Chest compression quality during CPR of potential contagious patients wearing personal protection equipment.

AIM OF THE STUDY: In this study we aimed to investigate whether changing rescuers wearing N95 masks every 1 min instead of the standard CPR change over time of 2 min would make a difference in effective chest compressions. METHODS: This study was a randomized controlled mannequin study. Participants were selected from healthcare staff. They were divided into two groups of two people in each group. The scenario was implemented on CPR mannequin representing patient with asystolic arrest, that measured compression depth, compression rate, recoil, and correct hand position. Two different scenarios were prepared. In Scenario 1, the rescuers were asked to change chest compression after 1 min. In Scenario 2, standard CPR was applied. The participants' vital parameters, mean compression rate, correct compression rate/ratio, total number of compressions, compression depth, correct recoil/ratio, correct hand position/ratio, mean no-flow time, and total CPR time were recorded. RESULTS: The study hence included 14 teams each for scenarios, with a total of 56 participants. In each scenario, 14 participants were physicians and 14 participants were women. Although there was no difference in the first minute of the cycles starting from the fourth cycle, a statistically significant difference was observed in the second minute in all cycles except the fifth cycle. CONCLUSION: Changing the rescuer every 1 min instead of every 2 min while performing CPR with full PPE may prevent the decrease in compression quality that may occur as the resuscitation time gets longer.

Effects of coordination training on the technical development in 10-/13-year-old football players.

BACKGROUND: The aim of this study was to determine the effects of coordination training carried out by 10- and 13-year-old male football players on the performance of dribbling, passing, shooting, ball bouncing and wall-volley skills. METHODS: A total of 45 male football players were divided into three 15 participants groups. The experimental group performed 30 min coordination training three days a week for ten weeks while the control group one continued their routine training and control group two performed unstructured football training. Measurements included Mor and Christian, Yeagley and Johnson football skill tests. Pre- and post-test measurements were compared by an ANOVA 2×3. A significant level of P<0.05 was established. RESULTS: All the skills, dribbling (P<0.001), passing (P<0.001), shooting (P<0.001), ball bouncing (P=0.047) and wall-volley (P<0.001), improved after ten weeks in the experimental group, while only passing (P=0.006), shooting (P=0.007) and wall-volley (P<0.001) improved in the control group one and none of the skills improved in the control group two (P>0.05). The improvement was significant in the experimental group in comparison with the control groups (P<0.001). CONCLUSIONS: The implementation of coordination exercises in combination with structured football training has been shown to be effective in improving general football ability among 10-13 male players.

Colostomy reversal after a Hartmann's procedure Effects of experience on mortality and morbidity.

AIM: Hartmann's procedure (HP) is mostly lifesaving procedure especially for obstructive colorectal carcinomas, but remains bothersome requiring staged operation and subsequent reversal colostomy. We aimed to investigate risk factors for unfavorable surgical outcome after Hartmann's reversal. MATERIALS AND METHODS: Between September 2003 and September 2014, all patients who underwent colostomy reversal surgery after HP were enrolled into the study. Retrospective data collection included demographics (age, gender, body mass index (BMI), ASA scores) primary pathologies, interval period, surgeon who performed procedure [general surgeon (GS)/colorectal specialty (CRS)], postoperative complications and hospital stay. RESULTS: There were 72 patients (49M/23F) with a median age of 64 (range: 29-83) years. The median BMI was 24 (21-44). Most of the patients (82%) had ASA score 3-4. Colorectal cancer was the primary diagnosis in 79% while others included diverticular perforation, volvulus, trauma and Crohn disease. Hartmann's procedure was performed as an emergency in three-fifths of patients. Median interval period between index surgery and reversal colostomy was 7 (1-24) months. The morbidity and mortality rates for colostomy reversal surgery were 34% and 8.3%, respectively. The most common postoperative complication was surgical site infection (22%) followed by anastomotic leak 5%. Mortality and morbidity rates were significantly higher in patients with higher BMI (p=0.031), higher ASA scores (p=0.028) and patients who underwent procedure not by a CRS. CONCLUSION: Reversal colostomy procedure resulted in significant morbidity and mortality, particularly in those with high BMI and ASA scores. Efforts to improve risk management and specialization in colorectal surgery may help to improve the outcome in reversal colostomy after Hartmann's procedure. KEY WORDS: Colorectal surgery, Experience, Hartmann's procedure, Reversal colostomy, Morbidity.

