Desulfovibrio vulgaris Hildenborough, a model organism for studies of sulfate-reducing bacteria, no genes for porins have been identified or proposed in its annotated genome. Results from initial biochemical studies suggested that the product of the DVU0799 gene, which is one of the most abundant proteins of the D. vulgaris Hildenborough OM and purified as a homotrimeric complex, was a strong porin candidate. To investigate this possibility, this protein was further characterized biochemically and biophysically. Structural analyses via electron microscopy of negatively stained protein identified trimeric particles with stain-filled depressions and structural modeling suggested a β-barrel structure for the monomer, motifs common among the known porins. Functional studies were performed in which crude OM preparations or purified DVU0799 was reconstituted into proteoliposomes and the proteoliposomes were examined for permeability against a series of test solutes. The results obtained establish DVU0799 to be a pore-forming protein with permeability properties similar to those observed for classical bacterial porins, such as those of Escherichia coli. Taken together, these findings identify this highly abundant OM protein to be the major porin of D. vulgaris Hildenborough. Classification of DVU0799 in this model organism expands the database of functionally characterized porins and may also extend the range over which sequence analysis strategies can be used to identify porins in other bacterial genomes.Due in large part to their ability to facilitate the diffusion of a diverse range of solutes across the outer membrane (OM) of Gram-negative bacteria, the porins represent one of the most prominent and important bacterial membrane protein superfamilies. Notably, for the Gram-negative bacterium IMPORTANCE Porins are membrane proteins that form transmembrane pores for the passive transport of small molecules across the outer membranes of Gram-negative bacteria. The present study identified and characterized the major porin of the model sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, observing its preference for anionic sugars over neutral ones. Its predicted architecture appears to be novel for a classical porin, as its core β-barrel structure is of a type typically found in solute-specific channels. Broader use of the methods employed here, such as assays for channel permeability and electron microscopy of purified samples, is expected to help expand the database of confirmed porin sequences and improve the range over which sequence analysis-based strategies can be used to identify porins in other Gram-negative bacteria. Functional characterization of these critical gatekeeping proteins from divergent Desulfovibrio species should offer an improved understanding of the physiological features that determine their habitat range and supporting activities. Desulfovibrio vulgaris Hildenborough has been one of the more extensively studied. This and other species of Gram-negative bacteria of the genus Desulfovibrio are widely distributed in the environment, and D. vulgaris Hildenborough has long served as a model for developing a better understanding of the biotic and abiotic factors that determine the distribution and activity of sulfate reducers , adding a second filter, in addition to the cytoplasmic membrane, for managing the passage of solutes into and out of the cell. Solute movement across the OM is facilitated by transmembrane channels, among which the porins form one of the most important families of bacterial membrane proteins  dodecyl ether (C12E9) or sodium lauroyl sarcosinate (Sarkosyl), to solubilize and remove the proteins of the IM. Following ultracentrifugation, a portion of the remaining OM-enriched pellet (∼0.1 g) was set aside for liposome incorporation, with the remainder being solubilized for protein purification using the detergent octyl glucoside (OG). Ion-exchange chromatography of the solubilized OM components yielded fractions enriched with DVU0799 eluting at the 350 mM NaCl step  for initiating protein transport across the inner membrane (IM) , while trough OM , 16 and aCl step . Sodium aCl step .To examine the native state characteristics of this protein, we subjected samples of the ion-exchange elution fractions containing DVU0799 to blue native PAGE (BN-PAGE). Operating under conditions found to support the preservation of membrane protein oligomeric states (in a cold room overnight at 70 V ), the BNhttps://zhanglab.ccmb.med.umich.edu/I-TASSER) (–http://www.cgl.ucsf.edu/chimera)  –19. The TASSER) –, a scoreTASSER) –. These mchimera) .Comparison of these predicted structures found them to be similar overall and to feature an 18-strand β-barrel core structure, varying predominately in the shape and length of the random coil and connecting loop regions at the extramembrane faces and within the cylinder. 21–www.uniprot.org [www.microbesonline.org [G2HC51, G2HB34, G2HC52, B2KPH3, and B7SKM7) are annotated as porins. Of these, only the assignments of the sequences with accession numbers B2KPH3 (Bilophila wadsworthia) and B7SKM7 (Desulfovibrio piger), each sharing about 43% sequence identity with DVU0799, are based on direct experimental studies  describline.org ) it is d studies .E. coli OmpF as a typical example of a classical porin (PDB accession number 1OPF) is shown in top view (Samples of ion-exchange chromatography fractions enriched with DVU0799 were negatively stained (2% uranyl acetate) and examined by transmission electron microscopy. Large patches or pseudoarrays of proteins associating to form honeycomb-like lattices were found throughout the sample preparations . Upon cltop view  21). So. SoE. cotop view . Collecttop view , 28.D. vulgaris Hildenborough OM proteins were reconstituted, using either crude OM preparations or purified protein. In this approach, the degree of membrane channel permeability is reflected in the rate of proteoliposome swelling occurring in response to the influx of water coupled with the movement of test solutes across the proteoliposome membranes. Swelling rates were experimentally determined by monitoring the decrease in the optical density (OD), measured at a source wavelength of 400 nm (OD400), of the proteoliposome and test solute mixtures over time. For plotting and comparisons of relative permeability, swelling rates were scaled by adjusting the value obtained for the relatively small uncharged sugar l-arabinose, at the upper limit of measureable permeability, to a value of 100 and scaling the values for the other solutes accordingly.To assess whether DVU0799 exhibits membrane channel properties characteristic of bacterial porins, we utilized the liposome swelling assay  to examiD. vulgaris Hildenborough OM preparations was conducted. Here, whole-cell membranes were subjected to only a relatively mild detergent step for the removal of IM components. In this way, OM proteins could remain within a lipid bilayer environment throughout the process, improving the likelihood that these proteins would retain their structure and function. This approach allowed us to assess the general suitability of the liposome swelling method for D. vulgaris Hildenborough OM protein characterization and permitted comparisons of the permeability of D. vulgaris Hildenborough crude OM to that of E. coli crude OM-based proteoliposomes prepared in a similar manner  were determined.As the native complex of DVU0799 was found to be more detergent sensitive than previously characterized porins, a set of preliminary experiments in which permeability could be evaluated using proteoliposomes prepared with crude r manner . The relD. vulgaris Hildenborough OM, at least with respect to this solute test set, is comparable to that of the E. coli OM. Of the candidate porin gene products in the D. vulgaris Hildenborough OM , DVU0799 is by far the most abundant  as the solubilizing detergent  and lactobionate (357 Da), whose molecular masses fall within the molecular mass range covered by the uncharged sugars examined  for removal of IM components, the resulting crude OM preparations (where OM proteins are maintained within lipid bilayers) were reconstituted into proteoliposomes and evaluated. The results obtained showed that the transmembrane channels of the OM remained functional. The permeability of the crude D. vulgaris Hildenborough OM was found to be similar to that of crude E. coli OM over the range of solutes tested. Next, DVU0799 was extracted from its native lipid bilayer environment in the crude OM using a relatively mild nonionic detergent, OG, and purified. The trimeric structure of DVU0799 was maintained using OG, as evidenced by blue native PAGE and electron microscopy results, and the complex was functionally preserved so that when it was reconstituted, the protein displayed the permeability characteristics expected for a porin-type transmembrane channel. Notably, sugar acids were found to permeate the channel more rapidly than uncharged sugars of similar size. In the case of D. vulgaris Hildenborough, having a high nutrient demand for sulfate, this preference for anions may be beneficial for growth under conditions where sulfate availability is low. Given the aforementioned suggestion that the DVU0799 gene is essential, DVU0799 may play a critical role in the uptake not only of sulfate but also of other solutes. The structural and functional results presented here establish DVU0799 as a pore-forming protein with size, shape, and permeability properties similar to those observed for classical bacterial porins. Given the relatively high abundance of this protein among all D. vulgaris Hildenborough OM proteins, these findings identify this protein as the major porin of this sulfate-reducing bacterium.In the present study, we investigated DVU0799 biochemically and biophysically, examining purified samples via blue native PAGE and 2D SDS-PAGE, imaging samples by the use of negative-stain electron microscopy and through sequence analysis and structural modeling. Structural modeling yielded predictions of an 18-strand β-barrel as the core motif, while electron microscopy of preparations enriched with DVU0799 homotrimeric complexes revealed trimers of stain-filled depressions approximately 8 nm in diameter, classic hallmarks of bacterial porins. We also examined DVU0799 for transmembrane channel function by reconstituting purified protein into proteoliposomes and monitoring