Structural remodeling by large-scale pruning occurs where relatively long neuronal branches are cut away from their parent neuron and removed by local degeneration. Until now, the molecular mechanisms executing such branch severing events have remained poorly understood. Here, we reveal a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery during neuronal remodeling. Our data show that a specific ESCRT pruning module, including members of the ESCRT-I and ESCRT-III complexes, but not ESCRT-0 or ESCRT-II, are required for the neurite scission event during pruning. Furthermore we show that this ESCRT module requires a direct,  Neuronal remodeling is a fundamental mechanism that is essential for building a functional nervous system2Drosophila have contributed greatly to our understanding of the cellular and molecular mechanisms of large-scale pruning. These studies have revealed that TGFβ and Ecdysone signaling pathways initiate pruning and that the Ubiquitin-Proteasome System, caspases, hdc and calcium signaling events are required, along with Kat60L, IKK and Mical for pruning to proceed to completionIn recent years, studies in To address this question we looked at how similar membrane bending and scission events occur within cells. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery has been shown to execute membrane-cutting events during intraluminal vesicular formation, viral budding, membrane repair and cytokinetic abscission5678Drosophila.Here we explore and identify a requirement for the ESCRTs during the developmental pruning of axons and dendrites in Drosophila  and ESCRT related proteins in the class IV dendritic arborizing (da) sensory neuron, ddaC 13. Theseosophila .The downregulation of five ESCRT related genes, TSG101, Shrub, Vps4, mop and UBPY, by expressing 9 different RNAi lines, lead to severe disruptions in pruning with dendrites still being attached to the cell body by 18 h APF, resulting from a lack of branch severing . These eHrs led to minor delays in pruning  displayed a pruning defect   . All theAlongside this, MARCM clones against Vps28  confirmeTogether multiple RNAi knockdown and MARCM clone data with null and strong alleles show that ESCRTs are required, in a cell autonomous manner, for pruning. The requirement of a specific subset of ESCRT including components of ESCRT-I, -III and Vps4 suggest the existence of an ESCRT pruning module.LL05494Alix and also found no disruption in pruning (n = 8) (Myopic (mop), the homolog of the human gene HD-PTP is another Bro-domain protein found in the fly genome. Mop is known to be involved in ESCRT-mediated receptor sorting20mop with two independent RNAi lines, we observed a robust block of dendrite severing in ddaC  . These d in ddaC  and 2k rshrub and mop are required for pruning, we next investigated how these two proteins interact with each other and whether a direct interaction between them is necessary for their function. Previous work identified that HD-PTP (the human homologue of mop) physically interacts with CHMP4B (the homologue of shrub) through the Bro1 domain of HD-PTP and that this interaction can be disrupted by introducing the point mutations L202D and I206DDrosophila Shrub and Mop using a yeast-two-hybrid assay  at similar levels , and Sox1424To determine whether the initiation of pruning takes place on schedule we also looked at the expression of the Taken together these data show that the gating of pruning proceeds normally and that the severing defects are the result of a requirement for the ESCRTs function during pruning rather than from some earlier developmental sequela.Mop and Shrub, like other components of the ESCRT machinery, are required for ubiquitinated endosomal cargos to be sorted into the Multivesicular Bodies (MVB) for degradation. One possibility is that the dendrite severing phenotypes we observe are the result of a disruption in this process. To address this question we examined levels of ubiquitinated proteins in ddaC neurons under different conditions.Hrs we observed that ubiquitinated proteins accumulate in the cell body but show no severing defects at 18 h APF and only a very mild delay in the clearance of severed branches  were amplified by PCR from cDNA ; HrsD28; HrsD28,Stam2L-2896 BL41806, SS6Vps22, A3Vps25 and B9Vps28 from Bloomington Stock Center. The RNAi lines UAS-Hrs-RNAi-BL33900, UAS-TSG101-RNAi-BL35710, UAS-Vps25-RNAi-BL26286, UAS-Vps36-RNAi-BL38286, UAS-Vps2-RNAi-BL38995, UAS-CHMP2B-RNAi-BL38375, UAS-Vps20-RNAi-BL40894, UAS- Vps24-RNAi-BL39281, UAS-mop-RNAi-BL32916 and UAS-ubpy-RNAi-BL38982 were obtained from Bloomington Stock Center. UAS-Dicer2 on the X chromosome was used with ppk-GAL4 for our screen. The RNAi lines UAS-Vps22-RNAi-21658GD; UAS-Alix-RNAi-32047GD and UAS-Vps60-RNAi-101422KK were obtained from the Vienna Drosophila RNAi Centre. The RNAi line UAS-Vps4-RNAi-6841R1 was obtained from the National Institute of Genetics (Japan). Appropriate genotypes of both sexes were used in this study.The following GAL4 driver strains were used: ppk1.9-GAL4 driver, expressed in ddaC neurons and occasionally in isolated epidermal cells; C161-GAL4, expressed in five dorsal da neurons; 201Y-GAL4, expressed in γ-MB neurons; and elavC155-GAL4, UAS-RedStinger, SOP-FLP; tub-GAL80, FRT40A/CyO crossed with ♂ w; D28Hrs, FRT40A/CyO; ppk-eGFP♀ w, elavC155-GAL4, UAS-RedStinger, SOP-FLP; tub-GAL80, FRT40A/CyO crossed with ♂ w; D28Hrs,Stam2L-2896 FRT40A/CyO; ppk-eGFP♀ w, elavC155-GAL4, UAS-RedStinger, SOP-FLP; FRT42B, tub- GAL80/CyO crossed with ♂ w; FRT42B shrub4/CyO; ppk-eGFP♀ w, elavC155-GAL4, UAS-RedStinger, SOP-FLP; FRT42D, tub-GAL80/CyO crossed with ♂ w; FRT42D, A3Vps25/CyO; ppk-eGFP♀ w, elavC155-GAL4, UAS-RedStinger, SOP-FLP; FRT42D, tub-GAL80/CyO crossed with ♂ w; FRT42D, B9Vps28/CyO; ppk-eGFP♀ w, elavC155-GAL4, UAS-RedStinger, SOP-FLP; ppk-eGFP; FRT82B, tubGAL80/TM6 b Tb Hu crossed with ♂ w; ppk-eGFP; FRT82B, LL05494Alix/Tm6 b Tb Hu♀ w, elavC155-GAL4, UAS-RedStinger, SOP-FLP; ppk-eGFP; FRT82B, tubGAL80/TM6 b Tb Hu crossed with ♂ w; ppk-eGFP; FRT82B, SS6Vps22/Tm6b Tb Hu♀ w, elavFor our modified mosaic analysis with a repressible cell marker (MARCM)  of da sensory neurons, clones were induced in the embryo by SOP-FLP. The following crosses were generated:C155 expresses strongly in neurons and also at low level in other tissues (including the epidermis) allowing us to see the number and location of ‘collateral clones’ generated. The neighbouring GFP-labelled non-MARCM ddaC neurons provided very important controls for non-cell autonomous phenotypes.All ddaC neurons were identified at 18 h after puparium formation (APF) by the expression of ppk-eGFP, MARCM clones expressed the RedStinger nuclear reporter protein. The GAL4 enhancer trap elavIndividual animals were collected at pupariation and maintained at 25°C in a Petri dish with moist filter paper. Staging was denoted as hours after puparium formation or APF.Immunocytochemistry was performed as described by Truman et al. (2004). Primary antibodies used were rabbit anti-GFP diluted 1/500 (Invitrogen), mouse anti-mop diluted 1/100 (Abcam) kindly provided by Dr Melissa Gilbert-Ross , rabbit anti-shrub diluted 1/1000 kindly provided by Dr Fen Biao Gao and mouse anti-Ubiquitin FK2 diluted 1/1000 (Biomol) and mouse anti-Flag M2 diluted 1:500 (Sigma). Secondary antibodies used were FITC donkey anti-rabbit or anti-mouse IgG diluted 1:500 from Jackson ImmunoResearch Laboratories and Cy3-conjugated donkey anti-rat or anti-mouse IgG diluted 1:500 from Stratech Scientific.http://rsb.info.nih.gov/ij/). Maximum projections generated. Images were adjusted only for brightness and contrast using Adobe Photoshop (Adobe Systems).The body walls of third-instar larvae, white pre-pupae, and pupae (until 12 h APF) were imaged directly using a Zeiss LSM 510 confocal microscope. After 12 h APF, pupae were peeled out of the pupal case. Between 15–40 optical sections at 1 μm intervals were taken for each neuron, and assembled in NIH ImageJ (All authors contributed to the conception and underlying ideas within the study. N.L. and M.A. performed experimental work. D.W. and N.L. prepared figures. D.W. and N.L. wrote the main manuscript text. All authors reviewed the manuscript.


