bluuebunny/crossref_metadata_2025 huggingface dataset:
Aesthetic Judgement of Orientation in Modern Art
Authors: Mather George
Published: January 2012

When creating an artwork, the artist makes a decision regarding the orientation at which the work is to be hung based on their aesthetic judgement and the message conveyed by the piece. Is the impact or aesthetic appeal of a work diminished when it is hung at an incorrect orientation? To investigate this question, Experiment 1 asked whether naïve observers can appreciate the correct orientation (as defined by the artist) of 40 modern artworks, some of which are entirely abstract. Eighteen participants were shown 40 paintings in a series of trials. Each trial presented all four cardinal orientations on a computer screen, and the participant was asked to select the orientation that was most attractive or meaningful. Results showed that the correct orientation was selected in 48% of trials on average, significantly above the 25% chance level, but well below perfect performance. A second experiment investigated the extent to which the 40 paintings contained recognisable content, which may have mediated orientation judgements. Recognition rates varied from 0% for seven of the paintings to 100% for five paintings. Orientation judgements in Experiment 1 correlated significantly with “meaningful” content judgements in Experiment 2: 42% of the variance in orientation judgements in Experiment 1 was shared with recognition of meaningful content in Experiment 2. For the seven paintings in which no meaningful content at all was detected, 41% of the variance in orientation judgements was shared with variance in a physical measure of image content, Fourier amplitude spectrum slope. For some paintings, orientation judgements were quite consistent, despite a lack of meaningful content. The origin of these orientation judgements remains to be identified.
Homeobox gene
distal-less
is required for neuronal differentiation and neurite outgrowth in the
Drosophila
olfactory system
Authors: Plavicki Jessica , Mader Sara , Pueschel Eric , Peebles Patrick , Boekhoff-Falk Grace
Published: January 2012

Vertebrate
Dlx
genes have been implicated in the differentiation of multiple neuronal subtypes, including cortical GABAergic interneurons, and mutations in
Dlx
genes have been linked to clinical conditions such as epilepsy and autism. Here we show that the single
Drosophila Dlx
homolog,
distal-less
, is required both to specify chemosensory neurons and to regulate the morphologies of their axons and dendrites. We establish that
distal-less
is necessary for development of the mushroom body, a brain region that processes olfactory information. These are important examples of
distal-less
function in an invertebrate nervous system and demonstrate that the
Drosophila
larval olfactory system is a powerful model in which to understand
distal-less
functions during neurogenesis.
Spatial organization of the extracellular matrix regulates cell–cell junction positioning
Authors: Tseng Qingzong , Duchemin-Pelletier Eve , Deshiere Alexandre , Balland Martial , Guillou Hervé , Filhol Odile , Théry Manuel
Published: January 2012

The organization of cells into epithelium depends on cell interaction with both the extracellular matrix (ECM) and adjacent cells. The role of cell–cell adhesion in the regulation of epithelial topology is well-described. ECM is better known to promote cell migration and provide a structural scaffold for cell anchoring, but its contribution to multicellular morphogenesis is less well-understood. We developed a minimal model system to investigate how ECM affects the spatial organization of intercellular junctions. Fibronectin micropatterns were used to constrain the location of cell–ECM adhesion. We found that ECM affects the degree of stability of intercellular junction positioning and the magnitude of intra- and intercellular forces. Intercellular junctions were permanently displaced, and experienced large perpendicular tensional forces as long as they were positioned close to ECM. They remained stable solely in regions deprived of ECM, where they were submitted to lower tensional forces. The heterogeneity of the spatial organization of ECM induced anisotropic distribution of mechanical constraints in cells, which seemed to adapt their position to minimize both intra- and intercellular forces. These results uncover a morphogenetic role for ECM in the mechanical regulation of cells and intercellular junction positioning.
Phosphatidylinositol 4-phosphate 5-kinase alpha (PIPKα) regulates neuronal microtubule depolymerase kinesin, KIF2A and suppresses elongation of axon branches
Authors: Noda Yasuko , Niwa Shinsuke , Homma Noriko , Fukuda Hiroyuki , Imajo-Ohmi Shinobu , Hirokawa Nobutaka
Published: January 2012

Neuronal morphology is regulated by cytoskeletons. Kinesin superfamily protein 2A (KIF2A) depolymerizes microtubules (MTs) at growth cones and regulates axon pathfinding. The factors controlling KIF2A in neurite development remain totally elusive. Here, using immunoprecipitation with an antibody specific to KIF2A, we identified phosphatidylinositol 4-phosphate 5-kinase alpha (PIPKα) as a candidate membrane protein that regulates the activity of KIF2A. Yeast two-hybrid and biochemical assays demonstrated direct binding between KIF2A and PIPKα. Partial colocalization of the clusters of punctate signals for these two molecules was detected by confocal microscopy and photoactivated localization microscopy. Additionally, the MT-depolymerizing activity of KIF2A was enhanced in the presence of PIPKα in vitro and in vivo. PIPKα suppressed the elongation of axon branches in a KIF2A-dependent manner, suggesting a unique PIPK-mediated mechanism controlling MT dynamics in neuronal development.
Cytoskeletal protein filamin A is a nucleolar protein that suppresses ribosomal RNA gene transcription
Authors: Deng Wensheng , Lopez-Camacho Cesar , Tang Jen-Yang , Mendoza-Villanueva Daniel , Maya-Mendoza Apolinar , Jackson Dean A. , Shore Paul
Published: January 2012