swelling rates following their introduction into solutions containing test solutes. It was initially unclear whether this method would be effective in evaluating the permeability of detergent-solubilized DVU0799, which, in contrast to porins previously studied, was not similarly stable in SDS. To examine whether Desulfovibrionaceae , D. piger and B. wadsworthia were found to be impermeable to even disaccharides , indicating a substantially smaller permeability limit and suggesting important differences in pore-defining structures. It should be noted that although all of these porins are from members of the family Desulfovibrionaceae, these proteins also come from bacteria cultivated from two widely different environments: D. vulgaris Hildenborough was isolated from soil  porin from the epsilonproteobacterium Campylobacter jejuni  establish a net negative charge on one side of the eyelet, while across the pore from this region, the inner β-barrel wall is lined with a predominance of positively charged residues. Through this separation of charge, a transverse electric field helping to shape channel selectivity is established across the eyelet. What sets MOMP apart, however, is an arrangement of negatively charged side chains and carbonyl groups on the loop segments of the constriction region that is able to bind a Ca2+ ion with a high affinity. With Ca2+ bound, channel permeability changes from what would be moderately cation selective to anion selective. Given the aforementioned similarities between MOMP and DVU0799, it is interesting to consider the possibility that some form of constriction region cation binding site also plays a role in establishing the preference for anionic sugars observed for DVU0799.Taking into consideration the potential for overinterpretation of a predicted model structure, even one with a relatively good confidence score, it remains worthwhile to note a further similarity with the Desulfovibrionaceae family will continue to expand the database of confirmed porin sequences and signatures and increase the range over which sequence analysis-based strategies can be used to identify porins in other Gram-negative bacteria. On a more specific level, the functional characterization of these critical gatekeeping proteins from divergent Desulfovibrio species should offer an improved understanding of the physiological features that determine their habitat range and supporting activities.High-resolution structural data remain an important tool for interpreting potential gene product function, especially when the homology of the amino acid sequence to the sequences of functionally related structures is low. However, the collection of such data continues to be challenging. By compiling sets of often more readily obtainable functional and lower-resolution structural data , gene descriptions can still be developed and assigned with confidence. It is hoped that broader use of the approach employed here and in the work of Avidan et al.  for the D. vulgaris Hildenborough (ATCC 29579) batch cultures were initiated from 15% glycerol freezer stocks inoculated into Balch tubes (18- by 150-mm glass tubes with a narrow opening to hold a 1-in.-thick rubber septum) containing coculture medium A  , and upon reaching the end of exponential growth, 3 ml of culture was removed and used to inoculate 3 liters of medium in two stoppered 2-liter bottles (each containing 1.5 liters of medium). These batch cultures were grown to stationary phase at 37°C and anoxically transferred into 500-ml sterile centrifuge bottles. The biomass was pelleted by centrifugation at 9,000 × g for 30 min at 4°C. Following centrifugation, the supernatant was removed and the cell pellet was resuspended in 50 ml of a basal salt solution. The resuspended cells were anoxically transferred to a 50-ml conical tube and subjected to centrifugation at 18,500 × g for 10 min at 4°C. Cell pellets were frozen in liquid nitrogen and stored at −80°C.Working within an anaerobic chamber (Coy Lab Products),  pH 7.2)  with 4.3E. coli K-12 JM101 (New England BioLabs), colonies grown on LB agar plates were used to inoculate LB medium (50 ml in 250-ml flasks), following which the flasks were placed in a shaker for growth overnight at 30°C. On the following day, flasks containing 1.5 liters LB medium were inoculated with 15 ml of the overnight culture and again placed in a shaker at 30°C. Upon reaching an OD600 of ∼1.3, cells were harvested from the culture medium by low-speed centrifugation . Cell pellets were frozen in liquid nitrogen and stored at −80°C.For the culturing of D. vulgaris Hildenborough cells were washed in cell wash buffer  prior to lysis. Cell pellets were resuspended in cell wash buffer  and gently stirred at 4°C until a uniform suspension was obtained. A broad-spectrum protease inhibitor  was added to the wash and lysis buffers. To obtain a washed cell pellet, the cell suspension was centrifuged at 10,000 × g for 10 min at 4°C. Washed cell pellets were resuspended in lysis buffer  and processed through a gas-driven cell disruptor  three times to break open the cells. To enhance the preservation of membrane protein complexes, the cell disruptor was chilled with ice. The broken cell suspension was centrifuged at low speed  to remove unbroken cells; the supernatant from this step was centrifuged at high speed  to isolate membranes. To reduce the presence of highly abundant soluble proteins, the membrane pellet was resuspended and washed several times in membrane wash buffer . After each wash, the membranes were pelleted by high-speed centrifugation . The membranes were either used immediately or quick-frozen and stored at −80°C. Cell membranes were prepared from E. coli K-12 JM101 cell pellets in a similar manner.To reduce the level of iron sulfide present as a consequence of growth in modified coculture medium A, D. vulgaris Hildenborough cell membranes in a two-step process, generally based on the protocol of Baldermann et al.  containing 0.1% polyoxyethylene (9) dodecyl ether (C12E9) or 0.5% sodium lauroyl sarcosinate (Sarkosyl), and homogenized on ice for 1 h. In general, detergents and buffers were adjusted to obtain a final detergent-to-protein ratio of about 1:1 while maintaining a protein concentration of at least 10 mg/ml. After solubilization, the detergent concentration was lowered by dilution with solubilization buffer, and the sample was centrifuged  to pellet unsolubilized membranes enriched in OM proteins. For our studies, proteoliposomes were prepared using either these enriched membranes directly or protein purified from these membranes. E. coli K-12 JM101 OM protein-enriched membranes were prepared in a similar fashion with the use of 0.5% Sarkosyl. To extract proteins of the D. vulgaris Hildenborough OM, the resulting D. vulgaris Hildenborough membrane pellets were processed in solubilization buffer (about 10 ml) containing a more active detergent, octyl glucoside , and homogenized on ice for 1 h. After solubilization, the detergent concentration was lowered by dilution with solubilization buffer and the sample was centrifuged . Proteins in the supernatant were then subjected to ion-exchange chromatography.To obtain cleaner preparations of outer membrane (OM) protein prior to chromatography, proteins were extracted from n et al. . In this3) containing 1% OG. Detergent-solubilized samples were loaded onto the column, and the column was washed to remove nonspecifically bound material using buffer A. Bound proteins were eluted in 8- to 12-ml steps at 50 mM NaCl increments over a range of from 50 to 400 mM NaCl, followed by a final step of elution with 1 M NaCl. The elution buffer was prepared by mixing buffer A with increasing levels of buffer B (buffer A with 1 M NaCl added).For purification of OM proteins from 0.3 to 0.5 g of treated membranes, a 2-ml bed of anion-exchange medium  was equilibrated in buffer A  of the ion-exchange chromatography fractions was performed on the basis of the protocol of Schägger et al.  with modSpecimens (4 μl) were placed on glow-discharged carbon-coated electron microscopy grids . After 5 min, the sample on the grid was blotted to less than 1 μl and the grid was placed on an ultrapure water droplet, sample side down, to reduce the level of salts on the grid. The water droplets used were 50 to 100 μl in volume and placed on a sheet of Parafilm. The grid was subsequently transferred to two more water droplets following wait periods of 2 min each. After the last water wash step, the grid was removed from the droplet and inverted. The water drop remaining on the grid was blotted to below 1 μl, after which 2 μl of 2% uranyl acetate was added. After 1 min the grid was blotted to dryness. Sample grids were examined in a JEOL JEM-1200x electron microscope operating at 100 kV. Images were recorded using a 2,000- by 2,000-pixel charge-coupled-device camera  controlled by the DigitalMicrograph software package.https://zhanglab.ccmb.med.umich.edu/I-TASSER) (–http://www.cgl.ucsf.edu/chimera) (A FASTA format amino acid sequence of DVU0799 was submitted to the I-TASSER server (-TASSER) –19 for mchimera) . Waters chimera) , 25 and chimera)  via the D. vulgaris Hildenborough OM , crude E. coli K-12 JM101 OM , or purified DVU0799 (0.5 to 1 μg protein). Control liposomes were prepared without the addition of protein. Portions (17 μl) of a liposome suspension were added to a cuvette containing 0.6 ml of a solution of about 20 mM uncharged sugar test solute in 10 mM Tris-HCl buffer, pH 8.0. The optical density at 400 nm was continuously recorded with a spectrophotometer . Test solute concentrations were adjusted as needed to ensure that the external solution was isotonic with the control liposomes. For experiments evaluating porin permeability for anionic as well as uncharged solutes, liposomes were made by combining 6.2 μmol of egg phosphatidylcholine and 0.2 μmol of dicetyl phosphate in 0.4 ml of a solution containing 12 mM stachyose (Sigma), 4 mM sodium NAD (Sigma), and 1 mM imidazole-NAD (Sigma), pH 6.0. For the preparation of proteoliposomes in this series of experiments, 10 to 25 μg of purified DVU0799 was included. For each measurement, 17 μl of the proteoliposome suspension was added to a cuvette containing either 0.6 ml of an approximately 20 mM solution of an uncharged sugar or a 9 mM solution of a monovalent anionic sugar in 4 mM sodium NAD (Sigma) and 1 mM imidazole-NAD (Sigma), pH 6.0; the optical density was recorded as described above. The uncharged sugars evaluated were l-arabinose (Sigma), d-glucose (Sigma), N-acetyl-d-glucosamine (Sigma), sucrose (Sigma), and d-raffinose (Sigma). The anionic sugars tested were sodium salts of d-gluconate (Sigma) and lactobionate (Glentham Life Sciences).The procedures used for the liposome swelling assay were based on earlier work by Nikaido and Rosenberg . In this