Eukaryotic cell membrane dynamics change in curvature during physiological and pathological processes. In the past ten years, a novel protein family, Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain proteins, has been identified to be the most important coordinators in membrane curvature regulation. The F-BAR domain family is a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily that is associated with dynamic changes in cell membrane. However, the molecular basis in membrane structure regulation and the biological functions of F-BAR protein are unclear. The pathophysiological role of F-BAR protein is unknown. This review summarizes the current understanding of structure and function in the BAR domain superfamily, classifies F-BAR family proteins into nine subfamilies based on domain structure, and characterizes F-BAR protein structure, domain interaction, and functional relevance. In general, F-BAR protein binds to cell membrane via F-BAR domain association with membrane phospholipids and initiates membrane curvature and scission via Src homology-3 (SH3) domain interaction with its partner proteins. This process causes membrane dynamic changes and leads to seven important cellular biological functions, which include endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, via distinct signaling pathways determined by specific domain-binding partners. These cellular functions play important roles in many physiological and pathophysiological processes. We further summarize F-BAR protein expression and mutation changes observed in various diseases and developmental disorders. Considering the structure feature and functional implication of F-BAR proteins, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating extracellular matrix degradation, and transmitting signaling for cell proliferation. Cell membrane curvature is a micro morphological change involved in many important cellular processes including endocytosis, phagocytosis, exocytosis, angiogenisis, and migration. The ability of cell membrane to achieve these dynamics is heavily determined by the collaboration between actin cytoskeleton and membrane-interacting proteins. Recently, a group of proteins named Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain family has emerged as the critical coordinators that regulate membrane curvature . F-BAR pRecently, research has started to reveal potential functions for F-BAR proteins. However, the structures and functional diversities of the mammalian F-BAR family have not been elucidated. The mechanisms underlying F-BAR proteins regulating cell membrane dynamic changes and the pathophysiological roles of F-BAR proteins in human diseases are not clear. We present here a comprehensive analysis of the current understanding for structure, signaling, biological function, and pathophysiological association of the mammalian F-BAR family, and its association with diseases and development disorders in human and experimental animal models.The F-BAR protein was originally identified in a yeast two-hybrid system screen as CDC42-interacting protein 4 (CIP4) [The F-BAR family belongs to the BAR superfamily, which also includes N-BAR and I-BAR families and possesses a similar N-terminal BAR domain. At the C-terminal region, the BAR superfamily contains various combinations of different domains, such as Src homology-3 (SH3) domain, Src homology-2 (SH2) domain, tyrosine kinase domain, Rho GTPase-activating protein (RhoGAP) domain, WW domain, protein kinase C-related kinase homology region 1 (HR1) domain, and μ-homology domain (μHD) , and transactivator of cytoskeletal assembly-1 (Toca-1).CIP4 recruits WASP and GTPase dynamin via SH3 domain to participate in the initiation and scission of clathrin-dependent endocytic vesicle . CIP4 haFBP17, similar to CIP4, mediates the endocytosis of clathrin-dependent vesicle by recruiting WASP and dynamin for the vesicle initiation and scission . FBP17 iToca-1 has been shown to induce both endocytic vesicle and filopodium depending on CDC42/N-WASP-mediated actin polymerization . Toca-1 FCHO include two members, FCHO1 and FCHO2, containing an N-terminal F-BAR and C-terminal μHD domain. FCHOs bind to the membrane via F-BAR domain and recruit the binding partners, Eps15 and intersectin, via its μHD domain to initiate a clathrin-dependent vesicle. FCHOs accumulate at the membrane before clathrin assembly and dissociate from the vesicle before it departs from the membrane Figure . Both FCsrGAP subfamily includes four members, srGAP1, srGAP2, srGAP3, and srGAP4, and are potentially involved in neuronal migration and angiogenesis. srGAP contains an N-terminal F-BAR domain, a central RhoGAP domain, and a C-terminal SH3 domain. SH3 domain binds to WASP in srGAP1, N-WASP in srGAP2, and WAVE1 in srGAP3 ,21. srGAPACSIN, also known as syndapins, have three isoforms including PACSIN1, PACSIN2, and PACSIN3, all containing an N-terminal F-BAR domain, a C-terminal SH3 domain, and NPF motif. PACSIN are associated with WASP or dynamin via their SH3 domain, leading to WASP-dependent activation, dynamin-dependent scission, and eventually endocytosis . The F-BPSTPIP subfamily consists of two members, PSTPIP1 and PSTPIP2. PSTPIP1 has an N-terminal F-BAR domain, PEST motifs , and a C-terminal SH3 domain, whereas PSTPIP2 lacks the PEST motifs and SH3 domain. PSTPIP1 was initially cloned as CD2-binding protein 1 . SimilarFCHSD subfamily has two members, FCHSD1 and FCHSD2, each containing an F-BAR domain and two SH3 domains. FCHSD1 and FCHSD2 are mammalian orthologs of drosophila nervous wreck (Nwk)2 and Nwk1, which interact with WASP via its first SH3 and cooperate with CDC42 to regulate endocytic actin assembly at drosophila larval neuromuscular junction . Nwk1 anFCHSD2 gene encodes a protein termed as Carom. Carom binds to membrane-associated guanylate kinase inverted 1 (MAGI-1) or calcium/calmodulin-dependent serine protein kinase (CASK), in a competitive manner, via its distinct sequences of the C-terminal region . CASK isThe FES/FER subfamily includes two members, FES and FER, containing an N-terminal F-BAR domain, a central SH2 domain, and a C-terminal tyrosine kinase domain. The region adjacent to the F-BAR domain also binds to phospholipids, thus named the F-BAR extension (FX) domain.The F-BAR and FX domains of FES/FER function as a membrane-binding module to bind to phospholipids and induce the membrane curvature for lamellipodium formation and cell motility . FER alsNOSTRIN contains an N-terminal F-BAR domain and a C-terminal SH3 domain. NOSTRIN binds to N-WASP through its C-terminal SH3 domain to facilitate the endocytosis and recruit dynamin-2 for vesicle scission .NOSTRIN is highly expressed in endothelial cells and highly vascularized organs, binding to the oxygenase domain of endothelial nitric oxide synthase (eNOS) . NOSTRINGAS7 possesses an SH3 domain and a WW domain in the N-terminal, with its F-BAR domain positioned in the center region. Human GAS7 binds to N-WASP via SH3 domain to induce cell filopodium and regulate neurite outgrowth in differentiated brain cells . Mouse GAs discussed above, F-BAR proteins may modulate seven cellular functions: endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation Figure . These sEndocytosis is the most common cellular function mediated by F-BAR proteins and their binding partners. In general, F-BAR protein binds to the membrane phospholipids via its F-BAR domain and recruits WASP/N-WASP and dynamin via SH3 domain to regulate the initiation and scission of endocytic vesicle  degradation and is associated with tumor metastasis. Tumor metastasis requires tumor cells to break through the basement membrane and invade through dense networks of interstitial ECM proteins. Filopodium and lamellipodium may also regulate ECM degradation as they connect the cytoskeleton to ECM via focal contact points [t points . It is rt points .Cytokinesis is a key process during cell proliferation which is a crucial process of development, tissue repair, and oncogenesis. In homeostasis status, normal cells accomplish a constant balance between cell growth and death to maintain proper tissue and organ size and patterning [tterning . During tterning . Thus, cCell adhesion is the binding of a cell to a surface or substrate, such as blood vessel wall or ECM, which can mediate immune response in different tissues. It is reported that endothelial cell adhesion molecules mediate the attachment of circulating inflammation cells to the blood vessel wall and subsequently their extravasation into perivascular tissues [ tissues .In summary, F-BAR proteins are involved in controlling endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, and are suggested to be relevant to various diseases and disorder conditions Figure . Based oAs indicated in Table CIP4, PACSIN3, and FCHO1 are associated with developmental disorders. CIP4 is reported to inhibit actin nucleation of embryonic morphogenesis in drosophila embryogenesis . OverexpInterestingly, NOSTRIN is reported to be necessary for proper vascular development in zebrafish and postnatal retinal angiogenesis in mice . KnockdoAltered expression and mutation of F-BAR family was associated with neurological disorders in human and mice. The expression of srGAP3 was found to be reduced in childhood-onset schizophrenia proband . srGAP3 Distal 3p25-p26 chromosome deletion syndrome (3p syndrome) is a rare contiguous gene disorder characterized by low birth weight, mental retardation, telecanthus, ptosis, and micrognathia. srGAP3 was disrupted and functionally inactivated by a translocation breakpoint in a patient with 3p syndrome . MicroarHuntington’s disease (HD) is caused by expansion of a polyglutamine repeat within the N-terminal region of huntingtin and present with severe neurodegenerative disorders. CIP4 interacted with huntingtin via SH3 domain, and overexpression of CIP4 induced the death of striatal neurons during HD pathogenesis . PACSIN1PACSIN1 complex with dynamin-1 may act as pivotal membrane anchoring factor for dynamin-1 during regeneration of synaptic vesicles. Gene deficiency of PACSIN1 or dynamin-1 in mice led to the development of epileptic seizures correlating with excessive hippocampal network activity .WAS is an X chromosome-linked immunodeficiency disorder, characterized by eczema, thrombocytopenia, immune deficiency, and bloody diarrhea. WAS patients lack expression of WASP which binds to F-BAR protein FBP17 and regulates membrane dynamic changes Figure . WAS patPAPA syndrome  is an autosomal dominant arising from PSTPIP1 gene mutation in the SH3 domain of PSTPIP1 . It impaChronic recurrent multifocal osteomyelitis (CRMO) is a human autoinflammatory disorder that primarily affects bone, skin, or gastrointestinal tract. A missense mutation (L98P) of PSTPIP2 in mice led to pathophysiological changes similar to CRMO, called chronic multifocal osteomyelitis in mice  with incFBP17 was found expressed in three bladder tumor cell lines and primary bladder tumor cells from patients . FBP17 kCIP4 suppressed Src-induced invasion in MDA-MB-231 breast tumor cells . CIP4 knRearrangement of the mixed lineage leukemia (MLL) gene at chromosome 11q23 is commonly detected in leukemia. FBP17 is a fusion partner of the MLL gene at 11q23 and may be related to MLL . FES/FERIt is suggested that CIP4 regulated intracellular hypertrophic signal transduction to control the growth of myocytes in heart disease. Knockdown of CIP4 inhibited myocyte hypertrophy and the inhibition could be rescued by expression of a recombinant CIP4 .CIP4 co-localized with glucose transporter (GLUT) 4 in L6 GLUT4 myc-expressing myoblasts . KnockdoCIP4 is highly expressed in tubular epithelia of 5/6-nephrectomized rat models and TGF-β1 treated human kidney (HK)-2 cells . OverexpFinally, we summarize the F-BAR protein expression changes and potential mechanisms in disease and pathophysiological conditions in Table F-BAR proteins can recruit different binding partners to regulate membrane dynamics and cellular functions. Considering the structure features and functional implications of F-BAR proteins described above, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating ECM degradation, and transmitting signaling for cell proliferation. The continued efforts to explore fundamental details of F-BAR family structure, partner, signaling, and regulation would provide important insights to our understanding and to the identification of their therapeutic potential.


