Spectroscopic investigations of a semi-synthetic [FeFe] hydrogenase with propane di-selenol as bridging ligand in the binuclear subsite: comparison to the wild type and propane di-thiol variants.

[FeFe] Hydrogenases catalyze the reversible conversion of H2 into electrons and protons. Their catalytic site, the H-cluster, contains a generic [4Fe-4S]H cluster coupled to a [2Fe]H subsite [Fe2(ADT)(CO)3(CN)2]2-, ADT = µ(SCH2)2NH. Heterologously expressed [FeFe] hydrogenases (apo-hydrogenase) lack the [2Fe]H unit, but this can be incorporated through artificial maturation with a synthetic precursor [Fe2(ADT)(CO)4(CN)2]2-. Maturation with a [2Fe] complex in which the essential ADT amine moiety has been replaced by CH2 (PDT = propane-dithiolate) results in a low activity enzyme with structural and spectroscopic properties similar to those of the native enzyme, but with simplified redox behavior. Here, we study the effect of sulfur-to-selenium (S-to-Se) substitution in the bridging PDT ligand incorporated in the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii using magnetic resonance (EPR, NMR), FTIR and spectroelectrochemistry. The resulting HydA1-PDSe enzyme shows the same redox behavior as the parent HydA1-PDT. In addition, a state is observed in which extraneous CO is bound to the open coordination site of the [2Fe]H unit. This state was previously observed only in the native enzyme HydA1-ADT and not in HydA1-PDT. The spectroscopic features and redox behavior of HydA1-PDSe, resulting from maturation with [Fe2(PDSe)(CO)4(CN)2]2-, are discussed in terms of spin and charge density shifts and provide interesting insight into the electronic structure of the H-cluster. We also studied the effect of S-to-Se substitution in the [4Fe-4S] subcluster. The reduced form of HydA1 containing only the [4Fe-4Se]H cluster shows a characteristic S = 7/2 spin state which converts back into the S = 1/2 spin state upon maturation with a [2Fe]-PDT/ADT complex.

Chalcogenide substitution in the [2Fe] cluster of [FeFe]-hydrogenases conserves high enzymatic activity.

[FeFe]-Hydrogenases efficiently catalyze the uptake and evolution of H2 due to the presence of an inorganic [6Fe-6S]-cofactor (H-cluster). This cofactor is comprised of a [4Fe-4S] cluster coupled to a unique [2Fe] cluster where the catalytic turnover of H2/H+ takes place. We herein report on the synthesis of a selenium substituted [2Fe] cluster [Fe2{µ(SeCH2)2NH}(CO)4(CN)2]2- (ADSe) and its successful in vitro integration into the native protein scaffold of [FeFe]-hydrogenases HydA1 from Chlamydomonas reinhardtii and CpI from Clostridium pasteurianum yielding fully active enzymes (HydA1-ADSe and CpI-ADSe). FT-IR spectroscopy and X-ray structure analysis confirmed the presence of structurally intact ADSe at the active site. Electrochemical assays reveal that the selenium containing enzymes are more biased towards hydrogen production than their native counterparts. In contrast to previous chalcogenide exchange studies, the S to Se exchange herein is not based on a simple reconstitution approach using ionic cluster constituents but on the in vitro maturation with a pre-synthesized selenium-containing [2Fe] mimic. The combination of biological and chemical methods allowed for the creation of a novel [FeFe]-hydrogenase with a [2Fe2Se]-active site which confers individual catalytic features.

Spin dynamics of light-induced charge separation in composites of semiconducting polymers and PC60BM revealed using Q-band pulse EPR.

