To identify novel antibiotic-producing microbial strains with unprecedented pertinence. We hypothesize that site-specific soil samples will contain a variety of antibiotic-producing species (APS) with diverse specificity of molecular elements.Laboratory of Microbiology, Division of Biological and Health Sciences, University of Pittsburgh, Bradford, PA-16701, USA, between August 2010 and May 2011.The environmental soil samples were collected from residential and recreational sites in Southern, PA, USA at longitude: −76 42 21.7116, latitude: 39 56 35.7252; approximately 201 meters above sea level. Over 70 natural antibiotic-producing soil bacteria were screened against 19 pathogenic microorganisms. Agar-plug assay was established to identify the antibiotics’ potency and pathogenic inhibitory index calculations were employed to measure the inhibitory potential of each isolate; 16S rRNA sequencing was used for microbial classification.S. aureus. We observed a highly bioactive residential soil microbiota compared to recreational soil.A total of 71 microorganisms from residential soil demonstrated zones of inhibition (ZOI), followed by 9 organisms from recreational soil sample. A total of 15 bioactive strains demonstrated convincing growth inhibitory properties against 16 clinically relevant pathogens; 40% revealed pDNA presence, of which 67% exhibited stringent potencies against Enterobacteriaceae, Xanthomonadaceae, and Bacillaceae. These findings may indicate a co-evolutionary biosynthesis of novel antibiotics driven by the increase of bioactive microbiota in residential environments.16S rRNA sequence analysis corroborated several of the species belonging to  The microbial products of secondary metabolism carry an important role in human health, providing roadmaps for the biosynthesis of many synthetic and semi-synthetic drugs. In nature, microbial bioactive products are present as mycotoxins or bacteriosins derived from filamentous and non-filamentous bacteria and fungi. The soil-based actinomycetes have been the source of countless drugs, from streptomycin and actinomycin, to erythromycin and vancomycin . NaturalPlasmid-mediated antibiotic producing genes can be acquired environmentally by transposition from one microorganism to another . The pheDue to their topographical inconvenience, little research has been thoroughly conducted on residential microenvironments. We aimed to explore the natural antibiotic biosynthesis potentials of microorganisms isolated from soil collected at residential and recreational sites and hypothesized that site-specific soil samples will contain a diversity of antibiotic-producing strains (APS) with a variety of molecular elements. The molecular characterization of several isolated growth-inhibitory species revealed plasmid DNA (pDNA) presence. We observed a higher antibiotic-producing community in residential soil compared to recreational soil. The diverse microbial population, approximately 105 microorganisms per gram of soil, displayed copious amounts of soil-dwelling APS against 16 clinically relevant pathogens; 16S rRNA sequencing revealed unique heterogeneity traits in these APS.Environmental soil samples were collected in Southern, PA, USA from 2010–2011 at longitude: −76 42 21.7116, latitude: 39 56 35.7252, approximately 201 meters above sea level. Two primary locations were selected for sample collection: a recreational area with high human traffic  and an authorized residential property with an existent human populace. Collected samples were placed in polyethylene bags and stored in dark at 4 °C until analyzed for antimicrobial properties. The soil type obtained at both locations was MT. Airy-Glenelg-Linganore soil .: Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus thermophilus, Listeria monocytogenes, Ochrobae anthropic, Staphylococcus sciuri, Kocuria kristinae, Klebsiella pneumoniae, Escherichia coli, Enterococcus saccharolyticus, Klebsiella oxytoca, Streptococcus zooepidemicus, Candida albicans, Enterococcus faecalis, Serratia marcescens, Saccharomyces cerevisiae, Micrococcus luteus, Bacillus cereus, and Pseudomonas fluorescens.Due to the heterogeneous nature of soil samples, three different growth medium, Tryptic Soy Agar (TSA), Potato Dextrose Agar (PDA), and Nutrient Agar (NA), were utilized for the emergence and isolation of microorganisms. The antibiotic-producing abilities of the collected microbiota was tested in triplicates and assayed by growth inhibition zone against 19 clinically relevant pathogenic microorganisms obtained from American Type Culture Collection (ATCC) All pathogenic microorganisms were spread on 90-mm polystyrene petri dishes filled with TSA, PDA, and NA medium, respectively. The collected bioactive soil samples (1–5g) from each 112 location were dried at 37°C and sprinkled (~ 80–100 particles) on dishes spread with each respective ATCC pathogen. Control plates were merely spread with ATCC pathogens and incubated at 37°C. After 48 h incubation period, microorganisms displaying clear zones of inhibition against the pathogens were sequestered onto plates of respective medium and assigned numbers. Repeated streaking purified the isolated microorganisms and single-cell colonies were picked up for further screening and temporary storage at 4°C in medium-agar-filled slants. Microorganisms displaying little or no pathogenic inhibitory properties were retested and discarded if scarce antibiotic biosynthesis persisted. Purified microorganisms obtained from this primary screening were further tested for growth-inhibitory properties by using the perpendicular cross-plate method (PCPM) [Due to their potential industrial significance, we aimed to thoroughly investigate antibiotic secreting abilities among the screened APSs. Secondarily screened APSs and blank controls were grown in 50 ml of respective liquid growth medium  at 37°C, 120 rpm for 24 h. Cells were pelleted (OD600 2.2–2.5) by centrifugation at 22°C, 10,000g for 10 min. The supernatant was collected and evaporated by sterile airflow incubation at 37°C. Desiccated supernatant was sterilized and reconstituted in 1 ml respective growth medium and stored at −20°C until analyzed for antibiotic properties through agar-plug assay.Agar-plug assay was adopted from Bechard et al.  with a 5™ Quick Plasmid Miniprep kit . The isolated pDNA was visualized on 1% ( w/v) agarose gel electrophoresis stained with ethidium bromide.The screened APSs were characterized for the presence of pDNA. pDNA was isolated from overnight grown APSs (OD600 2.2–2.5) using PureLink™ Genomic DNA MiniKit K1820-01 . The 16S rRNA gene sequences from these three APSs were then amplified using universal primer  and sequenced at Macrogen Service Center . All sequences were compared with closest relatives from GenBank and Ribosomal Database Project ( RDP) release 10 (http://rdp.cme.msu.edu/index.jsp). Phylogenetic trees were constructed by neighbor-joining method (NJ) with pairwise deletion of gaps in RDP database.Cellular DNA from three APSs: Y14p, Y16 and Y40p, which shared analogous morphologies and growth inhibitory properties as most soil isolates, was extracted using PureLinkThe sequences of all pure cultures have been deposited in GenBank database under accession numbers JQ956432, JQ956433, and JX121858 for Y14, Y16, and Y40, respectively.All experiments were carried out in triplicate, and the experimental results represent the mean of three identical sets of experiments. One-way ANOVAs followed by least significant difference (Tukey’s honestly significant difference) were performed to evaluate the potential significant differences.The primary screening of residential and recreational soils yielded a total of 71 and 9 promising APS with growth inhibitory properties, respectively . On NA mB. cereus, C. albicans, P. fluorescence, S. aureus, and S. zooepidemicus on NA medium (P = .05).All isolates selected from primary screening were re-screened by PCPM. Most isolates obtained from recreational sites showed insufficient inhibitory capacities against all nineteen pathogenic microorganisms during secondary screening. Only one isolate (L59) from recreational sites insistently exhibited growth inhibitory properties, inhibiting A medium . The broA medium . RecreatA medium , P = .05To determine the potential extracellular production of antibiotics from isolates screened by PCPM, agar plug assay was used. A total of eight , four  and two  isolates with growth inhibitory zone formations on NA, TSA, and PDA media respectively revealed extracellular antibiotic production abilities . The sinK. kristinae on PDA media. Isolate Y13 exhibited its maximum inhibitory perimeter against S. marcescess (9.40 mm). Y14p and Y44 displayed their maximum inhibitory perimeters against K. kristinae . Y16 and Y67p displayed their largest inhibitory perimeters against L. monocytogenes (2.54 mm and 2.54 mm). Y16a revealed maximum ZOI against O. anthropi (2.54 mm). The isolate Y17p exhibited its maximum inhibitory perimeter against P. fluorescence, C. albicans and S. aureus . Y29p displayed its largest inhibitory zone against P. fluorescence and S. aureus (2.54 mm and 2.54 mm). Similarly, Y39 had its greatest inhibitory perimeter against En. saccharolyticus (2.29 mm). Y40p greatest inhibitory perimeter was against P. fluorescence (2.54 mm). The isolates Y49 and L59 maximum inhibitory perimeter was against S. aureus (6.35 mm and 6.10 mm). Y63p largest ZOI was against S. zooepiderimicus (3.81 mm). Y64a maximum ZOI was against C. albicans (2.54 mm). Y64b largest ZOI was against L. monocytogenes (5.08 mm).Individual values of average zone of inhibition (ZOI) of each isolate are summarized in P. fluorescence (A.P. = 5) and M. luteus (A.P. = 5), respectively. Y63p however had the widest range of pathogenic inhibition, revealing growth suppressive capacities against more than 60% of the screened clinically-relevant pathogens L. monocytogenes, En. saccharolyticus, K. kristinae, K. faecalis, M. luteus, O. anthropi, S. cerevisiae, S. aureus, S. scuiri, and S. zooepiderimicus. Among the isolated microorganisms, isolates Y14p, Y63p, and Y13 had the greatest P.I.I , respectively, as the combination of their range of pathogenic inhibition, A.P, and ZOI was the greatest , ZOI , and the range of pathogens inhibition (P = .05)  and average range of growth inhibition of pathogenic microorganisms were determined. Isolates Y17p and Y14p demonstrated the greatest A.P, which was observed against greatest . IsolateP = .05) .L. monocytogenes and S. aureus, but the broadest inhibition was demonstrated against P. fluorescens by 83.3 % of pDNA-bearing APSs  were able to inhibit P. fluorescence. Some pDNA-free isolates , however displayed strong inhibitory properties against either S. sciuri, or S. cerevisiae, but the majority  demonstrated inhibition against K. kristinae , tetracyclic, oxytertracycline or other polycyclic compounds which certainly present potential for food-borne and nosocomial disease applications [pDNA-free APSs demonstrated potent antibiotic properties against S. sciuri and K. kristinae, gram-positive facultative anaerobes which are normal inhabitants of human tissue and have been cited to cause a wide range of infectious diseases [S. aureus, the pathogen responsible for Methicillin-Resistant Staphulococcus aureus (MRSA) development, which has been identified as the most common cause of nosocomial diseases, often acquired outside the household and brought back into residential communities by returning patients [S. aureus . The sequence analysis of 16S rRNA revealed the isolates Y14p, Y16, and Y40 to be closely related to family Enterobacteriaceae, Xanthomonadaceae, and Bacillaceae, respectively  release 10  revealed a higher antibiotic-producing community in residential soil compared to recreational soil, supporting the notion of a growing antibiotic resistant microbiota leading to a parallel upsurge of antibiotic-producing microorganisms in heterogeneous microenvironments [The newly emerging cases of antibiotic-resistance have been emphatically linked to the increased use of antibiotics, pharmaceutical drugs, and antibactericidal soaps in human communities. Frequent usage of fertilizing agents in residential environments has also been shown to promote horizontal gene transfer, therefore expanding antibiotic-resistance in these locations ,14. Our ronments ,16. We’vInterestingly, most of the 16S rRNA-sequenced isolates  have been previously associated with nosocomial infections ,20,21. TThe abundance of antibiotic-producing microorganisms in residential soils compared to recreational soils supports our hypothesis. This is likely due in large to antibiotics overuse and the release of unused or un-metabolized antibiotics in residential environments ,24, whicOur findings support previous studies demonstrating an increase in bioactive microbiota in places with high human activity, the limited number of sequenced soil isolates merely enables us to speculate on the possibility of a trend and does not permit us to confirm the frequency of antibiotic-producing microbial presence in residential microenvirons. Studies done on soils from various geographical locations would be needed to determine if this trend persists in most residential environments.


