bluuebunny/crossref_metadata_2025 huggingface dataset:
A dictionary of behavioral motifs reveals clusters of genes affecting
Caenorhabditis elegans
locomotion
Authors: Brown André E. X. , Yemini Eviatar I. , Grundy Laura J. , Jucikas Tadas , Schafer William R.
Published: December 2012

Visible phenotypes based on locomotion and posture have played a critical role in understanding the molecular basis of behavior and development in
Caenorhabditis elegans
and other model organisms. However, it is not known whether these human-defined features capture the most important aspects of behavior for phenotypic comparison or whether they are sufficient to discover new behaviors. Here we show that four basic shapes, or eigenworms, previously described for wild-type worms, also capture mutant shapes, and that this representation can be used to build a dictionary of repetitive behavioral motifs in an unbiased way. By measuring the distance between each individual's behavior and the elements in the motif dictionary, we create a fingerprint that can be used to compare mutants to wild type and to each other. This analysis has revealed phenotypes not previously detected by real-time observation and has allowed clustering of mutants into related groups. Behavioral motifs provide a compact and intuitive representation of behavioral phenotypes.
Laccase detoxification mediates the nutritional alliance between leaf-cutting ants and fungus-garden symbionts
Authors: De Fine Licht Henrik H. , Schiøtt Morten , Rogowska-Wrzesinska Adelina , Nygaard Sanne , Roepstorff Peter , Boomsma Jacobus J.
Published: December 2012

Leaf-cutting ants combine large-scale herbivory with fungus farming to sustain advanced societies. Their stratified colonies are major evolutionary achievements and serious agricultural pests, but the crucial adaptations that allowed this mutualism to become the prime herbivorous component of neotropical ecosystems has remained elusive. Here we show how coevolutionary adaptation of a specific enzyme in the fungal symbiont has helped leaf-cutting ants overcome plant defensive phenolic compounds. We identify nine putative laccase-coding genes in the fungal genome of
Leucocoprinus gongylophorus
cultivated by the leaf-cutting ant
Acromyrmex echinatior
. One of these laccases (
LgLcc1
) is highly expressed in the specialized hyphal tips (gongylidia) that the ants preferentially eat, and we confirm that these ingested laccase molecules pass through the ant guts and remain active when defecated on the leaf pulp that the ants add to their gardens. This accurate deposition ensures that laccase activity is highest where new leaf material enters the fungus garden, but where fungal mycelium is too sparse to produce extracellular enzymes in sufficient quantities to detoxify phenolic compounds. Phylogenetic analysis of
LgLcc1
ortholog sequences from symbiotic and free-living fungi revealed significant positive selection in the ancestral lineage that gave rise to the gongylidia-producing symbionts of leaf-cutting ants and their non–leaf-cutting ant sister group. Our results are consistent with fungal preadaptation and subsequent modification of a particular laccase enzyme for the detoxification of secondary plant compounds during the transition to active herbivory in the ancestor of leaf-cutting ants between 8 and 12 Mya.
M2 isoform of pyruvate kinase is dispensable for tumor maintenance and growth
Authors: Cortés-Cros Marta , Hemmerlin Christelle , Ferretti Stephane , Zhang Juan , Gounarides John S. , Yin Hong , Muller Alban , Haberkorn Anne , Chene Patrick , Sellers William R. , Hofmann Francesco
Published: December 2012

Many cancer cells have increased rates of aerobic glycolysis, a phenomenon termed the Warburg effect. In addition, in tumors there is a predominance of expression of the M2 isoform of pyruvate kinase (PKM2). M2 expression was previously shown to be necessary for aerobic glycolysis and to provide a growth advantage to tumors. We report that knockdown of pyruvate kinase in tumor cells leads to a decrease in the levels of pyruvate kinase activity and an increase in the pyruvate kinase substrate phosphoenolpyruvate. However, lactate production from glucose, although reduced, was not fully inhibited. Furthermore, we are unique in reporting increased serine and glycine biosynthesis from both glucose and glutamine following pyruvate kinase knockdown. Although pyruvate kinase knockdown results in modest impairment of proliferation in vitro, in vivo growth of established xenograft tumors is unaffected by PKM2 absence. Our findings indicate that PKM2 is dispensable for tumor maintenance and growth in vivo, suggesting that other metabolic pathways bypass its function.
Sirt1 suppresses RNA synthesis after UV irradiation in combined xeroderma pigmentosum group D/Cockayne syndrome (XP-D/CS) cells
Authors: Vélez-Cruz Renier , Zadorin Anton S. , Coin Frédéric , Egly Jean-Marc
Published: December 2012