Yersinia pestis. Plague dynamics within the Central Asian desert plague focus have been extensively modelled in recent years, but always with strong uniformity assumptions about the distribution of its primary reservoir host, the great gerbil (Rhombomys opimus). Yet, while clustering of this species’ burrows due to social or ecological processes could have potentially significant effects on model outcomes, there is currently nothing known about the spatial distribution of inhabited burrows. Here, we address this knowledge gap by describing key aspects of the spatial patterns of great gerbil burrows in Kazakhstan.The spatial structure of a population can strongly influence the dynamics of infectious diseases, yet rarely is the underlying structure quantified. A case in point is plague, an infectious zoonotic disease caused by the bacterium Kazakhstan.K statistic to determine whether and at what scale there was clustering of occupied burrows, and semi‐variograms to quantify spatial patterns in occupied burrows at scales of 250 m to 9 km.Burrows were classified as either occupied or empty in 98 squares of four different sizes: 200 m (side length), 250 m, 500 m and 590–1020 m. We used Ripley's Significant spatial clustering of occupied burrows occurred in 25% and 75% of squares of 500 m and 590–1020 m, respectively, but not in smaller squares. In clustered squares, the clustering criterion peaked around 250 m. Semi‐variograms showed that burrow density was auto‐correlated up to a distance of 7 km and occupied density up to 2.5 km.These results demonstrate that there is statistically significant spatial clustering of occupied burrows and that the uniformity assumptions of previous plague models should be reconsidered to assess its significance for plague transmission. This field evidence will allow for more realistic approaches to disease ecology models for both this system and for other structured host populations. Abundance thresholds exist here for both the pathogen's host  could occur by several mechanisms. First, exogenous factors such as climate and topography may result in higher or lower quality habitat patches for gerbils  marks and the recent clearance of burrow entrances (indicated by the presence of freshly dug sand).c. 10 km × 10 km  were monitored during six field sessions: April, June and September in 2011 and 2012. In one of the sectors the squares were also monitored in April 2013 and September 2013 . These 36 squares are spread evenly over six sectors (i.e. six per sector).Second, 38 squares of 250 m (side length) were mapped in April 2013 and September 2013 in one of the sectors. Placement of the squares was done in a similar way as the 200 m squares. Sixteen squares measuring 500 m were also visited: five in September 2011, five in June 2012, five in September 2012 and another one in May 2013. Nine of those squares were visited twice. Finally, eight squares, of which two had a rectangular shape, of varying sizes between 590 and 1020 m were monitored in May 2013.et al., In this study two types of point patterns are analysed: marked point patterns and unmarked point patterns , and dij is the Euclidean distance between point i and point j.We used Ripley's  Ripley, , hereaftK − πs2 was plotted instead of K  statistic for all burrows  using the R package splancs  was calculated discretely: every 5 m up to 175 m for the 200 m and 250 m squares and every 5 m up to 400 m for the larger squares. To assess which K‐values can be expected from a random distribution, we generated a complete spatial random distribution of burrows (csr) with the same burrow density  and lower (Kcsr_2.5) confidence interval curves from the ensemble of curves. If for a particular distance s Kall(s) is larger than Kcsr_97.5(s) ), this suggests that burrows within a distance s are aggregated . Next, for all distances, the results for all squares were summarized by calculating the percentages where Kall > Kcsr_97.5 or Kall < Kcsr_2.5. The procedure was repeated for the occupied and the empty burrows, denoting the K(s) statistics as Kocc and Kemp, respectively.First, we used the Kall, Kocc and Kemp patterns within an individual square were defined as being aggregated when the specific K was larger than Kcrs_97.5 for a distance of at least 35 m; this value was based on the mean nearest burrow neighbour distance (38.49 m) calculated using the 33 largest squares.To investigate the relationship between the size of the research area and spatial aggregation, first the frequencies of occurrence of aggregation for each size group of squares  were calculated. The occupied or empty burrows) to occur more closely together than would be expected from rs from within the ‘total population’   was calculated using only the occupied burrows (Kocc), and the confidence interval was simulated in a similar way as stated earlier, but now by randomly selecting (rs) the observed number of occupied burrows from the locations of all burrows. Because of the measure's sensitivity to small sample sizes  were then used to test whether there was a significant relationship between the presence/absence of spatial clustering of occupied burrows and square size, number of burrows, number of occupied burrows and occupancy level. Models were bootstrapped to account for the repeatedly visited sites: each model was run 100 times, each time using all research locations, but randomly drawing one value from the sites that were visited multiple times. The minimum and maximum et al., et al., GLMs were also used to test whether there was a relationship between clustering and landscape‐ecological variables, as well as variables related to the research design, such as the season of data collection . The presence or absence of clustering was used as a dependent variable, while explanatory variables included landscape type, presence of dunes  burrow point patterns up to a distance of 400 m, larger scale spatial patterns can also be important to incorporate into plague persistence models. Therefore, we used the density of burrows and the density of occupied burrows to construct semi‐variograms , and then only for s > 105 m  compared to completely random point patterns , however, did show aggregation: in 62.5% of the squares in the largest size group and in 8.3% of the squares in the 500 m size group, the patterns were classified as significantly aggregated (i.e. for a distance of at least 35 m Kocc is larger than Kcsr_97.5). For the empty patterns (Kemp), the percentages of squares that were aggregated were 50% and 4%, respectively.Characterizing Of a total of 74 tested squares, significant spatial clustering of occupied burrows  and that research areas should be at least 580 m (size length) to detect spatial clustering with a chance of more than 50% . The model was not improved by including either the number of burrows or the number of occupied burrows (Table S1), while bootstrapping the model resulted only in a very slight difference in the maximum and minimum P‐values. Hence, the model was run with all squares available.GLMs revealed that square size was significantly positively correlated with clustering  was smaller.Focusing on the squares classified as clustered Fig. , for disP = 0.065, Table To test whether absence or presence of spatial clustering could be explained by the landscape, several GLMs were constructed for the squares of the two largest size groups, because in those squares clustering proved to be possible to detect. Similar to the GLMs including all squares, the most parsimonious model here also included only square size, even though the 33 squares in the model were already selected on the basis of size (−1 (mean: 3.2 burrows ha−1); histograms are given in Appendix S1: Fig. S3. While burrows (irrespective of occupancy) showed no aggregation within the squares, differences in burrow density were apparent between squares. The semi‐variogram of burrow densities .In total, 133 data points were available in April 2013, from which a burrow density and an occupied burrow density semi‐variogram were constructed. The density values in the squares varied between 0.6 and 5.4 burrows haThe density of occupied burrows varied between 0 and 3.0 per hectare. The maximum distance at which the occupied burrow densities were auto‐correlated was 2.5 km .et al., et al., For plague in great gerbils, it has been established that a gerbil‐density threshold (percentage of burrows occupied) is required for plague to spread and persist , burrows often show significant spatial regularity, confirming earlier findings using nearest neighbourhood statistics  of occupied (and empty) burrows. This is also reflected in the patterns of clustering of occupied burrows, which is biologically more informative as these patterns represent the distribution of burrows selected by great gerbils from among those available to them. That there is clustering of occupied burrows indicates that the gerbils are indeed selective. Such clustering of occupied burrows was detected in 75% of the larger squares (> 590 m); empty burrows also showed evidence of clustering, although to a lesser extent. The basis for this selectivity is likely to be a combination of landscape‐ecological factors that influence habitat suitability, and aspects of gerbil dispersal behaviour linking currently occupied to newly colonized burrows. To explore these, it is necessary to understand the spatial scale at which clustering occurs.et al. (H=(K/π)−s, where K is Ripley's K and s is distance . Again following Kiskowski et al.  burrow densities than areas farther away. Previously, Wilschut et al. ,b studieet al., in prep.).Dealing first with habitat‐based explanation for the patterns, the landscape in the research area at small scales  can vary quite substantially, especially in terms of vegetation density and topography. These small‐scale variations in habitat can lead to differing conditions for burrows. The clustering of occupied burrows is likely to be related to both selection of the most optimal burrows, and increased survival chances of gerbils in the more optimal burrows. However, the absence or presence of clustering was not found to be related to any of the environmental variables measured. One explanation for this might be simply a lack of statistical power (too few squares that were classified as clustered). Another possibility is the scale at which we tested the relationship between clustering and environmental variables. It might be that the factors we tested do influence occupancy, but play a role at the scale of the individual burrow, averaged out in our analysis at the whole‐square level. For example, work in progress focuses on habitat at the level of the individual burrow and finds that occupancy tended to be less likely on alluvial substrata, but that this was counteracted by the presence of dunes (B. Levick −1 and also varied much in space and time. At larger scales, this variation can probably be explained by variations in vegetation density, which leads to differences in food availability.The explanation for the larger scale spatial structures in both burrow and occupied burrow density is likely to be related to large‐scale variations in the landscape. In the case of burrow density, they may be related to soil texture, as this is thought to be an important aspect determining burrow density (Naumov & Lobachev, et al., et al., et al. (Several aspects of great gerbil behaviour and ecology are also likely to contribute to the emergence of occupied burrow clusters. First, localized dispersal by great gerbil juveniles may reinforce local areas of high occupancy. Rates of dispersal by juvenile great gerbils are high, with one study reporting 42.8% of females and 100% of males changing burrows at least once within a year of their birth (Randall , et al. , for exa, et al. .The ability to detect clustering proved to be very dependent on the size of the research area. This is due to the fact that the clusters and the distances between them are fairly large, and it takes a large square to contain this pattern. Future research should focus on examining patterns in large research areas.et al., We have thus established that at a small scale, burrows occupied by great gerbils in the desert plague focus in south‐east Kazakhstan are clustered, and at a larger scale their densities are auto‐correlated. This population structure will probably influence the invasion and persistence of plague, suggesting in turn that exploring modifications to population structures in existing plague models is a priority. Many yet unexplained phenomena such as the long persistence of plague without being detected (Eisen Liesbeth Wilschut is a landscape epidemiologist interested in how the landscape factors interact with hosts and vectors. She has been working primarily on plague in Central Asia for the past 3 years.Author contributions: M.B., L.I.W. and A.L. initiated the research. L.I.W., A.L., N.K.H. and V.M.D. collected the data. L.I.W. carried out the analysis with help from S.E. and J.R. The writing of the article was led by L.I.W. and many contributions and edits were made by M.B., H.A.P., N.K.H., J.R., S.M.J., E.A.A., S.E., A.L. and V.M.D.Appendix S1 Examples of distribution patterns.Click here for additional data file.