Other types of RBC damage, such as eryptotic damage, failed to induce necroptosis when combined with hCD59 crosslinking. This work sheds light on the requirements for this recently discovered PCD in RBCs and provides a clear picture of the biochemical mechanism of induction of RBC necroptosis.Mature erythrocytes (red blood cells (RBCs)) undergo the programmed cell death (PCD) pathway of necroptosis in response to bacterial pore-forming toxins (PFTs) that target human CD59 (hCD59) but not hCD59-independent PFTs. Here, we investigate the biochemical mechanism of RBC necroptosis with a focus on the mechanism of induction and the minimal requirements for such RBC death. Binding or crosslinking of the hCD59 receptor led to Syk-dependent induction of vesiculated morphology (echinocytes) that was associated with phosphorylation of Band 3 and was required for Fas ligand (FasL) release. FasL-dependent phosphorylation of receptor-interacting protein kinase 1 (RIP1) in combination with plasma membrane pore formation was required for execution of RBC necroptosis. RIP1 phosphorylation led to the phosphorylation of RIP3, which was also critical for RBC necroptosis. Notably, RBC necroptosis was mediated by FasL and not by other candidate inducers, including tumor necrosis factor alpha (TNF- Nonetheless, RBC necroptosis proceeded in a manner similar to that observed in nucleated cells, requiring Fas and Fas ligand (Fas/FasL), mixed lineage kinase domain-like protein, and the phosphorylation of receptor-interacting protein kinase 1 (RIP1) kinase.1 RBC necroptosis was antagonized by caspase-8 and was associated with necrosome formation and conserved necroptosis effector pathways including acid sphingomyelinase-dependent ceramide formation, NADPH oxidase/iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products (AGEs). While RBC necroptosis shared many molecular steps with canonical necroptosis pathways, it was distinct from eryptosis, a RBC-specific programmed cell death (PCD) that functions as a trigger for RBC clearance by macrophages.2Recently, we demonstrated that bacterial pore-forming toxins (PFTs) that target human CD59 (hCD59) induce programmed necrosis (necroptosis) in primary erythrocytes .4 Pore formation may kill cells through osmotic lysis or may activate target cell signaling pathways including proinflammatory, membrane repair, and PCD modules.4 However, the specific mechanisms linking bacterial PFTs to RBC necroptosis have not been described.Proteins that induce plasma membrane pores are a common biological theme and may be pathogen produced, such as bacterial PFTs, or host produced, such as the membrane attack complex (MAC) of the complement system.α) and TNF-related apoptosis-inducing ligand (TRAIL).In this study, we sought to understand the mechanism of induction of RBC necroptosis by bacterial PFTs and the minimal requirements for such death in these target cells. In particular, we show that hCD59 signaling, induced by receptor crosslinking, results in Syk-dependent phosphorylation of Band 3 leading to vesicle (echinocyte) formation and release of FasL. Released FasL induces the phosphorylation of RIP1, which in turn leads to RIP3 phosphorylation. The FasL-dependent phosphorylation of RIP1/RIP3 produces RBC death by necroptosis only when combined with functional pore formation. The ability for necroptosis to proceed in RBCs differs depends on the size and nature of membrane pores. Additionally, we show that RBC necroptosis is mediated only by FasL and not by other known necroptotic mediators/stimuli, including tumor necrosis factor alpha (TNF-1 Recombinant FasL (rFasL) enhances RBC death by near sublytic quantities of PFTs in a necrostatin-1-inhibitable manner (1 and addition of rFasL (but not TNF-α or TRAIL) to RBCs induces the phosphorylation of RIP1 , to induce necroptosis in RBCs  and intermedilysin (ILY), is dependent on Fas-FasL signaling, as a monoclonal antibody targeting FasL (mAb NOK-1) significantly inhibited RBC death.e manner . The hCD of RIP1 . Due to  in RBCs .17 we asked whether two other defined stimuli of necroptosis, TNF-α and TRAIL,7 have a role in this PCD in RBCs. In contrast to rFasL, addition of rTNF-α or rTRAIL to RBCs failed to induce RIP1 phosphorylation  triggered RIP1 phosphorylation in RBCs (hCD59-specific PFTs (VLY and ILY) induce RBC necroptosis while hCD59-independent PFTs do not, in RBCs . Cross-l in RBCs . Thus, s1 To determine whether toxin-induced RIP1 phosphorylation depends entirely on PFT binding to hCD59, we generated binding domain-swapped mutant versions of these PFTs  in RBCs was retained when combined with the hCD59-independent PFTs, PLY or A-tox , or with1 RIP3, which is also critical for necroptosis,7 is phosphorylated in response to crosslinking of hCD59 or hCD59-specific PFTs, VLY and ILY in RBCs , or the polyene PFT of Streptococcus agalactiae β-hemolysin/cytolysin (4 in contrast to the large pores (≥10 nm) formed by cholesterol-dependent cytolysins9 and the MAC,10 CL-CD59 induced a much lower level of necroptosis in RBCs . RBC necroptosis induced by hCD59 signaling appears specific to damage by membrane pore formation, as CL-CD59 did not enhance death via eryptosis  in RBCs  and certain PFTs results in necroptosis of RBCs  may be o the MAC . This idPrimary human RBCs were obtained from healthy volunteers under a protocol approved by the Columbia University Institutional Review Board.μg/ml, ThermoScientific, Waltham, MA, USA) and goat anti-mouse IgG . hCD59 crosslinking was also achieved with a GFP and His-tagged version of the VLY binding domain  and mouse anti-His mAb  or chicken anti-GFP pAb . hCD55 crosslinking was achieved with anti-CD55 mAb  and goat anti-mouse IgG . As negative controls, His-tagged PLYD4 (10 μg/ml) and anti-His mAb  or rGFP and chicken anti-GFP pAb  was added to RBCs. For hemolysis assays, receptor crosslinking was performed concurrently with PFT treatment. For immunoprecipitations (IPs), receptor crosslinking was performed at 37 oC for 30 min followed by the IP procedure.Crosslinking of hCD59 was achieved with anti-CD59 mAb MEM-43 , J1D9 , or RIK-2 (Becton DIckenson), respectively, were used at 1 μg/mL. Inhibition of RIP1 kinase was achieved with 50 μM necrostatin-1 (EMD Millipore). Inhibition of RIP3 was achieved with 2 μM GSK'872 (EMD Millipore). Inhibition of acid sphingomyelinase was achieved with 20 μM desipramine hydrochloride . Iron chelation was achieved with 100 μM 2,2-bipyridyl . Inhibition of AGE formation was achieved with 1 mM pyridoxamine . For inhibition of Syk kinase, Syk kinase inhibitor IV, Bay 61-3606 (EMD Millipore) was used at 2 μM. Inhibition of Src family kinases was achieved with 1 μM PP1 (EMDf Millipore). Osmotic protection was performed with 10% dextran (500 000 MW) in DPBS.For neutralization of FasL, TNF-28β-Hemolysin was extracted from S. agalactiae according to the method of Liu et al.29 Hemolysis assays were performed in DPBS at 37 °C for 30 min with PFTs at the indicated hemolytic units (HU). 1 HU is defined as the concentration at which a PFT causes 50% hemolysis.1 When mAb or pharmacological inhibitors were used they were incubated with RBCs for 1 h at 37 °C before PFT addition. rFasL , rTNF-α , or rTRAIL  was added to RBCs at the same time as PFTs. Receptor crosslinking was performed concurrently with PFT treatment. Hemolysis was measured by relative quantification of hemoglobin release in a spectrophotometer at 415 nm.Recombinant PFTs were expressed and purified as previously described.Hemolysis by the MAC was induced by the classical complement pathway. RBCs were treated with rabbit anti-RBC mAb  for 1 h at 37  °C followed by treatment with several dilutions of rabbit complement serum  in DPBS.μg anti-RIP mAb  for RIP1 or 10 μg anti-RIP3 mAb  at 4 °C overnight following pre-clearing. Protein G Plus Agarose beads  were then added at room temperature for 2 h. After five washes with DPBS, agarose bead immunoprecipitates were suspended in 1 × NuPAGE LDS buffer (Invitrogen), boiled for 10 min, and run on SDS-PAGE followed by western blot transfer onto PVDF membranes. All PFT treatments were conducted by treating RBCs with 0.5 HU of PFT for 30 min at 37 °C before IP procedure. VLY-PLYD4 and PLY-VLYD4 were added to RBCs at a concentration of 1 μg/ml.IPs for RIP1 and RIP3 were performed on RBC sonicates with 10 30 For p-Band 3 or p-Syk detection, RBC lysates (5% (v/v)) were probed with anti-phospho Y21 Band 3 mAb  or anti-phospho Syk (Y525/526)  and blocked/washed with 5% BSA in TBS.For phosphorylated RIP1 (p-RIP1) and RIP3 (p-RIP3) detection, blots were blocked/washed with 5% BSA in TBS. A rabbit mAb  against phosphor-Ser/Thr was used at 1 : 1000 to detect p-RIP1 and p-RIP3 as previously.All other blots were blocked/washed with 5% milk in DPBS. Total RIP1 was detected with anti-RIP mAb  at 1 : 1000. Total RIP3 was detected with polyclonal anti-RIP3 (ThermoScientific) at 1 : 1000. Total Band 3 was detected with anti-Band 3 mAb  at 1 : 1000. Total Syk was detected with polyclonal anti-Syk  at 1 : 1000. FasL was detected with rabbit pAb against FasL  at 1 : 250. Anti-mouse or anti-rabbit secondary horseradish peroxidase conjugates  were used to detect primary antibodies. Western blots were developed with Lumi-Light substrate  for 5 min at room temperature and read in a FluorChem E imager .μg/ml of anti-CD59 mAb  and goat anti-mouse IgG  for 15 min at room temperature. Unstained RBCs were viewed under bright-field using an Axio Observer.Z1 microscope  and an AxioCam MRm digital camera (Zeiss).Human RBC suspensions (0.5% (v/v)) were treated with 0.2 HU of PFT or 1 1 or were treated with crosslinking mAbs against hCD59 as described above for 1 h at 37 °C. RBCs were centrifuged and supernatants were run on SDS-PAGE and transferred onto PVDF for western blot analysis or were analyzed via ELISA .FasL release assays were performed with 10% RBC suspensions. RBCs were treated with sub-hemolytic doses of PFTs2 for 24 h at 37 °C.Hyperosmotic eryptosis was induced by 24 h incubation of RBCs (1%) in DPBS containing 950 mM sucrose at 37 °C. Calcium-induced eryptosis was achieved by incubation of RBCs (1%) with 1 mM exogenous CaCl