Light-induced processes in composites of semiconducting polymers and fullerene derivatives have been widely studied due to their usage as active layers of organic solar cells. However the process of charge separation under light illumination - the key process of an organic solar cell is not well understood yet. Here we report a Q-band pulse electron paramagnetic resonance study of composites of the fullerene derivative PC60BM ([6,6]-phenyl-C61-butyric acid methyl ester) with different p-type semiconducting polymers regioregular and regiorandom P3HT (poly(3-hexylthiophene-2,5-diyl), MEH-PPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]), PCDTBT (poly[N-9'-heptadecanyl-2,7-carbazole-alt-5,5-(4',7'-di-2-thienyl-2',1',3'-benzothiadiazole)]), PTB7 (poly({4,8-bis[(2-ethylhexyl)oxy]benzo[1,2-b:4,5-b']dithiophene-2,6-diyl}{3-fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophenediyl}))), resulting in a detailed description of the in-phase laser flash-induced electron spin echo (ESE) signal. We found that in organic donor-acceptor composites the laser flash simultaneously induces species of two types: a polymer˙+/fullerene˙- spin-correlated polaron pair (SCPP) with an initial singlet spin state and (nearly) free polymer˙+ and fullerene˙- species with non-equilibrium spin polarization. Species of the first type (SCPP) are well-known for polymer/fullerene blends and are usually associated with a charge-separated state. Also, spin polarization of long-living free species (polarons in deep traps) is affected by the laser flash, which is the third contribution to the flash-induced ESE signal. A protocol for extracting the in-phase ESE signal of the SCPP based on the dependence of the microwave nutation frequency on the strength of the spin coupling within the polaron pair was developed. Nutation experiments revealed an unusual pattern of the SCPP in RR-P3HT/PC60BM composites, from which the strength of the exchange interaction between the polymer˙+ and fullerene˙- was extracted. In composites with low-efficient polymers the contribution of the SCPP to the in-phase ESE signal is high, while in composites with high-efficient polymers it is low. This finding can be used as a selection criterion of charge separation efficiency in the polymer/fullerene composites.

A cryogenic receiver for EPR.

Cryogenic probes have significantly increased the sensitivity of NMR. Here, we present a compact EPR receiver design capable of cryogenic operation. Compared to room temperature operation, it reduces the noise by a factor of ≈2.5. We discuss in detail the design and analyze the resulting noise performance. At low microwave power, the input noise density closely follows the emission of a cooled 50Ω resistor over the whole measurement range from 20K up to room temperature. To minimize the influence of the microwave source noise, we use high microwave efficiency (≈1.1-1.7mTW(-1/2)) planar microresonators. Their efficient conversion of microwave power to magnetic field permits EPR measurements with very low power levels, typically ranging from a few µW down to fractions of nW.

Biomimetic assembly and activation of [FeFe]-hydrogenases.

Hydrogenases are the most active molecular catalysts for hydrogen production and uptake, and could therefore facilitate the development of new types of fuel cell. In [FeFe]-hydrogenases, catalysis takes place at a unique di-iron centre (the [2Fe] subsite), which contains a bridging dithiolate ligand, three CO ligands and two CN(-) ligands. Through a complex multienzymatic biosynthetic process, this [2Fe] subsite is first assembled on a maturation enzyme, HydF, and then delivered to the apo-hydrogenase for activation. Synthetic chemistry has been used to prepare remarkably similar mimics of that subsite, but it has failed to reproduce the natural enzymatic activities thus far. Here we show that three synthetic mimics (containing different bridging dithiolate ligands) can be loaded onto bacterial Thermotoga maritima HydF and then transferred to apo-HydA1, one of the hydrogenases of Chlamydomonas reinhardtii algae. Full activation of HydA1 was achieved only when using the HydF hybrid protein containing the mimic with an azadithiolate bridge, confirming the presence of this ligand in the active site of native [FeFe]-hydrogenases. This is an example of controlled metalloenzyme activation using the combination of a specific protein scaffold and active-site synthetic analogues. This simple methodology provides both new mechanistic and structural insight into hydrogenase maturation and a unique tool for producing recombinant wild-type and variant [FeFe]-hydrogenases, with no requirement for the complete maturation machinery.

An improved coupling design for high-frequency TE011 electron paramagnetic resonance cavities.

In high-frequency electron paramagnetic resonance (EPR) spectroscopy the sample is usually accommodated in a single-mode cylindrical TE(011) microwave cavity. This cavity stands out in terms of flexibility for various types of EPR experiments due to convenient control of its resonance frequency and easy waveguide-to-cavity microwave coupling. In continuous wave and in pulsed EPR it is, however, essential to be able to vary the coupling efficiency over a large range. We present a new mechanical design to vary the microwave coupling to the cavity using a movable metal sphere. This coupling sphere is shifted in the plane of the iris wall inside the coupling waveguide. The design allows for a compact and robust construction of the EPR probehead that can be easily accommodated inside a limited space of helium flow cryostat. The construction details and characterization of the coupling element for 95 GHz (W-band) EPR as well as for 34 GHz (Q-band) are presented.

