Structural evidence for intermediates during O2 formation in photosystem II.

In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn4CaO5 cluster first stores four oxidizing equivalents, the S0 to S4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O-O bond formation chemistry1-3. Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok's photosynthetic water oxidation cycle, the S3→[S4]→S0 transition where O2 is formed and Kok's water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn4CaO5 cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom Ox, which was introduced as a bridging ligand between Ca and Mn1 during the S2→S3 transition4-6, disappears or relocates in parallel with Yz reduction starting at approximately 700 µs after the third flash. The onset of O2 evolution, as indicated by the shortening of the Mn1-Mn4 distance, occurs at around 1,200 µs, signifying the presence of a reduced intermediate, possibly a bound peroxide.

Intraoperative individualization of positive-end-expiratory pressure through electrical impedance tomography or esophageal pressure assessment: a systematic review and meta-analysis of randomized controlled trials.

PURPOSE: This systematic review of randomized-controlled trials (RCTs) with meta-analyses aimed to compare the effects on intraoperative arterial oxygen tension to inspired oxygen fraction ratio (PaO2/FiO2), exerted by positive end-expiratory pressure (PEEP) individualized trough electrical impedance tomography (EIT) or esophageal pressure (Pes) assessment (intervention) vs. PEEP not tailored on EIT or Pes (control), in patients undergoing abdominal or pelvic surgery with an open or laparoscopic/robotic approach. METHODS: PUBMED®, EMBASE®, and Cochrane Controlled Clinical trials register were searched for observational studies and RCTs from inception to the end of August 2022. Inclusion criteria were: RCTs comparing PEEP titrated on EIT/Pes assessment vs. PEEP not individualized on EIT/Pes and reporting intraoperative PaO2/FiO2. Two authors independently extracted data from the enrolled investigations. Data are reported as mean difference and 95% confidence interval (CI). RESULTS: Six RCTs were included for a total of 240 patients undergoing general anesthesia for surgery, of whom 117 subjects in the intervention group and 123 subjects in the control group. The intraoperative mean PaO2/FiO2 was 69.6 (95%CI 32.-106.4 ) mmHg higher in the intervention group as compared with the control group with 81.4% between-study heterogeneity (p < 0.01). However, at meta-regression, the between-study heterogeneity diminished to 44.96% when data were moderated for body mass index (estimate 3.45, 95%CI 0.78-6.11, p = 0.011). CONCLUSIONS: In patients undergoing abdominal or pelvic surgery with an open or laparoscopic/robotic approach, PEEP personalized by EIT or Pes allowed the achievement of a better intraoperative oxygenation compared to PEEP not individualized through EIT or Pes. PROSPERO REGISTRATION NUMBER: CRD 42021218306, 30/01/2023.

Water Networks in Photosystem II Using Crystalline Molecular Dynamics Simulations and Room-Temperature XFEL Serial Crystallography.

Structural dynamics of water and its hydrogen-bonding networks play an important role in enzyme function via the transport of protons, ions, and substrates. To gain insights into these mechanisms in the water oxidation reaction in Photosystem II (PS II), we have performed crystalline molecular dynamics (MD) simulations of the dark-stable S1 state. Our MD model consists of a full unit cell with 8 PS II monomers in explicit solvent (861 894 atoms), enabling us to compute the simulated crystalline electron density and to compare it directly with the experimental density from serial femtosecond X-ray crystallography under physiological temperature collected at X-ray free electron lasers (XFELs). The MD density reproduced the experimental density and water positions with high fidelity. The detailed dynamics in the simulations provided insights into the mobility of water molecules in the channels beyond what can be interpreted from experimental B-factors and electron densities alone. In particular, the simulations revealed fast, coordinated exchange of waters at sites where the density is strong, and water transport across the bottleneck region of the channels where the density is weak. By computing MD hydrogen and oxygen maps separately, we developed a novel Map-based Acceptor-Donor Identification (MADI) technique that yields information which helps to infer hydrogen-bond directionality and strength. The MADI analysis revealed a series of hydrogen-bond wires emanating from the Mn cluster through the Cl1 and O4 channels; such wires might provide pathways for proton transfer during the reaction cycle of PS II. Our simulations provide an atomistic picture of the dynamics of water and hydrogen-bonding networks in PS II, with implications for the specific role of each channel in the water oxidation reaction.

