Spatial connectivity of reef manta rays across the Raja Ampat archipelago, Indonesia.

The reef manta ray Mobula alfredi is present throughout most island groups that form the Raja Ampat archipelago, Indonesia. The species is protected regionally and nationally and is currently managed as a single homogeneous population within the 6.7 million ha archipelago. However, scientific evidence is currently lacking regarding the spatial connectivity and population structure of M. alfredi within this archipelago. Using network analysis and an array of 34 acoustic receivers deployed throughout Raja Ampat between February 2016 and September 2021, we examined the movements of 72 subadult and adult M. alfredi tagged in seven regions of Raja Ampat. A total of 1094 M. alfredi movements were recorded and were primarily concentrated between nearby receiver stations, highlighting frequent local movements within, and limited long-distance movements between regional acoustic receiver arrays. Network analysis revealed highly connected nodes acting as hubs important for M. alfredi movements. A community detection algorithm further indicated clusters within the network. Our results suggest the existence of a metapopulation comprising three demographically and geographically distinct subpopulations within the archipelago. They also reveal the importance of Eagle Rock as a critical node in the M. alfredi movement network, justifying the urgent inclusion of this site within the Raja Ampat marine protected area network.

Antibacterial, bacteriolytic, antibiofilm, and synergistic effects of the peel oils of Citrus microcarpa and Citrus x amblycarpa with tetracycline against foodborne Escherichia coli.

Citrus essential oils (EOs) have shown significant antibacterial activity. The present study was undertaken to evaluate the antibacterial activity of the peel oils of Citrus microcarpa and C. x amblycarpa against Escherichia coli. The minimum inhibition concentration (MIC) was determined by using the broth microdilution assay. The checkerboard method was used to identify synergistic effects of the EOs with tetracycline, while bacteriolysis was assessed by calculating the optical density of the bacterial supernatant, crystal violet assay was used to assess their antibiofilm. Ethidium bromide accumulation test was employed to assess efflux pump inhibition. Electron microscope analysis was performed to observe its morphological changes. The EOs of C. microcarpa and C. x amblycarpa were found to contain D-limonene major compound at 55.78% and 46.7%, respectively. Citrus microcarpa EOs exhibited moderate antibacterial against E. coli with a MIC value of 200 µg/mL. The combination of C. microcarpa oil (7.8 µg/mL) and tetracycline (62.5 µg/mL) exhibited a synergy with FICI of 0.5. This combination inhibited biofilm formation and disrupt bacterial cell membranes. Citrus microcarpa EOs blocked the efflux pumps in E. coli. Citrus microcarpa EOs demonstrated promising antibacterial activity, which can be further explored for the development of drugs to combat E. coli.

Directed evolution of glutathione transferases towards a selective glutathione-binding site and improved oxidative stability.

BACKGROUND: Glutathione transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic compounds. METHODS: A library of alpha class GSTs was constructed by DNA shuffling using the DNA encoding the human glutathione transferase A1-1 (hGSTA1-1) and the rat glutathione transferase A1-1 (rGSTA1-1). RESULTS: Activity screening of the library allowed the selection of a chimeric enzyme variant (GSTD4) that displayed high affinity towards GSH and GSH-Sepharose affinity adsorbent, higher kcat/Km and improved thermal stability, compared to the parent enzymes. The crystal structures of the GSTD4 enzyme in free form and in complex with GSH were determined to 1.6Å and 2.3Å resolution, respectively. Analysis of the GSTD4 structure showed subtle conformational changes in the GSH-binding site and in electron-sharing network that may contribute to the increased GSH affinity. The shuffled variant GSTD4 was further optimized for improved oxidative stability employing site-saturation mutagenesis. The Cys112Ser mutation confers optimal oxidative stability and kinetic properties in the GSTD4 enzyme. CONCLUSIONS: DNA shuffling allowed the creation of a chimeric enzyme variant with improved properties, compared to the parent enzymes. X-ray crystallography shed light on how recombination of a specific segment from homologous GSTA1-1 together with point mutations gives rise to a new functionally competent enzyme with improved binding, catalytic properties and stability. GENERAL SIGNIFICANCE: Such an engineered GST would be useful in biotechnology as affinity tool in affinity chromatography as well as a biocatalytic matrix for the construction of biochips or enzyme biosensors.

