Covalent organic frameworks are a class of crystalline porous polymers that integrate molecular building blocks into periodic structures and are usually synthesized using two-component [1+1] condensation systems comprised of one knot and one linker. Here we report a general strategy based on multiple-component [1+2] and [1+3] condensation systems that enable the use of one knot and two or three linker units for the synthesis of hexagonal and tetragonal multiple-component covalent organic frameworks. Unlike two-component systems, multiple-component covalent organic frameworks feature asymmetric tiling of organic units into anisotropic skeletons and unusually shaped pores. This strategy not only expands the structural complexity of skeletons and pores but also greatly enhances their structural diversity. This synthetic platform is also widely applicable to multiple-component electron donor–acceptor systems, which lead to electronic properties that are not simply linear summations of those of the conventional [1+1] counterparts. Covalent organic frameworks are crystalline porous polymers integrating molecular building blocks into periodic structures. Here, the authors report a general multiple-component condensation strategy that enables the use of one knot and two or three linkers to synthesize complex, anisotropic frameworks. As a result, MC-COFs exhibit greatly enhanced electronic properties that are not simple linear summation of these of the conventional [1+1] two-component COFs. Therefore, the multiple-component COFs provide a new platform that considerably expands the designability of structures and functions of porous organic materials.COFs are typically synthesized via topologically directed [1+1] condensation reactions between a knot component and another linker unit. As a result, for example, only a maximum of 10 different COFs can be synthesized from a library of one knot and 10 linkers. Under this conventional [1+1] design scheme, development of new COFs is largely dependent on the exploration of new knot and linkers, which is however, tedious and unproductive. Here we report a general multiple-component (MC) strategy that allows for the use of more than two components for the topological design and practical synthesis of MC-COFs, which are formed in a single phase with permanent porosity and high crystallinity, irrespective of their components and topologies. Notably, this MC strategy is exceptionally effective at increasing the structural diversity of COFs, and a collection of one C2+C2 topology diagram using one knot and two linkers enables the synthesis of imine-linked kagome-type COFs with triple pores2 g–1 with the total pore volume of 0.30 cm3 g–1 and SIOC-COF-2 has much low surface area of 46 m2 g–1 and decreased pore volume of only 0.09 cm3 g–1. This condensation reaction is interesting for creating triple pores, but the low porosity and limited crystallinity suggest that the tetraphenylethene knot-based reaction systems are likely very sensitive to the length of the linkers while the reason for the extremely low porosity remains unclear. On the other hand, the use of a bifunctional linker of 4-formylphenyl boronic acid allows for the synthesis of COFs with two different boronate and imine linkages in the skeletons that are not available for conventional [1+1] based COFs63+C2 and C4+C2 schemes for the synthesis of hexagonal and tetragonal COFs. We highlight that these MC-COFs cannot be predicted and synthesized by using the above three approaches  and tetragon (C4) have three and two pairs of same-sized parallel sides, respectively. From the perspective of polygon geometry, stretching or shrinking along one pair of parallel sides can produce C2-symmetric hexagons and tetragons while retaining original 120° and 90° angles  and C4-symmetric nickel phthalocyanine (NiPc) units are representative knots used for the synthesis of hexagonal and tetragonal COFs124512471) and a long linker (E7) at a molar ratio of 1:2 or 2:1 as the linkers to condense with 2, 3, 6, 7, 10, 11-hexahydroxytriphenylene (TP) as the knots . Their structures were characterized through various methods  measurements of MC-COF-TP-E11E72  revealedd 26.04° , which wd 26.44° . Notablyd 26.44° . The Pawd 26.44° . We useder pairs . These s1H nuclear magnetic resonance (NMR) spectra. Resonances with the predicted coupling patterns were observed in the expected regions for each of the linkers' unique protons. By integrating the resonance peak intensities, the E1 and E7 linkers were present in ratios of 1:2 for MC-COF-TP-E11E72   . The belctively) . These o11E72 and MC-COF-TP-E12E71 were highly porous and exhibited typical type-IV sorption isotherm profiles , (110), (210) and (001) facets, respectively , which are characteristic of the unique carbon atoms of the TP, E1, and E7 units , (200), (300) and (001) facets, respectively  This strategy provides a method for preparing tailor-made, specially shaped pores that are difficult to achieve with other porous materials and might have applications in shape-selective separation and catalysis24252627282944π-arrays may trigger strong electronic correlations among the latticed π-components. The [1+2] copolymerisation of electron-donating TP knots with an electron-accepting E2 linker and other electron-donating E1, E3, E4 and E7 linkers yielded a series of electron donor–acceptor MC-COFs, including MC-COF-TP-E11E22, MC-COF-TP-E12E21, MC-COF-TP-E22E31, MC-COF-TP-E22E41 and MC-COF-TP-E22E71  compared with the conventional [1+1] counterparts π-units within the MC-COFs may be useful for enhancing a specific property or achieving a new function.Along this line of study, we further explored the possibility for the integration of MC electron-donating and -accepting units into MC-COFs in which the sequenced P-E22E71 . These M spectra . Notably spectra . An enhaπ-columnar arrays and unconventionally shaped pores. At the same time, this strategy considerably increases the structural diversity of COFs while retaining high crystallinity and porosity. We envisage that the MC-COFs constitute an important step towards various unprecedented molecular systems for functional exploration with enhanced structural complexity and diversity that are hardly available for conventional COFs architectures and other porous materials.In summary, we have developed a general strategy for the design and synthesis of crystalline porous COFs that enable the integration of multiple components into lattice structures with sequenced alignment. The multiple-component COFs greatly expand the structural complexity via asymmetric tiling of building blocks, providing a new platform for constructing anisotropic A mesitylene/dioxane (0.5 ml/0.5 ml) mixture of TP  and two edge units  at different molar ratios of 1/2 and 2/1 in a Pyrex tube (10 ml) was degassed by using three freeze–pump–thaw cycles . The tubA mesitylene/dioxane (0.5 ml/0.5 ml) mixture of TP  and three edge units  at molar ratio of 1/1/1 in a Pyrex tube (10 ml) was degassed by using three freeze–pump–thaw cycles. The tube was sealed off using flame and heated at 120 °C for 3 days. The precipitate was collected via centrifuge and washed with anhydrous acetone for five times. The powder was dried at 120 °C under vacuum overnight to yield the corresponding MC-COFs. Yields between 82 and 94%.A mesitylene/dioxane (0.5 ml/0.5 ml) mixture of NiPc  and two edge units  at the ratio of 1/1 in a Pyrex tube (10 ml) was degassed by using three freeze–pump–thaw cycles. The tube was sealed off using flame and heated at 120 °C for 3 days. The precipitate was collected via centrifuge and washed with anhydrous acetone for five times. The powder was dried at 120 °C under vacuum overnight to yield the corresponding MC-COFs.d6-dimethyl sulfoxide (600 μl) was added to dissolve the resulting solid for 1H NMR spectroscopy. The proton signals and their integrations were used for the quantitative determination of the components of MC-COFs.In general, dried MC-COF samples (10 mg) were dissolved in 1.0 ml HCl solution (20 wt% aqueous HCl solution) on sonication and digested for 12 h at 25 °C. The mixture was dried under vacuum for 16 h. Then I–V curves were recorded at bias voltages from −10 to 10 V.MC-COFs samples (5 mg) were dispersed in 2 ml anhydrous dichloromethane and sonicated for 10 min. The highly dispersed MC-COFs solution was dropped on the center of conducting electrodes as a film. The measurement was conducted on MC-COF films between 10 μm platinum electrodes at 25 °C in Ar using a two-probe method with a subfemtoamp sourcemeter . 