Strikingly, while hSAA1.1 was not highly amyloidogenic in vitro, the addition of a single N-terminal methionine residue significantly enhanced the fibrillation propensity of hSAA1.1 and modulated its fibrillation pathway. A deeper understanding of the oligomerization and fibrillation pathway of hSAA1.1 may help elucidate its pathological role.The fibrillation of Serum Amyloid A (SAA) – a major acute phase protein – is believed to play a role in the disease Amyloid A (AA) Amyloidosis. To better understand the amyloid formation pathway of SAA, we characterized the oligomerization, misfolding, and aggregation of a disease-associated isoform of human SAA – human SAA1.1 (hSAA1.1) – using techniques ranging from circular dichroism spectroscopy to atomic force microscopy, fluorescence spectroscopy, immunoblot studies, solubility measurements, and seeding experiments. We found that hSAA1.1 formed alpha helix-rich, marginally stable oligomers  Reactive amyloidosis or Amyloid A (AA) amyloidosis is a condition in which amyloid deposits of the protein serum amyloid A (SAA) appear in organs like the spleen, liver, and kidney SAA is found in almost all vertebrates investigated and exhibits a high degree of similarity between species in vitro. We note that we have used delipidated (apo) hSAA1.1 for our studies. While physiologically, SAA is predominantly associated with HDL in vivo in lipid-free form in vitro studies.Despite the significant amount of work done to understand the physiological role of SAA E. coliin vivoInterestingly, most previous studies discussing the pathological importance of hSAA1.1 use a protein expressed in Based on size exclusion chromatography and circular dichroism spectroscopy, we found that both MetSAA1.1 and hSAA1.1 form alpha-helix-rich oligomers immediately upon refolding. While incubation of both the proteins at 37°C resulted in the formation of cross-beta-rich aggregates with similar aggregation kinetics, there were some striking differences between the fibrillation propensities and pathways of both the proteins, suggesting a critical role for the amino-terminal methionine. Aggregation of MetSAA1.1 resulted in the formation of soluble and spherical oligomers in the early stages and long and insoluble “rod-like” amyloid fibrils in the later stages. In contrast, hSAA1.1, even upon prolonged incubation at 37°C, existed as soluble spherical oligomers and short curvilinear aggregates, all of these aggregates being much smaller and having a different conformation relative to the aggregates formed by MetSAA1.1. Interestingly, the amyloid fibrils formed by MetSAA1.1 were able to seed the fibrillation of MetSAA1.1 while hSAA1.1 did not exhibit seeding properties further suggesting the importance of the methionine residue in the aggregation of the protein. This insight into the intrinsic oligomerization and fibrillation propensities of the AA Amyloidosis-relevant isoform of human SAA will provide a useful starting point in understanding the pathological activity of this protein.E. coli BL21 (DE3) pLysS-competent cells. The cells were grown, expressed, and lysed using standard procedures. Cation exchange buffer  was used as lysis buffer and the lysate was purified using SP Sepharose cation exchange column  on an AKTA purifier UPC 10 FPLC  using an elution buffer  to isolate the protein. The relevant fractions, based on analysis by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE), were pooled and desalted by dialyzing them against anion exchange buffer . This desalted fraction was then loaded on a DE52 anion exchange column and eluted with an anion exchange elution buffer  on an AKTA FPLC  using a 10−65% salt gradient. The relevant fractions, based on analysis by SDS-PAGE, were pooled and concentrated via several rounds of ultrafiltration and then loaded on a HiLoad 16/60 Superdex 200 preparative grade column  preequilibrated with size exclusion chromatography (SEC) buffer , and the relevant fractions were collected and concentrated using an Amicon ultrafiltration cell.MetSAA1.1 was purified following a procedure described previously for murine SAA 6–TEV-SAA) in E.coli BL21 (DE3) pLysS-competent cells and lysed the cells in lysis buffer . The ultracentrifuged and filtered lystate was then loaded on a Histrap column  and (His)6–TEV-SAA was eluted using immobilized metal affinity chromatography (IMAC) elution buffer . The relevant fractions containing (His)6–TEV-SAA were then pooled and loaded on a SEC column  pre-equilibrated with SEC buffer (8 M urea/20 mM Tris/200 mM NaCl) to isolate pure (His)6-TEV-SAA. Purified (His)6–TEV-SAA was then extensively dialyzed against Tris buffer  to remove urea from the protein solution. TEV protease was then used to cleave dialyzed (His)6–TEV-SAA (1∶20 TEV:SAA) resulting in the generation of cleaved protein (hSAA1.1) and uncleaved (His)6–TEV-SAA. Protein sequence of (His)6–TEV-SAA indicating TEV cleavage site has been included in the supporting information section. Figure S1 in 6-TEV-hSAA1.1 at a site between “Q” and “R” residues yielding hSAA1.1, sequence of which begins with “R” and with no residues from TEV remaining in the protein sequence. TEV proteolysis was quenched by adding a solution containing urea and NaCl to bring up the final concentration of the solution to 5 M urea and 500 mM NaCl. The protein solution containing hSAA1.1, uncleaved (His)6–TEV-SAA, and (His)6–TEV was then dialyzed against a buffer containing 8 M urea and 20 mM Tris, pH 8.3 to remove traces of EDTA. The resulting protein solution in IMAC buffer  was then passed through a Histrap column  and hSAA1.1 was collected in the eluant. The unbound protein was collected, concentrated, and further purified using a gel filtration column  preequilibrated with SEC buffer (8 M urea/20 mM Tris/200 Mm NaCl) to obtain hSAA1.1. Relevant fractions obtained after SEC were pooled and concentrated using Amicon ultrafiltration cells. The purity of MetSAA1.1 and hSAA1.1 was confirmed by SDS-PAGE and electrospray ionization−mass spectroscopy (ESI-MS). Protein concentration was determined by measuring the absorbance at 280 nm and using calculated values of the extinction coefficients.We used a protocol similar to one used in one of our previous studies to purify hSAA1.1 Both hSAA1.1 and MetSAA1.1 proteins were refolded by dialyzing purified protein in SEC buffer  against Tris buffer  using a 3 kDa MWCO membrane at 4°C. Dialysis was performed overnight with multiple buffer changes during the course of the experiment. Buffer used for dialysis was pre-cooled to 4°C prior to dialysis and the apparatus was maintained at 4°C at all times.All SEC studies were performed at 4°C on a Superdex 200 10/300 GL analytical grade column  using an AKTA purifier UPC 10 . Molecular weight (MW) standards were used to calibrate the SEC column. Approximately 30 µL of refolded proteins (20 µM) obtained after dialysis against Tris buffer, was loaded in the analytical SEC column pre-equilibrated with TBS . Elutions were performed at a flow rate of 0.5 mL/min. All the experiments involving characterization of the oligomeric state of refolded hSAA1.1 were performed at 4°C.Far-UV CD experiments were performed using a J-815 CD spectrometer (Jasco). Approximately 250 µL of SAA solution, diluted to a final concentration of 20 µM using Tris buffer , was used for the measurement of CD spectra and also for the thermal denaturation experiments. The diluted protein samples were loaded in a rectangular quartz cuvette (1 mm pathlength) and 10 spectra were collected from 200 nm to 260 nm and later averaged. Other experimental settings were as follows: Data pitch, 0.5 nm; Scanning mode, Continuous; Scanning speed, 50 nm/min; Bandwidth, 1 nm. Thermal denaturation experiments on hSAA1.1 were performed by measuring the molar ellipticity of hSAA1.1 with molar ellipticity at 222 nm as the reference. The experimental settings for thermal denaturation experiments were as follows: Temperature range, 4–80°C; Data pitch, 2°C; Slope, 1°C/min.Tryptophan fluorescence measurements were performed using a Spex FluoroLog Tau3 fluorometer (Horiba). Approximately 3 mL of SAA solution, diluted to a final concentration of 10 µg/mL using Tris buffer , was used for the measurement of fluorescence spectra and also for the tryptophan fluorescence-based thermal denaturation experiments. The diluted protein samples were loaded in a rectangular quartz cuvette (10 mm pathlength) and 10 spectra were collected from 320 nm to 400 nm and later averaged. Other experimental settings were as follows: Data pitch, 0.5 nm; Scanning mode, Continuous; Scanning speed, 50 nm/min; Bandwidth, 1 nm; Emission slit width, 5 nm; Excitation slit width; 3 nm. Thermal denaturation experiments on both proteins were performed by measuring the fluorescence intensity and emission maxima of the proteins with emission maxima of freshly refolded proteins used as the reference. The experimental settings for thermal denaturation experiments were as follows: Temperature range, 4–80°C; Data pitch, 2°C; Slope, 1°C/min.For ThT fluorescence assay, a solution containing freshly refolded protein (20 µM) in Tris buffer  was incubated at 37°C without agitation in an Eppendorf Mastercycler (Eppendorf). At desired time intervals, 5 µL of this solution were mixed with 5 µL ThT dye solution (260 µM) and 90 µL of glycine-NaOH buffer  and measurements were made on a Safire II microplate reader (Tecan). Analysis was done in triplicates. The experimental settings used were as follows: excitation wavelength, 440 nm; emission wavelength, 485 nm; excitation bandwidth, 5 nm; emission bandwidth, 5 nm; Number of scans, 6.4 cm−1 M−1). Two hundred microliters of the diluted Congo red solution (20 µM) were then mixed with 100 µL of a protein solution (that had been previously incubated for 24 h at 37°C at a starting concentration of 20 µM) and the resulting mixture was incubated for 30 min at RT. Congo red binding was confirmed by measuring a UV absorbance spectrum from 400–600 nm using a Lambda 35 UV spectrophotometer (Perkin – Elmer), with subtraction of the baseline spectrum of the Tris buffer. The final spectrum was compared to that of a solution containing Congo red (13.3 µM) but no protein.We followed the procedure described in Srinivasan et al. For assays involving the measurement of soluble protein in the protein sample, solutions containing freshly refolded protein (20 µM) in Tris buffer  were incubated at 37°C without agitation in an Eppendorf Mastercycler (Eppendorf). At desired time intervals, 50 µL of sample was centrifuged at 12,000 rpm  at 37°C for 10 min to separate the soluble fraction (supernatant) from the insoluble fraction (pellet). Soluble protein concentration was determined by measuring the absorbance of the supernatant at 280 nm and using the calculated value of the extinction coefficient.AFM measurements were performed as described previously Immunoblot assay using A11 and OC antibodies were performed as described previously Both versions of SAA were expressed, purified and refolded using the protocols described in the experimental procedures section . In order to have a consistent starting point for all the studies, the experiments were performed on freshly refolded protein. By doing so, we ensured that conformational changes associated with the storage of proteins for extended periods of time did not affect the consistency of the results in vitro “native-like” oligomers because they were obtained immediately after refolding the denatured protein. These conformations might not necessarily represent the oligomeric conformation of