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Herpes simplex virus 1 and 2 infections affect up to 50 million people in the United States, with a natural history of recurrent viral shedding with or without recurrence of symptoms. Although many patients remain asymptomatic or with mild symptoms, a spectrum of rare but significant nervous system complications have been reported. Although urinary retention and constipation associated with genital herpesvirus infections is often attributed to painful genital ulcerations, herpesvirus-associated lumbosacral myeloradiculitis has been reported in adults. Here, we report an 18-year-old man with constipation, urinary retention, perineal paresthesias, and erectile dysfunction in the setting of a genital herpes infection. His workup was notable for a cerebrospinal fluid pleocytosis and MRI with enhancement of the cauda equina and nerve roots, all of which are consistent with sacral myeloradiculitis. The patient was treated with a 3-week course of intravenous acyclovir with complete resolution of symptoms. Pediatric practitioners should be aware of this complication of anogenital herpes simplex virus infection because appropriate diagnosis has implications for treatment delivery and duration., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2019 by the American Academy of Pediatrics.)

Flavin-based electron bifurcation allows enzymes to redistribute energy among electrons by coupling endergonic and exergonic electron transfer reactions. Diverse bifurcating enzymes employ a two-flavin electron transfer flavoprotein (ETF) that accepts hydride from NADH at a flavin (the so-called bifurcating FAD, Bf-FAD). The Bf-FAD passes one electron exergonically to a second flavin thereby assuming a reactive semiquinone state able to reduce ferredoxin or flavodoxin semiquinone. The flavin that accepts one electron and passes it on via exergonic electron transfer is known as the electron transfer FAD (ET-FAD) and is believed to correspond to the single FAD present in canonical ETFs, in domain II. The Bf-FAD is believed to be the one that is unique to bifurcating ETFs, bound between domains I and III. This very reasonable model has yet to be challenged experimentally. Herein we used site-directed mutagenesis to disrupt FAD binding to the presumed Bf site between domains I and III, in the Bf-ETF from Rhodopseudomonas palustris ( Rpa ETF). The resulting protein contained only 0.80 ± 0.05 FAD, plus 1.21 ± 0.04 bound AMP as in canonical ETFs. The flavin was not subject to reduction by NADH, confirming absence of Bf-FAD. The retained FAD displayed visible circular dichroism (CD) similar to that of the ET-FAD of Rpa ETF. Likewise, the mutant underwent two sequential one-electron reductions forming and then consuming anionic semiquinone, reproducing the reactivity of the ET-FAD. These data confirm that the retained FAD in domain II corresponds the ET-FAD. Quantum chemical calculations of the absorbance and CD spectra of each of WT Rpa ETF's two flavins reproduced the observed differences between their CD and absorbance signatures. The calculations for the flavin bound in domain II agreed better with the spectra of the ET-flavin, and those calculated based on the flavin between domains I and III agreed better with spectra of the Bf-flavin. Thus calculations independently confirm the locations of each flavin. We conclude that the site in domain II harbours the ET-FAD whereas the mutated site between domains I and III is the Bf-FAD site, confirming the accepted model by two different tests., (This journal is © The Royal Society of Chemistry 2019.)

Electron bifurcation plays a key role in anaerobic energy metabolism, but it is a relatively new discovery, and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon Pyrobaculum aerophilum The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using nondenaturing MS, cross-linking, and homology modeling in which EtfA, -B, and -C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters. On the basis of analyses using transient absorption, EPR, and optical titrations with NADH or inorganic reductants with and without NAD + , we propose a catalytic cycle involving formation of an intermediary NAD + -bound complex. A charge transfer signal revealed an intriguing interplay of flavin semiquinones and a protein conformational change that gated electron transfer between the low- and high-potential pathways. We found that despite a common bifurcating flavin site, the proposed EtfABCX catalytic cycle is distinct from that of the genetically unrelated bifurcating NADH-dependent ferredoxin NADP + oxidoreductase (NfnI). The two enzymes particularly differed in the role of NAD + , the resting and bifurcating-ready states of the enzymes, how electron flow is gated, and the two two-electron cycles constituting the overall four-electron reaction. We conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETFs and can be applied to the large bifurcating ETF family.

Introduction: Procedural sedation for fracture reduction in the pediatric emergency department (ED) is a time-consuming process requiring multidisciplinary coordination. We implemented a quality improvement initiative aimed at (1) decreasing mean ED length of stay (LOS) for children with sedated long bone fracture reductions by 15% over 12 months and (2) improving interdisciplinary communication around procedural sedation., Methods: Pediatric emergency medicine fellows at a children's hospital designed and implemented an initiative targeting the efficiency of the sedation process. Interventions included a centralized sedation tracking board, a team member responsibility checklist, family handouts, early discharge initiatives, and postsedation review forms. We tracked progress via statistical process control charts and interdisciplinary communication by intermittent surveys., Results: Pediatric emergency medicine fellows performed 2,246 sedations during the study period. Mean LOS decreased from 361 to 340 minutes (5.8%) after implementation and demonstrated sustainability over the postintervention period. One hundred eight providers completed the preimplementation communication survey, with 58 and 64 completing surveys at 4 and 9 months postimplementation, respectively. The proportion reporting somewhat or strong satisfaction with communication increased from 68% at baseline to 86% at 4 months ( P = 0.02) and 92% at 9 months ( P < 0.001 versus baseline)., Conclusions: A quality improvement initiative created a sustainable process to reduce ED LOS for sedated reductions while improving satisfaction with interdisciplinary communication.

Recently, a variety of enzymes have been found to accept electrons from NAD(P)H yet reduce lower-potential carriers such as ferredoxin and flavodoxin semiquinone, in apparent violation of thermodynamics. The reaction is favorable overall, however, because these enzymes couple the foregoing endergonic one-electron transfer to exergonic transfer of the other electron from each NAD(P)H, in a process called "flavin-based electron bifurcation." The reduction midpoint potentials (E°s) of the multiple flavins in these enzymes are critical to their mechanisms. We describe methods we have found to be useful for measuring each of the E°s of each of the flavins in bifurcating electron transfer flavoproteins., (© 2019 Elsevier Inc. All rights reserved.)

We report a case of a patient presenting with fever, right lower leg swelling, and pain who was found to have a fluid collection between muscle planes noted on point-of-care ultrasound. Point-of-care ultrasound raised the clinician's concern for deep musculoskeletal infection, leading to prompt initiation of antibiotics and magnetic resonance imaging.

