Ack family non-receptor tyrosine kinases are unique with regard to their domain composition and regulatory properties. Human Ack1 (activated Cdc42-associated kinase) is ubiquitously expressed and is activated by signals that include growth factors and integrin-mediated cell adhesion. Stimulation leads to Ack1 autophosphorylation and to phosphorylation of additional residues in the C-terminus. The N-terminal SAM domain is required for full activation. Ack1 exerts some of its effects via protein-protein interactions that are independent of its kinase activity. In the basal state, Ack1 activity is suppressed by an intramolecular interaction between the catalytic domain and the C-terminal region. Inappropriate Ack1 activation and signaling has been implicated in the development, progression, and metastasis of several forms of cancer. Thus, there is increasing interest in Ack1 as a drug target, and studies of the regulatory properties of the enzyme may reveal features that can be exploited in inhibitor design. In the active conformation, the αC helix projects inward toward the ATP-binding site [ The mammalian nonreceptor tyrosine kinases (NRTKs) are divided into ten families: Src, Abl, Jak, Ack, Csk, Fak, Fes, Frk, Tec, and Syk . In addiing site . The coning site . The reging site , while Cing site .The contributions of the noncatalytic domains to enzyme regulation are well understood in the case of the Src-family of NRTKS. These enzymes have an N-terminal myristoylation site followed by an SH3 domain, an SH2 domain, and a kinase domain. Two tyrosine residues are important for their activity: Tyr 416 in the activation loop and Tyr 527 in the C-terminal tail. Structural studies showed the spatial arrangement of Src domains in the inactive state, in which the noncatalytic domains mediate intramolecular interactions that maintain the enzyme in a closed, inactive conformation. In this conformation, the SH3 domain is bound to a proline-rich helix located between the SH2 domain and the kinase domain, and the SH2 domain is bound to the phosphorylated Tyr 527 . ActivatThe mechanisms involved in enzymatic regulation vary among the different families of NRTKs. For example, Abl kinases have similar arrangements of domains as Src-family kinases, but the regulatory mechanisms are different; this has important implications for the design of pharmacological inhibitors . In bothAs new structures of NRTKs are solved, new molecular mechanisms for enzyme regulation are revealed. Structural studies have illuminated the contributions of noncatalytic domains to the regulation of Syk- , 5, Csk-The domain architecture of human Ack1  is shown in Drosophila melanogaster, and Caenorhabditis elegans  [Drosophila), and Kos1 (from mouse) lack CRIB domains [Ack1 belongs to a family of NRTKs that includes human Tnk1, and homologous proteins in mouse, cow, elegans  , 24. AltAck1, the first member of the Ack family, was cloned from a human hippocampal expression library by its ability to bind GTP-bound Cdc42 specifically and not Rac1 or RhoA . The genγ1 through its proline-rich region [κB activation and thereby promotes apoptosis mediated by the TNFα signaling pathway [Kos1 gene is located in chromosome 11 in mice and it is thought to be a murine splice variant of the human Tnk1 [Tnk1 (thirty-eight-negative kinase 1) was originally cloned from hematopoietic stem/progenitor cells from umbilical cord blood in humans . Tnk1 ish region . It has  pathway . Kos1 is pathway . The Kosman Tnk1 . Tnk1/Koman Tnk1 . Tnk1/Koman Tnk1 .Ack2 was first cloned from a bovine brain expression library. Ack2 has a predicted size of 83 kDa and contains the same basic domain structure as Ack1. Ack2 is thought to be an isoform of Ack1 that is generated by alternative splicing and is not encoded by a different gene . The AckDrosophila, there are two members of the Ack family: DACK and DPR2. The roles of DPR2 and DACK have not been dissected but they are thought to be different because DPR2 has a Cdc42-binding domain, while DACK lacks one. Cdc42 regulates the expression of DACK in Drosophila embryos [C. elegans, Ark-1 (A Ras-regulating Kinase 1), a cytoplasmic tyrosine kinase that is related to Ack1, acts as a negative regulator of EGFR signaling. In a yeast two-hybrid analysis, Ark-1 interacts with the Grb2 homologue, Sem5 [In  embryos . The tyr embryos . In C. eue, Sem5 .≈ 40% amino acid identity with other nonreceptor and receptor tyrosine kinases. Phylogenetic analyses of human tyrosine kinase catalytic domains place Ack1 near epidermal growth factor receptor (EGFR) family kinases [The degree of conservation of the full-length sequences varies among the family members. The common core that is conserved in all of the Ack family members spans from the N-terminus to the SH3 domain. The amino acid identity of this core (compared with Ack1) ranges between 100% for Ack2 and 29% for Kos1 . The Ack kinases , 33. As Expression of Ack1 has been demonstrated in several tissues in mammals, with the highest expression in spleen, thymus, and brain . A numbeIn vitro studies showed that Ack1 associates with several SH3 domains, including the SH3 domain from Grb2 [Upon EGF stimulation, Ack1 phosphorylates and activates the guanine exchange factor Dbl. Ack1 interacts with the Rho-family GTPase Cdc42 and the adaptor protein Grb2 and these interactions are essential for this pathway . The assrom Grb2 . Ack1 asrom Grb2 . β1-mediated signaling pathways that modulate processes such as cell migration, cell adhesion, and cell spreading. Ack2 is present in a complex with integrin β1; it is activated by cell adhesion to a fibronectin-coated surface in a Cdc42-dependent manner and it activates the phosphorylation of Jnk [Ack is involved in integrin n of Jnk . In respn of Jnk , 40. Ackn of Jnk . Cas appn of Jnk . Thus, o125I-EGF to the plasma membrane and reduces the degradation of 125I-EGF, suggesting that Ack1 promotes degradation of EGFR [Several studies have focused on the role of Ack1 in EGF receptor trafficking and dynamics. Ack1 is recruited to EGFR following EGF stimulation . EGFR st of EGFR . In Cos7 of EGFR . Increas of EGFR . In addition to the effect of Ack1 on receptor stability and dynamics, there is a reciprocal effect on Ack1 stability. After receptor stimulation by EGF, Ack1 is recruited to an EGFR complex , 47. ThiThere is currently no crystal structure of full-length Ack1. The crystal structure of the isolated kinase domain of Ack1 has been solved in both phosphorylated and unphosphorylated forms. In these structures, the activation loop conformations are similar and do not occlude the substrate-binding site, suggesting that the phosphorylation of the activation loop may not play a dramatic stimulatory role . The onlA major autophosphorylation site has been mapped to the Y284 in the activation loop . A constin vitro and can be used in enzymatic assays [in vivo does increase Ack1 Y284 phosphorylation [Relatively little information is available about the regulatory mechanisms of Ack1 and the roles that each domain plays in Ack1 regulation. The purified Nt-Kinase-SH3-CRIB polypeptide is active c assays . The pepc assays . In addic assays . Ligandsrylation . Residuerylation . One way of studying the role of each domain in Ack1 regulation is to assess the effects of point mutations on the enzyme's phosphorylation state . A mutatThe deletion of the N-terminal SAM domain reduced Ack1 autophosphorylation and kinase activity , 64. TheThe domain composition of Ack1 is complex and it is likely that in the future, additional interactions (both intra-and intermolecular) will be described that will explain in more detail the molecular mechanisms of Ack1 regulation and signaling.Ack1 has been implicated in several stages and several types of cancer; this topic has been reviewed recently in . Early eAck1 gene on chromosome 3. In addition, 42% of the aggressive lung tumors and 77% of metastatic hormone refractory prostate tumors showed overexpression of the Ack1 mRNA. Ack1 overexpression enhanced migration of human mammary epithelial cells (HMECs) and increased metastasis in the mouse mammary 4T1 breast cancer system [In a study of ~180 advanced-stage cancers, 9% of the lung tumors and 14% of the ovary tumors had an amplification of the r system . Moreover system . Thus, ar system . In addir system . This suThe mechanistic basis of Ack1's involvement in prostate tumorigenesis has been explored in detail. In the cultured prostate cancer cell line LNCaP, the tumor suppressor Wwox is phosphorylated and marked for degradation by Ack1 . The andA large-scale study that screened cancer tissues for somatic mutations identified four missense mutations in Ack1 . In lungThe functions of Ack1 in cancer cells appear to be of two types: kinase-independent and kinase-dependent. The effect of Ack1 on cell proliferation is independent of Ack1 activity . In a phA number of Ack1 substrates have been identified in normal cells as well as cancer cells. Phosphorylation of WASP by Ack1 promotes its actin remodeling activity . In addi A comprehensive list of known Ack1 substrates and interacting proteins was recently published , and it Nonreceptor tyrosine kinases share several common features. The NRTK catalytic domains never occur in isolation; they are tethered to noncatalytic modular domains that play important roles in enzymatic function. The biochemical properties of the individual noncatalytic domains are similar to those found in other signaling proteins. However, the regulatory mechanisms are diverse and vary between the different families of NRTKs. To date, 15 out of the 32 NRTKs in the human genome have been shown to have oncogenic potential . Moreove