1H NMR spectra were recorded on JEOL models JNM-LA400 NMR spectrometers, where chemical shifts (δ in p.p.m.) were determined with a residual proton of the solvent as standard. Solid-state 13C NMR spectra were recorded on JEOL model 920 MHz NMR spectrometer with a magnetic field of 21.62 Tesla. The frequency of the rotors was 15 kHz. For solid-state cross-polarization magic angle spinning 13C nuclear magnetic resonance (13C CP/MAS NMR), cross polarization with polarization inversion 1,808 scans (13C CPPI) and cross polarization with non-quaternary suppression (13C CPNQS) were performed with a delay time of 5 s. Ultraviolet–vis–infrared diffuse reflectance spectrum (Kubelka–Munk spectrum) was recorded on a JASCO model V-670 spectrometer equipped with integration sphere model IJN-727. Field-emission scanning electron microscopy was performed on a JEOL model JSM-6700 operating at an accelerating voltage of 5.0 kV. The sample was prepared by drop-casting a supersonicated solvents suspension onto mica substrate and then coated with gold. High-resolution transmission electron microscopy images were obtained on a JEOL model JEM-3200 microscopy. The sample was prepared by drop-casting a supersonicated tetrahydrofuran suspension of the COFs onto a copper grid. PXRD data were recorded on a Rigaku model RINT Ultima III diffractometer by depositing powder on glass substrate, from 2θ=1.5° up to 30° with 0.02° increment. Elemental analysis was performed on a Yanako CORDER MT-6 elemental analyser. Thermogravimetric analysis measurements were performed on a Mettler-Toledo model TGA/SDTA851 under N2, by heating to 800 °C at a rate of 10 °C min–1 with samples held in aluminium pans. Nitrogen sorption isotherms were measured at 77 K with Micromeritics Instrument Corporation model 3Flex surface characterization analyser. Before measurement, the samples were degassed in vacuum at 120 °C for more than 10 h. By using the non-local density functional theory (NLDFT) model, the pore volume was derived from the sorption curve.π-stacking interactions with starting structures created by AuToGraFSThe structures of COFs were calculated using the density-functional tight-binding (DFTB+) method including Lennard–Jones dispersion. The calculations were carried out with the DFTB+ program package version 1.2. DFTB is an approximate density functional theory method based on the tight-binding approach and utilizes an optimized minimal LCAO Slater-type all-valence basis set in combination with a two-center approximation for Hamiltonian matrix elements. The Coulombic interaction between partial atomic charges was determined using the self-consistent charge (SCC) formalism. Lennard–Jones type dispersion was employed in all calculations to describe van der Waals (vdW) and The data that support the findings of this study are available from the corresponding author on request.How to cite this article: Huang, N. et al. Multiple-component covalent organic frameworks. Nat. Commun. 7:12325 doi: 10.1038/ncomms12325 (2016).Supplementary Figures 1-193, Supplementary Tables 1-18, Supplementary Note 1 and Supplementary References


Almost all pre-miRNAs in eukaryotic cytoplasm are recognized and processed into double-stranded microRNAs by the endonuclease Dicer protein comprising of multiple domains. As a key player in the small RNA induced gene silencing pathway, the major domains of Dicer are conserved among different species with the exception of the N-terminal components. Human Dicer’s N-terminal domain has been shown to play an auto-inhibitory function of the protein’s dicing activity. Such an auto-inhibition can be released when the human Dicer protein dimerizes with its partner protein, such as TRBP, PACT through the N-terminal DExH/D (ATPase-helicase) domain. The typical feature of a pre-miRNA contains a terminal loop and a stem duplex, which bind to human Dicer’s DExH/D (ATPase-helicase) domain and PAZ domain respectively during the dicing reaction. Here, we show that pre-miRNA’s terminal loop can regulate human Dicer’s enzymatic activity by interacting with the DExH/D (ATPase-helicase) domain. We found that various editing products of pre-miR-151 by the ADAR1P110 protein, an A-to-I editing enzyme that modifies pre-miRNAs sequence, have different terminal loop structures and different activity regulatory effects on human Dicer. Single particle electron microscopy reconstruction revealed that pre-miRNAs with different terminal loop structures induce human Dicer’s DExH/D (ATPase-helicase) domain into different conformational states, in correlation with their activity regulatory effects.The online version of this article (doi:10.1007/s13238-014-0124-2) contains supplementary material, which is available to authorized users. MicroRNAs are emerging as an important regulator of almost all biological processes ranging from cell differentiation to senescence  3′ overhang. In a pre-miRNA maturation process, Dicer’s PAZ domain recognizes and binds with the 5′ phosphate and 2-nucleotide (nt) 3′ overhang of a pre-miRNA  domain in an open state. In comparison, the DExH/D (ATPase-helicase) domain in the hDicer-pre-miR-151A1I and hDicer-pre-miR-151A13I complexes appeared in a close state. The structural variation in good correlation with our dicing activity assay results indicated that the pre-miRNA’s terminal loop structure has a strong regulatory effect of hDicer’s activity via induced conformational change of the protein’s DExH/D (ATPase-helicase) domain.One of the ADAR family protein, ADAR1P110, was shown to induce the RNA editing of pre-miR-151 to generate three different products, pre-miR-151A1I, pre-miR-151A3I and pre-miR-151A13I by both We expressed and purified hDicer using the insect cell system to high purity Fig. A and 1B.ADAR enzymes deaminate the adenosine into inosine (A-to-I) within dsRNAs  domain plays an important auto-inhibition regulatory function on the enzyme’s dicing activity  after native gel electrophoresis and 5′-end-labeled with γ-32P-ATP (PerkinElmer) using T4 polynucleotide kinase (from TAKARA BIOTECHNOLOGY) before all related assays described in this work.All of our RNA substrates were synthesized by TAKARA BIOTECHNOLOGY. Human pre-let-7: 5′-UGAGGUAGUAGGUUGUAUAGUUUUAGGGUCACACCCACCACUGGGAGAUAACUAUACAAUCUACUGUCUUACC-3′, human pre-miR-151: 5′-CGAGGAGCUCACAGUCUAGUAUGUCUCAUCCCCUACUAGACUGAAGCUCCUUGAGG-3′, human pre-miR-151A1I: 5′-UCGAGGAGCUCACAGUCUAGUAUGUCUCAUCCCCUin vitro cleavage assay of different RNA substrates by hDicer were performed at 37°C for 1 h in 15 µL of reaction volume containing 60 nmol/L Dicer, 60 nmol/L γ-32p-labeled RNA substrates, 30 mmol/L Tris-HCl (pH 6.8), 50 mmol/L NaCl, 3 mmol/L MgCl2, 0.1% Triton X-100, 15% glycerol, 1 mmol/L DTT. Reactions were stopped with RNA loading buffer containing 8 mol/L Urea, 1× TBE, 0.05% Bromphenol blue and 0.05% Xylene cyanol, boiled for 4 min, then chilled on ice. RNA products were separated on an 18% polyacrylamide, 8 mol/L urea denaturing gel, visualized on a phosphor screen (Amersham Biosciences) with a Typhoon Trio Imager (Amersham Biosciences).The 2, 1 mmol/L DTT and 2 mmol/L EDTA. Samples were resolved through 6% native acrylamide gels in 0.5× Tris-glycine buffer under an electric field of 15 V/cm for 70 min visualized on a phosphor screen (Amersham Biosciences) with a Typhoon Trio Imager (Amersham Biosciences).For EMSA, RNA substrates were incubated with hDicer on ice for 40 min in a 20 µL volume containing 30 mmol/L Tris-HCl (Ph 6.8), 25 mmol/L NaCl, 2 mmol/L MgCl2, 1 mmol/L DTT and 2 mmol/L EDTA and 5% glycerol. All the samples were negatively stained on holey carbon grids covered by a thin layer of continuous carbon over holes with 2% (w/v) uranyl acetate solution. All micrographs were collected on a Tecnai F20 Twin transmission electron microscope (FEI) running at 200 kV or an Tecnai-12 Biotwin electron microscope (FEI) operated at 120 kV, using a nominal magnification of 50,000× or 49,000×, respectively. The micrographs were collected on Ultrascan 4000 CCD camera (Gatan Inc.) at specimen-level pixel sizes of 0.223 nm or 0.29 nm, respectively, using a dose of ~30 e− Å−2 and a nominal defocus range of −1 to −3 μm.Human Dicer-RNA complexes were assembled by pre-incubating 150 nmol/L hDicer with a 500% excess of unedited and edited pre-miR-151 in a specific volume containing 20 mmol/L Tris-HCl, pH 6.8, 25 mmol/L NaCl, 2 mmol/L MgClFor 3D classification of apo-hDicer and hDicer-RNA complexes, about 70,000 particles of each sample were picked together from each set of micrographs. EMAN2 Below is the link to the electronic supplementary material.