The effect of herbal extracts in an experimental mouthrinse on established plaque and gingivitis.

The purpose of the present study was to establish in vitro the inhibiting effect of a herbal extract mixture on a selected number of micro-organisms and to test in vivo the effect of a mouthwash containing 6.3 mg/ml herbal extract mixture on plaque and gingivitis as compared to a minus active control mouthrinse. The herbal extract was a mixture of: Juniperus communis (juniper), Urtica dioca (nettle), Achillaea millefolium (yarrow); 1:1:1. In the study, in-vitro, the effect of pure herbal extract mixture on acid production of Streptococcus mutans was tested and the minimum inhibitory concentrations (MIC) of the following micro-organisms were tested: Streptococcus mutans, Streptococcus mitis, Actynomyces viscosus, Actynomyces naeslundii, Actinobacillus actinomycetemcomitans, Prevotella intermedia, Campylobacter rectus, Fusobacterium nucleatum, Veillonella parvula. The MIC-values for A. viscosus and P. gingivalis were 100 mg/ml. The MIC-values for A. naeslundii and A. actinomycetemcomitans were considerably lower (10 mg/ml). S. mitis was the most susceptible of the tested organisms to the extract with a MIC value of 1 mg/ml. S. mutans, C. rectus, V. parvula, and F. nucleatum were not influenced by the extracts. No inhibitory effect of the 6.3 mg/ml herbal extract mixture was observed on the acid production of S. mutans. For the study in-vivo, 45 volunteers were selected on the basis of having moderate gingival inflammation. As efficacy parameters the plaque index, modified gingival index and angulated bleeding index were assessed. The subjects were randomly divided among 3 experimental groups (2x test and 1 'minus active' control). The participants were requested to rinse with 10 ml of mouthwash twice a day for a period of three months. After 6 weeks and 3 months, the same clinical indices as at baseline were recorded. The results show no difference between the two test groups and the control group. In conclusion, the results of the present study have shown that the mixture of the 3 herbal extracts, Juniperus communis, Urtica dioca and Achillaea millefolium when used in a mouthrinse has no effect on plaque growth and gingival health.

Identification of a putative histidine base and of a non-protein nitrogen ligand in the active site of Fe-hydrogenases by one-dimensional and two-dimensional electron spin-echo envelope-modulation spectroscopy.

The active H-cluster of the Fe-hydrogenases from Megasphaera elsdenii and Desulfovibrio vulgaris (strain Hildenborough) has been investigated with one- and two-dimensional pulsed EPR spectroscopy. In both complexes the coordination of a nitrogen-containing ligand was found. The unusual quadrupole interaction parameters (D. vulgaris: quadrupole coupling constant, K = 1.20 MHz, asymmetry parameter eta = 0.32, M. elsdenii: K = 1.23 MHz, eta = 0.25) indicate a non-protein type of nitrogen and are consistent with cyanide as ligand to the H-cluster. The additional interactions measured on the EPR signal of the inactivated H-cluster in D. vulgaris hydrogenase are consistent with an imidazole interaction similar to that found in Rieske-type iron-sulfur clusters. Since a His residue near the putative H-cluster binding motif of Cys residues, His371, is the only conserved His in Fe-hydrogenases, it is a likely candidate for the base that accepts the proton in the heterolytic cleavage of molecular hydrogen. The inactivation of the enzyme is accompanied by direct binding of the imidazole ring to the H-cluster.

Toothbrushing force in relation to plaque removal.