lipid-free or HDL-associated native SAA in vivo. Additionally, assignment of precise molecular size to the oligomers, based on SEC elution volumes, was made with the principal assumption that the eluting species were globular in shape.We characterized the quaternary structure of MetSAA1.1 and hSAA1.1 using size exclusion chromatography (SEC). The SEC chromatogram for MetSAA1.1 showed a prominent peak with an elution volume of ca. 15.6 mL and two smaller peaks corresponding to elution volumes of ca. 13.9 mL and ca. 18.1 mL . Based oWe then probed the oligomeric conformations of both the proteins refolded at different concentrations to study the effect of protein concentration on its oligomerization properties. Specifically, both the proteins were refolded at 75 µM, 20 µM, and 8.5 µM concentrations and their quaternary structure was analyzed by SEC at each of these concentrations. As shown in Figure S2A in m value for MetSAA1.1 was ca. 29°C. Low values of melting temperatures suggest marginal stability of these “native-like” MetSAA1.1 oligomers. Similar calculations for hSAA1.1 indicated that the melting temperature of this protein was ca. 35°C. Thus, addition of a hydrophobic methionine residue at the N-terminus of hSAA1.1 resulted in a slight decrease in the thermal stability of the protein. We note that the values of the melting temperature (Tm) obtained from these experiments are only for comparison purposes, as the thermal unfolding of the proteins was found to be irreversible (data not shown). We also observed a dependence of protein refolding concentration on the thermal stabilities of both the proteins. While MetSAA1.1 refolded at 8.5 µM concentration yielded a Tm value of 25°C  as a result of refolding at lower concentrations.Next, we used far-UV circular dichroism (CD) spectroscopy to characterize the secondary structure of these “native-like” SAA oligomers. As seen in m values for MetSAA1.1 and hSAA1.1 calculated by monitoring changes in the tryptophan emission maxima upon temperature increase was ca. 31°C and ca. 33°C respectively. To probe the influence of the concentration of the proteins on their tertiary structures, we measured tryptophan emission spectra for proteins refolded at concentrations of 20 µM and 8.5 µM. As shown in Figure S2E in Next we compared the tertiary structure of both hSAA1.1 and MetSAA1.1. Since both the proteins have three tryptophan residues each, comparison of tertiary structures was done by comparing the intrinsic fluorescence values of both the proteins by following tryptophan fluorescence. Protein solutions refolded at 20 µM concentrations were diluted in Tris buffer  to a final concentration of 10 µg/mL and fluorescence measurements were made at 4°C as described in the materials and methods section. As shown in We then performed structural studies to investigate whether the starting SAA proteins in 8 M urea prior to refolding against Tris buffer possessed any residual structure. Specifically, we monitored the secondary and quaternary structure of both the proteins in 8 M urea using far-UV CD and size exclusion chromatography techniques respectively. Quaternary structure analysis on SAA samples containing 8 M urea using SEC (column equilibrated in 8 M urea/20 mM Tris/200 mM NaCl) suggested that both the proteins eluted as a single peak with an elution volume of ca. 19.1 mL representing a monomeric species  using a sequential cation exchange chromatography, anion exchange chromatography, and size exclusion chromatography procedure, the purified protein in size exclusion chromatography buffer  was passed through a Histrap Ni-NTA column  and MetSAA1.1 proteins samples (referred to as “cross-purified” MetSAA1.1) were collected in the flowthrough. Similarly, following purification of hSAA1.1 (referred to as “regular” hSAA1.1) using the procedure described in the materials and methods section , the purified protein was further purified by using sequential cation exchange chromatography and anion exchange chromatography procedures to obtain “cross-purified” hSAA1.1. Relevant buffer exchanges and ultrafiltration steps were followed prior to each of the ion exchange chromatography steps. Both the “cross-purified” proteins were then refolded by using the same refolding protocol which has been described in the materials and methods section. “Cross-purification” therefore ensured that both the proteins, which we expressed using the same expression vector, same bacteria, and refolded using identical dialysis procedures, were also exposed to the same ions and resins during their purification and therefore had similar manipulation histories right from protein expression until protein refolding. We found that both “cross-purified” MetSAA1.1 and “regular” MetSAA1.1, upon refolding at 20 µM concentrations, formed a heterogeneous population consisting of tetramers (primary oligomer), octamers/hexamers, and monomers  at 37°C and monitored the intensity of ThT fluorescence as a function of time. As seen in To ascertain that the aggregates formed by both the proteins have cross-beta structures, we used the well-established Congo red binding assay Next, we monitored changes in the secondary structures of MetSAA1.1 and hSAA1.1 at different time intervals following incubation at 37°C. While the “native-like” oligomers formed by MetSAA1.1 were rich in alpha-helical content , the CD These studies were followed by experiments to check the solubilities of the aggregates formed during this 72 h period. Samples from both the proteins obtained after different incubation times were spun down at 12,000 rpm at 37°C for 10 min to separate the soluble protein fraction (supernatant) from the insoluble fraction (pellet) Collectively, these results suggest that the aggregation of MetSAA1.1 involved the conversion of the alpha helix - rich “native-like” oligomers to soluble oligomers in the early stages of aggregation and eventually into insoluble cross-beta-rich aggregates. On the other hand, aggregation of hSAA1.1 was characterized by the conversion of the “native-like” oligomers into soluble oligomers rich in cross-beta structure. These critical differences point to some fundamental differences between the aggregation pathways of the two proteins, although they differ by only a single methionine residue at the N-terminus.Intrigued by the observation that both versions of hSAA1.1 exhibited similar aggregation kinetics, but differed in the solubilities of their aggregates, we decided to further characterize the species formed during aggregation. We performed AFM studies for morphological characterization of the protein aggregates. We incubated the MetSAA1.1 “native’-like” oligomers  at 37°C and performed AFM on protein samples obtained after different time intervals. Aggregation of hSAA1.1 at 37°C resulted in the formation of spherical aggregates after ca. 3 h  that hadWe next tested whether MetSAA1.1 and hSAA1.1 aggregates shared any structural similarity with the soluble oligomers and amyloid fibrils formed by some commonly studied amyloid proteins. Glabe and co-workers have identified antibodies that recognize the soluble oligomers After analyzing the fibrillation pathways of the two proteins, we then tested the ability of the aggregates formed by both the proteins to seed aggregation. Specifically, we attempted to “seed” the “native-like” oligomers of both MetSAA1.1 and hSAA1.1 with amyloid aggregates formed by the same protein following 72 h of incubation at 37°C. The solutions for these seeding experiments contained 18 µM freshly refolded MetSAA1.1 or hSAA1.1 and 2 µM MetSAA1.1 or hSAA1.1 aggregates obtained after 72 h incubation at 37°C. The final concentration of proteins in each of these mixtures was thus 20 µM  and the percentage of “seed” was effectively 10% of the total protein concentration. We monitored the aggregation kinetics of the “seeded” protein solutions using the ThT fluorescence assay and compared them with those for protein solutions containing 20 µM MetSAA1.1 or hSAA1.1 alone. As shown in We observed that refolding of MetSAA1.1 and hSAA1.1 primarily resulted in the formation of “native-like” oligomers  and the in vitro may seem surprising given its pathogenicity in vivo. This result is, however, consistent with our recent studies of murine SAA, where the pathogenic isoform SAA1.1 was found to be less amyloidogenic than the non-pathogenic isoform SAA2.2 in vivo model system especially since recombinantly expressed proteins lack the post-translational modifications typical of mammalian proteins.Our AFM studies revealed that hSAA1.1 did not form full-length amyloid fibrils upon incubation at 37°C even after ca. 200 h . The lowin vitro, other factors may modulate the stability and fibrillation of SAA in vivo. In a previous study on a mouse isoform of SAA, we showed that Zn2+ binds to and stabilized the secondary, tertiary, and quaternary structure of mouse SAA2.2 while calcium destabilized SAA2.2 structure between 1 to 10 mM concentrations and induced its aggregation Finally, analogous to the observed effect of a single N-terminal methionine File S16-TEV-hSAA1.1 to hSAA1.1 by SDS-PAGE: (lane 1), TEV protease; (lane 2), (His)6-TEV-hSAA1.1 obtained post-IMAC and SEC; (lane 3–8), TEV cleavage of (His)6-TEV-hSAA1.1 to generate hSAA1.1 monitored as a function of time; (lane 9) hSAA1.1 post-SEC purification. Figure S2, Influence of concentration on the oligomerization behavior of MetSAA1.1 and hSAA1.1: (A and B) SEC elution profiles of MetSAA1.1 and hSAA1.1 respectively refolded at 75 µM (black solid line), 20 µM (blue solid line), and 8.5 µM (red solid line) concentrations; (C and D) Far-UV CD-based thermal denaturation profiles of MetSAA1.1 and hSAA1.1 respectively at 20 µM (blue solid line) and 8.5 µM (green solid line) concentrations; (E and F) Tryptophan fluorescence emission spectra of MetSAA1.1 and hSAA1.1 respectively at 20 µM (blue solid line) and 8.5 µM (green solid line) concentrations. Figure S3, Characterization of residual structure of MetSAA1.1 and hSAA1.1 in urea solution: SEC elution profiles of (A) MetSAA1.1 (blue solid line) and (B) hSAA1.1 (red solid line) in 8 M urea solution; (C) Far-UV CD of MetSAA1.1 in 0 M (black solid line), 1 M (blue solid line), and 2 M (red solid line) urea solutions; (D) Far-UV CD of hSAA1.1 in 0 M (black solid line), 1 M (blue solid line), and 2 M (red solid line) urea solutions. Concentration of proteins used for all the assays was 20 µM. Assays were performed at 4°C. Figure S4, “Cross-purification” studies to analyze the oligomerization behavior of MetSAA1.1 and hSAA1.1: SEC elution profiles of (A) “regular” MetSAA1.1 (blue solid line) and “cross-purified” MetSAA1.1 (red solid line); (B) “regular” hSAA1.1 (blue solid line) and “cross-purified” hSAA1.1 (red solid line). Concentration of proteins used was 20 µM. Assays were performed at 4°C. Figure S5, Biophysical characterization of “native-like” oligomers formed by MetSAA1.1 and hSAA1.1: AFM analysis of (A) MetSAA1.1 “native-like” oligomers; (B) hSAA1.1 “native-like” oligomers. All scale bars for AFM images represent 1 µm. Figure S6, Biophysical characterization of aggregates formed by MetSAA1.1 and hSAA1.1: AFM analysis of (A) MetSAA1.1, 6 h, 37°C; (B) MetSAA1.1, 24 h, 37°C; (C) hSAA1.1, 12 h, 37°C; (D) hSAA1.1, 48 h, 37°C. All scale bars for AFM images represent 1 µm. Figure S7, Characterization of “cross-seeding” properties of MetSAA1.1 by ThT fluorescence assay: ThT fluorescence intensity profile for freshly refolded hSAA1.1 only (black bars) and hSAA1.1+ MetSAA1.1 “seed” (DOC)Click here for additional data file.