The purpose of this study was to develop and evaluate two spinal cord (SC) diffusion tensor imaging (DTI) protocols, implemented at multiple sites (using scanners from two different manufacturers), one available on any clinical scanner, and one using more advanced options currently available in the research setting, and to use an automated processing method for unbiased quantification. DTI parameters are sensitive to changes in the diseased SC. However, imaging the cord can be technically challenging due to various factors including its small size, patient-related and physiological motion, and field inhomogeneities. Rapid acquisition sequences such as Echo Planar Imaging (EPI) are desirable but may suffer from image distortions. We present a multi-centre comparison of two acquisition protocols implemented on scanners from two different vendors (Siemens and Philips), one using a reduced field-of-view (rFOV) EPI sequence, and one only using options available on standard clinical scanners such as outer volume suppression (OVS). Automatic analysis was performed with the Spinal Cord Toolbox for unbiased and reproducible quantification of DTI metrics in the white matter. Images acquired using the rFOV sequence appear less distorted than those acquired using OVS alone. SC DTI parameter values obtained using both sequences at all sites were consistent with previous measurements made at 3T. For the same scanner manufacturer, DTI parameter inter-site SDs were smaller for the rFOV sequence compared to the OVS sequence. The higher inter-site reproducibility  of rFOV compared to the OVS sequence supports the idea that making research options such as rFOV more widely available would improve accuracy of measurements obtained in multi-centre clinical trials. Future multi-centre studies should also aim to match the rFOV technique and signal-to-noise ratios in all sequences from different manufacturers/sites in order to avoid any bias in measured DTI parameters and ensure similar sensitivity to pathological changes. By modelling the signal behaviour of water diffusion in tissue using DTI, it is possible to derive several indices that may be sensitive biomarkers for characterising tissue microstructural abnormalities [The spinal cord (SC) is a common site of involvement in neurological disorders such as multiple sclerosis, amyotrophic lateral sclerosis, spinal cord injury and neuromyelitis optica , and higmalities . These iin vivo due to its small cross-sectional size and the potential for SC motion  during scans [However, there are many technical challenges associated with making quantitative measurements in the SC ng scans , 9. The ng scans .The small physical dimensions of the cord contribute to partial volume effects with surrounding CSF, which may also result in errors in measured diffusion parameters and constrains the actual pixel size, especially in the plane orthogonal to the main axis of the spinal cord , 12.Additionally, susceptibility-induced distortions become more of a concern at higher image resolution. In fact, the FOV in the phase encoding direction per each echo-train readout and the time between the acquisition of two consecutive k-space lines both affect the bandwidth , and, therefore, the magnitude of image distortions.0 field induce dynamic distortions along the phase-encoding direction [Physiological motion in particular may bias MD measurements  and resuirection . It is p0) variation across the slice, especially if aligned with the centre of the intervertebral discs or of the vertebral bodies [Many artefacts in EPI result from the much smaller bandwidth per pixel (frequency difference between adjacent voxels) along the phase encoding (PE) direction compared with the frequency encoding axis. This means that chemically shifted signals from fat will be displaced by many pixels, and that small magnetic field differences will result in much greater geometric distortions along the PE axis, for example due to susceptibility differences of air, bone and soft tissue. Acquiring data oriented transverse to the cord may provide better image quality than sagittal or coronal images, as there will then be less main magnetic field .There is a trade-off between high spatial resolution and adequate SNR due to the small size of the cord; for reliable depiction of anatomical detail an in-plane resolution of at least 1x1mmIn order to minimize image distortions, the PE FOV per echo-train readout may be reduced, for example, by using parallel imaging, thereby reducing the acquisition window duration and hence image distortions. However, this may not be available along all imaging axes, depending on the type of receiver coil used . Ramp saHere we aimed to develop two different SC DTI protocols for use in multi-centre studies, one using research options to enable the use of rFOV techniques, and one using only standard options available on clinical scanners, but neither requiring pulse sequence programming. Below we give a brief introductory description of the techniques used in this study.For the first protocol we aimed to use a rFOV sequence   sequenceReduced FOV methods are often used in SC imaging to limit the extent of coverage in the PE direction, thereby reducing the required number of k-space lines, which in turn reduces off-resonance induced artefacts (at the cost of an SNR reduction the size of the square root of the PE FOV reduction factor).The ZOOM sequence has seen several implementations, all based on slightly different combinations of imaging gradients, and excitation and refocusing pulses. All ZOOM methods exploit the concept of inner volume imaging , and enaInstead of a 1D excitation pulse, 2D spatially selective RF pulses can be used to excite only a small FOV. These 2D-RF pulses have excitation profiles which are periodic in either the phase or the slice direction, followed by a 180° refocusing pulse which is used to refocus only the main lobe of the excitation.The 2DRF excitation excites a stripe of magnetization within the object, with the size of the stripe selected in such a way that it covers the desired FOV. The desired FOV is also oversampled to avoid aliasing of the transition region where the magnetization fades out.One possible disadvantage of 2DRF excitation, however, is that the 2DRF pulses may have long durations, causing the slice profiles to be sensitive to off resonance effects and lengthening the minimum echo time (TE) achievable, with consequences on the data SNR. Here, we used the implementation proposed in .The second method we chose to examine is a basic DTI sequence with outer volume suppression (OVS). This method uses OVS pulses prior to the excitation pulse, and can be implemented by positioning suppression ‘bands’ anteriorly and posteriorly (A/P) to the spinal cord  to avoid aliasing. This option is available on most commercial MRI scanners, and is often already utilized with other spinal cord anatomical sequences.We therefore aimed to develop two SC DTI protocols (neither requiring any pulse sequence programming): one which can be implemented on any standard clinical MRI scanner (i.e. OVS), and one utilising research options that are either not yet commercially available or that have just been released onto the market as specific packages, which therefore are not necessarily available clinically (i.e. ZOOM or 2DRF), the goal being to ascertain which protocol could enter into clinical trials. The sequences were tested on three scanners at three different sites  from two different manufacturers by quantitative assessment of MD, FA, AD , and RD  in five healthy subjects at each site. Intra- and inter-site reproducibilities were calculated for each sequence.Data were acquired on 3T scanners at three sites (1 (London)–Philips Achieva , 2 (Vanderbilt)–Philips Achieva , and 3 –TIM Trio ). The two Philips systems are equipped with 80 mT/m gradients with maximum 200 mT/m/ms slew rate and a 16-channel neurovascular receive coil. The Siemens system is equipped with 40 mT/m gradients with maximum 200 mT/m/ms slew rate and 12-channel head + 4-channel neck receive-only coils. This study was approved by the local ethics committee at each of the three sites ) and all subjects provided informed consent in writing prior to the commencement of data acquisition. Five subjects were scanned at each of the 3 different sites .Where possible, protocols for the two different scanner manufacturers utilised identical acquisition parameters, though some options required manufacturer-specific parameters.2, and the FOV centred at the level of the C3-4 intervertebral disc, spanning levels C1-C6 in all volunteers. The imaging plane was prescribed to be perpendicular to the cord at the C3-4 level. Cardiac gating via peripheral pulse oximetry was used in all protocols to reduce physiological motion. The diffusion weighting  was set to 750s/mm2 for all sequences. For the OVS sequences, saturation bands were positioned anteriorly and posteriorly to avoid aliasing. All other (manufacturer-specific) sequence details are given in Site-independent identical parameters: 21 axial slices of 5mm thickness were acquired (with phase encoding anterior/posterior (A/P)) using a single-shot EPI readout, with in-plane resolution 1x1mm2-weighted anatomical data were acquired at all sites to enable template-based analysis of diffusion-weighted data, in a two-step process with co-registration to the template performed via the anatomical images.Additionally, standard clinical T3, with isotropic 1x1x1mm3 resolution, no SENSE, halfscan = 0.64, bandwidth = 408.5Hz/voxel, and NSA = 1.For the two Philips sites a 3D Volume Isotropic Turbo Spin-echo Acquisition (VISTA) sequence was used, with the following acquisition parameters: TR = 2000ms, TE = 120ms, sagittal acquisition, FOV = 250x250x60mm3, with isotropic 1x1x1mm3 resolution, flip angle = 120°, bandwidth = 723 Hz/voxel).At the Siemens site (site 3), the equivalent sequence Sampling Perfection with Application optimized Contrasts using different flip angle Evolution (SPACE) was used, with a slab-selective fast spin echo , with manual adjustments to the whole cord regions of interest (ROIs) made where necessary. The ROI is traced as a continuous line and voxels are included in the final mask if at least 50% of them belong to the inner part of the ROI. These were applied to parameter maps to