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Background: Norovirus (NoV) is the most common cause of diarrheal episodes globally. Issues with in vitro cultivation systems, genetic variation, and animal models have hindered vaccine development. Plant-derived virus-like particles (VLPs) may address some of these concerns because they are highly immunogenic, can be administered by different routes, and can be rapidly produced to accommodate emerging viral strains., Methods: NoV VLPs (NoVLP) composed of the surface viral protein (VP) 1 of the GI and GII genogroups were produced in Nicotiana benthamiana using an Agrobacterium tumefaciens-based recombinant transient expression system. Leaves from infiltrated plants were harvested and NoVLPs were extracted and purified. The safety and immunogenicity of the GII.4 NoVLP, the genotype currently causing most human disease, were subsequently examined in rabbits and mice., Results: Fifteen GI and GII NoVLPs were successfully expressed in N. benthamiana and were structurally similar to NoV virions, as determined by cryogenic transmission electron microscopy. The NoVLP was well-tolerated, with no local or systemic signs of toxicity in rabbits. Three intramuscular doses of the GII.4 NoVLP adjuvanted with aluminum hydroxide induced robust IgG titers, IgG-secreting cells, histo-blood group antigen blocking titers, and IFNγ-secreting T cells in mice. In addition to circulating antibodies, oral administration of the NoVLP in mice induced significant IgA levels in feces, indicative of a mucosal response., Conclusions: The plant-made NoVLP vaccine was safe and immunogenic in mice and rabbits. Multi-modal vaccination, combining oral and intramuscular administration could be considered for future clinical development to maximize systemic and mucosal immune responses., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: GA, DB, MAD, BJW, MMC, MD, POL, SP, and ST are employees of Medicago Inc. JS is employee of the Center for Vaccine Preventable Diseases, Dalla Lana School of Public Health, University of Toronto, ON, Canada, where she is supported by a postdoctoral fellowship from the Canadian Immunization Research Network. HH is employee of Research Institute of the McGill University Health Centre (under MITACS Elevate post-doctorate fellowship), Montreal, QC, Canada. MB and CD are employees of Providence Therapeutics. NL is employee of PharmaJet, CO, USA., (Copyright © 2023. Published by Elsevier Ltd.)

Purpose: School staff who work with autistic students are at a high risk of exhaustion. More training and guidance are needed to enable them to respond to these students' needs. The purpose of this study was to design and evaluate a professional development program offered by an occupational therapist to help a team of school staff facilitate the participation of autistic students and their peers., Methods: A design-based research approach guided the development and evaluation of the modalities of the program. A case study was conducted in a school with nine school staff members using individual interviews, questionnaires, and a logbook., Results: The program comprised five modalities: regular presence of the occupational therapist, a needs analysis, content focused on the features of an inclusive school, individual coaching, and team coaching. The following elements emerged as particularly helpful for the participants: frequent interactions with the occupational therapist, opportunities to collaborate, personalized support, experimentation of new practices with the occupational therapist, and concrete training content that can benefit all students., Conclusion: The modalities of the program appear promising to help a school team facilitate the participation of all students, including those who are autistic.Implications for rehabilitationOccupational therapists can help school teams to facilitate the participation of autistic students and their peers.A combination of individual and team coaching sessions appears promising in helping school staff adopt practice changes to make their school more inclusive.Using a multi-tiered approach can help therapists collaborate with school staff members to first find solutions that will be applicable with all of their students.