X-ray diffraction, nitrogen sorption, thermogravimetric analysis, scanning electron microscopy, and energy-dispersive X-ray spectroscopy were used to characterize silicalite nanocrystals and silicalite-PFA composite membranes. The silicalite-PFA composite membrane with 20 wt.% silicalite loading exhibits good oxygen/nitrogen selectivity (4.15) and high oxygen permeability  at 50°C. Silicalite-PFA composite membranes are promising for the production of oxygen-enriched air for various applications.Silicalite-poly [PFA] composite membranes were prepared by solution casting of silicalite-furfuryl alcohol [FA] suspension on a porous polysulfone substrate and subsequent  In the work previously conducted by one of the authors , poly was used to prepare a zeolite 4A polyfurfuryl alcohol nanocomposite membrane by vapor deposition polymerization of furfuryl alcohol [FA] ,12. This× 5.5 Å) ,14.N,N'-methylenebisacrylamide [MBAM], and FA (98%) were purchased from Sigma-Aldrich Corporation . A polysulfone ultrafiltration membrane  was purchased from Sterlitech  and used as the support.One molar tetrapropylammonium hydroxide [TPAOH] aqueous solution and tetraethyl orthosilicate [TEOS] (99%), acrylamide [AM], 2:44 H2O. The synthesis solution was prepared in a 250-mL polypropylene bottle. First, 20 g of 1 M TPAOH solution was added dropwise into 20 g of TEOS under vigorous stirring. Strong magnetic stirring was maintained for 3 h. The solution was then heated in an oven at 80°C for 5 to 6 days for crystallization, resulting in a milky silicalite suspension. The solid product contained in the colloidal suspension was recovered by a repeated cycle of centrifugation with deionized water and ultrasonic redispersion in water until pH < 8. An organic polymer network was prepared from water soluble organic monomers, AM and MBAM, and the initiator (NH4)2S2O8 as a temporary barrier during calcination and carbonization to obtain highly redispersible template-free silicalite nanocrystals. Typically, 1.0 g of AM, 0.1 mg MBAM, and 25 mg of (NH4)2S2O8 were added under stirring into 10 g of silicalite colloidal suspension with 5 wt.% solid loading. After the monomers were dissolved, the mixture was ultrasonicated to ensure complete dispersion of silicalite nanocrystals for half an hour. The aqueous solution was then heated at 50°C for 30 min to be polymerized into an elastic hydrogel. This silicalite hydrogel polymer composite was dried at 80°C overnight. After drying, it was carbonized under nitrogen at 550°C for 2 h  and then calcined at 550°C for 3 h under air.Silicalite nanocrystals were synthesized according to the previously published procedures . In a tyw/w), 10% (w/w), 20% (w/w), and 30% (w/w) in PFA solution, respectively.Both plain PFA and silicalite-PFA composite membranes were hand-cast on commercial polysulfone ultrafiltration membranes.  A 25 mm X-ray diffraction [XRD] patterns were recorded on a Philips PW1140/90 diffractometer  with Cu Kα radiation (25 mA and 40 kV) at a scan rate of 2°/min with a step size of 0.02°. Nitrogen adsorption-desorption experiment was performed at 77 K and at room temperature with a Micrometritics ASAP 2020MC analyzer . To evaluate the thermal stability of the PFA, thermogravimetric analysis [TGA] was conducted using a thermogravimetric analyzer  in the temperature range of 20°C to 700°C under nitrogen gas and a heating rate of 5°C/min. All scanning electron microscopy [SEM] images were taken with a FEG-7001F microscope  operated at an accelerating voltage of 15 kV. Elemental analysis of samples was determined by energy dispersive X-ray spectroscopy [EDXS] on the FEG-7001F microscope.2. The pressure rise was recorded by a MKS 628D Baratron transducer . Membrane permeability, i P·cm·cm-2·s-1·cmHg-1), was defined as  membrane had an O2 permeability of 1,550 Barrers and an O2/N2 separation of 2.98 [2/N2 selectivity observed in the composite membrane with 30% silicalite loading should be due to silicalite-PFA interfacial defects [Table eference , the O2/ Barrers . Poly[1- of 2.98 . In our 2 and O2 gases and O2/N2 ideal selectivity for the 20 wt.% silicalite loading Sil-PFA composite membrane at different testing temperatures ranging from 20°C to 150°C. Single gas permeation experiments showed that as the temperature was increased from 20°C to 50°C, both permeabilities and selectivities increased; whereas the permeability of all gases still increased, but their selectivity decreased as the temperature further increased from 50°C to 150°C. For example, at a testing temperature of 20°C, the membrane had an oxygen permeability of 233.3 Barrers and an O2/N2 selectivity of 3.52. As the temperature increased to 50°C, oxygen permeability increased to 233.3 Barrers, and O2/N2 selectivity also increased to 4.15. At 150°C, the oxygen and nitrogen permeabilities were about 2.6 and 3.8 times higher than those at 20°C, respectively, but the O2/N2 selectivity was about 1.5 times lower. Furthermore, the nitrogen permeability at 150°C was also much higher than that at 20°C.Table 2 molecules require more energy to penetrate through the membranes than O2. In particular, the activation energy for N2 permeation through the composite membrane is only slightly smaller than that for the plain PFA membrane; however, the activation energy for O2 is much smaller for the composite membrane than that for the PFA membrane. This suggests that incorporating the silicalite particles into the PFA matrix can largely reduce the energy barrier for O2 permeation through the membrane, therefore improving O2 flux, and O2/N2 selectivity.The values of the apparent activation energy for nitrogen and oxygen permeation through PFA and 20% Sil-PFA membranes are presented in Table 2-enriched air containing 47.9 vol.% O2 was obtained when the 20% silicalite-PFA composite membrane was used.Figure 2 and N2 permeabilities and O2/N2 selectivity could be improved by incorporating silicalite nanoparticles into PFA. In particular, the Sil-PFA composite membrane with 20% silicalite loading had the highest O2/N2 selectivity and excellent O2 permeability, and an oxygen concentration of 47.9 vol.% was achieved in the single-pass air separation experiment at room temperature.Silicalite-PFA composite membranes were prepared for enrichment of oxygen from air. SEM results showed that silicalite nanoparticles were well dispersed in the PFA matrix. The gas permeation experiments indicated that OThe authors declare that they have no competing interests.LH carried out most of the experimental work including the membrane preparation, characterization, and gas permeation testing and drafted the manuscript. DL was involved in designing the gas permeation experiments, and KW helped with the electron microscopy experiments. HW revised the manuscript. AKS, JB, and TS were involved in the discussions of experimental results. All authors read and approved the manuscript.This work was supported by the Department of Innovation Industry, Science and Research of Australian Government through the Indo-Australian Science and Technology Fund and the Australian Research Council. The authors gratefully acknowledge the support and use of facilities in the Monash Centre for Electron Microscopy. Huanting Wang thanks the Australian Research Council for a Future Fellowship.


Within their litter, young altricial mammals compete for energy  but cooperate for warmth. The aim of this study was to examine the mechanisms by which huddling in altricial infants influences individual heat production and loss, while providing public warmth. Although considered as a textbook example, it is surprising to note that physiological mechanisms underlying huddling are still not fully characterised.Oryctolagus cuniculus) pups to huddle  and of their thermoregulatory capacities (non-insulated before 5 days old and insulated at ca. 10 days old). BAT contribution of pups exposed to cold was examined by combining techniques of infrared thermography (surface temperature), indirect calorimetry  and telemetry (body temperature). Through local heating, the huddle provided each pup whatever their age with an ambient “public warmth” in the cold, which particularly benefited non-insulated pups. Huddling allowed pups facing a progressive cold challenge to buffer the decreasing ambient temperature by delaying the activation of their thermogenic response, especially when fur-insulated. In this way, huddling permitted pups to effectively shift from a non-insulated to a pseudo-insulated thermal state while continuously allocating energy to growth. The high correlation between TEE and the difference in surface temperatures between BAT and back areas of the body reveals that energy loss for non-shivering thermogenesis is the major factor constraining the amount of energy allocated to growth in non-insulated altricial pups.The brown adipose tissue (BAT) contribution to energy output was assessed as a function of the ability of rabbit (By providing public warmth with minimal individual costs at a stage of life when pups are the most vulnerable, huddling buffers cold challenges and ensures a constant allocation of energy to growth by reducing BAT activation. In shared developmental environments, competition for limited resources is a major mechanism shaping phenotypic differences among siblings As a response to cold, heat is first produced in young mammals by non-shivering thermogenesis (NST), depending on the activation of brown adipose tissue (BAT) vs. non-insulated. In particular, we investigate how the huddle influences: 1) the thermal environment of the pup and 2) thermogenic responses of individuals as well as 3) how individual pups contribute to group thermoregulatory processes in the litter. In this context, we tested the physiological responses of rabbit pups in the cold from 23 to 11°C. By combining independent techniques to measure energy expenditure , body temperature (implanted sensors) and surface temperature  of a crossed strain, “Hyplus” from Grimaud . Four does and two males were housed individually in cages . Parturition occurred spontaneously after 30–31 days of gestation. The sliding door of a litter box  filled with fresh straw and hooked to the doe's cage was opened 3 days before parturition. Room temperature varied from 18 to 23°C, and a 16∶8-h light-dark cycle was maintained. The animals were kept and treated during experiments in accordance with the European Guidelines for Animal Care with full approvals from the French Government, the Centre National de la Recherche Scientifique and the Direction of the Veterinary Services (no. 67-188).The experiments were carried out on rabbits . Pups were placed in plastic boxes with an open top . Except during the experiment, all pups were housed in groups from their original litter in a room with a controlled ambient temperature of 23–24°C and continuous lighting. Ambient temperature set point was chosen to limit any mortality of the pups, based on a previous study by our team ca. 4 hours), in order to minimize thermogenic effects due to post-prandial digestion At birth, pups were left a few hours with the doe in the nesting box to allow them to suckle once without disturbance. Then pups were separated from the doe, color-marked on their back with an animal marking stick  for identification, and weighed  was designed to explore thermoregulatory responses of pups. Pups were placed in a room adjacent to the breeding room, and exposed to decreasing ambient temperatures of 23 (for 2 hours), 18, 15, and 11°C (for 1 hour). Room temperature after the cold exposure was again regulated at 23–24°C. Non-insulated pups aged 4 days old were placed in groups of six , and the subsequent day (at 5 days old), the same pups were separated in groups of two . The same procedure was repeated when pups were 15 days old  and 16 days old . After the experiments all pups were placed in the breeding room.Independently, Experiment 2 was designed to investigate brown adipose tissue activation using thermal imaging (n = 12 pups taken from two litters of 7–8 pups). Non-insulated pups aged 3 and 4 days old and insulated pups aged 10 and 11 days old were used. They were exposed to ambient temperatures of 23°C (for 2 hours) and 14°C (for 1 hour). On day 1, a group of six pups  