give whole cord (C1-C6) mean DTI parameter values for each subject and protocol.Standard DTI parameters were analysed by a single observer (RS) by fitting the data to a diffusion tensor model using the open-source Camino toolkit  ), and DTI parameters were estimated in the spinal cord [Additionally, all data were processed using the Spinal Cord Toolbox  to show [et al.  as imple [et al. , using anal cord  using a nal cord .Intra- and inter-site comparisons of whole-cord MD, FA, AD and RD values obtained in native space  were performed. Paired t-tests were used to look for differences between parameter values obtained with different protocols at the same site, and one-way ANOVA tests were used to examine differences between the 3 sites, for both the OVS and ZOOM protocols. All statistical analyses were performed for both semi-automatic whole cord ROI- and template-based data. Coefficients of Variation  %) were used to assess reproducibility both within and between sites. Statistical analysis was performed using SPSS version 21.0 .Average SNR measurements for each site were measured via the “difference method” , using t2-weighted template (top) and the white/grey matter probabilistic atlas (bottom). The white matter atlas was used here to compute DTI metrics while allowing the possibility of taking into account potential partial volume effects .Mean MD, FA, AD and RD values (± standard deviations (SDs)) for each site and protocol  parameters obtained using ZOOM had lower SDs, Vanderbilt (site 2) had similar parameter SDs for both sequences, and Montreal (site 3) had lower DTI parameter SDs for the OVS sequence than the 2DRF sequence.Site 3  shows a small difference in measured AD (p = 0.018) between the OVS and 2DRF sequences, but no other significant differences in measured parameters between sequences were observed. No significant differences in any DTI parameter were observed between measurements made using the two protocols on either of sites 1 and 2.Intra-vendor coefficients of variation (CVs (%)), measured considering only site 1 and 2, for MD, FA, AD and RD were 1.20, 0.61, 2.08 and 0.76 for OVS and 1.05, 0.59, 1.81 and 0.67 for ZOOM. Inter-site CVs measured considering all three sites, for MD, FA, AD and RD were 7.99, 3.01, 6.06 and 10.6 respectively for the OVS sequence and 12.3, 2.70, 15.4 and 8.11 for the ZOOM sequence.No significant differences between DTI parameters measured using the OVS sequence were observed (tested using a one-way ANOVA). However, significant differences between site 3 (2DRF sequence) and the 2 other sites (ZOOM sequence) were observed as follows:MD was found to be significantly different in site 3 from site 1 (p = 0.01) and site 2 (p = 0.018), and AD was found to be significantly different in site 3 from site 1 (p<0.001) and site 2 (p = 0.001).Similarly to the semi-automatic ROI analysis, intra-site SDs varied for all parameters measured using the two sequences at each site; In London (site 1) parameters obtained using ZOOM had lower SDs, Vanderbilt (site 2) had mostly lower parameter SDs for the ZOOM than the OVS sequence, and Montreal (site 3) had mostly lower DTI parameter SDs for the OVS sequence than the 2DRF sequence.However, almost all parameter values were found to be significantly different when measured via OVS compared to ZOOM/2DRF (tested using paired t-tests), with the exception of the MD and FA at site 3  and FA at site 2 (Vanderbilt).Intra-vendor coefficients of variation (CVs (%)), measured considering only site 1 and 2, for MD, FA, AD and RD were 9.53, 4.57, 5.24 and 16.1 for OVS and 9.13, 3.98, 5.40 and 14.8 for ZOOM. Inter-site CVs measured considering all three sites, for MD, FA, AD and RD were 12.7, 4.59, 11.4 and 18.9 respectively for the OVS sequence and 17.8, 4.01, 16.4, and 15.4 for the ZOOM sequence.Comparing the three sites using a one-way ANOVA, for the OVS sequence significant differences were observed for MD between sites 1 and 3 (p = 0.013), for RD between sites 1 and 3 (p = 0.018) and for AD between sites 1 and 3 (p = 0.007) and 2 and 3 (p = 0.05). For the rFOV sequence, significant differences were observed for MD (site 1 vs 3 (p = 0.002), and 2 vs 3 (p = 0.023)), FA (site 2 vs 3 (p = 0.023)), RD (site 2 vs 3 (p = 0.026)) and AD (site 1 vs 3 (p = 0.001), site 2 vs 3 (p<0.001)).Average (± SD) SNR measurements for each site , and one utilising research options not yet commercially available (reduced FOV technique).SC MD, FA, RD and AD values obtained using both acquisition protocols at all sites were consistent with previous measurements made at 3T –37.Some variations in contrast were observable in b = 0 images, in particular in the CSF for site 3, which are likely due to some remaining CSF pulsation effects. Cardiac gating implementations are different between the Siemens site and the Philips. It is possible that susceptibility artefacts close to vertebral discs may also have caused some signal dropouts.Lower CVs were demonstrated for rFOV than for OVS when only considering results from the two Philips sites (i.e. the ZOOM sequence only), via semi-automatic ROI analysis, and to a lesser extent via template-based analysis. However, some differences between measured DTI parameters were observed at site 3, using the 2DRF sequence compared to the ZOOM sequence implemented at the other two sites.It should, however, be acknowledged that the two implementations of the rFOV protocol are very different; for instance, the Siemens protocol has a longer TE (mainly due to the 40mT/m gradient strength vs. 80mT/m on the Philips) which has an impact on SNR, in addition to the use of a 2DRF pulse on the Siemens Trio system, and this lower SNR can in turn can affect the accuracy of the fitted DTI parameters, as well as possibly affecting the exact water compartment under investigation. Future work is also warranted to assess other acquisition issues such as shimming and cardiac gating for each vendor and each sequence implementation. Encouragingly, for the two Philips sites the intra-vendor CVs were reduced to extremely low values  (and similar to those observed in a previous multi-centre diffusion MRI study in the brain ), with lUnfortunately identical rFOV methods were not available on the scanners from both manufacturers used in this study. Since the goal of this study was to evaluate sequences not requiring any pulse programming , for potential future application in multi-centre trials, we did not compare additional in-house methods in order to match sequences more closely from both vendors.Standard deviations in SNR measurements across the 5 subjects were found to be large, in particular for site 1. It was not possible to measure SNR using measurements of signal and noise e.g. in a corner of the image, because of inhomogeneity correction algorithms used, and the fact that there are typically no regions of “air/no signal” in rFOV images. Therefore, it was necessary to use the “difference method”, with SNR values measured over the whole cord, meaning that it is possible that errors due to motion may have occurred and affected SNR measurements in all subjects. The SNR measured for the 2DRF sequence at site 3 was lower than for the OVS sequence at that site, whereas it was found to be higher for the ZOOM sequence than the OVS sequence for the 2 Philips sites, and the rFOV SNR was also lowest for site 3, which may be another explanation for the variability in measured DTI parameters observed between sites. As mentioned previously, the lower SNR at site 3 is mainly due to the lower maximum gradient strength on the Siemens Trio system, imposing a longer TE (81ms vs. 50ms for the Philips).The Philips scanner software used in this study (release 3.2.1) does not allow the possibility of interleaving b = 0 images within the diffusion-weighted images without the use of pulse programming, which may have led to less reliable motion correction of our data. This feature will be included in future releases, which may even further improve the reproducibility of the measured DTI indices on this system, by enabling more robust motion correction , 39, andAdditionally the ongoing development of a ZOOM EPI method for the Siemens scanner as well as the latest improvements on gradient strength (80mT/m for the Prisma scanner) may improve inter-scanner reproducibilities of measured DTI parameters.We also performed automatic template-based analysis of our data using SCT , which hIn particular the two methods of analysis deal with partial volume effects very differently, where the SCT tries to estimate partial volume, while the ASM method simply excludes voxels that contribute less than 50% to the outlined spinal cord ROI and all included voxels are averaged with the same weights. This difference means that the intrinsic inter-subject and inter-scanner variability could be affected by a different handling of partial volume with the surrounding CSF , potentiWe have demonstrated the feasibility of performing multi-centre SC DTI studies, but with the caveat that it is very important to try to match the sequence design as far as possible between sites, and in particular to ensure that sequences from different manufacturers/sites are SNR-matched, in order to avoid any bias in measured DTI parameters. The availability of different rFOV methods on different scanners was already a stepping stone for pushing spinal cord DTI towards clinical use, although the chapter has just been opened and requires international and joint efforts to speed up results.A larger multi-centre study including both scan-rescan tests, and possibly scanning of the same subjects at different sites is desirable to further evaluate the protocols developed in terms of precision of the measurements and sensitivity to changes.The higher inter-site reproducibility  of the rFOV sequence compared to the OVS sequence supports the suggestion that making research options such as rFOV more widely available on clinical systems may improve accuracy of measurements obtained in multi-centre clinical trials. This would enable optimal protocols to be used in such studies, and allow multiple sites and manufacturers to match different sequences more closely, both in terms of implementation, and SNR.S1 Dataset(XLSX)Click here for additional data file.