In Situ Structural Observation of a Substrate- and Peroxide-Bound High-Spin Ferric-Hydroperoxo Intermediate in the P450 Enzyme CYP121.

The P450 enzyme CYP121 from Mycobacterium tuberculosis catalyzes a carbon-carbon (C-C) bond coupling cyclization of the dityrosine substrate containing a diketopiperazine ring, cyclo(l-tyrosine-l-tyrosine) (cYY). An unusual high-spin (S = 5/2) ferric intermediate maximizes its population in less than 5 ms in the rapid freeze-quenching study of CYP121 during the shunt reaction with peracetic acid or hydrogen peroxide in acetic acid solution. We show that this intermediate can also be observed in the crystalline state by EPR spectroscopy. By developing an on-demand-rapid-mixing method for time-resolved serial femtosecond crystallography with X-ray free-electron laser (tr-SFX-XFEL) technology covering the millisecond time domain and without freezing, we structurally monitored the reaction in situ at room temperature. After a 200 ms peracetic acid reaction with the cocrystallized enzyme-substrate microcrystal slurry, a ferric-hydroperoxo intermediate is observed, and its structure is determined at 1.85 Å resolution. The structure shows a hydroperoxyl ligand between the heme and the native substrate, cYY. The oxygen atoms of the hydroperoxo are 2.5 and 3.2 Å from the iron ion. The end-on binding ligand adopts a near-side-on geometry and is weakly associated with the iron ion, causing the unusual high-spin state. This compound 0 intermediate, spectroscopically and structurally observed during the catalytic shunt pathway, reveals a unique binding mode that deviates from the end-on compound 0 intermediates in other heme enzymes. The hydroperoxyl ligand is only 2.9 Å from the bound cYY, suggesting an active oxidant role of the intermediate for direct substrate oxidation in the nonhydroxylation C-C bond coupling chemistry.

Tracking the first electron transfer step at the donor side of oxygen-evolving photosystem II by time-resolved infrared spectroscopy.

In oxygen-evolving photosystem II (PSII), the multi-phasic electron transfer from a redox-active tyrosine residue (TyrZ) to a chlorophyll cation radical (P680+) precedes the water-oxidation chemistry of the S-state cycle of the Mn4Ca cluster. Here we investigate these early events, observable within about 10 ns to 10 ms after laser-flash excitation, by time-resolved single-frequency infrared (IR) spectroscopy in the spectral range of 1310-1890 cm-1 for oxygen-evolving PSII membrane particles from spinach. Comparing the IR difference spectra at 80 ns, 500 ns, and 10 µs allowed for the identification of quinone, P680 and TyrZ contributions. A broad electronic absorption band assignable P680+ was used to trace largely specifically the P680+ reduction kinetics. The experimental time resolution was taken into account in least-square fits of P680+ transients with a sum of four exponentials, revealing two nanosecond phases (30-46 ns and 690-1110 ns) and two microsecond phases (4.5-8.3 µs and 42 µs), which mostly exhibit a clear S-state dependence, in agreement with results obtained by other methods. Our investigation paves the road for further insight in the early events associated with TyrZ oxidation and their role in the preparing the PSII donor side for the subsequent water oxidation chemistry.

Evolutionary diversity of proton and water channels on the oxidizing side of photosystem II and their relevance to function.