Filamin A (FLNA) is an actin-binding protein with a well-established role in the cytoskeleton, where it determines cell shape and locomotion by cross-linking actin filaments. Mutations in FLNA are associated with a wide range of genetic disorders. Here we demonstrate a unique role for FLNA as a nucleolar protein that associates with the RNA polymerase I (Pol I) transcription machinery to suppress rRNA gene transcription. We show that depletion of FLNA by siRNAs increased rRNA expression, rDNA promoter activity and cell proliferation. Immunodepletion of FLNA from nuclear extracts resulted in a decrease in rDNA promoter-driven transcription in vitro. FLNA coimmunoprecipitated with the Pol I components actin, TIF-IA, and RPA40, and their occupancy of the rDNA promoter was increased in the absence of FLNA in vivo. The FLNA actin-binding domain is essential for the suppression of rRNA expression and for inhibiting recruitment of the Pol I machinery to the rDNA promoter. These findings reveal an additional role for FLNA as a regulator of rRNA gene expression and have important implications for our understanding of the role of FLNA in human disease.
Toll-like receptor 5 (TLR5), IL-1β secretion, and asparagine endopeptidase are critical factors for alveolar macrophage phagocytosis and bacterial killing
Authors: Descamps Delphyne , Le Gars Mathieu , Balloy Viviane , Barbier Diane , Maschalidi Sophia , Tohme Mira , Chignard Michel , Ramphal Reuben , Manoury Bénédicte , Sallenave Jean-Michel
Published: January 2012

A deficit in early clearance of
Pseudomonas aeruginosa
(
P. aeruginosa
) is crucial in nosocomial pneumonia and in chronic lung infections. Few studies have addressed the role of Toll-like receptors (TLRs), which are early pathogen associated molecular pattern receptors, in pathogen uptake and clearance by alveolar macrophages (AMs). Here, we report that TLR5 engagement is crucial for bacterial clearance by AMs in vitro and in vivo because unflagellated
P. aeruginosa
or different mutants defective in TLR5 activation were resistant to AM phagocytosis and killing. In addition, the clearance of PAK (a wild-type
P. aeruginosa
strain) by primary AMs was causally associated with increased IL-1β release, which was dramatically reduced with PAK mutants or in WT PAK-infected primary TLR5
−/−
AMs, demonstrating the dependence of IL-1β production on TLR5. We showed that this IL-1β production was important in endosomal pH acidification and in inducing the killing of bacteria by AMs through asparagine endopeptidase (AEP), a key endosomal cysteine protease. In agreement, AMs from IL-1R1
−/−
and AEP
−/−
mice were unable to kill
P. aeruginosa
. Altogether, these findings demonstrate that TLR5 engagement plays a major role in
P. aeruginosa
internalization and in triggering IL-1β formation.
Critical role of a transmembrane lysine in aminophospholipid transport by mammalian photoreceptor P
4
-ATPase ATP8A2
Authors: Coleman Jonathan A. , Vestergaard Anna L. , Molday Robert S. , Vilsen Bente , Peter Andersen Jens
Published: January 2012

ATP8A2 is a P
4
-ATPase (“flippase”) located in membranes of retinal photoreceptors, brain cells, and testis, where it mediates transport of aminophospholipids toward the cytoplasmic leaflet. It has long been an enigma whether the mechanism of P
4
-ATPases resembles that of the well-characterized cation-transporting P-type ATPases, and it is unknown whether the flippases interact directly with the lipid and with counterions. Our results demonstrate that ATP8A2 forms a phosphoenzyme intermediate at the conserved aspartate (Asp
416
) in the P-type ATPase signature sequence and exists in
E
1
P and
E
2
P forms similar to the archetypical P-type ATPases. Using the properties of the phosphoenzyme, the partial reaction steps of the transport cycle were examined, and the roles of conserved residues Asp
196
, Glu
198
, Lys
873
, and Asn
874
in the transport mechanism were elucidated. The former two residues in the A-domain T/D-G-E-S/T motif are involved in catalysis of
E
2
P dephosphorylation, the glutamate being essential. Transported aminophospholipids activate the dephosphorylation similar to K
+
activation of dephosphorylation in Na
+
,K
+
-ATPase. Lys
873
mutants (particularly K873A and K873E) display a markedly reduced sensitivity to aminophospholipids. Hence, Lys
873
, located in transmembrane segment M5 at a “hot spot” for cation binding in Ca
2+
-ATPase and Na
+
,K
+
-ATPase, appears to participate directly in aminophospholipid binding or to mediate a crucial interaction within the ATP8A2-CDC50 complex. By contrast, Lys
865
is unimportant for aminophospholipid sensitivity. Binding of Na
+
, H
+
, K
+
, Cl
-
, or Ca
2+
to the
E
1
form as a counterion is not required for activation of phosphorylation from ATP. Therefore, phospholipids could be the only substrate transported by ATP8A2.
Scaffold protein Disc large homolog 1 is required for T-cell receptor-induced activation of regulatory T-cell function
Authors: Zanin-Zhorov Alexandra , Lin Jiqiang , Scher Jose , Kumari Sudha , Blair David , Hippen Keli L. , Blazar Bruce R. , Abramson Steven B. , Lafaille Juan J. , Dustin Michael L.
Published: January 2012

