Superoxide dismutases (SODs) are necessary antioxidant enzymes that protect cells from reactive oxygen species (ROS). Decreased levels of SODs or mutations that affect their catalytic activity have serious phenotypic consequences. SODs perform their bio-protective role by converting superoxide into oxygen and hydrogen peroxide by cyclic oxidation and reduction reactions with the active site metal. Mutations of SODs can cause cancer of the lung, colon, and lymphatic system, as well as neurodegenerative diseases such as Parkinson’s disease and amyotrophic lateral sclerosis. While SODs have proven to be of significant biological importance since their discovery in 1968, the mechanistic nature of their catalytic function remains elusive. Extensive investigations with a multitude of approaches have tried to unveil the catalytic workings of SODs, but experimental limitations have impeded direct observations of the mechanism. Here, we focus on human MnSOD, the most significant enzyme in protecting against ROS in the human body. Human MnSOD resides in the mitochondrial matrix, the location of up to 90% of cellular ROS generation. We review the current knowledge of the MnSOD enzymatic mechanism and ongoing studies into solving the remaining mysteries. Excessive amounts lead to a cascade of reactions causing damage to important biological macromolecules such as DNA, lipids, and proteins. Excess superoxide plays a role in the pathogenesis of many disease states including cancers, cardiovascular disorders, and neurodegenerative diseases . Wi. WiThermnd 5T30) ,64. A 10ry 1D5N) . This fuE. coli MnSOD with azide [E. coli MnSOD remains five-coordinate trigonal bipyramidal at physiological temperature (295 K). Upon substrate binding, an unidentified manganese ligand displaces, with protonated aspartate or the solvent molecule being the most likely candidates. At low-temperatures (275–280 K), the coordination becomes six. These spectroscopic observations are in conflict with the six-coordinate azide-MnSOD structure from T. thermophilus (PDB 1MNG), where the crystallization and data collection was at room temperature [T. thermophilus thrives in relatively high temperatures (320–350 K), room temperature may be a “cold” temperature for the thermophilic species. Whittaker and Whittaker do note that the six-coordinate complex is only marginally unstable at 295 K and suggest it could act as a kinetic intermediate [Whittaker and Whittaker proposed the “associative displacement” mechanism from their thermochromism  studies of th azide ,67. The perature . Since Trmediate ,67. ThusE. coli MnSOD indicate that the active site with azide adducts exists in a dynamic equilibrium between five and six coordinate states at 296 K. At 273 K, the coordination shifts to predominately six [E. coli FeSOD by the Miller group, azide did not bind to the reduced form of the active site metal, but instead was near Tyr34. These studies indicate that direct binding of superoxide to the active site metal for catalysis does not occur for at least some portions of the enzymatic mechanism [Density function theory (DFT) calculations performed by Jackson and co-workers with tely six . Howeverechanism .For MnSOD to perform its enzymatic function efficiently it must shuttle protons to the active site for proton-assisted electron transfer in a systematic manner. Extensive investigations through both experimental and theoretical approaches studied the proton-based mechanism and yielded several conflicting catalytic models ,75,76,77To date, insight into the proton-based mechanism has come from indirect observations. X-ray diffraction does not detect the hydrogen atom well, but analysis of X-ray crystal structures reveal a hydrogen bond network at the active site, consisting of His30–WAT2–Tyr34–Gln143–WAT1 . The extent of influence that manganese has on the pKas of molecules at the active site is dynamically changing through shuffling of its redox state . pKas are further determined by whether ionization would make a non-covalent interaction more favorable. Together, the net changes in ionization  at the active site must be energetically downhill and be able to regenerate through cycling of the redox state of the manganese cation.Theoretical studies of the proton-shuttling mechanism have been performed through quantum mechanical molecular modeling (QM/MM) and DFT ,75,80,81ransfers . The pos3+ state, the solvent ligand is hydroxide (OH−), with its ionization stabilized by the electrostatic interaction with Mn3+. Upon conversion to the Mn2+ state, the solvent ligand is protonated to H2O, counterbalancing the changes in the metal charge , which accounts for one of the two protons needed for conversion to H2O2. The source of the second proton to convert HO2− to H2O2 is poorly understood and is hypothesized to come from His30, WAT2, Tyr34, or bulk solvent [The metal-bound solvent molecule, WAT1, is probably a redox-linked proton accepter and perhaps the most studied component of the network ,71,83. Ql charge . The sec solvent ,70. Prot2+) is chemically unlikely in both QM/MM and DFT but its deprotonation (–O−) is plausible [Second sphere residues appear to play critical roles in catalysis. Working backwards from WAT1 in the hydrogen bond network, Gln143 and Tyr34 are evolutionarily conserved and mutation abolishes SOD activity. Yet, their exact functional role is unresolved . For Tyrlausible ,70. Howelausible . These iDFT calculations indicate His30 and WAT2 could relay protons . While p2•−–WAT1 or His30–O2•−–WAT1, could involve superoxide binding at the active site either directly to the manganese or by non-covalent interactions with Tyr34, Gln143, WAT2, and/or WAT1. QM/MM calculations by Srnec and colleagues suggest that superoxide binding differs between half-reactions, with superoxide directly binding to the manganese in the oxidized state but not in the reduced state [A superoxide-dependent proton shuttling network is thought to exist because superoxide binding to the active site allows different hydrogen-bonding and more energetically favorable conditions for proton transfers ,70 Figu. Such a ed state . These i2•−–Mn2+–(H2O), is converted to oxidized peroxide-bound manganese, O22−–Mn3+–(OH−), and peroxide does not disassociate [2−–Mn3+–(OH−), and hydroperoxyl radical disassociation is favorable. In both cases, an electron transfer occurs between the manganese and superoxide substrate, with the differences between these two outcomes being the fate of the WAT1 proton. During the active state, superoxide gains a proton from the solvent ligand to form hydroperoxyl radical, whereas the inactive complex forms due to proton donation from the solvent ligand to an alternative proton acceptor. The most probable acceptor is the carboxylate group (–COO−) of metal-bound Asp159 as it is within hydrogen-bond distance of the solvent ligand. Protonation of Asp159 would likely lead to disassociation from the metal. Mutagenesis and QM calculations support this inactivation mechanism [2•−–Mn2+(H2O), in conjunction with an electron transfer between the substrate and the metal. The reaction can then move forward to dismute superoxide.Studies of combined pulse radiolysis and metal visible absorption spectra experiments have shown that all MnSODs exhibit product-inhibition. An inactive complex can form when manganese is in the reduced state. Here, the superoxide-bound complex, Ossociate ,87,88. Iechanism ,90. To rE. coli MnSOD provides three-dimensional visualization of the putative product-inhibited complex (PDB entry 3K9S) [A cryotrapped hydrogen peroxide-soaked X-ray crystal structure of ry 3K9S) . A molecry 3K9S) . This puOf note, FeSODs, which have identical active site amino acid configurations that are nearly superimposable on those of MnSODs, do not exhibit a reversible inactive, product-inhibited complex . InsteadMnSOD is of great interest to the medical field because of its protection against the deleterious effects of excessive superoxide in disease states ,23,24,25The MnSOD mechanism includes three categories: (1) the means of highly efficient substrate and product diffusion; (2) the mode of superoxide binding to the active site; and (3) the proton-shuttling mechanism for proton-assisted electron transfer. An intricately fine-tuned electrostatic surface and long-range electrostatic vectors guide superoxide substrate to the active site and products away. Substrate-analog binding provides clues as to how superoxide binds to the active site of MnSOD, yet there is no consensus, with direct or indirect binding to the manganese with or without Mn-ligand displacement being possible modes. The difference in molecular length of superoxide and substrate analogs makes it possible that they do not bind exactly the same way. Two protonations occur during each redox cycle of catalysis that involve rapid catalytic turnover and a high catalytic efficiency . This poThe source of the second proton to convert hydroperoxyl radical to hydrogen peroxide is hypothesized to come from other players of the proton relay, His30 and WAT2, and/or bulk solvent ,70. EvenThe mechanistic SOD field would benefit from direct observation of the binding mode of superoxide and the proton relay. Observing the manner of superoxide binding has not been possible due to the exceedingly short half-life of superoxide and the fast catalytic rate of SODs. Visualization of protonation states through conventional structural biology techniques is difficult. NMR and X-ray crystallography are not very sensitive to revealing the positions of hydrogen, especially on solvent molecules. Recently, neutron diffraction experiments, which can reveal hydrogen positions, have been employed to investigate human MnSOD . The exp