Egbellichnus jordidegiberti igen et ispec. nov. and are interpreted as burrows produced by decapod crustaceans, specifically by a ghost shrimp of the family Callianassidae. This interpretation is based on two independent lines of evidence: environmental requirements of large bioturbators and the burrow morphology itself. The new ichnotaxon is distinguished from other related ichnotaxa by a combination of typically inclined (roughly at an angle of 45°) cylindrical burrows, absence of lining, and tunnels making loops or bends at approximately right angles. The burrow systems at Gbely document the survival of ghost shrimp long after the closure of all seaways and the origin of Lake Pannon. As today, no ghost shrimp are known from long-lived brackish lakes. Egbellichnus from Gbely is the only, although indirect, record of ghost shrimp from a brackish lake environment reported so far.Numerous trace fossils are described from the Late Miocene sediments of the Bzenec Formation exposed at the Gbely section . During deposition of the sediments the area was part of the large, long-lived brackish to freshwater Lake Pannon. Most of the trace fossils are attributed herein to  •Egbellichnus jordidegiberti igen et isp. nov is erected.A new ichnotaxon •A tracemaker is identified as a ghost shrimp .•Egbellichnus is the only record of ghost shrimps from the brackish lake environment reported so far. BrackisMany decapod taxa inhabiting brackish water environment produce burrows. Consequently, these can be identified in the sedimentological record as trace fossils with characteristic features. Numerous trace fossils found in the Late Miocene sediments of the Gbely section  are interpreted herein as burrows produced by decapod crustaceans, specifically by ghost shrimp of the Callianassidae. The first report on burrows made by “thalassinideans” from the Gbely section can be found in The objectives of this paper are 1) the report of the studied ichnoassemblage of the Gbely section; 2) description of a new ichnogenus and ichnospecies; 3) the interpretation of its tracemaker based on evidence from palaeoenvironmental reconstruction and burrow morphology; and 4) providing arguments for the presence of ghost shrimp in the long-lived brackish Lake Pannon.2The Paratethys was an epicontinental sea that developed in the Early Oligocene as a consequence of Africa’s northward movement and resulting European plate subduction. It was intermittently connected to the Mediterranean and the Indo-Pacific . The areThe Pannonian Basin formed in the late Early Miocene and in the Middle Miocene as a result of extension and rifting governed by thrusting in the surrounding Carpathian orogene . At the The depositional systems of Lake Pannon are heterogeneous, represented by alluvial and fluvial facies, ephemeral lake, swamp, and subaquatic delta plain deposits passing continually to offshore pelitic facies .2.1sensuThalassinoides minimus” and “Pectinaria burrows”; other common trace fossils are Spirisiphonella pannonica and Arenicolites isp. Trace fossils studied by Trace fossils of the deposits of the Pannon Lake were reported by several authors. sensuDiplocraterion and “Y-shaped trace fossils”.From the Szák Formation  represented by brown-grey clayey and silty beds has yielded completely and partially lithified trace fossils A–B. ThesThe upper bioturbated interval (BI 2 = FA 8B + FA 8C) represented by rhythmic deposition of silty clay, silt, and fine sand reflecting successive progradation of a prodelta (FA 8B) and laterally migrating distal bars (FA 8C) contains unlithified trace fossils C–D, 4.2.3Cyprideis, Euxinocythere, and Loxoconcha  was cleaned of clay by slow running water and a paintbrush. Wet casts were impregnated immediately by an acrylic sealer. The acrylic sealer hardened trace fossils and prevented further damage and cracking. Unlithified trace fossils were documented by taking photographs of vertical and horizontal serial sections in outcrops.Isolated burrow parts were oriented in the position they were found in the section and compared with extant and fossil burrow systems. This procedure provided clues to interpretation of the studied material (see chapter 4.1.1).sensuThe ichnoassemblage of the studied section seemingly consists of several different structures . After c3The material examined herein is deposited at the Natural History Museum of the Slovak National Museum in Bratislava (Slovakia) under the repository numbers SNM-Z 24165, SNM-Z 37735–37747.3.1Egbell, the Hungarian and German name for the town Gbely  and ιχνος, from the Greek, meaning trace.Etymology: after Diagnosis: Vertical, inclined, or horizontal cylindrical burrows of constant diameter without wall structure. The burrows are circular in cross-section. An inclined component typically slopes at ca. 45°; tunnels make loops resembling a spiral or bend at approximately right angles; the resulting burrow shape resembles a corkscrew or an irregular zigzag pattern depending on alternating directions. First to second and more orders of upward, downward or horizontal Y-shaped branching may occur on main burrows. Points of bifurcation and bending have usually an enlarged diameter.Egbellichnus jordidegiberti isp. nov. (only known ichnospecies).Type ichnospecies: Remarks: This ichnogenus is distinguished from other related ichnotaxa by a combination of typically inclined (roughly at 45°) cylindrical burrows, absence of lining, and tunnels making loops or bends.Thalassinoides Y- or T-branching is typical, thus, resembling Egbellichnus, however, a complex pattern of loops and bendings exhibitied by the latter ichnogenus is absent in Thalassinoides.For Gyrolithes, a tunnel forms a dextral or sinistral circular helix with rather constant radius of whorls ; these, however, usually lack branchings  burrows“. Starek, Pipík, and Hagarová. p. 379.Thalassinoides-like branching  animal(s). In some cases the diameter of side branches is noticeably smaller (8–20 mm) than the main burrow E.One burrow is evidently branched downward A. The buMaiakarichnus curraniMaiakarichnus the chamber is more regularly spherical and shafts radiate in all directions upward and downward; in Maiakarichnus thin shafts radiate mainly in stratigraphically upward direction from the upper part and from sides  were close to each other, the burrow walls nearly touch each other.E. jordidegiberti isp. nov.No trace fossil reported from the Lake Pannon previously  can be dEgbellichnus jordidegiberti n. igen. n. isp., several other components have been recognized in the studied section. In BI 1, vertical cylindrical shafts with knobby surface and a diameter of around 3–5 cm rarely occur. Knobs are oriented horizontally and they clearly represent bioglyphs  to larger burrows  in dark clayey sediment or as unlithified trace fossils in silty/sandy sediments. Associations of both preservation types are composed of very similar trace fossil assemblage; i.e. both bioturbated horizons are dominated by the newly recognized trace fossil  visible A. The fi visible C–D. Trac visible . The larLithification of trace fossils was probably caused by a fluid migration inside the burrow systems. Predisposition for easier fluid migration was supported by open burrow systems and coarse filling of burrows. Lithified silty/sandy burrow casts were cemented by ferruginous and calcareous compounds. Ferruginous concentric structures are visible on transverse cuts of burrows B, 6D, F.The light yellow/brown colour of the burrow walls produced in the Gbely section by early diagenetic pigmenation from pore water indicates more oxidizing conditions in the burrow than in the subsurface sediments. Such conditions are typical for ghost shrimp burrows, in which physico-chemical and microbial properties are more similar to the surface sediments than to the surrounding subsurface sediments . In this44.1Parmaichnus, Polykladichnus or Psilonichnus, especially on freshly excavated section wall. Removing the sediment, however, clearly showed that in our case the shafts and tunnels are parts of larger burrow system, and thus, they represent a compound structure. The tunnels are connected to each other and simultaneously they attain roughly the same diameter. In conclusion, the morphology of studied burrows strongly suggests that the tracemaker is a crustacean, possibly a malacostracan.The trace fossils described above show a suite of characters which is typically interpreted as arthropod burrows generally attributed to crustaceans e.g. . One of Among modern malacostracans there are basically two higher taxa, Decapoda and Stomatopoda, capable of producing large burrows comparable with those described herein. Stomatopods construct shallow and rather simple burrows . Thus, iin situ preservation is rather difficult; ichnofossils commonly attributed to decapod crustaceans usually do not contain any body fossils. Such associations are rare . In contrast, highly structured burrow systems are typical for axiidean and gebiidean shrimps. They are considered to be producers of the most complex burrow systems in the entire animal kingdom, and they construct species-specific burrows , spirally shaped tunnels when viewed from above, more-or-less constant diameter of most burrow-system tunnels with swellings interpreted in some taxa as turning chambers, are all characters typical for axiidean and gebiidean shrimps. Members of other decapod groups usually produce simpler burrows with limited branching  or rather complex horizontal mazes , Laomediidae (Jaxea), and Upogebiidae (Upogebia) are sufficiently known. Burrows of Axianassa australis are characterized by spiral vertical shafts (Gyrolithes-like) leading to wide horizontal galleries from which several evenly proportioned corkscrew-shaped spirals branch off and lead to further horizontal galleries at greater sediment depths  and Strahlaxiidae (Neaxius) seem to construct rather simple burrows without complex morphology . The burrow itself is more complex in vertical aspect with branching occurring mainly in horizontal aspect. Such morphologies , it is not known to make complex permanent burrows, and it reaches its greatest abundance at a depth of ca. 10 m , and they do not build their burrows below the fair-weather wave-base . Thus, we argue for an axiidean or gebiidean decapod crustacean as the tracemaker. Unfortunately, no detailed analysis of the distribution of axiidean and gebiidean species in relation to salinity exists, as this parameter is usually not recorded or mentioned in the literature. There are some species which prefer marine habitats, whereas others are found in brackish waters .Lepidophthalmus is the most extreme example as it is able to tolerate freshwater environments  and tidal influences. For instance, the Upogebiidae are able to tolerate a salinity range from fully marine (36 ‰) to salinity of 9 ‰ in freshwater-influenced areas . In thisronments . Generalh Africa . Lepidopt Africa .Egbellichnus jordidegiberti was an axiidean or gebiidean shrimp, most probably a member of the family Callianassidae.Thus, it seems most reasonable to assume that the tracemaker of 4.2Egbellichnus burrow system we refrain from inferring the main trophic mode of the tracemaker. Nevertheless, some phenomena commonly related with callianassid life strategies can be mentioned and briefly discussed.Among callianassid ghost shrimps, several life strategies have been adopted , but bec4.2.1Egbellichnus association exhibits burrows with variable diameter but the same morphological features. We interpret them here as a juvenile-adult association. Association of juvenile and adult burrows has been reported previously in Upogebia affinis . The tunnels with distinctly smaller diameter attached to larger Egbellichnus burrow system parts  e.g., . In sali4.2.3Endemic species are often documented from long-lived brackish The Late Miocene ichnoassemblage of the Bzenec Formation exposed at the Gbely section  is characterized by low diversity, relatively simple structures and presence of monospecific suites. These aspects are typical for brackish-water trace fossil assemblages; indeed, the previous study of the section interpreted the settings as a brackish-water environment influenced by freshwater input . At that(2)Egbellichnus jordidegiberti igen. et isp. nov., is described from the Gbely section. Egbellichnus is interpreted as part of a complex open burrow system and is distinguished from other related ichnotaxa by a combination of typically inclined cylindrical burrow parts, absence of lining, and tunnels making loops or bends at approximately right angles.A new ichnotaxon, (3)Egbellichnus is identified as a member of the family Callianassidae. Ghost shrimps of this family are able to tolerate high salinity fluctuations and construct very complex burrow systems.Based on evidence from palaeoenvironmental reconstruction and burrow morphology the tracemaker of (4)Egbellichnus, if interpreted correctly, is thus the only, although indirect, evidence for the persistence of a ghost shrimp in a brackish lake.Today, no ghost shrimps are known from long-lived brackish lakes.