One of the reasons for the high efficiency and selectivity of biological catalysts arise from their ability to control the pathways of substrates and products using protein channels, and by modulating the transport in the channels using the interaction with the protein residues and the water/hydrogen-bonding network. This process is clearly demonstrated in Photosystem II (PS II), where its light-driven water oxidation reaction catalyzed by the Mn4CaO5 cluster occurs deep inside the protein complex and thus requires the transport of two water molecules to and four protons from the metal center to the bulk water. Based on the recent advances in structural studies of PS II from X-ray crystallography and cryo-electron microscopy, in this review we compare the channels that have been proposed to facilitate this mass transport in cyanobacteria, red and green algae, diatoms, and higher plants. The three major channels (O1, O4, and Cl1 channels) are present in all species investigated; however, some differences exist in the reported structures that arise from the different composition and arrangement of membrane extrinsic subunits between the species. Among the three channels, the Cl1 channel, including the proton gate, is the most conserved among all photosynthetic species. We also found at least one branch for the O1 channel in all organisms, extending all the way from Ca/O1 via the 'water wheel' to the lumen. However, the extending path after the water wheel varies between most species. The O4 channel is, like the Cl1 channel, highly conserved among all species while having different orientations at the end of the path near the bulk. The comparison suggests that the previously proposed functionality of the channels in T. vestitus (Ibrahim et al., Proc Natl Acad Sci USA 117:12624-12635, 2020; Hussein et al., Nat Commun 12:6531, 2021) is conserved through the species, i.e. the O1-like channel is used for substrate water intake, and the tighter Cl1 and O4 channels for proton release. The comparison does not eliminate the potential role of O4 channel as a water intake channel. However, the highly ordered hydrogen-bonded water wire connected to the Mn4CaO5 cluster via the O4 may strongly suggest that it functions in proton release, especially during the S0 → S1 transition (Saito et al., Nat Commun 6:8488, 2015; Kern et al., Nature 563:421-425, 2018; Ibrahim et al., Proc Natl Acad Sci USA 117:12624-12635, 2020; Sakashita et al., Phys Chem Chem Phys 22:15831-15841, 2020; Hussein et al., Nat Commun 12:6531, 2021).

Anthocyanin and Lycopene Contents Do Not Affect ß-Carotene Bioefficacy from Multicolored Carrots (Daucus carota L.) in Male Mongolian Gerbils.

BACKGROUND: Anthocyanins and carotenoids are phytochemicals that may benefit health through provitamin A carotenoid (PAC), antioxidant, and anti-inflammatory activities. These bioactives may mitigate chronic diseases. Consumption of multiple phytochemicals may impact bioactivity in synergistic or antagonistic manners. OBJECTIVES: Two studies in weanling male Mongolian gerbils assessed the relative bioefficacy of ß-carotene equivalents (BCEs) to vitamin A (VA) with simultaneous consumption of the non-PAC lycopene or anthocyanins from multicolored carrots. METHODS: After 3-wk VA depletion, 5-6 gerbils were killed as baseline groups. The remaining gerbils were divided into 4 carrot treatment groups; the positive control group received retinyl acetate and the negative control group was given vehicle soybean oil (n = 10/group; n = 60/study). In the lycopene study, gerbils consumed feed varying in lycopene sourced from red carrots. In the anthocyanin study, gerbils consumed feed varying in anthocyanin content sourced from purple-red carrots, and positive controls received lycopene. Treatment feeds had equalized BCEs: 5.59 ± 0.96 µg/g (lycopene study) and 7.02 ± 0.39 µg/g (anthocyanin study). Controls consumed feeds without pigments. Serum, liver, and lung samples were analyzed for retinol and carotenoid concentrations using HPLC. Data were analyzed by ANOVA and Tukey's studentized range test. RESULTS: In the lycopene study, liver VA did not differ between groups (0.11 ± 0.07 µmol/g) indicating no effect of varying lycopene content. In the anthocyanin study, liver VA concentrations in the medium-to-high (0.22 ± 0.14 µmol/g) and medium-to-low anthocyanin (0.25 ± 0.07 µmol/g) groups were higher than the negative control (0.11 ± 0.07 µmol/g) (P < 0.05). All treatment groups maintained baseline VA concentrations (0.23 ± 0.06 µmol/g). Combining studies, serum retinol had 12% sensitivity to predict VA deficiency, defined as 0.7 µmol/L. CONCLUSIONS: These gerbil studies suggested that simultaneous consumption of carotenoids and anthocyanins does not impact relative BCE bioefficacy. Breeding carrots for enhanced pigments to improve dietary intake should continue.