This was a 2-part study. The purpose of the 1st part was to examine the relationship between brushing force and plaque removal efficacy comparing a regular manual toothbrush (M) with an electric toothbrush the Braun/Oral-B Plak Control (B). The study consisted of a single oral prophylaxis followed by 5 experiments which differed solely in respect to toothbrushing force. At baseline (after 24-h plaque accumulation), the amount of dental plaque was evaluated and subsequently, the subject's mouth was brushed by a dental hygienist. Brushing was carried out in a random split-mouth order. Either the 1st and 3rd quadrants or the 2nd and 4th quadrants were brushed with 1 toothbrush and the 2 remaining quadrants with the other. The available time for the brushing procedure was 2 min. After brushing, the amount of remaining dental plaque was assessed. The force used in experiment 1 through 5 was 100, 150, 200, 250, 300 g, respectively. The results show that when brushing force is increased, more plaque is removed with either of the two brushes. Except for the high brushing force (300 g), the electric toothbrush removed more plaque than the manual brush. The purpose of the 2nd part was to evaluate the habitual brushing force which individuals use with various toothbrushes. Besides a manual toothbrush (M), 3 electric toothbrushes were examined, the Rotadent (R), Interplak (I) and Braun (B). 20 subjects were selected on the basis of being 'good brushers' (plaque score at screening < 25%). At baseline, each subject randomly received 1 of the 4 brushes. They were allowed a training period of 3 weeks at the end of which they were asked to abstain from brushing for at least 24 h. The plaque (Turesky modification of the Quigley & Hein) was scored, after which the subjects brushed their teeth (2 min) with the assigned toothbrush equipped with a strain gauge. A computer set-up measured (100 Hz) and calculated the mean brushing force. After brushing, the amount of remaining plaque was assessed. The design of the study was a 4-way cross-over. The results show that with a manual brush, considerably more force is used than with the electric brushes (R = 96, I = 119, B = 146, M = 273). No significant relation between brushing force and plaque removal was demonstrated for any of the brushes.

Toothbrushing duration and plaque removing efficacy of electric toothbrushes.

PURPOSE: To test the plaque removing efficacy of three different toothbrushes in relation to duration of toothbrushing. MATERIALS AND METHODS: The brushes studied were the Sonicare, the Braun Plak Remover (D7), and a modified Plak Control with increased speed (D9). Part 1 of the study was divided into four experiments which differed only in respect to the brushing time. The available times were 15, 30, 45, 60 seconds per quadrant. After 48 hours plaque accumulation, all subjects (n = 22) were brushed professionally using at random a brush in one quadrant (without toothpaste). Part 2 was a 3-way cross-over design in which the same three brushes were tested. In total 49, subjects participated. They were given a brief instruction on the use of each toothbrush and were allowed to get familiarized with each brush during a 2-week period. After 48 hours plaque accumulation, they brushed in a random order the four quadrants, each with a different brushing time (15, 30, 45, 60 seconds). The amount of dental plaque was evaluated pre- and post-brushing by means of the Silness & Löe plaque index at six sites around each tooth investigated. RESULTS: The results of Part 1 and 2 showed an increase in efficacy for all brushes varying from 1 minute per quadrant to 4 minutes per quadrant. In Part 1, the D7 was more effective than the Sonicare brush at 15, 20, 45 seconds whereas the D9 was more effective at all four brushing times. In Part 2, both the D7 and D9 were more effective than the Sonicare at the 2-minute brushing time. These differences were mainly caused by a greater plaque removal from the approximal areas.

Comparison of an oscillating/rotating electric toothbrush and a 'sonic' toothbrush in plaque-removing ability. A professional toothbrushing and supervised brushing study.

The purpose of the present study was to compare the Braun/Oral-B Plak Control (D7) and Sonicare (S) in their plaque-removing efficacy. For this study, 35 university students (non-dental) were selected. This was a 2-part study. Prior to each appointment, all subjects were asked to abstain from all oral hygiene procedures for at least 24 h. In Part 1, the efficacy of the toothbrush was studied when one of the investigators brushed the teeth of the panellists. In Part 2, the efficacy of toothbrushing was studied after the students had received professional instruction. The available time for the brushing amounted to a total of 2 min per mouth. Dental plaque was evaluated by means of the Silness and Löe plaque index assessed at 6 sites per tooth. Results showed that both in Part 2 and Part 1 the D7 removed significantly more plaque than the S-electric toothbrush. The plaque reduction assessed according to Silness and Löe in Part 1 was for the D7, 1.28 and for the S, 1.12. In Part 2, the reductions were 0.90 and 0.80, respectively. The majority (n = 34) of subjects preferred the D7 over the S. In conclusion, the results of the present study show that the oscillating/rotating toothbrush (Braun/ Oral-B Plak Control) is more effective in plaque removal than the sonic electric toothbrush (Sonicare). In addition, given a choice, the panellists preferred the oscillating/rotating electric toothbrush.