Characterization and functional analysis of a recombinant tau class glutathione transferase GmGSTU2-2 from Glycine max.

The plant tau class glutathione transferases (GSTs) perform diverse catalytic as well as non-catalytic roles in detoxification of xenobiotics, prevention of oxidative damage and endogenous metabolism. In the present work, the tau class isoenzyme GSTU2-2 from Glycine max (GmGSTU2-2) was characterized. Gene expression analysis of GmGSTU2 suggested a highly specific and selective induction pattern to osmotic stresses, indicating that gene expression is controlled by a specific mechanism. Purified, recombinant GmGSTU2-2 was shown to exhibit wide-range specificity towards xenobiotic compounds and ligand-binding properties, suggesting that the isoenzyme could provide catalytic flexibility in numerous metabolic conditions. Homology modeling and phylogenetic analysis suggested that the catalytic and ligand binding sites of GmGSTU2-2 are well conserved compared to other tau class GSTs. Structural analysis identified key amino acid residues in the hydrophobic binding site and provided insights into the substrate specificity of this enzyme. The results established that GmGSTU2-2 participates in a broad network of catalytic and regulatory functions involved in the plant stress response.

Directed evolution of Tau class glutathione transferases reveals a site that regulates catalytic efficiency and masks co-operativity.

A library of Tau class GSTs (glutathione transferases) was constructed by DNA shuffling using the DNA encoding the Glycine max GSTs GmGSTU2-2, GmGSTU4-4 and GmGSTU10-10. The parental GSTs are >88% identical at the sequence level; however, their specificity varies towards different substrates. The DNA library contained chimaeric structures of alternated segments of the parental sequences and point mutations. Chimaeric GST sequences were expressed in Escherichia coli and their enzymatic activities towards CDNB (1-chloro-2,4-dinitrobenzene) and the herbicide fluorodifen (4-nitrophenyl α,α,α-trifluoro-2-nitro-p-tolyl ether) were determined. A chimaeric clone (Sh14) with enhanced CDNB- and fluorodifen-detoxifying activities, and unusual co-operative kinetics towards CDNB and fluorodifen, but not towards GSH, was identified. The structure of Sh14 was determined at 1.75 Å (1 Å=0.1 nm) resolution in complex with S-(p-nitrobenzyl)-glutathione. Analysis of the Sh14 structure showed that a W114C point mutation is responsible for the altered kinetic properties. This was confirmed by the kinetic properties of the Sh14 C114W mutant. It is suggested that the replacement of the bulky tryptophan residue by a smaller amino acid (cysteine) results in conformational changes of the active-site cavity, leading to enhanced catalytic activity of Sh14. Moreover, the structural changes allow the strengthening of the two salt bridges between Glu(66) and Lys(104) at the dimer interface that triggers an allosteric effect and the communication between the hydrophobic sites.

Catalytic features and crystal structure of a tau class glutathione transferase from Glycine max specifically upregulated in response to soybean mosaic virus infections.

The plant tau class glutathione transferases (GSTs) play important roles in biotic and abiotic stress tolerance in crops and weeds. In this study, we systematically examined the catalytic and structural features of a GST isoenzyme from Glycine max (GmGSTU10-10). GmGSTU10-10 is a unique isoenzyme in soybean that is specifically expressed in response to biotic stress caused by soybean mosaic virus (SMV) infections. GmGSTU10-10 was cloned, expressed in Escherichia coli, purified and characterized. The results showed that GmGSTU10-10 catalyzes several different reactions and exhibits wide substrate specificity. Of particular importance is the finding that the enzyme shows high antioxidant catalytic function and acts as hydroperoxidase. In addition, its Km for GSH is significantly lower, compared to other plant GSTs, suggesting that GmGSTU10-10 is able to perform efficient catalysis under conditions where the concentration of reduced glutathione is low (e.g. oxidative stress). The crystal structure of GmGSTU10-10 was solved by molecular replacement at 1.6Å resolution in complex with glutathione sulfenic acid (GSOH). Structural analysis showed that GmGSTU10-10 shares the same overall fold and domain organization as other plant cytosolic GSTs; however, major variations were identified in helix H9 and the upper part of helix H4 that affect the size of the active site pockets, substrate recognition and the catalytic mechanism. The results of the present study provide new information into GST diversity and give further insights into the complex regulation and enzymatic functions of this plant gene superfamily.