IGHG for six other genera: Bunolagus, Brachylagus, Lepus, Pentalagus, Romerolagus and Sylvilagus. The newly sequenced leporid IGHG gene has an organization and structure similar to that of the European rabbit IgG. A gradient in leporid IgG constant domain diversity was observed, with the CH1 being the most conserved and the CH3 the most variable domain. Positive selection was found to be acting on all constant domains, but with a greater incidence in the CH3 domain, where a cluster of three positively selected sites was identified. In the hinge region, only three polymorphic positions were observed. The same hinge length was observed for all leporids. Unlike the variation observed for the European rabbit, all 11 Lepus species studied share exactly the same hinge motif, suggesting its maintenance as a result of an advantageous structure or conformation.Immunoglobulin G (IgG) is the predominant serum immunoglobulin and has the longest serum half-life of all the antibody classes. The European rabbit IgG has been of significant importance in immunological research, and is therefore well characterized. However, the IgG of other leporids has been disregarded. To evaluate the evolution of this gene in leporids, we sequenced the complete  IgG participates directly and indirectly to immune responses through its effector functions, elicited through neutralization and binding to Fcγ receptors (FcγRs) and C1q. These functions include antibody-dependent cellular cytotoxicity, antibody-dependent cell phagocytosis and/or complement-dependent cytotoxicity . In mammd and e, each with two segregating alleles, d11/d12 and e14/e15, respectively , w, wd and mbering)   [Lepus is a polytypic, cosmopolitan genus, comprising 24–30 currently recognized species [Nesolagus, Pronoloagus and Sylvilagus, which comprise two, four and more than 16 species, respectively, are monotypic genera [Nesolagus, Poelagus and Pronolagus, and the second group includes the remaining eight genera. Within this second group, Romerolagus was the first genus to diverge, around 13 Ma, followed by Lepus, which diverged around 12 Ma. Brachylagus and Sylvilagus diverged from a group composed of Bunolagus, Caprolagus, Oryctolagus and Pentalagus around 10 Ma. The Brachylagus–Sylvilagus and Bunolagus–Caprolagus–Oryctolagus–Pentalagus splits are estimated at 9 Ma (approximate times [The family Leporidae comprises 11 genera: vilagus) ,24. Lepu species , and mos species ,28. The c genera –34. Somec genera ,28. MoleIGHG gene for six additional extant leporid genera: Bunolagus, Brachylagus, Lepus, Pentalagus, Romerolagus and Sylvilagus.In this study, we extend the knowledge on this immunoglobulin class in leporids by sequencing the complete 3.Bunolagus, Brachylagus, Lepus, Pentalagus, Romerolagus and Sylvilagus genera were extracted from frozen liver or ear tissue using an EasySpin Genomic DNA Minipreps Tissue Kit (Citomed). Additionally, six European rabbits were also analysed: two individuals of the subspecies Oryctolagus cuniculus algirus, three individuals of the subspecies Oryctolagus cuniculus cuniculus and a domestic rabbit belonging to the New Zealand White breed. PCR amplification of the four IGHG exons was conducted using primers designed on the basis of European rabbit IGHG available sequences . A fragAY386696  under thAY386696  and RG31Lepus granatensis, one Lepus europaeus and one Sylvilagus floridanus. Total RNA was extracted from spleen samples using RNeasy Mini Kit , following first strand cDNA synthesis using oligo(dT) as primer  and SuperScript III reverse transcription (Invitrogen) as recommended by the manufacturer. A mid-CH1 to mid-CH2 fragment was PCR amplified using primers FG1int2 (5′ CCAGTGACCGTGACTTGGAA 3′) and RG2int2 (5′ GGACTTTGCACTTGAACTCC 3′) designed in conserved regions of the leporid IGHG gene segment. A touchdown PCR was performed and the conditions were as follows: 3 min at 98°C followed by five cycles at 98°C (30 s), annealing starting at 66°C with a 1°C decrease/cycle until reaching 62°C (30 s) and 72°C (30 s), followed by 30 cycles at 98°C (30 s), 62°C (30 s) and 72°C (30 s), with a final extension at 72°C (5 min).To confirm the hinge length a fragment of the expressed IgG was also sequenced for one ioEdit software [ioEdit [naSP v. 5.10 [Sequences obtained in this study were edited and aligned using CLUSTAL W  as implesoftware  and the  [ioEdit . The obt [ioEdit . Amino a [ioEdit . Sequenc v. 5.10 .Table 1http://clavius.bc.edu/~clotelab/DiANNA/) [The secondary structure of the leporids IgG heavy chain was analysed using the DiAminoacid Neural Network Application (DiANNA) (DiANNA/) –42. DiANThe nucleotide sequences' alignment was screened for recombination as it can mislead positive selection analysis ,44. For ega 5 [To identify signatures of selection on leporid IgG, we compared the rate per site of non-synonymous substitution (dN) with the rate per site of synonymous substitutions (dS) in a maximum-likelihood (ML) framework, using six different methods. As each of the methods employs unique algorithms, and as done previously –49, we oega 5 , a neighega 5  were alswww.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml [The location within the IgG structure of the residues under positive selection was analysed by mapping the residues onto the solved crystal structures of rabbit IgG–Fab (PDB ID: 4HBC ) and Fc 3d.shtml ) was use4.IGHG gene newly sequenced for six leporid genera, Bunolagus, Brachylagus, Lepus, Pentalagus, Romerolagus and Sylvilagus, is similar to the European rabbit (Oryctolagus) IGHG, and thus the intron–exon organization was inferred from available published rabbit IGHG genes. Splicing signals were present at the intron boundaries and hence it is assumed that all studied leporids share the same IGHG exon organization as the European rabbit.The 4.1.Bunolagus IgG has a His for a Glu substitution at position 84.2 and Lepus IgG has a Thr for a Ser change at position 90. Only the substitution at position 84.2 involves changes in amino acid characteristics (a).The CH1 domain is the most conserved among leporid IGHG domains with only 14 amino acid variable positions, the majority of which involve one substitution, and half are conservative regarding the amino acid properties. The European rabbit IgG differs from the IgG of other leporids by the Asn/Ser change at position 45.3 and by the Thr/Pro change at position 92. eristics a.Figure4.2.Lepus, Brachylagus and Bunolagus, respectively. Romerolagus uniquely has Ala1.6, Leu45.4, Val82 and Leu85.2 residues (a).The CH2 domain of leporid IgGs, though fairly conserved, shows more diversity than the CH1 domain. For the CH2 domain, 15 amino acid variable positions were observed, 11 of which involve one substitution but, contrary to that observed for the CH1 domain, the majority of these polymorphic positions involve changes in amino acid properties. The European rabbit has specific Arg residues at positions 45.4 and 125. The residues Leu17, Pro45.4 and Leu84.1 are specific to residues a.4.3.IGHG domains with 36 variable positions, the majority involving changes in amino acid properties. It also has the highest number of diagnostic positions. In this domain, the European rabbit has specific Pro1.2 and Ala45.2 residues, Bunolagus uniquely has Val1.2, Arg3, Thr17 and Pro35 residues, and Brachylagus has an Asn for a Thr change at position 80. Three diagnostic positions were observed for each of three genera: Lepus at Asn15, Thr105 and Leu125 residues, Pentalagus at Arg1.3, Ser77 and Ala90, and Romerolagus at Lys11, Glu35 and Ala120 as distinctive. Only Sylvilagus lacked specific residues (b).This is the most diverse of the leporid residues b.4.4.Sylvilagus and Romerolagus, which share the same substitutions: Val1, Pro8 and Leu9 (a). For all studied leporids, an 11-residue hinge was observed, which, given the high variability observed in IgG hinge length in other mammals, is surprising. To check whether alternative splicing sites could be used by some leporids, the expressed hinge was sequenced for Lepus and Sylvilagus individuals, and each one had the same 11 residues.Three variable positions were found, all involving changes in amino acid properties. Changes at these positions define genera-specific motifs, with the exception of and Leu9 a. For al4.5.introns = 0.03704; Piexons = 0.03277). The major differences observed between leporid genera are insertions and deletions (indels), which reflect the accepted leporid phylogeny. Indeed, in the intron between CH1 and hinge exons, a 20 bp deletion is shared by Bunolagus, Oryctolagus and Pentalagus, whereas Romerolagus has a unique 10 bp deletion. Similarly, in the intron between CH2 and CH3 exons, a 1 bp deletion is shared by Brachylagus and Sylvilagus, whereas Oryctolagus has two characteristic 1 bp deletions and Romerolagus has an insertion of 13 bp .The intronic regions between CH1–hinge, hinge–CH2 and CH2–CH3 were fully sequenced. Overall nucleotide diversity for these regions is similar to that observed for the coding regions  for one L. granatensis allele. Disulfide bond prediction analysis indicates that the extra CH2 cysteine does not establish any bond. However, for the allele with the additional CH3 Cys, different bonds to those described above are predicted for the CH2 and CH3 domains. The CH2 domain Cys at position 23  is now predicted to bond with the CH3 domain Cys at position 104 , and the CH3 domain Cys at position 23  is predicted to bond with the CH3 domain Cys at position 124 . However, the physiological relevance of these predictions remains unclear.All immunoglobulin heavy chains are organized into globular domains, a structure stabilized by intra-chain disulfide bonds between conserved cysteines in each domain at positions 23 and 104 in each domain. As expected, all studied leporids share these cysteines. The European rabbit IgG further has two additional cysteines in the CH1 domain at positions 10 and 11  and a further two in the hinge, at positions 5 and 10. The CH1 domain Cys at position 10  bonds to the light chain Cys, the CH1 Cys at position 11  bonds to the Cys at position 5 in the hinge, and the other hinge Cys forms the inter-chain disulfide bridge with its equivalent in the other heavy chain. Again, conserved cysteines at these positions are shared by all studied leporids, suggesting that the IgG heavy chain structure is maintained across leporids. However, extra cysteines were found in the CH2 domain at position 1.5  of two Lepus and Bunolagus hinge position 8, and Pentalagus hinge positions 8 and 9, all occupied by Ser residues.Glycosylation is also important for protein structure and function. The European rabbit IgG Fc has an N-glycosylation site at CH2 84.4 , which was found to be conserved in all studied leporids, and indeed all vertebrate IgG. No other N-glycosylation sites were predicted. O-linked glycans (i.e. glycans linked to Ser/Thr residues in Ser/Thr/Pro-rich domains are known on human IgA1 and IgD hinges) and also on the rabbit IgG hinge, for which the Thr residue at hinge position 9 of d12 rabbits is O-glycosylated (d11 rabbits hinge position 9 have a Met residue) ,63,64. T4.7.