Foxp3+CD4+CD25highregulatory T cell (Treg) suppression of inflammation depends on T-cell receptor-mediated Nuclear Factor of Activated T cells c1 (NFATc1) activation with reduced Akt activity. We investigated the role of the scaffold protein Disc large homolog 1 (Dlgh1) in linking the T-cell receptor to this unique signaling outcome. The Treg immunological synapse (IS) recruited fourfold more Dlgh1 than conventional CD4+T-cell IS. Tregs isolated from patients with active rheumatoid arthritis, or treated with tumor necrosis factor-α, displayed reduced function and diminished Dlgh1 recruitment to the IS. Furthermore, Dlgh1 silencing abrogated Treg function, impaired NFATc1 activation, reduced phosphatase and tensin homolog levels, and increased Akt activation. Dlgh1 operates independently of the negative feedback pathway mediated by the related adapter protein Carma1 and thus presents an array of unique targets to selectively manipulate Treg function.
Glycerolipid signals alter mTOR complex 2 (mTORC2) to diminish insulin signaling
Authors: Zhang Chongben , Wendel Angela A. , Keogh Matthew R. , Harris Thurl E. , Chen Jie , Coleman Rosalind A.
Published: January 2012

Increased flux through the glycerolipid synthesis pathway impairs the ability of insulin to inhibit hepatic gluconeogenesis, but the exact mechanism remains unknown. To determine the mechanism by which glycerolipids impair insulin signaling, we overexpressed glycerol-3-phosphate acyltransferase-1 (GPAT1) in primary mouse hepatocytes. GPAT1 overexpression impaired insulin-stimulated phosphorylation of Akt-S473 and -T308, diminished insulin-suppression of glucose production, significantly inhibited mTOR complex 2 (mTORC2) activity and decreased the association of mTOR and rictor. Conversely, in hepatocytes from

Gpat1
−/−

mice, mTOR-rictor association and mTORC2 activity were enhanced. However, this increase in mTORC2 activity in

Gpat1
−/−

hepatocytes was ablated when rictor was knocked down. To determine which lipid intermediate was responsible for inactivating mTORC2, we overexpressed GPAT1, AGPAT, or lipin to increase the cellular content of lysophosphatidic acid (LPA), phosphatidic acid (PA), or diacylglycerol (DAG), respectively. The inhibition of mTOR/rictor binding and mTORC2 activity coincided with the levels of PA and DAG species that contained 16:0, the preferred substrate of GPAT1. Furthermore, di-16:0-PA strongly inhibited mTORC2 activity and disassociated mTOR/rictor in vitro. Taken together, these data reveal a signaling pathway by which phosphatidic acid synthesized via the glycerol-3-phosphate pathway inhibits mTORC2 activity by decreasing the association of rictor and mTOR, thereby down-regulating insulin action. These data demonstrate a critical link between nutrient excess, TAG synthesis, and hepatic insulin resistance.
Diabetes promotes an inflammatory macrophage phenotype and atherosclerosis through acyl-CoA synthetase 1
Authors: Kanter Jenny E. , Kramer Farah , Barnhart Shelley , Averill Michelle M. , Vivekanandan-Giri Anuradha , Vickery Thad , Li Lei O. , Becker Lev , Yuan Wei , Chait Alan , Braun Kathleen R. , Potter-Perigo Susan , Sanda Srinath , Wight Thomas N. , Pennathur Subramaniam , Serhan Charles N. , Heinecke Jay W. , Coleman Rosalind A. , Bornfeldt Karin E.
Published: January 2012

The mechanisms that promote an inflammatory environment and accelerated atherosclerosis in diabetes are poorly understood. We show that macrophages isolated from two different mouse models of type 1 diabetes exhibit an inflammatory phenotype. This inflammatory phenotype associates with increased expression of long-chain acyl-CoA synthetase 1 (ACSL1), an enzyme that catalyzes the thioesterification of fatty acids. Monocytes from humans and mice with type 1 diabetes also exhibit increased ACSL1. Furthermore, myeloid-selective deletion of ACSL1 protects monocytes and macrophages from the inflammatory effects of diabetes. Strikingly, myeloid-selective deletion of ACSL1 also prevents accelerated atherosclerosis in diabetic mice without affecting lesions in nondiabetic mice. Our observations indicate that ACSL1 plays a critical role by promoting the inflammatory phenotype of macrophages associated with type 1 diabetes; they also raise the possibilities that diabetic atherosclerosis has an etiology that is, at least in part, distinct from the etiology of nondiabetic vascular disease and that this difference is because of increased monocyte and macrophage ACSL1 expression.
BARHL2 transcription factor regulates the ipsilateral/contralateral subtype divergence in postmitotic dI1 neurons of the developing spinal cord
Authors: Ding Qian , Joshi Pushkar S. , Xie Zheng-hua , Xiang Mengqing , Gan Lin
Published: January 2012