Specific mutations in the
XPD
subunit of transcription factor IIH result in combined xeroderma pigmentosum (XP)/Cockayne syndrome (CS), a severe DNA repair disorder characterized at the cellular level by a transcriptional arrest following UV irradiation. This transcriptional arrest has always been thought to be the result of faulty transcription-coupled repair. In the present study, we showed that, following UV irradiation, XP-D/CS cells displayed a gross transcriptional dysregulation compared with “pure” XP-D cells or WT cells. Furthermore, global RNA-sequencing analysis showed that XP-D/CS cells repressed the majority of genes after UV, whereas pure XP-D cells did not. By using housekeeping genes as a model, we demonstrated that XP-D/CS cells were unable to reassemble these gene promoters and thus to restart transcription after UV irradiation. Furthermore, we found that the repression of these promoters in XP-D/CS cells was not a simple consequence of deficient repair but rather an active heterochromatinization process mediated by the histone deacetylase Sirt1. Indeed, RNA-sequencing analysis showed that inhibition of and/or silencing of Sirt1 changed the chromatin environment at these promoters and restored the transcription of a large portion of the repressed genes in XP-D/CS cells after UV irradiation. Our work demonstrates that a significant part of the transcriptional arrest displayed by XP-D/CS cells arises as a result of an active repression process and not simply as a result of a DNA repair deficiency. This dysregulation of Sirt1 function that results in transcriptional repression may be the cause of various severe clinical features in patients with XP-D/CS that cannot be explained by a DNA repair defect.
NONO couples the circadian clock to the cell cycle
Authors: Kowalska Elzbieta , Ripperger Juergen A. , Hoegger Dominik C. , Bruegger Pascal , Buch Thorsten , Birchler Thomas , Mueller Anke , Albrecht Urs , Contaldo Claudio , Brown Steven A.
Published: December 2012

Mammalian circadian clocks restrict cell proliferation to defined time windows, but the mechanism and consequences of this interrelationship are not fully understood. Previously we identified the multifunctional nuclear protein NONO as a partner of circadian PERIOD (PER) proteins. Here we show that it also conveys circadian gating to the cell cycle, a connection surprisingly important for wound healing in mice. Specifically, although fibroblasts from NONO-deficient mice showed approximately normal circadian cycles, they displayed elevated cell doubling and lower cellular senescence. At a molecular level, NONO bound to the
p16-Ink4A
cell cycle checkpoint gene and potentiated its circadian activation in a PER protein-dependent fashion. Loss of either NONO or PER abolished this activation and circadian expression of
p16-Ink4A
and eliminated circadian cell cycle gating. In vivo, lack of NONO resulted in defective wound repair. Because wound healing defects were also seen in multiple circadian clock-deficient mouse lines, our results therefore suggest that coupling of the cell cycle to the circadian clock via NONO may be useful to segregate in temporal fashion cell proliferation from tissue organization.
Mice with an adipocyte-specific lipin 1 separation-of-function allele reveal unexpected roles for phosphatidic acid in metabolic regulation
Authors: Mitra Mayurranjan S. , Chen Zhouji , Ren Hongmei , Harris Thurl E. , Chambers Kari T. , Hall Angela M. , Nadra Karim , Klein Samuel , Chrast Roman , Su Xiong , Morris Andrew J. , Finck Brian N.
Published: December 2012

Lipin 1 is a coregulator of DNA-bound transcription factors and a phosphatidic acid (PA) phosphatase (PAP) enzyme that catalyzes a critical step in the synthesis of glycerophospholipids. Lipin 1 is highly expressed in adipocytes, and constitutive loss of lipin 1 blocks adipocyte differentiation; however, the effects of
Lpin1
deficiency in differentiated adipocytes are unknown. Here we report that adipocyte-specific
Lpin1
gene recombination unexpectedly resulted in expression of a truncated lipin 1 protein lacking PAP activity but retaining transcriptional regulatory function. Loss of lipin 1-mediated PAP activity in adipocytes led to reduced glyceride synthesis and increased PA content. Characterization of the deficient mice also revealed that lipin 1 normally modulates cAMP-dependent signaling through protein kinase A to control lipolysis by metabolizing PA, which is an allosteric activator of phosphodiesterase 4 and the molecular target of rapamycin. Consistent with these findings, lipin 1 expression was significantly related to adipose tissue lipolytic rates and protein kinase A signaling in adipose tissue of obese human subjects. Taken together, our findings identify lipin 1 as a reciprocal regulator of triglyceride synthesis and hydrolysis in adipocytes, and suggest that regulation of lipolysis by lipin 1 is mediated by PA-dependent modulation of phosphodiesterase 4.
Predictability of evolution depends nonmonotonically on population size
Authors: Szendro Ivan G. , Franke Jasper , de Visser J. Arjan G. M. , Krug Joachim
Published: December 2012

To gauge the relative importance of contingency and determinism in evolution is a fundamental problem that continues to motivate much theoretical and empirical research. In recent evolution experiments with microbes, this question has been explored by monitoring the repeatability of adaptive changes in replicate populations. Here, we present the results of an extensive computational study of evolutionary predictability based on an experimentally measured eight-locus fitness landscape for the filamentous fungus
Aspergillus niger
. To quantify predictability, we define entropy measures on observed mutational trajectories and endpoints. In contrast to the common expectation of increasingly deterministic evolution in large populations, we find that these entropies display an initial decrease and a subsequent increase with population size
N
, governed, respectively, by the scales
Nμ
and
Nμ
2
, corresponding to the supply rates of single and double mutations, where
μ
denotes the mutation rate. The amplitude of this pattern is determined by
μ
. We show that these observations are generic by comparing our findings for the experimental fitness landscape to simulations on simple model landscapes.
Neddylation pathway regulates T-cell function by targeting an adaptor protein Shc and a protein kinase Erk signaling
Authors: Jin Hyung-seung , Liao Lujian , Park Yoon , Liu Yun-Cai
Published: December 2012