α < 0.01). Comparing the sites recognized by each of the six employed methods led to the identification of seven PSCs. One locates to the CH1 domain, two to the CH2 domain and four to the CH3 domain. All of these residues show changes in amino acid characteristics and occupy exposed positions on the European rabbit IgG structure. Interestingly, four of these codons locate near sites of interaction with ligands: the CH1 residue 1.3  is in the immediate vicinity of the VH domain, the CH2 residue 1.5  lies in the region of residues that interact with FcγRs, and the CH2 92 residue  and CH3 45.2 residue  locate on the region of residues that interact with FcRn. Of note, the CH3 domain residues at positions 98, 100 and 101  form an exposed cluster in the C-terminal portion of this domain  showing a significantly better fit than the model that did not (M7) . A gradient in constant domain diversity is observed with the CH1 domain being the most conserved of leporid IGHG domains and the CH3 domain the most variable. In fact, there are twice as many variable amino acid sites and number of diagnostic substitutions in the CH3 domain compared with the CH1 and CH2 domains, making the CH3 the most informative domain for leporid species identification. The CH3 domain has been identified as the most diagnostic domain to distinguish between IgG isotypes for swine [Oryctolagus and Romerolagus, genera that separated around 13 Ma [The results obtained in this study reveal that the leporids share considerable sequence similarity for their or swine  and primor swine . The divor swine  is similnd 13 Ma , and thuIGHG, hotspots of variability exist in the hinge and CH2 and CH3 domains, and we found evidence of positive selection acting on all IgG constant domains. Previous studies of leporid IGHG described the hinge position 9 and CH2 position 92 as hotspots of variability [Lepus-specific hotspot, with four different residues in this genus but only 2 in other leporids. Additionally, we can pinpoint as having high amino acid diversity the CH2 position 45.4  and CH3 position 100 , each having five different residues in the studied leporids. These hotspots of variability, and also the seven positions identified as positively selected, exhibit changes in amino acid physico-chemical properties, which may impact on the IgG conformation and structure. Changes at the positively selected CH1 1.3 residue  may impact on the antigen-binding site conformation. This would possibly improve leporid antigen-binding possibilities given that the usage of the VH1 gene in 90% of VDJ rearrangements in leporids confers a somewhat restricted diversity to the leporid VH domain [Despite the overall conservation found for leporid iability ,22. Our H domain –70. On tH domain ,71. As IH domain , consistH domain ) and theH domain ). IntereH domain , so it iIn contrast to what was found for mammalian IgA, where the Cα3 domain showed less evidence of having evolved under positive selection than Cα1 or Cα2 , the lepLepus IgG hinge is particularly interesting. Previous studies by Esteves et al. [Lepus, Bunolagus and Pentalagus hinge may protect the IgG from effects of proteases of pathogens and tumour cells. Despite this increased resistance against proteolytic attack of the d12 allotype, both allotypes interact with FcγR equally well . ,77. 10,7s et al.  indicates et al. ,79. The s et al. . Thus, tell (e.g.). We hav


Flavin-dependent monooxygenases play a variety of key physiological roles and are also very powerful biotechnological tools. These enzymes have been classified into eight different classes (A–H) based on their sequences and biochemical features. By combining structural and sequence analysis, and phylogenetic inference, we have explored the evolutionary history of classes A, B, E, F, and G and demonstrate that their multidomain architectures reflect their phylogenetic relationships, suggesting that the main evolutionary steps in their divergence are likely to have arisen from the recruitment of different domains. Additionally, the functional divergence within in each class appears to have been the result of other mechanisms such as a complex set of single-point mutations. Our results reinforce the idea that a main constraint on the evolution of cofactor-dependent enzymes is the functional binding of the cofactor. Additionally, a remarkable feature of this family is that the sequence of the key flavin adenine dinucleotide-binding domain is split into at least two parts in all classes studied here. We propose a complex set of evolutionary events that gave rise to the origin of the different classes within this family.  •Changes in domain architectures reflect the phylogeny of flavin monooxygenases.•Recruitment of different domains has been the main force driving its evolution.•A notable feature of flavin monooxygenases is that the flavin adenine dinucleotide-binding domain is split.•Classes of monooxygenases emerged from an ancestral domain by structural changes. Flavin-dependent enzymes are widespread in nature and perform a wide variety of redox reactions including hydroxylation, reduction, halogenation, monooxygenation, DNA repair, light emission, and cellular signaling Their cofactors, the flavins, are versatile redox compounds derived from vitamin B2 (riboflavin), the most common being flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). Flavins can receive up to two electrons from a reducing compound and then transfer them to electron acceptors. Two major groups of flavin-dependent enzymes react with molecular oxygen: the flavin-dependent oxidases and the flavin-dependent monooxygenases Flavin-dependent monooxygenases, which show a high degree of sequence divergence, have been classified into eight classes (A–H) et al.et al.All flavin-dependent monooxygenases belong to the Enzyme Commission (EC) oxidoreductase sub-subclasses: EC 1.14.13, EC 1.14.14, and EC 1.13.12 H. In flavin monooxygenases, it occurs in classes A, B, E, F, and G , where iThe FAD-binding domain occurs in different contexts in the flavin monooxygenases. In all cases it is split into two or three parts by the insertion of different domains or subdomains; that is, the FAD domain is formed from two noncontiguous regions of the protein sequence that are nevertheless able to maintain the domain's 3D structure and function. In the case of class B monooxygenases, the inserted domain is a second copy of the FAD-binding domain, as will be described later.This paper focuses on the classes having the FAD-binding domain in common , which we will refer to as Group 1 classes . Of the To get a preliminary insight into the evolutionary relationships between these enzymes, we compiled a dataset of 196 enzymes from Group 1 on the basis of their assigned EC numbers (Dataset S1). The sequence divergence among these proteins was too high to be able to determine homology, so a comparison of their 3D structures was undertaken. Structure provides a more sensitive measure of shared ancestry as it tends to be better conserved than sequence over evolutionary time To probe the evolutionary relationships among the Group 1 classes, we derived a phylogenetic tree based on the structural similarities of the proteins in these classes. Using the available 3D structures (Table S2), we performed an all-against-all structure similarity analysis with the help of the fold comparison program PDBeFold O13306, F2I9L3, O66402, K7STL0, and O48651) and zeaxanthin epoxidase We then focused on the FAD-binding domain common to our five Group 1 classes. Using the sequence similarities of just these domains (from proteins with and without 3D structures), we obtained the phylogenetic tree shown in A striking feature of our Group 1 flavin-dependent monooxygenases is that, as mentioned previously, the sequence of the FAD-binding domain is split into at least two noncontiguous segments. A closer look at In the second clade (classes G and B), major differences are observed. In Class G, the FAD-binding domain is also split into three parts—with lengths of around 120, 90, and 70 aa—but they are interspersed by CATH domain 3.90.660.10 here , which is typical of subunit A of the monoamine oxidases (MAOs). Additionally, the C-terminal region contains an α-helix domain (brown) of ~ 130 aa The above observations seem to suggest that the divergence of the Group 1 flavin-dependent monooxygenases into the five classes was principally driven at different evolutionary times by the divergence of their domain architectures b.As all the 3.50.50.60 domains in these enzymes are split , we examined the locations of the splits and any differences in lost/retained secondary structure elements. Considering the monophyly of classes A and B and that both are able to use FAD and NAD(P)H, further studies were performed to understand the biochemical differences on the basis of their evolutionary histories.p-hydroxybenzoate hydroxylase (PHBH) Class A is the archetypical flavin-dependent monooxygenases The other domain in the class A enzymes is the split 3.30.9.10 domain. It is difficult to envisage how two separate domains might have merged in this way, where each is separately split. A similar domain architecture is found in the D-amino acid oxidases, such as PDB entry: 1C0P m-hydroxybenzoate hydroxylase (PDB entry: 2DKH) and phenol hydroxylase (PDB entry: 1FOH), contain an extra C-terminal domain (CATH 3.40.30.20), which seems to be involved in protein–protein interactions and not in defining the catalytic site. This is also observed for the enzymes angucycline synthase , aklavinone 11-hydroxylase (PDB entry: 3IHG), and rebeccamycin synthase , which have C-terminal extensions that seem to be involved in quaternary interactions; these enzymes take part of multienzyme complexes such as the polyketide synthases Other class A monooxygenases, such as The unique domain architecture observed in class B, where a FAD-binding domain has been split in two by the insertion of a slightly truncated copy of the same domain, suggests a duplication event as the origin of this class. The first domain retained its ability to bind FAD, while the second evolved the ability to bind NAD(P)H. Superposition of the two domains places the two cofactors in the same but slightly shifted orientationrelative to one another. From the topology diagram , it was Of the three kinds of class B monooxygenases, the FMOs have a slightly shorter NAD(P)H-binding domain than the BVMOs and NMOs. The divergence of these three groups could have been the result of changes linked to the structure of the NAD(P)H-binding domain. The FAD-binding domains are virtually identical, in terms of 3D structure, within the class, while the NAD(P)H domains have extra embellishments, such as long interleaved α-helix structures in the BVMOs. In NMOs, the domain remains almost identical to the FAD-binding domain. It is evident that this domain has suffered several structural changes on its surface, while the core involved in the binding of the cofactor has remained conserved.Finally, we examined how the residues involved in binding have changed over evolutionary time and how this might have resulted in the enzymes' diverse functions. We first divided the proteins into two groups: those that have one copy of the FAD-binding domain  and those that have two, that is, class B. We then used the SAS (Sequence Annotated by Structure) server The second group of enzymes, the class B monooxygenases, gave 49 interacting residues. Phylogenetic analysis revealed that these residues contain the information determining their divergence into FMOs, BVMOs, and NMOs b.These results evidence the complex set of factors defining and constraining the evolution of flavin-dependent monooxygenases. As proposed above, it seems the main topology of these enzymes' phylogeny has been driven by changes in domain architecture, while the functionalization within the different classes has been driven by the accumulation of changes in the cofactor and substrate-binding sites.Enzyme evolution is extraordinarily complex, with many different evolutionary events yielding dissimilar chemistries and mechanisms within a superfamily Flavin-dependent monooxygenases are enzymes that display a remarkable versatility