Development and Validation of the Biotechnology Literacy Test.

In this study, a 'biotechnology literacy test' was developed to determine the biotechnology literacy of prospective science teachers, and its validity and reliability were determined. For this purpose, 42 items were prepared by considering Bybee's scientific literacy classifications (nominal, functional, procedural, and multidimensional). The experts were asked to rate each item for content validity. As a result of the experts' evaluations, the content validity of the test was confirmed. The draft test was applied to 494 science prospective teachers. Difficulty and discrimination coefficients of items within the test were calculated to provide evidence of construct validity. It was decided to exclude 13 items as a result of item analysis. It can be said that that the test was difficult (p = 0.40) and discriminative (D = 0.41) at medium level. Finally, a valid and reliable "biotechnology literacy test" consisting of 25 multiple-choice items each with five choices was developed in this research. © 2019 International Union of Biochemistry and Molecular Biology, 47(2): 179-188, 2019.

Exploring the Influence of Domain Architecture on the Catalytic Function of Diterpene Synthases.

Terpenoid synthases catalyze isoprenoid cyclization reactions underlying the generation of more than 80,000 natural products. Such dramatic chemodiversity belies the fact that these enzymes generally consist of only three domain folds designated as α, ß, and γ. Catalysis by class I terpenoid synthases occurs exclusively in the α domain, which is found with α, αα, αß, and αßγ domain architectures. Here, we explore the influence of domain architecture on catalysis by taxadiene synthase from Taxus brevifolia (TbTS, αßγ), fusicoccadiene synthase from Phomopsis amygdali (PaFS, (αα)6), and ophiobolin F synthase from Aspergillus clavatus (AcOS, αα). We show that the cyclization fidelity and catalytic efficiency of the α domain of TbTS are severely compromised by deletion of the ßγ domains; however, retention of the ß domain preserves significant cyclization fidelity. In PaFS, we previously demonstrated that one α domain similarly influences catalysis by the other α domain [ Chen , M. , Chou , W. K. W. , Toyomasu , T. , Cane , D. E. , and Christianson , D. W. ( 2016 ) ACS Chem. Biol. 11 , 889 - 899 ]. Here, we show that the hexameric quaternary structure of PaFS enables cluster channeling. We also show that the α domains of PaFS and AcOS can be swapped so as to make functional chimeric αα synthases. Notably, both cyclization fidelity and catalytic efficiency are altered in all chimeric synthases. Twelve newly formed and uncharacterized C20 diterpene products and three C25 sesterterpene products are generated by these chimeras. Thus, engineered αßγ and αα terpenoid cyclases promise to generate chemodiversity in the greater family of terpenoid natural products.

Structural Studies of Geosmin Synthase, a Bifunctional Sesquiterpene Synthase with αα Domain Architecture That Catalyzes a Unique Cyclization-Fragmentation Reaction Sequence.

Geosmin synthase from Streptomyces coelicolor (ScGS) catalyzes an unusual, metal-dependent terpenoid cyclization and fragmentation reaction sequence. Two distinct active sites are required for catalysis: the N-terminal domain catalyzes the ionization and cyclization of farnesyl diphosphate to form germacradienol and inorganic pyrophosphate (PPi), and the C-terminal domain catalyzes the protonation, cyclization, and fragmentation of germacradienol to form geosmin and acetone through a retro-Prins reaction. A unique αα domain architecture is predicted for ScGS based on amino acid sequence: each domain contains the metal-binding motifs typical of a class I terpenoid cyclase, and each domain requires Mg(2+) for catalysis. Here, we report the X-ray crystal structure of the unliganded N-terminal domain of ScGS and the structure of its complex with three Mg(2+) ions and alendronate. These structures highlight conformational changes required for active site closure and catalysis. Although neither full-length ScGS nor constructs of the C-terminal domain could be crystallized, homology models of the C-terminal domain were constructed on the basis of ∼36% sequence identity with the N-terminal domain. Small-angle X-ray scattering experiments yield low-resolution molecular envelopes into which the N-terminal domain crystal structure and the C-terminal domain homology model were fit, suggesting possible αα domain architectures as frameworks for bifunctional catalysis.