S. cerevisiae, and is a stress-inducible protein that functions in protein degradation through direct interaction with the proteasome and Cdc48. Here we report the solution structure of the Cuz1 AN1 ZnF which reveals a compact C6H2 zinc-coordinating domain that resembles a two-finger hand holding a tri-helical clamp. A central phenylalanine residue sits between the two zinc-coordinating centers. The position of this phenylalanine, just before the penultimate zinc-chelating cysteine, is strongly conserved from yeast to man. This phenylalanine shows an exceptionally slow ring-flipping rate which likely contributes to the high rigidity and stability of the AN1 domain. In addition to the zinc-chelating residues, sequence analysis of Cuz1 indicates a second highly evolutionarily conserved motif. This LDFLP motif is shared with three human proteins—Zfand1, AIRAP, and AIRAP-L—the latter two of which share similar cellular functions with Cuz1. The LDFLP motif, while embedded within the zinc finger domain, is surface exposed, largely uninvolved in zinc chelation, and not required for the overall fold of the domain. The LDFLP motif was dispensable for Cuz1's major known functions, proteasome- and Cdc48-binding. These results provide the first structural characterization of the AN1 zinc finger domain, and suggest that the LDFLP motif may define a sub-family of evolutionarily conserved AN1 zinc finger proteins.Zinc binding domains are common and versatile protein structural motifs that mediate diverse cellular functions. Among the many structurally distinct families of zinc finger (ZnF) proteins, the AN1 domain remains poorly characterized. Cuz1 is one of two AN1 ZnF proteins in the yeast  Zinc finger domains are among the most common protein structural motifs in eukaryotes. Sequence analysis in humans predicts over 1,000 distinct zinc finger-containing proteins . The funXenopus laevis egg [The AN1 domain represents a broadly distributed and evolutionarily conserved family of zinc finger proteins . The namevis egg –5. The Aevis egg . Among zS. cerevisiae contain two AN1-domain proteins, Cuz1 and Tmc1 [Budding yeast and Tmc1 –7. Recenand Tmc1 . The proand Tmc1 –7.We have undertaken a structural and functional analysis of Cuz1's AN1 domain. This represents the first reported structure of the AN1 ZnF, and reveals a novel mode of zinc coordination. Within Cuz1's ZnF, we identify a second highly conserved motif which appears to be largely uninvolved in zinc coordination and dispensable for the overall fold of the domain. We propose that this LDFLP motif defines a sub-family of evolutionarily conserved AN1 ZnF proteins.LDFL→AAAA was prepared by site-directed mutagenesis of pJH150, resulting in pJH171. The same mutation was introduced into pJH190, resulting in pJH219. Plasmids were verified by sequencing. Yeast were cultured at 30°C in YPD or selective media, as appropriate. YPD medium consisted of 1% yeast extract, 2% Bacto-peptone, and 2% dextrose.Several candidate expression plasmids for the Cuz1 (systematic name: Ynl155w) AN1 zinc finger domain were constructed and tested. Optimal yield and purity were obtained with plasmid pJH190. This pET45b-based plasmid encodes for Cuz1 amino acids 11–59 with an N-terminal 6x-Histidine tag for affinity purification. The GST-Cuz1 bacterial expression plasmid, pJH150, has been previously described . Full le15N-labeled NH4Cl and 13C-labeled glucose (Cambridge Isotope Laboratories) were used to generate 15N- and 15N/13C-labeled protein. Standard size exclusion chromatography for analysis was carried out with a Superdex 75 16/60 column .For structural analysis of the AN1 zinc finger domain, pJH190 (or pJH219) was expressed in BL21 (DE3) and cultured in M9 minimal media supplemented with zinc sulfate (50 μM) and carbenicillin (50 μg/mL). Logarithmic phase cultures were induced with IPTG (1 mM) and grown overnight at 20°C. Lysis buffer was phosphate buffered saline (PBS) pH 7.4, supplemented with imidazole (10 mM) and protease inhibitors (Roche). Lysates were prepared by French press, clarified by centrifugation in a SS-34 rotor for 25 min at 16,000xg, and filtered through cheesecloth. Protein was purified by Ni-NTA affinity chromatography (Qiagen), washed with PBS supplemented with NaCl (100 mM) and imidazole (20 mM), and eluted with PBS supplemented with imidazole (400 mM). The eluate was desalted using a PD-10 column  and then applied to a centrifugal filter with a 30 kDa cutoff (Millipore) to remove high molecular weight contaminants. Full length wild-type and mutant GST-Cuz1 proteins were prepared by standard glutathione sepharose affinity chromatography as previously described . 12xHis-2, 1 mM DTT, pH 7.5, with 10% D2O using centrifuge concentrators with a 3 kDa cutoff. Triple resonance experiments for backbone and sidechain assignments, as well as 15N and 13C edited 3D-NOESY experiments, were performed non-uniformly sampled on an Agilent dd2600 spectrometer at 25°C using a 0.7 mM 15N-13C labeled Cuz1 sample. 2D-NOESY and TOCSY data in D2O were acquired on a Bruker 750 spectrometer at 25°C using a 0.85 mM unlabeled Cuz1 sample. NMR data were processed using NMRPipe [15N/13C-chemical shift values. The manually assigned NOE distance constraints were first calibrated by default values and then by iteratively computed structure models. The zinc cluster constraints were modeled according to ZAFF (zinc amber force field) [MAHHHHHHVGTGSNDDDDKSPDPNWEL) was assigned but not included in the structural calculations and is not displayed in the figures shown. Structural coordinates have been deposited in the Protein Databank Bank (PDB: 5IJ4) and NMR chemical shifts in the Biological Magnetic Resonance Bank (BMRB: 30030).Cuz1 ZnF protein samples for NMR analysis were buffer-exchanged to 5 mM Tris, 50 mM NaCl, 0.2 mM ZnCl NMRPipe  and hmsI NMRPipe . The bac NMRPipe  softwaree field) , each wiProtein sequence analysis was performed using the ClustalW2 multiple protein sequence alignment tool –16.RPN11-TEV-ProA from the endogenous RPN11 locus [Interaction of GST-Cuz1 protein with proteasome or Cdc48 was as previously described . Metal c11 locus .Cuz1 is one of two AN1 zinc finger proteins in yeast. It is a 274 amino acid protein with two known motifs . Its AN1E. coli, and purified the protein by nickel-affinity chromatography. After subsequent efforts to improve purity (see 15N-HSQC (heteronuclear single quantum coherence) spectrum of the purified 15N-labeled Cuz1 AN1 domain sample showed well dispersed backbone amide resonance peaks indicating a stably formed structure  of Cuz1 representing the AN1 zinc finger domain with an N-terminal 6x-histidine tag. We expressed this construct in rity see , we obtarity see . We evalrity see , consistrity see  and the rity see  analysestructure . 15N-HSQ15N/13C labeled protein in Tris buffer (pH 7.5) at 25°C (The solution structure of the Cuz1 zinc finger (ZnF) domain was solved by NMR spectroscopy using  at 25°C ; Table 1 at 25°C . The sec at 25°C . The ove at 25°C  with twoThe overall tertiary fold of the AN1ZnF can be viewed as a two finger hand holding a tri-helical clamp, locked at both ends by two zinc binding clusters. Additional interactions at the back side of the Cuz1 ZnF domain further stabilize this compact structure . At the 15N-HMQC (Hetero-nuclear Multiple Quantum Coherence) spectrum , and in six of seven human AN1 proteins  . In addi protein –26. Seco protein , 27, 6–7 protein , 6–7. ItGiven the evolutionary conservation of the LDFLP motif, we turned our attention to its place within the AN1 domain. In contrast to the other evolutionarily conserved residues within the AN1 domain, the LDFLP residues were not involved in chelating zinc ions. Among these five residues, Asp25 and Pro28 might play some structural role, as the sidechain of Asp25 in the β2 strand could potentially form a hydrogen bond with the backbone amide of Ser40 at the beginning of the α1 helix, while Pro28 may help rigidify the β3/β4 loop. However, the three prominent hydrophobic residues Leu24, Phe26, and Leu27 are not involved in any clustering to stabilize the Cuz1 ZnF, but rather were largely surface exposed . This hyCuz1 has two known biochemical functions: it binds Cdc48 and the proteasome –7. We soCuz1 also interacts with the proteasome, although the precise residues within Cuz1 responsible for this interaction are not known –7. Previ15N-HSQC spectrum  of backbone superimposed NMR solution structure ensemble containing 15 conformers. The average backbone (red lines) RMSD is 0.13 Å in the twin zinc-finger core. Side-chains of cysteine and histidine residues involved in zinc atom (green spheres) coordination are shown with blue lines.(TIF)Click here for additional data file.S2 FigDue to the length of Zfand4, residues 1–459 were omitted. Note that the LDFLP motif in Cuz1 is not shared with any of these proteins. Asterisks, identical residues; double dots, highly similar residues; single dots, similar residues.(TIF)Click here for additional data file.S3 FigNote that the LDFLP motif in Cuz1 is not shared with Tmc1. Asterisks, identical residues; double dots, highly similar residues; single dots, similar residues.(TIF)Click here for additional data file.S4 FigA) Ribbon representation of Cuz1-ZnF (PDB: 5IJ4) and the second AN1-ZnF domain of mouse Zfand1 (PDB: 1WFE) superimposed by backbone (with an average RMSD of 1.4 Å). B) Ribbon representation of Cuz1-ZnF and the first AN1-ZnF domain of mouse Zfand1 (PDB: 1WYS) superimposed by backbone (with an average RMSD of 2.0 Å). The secondary structure elements of Cuz1-ZnF are colored in red and yellow, while those from mouse Zfand1 in blue and cyan. The DALI Z-score between Cuz1-ZnF and 1WFE is 4.3 with 30% identical sequence, and the Z-score between Cuz1-ZnF and 1WYS is 2.7 with 49% identical sequence.(TIF)Click here for additional data file.