in their cellular functions. Here, we have demonstrated that a subset of these enzymes  shares a common origin and that a complex set of events has taken place during their evolutionary history to result in the differences between them. Their common ancestor would appear to have been a single-domain protein consisting of just a FAD-binding domain. This is a very common, widely distributed fold found in an enormous variety of enzymes 4HL9), so this apparent subdomain of CATH 3.30.9.10 is actually a domain in its own right. The other two insertions—a beta-hairpin unit and a single alpha-helix— are very small fragments, or rather embellishments, that might have been inserted into the FAD-binding domain or evolved by decoration at different evolutionary times.Different evolutionary events led to the divergence of the ancestral and single-domain protein into the five classes. Probably, the divergence into clades I and II  was the The recruitment of domains has previously been reported as responsible for the different functionalities in other superfamilies—for example, the Ntn-type amide hydrolase superfamily A separate set of evolutionary events led to classes G and B . In the In this scenario, it seems reasonable to propose that class A and class B monooxygenases, which overlap in their taxonomic distribution but not in their reaction profiles, have emerged to solve different problems in nature, converging in their usage of cofactors. Class A monooxygenases are strongly dependent on the presence of substrate in order to enter into the catalytic cycle Our analysis of the origin of the diversity and enzyme function of the flavin-dependent monooxygenases demonstrates that a complex set of factors drove the evolution of this family of enzymes. The multidomain organization plays a key role in defining each class and conferring specific properties such as the ability to work with a reductase partner or form multienzymatic complexes. However, the accumulation of point changes on the shared FAD-binding domain has led to the functionalization of each class and, in the case of class B monooxygenases, has ultimately driven the divergence into the three groups of FMOs, BVMOs, and NMOs.Enzymes in the EC sub-subclasses 1.13.13, 1.14.13, and 1.14.14 were identified, analyzed, and collected from the enzyme information system BRENDAThe CATH hierarchical classification of protein domains Sequence-based MSAs were constructed by employing the MAFFT program version 7 via the online serverStructure-based alignments were constructed by employing the tool PDBeFoldThe initial evolutionary analysis was performed based on the MSAs of the amino acid sequences of the FAD-binding domain regions. The best fit model parameters were calculated using ProtTest version 3.4 The structure-based phylogeny employed the 3D alignments from PDBeFold and the same inference method as above. To test the evolutionary hypothesis, we also used Bayesian inference to construct a tree with Mr. Bayes software version 3.2 Topology diagrams were manually drawn based on the secondary and tertiary structure schemes obtained from PDBsum, as were the residues involved in the binding of ligands.Structures were visualized, compared, and analyzed using the PyMOL v1.7.6 molecular visualization system. PDB files were obtained from the RCSB PDB. The structure of the separate domains was obtained by manually editing the PDB files according to the information given by CATH.To identify the residues involved in the binding of ligands FAD, NADPH, and substrate, we used the SAS (Sequence Annotated by Structure) server


Priacma serrata (LeConte) (Coleoptera: Archostemata) were studied. The combination of these techniques allowed for studying mechanical properties of the headmandible- system without using living animals. The µCT analysis delivered precise volumetric data of the geometry of the system to be studied. Dimensions of the cuticle of the parts involved could be readily deduced from the µCT-data. Thus, an exact representation of the specimen without significant artifacts like deformations and misalignments, usually resulting from histological sectioning, could be reconstructed. A virtual 3D model built from these data allowed for investigating different stress scenarios with finite element analysis. Combining these methods showed that P. serrata most likely uses its robustly-built mandibles for cutting hard material. In combination with available information on its habitat, possible food sources are discussed.With the help of scanning electron microscopy, high resolution X-ray tomography (µCT), and finite element analysis, the mechanical and functional properties of the mandibles and associated muscles of the beetle  An important aspect for understanding nature and the interactions of organisms is to know the biological capabilities and requirements of as many species as possible. For example, knowledge of species' life cycles, modes of feeding, preferred habitats, etc., is crucial to understand how ecosystems are functioning. Unfortunately, this knowledge is still very scarce for the largest percentage of insect species.The best way to learn about the biology of a species is to observe its actions in its natural environment; this, unfortunately, often is not possible, because many insect species are very rare and difficult to find or they have not yet been observed alive at all, and often only the type specimens that were used to describe it are known. Under these circumstances, a helpful, indirect way to deduce biological capabilities of a species is to investigate its functional morphology.Priacma serrata (LeConte) (Coleoptera: Archostemata) is a characteristic example of a species whose biology is entirely unknown, although its morphology has been investigated in great detail  half of the head. The insertion on the mandible is mediated by a broad tendon that is nearly as long as the mandible itself. The abductor (M12) is significantly smaller. It originates from the ventro-lateral anterior half of the head capsule and inserts via a narrow tendon on the outer rim of the mandible, between the dorsal and ventral joints.Each mandible is attached to the head capsule via two joints. The posterior or ventral joint is the older one in evolutionary terms. It is composed of a ball on the posterior outer rim of the mandible and a corresponding socket in the head capsule. The anterior or dorsal joint is built vice-versa, i.e., the ball is on the head capsule and the socket on the mandible , 2C. TwoP. serrata are unknown. This lack of knowledge prevents studying this species in live experiments, even though it can be easily collected , high resolution X-ray tomography (µCT), and FEA allowed the mechanical and functional properties of the mandibles and associated muscles of Giving a detailed introduction into the basics of µCT and FEA here is far beyond the topic of this paper. However, a comprehensive account of µCT investigation of insects is give by Betz et al.  and examP. serrata and the associated muscles interact and how stress is distributed in the mandibles. Additionally, possible points of weakness in the system of mandibles, head capsule, and associated muscles were assessed. Behind these details stands the question on whether P. serrata uses its mandibles to process and/or access food or whether they are “only” there to impress other males in competitions for females as, for example, in the European stag beetle, Lucanus cervus. In combination with data available on its environment and behavior, possible food sources of P. serrata are discussed.A central aim of the virtual experiment was to learn how the mandibles of All 12 specimens used for the investigation were collected by T. Hörnschemeyer in the vicinity of Bozeman, Montana, USA, in June 1996. The specimens were fixed in Dubosq- Brasil  and tranwww.leica-microsystems.com), sputter-coated with gold , and investigated in a Leo 438VP SEM .For SEM investigation, the mandibles of 11 specimens were removed, dehydrated using absolute ethanol, dried at the critical point . The resulting dataset comprised 356 2D slices with a voxel size of 5.429 × 5.429 × 10.858 µm. Visualization of the resulting data was partly done with Amira 4.0 .The head of the specimen for the µCT investigation had a width of 2.1 mm and a single mandible measuring approximately 0.6 × 1.3 mm. It was dehydrated via absolute ethanol and dried at the critical point. The dry specimen was mounted on a standard SEM specimen holder with dental wax. The measurement was made with a Skyscan 1072 highresolution µCT system at 80 kV and 100 µA (http://www.simpleware.com/). The identification of cuticle and the differentiation between mandible and head capsule were done with a combination of automatic recognition of grey value distribution with subsequent manual correction. In order to remove errors within the preliminary 3D model, a floodfill operation was used to eliminate specified unconnected regions. The antennae of the specimen were manually removed from the model in order to reduce the number of elements present in the analysis. The voxel model was imported into +ScanFE  and smoothed with the following parameters: extra surface smoothing; curvature cutoff at 0.5; maximum iterations of 2; and a minimum quality target of 0.15. All other parameters remained at the default values.The 2D slices from the µCT data were combined into a 3D model using the program ScanIP , the model was divided into two separate material types: the mandibles and the rest of the head structure. To reduce the complexity of the model so that analysis was possible with the available computing power within a reasonable timeframe, the original data were resampled so that the resulting mesh consisted of fewer elements at the expense of model accuracy. A cubic resampling was done to a pixel spacing of 0.02169 mm (resample 50%), arriving at a tetrahedral mesh with node and element numbers as given in To prepare the data for the FEA with the program Abaqus , Poisson's ratio is the ratio of the contraction perpendicular to the applied load to the extension in the direction of the applied load. In the case of compression, it is the ratio of extension perpendicular to the load to compression in the direction of the load. Values for most materials are in the range of 0 to 0.5 .Principal strain: describes the deformation of an object in principal directions given as the amount of length change in relation to the original length .Total strain energy: describes the work that is utilized when deforming a structure .von Mises stress: Stress is a tensor quantity with nine components. The von Mises stress is a stress index especially suited for failure analysis and is a combination of these components .2 is used .In the case of The mandibles look quite strong, but in insects this does not necessarily mean that they are used for feeding. They could also be used as a kind of weapon or just for show when males compete for resources like females or territories, as in some stag beetles (Lucanidae).Nevertheless, the finite element analysis based on µCT data combined with SEM investigation of the mandible surfaces showed that the mandibles were built to withstand comparatively strong forces. The possible points of failure of the mandible structure were the mesal cutting edge posterior of the teeth and the narrow point of attachment of the tendon of the adductor muscle M11; . The strThis investigation illustrates that it is possible to simulate complex problems with a high degree of accuracy by combining µCT and image-based meshing techniques. FEA simulations of a model based on µCT data can be very helpful to understand the functional morphology of certain parts of an insect's body, especially where observations of living specimens are not possible.The additional SEM examination of the surfaces of the mandibles showed that there was heavy abrasion in certain areas, which indicates that the mandibles are used for cutting comparatively tough materials rather than just for impressing male competitors.vice versa. Furthermore, there always seemed to be some free space between the two mandibles, even when they were completely closed.The kind of abrasion observed in the mandibles also makes threatening behavior, like rubbing the mandibles together to produce sound for repelling possible predators or competitors (as other beetles are capable to do ), seem uTenomerga  and often have a more vertically oriented teethed cutting edge, as for example in Tenomerga , usually in the vicinity of Douglas Fir, Pseudotsuga menziesii, forests. If the beetle feeds on this plant, which seems to be very likely, there remain two possible food sources. The beetles might use their strong mandibles to cut into the bark and wood, probably of smaller twigs, to get to the sap of the tree, or they might open the fir cones to feed on the seeds. Since P. serrata does not have mouthparts specialized for taking up fluids ) or in Collembolans . Results