Overlapping protective roles for glutathione transferase gene family members in chemical and oxidative stress response in Agrobacterium tumefaciens.

In the present work, we describe the characterisation of the glutathione transferase (GST) gene family from Agrobacterium tumefaciens C58. A genome survey revealed the presence of eight GST-like proteins in A. tumefaciens (AtuGSTs). Comparison by multiple sequence alignment generated a dendrogram revealing the phylogenetic relationships of AtuGSTs-like proteins. The beta and theta classes identified in other bacterial species are represented by five members in A. tumefaciens C58. In addition, there are three "orphan" sequences that do not fit into any previously recognised GST classes. The eight GST-like genes were cloned, expressed in Escherichia coli and their substrate specificity was determined towards 17 different substrates. The results showed that AtuGSTs catalyse a broad range of reactions, with different members of the family exhibiting quite varied substrate specificity. The 3D structures of AtuGSTs were predicted using molecular modelling. The use of comparative sequence and structural analysis of the AtuGST isoenzymes allowed us to identify local sequence and structural characteristics between different GST isoenzymes and classes. Gene expression profiling was conducted under normal culture conditions as well as under abiotic stress conditions (addition of xenobiotics, osmotic stress and cold and heat shock) to induce and monitor early stress-response mechanisms. The results reveal the constitutive expression of GSTs in A. tumefaciens and a modulation of GST activity after treatments, indicating that AtuGSTs presumably participate in a wide range of functions, many of which are important in counteracting stress conditions. These functions may be relevant to maintaining cellular homeostasis as well as in the direct detoxification of toxic compounds.

Simulating face-to-face tracheal intubation of a trapped patient: a randomized comparison of the LMA Fastrach™, the GlideScope™, and the Airtraq™ laryngoscope.

BACKGROUND: We undertook a prospective randomized comparison of the LMA Fastrach™, Airtraq™ laryngoscope, and GlideScope™ used for face-to-face tracheal intubation simulated to mimic an entrapped patient. METHODS: Thirty senior emergency medicine physicians were trained in the use of the LMA Fastrach™, GlideScope™, and Airtraq™ laryngoscope with a standard airway trainer manikin (control). Participants were then asked to perform tracheal intubation in two difficult situations simulated on a difficult airway management manikin wearing a cervical collar. In Situation 1, the manikin was in the supine position with a difficult airway caused by stiffening the cervical spine. In Situation 2, the manikin was positioned to simulate face-to-face tracheal intubation. We measured intubation times, success rates for tracheal intubation, and the difficulty of tracheal intubation. Values are means (sd). RESULTS: In control and Situation 1, tracheal intubation details were similar. In Situation 2, face-to-face tracheal intubation success rate was increased with the Airtraq™ (100%), when compared with that of the GlideScope™ (70%, P<0.05) and LMA Fastrach™ (83%, P<0.05). Face-to-face tracheal intubation was less difficult (visual analogue scale: 0-100) with the Airtraq™ 11 (6) when compared with the GlideScope™ [33 (14) s, P<0.01)] and LMA Fastrach™ [22 (21) s, P<0.01]. The face-to-face tracheal intubation time was shorter with the Airtraq™ 14 (6) s than with the GlideScope™ [27 (18) s, P<0.01] and Fastrach™ [28 (10) s, P<0.01]. CONCLUSIONS: The Airtraq™ laryngoscope was superior to both the GlideScope™ and LMA Fastrach™ during simulated face-to-face difficult tracheal intubation.

[Cricothyrotomy in emergency context: assessment of a cannot intubate cannot ventilate scenario]. / Cricothyroïdotomie en situation d'urgence : évaluation d'un scénario dynamique associant intubation et ventilation impossibles.

