@article{9473, abstract = {Cytosine DNA methylation regulates the expression of eukaryotic genes and transposons. Methylation is copied by methyltransferases after DNA replication, which results in faithful transmission of methylation patterns during cell division and, at least in flowering plants, across generations. Transgenerational inheritance is mediated by a small group of cells that includes gametes and their progenitors. However, methylation is usually analyzed in somatic tissues that do not contribute to the next generation, and the mechanisms of transgenerational inheritance are inferred from such studies. To gain a better understanding of how DNA methylation is inherited, we analyzed purified Arabidopsis thaliana sperm and vegetative cells-the cell types that comprise pollen-with mutations in the DRM, CMT2, and CMT3 methyltransferases. We find that DNA methylation dependency on these enzymes is similar in sperm, vegetative cells, and somatic tissues, although DRM activity extends into heterochromatin in vegetative cells, likely reflecting transcription of heterochromatic transposons in this cell type. We also show that lack of histone H1, which elevates heterochromatic DNA methylation in somatic tissues, does not have this effect in pollen. Instead, levels of CG methylation in wild-type sperm and vegetative cells, as well as in wild-type microspores from which both pollen cell types originate, are substantially higher than in wild-type somatic tissues and similar to those of H1-depleted roots. Our results demonstrate that the mechanisms of methylation maintenance are similar between pollen and somatic cells, but the efficiency of CG methylation is higher in pollen, allowing methylation patterns to be accurately inherited across generations.}, author = {Hsieh, Ping-Hung and He, Shengbo and Buttress, Toby and Gao, Hongbo and Couchman, Matthew and Fischer, Robert L. and Zilberman, Daniel and Feng, Xiaoqi}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, number = {52}, pages = {15132--15137}, publisher = {National Academy of Sciences}, title = {{Arabidopsis male sexual lineage exhibits more robust maintenance of CG methylation than somatic tissues}}, doi = {10.1073/pnas.1619074114}, volume = {113}, year = {2016}, } @article{9477, abstract = {Cytosine methylation is a DNA modification with important regulatory functions in eukaryotes. In flowering plants, sexual reproduction is accompanied by extensive DNA demethylation, which is required for proper gene expression in the endosperm, a nutritive extraembryonic seed tissue. Endosperm arises from a fusion of a sperm cell carried in the pollen and a female central cell. Endosperm DNA demethylation is observed specifically on the chromosomes inherited from the central cell in Arabidopsis thaliana, rice, and maize, and requires the DEMETER DNA demethylase in Arabidopsis. DEMETER is expressed in the central cell before fertilization, suggesting that endosperm demethylation patterns are inherited from the central cell. Down-regulation of the MET1 DNA methyltransferase has also been proposed to contribute to central cell demethylation. However, with the exception of three maize genes, central cell DNA methylation has not been directly measured, leaving the origin and mechanism of endosperm demethylation uncertain. Here, we report genome-wide analysis of DNA methylation in the central cells of Arabidopsis and rice—species that diverged 150 million years ago—as well as in rice egg cells. We find that DNA demethylation in both species is initiated in central cells, which requires DEMETER in Arabidopsis. However, we do not observe a global reduction of CG methylation that would be indicative of lowered MET1 activity; on the contrary, CG methylation efficiency is elevated in female gametes compared with nonsexual tissues. Our results demonstrate that locus-specific, active DNA demethylation in the central cell is the origin of maternal chromosome hypomethylation in the endosperm.