Optimisation of composite flour comprising pearl millet, kidney beans and tigernut with xanthan gum was evaluated for rheological evaluations. The functional properties of the composite flour were optimized using optimal design of response surface methodology. The optimum blends, defined as blends with overall best functional characteristics were run 3 , run 7  and run 13 . The pasting characteristics and rheological evaluation of the optimized blends were further evaluated in rapid visco units (RVU). Run 7 had the overall best pasting characteristics; peak viscosity (462 RVU), trough (442 RVU), breakdown viscosity (20 RVU), final viscosity (975 RVU), setback (533 RVU), peak time (5.47 min) and pasting temperature (89.60 °C). These values were found to be better than several composite flours consisting mixture of wheat and non-wheat crops. In addition, the rheological characteristics (measured by Mixolab) showed that run 7 is the best in terms of dough stability, swelling, water absorption and shelf stability. Composite flour with 85% pearl millet flour in addition to kidney beans and tigernut flours could therefore serve as a viable alternative to 100% wheat flour in bread production. PartialPearl millet is a species of millet widely grown in Nigeria; Nigeria being the world’s third largest producer after India and Niger . MilletsKidney beans is an underutilized tropical legume rich in protein, ash, soluble and insoluble fibres, and linoleic acid . It has Mixolab measures dough development behaviour during mixing and heating, thereby enabling determination of contribution of protein and starch to dough rheology . It helpCurrently, there is little or no work on the pearl millet-kidney beans composite flour. In addition to utilizing pearl millet-kidney bean, this work further added tigernut flour and carry out the optimization of the functional, pasting and rheological properties of pearl millet-based composite flour as viable alternative to wheat flour in bread production.22.1(pennisetum glaucuml) was obtained from Mile 12 market, Lagos State; Xanthan gum was obtained from Payless Chemical Store, Ojota, Lagos, Nigeria; Tigernut (Cyperus esculentus) was obtained from Iddo market, Lagos; Kidney bean (Phaseolus vulgans) was purchased at Iddo market, Lagos State;Pearl Millet 2.2The method of 2.3Tigernut flour was produced using the method of 2.4The method of 2.5The experimental design was carried out using optimal mixture design of response surface methodology . The independent variables were pearl millet flour (75.00–85.00 g/100 g), kidney beans flour (10.00–20.00 g/100 g), and tigernut flour (5.00–10.00 g/100 g). Xanthan gum (0.5 g/100 g) was added to all the samples in order to enhance its viscoelastic characteristics. The dependent variables are the functional characteristics.2.63) was calculated using the formula:The method described by 2.7This was determined by the method described by 2.8Water absorption capacity (WAC) of sample was determined using the method outlined by 2.90) was weighed into pre-weighed 15 ml centrifuge tubes and thoroughly mixed with 10 ml (V1) of refined pure groundnut oil using vortex mixer. Samples were allowed to stand for 30 min. The sample-oil mixture was centrifuged at 3000 rpm for 20 min. Immediately after centrifugation, the supernatant was carefully poured into a 10 ml graduated cylinder, and the volume was recorded (V2). Oil absorption capacity (milliliter of oil per gram of sample) was calculated.Oil absorption capacity (OAC) was determined using the method of 2.101) of concentrate suspension was blended for 3 min using a high speed blender, poured into a 250 ml graduated cylinder, and the volume of foam (VF) was immediately recorded. Foaming capacity was calculated using;Foaming capacity (FC) was determined in triplicate using the method described by 2.11The method of 2.12The gel obtained from swelling index was used in calculating swelling capacity (SC) thus:2.13The pasting properties of the samples were assessed using the Rapid Visco-Analyser . A 3 g sample was dispersed in an aluminum canister containing 25 ml of distilled water. The samples were tested according to Standard Profile 1, where the flour-water suspension was held at 50 °C for 1 min and then heated to 95 °C, held for 10 min, and then cooled to 50 °C and held for another 2 min. The starch viscosity parameters measured were pasting temperature, peak viscosity, breakdown viscosity, final viscosity, trough viscosity setback viscosity and peak time. The results were expressed as RVU for all of the parameters with the exception of pasting temperature, which is expressed in °C.2.14Mixolab  was used according to standard methods (AACC 54–60.01). Mixolab consisted of heating/cooling cycle after constant mixing at 80 rpm. The cooling temperature was 50 °C while the maximum temperature was 90 °C at one heating/cooling cycle. The analyses were carried out at constant water absorption according to 2.15Statistical analyses were carried out using the Response Surface Methodology so as to fit the quadratic polynomial equation generated by the Design-Expert software version 8.0.3.1 . In order to correlate the response variable to the independent variables, multiple regressions was used to fit the coefficient of the polynomial model of the response. The quality of the fit of model was evaluated using analysis of variance (ANOVA).33.12 and adjusted R2 values of 0.6170 and 0.1792 respectively obtained buttressed the assertion. The results of the functional properties of the composite flour are presented in 2 and adjusted R2 values were 0.9230 and 0.8075 respectively, while the p-value (0.9733) the for lack of fit was not significant (p ≥ 0.05). A high adjusted R2 is an indication of less variations between the dependent and independent variables, hence, it is desirable (The dispersibility of the composite flour ranged from 71–80%. Kidney beans significantly (p ≤ 0.05) increase the dispersibility. The Resirable . In addiesirable . The p-vesirable  shows th2BC, AB2C, ABC2) were significant (p ≤ 0.05). The R2 and adjusted R2 values were 0.9753 and 0.9470 respectively. The WAC values obtained were higher than the values obtained for Wheat flour, Wheat-plantain-tigernut composite flour (The WAC ranged from 126–145%. Kidney beans significantly (p ≤ 0.05) increased the WAC. However, the ANOVA showed that the model  and model terms . The contour plot  were significant (P ≤ 0.05).The foaming capacity (0.96–2.95%) is very low. The combine effect of the raw materials must have been responsible for this. 2 and the adjusted R2 values were 0.9881 and 0.9703 respectively.The swelling capacity ranged between 4.93 and 5.69 g/g. Pearl millet and kidney beans flours conspicuously increased the swelling capacity of composite flour as shown in 3.2Three best blends based on the optimization of functional properties were run 3 , run 7 , and run 13 . There are 38 optimization results. The choice is subjective, based on individuals objectives. In this case, the three best combinations selected from the 38 possible combinations were based on high or low WAC, high OAC, high foaming (≥ 1.70%) and high swelling capacity.3.3The results of the pasting characteristics of the optimized composite flour samples  and the control are presented in The trough ranged from 354–442 RVU for the composite flour while it is 936 RVU for the control. The trend followed that of peak viscosity. Trough, which measures the ability of paste to withstand breakdown during cooling. The values obtained in this work are better than the values obtained by The breakdown viscosity had been shown to reflect the stability of the peak viscosity during processing . Starch The Final viscosity ranged from 782–940 RVU for the composite flour sample, while the final viscosity value for the control is 1906 RVU. Increased pearl millet content (as in run 7) had the increased the final viscosity of the composite flour. Final viscosity defines the particular quality of starch and stability of cooked paste. Lower final viscosity signifies reduced ability to form viscous pastes. The final viscosity obtained in this work is better than what were obtained by toasted maize-based composite flour  and wheaPasting temperature was lowest in the control (100% wheat), closely followed by composite flour with 85% pearl millet (run 7). The sample with highest pearl millet flour (run 7) has consistently displayed better pasting characteristics, though, unlike 100% wheat flour. Pasting temperature is defined as the minimum temperature at which starch granules in the composite flour swells. In addition, run 7 had the best pasting time.3.4The results of mixolab of the composite flours (runs 7 and 13) are presented in Stage one (C1) is the dough development which corresponds to farinograph curve . The water absorption for run 7  and run 13  were 51.3% and 46.3% respectively. Water absorption indicates the potential of protein molecules to absorb water, hence, an indicator of baking quality . In addiThe dough development time (DDT) which is the time the dough have optimum viscoelastic properties for gas retention  were 19.97 and 26.22 mins for runs 7 and 13 respectively. Run 7, therefore, had a faster dough development time. The stability time for runs 7 and 13 were 9.18 and 1.57 mins respectively. The longer stability period is an indication of better protein quality. Since the stage one had been shown to correspond to farinograph curve, run 7 therefore had better dough quality for bread making than run 13.et al., 2015). Wheat flour has C2 of 0.4Nm. So run 7 has C2 value close to wheat flour. High C2 value has been reported to indicate good protein quality.In stage two, the protein network weakening rate (α) was −0.064 and −0.026 Nm/min for runs 7 and 13 respectively. In addition, the minimum consistency (C2), which is the minimum values of torque produced as the dough was subjected to mechanical and thermal stress were 0.43 and 2.73 Nm for runs 7 and 13 respectively. Run 13 therefore had a better resistance to thermal and mechanical stresses. A C2 of 0.36 Nm was reported for wheat cassava flour (Ibidapo In the third stage (C3), the gelatinization rates (β) were 0.728 and 0.014 Nm/min for runs 7 and 13 respectively. The availability of dough heating and water from thermal denaturation of protein must have resulted in starch gelatinization . The maxIn stage four (C4), the cooking stability (γ) were −0.532 and −0.018 Nm/min respectively. Run 13 had better cooking stability. The breakdown torques (difference between C3 and C4) were 0.70 and 0.20 Nm. The last stage (C5) is the setback stage (retrogradation of starch molecules), which resulted from decrease in temperature. The setback torque (difference between C5 and C4) were 2.4 Nm and 1.7 Nm in runs 7 and 13 respectively. Run 7 was therefore better shelf stable. Mixolab characteristics were better in run 7 than in run 13. The presence of pearl millet could account for this.The Mixolab profiler indicating absorption, mixing index, gluten plus, viscosity, amylose and retrogradation for run 7 were one, six, four, one, nine and six respectively. Run 13 with 75% pearl millet had no result, indicating further that run 7 (85% pearl millet) sample had better Mixolab, hence, rheological behaviour. In comparison with 100% wheat flour, the results were absorption (2), mixing index (6), gluten plus (5), viscosity (8), amylose (6) and retrogradation (5) as reported by 4Pearl millet-based composite flour offered a viable alternative to wheat-based composite flour for bread production. The results of the functional, pasting and rheological properties confirmed it. While the functional properties of pearl millet – based composite flour was better than 100% wheat flour, the 100% wheat flour had better pasting and rheological characteristics. On the other hand, pearl millet – kidney beans – tigernut composite flour (85%:10%:5%) had excellent rheological behaviour comparable with 100% wheat flour.Olugbenga O. Awolu: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.The authors declare no conflict of interest.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.No additional information is available for this paper.