Background: This study is the first clinical trial for a parenteral non-replicating rotavirus vaccine developed using virus-like particle (VLP) technology., Methods: This open-labeled, randomized, placebo-controlled trial was conducted in two parts: Part A (a first-in-human study in Australian adults) and Part B (ascending dose and descending age in South African adults, toddlers and infants). In Part A, two cohorts of 10 adults were assigned to receive a single intramuscular injection of 1 of 2 escalating dose levels of the rotavirus VLP (Ro-VLP) vaccine (7 μg or 21 μg) or placebo. In Part B, one cohort of 10 adults was assigned to receive a single injection of the Ro-VLP vaccine (21 μg) or placebo, two cohorts of 10 toddlers were assigned to receive 2 injections of 1 of 2 escalating dose levels of the Ro-VLP vaccine (7 μg or 21 μg) or placebo 28 days apart, and three cohorts of 20 infants were assigned to receive 3 injections of 1 of 3 escalating dose levels of the Ro-VLP vaccine (2.5 μg, 7 μg or 21 μg) or placebo or 2 doses of oral Rotarix 28 days apart. Safety, reactogenicity and immunogenicity were assessed., Results: There were no safety or tolerability concerns after administration of the Ro-VLP vaccine. The Ro-VLP vaccine induced an anti-G1P[8] IgG response in infants 4 weeks after the second and third doses. Neutralizing antibody responses against homologous G1P[8] rotavirus were higher in all Ro-VLP infant groups than in the placebo group 4 weeks after the third dose. No heterotypic immunity was elicited by the Ro-VLP vaccine., Conclusions: The Ro-VLP vaccine was well tolerated and induced a homotypic immune response in infants, suggesting that this technology platform is a favorable approach for a parenteral non-replicating rotavirus vaccine., Clinical Trial Registration: NCT03507738., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: MM (Monica McNeal) has laboratory service agreements with Merck &Co., Inc, outside of the submitted work. No other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Virus-like particles (VLPs) are unable to replicate in the recipient but stimulate the immune system through recognition of repetitive subunits. Parenterally delivered rotavirus-VLP (Ro-VLP) vaccine could have the potential to overcome the weaknesses of licensed oral live-attenuated rotavirus vaccines, namely, low efficacy in low-income and high mortality settings and a potential risk of intussusception., Methods: A monovalent Ro-VLP composed of viral protein (VP) 7, VP6 and VP2 of G1 genotype specificity was produced in Nicotiana benthamiana using Agrobacterium tumefaciens infiltration-based transient recombinant expression system. Plants expressing recombinant G1 Ro-VLP were harvested, then the resultant biomass was processed through a series of clarification and purification steps including standard extraction, filtration, ultrafiltration and chromatography. The purified G1 Ro-VLP was subsequently examined for its immunogenicity and toxicological profile using animal models., Results: G1 Ro-VLP had a purity of ≥90% and was structurally similar to triple-layered rotavirus particles as determined by cryogenic transmission electron microscopy. Two doses of aluminum hydroxide-adjuvanted G1 Ro-VLP (1 μg, 5 μg or 30 μg), administered intramuscularly, elicited a robust homotypic neutralizing antibody response in rats. Also, rabbits administered G1 Ro-VLP (10 μg or 30 μg) four times intramuscularly with aluminum hydroxide adjuvant did not show any significant toxicity., Conclusions: Plant-derived Ro-VLP composed of VP7, VP6 and VP2 structural proteins would be a plausible alternative to live-attenuated oral rotavirus vaccines currently distributed worldwide., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Influenza virus-like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant-based biotechnology allows for the large-scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium-mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post-Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG-I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin-based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Medicago, Inc. has developed an efficient virus-like particle (VLP) vaccine production platform using the Nicotiana benthamiana expression system, and currently has influenza-based products targeting seasonal/pandemic hemagglutinin (HA) proteins in advanced clinical trials. We wished to generate a trackable HA-based VLP that would allow us to study both particle assembly in plants and VLP interactions within the mammalian immune system. To this end, a fusion protein was designed, composed of H5 (from influenza A/Indonesia/05/2005 [H5N1]) with enhanced green fluorescent protein (eGFP). Expression of H5-eGFP in N. benthamiana produced brightly fluorescent ∼160 nm particles resembling H5-VLPs. H5-eGFP-VLPs elicited anti-H5 serologic responses in mice comparable to those elicited by H5-VLPs in almost all assays tested (hemagglutination inhibition/IgG(total)/IgG1/IgG2b/IgG2a:IgG1 ratio), as well as a superior anti-GFP IgG response (mean optical density = 2.52 ± 0.16 sem) to that elicited by soluble GFP (mean optical density = 0.12 ± 0.06 sem). Confocal imaging of N. benthamiana cells expressing H5-eGFP displayed large fluorescent accumulations at the cell periphery, and draining lymph nodes from mice given H5-eGFP-VLPs via footpad injection demonstrated bright fluorescence shortly after administration (10 min), providing proof of concept that the H5-eGFP-protein/VLPs could be used to monitor both VLP assembly and immune trafficking. Given these findings, this novel fluorescent reagent will be a powerful tool to gain further fundamental insight into the biology of influenza VLP vaccines., (© FASEB.)

Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host-cell engineering approaches are being developed to ameliorate host-cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid-susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co-expression. The co-expression of a heterologous ion channel to stabilize acid-labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts., (Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The optimal age to begin CPR training is a matter of debate. This study aims to determine if elementary schoolchildren have the capacity to administer CPR efficiently., Methods: This quasi-experimental study took place in a Quebec City school. Eighty-two children 10 to 12 years old received a 6-hour CPR course based on the American Heart Association (AHA) Guidelines. A comparison group of 20 adults who had taken the same CPR course was recruited. After training, participants' performance was evaluated using a Skillreporter manikin. The primary outcome was depth of compressions. The secondary outcomes were compression rate, insufflation volume and adherence to the CPR sequence. Children's performance was primarily evaluated based on the 2005 AHA standards and secondarily compared to the adults' performance., Results: Schoolchildren did not reach the lower thresholds for depth (28.1 +/- 5.9 vs 38 mm; one-sided p = 1.0). The volume of the recorded insufflations was sufficient (558.6 +/222.8 vs 500 ml; one-sided p = 0.02), but there were a significant number of unsuccessful insufflation attempts not captured by the Skillreporter. The children reached the minimal threshold for rate (113.9 +/-18.3 vs 90/min; one-sided p < 0.001). They did not perform as well as the adults regarding compression depth (p < 0.001), but were comparable for insufflation volume (p = 0.83) and CPR sequence., Conclusions: In this study, schoolchildren aged 10-12 years old did not achieve the standards for compression depth, but achieved adequate compression rate and CPR sequence. When attempts were successful at generating airflow in the Skillreporter, insufflation volume was also adequate.

Objectives: This pilot study aimed to compare sensory processing, motor skills and adaptive behaviors in children with a double diagnosis of Autism Spectrum Disorder (ASD) and Attention Deficit Hyperactivity Disorder (ADHD) (ASD+ADHD) with children with ADHD alone and to examine the association of sensory processing and motor skills with adaptive behaviors (self-care)., Method: Thirty children aged 5-14 years diagnosed with ASD+ADHD (n = 13) or ADHD (n = 17) were evaluated on their sensory processing and motor skills and adaptive behaviors. Analysis of covariance compared the groups on these dimensions. Correlation analyses examined the association between sensory processing and motor skills and adaptive behaviors., Results: Compared to children with ADHD alone, children with ASD+ADHD had poorer skills in sensory processing (p < 0.001), motor (p = 0.001) and adaptive behaviors (p < 0.001). For all children, increased autonomy in self-care was correlated with better sensory processing (p < 0.001) and motor skills (p = 0.002)., Conclusion: Children with ASD+ADHD have poorer sensory processing, motor and adaptive skills than those with ADHD alone. Sensory processing and motor deficits were negatively associated with autonomy in self-care. Interventions aiming to improve sensory processing and motor skills and autonomy in self-care should become important targets for these children.