OBJECTIVE: The aim of this study was to assess airway management by emergency physicians in case of a simulated situation where intubation and ventilation were both impossible. STUDY DESIGN: Observational manikin study. METHODS: A manikin (Airman®; Laerdal) allowing simulating difficult airway situations was used. The scenario assessed concerned a patient needing tracheal intubation for severe traumatic brain injury. The manikin was settled to make tracheal intubation under direct laryngoscopy impossible at the first attempt and to make facemask ventilation impossible after the second attempt. Manikin could initially be ventilated through the intubating laryngeal mask Airway (ILMA) but became impossible few seconds after its insertion. With impossible ventilation through the ILMA, arterial oxygen saturation decreased during 2 minutes before an hypoxic cardiac arrest occurred. Physicians could use classic laryngoscope with Macintosh blade, a Gum Elastic Bougie, an ILMA and a cricothyrotomy set. Adhesion to the national airway management algorithm was assessed. Time to cricothyroidotomy decision after ventilation through ILMA became impossible was measured. RESULTS: Twenty-five emergency physicians were assessed. For 14 of them, national expert conference algorithm was perfectly followed. For ten physicians, cricothyroidotomy decision was taken after hypoxic cardiac arrest occurred. CONCLUSION: Simulation with a manikin is useful to assess the adhesion rate to difficult intubation algorithms. Our study shows that the decision making process for cricothyrotomy is too often delayed as soon as ventilation became impossible and oxygenation compromized.

[Performance of the glottiscopes: a randomized comparative study on difficult intubation simulation manikin]. / Performance des glottiscopes : une étude comparative randomisée menée sur simulateur d'intubation difficile.

INTRODUCTION: We have prospectively compared simulated-difficult tracheal intubation characteristics of four glottiscopes: Airtraq, GlideScope, McGrath, LMA CTrach with that of the conventional Macintosh laryngoscope. STUDY DESIGN: prospective with the airway devices proposed in a randomly assigned order. MATERIALS AND METHOD: Forty-two physicians, naïve to glottiscope handling accepted participating this study after the learning curve of each airway device was completed. Participants were requested to perform two series of five tracheal intubations on the manikin Airman, the first in standard situation and the second in difficult tracheal intubation simulation. The airway devices were chosen in a randomly assigned order. For each airway tool, the following tracheal intubation characteristics were recorded: laryngeal exposure quality, tracheal intubation and apnea durations. A performance index was calculated and a tracheal intubation difficulty was measured during simulation. RESULTS: More than 1600 supervised tracheal intubations were performed, including 1000 for the learning process of the glottiscopes, which was completed after 10 uses on the manikin. During standard situation, laryngeal exposure quality was similar with the five airway devices. As compared to the Macintosh laryngoscope, GlideScope, McGrath, tracheal intubation duration was shorter (p<0.05) with the Airtraq and longer (p<0.01) with the LMA CTrach. During difficult tracheal intubation simulation, laryngeal exposure and tracheal intubation duration was of better quality and shorter with the four glottiscopes as compared to that of LM, respectively. Performance index during difficult tracheal intubation simulation simulation was significantly more important (p<0.01) with the Airtraq and the LMA CTrach. Airtraq and Macintosh laryngoscope were respectively the simplest (p<0.01) and the most difficult (p<0.01) airway devices to manage a simulated difficult tracheal intubation. CONCLUSION: When difficult airway was simulated on the manikin, the four glottiscopes were superior to the Macintosh laryngoscope to improve laryngeal exposure quality and to reduce duration of tracheal intubation. Airtraq and the LMA CTrach both demonstrated remarkable advantage over GlideScope and McGrath for simulated difficult intubation management.

Sparing the larynx during gynecological laparoscopy: a randomized trial comparing the LMA Supreme and the ETT.