}, author = {Park, Kyunghyuk and Kim, M. Yvonne and Vickers, Martin and Park, Jin-Sup and Hyun, Youbong and Okamoto, Takashi and Zilberman, Daniel and Fischer, Robert L. and Feng, Xiaoqi and Choi, Yeonhee and Scholten, Stefan}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, keywords = {Multidisciplinary}, number = {52}, pages = {15138--15143}, publisher = {National Academy of Sciences}, title = {{DNA demethylation is initiated in the central cells of Arabidopsis and rice}}, doi = {10.1073/pnas.1619047114}, volume = {113}, year = {2016}, } @article{6118, abstract = {Carbon dioxide (CO2) gradients are ubiquitous and provide animals with information about their environment, such as the potential presence of prey or predators. The nematode Caenorhabditis elegans avoids elevated CO2, and previous work identified three neuron pairs called “BAG,” “AFD,” and “ASE” that respond to CO2 stimuli. Using in vivo Ca2+ imaging and behavioral analysis, we show that C. elegans can detect CO2 independently of these sensory pathways. Many of the C. elegans sensory neurons we examined, including the AWC olfactory neurons, the ASJ and ASK gustatory neurons, and the ASH and ADL nociceptors, respond to a rise in CO2 with a rise in Ca2+. In contrast, glial sheath cells harboring the sensory endings of C. elegans’ major chemosensory neurons exhibit strong and sustained decreases in Ca2+ in response to high CO2. Some of these CO2 responses appear to be cell intrinsic. Worms therefore may couple detection of CO2 to that of other cues at the earliest stages of sensory processing. We show that C. elegans persistently suppresses oviposition at high CO2. Hermaphrodite-specific neurons (HSNs), the executive neurons driving egg-laying, are tonically inhibited when CO2 is elevated. CO2 modulates the egg-laying system partly through the AWC olfactory neurons: High CO2 tonically activates AWC by a cGMP-dependent mechanism, and AWC output inhibits the HSNs. Our work shows that CO2 is a more complex sensory cue for C. elegans than previously thought, both in terms of behavior and neural circuitry.}, author = {Fenk, Lorenz A. and de Bono, Mario}, issn = {0027-8424}, journal = {Proceedings of the National Academy of Sciences}, number = {27}, pages = {E3525--E3534}, publisher = {National Academy of Sciences}, title = {{Environmental CO2 inhibits Caenorhabditis elegans egg-laying by modulating olfactory neurons and evokes widespread changes in neural activity}}, doi = {10.1073/pnas.1423808112}, volume = {112}, year = {2015}, } @article{8021, abstract = {Most excitatory inputs in the mammalian brain are made on dendritic spines, rather than on dendritic shafts. Spines compartmentalize calcium, and this biochemical isolation can underlie input-specific synaptic plasticity, providing a raison d'etre for spines. However, recent results indicate that the spine can experience a membrane potential different from that in the parent dendrite, as though the spine neck electrically isolated the spine. Here we use two-photon calcium imaging of mouse neocortical pyramidal neurons to analyze the correlation between the morphologies of spines activated under minimal synaptic stimulation and the excitatory postsynaptic potentials they generate. We find that excitatory postsynaptic potential amplitudes are inversely correlated with spine neck lengths. Furthermore, a spike timing-dependent plasticity protocol, in which two-photon glutamate uncaging over a spine is paired with postsynaptic spikes, produces rapid shrinkage of the spine neck and concomitant increases in the amplitude of the evoked spine potentials. Using numerical simulations, we explore the parameter regimes for the spine neck resistance and synaptic conductance changes necessary to explain our observations. Our data, directly correlating synaptic and morphological plasticity, imply that long-necked spines have small or negligible somatic voltage contributions, but that, upon synaptic stimulation paired with postsynaptic activity, they can shorten their necks and increase synaptic efficacy, thus changing the input/output gain of pyramidal neurons. }, author = {Araya, R. and Vogels, Tim P and Yuste, R.}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, number = {28}, pages = {E2895--E2904}, publisher = {Proceedings of the National Academy of Sciences}, title = {{Activity-dependent dendritic spine neck changes are correlated with synaptic strength}}, doi = {10.1073/pnas.1321869111}, volume = {111}, year = {2014}, } @article{10382, abstract = {Protein oligomers have been implicated as toxic agents in a wide range of amyloid-related diseases. However, it has remained unsolved whether the oligomers are a necessary step in the formation of amyloid fibrils or just a dangerous byproduct. Analogously, it has not been resolved if the amyloid nucleation process is a classical one-step nucleation process or a two-step process involving prenucleation clusters. We use coarse-grained computer simulations to study the effect of nonspecific attractions between peptides on the primary nucleation process underlying amyloid fibrillization. We find that, for peptides that do not attract, the classical one-step nucleation mechanism is possible but only at nonphysiologically high peptide concentrations. At low peptide concentrations, which mimic the physiologically relevant regime, attractive