In order to detect distributed ground surface deformation, an elastic helical structure Time Domain Reflectometry (TDR) sensing cable is shown in this paper. This special sensing cable consists of three parts: a silicone rubber rope in the center; a couple of parallel wires coiling around the rope; a silicone rubber pipe covering the sensing cable. By analyzing the relationship between the impedance and the structure of the sensing cable, the impedance model shows that the sensing cable impedance will increase when the cable is stretched. This specific characteristic is verified in the cable stretching experiment which is the base of TDR sensing technology. The TDR experiment shows that a positive reflected signal is created at the stretching deformation point on the sensing cable. The results show that the deformation section length and the stretching elongation will both affect the amplitude of the reflected signal. Finally, the deformation locating experiments show that the sensing cable can accurately detect the deformation point position on the sensing cable. Different kinds of ground deformation sensing technologies have been applied for detecting or measuring surface deformation, such as Global Positioning System (GPS) technology, Brillouin optical time domain reflectometer (BOTDR) and Differential Interferometric Synthetic Aperture Radar (D-InSAR).et al. built a comprehensive monitoring system including GPS InSAR and inclinometer to study the dynamic deformation process of the Jiaju landslide in Danba . With GPS displacement monitoring data, the FLAC3D numerical simulation method was adopted and the stress field, distribution of displacement and plastic zone in the dynamic deformation process were simulated. The simulation results were consistent with monitoring results. GPS deformation monitoring technology is also used in mine area safety monitoring applications. Zhao et al. established a systematical GPS monitoring network on the ground surface of the Longshou Opencast Mine in China. After five and half years of monitoring, a 3D numerical model was established to reveal the stress environment around the excavation and opencast slope rock mass, and the rock mass movement and deformation, stress distribution and failure mechanism were discussed. Combined with the monitoring results and field investigation, it pointed out that the overall slope stability is relatively good in the current stage. However, GPS technology can only measure the displacement of monitoring points with GPS observation stations. Rock masses are not rigid bodies. The deformation of different parts of the ground surface is different. One monitoring point's displacement result cannot represent other parts' displacement. In order to monitor ground deformation, the quantity of monitoring points should be large enough. In the Jiaju landslide monitoring system, there are 22 monitoring points. In the Longshou Opencast Mine, the number is nearly 300. In order to monitor so many deformation points, each deformation point has to be measured periodically with a limited amount of GPS equipment. The monitoring period can be several months, which limits the real-time performance of GPS deformation monitoring system.The GPS deformation monitoring system has been an important tool for studying surface deformation processes –6. It caBOTDR is a kind of distributed deformation monitoring technology –11. It set al. studied the Saint-Etienne-de-Tinee landslide in the south of France using D-InSAR technology with six interferometry pictures obtained from ERS-1 in 1995 and proved the consistency between the accuracy of D-InSAR technology and the accuracy of other ground monitoring methods. However, image coherence will seriously affect the application of D-InSAR in surface deformation monitoring. Especially at areas with a large amount of vegetation or when a large surface deformation happens in a short time, the coherence may be too low to obtain surface deformation data.D-InSAR is a wide area surface deformation sensing technology –15. InteAfter analyzing ground surface deformation characteristics and the present surface deformation sensing technologies, we put forward a new distributed surface deformation detection technology based on TDR using a special TDR sensing cable. This special TDR sensing cable can overcome BOTDR's intrinsic limit . It can detect large distributed deformations in geological hazards.2.TDR technology is something like radar . A TDR dTDR technology has been used in many fields. Using TDR technology, cable fault location equipment can point out where telephone cable is broken or short circuited. It helps workers fix communication networks. TDR is also used in measuring the water content of soils ,17. WateHowever, traditional TDR sensor cable is hard to apply in surface deformation measurements. Generally, there are two kinds of TDR sensing cables. One is the coaxial cable; the other is the parallel cable (like telephone cable). As Lin did, coaxial cable can measure shear deformation underground, but the TDR sensing cable should have the ability to measure stretch deformation (like BOTDR) in surface deformation monitoring . However3.As shown in The parallel wires have a plastic sheath as shown in 4.4.1.ρ = Reflection coefficient,where rU = Reflected signal voltage from TDR sensing cable,tU = Exciting signal voltage send by the TDR device,LZ = impedance at the deformation point on TDR sensing cable,CZ = TDR sensing cable's impedance.According to the transmission-line theory, transmission-line reflection coefficient is:LZ > CZ, a positive reflected signal will be found in the reflected signal waveform. If LZ < CZ, a negative signal will be received. Cables with different impedance characteristic can detect different parameters. In order to detect stretching ground surface deformation, a TDR sensing cable's impedance should change when the cable is stretched by ground surface deformation.According to L is the distributed inductance, C is the distributed capacitance and G is the distributed conductance.The TDR sensing cable can be modeled as the circuit shown in ω = frequency of the signal and j = complex unit.According to the transmission-line model, the impedance model of the TDR measurement cable is:ω is large enough, R and G can be ignored compared with jωL and jωC. The impedance can be changed to If Compared with coaxial cable and parallel cable, the electromagnetic fields of helical wires is much more complicatedand it is difficult to calculate L and C precisely. To simplify the analysis, we assume that the signal frequency is high enough and the electromagnetic wave along the helical structure is approximately a plane wave. Based on this assumption, the electromagnetic fields can be analyzed using static electromagnetic analysis methods.aE is the internal electric field of parallel wires. Besides the internal electric field aE, there is an electric field bE between adjacent helical wires as the helical wires are closely entwined.aC = internal distributed capacitance in parallel wires,where bC = distributed capacitance between adjacent helical wires.According to the surface electric field distribution, the distributed capacitance C consists of two parts:r = wire radius,where ad = distance of space a,bd = distance of space b,ε = permittivity.r is much smaller than ad and bd here.If the signal frequency is high enough, most of charge will be on the surface of the wires. According to the parallel wires capacitance calculation method, the distributed capacitance can be written as:v = propagation velocity and μ = permeability.When the signal frequency is high enough, there is a relationship between distributed capacitance and distributed inductance:ad = bd = d. According to Δd is the change value of bd. If the relative permeability is 1, the relative permittivity is 1.2, wires radius r is 0.2 mm and initial distance d is 1.27 mm. Δd and CZ.In the initial state, Δd increases. That means EHTSC can effectively detect the deformation in a certain range near Δd = 0.The relationship curve shows that EHTSC's impedance increases when the cable is stretched and the rate decreases when 4.2.Until now the relationship between elongation and impedance has been discussed through transmission-line theory. An impedance experiment is done here to verify the theoretical analysis. The impedance experiment system consists of three parts, namely a RIGOL DG3121A signal generator, Agilent MSO7054A oscilloscope and elastic helical TDR sensing cable. The RIGOL DG3121A can generate a fast-leading-edge (less than 2 ns) electrical step signal. Its output impedance is 50 Ω. The bandwidth of the Agilent MSO7054A is 500 MHz and its sampling frequency is 2 GHz. The signal generator and oscilloscope compose a TDR device. The sensing cable's initial length is 270 mm. Its diameter is about 10 mm.CZ is the impedance of EHTSC. Zs is signal generator's output impedance. SU and the transient voltage CU on CZ:SU created by the signal generator is 5 V. Because of the sensing cable impedance, the transient voltage is lower than the step signal voltage. However, the waveform is not a straight line. The change of the transient voltage can be caused by the non-uniformity of the cable's impedance. In order to find the relationship between the transient voltage and the elongation of the sensing cable, the average transient voltage is calculated.CU increases when EHTSC is stretched. And the rate decreases with the elongation increasing. According to The experiment results verify our conclusion that the impedance of EHTSC increases when it is stretched. There is a difference between 5.The last section shows that the impedance of an elastic helical structure TDR sensing cable increases when it is stretched. As The length of the deformation section is 25.0 mm. Then the deformation section is stretched to 54.6 mm. To obtain the local change of the transient voltage, a comparison method is applied in the TDR data processing. The difference waveform in In order to detect ground surface deformation, the relationship between reflected waveform spike and stretching deformation is researched. In this experiment, we select three different deformation sections—25 mm, 35 mm, 45 mm. With different deformation section, the waveforms show that the spike magnitude increase when the sensing cable is stretched from 4 mm to 24 mm with different deformation section lengths. It verifies the conclusion which is made according to the theoretical analysis above.However, the reflected waveform spike magnitude will be affected by the length of deformation section. From This problem had been put forward by Lin in quantification of coaxial cable deformation with TDR ,22. He s6.Although further research should be done in calculating out the cable's stretching length, EHTSC's ability to locate stretching deformation points is a useful characteristic. Because of the non-uniformity of the cable, the location of deformation points still uses the comparison method to deal with TDR data. Total length of the sensing cable in the experiment is 2 m. The deformation section length is 30 mm. The stretching length is 20 mm, which means the deformation section length changes from 30 mm to 50 mm. The distances shown in 7.There is a relationship between an elastic helical TDR sensing cable's structure and its impedance. This relationship is analyzed based on transmission-line model and shown as A positive pulse reflected signal will be generated at the deformation point on the sensing cable. Because of non-uniformity of the cable structure, it is difficult to obtain the reflected signal caused by deformation. To overcome this problem, we use a comparison method to deal with the reflected signal wave and get a clear positive pulse reflected signal from the noise.The reflected signal amplitude has a relationship with the deformation section length, stretching elongation and distance from TDR device. From experiments, we can see that the longer the deformation section length, the longer the stretching elongation and a short distance from TDR device will give a higher reflected signal amplitude.The sensing cable can locate the deformation point accurately. After demarcation, the elastic helical TDR sensing cable could effectively locate the position of the deformation point in the simulation ground surface deformation experiment. In further application, the sensing cable will be fixed on the surface of a landslide. When the landslide happens, the sensing cable will be stretched. Through the sensing cable, we can monitor any deformation on the landslide along the sensing cable.This research describes an elastic helical TDR sensing cable which is suitable for distributed ground surface deformation detection. From the research above, we can draw some conclusions as follows:


MGM101, is essential for yeasts that depend on mitochondrial DNA replication. Previously, in Saccharomyces cerevisiae, it has been found that the carboxy-terminal two-thirds of Mgm101p has a functional core. Furthermore, there is a high level of amino acid sequence conservation in this region from widely diverse species. By contrast, the amino-terminal region, that is also essential for function, does not have recognizable conservation. Using a bioinformatic approach we find that the functional core from yeast and a corresponding region of Mgm101p from the coral Acropora millepora have an ordered structure, while the N-terminal domains of sequences from yeast and coral are predicted to be disordered. To examine whether ordered and disordered domains of Mgm101p have specific or general functions we made chimeric proteins from yeast and coral by swapping the two regions. We find, by an in vivo assay in S.cerevisiae, that the ordered domain of A.millepora can functionally replace the yeast core region but the disordered domain of the coral protein cannot substitute for its yeast counterpart. Mgm101p is found in the mitochondrial nucleoid along with enzymes and proteins involved in mtDNA replication. By attaching green fluorescent protein to the N-terminal disordered domain of yeast Mgm101p we find that GFP is still directed to the mitochondrial nucleoid where full-length Mgm101p-GFP is targeted.The mitochondrial genome maintenance gene,  A distinctive feature of respiring eukaryotic cells is the mitochondrion. This organelle is the site for electron transport, oxygen consumption and ATP synthesis collectively termed oxidative phosphorylation. In most eukaryotes some components of oxidative phosphorylation are encoded by the organelle's genome that varies in size and may have from 3 to 67 protein-coding genes Saccharomyces cerevisiae the mitochondrial genome maintenance gene, MGM101, encodes a protein of 269 amino acids Kluyveromyces lactisS.cerevisiae.In Mgm101p has been implicated in recombination repair S.cerevisiae it has been shown that the carboxy-terminal two-thirds of the protein, termed the functional or active core of 165 amino acids, can restore growth at 35°C of a temperature sensitive mutant mgm101 null mutant indicating that for proper operation the active enzyme must be a dimer or multimer with input from the amino-terminus of the full-length protein. As the functional core of Mgm101p contains a large number of lysine and arginine residues it is reasonable to believe that this region is responsible for DNA binding and consequent activities. However, the role of the essential amino-terminus remains unknown.In MGM101 gene is widely distributed in fungi, some protists and cnidaria but it is not present in plants or the Bilateria. Alignment of amino acids from Mgm101p shows a high level of conservation in the carboxy-terminus The Intrinsically unstructured/disordered proteins (IDPs) do not adopt a well-defined structure in isolation, instead existing as a rapidly interchanging ensemble of conformations S.cerevisiae corresponds to an ordered domain that is preceded by an amino-terminal disordered region. To examine whether these two domains have specific or general functions we made chimeric proteins from S.cerevisiae and the coral Acropora millepora by swapping the two regions. By an in vivo assay in S.cerevisiae we find that the ordered domain of A.millepora can functionally replace the yeast core region but the disordered region of the coral protein cannot substitute for its yeast counterpart. In other words, operation of the disordered domain appears to be specific whereas activity of the ordered region is general. An implication from this result is that the disordered region functions by specific interaction with a component of the nucleoid whereas the core region is not so constrained.As described in this communication, it appears that the experimentally determined functional core of Mgm101p in S.cerevisiae Mgm101p are necessary for complementation of a temperature sensitive mutation, Mgm101-1. We termed this sequence a ‘functional core’ as it is preceded and followed by segments that are not needed for restoration. In the present report we use ‘core’ in a more general sense to include the 6 inessential carboxy-terminal amino acids. As the functional core region of A.millepora Mgm101p has not been determined, we use the more general terminology of ‘core’ in the first instance (see below), to include all amino acids downstream of the junction with the disordered domain.In a previous publication S.cerevisiae and A.millepora were studied from the viewpoint of protein disorder, domain content and mitochondrial target sequence. The alignment of the sequences confirmed earlier results, indicating a strong sequence conservation within the C-terminal region corresponding to the functional core, and the lack of apparent sequence conservation within the N-terminal regions , Trichoplax adhaerens (placozoan), Dictyostelium discoideum (slime mould) and Naegleria gruberi (amoebo-flagellate). These species were selected to represent the evolutionary diversity within this family. Their Mgm101p sequences show a very similar domain organization to S.cerevisiae and A.millepora . Apart from these three cases, the domain organization is common to all members of this sequence family, despite the fact that sequence and length of the disordered regions are not conserved. The lack of conservation in the N-terminal region suggests that the mitochondrial targeting signal and the disordered region operate in a species-specific manner.An extended analysis of the Mgm101 family included four additional sequences from illepora . The N-tsordered . An evenmgm101-1 P141S, located at the end of the first highly conserved region  of yeast Mgm101p joined to a putative core region of the coral gene and vice versa, were constructed in the vector pCXJ22 used in the previous study S.cerevisiae. The strain M2915-7C, containing the mgm101-1 temperature sensitive (ts) mutation was transformed with the three plasmids followed by selection for Ura+. Transformants were examined for restoration of growth at the restrictive temperature, 35°C  grows slower than the transformant containing the ID region of S.cerevisiae joined to the coral core domain (S.c.ID-A.m.C). Almost equal growth rates are displayed by the transformants containing the wild-type yeast gene and the ID region of the coral linked to the S.cerevisiae core domain (A.m.ID-S.c.C).In a previous study we used a recessive temperature-sensitive mutant, d region , to detere, 35°C . All thrura3 and heterozygous for disruption of MGM101 (mgm101::LEU2). The same pCXJ22 constructs, used before, were employed for transformation of the diploid. Transformants were sporulated, asci dissected and results from the four strains are summarised in + (the plasmid contains URA3 wild type) MGM101, indicated by Leu+ (disruption of MGM101 is by insertion of LEU2 wild type), segregates 2∶2. A demonstration that wild-type ScMGM101 can complement disruption of the chromosomal gene is shown in +,Leu+ spores can grow on glycerol. Growth on glycerol is an indication that respiration is present that in turn depends on a functional mitochondrial genome and an operational Mgm101p. By contrast, no complementation was found with the A.millepora gene or when the ID region of this gene is attached to the core region of S.cerevisiae mgm101p (A.m.ID-S.c.C). However, when the ID domain of the yeast gene is joined to the core region of the coral gene (S.c.ID-A.m.C), 10 out of 42 Ura+ Leu+ grow on glycerol  and disruption of MGM101 (Leu+).The above result led us to test whether restoration of respiration could occur in the absence of the Mgm101-1ts mutant protein. For this test we used a diploid strain, CS5/mL3, homozygous for glycerol . As show+,Leu+ cultures that failed to grow on glycerol revealed that mtDNA was no longer present. A possible explanation for failure to maintain mtDNA is that the chimeric protein has lower activity compared to the wild type and only in some cases is there sufficient plasmid to permit the remaining activity to function productively. Support for this view is that the expression of the MGM101 constructs relies on the relatively weak native promoter and that pCXJ22 depends on the ARS.CEN sequence for replication and is in low copy number as demonstrated by the limited frequency of Ura + (54–62%) in tetrads. This is also illustrated in + from some tetrads (2&9).Examination by DAPI staining of Uraura3 by cleavage at a single StuI site in the wild-type URA3 gene on the plasmid followed by selection for Ura+ transformants. All resulting isolates had a stable genotype that segregated 2∶2 for URA3:ura3 on tetrad dissection.In view of the variable presence of pCXJ22 in tetrad colonies, we hypothesized that integration of the constructs into a chromosomal location would improve frequency of growth on glycerol, especially of transformants containing the ID domain of the yeast gene joined to the coral core of Mgm101p. For this test we recloned the three constructs into the integrative vector pUC-n lacking an intrinsic origin of DNA replication. Such constructs can be integrated at S.cerevisiae MGM101 was integrated, all spores containing a Ura+,Leu+ phenotype grew on glycerol  have shown that Mgm101p is found in mitochondrial nucleoids S.cerevisiae Mgm101p is not disordered whereas the N-terminal region, as well as part of the mitochondrial signal sequence, is disordered. Sequence alignment of Mgm101 proteins shows that the N-terminal domain does not contain recognizable common elements while the functional core shares conserved sequences. We have used the predictions of the ordered and disordered domains of A.millepora and the conserved core of S.cerevisiae in constructing the chimeric proteins.Prediction by three different methods shows that the previously determined 165 amino acid functional core of Mgm101-1, has shown that the wild types and chimeras all restore temperature tolerance but at different levels  and reactions were run on an ABI 3730 DNA Analyzer at the Biomolecular Resource Facility .Total RNA was isolated from frozen coral tissue ground in liquid nitrogen using the RNAwiz reagent (Ambion) following manufacturer's instructions. cDNA was synthesized from mixed planula larva and adult RNA using an anchored oligo dT primer and PrimeScript reverse transcriptase (Takara). PCR primers based on the MGM101 sequence in the A.millepora