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Mean fundamental frequency (F(0)) values are often used in research on vocal load. In this study, we examine how the mean F(0) differs when evaluated through pronouncing a standard phrase as compared to the mean F(0) obtained in a real work/play environment. We also examine how the F(0) values change throughout the day. The study was performed in a preschool, nine adult female preschool teachers and 11 children participated. The participants wore a digital recorder equipped with an accelerometer, which was attached to the neck. In the study, the participant first pronounced a standard phrase in a controlled environment; thereafter, the voice was recorded in the environment where both children and adults normally reside throughout the day, denoted by the work/play environment. For each participant, the procedure was repeated four times throughout the day. Analyses showed that the F(0) values of the children's and adult's voices were significantly higher when recorded in the work/play environment as compared to the controlled environment. The average difference was 36 Hz for adults and 24 Hz for children. Previous studies have shown an increase of F(0) over the day for teachers. In this study, an increase between morning and afternoon values was found amounting to 8 Hz for adults and 24 Hz for children. For the child population, this increase was statistically significant. However, the total changes over the day revealed a somewhat more complex scheme, with an increase of F(0) in the morning, a decrease during lunch, and finally an increase in the afternoon. This pattern was verified statistically for the joint child-adult population. [ABSTRACT FROM AUTHOR]

Background: Sepsis is associated with substantial mortality rates. Antibiotic treatment is crucial, but global antibiotic resistance is now classified as one of the top ten global public health risks facing humanity. Ozone (O 3 ) is an inorganic molecule with no evident function in the body. We investigated the bactericide properties of ozone, using a novel system of extracorporeal ozone blood treatment. We hypothesized that ozone would decrease the concentration of viable Escherichia coli (E. coli) in human whole blood and that the system would be technically feasible and physiologically tolerable in a clinically relevant model of E. coli sepsis in swine., Methods: The E. coli strain B09-11822, a clinical isolate from a patient with septic shock was used. The in vitro study treated E. coli infected human whole blood (n = 6) with ozone. The in vivo 3.5-h sepsis model randomized swine to E. coli infusion and ozone treatment (n = 5) or E. coli infusion and no ozone treatment (n = 5). Live E. coli, 5 × 10 7 colony-forming units (CFU/mL) was infused in a peripheral vein. Ozone treatment was initiated with a duration of 30 min after 1.5 h., Results: The single pass in vitro treatment decreased E. coli by 27%, mean 1941 to 1422 CFU/mL, mean of differences - 519.0 (95% CI - 955.0 to - 82.98, P = 0.0281). pO 2 increased (95% CI 31.35 to 48.80, P = 0.0007), pCO 2 decreased (95% CI - 3.203 to - 1.134, P = 0.0069), oxyhemoglobin increased (95% CI 1.010 to 3.669, P = 0.0113). Methemoglobin was not affected. In the sepsis model, 9/10 swine survived. One swine randomized to ozone treatment died from septic shock before initiation of the treatment. Circulatory, respiratory, and metabolic parameters were not affected by the ozone treatment. E. coli in arterial blood, in organs and in aerobic and anaerobic blood cultures did not differ. Hemoglobin, leucocytes, and methemoglobin were not affected by the treatment., Conclusions: Ozone decreased the concentration of viable E. coli in human whole blood. The system was technically feasible and physiologically tolerable in porcine sepsis/septic shock and should be considered for further studies towards clinical applications., (© 2022. The Author(s).)

Denitrification is a microbial pathway that constitutes an important part of the nitrogen cycle on earth. Denitrifying organisms use nitrate as a terminal electron acceptor and reduce it stepwise to nitrogen gas, a process that produces the toxic nitric oxide (NO) molecule as an intermediate. In this work, we have investigated the possible functional interaction between the enzyme that produces NO; the cd 1 nitrite reductase (cd 1 NiR) and the enzyme that reduces NO; the c-type nitric oxide reductase (cNOR), from the model soil bacterium P. denitrificans. Such an interaction was observed previously between purified components from P. aeruginosa and could help channeling the NO (directly from the site of formation to the side of reduction), in order to protect the cell from this toxic intermediate. We find that electron donation to cNOR is inhibited in the presence of cd 1 NiR, presumably because cd 1 NiR binds cNOR at the same location as the electron donor. We further find that the presence of cNOR influences the dimerization of cd 1 NiR. Overall, although we find no evidence for a high-affinity, constant interaction between the two enzymes, our data supports transient interactions between cd 1 NiR and cNOR that influence enzymatic properties of cNOR and oligomerization properties of cd 1 NiR. We speculate that this could be of particular importance in vivo during metabolic switches between aerobic and denitrifying conditions.