interpeptide interactions are essential for fibril formation. Nucleation then inevitably takes place through a two-step mechanism involving prefibrillar oligomers. We show that oligomers not only help peptides meet each other but also, create an environment that facilitates the conversion of monomers into the β-sheet–rich form characteristic of fibrils. Nucleation typically does not proceed through the most prevalent oligomers but through an oligomer size that is only observed in rare fluctuations, which is why such aggregates might be hard to capture experimentally. Finally, we find that the nucleation of amyloid fibrils cannot be described by classical nucleation theory: in the two-step mechanism, the critical nucleus size increases with increases in both concentration and interpeptide interactions, which is in direct contrast with predictions from classical nucleation theory.}, author = {Šarić, Anđela and Chebaro, Yassmine C. and Knowles, Tuomas P. J. and Frenkel, Daan}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, keywords = {multidisciplinary}, number = {50}, pages = {17869--17874}, publisher = {National Academy of Sciences}, title = {{Crucial role of nonspecific interactions in amyloid nucleation}}, doi = {10.1073/pnas.1410159111}, volume = {111}, year = {2014}, } @article{9479, abstract = {Centromeres mediate chromosome segregation and are defined by the centromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A). Removal of CenH3 from centromeres is a general property of terminally differentiated cells, and the persistence of CenH3 increases the risk of diseases such as cancer. However, active mechanisms of centromere disassembly are unknown. Nondividing Arabidopsis pollen vegetative cells, which transport engulfed sperm by extended tip growth, undergo loss of CenH3; centromeric heterochromatin decondensation; and bulk activation of silent rRNA genes, accompanied by their translocation into the nucleolus. Here, we show that these processes are blocked by mutations in the evolutionarily conserved AAA-ATPase molecular chaperone, CDC48A, homologous to yeast Cdc48 and human p97 proteins, both of which are implicated in ubiquitin/small ubiquitin-like modifier (SUMO)-targeted protein degradation. We demonstrate that CDC48A physically associates with its heterodimeric cofactor UFD1-NPL4, known to bind ubiquitin and SUMO, as well as with SUMO1-modified CenH3 and mutations in NPL4 phenocopy cdc48a mutations. In WT vegetative cell nuclei, genetically unlinked ribosomal DNA (rDNA) loci are uniquely clustered together within the nucleolus and all major rRNA gene variants, including those rDNA variants silenced in leaves, are transcribed. In cdc48a mutant vegetative cell nuclei, however, these rDNA loci frequently colocalized with condensed centromeric heterochromatin at the external periphery of the nucleolus. Our results indicate that the CDC48ANPL4 complex actively removes sumoylated CenH3 from centromeres and disrupts centromeric heterochromatin to release bulk rRNA genes into the nucleolus for ribosome production, which fuels single nucleus-driven pollen tube growth and is essential for plant reproduction.}, author = {Mérai, Zsuzsanna and Chumak, Nina and García-Aguilar, Marcelina and Hsieh, Tzung-Fu and Nishimura, Toshiro and Schoft, Vera K. and Bindics, János and Ślusarz, Lucyna and Arnoux, Stéphanie and Opravil, Susanne and Mechtler, Karl and Zilberman, Daniel and Fischer, Robert L. and Tamaru, Hisashi}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, number = {45}, pages = {16166--16171}, publisher = {National Academy of Sciences}, title = {{The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes}}, doi = {10.1073/pnas.1418564111}, volume = {111}, year = {2014}, } @article{6133, abstract = {cGMP signaling is widespread in the nervous system. However, it has proved difficult to visualize and genetically probe endogenously evoked cGMP dynamics in neurons in vivo. Here, we combine cGMP and Ca2+ biosensors to image and dissect a cGMP signaling network in a Caenorhabditis elegans oxygen-sensing neuron. We show that a rise in O2 can evoke a tonic increase in cGMP that requires an atypical O2-binding soluble guanylate cyclase and that is sustained until oxygen levels fall. Increased cGMP leads to a sustained Ca2+ response in the neuron that depends on cGMP-gated ion channels. Elevated levels of cGMP and Ca2+ stimulate competing negative feedback loops that shape cGMP dynamics. Ca2+-dependent negative feedback loops, including activation of phosphodiesterase-1 (PDE-1), dampen the rise of cGMP. A different negative feedback loop, mediated by phosphodiesterase-2 (PDE-2) and stimulated by