Recent single molecule experiments, using either atomic force microscopy (AFM) or Förster resonance energy transfer (FRET) have shown that multidomain proteins containing tandem repeats may form stable misfolded structures. Topology-based simulation models have been used successfully to generate models for these structures with domain-swapped features, fully consistent with the available data. However, it is also known that some multidomain protein folds exhibit no evidence for misfolding, even when adjacent domains have identical sequences. Here we pose the question: what factors influence the propensity of a given fold to undergo domain-swapped misfolding? Using a coarse-grained simulation model, we can reproduce the known propensities of multidomain proteins to form domain-swapped misfolds, where data is available. Contrary to what might be naively expected based on the previously described misfolding mechanism, we find that the extent of misfolding is not determined by the relative folding rates or barrier heights for forming the domains present in the initial intermediates leading to folded or misfolded structures. Instead, it appears that the propensity is more closely related to the relative stability of the domains present in folded and misfolded intermediates. We show that these findings can be rationalized if the folded and misfolded domains are part of the same folding funnel, with commitment to one structure or the other occurring only at a relatively late stage of folding. Nonetheless, the results are still fully consistent with the kinetic models previously proposed to explain misfolding, with a specific interpretation of the observed rate coefficients. Finally, we investigate the relation between interdomain linker length and misfolding, and propose a simple alchemical model to predict the propensity for domain-swapped misfolding of multidomain proteins. stability of domains present in the intermediates on the folding and misfolding pathways, consistent with the energy landscape view of protein folding. Based on these observations, we propose a simplified model that can be used to predict misfolding propensity for other multidomain proteins.Multidomain proteins with tandem repeats are abundant in eukaryotic proteins. Recent studies have shown that such domains may have a propensity for forming domain-swapped misfolded species which are stable for long periods, and therefore a potential hazard in the cell. However, for some types of tandem domains, no detectable misfolding was observed. In this work, we use coarse-grained structure-based folding models to address two central questions regarding misfolding of multidomain proteins. First, what are the possible structural topologies of the misfolds for a given domain, and what determines their relative abundance? Second, what is the effect of the topology of the domains on their propensity for misfolding? We show how the propensity of a given domain to misfold can be correlated with the  Protoligomer .β-sheets. Theoretical work in fact made the prediction that such species would be formed in tandem repeats of titin domains [In addition to domain-swapped misfolding, an alternative type of misfolded state is conceivable for polyproteins in which the sequences of adjacent domains are similar, namely the formation of amyloid-like species with parallel  domains . Recentl domains . HoweverA simplified illustration of the mechanism for folding and misfolding, based on both coarse-grained simulations as well as single-molecule and ensemble kinetics , 12, is While domain-swapped misfolding of tandem repeats has been identified in a number of proteins to date, there are several other proteins for which it does not occur to a detectable level. For instance, extensive sampling of repeated unfolding and folding of a polyprotein of Protein G (GB1) by AFM revealed no indication of misfolded states, in contrast to Titin . SimilarIn order to investigate the misfolding propensity of different types of domains, we have chosen seven domains, based on (i) the superfamilies with the largest abundance of repeats in the human genome , (ii) prTandem Src homology 3 (SH3) domains  are wideWith a similar function to the SH3 domains, Src homology 2 (SH2) domains  are alsoFibronectin type III (fn3) domains  are highPDZ domains  are one Titin  is a gia8 folding correctly in repetitive stretching–relaxation cycles [In contrast, with the above misfolding-prone systems, there are certain polyprotein chains have been shown be resistant to misfolding, according to pulling experiments. For instance little evidence for misfolding was identified in a polyprotein of GB1  Fig 2g)Fig 2g), n cycles .Lastly, we consider polyUbiquitin , for whiA coarse grained structure-based (Go-like) model similar to the earlier work is employed for the study here , 41. EacL amino acids, given any pair of residues (with indices i and j) that are the native interactions within a single domain, the interaction energy for the intradomain interaction ) is the same as the interdomain interaction between the residue (i or j) and the corresponding residue (j + L or i + L) in the adjacent domain, i.e. Ei,j(r) = Ei+L,j(r) = Ei,j+L(r) = Ei+L,j+L(r).In order to allow for domain-swapped misfolding, the native contact potentials within a single domain are also allowed to occur between corresponding residues in different domains, with equal strength. Specifically, considering each single repeat of the dimeric tandem that has −1 and a time step of 10 fs.To investigate the folding kinetics of the dimeric tandem, a total of 1024 independent simulations are performed on each system for a duration of 12 microseconds each. Different misfolding propensities are observed at the end of the simulations. With the exception of Ubiquitin and GB1, the vast majority of the simulations reached stable native states with separately folded domains. A small fraction of simulations form stable domain-swapped misfolded states. All the simulations are started from a fully extended structure, and run using Langevin dynamics with a friction of 0.1 psK in sequence where the native domain would be cut to form the circular permutant (K = 0 corresponds to the native fold). If a native contact Cnative =  exists between residues i and j in the native fold, the corresponding native-like contacts for the central (Cin(K)) and terminal domains (Cout(K)) of the domain swapped conformation are generated asx) is the Heaviside step function and L is the length of each single domain (plus interdomain linker). SKin, is the set of native-like contacts Cin of the central domain, and SKout, is the set of all the native-like contacts Cout of the terminal domain. SKin, and SKout, can be used to define a contact-based reaction coordinate to analyze the kinetics of the dimeric tandem misfolding. The corresponding fraction of contacts for the central domain could be calculated by:N is the total number of domain swapped contacts, SK = SKin, ∪ SKout, , rij(χ) is the distance between residue i and j in the protein configuration χ. β = 50 nm−1 and λ = 1.2 is used to account for fluctuations about the native contact distance.We note that all the generated domain-swapped misfolding structures, containing the central and terminal domains, can be monitored by a reaction coordinate based on circular permutated native-like contact sets. Each circularly permuted misfold can be characterized according to the loop position Q as the reaction coordinate. The obtained melting temperature of each system is listed in Table A in Gf of approximately ∼ 2.5 kBT is chosen for the 2-domain tandem simulations for reasons described below. The stability ΔGs is calculated askB and T are the Boltzmann constant and temperature respectively. Q‡ is the position of the barrier top in F(Q), separating the folded and unfolded states and F(Q) represents the free energy profile on Q. Barrier heights ΔGf were simply defined as ΔGf = G(Q‡) − G(Qu), where Qu is the position of the unfolded state free energy minimum on Q.The equilibrium properties of a single domain of each system are obtained from umbrella sampling along the native contacts RCOK of different circular permutants K viaL is the length of the single domain, and N is the total number of the native like contacts (the same for different K). Sin,K is the contacts set of the circular permutant corresponding to the “central domain” of the misfolded state. Note that the contact order calculation here is using residue-based native contacts (the same ones defined as attractive in the Gō model), instead of all atom native contacts.We calculated the relative contact order , RCOK ofS, while all possible native contacts involving residues within the native segment are considered to be formed, each with a favourable energy of contact formation ϵ.An Ising-like model was built based on the native contact map, in which each residue is considered either folded or unfolded and so any individual configuration can be specified as a binary sequence, in a similar spirit to earlier work –52. IntekB and T are the Boltzmann constant and temperature. G(χ) is the free energy determined by the number of native contacts n(χ) in the configuration χ, and the number of native residues, Nf(χ). The distribution of the microstates (χ) can be efficiently generated by the Metropolis-Hastings method with Monte Carlo simulation. In each iteration, the state of one randomly chosen residue (among the residues at the two ends of the native fragment and their two neighbouring residues) is perturbed by a flip, from native to unfolded or from unfolded to native, taking the system from a microstate χ1 with energy E1 to a microstate χ2 with energy E2. The new microstate is subject to an accept/reject step with acceptance probabilityThe partition function for such a model can be enumerated as:Ep has been added whenever the native fragment crosses the midpoint of the sequence from either side (the function θ(χ) above is 1 if this is true, otherwise zero). That situation corresponds to formation of a domain-swapped structure, in which there is additional strain energy from linking the termini, represented by Ep. We only use the Ising model here to investigate formation of the first domain (either native or circular permutant), by rejecting any proposed Monte Carlo step that would make the native segment longer than the length of single domain, L.To mimic the folding stability difference between native and circular permutant folds, a penalty energy term ϕ-values [In order to characterize the potential misfolding properties of each type of domain, we have used a Gō-type energy function based on the native structure. Such models have successfully captured many aspects of protein folding, including ϕ-values , 54, dimϕ-values , 56, domϕ-values –60, and ϕ-values , 62. Morϕ-values , 12. We μs each) such that the vast majority of them reached either the correctly folded tandem dimer, or a domain-swapped misfolded state similar to that shown in K, in sequence after which the central domain begins. Thus, the native fold has K = 0, and all the misfolded states have K > 0. Typically, because of the nature of domain swapping, K must fall within a loop. Of course, there is a range of residues within the loop in question that could be identified as K and we have merely chosen a single K close to the centre of the loop. This position, and the central domain, are indicated for the Src SH3 misfolded structures in K.For each of the folds shown in With this nomenclature in hand, we can more easily describe the outcome of the folding simulations for the seven domain types considered in terms of the fraction of the final frames that belonged to the native fold, versus each of the possible misfolded states. These final populations are shown in The ability to capture domain-swapped misfolds with simple coarse-grained simulations potentially allows us to investigate the origin of the misfolding, and its relation, if any, to the topology of the domain in question. However, we