and two isolated pups  were tested. On day 2, a group of six pups  and two isolated pups  were tested.2) consumption and carbon dioxide (CO2) production were measured using an open-circuit respirometry system . The concentrations of O2 and CO2 in the outgoing air were measured in one experimental chamber for G6 , and in three chambers for the three G2 . The two different sizes of chambers were defined after theoretical calculations taking into account the air flow and the energy expenditure of pups, to ensure robust respirometry measurements. Measurements were performed continuously over the cold challenge (from 23 to 11°C). Calibration of O2 and CO2 analyzers were undertaken before and after each experiment.Oxygen  at a flow rate of 1 L.min−1 every 8 min. For both experiments, final values of O2 and CO2 concentrations were the mean of values recorded during 60 and 90 s, respectively. The system was flushed with air for 120 s between each cycle. Energy expenditure was calculated using an energy equivalent of 16.47 J.ml−1 of O2 consumed and 4.62 J.ml−1 of CO2 produced, according to Weir's equations −1.g−1. The mean body mass of rabbits was determined from a weight before measurements and a weight the day after the experiment, before subsequent suckling.For the “G6-procedure”, the cage was sampled for 120 s (1 sample per second) at a flow rate of 1 L.min2 and CO2 in the outgoing air were measured simultaneously in the three chambers. Measurements were performed continuously over the cold challenge (at 23°C for 2 hours and 14°C for 1 hour). Samplings and calculations were similar to Experiment 1, according to “G2-procedure”.For Experiment 2, the G6 was placed in a chamber of 25×36×16 cm, while the two G1 rabbit pups were placed in cages of 17×24×15 cm. The concentrations of Ohttp://www.sensoray.com). Thermoresistive devices were calibrated in a thermostatic bath from 20 to 40°C, with 5°C increments before and after the experiments.In order to evaluate any local heating effect, in particular in the chambers for G6 pups compared with G2 pups, temperatures were also measured in each calorimetric chamber. The ambient temperature within chambers was continuously monitored by a thermoresistive device  placed in the centre of the chamber (see above for box dimensions). The thermal probes were connected to a Smart A/D computer card (model no. 619), and data were recorded every 30 s using Sensoray Quicksense software . TA10TA-F20 transmitters  were placed intraperitoneally, under gaseous anesthesia  and strictly aseptic surgical conditions. The surgery took place at least 5 h after suckling, and a heating pad was used to prevent hypothermia. Antibiotics  and anti-inflammatory molecules  were injected at the end of surgery and the pup was returned to its littermates within 1–2 h. Transmitters recorded body temperature at 30-s intervals during the experiments. Because of radio frequency interference, only one pup per group could be monitored . In Experiment 2, 4 randomly selected pups were implanted with transmitters following a similar protocol.In Experiment 1, 11 randomly selected pups were implanted with transmitters at 2.5 days of age (mean body mass of ear), the pinna being the principal site of heat dissipation for rabbits BAT). The back temperature (Tback) therefore represented a reference surface temperature, from a neutral area with respect to heat flow. The gradient (TBAT - Tback) allowed the comparison of BAT activation independently of ambient temperatures (23 or 14°C). For G1 pups, surface temperatures  were calculated as the mean (± SD) of two individuals for each age group. For G6 pups, the mean (± SD) surface temperatures  were determined from three individuals within the group as positions of pups prevented a clear view of all pups within the huddle. The back region was defined using a circle (diameter = 20 pixels) positioned on the dorsal surface in the centre of the hips. Ear temperatures were determined by fitting a polygon around the outer edge of both left and right ears. As it was not possible to precisely define the surface overlying BAT, a circle (diameter = 20 pixels) was positioned on the image between scapulae  using a thermal imaging camera PM595 . The lid of the respirometry chamber was removed and images were recorded at a height of 1 m above the chamber. Thermal images were analysed using Thermacam Reporter 7.0  using an emissivity for fur of 0.98 Prior to analyses, we verified on raw data the absence of any difference between litters and categories of pups.Cold challenges in the chambers were tested independently for the four categories of pups, between the four ambient temperatures, using ANOVAs and Kruskal-Wallis ANOVAs when distribution was not normal. In order to compare local heating between G6 and G2 pups, insulated and non-insulated pups were pooled and Mann-Whitney tests were used for each room temperature. Body mass of pups were compared with paired t-tests between the two sessions: G6 pups at day 4, and G2 pups at day 5; G6 pups at day 15, and G2 pups at day 16. To assess the effects of cold challenge, huddling (G2 vs. G6), and age (non-insulated vs. insulated pups) on either energy expenditure or body temperature, and to take into account repeated measurement nature of the data, multivariate linear mixed-effects regression models were implemented using the SAS PROC MIXED procedure . Independent variables included external temperature, group (G2 vs. G6), and age (insulated vs. non-insulated). Body mass was also included as a potential confounder. These models were fitted to the data with the intercept as the random effect.Surface temperature of the different tissues  were compared with one-way ANOVAs on repeated measures for each category (post-hoc Tukey tests).Room temperature during the cold challenge was decreased successively from 22.8±0.8 to 18.2±0.8, 15.0±0.8, and 10.6±1.3°C Table 1.Between the two sessions (at day 4 and day 5), pups gained on average 13.4±5.3 g, corresponding to a 14% increase in body mass . In the same way, between day 15 and day 16, pups gained 15.2±7.0 g, i.e. 6% of their total body mass . Hence, G2 pups, one day older than when placed in groups of six, possessed a higher body mass, i.e. a lower surface area to volume ratio than G6 pups. This may have minimised any physiological variations and effects linked to huddling.vs. 0.49±0.06 kJ.g−1.day−1; p<0.001 after adjustment for cold, group and body mass). In addition, TEE of G6 pups was 14% lower than G2 pups .For all ambient temperatures, non-insulated pups showed a total energy expenditure (TEE) more than twofold higher than insulated pups . In the same way, TEE of non-insulated G2 pups increased significantly by 38% in response to cold, while their body temperature decreased by 1.2°C (both p<0.0001 after adjustment for body mass).Considering insulated pups . By contrast, TEE of G2 insulated pups increased by 23% in response to cold, while their body temperature decreased by 0.3°C (both p<0.0001 after adjustment for body mass).Body temperature and energy expenditure responses of G6 and G1 pups, at 23°C (23.0±0.1°C) and 14°C (14.0±0.7°C) for non-insulated (at 3 and 4 days old) and insulated pups (at 10 and 11 days old), were consistent with Experiment 1 Figure 2vs. 23.6±0.3°C for 4-day G1 pups) and 2.1°C for insulated pups (25.6±0.6°C for 11-day G6 pups vs. 23.5±0.1°C for 10-day G1 pups). In the same way, at cold ambient temperature, local heating was 3.7°C for G6 non-insulated pups (18.0±0.2°C for 3-day G6 pups vs. 14.3±0.4°C for 4-day G1 pups) and 2.6°C for G6 insulated pups (18.2±0.3°C for 11-day G6 pups vs. 15.6±0.3°C for 10-day G1 pups). Cold challenges were therefore 6.4°C for 3-day G6 pups, 9.3°C for 4-day G1 pups, 7.4°C for 11-day G6 pups, and 7.9°C for 10-day G1 pups.Ambient temperatures were monitored in each experimental chamber. At warm ambient temperature, local heating in the chambers containing G6 pups compared with isolated pups was 0.8°C for non-insulated pups . At cold ambient temperatures, the mean surface temperature of the huddle was 4.7°C higher than the mean temperature of isolated pups (35.6±0.4 vs. 30.9±0.9°C).Considering non-insulated pups, at warm ambient temperatures, the mean surface temperature of the huddle (i.e. group of 6 pups) was 1.9°C higher than the mean temperature of isolated pups . At 14°C, the mean surface temperature of the huddle was 0.9°C higher than the mean temperature of isolated pups (26.7±0.4 vs. 25.8±1.0°C).Considering insulated pups, at 23°C, the mean surface temperature of the huddle was 1.2°C higher than the mean temperature of isolated pups  and TBAT - Tback, at warm and cold temperatures was examined (BAT - Tback (TEE = 0.534+0.354.(TBAT - Tback), r2 = 0.991, F1,3 = 223.9, p = 0.004; BAT - Tback  were designed to be significant for developing rabbit pups Microtus agrestis) accounts for 55% of the energetic benefits of huddling et al. The chambers were closed for respirometry measurements, and therefore local heating in the chambers containing G6 huddling pups was important in comparison to isolated or G2 pups. In warm conditions (23°C), local heating was 0.8°C higher for huddling non-insulated pups (aged 3–4 days), and 2.1°C higher for huddling insulated pups (aged 10–11 days) compared with isolated littermates. Local heating was even more pronounced in a cold environment (14°C): surrounding temperatures of huddling pups were 3.7 and 2.6°C higher compared with isolated pups . Warming of the local microclimate is part of the thermal benefits of huddling When exposed to cold, rabbit pups increase their energy expenditure by a rise in metabolic heat production but when overwhelmed by heat loss body temperature decreases et al.In endotherms, physiological responses to cold involve vasoconstriction to reduce skin surface temperature and thermoregulatory thermogenesis to maintain homeostasis Within their litter, altricial mammal pups cooperate for warmth, but compete for energy, obtained from maternal milk and allocated to growth and thermogenesis. Pup growth in rabbits is hence primarily dependent on maternal factors and litter size BAT was higher than Tback in all cases, indicating that heat production was largely due to BAT activation. Pups also reduced their heat loss by vasoconstriction, as seen by a drop in ear temperature  and isolated pups produced extra heat when exposed to cold: their energy expenditure increased, while their body temperature decreased Figure 2BAT - Tback. However, when comparing isolated insulated pups at warm and cold temperatures, they seem to present thermogenic responses similar to non-insulated pups  of non-insulated huddling pups were equivalent to insulated isolated individuals (23°C, As energy supply is limited for pups, extra energy required for thermally challenged pups will reduce energy available for growth and possibly influence their short or long-term survival and their adult performances ls 23°C, Figure 6Huddling reduces BAT thermogenesis, allowing pups to lower the amount of “private heat” given to the “public good”, essential for the maximum allocation of energy for growth. When ambient temperatures were challenging, the pattern of thermogenic responses was equivalent when pups were exposed to warm or cold temperatures in our study, but differed considering the extent of the response Figure 5Proximal thermoregulatory constraints that ultimately govern development and survival of rabbit pups were examined. Huddling, through “public warmth”, drastically modifies thermal constraints of pups, reducing cold challenges and effectively increasing their developmental stage of thermoregulation. Since energy is limited for an organism, variations in its thermoregulatory requirements impact on the amount of energy allocated to growth. By providing public warmth when pups are the most vulnerable, huddling buffers cold challenges and ensures a constant allocation of energy to growth by delaying non-shivering thermogenesis. This study may stimulate further research focusing on the energetic implications of cold challenges in other altricial infants, and more particularly by investigating individual thermogenic constraints within a litter on the early development of personality differences between siblings.