A recent strategy that has emerged for the design of increasingly functional hydrogels is the incorporation of nanofillers in order to exploit their specific properties to either modify the performance of the hydrogel or add functionality. The emergence of carbon nanomaterials in particular has provided great opportunity for the use of graphene derivatives (GDs) in biomedical applications. The key challenge when designing hybrid materials is the understanding of the molecular interactions between the matrix (peptide nanofibers) and the nanofiller (here GDs) and how these affect the final properties of the bulk material. For the purpose of this work, three gelling β-sheet-forming, self-assembling peptides with varying physiochemical properties and five GDs with varying surface chemistries were chosen to formulate novel hybrid hydrogels. First the peptide hydrogels and the GDs were characterized; subsequently, the molecular interaction between peptides nanofibers and GDs were probed before formulating and mechanically characterizing the hybrid hydrogels. We show how the interplay between electrostatic interactions, which can be attractive or repulsive, and hydrophobic (and π-π in the case of peptide containing phenylalanine) interactions, which are always attractive, play a key role on the final properties of the hybrid hydrogels. The shear modulus of the hydrid hydrogels is shown to be related to the strength of fiber adhesion to the flakes, the overall hydrophobicity of the peptides, as well as the type of fibrillar network formed. Finally, the cytotoxicity of the hybrid hydrogel formed at pH 6 was also investigated by encapsulating and culturing human mesemchymal stem cells (hMSC) over 14 days. This work clearly shows how interactions between peptides and GDs can be used to tailor the mechanical properties of the resulting hydrogels, allowing the incorporation of GD nanofillers in a controlled way and opening the possibility to exploit their intrinsic properties to design novel hybrid peptide hydrogels for biomedical applications.

Self-assembling peptide hydrogels offer a novel 3-dimensional platform for many applications in cell culture and tissue engineering but are not compatible with current methods of RNA isolation; owing to interactions between RNA and the biomaterial. This study investigates the use of two techniques based on two different basic extraction principles: solution-based extraction and direct solid-state binding of RNA respectively, to extract RNA from cells encapsulated in four β-sheet forming self-assembling peptide hydrogels with varying net positive charge. RNA-peptide fibril interactions, rather than RNA-peptide molecular complexing, were found to interfere with the extraction process resulting in low yields. A column-based approach relying on RNA-specific binding was shown to be more suited to extracting RNA with higher purity from these peptide hydrogels owing to its reliance on strong specific RNA binding interactions which compete directly with RNA-peptide fibril interactions. In order to reduce the amount of fibrils present and improve RNA yields a broad spectrum enzyme solution-pronase-was used to partially digest the hydrogels before RNA extraction. This pre-treatment was shown to significantly increase the yield of RNA extracted, allowing downstream RT-qPCR to be performed., Competing Interests: A. Saiani and A.F. Miller have financial interests in the company PeptiGelDesign which they co-founded. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

A newly recognized third fundamental mechanism of energy conservation in biology, electron bifurcation, uses free energy from exergonic redox reactions to drive endergonic redox reactions. Flavin-based electron bifurcation furnishes low-potential electrons to demanding chemical reactions, such as reduction of dinitrogen to ammonia. We employed the heterodimeric flavoenzyme FixAB from the diazotrophic bacterium Rhodopseudomonas palustris to elucidate unique properties that underpin flavin-based electron bifurcation. FixAB is distinguished from canonical electron transfer flavoproteins (ETFs) by a second FAD that replaces the AMP of canonical ETF. We exploited near-UV-visible CD spectroscopy to resolve signals from the different flavin sites in FixAB and to interrogate the putative bifurcating FAD. CD aided in assigning the measured reduction midpoint potentials ( E ° values) to individual flavins, and the E ° values tested the accepted model regarding the redox properties required for bifurcation. We found that the higher- E ° flavin displays sequential one-electron (1-e - ) reductions to anionic semiquinone and then to hydroquinone, consistent with the reactivity seen in canonical ETFs. In contrast, the lower- E ° flavin displayed a single two-electron (2-e - ) reduction without detectable accumulation of semiquinone, consistent with unstable semiquinone states, as required for bifurcation. This is the first demonstration that a FixAB protein possesses the thermodynamic prerequisites for bifurcating activity, and the separation of distinct optical signatures for the two flavins lays a foundation for mechanistic studies to learn how electron flow can be directed in a protein environment. We propose that a novel optical signal observed at long wavelength may reflect electron delocalization between the two flavins.

Nitroreductases (NRs) hold promise for converting nitroaromatics to aromatic amines. Nitroaromatic reduction rate increases with Hammett substituent constant for NRs from two different subgroups, confirming substrate identity as a key determinant of reactivity. Amine yields were low, but compounds yielding amines tend to have a large π system and electron withdrawing substituents. Therefore, we also assessed the prospects of varying the enzyme. Several different subgroups of NRs include members able to produce aromatic amines. Comparison of four NR subgroups shows that they provide contrasting substrate binding cavities with distinct constraints on substrate position relative to the flavin. The unique architecture of the NR dimer produces an enormous contact area which we propose provides the stabilization needed to offset the costs of insertion of the active sites between the monomers. Thus, we propose that the functional diversity included in the NR superfamily stems from the chemical versatility of the flavin cofactor in conjunction with a structure that permits tremendous active site variability. These complementary properties make NRs exceptionally promising enzymes for development for biocatalysis in prodrug activation and conversion of nitroaromatics to valuable aromatic amines. We provide a framework for identifying NRs and substrates with the greatest potential to advance., Competing Interests: The authors declare no conflict of interest. “The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results”.

Objectives: Describe the trends in pediatric sedation use over time and determine variation in use of procedural sedation across children's hospital emergency departments (EDs)., Methods: We analyzed ED data from 35 hospitals within the Pediatric Health Information System for patients <19 years old who received sedation medications and were discharged from 2009 to 2014. Patients with chronic comorbidities or undergoing intubation were excluded. We determined frequency and trends in use of sedation and compared these between EDs. Descriptive statistics with appropriate weighting were used., Results: Of the 1 448 011 patients potentially requiring sedation who presented to the ED, 99 951 (7.9%) underwent procedural sedation. Medication usage in 2014 included ketamine (73.7%), fentanyl and midazolam (15.9%), ketofol (7.3%), and propofol (2.7%). Use of fentanyl and midazolam increased, whereas use of ketamine, pentobarbital, etomidate, chloral hydrate, and methohexital decreased over time. Significant variation exists in the use of sedation across hospitals; in 2014, the sedation rate ranged 0.2% to 32.0%, with a median of 8.0%. The diagnosis with the largest variation in procedural sedation use was dislocation, with sedation rates ranging from 2% to 35%., Conclusions: There is significant variability across pediatric EDs in the use of procedural sedation, suggesting sedations may be performed too often or too little in some hospitals., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2018 by the American Academy of Pediatrics.)