Massive integration of biosensors into design of Internet-of-Things (IoT) is vital for progress of healthcare. However, the integration of biosensors is challenging due to limited availability of battery-less biosensor designs. In this work, a combination of nanomaterials for wireless sensing of biological redox reactions is described. The design exploits silver nanoparticles (AgNPs) as part of the RFID tag antenna. We demonstrate that a redox enzyme, particularly, horseradish peroxidase (HRP), can convert AgNPs into AgCl in the presence of its substrate, hydrogen peroxide. This strongly changes the impedance of the tag. The presented example exploits gold nanoparticle (AuNP)-assisted electron transfer (ET) between AgNPs and HRP. We show that AuNP is a vital intermediate for establishing rapid ET between the enzyme and AgNPs. As an example, battery-less biosensor-RFID tag designs for H 2 O 2 and glucose are demonstrated. Similar battery-less sensors can be constructed to sense redox reactions catalysed by other oxidoreductase enzymes, their combinations, bacteria or other biological and even non-biological catalysts. In this work, a fast and general route for converting a high number of redox reaction based sensors into battery-less sensor-RFID tags is described.

The Saccharomyces cerevisiae respiratory supercomplex factor 1 (Rcf1) protein is located in the mitochondrial inner membrane where it is involved in formation of supercomplexes composed of respiratory complexes III and IV. We report the solution structure of Rcf1, which forms a dimer in dodecylphosphocholine (DPC) micelles, where each monomer consists of a bundle of five transmembrane (TM) helices and a short flexible soluble helix (SH). Three TM helices are unusually charged and provide the dimerization interface consisting of 10 putative salt bridges, defining a "charge zipper" motif. The dimer structure is supported by molecular dynamics (MD) simulations in DPC, although the simulations show a more dynamic dimer interface than the NMR data. Furthermore, CD and NMR data indicate that Rcf1 undergoes a structural change when reconstituted in liposomes, which is supported by MD data, suggesting that the dimer structure is unstable in a planar membrane environment. Collectively, these data indicate a dynamic monomer-dimer equilibrium. Furthermore, the Rcf1 dimer interacts with cytochrome c , suggesting a role as an electron-transfer bridge between complexes III and IV. The Rcf1 structure will help in understanding its functional roles at a molecular level., Competing Interests: The authors declare no conflict of interest.

We have investigated the effect of lipid composition on interactions between cytochrome bo 3 and ATP-synthase, and the ATP-synthesis activity driven by proton pumping. The two proteins were labeled by fluorescent probes and co-reconstituted in large (d ≅ 100 nm) or giant (d ≅ 10 µm) unilamellar lipid vesicles. Interactions were investigated using fluorescence correlation/cross-correlation spectroscopy and the activity was determined by measuring ATP production, driven by electron-proton transfer, as a function of time. We found that conditions that promoted direct interactions between the two proteins in the membrane (higher fraction DOPC lipids or labeling by hydrophobic molecules) correlated with an increased activity. These data indicate that the ATP-synthesis rate increases with decreasing distance between cytochrome bo 3 and the ATP-synthase, and involves proton transfer along the membrane surface. The maximum distance for lateral proton transfer along the surface was found to be ~80 nm.