MGM101 cDNA insert was excised with SpeI and EcoRI and inserted between the SpeI and EcoRI sites of pCXJ22-ScMGM101 A.millepora intrinsically disordered region and Mgm101p core downstream of the S.cerevisiae mitochondrial import signal sequence. To make a construct with an ORF consisting of the S.cerevisiae intrinsically disordered amino terminal region fused to the A.millepora Mgm101p core the primers, 5′ GTTAGCGGAATTCACTGGCCGTCGTTTTACAACGTC 3′ and 5′ CCAGTCGGTACCTCCCTTGACTTGCTTATAACTATTG 3′ were used to amplify a product from pCXJ22-ScMGM101, and the primers 5′ TCATATGGTACCCTATCAGAAGACTTTTCTGGAGCTTC 3′ and 5′ GGCCACATGAATTCTCTTTCTCACACTGGATGGCAAG 3′ were used to amplify a product from the A.millepora MGM101 cDNA clone. The products were digested with Acc651 and EcoRI and ligated together to give pScAmMGM101. To make a construct with an ORF consisting of the A.millepora intrinsically disordered amino terminal region fused to the S.cerevisiae Mgm101p core the primers, 5′ GTTAGCGGAATTCACTGGCCGTCGTTTTACAACGTC 3′ and 5′ CCAGAAAAGGTACCTGATAGCCTATCATATGAATTATTCG 3′ were used to amplify a product from pCXJ22-AmMGM101 and primers 5′ GACTGGGGTACCTCATGGTATGGCCTAGGTATGAAGC 3′ and 5′ GGCCAGTGAATTCCGCTAACCCTGAAATAGAAGGCG 3′ were used to amplify a product from pCXJ22-ScMGM101. The products were digested with Acc651 and EcoRI and ligated together to give pAmScMGM101. The amino acid sequences of the Mgm101 proteins coded by these constructs are shown in The SpeI and EcoRI and ligated into the SpeI and EcoRI sites of pUC-n-MGM101His To make the integrative constructs, fragments containing the intrinsically disordered and core domains were excised from pCXJ22-AmMGM101, pScAmMGM101, and pAmScMGM101 with S.cerevisiae Mgm101p intrinsically disordered region fused to GFP in a pCXJ8 vector , an EcoRI fragment containing the S.cerevisiae MGM101 open reading frame fused to GFP was excised from pCXJ8-MGM101GFP EcoRI site of pEMBL8+ Pfu Ultra II Fusion HS DNA polymerase (Agilent Technologies) with a method modified from that described in 5′ CAAGTCAAGGGAGACAGTAAAGGAGAAGAACTTTTCAC 3′; 5′ TTATAACTATTGTTCAAAGAATC 3′ and 5′ TTCTTCTCCTTTACTGTCTCCCTTGACTTGCTTATAAC 3′; 5′ CTTTTCACTGGAGTTGTCCC 3′. PCR reactions were digested with DpnI (New England Biolabs) to remove template DNA and purified with the QIAquick PCR Purification Kit (Qiagen). 50 ng of each PCR product were combined in 10 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA heated to 95°C for 3 minutes and annealed at 65°C for 2 minutes followed by 15 minutes at 25°C. Colonies harbouring the desired deletion construct were recovered following transformation of JM109 (Promega) with the annealed products. The resulting plasmid, pEMBLMGM101Δ99-269GFP, was digested with EcoRI and the purified insert was ligated into the EcoRI site of pCXJ8 to give pCXJ8-MGM101Δ99-269GFP. The domain structure of the wild-types and chimeric constructs is illustrated in To make a construct containing the Transformation of yeast was performed by the lithium acetate-dimethyl sulfoxide method Digital images of yeast cells were captured with a Spot Camera mounted on a Leica DM6000B microscope with 63× or 100× NA1.4 oil immersion objectives.Figure S1S.cerevisiae and the corresponding region determined from the sequence alignment for A.millepora. The orange bars represent predicted disordered regions. Disorder predictions were carried out using three independent methods, IUPred, PONDR VSL2, and DISOPRED2, indicated by different shades. The sequences were aligned so that the beginning of the core region is in the same vertical position.Domain organization for the six representative members of Mgm101p sequence family. The thick black line represents the predicted mitochondrial target signal. The green bar indicates the experimentally determined core region for (EPS)Click here for additional data file.Figure S2S.cerevisiae and A.millepora. Based on these regions three constructs were designed. Construct 1 (A.m.ID-A.m.C) has the A.millepora disordered and A.millepora core regions fused to the S.cerevisiae mitochondrial import signal. Construct 2 (S.c.ID-A.m.C) has the S.cerevisiae mitochondrial import signal and disordered region fused to the A.millepora core. Construct 3 (A.m.ID-S.c.C) has the A.millepora disordered region and the S.cerevisiae core fused to the S.cerevisiae mitochondrial import signal. In the last two cases the dipeptide GlyThr was introduced by the restriction site (Acc651) that was used to facilitate the cloning.Structure of the Mgm101p constructs. The sequence regions for Mgm101p, indicating the mitochondrial target sequence (S.c.T/A.m.T) in blue, the disordered region (S.c.ID/A.m.ID) in red and orange respectively, and the core region (S.c.C/A.m.C) in green for (EPS)Click here for additional data file.Figure S3A.millepora MGM101 protein derived from Accession JR994989 and the A.millepora genome sequence . ScMgm101p. The sequence of the S.cerevisiae MGM101 protein (Accession NP_012678) A.m.ID-A.m.C. (pCXJ22-AmMGM101). This construct consists of the S.cerevisiae mitochondrial targeting signal and the A.millepora ID and core regions. S.c.ID-A.m.C. (pScAmMGM101). This construct consists of the S.cerevisiae mitochondrial targeting signal and ID region and the A.millepora core region. A.m.ID-S.c.C. (pAmScMGM101). This construct consists of the S.cerevisiae mitochondrial targeting signal, the A.millepora ID region and the S.cerevisiae core. S.c.MGM101GFP. The sequence of the MGM101-GFP fusion protein from pCXJ8-MGM101GFP. S.c.ID-GFP. (pCXJ8MGM101Δ99-269GFP). The sequence of the S.cerevisiae ID region fused to GFP. It represents a deletion of amino acids 99–272 from S.c.MGM101GFP Click here for additional data file.Figure S4S.cerevisiae MGM101 open reading frame fused in-frame to GFP . Expression of the fusion protein is under the control of the S.cerevisiae ADH  promoter.Map of plasmid pCXJ8-MGM101GFP. This plasmid contains the full-length (TIF)Click here for additional data file.Table S1MGM101 constructs integrated at ura3Distribution of phenotypes in segregants from pUC-N (DOC)Click here for additional data file.


Pseudomonas syringae and Xanthomonas species, but so far their mode of action remains unrevealed. The crystal structure of LlpABW, the prototype lectin-like bacteriocin from Pseudomonas putida, reveals an architecture of two monocot mannose-binding lectin (MMBL) domains and a C-terminal β-hairpin extension. The C-terminal MMBL domain (C-domain) adopts a fold very similar to MMBL domains from plant lectins and contains a binding site for mannose and oligomannosides. Mutational analysis indicates that an intact sugar-binding pocket in this domain is crucial for bactericidal activity. The N-terminal MMBL domain (N-domain) adopts the same fold but is structurally more divergent and lacks a functional mannose-binding site. Differential activity of engineered N/C-domain chimers derived from two LlpA homologues with different killing spectra, disclosed that the N-domain determines target specificity. Apparently this bacteriocin is assembled from two structurally similar domains that evolved separately towards dedicated functions in target recognition and bacteriotoxicity.Lectin-like bacteriotoxic proteins, identified in several plant-associated bacteria, are able to selectively kill closely related species, including several phytopathogens, such as  BW from a saprophytic plant-associated Pseudomonas whose genus-specific target spectrum includes several phytopathogenic pseudomonads. By determining the 3D structure of this protein, we could assign LlpA to the so-called monocot mannose-binding lectin (MMBL) family, representing its first prokaryotic member, and also add a new type of protective function, as the eukaryotic MMBL members have been linked with antiviral, antifungal, nematicidal or insecticidal activities. For the protein containing two similarly folded domains, we constructed site-specific mutants affected in carbohydrate binding and domain chimers from LlpA homologues to show that mannose-specific sugar binding mediated by one domain is required for activity and that the other domain determines target strain specificity. The strategy that evolved for these bacteriocins is reminiscent of the one used by mammalian bactericidal proteins of the RegIII family that recruited a C-type lectin fold to kill bacteria.In their natural environments, microorganisms compete for space and nutrients, and a major strategy to assist in niche colonization is the deployment of antagonistic compounds directed at competitors, such as secondary metabolites (antibiotics) and antibacterial peptides or proteins (bacteriocins). The latter selectively kill closely related bacteria, which is also the case for members of the LlpA family. Here, we investigate the structure-function relationship for the prototype LlpA In most natural settings, complex interactions occur among microorganisms, ranging from nutritional co-operation to warfare among competitors. Examples of such interplay have been reported not only between unrelated microorganisms , first identified in Pseudomonas putida BW11M1 Pseudomonas-specific antibacterial proteins not related to any known bacteriocin. Additional llpA-like genes encoding functional bacteriocins were identified by genome mining in the biocontrol strain Pseudomonas fluorescens Pf-5 Pseudomonas syringae pv. syringae 642 Xanthomonas pathovars extended its occurrence as a genus-specific killer protein Xanthomonas LlpA precursor is proteolytically processed by removal of a characteristic Type II secretion signal peptide, whereas such N-terminal sequence is lacking in Pseudomonas homologues, indicating that secretory routes may differ among LlpA producers.Bacteriocins are structurally and mechanistically very diverse. This is reflected in the bacteriocinogenic potential of the genus Ruminococcus species, Gram-positive bacteria colonizing the rumen, was demonstrated The amino acid sequence of LlpA suggests the presence of two related domains belonging to the ‘monocot mannose-binding lectin’ (MMBL) family BW as the prototype of a novel family of antibacterial proteins and explore how domain architecture and specific structural elements contribute to its activity and specificity.Here we report on the crystal structure of LlpABW from P. putida BW11M1 (LlpABW) shows it contains two β-prism MMBL domains, referred to as the N-domain and the C-domain following their position in the amino acid sequence  of 0.34 Å for 270 Cα atoms. This RMSD value does not change significantly when the individual domains are fitted separately (0.32 Å for 120 Cα's of the N-domain and 0.22 Å for 115 Cα's of the C-domain), indicating that the inter-domain orientation is fixed. This stems from three sets of interactions . Strand β1′ is a short piece of β-strand that is part of the long N-terminus and forms a parallel extension on the opposite site of sheet IIN (hydrogen bonding to strand β2), making this β-sheet a mixed type six-stranded one rather than the