Objectives: Data regarding clinical predictors of orbital fractures in children are limited. We sought to describe the epidemiology of pediatric orbital fractures and identify the signs and symptoms of orbital fractures in children., Methods: We performed a retrospective cohort study of children younger than 22 years who underwent a facial or orbital computed tomography (CT) scan to evaluate for orbital fracture. We included patients presenting to an emergency department of a tertiary care children's hospital between January 2009 and May 2013. The presence of an orbital fracture was assessed using the final interpretation of the CT by an attending radiologist in the electronic medical record., Results: Among 326 children who underwent facial or orbital CT during the study period, 133 (41%) had an orbital fracture. The presence of nausea or vomiting, orbital tenderness, swelling, or ecchymosis and limitation of extraocular movement were each associated with the presence of an orbital fracture (P < 0.05 for each). Twenty-two children (6.7% of cohort and 16.5% of children with orbital fractures) underwent surgical repair for an orbital fracture. In the absence of orbital tenderness, swelling, or ecchymosis, there was 1 child with an orbital fracture who underwent operative intervention., Conclusions: Fewer than half of children undergoing CT have an orbital fracture identified, and fewer than 7% have a fracture that requires operative intervention. In the absence of orbital tenderness, swelling, or ecchymosis, only 1 child had an orbital fracture requiring surgical repair.

Continuous optimization of in vitro analytical techniques is ever more important, especially given the development of new materials for tissue engineering studies. In particular, isolation of cellular components for downstream applications is often hindered by the presence of biomaterials, presenting a major obstacle in understanding how cell-matrix interactions influence cell behavior. Here, we describe an approach for western blot analysis of cells that have been encapsulated in self-assembling peptide hydrogels (SAPHs), which highlights the need for complete solubilization of the hydrogel construct. We demonstrate that both the choice of buffer and multiple cycles of sonication are vital in obtaining complete solubilization, thereby enabling the detection of proteins otherwise lost to SAP aggregation. Moreover, we show that the presence of self-assembling peptides (SAPs) does not interfere with the standard immunoblotting technique, offering the potential for use in more full-scale proteomic studies.

Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low- and high-potential electrons. It is the third recognized form of energy conservation in biology and was recently described for select electron-transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via Etf-quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer, as well as a non-redox-active AMP. However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatic and structural analyses. Etfs were identified in diverse archaea and bacteria, and they clustered into five distinct well-supported groups, based on their amino acid sequences. Gene neighborhood analyses indicated that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting that distinct conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH- and FAD-binding regions of bifurcating Etfs. Collectively, a new classification scheme for Etf proteins that delineates putative bifurcating versus nonbifurcating members is presented and suggests that Etf-mediated bifurcation is associated with surprisingly diverse enzymes. IMPORTANCE Electron bifurcation has recently been recognized as an electron transfer mechanism used by microorganisms to maximize energy conservation. Bifurcating enzymes couple thermodynamically unfavorable reactions with thermodynamically favorable reactions in an overall spontaneous process. Here we show that the electron-transferring flavoprotein (Etf) enzyme family exhibits far greater diversity than previously recognized, and we provide a phylogenetic analysis that clearly delineates bifurcating versus nonbifurcating members of this family. Structural modeling of proteins within these groups reveals key differences between the bifurcating and nonbifurcating Etfs., (Copyright © 2017 American Society for Microbiology.)

We report a case of a patient presenting with abdominal pain after cardiac surgery who was noted on point-of-care ultrasound (POCUS) to have pericardial and pleural effusion, in addition to ascites. The most notable findings were pleural and pericardial effusions, which combined with symptomatology met criteria for postpericardiotomy syndrome. Point-of-care ultrasound expedited the diagnosis of a pericardial effusion with impending tamponade and transfer for pericardiocentesis and placement of pericardial drain.

Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential ( i.e. intermediate reducing power), and each electron is passed to a different acceptor, one with lower and the other with higher reducing power, leading to "bifurcation." It is believed that a strongly reducing semiquinone species is essential for this process, and it is expected that this species should be kinetically short-lived. We now demonstrate that the presence of a short-lived anionic flavin semiquinone (ASQ) is not sufficient to infer the existence of bifurcating activity, although such a species may be necessary for the process. We have used transient absorption spectroscopy to compare the rates and mechanisms of decay of ASQ generated photochemically in bifurcating NADH-dependent ferredoxin-NADP + oxidoreductase and the non-bifurcating flavoproteins nitroreductase, NADH oxidase, and flavodoxin. We found that different mechanisms dominate ASQ decay in the different protein environments, producing lifetimes ranging over 2 orders of magnitude. Capacity for electron transfer among redox cofactors versus charge recombination with nearby donors can explain the range of ASQ lifetimes that we observe. Our results support a model wherein efficient electron propagation can explain the short lifetime of the ASQ of bifurcating NADH-dependent ferredoxin-NADP + oxidoreductase I and can be an indication of capacity for electron bifurcation.

The biological reduction of dinitrogen (N 2 ) to ammonia (NH 3 ) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (E m = -320 mV) coupled to reduction of flavodoxin semiquinone (E m = -460 mV) and reduction of coenzyme Q (E m = 10 mV). Knocking out fix genes rendered Δrnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.

Positron emission tomography (PET) and fluorescence labelling have been used to assess the pharmacokinetics, biodistribution and eventual fate of a hydrogel-forming nonapeptide, FEFKFEFKK (F9), in healthy mice, using 18 F-labelled and fluorescein isothiocyanate (FITC)-labelled F9 analogues. F9 was site-specifically radiolabelled with 2-[ 18 F]fluoro-3-pyridinecarboxaldehyde ([ 18 F]FPCA) via oxime bond formation. [ 18 F]FPCA-F9 in vivo fate was evaluated both as a solution, following intravenous administration, and as a hydrogel when subcutaneously injected. The behaviour of FITC-F9 hydrogel was assessed following subcutaneous injection. [ 18 F]FPCA-F9 demonstrated high plasma stability and primarily renal excretion; [ 18 F]FPCA-F9 when in solution and injected into the bloodstream displayed prompt bladder uptake (53.4 ± 16.6 SUV at 20 minutes postinjection) and rapid renal excretion, whereas [ 18 F]FPCA-F9 hydrogel, formed by co-assembly of [ 18 F]FPCA-F9 monomer with unfunctionalised F9 peptide and injected subcutaneously, showed gradual bladder accumulation of hydrogel fragments (3.8 ± 0.4 SUV at 20 minutes postinjection), resulting in slower renal excretion. Gradual disaggregation of the F9 hydrogel from the site of injection was monitored using FITC-F9 hydrogel in healthy mice (60 ± 3 over 96 hours), indicating a biological half-life between 1 and 4 days. The in vivo characterisation of F9, both as a gel and a solution, highlights its potential as a biomaterial., (Copyright © 2017 The Authors Journal of Labelled Compounds and Radiopharmaceuticals Published by John Wiley & Sons Ltd.)