The tyrosine residue D2-Tyr160 (Tyr D ) in photosystem II (PSII) can be oxidized through charge equilibrium with the oxygen evolving complex in PSII. The kinetics of the electron transfer from Tyr D has been followed using time-resolved EPR spectroscopy after triggering the oxidation of pre-reduced Tyr D by a short laser flash. After its oxidation Tyr D is observed as a neutral radical (Tyr D • ) indicating that the oxidation is coupled to a deprotonation event. The redox state of Tyr D was reported to be determined by the two water positions identified in the crystal structure of PSII [Saito et al. (2013) Proc. Natl. Acad. Sci. USA 110, 7690]. To assess the mechanism of the proton coupled electron transfer of Tyr D the oxidation kinetics has been followed in the presence of deuterated buffers, thereby resolving the kinetic isotope effect (KIE) of Tyr D oxidation at different H/D concentrations. Two kinetic phases of Tyr D oxidation - the fast phase (msec-sec time range) and the slow phase (tens of seconds time range) were resolved as was previously reported [Vass and Styring (1991) Biochemistry 30, 830]. In the presence of deuterated buffers the kinetics was significantly slower compared to normal buffers. Furthermore, although the kinetics were faster at both high pH and pD values the observed KIE was found to be similar (~2.4) over the whole pL range investigated. We assign the fast and slow oxidation phases to two populations of PSII centers with different water positions, proximal and distal respectively, and discuss possible deprotonation events in the vicinity of Tyr D ., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Tyrosine D (TyrD) is one of two well-studied redox active tyrosines in Photosystem II. TyrD shows redox kinetics much slower than that of its homologue, TyrZ, and is normally present as a stable deprotonated radical (TyrD(•)). We have used time-resolved continuous wave electron paramagnetic resonance and electron spin echo envelope modulation spectroscopy to show that deuterium exchangeable protons can access TyrD on a time scale that is much faster (50-100 times) than that previously observed. The time of H/D exchange is strongly dependent on the redox state of TyrD. This finding can be related to a change in position of a water molecule close to TyrD.

We have earlier shown that all electron transfer reactions in Photosystem II are operational up to 800 nm at room temperature [Thapper, A., et al. (2009) Plant Cell 21, 2391-2401]. This led us to suggest an alternative charge separation pathway for far-red excitation. Here we extend these studies to a very low temperature (5 K). Illumination of Photosystem II (PS II) with visible light at 5 K is known to result in oxidation of almost similar amounts of YZ and the Cyt b559/ChlZ/CarD2 pathway. This is reproduced here using laser flashes at 532 nm, and we find the partition ratio between the two pathways to be 1:0.8 at 5 K [the partition ratio is here defined as (yield of YZ/CaMn4 oxidation):(yield of Cyt b559/ChlZ/CarD2 oxidation)]. The result using far-red laser flashes is very different. We find partition ratios of 1.8 at 730 nm, 2.7 at 740 nm, and >2.7 at 750 nm. No photochemistry involving these pathways is observed above 750 nm at this temperature. Thus, far-red illumination preferentially oxidizes YZ, while the Cyt b559/ChlZ/CarD2 pathway is hardly touched. We propose that the difference in the partition ratio between visible and far-red light at 5 K reflects the formation of a different first stable charge pair. In visible light, the first stable charge pair is considered to be PD1+QA-. In contrast, we propose that the electron hole is residing on the ChlD1 molecule after illumination by far-red light at 5 K, resulting in the first stable charge pair being ChlD1+QA-. ChlD1 is much closer to YZ (11.3 Å) than to any component in the Cyt b559/ChlZ/CarD2 pathway (shortest ChlD1-CarD2 distance of 28.8 Å). This would then explain that far-red illumination preferentially drives efficient electron transfer from YZ. We also discuss mechanisms for accounting for the absorption of the far-red light and the existence of hitherto unobserved charge transfer states. The involvement of two or more of the porphyrin molecules in the core of the Photosystem II reaction center is proposed.