BACKGROUND: We designed a prospective randomized single-blind study to compare efficiency and post-operative upper airway morbidity when the laryngeal mask airway (LMA) Supreme is used as an alternative to the endotracheal tube (ETT). METHODS: One hundred and thirty-eight elective pelvic laparoscopic ASA I-II female patients were assigned to receive either the LMA Supreme or the ETT for airway management. Balanced anesthesia and ventilation techniques were standardized to control end-tidal CO(2) and BIS value in the range 4.5-5 kPa and 40-50, respectively, and to maintain adequate hemodynamic stability. A single surgeon blinded to the airway management technique performed all surgical procedures. The ventilation efficiency of each airway was evaluated. Anesthesia- and surgery-related times were calculated and anesthesia details were recorded. Post-operative pain and pharyngolaryngeal morbidity were measured in a blind fashion using a numerical rating scale (NRS) (0-100). RESULTS: Surgery duration was similar in both groups. Airway management duration was shorter with the LMA Supreme. Post-operative pharyngolaryngeal morbidity incidence and all symptoms' intensity were significantly increased after ETT as compared with LMA Supreme anesthesia. At the end of the PACU stage, the incidence and mean NRS of post-operative hoarseness were reduced when LMA Supreme was used as an alternative to the ETT (16% vs. 47%; P<0.01 and 9 vs. 19, P<0.01, respectively). CONCLUSION: We demonstrated that choosing an LMA Supreme was an efficient pharyngolaryngeal morbidity-sparing strategy. Moreover, we showed that the LMA Supreme and the ETT were equally effective airways for a routine gynecological laparoscopy procedure.

Optimising tracheal intubation success rate using the Airtraq laryngoscope.

In this study we have used a video-recording, retrospective analysis technique to evaluate the influence of the Airtraq laryngoscope manipulations and the resulting changes in position of the glottic opening and inter-arytenoids cleft, on the success rate of tracheal intubation. The video recordings of the internal views of 109 tracheal intubation attempts, in 50 anaesthetised patients were analysed. We demonstrated that successful tracheal intubation using the Airtraq laryngoscope require the glottic opening to be centred in the view, and positioning the inter-arytenoid cleft medially below the horizontal line in the centre of the view. We also demonstrated that repositioning of the Airtraq laryngoscope following a failed tracheal intubation attempt required the performance of a standard series of manoeuvres.

Evidence of pulmonary aspiration during difficult airway management of a morbidly obese patient with the LMA CTrach.

We describe a pulmonary aspiration that occurred during tracheal intubation with the LMA CTrach (SEBAC, Pantin, France) in a male morbidly obese patient (178 cm height, BMI=48 kg m(-2)) admitted for elective gastric banding. Our report suggests that manipulations of the CTrach such as Up-manoeuvre may lead to pulmonary aspiration in the case of regurgitated gastric content.

Sampling variability on percutaneous liver biopsy.

Sampling variability of liver biopsy was determined in three consecutive biopsy specimens obtained from each of 118 patients immediately prior to autopsy. No sampling variability was found for fatty liver, alcoholic hepatitis, nonspecific hepatitis, fulminant hepatitis, leukemic infiltrate, and venous congestion. Cirrhosis was diagnosed in 80% of cases at the first biopsy but in all cases after three biopsies. Chronic aggressive and chronic persistent hepatitis were diagnosed correctly in two of three cases each at the first biopsy, and in all cases after three biopsies. Metastatic carcinoma was detected in 46% of cases at the first biopsy and in 69% after three biopsies. Granulomas were missed once on the first biopsy, but found on a subsequent biopsy. The amounts of fat and fibrosis in the biopsy specimens often were not representative of the amounts present at autopsy.

Changes in hepatic collagen metabolism in rats produced by chronic ethanol feeding.

The effect of ethanol feeding for a period of 6 months on parameters of hepatic collagen metabolism was studied in the rat. Ethanol feeding resulted in small increases in the fibrous and ground substance components of hepatic collagen as measured by increases in collagen-bound hydroxyproline and hexosamine, respectively. Liver histology revealagen proline hydroxylase and the incorporation of labeled proline into collagen by liver slices, both of which are associated with collagen synthesis, were not changed. Ethanol feeding resulted in increases in the concentration of protein and deoxyribonucleic acid in the Kupffer cells, but in no changes in collagenase activity. An increase in collagen degradation was suggested, however, by the increase in the urinary excretion of hydroxyproline and glycosaminoglycans found after 2 and 6 months of ethanol feeding, respectively. This study demonstrates that fatty infiltration of the liver in the rat, after prolonged ethanol feeding, is associated with increased deposition of chemically detectable collagen and evidence of increased collagen degradation, although no significant changes in parameters associated with hepatic collagen synthesis were found.