The catalytic active site of Mn-specific superoxide dismutase (MnSOD) is organized around a redox-active Mn ion. The most highly conserved difference between MnSODs and the homologous FeSODs is the origin of a Gln in the second coordination sphere. In MnSODs it derives from the C-terminal domain whereas in FeSODs it derives from the N-terminal domain, yet its side chain occupies almost superimposable positions in the active sites of these two types of SODs. Mutation of this Gln69 to Glu in Escherichia coli FeSOD increased the Fe 3+/2+ reduction midpoint potential by >0.6 V without disrupting the structure or Fe binding [ Yikilmaz, E., Rodgers, D. W., and Miller, A.-F. ( 2006 ) Biochemistry 45 ( 4 ), 1151 - 1161 ]. We now describe the analogous Q146E mutant of MnSOD, explaining its low Mn content in terms increased stability of the apo-Mn protein. In 0.8 M guanidinium HCl, Q146E-apoMnSOD displays an apparent melting midpoint temperature (T m ) 35 °C higher that of wild-type (WT) apoMnSOD, whereas the T m of WT-holoMnSOD is only 20 °C higher than that of WT-apoMnSOD. In contrast, the T m attributed to Q146E-holoMnSOD is 40 °C lower than that of Q146E-apoMnSOD. Thus, our data refute the notion that the WT residues optimize the structural stability of the protein and instead are consistent with conservation on the basis of enzyme function and therefore ability to bind metal ion. We propose that the WT-MnSOD protein conserves a destabilizing amino acid at position 146 as part of a strategy to favor metal ion binding.

Nitroreductase (NR) from Enterobacter cloacae reduces diverse nitroaromatics including herbicides, explosives, and prodrugs, and holds promise for bioremediation, prodrug activation, and enzyme-assisted synthesis. We solved crystal structures of NR complexes with bound substrate or analog for each of its two half-reactions. We complemented these with kinetic isotope effect (KIE) measurements elucidating H-transfer steps essential to each half-reaction. KIEs indicate hydride transfer from NADH to the flavin consistent with our structure of NR with the NADH analog nicotinic acid adenine dinucleotide (NAAD). The KIE on reduction of p-nitrobenzoic acid (p-NBA) also indicates hydride transfer, and requires revision of prior computational mechanisms. Our mechanistic information provided a structural restraint for the orientation of bound substrate, placing the nitro group closer to the flavin N5 in the pocket that binds the amide of NADH. KIEs show that solvent provides a proton, enabling accommodation of different nitro group placements, consistent with the broad repertoire of NR., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The recently realized biochemical phenomenon of energy conservation through electron bifurcation provides biology with an elegant means to maximize utilization of metabolic energy. The mechanism of coordinated coupling of exergonic and endergonic oxidation-reduction reactions by a single enzyme complex has been elucidated through optical and paramagnetic spectroscopic studies revealing unprecedented features. Pairs of electrons are bifurcated over more than 1 volt of electrochemical potential by generating a low-potential, highly energetic, unstable flavin semiquinone and directing electron flow to an iron-sulfur cluster with a highly negative potential to overcome the barrier of the endergonic half reaction. The unprecedented range of thermodynamic driving force that is generated by flavin-based electron bifurcation accounts for unique chemical reactions that are catalyzed by these enzymes.

Functionalized membranes provide versatile platforms for the incorporation of biocatalysts and nanostructured materials for efficient and benign environmental remediation. The existing techniques for remediating chloro-organics in water consist of both physical and chemical means mostly using metal oxide-based catalysts, despite associated environmental concerns. To offer bioinspired remediation as an alternative, we herein demonstrate a layer-by-layer approach to immobilize laccase enzyme onto pH-responsive functionalized membranes for the degradation of chloro-organics in water. The efficacy of these bioinspired membranes toward dechlorination of 2,4,6-trichlorophenol (TCP) is demonstrated under a pressure-driven continuous flow mode (convective flow) for the first time to the best of our knowledge. Over 80% of the initial TCP was degraded at an optimum flow rate under an applied air pressure of about 0.7 bar or lower. This corresponds to degradation of a substantial amount of the initial substrate in only 36 s residence time, whereas it takes hours for degradation in a batch reaction. This, in fact, demonstrates an energy efficient flow-through system with potentially large-scale applications. Comparison of the stability of the enzyme in the solution phase versus immobilized on the membrane phase showed a loss of some 65% of enzyme activity in the solution phase after 22 d, whereas the membrane-bound enzyme lost only a negligible percentage of the activity in a comparable time span. Finally, the membrane was exposed to rigorous cycles of TCP degradation trials to study its reusability. The primary results reveal a loss of only 14% of the initial activity after 4 cycles of use in a period of 25 d, demonstrating its potential to be reused. Regeneration of the functionalized membrane was also validated by dislodging the immobilized enzyme, followed by immobilization of fresh enzyme onto the membrane.

Self-assembling peptide-based hydrogels have encountered increasing interest in the recent years as scaffolds for 3D cell culture or for controlled drug delivery. One of the main challenges is the fine control of the mechanical properties of these materials. The bulk properties of hydrogels not only depend on the intrinsic properties of the fibers but also on the network topology formed. In this work we show how fiber-fiber interactions can be manipulated by design to control the final hydrogel network topology and therefore control the final properties of the material. This was achieved by exploiting the design features of β-sheet forming peptides based on hydrophobic and hydrophilic residue alternation and exploiting the ability of the arginine's guanidine side group to interact with itself and with other amino acid side groups. By designing octa-peptides based on phenylalanine, glutamic acid, lysine, and arginine, we have investigated how fiber association and bundling affect the dynamic shear modulus of hydrogels and how it can be controlled by design. This work opens the possibility to fine-tune by design the bulk properties of peptide hydrogels.