NEDD8 (neural precursor cell expressed, developmentally down-regulated 8) is a ubiquitin-like molecule whose action on modifying protein substrates is critical in various cellular functions but whose importance in the immune system is not well understood. Here we investigated the role of protein neddylation in regulating T-cell function using an in vivo knockdown technique. We found that reduced expression of Ubc12 in CD4
+
T cells led to impaired T-cell receptor/CD28-induced proliferation and cytokine production both in vitro and in vivo, accompanied by reduced Erk activation. These findings were recapitulated by treatment with MLN4924, an inhibitor of NEDD8-activating enzyme. Furthermore, Shc, an adaptor molecule between antigen receptors and the Ras/Erk pathway, was identified as a target for neddylation. Importantly, mice adoptively transferred with Ubc12 knockdown CD4
+
T cells showed markedly ameliorated allergic responses. This study thus identifies an important role for protein neddylation in T-cell function, which may serve as a therapeutic target for inflammatory diseases.
Probing kinase and phosphatase activities of two-component systems in vivo with concentration-dependent phosphorylation profiling
Authors: Gao Rong , Stock Ann M.
Published: December 2012

Quantitative analyses of protein concentrations, modifications and activities in their native environments are playing an increasingly vital role in unraveling the general principles underlying signal transduction pathways. The prevalent bacterial two-component systems (TCSs) use a central phosphotransfer for signaling; however, in vivo characterization of the kinase and phosphatase activities of TCS proteins is often limited by traditional transcriptional reporter assays and complicated by simultaneous actions of multiple TCS activities. Here, we report a strategy that combines concentration-dependent phosphorylation profiling and mathematical modeling to characterize the cellular activities of the archetype
Escherichia coli
PhoR/PhoB system. Phosphorylation of the response regulator (RR) PhoB has been found to be dependent on the total concentrations of PhoB/PhoR and saturated at high concentrations. The relationship between RR phosphorylation and total concentrations has been defined by the modeling of the kinase and phosphatase reactions and quantified to derive the biochemical parameters of the PhoR/PhoB system in vivo. In a further test of this approach on a PhoB mutant, PhoB
F20D
, it proved highly effective in exploring the mechanistic differences of TCSs that are not revealed by traditional reporter assays. Measurement of biochemical parameters for PhoB
F20D
led to the discovery that a weaker interaction between the histidine sensor kinase and RR could result in a higher and nonrobust phosphorylation due to diminished phosphatase activities.
Blink-related momentary activation of the default mode network while viewing videos
Authors: Nakano Tamami , Kato Makoto , Morito Yusuke , Itoi Seishi , Kitazawa Shigeru
Published: December 2012

It remains unknown why we generate spontaneous eyeblinks every few seconds, more often than necessary for ocular lubrication. Because eyeblinks tend to occur at implicit breakpoints while viewing videos, we hypothesized that eyeblinks are actively involved in the release of attention. We show that while viewing videos, cortical activity momentarily decreases in the dorsal attention network after blink onset but increases in the default-mode network implicated in internal processing. In contrast, physical blackouts of the video do not elicit such reciprocal changes in brain networks. The results suggest that eyeblinks are actively involved in the process of attentional disengagement during a cognitive behavior by momentarily activating the default-mode network while deactivating the dorsal attention network.
Molecular structures of trimeric HIV-1 Env in complex with small antibody derivatives
Authors: Meyerson Joel R. , Tran Erin E. H. , Kuybeda Oleg , Chen Weizao , Dimitrov Dimiter S. , Gorlani Andrea , Verrips Theo , Lifson Jeffrey D. , Subramaniam Sriram
Published: December 2012

The extensive carbohydrate coat, the variability of protein structural features on HIV-1 envelope glycoproteins (Env), and the steric constraints of the virus-cell interface during infection, present challenges to the elicitation of effective full-length (∼150 kDa), neutralizing antibodies against HIV. These hurdles have motivated the engineering of smaller antibody derivatives that can bind Env and neutralize the virus. To further understand the mechanisms by which these proteins neutralize HIV-1, we carried out cryoelectron tomography of native HIV-1 BaL virions complexed separately to two small (∼15 kDa) HIV-neutralizing proteins: A12, which binds the CD4-binding site on Env, and m36, whose binding to Env is enhanced by CD4 binding. We show that despite their small size, the presence of these proteins and their effects on the quaternary conformation of trimeric Env can be visualized in molecular structures derived by cryoelectron tomography combined with subvolume averaging. Binding of Env to A12 results in a conformational change that is comparable to changes observed upon its binding to the CD4-binding site antibody, b12. In contrast, binding of Env to m36 results in an “open” quaternary conformation similar to that seen with binding of soluble CD4 or the CD4i antibody, 17b. Because these small neutralizing proteins are less sterically hindered than full-length antibodies at zones of virus-cell contact, the finding that their binding has the same structural consequences as that of other broadly neutralizing antibodies highlights their potential for use in therapeutic applications.
Cell cycle-dependent deposition of CENP-A requires the Dos1/2–Cdc20 complex
Authors: Gonzalez Marlyn , He Haijin , Sun Siyu , Li Chen , Li Fei
Published: December 2012