canonical four-stranded anti-parallel sheet.A superposition of the Cα-trace of the N- and C-domain of LlpAelements . A threeDespite these additions to the β-prism fold, the common core of the N-domain more closely resembles that of the well-studied and highly conserved monocot lectins (e.g. RMSD of 1.35 Å with snowdrop lectin compared to 1.82 Å for the C-domain). This structural divergence is in contrast with the degree of conservation of the carbohydrate-binding motif characteristic of the monocot lectins (QxDxNxVxY) in each of the three subdomains. In the N-domains of LlpA homologues, the surface-exposed motifs III and II are not well conserved and likely lost their function during evolution. In contrast they seem to be better conserved in the C-domains . ApparenBW further extends into a β-hairpin that helps to define the relative orientations of its two MMBL domains. This β-hairpin is highly bent due to a β-bulge inserted into its second β-strand  of an active MMBL mannose-binding site (BW with 200 mM methyl-α-D-mannopyranoside (Me-Man) led to clear electron density of a single Me-Man in site IIIC of each of the two LlpABW monomers in the asymmetric unit Man and the pentasaccharide GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man ]Man ,C. In th1–6)]Man . In the 1–6)]Man .C of both LlpABW molecules in the asymmetric unit is involved in crystal packing interactions and the presence of Me-Man is therefore sterically excluded. All residues that form specific hydrogen bonds with Me-Man are retained but substitutions occur for three side chains that provide van der Waals contacts .The putative carbohydrate-binding sites in the N-domain of LlpAractions . In the as Gln82 . All othas Gln82 . The potonserved  and contBW motifs IIIN, IIIC and IIC create potential carbohydrate binding sites that may be involved in bacteriotoxicity of the protein. We therefore examined the role of carbohydrate binding in the bactericidal function of LlpABW. The presence of methyl-α-D-mannopyranoside up to 500 mM in the medium did not influence the activity of LlpABW on P. syringae GR12-2R3. Glycan array profiling did not highlight any specific oligosaccharide structure that could represent a natural ligand of LlpABW . A minor negative effect of the IIC mutation is only apparent in a double mutant, when combined with a modified IIIN motif.To assess whether the mannose-recognizing QxDxNxVxY motifs in LlpAng sites . Semi-quGR12-2R3 . In contBW, indicating that the mutations do not affect the overall structure of the protein. Isothermal titration calorimetry (ITC) showed that LlpABW has an affinity of 2.1 mM for the pentasaccharide GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man, the highest among all the tested oligo-mannosides (See BW-carbohydrate interactions). This is in agreement with the crystal structures of the different complexes since this sugar is the one with the largest binding interface , LlpAV208Y (a site IIC knockout) and of the double mutant LlpAV177Y-V208Y with α-methyl mannoside clearly pinpoint site IIIC as the only responsible for the sugar binding activity. Point mutations in both sites or IIIC (V177Y) alone, completely abrogate sugar binding. However knocking out site IIC (V208Y) has little effect in binding and the affinities of LlpAV208Y for α-methyl mannoside and Manα(1–3)Man are very close to the ones measured for the wild-type protein . For LlpAV177Y this value was reduced to 6.1%, significantly lower than for LlpA. Killing activity was even further reduced for LlpAV177Y-V208Y (3.7%). These results are consistent with the MIC determination and ITC data, indicating that an active site IIIC is required to generate a fully active LlpA bacteriocin. The difference in bacteriotoxicity between LlpAV177Y and LlpAV177Y-V208Y suggests that site IIC has a supporting role in the LlpABW bacteriotoxicity.While the V208Y mutation in the IIe in MIC . The mutanalysis . ProportBW toxicity. Considering the increased binding motif degeneration in the N-domain and the fact that a Ruminococcus bacteriocin composed of only a single MMBL domain fused to an unknown domain has been identified BWllpA were engineered to potentially encode proteins lacking the first or second MMBL domain, a gene product devoid of the C-terminal hairpin, or a protein retaining only an individual domain   . To takeE. coli  and in the C-domain (IIIC and IIC) of LlpABW, revealed that a non-occluded IIIC pocket is required to obtain a fully active LlpABW molecule. A negative co-operative effect on activity resulted when the IIC site was additionally modified. Although mannose-containing carbohydrates can bind to the IIIC pocket of LlpABW, it remains unclear if these are part of or mimic the natural ligand required for biological activity since bacteriocin activity is not impaired in the presence of excess mannose.Structure elucidation of LlpAN site did not provoke a negative effect on antibacterial activity. However, the N-domain appears to play a major role in target selection. This was demonstrated by assessing the differential activity of domain chimers against two target strains, diagnostic for the LlpABW- and LlpA1-specific killing.A mutated IIIBW. An equivalent C-terminal stretch is absent from the Xanthomonas citri LlpA-like bacteriocin E. coli and a bacteriocin assay with permeabilized cells, it cannot be excluded that this structural element may play a role in the way an LlpA protein is exported by its native producer cells.The β-hairpin does not appear to be a specificity determinant, although it constitutes the most variable region among LlpA-like bacteriocins. Possibly, it is required for thermodynamic stability since it needs to be intact in LlpABW as a bactericidal protein fits within this picture of MMBL domains being involved in general defense mechanisms. Since no antibacterial activity has been assigned to the eukaryotic MMBL proteins, it is challenging to identify structural features that confer the intragenus-specific bacteriocin activity of LlpA, as shown for proteins from P. putidaP. fluorescensP. syringaeXanthomonas citriIn general, a defensive role has been proposed for the (oligo)mannose-binding MMBL lectins based on insecticidal, nematicidal, antifungal, or even antiviral activities demonstrated for several of these proteins that are abundantly found in monocot plants BW and its homologues in other Pseudomonas species is intriguing in view of their extracellular location P. aeruginosa, that also lacks such signal sequence The absence of any known secretory signal sequence in LlpAEscherichia coli was routinely grown in shaken Luria-Bertani  broth at 37°C. Pseudomonas strains were grown in Tryptic Soy Broth (BD Biosciences) at 30°C with shaking. Media were solidified with 1.5% agar (Invitrogen) and supplemented with filter-sterilized antibiotics as required at following concentrations: ampicillin (Sigma-Aldrich), 100 µg/ml or kanamycin (Sigma-Aldrich), 50 µg/ml. Isopropyl β-D-thiogalactoside  and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside  were added for blue/white screening of pUC18-derived plasmids in E. coli.Bacterial strains and plasmids used in this study are listed in E. coli TOP10F′ (Invitrogen). E. coli BL21(DE3) (Novagen) was used as a host for plasmids driving recombinant protein expression. Genomic DNA from P. putida BW11M1 was isolated using the Puregene Yeast/Bact. Kit B (Qiagen). Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen). Stocks were stored at −80°C in the appropriate medium in 25% (v/v) glycerol.Plasmids used for antibacterial testing and sequencing were propagated in E. coli cells, heat shock transformation of E. coli and DNA electrophoresis Standard methods were used for preparation of competent BWllpA was amplified by PCR with Platinum Pfx DNA polymerase (Invitrogen), using a C1000 Thermal Cycler (Bio-Rad). P. putida BW11M1 genomic DNA was taken as a template, and combined with primers PGPRB-3155 and PGPRB-3156. The amplicon was purified using the QIAquick PCR Purification Kit (Qiagen), digested with KpnI and BamHI, ligated in pUC18, and transformed to E. coli TOP10F′. Transformants were verified for the presence of the insert by PCR using Taq Polymerase (BIOKÉ) with primers PGPRB-2545 and PGPRB-2546. The cloned construct (pCMPG6129) was purified and its insert confirmed by sequencing.A 921-bp fragment containing BWllpA in pUC18 (pCMPG6129) and N-terminal His-tagged BWllpA in pET28a . Chimeric constructs were obtained using pCMPG6129 and pCMPG6053 , carrying expression constructs pCMPG6056, pCMPG6149, pCMPG6150 and pCMPG6151 respectively, were performed as described by Parret and collaborators 6  as primary antibody. Fractions free of other proteins, as verified by SDS-PAGE and subsequent Coomassie Blue staining, were dialyzed against bis-TRIS propane buffer . Concentrations of purified proteins were determined by absorbance measurement at 280 nm using molar extinction coefficients of 62910 M−1 cm−1 for LlpABW, 64400 M−1 cm−1 for LlpAV177Y and LlpAV208Y, and 65890 M−1 cm−1 for LlpAV177Y-V208Y. Extinction coefficients were calculated according to Pace and collaborators Protein isolation and purification of N-terminal HisE. coli cells carrying pUC18-derived constructs was assayed as follows: 2-µl drops of an overnight stationary-phase culture were spotted onto LB agar plates and incubated for 8 h at 37°C. Next, plates were exposed to chloroform vapor (30 min), aerated and subsequently overlaid with 5 ml of soft agar (0.5%), seeded with 200 µl of a cell culture of an indicator strain (∼108 CFU/ml), followed by overnight incubation at 30°C. Next day, plates were scored for the presence of halos in the confluently grown overlay.Bacteriocin production by 6-tagged proteins were performed as described by Ghequire and collaborators Antibacterial activity assays with purified recombinant His4–105 CFU/ml and incubated at 30°C, with a two-fold dilution series of recombinant His6-tagged LlpABW or mutant LlpABW. Bis-TRIS propane buffer was used as control. The MIC value was determined as the minimum concentration of protein at which no growth of the indicator strain (OD600<0.2) occurred after 24 h. At least three independent repeats, each with three replicates, were carried out.A Bioscreen C apparatus  was used to determine the minimum inhibitory concentration (MIC). An overnight culture (16 h) of the indicator strain was diluted to 106-tagged LlpABW was lyophilized and verified for antibacterial activity. After re-dissolving in MilliQ water, recombinant LlpABW was diluted to 200 µg/ml with binding buffer , and used to probe the printed glycan arrays http://www.functionalglycomics.org/). Monoclonal anti-His6 antibodies (Roche Diagnostics) were used as primary antibodies, and fluorescently labeled anti-mouse IgG as secondary antibodies.HisCD spectra were acquired on a Jasco J-715 spectropolarimeter. Curves were averaged over 8 scans, taken at 25°C using a 1 mm cuvette. Samples were dialyzed against bis-TRIS propane buffer , filtered