cGMP-dependent kinase (PKG), unexpectedly promotes cGMP accumulation following a rise in O2, apparently by keeping in check gating of cGMP channels and limiting activation of Ca2+-dependent negative feedback loops. Simultaneous imaging of Ca2+ and cGMP suggests that cGMP levels can rise close to cGMP channels while falling elsewhere. O2-evoked cGMP and Ca2+ responses are highly reproducible when the same neuron in an individual animal is stimulated repeatedly, suggesting that cGMP transduction has high intrinsic reliability. However, responses vary substantially across individuals, despite animals being genetically identical and similarly reared. This variability may reflect stochastic differences in expression of cGMP signaling components. Our work provides in vivo insights into the architecture of neuronal cGMP signaling.}, author = {Couto, A. and Oda, S. and Nikolaev, V. O. and Soltesz, Z. and de Bono, Mario}, issn = {0027-8424}, journal = {Proceedings of the National Academy of Sciences}, number = {35}, pages = {E3301--E3310}, publisher = {Proceedings of the National Academy of Sciences}, title = {{In vivo genetic dissection of O2-evoked cGMP dynamics in a Caenorhabditis elegans gas sensor}}, doi = {10.1073/pnas.1217428110}, volume = {110}, year = {2013}, } @article{9481, abstract = {Arabidopsis thaliana endosperm, a transient tissue that nourishes the embryo, exhibits extensive localized DNA demethylation on maternally inherited chromosomes. Demethylation mediates parent-of-origin–specific (imprinted) gene expression but is apparently unnecessary for the extensive accumulation of maternally biased small RNA (sRNA) molecules detected in seeds. Endosperm DNA in the distantly related monocots rice and maize is likewise locally hypomethylated, but whether this hypomethylation is generally parent-of-origin specific is unknown. Imprinted expression of sRNA also remains uninvestigated in monocot seeds. Here, we report high-coverage sequencing of the Kitaake rice cultivar that enabled us to show that localized hypomethylation in rice endosperm occurs solely on the maternal genome, preferring regions of high DNA accessibility. Maternally expressed imprinted genes are enriched for hypomethylation at putative promoter regions and transcriptional termini and paternally expressed genes at promoters and gene bodies, mirroring our recent results in A. thaliana. However, unlike in A. thaliana, rice endosperm sRNA populations are dominated by specific strong sRNA-producing loci, and imprinted 24-nt sRNAs are expressed from both parental genomes and correlate with hypomethylation. Overlaps between imprinted sRNA loci and imprinted genes expressed from opposite alleles suggest that sRNAs may regulate genomic imprinting. Whereas sRNAs in seedling tissues primarily originate from small class II (cut-and-paste) transposable elements, those in endosperm are more uniformly derived, including sequences from other transposon classes, as well as genic and intergenic regions. Our data indicate that the endosperm exhibits a unique pattern of sRNA expression and suggest that localized hypomethylation of maternal endosperm DNA is conserved in flowering plants.}, author = {Rodrigues, Jessica A. and Ruan, Randy and Nishimura, Toshiro and Sharma, Manoj K. and Sharma, Rita and Ronald, Pamela C and Fischer, Robert L. and Zilberman, Daniel}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, keywords = {Multidisciplinary}, number = {19}, pages = {7934--7939}, publisher = {National Academy of Sciences}, title = {{Imprinted expression of genes and small RNA is associated with localized hypomethylation of the maternal genome in rice endosperm}}, doi = {10.1073/pnas.1306164110}, volume = {110}, year = {2013}, } @article{14305, abstract = {Understanding the mechanism of protein folding requires a detailed knowledge of the structural properties of the barriers separating unfolded from native conformations. The S-peptide from ribonuclease S forms its α-helical structure only upon binding to the folded S-protein. We characterized the transition state for this binding-induced folding reaction at high resolution by determining the effect of site-specific backbone thioxylation and side-chain modifications on the kinetics and thermodynamics of the reaction, which allows us to monitor formation of backbone hydrogen bonds and side-chain interactions in the transition state. The experiments reveal that α-helical structure in the S-peptide is absent in the transition state of binding. Recognition between the unfolded S-peptide and the S-protein is mediated by loosely packed hydrophobic side-chain interactions in two well defined regions on the S-peptide. Close packing and helix formation occurs rapidly after binding. Introducing hydrophobic residues at positions outside the recognition region can drastically slow down association.