E. coli. BC chimeras function on native att-sites and on att-sites that are hybrids between those of the two donor enzymes, while CT chimeras only act on the latter att-sites. A BC hybrid, BC{−1}, was also active in human HeLa cells. Our work is the first to demonstrate chimeric serine integrase activity. This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies.In recent years, application of serine integrases for genomic engineering has increased in popularity. The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms. In order to generate information that might be useful for altering the specificity of serine integrases and to improve their efficiency, we tested a hybridization strategy that has been successful with several small serine recombinases. We created chimeras derived from three characterized members of the serine integrase family, phiC31, phiBT1, and TG1 integrases, by joining their amino- and carboxy-terminal portions. We found that several phiBT1-phiC31 (BC) and phiC31-TG1 (CT) hybrid integrases are active in  The N-terminal domain is principally involved in catalysis, but also imparts some sequence specificity, and the C-terminal domain appears to be primarily responsible for DNA-binding and directionality control  or selection (e.g. G418-resistance) to isolate candidate clones. To identify desirable lines, additional screens are usually performed after outgrowth, e.g. for att-site platform integrity, copy number, integration locus, etc. So, in addition to precluding the application of serine integrases for in vivo applications, att-site pre-introduction often consumes a considerable amount of labor and time.Insertion of a wild-type egration . Homologtt-sites , and thitt-sites . Most atatt-site introduction work could be avoided. This development would reduce the time and resources needed for tasks like site-directed integration and would make it feasible to use serine integrases for tasks like in vivo gene therapy. Attempts have been made to realize this goal via directed evolution of phiC31 integrase, but were ultimately not able to achieve significant alteration of specificity , phiBT1-phiC31 (BC), phiC31-TG1 (CT) and TG1-phiC31 (TC) chimeras.Hybridization has yet to be attempted with serine integrases, so we decided to pursue this approach to explore enzyme function and modularity and as a method to alter specificity. We focused on the phiC31 family of enzymes – phiC31, phiBT1 and TG1 integrases – because they are currently the best characterized set of closely related serine integrases . We consE. coli on hybrid and/or parental att-sites. We also show that BC hybrids can function efficiently in HeLa cells, in both extra-chromosomal and pseudo site assays. Our study is the first to describe active serine integrase chimeras and sheds light on the structure–function relationships of these enzymes. The work also lays the groundwork for a more tractable means to sample the specificity of the putative large serine recombinases that have been identified by genome sequencing, which currently number in the thousands  . Jpred 3   for eachx E (αE) . For fur Jpred 3  was usedatt-sites were named using the format “WvW QR”. In this scheme, W and v refer to the source of the outer and inner att-site sequence, respectively . The majority of these CT hybrids formed aggregates and/or were not active on the tested parental or chimeric att-sites . However, three chimeras, CT{−75}, CT{−41} and CT{8,8} , showed clear signs of activity in our E. coli assay . These hybrids seemed to function only on chimeric TcT att-sites . The CT{8,8} hybrid had the broadest activity of the three and was specifically able to recombine TcT P3 × B3 and TcT P6 × B3  hybrid integrases, we constructed many fusions between the two enzymes. In total, eleven CT chimeras were constructed . TC hybrids with up to ∼70% of the TG1 domain – TC{−135}, TC{−112} and TC{−80} – were active on wild-type phiC31 att-sites in our E. coli assay . However, chimeras with larger swaps, like TC{−43}, TC{−7}, etc., were unable to recombine wild-type phiC31 or hybrid CtC att-sites .Despite our successes with CT hybrids and the strong sequence similarity of phiC31 and TG1 integrase , we did supplementary material Table S3). Fusions were made in this region to mirror the precedent set by zinc-finger small-serine recombinase chimeras  with various combinations of hybrid and wildtype att-sites. To express the chimeric and parental integrases, we utilized a tetracycline inducible (tet) promoter .To detect recombination in HeLa, we cloned several CMV-driven EGFP-inversion plasmids . Representative flow-cytometry plots of cells that have been exposed to control and experimental plasmid combinations have been provided in the supplement .To facilitate comparison of integrase expression plasmid performance, results from the inversion activity assays performed in HeLa have been normalized to the transfection efficiency of the positive-control plasmid pMF-CLCR . Nevertheless, even if either of these normalization assumptions proves to not be the case, our measurements in HeLa will still serve as the first qualitative confirmation of hybrid serine integrase activity in a mammalian cell line.The normalization that we have applied is only valid if the following assumptions are correct: (i) the unflipped pMF plasmids transfect with similar efficiencies as the flipped pMF-CLCR vector and (ii) EGFP transcripts on flipped pMF plasmids are transcribed and translated with similar efficiencies as the pMF-CLCR EGFP transcript. We believe that both of these assumptions are likely to be true because the tested pMF plasmids share at least ∼98% sequence identity with pMF-CLCR . This variation stems completely from sequence differences between the integrase ORFs, as the promoter, untranslated regions and plasmid backbone employed in these plasmids are all identical. Thus, despite the fact that we have transfected equimolar amounts of these vectors, the integrase sequence differences may result in different transcription efficiencies, transcript stabilities and/or translation efficiencies. Therefore, it is our intention that the efficiencies that we report here be interpreted as integrase plasmid efficiencies, and not intrinsic enzyme efficiencies.While the pMF plasmids consist of nearly identical sequence segments, the integrase expression vector sequences differ by ∼5–25% , and led to recombination of all tested hybrid att-site pairings at the ∼20% efficiency level or above: P0 × B3 (25%), P3 × B0 (29%), P3 × B3 (21%).To assess hybrid serine integrase activity in a mammalian environment, we tested a BC hybrid, BC{−1}, in HeLa cells. As was the case in  in HeLa . The BC{E. coli  into pseudo attP sites  into the genome  and attP sites , the only active full-domain-swap hybrids that we could produce were of the CT variety. TC chimeras with a full TG1 integrase catalytic domain were not active on any of the tested parental or hybrid att-site pairings. This was an unexpected result because, as evidenced by certain BC hybrids, the C-terminal domain of phiC31 integrase is able to function with the catalytic domain of phiBT1 integrase, a much more distant relative . One possible explanation for these results is that a structurally-incompatible mutation has arisen in the TG1 integrase catalytic domain, because the phiC31 counterpart can still function with the phiC31 and TG1 C-terminal domains. Another possibility is that a change in the phiC31 C-terminal domain is responsible, but we feel that this is less likely, as the distantly-related phiBT1 catalytic domain remains compatible with it (e.g. BC hybrids can function).Active fusions between the phiC31 and TG1 integrases proved to be surprisingly difficult to make. Despite the high similarity of their catalytic domains  induced valid GFP signal above background for all tested TcT att-site pairings in HeLa , we were only able to confirm the expected CT{8,8} TcT P3 × B3 activity in HeLa via PCR amplification and sequencing of the attL and attR junctions . GFP fluorescence was never detected above background for the CT{8,8} reaction, so it is likely to have only been mediated at trace levels in HeLa.While we were able to demonstrate clear CT{8,8} activity in E. coli and the vast majority of eukaryotes, metazoans completely lack a ClpB/Hsp104 homolog or functional equivalent (The reason(s) for this severe inhibition of CT{8,8} activity in mammalian cells are not clear. We investigated three potential problems that can be encountered when expressing prokaryotic proteins in mammals – aberrant mRNA splicing/stability, protein mis-localization and protein insolubility – all to no avail. Neither codon optimization nor addition of an SV40 NLS managed to rescue activity, and solubility of an HA-tagged CT{8,8} was confirmed via a western blot (data not shown). Several potential sources of CT{8,8} inhibition remain, but are outside the scope of our serine integrase chimera study. One possibility is that the majority of synthesized CT{8,8} protein is mis-folded in mammals due to lack of ClpB/Hsp104 chaperone activity. While present in uivalent , so it iuivalent , so it iE. coli, and one BC hybrid also performed well in HeLa .Chimeras produced from the phiBT1 and phiC31 integrases also proved to be surprising. While we were unable to produce any functional CB hybrids, a few of our BC chimeras exhibited activity in  in HeLa , Fig. 5BE. coli, BC{−1} exhibited a broader activity profile than the CT hybrids, since it was able to react both wild-type and hybrid att-sites (att-sites with less efficiency than the wild-type phiC31 P0 × B0 reaction (∼20–30% vs ∼37%), but its hybrid-site performance was still superior to that of the phiC31 vector for the three tested combinations . Recognition of both pseudo site types may preclude usage of BC{−1} for genomic engineering due to safety concerns, as recombination between said sites could lead to unwanted inversions, deletions and/or translocations. However, we have evidence that the pseudo-site reactivity of phiC31 integrase can be lowered to background levels in mammalian cells without also inhibiting wild-type recombination efficiency , so it might be possible to create an improved variant of BC{−1} integrase for use in genomic engineering applications.Like its parents, BC{−1} was also able to mediate recombination into endogenous pseudo-sites in HeLa . The psetB sites . This feWith this study, we have shed light on the degree of structural compatibility that exists between the catalytic and C-terminal domains of the phiC31, phiBT1 and TG1 integrases. Furthermore, we have laid the groundwork for the use of hybridization to create serine integrases with novel specificities. We describe chimeras with rationally altered and/or broadened reactivity relative to their