Illuminating a photosystem II sample at low temperatures (here 5-10 K) yields so-called split signals detectable with continuous wave-electron paramagnetic resonance (CW-EPR). These signals reflect the oxidized, deprotonated radical of D1-Tyr161 (YZ(•)) in a magnetic interaction with the CaMn4 cluster in a particular S state. The intensity of the split EPR signals are affected by the addition of the water substrate analogue methanol. This was previously shown by the induction of split EPR signals from the S1, S3, and S0 states [Su, J.-H. et al. (2006) Biochemistry 45, 7617-7627.]. Here, we use two split EPR signals induced from photosystem II trapped in the S2 state to further probe the binding of methanol in an S state dependent manner. The signals are induced with either visible or near-infrared light illumination provided at 5-10 K where methanol cannot bind or unbind from its site. The results imply that the binding of methanol not only changes the magnetic properties of the CaMn4 cluster but also the hydrogen bond network in the oxygen evolving complex (OEC), thereby affecting the relative charge of the S2 state. The induction mechanisms for the two split EPR signals are different resulting in two different redox states, S2YZ(•) and S1YZ(•) respectively. The two states show different methanol dependence for their induction. This indicates the existence of two binding sites for methanol in the CaMn4 cluster. It is proposed that methanol binds to MnA with high affinity and to MnD with lower affinity. The molecular nature and S-state dependence of the methanol binding to each respective site are discussed.

Two-photon excitation laser-induced fluorescence of carbon monoxide suffers from interference from mainly C2 and strong pressure quenching. This paper presents an investigation of three excitation/detection schemes for two-photon excitation laser-induced fluorescence on carbon monoxide. The schemes are evaluated for pressure and quenching partner dependencies and C2 interference. Three different emission bands lie in the Hopefield-Birge system: The Ångström B(1)Σ(+)→A(1)Πu band, with two-photon excitation through B(1)Σ(+)←X(1)Π around 230 nm; the Herzberg band C(1)Σ(+)→A(1)Πu, with two-photon excitation through, C(1)Σ(+)←X(1)Π, around 217 nm; and the third positive group b(3)Σ→a(3)Π, also with excitation of B(1)Σ(+)←X(1)Π around 230 nm. The measurements are performed in laminar premixed flames with various equivalence ratios as well as in a high-pressure cell, where pressure and species concentrations are varied in order to investigate the fluorescence quenching dependence.

Cryogenic illumination of Photosystem II (PSII) can lead to the trapping of the metastable radical Y(Z)(•), the radical form of the redox-active tyrosine residue D1-Tyr161 (known as Y(Z)). Magnetic interaction between this radical and the CaMn(4) cluster of PSII gives rise to so-called split electron paramagnetic resonance (EPR) signals with characteristics that are dependent on the S state. We report here the observation and characterization of a split EPR signal that can be directly induced from PSII centers in the S(2) state through visible light illumination at 10 K. We further show that the induction of this split signal takes place via a Mn-centered mechanism, in the same way as when using near-infrared light illumination [Koulougliotis, D., et al. (2003) Biochemistry 42, 3045-3053]. On the basis of interpretations of these results, and in combination with literature data for other split signals induced under a variety of conditions (temperature and light quality), we propose a unified model for the mechanisms of split signal induction across the four S states (S(0), S(1), S(2), and S(3)). At the heart of this model is the stability or instability of the Y(Z)(•)(D1-His190)(+) pair that would be formed during cryogenic oxidation of Y(Z). Furthermore, the model is closely related to the sequence of transfers of protons and electrons from the CaMn(4) cluster during the S cycle and further demonstrates the utility of the split signals in probing the immediate environment of the oxygen-evolving center in PSII.

