Investigating the Kinetic Competency of CrHydA1 [FeFe] Hydrogenase Intermediate States via Time-Resolved Infrared Spectroscopy.

Hydrogenases are metalloenzymes that catalyze the reversible oxidation of H2. The [FeFe] hydrogenases are generally biased toward proton reduction and have high activities. Several different catalytic mechanisms have been proposed for the [FeFe] enzymes based on the identification of intermediate states in equilibrium and steady state experiments. Here, we examine the kinetic competency of these intermediate states in the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1), using a laser-induced potential jump and time-resolved IR (TRIR) spectroscopy. A CdSe/CdS dot-in-rod (DIR) nanocrystalline semiconductor is employed as the photosensitizer and a redox mediator efficiently transfers electrons to the enzyme. A pulsed laser induces a potential jump, and TRIR spectroscopy is used to follow the population flux through each intermediate state. The results clearly establish the kinetic competency of all intermediate populations examined: Hox, Hred, HredH+, HsredH+, and Hhyd. Additionally, a new short-lived intermediate species with a CO peak at 1896 cm-1 was identified. These results establish a kinetics framework for understanding the catalytic mechanism of [FeFe] hydrogenases.

Direct Detection of the Terminal Hydride Intermediate in [FeFe] Hydrogenase by NMR Spectroscopy.

Hydride state intermediates are known to occur in various hydrogen conversion enzymes, including the highly efficient [FeFe] hydrogenases. The intermediate state involving a terminal iron-bound hydride has been recognized as crucial for the catalytic mechanism, but its occurrence has up to now eluded unequivocal proof under (near) physiological conditions. Here we show that the terminal hydride in the [FeFe] hydrogenase from Chlamydomonas reinhardtii can be directly detected using solution 1H NMR spectroscopy at room temperature, opening new avenues for detailed in situ investigations under catalytic conditions.

1H NMR Spectroscopy of [FeFe] Hydrogenase: Insight into the Electronic Structure of the Active Site.

The [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii has been studied using 1H NMR spectroscopy identifying the paramagnetically shifted 1H resonances associated with both the [4Fe-4S]H and the [2Fe]H subclusters of the active site "H-cluster". The signal pattern of the unmaturated HydA1 containing only [4Fe-4S]H is reminiscent of bacterial-type ferredoxins. The spectra of maturated HydA1, with a complete H-cluster in the active Hox and the CO-inhibited Hox-CO state, reveal additional upfield and downfield shifted 1H resonances originating from the four methylene protons of the azadithiolate ligand in the [2Fe]H subsite. The two axial protons are affected by positive spin density, while the two equatorial protons experience negative spin density. These protons can be used as important probes sensing the effects of ligand-binding to the catalytic site of the H-cluster.

Unique Spectroscopic Properties of the H-Cluster in a Putative Sensory [FeFe] Hydrogenase.

Sensory type [FeFe] hydrogenases are predicted to play a role in transcriptional regulation by detecting the H2 level of the cellular environment. These hydrogenases contain the hydrogenase domain with distinct modifications in the active site pocket, followed by a Per-Arnt-Sim (PAS) domain. As yet, neither the physiological function nor the biochemical or spectroscopic properties of these enzymes have been explored. Here, we present the characterization of an artificially maturated, putative sensory [FeFe] hydrogenase from Thermotoga maritima (HydS). This enzyme shows lower hydrogen conversion activity than prototypical [FeFe] hydrogenases and a reduced inhibition by CO. Using FTIR spectroelectrochemistry and EPR spectroscopy, three redox states of the active site were identified. The spectroscopic signatures of the most oxidized state closely resemble those of the Hox state from the prototypical [FeFe] hydrogenases, while the FTIR spectra of both singly and doubly reduced states show large differences. The FTIR bands of both the reduced states are strongly red-shifted relative to the Hox state, indicating reduction at the diiron site, but with retention of the bridging CO ligand. The unique functional and spectroscopic features of HydS are discussed with regard to the possible role of altered amino acid residues influencing the electronic properties of the H-cluster.

Engineering an [FeFe]-Hydrogenase: Do Accessory Clusters Influence O2 Resistance and Catalytic Bias?