In the dorsal spinal cord, distinct interneuron classes relay specific somatosensory information, such as touch, heat, and pain, from the periphery to higher brain centers via ipsilateral and contralateral axonal pathways. The transcriptional mechanisms by which dorsal interneurons choose between ipsilateral and contralateral projection fates are unknown. Here, we show that a single transcription factor (TF), BARHL2, regulates this choice in proprioceptive dI1 interneurons by selectively suppressing cardinal dI1contra features in dI1ipsi neurons, despite expression by both subtypes. Strikingly, dI1ipsi neurons in
Barhl2
-null mice exhibit a dI1contra cell settling pattern in the medial deep dorsal horn, and, most importantly, they project axons contralaterally. These aberrations are preceded by ectopic dI1ipsi expression of the defining dI1contra TF, LHX2, and down-regulation of the dI1ipsi-enriched TF, BARHL1. Taken together, these results elucidate BARHL2 as a critical postmitotic regulator of dI1 subtype diversification, as well as its intermediate position in the dI1 genetic hierarchy.
Surface multiheme
c
-type cytochromes from
Thermincola potens
and implications for respiratory metal reduction by Gram-positive bacteria
Authors: Carlson Hans K. , Iavarone Anthony T. , Gorur Amita , Yeo Boon Siang , Tran Rosalie , Melnyk Ryan A. , Mathies Richard A. , Auer Manfred , Coates John D.
Published: January 2012

Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they may be the dominant species in some environments.
Thermincola potens s
train JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that
T. potens
coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or anthraquinone-2,6-disulfonate (AQDS), an analog of the redox active components of humic substances. The genome of
T. potens
was recently sequenced, and the abundance of multiheme
c
-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin-shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during
T. potens
growth on either HFO or AQDS, and that several MHCs are localized to the cell wall or cell surface. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants, suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results provide unique direct evidence for cell wall-associated cytochromes and support MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium.
Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)
Authors: Girard Martine , Larivière Roxanne , Parfitt David A. , Deane Emily C. , Gaudet Rebecca , Nossova Nadya , Blondeau Francois , Prenosil George , Vermeulen Esmeralda G. M. , Duchen Michael R. , Richter Andrea , Shoubridge Eric A. , Gehring Kalle , McKinney R. Anne , Brais Bernard , Chapple J. Paul , McPherson Peter S.
Published: January 2012

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset neurological disease resulting from mutations in the
SACS
gene encoding sacsin, a 4,579-aa protein of unknown function. Originally identified as a founder disease in Québec, ARSACS is now recognized worldwide. Prominent features include pyramidal spasticity and cerebellar ataxia, but the underlying pathology and pathophysiological mechanisms are unknown. We have generated an animal model for ARSACS, sacsin knockout mice, that display age-dependent neurodegeneration of cerebellar Purkinje cells. To explore the pathophysiological basis for this observation, we examined the cell biological properties of sacsin. We show that sacsin localizes to mitochondria in non-neuronal cells and primary neurons and that it interacts with dynamin-related protein 1, which participates in mitochondrial fission. Fibroblasts from ARSACS patients show a hyperfused mitochondrial network, consistent with defects in mitochondrial fission. Sacsin knockdown leads to an overly interconnected and functionally impaired mitochondrial network, and mitochondria accumulate in the soma and proximal dendrites of sacsin knockdown neurons. Disruption of mitochondrial transport into dendrites has been shown to lead to abnormal dendritic morphology, and we observe striking alterations in the organization of dendritic fields in the cerebellum of knockout mice that precedes Purkinje cell death. Our data identifies mitochondrial dysfunction/mislocalization as the likely cellular basis for ARSACS and indicates a role for sacsin in regulation of mitochondrial dynamics.
Retinotopic memory is more precise than spatiotopic memory
Authors: Golomb Julie D. , Kanwisher Nancy
Published: January 2012

Successful visually guided behavior requires information about spatiotopic (i.e., world-centered) locations, but how accurately is this information actually derived from initial retinotopic (i.e., eye-centered) visual input? We conducted a spatial working memory task in which subjects remembered a cued location in spatiotopic or retinotopic coordinates while making guided eye movements during the memory delay. Surprisingly, after a saccade, subjects were significantly more accurate and precise at reporting retinotopic locations than spatiotopic locations. This difference grew with each eye movement, such that spatiotopic memory continued to deteriorate, whereas retinotopic memory did not accumulate error. The loss in spatiotopic fidelity is therefore not a generic consequence of eye movements, but a direct result of converting visual information from native retinotopic coordinates. Thus, despite our conscious experience of an effortlessly stable spatiotopic world and our lifetime of practice with spatiotopic tasks, memory is actually more reliable in raw retinotopic coordinates than in ecologically relevant spatiotopic coordinates.
Using targeted transgenic reporter mice to study promoter-specific p53 transcriptional activity
Authors: Goh Amanda M. , Lim Chin Yan , Chiam Poh Cheang , Li Ling , Mann Michael B. , Mann Karen M. , Menendez Sergio , Lane David P.
Published: January 2012

The p53 transcription factor modulates gene expression programs that induce cell cycle arrest, senescence, or apoptosis, thereby preventing tumorigenesis. However, the mechanisms by which these fates are selected are unclear. Our objective is to understand p53 target gene selection and, thus, enable its optimal manipulation for cancer therapy. We have generated targeted transgenic reporter mice in which EGFP expression is driven by p53 transcriptional activity at a response element from either the
p21
or
Puma
promoter, which induces cell cycle arrest/senescence and apoptosis, respectively. We demonstrate that we could monitor p53 activity in vitro and in vivo and detect variations in p53 activity depending on the response element, tissue type, and stimulus, thereby validating our reporter system and illustrating its utility for preclinical drug studies. Our results also show that the sequence of the p53 response element itself is sufficient to strongly influence p53 target gene selection. Finally, we use our reporter system to provide evidence for p53 transcriptional activity during early embryogenesis, showing that p53 is active as early as embryonic day 3.5 and that p53 activity becomes restricted to embryonic tissue by embryonic day 6.5. The data from this study demonstrate that these reporter mice could serve as powerful tools to answer questions related to basic biology of the p53 pathway, as well as cancer therapy and drug discovery.
Infants deploy selective attention to the mouth of a talking face when learning speech
Authors: Lewkowicz David J. , Hansen-Tift Amy M.
Published: January 2012