Peptide-based hydrogels have attracted significant interest in recent years as these soft, highly hydrated materials can be engineered to mimic the cell niche with significant potential applications in the biomedical field. Their potential use in vivo in particular is dependent on their biocompatibility, including their potential to cause an inflammatory response. In this work, we investigated in vitro the inflammatory potential of a β-sheet forming peptide (FEFEFKFK; F: phenylalanine, E: glutamic acid; K: lysine) hydrogel by encapsulating murine monocytes within it (3D culture) and using the production of cytokines, IL-β, IL-6 and TNFα, as markers of inflammatory response. No statistically significant release of cytokines in our test sample (media + gel + cells) was observed after 48 or 72 h of culture showing that our hydrogels do not incite a pro-inflammatory response in vitro. These results show the potential biocompatibility of these hydrogels and therefore their potential for in vivo use. The work also highlighted the difference in monocyte behaviour, proliferation and morphology changes when cultured in 2D vs. 3D. © 2016 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd., (© 2016 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

Nuclear magnetic resonance is applied to investigate the relative positions and interactions between cationic and non-ionic carbohydrate-based surfactants in mixed micelles with D 2 O as the solvent. This is accomplished by using relaxation measurements [spin-lattice (T 1 ) and spin-spin (T 2 ) analysis] and nuclear Overhauser effect spectroscopy (NOESY). This study focuses on the interactions of n-octyl β-d-glucopyranoside (C8G1) and β-d-xylopyranoside (C8X1) with the cationic surfactant hexadecyltrimethylammonium bromide (C 16 TAB). Whereas the interactions between carbohydrate and cationic surfactants are thermodynamically favorable, the NOESY results suggest that both of the sugar head groups are located preferentially at the interior core of the mixed micelles, so that they are not directly exposed to the bulk solution. The more hydrophilic sugar headgroups of C8G1 have more mobility than sugar heads of C8X1 owing to increased hydration. Herein, an inverted carbohydrate configuration in mixed micelles is proposed for the first time and supported by fluorescence spectroscopy experiments. This inverted carbohydrate headgroup configuration would limit the use of these mixed surfactants when access to the carbohydrate headgroup is important, but may present new opportunities where the carbohydrate-rich core of the micelles can be exploited., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An attractive strategy for the regeneration of tissues has been the use of extracellular matrix analogous biomaterials. Peptide-based fibrillar hydrogels have been shown to mimic the structure of extracellular matrix offering cells a niche to undertake their physiological functions. In this study, the capability of an ionic-complementary peptide FEFEFKFK (F, E, and K are phenylalanine, glutamic acid, and lysine, respectively) hydrogel to host human mesenchymal stem cells in three dimensions and induce their osteogenic differentiation is demonstrated. Assays showed sustained cell viability and proliferation throughout the hydrogel over 12 days of culture and these human mesenchymal stem cells differentiated into osteoblasts simply upon addition of osteogenic stimulation. Differentiated osteoblasts synthesized key bone proteins, including collagen-1 (Col-1), osteocalcin, and alkaline phosphatase. Moreover, mineralization occurred within the hydrogel. The peptide hydrogel is a naturally biodegradable material as shown by oscillatory rheology and reversed-phase high-performance liquid chromatography, where both viscoelastic properties and the degradation of the hydrogel were monitored over time, respectively. These findings demonstrate that a biodegradable octapeptide hydrogel can host and induce the differentiation of stem cells and has the potential for the regeneration of hard tissues such as alveolar bone.

Fabrication of bio-electrode systems decorated with redox active biomolecules, flavins, is demonstrated. Exploiting the photochemistry and electrochemistry of flavins, we explored the photo-electrochemical activity of flavin-functionalized electrode systems to assess their potential utility for sustainable energy production. As model systems, lumiflavin and flavin adenine dinucleotide were immobilized on carbon electrodes by dropcasting and covalent grafting techniques. Activity of these bio-electrodes towards generation of O2 from H2O in 0.5 M potassium phosphate buffer at pH 7.1 was demonstrated. Irradiation of the electrode system with visible light led to increased activity of the electrodes with a 3-fold enhancement of oxidation of H2O.

β-Sheet forming peptides have attracted significant interest for the design of hydrogels for biomedical applications. One of the main challenges is the control and understanding of the correlations between peptide molecular structure, the morphology, and topology of the fiber and network formed as well as the macroscopic properties of the hydrogel obtained. In this work, we have investigated the effect that functionalizing these peptides through their hydrophobic face has on their self-assembly and gelation. Our results show that the modification of the hydrophobic face results in a partial loss of the extended β-sheet conformation of the peptide and a significant change in fiber morphology from straight to kinked. As a consequence, the ability of these fibers to associate along their length and form large bundles is reduced. These structural changes (fiber structure and network topology) significantly affect the mechanical properties of the hydrogels (shear modulus and elasticity).

We report here the first experimental evidence of a self-assembling three-dimensional (3D) peptide hydrogel, with recognition motifs immobilized on the surface of fibres capable of sequence-specific oligonucleotide detection. These systems have the potential to be further developed into diagnostic and prognostic tools in human pathophysiology.

Electron bifurcation is the recently recognized third mechanism of biological energy conservation. It simultaneously couples exergonic and endergonic oxidation-reduction reactions to circumvent thermodynamic barriers and minimize free energy loss. Little is known about the details of how electron bifurcating enzymes function, but specifics are beginning to emerge for several bifurcating enzymes. To date, those characterized contain a collection of redox cofactors including flavins and iron-sulfur clusters. Here we discuss the current understanding of bifurcating enzymes and the mechanistic features required to reversibly partition multiple electrons from a single redox site into exergonic and endergonic electron transfer paths., (Copyright © 2016. Published by Elsevier Ltd.)

Two complementary β-sheet-forming decapeptides have been created that form binary self-repairing hydrogels upon combination of the respective free-flowing peptide solutions at pH 7 and >0.28 wt%. The component peptides showed little structure separately but formed extended β-sheet fibres upon mixing, which became entangled to produce stiff hydrogels. Microscopy revealed two major structures; thin fibrils with a twisted or helical appearance and with widths comparable to the predicted lengths of the peptides within a β-sheet, and thicker, longer, interwoven fibres that appear to comprise laterally-packed fibrils. A range of gel stiffnesses (G' from 0.05 to 100 kPa) could be attained in this system by altering the assembly conditions, stiffnesses that cover the rheological properties desirable for cell culture scaffolds. Doping in a RGD-tagged component peptide at 5 mol% improved 3T3 fibroblast attachment and viability compared to hydrogel fibres without RGD functionalisation.