The Janus kinases (JAKs) are non-receptor tyrosine kinases essential for signaling in response to cytokines and interferons and thereby control many essential functions in growth, development, and immune regulation. JAKs are unique among tyrosine kinases for their constitutive yet non-covalent association with class I and II cytokine receptors, which upon cytokine binding bring together two JAKs to create an active signaling complex. JAK association with cytokine receptors is facilitated by N-terminal FERM and SH2 domains, both of which are classical mediators of peptide interactions. Together, the JAK FERM and SH2 domains mediate a bipartite interaction with two distinct receptor peptide motifs, the proline-rich “Box1” and hydrophobic “Box2,” which are present in the intracellular domain of cytokine receptors. While the general sidechain chemistry of Box1 and Box2 peptides is conserved between receptors, they share very weak primary sequence homology, making it impossible to posit why certain JAKs preferentially interact with and signal through specific subsets of cytokine receptors. Here, we review the structure and function of the JAK FERM and SH2 domains in light of several recent studies that reveal their atomic structure and elucidate interaction mechanisms with both the Box1 and Box2 receptor motifs. These crystal structures demonstrate how evolution has repurposed the JAK FERM and SH2 domains into a receptor-binding module that facilitates interactions with multiple receptors possessing diverse primary sequences. Yet inThe overall structure of the JAK family SH2 domain is reminiscent of a canonical phosphotyrosine (pTyr)-binding SH2 domain, consisting of a central β-sheet flanked by two α-helices. In canonical SH2 domains, two loops flanking the SH2-αB helix form a hydrophobic groove that is the binding site for specificity determining residues at positions +3 and +5 relative to the pTyr . In the Structural studies of the JAKs bound to their receptors have historically been hampered by poor expression and solubility of the JAKs as well as weak affinities for their receptors. To overcome these biochemical hurdles, a construct containing a juxtamembrane peptide from IFNAR1 fused to the C-terminus of the TYK2 FERM and SH2 domains was used to generate the TYK2–IFNAR1 complex . Prior sThe overall topology of the IFNAR1 Box2 interaction with TYK2 resembles that of a pTyr-containing peptide bound to an SH2 domain and can be broken down into three sections, all of which are important for stable interaction with TYK2 . At the The structure of TYK2 bound to IFNAR1 only revealed the binding site for residues near the Box2 peptide; therefore, further structural characterization was required to elucidate the JAK binding site for the Box1 motif. To this end, crystal structures of the JAK1 FERM–SH2 bound to the Box1 of IFNLR1 and IL10R1 were determined , utilizi10 helix that facilitates the insertion of Trp257 into a deep groove formed by the F2-α2 and α3 helices  of 70 nM. The isolated IFNLR1 Box1 binds to JAK1 with an affinity of 1.2 µM, while the isolated Box2 does not measurably interact . T. T46). TAs mentioned above, the structure of the IFNLR1 Box1 region is nearly identical to that of previously solved structures of JAK1–IFNLR1 and JAK1–IL10R1 Box1 peptides . Five adSimilarities between JAK1 and TYK2 peptide recognition extend to other sites identified in the TYK2–IFNAR1 interaction. The key “segment 1” leucine triplet found in IFNAR1 is changed to an Ala–Thr–Phe sequence in IFNLR1, yet it maintains a similar binding orientation. IFNLR1 Ala277 and Thr278 pack into the F2/L1 pocket in a similar conformation to IFNAR1 Leu490 and Leu491, while Leu492 of IFNAR1 is replaced by Phe279 in IFNLR1 and binds to the same pocket at the interface of the F1 and SH2 domains. While these binding triplets have a similar interaction phenotype, the gap between the IFNAR1 Leu492/IFNLR1 Phe279 residue and the SH2-interacting glutamate is six residues in IFNLR1 rather than the five residues seen in IFNAR1. This has profound effects on the backbone positioning of the peptide, which bulges out to accommodate the extra amino acid .via a number of backbone hydrogen bonds, in a similar manner to that seen for the IFNAR1 Box2 peptide . It. Itbona via main chain hydrogen bonding and aliphatic contacts.With regards to the SH2 domain, the two structures that define our understanding of SH2–Box2 interactions (TYK2–IFNAR1 and JAK1–IFNLR1) show a remarkable level of conservation in their interaction mechanisms given the low sequence homology between receptors Figure . Both reIn recent years, significant progress has been made in our understanding of the structure of JAK kinases. In addition to structures of the FERM–SH2 modules and the mechanism of interaction with the receptors described in this review, structural models for the pseudokinase–kinase modules from TYK2 and JAK2 have also been revealed, providing an explanation for the mechanism of negative regulation by the pseudokinase as well as mutations linked to myeloproliferative diseases present in the psKD , 36. AddA second question is what role the enormous variation in cytokine receptor intracellular sequences plays in JAK activation Figure . Even thvia its F2 domain (The JAKs are not unique in their utilization of a FERM domain to mediate partner interactions. The FERM domain is a remarkably prolific protein interaction module, having evolved at least four separate interaction sites for both proteins and lipids. The FERM F3 subdomain, in particular, utilizes multiple surfaces to interact with a wide variety of binding partners. The ERM family members radixin and merlin along with myosin X use a shallow groove between F3-β5 and F3-α1 to interact with their partner molecules –70, whil2 domain . LATS1/22 domain , 74. WhiRF and PL wrote and edited the manuscript.The authors are full-time employees of Genentech, Inc.


Product-releasing enzyme (PRE) domains in fungal non-reducing polyketide synthases (NR-PKSs) play a crucial role in catalysis and editing during polyketide biosynthesis, especially accelerating final biosynthetic reactions accompanied with product offloading. However, up to date, the systematic knowledge about PRE domains is deficient. In the present study, the relationships between sequences, structures, and functions of PRE domains were analyzed with 574 NR-PKSs of eight groups (I–VIII). It was found that the PRE domains in NR-PKSs could be mainly classified into three types, thioesterase (TE), reductase (R), and metallo-β-lactamase-type TE (MβL-TE). The widely distributed TE or TE-like domains were involved in NR-PKSs of groups I–IV, VI, and VIII. The R domains appeared in NR-PKSs of groups IV and VII, while the physically discrete MβL-TE domains were employed by most NR-PKSs of group V. The changes of catalytic sites and structural characteristics resulted in PRE functional differentiations. The phylogeny revealed that the evolution of TE domains was accompanied by complex functional divergence. The diverse sequence lengths of TE lid-loops affected substrate specificity with different chain lengths. The volume diversification of TE catalytic pockets contributed to catalytic mechanisms with functional differentiations. The above findings may help to understand the crucial catalysis of fungal aromatic polyketide biosyntheses and govern recombination of NR-PKSs to obtain unnatural target products. A great variety of fungal aromatic polyketides have an important impact on the pharmaceutical industry and agricultural production due to a wide range of biological products including clinical drugs as well as undesirable toxins and virulence factors . Fungal Aspergillus parasiticus PksA in group IV, which is a TE domain participating in aflatoxin B1 biosynthesis .As a class of crucial domains, the product-releasing related domains have been demonstrated to dominate the final step reaction to synthesize and release the products in many NR-PKSs . It is eynthesis . GeneralAspergillus nidulans, the product-releasing related domains including R domains were marked corresponding to NR-PKSs in groups I, III–VII  domains. The differentiations of PRE domains were labeled in the phylogenetic tree of 574 NR-PKSs involved in groups I–VIII. The three-dimensional structures of different PRE domains were modeled, and the relationships between catalytic sites, structural characteristics, and catalytic mechanisms were analyzed and compared. Specifically, the reasons that TE domains perform diverse functions were discussed with variations on the differentiations of sequences and structures. The complex and interesting evolution tracks of PRE domains in fungal NR-PKSs were also described according to the relationships between sequences, structures, and functions.The amino acid sequences of 58 fungal NR-PKSs with known product release mechanisms were collected from NCBI database GenBank;  with accThe NR-PKS sequences and PRE sequences were aligned with MAFFT . PhylogeThe three-dimensional models of PRE domains were constructed using comparative protein modeling method by I-TASSER . All theThe structural mapping and pocket architecture visualization were displayed using VMD . The lidThe evolutionary conservation of amino acid positions in the PRE sequences was estimated by using ConSurf algorithm . The LG Figure 2A) clearly classified the collected sequences into eight major groups with NR-PKSs from basidiomycetes only appeared in group VIII, which was consistent with our previous study . The NR-PKS phylogenetic tree was constructed on the basis of the phylogeny and domain architectures. The resulting NR-PKS phylogenetic tree . It could be found that there are mainly three types of PRE domains, TE, R, and MβL-TE. The majority of TE and R domains located at the C-terminus of NR-PKSs, with 304 sequences containing TE domains and 125 sequences including R domains. Most PRE domains of NR-PKSs in groups I–III, VI, and VIII were characterized as TE or TE-like domains . The sequence lengths of PRE domains of NR-PKSs in group VI, approximately 350 amino acid residues, were obviously longer than that of NR-PKSs in groups I–IV and VIII containing 250–300 residues. Considering the unusual sequence lengths, we proposed that the PRE domains of NR-PKSs in group VI could be defined as “TE-like” domains. About two-thirds PRE domains of NR-PKSs in group IV were TE domains, while the others were R domains. All PRE domains of NR-PKSs in group VII were identified with R domains. In addition, the physically discrete MβL-TE domains were employed by most NR-PKSs in group V, which do not possess the C-terminal fused PRE domains.The various PRE domains were labeled on the derived NR-PKSs in phylogenetic tree of fungal NR-PKSs . In order to describe the phylogeny of PRE domains, the PRE phylogenetic tree was constructed with the PRE sequences with the product release functions, mainly including TE and R domains. Besides these two domains, MβL-TE was also involved due to its product release functions, despite it evolved independently. The resulting PRE