We have recently demonstrated the benefits of a radially undersampled 4D-MR flow acquisition (PC-VIPR) ,2 for anEight healthy volunteers (mean age=26.9±2.5years) were scanned on a 3TMR scanner with a 32-channel phased-array torso coil. Subjects refrained from eating for a minimum of four hours prior to the MR examination. 2DPC scan planes were prescribed in the supra- and infrarenal aorta and in each renal artery Fig. with a din) and the sum of flow measurements in the renal arteries and infrarenal aorta (Qout); differences were normalized by the average of Qin and Qout. 2D and 4D percent differences were compared with a Student's t-test. The repeatability of flow measurements was assessed with Pearson correlation and Bland-Altman analysis.Total flow over the cardiac cycle was measured with in-house-developed software. To test for internal consistency, differences were computed between the suprarenal aortic flow (Qin (2404.8±632.5ml/min) was similar to average Qout (2259.1±449.4ml/min). Similarly, for all 16 sets of 4D measurements, average Qin (1517.5±585.2ml/min) was similar to average Qout (1343.9±410.8ml/min). 4D flow measurements tended to be lower in magnitude than 2D flow measurements. Normalized, the total flow difference for all 16 2D sets of measurements (18.0±16.5%) was greater than the mean percent difference of all 16 4D measurements  and 4D  flow measurements was strongly correlative. Bland-Altman analysis demonstrated small overall flow differences for both the 2D (mean±2SD=-24.8±1865ml/min) and 4D (mean±2SD=56.9±708ml/min) techniques (Fig.For all 16 sets of 2D measurements, average Qques Fig..Flow measurements in healthy volunteers revealed that 4D measurements tended to be more internally consistent than 2D measurements, with average differences slightly greater than 10%. The repeatability of the 2D and 4D data were similar. These results are favorable compared to a previous report  comparin


The PdII complex shows a square-planar coordination with significant distortions of the Cl–Pd–Cl moiety in the solid state. In contrast, the phosphane ligand exhibits a large flexibility in the trigonal-planar coordination of the Cu salt, as proven by X-ray crystallography. C–C cross-coupling reactions and 1,3-dipolar cycloadditions have been tested for the PdII and CuI complexes, respectively. Whereas the reactivity of the PdII complex is good at low temperature, the CuI complex reveals remarkable reaction rates at temperatures up to 130 °C. Furthermore, the CuI-catalyzed azide/alkyne 1,3-dipolar cycloaddition was successfully adapted for flow conditions.A facile large-scale synthesis of the “push–pull”-substituted ligand bis(diethylamino)(pentafluorophenyl)phosphane is reported. A selenophosphorane as well as complexes with CuI and PdCl Over the last decades palladium has achieved a prominent role in catalysis and organic synthesis. Different types of reaction protocols have been developed for the formation of C–C bonds by the use of palladium catalysts.86F5)3P in 196014The opposite approach, that is the design of electron-deficient phosphane ligands also had a profound impact on catalyst design. Since the first preparation of , for example CuI, have also attracted significant attention in catalysis. In particular, CuI-catalyzed Huisgen azide/alkyne 1,3-dipolar cycloaddition (CuAAC)I species involved.24It is generally accepted that in palladium-catalyzed cross-coupling reactions 6F5P(NEt2)2 was reported already in the 1960s2)2. Here we report a straightforward and practical alternative to the original synthetic procedure and further investigations into the coordination and catalytic chemistry of C6F5P(NEt2)2 in standard model reactions. Our findings are corroborated by X-ray crystallography, spectroscopic methods, and ab initio calculations.C1) combines electron-releasing amino groups with an electron-withdrawing fluoroaryl unit at the same phosphorus atom. Therefore, we were interested in the properties of this “push–pull”-substituted phosphane and in further exploring the reactivity of this class of compounds. Compound 1 was synthesized by the straightforward and cost-efficient reaction of LiC6F5 with ClP(NEt2)2 at low temperature yielding the pure product after distillation (1 are in agreement with the available literature data and have been further amended.28Bis(diethylamino)(pentafluorophenyl)phosphane (illation . Spectro1 in which ethyl groups were replaced by isopropyl groups was reported in 2003.6F5P(NiPr2)2 neither formation of a selenophosphorane nor coordination to (carbonyl)Rh complexes could be achieved, which was attributed to steric and electronic factors.6F4P(NEt2)2, in which the amino groups are identical but the fluoroaryl substituent differs by one fluorine atom, was published very recently.1 would be unavailable for reactions at the lone pair, just like its isopropyl-substituted analog, or prone to oxidation and coordination like its partially fluorinated congener. In an initial attempt to explore the reactivity of the phosphorus lone pair in this compound we investigated the oxidation of 1 with gray selenium (2 is easily obtained at room temperature in quantitative yield. In the 31P NMR spectra a single resonance at δ = +49.4 ppm is observed showing the expected 77Se satellites (1JPSe = 800 Hz), which is shifted to high field compared with 1. In general, the 1JPSe coupling constant in selenophosphoranes is a rough estimate for the overall electron-withdrawing property of the substituents at the phosphorus atom featuring larger values for more electron-withdrawing substituents.2 is in the typical range for trisselenophosphoranes,77Se NMR signal of 2 resonates at lower field compared to (Me2N)3PSe, while the 31P NMR signal is observed at higher field {δSe = –136.9 ppm, δP = 49.4 ppm (2) vs. δSe = –366 ppm, δP = 81.8 ppm [(Me2N)3PSe]}. Selenophosphorane 2 shows similar spectroscopic data as its partially fluorinated analog HC6F4P(=Se)(NEt2)2.1 with elemental selenium is in marked contrast to the reactivity of the analogous iPr2N-substituted phosphane reported by Dyer et al.iPr in C6F5P(NR2)2. The selenophosphorane 2 could also be characterized by means of X-ray diffraction and by EI-MS showing the characteristic pattern for the selenium-containing species. Geometric parameters are similar as for HC6F4P(=Se)(NEt2)2.···N contacts [2.767(7) Å and 2.844(7) Å] for both molecules in the asymmetric unit. Furthermore, weak π–π and π–Se interactions can be concluded from the solid-state packing. A graphical representation of 2 is depicted in 1 should open the way to the coordination chemistry of this class of phosphane ligands.The closely related analog of selenium . The env1 we investigated its reactivity towards late transition metals like Pd and Cu. The corresponding Pd complex (3) can be synthesized from a suitable precursor, that is Cl2Pd(MeCN)2, and ligand 1 in nearly quantitative yield within a short time  have been studied by Trzeciak et al. showing a correlation between Δδ, the phosphorus cone angleδ) in the 31P NMR spectra of this phosphane is quite small . A single crystal structure analysis of Pd complex 3 confirms that in the solid state the ligand coordinates through the phosphorus atom to the metal center. Two independent molecules of complex 3 with very similar structural parameters are present in the unit cell. One is located near the center of inversion and shows disorder over two orientations, whereas the second molecule is not disordered. Consequently, the geometric parameters are discussed only for the molecule without disorder. The bis(diethylamino)(pentafluorophenyl)phosphane ligands in 3 are bonded to the Pd atoms in trans orientation. The pentafluorophenyl rings adopt orientations where an N atom of a diethylamino group is coplanar to the ring [N–P–C–C smaller than 1.8(2)°]. The most remarkable structural feature of 2 is the significant distortion from planarity around the central Pd atom. Whereas the P–Pd–P angle is almost linear [178.27(2)°], the Cl–Pd–Cl angle deviates strongly from linearity with 164.96(2)°.Coordination shifts (Δ3 the least-squares (l.s.) plane through P1, Pd1, P3, and Cl1 encloses an angle with the Pd1–Cl3 bond of 14.86(3)°. This motif is best described as a distorted planar geometry, because the Cl–Pd–P angles are close to a right angle [87.87(2)° to 91.81(2)°]. The Pd–Cl distances [2.309(1) Å and 2.320(1) Å] are rather long for dichloridobis(phosphane)palladium complexes, with the exception of bridging chlorine atoms, that is Pd–Cl–Pd, which can have Pd–Cl distances up to 2.407(5) Å.3 is 0.11 Å longer than for example in PdCl2(PPh3)2 (= M2). The Pd–P contacts are 2.322(1) Å and 2.355(1) Å and thus in the upper range of Pd–P distances for this class of compounds.6F5P(NiPr2)2 [1.683(3) Å/1.669(4) Å]p-C6F4[P(NEt2)2]2 [1.675(2) Å/1689(2) Å]3 are shortened upon coordination to the metal center [1.646(2) Å–1.662(2) Å], whereas the P–C bond is essentially unaffected. In contrast, the crystal structure of tris(pentafluorophenyl)phosphane derivative[(C6F5)3P]2PdCl2 shows much shorter Pd–P and Pd–Cl distances , and the palladium center shows an almost planar coordination geometry.3 is depicted in FH+, ArF+–NEt2, C6F5PH+). In order to test the thermal stability of 3, we performed 31P variable-temperature (VT) NMR experiments. Measurements of 3 from room temperature (room temp.) up to 70 °C in C6D6 solution showed that no decomposition occurs, and the complex is stable at these temperatures for at least 30 min. The integrity of the complex is further corroborated by the persistence of the two distinctive multiplets for the methylenic protons in the 1H NMR spectra at elevated temperatures (70 °C). Formation of a second isomer  could not be detected within the limits of resolution and quantification in 31P NMR spectroscopy.There are only a few examples in the literature with a bent linear arrangement of the phosphane ligands across the metal atom with a Cl–Pd–Cl angle significantly smaller than 180°. In these cases, such bending was imposed by the use of rigid bidentate ligands,3 we investigated two model compounds by means of DFT calculations. In our model compounds M1 {[C6F5(Me2N)2P]2PdCl2} the ethyl groups of 3 were replaced by methyl groups to avoid nonessential rotations around the CH2–CH3 bond. The DFT studies (B3LYP/6-31G*/LANL2DZ) were performed on two isomers of M1, namely cis- and trans-M1, which are compared to computational results for the structurally well-characterized (Ph3P)2PdCl2 (= M2). A detailed analysis of the molecular orbitals was carried out in order to understand the bonding situation of these complexes. A comparison of measured and calculated geometric parameters as well as a natural bond orbital (NBO) analysis should reveal the differences and commonalities of both complexes. The P–Pd–P and Cl–Pd–Cl angles in trans-M1 are 178.9° and 168.8°, respectively, which differs only slightly from the experimental values obtained by X-ray analysis.M1 with the well-known M2 should reveal the effect of the electron-withdrawing group of the phosphane ligand. An NBO charge analysis on the C6F5-substituted phosphane ligand in M1 results in a net charge of 1.46/1.48 a.u. on P and 0.39 a.u. on Pd. In complex M2 a quite similar charge for Pd (0.40 a.u.) is found but a less positive P-center (1.09 a.u.), which is in line with the electron-withdrawing properties of the C6F5 group of ligand 1. The natural charge of the chlorine atoms seems to be unaffected by the phosphane ligands . A comparison of the absolute gas phase energies of cis-/trans-M1 shows that the trans isomer is just 15.3 kcal mol–1 lower in energy (ΔG298). Thus, formation of the cis isomer may occur easily with suitable reaction partners. Owing to the distorted geometry in trans-M1 and 3, the isomerization barrier is expected to be rather low. From our calculations the experimentally observed bending of the Cl–Pd–Cl unit can be reasonably reproduced suggesting that secondary interactions of the ligand with the Pd–Cl moiety are responsible for this distorted geometry. Furthermore, it can be excluded that the significant distortion from planarity results from crystal packing effects.For a better understanding of the unusual geometry around the palladium center in complex 3 are, we set out to explore the coordination behavior of phosphane ligand 1 towards a d10 metal ion where little backbonding can be expected. We have chosen CuI to extend our study owing to its relevance in “click” chemistry. The CuI complex 4 of phosphane 1 was obtained by direct reaction of the ligand with CuI in a 2:1 ratio in chloroform or dichloromethane . Similar to complex 3, the methylenic protons are diastereotopic. Slow evaporation of chloroform or dichloromethane under ambient conditions gave colorless hexagonal crystals of 4a. Crystal structure analysis of the complex shows a trigonal-planar surrounding of the central copper atom with almost ideal parameters. The P–Cu–P angle [129.60(1)°] is distinctly larger than the I–Cu–P angles [113.62(1)° and 116.77(1)°]. The C6F5 groups are almost coplanar [7.41(7)°] suggesting that π-stacking may govern the relative orientation of the two phosphane ligands. Thus, the inter-aryl distance is as short as 3.074(2) Å, which is 0.10 Å shorter than the sum of their van der Waals (vdW) radii. The