We have investigated the effect of doping the self-assembling octapeptide FEFEFKFK (F, phenylalanine; E, glutamic acid; K, lysine) hydrogels with various amounts of thermoresponsive conjugate of FEFEFKFK and poly(N-isopropylacrylamide) (PNIPAAm) in order to create novel hydrogels. The samples were characterized using a range of techniques including microdifferential scanning calorimetry (μDSC), oscillatory rheology, transmission electron microscopy (TEM), atomic force microscopy (AFM), and small angle neutron scattering (SANS). The peptide from the conjugate was shown to be incorporated into the peptide fiber, resulting in the polymer being anchored to the peptide fiber. The conjugation of the polymer to the peptide and its anchoring to the peptide fibers did not affect its lower critical solution temperature (LCST). On the other hand, it did result in a decrease in the LCST enthalpy and a significant increase in the G' of the hydrogels, suggesting the presence of hydrogen bond interactions between the peptide and the polymer. As a result, the polymer was found to adopt a fibrillar arrangement tightly covering the peptide fiber. The polymer was still found to go through a conformational change at the LCST, suggesting that it collapses onto the peptide fiber. On the other hand, the fibrillar network was found to be mainly unaffected by the polymer LCST. These changes at the LCST were also found to be fully reversible. The nature of the interaction between the polymer and the peptide was shown to have a significant effect on the conformation adopted by the polymer around the fibers and the mechanical properties of the hydrogels.

Biomaterials that provide three-dimensional support networks for the culture of cells are being developed for a wide range of tissue engineering applications including the regeneration of bone. This study explores the potential of the versatile ionic-complementary peptide, FEFEFKFK, for such a purpose as this peptide spontaneously self-assembles into β-sheet-rich fibres that subsequently self-associate to form self-supporting hydrogels. Via simple live/dead cell assays, we demonstrated that 3 wt% hydrogels were optimal for the support of osteoblast cells. We went on to show that these cells are not only viable within the three-dimensional hydrogel but they also proliferate and produce osteogenic key proteins, that is, they behave like in vivo bone cells, over the 14-day period explored here. The gel elasticity increased over time when cells were present - in comparison to a decrease in control samples - indicating the deposition of matrix throughout the peptide scaffold. Moreover, significant quantities of calcium phosphate were deposited. Collectively, these data demonstrate that ionic-complementary octapeptides offer a suitable three-dimensional environment for osteoblastic cell function.

We have investigated the possibility of using enzymatically triggered peptide hydrogels for the encapsulation and culture of cells. Based on recent work done on the enzymatically triggered gelation of FEFK (F, phenylalanine; E, glutamic acid; K, lysine) using thermolysin, a protease enzyme from Bacillus Thermoproteolyticus Rokko, we have investigated the possibility of using this gelation triggering mechanism to encapsulate cells within a 3D hydrogel matrix. First, the properties of enzymatically triggered hydrogels prepared in phosphate buffer solution were investigated and compared with the properties of hydrogels prepared in HPLC grade water from our previous work. We showed that the use of phosphate buffer solution allowed the production of hydrogels with very high shear moduli (>1 MPa). The gelation kinetics was also investigated, and the mechanical properties of the system were shown to closely follow the synthesis of the octapeptide by the enzyme through reverse hydrolysis. In a second phase, we developed, on the basis of information acquired, a facile protocol for the encapsulation of cells and plating of the hydrogel. Human dermal fibroblasts were then used to exemplify the use of these materials. FEFEFKFK octapeptide hydrogels prepared under the same conditions and with the same mechanical properties were used as a control. We showed that no significant differences were observed between the two systems and that after a decrease in cell number on day 1, cells start to proliferate. After 5 days of culture, the cells can be seen to start to adopt a stretched morphology typical of fibroblasts. The results clearly show that the protocol developed minimises the potential detrimental effect that thermolysin can have on the cells and that these enzymatically triggered hydrogels can be used for the 3D encapsulation and culture of cells., (Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.)

Oligopeptides are well-known to self-assemble into a wide array of nanostructures including β-sheet-rich fibers that when present above a critical concentration become entangled and form self-supporting hydrogels. The length, quantity, and interactions between fibers influence the mechanical properties of the hydrogel formed and this is typically achieved by varying the peptide concentration, pH, ionic strength, or the addition of a second species or chemical cross-linking agent. Here, we outline an alternative, facile route to control the mechanical properties of the self-assembling octa-peptide, FEFEFKFK (FEKII); simply doping with controlled quantities of its double length peptide, FEFEFKFK-GG-FKFKFEFE (FEKII18). The structure and properties of a series of samples were studied here (0–100 M% of FEKII18) using Fourier transform infrared, small angle X-ray scattering, transmission electron microscopy, and oscillatory rheology. All samples were found to contain elongated, flexible fibers and all mixed samples contained Y-shaped branch points and parallel fibers which is attributed to the longer peptide self-assembling within two fibers, thus creating a cross-link in the network structure. Such behavior was reflected in an increase in the elasticity of the mixed samples with increasing quantity of double peptide. Interestingly the elastic modulus increased up to 30 times the pure FEKII value simply by adding 28 M% of FEKII18. These observations provide an easy, off-the-shelf method for an end-user to control the cross-linked network structure of the peptide hydrogel, and consequently strength of the hydrogel simply by physically mixing pre-determined quantities of two similar peptide molecules.

The oxygen-insensitive nitroreductase from Enterobacter cloacae (NR) catalyzes two-electron reduction of nitroaromatics to the corresponding nitroso compounds and, subsequently, to hydroxylamine products. NR has an unusually broad substrate repertoire, which may be related to protein dynamics (flexibility) and/or a simple non-selective kinetic mechanism. To investigate the possible role of mechanism in the broad substrate repertoire of NR, the kinetics of oxidation of NR by para-nitrobenzoic acid (p-NBA) were investigated using stopped-flow techniques at 4 °C. The results revealed a hyperbolic dependence on the p-NBA concentration with a limiting rate of 1.90 ± 0.09 s(-1), indicating one-step binding before the flavin oxidation step. There is no evidence for a distinct binding step in which specificity might be enforced. The reduction of p-NBA is rate-limiting in steady-state turnover (1.7 ± 0.3 s(-1)). The pre-steady-state reduction kinetics of NR by NADH indicate that NADH reduces the enzyme with a rate constant of 700 ± 20 s(-1) and a dissociation constant of 0.51 ± 0.04 mM. Thus, we demonstrate simple transient kinetics in both the reductive and oxidative half-reactions that help to explain the broad substrate repertoire of NR. Finally, we tested the ability of NR to reduce para-hydroxylaminobenzoic acid, demonstrating that the corresponding amine does not accumulate to significant levels even under anaerobic conditions. Thus E. cloacae NR is not a good candidate for enzymatic production of aromatic amines., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