}, author = {Bachmann, Annett and Wildemann, Dirk and Praetorius, Florian M and Fischer, Gunter and Kiefhaber, Thomas}, issn = {1091-6490}, journal = {PNAS}, keywords = {Multidisciplinary}, number = {10}, pages = {3952--3957}, publisher = {Proceedings of the National Academy of Sciences}, title = {{Mapping backbone and side-chain interactions in the transition state of a coupled protein folding and binding reaction}}, doi = {10.1073/pnas.1012668108}, volume = {108}, year = {2011}, } @article{6137, abstract = {Variation in food quality and abundance requires animals to decide whether to stay on a poor food patch or leave in search of better food. An important question in behavioral ecology asks when is it optimal for an animal to leave a food patch it is depleting. Although optimal foraging is central to evolutionary success, the neural and molecular mechanisms underlying it are poorly understood. Here we investigate the neuronal basis for adaptive food-leaving behavior in response to resource depletion in Caenorhabditis elegans, and identify several of the signaling pathways involved. The ASE neurons, previously implicated in salt chemoattraction, promote food-leaving behavior via a cGMP pathway as food becomes limited. High ambient O2 promotes food-leaving via the O2-sensing neurons AQR, PQR, and URX. Ectopic activation of these neurons using channelrhodopsin is sufficient to induce high food-leaving behavior. In contrast, the neuropeptide receptor NPR-1, which regulates social behavior on food, acts in the ASE neurons, the nociceptive ASH neurons, and in the RMG interneuron to repress food-leaving. Finally, we show that neuroendocrine signaling by TGF-β/DAF-7 and neuronal insulin signaling are necessary for adaptive food-leaving behavior. We suggest that animals integrate information about their nutritional state with ambient oxygen and gustatory stimuli to formulate optimal foraging strategies.}, author = {Milward, K. and Busch, K. E. and Murphy, R. J. and de Bono, Mario and Olofsson, B.}, issn = {0027-8424}, journal = {Proceedings of the National Academy of Sciences}, number = {51}, pages = {20672--20677}, publisher = {National Academy of Sciences}, title = {{Neuronal and molecular substrates for optimal foraging in Caenorhabditis elegans}}, doi = {10.1073/pnas.1106134109}, volume = {108}, year = {2011}, } @article{9483, abstract = {Imprinted genes are expressed primarily or exclusively from either the maternal or paternal allele, a phenomenon that occurs in flowering plants and mammals. Flowering plant imprinted gene expression has been described primarily in endosperm, a terminal nutritive tissue consumed by the embryo during seed development or after germination. Imprinted expression in Arabidopsis thaliana endosperm is orchestrated by differences in cytosine DNA methylation between the paternal and maternal genomes as well as by Polycomb group proteins. Currently, only 11 imprinted A. thaliana genes are known. Here, we use extensive sequencing of cDNA libraries to identify 9 paternally expressed and 34 maternally expressed imprinted genes in A. thaliana endosperm that are regulated by the DNA-demethylating glycosylase DEMETER, the DNA methyltransferase MET1, and/or the core Polycomb group protein FIE. These genes encode transcription factors, proteins involved in hormone signaling, components of the ubiquitin protein degradation pathway, regulators of histone and DNA methylation, and small RNA pathway proteins. We also identify maternally expressed genes that may be regulated by unknown mechanisms or deposited from maternal tissues. We did not detect any imprinted genes in the embryo. Our results show that imprinted gene expression is an extensive mechanistically complex phenomenon that likely affects multiple aspects of seed development.}, author = {Hsieh, Tzung-Fu and Shin, Juhyun and Uzawa, Rie and Silva, Pedro and Cohen, Stephanie and Bauer, Matthew J. and Hashimoto, Meryl and Kirkbride, Ryan C. and Harada, John J. and Zilberman, Daniel and Fischer, Robert L.