and degassed prior to data acquisition. All proteins were assayed at 0.4 mg/ml.BW, LlpAV177Y, LlpAV208Y and LlpAV177Y-V208Y was dialyzed to bis-TRIS propane buffer. Sugars were directly dissolved into the same buffer. The samples were filtered and degassed for 10 min at 25°C before being examined in the calorimeter. The titrations were carried out at 25°C, injecting the sugars Man, Manα(1–3)Man, Manα(1–6)Man, Manα(1–3)[Manα(1–6)]Man and GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man) into a protein solution (protein concentrations ranged from 2 mM to 4 mM depending on protein availability). All data were analyzed using the MicroCal Origin ITC 7.0 software. Binding affinities and thermodynamic parameters from all ITC titrations are reported in ITC titrations were carried out on an ITC200 apparatus . Prior to the measurement, LlpABW have been described Expression, purification and crystallization of recombinant His-tagged LlpABW was solved combining single isomorphous replacement with anomalous scattering (SIRAS strategy) from a p-chloromercurybenzoate derivative. The heavy-atom substructure was determined with SHELXD phenix.refineThe crystal structure of free LlpABW were transferred to artificial mother liquor  enriched with either 200 mM methyl-α-D-mannopyranoside (Me-Man), Manα(1–2)Man, GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man (M592), D-galactose, L-fucose or N-acetyl-D-glucosamine and allowed to equilibrate overnight . Data were collected at room temperature on EMBL beamline X13, except for the N-acetyl-D-glucosamine soak collected at the PROXIMA-1 beamline of the SOLEIL synchrotron  and the D-galactose soak collected at ESRF beam line ID14-1 . All data were scaled and merged using the HKL package. Refinement was started from the coordinates of the ligand-free structure using phenix.refine. Manual rebuilding, including the introduction of the carbohydrate ligand if present, was done with Coot BW from P. putida BW11M1 (PDB entry 3M7H) and LlpABW in complex with methyl-α-D-mannoside (PDB entry 3M7J), with Manα(1–2)Man (PDB entry 4GC1), and with GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man (PDB entry 4GC2) have been deposited at the PDB.Crystals of LlpAP. syringae GR12-2R3 (16 h) were diluted to OD600 0.5 and washed twice with phosphate-buffered saline (PBS). Cells were treated with LlpA, mutant proteins or buffer , at a final concentration of 50 µg/ml for 1 h, at 20°C. Next, PBS-washed bacteria were stained using the Live/Dead BacLight bacterial viability kit (Invitrogen), incubated for 15 minutes, and analyzed on a BD Influx (BD Biosciences). Excitation of the dyes was done at 488 nm, and fluorescence measured at 530 nm for SYTO 9 and at 610 nm for propidium iodide. Results were processed with FlowJo 10.0.4 software Click here for additional data file.Figure S2Quaternary structures and domain organization of various MMBL family members. Individual domains or protomers are shown in different colours. The domain or protomer colored green  is always shown in the same orientation. Bound carbohydrates are shown in black stick representation. For LlpA a single pentasaccharide is bound to site IIIC. In the case of Galanthus nivalis (snowdrop) lectin (PDB entry 1JPC), twelve trimannosides are bound to all QxDxNxVxY motifs (three on each monomer of the homotetrameric protein). The snowdrop lectin tetramer consists of the association of two domain-swapped dimers (green-blue and pink-yellow). In the case of Allium sativum (garlic lectin ASA I - PDB entry 1KJ1), again each QxDxNxVxY motif has a dimannose bound while an additional sugar (shown in red) is bound to a non-canonical site. The protein is synthesized as a single chain precursor and post-translationally cleaved into two MMBL domains that adopt the same domain-swapped dimer as found in snowdrop lectin. Gastrodianin is a monomeric MMBL family member from the orchid Gastrodia elata (PDB entry 1XD5). The location(s) of its carbohydrate-binding site(s) is (are) not known. The fetuin-binding tandem-MMBL SCAfet from Scilla campanulata (PDB entry 1DLP) consists of two covalently attached MMBL domains, whereas in LlpA the swap of the C-terminal β-strands is retained. The relative orientation in the two domains is as in ASA I. This lectin binds fetuin rather than oligomannosides, but the locations of the binding sites are not known.(JPG)Click here for additional data file.Figure S3BW (red), C-domain of LlpABW (blue) and Stereo view of the superpositions (Cα representations) of the N-domain of LlpAGalanthus nivalis lectin . The superposition is shown in two orientations rotated by 90°.(JPG)Click here for additional data file.Figure S4Sequence alignment of potential mannose-binding motifs in prokaryotic tandem MMBL proteins. The sequences corresponding to the consensus motif QxDxNxVxY, extracted from the N-domain and the C-domain of P. putida LlpABW and its homologues, are aligned per domain. Sequence conservation is visualized by differential shading. The sequence logo representation visualizes the degree of consensus for each residue. LlpA proteins with proven bacteriotoxic activity are labeled with an asterisk. Accession numbers: Arthrobacter sp. FB24 (YP_829274), Burkholderia ambifaria MEX-5 (ZP_02905572), Burkholderia cenocepacia AU 1054 (Pseudomonas chlororaphis subsp. aureofaciens 30–84 (EJL08681), Pseudomonas putida BW11M1 (AAM95702), Pseudomonas fluorescens Pf-5 (LlpA1 Pseudomonas sp. GM80 (Pseudomonas syringae pv. aptata DSM 50252 (EGH77666), Pseudomonas syringae pv. syringae 642 (ZP_07263221), Xanthomonas axonopodis pv. citri str. 306 (AAM35756).(TIF)Click here for additional data file.Figure S5Sequence alignment of the carboxy-terminal sequences of LlpA-like proteins. The P. putida LlpABW sequence adopting a β-hairpin fold is delineated in C (ted in C . The seq(TIF)Click here for additional data file.Figure S6Electron density for (A) Methyl-α-D-Man, (B) Manα(1–2)Man and (C) GlcNAcβ(1–2)Manα(1–3)[GlcNAcβ(1–2)Manα(1–6)]Man. Difference electron-density maps are calculated by removing the sugar residues from the final coordinates and applying one round of slow-cool simulated annealing refinement to remove potential bias. The atomic model is superimposed in each case.(JPG)Click here for additional data file.Figure S7BW and garlic lectin.Mannose binding to LlpA (A) Cartoon representation of subdomain IIIC of LlpABW (green) with residues implicated in carbohydrate binding showing in ball-and-stick representation and labeled . The Me-Man residue is shown in red. Selected hydrogen bonds are shown as black dotted lines. (B) Stereoview of the superposition of subdomain IIIC of LlpABW (green) on the equivalent subdomain of garlic lectin (blue). The Me-Man residue bound to LlpABW is shown in red, the mannose bound to garlic lectin in blue. (C) Stereoview of the superposition of subdomain IIIC of LlpABW (green) identical as in panel A, but emphasizing the location of Val177 (shown as black sticks). The modeled Val177Tyr mutation is shown as orange sticks. Tyr177 makes a steric clash with the bound mannose (red) and is therefore expected to prevent binding, in agreement with our ITC experiments.(JPG)Click here for additional data file.Figure S8BW C-domain.Sites II and I of the LlpA (A) Stereoview of site IIC of the C-domain (colored according to atom type) superimposed on site IIIC of the C-domain (dark gray). The Me-Man bound in site IIIC is shown in red. This site is very similar to site IIIC but in the crystal it is inaccessible due to crystal lattice interactions. Residue labels correspond to residues of site IIC. (B) Similar view showing site IC of the C-domain (colored according to atom type) superimposed on site IIIC of the C-domain (dark gray). The Me-Man bound in site IIIC is shown in red. The stretch of Ile137-Leu139 that provides a steric conflict preventing Me-Man binding in site IC, is highlighted with carbon atoms drawn in green. Residue labels correspond to residues of site IC.(JPG)Click here for additional data file.Figure S9BW N-terminal domain.Sites of the LlpA (A) Stereoview of site IN of the N-domain (colored according to atom type) superimposed on site IIIC of the C-domain (dark gray). The Me-Man bound in site IIIC is shown in red. The stretch of Ile271-Trp274 that provides a steric conflict preventing Me-Man binding in site IN is highlighted with carbon atoms drawn in green. Residue numbering corresponds to residues of site IN. (B) Similar superposition for site IIN of the N-domain. Phe86 that prevents Me-Man binding to this site through a steric conflict is highlighted in green. Other residues belonging to site IIN are labeled in teal. Three residues of site IIIC for which site IIN has no structural equivalent are labeled in black. (C) Similar superposition for site IIIN of the N-domain. Residues belonging to site IIIN are labeled in teal. One residue of site IIIC for which site IIIN has no structural equivalent, is labeled in black. For this site there are no obvious steric conflicts that would prevent positioning of a Me-Man residue although none is observed experimentally.(JPG)Click here for additional data file.Figure S10Quantification of live and dead cells by flow cytometry.P. syringae GR12-2R3 cells were treated with LlpA (A), LlpAV177Y (B), LlpAV208Y (C), LlpAV177Y-V208Y (D), or buffer (E) at a final concentration of 50 µg/ml for 1 h at 20°C. After live/dead staining, cells were analysed by flow cytometry. Data processing allowed to distinguish populations of dead (left) and live (right) cells. Spot densities ranging from high to low are differentiated by a color gradient from red, yellow, green, teal to blue. Representative samples for LlpA, mutant proteins and buffer control are shown in panels A–E.(TIF)Click here for additional data file.Figure S11BW deletants and inactive LlpABW/LlpA1 chimers.Overview of inactive LlpA The equivalent domains of LlpA1 are delineated based on pairwise sequence alignment with LlpABW: N-domain (orange), C-domain (purple), C-terminal extension (grey). No bacteriocin activity was conferred by these constructs upon recombinant E. coli cells tested against P. syringae GR12-2R3 (indicator strain for native LlpABW) and P. fluorescens LMG 1794 (indicator strain for native LlpA1). The small black rectangle represents an artificial linker sequence (DASRS).(TIF)Click here for additional data file.Table S1BW as measured by fluorescence intensity.Glycan array profile of LlpA Results including a comprehensive list of oligosaccharides (array version PA_v5) are available from the Consortium of Functional Glycomics .(XLS)Click here for additional data file.Table S2Bacterial strains and plasmids used in this study.(DOC)Click here for additional data file.Table S3PCR primers used in this study.(DOCX)Click here for additional data file.Table S4BW deletants and LlpABW/LlpA1 chimers.Protein sequences of LlpA(DOCX)Click here for additional data file.Table S5Structure determination and refinement.(DOCX)Click here for additional data file.


