recognition, adaptor function, and ATP-fueled unfolding and translocation. The unfolding activities of these and related AAA+ machines can also be used to disassemble or remodel macromolecular complexes and to resolubilize aggregates.
Assembly of Bacterial Inner Membrane Proteins
Authors: Dalbey Ross E. , Wang Peng , Kuhn Andreas
Published: March 2011

Numerous membrane proteins form multisubunit protein complexes, which contain both integral and peripheral subunits, in addition to prosthetic groups. Bacterial membrane proteins are inserted into the inner membrane by the Sec translocase and YidC insertase. Their folding can be facilitated by YidC and the phospholipid phosphatidylethanolamine (PE). Glycine zippers and other motifs promote transmembrane-transmembrane (TM-TM) helix interactions that may lead to the formation of α-helical bundles of membrane proteins. During or after membrane insertion, the subunits of oligomeric membrane proteins must find each other to build the homo-oligomeric and the hetero-oligomeric membrane complexes. Although chaperones may function as assembly factors in the formation of the oligomer, many protein oligomers appear to fold and oligomerize spontaneously. Current studies show that most subunits of hetero-oligomers follow a sequential and ordered pathway to form the membrane protein complex. If the inserted protein is misfolded or the membrane protein is misassembled, quality control mechanisms exist that can degrade the proteins.
Applications of Mass Spectrometry to Lipids and Membranes
Authors: Harkewicz Richard , Dennis Edward A.
Published: March 2011

Lipidomics, a major part of metabolomics, constitutes the detailed analysis and global characterization, both spatial and temporal, of the structure and function of lipids (the lipidome) within a living system. As with proteomics, mass spectrometry has earned a central analytical role in lipidomics, and this role will continue to grow with technological developments. Currently, there exist two mass spectrometry–based lipidomics approaches, one based on a division of lipids into categories and classes prior to analysis, the “comprehensive lipidomics analysis by separation simplification” (CLASS), and the other in which all lipid species are analyzed together without prior separation, shotgun. In exploring the lipidome of various living systems, novel lipids are being discovered, and mass spectrometry is helping characterize their chemical structure. Deuterium exchange mass spectrometry (DXMS) is being used to investigate the association of lipids and membranes with proteins and enzymes, and imaging mass spectrometry (IMS) is being applied to the in situ analysis of lipids in tissues.
Biological Phosphoryl-Transfer Reactions: Understanding Mechanism and Catalysis
Authors: Lassila Jonathan K. , Zalatan Jesse G. , Herschlag Daniel
Published: March 2011

Phosphoryl-transfer reactions are central to biology. These reactions also have some of the slowest nonenzymatic rates and thus require enormous rate accelerations from biological catalysts. Despite the central importance of phosphoryl transfer and the fascinating catalytic challenges it presents, substantial confusion persists about the properties of these reactions. This confusion exists despite decades of research on the chemical mechanisms underlying these reactions. Here we review phosphoryl-transfer reactions with the goal of providing the reader with the conceptual and experimental background to understand this body of work, to evaluate new results and proposals, and to apply this understanding to enzymes. We describe likely resolutions to some controversies, while emphasizing the limits of our current approaches and understanding. We apply this understanding to enzyme-catalyzed phosphoryl transfer and provide illustrative examples of how this mechanistic background can guide and deepen our understanding of enzymes and their mechanisms of action. Finally, we present important future challenges for this field.
Reporting from the Field: Genetically Encoded Fluorescent Reporters Uncover Signaling Dynamics in Living Biological Systems
Authors: Mehta Sohum , Zhang Jin
Published: March 2011

Real-time visualization of a wide range of biochemical processes in living systems is being made possible through the development and application of genetically encoded fluorescent reporters. These versatile biosensors have proven themselves tailor-made to the study of signal transduction, and in this review, we discuss some of the unique insights that they continue to provide regarding the spatial organization and dynamic regulation of intracellular signaling networks. In addition, we explore the more recent push to expand the scope of biological phenomena that can be monitored using these reporters, while also considering the potential to integrate this highly adaptable technology with a number of emerging techniques that may significantly broaden our view of how networks of biochemical processes shape larger biological phenomena.
Cross Talk Between O-GlcNAcylation and Phosphorylation: Roles in Signaling, Transcription, and Chronic Disease
Authors: Hart Gerald W. , Slawson Chad , Ramirez-Correa Genaro , Lagerlof Olof
Published: March 2011