}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, number = {5}, pages = {1755--1762}, publisher = {National Academy of Sciences}, title = {{Regulation of imprinted gene expression in Arabidopsis endosperm}}, doi = {10.1073/pnas.1019273108}, volume = {108}, year = {2011}, } @article{9485, abstract = {Cytosine methylation silences transposable elements in plants, vertebrates, and fungi but also regulates gene expression. Plant methylation is catalyzed by three families of enzymes, each with a preferred sequence context: CG, CHG (H = A, C, or T), and CHH, with CHH methylation targeted by the RNAi pathway. Arabidopsis thaliana endosperm, a placenta-like tissue that nourishes the embryo, is globally hypomethylated in the CG context while retaining high non-CG methylation. Global methylation dynamics in seeds of cereal crops that provide the bulk of human nutrition remain unknown. Here, we show that rice endosperm DNA is hypomethylated in all sequence contexts. Non-CG methylation is reduced evenly across the genome, whereas CG hypomethylation is localized. CHH methylation of small transposable elements is increased in embryos, suggesting that endosperm demethylation enhances transposon silencing. Genes preferentially expressed in endosperm, including those coding for major storage proteins and starch synthesizing enzymes, are frequently hypomethylated in endosperm, indicating that DNA methylation is a crucial regulator of rice endosperm biogenesis. Our data show that genome-wide reshaping of seed DNA methylation is conserved among angiosperms and has a profound effect on gene expression in cereal crops.}, author = {Zemach, Assaf and Kim, M. Yvonne and Silva, Pedro and Rodrigues, Jessica A. and Dotson, Bradley and Brooks, Matthew D. and Zilberman, Daniel}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, number = {43}, pages = {18729--18734}, publisher = {National Academy of Sciences}, title = {{Local DNA hypomethylation activates genes in rice endosperm}}, doi = {10.1073/pnas.1009695107}, volume = {107}, year = {2010}, } @article{6146, abstract = {Homeostasis of internal carbon dioxide (CO2) and oxygen (O2) levels is fundamental to all animals. Here we examine the CO2 response of the nematode Caenorhabditis elegans. This species inhabits rotting material, which typically has a broad CO2 concentration range. We show that well fed C. elegans avoid CO2 levels above 0.5%. Animals can respond to both absolute CO2 concentrations and changes in CO2 levels within seconds. Responses to CO2 do not reflect avoidance of acid pH but appear to define a new sensory response. Sensation of CO2 is promoted by the cGMP-gated ion channel subunits TAX-2 and TAX-4, but other pathways are also important. Robust CO2 avoidance in well fed animals requires inhibition of the DAF-16 forkhead transcription factor by the insulin-like receptor DAF-2. Starvation, which activates DAF-16, strongly suppresses CO2 avoidance. Exposure to hypoxia (<1% O2) also suppresses CO2 avoidance via activation of the hypoxia-inducible transcription factor HIF-1. The npr-1 215V allele of the naturally polymorphic neuropeptide receptor npr-1, besides inhibiting avoidance of high ambient O2 in feeding C. elegans, also promotes avoidance of high CO2. C. elegans integrates competing O2 and CO2 sensory inputs so that one response dominates. Food and allelic variation at NPR-1 regulate which response prevails. Our results suggest that multiple sensory inputs are coordinated by C. elegans to generate different coherent foraging strategies.}, author = {Bretscher, A. J. and Busch, K. E. and de Bono, Mario}, issn = {0027-8424}, journal = {Proceedings of the National Academy of Sciences}, number = {23}, pages = {8044--8049}, publisher = {Proceedings of the National Academy of Sciences}, title = {{A carbon dioxide avoidance behavior is integrated with responses to ambient oxygen and food in Caenorhabditis elegans}}, doi = {10.1073/pnas.0707607105}, volume = {105}, year = {2008}, } @article{8483, abstract = {Atom-resolved real-time studies of kinetic processes in proteins have been hampered in the past by the lack of experimental techniques that yield sufficient temporal and atomic resolution. Here we present band-selective optimized flip-angle short transient (SOFAST) real-time 2D NMR spectroscopy, a method that allows simultaneous observation of reaction kinetics for a large number of nuclear sites along the polypeptide chain of a protein with an unprecedented time resolution of a few seconds. SOFAST real-time 2D NMR spectroscopy combines fast NMR data acquisition techniques with rapid sample mixing inside the NMR magnet to initiate the kinetic event. We demonstrate the use of SOFAST real-time 2D NMR to monitor the conformational transition of α-lactalbumin from a molten globular to the native state for a large number of amide sites along the polypeptide chain. The kinetic behavior observed for the disappearance of the molten globule and the appearance of the native state is monoexponential and uniform along the polypeptide chain. This observation confirms previous findings that a single transition state ensemble controls folding of α-lactalbumin from the molten globule to the native state. In a second application, the spontaneous unfolding of native ubiquitin under nondenaturing conditions is characterized by amide hydrogen exchange rate constants measured at high pH by using SOFAST real-time 2D NMR. Our data reveal that ubiquitin unfolds in a gradual manner with distinct unfolding regimes.