parents, all produced via catalytic domain swaps.While this manuscript was in preparation, a new serine integrase structure, PDB ID 4KIS, was published by Rutherford et al. The structure is of an N-terminal truncation mutant of LI integrase (LI-int) bound to an A118 integrase (A118-int) attP half-site .This structure reveals the presence of two novel DNA binding domains in the C-terminal domain of LI-int: a ∼100 residue mixed α/β “recombinase” domain (RD) that is adjacent to αE and a ∼180 residue zinc-beta ribbon domain (ZBRD) downstream of the RD. While the region corresponding to the LI-int RD in C31-int has been previously identified as a DNA binding domain , Rutherfsupplementary material Fig. S5; e.g. CB{2}, CT{0}, TC{−1}, etc.).In addition to identifying the LI-int DNA binding domains, Rutherford et al. have demonstrated that the two linker segments that flank the recombinase domain are likely to be involved in att-site interaction. If these linkers also play this role in the phiC31 family of integrases that we used to make hybrids, then this revelation by Rutherford et al. offers a potential explanation for why many of the hybrids that were fused in the αE-RD linker region did not function .While avoidance of these linkers may increase the likelihood of obtaining an active hybrid, it is clear that other barriers remain, as several hybrids that were fused in other regions were also inactive . These vectors were cloned via a series of PCR, exonuclease, restriction digest and ligation steps. The PCR-amplified functional elements that are common to all pFlip plasmids were sourced from pET-50b (Novagen). These elements include the repressor of primer (rop) cassette, ColE1 replication origin, kanamycin-resistance (kanR) cassette, and lac repressor (lacI) ORF. All att-sites and remaining bacterial regulatory sequences were synthesized in vitro (oligos from Invitrogen). Phusion polymerase, lambda exonuclease, T4 DNA ligase and all restriction enzymes used for pFlip cloning were obtained from NEB. Optikinase was purchased from USB. Sequences of the 50 nt att-sites and hybrid integrase cloning primers have been provided in the supplementary material .Integrase activity tests in supplementary material Fig. S1B) – were constructed in a manner similar to the pFlip vectors, i.e. a series of PCR, exonuclease, restriction digest and ligation steps. All PCR-amplified functional elements used in the pMF plasmids were derived from pEGFP-C1 (Clontech). These elements include the CMV promoter, enhanced green fluorescent protein ORF (EGFP) and kanamycin/neomycin resistance cassette. All cloned att-sites are 50 nt in length  and were synthesized in vitro (oligos form Invitrogen).In HeLa cells, we used separate vectors for inversion detection and protein expression. Our inversion plasmids – given the prefix “Pmf”  – were constructed via standard PCR amplification, restriction digest and ligation steps. The functional elements common to all pN1t8 vectors include a tet promoter, SV40 early 3′ UTR and kanamycin-resistance cassette, which were PCR-amplified from the PB-TET-MKOS  cloned in place of phiC31 attB.Pseudosite integration donor vectors  were cloE. coli, we assembled pFlip vectors with the desired integrase and att-site combinations via in vitro ligation, and then transformed these ligations into NEB 10β cells (C3019H) using their high-efficiency protocol. The transformed cells were plated on X-gal (70 µg/ml) IPTG (80 µM) kanamycin (50 µg/ml) LB agar plates after 1 hour of outgrowth at 37°C in SOC. Plates were incubated at 37°C for 16 hours and were then visually assessed for the presence of blue colonies. For each integrase-att-site pairing, minipreps from at least two separate colony outgrowths were sequenced  to confirm the validity of all PCR-amplified integrase and/or att-site segments. In addition, all recombined junctions were confirmed to be as expected via sequencing of plasmids from at least two colonies (Sequetech).To perform activity tests in To perform the transient recombination assay in HeLa (ATCC CCL-2), the cells were grown to sub-confluence (60–80%) in DMEM (Cellgro 10-013-CV) supplemented with 9% FBS (Gemini Benchmark 100-106), 1× GlutaMAX (Invitrogen 35050-061), 1% penicillin/streptomycin (Invitrogen 15140-122), and 1.5 µg/ml doxycycline (Sigma D9891) in 24-well plates. Transfection was performed in triplicate, overnight, with 3 µg of DNA per well using Xfect (Clontech) at a 0.3 µl:1 µg polymer to DNA ratio. For all transfections, 500 ng of the PB-CA-rtTA tet-promoter activation plasmid , and 580attL and attR junctions, they were PCR amplified  and sequenced using our previously described method  using the same medium described for the plasmid inversion assay, and were then transfected in triplicate with a ternary mixture consisting of polyethylenimine (PEI), γ-polyglutamic acid (PGA) and plasmid (pDNA) . To formmid e.g. , 63 ng Pmid e.g. , ranged


For long time, plasticity of brain circuits has been hypothesized to mainly rely on the flexibility of glutamatergic excitatory synapses, whereas inhibitory synapses have been assumed to be relatively invariant. Based on this view, inhibition should be exclusively modulated by the differential glutamatergic-driven activation of a highly diverse population of inhibitory interneurons displaying specific temporal dynamics and selective innervation patterns. However, it has been demonstrated that inhibitory synapses undergo several forms of plasticity, thus providing an additional source of versatility to the regulation of the neuronal network and the emergence of complex brain states.The cellular and molecular mechanisms occurring at inhibitory synapses during the induction/expression of inhibitory short- and long-term synaptic plasticity are now beginning to be unraveled. At the presynaptic side, retrograde synaptic messengers modulate GABA release  cover the mechanisms of inhibitory synaptic plasticity, at the molecular and microcircuit levels. Zacchi et al.  focus onSynaptic signaling does not only depend on pre- or post-synaptic determinants but is also shaped by the dynamics of neurotransmitter in the synaptic cleft. The minireview and the hypothesis and theory by Scimemi ,b proposIn addition to synaptic inhibition, tonic inhibition produced by the persistent activation of extrasynaptic GABAA receptors is crucial for the tuning of neuronal excitability. Recent evidence demonstrates that also tonic inhibition is plastic. The original article by Barth et al.  illustraThe ability of inhibitory synapses to undergo plasticity emphasized in this ebook raises important questions. First, what are the specific molecular mechanisms of inhibitory plasticity at synapses formed by different interneuron subtypes? Second: how is plasticity orchestrated at both excitatory and inhibitory synapses? In keeping with this, how do different forms of excitatory and inhibitory plasticity co-exist? Do variations of both excitation and inhibition strength occur in parallel/homestatic  as a “dynamic” activity-dependent determinant for the functioning of brain microcircuits.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli and Staphylococcus aureus [Cordia nodosa specie has proven antifungal activity but the work as they relate to the antibacterial activity are scarce [Cordia nodosa.Increasingly infectious agents change, which is why there is a failure in the treatment of microbial infections, above all, the emergence of resistance to existing antibiotics . Therefos aureus . Phytoche scarce . The aimCordia nodosa, A, B, C and D. Antimicrobial activity was determined by microbial sensitivity tests, the method of disk diffusion (DD) and broth microdilution method for determination of minimum inhibitory concentration (MIC). The percentage inhibition of bacterial disk diffusion test was calculated by dividing the average inhibition hundred times the sample by the average zone of inhibition positive control [Artemia salina.The experimental research was conducted in the Research Laboratory of Wound Care, Federal University of Alagoas. Four fractions were tested in different parts of  control . 8 were Staphylococcus aureus, with a percent inhibition of 29.2% (inhibition = 8 mm) and 55.6% (inhibition = 15 mm), respectively, when compared with the mean of the positive control used Gentamicin (about 27 mm). Both samples were also moderately active against the strains of S. epidermidis, involving inhibition percentages of 33.4%. The sample C presented little active front of the line of P. aeruginosa as the percentage of inhibition was less than 25%. The results obtained with the MIC determination showed that the sample B is the fraction of better antimicrobial activity inhibiting the growth of the strain of S. epidermidis concentration of 1000 to 125 mg mL- 1. Identified the absence of toxicity in all samples, because the mortality of Artemia salina was ≤ 30%. This finding dismissed the test quantitative, and therefore the LC50 determination, because it means that the concentrations tested were below the LC50 of each sample (LC50 ≥ 1000 mg mL-1). These results represent the first evidence of safety of plant species that are performed in sequence in vivo bioassays.Samples B and C were considered moderately active against the strain of