The mechanisms underlying the acquisition of speech-production ability in human infancy are not well understood. We tracked 4–12-mo-old English-learning infants’ and adults’ eye gaze while they watched and listened to a female reciting a monologue either in their native (English) or nonnative (Spanish) language. We found that infants shifted their attention from the eyes to the mouth between 4 and 8 mo of age regardless of language and then began a shift back to the eyes at 12 mo in response to native but not nonnative speech. We posit that the first shift enables infants to gain access to redundant audiovisual speech cues that enable them to learn their native speech forms and that the second shift reflects growing native-language expertise that frees them to shift attention to the eyes to gain access to social cues. On this account, 12-mo-old infants do not shift attention to the eyes when exposed to nonnative speech because increasing native-language expertise and perceptual narrowing make it more difficult to process nonnative speech and require them to continue to access redundant audiovisual cues. Overall, the current findings demonstrate that the development of speech production capacity relies on changes in selective audiovisual attention and that this depends critically on early experience.
Qa-SNAREs localized to the
trans
-Golgi network regulate multiple transport pathways and extracellular disease resistance in plants
Authors: Uemura Tomohiro , Kim Hyeran , Saito Chieko , Ebine Kazuo , Ueda Takashi , Schulze-Lefert Paul , Nakano Akihiko
Published: January 2012

In all eukaryotic cells, a membrane-trafficking system connects the post-Golgi organelles, such as the
trans
-Golgi network (TGN), endosomes, vacuoles, and the plasma membrane. This complex network plays critical roles in several higher-order functions in multicellular organisms. The TGN, one of the important organelles for protein transport in the post-Golgi network, functions as a sorting station, where cargo proteins are directed to the appropriate post-Golgi compartments. Unlike its roles in animal and yeast cells, the TGN has also been reported to function like early endosomal compartments in plant cells. However, the physiological roles of the TGN functions in plants are not understood. Here, we report a study of the SYP4 group (SYP41, SYP42, and SYP43), which represents the plant orthologs of the Tlg2/syntaxin16 Qa-SNARE (soluble
N
-ethylmaleimide sensitive factor attachment protein receptor) that localizes on the TGN in yeast and animal cells. The SYP4 group regulates the secretory and vacuolar transport pathways in the post-Golgi network and maintains the morphology of the Golgi apparatus and TGN. Consistent with a secretory role, SYP4 proteins are required for extracellular resistance responses to a fungal pathogen. We also reveal a plant cell-specific higher-order role of the SYP4 group in the protection of chloroplasts from salicylic acid-dependent biotic stress.
Automated selection of stabilizing mutations in designed and natural proteins
Authors: Borgo Benjamin , Havranek James J.
Published: January 2012

The ability to engineer novel protein folds, conformations, and enzymatic activities offers enormous potential for the development of new protein therapeutics and biocatalysts. However, many de novo and redesigned proteins exhibit poor hydrophobic packing in their predicted structures, leading to instability or insolubility. The general utility of rational, structure-based design would greatly benefit from an improved ability to generate well-packed conformations. Here we present an automated protocol within the RosettaDesign framework that can identify and improve poorly packed protein cores by selecting a series of stabilizing point mutations. We apply our method to previously characterized designed proteins that exhibited a decrease in stability after a full computational redesign. We further demonstrate the ability of our method to improve the thermostability of a well-behaved native protein. In each instance, biophysical characterization reveals that we were able to stabilize the original proteins against chemical and thermal denaturation. We believe our method will be a valuable tool for both improving upon designed proteins and conferring increased stability upon native proteins.
Transcriptional interpretation of the EGF receptor signaling gradient
Authors: Fuchs Alisa , Cheung Lily S. , Charbonnier Enrica , Shvartsman Stanislav Y. , Pyrowolakis George
Published: January 2012

Epidermal growth factor receptor (EGFR) controls a wide range of developmental events, from body axes specification in insects to cardiac development in humans. During
Drosophila
oogenesis, a gradient of EGFR activation patterns the follicular epithelium. Multiple transcriptional targets of EGFR in this tissue have been identified, but their regulatory elements are essentially unknown. We report the regulatory elements of
broad
(
br
) and
pipe
(
pip
), two important targets of EGFR signaling in
Drosophila
oogenesis.
br
is expressed in a complex pattern that prefigures the formation of respiratory eggshell appendages. We found that this pattern is generated by dynamic activities of two regulatory elements, which display different responses to Pointed, Capicua, and Mirror, transcription factors involved in the EGFR-mediated gene expression. One of these elements is active in a pattern similar to
pip
, a gene repressed by EGFR and essential for establishing the dorsoventral polarity of the embryo. We demonstrate that this similarity of expression depends on a common sequence motif that binds Mirror in vitro and is essential for transcriptional repression in vivo.
IL-33 is processed into mature bioactive forms by neutrophil elastase and cathepsin G
Authors: Lefrançais Emma , Roga Stephane , Gautier Violette , Gonzalez-de-Peredo Anne , Monsarrat Bernard , Girard Jean-Philippe , Cayrol Corinne
Published: January 2012