Room temperature X-ray absorption spectroscopy of metalloenzymes with drop-on-demand sample delivery at XFELs.

X-ray crystallography and X-ray spectroscopy using X-ray free electron lasers plays an important role in understanding the interplay of structural changes in the protein and the chemical changes at the metal active site of metalloenzymes through their catalytic cycles. As a part of such an effort, we report here our recent development of methods for X-ray absorption spectroscopy (XAS) at XFELs to study dilute biological samples, available in limited volumes. Our prime target is Photosystem II (PS II), a multi subunit membrane protein complex, that catalyzes the light-driven water oxidation reaction at the Mn4CaO5 cluster. This is an ideal system to investigate how to control multi-electron/proton chemistry, using the flexibility of metal redox states, in coordination with the protein and the water network. We describe the method that we have developed to collect XAS data using PS II samples with a Mn concentration of <1 mM, using a drop-on-demand sample delivery method.

Untangling the sequence of events during the S2 → S3 transition in photosystem II and implications for the water oxidation mechanism.

In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S2 → S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.

Genetic characterization of carrot root shape and size using genome-wide association analysis and genomic-estimated breeding values.

KEY MESSAGE: The principal phenotypic determinants of market class in carrot-the size and shape of the root-are under primarily additive, but also highly polygenic, genetic control. The size and shape of carrot roots are the primary determinants not only of yield, but also market class. These quantitative phenotypes have historically been challenging to objectively evaluate, and thus subjective visual assessment of market class remains the primary method by which selection for these traits is performed. However, advancements in digital image analysis have recently made possible the high-throughput quantification of size and shape attributes. It is therefore now feasible to utilize modern methods of genetic analysis to investigate the genetic control of root morphology. To this end, this study utilized both genome wide association analysis (GWAS) and genomic-estimated breeding values (GEBVs) and demonstrated that the components of market class are highly polygenic traits, likely under the influence of many small effect QTL. Relatively large proportions of additive genetic variance for many of the component phenotypes support high predictive ability of GEBVs; average prediction ability across underlying market class traits was 0.67. GWAS identified multiple QTL for four of the phenotypes which compose market class: length, aspect ratio, maximum width, and root fill, a previously uncharacterized trait which represents the size-independent portion of carrot root shape. By combining digital image analysis with GWAS and GEBVs, this study represents a novel advance in our understanding of the genetic control of market class in carrot. The immediate practical utility and viability of genomic selection for carrot market class is also described, and concrete guidelines for the design of training populations are provided.

Evaluating prediction methods for glomerular filtration to optimise drug doses in obese and nonobese patients.

AIMS: The most suitable method for predicting the glomerular filtration rate (GFR) in obesity is currently debated. Therefore, multiple GFR/creatinine clearance prediction methods were applied to (morbidly) obese and nonobese patients ranging from moderate renal impairment to glomerular hyperfiltration and their predictions were rated based on observed fosfomycin pharmacokinetics, as this model drug is exclusively eliminated via glomerular filtration. METHODS: The GFR/creatinine clearance predictions via the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI), Modification of Diet in Renal Disease (MDRD; indexed and de-indexed by body surface area) and creatinine clearance via the Cockcroft-Gault formula (CLCRCG ) using different body size descriptors were compared to the fosfomycin clearance (CLFOF ) from 30 surgical patients (body mass index = 20.1-52.0 kg m-2 ), receiving 8000 mg as intravenous infusion. RESULTS: The concordance between CLFOF and creatinine clearance predictions was highest for CLCRCG employing either ideal body weight or adjusted body weight (if body mass >1.3 ideal body weight; CLCRCG_ABW-Schwartz , concordance-correlation coefficient [95% confidence interval] = 0.474 [0.156; 0.703], CCC) and GFR predictions via the de-indexed MDRD equation (concordance-correlation coefficient = 0.452 [0.137; 0.685]). The proportion of predicted GFR values within ±30% of the observed CLFOF (P30 = 72.3-76.7%) was only marginally lower than the reported P30 in the original CKD-EPI and MDRD publications (P30 = 84.1-90.0%). CONCLUSION: This analysis represents a successful proof-of-concept for evaluating GFR/creatinine clearance prediction methods: Across all body mass index classes CLCRCG_ABW-Schwartz or the de-indexed MDRD were most suitable for predicting creatinine clearance/GFR also in (morbidly) obese, CKD stage <3B individuals in therapeutic use. Their application is proposed in optimising doses for vital therapies in obese patients requiring monitoring of renal function (e.g. methotrexate dosing).