also need to benchmark the accuracy of the results against experiment as far as possible, in order to show that they are relevant. There are two main sources of information to validate our results. The first is the overall degree of domain-swapped misfolding for those proteins where it has been characterized, for example by single molecule AFM or FRET experiments. Qualitatively we do observe good agreement, where data is available: in experiment, domains which have been shown to misfold are TNfn3 (AFM) and Titin I27 , which are both found to misfold here, while there is no detectable misfolded population for protein G (AFM), again consistent with our results. We also do not observe any misfolding for Ubiquitin, consistent with the lack of experimental evidence for fully domain-swapped species for this protein –23.8) is extrememly low (< 0.2%) [Quantitatively, the fractional misfolded population is also consistent with the available experimental data. For instance, the frequency of misfolded domains in native tenascin is ∼ 4% as shown by previous AFM experiments , the mis(< 0.2%) . Even th(< 0.2%) . Recent (< 0.2%) .K = 37 . The domain swapped dimers solved obtained from experiments  are strins) = 37 , SH2 wit , we set out to establish some general principles relating the properties of each domain to its propensity to misfold in this way. We can start to formulate a hypothesis based on the alternative folding and misfolding pathways illustrated in QK. QK is exactly analogous to the conventional fraction of native contacts coordinate Q [K indicates the position along the sequence of the WT where the cut is made in order to convert to the circular permutant.We investigate the role of the central domain by characterizing the free energy landscape of the single domain of each system, as well as all of its possible circular permutants, using umbrella sampling along the reaction coordinate dinate Q , but defF(QK) of two representative systems, SH3 and Ubiquitin, are shown in Gf and the stability ΔGs are listed in the The free energy surfaces Gf projected onto Q (for native) or QK (for circular permutants) show little correlation to the relative frequency of the corresponding folded or misfolded state, when considering all proteins (Gf (Table B in Gs) of the folded single domains in our simulations are generally comparable to those in experiment  [In contrast to the situation for GB1 and Ubiquitin, all of the circular permutants of the SH3 domain in proteins . Since tcal/mol) , 69–72.stability ΔGs of the corresponding intermediate. Spearman rank correlation coefficients between the folded stability ΔGs of the intermediate structure and the frequency of folded/misfolded states were 0.63, 0.94, 0.74, 0.81, 0.86 for the SH3, PDZ, TNfn3, SH2 and Titin I27 domains respectively. We note that there is also a reasonable correspondence between the relative stabilities of circular permutants in simulation and experiment, where data are available [in the context of the full sequence. This is important because a large fraction of native (or native-like) contacts are shared between the native fold and the various misfolded domains. As such, the native and misfolded states can be considered as belonging to the same folding funnel, with differentiation between the two occurring at a late stage of folding. This scenario is illustrated schematically in β-strands [On the other hand, we did note that there was a significant, and unexpected, correlation between the population of the final folded or misfolded states and the vailable , 26. How-strands , rather K = 18. Each event is projected onto two different reaction coordinates,QK, for K = 0 (standard native Q) and K = 18 (the Q when the circular permutant for K = 18 is considered as “native”). As is evident, a large fraction of the transition path looks very similar in Q0 ≈ Q18 ≈ 0.5, the first trajectory moves toward the native structure, where it terminates  is the native or native-like contact formed by the residues i and j in the configuration χ, and S(χ) is the set of all such contacts which are formed in χ. We can locate the position of nascent structure in the sequence by plotting the distributions of χ drawn from the equilibrium distribution at selected values of the global coordinate Q, defined as the fraction of native contacts in the native dimer structure . Q values . We have constructed an even simpler simulation model for formation of the first intermediate , by using a simulation of a Wako-Saito-Muñoz-Eaton Ising-like model , 76. In Qres, and Qres equals to the number of residues which are in the native-like state divided by the total number of residues of one domain (L). The projection of a trajectory for the model onto Ep > 0 was used in this instance. The effect of the stability penalty for the circular permutants is illustrated by the two-dimensional free energy surfaces Qres corresponding to unfolded structures and at high Qres for folded (native or circular permutants). If the penalty Ep = 0  . Qres eqy Ep = 0 , in addiecreases , as expea priori [GJ). Note that this step is the same for all circular permutant folds. The second step is to cut different loops on the cyclic configuration I to form a circular permutant CP (GC):Knowing that misfolding correlates with the relative stability of the native single domain and its circular permutants is useful because it suggests a means to predict the likelihood of misfolding, provided one has an estimate of the circular permutant stability. While one could attempt to determine this experimentally, by synthesizing the circular permutants, or computationally, as we have done, it would be very helpful to have a quick method to estimate this stability a priori . Here, wutant CP  with theAssuming the native and circular permutant unfolded states to have the same free energy, the overall change in stability between the circular permutant (CP) and the wild-type (WT) is:i at the N-terminus and j at the C-terminus remains native, then the change in free energy for linking the termini for cyclization, ΔGJ, can be split into energetic and entropic components:For the first step , in ordep ∈ {0 .. i − 1} and q ∈ {j + 1 .. L}, which are on the N- and C- termini respectively, change from the native state to non-native state (joint loop), the total energy increase will be ∑x ∈ σ(p)ϵpx + ∑x ∈ σ(p)ϵqx, which is the summation of all the native contact energy, in the Go model over the sets of residues σ(p), σ(q) involving residues p and q respectively. x represents the residues that form the native contacts with either p or q. We approximate the entropy gained per residue as δs, where δs = ∑i,j)native(ϵij/(TN), where N is the number of residues and T is the folding temperature. The gained entropy is set to 0 if residue p or q does not have any contact with other part of the protein except for the neighboring residues, and the number of such residues is denoted by κ. The average length contribution (r0) of peeling off each residue from the native structure is set to 3.5 Å here. The topological requirement of joining the two termini by peeling off residues 1 to i − 1, and j + 1 to L from the native state is that the linear distance between the residues i and j ) on the native structure is shorter than the effective length contributed by the joint parts:GJ by adjusting i and j could be given by:Assuming the states of the residues  domains , around  domains , whose N domains , the minK and K + 1, the states of the residues on each side of the cutting point p′ ∈ {i′,.., K} and q′ ∈ {K + 1,.. j′}, will change from the native state to the non-native state. The gained entropy per residue is δs. ∑x ∈ σ(p′)ϵp′x and ∑x ∈ σ(q′)ϵq′x are the summation of all the energy of the native contacts which are broken due to the cutting. Therefore, by comparing different combinations of i′ and j′, the minimum change of stabilities ΔGC in this step is:Analogously, for the second step , assume Gtot calculated using this alchemical free energy method is very well correlated with the stability of the circular permutant ΔGs obtained by umbrella sampling simulations  is relatively small and is similar for all the circular permutants during the transformation . This extra peptide corresponds to the linker between the tandem domains. The stability of circular permutants, obtained from simulations using umbrella sampling, as a function of linker length (ll) is shown in QK in Figs C and D in ll = 0 to ll = 20 is roughly the same for all circular permutants of a given protein, as expected since the change in all cases is the same loop extension. Note, however, that the effect is much larger for ubiquitin than for TNfn3. To investigate the consequences of the change of central domain stability for the misfolding propensity of tandem repeats, we carried out first passage simulations of a tandem dimer of Ubiquitin with linker lengths of 5,8 and 10 residues respectively. The setup of the dimer simulations was the same previously. For each linker length, 1024 independent simulations were run from fully extended structures. No domain-swapped misfolding was found for ll = 5 and ll = 10, however, we indeed obtained three domain-swapped misfolding events for ll = 8. Two of the misfolds belong to the K = 61  occurs in the background of the full-length sequence. In this context, while folding may be initiated at any point in the chain, the nascent structure will tend to migrate towards the N- or C-terminus because of the free energy bias towards the native fold; circular permutants invariably pay a cost in stability for joining the protein termini. Thus the folding rate of isolated circular permutants relative to wild-type protein may not be a good proxy for these rates in the context of the full length sequence, whilst the domain stability is a better guide as to the free energy bias towards a particular structure. This suggests that the rates of formation of these domains inferred from single-molecule experiments  should bS1 TextThe melting temperature of single domain folding from umbrella sampling simulations (Table A). The mean first passage time of the folding simulation of the central domains (Table B). Free energy profile of WT (single domain) and its circular permutants . The spread of native contacts formed at a given Q . Free energy profile of the circular permutant of Ubiquitin with different linker lengths . Free energy profile of the circular permutant of TNfn3 withdifferent linker lengths .(PDF)Click here for additional data file.