[FeFe]-hydrogenases, HydAs, are unique biocatalysts for proton reduction to H2. However, they suffer from a number of drawbacks for biotechnological applications: size, number and diversity of metal cofactors, oxygen sensitivity. Here we show that HydA from Megasphaera elsdenii (MeHydA) displays significant resistance to O2. Furthermore, we produced a shorter version of this enzyme (MeH-HydA), lacking the N-terminal domain harboring the accessory FeS clusters. As shown by detailed spectroscopic and biochemical characterization, MeH-HydA displays the following interesting properties. First, a functional active site can be assembled in MeH-HydA in vitro, providing the enzyme with excellent hydrogenase activity. Second, the resistance of MeHydA to O2 is conserved in MeH-HydA. Third, MeH-HydA is more biased toward proton reduction than MeHydA, as the result of the truncation changing the rate limiting steps in catalysis. This work shows that it is possible to engineer HydA to generate an active hydrogenase that combines the resistance of the most resistant HydAs and the simplicity of algal HydAs, containing only the H-cluster.

A [RuRu] Analogue of an [FeFe]-Hydrogenase Traps the Key Hydride Intermediate of the Catalytic Cycle.

The active site of the [FeFe]-hydrogenases features a binuclear [2Fe]H sub-cluster that contains a unique bridging amine moiety close to an exposed iron center. Heterolytic splitting of H2 results in the formation of a transient terminal hydride at this iron site, which, however is difficult to stabilize. We show that the hydride intermediate forms immediately when [2Fe]H is replaced with [2Ru]H analogues through artificial maturation. Outside the protein, the [2Ru]H analogues form bridging hydrides, which rearrange to terminal hydrides after insertion into the apo-protein. H/D exchange of the hydride only occurs for [2Ru]H analogues containing the bridging amine moiety.

Proton Coupled Electronic Rearrangement within the H-Cluster as an Essential Step in the Catalytic Cycle of [FeFe] Hydrogenases.

The active site of [FeFe] hydrogenases, the H-cluster, consists of a [4Fe-4S] cluster connected via a bridging cysteine to a [2Fe] complex carrying CO and CN- ligands as well as a bridging aza-dithiolate ligand (ADT) of which the amine moiety serves as a proton shuttle between the protein and the H-cluster. During the catalytic cycle, the two subclusters change oxidation states: [4Fe-4S]H2+ ⇔ [4Fe-4S]H+ and [Fe(I)Fe(II)]H ⇔ [Fe(I)Fe(I)]H thereby enabling the storage of the two electrons needed for the catalyzed reaction 2H+ + 2e- ⇄ H2. Using FTIR spectro-electrochemistry on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) at different pH values, we resolve the redox and protonation events in the catalytic cycle and determine their intrinsic thermodynamic parameters. We show that the singly reduced state Hred of the H-cluster actually consists of two species: Hred = [4Fe-4S]H+ - [Fe(I)Fe(II)]H and HredH+ = [4Fe-4S]H2+ - [Fe(I)Fe(I)]H (H+) related by proton coupled electronic rearrangement. The two redox events in the catalytic cycle occur on the [4Fe-4S]H subcluster at similar midpoint-potentials (-375 vs -418 mV); the protonation event (Hred/HredH+) has a pKa ≈ 7.2.

Reaction Coordinate Leading to H2 Production in [FeFe]-Hydrogenase Identified by Nuclear Resonance Vibrational Spectroscopy and Density Functional Theory.

[FeFe]-hydrogenases are metalloenzymes that reversibly reduce protons to molecular hydrogen at exceptionally high rates. We have characterized the catalytically competent hydride state (Hhyd) in the [FeFe]-hydrogenases from both Chlamydomonas reinhardtii and Desulfovibrio desulfuricans using 57Fe nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT). H/D exchange identified two Fe-H bending modes originating from the binuclear iron cofactor. DFT calculations show that these spectral features result from an iron-bound terminal hydride, and the Fe-H vibrational frequencies being highly dependent on interactions between the amine base of the catalytic cofactor with both hydride and the conserved cysteine terminating the proton transfer chain to the active site. The results indicate that Hhyd is the catalytic state one step prior to H2 formation. The observed vibrational spectrum, therefore, provides mechanistic insight into the reaction coordinate for H2 bond formation by [FeFe]-hydrogenases.

General Movements in preterm infants undergoing craniosacral therapy: a randomised controlled pilot-trial.