Tricholoma matsutakei exerted an antihypertensive action on spontaneously hypertensive rats (SHRs) at a dosage of 400 mg/kg. An angiotensin converting enzyme (ACE) inhibitory peptide with an IC50 of 0.40 μM was purified from the extract and designated as TMP. Its amino acid sequence was elucidated to be WALKGYK through LC-MS/MS analysis. The Lineweaver-Burk plot suggested that TMP was a non-competitive inhibitor of ACE. A short-term assay of antihypertensive activity demonstrated that TMP at the dosage of 25 mg/kg could significantly lower the systolic blood pressure (SBP) of SHRs. TMP exhibited remarkable stability over a wide range of temperatures and pH values. It also demonstrated 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. The aforementioned activities of TMP were corroborated by utilizing the synthetic peptide. Hence T. matsutake can be used as a functional food to help prevent hypertension- associated diseases.Hypertension is a major risk factor for cardiovascular disease. A crude water extract of the fruiting bodies of a highly prized mushroom  Cardiovascular disease is one of the main causes of death worldwide, and hypertension (high blood pressure) is a leading risk factor for cardiovascular disease. According to a survey conducted by the World Health Organization, hypertension currently kills nine million people every yearAngiotensin converting enzyme ACE, EC.3.4.15.1), a Zn-metallopeptidase, plays a key role in the regulation of peripheral blood pressure mainly through the renin-angiotensin (RAS) and kallikrain-kinin systems (KKS).4.15.1, Grifola frondosaPholiota adiposaPleurotus cornucopiaePleurotus cystidiosusTricholoma giganteumHypsizygus marmoreusAgaricus bisporusLeucopaxillus tricolor and Ganoderma lucidumRecently, many reports on bioactive natural ACE inhibitors from food sources, e.g., milk, turtle egg white, soybean, sweet potato etc have appeared7Tricholoma matsutake, a well-known wild edible mushroom, is an ectomycorrhizal basidiomycete which grows in a symbiotic relationship with Pinaceae in the Northern hemisphere and produces prized “matsutake” mushroomT. matsutake such as nucleaseT. matsutake, and to investigate the inhibitory effect of the purified ACE inhibitory peptide on the spontaneously hypertensive rats. This work would provide new evidence for further advocating T. matsutake as a functional food to prevent chronic diseases.The purpose of this study was to isolate and identify the ACE inhibitory peptide from Tricholoma were prepared for examination of their respective inhibitory activity on ACE. As shown in Tricholoma mushrooms were in the range of 2.4–95.0%, with the water extract of T. matsutake displaying the most potent inhibitory activity. This was followed by T. mongolicum and T. saponaceum extracts, which brought about 63.9% and 38.2% inhibition of ACE activity, respectively. The lowest ACE inhibitory activity  was shown by extracts of T. pessundatum and T. bakamatsutake Hongo.Extracts from the fruit bodies of eight mushrooms that all belong to the genus T. matsutake ACE inhibitory peptide. The water extract was subjected to ultrafiltration through a 5-kDa molecular weight cut-off membrane. Filtrate with a molecular weight (M.W.) over 5 kDa elicited 27% inhibition of ACE activity while filtrate with a M.W. below 5 kDa showed 63% inhibition of ACE activity. Thus the active filtrate with a M.W. below 5 kDa was employed for isolation of ACE inhibitory peptide. After a series of purification procedures, three peaks were observed upon FPLC-gel filtration on a Superdex Peptide 10/300 GL column. Among these peaks, fraction P3 expressed the strongest ACE inhibitory activity  on a Superdex Peptide column. The procedure was efficacious for purifying activity .50 = 0.40 μM) and was further studied.Five peptides were obtained in P3 by linear trap quadrupole (LTQ) LC-MS/MS analysis . To confInterestingly, it was found that the isolated ACE inhibitory peptide TMP demonstrated DPPH radical scavenging activity . In thisAs shown in The mode of inhibition of the isolated inhibitory peptide WALKGYK on ACE was ascertained from the Lineweaver-Burk plot. According to the plot presented in The antihypertensive action of the isolated inhibitory peptide TMP was investigated by measuring the change of SBP in SHRs at 0.5, 2, 4, 6 and 8 h after oral administration of TMP as shown in T. matsutake which inhibited ACE with an IC50 value of 0.4 μM and exhibited a clear antihypertensive effect on spontaneously hypertensive rats. A tripeptide GQP from T. giganteum belonging to the same genus manifested ACE inhibitory activityThe present account represents the first report on the isolation of the peptide WALKGYK from the renowned edible mushroom Tricholoma. Hence the ACE inhibitory activities of eight mushrooms belonging to the genus of Tricholoma were examined in the present study  engendered a 19% decline in blood pressure in SHRs from 190 mm Hg to 154 mm Hg while the water extract of H. marmoreus at the dosage of 800 mg/kg lowered the blood pressure by 14.4%, less potently than T. matsutake. The water extract of P. coruncopiae displayed a more pronounced effect on suppressing the blood pressure than T. matsutake and H. marmoreus. There are many bioactive substances for lowering the blood pressure encompassing various peptides, phenolic compounds, proteins, polysaccharides, saponins, sterols, pigments, fiber as well as minerals50 values of ACE inhibitory activity of peptides from H. marmoreus and T. matsutake were almost identical while the crude water extract of T. matsutake was superior to H. marmoreus in reducing blood pressureT. matsutake was 400 mg/kg, which corresponded to 14.5 g of fruiting bodies of T. matsutake. The peptide TMP from T. matsutake evinced good thermostability, which ensures preservation of the ACE inhibitory activity when T. matsutake is employed in tea or other food items. All this may furnish evidence for T. matsutake being a functional food to help prevent hypertension- associated diseases.ACE inhibitory activity Tricholoma matsutake were collected from Yunnan Province in China. Q-Sepharose, Mono-Q, Superdex peptide 10/300 GL and AKTA Purifier were purchased from GE Healthcare. Hippuryl-L-histidyl-L-leucine (HHL), 2,2-diphenyl-1-picrylhydrazyl(DPPH), and phenyl-methylsulfonyl fluoride (PMSF) were purchased from Sigma-Aldrich . All solvents and chemicals used in this study were of analytical and HPLC grade.Fresh fruiting bodies of the mushroom Twelve-week-old male spontaneously hypertensive rats (SHRs) were purchased from Beijing Vital River Experimental Animal Technical co., Ltd.et al.ACE was prepared using fresh rabbit lungs according to the protocol reported by Hayakari The ACE inhibitory activity was assayed by using a spectrophotometric method following the protocol described by Cushman50.where A was the absorbance of the solution containing ACE and inhibitor, B was the absorbance of solution containing ACE and inhibitor but terminated in advance, C was the absorbance of the solution containing ACE without inhibitor, and D was the absorbance of the solution containing ACE without inhibitor but terminated in advance. The concentration of the inhibitor required to inhibit 50% of the ACE activity under the above assay conditions was defined as ICT. matsutake (stored at −20 °C) were soaked in distilled water (weight: volume = 1:2) at 4 °C for 1 hour, and then homogenized in a Waring blender. The homogenate was extracted overnight at 4 °C before centrifugation at 9,000 rpm for 20 min at 4 °C. The supernatant was centrifuged again at 9,000 rpm for 20 min after boiling for 10 min. Then the supernatant was subjected to ultrafiltration with a 5 kDa M.W. cut-off membrane. The filtrate and solution of the filter-cake were assayed for ACE inhibitory activity as described above. The active fraction was applied on an anion exchange Q-Sepharose column (2.5 cm × 20 cm) in Tris-HCl buffer  with a flow rate of 2 mL/min. After removal of the unadsorbed fraction Q1, adsorbed material was fractionated using a linear concentration gradient of 0–1 M NaCl in the same buffer. The fraction with ACE inhibitory activity was loaded on another strong anion exchange Mono-Q column (4.6 cm × 10 cm) in Tris-HCl buffer  with a flow rate of 1 mL/min. Following removal of the unadsorbed fraction, the column was further eluted with a linear gradient of 0–1 M NaCl in the same buffer. Fractions with ACE inhibitory activity were pooled, and final purification by fast protein liquid chromatography (FPLC) on a Superdex peptide gel filtration column at a flow rate of 0.8 mL/min using an AKTA Purifier was carried out. The most active fraction was lyophilized for further research.Fresh fruiting bodies of the mushroom After lyophilization, the most active fraction was dissolved in 0.1% formic acid and 2% acetonitrile for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using a LTQ-Orbitrap mass spectrometer . Samples were loaded onto a reversed-phase C18 column with a diameter of 0.1 mm × 10 cm and a particle size of 3 μm. Separation of the peptides was achieved by using a gradient of 5 to 35% acetonitrile in 0.1% formic acid over 120 min. The acquired MS/MS data were transformed to MGF by BIWORKS, and the data were searched against the NCBI database using MASCOAT software.50 value of the peptide was investigated according to the method described above.To investigate antihypertensive activity on SHRs, antioxidant capacity and mode of inhibition of ACE, the purified peptide was synthesized by Shanghai Bootech BioScience & Technology Co., Ltd. The purity of the synthetic peptide was 99% as evidenced by HPLC analysis. The ICet al.The DPPH radical scavenging activity of the inhibitor was determined according to the method described by Cheng control was the absorbance in the absence of sample, Asample was the absorbance in the presence of the sample.where AThe inhibitory peptide was dissolved in distilled water and subsequently incubated for 2 h at different pH values ranging from 2 to 11. The pH value was then adjusted to 7.0. ACE inhibitory activity was determined with the abovementioned method.The inhibitory peptide was incubated for 2 h at different temperatures covering a range from 40 °C to 90 °C. After acclimation to room temperature, the ACE inhibitory activity was assayed at 37 °C as mentioned above.The mode of inhibition of ACE by the peptide was determined spectrophotometrically using HHL as the substrate to distinguish between competitive, non-competitive and uncompetitive inhibition. Basically, a series of concentrations  of HHL were incubated with ACE in the presence or absence of the peptide . The ACE inhibitory activity was determined according to the aforementioned method. The kinetic parameters Vmax (maximum velocity) and Km (Michaelis constant) were investigated by constructing Lineweaver-Burk plots.T. matsutake received the extract at two different concentrations: 200 mg/kg body weight and 400 mg/kg body weight. SBP was measured thrice at each of the following time points: 0, 0.5, 2, 4, 6 and 8 h after drug/sample administration, using a tail-cuff method with a programmable BP-100A electro-sphygmomanometer .All experiments were performed in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China and also with the approval of the Animal Experimentation Ethics Committee of The Chinese University of Hong Kong which had been procured prior to the animal experiments. SHRs with systolic blood pressure (SBP) higher than 180 mmHg were randomly divided into six groups after acclimation for 1 week. During the acclimatization period prior to the test, the blood pressures of the rats were measured four times weekly. Each group containing four rats. A blank control group receiving oral administration of 0.9% saline solution and a positive control group receiving oral administration of captopril (100 mg/kg body weight) were included. The ACE inhibitory peptide-treated group received the peptide at two different concentrations: 25 mg/kg body weight and 50 mg/kg body weight. The group treated with crude water extract of How to cite this article: Geng, X. et al. A Tricholoma matsutake Peptide with Angiotensin Converting Enzyme Inhibitory and Antioxidative Activities and Antihypertensive Effects in Spontaneously Hypertensive Rats. Sci. Rep.6, 24130; doi: 10.1038/srep24130 (2016).