Centromeric histone CENP-A, a variant of canonical histone H3, plays a central role in proper chromosome segregation. Loading of CENP-A at centromeres is cell cycle-regulated: parental CENP-A is deposited at centromeres during S phase, whereas newly synthesized CENP-A is deposited during later stages of the cell cycle. The mechanisms involved in deposition of CENP-A at centromeres during S phase remain poorly understood. In fission yeast, loading of CENP-A during S phase is regulated by the GATA-type factor, Ams2. Here we show that the Dos1/2-Cdc20 complex, previously characterized as a silencing complex essential for inheritance of H3K9 methylation during S phase, is also required for localization of CENP-A
cnp1
at centromeres at this stage. Disruption of Dos1 (also known as Raf1/Clr8/Cmc1), Dos2 (also known as Raf2/Clr7/Cmc2), or Cdc20, a DNA polymerase epsilon subunit, results in dissociation of CENP-A from centromeres and mislocalization of the protein to noncentromeric sites. All three mutants display spindle disorganization and mitotic defects. Inactivation of Dos1 or Cdc20 also results in accumulation of noncoding RNA transcripts from centromeric cores, a feature common to mutants affecting kinetochore integrity. We further find that Dos1 physically associates with Ams2 and is required for the association of Ams2 with centromeric cores during S phase. Finally, we show that Dos2 associates with centromeric cores during S phase and that its recruitment to centromeric cores depends on Cdc20. This study identifies a physical link between DNA replication and CENP-A assembly machinery and provides mechanistic insight into how CENP-A is faithfully inherited during S phase.
Retinol dehydrogenase 10 is indispensible for spermatogenesis in juvenile males
Authors: Tong Ming-Han , Yang Qi-En , Davis Jeffrey C. , Griswold Michael D.
Published: December 2012

Retinoic acid (RA), an active vitamin A derivative, is essential for mammalian spermatogenesis. Genetic studies have revealed that oxidation of vitamin A to retinal by retinol dehydrogenase 10 (RDH10) is critical for embryonic RA biosynthesis. However, physiological roles of RDH10 in postnatal RA synthesis remain unclear, given that
Rdh10
loss-of-function mutations lead to early embryonic lethality. We conducted in vivo genetic studies of
Rdh10
in postnatal mouse testes and found that an RDH10 deficiency in Sertoli cells, but not in germ cells, results in a mild germ cell depletion phenotype. A deficiency of RDH10 in both Sertoli and germ cells in juvenile mice results in a blockage of spermatogonial differentiation, similar to that seen in vitamin A-deficient animals. This defect in spermatogenesis arises from a complete deficiency in juvenile testicular RA synthesis and can be rescued by retinoid administration. Thus, in juvenile mice, the primary, but not exclusive, source of RA in the testes is Sertoli cells. In contrast, adult
Rdh10
-deficient mice exhibit phenotypically normal spermatogenesis, indicating that during development a change occurs in either the cellular source of RA or the retinaldehyde dehydrogenase involved in RA synthesis.
Transcriptional corepressor TOPLESS complexes with pseudoresponse regulator proteins and histone deacetylases to regulate circadian transcription
Authors: Wang Lei , Kim Jeongsik , Somers David E.
Published: December 2012

Circadian clocks are ubiquitous molecular time-keeping mechanisms that coordinate physiology and metabolism and provide an adaptive advantage to higher plants. The central oscillator of the plant clock is composed of interlocked feedback loops that involve multiple repressive factors acting throughout the circadian cycle. PSEUDO RESPONSE REGULATORS (PRRs) comprise a five-member family that is essential to the function of the central oscillator. PRR5, PRR7, and PRR9 can bind the promoters of the core clock genes
CIRCADIAN CLOCK ASSOCIATED 1
(
CCA1
) and
LATE ELONGATED HYPOCOTYL
(
LHY
) to restrict their expression to near dawn, but the mechanism has been unclear. Here we report that members of the plant Groucho/Tup1 corepressor family, TOPLESS/TOPLESS-RELATED (TPL/TPR), interact with these three PRR proteins at the
CCA1
and
LHY
promoters to repress transcription and alter circadian period. This activity is diminished in the presence of the inhibitor trichostatin A, indicating the requirement of histone deacetylase for full TPL activity. Additionally, a complex of PRR9, TPL, and histone deacetylase 6, can form in vivo, implicating this tripartite association as a central repressor of circadian gene expression. Our findings show that the TPL/TPR corepressor family are components of the central circadian oscillator mechanism and reinforces the role of this family as central to multiple signaling pathways in higher plants.
Agrobacterium tumefaciens
recognizes its host environment using ChvE to bind diverse plant sugars as virulence signals
Authors: Hu Xiaozhen , Zhao Jinlei , DeGrado William F. , Binns Andrew N.
Published: December 2012

Agrobacterium tumefaciens
is a broad host range plant pathogen that combinatorially recognizes diverse host molecules including phenolics, low pH, and aldose monosaccharides to activate its pathogenic pathways. Chromosomal virulence gene
E
(
chvE
) encodes a periplasmic-binding protein that binds several neutral sugars and sugar acids, and subsequently interacts with the VirA/VirG regulatory system to stimulate virulence (
vir
) gene expression. Here, a combination of genetics, X-ray crystallography, and isothermal calorimetry reveals how ChvE binds the different monosaccharides and also shows that binding of sugar acids is pH dependent. Moreover, the potency of a sugar for
vir
gene expression is modulated by a transport system that also relies on ChvE. These two circuits tune the overall system to respond to sugar concentrations encountered in vivo. Finally, using
chvE
mutants with restricted sugar specificities, we show that there is host variation in regard to the types of sugars that are limiting for
vir
induction.
Diagnostic power of diffuse reflectance spectroscopy for targeted detection of breast lesions with microcalcifications
Authors: Soares Jaqueline S. , Barman Ishan , Dingari Narahara Chari , Volynskaya Zoya , Liu Wendy , Klein Nina , Plecha Donna , Dasari Ramachandra R. , Fitzmaurice Maryann
Published: December 2012