Adverse effects of delayed antimicrobial treatment and surgical source control in adults with sepsis: results of a planned secondary analysis of a cluster-randomized controlled trial.

BACKGROUND: Timely antimicrobial treatment and source control are strongly recommended by sepsis guidelines, however, their impact on clinical outcomes is uncertain. METHODS: We performed a planned secondary analysis of a cluster-randomized trial conducted from July 2011 to May 2015 including forty German hospitals. All adult patients with sepsis treated in the participating ICUs were included. Primary exposures were timing of antimicrobial therapy and delay of surgical source control during the first 48 h after sepsis onset. Primary endpoint was 28-day mortality. Mixed models were used to investigate the effects of timing while adjusting for confounders. The linearity of the effect was investigated by fractional polynomials and by categorizing of timing. RESULTS: Analyses were based on 4792 patients receiving antimicrobial treatment and 1595 patients undergoing surgical source control. Fractional polynomial analysis identified a linear effect of timing of antimicrobials on 28-day mortality, which increased by 0.42% per hour delay (OR with 95% CI 1.019 [1.01, 1.028], p ≤ 0.001). This effect was significant in patients with and without shock (OR = 1.018 [1.008, 1.029] and 1.026 [1.01, 1.043], respectively). Using a categorized timing variable, there were no significant differences comparing treatment within 1 h versus 1-3 h, or 1 h versus 3-6 h. Delays of more than 6 h significantly increased mortality (OR = 1.41 [1.17, 1.69]). Delay in antimicrobials also increased risk of progression from severe sepsis to septic shock (OR per hour: 1.051 [1.022, 1.081], p ≤ 0.001). Time to surgical source control was significantly associated with decreased odds of successful source control (OR = 0.982 [0.971, 0.994], p = 0.003) and increased odds of death (OR = 1.011 [1.001, 1.021]; p = 0.03) in unadjusted analysis, but not when adjusted for confounders (OR = 0.991 [0.978, 1.005] and OR = 1.008 [0.997, 1.02], respectively). Only, among patients with septic shock delay of source control was significantly related to risk-of death (adjusted OR = 1.013 [1.001, 1.026], p = 0.04). CONCLUSIONS: Our findings suggest that management of sepsis is time critical both for antimicrobial therapy and source control. Also patients, who are not yet in septic shock, profit from early anti-infective treatment since it can prevent further deterioration. Trial registration ClinicalTrials.gov ( NCT01187134 ). Registered 23 August 2010, NCT01187134.

Perioperative administration of cefazolin and metronidazole in obese and non-obese patients: a pharmacokinetic study in plasma and interstitial fluid.