}, author = {Schanda, Paul and Forge, V. and Brutscher, B.}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, keywords = {Multidisciplinary}, number = {27}, pages = {11257--11262}, publisher = {National Academy of Sciences}, title = {{Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy}}, doi = {10.1073/pnas.0702069104}, volume = {104}, year = {2007}, } @article{13425, abstract = {Nanoparticles (NPs) decorated with ligands combining photoswitchable dipoles and covalent cross-linkers can be assembled by light into organized, three-dimensional suprastructures of various types and sizes. NPs covered with only few photoactive ligands form metastable crystals that can be assembled and disassembled “on demand” by using light of different wavelengths. For higher surface concentrations, self-assembly is irreversible, and the NPs organize into permanently cross-linked structures including robust supracrystals and plastic spherical aggregates.}, author = {Klajn, Rafal and Bishop, Kyle J. M. and Grzybowski, Bartosz A.}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, keywords = {Multidisciplinary}, number = {25}, pages = {10305--10309}, publisher = {Proceedings of the National Academy of Sciences}, title = {{Light-controlled self-assembly of reversible and irreversible nanoparticle suprastructures}}, doi = {10.1073/pnas.0611371104}, volume = {104}, year = {2007}, } @article{9487, abstract = {Cytosine DNA methylation is considered to be a stable epigenetic mark, but active demethylation has been observed in both plants and animals. In Arabidopsis thaliana, DNA glycosylases of the DEMETER (DME) family remove methylcytosines from DNA. Demethylation by DME is necessary for genomic imprinting, and demethylation by a related protein, REPRESSOR OF SILENCING1, prevents gene silencing in a transgenic background. However, the extent and function of demethylation by DEMETER-LIKE (DML) proteins in WT plants is not known. Using genome-tiling microarrays, we mapped DNA methylation in mutant and WT plants and identified 179 loci actively demethylated by DML enzymes. Mutations in DML genes lead to locus-specific DNA hypermethylation. Reintroducing WT DML genes restores most loci to the normal pattern of methylation, although at some loci, hypermethylated epialleles persist. Of loci demethylated by DML enzymes, >80% are near or overlap genes. Genic demethylation by DML enzymes primarily occurs at the 5′ and 3′ ends, a pattern opposite to the overall distribution of WT DNA methylation. Our results show that demethylation by DML DNA glycosylases edits the patterns of DNA methylation within the Arabidopsis genome to protect genes from potentially deleterious methylation.}, author = {Penterman, Jon and Zilberman, Daniel and Huh, Jin Hoe and Ballinger, Tracy and Henikoff, Steven and Fischer, Robert L.}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, number = {16}, pages = {6752--6757}, publisher = {National Academy of Sciences}, title = {{DNA demethylation in the Arabidopsis genome}}, doi = {10.1073/pnas.0701861104}, volume = {104}, year = {2007}, } @article{11877, abstract = {The World Wide Web provides a unprecedented opportunity to automatically analyze a large sample of interests and activity in the world. We discuss methods for extracting knowledge from the web by randomly sampling and analyzing hosts and pages, and by analyzing the link structure of the web and how links accumulate over time. A variety of interesting and valuable information can be extracted, such as the distribution of web pages over domains, the distribution of interest in different areas, communities related to different topics, the nature of competition in different categories of sites, and the degree of communication between different communities or countries.