A 15-year-old boy was admitted to our hospital with abdominal pain and a surgical history of video-assisted transumbilical appendectomy for a gangrenous appendicitis 7 years back. The small stump was closed without purse-string suture. The postoperative period was complicated by an intraperitoneal abscess treated with antibiotics. Later on he experienced multiple episodes (thrice a year) of self limiting intermittent abdominal pain with pallor, sweating and diarrhea. During current admission, the abdominal pain started 3 days back. It was described as colic, getting worse and associated with pallor and sweating. There was no fever, vomiting, diarrhea, or urinary symptoms. Vital signs were normal. Physical examination revealed a subumbilical scar and peritoneal irritation in the right lower quadrant; no palpable masses were appreciated and genitourinary examination was unremarkable. The abdomen was not distended but bowel sounds were decreased. Laboratory data showed an increased white blood cell count of 10,570 cells/mm with 74.2% neutrophils, CRP 5.41 mg/dl and ESR 4 mm/h. Electrolytes, urinalysis, transaminases and amylase were all in the normal range. Abdominal ultrasound demonstrated ileocecal wall thickening (3-6 mm) with increased signals in the vascular color-doppler and hyperplasia of the surrounding fatty tissue. A small amount of corpuscular pericecal free fluid extending to Douglas pouch was noticed . CT scan showed a mixed cystic and solid mass (6.5 cm x 6 cm x 4.2 cm) in the right lower quadrant with a calcified fecolith . The presumptive preoperative diagnosis was stump appendicitis. At operation, a 5-cm-long appendiceal stump with a perforation at the base and a fecolith outside of the lumen was found and removed. A purse-string suture was placed to close the cecal wall at the base of the removed stump. Histopathology showed acute inflammation and patchy necrosis of the appendiceal stump. The post-operative course was uneventful.Stump appendicitis is the re-inflammation of the residual appendiceal tissue after an appendectomy. It represents a rare delayed complication of appendectomy the exact incidence of which is not known. In the cases described in literature the time from appendectomy to the development of stump appendicitis ranges from few weeks to decades. Incomplete appendectomy leaving a stump longer than 5 mm, severe local inflammation preventing adequate identification of the appendiceal base, retrocecal or subserosal appendix, and, last but not least, the insufficient experience of the surgeon may influence the occurrence of this condition.[1-5] Moreover some sources have suggested that the growing use of laparoscopic appendectomy may increase the frequency of stump appendicitis. This may be the result of leaving a longer stump, secondary to a smaller field of vision, lack of three-dimensional perspective, and the absence of tactile feedback. The incidence of stump appendicitis can be minimized simply by adequately visualizing the base of the appendix and creating a stump less than 3 mm in length. Therefore, if performed properly, there is no reason why laparoscopic appendectomy should lead to a higher incidence of stump appendicitis.Clinical features of stump appendicitis do not differ from that of acute appendicitis, even though the history of a previous appendectomy can be misleading causing a delay in diagnosis. Ultrasonography can be useful in identifying inflammatory changes. The abdominal CT is reported as the gold standard for the diagnosis and should be considered as the initial diagnostic study in patients with right lower quadrant symptoms after appendectomy. It allows the recognition of retained fecolith or postoperative abscess as well as cases of stump appendicitis.[1] Stump appendicitis has a higher risk of complications with perforation being reported in nearly 70% of the cases. It is therefore imperative that patients with stump appendicitis undergo complete appendectomy as soon as possible. Stump appendicitis should be considered in any patient with a previous history of appendectomy and a convincing history of acute appendicitis. Treatment by re-surgery and complete removal of the appendix will resolve the case.Source of Support: NilConflict of Interest: None declared


Cryopreservation of three endangered Belgian sheep breeds required to characterize their intra-breed genetic diversity. It is assumed that the genetic structure of a livestock breed depends mostly on gene flow due to exchanges between herds. To quantify this relation, molecular data and analyses of the exchanges were combined for three endangered Belgian breeds.Structure). Exchanges of animals between 20, 46 and 95 herds according to breed were identified via semi-directed interviews and were analyzed using the concepts of the network theory to calculate average degrees and shortest path lengths between herds. Correlation between the Reynolds’ genetic distances and the shortest path lengths between each pair of herds was assessed by a Mantel test approach.For each breed, between 91 and 225 sheep were genotyped with 19 microsatellites. Genetic differentiations between breeds and among herds within a breed were evaluated and the genetic structure of the breeds was described using Bayesian clustering (Genetic differentiation between breeds was high (0.16). Overall Fst values among herds were high in each breed . Use of the Bayesian approach made it possible to identify genetic groups of herds within a breed. Significant correlations between the shortest path lengths and the Reynolds’ genetic distances were found in each breed , which demonstrate the influence of exchanges between herds on the genetic diversity. Correlation differences between breeds could be explained by differences in the average degree of the animal exchange networks, which is a measure of the number of exchanges per herd. The two breeds with the highest average degree showed the lowest correlation. Information from the exchange networks was used to assign individuals to the genetic groups when molecular information was incomplete or missing to identify donors for a cryobank.A fine-scale picture of the population genetic structure at the herd level was obtained for the three breeds. Network analysis made it possible to highlight the influence of exchanges on genetic structure and to complete or replace molecular information in establishing a conservation program. Local livestock breeds play an important role in providing food products and environmental services and are part of the cultural heritage. In 2006, 179 of 1409, i.e. the total number of sheep breeds in the world were listed as “endangered” or “critical” and 417 other breeds had an unknown status [.[.[.[Characterization of the intra-breed genetic diversity is a key element to select donor animals in view of ex situ conservation in a cryopreservation program. For the Belgian sheep breeds, little information is available on the pedigrees, which may result in poor evaluation of the intra-breed genetic diversity . Therefo. have hig.[. have dem.[.[. have meaTools such as network techniques can be useful to describe exchange practices. Network analysis has been used in veterinary epidemiology studies to analyze the impact of animal movements between herds on how diseases spread through a population ,15. In wIn this study, we have investigated the intra-breed diversity at the herd level by combining two approaches: genetic markers (microsatellites) and animal exchanges between farmers. For each breed, we have correlated the genetic differentiation between herds and the network of animal exchanges between herds. Thus, this study is aimed at: (i) determining the finest genetic structure of each breed by identifying genetic groups within breeds; (ii) determining whether the network of animal exchanges between herds is linked to genetic differentiation. The results are then used to identify which individuals should be sampled to provide a good representation of the genetic diversity in the cryobank.The Entre-Sambre-et-Meuse (ESM) and the Ardennais Roux (AR) sheep breeds are both bred for meat production and for the management of natural reserves. The Mouton Laitier Belge (MLB) is bred for milk production. The lower number of rams available in the MLB breed and the absence of any selection program have led some breeders to carry out outcross breeding with other breeds such as Zealand or Dutch and German Friesian breeds. For each of the three studied breeds, a first list of breeders was provided by the breeder’s associations. Other breeders were identified during farm investigations. Five hundred and fourteen sheep belonging to 53 herds and born in 84 different herds  were calculated with the same software and methods as mentioned above. In addition Fst over genetic groups were evaluated for each breed.Dr[Genetix version 4.05.2 software [Finally, the Reynolds’ genetic distances Dr between software  for eachsoftware .i and j, the ijth entry of the matrix is 1 if there are one or more animal exchanges between them and 0 if there are none. From this matrix, a visual representation of the network can be obtained, where herds are represented as vertices and the exchanges as edges. For the calculation of network metrics, the direction of the exchanges was not taken into account (undirected networks). Since genetic distances between herds depend on the animal exchanges between the herds, networks of animal exchanges for the three breeds were compared by evaluating the average degree (AD) of the network of each breed. The average degree measures the number of exchanges between herds relatively to the number of herds and is expressed as AD = 2e/n where n is the number of vertices and e, the number of edges [Investigations were carried out on the breeders for each breed. We identified animal exchanges between 20, 46 and 95 herds, respectively for the ESM, MLB and AR populations. For each breed, an adjacency matrix was constructed in which for each pair of herds of edges . The aveGenetic structure was expected to be partially explained by animal exchanges between herds. To verify this assumption, a Mantel test  was perfevaluation of all possible pathways (succession of edges) between two herds for each pair of herds;identification of the shortest path(s) between each pair of herds.igraph package from the R statistical program [The value of the distance between each pair of herds in the matrix corresponds to the number of edges separating the two herds along the shortest path(s). The shortest path lengths were calculated with the  program . All theZT software [5 permutations.Shortest path lengths and Reynolds’ genetic distances were calculated for each pair of herds with at least five sampled individuals, i.e. 8 ESM, 17 AR and 17 MLB herds. The Mantel tests were performed with the software  to evaluOarAE129 marker in the AR breed. Thus, this marker was not taken into account for the joint analysis of the three breeds . Since not all the 205 breeders known for the AR breed could be contacted, interviews were restricted to 58 breeders, i.e. all breeders with more than twenty sheep registered in the flock-book Table . Null alThe average genetic differentiation (Fst) among the three breeds was 0.16. The overall Fst value of pair-wise comparisons among the herds was highest for the ESM population (0.17), indicating a genetic differentiation between herds higher than in the MLB (0.11) and AR (0.10) populations..