T = 3 Grouper nervous necrosis virus-like particle (GNNV-LP) is determined by the ab initio method with non-crystallographic symmetry averaging at 3.6 Å resolution. Each capsid protein (CP) shows three major domains: (i) the N-terminal arm, an inter-subunit extension at the inner surface; (ii) the shell domain (S-domain), a jelly-roll structure; and (iii) the protrusion domain (P-domain) formed by three-fold trimeric protrusions. In addition, we have determined structures of the T = 1 subviral particles (SVPs) of (i) the delta-P-domain mutant (residues 35−217) at 3.1 Å resolution; and (ii) the N-ARM deletion mutant (residues 35−338) at 7 Å resolution; and (iii) the structure of the individual P-domain (residues 214−338) at 1.2 Å resolution. The P-domain reveals a novel DxD motif asymmetrically coordinating two Ca2+ ions, and seems to play a prominent role in the calcium-mediated trimerization of the GNNV CPs during the initial capsid assembly process. The flexible N-ARM  appears to serve as a molecular switch for T = 1 or T = 3 assembly. Finally, we find that polyethylene glycol, which is incorporated into the P-domain during the crystallization process, enhances GNNV infection. The present structural studies together with the biological assays enhance our understanding of the role of the P-domain of GNNV in the capsid assembly and viral infection by this betanodavirus.Betanodaviruses cause massive mortality in marine fish species with viral nervous necrosis. The structure of a  Nodaviridae and cause the mortality of numerous larval-stage fish species. Here we report protein crystal structures of a piscine betanodavirus, the Grouper nervous necrosis virus (GNNV), in four different forms. Highlights are two structural features that contribute to the viral molecular mechanisms of the T = 3 and T = 1 capsid assembly: a calcium-associated protrusion domain and a functional arginine-rich motif. These results also shed insights into the structural basis for evolutionary lineage of the family Nodaviridae.Betanodaviruses belong to the family  Nodaviridae is a family of positive-sense single-stranded RNA viruses with a non-enveloped T = 3 capsid. These viruses are characterized by a viral genome comprising two RNA molecules–RNA1 and RNA2. RNA1 (3.1 kb) encodes protein A, which is a RNA-dependent RNA polymerase (RdRp) responsible for viral RNA replication [lication .Nodaviridae [Macrobrachium rosenbergii Nodavirus (MrNV) and Penaeus vannamei Nodavirus (PvNV), which cause muscle necrosis in prawns [Nodaviridae includes not only the known types but also other members with a wide distribution.Alphanodaviruses and betanodaviruses are the major genera in the family aviridae . Alphanoaviridae . VNN is aviridae . Betanodaviridae . Based oaviridae . One repn prawns . These fNodaviridae, an assemblage of 180 CPs form a T = 3 capsid of diameter ~29−35 nm. CP is typically composed of the core jelly-roll topology, forming a face-to-face β-sandwich with two pairs of anti-parallel β-sheets [In the family β-sheets . During β-sheets . Proteinβ-sheets ,16; it aβ-sheets . The strβ-sheets . HoweverT = 3 GNNV-like particle (GNNV-LP) at 3.6 Å resolution; (ii) T = 1 subviral particles (SVPs) of the delta-P-domain mutant at 3.1 Å; (iii) the N-ARM deletion mutant at 7.0 Å; and (iv) the individual P-domain of GNNV CP at 1.2 Å. The crystal structure of GNNV-LP demonstrates several significant and distinct variations in capsid architecture and molecular mechanisms of capsid assembly compared to the genus alphanodavirus and other RNA viruses. In particular, we have identified the conserved structural characteristics of the shell domain on GNNV. Various forms of the T = 3 and T = 1 GNNV capsids show that the N-terminal arginine-rich motif (N-ARM) acts as a molecular switch. Second, the P-domain, with its DxD motif together with two bound Ca2+ ions, plays a pivotal role in the trimerization of the GNNV CP and the particle assembly. These high-resolution structural details contribute further to our in-depth understanding of the molecular mechanisms of viral assembly and infection, and should provide the structural basis for studying the evolution of the family Nodaviridae.In this report, we describe the crystal structure of the grouper nervous necrosis virus (GNNV) of the genus betanodavirus in various forms: (i) a complete Escherichia coli (E. coli) and the GNNV-LPs are self-assembled in vitro. Based on the EM images, the morphology of GNNV-LP shows a T = 3 capsid with a diameter of 30~35 nm  averaging and refine the structure to 3.6 Å  (residues 34−51), the shell domain (S-domain) (residues 52−213), the linker region (residues 214−220) and the protrusion domain (P-domain) (residues 221−338) . The ordquasi three-fold (Q3) axes to form 60 protrusions on the particle surface  of the family Caliciviridae also contains three ordered N-arms from the C1, C10 and C12 subunits located near the I3 axis similar to GNNV, but oriented toward another direction  axis and the subunit-C/C dimer along the I2 axis during T = 3 particle assembly [T = 3 quaternary organization [6 through hydrogen bonds to stabilize the subunit-C/C dimer.The CP subunits adapt to the assembly . The bennization ,16. The 2+ ions located at interfaces between pairs of subunits within each of the S-domains, which are coordinated with side chains of Asp130 and Asp133 to form the 130DxxDxD135 motif at the E-F loop, Gln100 at the C-D loop, Ser170 at the G-H loop and Glu213 near the linker region from the neighboring subunit (DxDxxD) [DxxD) [DxExxD motif and incorporate one or two Ca2+ along the Q3 axis in its CP [Divalent metal ions, such as calcium, are typically associated with metal-coordinating residues for particle formation, stability and infectivity . The GNN subunit . There a(DxDxxD) –30 and S) [DxxD) . In contn its CP ,32.T = 3 PaV  of alphanodavirus . Our cryTo gain more complete and detailed structural information, we have determined the crystal structure of the truncated P-domain (residues 214−338) at high resolution 1.2 Å; . Since i2+ ions near the non-crystallographic three-fold axis, which are coordinated with the C’-D’ loop to stabilize the trimeric structural fold  to bind to the cell surface and to enhance virus infection [An inspection of the structure of the truncated P-domains reveals additional electron densities in several pockets on the surface. We consistently find three glycerol (GOL) molecules located between the B’-C’ and F’-G’ loops of the three P-domains, and one polyethylene glycol (PEG) molecule at the interface between the two F’-G’ loops from neighboring subunits . The B-fnfection . On the T = 3 GNNV-LP, the N-terminus of the CP contains the disordered N-ARM for putative RNA interactions at the inner cavity of the particles. The next N-arm is ordered along the I2 interface only on subunit C. The P-domains of GNNV-LP show an independent trimeric organization, which is different from that of the S-domains. We therefore speculate that the N-ARM or P-domain of GNNV-LP might act as a major molecular switch in regulating T = 3 or T = 1 assembly. To address this issue, we have constructed two sub-clones, including (i) the delta-P-domain mutant (residues 35−217) and (ii) the N-ARM deletion mutant (residues 35−338), and have determined their structures . Based on the failure of VLP formation in the presence of single mutations of either C115A or C201A, the existence of a disulfide-bond linkage between Cys115 and Cys201 was previously postulated [Despite significant variations in amino-acid sequences, the structure of the GNNV S-domain exhibits a jelly-roll topology, similar to other structural viral CPs . Divalenomavirus , rotaviromavirus , tombusvomavirus –30, sobeomavirus ,31,40 anomavirus ,32. In G viruses –31,40,43 viruses . Investistulated . Howeverstulated  but diffstulated   are negatively charged components of the cell surface and play a role in virion attachment to host cells and binding to secondary host receptors during viral infection. For instance, the surface L-protein of HBV is reported to bind to glycosaminoglycans (GAGs) on the host-cell surface; its GAG-dependent binding is enhanced by PEG to facilitate viral infection . Our stuT = 3 capsid assembly. However, the bent conformation of the subunit dimer leads to the disordered N-arm lying at the inner cavity of the T = 1 delta-P-domain mutant without β-annulus formation. This observation indicates that the N-ARM of GNNV CP makes an essential contribution to the organization of the β-annulus along the I3 axes. We propose that the β-annulus on T = 3 GNNV might be an outcome of T = 3 capsid assembly rather than a profound effect on switching structural symmetries.The organization of the N-terminus and encapsidated RNA have been implicated in providing a dynamic equilibrium of the dimeric subunits between “bent” and “flat” conformations during viral assembly . The N-tT = 1 architecture. Comparatively, in alphanodavirus, a complete N-ARM deletion (delta residues 1−54) leads to the inhibition of particle assembly. Conversely, the partial N-ARM deletion (delta residues 1−31) was shown to cause the formation of highly heterogeneous particles, including small bacilliform-like and irregular structures [T = 3 VLPs and two SVPs of T = 2 and T = 1 architectures in vitro [Nodaviridae shows that the Orsay virus CP contains a basic-charged N-terminus but lacks the N-ARM  of National Cheng Kung University (IACUC #100065).E. coli BL21-CodonPlus(DE3)-RIL (Stratagene), and the cells were cultured in Luria Bertani (LB) broth (Merck) containing chloramphenicol (34 μg/ml) and ampicillin (100 μg/ml) until the OD reached 0.6–0.7 at 600 nm at 37°C. IPTG  (Bioshop) was added to a final concentration of 0.5 mM and cultures were incubated overnight at 18°C. The cells were harvested and disrupted by sonication in lysis buffer (50 mM Tris HCl (pH 8.0), 0.25 M NaCl, 20 mM imidazole, 5 mM β-mercaptoethanol and 1 mM EGTA). CP was purified through a Ni-NTA column . The SUMO-tag was cleaved using SUMO protease that was later removed with a Ni-NTA column.A consensus CP DNA sequence from the orange-spotted grouper nervous necrosis virus (OSGNNV) RNA2 (GenBank accession no KT071606) was amplified by PCR and cloned into a modified pET32-Xa/LIC vector carrying 6×histidine residues and yeast SUMO (SMT3) as the N-terminal fusion tag . This co4)2SO4 (750 mM) was added to the dialysis, and GNNV CP was finally dialyzed against the GNNV-LP formation buffer (20 mM Tris HCl (pH 8.0), 0.2 M NaCl, 1% (v/v) glycerol and 2 mM CaCl2). The size of GNNV-LP was measured by size-exclusion chromatography on a Superose 6 10/300 GL column . The purified GNNV-LP was concentrated to 30 mg/ml and stored at 4°C.The purified GNNV CP was diluted to a concentration of 0.3 mg/ml and dialyzed overnight at 4°C against lysis buffer without EGTA or β-mercaptoethanol at a ratio of 1:150. (NHThe truncated P-domain (residues 214−338), delta-P-domain mutant (residues 35−217) and N-ARM deletion mutant (residues 35−338) proteins were prepared using the same methods described above for GNNV CP. The truncated P-domain (20 mg/ml) and S-domain (30 mg/ml) proteins were stored in a buffer containing 300 mM NaCl and 50 mM Tris HCl (pH 7.5), whereas the N-ARM deletion mutant (30 mg/ml) was stored in the GNNV-LP formation buffer at 4°C.The purified GNNV-LP, the N-ARM deletion mutant and the delta-P-domain mutant were all diluted to a final concentration of 50 μg/ml and blotted on freshly glow-discharged, carbon-coated 200 mesh copper grids . Grids were negatively stained with 5 μl of 2% (w/v) uranyl acetate solution and screened using the H-7650 transmission electron microscope (Hitachi High-Technologies Co.) operated at 80 kV. All images were acquired using a 1024 x 1024 pixels CCD camera  and recorded at a magnification of 100,000 ×.3). All diffraction data were processed with HKL2000 [C2 space group with unit-cell dimensions of a = 477 Å, b = 422 Å, c = 337 Å, and β = 134°. The diffraction data of GNNV-LP crystals contained 499,184 reflections and was 98% complete at a resolution range from 50 to 3.6 Å. To help initial phase determination by ab initio phasing [The initial GNNV-LP crystallization experiment was performed at 18°C with the hanging-drop vapor-diffusion method. A Mosquito liquid-handling robot (TTP Labtech) was used for high-throughput crystallization condition screening. The initial condition of 0.2 M sodium formate (pH 7.2) and 20% (w/v) PEG3350 was obtained from the PEG/Ion Screen I kit (Hampton Research). This condition was further optimized to improve the diffraction quality and resolution of the crystals. Crystals appeared within 1−2 weeks. All crystals were cryoprotected with 25~30% (w/v) PEG3350 and frozen in liquid nitrogen before data collection. X-ray diffraction data were collected on BL44XU at SPring-8  with a CCD detector  using X-ray wavelength of 0.9 Å. All images were collected with an oscillation angle of 0.3° per frame with an exposure time of 3 s and a crystal-to-detector distance of 600 mm. A total of 600 frames were recorded on different positions from one crystal , 30% (w/v) PEG400; and 0.1 M NaCl, 0.1M lithium sulfate, 0.1 M MES (pH 6.5), 30% (w/v) PEG400, respectively. All crystals appeared within one week. X-ray diffraction data of the truncated P-domain and delta-P-domain mutants were collected on BL15A1 with a CCD detector  of NSRRC in Taiwan at a wavelength of 1.0 Å. The N-ARM deletion mutant was collected on BL44XU at SPring-8  with a CCD detector  at a wavelength of 0.9 Å. The diffraction data were