Interleukin-33 (IL-33) (NF-HEV) is a chromatin-associated nuclear cytokine from the IL-1 family, which has been linked to important diseases, including asthma, rheumatoid arthritis, ulcerative colitis, and cardiovascular diseases. IL-33 signals through the ST2 receptor and drives cytokine production in type 2 innate lymphoid cells (ILCs) (natural helper cells, nuocytes), T-helper (Th)2 lymphocytes, mast cells, basophils, eosinophils, invariant natural killer T (iNKT), and natural killer (NK) cells. We and others recently reported that, unlike IL-1β and IL-18, full-length IL-33 is biologically active independently of caspase-1 cleavage and that processing by caspases results in IL-33 inactivation. We suggested that IL-33, which is released upon cellular damage, may function as an endogenous danger signal or alarmin, similar to IL-1α or high-mobility group box 1 protein (HMGB1). Here, we investigated the possibility that IL-33 activity may be regulated by proteases released during inflammation. Using a combination of in vitro and in vivo approaches, we demonstrate that neutrophil serine proteases cathepsin G and elastase can cleave full-length human IL-331–270and generate mature forms IL-3395–270, IL-3399–270, and IL-33109–270. These forms are produced by activated human neutrophils ex vivo, are biologically active in vivo, and have a ∼10-fold higher activity than full-length IL-33 in cellular assays. Murine IL-33 is also cleaved by neutrophil cathepsin G and elastase, and both full-length and cleaved endogenous IL-33 could be detected in the bronchoalveolar lavage fluid in an in vivo model of acute lung injury associated with neutrophil infiltration. We propose that the inflammatory microenvironment may exacerbate disease-associated functions of IL-33 through the generation of highly active mature forms.
Carnivorous dinocephalian from the Middle Permian of Brazil and tetrapod dispersal in Pangaea
Authors: Cisneros Juan Carlos , Abdala Fernando , Atayman-Güven Saniye , Rubidge Bruce S. , Şengör A. M. Celâl , Schultz Cesar L.
Published: January 2012

The medial Permian (∼270–260 Ma: Guadalupian) was a time of important tetrapod faunal changes, in particular reflecting a turnover from pelycosaurian- to therapsid-grade synapsids. Until now, most knowledge on tetrapod distribution during the medial Permian has come from fossils found in the South African Karoo and the Russian Platform, whereas other areas of Pangaea are still poorly known. We present evidence for the presence of a terrestrial carnivorous vertebrate from the Middle Permian of South America based on a complete skull.Pampaphoneus biccaigen. et sp. nov. was a dinocephalian “mammal-like reptile” member of the Anteosauridae, an early therapsid predator clade known only from the Middle Permian of Russia, Kazakhstan, China, and South Africa. The genus is characterized, among other features, by postorbital bosses, short, bulbous postcanines, and strongly recurved canines. Phylogenetic analysis indicates that the Brazilian dinocephalian occupies a middle position within the Anteosauridae, reinforcing the model of a global distribution for therapsids as early as the Guadalupian. The close phylogenetic relationship of the Brazilian species to dinocephalians from South Africa and the Russian Platform suggests a closer faunistic relationship between South America and eastern Europe than previously thought, lending support to a Pangaea B-type continental reconstruction.
Dickkopf-3, an immune modulator in peripheral CD8 T-cell tolerance
Authors: Papatriantafyllou Maria , Moldenhauer Gerhard , Ludwig Julia , Tafuri Anna , Garbi Natalio , Hollmann Gorana , Küblbeck Günter , Klevenz Alexandra , Schmitt Sabine , Pougialis Georg , Niehrs Christof , Gröne Hermann-Josef , Hämmerling Gunter J. , Arnold Bernd , Oelert Thilo
Published: January 2012

In healthy individuals, T cells react against incoming pathogens, but remain tolerant to self-antigens, thereby preventing autoimmune reactions. CD4 regulatory T cells are major contributors in induction and maintenance of peripheral tolerance, but a regulatory role has been also reported for several subsets of CD8 T cells. To determine the molecular basis of peripheral CD8 T-cell tolerance, we exploited a double transgenic mouse model in which CD8 T cells are neonatally tolerized following interaction with a parenchymal self-antigen. These tolerant CD8 T cells have regulatory capacity and can suppress T cells in an antigen-specific manner during adulthood. Dickkopf-3 (DKK3) was found to be expressed in the tolerant CD8 T cells and to be essential for the observed CD8 T-cell tolerance. In vitro, genetic deletion of DKK3 or blocking with antibodies restored CD8 T-cell proliferation and IL-2 production in response to the tolerizing self-antigen. Moreover, exogenous DKK3 reduced CD8 T-cell reactivity. In vivo, abrogation of DKK3 function reversed tolerance, leading to eradication of tumors expressing the target antigen and to rejection of autologous skin grafts. Thus, our findings define DKK3 as a immune modulator with a crucial role for CD8 T-cell tolerance.
Crystal structure of a Tankyrase-Axin complex and its implications for Axin turnover and Tankyrase substrate recruitment
Authors: Morrone Seamus , Cheng Zhihong , Moon Randall T. , Cong Feng , Xu Wenqing
Published: January 2012