Microcalcifications geographically target the location of abnormalities within the breast and are of critical importance in breast cancer diagnosis. However, despite stereotactic guidance, core needle biopsy fails to retrieve microcalcifications in up to 15% of patients. Here, we introduce an approach based on diffuse reflectance spectroscopy for detection of microcalcifications that focuses on variations in optical absorption stemming from the calcified clusters and the associated cross-linking molecules. In this study, diffuse reflectance spectra are acquired ex vivo from 203 sites in fresh biopsy tissue cores from 23 patients undergoing stereotactic breast needle biopsies. By correlating the spectra with the corresponding radiographic and histologic assessment, we have developed a support vector machine-derived decision algorithm, which shows high diagnostic power (positive predictive value and negative predictive value of 97% and 88%, respectively) for diagnosis of lesions with microcalcifications. We further show that these results are robust and not due to any spurious correlations. We attribute our findings to the presence of proteins (such as elastin), and desmosine and isodesmosine cross-linkers in the microcalcifications. It is important to note that the performance of the diffuse reflectance decision algorithm is comparable to one derived from the corresponding Raman spectra, and the considerably higher intensity of the reflectance signal enables the detection of the targeted lesions in a fraction of the spectral acquisition time. Our findings create a unique landscape for spectroscopic validation of breast core needle biopsy for detection of microcalcifications that can substantially improve the likelihood of an adequate, diagnostic biopsy in the first attempt.
Eye opening and PSD95 are required for long-term potentiation in developing superior colliculus
Authors: Zhao Jian-Ping , Murata Yasunobu , Constantine-Paton Martha
Published: December 2012

The only major glutamate receptor membrane-associated guanylate kinase scaffolds expressed in the young superficial superior colliculus (SC) are synapse-associated protein 102 (SAP102) and postsynaptic density protein 95 (PSD95). In this, as in all visual brain regions examined, synaptic PSD95 increases rapidly following simultaneous eyelid opening (EO). We show that EO and PSD95 are necessary for SC NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) and this LTP is eliminated or reinstated by manipulating EO. PSD95 knockdown (KD) in vivo blocks this LTP, but not long-term depression, and reduces frequencies of miniature AMPA receptor and NMDAR currents with no change in presynaptic release. Furthermore, miniature NMDAR currents after PSD95 KD show an activity-triggered calcineurin sensitivity that is normally only found in the pre-EO period when SAP102 binds mixed GluN2A/GluN2B NMDARs. These data indicate that young SC LTP arises from PSD95 unsilencing of silent synapses, that unsilencing is labile in young brain, and that even though SAP102 and PSD95 can bind the same NMDARs, only PSD95 enables SC synaptic maturation.
Population genomic and genome-wide association studies of agroclimatic traits in sorghum
Authors: Morris Geoffrey P. , Ramu Punna , Deshpande Santosh P. , Hash C. Thomas , Shah Trushar , Upadhyaya Hari D. , Riera-Lizarazu Oscar , Brown Patrick J. , Acharya Charlotte B. , Mitchell Sharon E. , Harriman James , Glaubitz Jeffrey C. , Buckler Edward S. , Kresovich Stephen
Published: December 2012

Accelerating crop improvement in sorghum, a staple food for people in semiarid regions across the developing world, is key to ensuring global food security in the context of climate change. To facilitate gene discovery and molecular breeding in sorghum, we have characterized ∼265,000 single nucleotide polymorphisms (SNPs) in 971 worldwide accessions that have adapted to diverse agroclimatic conditions. Using this genome-wide SNP map, we have characterized population structure with respect to geographic origin and morphological type and identified patterns of ancient crop diffusion to diverse agroclimatic regions across Africa and Asia. To better understand the genomic patterns of diversification in sorghum, we quantified variation in nucleotide diversity, linkage disequilibrium, and recombination rates across the genome. Analyzing nucleotide diversity in landraces, we find evidence of selective sweeps around starch metabolism genes, whereas in landrace-derived introgression lines, we find introgressions around known height and maturity loci. To identify additional loci underlying variation in major agroclimatic traits, we performed genome-wide association studies (GWAS) on plant height components and inflorescence architecture. GWAS maps several classical loci for plant height, candidate genes for inflorescence architecture. Finally, we trace the independent spread of multiple haplotypes carrying alleles for short stature or long inflorescence branches. This genome-wide map of SNP variation in sorghum provides a basis for crop improvement through marker-assisted breeding and genomic selection.
Selectivity and self-assembly in the control of a bacterial toxin by an antitoxic noncoding RNA pseudoknot
Authors: Short Francesca L. , Pei Xue Y. , Blower Tim R. , Ong Shue-Li , Fineran Peter C. , Luisi Ben F. , Salmond George P. C.
Published: December 2012