phylogenetic tree clearly classified the PRE sequences into mainly three distinct types corresponding with TE, R, and MβL-TE. The phylogenetic relationships of diverse TE and TE-like domains were corresponding with that of their derived NR-PKSs . The TE-like domains of NR-PKSs in group VI were arisen in early evolution from TE domains. Notably, the R domains mostly from NR-PKSs in groups IV and VII converged into a separate clade and the MβL-TE recruited by NR-PKSs in group V formed another one. The product release functions of PRE domains and their derived NR-PKS groups were precisely labeled in Figure 3B and Supplementary Figure S1. It should be clarified that different from the previous reports (The amino acid sequences of 58 NR-PKSs were selected according to the criterion of NR-PKS whose product release modes has been reported in the literature (Supplementary Table S2). These 58 NR-PKSs were found to cover all known groups (groups I–VIII) of fungal NR-PKSs. Successively, the PRE phylogenetic tree from these 58 NR-PKSs was established based on their product release modes . It should be noted that TE and TE-like domains from these six NR-PKS groups are characterized not only various release mechanisms but also different chain lengths of polyketide intermediates .The TE and TE-like domains, involved in NR-PKSs of six groups , present more complex phylogenetic relationships and various product release mechanisms than other two types. The product release functions of TE and TE-like domains could be generally differentiated into two manners, O–C bond formation and C–C bond formation . The R domains reductively release the different polyketide chains through catalyzing an NAD(P)H-dependent two-electron reduction of the acyl thioester to an aldehyde intermediate. The R domain of Acremonium strictum MOS producing xenovulene is the first identified PRE domain with reductive release mechanism during polyketide biosynthesis . In addition, the catalytic sites of PRE domains in three types are also distinguishing significantly.In order to illustrate the PRE structures corresponding to various product release mechanisms, the three-dimensional structures of all PRE domains from the selected 58 NR-PKSs with known functions were modeled and analyzed. The three-dimensional PRE structures of NR-PKSs in groups I–VIII were built by comparative protein modeling method. According to quality assessment of Ramachandran plot, the accuracy of all the PRE protein model structures was acceptable (Supplementary Table S3). Based on the above simulated structures, the structure and sequence analyses of PRE domains showed obvious differences between TE, R, and MβL-TE. In the whole, the TE and TE-like proteins show the α/β-hydrolase folds with different lid structures, while the R and MβL-TE proteins display α/β Rossmann fold and typical MβL fold, respectively . The residues of catalytic triad (Ser-Asp-His) are highly conserved in diverse TE domains, as previous mutation experiments reported .Our analysis of model structures indicated that the TE proteins of NR-PKSs in groups I–IV and VIII display an α/β-hydrolase fold and a loop between two helices of lid, similar to that of PksA . The two flexible ends of lid suggested that the integral lid region could open and close within a certain space. The highest flexible loop in the middle of lid may be the entrance for substrate to move into the chamber.In order to study the different TE domains corresponding to various release mechanisms, the structure and sequence differentiations of TE domains of NR-PKSs in groups I–IV and VIII were analyzed and compared. The structural differentiation analyses were focused on the lid regions and catalytic pockets of TE domains. The results indicated that all TE domains of NR-PKSs in groups I–IV and VIII possess a similar loop region between two lid helices. B factor is a measure to capture the atomic vibrational motion. Analysis of the B factor for lid region in each TE structure showed that there are three regions of high disorder, corresponding with loop and two ends of lid . It should be noted that there is an interesting relationship between loop sequence lengths and synthesized compound sizes. The average sequence length of TE lid-loops of NR-PKSs in group IV is the longest (∼19 residues) corresponding with the products of decaketides, while shortest (∼12 residues) in group VIII with tetraketides. It is probable that the loop regions of TE domains could play a crucial role in substrate recognition. To better understand their relationship, the statistical data has been calculated (Supplementary Table S4). The statistical data of lid-loop sequence lengths and chain lengths of polyketide intermediates of TE domains in NR-PKSs (groups I–IV and VIII) showed a high degree of positive correlation .By comparing the lid and loop sequence lengths of TE domains of NR-PKSs in groups I–IV and VIII, it could be found that the lid sequence length differences mainly ascribe to the loop sequence length differences . The average cavity volumes of O–C bond-forming pockets in groups I and VIII are significantly bigger than that of C–C bond-forming pockets in groups II–IV. However, the cavity volumes of TE catalytic pockets with C–C ring closure reactions are similar despite the different chain lengths of polyketide intermediates synthesized by NR-PKSs in groups II–IV.The cavity volumes of TE catalytic pockets were calculated and analyzed by CASTp and CAVER. The average cavity volumes of TE catalytic pockets of NR-PKSs in groups I–IV and VIII are about 500–650 ÅFigure 8A and Supplementary Table S5). Therefore, based on the TE crystal structure of PksA, 27 CLR sites of TE domains were predicted by using CASTp and CAVER (Supplementary Table S5). Among them, 11 CLR sites locate at the lid regions of TE domains. It was found that there are nine most conserved CLR sites showing the same or similar residues in the predicted CLR sites of TE domains in NR-PKSs of groups I–IV and VIII. These CLR sites in the catalytic pockets present as Asp1873, Gly1874, Trp1936, Phe2056, Phe2089, and Ser1937-Asp1964-His2088 , while in the lid regions only Pro1968 occurs (amino acid numbering as in Q12053). It should be noted that the results revealed that the small side chain residues Gly and Leu in the conserved CLRs in the TE catalytic pockets in group I were replaced by the conserved CLRs Ser1938 and Phe2010 (amino acid numbering as in Q12053) in groups II–IV, respectively, resulting in the decrease of pocket volumes. The positively charged residue Arg in group I was substituted by the conserved Pro1966 in groups II–IV. The conserved Leu1974 identical in TE lid regions of groups II–IV are not conserved in groups I and VIII. In a previous study of TE crystal structure of PksA, it was proposed that Trp1936 and Phe2010 together with other two residues envelope the hexyl portion of the substrate to cause the conformational change of the hexyl group, and backbone nitrogens of Gly1874 and Ser1938 act as the oxyanion hole to stabilize the transacylation reaction . The conservation of residue sites in the loop is obviously lower than those of helixes at two sides. Specifically, the Weblogo was generated to visualize the sequence diversity at each position of TE lid regions . It was found that the highly conserved aromatic amino acids and charged amino acids of TE lid regions may be potential functional sites.Due to the structures are confined by sequences, the TE sequence differentiations of NR-PKSs in groups I–IV and VIII were also analyzed. The conservation analyses of all amino acid residue sites were performed with the evolutionary conservation scores calculated by ConSurf. The results showed that the majority of conserved residue sites (ConSurf grade 7–9) in TE sequences of NR-PKSs in groups I–IV and VIII locate at the catalytic pockets . Multiple sequence alignment revealed that the average lid length of TE-like domains is more than 67 amino acid residues, comparing with those of TE domains (45–52 residues) of NR-PKSs in groups I–IV and VIII. In addition, it is worth noting that the residue Asp of catalytic triad is not conserved in TE-like domains. According to multiple sequence alignment, it could be deduced that the catalytic triad residues in TE-like domains should be Ser2251, Tyr2279, and His2445 (amino acid numbering as in XP_681652).The structural and functional differentiations of TE-like, R, and MβL-TE domains are relatively simple compared to TE domains according to our analyses. The structure types of TE-like, R, and MβL-TE domains were obviously different from the TE domains. The TE-like structures of NR-PKSs in group VI are different from TE structures of NR-PKSs in groups I–IV and VIII in the lid regions. The lid regions of TE-like domains could not be finely modeled, despite the α/β-hydrolase core of TE-like domains are similar to those of TE domains . The residues His97, His99, Asp101, His102, His153, Asp172, and His207 of MβL-TE domains (amino acid numbering as in CBF70385) are critical for binding substrates and catalyzing hydrolytic activities reported by the previous biochemical experiment with MβL-TE domain of A. nidulans AptB  and surfactin synthase TE . The relationship of catalytic sites between TE, R, and TE-like domains is similar to that of phylogenetic tree of NR-PKSs and PRE domains. Considering distantly phylogenetic relationships of NR-PKSs between groups IV and VII, we deduced that the R domains of NR-PKSs in group IV should derive from separate gene fusion events and domain replacements. It could be further implied that the PRE domains at the C-terminus of NR-PKSs should originate from separate gene fusion events.Besides the diverse TE domains, our interesting findings also involve TE-like and R domains. The catalytic sites of TE-like domains display the intermediate formation between TE and R domains, as Ser-Asp-His in TE domains, Ser-Tyr-Lys in R domains, while Ser-Tyr-His in TE-like domains. Based on the phylogenetic tree of NR-PKSs and PRE domains, the evolutionary status of TE-like domains also exist between TE and R domains (In the past decades, the PRE domains have been reported as TE, R, and MβL-TE domains in literature . In our The above results by our study may help to predict the structures of fungal secondary metabolites by genome mining. Based on the phylogeny of PRE domains of fungal NR-PKSs, the structures of secondary metabolites could be estimated primarily. The cavity volumes of TE catalytic pockets could help us to understand the bond formation types of polyketide intermediates. According to the sequence lengths of TE lid-loops, the possible chain lengths of polyketide intermediates of TE domains could be recognized.Until now, the chain-release mechanisms of several NR-PKSs in group V are still mysterious, which may exceed the known mechanisms. These issues indicated that besides C-terminal PRE domains, NR-PKSs may recruit MβL-TE and other possible types of domains, which demonstrate the versatility, flexibility and complexity of PRE domains. The unknown product release mechanisms should be revealed in the future and may lead to new insights.In the present study, the relationships between sequences, structures, and functions of PRE domains in fungal NR-PKSs were systematically analyzed and elucidated. Our investigation revealed that the PRE domains could be mainly classified into three types, TE, R, and MβL-TE, with distinct catalytic modes caused by different structural characteristics as well as catalytic sites. The evolution routes of TE domains display complicated functional divergences. Furthermore, the functional differentiations of various TE domains are attributed to the structural changes, such as the lid-loop sequence lengths and catalytic cavity volumes, arising from the sequence variations. The specific catalytic sites and structural characteristics of TE-like and R domains were described for the first time. The above findings may provide the novel mechanistic insights for PRE domains and may help to predict the structural characteristics of the aromatic polyketides biosynthesized by fungal NR-PKSs. It could be prospected to regulate the biosynthetic pathways and further apply the combinatorial biosynthesis to obtain new target products.LL and ZZ designed the experiments, analyzed the data, and wrote the paper; C-LS and C-YW directed the experiments, and wrote and revised the manuscript. All authors gave final approval for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