}, author = {Henzinger, Monika H and Lawrence, Steve}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, number = {suppl_1}, pages = {5186--5191}, publisher = {Proceedings of the National Academy of Sciences}, title = {{Extracting knowledge from the World Wide Web}}, doi = {10.1073/pnas.0307528100}, volume = {101}, year = {2004}, } @article{3992, abstract = {Computing the volume occupied by individual atoms in macromolecular structures has been the subject of research for several decades. This interest has grown in the recent years, because weighted volumes are widely used in implicit solvent models. Applications of the latter in molecular mechanics simulations require that the derivatives of these weighted volumes be known. In this article, we give a formula for the volume derivative of a molecule modeled as a space-filling diagram made up of balls in motion. The formula is given in terms of the weights, radii, and distances between the centers as well as the sizes of the facets of the power diagram restricted to the space-filling diagram. Special attention is given to the detection and treatment of singularities as well as discontinuities of the derivative.}, author = {Edelsbrunner, Herbert and Koehl, Patrice}, issn = {0027-8424}, journal = {PNAS}, number = {5}, pages = {2203 -- 2208}, publisher = {National Academy of Sciences}, title = {{The weighted-volume derivative of a space-filling diagram}}, doi = {10.1073/pnas.0537830100}, volume = {100}, year = {2003}, } @article{885, abstract = {We study fitness landscape in the space of protein sequences by relating sets of human pathogenic missense mutations in 32 proteins to amino acid substitutions that occurred in the course of evolution of these proteins. On average, ≈10% of deviations of a nonhuman protein from its human ortholog are compensated pathogenic deviations (CPDs), i.e., are caused by an amino acid substitution that, at this site, would be pathogenic to humans. Normal functioning of a CPD-containing protein must be caused by other, compensatory deviations of the nonhuman species from humans. Together, a CPD and the corresponding compensatory deviation form a Dobzhansky-Muller incompatibility that can be visualized as the corner on a fitness ridge. Thus, proteins evolve along fitness ridges which contain only ≈10 steps between sucessive corners. The fraction of CPDs among all deviations of a protein from its human ortholog does not increase with the evolutionary distance between the proteins, indicating that subtitutions that carry evolving proteins around these corners occur in rapid succession, driven by positive selection. Data on fitness of interspecies hybrids suggest that the compensatory change that makes a CPD fit usually occurs within the same protein. Data on protein structures and on cooccurrence of amino acids at different sites of multiple orthologous proteins often make it possible to provisionally identify the substitution that compensates a partiCUlar CPD.}, author = {Kondrashov, Alexey and Sunyaev, Shamil and Kondrashov, Fyodor}, issn = {0027-8424}, journal = {PNAS}, number = {23}, pages = {14878 -- 14883}, publisher = {National Academy of Sciences}, title = {{Dobzhansky-Muller incompatibilities in protein evolution}}, doi = {10.1073/pnas.232565499}, volume = {99}, year = {2002}, } @article{3800, abstract = {Networks of GABAergic interneurons are of critical importance for the generation of gamma frequency oscillations in the brain. To examine the underlying synaptic mechanisms, we made paired recordings from "basket cells" (BCs) in different subfields of hippocampal slices, using transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of the parvalbumin promoter. Unitary inhibitory postsynaptic currents (IPSCs) showed large amplitude and fast time course with mean amplitude-weighted decay time constants of 2.5, 1.2, and 1.8 ms in the dentate gyrus, and the cornu ammonis area 3 (CA3) and 1 (CA1), respectively (33-34 degrees C). The decay of unitary IPSCs at BC-BC synapses was significantly faster than that at BC-principal cell synapses, indicating target cell-specific differences in IPSC kinetics. In addition, electrical coupling was found in a subset of BC-BC pairs. To examine whether an interneuron network with fast inhibitory synapses can act as a gamma frequency oscillator, we developed an interneuron network model based on experimentally determined properties. In comparison to previous interneuron network models, our model was able to generate oscillatory activity with higher coherence over a broad range of frequencies (20-110 Hz). In this model, high coherence and flexibility in frequency control emerge from the combination of synaptic properties, network structure, and electrical coupling.}, author = {Bartos, Marlene and Vida, Imre and Frotscher, Michael and Meyer, Axel and Monyer, Hannah and Geiger, Jörg and Jonas, Peter M}, issn = {0027-8424}, journal = {PNAS}, number = {20}, pages = {13222 -- 13227}, publisher = {National Academy of Sciences}, title = {{Fast synaptic inhibition promotes synchronized gamma oscillations in hippocampal interneuron networks}}, doi = {10.1073/pnas.192233099}, volume = {99}, year = {2002}, }