pentafluorophenyl rings adopt orientations in which the N atom of one diethylamino group is coplanar relative to the aryl ring [torsion angles N–P–C–C smaller than 4.86(15)°]. The N atoms are consequently below/above the least-squares plane spanned by I1, Cu1, P1, P2 . The P–N bonds are slightly longer [1.651(1) Å–1.668(1) Å] than in complex 3. The observed Cu–I distance [2.5476(2) Å] is in the normal range for this class of compounds. On the basis of the structure analysis, we conclude that the nitrogen atoms do not contribute to the stabilization of the copper atom.To check how general the unusual geometric features in Pd complex omethane . As prev4 is completely air-stable and shows no formation of CuII species over months under ambient conditions. Its stability is, however, limited to nonacidic media owing to the lability of the P–N bonds toward acid-mediated bond cleavage. The EI mass spectra of complex 4 exclusively show the ligand and its fragments, whereas in the ESI spectra also some fragments (L+ – NEt2) and adducts of the complex (+4 – I and +4 + Cu) could be observed.Interestingly, compound 4 with those obtained for the free ligand  measurements and compared the results of e ligand . Ligand of water .31P NMR spectra can be observed. We were able to grow single crystals from both solvents. Although the copper atom still has a trigonal-planar coordination environment, the two phosphorus ligands show significantly different orientations. Whereas in 4a both C6F5 groups of the ligands are in trans orientation to the iodide [Cu–I–P–C –172.02(5)° and –177.59(5)°] in compound 4b  only one of the C6F5 rings adopts this trans orientation [Cu–I–P–C 175.00(4)°], and the other one is in a cis orientation [Cu–I–P–C –16.67(4)°]. The P–Cu–P angle is also slightly larger than in 4a, but the sum of the angles around the Cu atom is for all complexes ca. 360° . The Cu–I bond is also slightly longer in 4b. The structure obtained by recrystallization from THF (4c) shows very similar structural parameters. Details of the structure solution for 4a,b,c are given in the Supporting Information. Phosphane ligand 1 entails a high spatial flexibility in these copper complexes, which therefore should be predestined to adapt to a large variety of substrates in catalytic reactions.Since CuAAC (“click”) cycloaddition reactions are often carried out in donating/coordinating solvents, we wanted to explore the behavior of the complex towards solvents having oxygen (THF) and nitrogen (MeCN) donor sites. In solution no significant differences in the 3 was studied with a Suzuki–Miyaura-type C–C cross-coupling reaction (2(PPh3)2. Reactions were carried out under ambient conditions in 1,4-dioxane or toluene. The reaction conditions and achieved conversions are summarized in the Supporting Information. Neither solvents nor other reaction conditions  were optimized for these catalytic studies. The stability of the complex against oxidation to PV under ambient conditions is sufficient, and short exposure of 3 to air does not reduce its reactivity. At elevated temperatures (110 °C) under microwave heating palladium catalyst 3 shows an activity comparable to PdCl2(PPh3)2 and reactions without addition of ligand to PdCl2. Conducting these cross-couplings at ambient temperature shows superior conversion rates using 3  compared to the ones of the control experiments [PdCl2 (48 h/34 % conv.) and (PPh3)2PdCl2 (48 h/43 % conv.)]. The higher activity of 3 compared with PdCl2 and (PPh3)2PdCl2 is probably a result of the structural distortion discussed before associated with longer and consequently weaker and more reactive Pd–P and Pd–Cl bonds.In order to test the catalytic scope of our push–pull phosphane complexes, we carried out two types of reactions. The catalytic performance of Pd complex reaction  and compI species are often used as catalysts to perform CuAAC “click” reactions. Suitable catalysts are (amine-)stabilized CuI halidesII precursors and a reducing agent, that is CuSO4 and sodium ascorbate,0 and CuII. Elemental copper exhibits much better performance upon pretreatment with H2O2, suggesting Cu2O as the active species.4 as catalyst. Initial studies in a microwave reactor on a model reaction . Full conversion is reached at 60 °C after 90 min (Entry 6). Catalyst 4 or in situ generated catalyst give comparable results for conventional heating at 65 °C . Full conversion at higher temperatures is already reached after 15 min (entry 11). Preliminary attempts to detect adducts of the copper catalyst 4 and either the alkyne or the azide by means of NMR spectroscopy have been unsuccessful. In the absence of azide, Glaser-type coupling reactions have been observed at ambient conditions with formation of the corresponding bis, which could be proven by X-ray analysis. This suggests a competitive reactivity between Glaser-type and “click” reactions, which seems much faster for the latter, as evidenced by the complete lack of homocoupling products in the presence of azide.As outlined before, Cureaction , II demo process , whereas4 for this model reaction at higher temperatures, we explored the possibility to adapt this catalyst system for continuous-flow conditions. Previous studies on CuAAC chemistry performed in flow exclusively employed immobilized CuI species.–1 flow rate, in which case a conversion of 80 % and a similar isolated yield was obtained (75 %). Hence, CuI complex 4 seems to be an excellent catalyst for CuAAC reactions over a wide range of temperatures suitable for continuous-flow applications in high-throughput or combinatorial chemistry. As an additional advantage, simple removal of the catalyst can be envisaged by acidic cleavage of the amino groups and subsequent aqueous extraction,52Owing to the stability of the catalyst at temperatures up to 100 °C, we envisaged to adapt the CuAAC process , II for I species. This is also underlined by the fact that solid complex 4 can be stored at ambient conditions in air over months, and neither decomposition nor disproportionation reactions are observed.The good activity of the complex is mainly attributed to the excellent stabilization of the Cu1, which is available for reactions at the phosphorus lone pair. Besides the selenation also the coordination of 1 towards PdII and CuI centers has been accomplished, which is in marked contrast to its isopropyl analogue as described in the literature. On the basis of the distorted geometry of the PdII complex, which was confirmed by X-ray and theoretical studies, we concluded good reactivity in C–C coupling reactions. This was confirmed for Suzuki–Miyaura-type cross-coupling reactions at ambient conditions. Therefore, this catalyst is a promising candidate for C–C coupling reactions of temperature-sensitive compounds. The analogous CuI complex shows a trigonal-planar conformation for the central atom, whereas orientation of the ligands strongly depends on the coordination properties of the solvent. The catalytically active species was either added directly or prepared in situ showing equally good performance in CuAAC reactions, conducted under batch or continuous-flow conditions. Further studies must be carried out to investigate the scope and the limitations on substrates and reaction conditions of this push–pull phosphane ligand system.We have demonstrated the cost-efficient synthesis of the pentafluorophenyl-substituted aminophosphane ligand General: NMR spectroscopic data were collected either with a Varian INOVA 400, or a Bruker Avance 300 operating at a proton frequency of 400 or 300 MHz, respectively; 1H and 13C NMR spectra were referenced internally to residual solvent signals; 31P and 19F NMR spectra were referenced externally to H3PO4 (85 %) and C6H5CF3 (δ = –63.7 ppm), respectively. Compound 1 was prepared according to a modified literature procedure.2O3 columns by the PureSolv solvent purification system. All chemicals were purchased from Aldrich or ABCR (C6F5H) and used without further purification. Owing to the evolution of HF, the determination of conventional combustion analyses was not possible with the equipment available to us. Therefore, the purity of products was determined with NMR, HPLC, or GC, and relevant spectra are depicted in the Supporting Information.Electrochemical Studies: CV spectra were recorded with a Gamry Reference 600 device in CH3CN solution (Bu4NBF4 0.06 m) of the respective substance under argon. A standard three-electrode system .MS Measurements: DI-EI measurements were performed with an Agilent 5975C quadrupole mass spectrometer equipped with a direct injection unit. HRMS measurements: Samples were dissolved in CH3CN and directly infused into the ESI ion source of a Synapt HDMS Q-TOF mass spectrometer . Samples were analyzed in positive-ion mode with a capillary voltage of 2.50 kV and a source temperature of 100 °C. The [MH]+ peak at m/z = 556.2771 of Leu-Enk  was used as the reference signal. A table containing selected HRMS data is given in the Supporting Information.Preparation of Bis(diethylamino)(pentafluorophenyl)phosphane (1):CAUTION! The lithio intermediate in the preparation of compound 1 is highly reactive and prone to LiF elimination at temperatures above –50 °C under the formation of very reactive aryne species! To a cooled (–50 °C) solution of C6F5H  in THF (120 mL) was added nBuLi  over a period of 15 min, and stirring was continued at this temperature for 1 h. The suspension was cooled to –80 °C, and ClP(NEt2)2  was added slowly. The mixture was allowed to reach room temp. and stirred overnight. After removal of the solvent, the residue was extracted with pentane yielding crude 1. Distillation under reduced pressure  yielded 80 % pure product . 1H NMR : δ = 0.97 , 2.93  ppm. 13C NMR : δ = 14.94 , 44.69 , 117.11 , 137.63 , 140.95 , 146.21  ppm. 19F NMR : δ = –136.96, –156.48, –163.02 ppm. 31P NMR : δ = 82.0 ppm.Preparation of Bis(diethylamino)(pentafluorophenyl)(seleno)phosphorane (2): To a solution of 1  in chloroform (2 mL) a slight excess of gray selenium  was added. The suspension was stirred at ambient temperature for 15 min. After filtration and removal of all volatiles, pure product was obtained in almost quantitative yield as a colorless solid . M.p. 56–58 °C (CHCl3). 1H NMR : δ = 1.18 , 3.28 . 13C NMR : δ = 14.08 , 44.69 , 113.32 , 137.84 , 142.85 , 146.09  ppm. 19F NMR : δ = –133.72 , –150.52 , –160.65  ppm. 31P NMR : δ = 48.4  ppm. 77Se NMR : δ = –136.9  ppm.trans-Dichloridobis[bis(diethylamino)(pentafluorophenyl)phosphane]palladium(II), trans-Pd[P(C6F5)(NEt2)2]2Cl2 (3):Preparation of  A mixture of PdCl2  and acetonitrile  was refluxed, until the brown residue was completely dissolved and the (MeCN)2PdCl2 complex formed. The solution was allowed to reach room temperature and subsequently added to a solution of 1  in acetonitrile (3 mL); the mixture was stirred at room temperature for 2 h. The solvent was removed and the residue extracted with dichloromethane to yield 3 as an oily material. Recrystallization from cold MeCN (–28 °C) yielded 95 %  spectroscopically pure 3. 1H NMR : δ = 1.04 , 3.19 , 3.30  ppm. 13C NMR : δ = 14.11 , 43.23 , 111.13 , 137.54 , 142.22  146.80  ppm. 19F NMR : δ = –132.28 , –151.46 , –162.43 (br. m) ppm. 31P NMR : δ = 78.4 (br. s) ppm.6F5)(NEt2)2]2I (4):Preparation of Bis[bis(diethylamino)(perfluorophenyl)phosphane]iodidocopper(I), Cu with the following physical properties: M.p. 132 °C : δ = 8.67 , 7.86 , 7.52 , 5.65  ppm. 13C NMR : δ = 147.1, 136.5, 131.1, 129.4, 129.3, 128.6, 128.4, 125.6, 122.0, 53.5 ppm.General Procedure for the Copper-Catalyzed Cycloaddition :3  was weighed into a 10-mL Biotage vial. 1,4-Dioxane (2.0 mL), benzyl azide , and phenyl acetylene  were added, and the vial was capped with a Teflon-coated rubber septum and crimped. The solution was stirred for the indicated time at the respective temperature , benzyl azide , phenyl acetylene , and 1,4-dioxane (2.0 mL) were combined in a 5-mL vial. As the reactor reached the reaction conditions, the mixture was introduced into the flow system. After the introduction, the pumps were switched back to the solvent reservoir, and 64 mL of solvent was collected in a 100-mL round-bottomed flask. The experiment was stopped, the solvent was removed under reduced pressure, and a sample of the remaining solid was subjected to NMR analysis to determine the conversion (80 %). The residual reaction mixture was taken up in acetone (2 mL), the product was precipitated by the addition of water (6 mL), and the mixture was filtered through a small frit (D3) to give 1-benzyl-4-phenyl-1,2,3-triazole (9) as a pale yellow solid  with identical physical properties as reported above.Crystal Structure Determination: The crystal structures were determined with a Bruker APEX-II CCD diffractometer by using graphite-monochromatized Mo-Kα radiation (0.71073 Å). Structures were solved by direct methods and refined by using the SHELXL and SHELXS suite of programs.2, 3, 4a, 4b, and 4c are given in 2), and-CCDC-771838 (for 3), -CCDC-771839 (for 4a), -CCDC-771840 (for 4b), and -CCDC-771841 (for 4c) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.Supporting Information (see footnote on the first page of this article): Further experimental details including spectra, crystallographic information, information on catalytic studies, as well as computational details.