Photosystem II (PSII), the thylakoid membrane enzyme which uses sunlight to oxidize water to molecular oxygen, holds many organic and inorganic redox cofactors participating in the electron transfer reactions. Among them, two tyrosine residues, Tyr-Z and Tyr-D are found on the oxidizing side of PSII. Both tyrosines demonstrate similar spectroscopic features while their kinetic characteristics are quite different. Tyr-Z, which is bound to the D1 core protein, acts as an intermediate in electron transfer between the primary donor, P(680) and the CaMn₄ cluster. In contrast, Tyr-D, which is bound to the D2 core protein, does not participate in linear electron transfer in PSII and stays fully oxidized during PSII function. The phenolic oxygens on both tyrosines form well-defined hydrogen bonds to nearby histidine residues, His(Z) and His(D) respectively. These hydrogen bonds allow swift and almost activation less movement of the proton between respective tyrosine and histidine. This proton movement is critical and the phenolic proton from the tyrosine is thought to toggle between the tyrosine and the histidine in the hydrogen bond. It is found towards the tyrosine when this is reduced and towards the histidine when the tyrosine is oxidized. The proton movement occurs at both room temperature and ultra low temperature and is sensitive to the pH. Essentially it has been found that when the pH is below the pK(a) for respective histidine the function of the tyrosine is slowed down or, at ultra low temperature, halted. This has important consequences for the function also of the CaMn₄ complex and the protonation reactions as the critical Tyr-His hydrogen bond also steer a multitude of reactions at the CaMn₄ cluster. This review deals with the discovery and functional assignments of the two tyrosines. The pH dependent phenomena involved in oxidation and reduction of respective tyrosine is covered in detail. This article is part of a Special Issue entitled: Photosystem II., (© 2011 Elsevier B.V. All rights reserved.)

The electrons extracted from the CaMn(4) cluster during water oxidation in photosystem II are transferred to P(680)(+) via the redox-active tyrosine D1-Tyr161 (Y(Z)). Upon Y(Z) oxidation a proton moves in a hydrogen bond toward D1-His190 (His(Z)). The deprotonation and reprotonation mechanism of Y(Z)-OH/Y(Z)-O is of key importance for the catalytic turnover of photosystem II. By light illumination at liquid helium temperatures (∼5 K) Y(Z) can be oxidized to its neutral radical, Y(Z)(•). This can be followed by the induction of a split EPR signal from Y(Z)(•) in a magnetic interaction with the CaMn(4) cluster, offering a way to probe for Y(Z) oxidation in active photosystem II. In the S(3) state, light in the near-infrared region induces the split S(3) EPR signal, S(2)'Y(Z)(•). Here we report on the pH dependence for the induction of S(2)'Y(Z)(•) between pH 4.0 and pH 8.7. At acidic pH the split S(3) EPR signal decreases with the apparent pK(a) (pK(app)) ∼ 4.1. This can be correlated to a titration event that disrupts the essential H-bond in the Y(Z)-His(Z) motif. At alkaline pH, the split S(3) EPR signal decreases with the pK(app) ∼ 7.5. The analysis of this pH dependence is complicated by the presence of an alkaline-induced split EPR signal (pK(app) ∼ 8.3) promoted by a change in the redox potential of Y(Z). Our results allow dissection of the proton-coupled electron transfer reactions in the S(3) state and provide further evidence that the radical involved in the split EPR signals is indeed Y(Z)(•).

Water oxidation in photosystem II is catalyzed by the CaMn(4) cluster. The electrons extracted from the CaMn(4) cluster are transferred to P(680)(+) via the redox-active tyrosine residue D1-Tyr161 (Y(Z)). The oxidation of Y(Z) is coupled to a deprotonation creating the neutral radical Y(Z)(*). Light-induced oxidation of Y(Z) is possible down to extreme temperatures. This can be observed as a split EPR signal from Y(Z)(*) in a magnetic interaction with the CaMn(4) cluster, offering a way to probe for Y(Z) oxidation in active PSII. Here we have used the split S(0) EPR signal to study the mechanism of Y(Z) oxidation at 5 K in the S(0) state. The state of the hydrogen bond between Y(Z) and its proposed hydrogen bond partner D1-His190 is investigated by varying the pH. The split S(0) EPR signal was induced by illumination at 5 K between pH 3.9 and pH 9.0. Maximum signal intensity was observed between pH 6 and pH 7. On both the acidic and alkaline sides the signal intensity decreased with the apparent pK(a)s (pK(app)) approximately 4.8 and approximately 7.9, respectively. The illumination protocol used to induce the split S(0) EPR signal also induces a mixed radical signal in the g approximately 2 region. One part of this signal decays with similar kinetics as the split S(0) EPR signal ( approximately 3 min, at 5 K) and is easily distinguished from a stable radical originating from Car/Chl. We suggest that this fast-decaying radical originates from Y(Z)(*). The pH dependence of the light-induced fast-decaying radical was measured in the same pH range as for the split S(0) EPR signal. The pK(app) for the light-induced fast-decaying radical was identical at acidic pH ( approximately 4.8). At alkaline pH the behavior was more complex. Between pH 6.6 and pH 7.7 the signal decreased with pK(app) approximately 7.2. However, above pH 7.7 the induction of the radical species was pH independent. We compare our results with the pH dependence of the split S(1) EPR signal induced at 5 K and the S(0) --> S(1) and S(1) --> S(2) transitions at room temperature. The result allows mechanistic conclusions concerning differences between the hydrogen bond pattern around Y(Z) in the S(0) and S(1) states.