O-GlcNAcylation is the addition of β-D-N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. O-linked N-acetylglucosamine (O-GlcNAc) was not discovered until the early 1980s and still remains difficult to detect and quantify. Nonetheless, O-GlcNAc is highly abundant and cycles on proteins with a timescale similar to protein phosphorylation. O-GlcNAc occurs in organisms ranging from some bacteria to protozoans and metazoans, including plants and nematodes up the evolutionary tree to man. O-GlcNAcylation is mostly on nuclear proteins, but it occurs in all intracellular compartments, including mitochondria. Recent glycomic analyses have shown that O-GlcNAcylation has surprisingly extensive cross talk with phosphorylation, where it serves as a nutrient/stress sensor to modulate signaling, transcription, and cytoskeletal functions. Abnormal amounts of O-GlcNAcylation underlie the etiology of insulin resistance and glucose toxicity in diabetes, and this type of modification plays a direct role in neurodegenerative disease. Many oncogenic proteins and tumor suppressor proteins are also regulated by O-GlcNAcylation. Current data justify extensive efforts toward a better understanding of this invisible, yet abundant, modification. As tools for the study of O-GlcNAc become more facile and available, exponential growth in this area of research will eventually take place.
Biochemistry of Mobile Zinc and Nitric Oxide Revealed by Fluorescent Sensors
Authors: Pluth Michael D. , Tomat Elisa , Lippard Stephen J.
Published: March 2011

Biological mobile zinc and nitric oxide (NO) are two prominent examples of inorganic compounds involved in numerous signaling pathways in living systems. In the past decade, a synergy of regulation, signaling, and translocation of these two species has emerged in several areas of human physiology, providing additional incentive for developing adequate detection systems for Zn(II) ions and NO in biological specimens. Fluorescent probes for both of these bioinorganic analytes provide excellent tools for their detection, with high spatial and temporal resolution. We review the most widely used fluorescent sensors for biological zinc and nitric oxide, together with promising new developments and unmet needs of contemporary Zn(II) and NO biological imaging. The interplay between zinc and nitric oxide in the nervous, cardiovascular, and immune systems is highlighted to illustrate the contributions of selective fluorescent probes to the study of these two important bioinorganic analytes.
Quantitative, High-Resolution Proteomics for Data-Driven Systems Biology
Authors: Cox Jürgen , Mann Matthias
Published: March 2011

Systems biology requires comprehensive data at all molecular levels. Mass spectrometry (MS)-based proteomics has emerged as a powerful and universal method for the global measurement of proteins. In the most widespread format, it uses liquid chromatography (LC) coupled to high-resolution tandem mass spectrometry (MS/MS) to identify and quantify peptides at a large scale. This peptide intensity information is the basic quantitative proteomic data type. It is used to quantify proteins between different proteome states, including the temporal variation of the proteome, to determine the complete primary structure of proteins including posttranslational modifications, to localize proteins to organelles, and to determine protein interactions. Here, we describe the principles of analysis and the areas of biology where proteomics can make unique contributions. The large-scale nature of proteomics data and its high accuracy pose special opportunities as well as challenges in systems biology that have been largely untapped so far.
β-Barrel Membrane Protein Assembly by the Bam Complex
Authors: Hagan Christine L. , Silhavy Thomas J. , Kahne Daniel
Published: March 2011