Pseudomonas aeruginosa that shares functional similarity with related proteins in infectious Neisseria and Kingella species. Previous data have shown that PilY1 acts as a calcium-dependent pilus biogenesis factor necessary for twitching motility with a specific calcium binding site located at amino acids 850–859 in the 1,163 residue protein. In addition to motility, PilY1 is also thought to play an important role in the adhesion of P. aeruginosa tfp to host epithelial cells. Here, we show that PilY1 contains an integrin binding arginine-glycine-aspartic acid (RGD) motif located at residues 619–621 in the PilY1 from the PAK strain of P. aeruginosa; this motif is conserved in the PilY1s from the other P. aeruginosa strains of known sequence. We demonstrate that purified PilY1 binds integrin in vitro in an RGD-dependent manner. Furthermore, we identify a second calcium binding site (amino acids 600–608) located ten residues upstream of the RGD. Eliminating calcium binding from this site using a D608A mutation abolished integrin binding; in contrast, a calcium binding mimic (D608K) preserved integrin binding. Finally, we show that the previously established PilY1 calcium binding site at 851–859 also impacts the protein's association with integrin. Taken together, these data indicate that PilY1 binds to integrin in an RGD- and calcium-dependent manner in vitro. As such, P. aeruginosa may employ these interactions to mediate host epithelial cell binding in vivo.PilY1 is a type IV pilus (tfp)-associated protein from the opportunistic pathogen  Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen prevalent in immunocompromised patients, burn victims, and people with cystic fibrosis. P. aeruginosa can infect any part of the body, but typically targets the respiratory tissues, skin abrasions, and the urinary tract P. aeruginosa uses a range of methods to resist the effects of antibiotics, including efflux pumps, adaptive mutagenesis, and protective biofilms Pseudomonas aeruginosa presents a significant challenge to human health.P. aeruginosa employs type IV pili (tfp) for twitching motility and infection. The precise mechanism of host cell attachment has remained unclear, although evidence exists that loops of the major pilus structural subunit PilA exposed at the tip of the pilus fiber bind to gangliosides GM1 and GM2 P. aeruginosa clinical isolates, as well as laboratory strains, do not employ GM1 and GM2 during host cell attachment Pseudomonas tfp are involved in binding to target cells Pseudomonas infection. Anti-integrin antibodies were shown to reduce P. aeruginosa attachment to host cells P. aeruginosa binding to host cells, with antibodies to αVβ5 integrin having the most pronounced effect P. aeruginosa; indeed, αVβ5 integrin is highly expressed in the lungs P. aeruginosa has also been shown to increase the expression of integrin subunits αV, α5, and β1 in epithelial cells P. aeruginosa infection e.g., leucine-aspartic acid-valine or LDV).Recent studies have suggested that host cell integrin proteins play a role in Neisseria gonorrhoeae, Neisseria meningitidis, and Kingella kingae have been characterized as tfp biogenesis factors and as proteins involved in adhering to target tissues P. aeruginosa PilY1 shares sequence homology with the C-terminal regions of the PilCs proteins, and this domain in PilY1 has been shown to be a calcium-mediated tfp biogenesis factor  P. aeruginosa contained an RGD or other putative integrin binding sequence P. aeruginosa strains of known sequence. For example, in the PilY1 from P. aeruginosa strain K (PAK), the RGD is located at residues 619–621; however, in the PAO1 P. aeruginosa strain PilY1, the RGD is at 657–659  and tested the ability of 532–1163 PilY1 to bind to αVβ5 using an integrin binding assay. Titration of increasing concentrations of 532–1163 PilY1 showed that mid-nanomolar concentrations of this purified protein binds specifically to αVβ5 . Next, wintegrin . Importaintegrin . Taken tP. aeruginosa PilY1 between residues 600 and 608, which is in close proximity to the RGD motif (619–621) described above  into the other site (600–608). To test this hypothesis, we created two triple mutant forms of 532–1163 PilY1, D608A/D851A/D859A and D608A/D855A/D859A, and one quadruple mutant form, D608A/D851A/D855A/D859A. Both triple mutants and the quadruple mutant show wild type levels of binding  to the RGD sequence, would impact integrin binding. In fact, D859A can be seen as a dominant positive with respect to integrin binding . For exaP. aeruginosa itself up-regulates the expression levels of the αV integrin subunit Bordetella pertussis protein pertactin, containing two RGD motifs, has been shown to adhere to Chinese hamster ovary cells in an RGD-dependent manner in vitro and in vivo studies have shown that the following bacteria bind integrin in an RGD-dependent manner: Pyrenophora tritici-repentis (a wheat pathogen), Mycoplasma conjunctivae (sheep pathogen), and the mammalian pathogens Klebsiella pneumoniae and Helicobacter pyloriP. aeruginosa.Integrins and integrin-like proteins are present in plants, insects, and the animal kingdom. They are up-regulated during cellular stress and recovery Interestingly, calcium has also been shown to play a vital role in host RGD substrate:integrin binding events . CalciumP. aeruginosa adhesion to host cells by functional studies Neisseriain vitro advance in our understanding of PilY1 structure and function. It will be of interest to determine in future studies whether PilY1 and integrins are involved in P. aeruginosa attachment to host cells in vivo.PilY1 is an established pilus biogenesis factor 600 reached 0.6–0.8. Temperature was reduced to 18°C and protein expression was induced with 0.5 mM IPTG. Cells were grown overnight and harvested by centrifugation 6000×g at for 15 minutes at 4°C, and pellets were stored at −80°C.Site-directed mutagenesis was performed to produce D608A, D608K, D621A, Δ619–621, D859A, D859K, D608A/D859A, D608A/D859K, D608K/D859A, D608K/D859K, D608A/D851A/D859A, D608A/D855A/D859A, and D608A/D851A/D855A/D859A mutants in a pDONR vector containing PilY1. Amino acids 532–1163 were cloned out of the pDONR vector for entry into pMCSG7 for protein expression. Vectors were transformed into BL21 Gold (Stratagene) on ampicillin plates overnight and a single colony was used to inoculate a 100 mL LB flask overnight containing 50 µM/mL ampicillin. Cell cultures were centrifuged at 3000×g and the supernatant was discarded. The resultant pellet was used to inoculate a 1.5 L shaker flask of Terrific Broth with 50 µL of antifoam (Sigma-Aldrich) and 50 µM/mL ampicillin. Cells were grown at 37°C until ODCells pellets were thawed using buffer consisting of 25 mM HEPES pH 7.5, 150 mM NaCl, 10 mM imidazole, 5% glycerol, DNase and protease inhibitor tablets (Roche). Cells were sonicated and cell lysate was separated into soluble and insoluble fractions using high-speed centrifugation. The soluble fraction was filtered then nickel purified, buffer exchanged, and separated using an S200 gel filtration column on an ÄKTAxpress™ . If necessary, protein and storage buffers were chelated by Chelex-100 to remove bound calcium (Bio-Rad Laboratories). Purified proteins were concentrated to ∼100 µM, frozen, and stored at −80°C.2, 1 mM MgCl2, 1 mM MnCl2, and 0.1% Triton) was added to each well including the BSA control wells for 6 hours at 16°C with or without RGDS peptide (inhibitor), GRADSP peptide (control), or no peptide. Metals are needed in the binding buffer for integrin structural integrity. Plates were then washed 3× with PBST (0.1% Tween-20) and 3× with PBS before adding rabbit αPilY1 polyclonal antibody 1∶500 per well in 1% BSA in PBS overnight for 16 hours at 4°C. Plates were then washed 3 times with PBST and 3× with PBS before adding the Alexa fluor 488 f(ab′)2 fragment goat anti-rabbit (Invitrogen) 1∶5000 in 1% BSA in PBS protected from light at room temperature for 1 hour. Plates were then washed 3× with PBST, 3× with PBS, then dried. Plates were read on a PHERAstar (BMG LabTech). The experimental and control well averages and standard error of the mean (SEM) were calculated, and then the control average was subtracted from the experimental values and the errors were compounded using the equation √2+(control SEM))2.Costar EIA/RIA stripwell high binding plates were coated with either 2 µg of purified αVβ5 integrin or 2 µg of purified αVβ3 integrin (Millipore) for the experimental wells, or the molar equivalent of BSA for the control wells, at 100 µL in 100 mM sodium bicarbonate buffer pH 9.6 (Abcam) for 16 hours at 4°C. Plates were washed 1× with 300 µL of PBS. Wells were then blocked with 1% BSA in PBS for one hour at room temperature. Plates were washed 1× with PBS and 100 µg/mL of PilY1  in binding buffer  in 25 mM HEPES pH 7.5, 250 mM NaCl, 10 mM imidazole, 5% glycerol was measured on a PHERAstar (BMGLabtech) at 488 nm. 20 µM Oregon Green, either 2 µM CaClxHxPO4 50 mM NaF pH 7.7 buffer and brought to 5 µM with or without the addition of CaCl2. A wavelength scan from 200–260 λ was performed on a Circular Dichroism Spectrometer 62 DS (Aviv) at 16°C with a 10 second averaging time. Melting temperatures were measures at λ 214 from 3°C to 95°C at one degree increments with a 10 second averaging time.Protein samples were buffer exchanged into chelated 10 mM KFigure S1Circular dichroism for calcium binding mutants. A–E Molar ellipticity values were calculated for the respective wavelength scans and compared.(TIFF)Click here for additional data file.Figure S2PilY1 mutation binding curves. (a) ΔRGD and R619A were modeled to one-site binding. (b) Double calcium binding site mutants D608A/D859K, D608K/D859A, and D608K/D859K. Error represents standard error of the mean.(TIFF)Click here for additional data file.Figure S3PilY1 alternate metal binding curves. (a) Oregon Green binding curves were made by titrating magnesium chloride, manganese chloride, or zinc chloride. Curves were fit to one-site saturation (magnesium chloride and manganese chloride) to determine Kd.  Zinc and mangansese curves were fit to one-site binding. (d) Magnesium was titrated against Oregon Green, calcium, and PilY1 at the Kd to determine affinity.(TIFF)Click here for additional data file.Figure S4Mutational effects on PilY1:Integrin binding. As in (TIFF)Click here for additional data file.Table S1P. aeruginosa PilY1 is homologous to PilC family of bacterial adhesin and pilus biogenesis proteins. PilY1 was individually aligned to K. Kingae (Kk) PilC1 and PilC2, N. gonorrhea (Ng) PilC1 and PilC2, and N. meningitidis (Nm) PilC1 and PilC2 using http://blast.ncbi.nlm.nih.gov/. C-terminal homology indicates residues that are similar (e.g. leucine and isoleucine) while c-terminal identity corresponds to identical residues. Strains used for each protein are as follows: ACF19883 for Kingella kingae PilC1, ACF19886.1 for Kingella kingae PilC2, O05924_NEIME for Neisseria meningitidis PilC1 O05925_NEIME for Neisseria meningitidis PilC2, O05923_NEIGO for Neisseria gonorrhea PilC1, and Q51019_NEIGO for Neisseria gonorrhea PilC2.(TIFF)Click here for additional data file.Table S2M) for mutations of PilY1.Melting temperatures Click here for additional data file.