Viral infections result in millions of deaths in the world today. A thorough analysis of virus-host interactomes may reveal insights into viral infection and pathogenic strategies. In this study, we presented a landscape of virus-host interactomes based on protein domain interaction. Compared to the analysis at protein level, this domain-domain interactome provided a unique abstraction of protein-protein interactome. Through comparisons among DNA, RNA, and retrotranscribing viruses, we identified a core of human domains, that viruses used to hijack the cellular machinery and evade the immune system, which might be promising antiviral drug targets. We showed that viruses preferentially interacted with host hub and bottleneck domains, and the degree and betweenness centrality among three categories of viruses are significantly different. Further analysis at functional level highlighted that different viruses perturbed the host cellular molecular network by common and unique strategies. Most importantly, we creatively proposed a viral disease network among viral domains, human domains and the corresponding diseases, which uncovered several unknown virus-disease relationships that needed further verification. Overall, it is expected that the findings will help to deeply understand the viral infection and contribute to the development of antiviral therapy. Viral infections result in millions of deaths each year. AIDS has become one of the leading killers worldwide and the influenza has always been a headache of public health organizations. Governments around the world annually invest billions of dollars to investigate the mechanism of viral infections, potential targets for treatment, and innovative vaccines. It is widely accepted that viral infection and pathogenesis mainly depend on their ability to interact with human proteins through a complex network of protein-protein interactions (PPIs). For humans, eukaryotic cells express a large group of proteins to develop normal function through a highly connected and two-side network, which exhibits robustness against random attack and a high sensitivity to targeted subversion , 2. The It is well established that many PPIs are mediated by protein domain pairs. The domain, a stable part of protein structure, evolves and functions independently. The domain is usually used to combine with other domains to form a multidomains protein , which fIn this study, we developed an integrated pipeline to construct a virus-host interactome based on protein domain pair, where we hoped to present novel insights that might not be provided in the binary protein interaction networks. Therefore, we performed topological and functional analysis of this interactome. Moreover, we attempted to map somatic mutations to human domains and gain novel associations between viruses and diseases.http://dx.doi.org/10.1155/2014/867235).We downloaded literature-curated binary PPIs in July 2012, from ten public databases: the Biomolecular Interaction Network Database (BIND) , the DatWe downloaded the DDI dataset from Integrated Domain-Domain Interaction Database (IDDI) , which iAll proteins in the PPI dataset were scanned by the Pfam scan utility and HMMER 3.0 with default parameters against Pfam-A models obtained from Pfam (v.26.0) . Then, wWe integrated all the structure-derived DDIs and top 30% of predicted DDIs in consideration of the reliability and quantity. Additionally, DDIs were further filtered to exclude those domain pairs that were reported as non-interacting by the Negatome database . Therefov in a graph is a global centrality measure which is defined by the number of shortest paths going through this node between any pairs and normalized by twice the total number of protein pairs in the graph (n∗(n − 1)/2, supposing there are n nodes in the graph). The equation used to calculate the betweenness centrality of the node v, b(v), is as follows:gij is the number of shortest paths going from node i to j and gij(v) is the number of shortest paths from i to j that pass through the node v. A domain with high degree or betweenness centrality is characteristic of a hub or bottleneck in an interaction network and often is critical to this network [The degree or connectivity of one domain node in a graph is the number of edges that are linked to this domain node, which stands for a local centrality measure. The betweenness of one domain node  network . In the v is calculated asE is the edges between neighbors of v and k is the degree of v. Thus, a large clustering coefficient means that neighbors of a node tend to cluster together. We used the R package igraph [The average shortest path length, also called characteristic path length, is defined as the average of all the shortest path lengths between the nodes in the graph. It measures the efficiency of information transport on a network. The local clustering coefficient measures the probability that the adjacent vertices of a vertex are connected, and the clustering coefficient of a network is the average local clustering coefficient of all vertices of the network. Simply, the local clustering coefficient of a node e igraph  to compuhttp://www.geneontology.org/external2go/pfam2go). Using all Pfam-A domains of the human's proteome as universe, we sought to find enriched functional terms associated with viral infection and infection mechanisms using R and Bioconductors topGO package [To analyze the functional impact of host domains targeted by viruses, we conducted an enrichment analysis of GO  terms. T package . We usedThe Ensembl variation database imports variation data  from a variety of sources  . For humLikewise, by mapping all disease-associated mutations to the corresponding domains after Pfam scan utility scanning, a total of 210,887 mutations, including 177,493 missense and 33,394 truncating mutations, were obtained in 3,906 domains associated with 3,531 clinically distinct disorders. Insertions and deletions were mapped to domains using the starting position of the mutations. We also ranked these domains according to their normalized cumulative score, where the domain was normalized by dividing by the cumulative length of all occurrences of the domain within proteins, with VHL, NOD, and P53 having the highest scores. Among these, 118 domains from 220 proteins with 3,832 mutations were targeted by viruses.We built the virus-host interactomes by screening domain interactions between virus-host PPIs, and then we studied the network distribution, performed topological and function analysis, and speculated the association between viruses and diseases . AccordiFirst, we examined the distribution of domains and DDIs in virus-host interactomes. Then, we put the virus-host DDIs in the context of human interactome and investigated that some conserved DDIs not only mediate interactions within host but also in the virus-host interface. Many studies have showed that viral proteins tend to interact preferentially with hub and bottleneck proteins in the human interactome network , 51, 52.For the viral target domains, we examined top ten hub and bottleneck domains with highest degrees and betweenness centralities . All theIn addition, we compared the degree of viral domains in virus-host DDI network. We divided host domains into the following sets: domains targeted by any virus , domains by DNA viruses (DNA set), domains by RNA viruses (RNA set), and domains by retrotranscribing viruses (retro set), as well as domains by different families, genera, and species. We computed overrepresented GO terms of host domains in above sets. Overall, we found 19 unique enriched GO  terms . All enr In vitro assay shows DNA-dependent protein kinase (DNA-PK), which plays an important role in NHEJ [Our analysis also highlighted an interesting mechanism “double-strand break repair via nonhomologous end joining” , which w in NHEJ . Our dat in NHEJ . Other GComplex biological systems and cellular networks may underlie most relationships from genotype to phenotype. Understanding genotype-phenotype relationships requires that phenotypes be viewed as manifestations of network properties, rather than simply as the result of individual gene variations. Some studies have proved that missense point mutations and in-frame short indels associated with the corresponding human disorders are enriched on the interaction interfaces of proteins . The ideP < 2.2e − 16, odds ratio = 3.95; Padj = 4.8 × 10−9). Other GO terms also covered plausible contributors to cancer pathogenesis, such as “regulation of cell proliferation.” Consequently, many human cancers might not only arise from mutations of disease susceptibility genes, but also from viral infections, which just used these disease susceptibility genes as their direct or indirect targets.To systematically investigate the extent to which viral proteins globally targeted host proteins causally implicated in cancer, we first compared viral target proteins against a gold standard set of 487 high-confidence causal human cancer genes in the COSMIC Classic Genes set identified by Cancer Gene Census . Viral tP = 3.015 × 10−9 with Wilcoxon rank sum test). Undoubtedly, viruses tended to target cancer-related domains. Compared with overall disease-related domains, it was shown that the hub and/bottleneck domains  ranked higher by their normalized cumulative score , meaning that hub or bottleneck domains might preferentially relate with human cancers, and cancer-related domains equally had more interaction partners than noncancer domains in DDI networks.Further, we constructed a domain-cancer network with 3,906 domains and 3,531 relevant diseases by mapping variations to domains on the corresponding disease proteins. And these domains containing at least one nonsynonymous somatic mutations were also ranked according to their normalized cumulative scores (Supplementary Table 4). We examined that viral target domains were enriched in cancer-related domains . The supplementary materials also include GO enrichment results for different host domain sets by topGO, including BP, MF and CC. We computed enriched GO terms for each species, genera, family, type and all of viruses, and “1” in each cell means the corresponding GO term is enriched (P<0.05), otherwise 0 (Supplementary Table 2). Supplementary Table 3 and 4 are ranked gene and domain lists according to their cumulative normalized scores.