A combined experimental and computational study, using mechanically controlled break junction measurements and density functional theory calculations, demonstrates consistently higher conductance in the para series compared to the meta series: this is in agreement with increased conjugation of the π–system in the para series. Within the para series conductance increases in the order of decreasing heteroaromaticity (dibenzothiophene < carbazole < dibenzofuran). However, the sequence is very different in the meta series, where dibenzothiophene ≈ dibenzofuran < carbazole. Excellent agreement between theoretical and experimental conductance values is obtained. Our study establishes that both quantum interference and heteroaromaticity in the molecular core units play important and inter-related roles in determining the conductance of single molecular junctions.Is there a correlation between the (hetero)aromaticity of the core of a molecule and its conductance in a single molecular junction? To address this question, which is of fundamental interest in molecular electronics, oligo(arylene-ethynylene) (OAE) molecular wires have been synthesized with core units comprising dibenzothiophene, carbazole, dibenzofuran and fluorene. The biphenyl core has been studied for comparison. Two isomeric series have been obtained with 4-ethynylpyridine units linked to the core either at  Many studies have addressed correlations between molecular structure and transport properties of molecules wired into metal–molecule–metal nanoscale junctions8. Several experimental approaches are well established for measuring transport through single (or a few) molecules, notably the mechanically controlled break junction (MCBJ)9 and scanning tunnelling microscopy-break junction (STM-BJ) techniques10. Combined experimental and theoretical studies11 have established that charge transport through molecular junctions is controlled by the intrinsic properties of the molecular backbone, the terminal anchoring group, and the metal leads. Key features are the molecular length, the molecular conformation, the gap between the highest occupied and the lowest unoccupied molecular orbitals (the HOMO-LUMO gap), the alignment of this gap to the Fermi level of the metal electrodes, and the coordination geometry at the metal-molecule contacts. Oligo(arylene-ethynylene) (OAE)-type molecular wires have been widely explored in single molecular junctions16. They are π–conjugated, rod-like molecules and their functional properties can be systematically tuned over a wide range of parameters by chemical synthesis17.The measurement and understanding of charge transport in single molecules is of fundamental interest and is relevant to the proposed future applications of molecules in electronic devices1–10 whose structures are shown in Fig. para-para or meta-meta conjugation through the core unit, providing two isomeric series. The dibenzothiophene , N-ethylcarbazole , dibenzofuran  and 9,9-dimethylfluorene cores  are rigid and planar. Heteroaromaticity, i.e., the resonance energy, will decrease in the sequence dibenzothiophene > carbazole > dibenzofuran, reflecting the extent of delocalization of a lone pair from the heteroatom into the π–system of the central ring (S > N > O)18. Fluorene, with no heteroatom and a bridging sp3 carbon atom instead, has a non-aromatic central ring. In contrast to the other molecules in Fig. 5 and 10 possess a flexible and twisted core. It is well known that increasing the torsion angle within a biphenyl unit leads to reduced single-molecule conductance23, therefore, 5 and 10 are studied here as model compounds.In the present work we investigate a series of ten OAE molecules 11–13, Fig. 13 has the highest conductance, while the most aromatic thiophene derivative 11 has the lowest conductance24. This work did not consider the linkage of the anchor units to different positions on the core.We are aware of only two related reports on the effect of heteroaromaticity on single-molecule conductance. Venkataraman, Breslow and co-workers studied three amine-terminated molecules comprising thiophene, furan and dimethylcyclopentadiene cores  connectivity through a core unit results in enhanced conductance compared to the isomeric meta (reduced conjugation) connectivity. This is ascribed to quantum interference and has been observed experimentally and theoretically in aromatic rings such as benzene29, naphthalene14, anthracene14, pyrene30 and anthanthrene31.A second study concerns multiple pathways through a molecular wire based on fluorene-like moleculespara versus meta conjugation through the core unit.The motivation for the present work is to study for the first time the combined effects of two important molecular parameters on the single-molecule conductance of molecular wires: (i) heteroaromaticity in the core of the wire, and (ii) para-linked 1–5 and meta-linked 6–10 molecules (Scheme 1) a tandem two-pot reaction sequence was followed. To enable the Sonogashira cross coupling of the corresponding aryldibromide central units (structures 14–2339 in SI) at elevated temperatures, desilylation of 4-((trimethylsilyl)ethynyl)pyridine40 with TBAF (1 M in THF) was performed in 1,4-dioxane (room temperature for 30 min). Since the light-sensitive 4-ethynylpyridine is not stable in air, the mixture was directly subjected to the aryldibromide in the presence of PdCl2(PhCN)2, CuI, t-Bu3P, and (i-Pr)2NH, to give the target structures 1–10 in very good yields  were calculated from the onset of the absorption and are listed in Supplementary Table 1–5, with the anchor groups attached in the para positions, show a smaller HOMO–LUMO gap compared to the meta isomers 6–10. This is consistent with reduced conjugation in the meta series.For the synthesis of the 1–10 in molecular junctions were performed using a home-built mechanically controllable break junction (MCBJ) setup at a bias Vbias = 0.1 V. Figure G (in units of quantum point conductance G0 = 2e2/h) versus distance (Δz) stretching traces in the measurement of Np. The conductance in the molecule-free traces (black line) reveals exponential decrease characteristics upon the stretching process. When molecule Np is present a pronounced conductance plateau around 10−5 G0 could be detected (green line) after the Au–Au contact breaks, which is assigned to the gold–molecule–gold junction. Since the break junction method can create a large number of molecular junctions with different molecule–electrode contact geometries, more than 1000 curves were recorded for statistical analysis to determine the most probable conductance of the molecular junctions. We further introduced a relative distance (Δz) and defined Δz = 0 at 0.5 G0 to align all the traces. This procedure leads to an accurate alignment of the conductance–distance traces because of the sharp drop in conductance at G < G0. The electrode separation zexp is then estimated by zexp = Δz + Δzcorr, where Δzcorr = 0.5 ± 0.1 nm corresponds to the “snap-back” nanogap which forms immediately upon breaking of the gold-gold atomic contact41. The all–data two–dimensional (2D) histogram  and without any bridging atom (5-2Hp and 10-2Hm) were also conducted. The conductance of 2Hm could not be measured within the detection range of our setup. This can be explained by the meta coupling combined with a non-planar biphenyl core giving a conductance value below the direct tunneling conductance29.Single-molecule conductance measurements of ram Fig.  exhibits42. After a linear fitting, the Np junction conductance with a fully stretched molecular conformation before the junction rupture can be deduced as 10−5.74±0.17 G0, which should be closer to the theoretical predicted configurations.As the junction configuration is known to have a significant effect on the single–molecule conductance, we further explored the master curves composed of the fitted conductance with standard variation at each cross-sectional distance pointOm shows lower conductance than Sm for the conductance of the fully-stretched configurations, while Om shows a slightly higher conductance for the most probable conductance extracted from conductance histogram.It is found that there is some difference in the conductance comparison among different molecules: 3-Op and 8-Om. The lower peaks covered by the grey area below 10−7 G0 is the noise level of the MCBJ experiments  effect. On the contrary, in the meta-anchored isomers the waves are out of phase leading to destructive quantum interference. The conductance relationship of the para and meta molecules is consistent with that of molecules with a central single benzene ring43, indicating the quantum interference effect can still operate in polycyclic compounds. Secondly, the structure of the central core plays an important role in the conductance of QI molecules. It is noted that the largest difference between the para– and meta–anchored molecules (ΔG) is for the dibenzofuran pair 3-Op and 8-Om, 1.37 log(G/G0). However, as we reported previously, the differences between para and meta linked molecules are nearly 1.50 log(G/G0) in benzene-cored analogs29. The lower experimental differences in the present study demonstrate that the quantum interference effect has not been amplified, and is even slightly reduced, by bridging the two benzene rings with a five-membered ring. Additionally, differences of the conductance in fully-stretched conformations between para– and meta–anchored molecules follow the increasing order of ΔGC < ΔGN < ΔGS < ΔGO, illustrating that the heteroatom can also contribute to the expression of the quantum interference. The electrode separations  sterically hinder the interaction between the electrode and the core of the molecule as well as restricting any π-π interaction of two molecules. Thirdly, molecules with similar core structures have been reported23 to exhibit only one conductance statistical peak, suggesting that the junction formed by the core of the molecule is not robust enough during the elongation process. In the control experiments with 