During the last decade, the spectre of an influenza pandemic of avian origin has led to a revision of national and global pandemic preparedness plans and has stressed the need for more efficient influenza vaccines and manufacturing practices. The 2009 A/H1N1 (swine flu) outbreak has further emphasized the necessity to develop new solutions for pandemic influenza vaccines. Influenza virus-like particles (VLPs)-non-infectious particles resembling the influenza virus-represent a promising alternative to inactivated and split-influenza virions as antigens, and they have shown uniqueness by inducing a potent immune response through both humoral and cellular components of the immune system. Our group has developed a plant-based transient influenza VLP manufacturing platform capable of producing influenza VLPs with unprecedented speed. Influenza VLP expression and purification technologies were brought to large-scale production of GMP-grade material, and pre-clinical studies have demonstrated that low doses of purified, plant-produced influenza VLPs induce a strong and broad immune response in mice and ferrets. This review positions the recent developments towards the successful production of influenza VLPs in plants, including the production of VLPs from other human viruses and other forms of influenza antigens. The platform developed for large-scale production of VLPs is also presented along with an assessment of the speed of the platform to produce the first experimental vaccine lots from the identification of a new influenza strain.

A strain-specific vaccine represents the best possible response to the threat of an influenza pandemic. Rapid delivery of such a vaccine to the world's population before the peak of the first infection wave seems to be an unattainable goal with the current influenza vaccine manufacturing capacity. Plant-based transient expression is one of the few production systems that can meet the anticipated surge requirement. To assess the capability of plant agroinfiltration to produce an influenza vaccine, we expressed haemagglutinin (HA) from strains A/Indonesia/5/05 (H5N1) and A/New Caledonia/20/99 (H1N1) by agroinfiltration of Nicotiana benthamiana plants. Size distribution analysis of protein content in infiltrated leaves revealed that HA was predominantly assembled into high-molecular-weight structures. H5-containing structures were purified and examination by transmission electron microscopy confirmed virus-like particle (VLP) assembly. High-performance thin layer chromatography analysis of VLP lipid composition highlighted polar and neutral lipid contents comparable with those of purified plasma membranes from tobacco plants. Electron microscopy of VLP-producing cells in N. benthamiana leaves confirmed that VLPs accumulated in apoplastic indentations of the plasma membrane. Finally, immunization of mice with two doses of as little as 0.1 microg of purified influenza H5-VLPs triggered a strong immune response against the homologous virus, whereas two doses of 0.5 microg of H5-VLPs conferred complete protection against a lethal challenge with the heterologous A/Vietnam/1194/04 (H5N1) strain. These results show, for the first time, that plants are capable of producing enveloped influenza VLPs budding from the plasma membrane; such VLPs represent very promising candidates for vaccination against influenza pandemic strains.

The Rieske iron-sulfur proteins have reduction potentials ranging from -150 to +400 mV. This enormous range of potentials was first proposed to be due to differing solvent exposure or even protein structure. However, the increasing number of available crystal structures for Rieske iron-sulfur proteins has shown this not to be the case. Colbert and colleagues proposed in 2000 that differences in the electrostatic environment, and not structural differences, of a Rieske proteins are responsible for the wide range of reduction potentials observed. Using computational simulation methods and the newly determined structure of Pseudomonas sp. NCIB 9816-4 naphthalene dioxygenase Rieske ferredoxin (NDO-F9816-4), we have developed a model to predict the reduction potential of Rieske proteins given only their crystal structure. The reduction potential of NDO-F9816-4, determined using a highly oriented pyrolytic graphite electrode, was -150+/-2 mV versus the standard hydrogen electrode. The predicted reduction potentials correlate well with experimentally determined potentials. Given this model, the effect of protein mutations can be evaluated. Our results suggest that the reduction potential of new proteins can be estimated with good confidence from 3D structures of proteins. The structure of NDO-F9816-4 is the most basic Rieske ferredoxin structure determined to date. Thus, the contributions of additional structural motifs and their effects on reduction potential can be compared with respect to this base structure.