1.55Å-resolution structure of ent-copalyl diphosphate synthase and exploration of general acid function by site-directed mutagenesis.

BACKGROUND: The diterpene cyclase ent-copalyl diphosphate synthase (CPS) catalyzes the first committed step in the biosynthesis of gibberellins. The previously reported 2.25Å resolution crystal structure of CPS complexed with (S)-15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (1) established the αßγ domain architecture, but ambiguities regarding substrate analog binding remained. METHOD: Use of crystallization additives yielded CPS crystals diffracting to 1.55Å resolution. Additionally, active site residues that hydrogen bond with D379, either directly or through hydrogen bonded water molecules, were probed by mutagenesis. RESULTS: This work clarifies structure-function relationships that were ambiguous in the lower resolution structure. Well-defined positions for the diphosphate group and tertiary ammonium cation of 1, as well as extensive solvent structure, are observed. CONCLUSIONS: Two channels involving hydrogen bonded solvent and protein residues lead to the active site, forming hydrogen bonded "proton wires" that link general acid D379 with bulk solvent. These proton wires may facilitate proton transfer with the general acid during catalysis. Activity measurements made with mutant enzymes indicate that N425, which donates a hydrogen bond directly to D379, and T421, which hydrogen bonds with D379 through an intervening solvent molecule, help orient D379 for catalysis. Residues involved in hydrogen bonds with the proton wire, R340 and D503, are also important. Finally, conserved residue E211, which is located near the diphosphate group of 1, is proposed to be a ligand to Mg(2+) required for optimal catalytic activity. GENERAL SIGNIFICANCE: This work establishes structure-function relationships for class II terpenoid cyclases.

Unexpected reactivity of 2-fluorolinalyl diphosphate in the active site of crystalline 2-methylisoborneol synthase.

The crystal structure of 2-methylisoborneol synthase (MIBS) from Streptomyces coelicolor A3(2) has been determined in its unliganded state and in complex with two Mg(2+) ions and 2-fluoroneryl diphosphate at 1.85 and 2.00 Å resolution, respectively. Under normal circumstances, MIBS catalyzes the cyclization of the naturally occurring, noncanonical 11-carbon isoprenoid substrate, 2-methylgeranyl diphosphate, which first undergoes an ionization-isomerization-ionization sequence through the tertiary diphosphate intermediate 2-methyllinalyl diphosphate to enable subsequent cyclization chemistry. MIBS does not exhibit catalytic activity with 2-fluorogeranyl diphosphate, and we recently reported the crystal structure of MIBS complexed with this unreactive substrate analogue [ Köksal, M., Chou, W. K. W., Cane, D. E., Christianson, D. W. (2012) Biochemistry 51 , 3011-3020 ]. However, cocrystallization of MIBS with the fluorinated analogue of the tertiary allylic diphosphate intermediate, 2-fluorolinalyl diphosphate, reveals unexpected reactivity for the intermediate analogue and yields the crystal structure of the complex with the primary allylic diphosphate, 2-fluoroneryl diphosphate. Comparison with the structure of the unliganded enzyme reveals that the crystalline enzyme active site remains partially open, presumably due to the binding of only two Mg(2+) ions. Assays in solution indicate that MIBS catalyzes the generation of (1R)-(+)-camphor from the substrate 2-fluorolinalyl diphosphate, suggesting that both 2-fluorolinalyl diphosphate and 2-methyllinalyl diphosphate follow the identical cyclization mechanism leading to 2-substituted isoborneol products; however, the initially generated 2-fluoroisoborneol cyclization product is unstable and undergoes elimination of hydrogen fluoride to yield (1R)-(+)-camphor.