BACKGROUND: The objective of this study was to investigate neurological short-term effects of craniosacral therapy as an ideal form of osteopathic manipulative treatment (OMT) due to the soft kinaesthetic stimulation. METHODS: Included were 30 preterm infants, with a gestational age between 25 and 33 weeks, who were admitted to the neonatal intensive care unit of the University Hospital of Graz, Austria. The infants were randomized either into the intervention group (IG) which received standardised craniosacral therapy, or the control group (CG) which received standard care. To guarantee that only preterm infants with subsequent normal neurodevelopment were included, follow up was done regularly at the corrected age (= actual age in weeks minus weeks premature) of 12 and 24 months. After 2 years 5 infants had to be excluded (IG; n = 12; CG: n = 13). General Movements (GMs) are part of the spontaneous movement repertoire and are present from early fetal life onwards until the end of the first half year of life. To evaluate the immediate result of such an intervention, we selected the General Movement Assessment (GMA) as an appropriate tool. Besides the global GMA (primary outcome) we used as detailed GMA, the General Movement Optimality Score (GMOS- secondary outcome), based on Prechtl's optimality concept. To analyse GMOS (secondary outcome) a linear mixed model with fixed effects for session, time point (time point refers to the comparisons of the measurements before vs. after each session) and intervention (IG vs. CG), random effect for individual children and a first order autoregressive covariance structure was used for calculation of significant differences between groups and interactions. Following interaction terms were included in the model: session*time point, session*intervention, time point*intervention and session*time point*intervention. Exploratory post hoc analyses (interaction: session*time point*intervention) were performed to determine group differences for all twelve measurement (before and after all 6 sessions) separately. RESULTS: Between groups no difference in the global GMA (primary outcome) could be observed. The GMOS (secondary outcome) did not change from session to session (main effect session: p = 0.262) in the IG or the CG. Furthermore no differences between IG and CG (main effect group: p = 0.361) and no interaction of time*session could be observed (p = 0.658). Post hoc analysis showed a trend toward higher values before (p = 0.085) and after (p = 0.075) the first session in CG compared to IG. At all other time points GMOS were not significantly different between groups. CONCLUSION: We were able to indicate that a group of "healthy" preterm infants undergoing an intervention with craniosacral therapy (IG) showed no significant changes in GMs compared to preterm infants without intervention (CG). In view of the fact that the global GMA (primary outcome) showed no difference between groups and the GMOS (detailed GMA-secondary outcome) did not deteriorate in the IG, craniosacral therapy seems to be safe in preterm infants. TRIAL REGISTRATION: German Clinical Trials Register DRKS00004258 .

Safety and toxicity of intrathecal liposomal cytarabine (Depocyte) in children and adolescents with recurrent or refractory brain tumors: a multi-institutional retrospective study.

This retrospective study aimed to evaluate the safety and toxicity of intrathecal liposomal cytarabine (Depocyte) in children and adolescents with refractory or recurrent brain tumors. Nineteen heavily pretreated patients (males, n = 14; females, n = 5; median age at diagnosis 8.5 years; range, 1.4-22 years) were given intrathecal liposomal cytarabine on a compassionate use basis for recurrent refractory medulloblastoma (n = 12), mixed germ cell tumor (n = 2), central nervous system primitive neuroectodermal tumors of the pons (n = 1), anaplastic ependymoma (n = 1), anaplastic oligodendroglioma (n = 1), atypical teratoid rhabdoid tumor (n = 1), or rhabdoid papillary meningioma (n = 1). Eighteen patients received concomitant systemic radiochemotherapy. A total of 88 intrathecal injections of liposomal cytarabine (dose range, 20-50 mg) were administered with concomitant dexamethasone prophylaxis. The median number of doses per patient was four (range, 1-10). Duration of treatment ranged from (1/2) to 10 months. Eleven patients (57.9%) did not show any side effects, whereas eight patients (42.1%) developed side effects related to either chemical arachnoiditis (n = 4) or neurological progression (n = 2). Less typical treatment-related symptoms (e.g. lethargy, ataxia, and slurred speech) were observed in two patients. Treatment with intrathecal liposomal cytarabine was discontinued twice because of side effects. In conclusion, although intrathecal liposomal cytarabine was generally well tolerated, it should be used cautiously and only with dexamethasone prophylaxis in extensively pretreated patients with recurrent brain tumors. Proof of efficacy requires a prospective single-agent phase II study.

Neuroophthalmological side effects following intrathecal administration of liposomal cytarabine for central nervous system prophylaxis in three adolescents with acute myeloid leukaemia.