Bacterial small RNAs perform numerous regulatory roles, including acting as antitoxic components in toxin–antitoxin systems. In type III toxin–antitoxin systems, small processed RNAs directly antagonize their toxin protein partners, and in the systems characterized the toxin and antitoxin components together form a trimeric assembly. In the present study, we sought to define how the RNA antitoxin, ToxI, inhibits its potentially lethal protein partner, ToxN. We show through cross-inhibition experiments with the ToxIN systems from
Pectobacterium atrosepticum
(ToxIN
Pa
) and
Bacillus thuringiensis
(ToxIN
Bt
) that ToxI RNAs are highly selective enzyme inhibitors. Both systems have an “addictive” plasmid maintenance phenotype. We demonstrate that ToxI
Pa
can inhibit ToxN
Pa
in vitro both in its processed form and as a repetitive precursor RNA, and this inhibition is linked to the self-assembly of the trimeric complex. Inhibition and self-assembly are both mediated entirely by the ToxI
Pa
RNA, with no requirement for cellular factors or exogenous energy. Finally, we explain the origins of ToxI antitoxin selectivity through our crystal structure of the ToxIN
Bt
complex. Our results show how a processed RNA pseudoknot can inhibit a deleterious protein with exquisite molecular specificity and how these self-contained and addictive RNA-protein pairs can confer different adaptive benefits in their bacterial hosts.
TAWAWA1
, a regulator of rice inflorescence architecture, functions through the suppression of meristem phase transition
Authors: Yoshida Akiko , Sasao Masafumi , Yasuno Naoko , Takagi Kyoko , Daimon Yasufumi , Chen Ruihong , Yamazaki Ryo , Tokunaga Hiroki , Kitaguchi Yoshinori , Sato Yutaka , Nagamura Yoshiaki , Ushijima Tomokazu , Kumamaru Toshihiro , Iida Shigeru , Maekawa Masahiko , Kyozuka Junko
Published: December 2012

Inflorescence structures result from the activities of meristems, which coordinate both the renewal of stem cells in the center and organ formation at the periphery. The fate of a meristem is specified at its initiation and changes as the plant develops. During rice inflorescence development, newly formed meristems acquire a branch meristem (BM) identity, and can generate further meristems or terminate as spikelets. Thus, the form of rice inflorescence is determined by a reiterative pattern of decisions made at the meristems. In the dominant gain-of-function mutant
tawawa1-D
, the activity of the inflorescence meristem (IM) is extended and spikelet specification is delayed, resulting in prolonged branch formation and increased numbers of spikelets. In contrast, reductions in
TAWAWA1
(
TAW1
) activity cause precocious IM abortion and spikelet formation, resulting in the generation of small inflorescences.
TAW1
encodes a nuclear protein of unknown function and shows high levels of expression in the shoot apical meristem, the IM, and the BMs.
TAW1
expression disappears from incipient spikelet meristems (SMs). We also demonstrate that members of the
SHORT VEGETATIVE PHASE
subfamily of MADS-box genes function downstream of
TAW1
. We thus propose that
TAW1
is a unique regulator of meristem activity in rice and regulates inflorescence development through the promotion of IM activity and suppression of the phase change to SM identity.
Adaptation of the genetically tractable malaria pathogen
Plasmodium knowlesi
to continuous culture in human erythrocytes
Authors: Moon Robert W. , Hall Joanna , Rangkuti Farania , Ho Yung Shwen , Almond Neil , Mitchell Graham H. , Pain Arnab , Holder Anthony A. , Blackman Michael J.
Published: December 2012

Research into the aetiological agent of the most widespread form of severe malaria,
Plasmodium falciparum
, has benefitted enormously from the ability to culture and genetically manipulate blood-stage forms of the parasite in vitro. However, most malaria outside Africa is caused by a distinct
Plasmodium
species,
Plasmodium vivax
, and it has become increasingly apparent that zoonotic infection by the closely related simian parasite
Plasmodium knowlesi
is a frequent cause of life-threatening malaria in regions of southeast Asia. Neither of these important malarial species can be cultured in human cells in vitro, requiring access to primates with the associated ethical and practical constraints. We report the successful adaptation of
P. knowlesi
to continuous culture in human erythrocytes. Human-adapted
P. knowlesi
clones maintain their capacity to replicate in monkey erythrocytes and can be genetically modified with unprecedented efficiency, providing an important and unique model for studying conserved aspects of malarial biology as well as species-specific features of an emerging pathogen.
Anatomical enablers and the evolution of C
4
photosynthesis in grasses
Authors: Christin Pascal-Antoine , Osborne Colin P. , Chatelet David S. , Columbus J. Travis , Besnard Guillaume , Hodkinson Trevor R. , Garrison Laura M. , Vorontsova Maria S. , Edwards Erika J.
Published: December 2012

C
4
photosynthesis is a series of anatomical and biochemical modifications to the typical C
3
pathway that increases the productivity of plants in warm, sunny, and dry conditions. Despite its complexity, it evolved more than 62 times independently in flowering plants. However, C
4
origins are absent from most plant lineages and clustered in others, suggesting that some characteristics increase C
4
evolvability in certain phylogenetic groups. The C
4
trait has evolved 22–24 times in grasses, and all origins occurred within the PACMAD clade, whereas the similarly sized BEP clade contains only C
3
taxa. Here, multiple foliar anatomy traits of 157 species from both BEP and PACMAD clades are quantified and analyzed in a phylogenetic framework. Statistical modeling indicates that C
4
evolvability strongly increases when the proportion of vascular bundle sheath (BS) tissue is higher than 15%, which results from a combination of short distance between BS and large BS cells. A reduction in the distance between BS occurred before the split of the BEP and PACMAD clades, but a decrease in BS cell size later occurred in BEP taxa. Therefore, when environmental changes promoted C
4
evolution, suitable anatomy was present only in members of the PACMAD clade, explaining the clustering of C
4
origins in this lineage. These results show that key alterations of foliar anatomy occurring in a C
3
context and preceding the emergence of the C
4
syndrome by millions of years facilitated the repeated evolution of one of the most successful physiological innovations in angiosperm history.
Two methods for full-length RNA sequencing for low quantities of cells and single cells
Authors: Pan Xinghua , Durrett Russell E. , Zhu Haiying , Tanaka Yoshiaki , Li Yumei , Zi Xiaoyuan , Marjani Sadie L. , Euskirchen Ghia , Ma Chao , LaMotte Robert H. , Park In-Hyun , Snyder Michael P. , Mason Christopher E. , Weissman Sherman M.
Published: December 2012