5-2Hp and 10-2Hm, however, we observed such an obvious difference that the conductance of 5-2Hp is higher than that of 10-2Hm by almost two orders of magnitude.Moreover, there is no distinct correlation between the plateau length (or JFP) and the nature of the bridging atoms , demonstrating that these atoms have no significant influence on the conformation of the molecular junction. Furthermore, we did not observe any additional conductance group during the experiments for the ten molecules. We attributed this fact to three reasons. Firstly, pyridyl-terminated compounds have been reported to show well-defined peaks in the conductance histograms resulting from the high directionality of the donor-acceptor binding between N lone pair and AuTo understand the effect of pendant groups on quantum interference in the molecules of Fig. α = 0, the pendant orbital is decoupled from the central core. Since the latter is a bipartite lattice, in the meta case, destructive interference should occur at the centre of the HOMO–LUMO gap (i.e. E = 0)31. The black curves in Fig. T (E), when εb = 0. The other curves in Fig. α to the pendant orbital is increased from zero (black curves) to unity (red curves).The tight binding model is introduced to illustrate the underlying trends in the transmission function and to allow us to obtain an analytic formula. Figure α = 0) the meta case shows a sharp transmission dip due to destructive interference at the gap centre, which is absent in the para case. In the presence of pendant groups (i.e. when α is non-zero) this destructive interference is alleviated in the meta case. In the para case, the non-zero coupling to the pendant group introduces a new conductance pathway, which can cause destructive interference within the gap, signaled by the Fano lineshape just below E = 0. Further examples of this evolution for different choices of εb are presented in the SI. For the values of εb shown in Supplementary Table 2Hm, which does not possess a pendant orbital, shows a pronounced transmission dip near E = 0, which is reflected in the low transmission coefficient predicted by DFT. The above results show that in the absence of pendant groups 47.The electronic interference structure calculations leading to transmission curves Fig.  were perpara and meta shown in Fig. T (E) were calculated using the Gollum code48. An excellent agreement between theoretical and experimental conductance values has been obtained  there appear sharp transmission dips due to destructive interference in the meta case, which are alleviated by the presence of pendant groups. In contrast, in the para case, constructive interference in the pi-channel is preserved in the presence of bridging atoms. On the other hand, in the DFT-based transmission curves perfect destructive interference is masked by the presence of sigma orbitals, which provide a parallel path for conductance.To calculate the conductance through these two groups of molecules, para series 1–4 and the meta series 6–9, that there is a clear correlation between aromaticity of the central ring when heteroatoms are present and the single-molecule conductance value in the para series. Aromaticity follows the sequence: S > NEt > O > CMe2. Our conductance trend for the heterocyclic para series, i.e. O > NEt > S is in agreement with a previous experimental study by Venkataraman, Breslow et al.24 on monocyclic core units (furan > thiophene)  shows a lower conductance than 2-Np and 3-Op although it exhibits a non-aromatic core. The reason for this exceptional behaviour of 4-Cp is not clear. However, we note that other workers23 have observed that the single-molecule conductance of fluorene-based cores do not follow expected trends.We conclude, therefore, from our experimental and theoretical data for the 1–5 exhibits a clear difference in comparison to the monocyclic series shown in Scheme 2. However, our data show that the sequence is very different in the meta series where dibenzothiophene ≈ dibenzofuran < carbazole. Multiple factors  and their composite effects should be taken into consideration in explaining the trends in the conductance. Our results show that bridging heteroatoms alleviate destructive quantum interference in the meta-connected molecules. The contribution of electronegativity of the bridging atoms should not be ignored. For the meta series dibenzothiophene 6-Sm and dibenzofuran 8-Om represent both extremes. Dibenzothiophene Sm is the most aromatic and therefore it lowers the conductance. As was mentioned above, dibenzofuran 8-Om is the least aromatic core unit. Therefore, based on the conclusions of the series shown in Fig. 8-Om should be the most conductive molecule. However, the lone pair of oxygen is tightly bonded due to oxygen’s high electronegativity, which hinders the delocalization of electrons and decreases the electron density of the conjugated π system in 8-Om. The carbazole derivative 7-Nm is the most conductive in this meta series because it is less aromatic than dibenzothiophene, but also bears a lone pair which allows transmission through the molecule. The conductance of model non-bridged compound 5–2Hp is reduced because of the dihedral angle between the two phenyl units. Model compound 10–2Hm shows no conductance within the detection limit of the MCBJ setup. This is consistent with the bridging atom of 6–9 planarizing the core, which is essential for raising the conductance in the meta-series.These results demonstrates that a non-aromatic core unit does not necessarily lead to higher conductance since the polycyclic series para-para or meta-meta conjugation through the core unit. In all cases molecules with para connectivity present larger conductances than their meta isomers, regardless of the bridging unit. We have experimentally and theoretically observed clear and distinct trends in the para and meta series. In the para series there is a clear correlation between aromaticity of the central ring and the single-molecule conductance values in the sequence dibenzofuran > carbazole > dibenzothiophene, in agreement with a previous experimental study on monocyclic core units (furan > thiophene). However, in the meta series the carbazole derivative is the most conductive: the sequence dibenzothiophene ≈ dibenzofuran < carbazole. It is concluded that the nitrogen lone pair facilitates transmission through the molecule. Overall, we find that constructive quantum interference in the para-connected molecules persists in the presence of bridging atoms and is partly masked by the presence of sigma channels, whereas bridging atoms alleviate destructive quantum interference in the meta-connected molecules. Our comprehensive study establishes that both quantum interference and heteroaromaticity in the molecular core units play important and inter-related roles in determining the conductance of single molecular junctions. These results should assist in future research in the development of new molecules for incorporation into nanoscale molecular circuits.We have studied the single-molecule conductance of ten oligo(arylene-ethynylene) derivatives with five different core units  attached to gold electrodes by pyridyl anchoring groups. Within the two series there is either Details of the synthesis and molecular characterization are in the Supplementary Notes 1–10 molecules in a mixture of 1,3,5-trimethylbenzene  and tetrahydrofuran , 4:1 (v/v). Single-molecule conductance experiments were performed during the formation and breaking of a nanogap on a notched, freely suspended gold wire  fixed on spring steel sheets  with a two-component epoxy glue . A Kel-F liquid cell with a Kalrez O-ring was mounted onto the sample sheet fixed by two holders. During the measurements, the steel sample could be bent with a pushing rod controlled by a stepper motor (or a piezo motor) and a piezo stack. The stepper motor initialized the bending process. Once the measured current reached a value corresponding to 15 G0, the stepper motor paused and the piezo stack was activated. This strategy managed to decrease environmental noise significantly from the operation of the stepper motor. After the junction was completely opened, the piezo stack was reset and the stepper motor drove down the pushing rod. The movement of the piezo stack controlled the breaking and the reformation of nanoscale contacts and the stretching rate of the two gold leads controlled by the piezo stack is about 5–20 nm/s. Molecular junctions could form upon breaking the gold–gold nanocontacts. More than 1000 conductance–distance curves were recorded for statistically relevant data analysis during the repeated cycles.Electron transport characteristics in single-molecule junctions were studied by MCBJ measurements in solution at room temperature. The molecular solution contained typically 0.1 mM of the −7 G0. The tunneling current between the two ends of the ‘broken wire’ (taken as WE1 and WE2) could be recorded as a feedback signal at a given bias voltage 0.1 V. The distance between the two gold electrodes in the MCBJ setup is calibrated by the STM-BJ setup with the assumption that the tunneling decay is identical under the same experimental conditions. After breaking a gold-gold contact, the conductance of the junction drops to approximately 10−3 G0. Due to the so-called “snap-back” effect, the gap between the two gold electrodes increases instantaneously to a certain distance Δzcorr. A perfect linear atomic chain of gold has a conductance of G0. Due to the tunneling theory, we assume that log (G/G0) = −αzexp, where zexp = 0 corresponds to the point where the distance between the terminating gold atoms is equal to the equilibrium gold-gold separation. Our measured separation is Δz = zexp − Δzcorr, where Δzcorr is the snap-back distance. Then, the relationship between log (G/G0) and Δz is log (G/G0) = −αΔz − αΔzcorr. The log(G/G0) plot has a slope of −α and an intercept of −αΔzcorr. To calibrate the stretching distance in the absence of molecules, we measured the conductance G versus Δz for conductances ranging from 10−4 to 10−6 G0 and extracted the slope and intercept. From repeated measurements, we obtained a distribution of slopes and intercepts and from the most probable slopes and intercepts obtained the most probable values of α and Δzcorr. We concluded from individual experiments that in TMB: THF (v:v = 4:1), the Δzcorr is determined as 0.5 nm and the addition of a low concentration of molecules in solution does not influence the snap-back distance. Further technical details and data evaluation methods have been described in our previous article by Hong et al.49.The MCBJ controller is based on a laboratory-built bipotentiostat. All current measurements were performed with two custom-designed bipolar and tunable logarithmic I–V converters operating in a wide dynamic range from 10 to below 10Details of the theory and simulations are in Supplementary Notes SI