Three adolescents with central nervous system (CNS) negative acute myeloid leukaemia (AML) refused cranial irradiation for CNS prophylaxis. Instead, these patients received four doses of 50 mg of intrathecal (IT) liposomal cytarabine on day 1, 15, 43 and 71 of maintenance therapy. Corticosteroids were given to prevent chemical arachnoiditis. All patients developed bilateral papilloedema after the third or fourth dose of liposomal cytarabine, local side effects were observed in two patients. Under prolonged dexamethasone therapy, side effects resolved completely. As reversible neuroophthalmological side effects caused by intrathecal liposomal cytarabine can also be observed under low-dose maintenance therapy in patients with CNS negative AML, it should be used with caution in children and adolescents.

Light-Induced Conformational Changes in the Plant Cryptochrome Photolyase Homology Region Resolved by Selective Isotope Labeling and Infrared Spectroscopy.

Plant cryptochromes are photoreceptors that regulate flowering, circadian rhythm and photomorphogenesis in response to blue and UV-A light. It has been demonstrated that the oxidized flavin cofactor is photoreduced to the neutral radical state via separate electron and proton transfer. Conformational changes have been found in the C-terminal extension, but few studies have addressed the changes in secondary structure in the sensory photolyase homology region (PHR). Here, we investigated the PHR of the plant cryptochrome from the green alga Chlamydomonas reinhardtii by light-induced infrared difference spectroscopy in combination with global 13 C and 15 N isotope labeling. Assignment of the signals is achieved by establishing a labeling strategy for cryptochromes that preserves the flavin at natural abundance. We demonstrate by UV/vis spectroscopy that the integrity of the sample is maintained and by mass spectrometry that the global labeling was highly efficient. As a result, difference bands are resolved at full intensity that at natural abundance are compensated by the overlap of flavin and protein signals. These bands are assigned to prominent conformational changes in the PHR by blue light illumination. We postulate that not only the partial unfolding of the C-terminal extension but also changes in the PHR may mediate signaling events.

Following [FeFe] Hydrogenase Active Site Intermediates by Time-Resolved Mid-IR Spectroscopy.

Time-resolved nanosecond mid-infrared spectroscopy is for the first time employed to study the [FeFe] hydrogenase from Chlamydomonas reinhardtii and to investigate relevant intermediates of the enzyme active site. An actinic 355 nm, 10 ns laser flash triggered photodissociation of a carbonyl group from the CO-inhibited state Hox-CO to form the state Hox, which is an intermediate of the catalytic proton reduction cycle. Time-resolved infrared spectroscopy allowed us to directly follow the subsequent rebinding of the carbonyl, re-forming Hox-CO, and determine the reaction half-life to be t1/2 ≈ 13 ± 5 ms at room temperature. This gives direct information on the dynamics of CO inhibition of the enzyme.

Near-infrared spectroscopy for objectifying cerebral effects of laser acupuncture in term and preterm neonates.

Laser acupuncture (LA) becomes more and more relevant in neonates and infants. With near-infrared spectroscopy (NIRS), a continuous and noninvasive measurement of tissue oxygenation is possible. Aim was to investigate, whether the application of LA was associated with any changes in regional cerebral oxygen saturation (rcSO2) in term and preterm neonates. The study included 20 neonates (12 males, 8 females). The Large Intestine 4 acupuncture point (LI 4, Hegu) was stimulated by a microlaser needle (10 mW, 685 nm laser needle EG GmbH, Germany) for 5 minutes, bilaterally. All neonates underwent polygraphic recording during undisturbed daytime sleep, including heart rate (HR), peripheral oxygen saturation (SpO2), and measurement of nasal flow. Using NIRS, rcSO2 was measured continuously. Cerebral fractional tissue oxygen extraction (cFTOE) was calculated. We did not observe any significant changes in SpO2 and HR values during the whole observation period. However, there was a significant decrease in rcSO2 (P = 0.003) within postintervention period, accompanied by a significant increase in cFTOE (P = 0.010) in postintervention period.

Cost-effectiveness of palivizumab for respiratory syncytial virus infection in high-risk children, based on long-term epidemiologic data from Austria.