OBJECTIVES: To assess plasma and tissue pharmacokinetics of cefazolin and metronidazole in obese patients undergoing bariatric surgery and non-obese patients undergoing intra-abdominal surgery. PATIENTS AND METHODS: Fifteen obese and 15 non-obese patients received an IV short infusion of 2 g cefazolin and 0.5 g metronidazole for perioperative prophylaxis. Plasma and microdialysate from subcutaneous tissue were sampled until 8 h after dosing. Drug concentrations were determined by HPLC-UV. Pharmacokinetic parameters were calculated non-compartmentally. RESULTS: In obese patients (BMI 39.5-69.3 kg/m2) compared with non-obese patients (BMI 18.7-29.8 kg/m2), mean Cmax of total cefazolin in plasma was lower (115 versus 174 mg/L) and Vss was higher (19.4 versus 14.2 L). The mean differences in t½ (2.7 versus 2.4 h), CL (5.14 versus 4.63 L/h) and AUC∞ (402 versus 450 mg·h/L) were not significant. The influence of obesity on the pharmacokinetics of metronidazole was similar (Cmax 8.99 versus 14.7 mg/L, Vss 73.9 versus 51.8 L, t½ 11.9 versus 9.1 h, CL 4.62 versus 4.13 L/h, AUC∞ 116 versus 127 mg·h/L). Regarding interstitial fluid (ISF), mean concentrations of cefazolin remained >4 mg/L until 6 h in both groups, and those of metronidazole up to 8 h in the non-obese group. In obese patients, the mean ISF concentrations of metronidazole were between 3 and 3.5 mg/L throughout the measuring interval. CONCLUSIONS: During the time of surgery, cefazolin concentrations in plasma and ISF of subcutaneous tissue were lower in obese patients, but not clinically relevant. Regarding metronidazole, the respective differences were higher, and may influence dosing of metronidazole for perioperative prophylaxis in obese patients.

The influence of the Or and Carotene Hydroxylase genes on carotenoid accumulation in orange carrots [Daucus carota (L.)].

KEY MESSAGE: The Or and CH genes are necessary for the accumulation of high amounts of ß-carotene and other carotenoid pigments in carrot roots, in addition to the Y and Y2 genes. Carrot taproot color results from the accumulation of various carotenoid and anthocyanin pigments. Recently, the Or gene was identified as a candidate gene associated with the accumulation of ß-carotene and other provitamin A carotenoids in roots. The specific molecular mechanisms involved with this process, as well as the interactions between Or and the other genes involved in this process are not well understood. In order to better characterize the effect that Or alleles have on conditioning the accumulation of carotenoids in roots, we analyzed an F3 family fixed homozygous recessive for y and y2, derived from a cross between an orange carrot and a white wild carrot, segregating for the two known Or alleles, which we name Orc and Orw. QTL mapping across three different environments revealed that the accumulation of several carotenoids was associated with the Orc allele, with consistent patterns across environments. A second QTL on chromosome 7, harboring a carotene hydroxylase gene homologous to Lut5 in Arabidopsis, was also associated with the accumulation of several carotenoids. Two alleles for this gene, which we name CHc and CHw, were discovered to be segregating in this population. Our study provides further evidence that Or and CH are likely involved with controlling the accumulation of ß-carotene and may be involved with modulating carotenoid flux in carrot, demonstrating that both were important domestication genes in carrot.

Individualized versus Fixed Positive End-expiratory Pressure for Intraoperative Mechanical Ventilation in Obese Patients: A Secondary Analysis.