DitA3, a small soluble ferredoxin, is a component of a ring-hydroxylating dioxygenase involved in the microbial degradation of the diterpenoid, dehydroabietic acid. The anaerobic purification of a heterologously expressed his-tagged DitA3 yielded 20 mg of apparently homogeneous recombinant protein, rcDitA3, per liter of cell culture. Each mole of purified rcDitA3 contained 2.9 equivalents of iron and 4.2 equivalents of sulfur, indicating the presence of a single [Fe(3)S(4)] cluster. This conclusion was corroborated by UV-Visible absorption (epsilon(412)=13.4 mM(-1) cm(-1)) and EPR (g(x,y)=2.00 and g(z)=2.02) spectroscopies. The reduction potential of rcDitA3, determined using a highly oriented parallel graphite (HOPG) electrode, was -177.0+/-0.5 mV vs. the standard hydrogen electrode (SHE) (20 mM MOPS, 80 mM KCl, pH 7.0, 22 degrees C). This potential is similar to those of small, soluble Rieske-type ferredoxin components of aromatic-ring dihydroxylating dioxygenases. In contrast to these Rieske-type ferredoxins, DitA3 appears to exist as a dimer in solution. The dimeric ferredoxin may be more stable or may increase the catalytic efficiency of the dioxygenase by delivering the two reducing equivalents required for turnover of the oxygenase.

Sulfur K-edge X-ray absorption spectroscopy (XAS) is reported for [Fe(4)S(4)](1+,2+,3+) clusters. The results are quantitatively and qualitatively compared with DFT calculations. The change in covalency upon redox in both the [Fe(4)S(4)](1+/2+) (ferredoxin) and the [Fe(4)S(4)](2+/3+) (HiPIP) couple are much larger than that expected from just the change in number of 3d holes. Moreover, the change in the HiPIP couple is higher than that of the ferredoxin couple. These changes in electronic structure are analyzed using DFT calculations in terms of contributions from the nature of the redox active molecular orbital (RAMO) and electronic relaxation. The results indicate that the RAMO of HiPIP has 50% ligand character, and hence, the HiPIP redox couple involves limited electronic relaxation. Alternatively, the RAMO of the ferredoxin couple is metal-based, and the ferredoxin redox couple involves extensive electronic relaxation. The contributions of these RAMO differences to ET processes in the different proteins are discussed.

Rieske [2Fe-2S] clusters can be classified into two groups, depending on their reduction potentials. Typical high-potential Rieske proteins have pH-dependent reduction potentials between +350 and +150 mV at pH 7, and low-potential Rieske proteins have pH-independent potentials of around -150 mV at pH 7. The pH dependence of the former group is attributed to coupled deprotonation of the two histidine ligands. Protein-film voltammetry has been used to compare three Rieske proteins: the high-potential Rieske proteins from Rhodobacter sphaeroides (RsRp) and Thermus thermophilus (TtRp) and the low-potential Rieske ferredoxin from Burkholderia sp. strain LB400 (BphF). RsRp and TtRp differ because there is a cluster to serine hydrogen bond in RsRp, which raises its potential by 140 mV. BphF lacks five hydrogen bonds to the cluster and an adjacent disulfide bond. Voltammetry measurements between pH 3 and 14 reveal that all the proteins, including BphF, have pH-dependent reduction potentials with remarkably similar overall profiles. Relative to RsRp and TtRp, the potential versus pH curve of BphF is shifted to lower potential and higher pH, and the pK(a) values of the histidine ligands of the oxidized and reduced cluster are closer together. Therefore, in addition to simple electrostatic effects on E and pK(a), the reduction potentials of Rieske clusters are determined by the degree of coupling between cluster oxidation state and histidine protonation state. Implications for the mechanism of quinol oxidation at the Q(O) site of the cytochrome bc(1) and b(6)f complexes are discussed.