OBJECTIVE: To assess the cost-effectiveness of palivizumab, a monoclonal antibody against respiratory syncytial virus (RSV), in infants at high risk for severe RSV lower respiratory tract infection, such as premature infants, infants with bronchopulmonary dysplasia, and those with congenital heart disease, based on long-term epidemiologic data from Austria. METHODS: A decision-tree model was used, and the analysis was based on a lifetime follow-up investigating cost-effectiveness of palivizumab versus no RSV infection prevention. The primary perspective of the study was that of the healthcare system, the second that of society. Cost and effects were discounted by 5%. The base case analysis included only direct medical costs, and a scenario analysis included various indirect costs. RESULTS: Analyses were based on epidemiologic data on a total of 1579 children hospitalized because of RSV lower respiratory tract infection during 16 seasons. The incremental cost-effectiveness ratio for the first outcome measure (life years gained) amounted to discounted costs of €34,956 (for all preterm infants), €35,056 (for < 33 weeks' gestational age [wGA] infants), €35,233 (for 33-35 wGA infants), €35,611 (for infants with bronchopulmonary dysplasia), and €8956 (for infants with congenital heart disease). Use of palivizumab compared with no prophylaxis had an incremental cost-utility ratio of €26,212, €26,292, €24,392, €24,654, and €8484, respectively, per quality-adjusted life years. Results from the society perspective were more cost-effective in all study populations. An additional scenario analysis with 7 injections for the 33 to 35 wGA group revealed cost-effectiveness as well. CONCLUSIONS: Our results based on nationwide long-term epidemiologic data suggest that palivizumab is cost-effective in prevention of RSV disease in high-risk infants.

Risk factors for severe respiratory syncytial virus lower respiratory tract infection.

RSV infection is a leading cause of lower respiratory tract infection, especially in High-risk infants with a history of prematurity, bronchopulmonary dysplasia (BPD), congenital heart disease (CHD), neuromusculair impairment, immunodeficiency, and Down syndrome. Host related risk factors that have been identified to be associated with severe RSV related lower respiratory tract infection include young age below 6 months at the beginning of RSV season, multiple birth, male sex, low socioeconomic status and parental education, crowded living conditions, young siblings, maternal smoking and indoor smoke pollution, malnutrition/small for gestational age, family history of atopy or asthma, low cord serum RSV antibody titers, and living at altitude.Risk factors increasing the risk of acquisition of RSV have been identified to be birth before and/or during RSV season, day care attendance, presence of older siblings in school or day-care, and lack of breast feeding. Some of these risk factors are discussed controversially and some of them are found continuously throughout the literature.Given the high cost of RSV prophylaxis, especially for the large population of late preterm infants, algorithms and risk score systems have been published that could identify high-risk infants for treatment with palivizumab out of this gestational age group. Several models reported on an average sensitivity and specificity of 70 percent and, thus, are helpful to identify infants at high risk for severe RSV infection and need for prophylaxis with palivizumab.

Prognostic significance of amplitude-integrated EEG during the first 72 hours after birth in severely asphyxiated neonates.

Amplitude-integrated EEG (aEEG) is used to select patients for neuroprotective therapy after perinatal asphyxia because of its prognostic accuracy within several hours after birth. We aimed to determine the natural course of aEEG patterns during the first 72 h of life, in relation to neurologic outcome, in a group of severely asphyxiated term infants. Thirty infants, admitted to our neonatal intensive care unit from October 1998 until February 2001, were studied retrospectively. The aEEG traces obtained during the first 72 h after birth were assessed by pattern recognition: continuous normal voltage (CNV), discontinuous normal voltage (DNV), burst suppression (BS), continuous low voltage, and flat trace. Epileptic activity was also determined. The course of aEEG patterns was examined in relation to neurologic findings at 24 mo. Initially, 17 of 30 infants had severely abnormal aEEG patterns (BS or worse), which changed spontaneously to normal voltage patterns (CNV, DNV) in 7 within 48 h. The sooner the abnormalities on aEEG disappeared, the better the prognosis. The likelihood ratio of BS or worse for adverse outcome was 2.7 (95% confidence interval 1.4-5.0) between 0 and 6 h and increased to a highest value of 19 (95% confidence interval 2.8-128) between 24 and 36 h; after 48 h, it was not significant. Normal voltage patterns (CNV and DNV) up to 48 h of life were predictive for normal neurologic outcomes (negative likelihood ratios <0.3). Our findings indicate that the course of aEEG patterns adds to the prognostic value of aEEG monitoring in asphyxiated infants. Spontaneous recovery of severely abnormal aEEG patterns is not uncommon.