The ability to determine the gene expression pattern in low quantities of cells or single cells is important for resolving a variety of problems in many biological disciplines. A robust description of the expression signature of a single cell requires determination of the full-length sequence of the expressed mRNAs in the cell, yet existing methods have either 3′ biased or variable transcript representation. Here, we report our protocols for the amplification and high-throughput sequencing of very small amounts of RNA for sequencing using procedures of either semirandom primed PCR or phi29 DNA polymerase-based DNA amplification, for the cDNA generated with oligo-dT and/or random oligonucleotide primers. Unlike existing methods, these protocols produce relatively uniformly distributed sequences covering the full length of almost all transcripts independent of their sizes, from 1,000 to 10 cells, and even with single cells. Both protocols produced satisfactory detection/coverage of the abundant mRNAs from a single K562 erythroleukemic cell or a single dorsal root ganglion neuron. The phi29-based method produces long products with less noise, uses an isothermal reaction, and is simple to practice. The semirandom primed PCR procedure is more sensitive and reproducible at low transcript levels or with low quantities of cells. These methods provide tools for mRNA sequencing or RNA sequencing when only low quantities of cells, a single cell, or even degraded RNA are available for profiling.
Amphipathic antenna of an inward rectifier K
+
channel responds to changes in the inner membrane leaflet
Authors: Iwamoto Masayuki , Oiki Shigetoshi
Published: December 2012

Membrane lipids modulate the function of membrane proteins. In the case of ion channels, they bias the gating equilibrium, although the underlying mechanism has remained elusive. Here we demonstrate that the N-terminal segment (M0) of the KcsA potassium channel mediates the effect of changes in the lipid milieu on channel gating. The M0 segment is a membrane-anchored amphipathic helix, bearing positively charged residues. In asymmetric membranes, the M0 helix senses the presence of negatively charged phospholipids on the inner leaflet. Upon gating, the M0 helix revolves around the axis of the helix on the membrane surface, inducing the positively charged residues to interact with the negative head groups of the lipids so as to stabilize the open conformation (i.e., the “roll-and-stabilize model”). The M0 helix is thus a charge-sensitive “antenna,” capturing temporary changes in lipid composition in the fluidic membrane. This unique type of sensory device may be shared by various types of membrane proteins.
Aliphatic peptides show similar self-assembly to amyloid core sequences, challenging the importance of aromatic interactions in amyloidosis
Authors: Lakshmanan Anupama , Cheong Daniel W. , Accardo Angelo , Di Fabrizio Enzo , Riekel Christian , Hauser Charlotte A. E.
Published: December 2012

The self-assembly of abnormally folded proteins into amyloid fibrils is a hallmark of many debilitating diseases, from Alzheimer’s and Parkinson diseases to prion-related disorders and diabetes type II. However, the fundamental mechanism of amyloid aggregation remains poorly understood. Core sequences of four to seven amino acids within natural amyloid proteins that form toxic fibrils have been used to study amyloidogenesis. We recently reported a class of systematically designed ultrasmall peptides that self-assemble in water into cross-β–type fibers. Here we compare the self-assembly of these peptides with natural core sequences. These include core segments from Alzheimer’s amyloid-β, human amylin, and calcitonin. We analyzed the self-assembly process using circular dichroism, electron microscopy, X-ray diffraction, rheology, and molecular dynamics simulations. We found that the designed aliphatic peptides exhibited a similar self-assembly mechanism to several natural sequences, with formation of α-helical intermediates being a common feature. Interestingly, the self-assembly of a second core sequence from amyloid-β, containing the diphenylalanine motif, was distinctly different from all other examined sequences. The diphenylalanine-containing sequence formed β-sheet aggregates without going through the α-helical intermediate step, giving a unique fiber-diffraction pattern and simulation structure. Based on these results, we propose a simplified aliphatic model system to study amyloidosis. Our results provide vital insight into the nature of early intermediates formed and suggest that aromatic interactions are not as important in amyloid formation as previously postulated. This information is necessary for developing therapeutic drugs that inhibit and control amyloid formation.
Learning to detect and combine the features of an object
Authors: Suchow Jordan W. , Pelli Denis G.
Published: December 2012

To recognize an object, it is widely supposed that we first detect and then combine its features. Familiar objects are recognized effortlessly, but unfamiliar objects—like new faces or foreign-language letters—are hard to distinguish and must be learned through practice. Here, we describe a method that separates detection and combination and reveals how each improves as the observer learns. We dissociate the steps by two independent manipulations: For each step, we do or do not provide a bionic crutch that performs it optimally. Thus, the two steps may be performed solely by the human, solely by the crutches, or cooperatively, when the human takes one step and a crutch takes the other. The crutches reveal a double dissociation between detecting and combining. Relative to the two-step ideal, the human observer’s overall efficiency for unconstrained identification equals the product of the efficiencies with which the human performs the steps separately. The two-step strategy is inefficient: Constraining the ideal to take two steps roughly halves its identification efficiency. In contrast, we find that humans constrained to take two steps perform just as well as when unconstrained, which suggests that they normally take two steps. Measuring threshold contrast (the faintness of a barely identifiable letter) as it improves with practice, we find that detection is inefficient and learned slowly. Combining is learned at a rate that is 4× higher and, after 1,000 trials, 7× more efficient. This difference explains much of the diversity of rates reported in perceptual learning studies, including effects of complexity and familiarity.
Activated protein C ameliorates diabetic nephropathy by epigenetically inhibiting the redox enzyme p66
Shc
Authors: Bock Fabian , Shahzad Khurrum , Wang Hongjie , Stoyanov Stoyan , Wolter Juliane , Dong Wei , Pelicci Pier Giuseppe , Kashif Muhammed , Ranjan Satish , Schmidt Simone , Ritzel Robert , Schwenger Vedat , Reymann Klaus G. , Esmon Charles T. , Madhusudhan Thati , Nawroth Peter P. , Isermann Berend
Published: December 2012