In this study, we develop an accessible nonlinear and discrete probabilistic model to study simple generalizations of lateral activation and inhibition. By doing so, we identify a range of modes of morphogenesis beyond the familiar Turing-type modes; notably, beyond stripes, hexagonal nets, pores and labyrinths, we identify labyrinthine highways, Kagome lattices, gyrating labyrinths and multi-colour travelling waves and spirals. The results are discussed within the context of Turing's original motivating interest: the mechanisms which underpin the morphogenesis of living organisms.The combination of laterally activating and inhibiting feedbacks is well known to spontaneously generate spatial organization. It was introduced by Gierer and Meinhardt as an extension of Turing's great insight that two reacting and diffusing chemicals can spontaneously drive spatial morphogenesis  Turing was among the first to present a powerful idea pertaining to this phenomenon, when he realized that a spatially homogeneous soup of just two chemically reacting species can spontaneously morph into a structured pattern owing to nothing more than the diffusion of these species, so long as the reaction kinetics are of the appropriate activatory or inhibitory nature and the diffusivities are sufficiently different . Later GGroups of cells can effect lateral activation/inhibition by, for example, secreting ligands that diffuse on average a few cell lengths before degradation or binding to receptors; this binding triggers an intra-cellular signalling cascade which in turn increases/decreases the ligand's expression. Given the vast number of intra- and inter-cellular signals operating within and between cells, it seems probable that other types of lateral feedback beside lateral activation and inhibition also operate during development; furthermore, the structural diversity among living organisms is immense, whereas lateral activation and inhibition alone generate a limited range of patterns. What other types of lateral feedback may be operating to bring about the development of a multicellular organism? What patterns can be generated by recombinations of these feedbacks and what feedbacks are necessary and sufficient to generate a particular pattern?Here, we study a model of interacting lattice sites that laterally activate and inhibit one another . The lat2.2.1.The core model of discrete and probabilistic lateral activation and inhibition introduced in  is now dB) or white (W). From an initial configuration, lattice sites flip their colour, from black to white or white to black, one at a time. The rate at which a particular site flips its colour is determined by the following three rules:activation at the interface);(i) a site can flip to a colour only if a neighbouring site has that colour (this is rL (i.e. long-range inhibition), where (ii) the likelihood of flipping to a colour decreases with the density of sites of that colour within a particular long range rS (i.e. short-range activation).(iii) the likelihood of flipping to a colour increases with the density of sites of that colour within a particular short range The model runs on a two-dimensional square grid of lattice sites that are either black  below). Perturbing β away from zero, then rather colour configurations accumulate an excess of one particular colour—white is more abundant for β > 0, while black is more abundant for β < 0—and so β is called a symmetry breaking parameter (a). To interpret the rules (i)–(iii) in the context of cellular dynamics, each lattice site would represent a cell that can be in one of a number of discrete states. Its ability to change from a state A to a different state B would be dependent on contact with another cell of that particular state. The rate at which the transition A → B happens would be regulated by two ligands produced by B cells, one activator of the transition that is degraded within an average length rL (long-range inhibition of type A), and one inhibitor of the transition that is degraded within a length rS (short-range activation of type A).A noise level arameter a. To intx to colour C = W or B is non-zero if and only if any of the eight neighbours of x has colour C  over every non-zero probability colour flip. This completely specifies the model's reaction kinetics, except for initial and boundary conditions which are described in the next section. For the corresponding continuous time definition and a partial derivation of the mean-field equations .Precisely, from a given colour configuration, the probability that the next colour flip is at site b,c; electronic supplementary material, Movies S1 and S2, [When the model is run, invariably it converges to a macroscopically stationary attractor that is either patterned, or homogeneously noisy, or uniform block colour . PatterTS = ∞, low noise of the long range β = 0), from (2.2) stationary attractors have β = 0) then we have β for lateral inhibition only (TS = ∞ in (2.2)).For lateral inhibition only and no symmetry break (high noise on the short range 2.2.l + 2rL) × (l + 2rL) sites. Initially, all sites of the simulation are equally and independently likely to be one colour among a list of permitted colours . Thereafter, sites within the central square domain of l × l lattice sites flip their colour according to the reaction kinetics specified in each section. Outside of this l × l domain, colours remain fixed for all time—this constitutes the boundary condition.Each model was simulated on a two-dimensional square grid of (β = 0), attractors are approximately straight stripes, whereas curved labyrinths are generated by lateral activation and inhibition together  [l × l appears to have no effect on this wavelength. In all simulations, the domain size was set such that the wavenumber Simulations of the core model show that the local solutions predicted by the mean-field approximation (2.2) are invariably realized: for colour symmetry and lateral inhibition only . Now brttractor . Near toaterial) ; increaslateral feedback module. Each lateral feedback module has a corresponding free parameter.We have seen, then, that in this model the combination of the three rules (i) activation at the interface, (ii) short-range activation, and (iii) long-range inhibition generates a range of Turing-type patterns—stripes, labyrinths, hexagonal nets and spots—and, moreover, that in several cases the correspondence between stationary patterning modes and parameter values, i.e. the bifurcation diagrams, can be roughly anticipated simply by inspecting the mean-field approximation (2.2). Therefore, we searched for interesting new attractors by retaining the three rules (i)–(iii) while systematically generalizing the model in different simple ways. Importantly, all generalizations in this study introduce new linearly independent terms into the mean-field approximation for stationary interfaces: we hypothesized that only such generalizations can produce attractors with interesting new patterns . We consider each linearly independent term in the mean-field approximation to be a β is varied from zero, are quantitatively represented by the variation in fb which represents the fractional excess in white over black sites in the l × l domain, as shown in the plots in figure 1c. In order to generate these plots, the simulation was run five times for each value of β with all other parameters held constant. The cross-hairs are fb computed for each instance of the simulation; the line-graph connects the averages of fb for different values of β. A similar format is followed for every plot in the article –(iii), includes in the exponent symmetry breaking nonlinear terms b and electronic supplementary material, Movie S3, increasing b, denoted by fS, measures the prevalence of the short-range spots within the labyrinth tracks . Values of fS appear to be unaltered when l, rL and rS are simultaneously rescaled while other parameters are held constant, indicating that discretization effects of the lattice are small (compare the blue and black curves which are superposed in the upper plot of figure 2b). At least for For colour symmetry  as depicted in figure 2c; electronic supplementary material, Movies S4 and S5. When the short-range symmetry breaking term is perturbed βS≠0, labyrinthine highways transit to train tracks. As both β and βS are varied, the transitions between patterns are apparently gradual and smooth, as demonstrated by the plots of figure 2c which show gradual and smooth increases of fb .When colour symmetry is broken by perturbing see e.g. ,17. Wher3.2.βL enhances the black colour flipping rate wherever there is an imbalance of colour on the long range; increasing Z is the colour configuration-dependent normalizing factor. Again, we focus on perturbations to labyrinths: in all simulations A second natural extension of the model, analogous to the extension of §3.1, includes in the exponent symmetry breaking nonlinear terms a and electronic supplementary material, Movie S6, stationary labyrinths bifurcate to continually gyrating labyrinths as a by the summary statistic fL which represents the time-averaged speed of movement of the interface . fL appears to be unaltered when all length-scales are simultaneously rescaled while other parameters are held constant (compare the blue and green curves in the plot of figure 3a), indicating that the structure of the bifurcation diagram is unaffected by discretization effects of the lattice. The dependency of the bifurcation point on rS/rL may be due to more varied interface geometries that are got by increasing rS/rL, since some variations may be more likely to creep into unstable configurations that must then gyrate to new configurations that are locally stable. For σL = 2, the value of rS/rL must exceed a threshold of approximately 1/3 in order for this bifurcation to occur (not shown). An argument precisely analogous to that in the previous section explains why the bifurcation point for For colour symmetry . The bifurcation is similar when perturbing βL ≠ 0 but appears to be continuous.When colour symmetry is broken by perturbing 3.3.iC within range Jr. We simulated three and four colours with activation at the interface and (i) long-range inhibition only; (ii) short-range activation only; (iii) both long-range inhibition and short-range activation; and (iv) symmetry preserving nonlinear terms that compete with the short-range activation . However, increasing the long-range symmetry preserving nonlinear competition When multi-colour labyrinths are perturbed by increasing the short-range symmetry preserving nonlinear competition  colours b. Howeve3.4.jth element of the vectorM can be defined similarly for n colours. The circulancy of M drives the dynamics in a symmetry breaking cyclic colour ordering a priori propensity for one particular colour to accumulate. Reversing the sign of γS (or γL) drives the cycle in the opposite direction on the short (or long) range. This circulancy condition could correspond to a scenario where cells transit between states as partly governed by a non-transitive rock–paper–scissors-like dynamic; for example, high local density of state C1 enhances the likelihood of state C2 possibly via the diffusivity of some C1-produced species, similarly high local density of state C2 enhances the likelihood of state C3 and high local density of state C3 enhances the likelihood of state C1. A similar mode of symmetry breaking has been called cyclic dominance and studied in a spatial and probabilistic context within [When the model is extended to more than two colours, a new mode of symmetry breaking is possible which, unlike in §3.1 and 3.2, does not enforce an accumulation of one particular colour. As in §3.3, because there are no symmetry breaking parameters that are associated with particular colours, the reaction kinetics can be described by a single expression:t within .N varies linearly with N appears to be unaffected by the simulation's initial condition  a master regulator that once expressed initiates the expression of two diffusing ligands and a membrane-junction signal; (ii) receiving the membrane-junction signal from a neighbouring cell is an absolute requirement for the expression of the master regulator; (iii) the first ligand diffuses in excess of a few cell lengths on average before being degraded; or it binds to cell-surface receptors to trigger a signal that decreases the likelihood of expression of the master regulator; (iv) the second diffusing ligand, which tends to be degraded within a range that is short compared with the range of the first diffusing ligand, upon binding to a receptor either increases or decreases the likelihood of expression of the master regulator depending on whether the number of bound receptors is below or above a threshold. However, it must be recognized that our model is only an abstract representation of (i)–(iv); in particular, it represents only cases where the time-scales of diffusion, degradation and membrane-junction signalling are much faster than the time-scale of the master regulator's response to such signals. There are other possible long-range signalling mechanisms besides diffusion, operating over lengths which span multiple cells, that can account for lateral feedbacks: in animals, these include the active migration of cells, e.g. , and theβ = 0)—a prerequisite for e.g. stripes and labyrinths—that region of parameter space is comparatively rich in the diversity of final patterns that are generated; consequently, the final pattern responds sensitively and flexibly to sustained changes in parameter values. The broad range of natural patterns suggests that this might be evolutionarily advantageous. While genetic drift would tend to take the system away from colour symmetry, we expect evolution to act as a tuning force which maintains the sensitivity and flexibility of developmental programs.1Secondly, our toy model motivates us to speculate about a possible dynamical feature of some developmental programs. In the model, when parameters enforce what we have called colour symmetry (