By calibration of the AFM signal, an upper estimate of ∼1 × 1016 cm−3 is calculated for the mobile carrier density in the wall, around four orders of magnitude below that required for complete screening of the polar discontinuity. A carrier mobility of∼50 cm2V−1s−1 is calculated, about an order of magnitude below equivalent carrier mobilities in p-type silicon, but sufficiently high to preclude carrier-lattice coupling associated with small polarons.Enhanced conductivity at specific domain walls in ferroelectrics is now an established phenomenon. Surprisingly, however, little is known about the most fundamental aspects of conduction. Carrier types, densities and mobilities have not been determined and transport mechanisms are still a matter of guesswork. Here we demonstrate that intermittent-contact atomic force microscopy (AFM) can detect the Hall effect in conducting domain walls. Studying YbMnO et al. report the type, density and mobility of carriers in conducting domain walls in ytterbium manganite using nanoscale Hall effect measurements.Conduction in ferroelectric domain walls is now an established phenomenon, yet fundamental aspects of transport physics remain elusive. Here, Campbell  Domain wall conduction has now been seen in numerous ferroelectric systems , (Bi0.9La0.1)FeO3 2O7 ref. , Cu3B7O1Unfortunately, the mechanisms associated with charge transport are not yet clear. There is an expectation that domain walls should have electronic band-structures that differ from domains1958910111221223, for example, which is known to have acceptor dopant states in bulk and where conduction is only seen at tail-to-tail wallsSeveral studies on domain wall conduction have noted the importance of polar discontinuities in determining or enhancing conduction589101124Here we perform local measurement of the Hall effect on mobile charge carriers in domain walls using a novel scanning probe technique. We validate that conduction in tail-to-tail walls is associated with p-type carriers and use quantitative values of the Hall voltage to give an estimate of the carrier mobility and an upper estimate of carrier density.3. As with other members of the hexagonal rare earth manganite family, YbMnO3 is an improper ferroelectric. Structural collapse from the high symmetry, high temperature state at a temperature somewhat above 1,250 K . The combination of the two equivalent ferroelectric states with dipoles of opposite orientations along the [001] direction and three equivalent senses of structural distortion designated as α, β and γ leads to six discrete, but energetically equivalent possible domains . Domain walls are not crystallographically confined and so meander quite strongly before converging at characteristic sixfold junctions, as imaged in 3 . The local development of the Hall voltage on conducting domain walls was manifested in the measured topographic image.For measurement of the Hall effect, gold electrodes were sputtered onto polished surfaces of the YbMnO3 crystal within the interelectrode gap, between the sputtered gold electrodes, was first mapped using standard c-AFM. As expected  of the polar discontinuity. Meier et al.3 domain wall conduction.Topographic images obtained on the YbMnOckground . Using tckground . The posckground . We weren) in a domain wall can be related to the Hall voltage (VH) as follows:Using the simplest approximations, the carrier density . Given that VH∼30 mV, |B|∼0.3T, assuming that the carriers are singly charged and that the current of |I|∼22 μA is shared among ∼10 domain walls acting in parallel, each of width ∼10 nm, a carrier density of order n∼1 × 1016 cm−3 can be obtained. Since current is not entirely restricted to the domain walls, this is likely to be a significant overestimate.where |nscreen) found at tail-to-tail walls in YbMnO3 can be estimated by:The charge density required to screen the polarization discontinuity  gives a value for nscreen∼1 × 1020cm−3, consistent with estimates for BaTiO3 and PbTiO3 made previouslywhere |The effective carrier mobility can be estimated by re-expressing the current as:V is the potential difference between source and drain electrodes and R is approximated to be the resistance of the percolating domain walls; |E| is the estimated uniform electric field in the interelectrode gap, σ is the conductivity of the walls and A is the effective cross-sectional area of the walls. Using the relationship:where μ is the carrier mobility, the Hall voltage can be expressed as:where D), the carrier mobility can then be given as:and taking the cross-sectional area of the domain wall as the width multiplied by the depth from the top surface , p-type carrier mobilities of ∼50 cm2V−1s−1 can be estimated. This is about an order of magnitude lower than that typical of p-type silicon with a similar carrier density34Given a Hall voltage of ∼30 mV, an effective electric field of 1 × 103 reveal that current is due to p-type carriers, which in this case are very likely to be holes. In addition, quantitative estimates for carrier mobilities and densities have been made. Further investigation is under way to fully elucidate the contrast mechanism and facilitate greater precision in measurement of the Hall potential. Nonetheless, our measurements represent the first explicit characterization of the carriers involved in conducting ferroelectric domain walls.A novel scanning probe microscopy technique has been demonstrated to allow measurement of the Hall effect, with spatial resolution on the order of tens of nanometers. Measurements on conducting domain walls in YbMnO2O3 and MnO2 powders were mixed and calcined in air at 1,100 °C and 1,200 °C for 36 h each with intermediate grinding. The reacted powder was then formed into cylindrical feed rod shapes, 10 mm in diameter and 10 cm in length, and sintered at 1,250 °C for 24 h in air. Single crystals of YbMnO3 were then grown using an optical floating-zone technique . The growth was carried out at a rate of 2–3 mm h−1 in a flow (200 cc min−1) of Ar/O2 mixed gas atmosphere with a feed and seed rod rotation at 20 r.p.m.Stoichiometric amounts of high purity (>99.99%) YbRMS with a frequency of 20 kHz was applied to the base of the sample via a silver paste bottom electrode. Current maps were obtained on the same system using an additional Bruker Tunnelling AFM (TUNA) module. Here a DC bias was applied to a silver paste bottom electrode and a lateral gold electrode, and the tip grounded, for data in −1.PFM measurements were carried out with a Veeco Dimension 3100 AFM system, modified for PFM, with a Nanoscope IIIa controller using a EG&G 7256 lock-in amplifier. Here an AC bias of 5 VHall measurements were carried out with a Bruker Dimension 5000, modified to include a reversible electromagnet with field strengths of up to 0.4 T, in conjunction with a Nanoscope V controller. Nanosensor model PPP-EFM probes were used. A constant DC current of up to 22 μA was driven across the interelectrode gap, orthogonal to the applied magnetic field, using a Keithley 6221 current source. Tapping mode topography scans were carried out, at a scan rate of 0.1 Hz, at a range of applied currents and magnetic field strengths. The resulting interaction between the probe and established Hall potential led to deflection of the tip which was monitored with nanoscale spatial resolution such that domain wall-specific signals could be identified.The data supporting the findings of this study are available from the corresponding author upon request.How to cite this article: Campbell M. P. et al. Hall effect in charged conducting ferroelectric domain walls. Nat. Commun.7, 13764 doi: 10.1038/ncomms13764 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-3


The structure belongs to the alluaudite structural type and thus, it obeys the X(2)X(1)M(1)M(2)2(PO4)3 general formula. Both the X(2) and X(1) sites are fully occupied by sodium, while M(1) is occupied by calcium and M(2) exhibits a statistical distribution of iron and manganese.Disodium calcium manganese iron tris(phosphate), Na 2CaMnFe(PO4)3, has been synthesized as single crystals by the flux technique. This compound crystallizes in the monoclinic space group C2/c. The structure belongs to the alluaudite structural type and thus, it obeys the X(2)X(1)M(1)M(2)2(PO4)3 general formula. Both the X(2) and X(1) sites are fully occupied by sodium, while M(1) is occupied by calcium and M(2) exhibits a statistical distribution of iron and manganese.A new iron phosphate, disodium calcium manganese(II) iron(III) tris(phosphate), Na The M sites are fully occupied while the X sites can be empty or partially occupied. In this paper, we report a structural study of a new composition of alluaudite-like iron phosphate Na2CaMnFe(PO4)3. In this compound the M(1) and M(2) sites are occupied by Ca and (0.5Mn + 0.5Fe), respectively, while the X(1) and X(2) sites are fully occupied by Na atoms.A promising line of research in the materials science field is the creation of materials based on inorganic phosphates, which have considerable potential for use in laser engineering, optics and electronics owing to their non-linear optical, electrical and luminescent properties. In recent years, iron monophosphates have assumed great importance for their promising applications in several fields such as catalysis  site is often preferentially occupied by iron with oxidation state +III. Consequently, and on basis of the Mössbauer spectroscopy results observed in similar compounds, the presence of FeII and MnIII in the M(2) site was not considered in the Na2CaMnFe(PO4)3 compound. Indeed, in Na2Mn2Fe(PO4)3 3 O6 octahedra linked together by common edges and