β-barrel membrane proteins perform important functions in the outer membranes (OMs) of Gram-negative bacteria and of the mitochondria and chloroplasts of eukaryotes. The protein complexes that assemble these proteins in their respective membranes have been identified and shown to contain a component that has been conserved from bacteria to humans. β-barrel proteins are handled differently from α-helical membrane proteins in the cell in order to efficiently transport them to their final locations in unfolded but folding-competent states. The mechanism by which the assembly complex then binds, folds, and inserts β-barrels into the membrane is not well understood, but recent structural, biochemical, and genetic studies have begun to elucidate elements of how the complex provides a facilitated pathway for β-barrel assembly. Ultimately, studies of the mechanism of β-barrel assembly and comparison to the better-understood process of α-helical membrane protein assembly will reveal whether there are general principles that guide the folding and insertion of all membrane proteins.
STIM Proteins and the Endoplasmic Reticulum-Plasma Membrane Junctions
Authors: Carrasco Silvia , Meyer Tobias
Published: March 2011

Eukaryotic organelles can interact with each other through stable junctions where the two membranes are kept in close apposition. The junction that connects the endoplasmic reticulum to the plasma membrane (ER-PM junction) is unique in providing a direct communication link between the ER and the PM. In a recently discovered signaling process, STIM (stromal-interacting molecule) proteins sense a drop in ER Ca2+ levels and directly activate Orai PM Ca2+ channels across the junction space. In an inverse process, a voltage-gated PM Ca2+ channel can directly open ER ryanodine-receptor Ca2+ channels in striated-muscle cells. Although ER-PM junctions were first described 50 years ago, their broad importance in Ca2+ signaling, as well as in the regulation of cholesterol and phosphatidylinositol lipid transfer, has only recently been realized. Here, we discuss research from different fields to provide a broad perspective on the structures and unique roles of ER-PM junctions in controlling signaling and metabolic processes.
Advances in the Mass Spectrometry of Membrane Proteins: From Individual Proteins to Intact Complexes
Authors: Barrera Nelson P. , Robinson Carol V.
Published: March 2011

Rapid advances in structural genomics and in large-scale proteomic projects have yielded vast amounts of data on soluble proteins and their complexes. Despite these advances, progress in studying membrane proteins using mass spectrometry (MS) has been slow. This is due in part to the inherent solubility and dynamic properties of these proteins, but also to their low abundance and the absence of polar side chains in amino acid residues. Considerable progress in overcoming these challenges is, however, now being made for all levels of structural characterization. This progress includes MS studies of the primary structure of membrane proteins, wherein sophisticated enrichment and trapping procedures are allowing multiple posttranslational modifications to be defined through to the secondary structure level in which proteins and peptides have been probed using hydrogen exchange, covalent, or radiolytic labeling methods. Exciting possibilities now exist to go beyond primary and secondary structure to reveal the tertiary and quaternary interactions of soluble and membrane subunits within intact assemblies of more than 700 kDa.
Assembly of Bacterial Ribosomes
Authors: Shajani Zahra , Sykes Michael T. , Williamson James R.
Published: March 2011

The assembly of ribosomes from a discrete set of components is a key aspect of the highly coordinated process of ribosome biogenesis. In this review, we present a brief history of the early work on ribosome assembly in Escherichia coli, including a description of in vivo and in vitro intermediates. The assembly process is believed to progress through an alternating series of RNA conformational changes and protein-binding events; we explore the effects of ribosomal proteins in driving these events. Ribosome assembly in vivo proceeds much faster than in vitro, and we outline the contributions of several of the assembly cofactors involved, including Era, RbfA, RimJ, RimM, RimP, and RsgA, which associate with the 30S subunit, and CsdA, DbpA, Der, and SrmB, which associate with the 50S subunit.
Development of Probes for Cellular Functions Using Fluorescent Proteins and Fluorescence Resonance Energy Transfer
Authors: Miyawaki Atsushi
Published: March 2011

Many genetically encoded probes that employ fluorescent proteins and fluorescence resonance energy transfer (FRET) have been developed to better understand the spatiotemporal regulation of various cellular processes. The different types of FRET and measurement techniques necessitate characterization of their specific features. Here I provide theoretical and practical comparisons of bimolecular and unimolecular FRET constructs, intensity-based and lifetime-based FRET measurements, FRET imaging using live- and fixed-cell samples, green fluorescent protein–based and chemical fluorophore-based FRET, and FRET efficiency and indices. The potential benefits and limitations of a variety of features in the technologies using fluorescent proteins and FRET are discussed.