To investigate the efficacy, key technical points, and complication management of the transabdominal preperitoneal (TAPP) approach for incarcerated inguinal hernia repair. Seventy-three patients with incarcerated inguinal hernias underwent TAPP surgery in our department between Jan 2010 and Dec 2015. A retrospective review was performed by analyzing the perioperative data from these patients. The operation was successfully completed in all 73 patients. Operation time was 54.0 ± 18.8 minutes . Length of stay was 3.9 ± 1.1 days . There was 1 case of incisional infection, 32 cases of seroma, and 3 cases of postoperative pain during follow-up. All patients recovered after the appropriate treatment. No recurrence or fistula was observed. The TAPP approach represents a safe and effective technique for incarcerated inguinal hernia repair because of its potential in assessment of hernia content and decreasing incisional infection rate. However, it requires experienced surgeons to ensure safety with special attention paid to the key technical points as well as complication management. Therefore, regardless of the site and size, emergency surgery is usually the first choice for an incarcerated hernia. However, there remain many uncertainties, and surgical options are still controversial. Since the transabdominal preperitoneal (TAPP) approach has advantages in assessing hernia content and decreasing incision infection rate, some researchers have started to use the TAPP approach for incarcerated inguinal hernia repair. However, therapeutic outcomes have not been validated. In the present report, we conducted a retrospective analysis to evaluate the efficacy of the TAPP approach for incarcerated inguinal hernia repair, and further discuss key technical points and complication management.Incarcerated inguinal hernia is a life-threatening clinical emergency.2From Jan 2010 to Dec 2015, data from 73 patients diagnosed with incarcerated inguinal hernia  I or ASA II.Case exclusion criteria were as follows: the presence of a recurrent hernia or previous treatment with preperitoneal tissue adhesives.2 pressure was set at 14 mm Hg. A 10-mm optical trocar was inserted directly from the skin (just above the umbilicus) into the abdomen without dermal incision, and 2 5-mm operating trocars were inserted at each midclavicular line (1 or 2 cm under the umbilicus). Then, an abdominal exploration was performed to identify the superficial anatomical landmarks  as well as the site and type of hernia  and soft  types. Biomaterial patches  were used for 2 young patients. All meshes were fixed using medical glue . The peritoneum was sutured continuously using 3-0 Prolene . For strangulated hernia , open intestinal resection was performed and a soft mesh was used with a latex drainage catheter placed in the pelvic cavity at the end of surgery.All surgeries were performed under complete general anesthesia by a single surgeon. The clinical conditions and surgical risks were preoperatively evaluated by the same anesthesiologist. Prophylactic antibiotics were administered in all cases, and a postoperative antibiotic treatment was performed only for strangulated hernias. The antibiotics used were cefoxitin or alternatively levofloxacin, if the patients were allergic to cefoxitin. The TAPP approach was applied in all cases. A pneumoperitoneum was built via a Veress needle, and COData are expressed as mean ± standard deviation.3All surgeries were successful. The operation time was 54.0 ± 18.8 minutes . The length of hospital stay was 3.9 ± 1.1 days . The follow-up duration was 18.2 ± 13.1 months  after the operation with a 100% follow-up rate.During surgery, of 73 patients, 19 experienced an automatic hernia content reduction after initiation of anesthesia with the usage of muscle relaxants. Forceps-assisted pulling was needed in 45 patients, and hernia sac incision was performed in 9 patients. Two patients with strangulated hernia underwent partial small bowel resection due to necrosis; 1 of these patients had wound fat liquefaction, and the wound eventually healed after repeated dressing changes. Postoperative hematoma or seroma within the groin area was observed in 32 patients subsequent to indirect hernia repair. The seromas were absorbed without intervention within 2 weeks in 20 patients. Twelve patients showed swelling of the scrotum with a hydrocele over 30 mL (confirmed by ultrasound) at 2 weeks after surgery and were treated by puncturing the hydrocele. Three patients experienced postoperative pain , which was relieved within 2 months after using low-dose painkillers (ibuprofen or aspirin) or undergoing physiotherapy. No recurrence or bowel fistula was observed (Table 4The TAPP approach is a laparoscopic technique with superior achievement of fast postoperative recovery compared to open surgery and is utilized in simultaneous bilateral hernia treatment as well as for assessing hernia content. However, conventional wisdom may suggest that the TAPP approach not be applicable to incarcerated hernia or strangulated hernia, because of its long learning curve and demanding skill requirements. It can be successfully performed in most cases of inguinal hernia. However, for incarcerated hernia, there are some differences because of its particular pathophysiology. Based on the experience of our department, we can provide some guidance on applying this surgical technique.Currently, with a growing need for incessant improvement of surgical skills, we have started to use the TAPP approach for incarcerated hernia repair, because of its superiority in assessing gut viability. In open surgery, the incarcerated hernia content might return to the abdominal cavity due to the usage of narcotic drugs and muscle relaxants, and, accordingly, the incarcerated content always causes a dilemma for surgeons: should exploratory laparotomy be performed or not? Exploratory laparotomy searching for the incarcerated content might increase the risk of infection and extensive surgical trauma; however, if it is not performed, the potentially necrotic content might lead to bowel perforation, abdominal infection, and even deadly sepsis. Using the TAPP approach avoids this conundrum.4.14.1.1 For those requiring intraoperative intervention, atraumatic forceps are strongly recommended to grasp the nonedematous mesenterium and pull back the incarcerated content, avoiding any bleeding or damage to the bowel. In case the incarcerated content cannot be reduced by simple pulling, the hernia sac should be instantly opened. For direct hernia, it is better to open the conjoined tendon or the outer rim of abdominal rectus muscle; for indirect hernia, we suggest dissecting from the upper lateral, which is safe and away from the inferior epigastric vessels as well as the triangle of doom; for femoral hernia, it is easier to open the inguinal ligament in the upper middle (Fig. During the TAPP procedure, most of the incarcerated content might have returned to the abdominal cavity automatically due to the use of narcotic drugs, muscle relaxants, or the force of gravity.dle Fig. . It shoudle Fig. .4.1.2 For indirect hernia, we recommend resecting the sac 1 to 2 cm away from the inner ring, which may shorten the operative time.The TAPP approach procedure recommends entering the peritoneum 1 to 2 cm above the inner ring. Nevertheless, in incarcerated cases, it is required to enter the peritoneum away from the inner ring, as the edematous tissues could lead to tardive rupture.4.24.2.1 an impressive body of evidence indicates that a tension-free herniorrhaphy with a full-size light mesh implantation is safe, based on an acceptable rate of infection and lower rate of recurrence. In the present report, of all 73 cases, incisional liquefaction only occurred in 1 strangulated case after converting to open surgery, and only 1 infection case was observed. These outcomes favor the feasibility and safety of the TAPP approach with mesh implantation for incarcerated inguinal hernia repair.A mesh implantation for incarcerated hernia was previously considered as a contraindication, as the prosthesis may cause severe infection. In recent years, with further understanding of the anatomy of the groin area, improvement in surgical techniques, and better tissue compatibility of the hernia-repairing materials,4.2.2 The distal sac of the indirect hernia was challenging to dissect completely and might leave small cavities, which could explain the high rate of hematoma or seroma after indirect hernia repair in the present report. It should be noted that careful observation of dynamic changes within the groin area and proper antibiotic treatment in some cases are necessary.Hematoma or seroma generally refers to a mass around the groin area, which may or may not extend to the scrotum, diagnosed by ultrasound, and fully absorbable in most cases.4.2.3 Regarding the former, using medical glue or a self-gripping mesh might provide better outcomes; for the latter, using a light-weight mesh might assist in reducing postoperative pain. Furthermore, tissue damage or postoperative edema could also result in postoperative pain. Therefore, specific attention should be paid to tissue dissection, particularly when resecting ligaments or dissecting a hernia sac.Postoperative pain results from one or both of the following: nerve damage, contracture, and cicatrization of the implanted mesh.It should be noted that the present study is a case series, based on experiences from a single institution. Further randomized controlled trials investigating TAPP approach for incarcerated inguinal hernia repair are still warranted.5The TAPP approach represents a safe and effective technique for incarcerated inguinal hernia repair because of its potential advantages in assessing hernia content and decreasing incisional infection rate. However, it requires experienced surgeons to ensure safety with special attention paid to the key technical points as well as complication management.