Azospirillum is a plant growth-promoting rhizobacterium living in the rhizosphere of many important crops. Despite numerous studies about its plant beneficial properties, little is known about how the bacterium senses and responds to its rhizospheric environment. The availability of complete genome sequenced from four Azospirillum strains  offers the opportunity to conduct a comprehensive comparative analysis of the TCS gene family.Two-component systems (TCS) play critical roles in sensing and responding to environmental cues. Azospirillum genomes harbour a very large number of genes encoding TCS, and are especially enriched in hybrid histidine kinases (HyHK) genes compared to other plant-associated bacteria of similar genome sizes. We gained further insight into HyHK structure and architecture, revealing an intriguing complexity of these systems. An unusual proportion of TCS genes were orphaned or in complex clusters, and a high proportion of predicted soluble HKs compared to other plant-associated bacteria are reported. Phylogenetic analyses of the transmitter and receiver domains of A. lipoferum 4B HyHK indicate that expansion of this family mainly arose through horizontal gene transfer but also through gene duplications all along the diversification of the Azospirillum genus. By performing a genome-wide comparison of TCS, we unraveled important ‘genus-defining’ and ‘plant-specifying’ TCS.Azospirillum TCS which may confer important regulatory flexibility. Collectively, these findings highlight that Azospirillum genomes have broad potential for adaptation to fluctuating environments.This study shed light on The online version of this article (doi:10.1186/s12864-015-1962-x) contains supplementary material, which is available to authorized users. Mbp encormation) , 33. Addmotility . Althougreported  and thisnditions . This suAzospirillum TCS genes according to their genetic context as either (i) orphan when no TCS gene was found adjacent to another TCS gene, (ii) paired when two TCS genes, whatever their nature (HK or RR), were found adjacent and (iii) complex when more than two genes were contiguous . Th. ThAzospmotility .A. brasilense strains but present in A. lipoferum 4B and Azospirillum sp. B510, such as cNMP binding domain (PF00027) and S_TK (PF00069), that are respectively involved in cyclic nucleotide monophosphate binding detection and crosstalk between Ser/Thr signalling process. Conversely, one PBPb (Periplasmic solute-Binding Protein) domain (PF12974) was found associated only with the two A. brasilense strains (AZOBR_p230031 and AZCBG_p2110061). Bacterial periplasmic solute-binding proteins often function as receptors regulating chemotaxis, transport systems and/or signal transduction pathways [Azospirillum strain. This is the case for the NIT (Nitrate- and nitrite- sensing) domain (PF08376) that was found only in a single Azospirillum sp. B510 classic HK (AZL_012680). Its association with two other PAS domains suggests that it could detect change in nitrate or nitrite concentration [Some sensing domains were found to be absent in the two pathways . Furtherntration . HoweverAzospirillum strains have evolved a multitude of sensing strategies that may favor adaptation to particular ecological niches.Altogether, it seems that Azospirillum showed a wide array of domain combinations among HyHKs compared to their classical counterparts . On the contrary, a remarkable diversity of domain architectures was observed when considering HyHKs  (Table Azospirillum genomes (67 ± 2 %), whereas the remaining 33 % of HyHKs exhibit atypical domain architectures with multiple transmitter and receiver domains. Such complex domain architectures are puzzling as they may give rise to complex phosphotransfer pathways within each protein. Secondly, while around 25 % of HyHKs consist solely of transmitter and receiver domains without any input domain, the majority of HyHKs seem to have accumulated multiple sensing domains, with up to 10 sensing domains (including nine tandem repeated HAMP-GAF) in two A. lipoferum 4B and Azospirillum sp. B510 orphan HyHKs .Domain architecture has been proven to be very informative regarding the putative function of signalling proteins . AnalysiShewanella oineidensis, a HyHK comprising one extracellular Cache domain, two PAS domains, a transmitter domain and two REC domains [in vitro phosphotransfer studies [Azospirillum may have adapted to their environment by retaining new combinations of existing domains, a process that eventually contributed to increase the genetic diversity of HyHKs.The existence of multi-domain proteins harbouring several sensing domains suggests their ability to detect a wider range of signals. This was functionally validated by deleting the PAS1 or the PAS2 domains of ArcS of  domains ; deletio studies . AltogetAzospirillum genomes prompted us to investigate how this expansion of HyHKs arose. To this end, phylogenetic analysis of the 49 transmitter and 62 REC domains from the 49 HyHKs of A. lipoferum 4B  has been performed . Although phylogenies of the REC domains were overall less resolved, due to the low number of conserved positions for phylogenetic reconstruction, the same trend could be observed. A clear illustration of such HGT lies in AZOLI_p30008, one HyHK specific to the A. lipoferum species  and AZOLI_p40210 (YP_004975186.1) likely originated from the duplication of an ancestral gene that was present in the common ancestor shared by 569.1 andSome HyHKs may have also arisen through duplication of a classical HK and fusion with a REC domain of a RR, as previously described , 39; sucA. lipoferum 4B is complex, involving HGT (mainly from proteobacteria), duplications events as well as recombination events allowing domain shuffling. Domain shuffling often goes hand in hand with lineage specific extension (LSE), and LSE genes are much more likely to be present as orphans as depicted above [Azospirillum HyHKs might have evolved by lateral recruitment of additional sensing domains, thus enabling the detection of a large panel of signals [Azospirillum may have arisen in response to unique challenges that members of this genus faced.In summary, the evolutionary history of HyHKs in ed above . Genome-ed above . Azospir signals . AltogetAzospirillum RRs were classified according to their output domains as shown in Table Azospirillum TCS . In comparison, genes encoding RRs consisting of solely a REC domain represent respectively 10.1 % and 13.8 % of P. protegens CHA0 and P. fluorescens SBW25 total RRs (data not shown). Those genes represent nearly one third of total RRs encoded within the R. leguminosarum bv. trifolii WSM 1325 genome, while they represent 23.3 % of R. tropici CIAT 899 total RRs. Intriguingly, overrepresentation of RRs with standalone REC domains was found in N. punctiforme and M. xanthus that respectively represent 36 % and 34 % of the total of their RRs [E. coli, but their roles go far beyond as they can also act as connectors between TCS partners thereby facilitating crosstalk, feedback, and phosphorelays within the two-component phosphorylation network [A. brasilense Sp245 genome harbours one gene encoding a FrzZ-like RR (AZOBR_p330125), that consists of two REC domains arranged in tandem. In M. xanthus, FrzZ was shown to act as a major RR of the chemotaxis system, controlling cell motility and fruiting body formation [A. lipoferum 4B interacting with rice roots revealed that CheY-like standalone RRs were all differentially expressed, strongly suggesting that they may be involved in Azospirillum-plant interaction [Contrarily to HK transmitter domains, classification of RRs based on sequence similarity cannot help fully determine a putative biological function. However, classification of RRs based on output domain and architecture has been proven to be useful to decipher putative RR functions . Therefoheir RRs , 33. Che network . Interesormation . Surpriseraction . HoweverAzospirillum RRs with known output domains (64 % ± 1 %) contain a DNA-binding domain (39 % ± 2 %) and may serve as transcriptional regulators, no RNA-binding domain was detected among Azospirillum RRs, suggesting that no control of gene expression through antitermination is mediated by RRs. Furthermore, enzymatic output domains are common among RRs as 17 % (±1) of them are involved in the modulation of the secondary messenger c-di-GMP or Ser/Thr phosphorylation, suggesting interconnection of Azospirillum TCS with other signal transduction pathways.While most of Azospirillum strains was identified based on a reciprocal best BLAST hit criterion, as previously described [Azospirillum TCS gene content  and prevents flagellum biosynthesis [Azospirillum plays a role in the plant-bacteria interaction has never been elucidated.Homologues of the es Table . This TCefaciens  whereas ynthesis . HoweverAzospirillum TCS belongs to the family of LytS/LytR TCS, shown to be involved in the regulation of bacterial virulence genes  showing