Axin is a tumor suppressor and a key negative regulator of the Wnt/β-catenin signaling pathway. Axin turnover is controlled by its poly-ADP-ribosylation catalyzed by tankyrase (TNKS), which requires the direct interaction of Axin with TNKS. This interaction is thus an attractive drug target for treating cancers, brain injuries, and other diseases where β-catenin is involved. Here we report the crystal structure of a mouse TNKS1 fragment containing ankyrin-repeat clusters 2 and 3 (ARC2-3) in a complex with the TNKS-binding domain of mouse Axin1. Surprisingly, we found that Axin contains two discrete TNKS-binding segments, both of which bind simultaneously to the two ARC2 domains in the ARC2-3 homodimer. Our crystal structure shows that in each TNKS-binding segment of Axin there is a conserved glycine residue that lies in the bottom of a narrow “gate” formed by two parallel tyrosine side chains on the TNKS surface. This glycine-selection gate is crucial for TNKS-Axin interactions, as mutation of the TNKS gate-forming residues, or mutation of either glycine residue in the two Axin segments, completely abolishes the binding of the corresponding Axin segment to TNKS. The bivalent binding of Axin to TNKS is required for Axin turnover, since mutations in either gate-binding glycine residue in Axin lead to Axin stabilization in the cell. In addition, our analyses also reveal the structural basis for TNKS substrate recruitment, and shed light on the overall structure of TNKS that should help in developing specific inhibitors of Wnt/β-catenin signaling.
Ca
2+
signaling amplification by oligomerization of L-type Ca
v
1.2 channels
Authors: Dixon Rose E. , Yuan Can , Cheng Edward P. , Navedo Manuel F. , Santana Luis F.
Published: January 2012

Ca
2+
influx via L-type Ca
v
1.2 channels is essential for multiple physiological processes, including gene expression, excitability, and contraction. Amplification of the Ca
2+
signals produced by the opening of these channels is a hallmark of many intracellular signaling cascades, including excitation-contraction coupling in heart. Using optogenetic approaches, we discovered that Ca
v
1.2 channels form clusters of varied sizes in ventricular myocytes. Physical interaction between these channels via their C-tails renders them capable of coordinating their gating, thereby amplifying Ca
2+
influx and excitation-contraction coupling. Light-induced fusion of WT Ca
v
1.2 channels with Ca
v
1.2 channels carrying a gain-of-function mutation that causes arrhythmias and autism in humans with Timothy syndrome (Ca
v
1.2-TS) increased Ca
2+
currents, diastolic and systolic Ca
2+
levels, contractility and the frequency of arrhythmogenic Ca
2+
fluctuations in ventricular myocytes. Our data indicate that these changes in Ca
2+
signaling resulted from Ca
v
1.2-TS increasing the activity of adjoining WT Ca
v
1.2 channels. Collectively, these data support the concept that oligomerization of Ca
v
1.2 channels via their C termini can result in the amplification of Ca
2+
influx into excitable cells.
Plasma membrane recruitment and activation of the AGC kinase Ypk1 is mediated by target of rapamycin complex 2 (TORC2) and its effector proteins Slm1 and Slm2
Authors: Niles Brad J. , Mogri Huzefa , Hill Andrew , Vlahakis Ariadne , Powers Ted
Published: January 2012

The yeast AGC kinase orthologs Ypk1 and Ypk2 control several important cellular processes, including actin polarization, endocytosis, and sphingolipid metabolism. Activation of Ypk1/2 requires phosphorylation by kinases localized at the plasma membrane (PM), including the 3-phosphoinositide-dependent kinase 1 orthologs Pkh1/Pkh2 and the target of rapamycin complex 2 (TORC2). Unlike their mammalian counterparts SGK and Akt, Ypk1 and Ypk2 lack an identifiable lipid-targeting motif; therefore, how these proteins are recruited to the PM has remained elusive. To explore Ypk1/2 function, we constructed ATP analog-sensitive alleles of both kinases and monitored global changes in gene expression following their inhibition, where we observed increased expression of stress-responsive target genes controlled by Ca
2+
-dependent phosphatase calcineurin. TORC2 has been shown previously to negatively regulate calcineurin in part by phosphorylating two related proteins, Slm1 and Slm2, which associate with the PM via plextrin homology domains. We therefore investigated the relationship between Slm1 and Ypk1 and discovered that these proteins interact physically and that Slm1 recruits Ypk1 to the PM for phosphorylation by TORC2. We observed further that these steps facilitate subsequent phosphorylation of Ypk1 by Pkh1/2. Remarkably, a requirement for Slm1, can be bypassed by fusing the plextrin homology domain of Slm1 alone onto Ypk1, demonstrating that the essential function of Slm1 is largely attributable to its role in Ypk1 activation. These findings both extend the scope of cellular processes regulated by Ypk1/2 to include negative regulation of calcineurin and broaden the repertoire of mechanisms for membrane recruitment and activation of a protein kinase.
Nonlinear Laplacian spectral analysis for time series with intermittency and low-frequency variability
Authors: Giannakis Dimitrios , Majda Andrew J.
Published: January 2012