The developed system was evaluated for analysis of disinfectant samples and showed good agreement between the results obtained by the titration method without significant differences at the 0.05 significance level. The proposed strategy based on chitosan-gelatin composite biopolymer nanofibers for the immobilization of enzymes can be extended for the development of other enzyme-based biosensors.A biosensor based on chitosan-gelatin composite biopolymers nanofibers is found to be effective for the immobilization of horseradish peroxidase to detect hydrogen peroxide. The biopolymer nanofibers were fabricated by an electrospining technique. Upon optimization of synthesis parameters, biopolymers nanofibers, an average of 80 nm in diameter, were obtained and were then modified on the working electrode surface. The effects of the concentration of enzyme, pH, and concentration of the buffer and the working potential on the current response of the nanofibers-modified electrode toward hydrogen peroxide were optimized to obtain the maximal current response. The results found that horseradish peroxidase immobilization on chitosan-gelatin composite biopolymer nanofibers had advantages of fast response, excellent reproducibility, high stability, and showed a linear response to hydrogen peroxide in the concentration range from 0.1 to 1.7 mM with a detection limit of 0.05 mM and exhibited high sensitivity of 44 µA∙mM Recently, biosensors have become promising devices for analytical methods in several fields, including clinical detection ,2, the fElectrospinning is the most effective method which produces fibers in the range of nanometer in diameter with high voltage electrostatic field. Electrically-charged jets were obtained when applying a high voltage to polymer solution. During the time that these jets moved toward the collector, the solvent evaporated and the nanofibers were obtained on the collector . ElectroIn the present work, we described the fabrication of novel hybrid biopolymer nanofibers for hydrogen peroxide biosensor detection. The nanofibers were characterized with scanning electron microscopy and electrochemical methods. To the best of our knowledge, this is the first fabrication of chitosan-gelatin composite biopolymer nanofibers for a hydrogen peroxide biosensor based on horseradish peroxidase. In addition, the experimental conditions for fabrication and analytical performance of the biosensor based on nanofibers were optimized. Finally, we have investigated the performance of the nanofiber biosensor, based on HRP immobilized on a chitosan-gelatin composite biopolymer nanofibers-modified electrode, for the determination of hydrogen peroxide in real samples.2HPO4 and NaH2PO4. All other reagents were of analytical grade. All aqueous solutions were prepared with deionized water.Horseradish peroxidase (HRP), chitosan (medium molecular weight), hydrogen peroxide, and gelatin were purchased from Sigma-Aldrich ). Acetic acid was purchased from Ajex . Phosphate buffer solution (0.1 M) was prepared with NaA schematic drawing of the electrospinning setup for this work is presented in v/v of glutaraldehyde for 20 min to make the covalent binding with enzyme. After washing, the electrode was immersed in HRP for 12 h to be immobilized on the nanofibers. The electrode was finally washed with phosphate buffer to remove unbound enzyme. Finally, 1% (v/v) nafion solution was dropped onto the electrode and dried at room temperature. Hence, the modified electrode was successfully achieved. The prepared electrodes were stored at 4 °C in a pH 7.00 phosphate buffer solution until further use.The biosensor was prepared as follows: Firstly, nanofibers modified electrode was immersed into 2.5% The morphology and diameter of nanofibers samples were determined using scanning electron microscopy . For SEM sample preparation, the sample was attached to a SEM stub and then was sputtered with a thin layer of gold. SEM was performed under an accelerating voltage of 5 kV.3Fe(CN)6/K4Fe(CN)6 and 10 mM KCl solution to characterize the immobilization steps on the electrode surface. Amperometric measurements were carried out under stirred phosphate buffer solution  by applying the potential of −0.3 V at room temperature (25 °C).The electrochemical experiments were performed with a CHI660a electrochemical workstation . All experiments were carried out with a three-electrode system with an enzyme nanofibers-modified graphite electrode as the working electrode, a platinum wire as the auxiliary electrode, and an Ag/AgCl/3.0 M KCl electrode as the reference electrode. Cyclic voltammetric was performed in 10 mM KThe performance of this biosensor was dependent on various factors, such as the concentration of the enzyme, the pH and concentration of phosphate buffer, and the applied potential for the detection of hydrogen peroxide. Therefore, these parameters were studied to obtain a high analytical signal.Under optimum conditions, the performance of the biosensor system was evaluated by studying the precision, linearity range, detection limit, storage stability, and application in real samples.3Fe(CN)6/K4Fe(CN)6 at a scan rate of 50 mV/s. As shown in  follows :HRP(red)The generated current was measured by the amperometric technique. The amperometric responses of the nanofibers-modified electrode at a working potential of −0.2 V for each successive addition of various amount of hydrogen peroxide are presented in There are many factors affecting the morphologies of electrospun nanofibers, such as solvent polarity, applied voltage, feed rate, needle diameter, and collection distance. All of thsse factors significantly affect the quality of the nanofibers. Therefore, these parameters were studied in this present work.In order to best optimize the nanofiber, the acetic acid concentrations were varied, ranging from 1 to 90%, whereas all other parameters were kept constant. Decreasing the acetic acid concentration in the solvent increases the mean diameter of the nanofibers. An optimum size of nanofibers was achieved and 60% acetic acid was found to be most suitable for the formation of uniform nanofibers.Once an electric field is applied, the surface of the polymer solution becomes charged. Thus, the effect of the applied electric field was studied from 15 to 20 kV. The voltage was gradually increased up to 20 kV until the liquid came out through the needle and split into a web of fibers collected on the aluminum foil. Twenty kilovolts was selected.The flow rate of the polymer solution within the syringe is another important process parameter. The flow rate was optimized in the range of 0.8–1.6 mL/h. At a low flow rate (0.8–1.4 mL/h) the electrospun fiber is cylindrical and uniform. The results found that the continuous fibers were obtained at the flow rate of 1.6 mL/h.The effect of the diameter of the syringe needle was investigated from 0.5–0.7 mm. The increase of the needle diameter from 0.5 to 0.6 mm significantly increased the average nanofiber diameter. The nanofiber obtained with a larger needle diameter showed a more uniform fiber size distribution. The optimum diameter of the syringe was 0.6 mm.The distance between the collector and the tip of the syringe can also affect the fiber diameter and morphologies. Thus, the collection distance was studied by varying it from 20 to 25 cm. At a distance of 15 cm, the fiber will not have enough time to solidify before reaching the collector, whereas at a distance of 25, fiber beads can be obtained. The optimum collection distance was at 20 cm.The optimum conditions resulting in a continuous fiber with an average diameter of 80 nm are featured in The nanofiber-modified electrode was immobilized with HRP and was then applied in a biosensor for hydrogen peroxide detection. In order to obtain the highest sensitivity of the proposed biosensor system, several parameters, including enzyme concentration, applied voltage, buffer concentration, and pH of solution buffer, were studied.First, the immobilizations of different amounts of HRP were optimized in the range of 2.0–8.0 mg/mL. Results obtained from the experiments are shown in The influence of applied potential on the nanofiber-modified working electrode for amperometric measurement, from −0.05 to −0.4 Vvs. Ag/AgCl onto the enzyme biosensor system was investigated. The concentrations and pH of buffer, which were variable in the proposed work, could affect the enzymatic biosensor for hydrogen peroxide detection. The effect of the different buffer concentrations on the response of the nanofiber-enzyme electrode is shown in The pH value of the buffer is another variable that affects the biosensor measurement. The pH effect on the biosensor performance was studied using a 0.15 M phosphate buffer solution, and pH varied between 5.00 and 8.00 D. The maUnder the optimized conditions, the biosensor was evaluated by studying the analytical precision, linear range, limit of detection, and storage stability.Analytical precision was carried out by measuring the current response of the same nanofibers-enzyme electrode in the presence of 1 mM hydrogen peroxide. The average mean steady-state of the current response was 1100 nA with the relative standard deviation (RSD) of 3.3% for twelve successive assays. Therefore, the nanofiber-modified electrode showed high precision.2 and the response time of the proposed method was around 10 s. A detection limit of 0.05 mM (S/N = 3) was obtained.2O2. Therefore, the proposed sensor was selective to H2O2 detection.The selectivity of the fabricated sensor was tested with possible interferences, such as ascorbic acid, glucose, and uric acid. It was found that that there was no significant change of the current responses generated from ascorbic acid, glucose, and uric acid compared to the response of H2O2. No significant decrease of current was measured after the electrode was studied 20 times continuously. Additionally, the storage stability of the nanofibers enzyme-modified electrode was studied by monitoring the current response of 1 mM of hydrogen peroxide every week and it was stored at 4 °C in a refrigerator after use. The results show that there was only a 14% loss from its initial current response after two months of use, indicating that the immobilized HRP on gelatin/chitosan nanofibers possessed good bioactivity.Additionally, the stability of the fabricated sensor was evaluated by amperometric measurements in the presence of Hv/v of glutaraldehyde for 20 min and was immersed in HRP for 12 h. Finally, the electrode surface was cover with 1% (v/v) nafion solution. The results found that the nanofibers-modified electrodes provided higher response than the thin film-modified electrodes. This confirmed that nanofibers were successfully applied for use as an enzyme biosensor.The generated current was measured by the amperometric technique. 4) by hydrogen peroxide in sulfuric acid. KMnO4 solution (0.02 M) was employed for the standard solution. The endpoint of the titration is the point at which the last drop of KMnO4 added to the solution causes it to turn pink. Results obtained with the proposed method were compared statistically with those obtained with the titration method (To investigate the feasibility of the nanofiber biosensor system for analysis of hydrogen peroxide in different brands of disinfectant was evaluated. The dilution of samples was required with phosphate buffer in order to fit the linear range. All samples were analyzed by the proposed method and the titration method. The redox titration was used as the standard method. This method utilizes the reduction of potassium permanganate (KMnOn method . There w2, respectively. This present work had advantages, such as easy electrode preparation, long-term stability, and good repeatability compared to other previous works [From the above results, we have concluded that the nanofibers-modified electrodes exhibited good efficiency for immobilization of enzyme on the electrode surface resulting in enhancing the sensitivity of biosensor system for hydrogen peroxide detection. The detection limit and sensitivity of these electrodes was found to be 0.05 mM and 44 µA/mM/cmus works ,22. In a