Probing the mechanism of 1,4-conjugate elimination reactions catalyzed by terpene synthases.

The reaction mechanisms of (E)-ß-farnesene synthase (EBFS) and isoprene synthase (ISPS), enzymes that catalyze a formal regiospecific 1,4-conjugate elimination of hydrogen diphosphate from (E,E)-farnesyl and dimethylallyl diphosphate (FDP and DMADP) to generate the semiochemicals (E)-ß-farnesene and isoprene, respectively, were probed with substrate analogs and kinetic measurements. The results support stepwise reaction mechanisms through analogous enzyme-bound allylic cationic intermediates. For EBFS, we demonstrate that the elimination reaction can proceed via the enzyme-bound intermediate trans-nerolidyl diphosphate, while for ISPS the intermediacy of 2-methylbut-3-enyl 2-diphosphate can be inferred from the product outcome when deuterated DMADPs are used as substrates. Possible implications derived from the mechanistic details of the EBFS-catalyzed reaction for the evolution of sesquiterpene synthases are discussed.

Differential binding between volatile ligands and major urinary proteins due to genetic variation in mice.

Two different structural classes of chemical signals in mouse urine, i.e., volatile organic compounds (VOCs) and the major urinary proteins (MUPs), interact closely because MUPs sequester VOCs. Although qualitative and/or quantitative differences in each chemical class have been reported, previous studies have examined only one of the classes at a time. No study has analyzed these two sets simultaneously, and consequently binding interactions between volatile ligands and proteins in urines of different strains have not been compared. Here, we compared the release of VOCs in male urines of three different inbred strains (C57BL/6J, BALB/b and AKR) before and after denaturation of urinary proteins, mainly MUPs. Both MUP and VOC profiles were distinctive in the intact urine of each strain. Upon denaturation, each of the VOC profiles changed due to the release of ligands previously bound to MUPs. The results indicate that large amounts of numerous ligands are bound to MUPs and that these ligands represent a variety of different structural classes of VOCs. Furthermore, the degree of release in each ligand was different in each strain, indicating that different ligands are differentially bound to proteins in the urines of different strains. Therefore, these data suggest that binding interactions in ligands and MUPs differ between strains, adding yet another layer of complexity to chemical communication in mice.

Structure of geranyl diphosphate C-methyltransferase from Streptomyces coelicolor and implications for the mechanism of isoprenoid modification.

Geranyl diphosphate C-methyltransferase (GPPMT) from Streptomyces coelicolor A3(2) is the first methyltransferase discovered that modifies an acyclic isoprenoid diphosphate, geranyl diphosphate (GPP), to yield a noncanonical acyclic allylic diphosphate product, 2-methylgeranyl diphosphate, which serves as the substrate for a subsequent cyclization reaction catalyzed by a terpenoid cyclase, methylisoborneol synthase. Here, we report the crystal structures of GPPMT in complex with GPP or the substrate analogue geranyl S-thiolodiphosphate (GSPP) along with S-adenosyl-L-homocysteine in the cofactor binding site, resulting from in situ demethylation of S-adenosyl-L-methionine, at 2.05 or 1.82 Å resolution, respectively. These structures suggest that both GPP and GSPP can undergo catalytic methylation in crystalline GPPMT, followed by dissociation of the isoprenoid product. S-Adenosyl-L-homocysteine remains bound in the active site, however, and does not exchange with a fresh molecule of cofactor S-adenosyl-L-methionine. These structures provide important clues about the molecular mechanism of the reaction, especially with regard to the face of the 2,3 double bond of GPP that is methylated as well as the stabilization of the resulting carbocation intermediate through cation-π interactions.

Structure of 2-methylisoborneol synthase from Streptomyces coelicolor and implications for the cyclization of a noncanonical C-methylated monoterpenoid substrate.