Comparison of 2 electric toothbrushes in plaque-removing ability. Professional and supervised brushing.

The purpose of the present study was to compare the Braun Plak Control (D7) and Philips HP 500 (MM) in their plaque removing efficacy. Furthermore the brushing force applied by the panelist on the brush-head with both these brushes was evaluated. For this study, 35 university students (non-dental) were selected. The study consisted of 3 experiments. Prior to each experiment, all subjects were asked to abstain from all oral hygiene procedures for at least 48 h. In Exp. I, the efficacy of toothbrushing was studied when one of the investigators brushed the teeth of the panellists. In Exp. II, the efficacy of the brushing was studied after the students had received a professional instruction. In Exp. III, the brushing force used during a single brushing exercise was evaluated. The available time for the brushing amounted to a total of 2 min per mouth. Dental plaque was evaluated by means of the Silness and Löe and Quigley and Hein plaque indices assessed at 6 sites per tooth. Results showed that both in Exp. I and Exp. II, the D7 removed significantly more plaque than the MM electric toothbrush. The plaque reduction assessed according to Silness and Löe in Exp. I was for the D7, 1.07 and for the MM, 0.78. In Exp. II, the reduction was 1.08 and 0.88, respectively. The results of Exp. III show that the mean brushing force was comparable for both brushes (D7 = 173 g, MM = 175 g). All 35 subjects were allowed to choose one electric toothbrush as compensation for their participation.(ABSTRACT TRUNCATED AT 250 WORDS)

The effectiveness of an electronic toothbrush in the removal of established plaque and treatment of gingivitis.

The purpose of the present study was to evaluate the effect of an electronic toothbrush on established plaque and gingivitis in a 5-month clinical trial. This electronic toothbrush sends approximately a 0.15 mA current through the brushhead which presumedly enhances the efficacy of the brush in plaque elimination. For this study, 80 volunteers (non-dental University students) were selected on the basis of having moderate gingival inflammation. At baseline, plaque and bleeding upon probing were assessed on the vestibular, mesio-vestibular, distovestibular and lingual surfaces using the 'half-mouth' design. After 2-months and 5-months, these clinical indices were again recorded. At each assessment, the participants received a new brushhead. All baseline indices appeared to be well-balanced. A mean Silness and Löe plaque score of 1.70 and 1.64 were found in the control and test groups, respectively. Little change was observed from baseline to 5-months. The Quigley and Hein plaque index behaved comparably. The mean bleeding upon probing score at baseline was 1.43 and 1.39 for the control and test group, respectively. Little to no improvement was observed in the course of this study. No beneficial effect could be shown for this 'electronic' design of manual toothbrush.

The prevalence of A. actinomycetemcomitans, P. gingivalis and P. intermedia in selected subjects with periodontitis.

The present study was carried out in a private periodontal practice. 8 clinical criteria were chosen to select patients for microbiological examination. These criteria characterize prominent features of disease which at the start, during, or in the maintenance phase of treatment suggest difficulties in the progress of the periodontal therapy. Based on these clinical characteristics, an inventory was made of the prevalence of the 3 putative periodontal pathogens: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia. Out of 320 referred patients 91 patients did meet > or = 1 of the clinical criteria and consequently were selected for microbiological examination. Results showed that young patients (19-30 years) with periodontitis demonstrated the presence of A. actinomycetemcomitans more often (69%: p < 0.005) compared to older age cohorts. A. actinomycetemcomitans was found more often when localized breakdown (in particular in the 1st molar and incisor region) was observed and in patient who responded poorly to scaling and rootplaning. P. gingivalis and P. intermedia were most frequently recovered in patients showing primarily a bone loss pattern of angular defects of > or = 5 mm. Retrospectively, these patients were further categorized, based on full mouth radiographs, in (1) patients with primarily horizontal bone loss and (2) patients with primarily angular bony defects. Results showed that A. actinomycetemcomitans occurred less frequently in patients showing horizontal bone loss (p < 0.05).