BphF is a small, soluble, Rieske-type ferredoxin involved in the microbial degradation of biphenyl. The rapid, anaerobic purification of a heterologously expressed, his-tagged BphF yielded 15 mg of highly homogeneous recombinant protein, rcBphF, per liter of cell culture. The reduction potential of rcBphF, determined using a highly oriented pyrolytic graphite (HOPG) electrode, was -157+/- 2 mV vs the standard hydrogen electrode (SHE) (20 mM MOPS, 80 mM KCl, and 1 mM dithiothreitol, pH 7.0, 22 degrees C). The electron paramagnetic resonance spectrum of the reduced rcBphF is typical of a Rieske cluster while the close similarity of the circular dichroic (CD) spectra of rcBphF and BedB, a homologous protein from the benzene dioxygenase system, indicates that the environment of the cluster is highly conserved in these two proteins. The reduction potential and CD spectra of rcBphF were relatively independent of pH between 5 and 10, indicating that the pK(a)s of the cluster's histidinyl ligands are not within this range. Gel filtration studies demonstrated that rcBphF readily oligomerizes in solution. Crystals of rcBphF were obtained using sodium formate or poly(ethylene glycol) (PEG) as the major precipitant. Analysis of the intermolecular contacts in the crystal revealed a head-to-tail interaction that occludes the cluster, but is very unlikely to be found in solution. Oligomerization of rcBphF in solution was reversed by the addition of dithiothreitol and is unrelated to the noncovalent crystallographic interactions. Moreover, the oligomerization state of rcBphF did not influence the latter's reduction potential. These results indicate that the 450 mV spread in reduction potential of Rieske clusters of dioxygenase-associated ferredoxins and mitochondrial bc(1) complexes is not due to significant differences in their solvent exposure.

Background: Ring-hydroxylating dioxygenases are multicomponent systems that initiate biodegradation of aromatic compounds. Many dioxygenase systems include Rieske-type ferredoxins with amino acid sequences and redox properties remarkably different from the Rieske proteins of proton-translocating respiratory and photosynthetic complexes. In the latter, the [Fe2S2] clusters lie near the protein surface, operate at potentials above +300 mV at pH 7, and express pH- and ionic strength-dependent redox behavior. The reduction potentials of the dioxygenase ferredoxins are approximately 150 mV and are pH-independent. These distinctions were predicted to arise from differences in the exposure of the cluster and/or interactions of the histidine ligands., Results: The crystal structure of BphF, the Rieske-type ferredoxin associated with biphenyl dioxygenase, was determined by multiwavelength anomalous diffraction and refined at 1.6 A resolution. The structure of BphF was compared with other Rieske proteins at several levels. BphF has the same two-domain fold as other Rieske proteins, but it lacks all insertions that give the others unique structural features. The BphF Fe-S cluster and its histidine ligands are exposed. However, the cluster has a significantly different environment in that five fewer polar groups interact strongly with the cluster sulfide or the cysteinyl ligands., Conclusions: BphF has structural features consistent with a minimal and perhaps archetypical Rieske protein. Variations in redox potentials among Rieske clusters appear to be largely the result of local electrostatic interactions with protein partial charges. Moreover, it appears that the redox-linked ionizations of the Rieske proteins from proton-translocating complexes are also promoted by these electrostatic interactions.

The role of a flattened, relatively hydrophobic surface patch in the self-association of Chromatium vinosum HiPIP was assessed by substituting phenylalanine 48 with lysine. The reduction potential of the F48K variant was 26 mV higher than that of the wild-type (WT) recombinant (rc) HiPIP, consistent with the introduction of a positive charge close to the cluster. Nuclear magnetic resonance spectroscopy (NMR) revealed that the electronic structure of the oxidized cluster in these two proteins is very similar at 295 K. In contrast, the electron transfer self-exchange rate constant of F48K was at least 15-fold lower than that of the WT rcHiPIP, indicating that the introduction of a positive charge at position 48 diminishes self-association of the HiPIP in solution. Moreover, the substitution at position 48 abolished the fine structure in the g(z) region of the electron paramagnetic resonance (EPR) spectrum of oxidized C. vinosum rcHiPIP recorded in the presence of 1 M sodium chloride. These results support the hypothesis that the flattened, relatively hydrophobic patch mediates interaction between two molecules of HiPIP and that freezing-induced dimerization of the HiPIP mediated by this patch is responsible for the unusual fine structure observed in the EPR spectrum of the oxidized C. vinosum HiPIP.