In order to properly evaluate the ecological and human health risks of ZnO manufactured nanomaterials (MNMs) released to the environment, it is critical to understand the likely transformation products in various environments, such as soils, surface and ground waters, and wastewater treatment processes. To address this knowledge gap, we examined the transformation of 30 nm ZnO MNMs in the presence of different concentrations of phosphate as a function of time and pH using a variety of orthogonal analytical techniques. The data reveal that ZnO MNMs react with phosphate at various concentrations and transform into two distinct morphological/structural phases: a micrometer scale crystalline zinc phosphate phase (hopeite-like) and a nanoscale phase that likely consists of a ZnO core with an amorphous Zn3(PO4)2 shell. The P species composition was also pH dependent, with 82% occurring as hopeite-like P at pH 6 while only 15% occurred as hopeite-like P at pH 8. These results highlight how reactions of ZnO MNMs with phosphate are influenced by environmental variables, including pH, and may ultimately result in structurally and morphologically heterogeneous end products.

Enzymes are attractive, "green" alternatives to chemical catalysts within the industrial sector, but their robustness to environmental conditions needs optimizing. Here, an enzyme is tagged chemically and recombinantly with a self-assembling peptide that allows the conjugate to spontaneously assemble with pure peptide to form β-sheet-rich nanofibers decorated with tethered enzyme. Above a critical concentration, these fibers entangle and form a 3D hydrogel. The immobilized enzyme catalyzes chemical transformations and critically its stability is increased significantly where it retains activity after exposure to high temperatures (90 °C) and long storage times (up to 12 months)., (© 2014 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We have investigated the possibility of using self-assembling peptide-based viscous solutions and hydrogels as mucoadhesives for the improved delivery of drugs to local mucosal surfaces. The stability of the samples under flow after deposition on a mucosal surface mimic was studied using a simplified in vitro model. Subsequently lidocaine and flurbiprofen, two commercial drugs, were incorporated into the viscous solutions and hydrogels and their release properties investigated using the same model. Peptide-based hydrogels showed a good resistance to erosion under flow conditions. Addition of the soluble drug (lidocaine at low pH) resulted in a stiffening of the samples but did not affect the overall peptide release. Although for this drug the conditions were not favourable, improved retention of the drug was observed for the stiffest samples tested. In the case of the insoluble drug (flurbiprofen) the samples mechanical properties were not altered when the drug was incorporated, however the sample stability and peptide release were. For mechanically weaker samples the presence of the drug as insoluble small particles resulted in an increase in their susceptibility to physically erode when a flow of medium was applied over its surface. On the other hand mechanically stronger samples showed an improved resistance to erosion, which resulted in enhanced drug retention., (Copyright © 2014. Published by Elsevier B.V.)

Lolines are potent insecticidal agents produced by endophytic fungi of cool-season grasses. These alkaloids are composed of a pyrrolizidine ring system and an uncommon ether bridge linking carbons 2 and 7. Previous results indicated that 1-aminopyrrolizidine was a pathway intermediate. We used RNA interference to knock down expression of lolO, resulting in the accumulation of an alkaloid identified as exo-1-acetamidopyrrolizidine based on high-resolution MS and NMR. Genomes of endophytes differing in alkaloid profiles were sequenced, revealing that those with mutated lolO accumulated exo-1-acetamidopyrrolizidine but no lolines. Heterologous expression of wild-type lolO complemented a lolO mutant, resulting in the production of N-acetylnorloline. These results indicated that the non-heme iron oxygenase, LolO, is required for ether bridge formation, probably through oxidation of exo-1-acetamidopyrrolizidine., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Why apply solid-state NMR (SSNMR) to flavins and flavoproteins? NMR provides information on an atom-specific basis about chemical functionality, structure, proximity to other groups, and dynamics of the system. Thus, it has become indispensable to the study of chemicals, materials, catalysts, and biomolecules. It is no surprise then that NMR has a great deal to offer in the study of flavins and flavoenzymes. In general, their catalytic or electron-transfer activity resides essentially in the flavin, a molecule eminently accessible by NMR. However, the specific reactivity displayed depends on a host of subtle interactions whereby the protein biases and reshapes the flavin's propensities to activate it for one reaction while suppressing other aspects of this cofactor's prodigious repertoire (Massey et al., J Biol Chem 244:3999-4006, 1969; Müller, Z Naturforsch 27B:1023-1026, 1972; Joosten and van Berkel, Curr Opin Struct Biol 11:195-202, 2007). Thus, we are fascinated to learn about how the flavin cofactor of one enzyme is, and is not, like the flavin cofactor of another. In what follows, we describe how the capabilities of SSNMR can help and are beginning to bear fruit in this exciting endeavor.

The site-specific conjugation of DNA-binding protein (Tus) to self-assembling peptide FEFEFKFKK was demonstrated. Rheology studies and TEM of the corresponding hydrogels (including PNIPAAm-containing systems) showed no significant variation in properties and hydrogel morphology compared to FEFEFKFKK. Critically, we demonstrate that Tus is accessible within the gel network displaying DNA-binding properties.

We have recently shown that thermolysine, a protease enzyme obtained from Bacillus thermoproteolyticus rokko , can be used to trigger the gelation of FEFK (F, phenylalanine; E, glutamic acid; K, lysine) tetrapeptides through reverse hydrolysis and formation of longer peptide sequences, mainly octapeptides, that self-assemble readily. In this article we investigate the effect of enzyme concentration on the morphology and properties of enzymatically triggered peptide hydrogels using HPLC, FTIR, real-time SAXS, TEM, and shear rheology. We have shown that the enzyme concentration, Cenz, does not affect the final composition of the samples. Instead, this is dictated by the initial tetrapeptide concentration, C0, suggesting the existence of a chemical equilibrium. We went on to show that Cenz does not affect the self-assembly of these peptides at a molecular level either nor the structure of the fibrillar network formed at the nanometer scale. Interestingly, the mechanical properties were found to be affected by Cenz, where the shear moduli of the hydrogels were found to increase with increasing Cenz. These results suggest that morphological differences between the hydrogels at the microscale are at the origin of their difference in mechanical properties. In this paper, we propose a morphological model in which denser network regions are found around the enzymes, resulting in the creation of heterogeneous networks. These were confirmed by TEM measurements. The existence of these denser network regions will result in the reinforcement of the hydrogels, thus, explaining the high shear moduli obtained increasing Cenz.