BACKGROUND: General anesthesia may cause atelectasis and deterioration in oxygenation in obese patients. The authors hypothesized that individualized positive end-expiratory pressure (PEEP) improves intraoperative oxygenation and ventilation distribution compared to fixed PEEP. METHODS: This secondary analysis included all obese patients recruited at University Hospital of Leipzig from the multicenter Protective Intraoperative Ventilation with Higher versus Lower Levels of Positive End-Expiratory Pressure in Obese Patients (PROBESE) trial (n = 42) and likewise all obese patients from a local single-center trial (n = 54). Inclusion criteria for both trials were elective laparoscopic abdominal surgery, body mass index greater than or equal to 35 kg/m2, and Assess Respiratory Risk in Surgical Patients in Catalonia (ARISCAT) score greater than or equal to 26. Patients were randomized to PEEP of 4 cm H2O (n = 19) or a recruitment maneuver followed by PEEP of 12 cm H2O (n = 21) in the PROBESE study. In the single-center study, they were randomized to PEEP of 5 cm H2O (n = 25) or a recruitment maneuver followed by individualized PEEP (n = 25) determined by electrical impedance tomography. Primary endpoint was Pao2/inspiratory oxygen fraction before extubation and secondary endpoints included intraoperative tidal volume distribution to dependent lung and driving pressure. RESULTS: Ninety patients were evaluated in three groups after combining the two lower PEEP groups. Median individualized PEEP was 18 (interquartile range, 16 to 22; range, 10 to 26) cm H2O. Pao2/inspiratory oxygen fraction before extubation was 515 (individual PEEP), 370 (fixed PEEP of 12 cm H2O), and 305 (fixed PEEP of 4 to 5 cm H2O) mmHg (difference to individualized PEEP, 145; 95% CI, 91 to 200; P < 0.001 for fixed PEEP of 12 cm H2O and 210; 95% CI, 164 to 257; P < 0.001 for fixed PEEP of 4 to 5 cm H2O). Intraoperative tidal volume in the dependent lung areas was 43.9% (individualized PEEP), 25.9% (fixed PEEP of 12 cm H2O) and 26.8% (fixed PEEP of 4 to 5 cm H2O) (difference to individualized PEEP: 18.0%; 95% CI, 8.0 to 20.7; P < 0.001 for fixed PEEP of 12 cm H2O and 17.1%; 95% CI, 10.0 to 20.6; P < 0.001 for fixed PEEP of 4 to 5 cm H2O). Mean intraoperative driving pressure was 9.8 cm H2O (individualized PEEP), 14.4 cm H2O (fixed PEEP of 12 cm H2O), and 18.8 cm H2O (fixed PEEP of 4 to 5 cm H2O), P < 0.001. CONCLUSIONS: This secondary analysis of obese patients undergoing laparoscopic surgery found better oxygenation, lower driving pressures, and redistribution of ventilation toward dependent lung areas measured by electrical impedance tomography using individualized PEEP. The impact on patient outcome remains unclear.

Fever and hypothermia represent two populations of sepsis patients and are associated with outside temperature.

BACKGROUND: Fever and hypothermia have been observed in septic patients. Their influence on prognosis is subject to ongoing debates. METHODS: We did a secondary analysis of a large clinical dataset from a quality improvement trial. A binary logistic regression model was calculated to assess the association of the thermal response with outcome and a multinomial regression model to assess factors associated with fever or hypothermia. RESULTS: With 6542 analyzable cases we observed a bimodal temperature response characterized by fever or hypothermia, normothermia was rare. Hypothermia and high fever were both associated with higher lactate values. Hypothermia was associated with higher mortality, but this association was reduced after adjustment for other risk factors. Age, community-acquired sepsis, lower BMI and lower outside temperatures were associated with hypothermia while bacteremia and higher procalcitonin values were associated with high fever. CONCLUSIONS: Septic patients show either a hypothermic or a fever response. Whether hypothermia is a maladaptive response, as indicated by the higher mortality in hypothermic patients, or an adaptive response in patients with limited metabolic reserves under colder environmental conditions, remains an open question. Trial registration The original trial whose dataset was analyzed was registered at ClinicalTrials.gov (NCT01187134) on August 23, 2010, the first patient was included on July 1, 2011.

High-Resolution XFEL Structure of the Soluble Methane Monooxygenase Hydroxylase Complex with its Regulatory Component at Ambient Temperature in Two Oxidation States.

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O2 activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of the enzyme in pure oxidation states. Here, microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the ≤35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage-free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. This study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.

Overlapping Vitamin A Interventions with Provitamin A Carotenoids and Preformed Vitamin A Cause Excessive Liver Retinol Stores in Male Mongolian Gerbils.