The coagulation protease activated protein C (aPC) confers cytoprotective effects in various in vitro and in vivo disease models, including diabetic nephropathy. The nephroprotective effect may be related to antioxidant effects of aPC. However, the mechanism through which aPC may convey these antioxidant effects and the functional relevance of these properties remain unknown. Here, we show that endogenous and exogenous aPC prevents glomerular accumulation of oxidative stress markers and of the redox-regulating protein p66
Shc
in experimental diabetic nephropathy. These effects were predominately observed in podocytes. In vitro, aPC inhibited glucose-induced expression of p66
Shc
mRNA and protein in podocytes (via PAR-1 and PAR-3) and various endothelial cell lines, but not in glomerular endothelial cells. Treatment with aPC reversed glucose-induced hypomethylation and hyperacetylation of the p66
Shc
promoter in podocytes. The hyperacetylating agent sodium butyrate abolished the suppressive effect of aPC on p66
Shc
expression both in vitro and in vivo. Moreover, sodium butyrate abolished the beneficial effects of aPC in experimental diabetic nephropathy. Inhibition of p66
Shc
expression and mitochondrial translocation by aPC normalized mitochondrial ROS production and the mitochondrial membrane potential in glucose-treated podocytes. Genetic ablation of p66
Shc
compensated for the loss of protein C activation in vivo, normalizing markers of diabetic nephropathy and oxidative stress. These studies identify a unique mechanism underlying the cytoprotective effect of aPC. Activated PC epigenetically controls expression of the redox-regulating protein p66
Shc
, thus linking the extracellular protease aPC to mitochondrial function in diabetic nephropathy.
Interleukin-7 receptor controls development and maturation of late stages of thymocyte subpopulations
Authors: Tani-ichi Shizue , Shimba Akihiro , Wagatsuma Keisuke , Miyachi Hitoshi , Kitano Satsuki , Imai Kumiko , Hara Takahiro , Ikuta Koichi
Published: December 2012

Interleukin (IL)-7 is a cytokine essential for T lymphocyte development and homeostasis. However, little is known about the roles of IL-7 receptor α-chain (IL-7Rα) in late stages of T-cell development. To address this question, we established IL-7Rα–floxed mice and crossed them with CD4-Cre transgenic mice. Resultant IL-7R conditional knockout (IL-7RcKO) mice exhibited marked reduction in CD8 single positive (SP) T cells, regulatory T cells (Tregs), and natural killer T (NKT) cells in thymus. The proportion and proliferation of both mature CD4SP and CD8SP thymocytes were decreased without affecting Runx expression. In addition, expression of the glucocorticoid-induced TNF receptor was reduced in CD4SP and CD8SP thymocytes, and expression of CD5 was decreased in CD8SP thymocytes. IL-7RcKO mice also showed impaired Treg and NKT cell proliferation and inhibition of NKT cell maturation. Bcl-2 expression was reduced in CD4SP and CD8SP thymocytes but not in Tregs and NKT cells, and introduction of a Bcl-2 transgene rescued frequency and CD5 expression of CD8SP thymocytes. Furthermore, IL-7RcKO mice exhibited greatly increased numbers of B cells and, to a lesser extent, γδ T and dendritic cells in thymus. Overall, this study demonstrates that IL-7Rα differentially controls development and maturation of thymocyte subpopulations in late developmental stages and suggests that IL-7R expression on αβ T cells suppresses development of other cell lineages in thymus.
pHLIP peptide targets nanogold particles to tumors
Authors: Yao Lan , Daniels Jennifer , Moshnikova Anna , Kuznetsov Sergey , Ahmed Aftab , Engelman Donald M. , Reshetnyak Yana K. , Andreev Oleg A.
Published: December 2012

Progress in nanomedicine depends on the development of nanomaterials and targeted delivery methods. In this work, we describe a method for the preferential targeting of gold nanoparticles to a tumor in a mouse model. The method is based on the use of the pH Low Insertion Peptide (pHLIP), which targets various imaging agents to acidic tumors. We compare tumor targeting by nonfunctionalized nanogold particles with nanogold–pHLIP conjugates, where nanogold is covalently attached to the N terminus of pHLIP. Our most important finding is that both intratumoral and i.v. administration demonstrated a significant enhancement of tumor uptake of gold nanoparticles conjugated with pHLIP. Statistically significant reduction of gold accumulation was observed in acidic tumors and kidney when pH-insensitive K-pHLIP was used as a vehicle, suggesting an important role of pH in the pHLIP-mediated targeting of gold nanoparticles. The pHLIP technology can substantially improve the delivery of gold nanoparticles to tumors by providing specificity of targeting, enhancing local concentration in tumors, and distributing nanoparticles throughout the entire tumor mass where they remain for an extended period (several days), which is beneficial for radiation oncology and imaging.