sharing edges with a strongly distorted M(1)O8 polyhedron. Connected equivalent chains through the PO4 tetrahedra lead to the formation of sheets stacked parallel to the ac plane  and   Fig. 3 and occuM(2)—O distances and the O—M(2)—O angles range from 2.027 (2) to 2.246 (2) Å and from 80.11 (9) to 174.29 (9)°, respectively. This dispersion evidences an important distortion of the M(2)O6 octahedron due to edge-sharing. The M(1)O8 polyhedron is also very distorted as indicated by the M(1)—O distances and the O—M(1)—O angles which vary from 2.336 (2) to 2.951 (3) Å and from 54.00 (8) to 161.85 (8)°, respectively. In the P1O4 and P2O4 tetrahedra, the P—O distances vary between 1.521 (2) and 1.547 (2) Å. Their mean distances 〈P1—O〉= 1.538 (2) Å and 〈P2—O〉= 1.537 (2) Å are in a good accordance with the value of 1.537 Å calculated by Baur (1974The Baur 1974 for monoX(2) and X(1) sites are eightfold coordinated, such as for example in Na2Mn2Fe(PO4)3 and Na2Cd2Fe(PO4)3 6  and X(2) sites are eight and six, respectively. The decrease of the X(2) coordination number seems to be related to the presence of calcium (0.5 Ca + 0.5 Na) in the M(1) site. In the title compound, the decrease of the coordination numbers from eight to six for both the X(1) and X(2) sites is probably related to the increase of the calcium content in the M(1) site, which becomes exclusively occupied by calcium.Assuming sodium–oxygen distances below 3.0, both the Na1 and Na2 sites are surrounded by six oxygen atoms. Their environments approximate strongly distorted octahedra Fig. 5. Note th2Mo2O7. A starting mixture of appropriate amounts of Fe(NO3)3·9H2O (3.999 g); Mn(NO3)2·6H2O (2.472 g); CaCO3 (0.985 g); (NH4)2HPO4 (3.921 g); Na2CO3 (1.845 g) and MoO3 (2.148 g) was dissolved in nitric acid and then dried for 24 h at 353 K. The dry residue was well ground in an agate mortar and was gradually heated up to 873 K in a platinum crucible to evacuate the decomposition products NH3, CO2 and H2O. Then, the obtained product was melted for 1 h at 1073 K and was cooled slowly to 473 K at a rate of 10 K h−1. Finally, hexagonally shaped brown crystals of Na2CaMnFe(PO4)3 were obtained after washing the mixture with boiling water.Single crystals of the title compound were obtained in a flux of sodium dimolybdate NaM(2), statistically occupied by the Fe3+ and Mn2+ ions. This distribution was supported by the M(2)—O distances, which range between those of Mn—O and Fe—O observed in similar environments.Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2056989016017771/br2263sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989016017771/br2263Isup2.hklStructure factors: contains datablock(s) I. DOI: 1515407CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The data given below relates to the research paper entitled: “Eco-friendly films prepared from plantain flour/PCL blends under reactive extrusion conditions using zirconium octanoate as a catalyst”, recently published by our research group  Specifications TableValue of the data•4 as a catalyst under REx conditions.The data gives detailed descriptions of the physicochemical properties of active eco-friendly films based on plantain flour/PCL blends using Zr(Oct)•The data provides information to researchers about the effects of the catalyst and the PCL-containing blends on the opacity of the materials.14 as a catalyst. Thickness, density, opacity, moisture content as well as surface moisture were evaluated.The data given in this study  shows thp ≤ 0.05).Equal letters in the same row indicate no statistically significant differences  ), plantain flour + PCL  + catalyst +CAT), plantain flour + PCL  ) and plantain flour + PCL  + catalyst +CAT).Film systems: plantain flour (TPPF), plantain flour + PCL , plantain flour + PCL  ), plantain flour + PCL  + catalyst +CAT), plantain flour + PCL  ) and plantain flour + PCL  + catalyst +CAT). The resulting materials were conditioned with a saturated NaBr solution (aw ∼0.575 at 25 °C) for seven days prior to each test.The systems characterized here are the active eco-friendly films that were studied in 2.12.1.1®, Mitutoyo, USA) with 0.001 mm accuracy. Measurements were taken from eighteen random positions on each sample. Results were reported as the average thickness ± standard deviation (SD). These mean values were used to determine the density and opacity as described in the following sections.Film thickness was determined using a digital micrometer (Micromaster2.1.2ρ) was determined by cutting samples of each film type into 6 mm radius (r) discs. The thickness (e) of the discs was then determined by taking 18 random measurements of each one. Afterwards, the discs were dried at 105 °C for 24 h and weighed. The density was then calculated as the ratio between the weight and volume (thickness × area) of each disc using the equation proposed by Gutiérrez Wi is the initial dry weight, and Wf the final dry weight.Film density (Results were reported as the density ± SD from three measurements.2.1.3A600 = the absorbance at 600 nm and “e” = film thickness (mm).The opacity was determined according to the method proposed by Sukhija et al. 2.1.42) were weighed using an analytical balance  in order to determine their initial mass (mi). The samples were then placed in an oven at 105 ± 1 °C  for 24 h until constant weight  was reached. All the tests were performed in triplicate, and the means ± SD were reported. Moisture content was determined by the following equation:Moisture content of the films was calculated gravimetrically. For this, square-shaped samples . Square shaped samples (2 × 2 cm) were dried at 105 °C for 120 s. Measurements were then conducted in triplicate for each film and the results were reported as % of average surface moisture ± SD.


Escherichia coli non-O157 isolates with a matching pulsed-field gel electrophoresis (PFGE) pattern combination that was new to the PulseNet Canada database. The patients resided in three geographically distinct provinces. In January 2017, the Public Health Agency of Canada (PHAC) initiated an investigation with local, provincial, and federal partners to investigate the source of the outbreak.On December 29, 2016, PulseNet Canada identified a cluster of six E. coli non-O157 with the outbreak PFGE pattern or closely related by whole genome sequencing (WGS) in a Canadian resident or visitor with onset of symptoms of gastroenteritis on or after November 1, 2016. Patients’ illness onset dates ranged from November 2016 to April 2017 (E. coli O121:H19 (one case was typed as E. coli O121:H undetermined) with Shiga toxin 2–producing genes by in silico toxin testing and had closely related PFGE patterns and WGS.A case was defined as isolation of ril 2017 . As of ME. coli O121 flour-associated outbreak prompted interviewers to ask about baking and exposure to raw flour or dough  reported that the implicated brand of flour was used or probably used in the home during the exposure period. Comparison data on the expected proportion with exposure to this brand of flour were not available. Eleven of these sixteen patients reported they ate or probably ate raw dough during their exposure period.E. coli infections identified in Canada and the first Canadian outbreak linked to flour. An open-ended interview approach and flour sampling were used to implicate flour as the source. Because of the recent emergence of E. coli outbreaks linked to flour, public health professionals should consider flour as a possible source in E. coli outbreaks and communicate the risk associated with exposure to flour, raw batter, and dough in public health messaging.This is the first national outbreak of non-O157 Shiga toxin–producing


Autophagy is a highly conserved lysosomal degradation pathway with major impact on diverse human pathologies. Despite the development of different methodologies to detect autophagy both in vitro and in vivo, monitoring autophagy in tissue via immunohistochemical techniques is hampered due to the lack of biomarkers. Immunohistochemical detection of a punctate pattern of ATG8/MAP1LC3 proteins is currently the most frequently used approach to detect autophagy in situ, but it depends on a highly sensitive detection method and is prone to misinterpretation. Moreover, reliable MAP1LC3 immunohistochemical staining requires correct tissue processing and high-quality, isoform-specific antibodies. Immunohistochemical analysis of other autophagy-related protein targets such as SQSTM1, ubiquitin, ATG5 or lysosomal proteins is not recommended as marker for autophagic activity in tissue for multiple reasons including aspecific labeling of cellular structures and a lack of differential protein expression during autophagy initiation. To better understand the role of autophagy in human disease, novel biomarkers for visualization of the autophagic process with standard histology techniques are urgently needed.  AuTophaGy-related (ATG) genes [Macroautophagy (hereafter referred to as autophagy) is a normal physiological process in the body that maintains intracellular homeostasis by degrading unnecessary or dysfunctional cellular components in lysosomes . The proG) genes .A large body of evidence indicates that autophagy is involved in the prevention of a wide range of human pathological conditions such as cardiovascular disease , neurodeMammalian autophagy-related 8 (ATG8) proteins are ubiquitin-like proteins that are ubiquitously expressed, although some subfamily members are expressed at increased levels in certain tissues, where they participate in multiple cellular processes such as intracellular membrane trafficking and autophagy . They coIn mammalian cells, at least three different MAP1LC3 isoforms are known: MAP1LC3A, MAP1LC3B, and MAP1LC3C. They are produced as a C-terminally extended precursor, which is rapidly cleaved by the cysteine protease ATG4B to yield the cytosolic form MAP1LC3-I ,20. AfteIn most mammalian tissues—including liver, heart, spleen, and lung—MAP1LC3 is a fragile low-abundance protein making its immunohistochemical detection quite challenging. Fortunately, many tumor cells tend to express higher MAP1LC3 levels as compared to adjacent nonmalignant tissue ,28,29, bFor several reasons, immunohistochemical analysis of MAP1LC3 rapidly leads to false conclusions as outlined in more detail below, and thus should be interpreted with caution. Firstly, detection of autophagosomes by immunostaining of MAP1LC3 is technically challenging because of the high background from MAP1LC3-I unassociated with autophagosomes. MAP1LC3-I is diffusely spread in the cell and may yield cytoplasmic staining without clear formation of MAP1LC3-positive puncta. Therefore, diffuse cellular staining may not reflect autophagosome staining, but simply represents MAP1LC3-I protein expression. Granular MAP1LC3 staining is sometimes falsely interpreted as autophagosome formation, but may result from nonspecific staining of cytoplasmic components or from the incorporation of MAP1LC3 into lipofuscin or other lysosomal residual bodies . SecondlFor optimal staining results, tissue samples should be fixed in neutral-buffered formalin (4–10%) for a minimum of 24 h prior to paraffin embedding ,24. SampSequestosome 1 (SQSTM1), also known as p62, is a ubiquitously expressed cellular protein that serves as an autophagic cargo adaptor. It is capable of binding to both ubiquitinated and MAP1LC3 proteins, via the ubiquitin-associated (UBA) domain and MAP1LC3-interacting region (LIR), respectively . BecausePolyubiquitinated aggregates of misfolded or damaged proteins in the cytosol are recruited to autophagosomes for subsequent degradation via autophagy and were therefore frequently used in the early 2000s as markers for autophagic cell death during heart failure ,63 or inHitherto, detection of MAP1LC3 punctae remains the most reliable method to monitor autophagy via immunohistochemistry B. Howeve