The reduced high-potential iron sulfur protein I from Ectothiorhodospira halophila which contains the [4Fe-4S]2+ polymetallic center has been fully labeled with 15N and 13C. The protein is paramagnetic, the nuclear relaxation times of nuclei close to the paramagnetic ion are drastically shortened and some strategic dipolar connectivities are lost. Notwithstanding, the solution structure has been reported [Banci, L., Bertini, I., Eltis, L. D., Felli, I. C., Kastrau, D. H. W., Luchinat, C., Piccioli, M., Pierattelli, R. & Smith, M. (1994) Eur. J. Biochem. 225, 715-725]. We have performed classical HNHA, HNCA soft-COSY, soft-HCCH E. COSY and 15N-1H correlated NOESY experiments in order to obtain a set of 3J scalar coupling constants. Some experiments have been optimized to counterbalance the effect of paramagnetism. From heteronuclear single-quantum experiments preceded by a 180 degrees pulse and variable delay times, the non-selective magnetization recovery has been followed from which the contribution to dipolar relaxation of nuclei due to the interaction with the paramagnetic metal ions (rho para) has been estimated. Finally, the intensities of NOEs have been corrected for the presence of paramagnetic metal ions and these corrected values together with 3J values and rho para data have been used to obtain a well defined solution structure. The aim is that of obtaining a structure with enough constraints to be well resolved all over the protein, including the vicinity of the paramagnetic metal cluster, which is anchored to the protein through the rho para constraints. In total, 1226 corrected NOESY crosspeaks (of which 945 were found to be meaningful), 37 one-dimensional NOEs, 39 3JHNH alpha and 37 3JHNC' (providing 45 phi dihedral angle constraints) 54 3JH alpha H beta and 31 3JNH beta (providing 26 chi 1 dihedral angle constraints), 4 chi 2 dihedral angle constraints of the coordinated cysteines, obtained from the hyperfine shifts of the beta CH protons, and 58 rho para constraints, have been used for structure calculation. Restrained molecular dynamics simulations have also been performed to provide the final family of structures. This research demonstrates that stable isotope labeling provides specific advantages for the NMR investigation of paramagnetic molecules, as the small magnetic moment of heteronuclei minimizes the paramagnetic influence of unpaired electrons.

Many types of materials are available for the regeneration of the periodontium destroyed by periodontal disease. In view of the fact that there are not many properly structured classical studies relative to the same, one cannot conclude that these materials regenerate bone, the periodontal ligament and the cementum which make up the periodontium. In this article, many classical studies relative to the use of bone grafts are carefully examined and analyzed.

A review of the evaluation, treatment, and end results for 52 patients with thymoma treated at The University of Texas M.D. Anderson Cancer Center (1950-1984) is presented. The objective of the study was to examine the influence of a number of clinical characteristics on survival, including histologic and staging classifications, associated diseases, symptom status, and treatment. Forty-nine patients (94%) underwent surgical exploration; 13 were stage I, 12 were stage II, and 24 were stage III. Complete resection was accomplished in all of the stage I and II groups and in 6 of the stage III patients. An asymptomatic history, surgical stage I disease, lymphocytic thymoma cell type, and complete resection favorably influenced prognosis. The cumulative 5-year survival rate for all patients was 40%. No patient with stage I thymoma had recurrent disease, however, there were 9 recurrences (50%) in the completely resected stage II/III patients, six of whom remained disease-free following treatment with radiotherapy, chemotherapy, or a combined approach.