[The high Fst values within each breed suggested that the level of genetic differentiation was high among herds and motivated further investigation. According to the criterion proposed by Evanno et al., the mosIn the ESM breed, the G3 group comprised two source herds originating from the splitting of a single ancient herd. The last herds  included two source herds. The next 10 MLB herds  comprised pairs of herds with the highest average Reynolds’ distances Figure . This caIn addition to the correlation analysis, a graphical method is proposed  to the hypothetical clusters higher than 0.7). Secondly, 20 non-genotyped animals with genotyped related animals and for which information on the animal exchanges from the original herd with the other herds was available were genetically characterized (17 putatively assigned to the genetic groups and 3 unassigned) using the genotypic information on their dam and sire (17) or on their grandparents (3). Thirdly, two animals were putatively assigned to the genetic groups based only on the information about networks of exchanges  and within the breeds (intra-breed diversity). This latter was firstly evaluated by determining the heterozygosity and the allelic richness. Secondly, the intra-breed diversity was analysed by evaluating the genetic differentiation between herds (inter-herd diversity) and between the genetic groups of herds (inter-group diversity) highlighted with Bayesian clustering in each breed..[.[The observed and expected genetic heterozygosities are smaller than the average values detected in other studies of European sheep breeds -6,10. Th. in a stu.[. were in .[.[Genetic differentiation within the ESM, MLB and AR breeds, respectively 0.17, 0.11 and 0.10, was higher than those obtained by Berthouly et al. and Serr.[. in their.[., 57 Euro.[., nine Al.[. and five.[This high differentiation allowed us to identify genetic groups of herds with similar sheep in each studied breed using clustering methods. Fst values between genetic groups of the MLB and AR breeds are smaller than Fst values between herds, indicating that intra-group variation is higher than intra-herd variation. In the ESM breed, intra-herd variation is higher than intra-group variation. In comparison with the value of 0.12 observed by Guastella et al. among niInformation from the breeders allowed us to explain the observed substructure. Indeed, the genetic homogeneity between herds of the same group can be related to a common origin of the animals or to exchanges between herds. Moreover, suspected events of crossbreeding were confirmed for the MLB breed in which crossbred animals belong to unassigned herds or herds classified in a single group with the Friesian and Zealand sheep..[.[The lower connectivity assessed by the smaller average degree detected in the ESM breed indicates that on average an ESM breeder exchanges animals with fewer breeders than the MLB and AR breeders. This implies a lower gene flow between herds in the ESM breed, which can explain the higher average Reynolds’ distance between herds and a higher inbreeding. For a comparison, Ortiz et al. observed.[. did not .[AD) observed for the ESM breed could explain the higher correlation. A higher AD means that more exchanges occurred between herds, implying more connectivity between them. Thus, two herds in a network with a higher AD are in general linked by a bigger number of shortest paths between them than in a network with a lower AD, for the same value of the shortest path length for the two networks. This can explain the lower Reynolds’ distances observed in the AR and MLB breeds in comparison with the ESM breed, for the networks of herds with a shortest path length value of 3 and 4. The lower increase of Reynolds’ distances in relation with the shortest path length for the MLB and AR breeds is due to a higher connectivity between herds (higher AD) in these two breeds. This could explain the higher correlation between Reynolds’ distances and shortest paths lengths observed in ESM.The impact of farmers’ practices and more specifically of the animal exchanges on the genetic differentiation was confirmed by the significant correlations observed between genetic distances and distances based on the animal exchanges between herds, for the three breeds. This is in accordance with the study of Berthouly et al.. The higOur results show that information about animal exchanges can be used in combination with molecular data. These two types of information were used to characterize and identify herds and animals of the three Belgian sheep breeds to be integrated in a cryopreservation program. Firstly, this fine-scale study of the intra-breed diversity at the herd level allowed us to identify genetic groups and to select genotyped animals, representative of each group in each breed. Secondly, when molecular information was partial (only for relatives) or missing, information about animal exchanges was useful to assign the donor to the identified genetic groups using cluster analysis (presumed assignment). The relevance of molecular data to guide the choice of donors is higher than the relevance of network data because the network information is determined at the herd level only, is based on interviews and thus is heterogeneous in quality and depicts exchanges for a shorter span of time than the molecular information. Nevertheless, this approach was preferred to a method based on a random choice of donors when genetic information is missing and because it is neither time nor money consuming.The use of network techniques was very useful to depict animal exchanges between herds and to evaluate their level of relationship due to animal exchanges. This was necessary to calculate the correlation between genetic distances and distances based on exchanges. Moreover, differences in connectivity of the herds  between breeds in relation with the level of genetic differentiation could also be highlighted with these techniques. However, until now and to our knowledge, no other study has used the networks’ techniques in combination with the analysis of genetic diversity in livestock science. Such techniques could be applied to study the diversity of livestock breeds when other information like molecular, pedigree or phenotypical data is unavailable or not reliable. Many network descriptors are available and could be useful for genetic diversity studies ,38. SincMoreover, network information can be valuable when molecular information is unavailable or incomplete to establish a conservation program (in situ or ex situ). A methodology to choose donors for a cryobank that are representative of the genetic diversity of a given breed based on results from both genetic and the network analyses was developed. Such an approach could be used to establish conservation programs for endangered breeds.The authors declare that they have no competing interests.CD designed the study, collected data and performed statistical analyses for the ESM breed. ID and PVB participated in the design and the coordination of the study. They contributed to data analyses, critically reviewed and helped to draft the manuscript. JFD was responsible for all the steps of the study conception, the collection, the organization and the analyses of the data, and the drafting of the manuscript. All the authors read and approved the final manuscript.Genetic diversity measures for each locus for the three breeds. The file contains the number of samples, the number of alleles, the observed and the expected heterozygosity, the Fis statistic and the result of the test for deviation from Hardy-Weinberg equilibrium for each breed and each locus.Click here for fileRelation between Delta K and the number of clusters K for each breed. The file contains the graphs representing the relation between the Delta K criterion proposed by Evanno et al.[no et al. and the Click here for fileGenetic structure of the ESM, MLB and AR populations for K = 2 and K = 3. The file contains the graphical representation of the Structure results with the Distruct software for the three breeds for K = 2 and K = 3. Each color represents a cluster. Numbers in brackets: number of assigned herds in the genetic groups; numbers below the figures: herds with at least five sampled animals and source herds (*); G1, G2 and G3: genetic groups; GF: German Friesian; DF: Dutch Friesian; Z: Zealand; UA: unassigned individuals.Click here for fileRepresentation of the directed network of exchanges of ESM animals. The file contains a representation of the directed network of animal exchanges between herds of the ESM breed. Each number represents a herd. Orange circles: herds of genetic group G1; blue circles: herds of genetic group G3; green circles: herds of genetic group G2.Click here for fileRepresentation of the directed network of exchanges of MLB animals. The file contains a representation of the directed network of animal exchanges between herds of the MLB breed. Each number represents a herd. Blue circles: herds of the genetic group including animals from the Friesian and Zealand breeds; green circles: herds of genetic group G1; orange circles: herds of genetic group G2.Click here for fileRepresentation of the directed network of exchanges of AR animals. The file contains a representation of the directed network of animal exchanges between herds of the AR breed. Each number represents a herd. Green circles: herds of genetic group G1; blue circles: herds of genetic group G2; orange circles: herds of genetic group G3; dotted edge: link between the small network and the other herds corresponding to the smallest Reynolds’ distance between herds of the small network and the other herds.Click here for fileNumber of herds and exchanges and average degree of networks for each breed. The file contains the number of herds and exchanges and the average degree of the networks for herds with at least five sampled animals and the networks for all herds with identified exchanges for each breed.Click here for fileComparison of genetic and exchange networks. The file contains a comparison between the animal exchange networks and the networks based only on Reynolds’ distances (genetic networks) for the three breeds.Click here for fileMethod of characterization of donors for a cryobank. The file contains a detailed description of the method for characterizing donors for a cryobank.Click here for file