MTOCs) that contain centrioles. Meiotic spindles in oocytes, however, lack centrioles. In mouse oocytes, spindle microtubules are nucleated from multiple acentriolar MTOCs that are sorted and clustered prior to completion of spindle assembly in an “inside‐out” mechanism, ending with establishment of the poles. We used HSET (kinesin‐14) as a tool to shift meiotic spindle assembly toward a mitotic “outside‐in” mode and analyzed the consequences on the fidelity of the division. We show that HSET levels must be tightly gated in meiosis I and that even slight overexpression of HSET forces spindle morphogenesis to become more mitotic‐like: rapid spindle bipolarization and pole assembly coupled with focused poles. The unusual length of meiosis I is not sufficient to correct these early spindle morphogenesis defects, resulting in severe chromosome alignment abnormalities. Thus, the unique “inside‐out” mechanism of meiotic spindle assembly is essential to prevent chromosomal misalignment and production of aneuploidy gametes.Mitotic spindles assemble from two centrosomes, which are major microtubule‐organizing centers ( Drosophila that maintaining functional supernumerary centrioles during female meiotic divisions leads to abnormal meiosis and aborted embryonic development Animal cells generally assemble mitotic spindles using an “outside‐in” mechanism that relies on centrosomes acting as dominant microtubule‐nucleating centers (MTOCs). The two centrosomes define the spindle poles and thus the spindle axis along which chromosome segregation will take place at anaphase Spindle assembly in oocytes is a very slow process. Spindle bipolarization is achieved by 4 h in mice To modify spindle morphogenesis, we developed an HSET gain‐of‐function approach. The localization of endogenous HSET was first analyzed in mouse oocytes, by performing immunofluorescence experiments on fixed oocytes. We found that endogenous HSET is localized on the spindle in meiosis I Fig . We therThe gain‐of‐function analysis was complemented with an HSET loss‐of‐function approach. To do so, oocytes were treated with AZ82, a small molecule inhibitor of HSET To understand how HSET drastically impacts the timing of spindle bipolarization, we took advantage of a GFP‐HSET mutant N593K (HSET N593K) that can cross‐link but does not slide microtubules Because spindle bipolarization occurs precociously in oocytes overexpressing HSET WT, we next analyzed the consequences of its overexpression on sorting of the aMTOCs. This process occurs concomitant with spindle bipolarization and is followed by aMTOC clustering, which allows spindle pole focusing. To do this, the behavior of aMTOCs was followed by time‐lapse microscopy, using mCherry‐Plk4 (Polo‐like kinase 4) as a marker At NEBD+1h30, microtubules form a ball, with aMTOCs dispersed around it We first analyzed aMTOC sorting in controls and HSET WT oocytes Figs B, schemeWe then analyzed aMTOC clustering in controls and oocytes overexpressing HSET WT Figs A and D. We next assessed whether microtubule dynamics was altered in the hyper‐clustered spindle poles of oocytes overexpressing HSET WT. To compare microtubule dynamics, we performed FRAP of SiR‐Tubulin at spindle poles at NEBD+6h30 Fig F. EssentThese results thus show that the timing of spindle morphogenesis is accelerated in oocytes overexpressing HSET WT: Spindle bipolarization is established precociously together with more efficient sorting and clustering of aMTOCs, markers of spindle pole assembly. We then analyzed the impact of accelerated kinetics on spindle shape.To determine whether accelerating bipolarization and spindle pole formation affected global spindle shape, spindle length, central spindle width, and spindle pole width were measured at the same time points used to analyze aMTOC behavior. In oocytes overexpressing HSET WT, the spindle was already bipolar at NEBD+1h30. Strikingly, the spindle at this stage was extraordinarily long Fig A. HoweveEven though spindles in oocytes overexpressing HSET WT did recover in length Fig D, we obsIn an effort to understand how minor deregulation of HSET level impacts the entire process of spindle morphogenesis, endogenous HSET expression levels were measured before, throughout and after meiosis. Levels of HSET were low in Prophase I  have no effect on spindle shape. This strongly argues that the mitotic‐like spindle shape observed after perturbing HSET levels early on are not due to a late and cumulative effect of HSET overexpression throughout meiosis I, but are strictly attributable to increasing HSET levels during early stages of meiosis I. In addition, we did not observe similar defects in spindle bipolarization, pole assembly or chromosome alignment in oocytes overexpressing HSET N593K that can cross‐link but not slide microtubules We were surprised that these early spindle defects were not fully rescued over the extremely long duration of meiosis I. In particular, more highly focused poles were shown to be associated with fewer chromosome alignment defects in meiosis when the microtubule ball stage was not skipped In conclusion, forcing meiosis I spindle morphogenesis to be more mitotic‐like leads to chromosome alignment abnormalities that cannot be fully reversed. In an unexpected manner, the unusual length of meiosis I (8 h) is not sufficient to correct early spindle morphogenesis defects, contributing to chromosome misalignment and segregation. This could be relevant to other systems as well, spindle formation being even slower in human oocytes, taking ~15 h Ovaries were collected from 11‐week‐old OF1 (wt) female mice. Fully‐grown oocytes were extracted by shredding the ovaries 2 and supplemented with 10 mM SrCl2. Activated oocytes were then cultured in M2 + BSA medium for 6 h until pronuclear formation.Oocytes in metaphase of meiosis II were incubated 2 h in M2 + BSA medium lacking CaClin vitro cRNA transcription. The hHSET WT and N593K expressing plasmids were amplified using One shot Top 10 competent bacteria (Invitrogen), subsequently extracted and purified using mini and midi prep kits (Qiagen).hHSET WT and hHSET N593K were subcloned from plasmids provided by Claire E. Walczak  into a pRN3 plasmid suitable for We used the following constructs: pRN3‐GFP‐hHSET, pRN3‐hHSET, pSpe3‐GFP‐hHSET‐N593K, pRN3‐GFP‐EB3 Plasmids were linearized using appropriate restriction enzymes. cRNAs were synthesized with the mMessage mMachine kit (Ambion) and subsequently purified using the RNAeasy kit (Qiagen). Their concentration was measured using NanoDrop 2000 from ThermoScientific. cRNAs were centrifuged at 4°C during 45 min prior to microinjection into the cytoplasm of oocytes blocked in Prophase I in M2 + BSA medium supplemented with 1 μM milrinone at 37°C. cRNAs were microinjected using an Eppendorf Femtojet microinjector Oocytes were microinjected with 150 ng/μl of hHSET WT cRNAs or 250 ng/μl of GFP‐hHSET WT cRNAs. We have observed that this is the optimal concentration to detect interpretable phenotypes whereas lower or higher concentrations gave, respectively, no phenotypes or induced spindle collapse and mono‐aster formation. Oocytes were microinjected with 250 ng/μl of GFP‐hHSET N593K. After microinjection, cRNA translation was allowed for 1 h and oocytes were transferred into milrinone‐free M2 + BSA medium to allow meiosis resumption.The AZ82 inhibitor of HSET was a gift from AstraZeneca (USA) [29–30]. AZ82 was stored diluted in DMSO at 100 μM and further diluted in M2 medium to a final concentration of 10 μM. Control experiments were done in M2 + BSA medium supplemented with equivalent concentrations of DMSO.The CW069 inhibitor of HSET was a gift from Fanni Gergely  Spinning disk movies were acquired using a Plan‐APO 40×/1.25NA objective on a Leica DMI6000B microscope enclosed in a thermostatic chamber (Life Imaging Service) equipped with a CoolSnap HQ2/CCD camera coupled to a Sutter filter wheel (Roper Scientific) and a Yokogawa CSU‐X1‐M1 spinning disk. Metamorph Software  was used to collect data.For SIM super‐resolution microscopy of aMTOCs, image acquisition was performed in 3D SIM mode, with a N‐SIM Nikon microscope (Nikon Imaging Centre @ Institut Curie‐CNRS) before image reconstruction using the NISElements software in vitro culture of oocytes, their zona pellucida was removed by incubation in acid Tyrode's medium (pH = 2.3). Prophase I‐arrested oocytes were incubated in M2 + BSA medium supplemented with 0.4% pronase to remove the zona pellucida.After To visualize aMTOCs by SIM, oocytes were fixed 30 min at 30°C in 4% formaldehyde at NEBD+6h30 on coverslips treated with gelatin and polylysine. Permeabilization was achieved by incubating oocytes in 0.5% Triton X‐100 in PBS for 10 min at room temperature. Mouse anti‐pericentrin antibody (BD Transduction Laboratories) was used at 1:2,000. As secondary antibody, anti‐mouse Cy3 (Molecular Probes) was used at 1:600. Slides were mounted in ProLong Gold.To visualize endogenous HSET and exogenous microinjected hHSET, oocytes were fixed 30 min at 30°C in 3.7% formaldehyde and permeabilized 10 min at room temperature in 0.25% Tween‐20–PBS. The HSET antibody was a gift from Renata Basto . As secondary antibody, anti‐rabbit Cy2 (Molecular Probes) was used at 1:200. Chromosomes were stained with Prolong‐DAPI .Images were acquired using a Plan‐APO 60×/1.4NA objective on a Ti Nikon microscope enclosed in a thermostatic chamber (Life Imaging Service) equipped with a Flash4.0 V2 CMOS camera (Hamamatsu) coupled to a Yokogawa CSU‐X1 spinning disk. Metamorph Software  was used to collect data. All oocytes expressed SiR‐Tubulin . For all oocytes, an identical region of interest (diameter of 5 μm) was bleached at spindle poles. Images were acquired every 5 s for 125 s. One image was taken before bleaching. The SiR‐Tubulin fluorescence intensity quantification was performed using the Metamorph software . Normalization of the measured fluorescence intensities was performed using the Microsoft Excel software. As the expression levels of SiR‐Tubulin vary from one experiment to another, the signal intensity was normalized so that the prebleached value was 1 and the value at the first time point after bleaching was 0.Metamorph , Imaris (Oxford Instruments), and Fiji (NIH) software were used to analyze and process data.The timing of spindle bipolarization was measured on oocytes expressing GFP‐EB3 or SiR‐Tubulin  using Metamorph software; bipolarity was scored when two poles were distinguishable  was first filtered using 3D Gaussian filter (radius = 2) to reduce noise and then thresholded using MaxEntropy method. aMTOCs (RFP channel) were first filtered using difference of Gaussians  to increase spot‐like signals, then thresholded using MaxEntropy method. For each channel, 3D objects were segmented using the 3D ImageJ suite For the aMTOCs 3D analysis see Fig A–C, the The spindle length, central spindle width, and spindle pole measurements were performed in 3D using Imaris software see Figs D. The spThe aMTOCs volume measurements were performed in 3D using Imaris software see Fig D and E. www.graphpad.com. For comparisons between two groups, the normality of the variables was checked  and parametric Student's t‐tests (with Welch correction when indicated) or non‐parametric comparison tests were performed with a confidence interval of 95%. For chromosome alignment experiments, repartitions were analyzed for statistical significance using Fisher's test used with a confidence interval of 95%. All error bars are expressed as standard deviation (SD). Values of P < 0.05 were considered significant. In all figures, * corresponds to a P‐value < 0.05, ** to a P‐value < 0.005, *** to a P‐value < 0.0001. n.s.: not statistically significant.Experiments were repeated at least three times, and a sample of sufficient size was used. The statistical analysis was performed using GraphPad Prism version 7.00 for MacOS, GraphPad Software, La Jolla, California, USA, IB, AK, M‐HV, and M‐ET designed the experiments, interpreted the results, and wrote the manuscript. IB, IQ, AK, and M‐ET carried out the experiments. TB and PM designed the Fiji plug‐in. M‐ET and M‐HV supervised the project.The authors declare that they have no conflict of interest.Expanded View Figures PDFClick here for additional data file.Review Process FileClick here for additional data file.