Lyngbya majuscula CCAP 1446/4 is a filamentous cyanobacterium possessing both an uptake and a bi-directional hydrogenase. The presence of a single copy of the hyp operon in the cyanobacterial genomes suggests that these accessory genes might be responsible for the maturation of both hydrogenases. We investigated the concomitant transcription of hypFCDEAB with the hydrogenases structural genes--hup and hox. RT-PCRs performed with L. majuscula cells grown under different physiological conditions showed a substantial decrease in the relative amount of hupL transcript under non-N2-fixing conditions. In contrast, no significant differences were observed for the transcript levels of hypFCDEAB in all conditions tested, while minor fluctuations could be discerned for hoxH. Previously, it was demonstrated that the transcriptional regulators NtcA and LexA interact with the promoter regions of hup and hox, respectively, and that putative binding sites for both proteins are present in the hyp promoter of L. majuscula. Therefore, a putative involvement of NtcA and LexA in the regulation of the hyp transcription was investigated. Electrophoretic mobility shift assays resulted in NtcA or LexA-bound retarded fragments, suggesting the involvement of these proteins in the transcriptional regulation of hypFCDEAB.

The filamentous, heterocystous cyanobacterium Nostoc sp. strain PCC 7120 (Anabaena sp. strain PCC 7120) possesses an uptake hydrogenase and a bidirectional enzyme, the latter being capable of catalyzing both H2 production and evolution. The completely sequenced genome of Nostoc sp. strain PCC 7120 reveals that the five structural genes encoding the bidirectional hydrogenase (hoxEFUYH) are separated in two clusters at a distance of approximately 8.8 kb. The transcription of the hox genes was examined under nitrogen-fixing conditions, and the results demonstrate that the cluster containing hoxE and hoxF can be transcribed as one polycistronic unit together with the open reading frame alr0750. The second cluster, containing hoxU, hoxY, and hoxH, is transcribed together with alr0763 and alr0765, located between the hox genes. Moreover, alr0760 and alr0761 form an additional larger operon. Nevertheless, Northern blot hybridizations revealed a rather complex transcription pattern in which the different hox genes are expressed differently. Transcriptional start points (TSPs) were identified 66 and 57 bp upstream from the start codon of alr0750 and hoxU, respectively. The transcriptions of the two clusters containing the hox genes are both induced under anaerobic conditions concomitantly with the induction of a higher level of hydrogenase activity. An additional TSP, within the annotated alr0760, 244 bp downstream from the suggested translation start codon, was identified. Electrophoretic mobility shift assays with purified LexA from Nostoc sp. strain PCC 7120 demonstrated specific interactions between the transcriptional regulator and both hox promoter regions. However, when LexA from Synechocystis sp. strain PCC 6803 was used, the purified protein interacted only with the promoter region of the alr0750-hoxE-hoxF operon. A search of the whole Nostoc sp. strain PCC 7120 genome demonstrated the presence of 216 putative LexA binding sites in total, including recA and recF. This indicates that, in addition to the bidirectional hydrogenase gene, a number of other genes, including open reading frames connected to DNA replication, recombination, and repair, may be part of the LexA regulatory network in Nostoc sp. strain PCC 7120.