processed with HKL2000 [The initial crystallizations of the truncated P-domain, delta-P-domain mutant and N-ARM deletion mutant proteins were performed with similar approaches as for the GNNV-LP. The initial crystallization conditions of the truncated P-domain, the delta-P-domain mutant and the N-ARM deletion mutant were 0.2 M Ca acetate, 0.1 M MES (pH 6.5), 10% (w/v) PEG8000; 0.2 M MgCl HKL2000 . All datT = 3 GNNV-LP were determined by the ab initio method using icosahedral non-crystallographic symmetry (NCS) averaging [κ = 72°, 120° and 180° hemispheres were analyzed with Molrep [T = 3 GNNV-LP particles in the monoclinic unit cell with one two-fold NCS axis of the virus particle coinciding with the crystallographic two-fold axis. The asymmetric unit contained half of the particle or 30 copies of the icosahedrally-related trimeric CPs. The spherical-shell model with uniform density was used as the starting model. The inner and outer radii of 119 and 159 Å, respectively, were chosen as initial parameters of the model [ab initio method, which used NCS-averaging (NCSA) with phase extension, a proper mask was necessary for dividing two regions: the protein region to be NCS-averaged and the solvent region to be flattened. The initial mask for NCSA and solvent flattening was created from the atomic structure of T = 3 FHV (PDB ID: 4FSJ) with a large mask-radius of 11~13 Å around each atoms (RAVE [CCP4 [1/a) (This process is referred as “procedure” hereafter). During cycles of iterations, the R factor and correlation coefficient comparing Fobs and Fcalc were monitored. The interpretable electron density map was successfully obtained . The complete models of the S-domain and the linker region were subsequently built up based on the amino-acid sequence of the GNNV CP manually .For structure determination of the on by DM  was used REFMAC5  and manuC5 [Coot  to fit th MAPROT , the modARP/wARP . Structu [PHENIX . Resolut [PHASER . The cryEpinephelus coioides) collected in Taiwan. The isolated virus was propagated in GF-1 cells and collected when 90% of the cells displayed a cytopathic effect (CPE). GF-1 cells were seeded in 12-well plates at a density of 1 x 105 per well in 2 ml L-15 medium supplemented with 5% (v/v) FBS, and cultured to 80−90% confluence. For infection, GF-1 cells were washed with PBS three times and subsequently infected with GNNV at a titer of 104 TCID50/ml in serum-free medium, 4% (w/v) PEG3000- and 4% (w/v) PEG8000-containing serum-free medium, respectively. After incubation with the virus for 30 min, the cells were washed thrice with PBS and L-15 medium (2 ml) supplemented with 1% (v/v) FBS in the presence or absence of PEG3000 and PEG8000 was added.The GF-1 grouper cell line  was cultAfter infection, GF-1 cells were washed with PBS, and RNA was extracted with the TRIzol reagent (Invitrogen). Reverse transcription and real-time quantitative PCR were performed as previously described . All datT = 3 GNNV-LP, the truncated P-domain (214−338) and the T = 1 delta-P-domain mutant (35−217) have been deposited at the Protein Data Bank (PDB) with accession codes 4WIZ, 4RFU and 4RFT, respectively.Nucleotide sequences of PCR-amplified fragments of OSGNNV RNA2 from have been deposited in the GenBank nucleotide database under the accession code KT071606. Atomic coordinates and diffraction data of the S1 FigT = 3 GNNV-LP  and FHV  and the first  interpretable electron density map at 3.7 Å and the ‘initial’ mask made from the T = 3 FHV (orange). (B) Inner surface view of the density map at 3.7 Å corresponding to the iASU, labeled accordingly with the Cα carbon skeletons of CP of GNNV (blue) and FHV (green) superimposed. RNA-binding α-helices of FHV are removed for clear view. (C) A stereo-view of the α-helix (residues 90–99) in the S-domain directed towards the corresponding ‘initial’ density map at 3.7 Å. (D) Progress of the R factors and correlation coefficients during phase extension. The ‘initial’ means the first interpretable trial by the ab initio method. The ‘final’ means the progress during the phase extension with the revised mask and the refined NCSA matrices.(A) Lateral view of the iASU of (TIFF)Click here for additional data file.S2 FigT = 3 GNNV-LPs; (B) T = 1 SVPs of the N-ARM deletion mutant; (C) the delta-P-domain mutant. Bar: 100 nm.Electron micrographs of negatively stained VLPs used for crystallization. (A) (TIFF)Click here for additional data file.S3 FigT = 1 assembly is well arranged in the corresponding unit cell with dimensions of a = b = 260.8 Å, c = 250.5 Å and γ = 120° in space group P6322. Five neighboring subunits of CP along the I5 axis are labeled as green, wheat, cyan, yellow and magenta colors, respectively. (B) Analyses of self-rotation functions of the T = 1 N-ARM deletion mutant. The NCS relationship was corroborated by the self-rotation functions of κ = 72°, 120° and 180° hemispheres, and calculated with Molrep [(A) Crystal packing of N-ARM deletion mutant in h Molrep .(TIFF)Click here for additional data file.S4 Fig2+ ions (yellow spheres) are incorporated at the interfaces of neighboring subunits A (cyan), B (green) and C (magenta), where Gln100, Asp130, Asp133, Ser170 and Glu213 (orange sticks) participate in Ca2+ coordination. (B) An illustration of the geometric and electrostatic differences on the inner surface between GNNV (left) and PaV (right). The charge distribution around the inner surface of the pentameric capsomers of empty GNNV-LP and PaV has a positive electrostatic potential (blue), and five short ordered encapsidated RNA duplexes are shown along the I2 axes. (C) The distribution of cysteine residues in the S-domains. A superimposition of S-domain structures from GNNV (magenta), SeMV  and Orsay virus  is shown. Cysteine residues are shown in stick, and Cys107−Cys195 of Orsay virus represents the disulfide linkage.(A) S-domains of three subunits per iASU. Three Ca(TIFF)Click here for additional data file.S5 FigThe crystal structures of the P-domain of the GNNV and Orsay viruses (PDB ID: 4NWV), and P1-domain of HEV (PDB ID: 2ZTN) and Calicivirus (PDB ID: 2GH8) are shown as ribbon diagrams. The N’ and C’ terminus of Calicivirus indicate the connecting regions between the P1 and P2 domains.(TIFF)Click here for additional data file.S6 Fig2+ using size-exclusion chromatography (SEC) on a Superdex 75 10/300 GL column . These data were compared to protein standards . (C) SDS-PAGE analysis of oligomerization of three CPs in solution. Full-length GNNV CP (lane 1) and the N-ARM deletion mutant (residues 35−338) (lane 2) exhibit monomeric and trimeric forms concurrently; and monomeric and dimeric forms of the delta-P-domain mutant (residues 35−217) (lane 3) are shown compared to protein standards (lane M).(A) Conservation of water molecules at the interfaces of neighboring subunits in the truncated P-domain. Three sets of two water molecules (blue spheres), at the subunit-interface regions, are coordinated with the conserved residues shown from two neighboring subunits in yellow and green, respectively. (B) The equilibrium properties of the monomeric and trimeric P-domains. The purified GNNV P-domain was analyzed in the absence (solid line) or presence (dashed line) of Ca(TIFF)Click here for additional data file.S7 Fig2+ ions and water molecules bound at the DxD motif. Two residues  coordinating two Ca2+ ions (yellow spheres) and water molecules (blue spheres) are conserved on the P-domain of each neighboring subunit. The |2Fo–Fc| map (orange mesh) around the binding site is shown with a contour level at 3σ. (B) There are three conserved GOL-binding pockets and one PEG-binding site on the electrostatic surface of the trimeric P-domains. Electron density maps of PEG (blue) and GOL (green) are shown with the |2Fo–Fc| coefficient and contour at 1σ around the interaction site. (C) A comparison of the linker regions between the truncated P-domain and the T = 3 GNNV-LP. Each hinge region is identified and color-coded as a rainbow gradient with respect to B-factor values  to depict the relatively rigid and flexible area. The three subunits  are shown with magenta, green and cyan, respectively.(A) A stereo view of the trimeric P-domains with Ca(TIFF)Click here for additional data file.S8 FigNodaviridae. The neighbor-joining phylogenetic unrooted tree was built using Mega5 [Nodaviridae are identified as alphanodavirus, betanodavirus, unassigned nodavirus and Orsay virus, respectively. (B) The sequence alignment of RNA2-encoded CP from different genotypes of betanodavirus. Multiple sequence alignment was performed with sequences of the CPs from OSGNNV, DGNNV, RGNNV, BFNNV, SJNNV and TPNNV using ClustalW. Each domain of GNNV CP is indicated on the top of alignment with colors as in DxxDxD and DxD motifs of GNNV CP are identified in the orange boxes. (C) N-terminal sequence identification of the CPs from different strains of the family Nodaviridae. Basic- and acidic-charged residues are colored in blue and red, respectively.(A) The phylogenetic tree of the family ng Mega5  with a m(TIFF)Click here for additional data file.


Three years after beginning employment at a bakery, a 32-year-old Japanese man began experiencing acute asthma exacerbations after exposure to rye flour. Antigen-specific serum IgE antibodies were detected to the albumin and globulin, gliadin, prolamin, and glutenin protein fractions of rye flour purified from the crude antigen, but only to the albumin and globulin fraction of wheat flour. The histamine concentration producing one-half maximal effect was lower for all four rye flour fractions than for the wheat flour fractions. After inhalation of the albumin and globulin fraction of rye flour, forced expiratory volume in 1 sec decreased to 77.7% of that pre-provocation. To our knowledge, this is the first report of baker’s asthma due to rye flour in Japan. Baker’s asthma is caused by inhalation of cereal flours and is a common occupational allergic disease in Europe and the United States, where bread is a staple food , 2]. Wh. Wh2]. WA 32-year-old Japanese male ex-smoker (Brinkman index: 390) presented with a history of allergic seasonal rhinoconjunctivitis since the age of 20, but with no history of asthma, although his younger brother did have bronchial asthma. The patient had worked as a baker since the age of 25. Since the age of 27, he had experienced rhinoconjunctivitis exacerbations while handling rye flour but not wheat flour. At the age of 28, he experienced the first episode of chest tightness, cough, and wheezing while handling rye flour; the episode persisted until the next day. At age 32, 30 min after eating 200 g of bread that included 50% rye flour, the patient experienced anaphylactic symptoms, including upper body urticaria, facial swelling, angioedema of the eyelids, and dyspnea, due to narrowing of the respiratory tract.We purified crude antigen extracted from wheat or rye flour into four protein fractions – albumin and globulin, gliadin, prolamin, and glutenin – as described . We perfThere were no abnormal chest radiograph findings. The patient had airway obstruction. His spirometry parameters when not experiencing acute asthma exacerbation were: forced vital capacity (FVC), 4.76 L; percent-predicted FVC, 115.0%; forced expiratory volume in 1 sec, 3.87 L; percent forced expiratory volume in 1 sec, 100.3%; maximum expiratory flow rate at 50% FVC, 3.86 L/sec; percent maximum expiratory flow rate at 50% FVC, 66.0%; maximum expiratory flow rate at 25% FVC, 1.48 L/sec; and percent maximum expiratory flow rate at 25% FVC, 47.9% (Table The incidence of cereal-induced occupational asthma is 0.081/1000 person-years in the UK and 3.4/1000 person-years in Norway [Recently, the demand for bread made with rye flour has increased in Japan because of the diversification of eating habits and greater concern for a healthy lifestyle. This is the first report of baker’s asthma caused by exposure to rye flour in a Japanese patient. This report will contribute to progress in the diagnosis and management of occupational asthma.