Interest in addressing antimicrobial resistance (AMR) has grown recently but little effort has been made to consider how existing and new vaccines could impact on AMR. A 2017 Chatham House meeting considered the role of vaccines and how to demonstrate their value through their impact on AMR. Ways existing vaccines have reduced antibiotic prescribing and the prevalence of some resistant organisms were reviewed. Other new vaccines could have a similar impact. In gonorrhoea, where complete resistance has developed, vaccine may be the best option. Valuing the impact of vaccines on AMR was challenging: there were difficult methodological issues and a lack of data for modelling. A participant poll suggested priorities for accelerated vaccine development were tuberculosis, typhoid, influenza, RSV and gonorrhoea. More evidence is needed to convince policymakers but that vaccine development projects should be considered by funders on the same basis as those for new antibiotics or diagnostics. It has been estimated that about $500 million in new funding had been allocated in 2016 from 13 existing or new initiatives whose principal purpose is to accelerate the development of new antibiotics There is no doubt that addressing a threat as significant and complex as AMR requires a portfolio of solutions – including new antimicrobials, better diagnostics and better stewardship. It also requires that more attention be paid to a range of measures that prevent infection and reduce the use of antimicrobials including improved sanitation and the wider use of vaccines. For diseases where AMR has become rampant, vaccines may be the best or only way to save lives.While the role of vaccines in combatting antimicrobial resistance has been mentioned or considered in recent reports and plans on AMR, much less effort has been put into supporting the greater use of existing vaccines and the development of new ones to address AMR. An exception was the UK’s 2016 Review on Antimicrobial Resistance (chaired by Lord O’Neill) which argued in its main report 2To investigate the potential for vaccines further, the Centre on Global Health Security at Chatham House convened a meeting in London in March this year of vaccine experts and stakeholders, including international and regional organizations, economists, modellers and scientists from several vaccine-producing companies. The purpose of the meeting was to review current knowledge and activities on the role of vaccines in combatting AMR, and to consider the issues involved in modelling how their value in this role could be established. This was with a view, in particular, to ensuring that national and international policies on their use and support for R&D efforts properly recognized the contribution of vaccines in mitigating the growth of AMR The general case for using existing vaccines more widely and developing new ones to combat AMR is quite easy to make in principle. The development of AMR is inherent in the use of antibiotics. No new classes of antibiotic have been discovered in the last 25 years and AMR continues to grow. Even if recent initiatives are successful in stimulating the development of new classes of antibiotic, they will most likely encounter resistance. In gonorrhoea, resistance has developed to each successive antibiotic used to the extent that we are now nearly at the stage where it is becoming untreatable. Antibiotic development is therefore comparable to the work of Sisyphus, continually rolling his boulder up to the top of the hill only to see it roll down again. Because AMR is intrinsic to antibiotic use, the only long term solution to AMR is preventing the infections that necessitate their use. There are many factors that contribute to prevention, such as improved living standards and water and sanitation, but vaccines have been a key element in disease prevention globally for almost two centuries. Vaccines should therefore be considered as key weapons in the fight against AMR.The meeting provided some powerful examples where existing vaccines have reduced antibiotic use. The pneumococcal conjugate vaccine has reduced overall antibiotic prescribing and also the prevalence of resistant organisms However, there are many factors, including finance, that inhibit the wider use of existing vaccines  and there are scientific as well as commercial challenges in developing several potential new vaccines  which would impact AMR. However, convincing decision makers in ministries of health and finance as well as in the scientific community and donors, to invest more in vaccine distribution and development requires hard evidence of cost-effectiveness. The meeting grappled with the problem of how a value could be put on the contribution of vaccines to combat AMR.Three scenarios can be envisaged for modelling the impact. First, there is the direct impact of vaccination on reducing antibiotic use through fewer cases of infection translating into fewer antibiotics used and therefore fewer opportunities for resistance to occur. But the AMR impact for any individual vaccine may be fairly small compared to the ‘conventional’ health and economic benefits associated with reducing illness or death. For example, a flu vaccine by itself might not reduce inappropriate antibiotic use when other viral infections could be the cause of flu-like symptoms, demonstrating the need for reliable, fast and cheap point of care diagnostics to make the most of vaccines in reducing AMR. Secondly, there is the ‘herd effect’ of vaccines that reduces infections in the non-vaccinated thereby reducing antibiotic use. Again, the benefits attributable to AMR reduction may be relatively small. The third scenario is when there are no effective antibiotics for a particular disease (as is nearly the case with gonorrhoea). Because the health and economic costs of gonorrhoea are small relative to some other diseases (e.g. infertility rather than death), and the challenges of vaccine development are considerable, decision makers may not be swayed. At the extreme, there could be a level of AMR that makes a wide range of surgery and treatments extremely risky in the absence of antibiotics e.g. bone marrow transplants, chemotherapy, and bowel and orthopaedic surgery. Because the potential health and economic costs of the latter are potentially astronomic, this could make the case for vaccines seem unanswerable, but decision makers will not take action unless convinced that the risk is (very) high and there are optimistic prospects for new vaccine development. The meeting considered that modelling the impact of vaccines on AMR was complex and more work was needed both in clarifying methodologies for modelling the AMR impact of vaccines and in generating more data to populate the models. Extensive assumptions would be needed to link the impact of vaccines in preventing infections to the reductions of antimicrobial use that would result and the latter’s impact on the development of resistance.As an exercise in prioritisation participants at the meeting were invited to consider what vaccines would have the greatest impact on AMR. The top five organisms chosen for accelerated vaccine development were tuberculosis, typhoid, influenza, RSV and gonorrhoea. This choice is significantly different to the prioritisation undertaken recently by the World Health Organization of infections for which new antibiotics are needed Several argued that vaccination to reduce the use of antibiotics in animals could be as important as, if not more important than, in humans. More than 70 percent of the antibiotics deemed medically important for human health by the FDA sold in the United States (and over 50 percent in most countries in the world) are used in livestock 3Policy makers at national and international level need to be presented with more evidence, underpinned by economic modelling, on the value of vaccines in combatting AMR so that the latter’s development and use are encouraged and supported. Those responsible for vaccine research, international organisations that support vaccine research and vaccination, and national governments need to be persuaded that investment in vaccines will play a significant role in the reduction of AMR. Researchers and manufacturers need to be offered appropriate incentives, in particular for vaccines that could have a high impact on AMR but where the commercial market prospects are uncertain. At the very least new and potential future AMR initiatives should consider vaccine projects on the same basis as proposals for new antibiotics or diagnostics. There should be regular reviews of progress in vaccine development and promotion such that the ‘vaccine AMR value’ concept is prominent whenever strategies for combatting AMR are being considered. And there needs to be a similar initiative in livestock production, to consider measures necessary to reduce antibiotic use, including enhanced use of vaccination.