The active-site structures of the oxidized and reduced forms of manganese-substituted iron superoxide dismutase (Mn(Fe)SOD) are examined, for the first time, using a combination of spectroscopic and computational methods. On the basis of electronic absorption, circular dichroism (CD), magnetic CD (MCD), and variable-temperature variable-field MCD data obtained for oxidized Mn(Fe)SOD, we propose that the active site of this species is virtually identical to that of wild-type manganese SOD (MnSOD), with both containing a metal ion that resides in a trigonal bipyramidal ligand environment. This proposal is corroborated by quantum mechanical/molecular mechanical (QM/MM) computations performed on complete protein models of Mn(Fe)SOD in both its oxidized and reduced states and, for comparison, wild-type (WT) MnSOD. The major differences between the QM/MM optimized active sites of WT MnSOD and Mn(Fe)SOD are a smaller (His)N-Mn-N(His) equatorial angle and a longer (Gln146(69))NH···O(sol) H-bond distance in the metal-substituted protein. Importantly, these modest geometric differences are consistent with our spectroscopic data obtained for the oxidized proteins and high-field electron paramagnetic resonance spectra reported previously for reduced Mn(Fe)SOD and MnSOD. As Mn(Fe)SOD exhibits a reduction midpoint potential (E(m)) almost 700 mV higher than that of MnSOD, which has been shown to be sufficient for explaining the lack of SOD activity displayed by the metal-subtituted species (Vance, C. K.; Miller, A. F. Biochemistry 2001, 40, 13079-13087), E(m)'s were computed for our experimentally validated QM/MM optimized models of Mn(Fe)SOD and MnSOD. These computations properly reproduce the experimental trend and reveal that the drastically elevated E(m) of the metal substituted protein stems from a larger separation between the second-sphere Gln residue and the coordinated solvent in Mn(Fe)SOD relative to MnSOD, which causes a weakening of the corresponding H-bond interaction in the oxidized state and alleviates steric crowding in the reduced state.

Nature has evolved a variety of creative approaches to many aspects of materials synthesis and microstructural control. Molecular self-assembly is a simple and efficient way to fabricate complex nanostructures such as hydrogels. We have recently investigated the gelation properties of a series of ionic-complementary peptides based on the alternation of non-polar hydrophobic and polar hydrophilic residues. In this work we focus on one specific octapeptide, FEFEFKFK (F, phenylalanine; E, glutamic acid; K, lysine). This peptide was shown to self-assemble in solution and form β-sheet-rich nanofibres which, above a critical gelation concentration, entangle to form a self-supporting hydrogel. The fibre morphology of the hydrogel was analysed using transmission electron microscopy and cryo-scanning electron microscopy illustrating a dense fibrillar network of nanometer size fibres. Oscillatory rheology results show that the hydrogel possesses visco-elastic properties. Bovine chondrocytes were used to assess the biocompatibility of the scaffolds over 21 days under two-dimensional (2-D) and three-dimensional (3-D) cell culture conditions, particularly looking at cell morphology, proliferation and matrix deposition. 2-D culture resulted in cell viability and collagen type I deposition. In 3-D culture the mechanically stable gel was shown to support the viability of cells, the retention of cell morphology and collagen type II deposition. Subsequently the scaffold may serve as a template for cartilage tissue engineering., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The thermal aggregation of the biopharmaceutical protein recombinant protective antigen (rPA) has been explored, and the associated kinetics and thermodynamic parameters have been extracted using optical and environmental scanning electron microscopies (ESEMs) and ultraviolet light scattering spectroscopy (UV-LSS). Visual observations and turbidity measurements provided an overall picture of the aggregation process, suggesting a two-step mechanism. Microscopy was used to examine the structure of aggregates, revealing an open morphology formed by the clustering of the microscopic aggregate particles. UV-LSS was used and developed to elucidate the growth rate of these particles, which formed in the first stage of the aggregation process. Their growth rate is observed to be high initially, before falling to converge on a final size that correlates with the ESEM data. The results suggest that the particle growth rate is limited by rPA monomer concentration, and by obtaining data over a range of incubation temperatures, an approach was developed to model the aggregation kinetics and extract the rate constants and the temperature dependence of aggregation. In doing so, we quantified the susceptibility of rPA aggregation under different temperature and environmental conditions and moreover demonstrated a novel use of UV spectrometry to monitor the particle aggregation quantitatively, in situ, in a nondestructive and time-resolved manner.

Water decoction made from the seed of Hunteria umbellata is widely used in the traditional management of diabetes mellitus by Nigerian herbalists, particularly, in the southwest region of the country. Recently, a new bisindole alkaloid, erinidine, was isolated but its antihyperglycemic profile remains largely un-investigated scientifically. This forms the basis for the current study which is primarily designed at investigating the antihyperglycemic profile of erinidine and other fractions in both in vitro and in vivo models of diabetes mellitus. In the present study, erinidine was isolated and purified using the earlier described methods and its antihyperglycemic potentials tested in in vitro models such as dipeptidylpeptidase (IV), glycogen phosphorylase, HIT-T15 cell insulin secretion, glucose uptake activity, aldose reductase assays and α-glucosidase inhibition assay testings. In addition, 50 mg/kg of erinidine and that of other fractions were evaluated in in vivo models of normal and chemically-induced hyperglycemic rats. Results showed that erinidine was a light yellow, amorphous solid with UV (CHCl3) λ max 256 nm, HRESIMS m/z 382.1881 [(M+H)(+)] (calculated for C22H26N4O2, 382.1876) and melting point of 230 °C. The in vitro study showed the antihyperglycemic action of erinidine to be weakly mediated via α-glucosidase inhibition mechanism as the results for other in vitro tests such as dipeptidylpeptidase (IV), glycogen phosphorylase, HIT-T15 cell insulin secretion, glucose uptake activity and aldose reductase assays were all negative. However, the in vivo results showed 50 mg/kg erinidine given per os to normal and alloxan-induced hyperglycemic rats to significantly (p<0.05, p<0.001) attenuate an increase in their post-absorptive blood glucose concentrations after 3 g/kg glucose loading in the rats, suggesting its antihyperglycemic mechanism to be via α-glucosidase inhibition. This result, although, further corroborated the in vitro findings but also suggests that erinidine needs to be biotransformed in vivo for its inhibitory activity on intestinal glucose absorption to become evident. Thus, the present study suggests erinidine to be the possible antihyperglycemic agent in Hunteria umbellata seed extract mediating its antihyperglycemic action via intestinal glucose uptake inhibition.