a significant identity (41 %) over the PAS and GGDEF domains with a protein from psychrophile Colwellia psychrerythraea belonging to the gammaproteobacteria lineage . Therefore, tight regulation of this metabolic versatility may be relevant in specific environments such as rice paddy fields, from which A. lipoferum 4B was isolated.However, there are differences in TCS proteins among the four Azospirillum TCS regulatory networks. The existence of large numbers of TCS and especially HyHKs reinforces the idea that these genes can support traits needed for thriving in heterogeneous, fluctuating and highly competitive environments, such as the rhizosphere. Our phylogenetic analyses indicate that the expansion of the HyHK family in Azospirillum has occurred mainly through HGT, even if gene duplication and domain shuffling also played an important role. We further gained insights into the core of orthologues TCS proteins and differences between TCS repertoire of the four strains that are likely to facilitate Azospirillum adaptation to different host plants. Future studies focused on the functional analysis of these TCS might shed light on the complex events occurring during Azospirillum soil survival and plant growth-promotion in different environmental settings. Ultimately, focusing on mechanisms by which beneficial plant-associated bacteria persist and compete in soil would be useful for improving plant growth-promotion strategies.In summary, here we provide evidence of the incredible complexity of


IIbβ3 constructs, we discovered that an αIIb cytoplasmic domain truncation led to integrin activation but not defective outside-in signaling. In contrast, preventing transmembrane domain separation abolished both inside-out and outside-in signaling regardless of removing the αIIb cytoplasmic tail. Truncation of the αIIb cytoplasmic tail did not obviously affect adhesion-induced outside-in signaling. Our research revealed that transmembrane domain separation is a downstream conformational change after the cytoplasmic domain dissociation in inside-out activation and indispensable for ligand-induced outside-in signaling. The result implicates that the β TM helix rearrangement after dissociation is essential for integrin transmembrane signaling. Furthermore, we discovered that the PI3K/Akt pathway is not essential for cell spreading but spreading-induced Erk1/2 activation is PI3K dependent implicating requirement of the kinase for cell survival in outside-in signaling.Integrins play an essential role in hemostasis, thrombosis, and cell migration, and they transmit bidirectional signals. Transmembrane/cytoplasmic domains are hypothesized to associate in the resting integrins; whereas, ligand binding and intracellular activating signals induce transmembrane domain separation. However, how this conformational change affects integrin outside-in signaling and whether the α subunit cytoplasmic domain is important for this signaling remain elusive. Using Chinese Hamster Ovary (CHO) cells that stably expressed different integrin α Integrins are single transmembrane (TM) α-β heterodimeric cell adhesion receptors with each subunit comprised of a large extracellular domain, a single TM helix, and a short cytoplasmic domain –4; integThe integrin TM/cytoplasmic domains regulate integrin affinity and mediate downstream signal transduction. Association of the TM/cytoplasmic domains between the α and β subunits is critical for maintaining integrins in the low-affinity state. Intracellular signals that impinge on the cytoplasmic domain destabilize αβ association and result in integrin activation –15. Rece4 and α9 integrin cytoplasmic tails negatively affects cell spreading but can promote cell migration . Lysates were subjected to reducing SDS-PAGE and transferred to PVDF membrane. The total amount of the kinases was determined by mouse monoclonal anti-FAK , rabbit monoclonal anti-Akt, or anti-Erk1/2  antibody, respectively, and activation of the kinases was tested by rabbit monoclonal anti-FAKp397, anti-Aktp473, and anti-Erk1/2p202/204 respectively . Mouse monoclonal GAPDH antibody  was used to determine total protein concentration.Six-well plates were pre-coated with 20 μg/mL fibrinogen followed by blocking with 1% BSA. Stable transfectants were then seeded in the wells, cultured to a confluent monolayer and serum starved overnight at 37°C. Artificial wounds were carefully created with sterilized micropipette tips. Cells were then washed twice with serum-free MEM-α medium and cultured in complete medium at 37°C. Closure of the artificial wounds was observed and recorded every 4 h for 12 h using the 10 × lens of an Olympus IX81 microscope. The wound closure rates were normalized to the original wound areas.IIb_W968C and β3_I693C reversed inside-out activation and kept integrin in the low affinity state [IIb cytoplasmic domain with or without the TM disulfide clasp. We established CHO cells stably transfected with each of the four different αIIbβ3 constructs , truncated mutant , and disulfide-bonded truncated mutant . Four cell lines were generated after single-cell sorting and the integrin αIIbβ3 expression levels were determined by staining with three specific mAbs: anti-β3 mAbs AP3 and 7E3, and an anti-αIIb mAb 10E5 mAb 10E5 . Flow cymAb 10E5  confirmeIIbβ3 mutants to bind ligands including PAC-1, a ligand mimetic mAb, and fibrinogen  and the disulfide-bonded mutant (B2) showed similar low ligand binding, whereas the α-truncated mutant (B3) exhibited much higher binding. However, the TM disulfide-bridge clasp of the mutant B4 reduced ligand binding to basal level. Reduction of the disulfide bond with DTT drastically increased ligand binding of the mutant. These results indicated that TM domain separation is required for cytoplasmic domain dissociation induced high affinity integrin.We determined the ability of the integrin αbrinogen . In the The binding of integrin to immobilized multivalent ligands can induce integrin clustering and outside-in signaling. To dissect the role of the TM and cytoplasmic domains in outside-in signaling, we used these integrin mutants to perform a cell spreading assay on immobilized fibrinogen . Claspinin vitro wound healing assay  to FAs (IIb integrin cytoplasmic tail is dispensable for paxillin recruitment and phosphorylation. Because both disulfide-bonded mutants (B2 and B4) exhibited defective phospho-paxillin recruitment, which was restored upon DTT treatment  to FAs  in both reatment , we concreatment . This prIIb cytoplasmic domain truncation restored FAK activation in both disulfide-bonded mutants, suggesting that the TM separation rather than αIIb cytoplasmic domain is essential in regulating FAK activity.In our study, FAK activation  FAs in cells expressing α-truncated integrin were also observed. The fact indicates that, though α integrin is not required for cell interaction and spreading on ECM ligands, this subunit may be involved in FA turnover and directional cell migration. Our preliminary study suggests defective cell migration in α cytoplasmic domain truncation mutant. However, it is yet to know if this phenomenon is caused by the mutation since a high ligand binding affinity induced by α-truncation may also lead to reduced cell motility. We are planning further studies to better understand these events.Studies of integrin signaling have largely focused on β integrin, thus the role of the α subunit cytoplasmic domain has remained elusive. It has also been reported that the α integrin cytoplasmic tail participates in outside-in signaling ,55. ThisKinases have long been considered important in integrin signaling. Akt activation in platelets during outside-in signaling has been reported ,33. HoweS1 FigFA counts and area measurement were achieved by ImageJ, as described. Error bars represent FA counts and area measurements from 50 randomly chosen adherent cells.(TIF)Click here for additional data file.S2 Fig(A) Cells were seeded on 20μg/mL Fbg- coated 6-well plates and cultured to confluence. Artificial wounds were then created with sterilized tips. Photos were taken every 4 hours, white bar: 50μm. The results represent five independent assays. (B) Normalized quantification of wound closures were done by ImageJ. Error bars represent five independent assays  cells after 12 hrs culture).(TIF)Click here for additional data file.S3 FigIIb truncation.Cells were seeded on Fbg (20μg/mL)-coated dishes and cultured at 37°C for 1 h and then lysed and subjected to western blot. Activation of Erk1/2 was suppressed by TM-clasping (B2) and was not restored by α(TIF)Click here for additional data file.S4 FigCells were serum starved overnight and treated with PP2 (15μM/mL) for 2 h prior to seeding on fibrinogen pre-coated plates and during the spreading assay. The treatment did not affect FA formation (blue), recruitment, or activation of FAK  or paxillin  and actin organization (red). White bar: 10μm.(TIF)Click here for additional data file.