The results indicated that CCNPs had relatively regular chain-like structure, and the size of the crystallites was around 62.5 nm. FT-IR analysis indicated that the GS could effectively load onto CCNPs. Meanwhile, the dosage of CCNPs would affect the drug loading and entrapment efficiency of GS. CCNPs could prolong the release of GS, and the complete release of GS from CCNPs was extended up to 24 h. Additionally, CCNPs could obviously increase the antibacterial effect of GS. The zeta potential analysis and microscopic investigations indicated that the adsorbed CCNPs could increase the damage level of bacterial cell wall and enhance the permeability of cell membranes, leading to increased bacterial death.In the present study, we used CaCO As a water-soluble aminoglycoside antibiotic with potent broad-spectrum antibacterial activity  mainly consists of three polymorphs, named calcite, aragonite, and vaterite  are widely used in coating, plastic, paper, cosmetics, medicine, and food industries because of its characteristics of small size and high specific surface area , and the physicochemical properties and antimicrobial effect have been evaluated  was selected as the target bacterium. Our data provided valuable insights into the real application of CaCO3 as drug carrier.Previous studies have incorporated two antibiotics (metronidazole and gentamicin) into the non-sintered porous synthetic carbonate as the drug-carrier without alteration or degradation  and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. All the chemicals were of reagent grade.GS was supplied from J&K Co. . Calcium hydroxide [Ca(OH)3 particles and reduce the reaction time The morphology of synthesized CCNPs was characterized on a Hitachi S-4800 scanning electron microscope (SEM) and a JEM-2010 transmission electron microscope (TEM). Meanwhile, the synthesized CCNPs were identified by X-ray powder diffraction (XRD) analysis, and the patterns of samples were recorded using a PANalytical X'Pert PRO diffractometer with Cu-Kα radiation  in a continuous scanning mode. The 2θ scanning ranged from 5 to 85° in steps of 0.008° with a collection time of 50 s per step. The average size of NPs was calculated from the peak broadening using the Scherrer equation:d is the crystallite size; λ is the X-ray wavelength; Cu Kα λ = 0.15418 nm; B is the width of the line at the half-maximum intensity (in radians); and θ is the half of the diffraction angle  was added to the solution, and the mixture was mildly stirred at 300 rpm. The resultant CGPs were obtained by centrifugation at 12,000 rpm for 5 min.−1 at a spectral resolution of 4 cm−1 , and the baseline was corrected.Moreover, the prepared CCNPs, GS, and CGPs were analyzed by Fourier-transform infrared spectroscopy (FT-IR). Briefly, the samples were prepared in a KBr pellet with a sample/KBr ratio of 2:100, a thin pellet was placed in the IR beam on FT-IR spectrometer  within a range of 4,000–500 cm2O was then added to the flask, and the mixture was placed in water bath at 37°C for 10 min and finally diluted with water to volume. The GS had the maximum absorbance at 232 nm, and the standard curve was shown in Figure The standard curve of GS dispersion in distilled water was examined by using UV/VIS spectrophotometer (Perkin-Elmer Lamda 35). The concentration of GS ranged from 0.25 to 2.0 g/L, and the sample was completely dissolved using magnetic stirrer. Then 1 mL solution was transferred into a 50-mL volumetric flask, 0.5 mL HBriefly, 1 mL of sulfuric acid was added into CGPs, the CGPs was completely dissolved after incubation in water bath for 10 min, and then the amount of loaded GS was detected by a UV/VIS spectrophotometer at 232 nm and quantified from standard curve.The percentages of drug loading and entrapment efficiency were calculated according to the following equations:m0 is the actual mass of the added GS; m1 is the mass of the GS on CGPs; and m2 is the mass of CCNP carrier.Where B. subtillis used in whole experiment was stocked in our laboratory. The liquid Luria–Bertani medium  consisted of 10 g/L NaCl, 10 g/L tryptone, and 5 g/L yeast extract, and the solid LB medium contained extra 20 g/L agar powder in addition to liquid LB medium. Subsequently, 1% B. subtillis in glycerine was inoculated into liquid LB medium at 37°C. After shaking at 180 rpm for 24 h, bacterial suspension was centrifuged at 6,000 rpm for 5 min and then washed with Tris-HCl twice to remove excess reactants.The 2O) was prepared as control, and bacteria-absent groups with the same other parameters were used as background in order to remove turbidity from CCNPs and GS. The mixture was incubated at 37°C on a shaker at 160 rpm. The OD-value at a wavelength of 600 nm was recorded by using UV/VIS spectrophotometer in order to detect the bacterial turbidity after 24-h incubation, and the inhibition rate (η) was calculated as follows:The antibacterial activity of CCNPs, GS, and CGPs was determined at a GS concentration of 0.1 g/L. GS, CCNPs, and GCPs were added to the solution containing the same volume of bacteria. Double distilled water  for 24 h and washed in the phosphate buffer  for three times (15 min for each). Then samples were post-fixed with 1% OsOMoreover, the confocal laser scanning microscopy was also applied to observe the bacteria. LIVE/DEAD Bac Light Bacterial Viability Kit (L7012) was used in our experiment. After exposure to CCNPs, GS, and CGPs for 12 h, the bacteria were collected and incubated with 10 μL Syto 9 at 37°C for 10 min . Subsequently, the bacterial suspension was centrifuged at 5,000 g for 5 min at 4°C, and the supernatant was discarded. Then the samples were resuspended in 1.0 mL PBS and incubated with 10 μL propidium iodide (PI) for 10 min in the dark . The unincorporated dyes were removed by washing with PBS. One droplet of cell suspension (5 μL) was dropped on the freshly treated glass slide, and then it was covered with coverslip. Finally, the glass piece was sealed using the petroxolin. The samples were recorded using a confocal laser scanning microscope (Leica SP8).p-value of < 0.05 was considered as statistically significant.All data were expressed as mean ± standard deviation (SD). Data were arranged with Microsoft Excel 2010 and analyzed with SPSS 18.0 by one-way ANOVA. A 3 as a nanometer-level particle. Such structure could help particles disperse GS more evenly.Rhombohedron is the dominant morphology of calcite polymorph, and truncated prismatic, scalenohedral, spherical, or chain-like agglomerates also exist in calcite  . The spectrum of CGPs showed most characteristic bands of GS and CCNPs, which indicated the effective loading of drug onto CCNPs. It was noticed that the bands at 1,631 and 1,535 cm−1 were gradually disappeared and a new band at 1,421 cm−1 was appeared, thus we speculated that hydrogen bonding interaction might exist between the drug and carrier during the preparation process.The possible interaction between GS and CCNPs was investigated by FT-IR spectroscopy. Figure In order to evaluate the usability of CCNPs as the nano-based carriers, we assessed the drug loading and entrapment efficiency in our drug delivery system. Table The drug release was determined by the dissolution testing under the controlled conditions. Figure B. subtillis with an inhibition rate of 22.84%. However, GS significantly inhibited B. subtillis after loading onto CCNPs, showing an inhibition rate of 69.79%. This finding indicated that GS was successfully incorporated into the matrix of CCNPs, and such incorporation not only maintained its biological effect, but also increased the antibacterial activity to a great extent. Additionally, the solid agar plate experiment also confirmed that CGPs had a significant antibacterial efficiency (Figure B. subtillis could be ranked as follows: GS:CCNPs = 2:3>GS:CCNPs = 1:2 ≈GS:CCNPs = 1:1>GS>CCNPs. Taken together, CCNPs could increase the antibacterial activity of GS.Moreover, we evaluated the antibacterial activity of GS, CCNPs, and CGPs in liquid medium or solid agar plate. Table B. subtillis for 12 h, all the three mixtures still carried negative charges. This result indicated that the binding of CCNPs, GS, and CGPs with bacteria could not be driven by the electrostatic interactions, but might by intermolecular forces.In order to explore the synergistic antibacterial mechanism of CCNPs, we further examined the ZPs of CCNPs, GS, and CGPs before and after exposure to bacteria. Table Although previous studies have also incorporated the drugs into CCNPs to develop the efficient delivery system, researchers focus on the physicochemical characterization and antimicrobial evaluation, while the microscopic investigations are rarely conducted. In the present study, we performed TEM and confocal microscopy in order to explore the detailed antibacterial mechanism. Figure Meanwhile, the bacteria were also examined by fluorescence microscope. Both Syto-9 and PI were used to reveal the changes of the viability and membrane integrity of bacterial cells. PI can only penetrate cells with disrupted membranes, showing intracellular staining of red fluorescence (George et al., Based on the above-mentioned analyses, we proposed the possible synergistic antibacterial mechanism of CGPs Figure . First, 3.It is critical to develop an effective targeted delivery of antibiotics in recent years. In the present study, CCNPs were used as carriers to incorporate GS, and physicochemical characterization and antibacterial effect of CGPs were evaluated. Our results indicated that CCNPs could prolong the release of GS and increase the antibacterial activity of GS. Moreover, the synergistic antibacterial mechanism of CGPs was proposed through microscopic investigations. Taken together, CCNPs could be used as potential carriers in drug delivery system, and our findings offered valuable insights into the real application of nano-CaCOXP designed the study, analyzed the data, and wrote the text. SC participated in the manuscript preparation. DL, WR, YZ, ZY, and LL performed the related experiments, and the sequential arrangement reflects their workload. XG and ZC participated in the manuscript preparation. All authors contributed to its final form and gave final approval for its publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.