To validate the role of high mobility group box-1(HMGB1) in diagnosis of acute appendicitis (AA) with different pathological severity.According to the pathologically diagnosis, 150 patients underwent appendectomies between Jan. 2007 and Dec, 2010 were divided into acute simple, acute suppurative and acute gangrenous appendicitis as group 1, 2 and 3, respectively. Each patient group contains 50 sex and age matched cases to make comparison with 50 healthy volunteers. The mRNA and protein expression levels of serum HMGB1 were determined by real-time quantitative PCR and enzyme linked immunosorbent assay (ELISA). Serum High-sensitivity C-reactive protein (hs-CRP) levels were determined by rate nephelometric immunoassay.In comparison with health volunteers, relative HMGB1 mRNA levels in group 1, 2 and 3 were significantly increased 3.05 ± 0.51,8.33 ± 0.75 and 13.74 ± 1.09 folds, reflecting a tendency of augmented severity. In accordance, serum protein levels of HMGB1 were 10.97 ± 1.64, 14.42 ± 1.56 and 18.08 ± 2.41 ng/ml in 3 patient groups, which are significantly higher than that of healthy volunteers’ 5.47 ± 0.73 ng/ml. hs-CRP levels were 12.85 ± 3.41, 21.04 ± 1.98 and 31.07 ± 5.46 ng/ml in 3 patients groups compared with 2.06 ± 0.77 ng/ml in controls. The concentrations of HMGB1 and hs-CRP were both positively correlated with disease severity.Serum HMGB1 constitutes as a valuable marker in diagnosis of AA. Positively correlated with hs-CRP level, mRNA and protein expression of HMGB1 to a certain extent reflected the severity of AA. Acute appendicitis is the most common acute abdominal disease in general surgeries of patients with typical clinical symptoms. The stages of AA can be divided into simple, suppurative, gangrenous, perforated, recurrent, and chronic etc. according to the severity of the diseases. Although AA is not a life-threatening disease, mortality is still reported as high as 1%. Diagnosis of AA is often a clinical challenge because it can mimic several abdominal pain conditions. So far no single symptom or diagnostic test can accurately confirm the diagnosis of AA in all cases. The emergencies are sometimes misdiagnosed or missed. Although the surgery appendectomy itself is a relatively safe surgical procedure, rates of negative appendectomy and perforated appendicitis remain fairly high, around 10–30 percent, with a higher rate of ~40% in high risk populations such as women of reproductive age. For a long time, the preoperative diagnosis of AA is largely relied on the clinical assessment, previous experience, total white blood cell (WBC) counts, neutrophil percentage or a combination of several examinations. These traditional methods are rather nonspecific. Recently ultrasonography and computed tomography were applied in diagnosis of AA. However, it is still controversial if these imaging aided applications have actually resulted in significantly reduced cases of negative appendectomy and perforated appendicitis . TherefoHMGB1 is recently identified inflammatory factor which plays a crucial role in the late stage of severe sepsis. It is highly expressed in severe sepsis, acute pancreatitis, hemorrhagic shock, severe trauma and acute lung injury. And its expression is closely correlated with the severity of these symptoms -5. RecenIn this study, we have evaluated the mRNA and protein expression level of HMGB1 in AA patients. In addition, HMGB1 was assessed to be a diagnostic marker for AA patients with differential pathological severity of the disease, aiming to provide a valuable marker to improve the preoperative diagnosis of AA for clinical use.According to the pathologically diagnosis, 150 patients underwent appendectomies between Jan. 2007 and Dec 2010 were divided into acute simple, acute suppurative and acute gangrenous appendicitis as group 1, 2 and 3, respectively. All experimental protocols were approved by the local research ethics committee of the Second Affiliated Hospital of Chongqing Medical University and complied with the China government guidelines. There are randomized chosen 25 males and 25 females in each AA group. The average age for acute simple, acute suppurative and acute gangrenous appendicitis groups are 37.63 ± 14.68, 34.72 ± 15.45 and 36.63 ± 15.98 respectively. 50 healthy volunteers  without any abdominal syndromes were used as a control group. Blood samples of the patients were withdrawn when admitted to the hospital. White blood cells were counted and the blood samples were stored for future use. No significant differences were observed in age, family history and other medical conditions between groups.Serum HMGB1 concentrations were measured by ELISA with human HMGB1 detection ELISA kit (SUNBIO (Cat# HE017), Beijing, China) according to the manufacturer’s instruction.-▽▽CT > 2, were considered to be statistically significant.Peripheral blood total RNA was isolated with total RNA isolation kit purchased from Bioteke, Beijing, China, according to manufacturers’ instruction. RNA was reverse transcribed using PrimeScript® RT reagent Kit  from TaKaRa biotech, Dalian, China. SYBR® Premix Ex Taq™  from TaKaRa was used for quantitative real-time PCR. The primers for human HMGB1 were designed with Primier4.1 software, GADPH was used as a reference gene. Both primers were synthesized by TaKara. Primers used for human HMGB1 are, forward: 5’-GCG GAC AAG GCC CGT TA-3’; reverse: 5’-AGA GGA AGA AGG CCG AAG GAA-3’; Amplicon size 119 bp. Human GADPH primers are Forward: 5’- TGC CAA ATA TGA TGA CAT CAA GAA-3’; Reverse: 5’- GGA GTG GGT GTC GCT GTT G-3’; Amplicon size 121 bp. Relative gene expression was calculated to that of GAPDH on the basis of ΔCt value. Data with 2Serum Hs-CRP concentrations were determined locally in clinical laboratory of the hospital with rate nephelometric immunoassay.Values were expressed as the standard deviations of the mean. Data were analyzed with SPSS 13.0 software. Group means did not exhibited as normal distribution and were examined using Kruskal-Wallis test. The P values of less than 0.05 were considered to be statistically significant.P <0.01). The HMGB1 concentration in acute simple appendicitis group is almost doubled compared with healthy controls. And acute gangrenous appendicitis is about 4 folds higher in HMGB1 secretion. Importantly, HMGB1 concentration in group 2 is significantly higher than that of group 1, and group 3 is much higher than group 2 , acute suppurative (group 2) and acute gangrenous appendicitis (group 3) are 10.97 ± 1.64 ng/ml, 14.42 ± 1.56 ng/ml and 18.08 ± 2.41 ng/ml,which is significantly higher than that of health volunteer group at 5.47 ± 0.73 ng/ml , and group 3 is significantly higher than group 2 (P < 0.01), indicating that the increased HMGB1 level is positively correlated with the severity of the AA conditions.In comparison with that of health volunteers, the HMGB1 mRNA expression level of patients in acute simple (group 1), acute suppurative group 2) and acute gangrenous appendicitis (group 3) are 3.05 ± 0.51,8.33 ± 0.75 and 13.74 ± 1.09 . Hs-CRP concentration in group 2 is significantly higher than that of group 1 and group 3 is higher than group 2 , acute suppurative (group 2) and acute gangrenous appendicitis group 3) are 12.85 ± 3.41 ng/ml, 21.04 ± 1.98 and 31.07 ± 5.46 ng/ml, which are all significantly higher than that of healthy volunteer group at 2.06 ± 0.77 ng/ml at 8.73 ± 3.49 × 109 and healthy volunteer group at 7.45 ± 2.98 × 109(P <0.01). Importantly, we did not observe a significant increase in simple appendicitis patients group and healthy volunteer group (P >0.05), demonstrating that WBC is not a bona fide diagnostic marker for AA, especially at the early stage of AA progression. We also did not observe a significant difference in WBC counts between group 1 and 2, indicating that WBC counts are not very sensitive to reflect the severity of AA  and acute gangrenous appendicitis (group 3) are 12.90 ± 3.69 × 10Statistical analysis demonstrated that in AA patients, serum HMGB1 and Hs-CRP are positively correlated. Both factors are increased with the development of AA and in accordance with the severity of the disease  declare that they have no competing interests.WC and SH designed and performed the study, acquired and analyzed the data, and drafted the manuscript. WH and CJ selected the patients and collected specimens. GH carried out the immunoassays. GJ and LQ designed the study and revised the manuscript critically. All authors read and approved the final manuscript.This study was financially supported by grants from the National Natural Science Foundation of China  and the Traditional Chinese Medicine Science and Technology Research Project of Chongqing Health Bureau (2009-2-148).