Many processes in science and engineering develop multiscale temporal and spatial patterns, with complex underlying dynamics and time-dependent external forcings. Because of the importance in understanding and predicting these phenomena, extracting the salient modes of variability empirically from incomplete observations is a problem of wide contemporary interest. Here, we present a technique for analyzing high-dimensional, complex time series that exploits the geometrical relationships between the observed data points to recover features characteristic of strongly nonlinear dynamics (such as intermittency and rare events), which are not accessible to classical singular spectrum analysis. The method employs Laplacian eigenmaps, evaluated after suitable time-lagged embedding, to produce a reduced representation of the observed samples, where standard tools of matrix algebra can be used to perform truncated singular-value decomposition despite the nonlinear geometrical structure of the dataset. We illustrate the utility of the technique in capturing intermittent modes associated with the Kuroshio current in the North Pacific sector of a general circulation model and dimensional reduction of a low-order atmospheric model featuring chaotic intermittent regime transitions, where classical singular spectrum analysis is already known to fail dramatically.
Efficient coupling of ATP hydrolysis to translocation by RecQ helicase
Authors: Rad Behzad , Kowalczykowski Stephen C.
Published: January 2012

Helicases are ubiquitous enzymes that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) during essential processes such as replication, transcription, or repair. TheEscherichia coliRecQ protein is a 3′ to 5′ helicase, which functions in the processes of homologous recombination and replication fork restart. Here, we analyzed the relationship between ATP hydrolysis by RecQ and its translocation on ssDNA. We monitored a single round of RecQ translocation on ssDNA by measuring the rates of inorganic phosphate release during translocation, and the dissociation of RecQ from ssDNA. We find that RecQ translocates with a rate of 16( ± 4) nucleotides/s and moves on average only 36( ± 2) nucleotides before dissociating. Fitting to an n-step kinetic model suggests that the helicase displays a nonuniform translocation mechanism in which it moves approximately five nucleotides rapidly before undergoing a rate-limiting kinetic slow step. Unexpectedly, RecQ requires a length of 34( ± 3) nucleotides to bind and translocate on ssDNA. This large site size suggests that several monomers are required to bind DNA prior to translocation. Energetically, the RecQ helicase couples the hydrolysis of one ATP molecule to the translocation of more than one nucleotide (1.6 ± 0.3). Thus, our data show that RecQ translocates on ssDNA by efficiently coupling the hydrolysis of one ATP molecule into structural alterations that result in movement of approximately two nucleotides, presumably by an inchworm mechanism. These attributes are consistent with the function of RecQ in recombination and replication.
In-feed antibiotic effects on the swine intestinal microbiome
Authors: Looft Torey , Johnson Timothy A. , Allen Heather K. , Bayles Darrell O. , Alt David P. , Stedtfeld Robert D. , Sul Woo Jun , Stedtfeld Tiffany M. , Chai Benli , Cole James R. , Hashsham Syed A. , Tiedje James M. , Stanton Thad B.
Published: January 2012

Antibiotics have been administered to agricultural animals for disease treatment, disease prevention, and growth promotion for over 50 y. The impact of such antibiotic use on the treatment of human diseases is hotly debated. We raised pigs in a highly controlled environment, with one portion of the littermates receiving a diet containing performance-enhancing antibiotics [chlortetracycline, sulfamethazine, and penicillin (known as ASP250)] and the other portion receiving the same diet but without the antibiotics. We used phylogenetic, metagenomic, and quantitative PCR-based approaches to address the impact of antibiotics on the swine gut microbiota. Bacterial phylotypes shifted after 14 d of antibiotic treatment, with the medicated pigs showing an increase in
Proteobacteria
(1–11%) compared with nonmedicated pigs at the same time point. This shift was driven by an increase in
Escherichia coli
populations. Analysis of the metagenomes showed that microbial functional genes relating to energy production and conversion were increased in the antibiotic-fed pigs. The results also indicate that antibiotic resistance genes increased in abundance and diversity in the medicated swine microbiome despite a high background of resistance genes in nonmedicated swine. Some enriched genes, such as aminoglycoside O-phosphotransferases, confer resistance to antibiotics that were not administered in this study, demonstrating the potential for indirect selection of resistance to classes of antibiotics not fed. The collateral effects of feeding subtherapeutic doses of antibiotics to agricultural animals are apparent and must be considered in cost-benefit analyses.
Soluble factors produced by activated CD4+T cells modulate EBV latency
Authors: Nagy Noémi , Ádori Mónika , Rasul Abu , Heuts Frank , Salamon Daniel , Ujvári Dorina , Madapura Harsha S. , Leveau Benjamin , Klein George , Klein Eva
Published: January 2012

Following infection with Epstein–Barr virus (EBV), the virus is carried for life in the memory B-cell compartment in a silent state (latency I/0). These cells do not resemble the proliferating lymphoblastoid cells (LCLs) (latency III) that are generated after infection. It is of fundamental significance to identify how the different EBV expression patterns are established in the latently infected cell. In view of the prompt activatability of CD4+T cells in primary EBV infection, and their role in B-cell differentiation, we studied the involvement of CD4+T cells in the regulation of EBV latency. Lymphoblastoid cell lines (LCLs) were cocultured with autologous or allogeneic CD4+T cells. Activated T cells influenced the expression of two key viral proteins that determine the fate of the infected B cell. EBNA2 was down-regulated, whereas LMP1 was unregulated and the cells proliferated less. This was paralleled by the down-regulation of the latency III promoter (Cp). Experiments performed in the transwell system showed that this change does not require cell contact, but it is mediated by soluble factors. Neutralizing experiments proved that the up-regulation of LMP1 is, to some extent, mediated by IL21, but this cytokine was not responsible for EBNA2 down-regulation. This effect was partly mediated by soluble CD40L. We detected similar regulatory functions of T cells in in vitro-infected lymphocyte populations. In conclusion, our results revealed an additional mechanism by which CD4+T cells can control the EBV-induced B-cell proliferation.