BACKGROUND: Vitamin A (VA) deficiency is a public health problem in some countries. Fortification, supplementation, and increased provitamin A consumption through biofortification are efficacious, but monitoring is needed due to risk of excessive VA intake when interventions overlap. OBJECTIVES: Two studies in 28-36-d-old male Mongolian gerbils simulated exposure to multiple VA interventions to determine the effects of provitamin A carotenoid consumption from biofortified maize and carrots and preformed VA fortificant on status. METHODS: Study 1 was a 2 × 2 × 2 factorial design (n = 85) with high-ß-carotene maize, orange carrots, and VA fortification at 50% estimated gerbil needs, compared with white maize and white carrot controls. Study 2 was a 2 × 3 factorial design (n = 66) evaluating orange carrot and VA consumption through fortification at 100% and 200% estimated needs. Both studies utilized 2-wk VA depletion, baseline evaluation, 9-wk treatments, and liver VA stores by HPLC. Intestinal scavenger receptor class B member 1 (Scarb1), ß-carotene 15,15'-dioxygenase (Bco1), ß-carotene 9',10'-oxygenase (Bco2), intestine-specific homeobox (Isx), and cytochrome P450 26A1 isoform α1 (Cyp26a1) expression was analyzed by qRT-PCR in study 2. RESULTS: In study 1, liver VA concentrations were significantly higher in orange carrot (0.69 ± 0.12 µmol/g) and orange maize groups (0.52 ± 0.21 µmol/g) compared with baseline (0.23 ± 0.069 µmol/g) and controls. Liver VA concentrations from VA fortificant alone (0.11 ± 0.053 µmol/g) did not differ from negative control. In study 2, orange carrot significantly enhanced liver VA concentrations (0.85 ± 0.24 µmol/g) relative to baseline (0.43 ± 0.14 µmol/g), but VA fortificant alone (0.42 ± 0.21 µmol/g) did not. Intestinal Scarb1 and Bco1 were negatively correlated with increasing liver VA concentrations (P < 0.01, r2 = 0.25-0.27). Serum retinol concentrations did not differ. CONCLUSIONS: Biofortified carrots and maize without fortification prevented VA deficiency in gerbils. During adequate provitamin A dietary intake, preformed VA intake resulted in excessive liver stores in gerbils, despite downregulation of carotenoid absorption and cleavage gene expression.

Individualised positive end-expiratory pressure guided by electrical impedance tomography for robot-assisted laparoscopic radical prostatectomy: a prospective, randomised controlled clinical trial.

BACKGROUND: Robot-assisted laparoscopic radical prostatectomy requires general anaesthesia, extreme Trendelenburg positioning and capnoperitoneum. Together these promote impaired pulmonary gas exchange caused by atelectasis and may contribute to postoperative pulmonary complications. In morbidly obese patients, a recruitment manoeuvre (RM) followed by individualised PEEP improves intraoperative oxygenation and end-expiratory lung volume (EELV). We hypothesised that individualised PEEP with initial RM similarly improves intraoperative oxygenation and EELV in non-obese individuals undergoing robot-assisted prostatectomy. METHODS: Forty males (age, 49-76 yr; BMI <30 kg m-2) undergoing prostatectomy received volume-controlled ventilation (tidal volume 8 ml kg-1 predicted body weight). Participants were randomised to either (1) RM followed by individualised PEEP (RM/PEEPIND) optimised using electrical impedance tomography or (2) no RM with 5 cm H2O PEEP. The primary outcome was the ratio of arterial oxygen partial pressure to fractional inspired oxygen (Pao2/Fio2) before the last RM before extubation. Secondary outcomes included regional ventilation distribution and EELV which were measured before, during, and after anaesthesia. The cardiovascular effects of RM/PEEPIND were also assessed. RESULTS: In 20 males randomised to RM/PEEPIND, the median PEEPIND was 14 cm H2O [inter-quartile range, 8-20]. The Pao2/Fio2 was 10.0 kPa higher with RM/PEEPIND before extubation (95% confidence interval [CI], 2.6-17.3 kPa; P=0.001). RM/PEEPIND increased end-expiratory lung volume by 1.49 L (95% CI, 1.09-1.89 L; P<0.001). RM/PEEPIND also improved the regional ventilation of dependent lung regions. Vasopressor and fluid therapy was similar between groups, although 13 patients randomised to RM/PEEPIND required pharmacological therapy for bradycardia. CONCLUSION: In non-obese males, an individualised ventilation strategy improved intraoperative oxygenation, which was associated with higher end-expiratory lung volumes during robot-assisted laparoscopic prostatectomy. CLINICAL TRIAL REGISTRATION: DRKS00004199 (German clinical trials registry).