Comparison of different approaches to assess bleeding on probing as indicators of gingivitis.

The purpose of this study was to evaluate different methods of eliciting gingival bleeding as indicators of gingival inflammation in the experimental gingivitis model. Following a period of stringent oral hygiene, 103 dental students were scored for plaque and gingival bleeding assessed by 4 methods. From this group, 41 volunteers were randomly allocated to 2 treatment groups. Dental students with clean teeth and healthy gingivae were asked to abolish all mechanical tooth cleaning in the lower jaw for a period of 3-weeks. During the 21-day experimental period, chlorhexidine (Peridex) or a placebo mouthrinse was applied to the lower jaw. Subjects brushed the upper jaw with a standard toothpaste. In principal, 2 different methods were employed to provoke bleeding: (1) at the marginal gingival tissue by running a probe along the soft tissue wall at the orifice of the pocket, and (2) by probing to the "bottom" of the pocket. Variations in the methods were based on angulation (AngBI, ParBI) of the probe in relation to the tooth surface and to the probing force (PPBI.25N, PPBI.75N). 1 randomly selected quadrant in the lower jaw was scored using the AngBI. The opposing quadrant was scored with a randomly-allocated bleeding index, either ParBI, PPBI.25N or PPBI.75N. The results of this study confirm earlier findings that the angulation of the probe determines the number of sites with bleeding observed. It also indicates that bleeding as elicited by probing to the bottom of the pocket is a poor indicator of early gingivitis. It is recommended that gingivitis should be assessed by probing the marginal gingiva.

The long-term effect of an oscillating/rotating electric toothbrush on gingivitis. An 8-month clinical study.

The purpose of this study was to evaluate the safety and efficacy of the Braun Plak Control for the removal of supragingival plaque and improving gingival health in a long-term clinical trial, and to compare it to regular manual toothbrush. Assessed were plaque accumulation, amount of gingival inflammation, gingival bleeding on probing, and calculus. In total, 77 young individuals were selected on the basis of having 'moderate gingivitis'. They were monitored over 8 months and divided among 2 groups; a control group that used a manual toothbrush and a test group that used the Braun Plak Control. The clinical assessments were repeated after 1, 2, 5, and 8 months. At baseline, subjects were handed their assigned toothbrushes together with written oral hygiene instructions. They were instructed to brush for at least 2 min. 1 month after baseline examinations, all subjects received a professional prophylaxis and oral hygiene instruction from an experienced dental hygienist. Plaque removal was reinforced at the 2-and 5-month examination. In conclusion, results indicate that the Braun Plak Control is a safe and efficient home care device. At the end of this trial, this electric toothbrush proved to be more effective than a regular manual toothbrush.

Conformational changes of yeast tRNAphe as monitored by 31P NMR.

The 31P NMR spectra of tRNAs contain approximately 17 resonances resolved from the main resonance which consists of about 80% of the total resonance intensity arising from the sugar phosphate backbone. In the present paper we study the behavior of the 31P resonances of yeast tRNAPhe as a function of temperature and of solution conditions. By comparison with other melting experiments we show that three resonances (called c, e and j2) belong to phosphates in the anticodon loop, while the remaining resolved 31P resonances come from phosphates in specific conformations in the central part of the molecule imposed by the tertiary structure. These conformations are different from the normal g-,g- conformation found in A-RNA double helices. The assignments are in good agreement with those previously made on the basis of chemical and enzymatic modification experiments [P. J. M. Salemink, T. Swarthof & C. W. Hilbers (1979) Biochemistry, 18, 3477-3485]. AT high Mg2+ concentrations the anticodon loop is found to be present in two different conformations. For all solution conditions studied loss of the anticodon loop structure takes place before the tertiary structure is melted out. The melting of the tertiary structure is not strictly an all- or-none process. The lifetimes of phosphate conformations involved in the tertiary structure may differ by at least a factor of two. It can also be concluded that the range of chemical shifts observed for phosphodiesters cannot at the moment be accounted for by theoretical calculations.