The crystal structure of 2-methylisoborneol synthase (MIBS) from Streptomyces coelicolor A3(2) has been determined in complex with substrate analogues geranyl-S-thiolodiphosphate and 2-fluorogeranyl diphosphate at 1.80 and 1.95 Å resolution, respectively. This terpenoid cyclase catalyzes the cyclization of the naturally occurring, noncanonical C-methylated isoprenoid substrate, 2-methylgeranyl diphosphate, to form the bicyclic product 2-methylisoborneol, a volatile C(11) homoterpene alcohol with an earthy, musty odor. While MIBS adopts the tertiary structure of a class I terpenoid cyclase, its dimeric quaternary structure differs from that previously observed in dimeric terpenoid cyclases from plants and fungi. The quaternary structure of MIBS is nonetheless similar in some respects to that of dimeric farnesyl diphosphate synthase, which is not a cyclase. The structures of MIBS complexed with substrate analogues provide insights regarding differences in the catalytic mechanism of MIBS and the mechanisms of (+)-bornyl diphosphate synthase and endo-fenchol synthase, plant cyclases that convert geranyl diphosphate into products with closely related bicyclic bornyl skeletons, but distinct structures and stereochemistries.

Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase.

The structure of ent-copalyl diphosphate synthase reveals three α-helical domains (α, ß and γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the ßγ domains in ent-copalyl diphosphate synthase but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

Taxadiene synthase structure and evolution of modular architecture in terpene biosynthesis.

With more than 55,000 members identified so far in all forms of life, the family of terpene or terpenoid natural products represents the epitome of molecular biodiversity. A well-known and important member of this family is the polycyclic diterpenoid Taxol (paclitaxel), which promotes tubulin polymerization and shows remarkable efficacy in cancer chemotherapy. The first committed step of Taxol biosynthesis in the Pacific yew (Taxus brevifolia) is the cyclization of the linear isoprenoid substrate geranylgeranyl diphosphate (GGPP) to form taxa-4(5),11(12)diene, which is catalysed by taxadiene synthase. The full-length form of this diterpene cyclase contains 862 residues, but a roughly 80-residue amino-terminal transit sequence is cleaved on maturation in plastids. We now report the X-ray crystal structure of a truncation variant lacking the transit sequence and an additional 27 residues at the N terminus, hereafter designated TXS. Specifically, we have determined structures of TXS complexed with 13-aza-13,14-dihydrocopalyl diphosphate (1.82 Å resolution) and 2-fluorogeranylgeranyl diphosphate (2.25 Å resolution). The TXS structure reveals a modular assembly of three α-helical domains. The carboxy-terminal catalytic domain is a class I terpenoid cyclase, which binds and activates substrate GGPP with a three-metal ion cluster. The N-terminal domain and a third 'insertion' domain together adopt the fold of a vestigial class II terpenoid cyclase. A class II cyclase activates the isoprenoid substrate by protonation instead of ionization, and the TXS structure reveals a definitive connection between the two distinct cyclase classes in the evolution of terpenoid biosynthesis.

Structure of isoprene synthase illuminates the chemical mechanism of teragram atmospheric carbon emission.

The X-ray crystal structure of recombinant PcISPS (isoprene synthase from gray poplar hybrid Populus×canescens) has been determined at 2.7 Å resolution, and the structure of its complex with three Mg(2+) and the unreactive substrate analogue dimethylallyl-S-thiolodiphosphate has been determined at 2.8 Å resolution. Analysis of these structures suggests that the generation of isoprene from substrate dimethylallyl diphosphate occurs via a syn-periplanar elimination mechanism in which the diphosphate-leaving group serves as a general base. This chemical mechanism is responsible for the annual atmospheric emission of 100 Tg of isoprene by terrestrial plant life. Importantly, the PcISPS structure promises to guide future protein engineering studies, potentially leading to hydrocarbon fuels and products that do not rely on traditional petrochemical sources.