Identification of the 11-cis-specific retinyl-ester synthase in retinal Müller cells as multifunctional O-acyltransferase (MFAT)

Absorption of a photon by a rhodopsin or cone-opsin pigment isomerizes its 11-cis-retinaldehyde (11-cis-RAL) chromophore to all-trans-retinaldehyde (all-trans-RAL), which dissociates after a brief period of activation. Light sensitivity is restored to the resulting apo-opsin when it recombines with another 11-cis-RAL. Conversion of all-trans-RAL to 11-cis-RAL is carried out by an enzyme pathway called the visual cycle in cells of the retinal pigment epithelium. A second visual cycle is present in Müller cells of the retina. The retinol isomerase for this noncanonical pathway is dihydroceramide desaturase (DES1), which catalyzes equilibrium isomerization of retinol. Because 11-cis-retinol (11-cis-ROL) constitutes only a small fraction of total retinols in an equilibrium mixture, a subsequent step involving selective removal of 11-cis-ROL is required to drive synthesis of 11-cis-retinoids for production of visual chromophore. Selective esterification of 11-cis-ROL is one possibility. Crude homogenates of chicken retinas rapidly convert all-trans-ROL to 11-cis-retinyl esters (11-cis-REs) with minimal formation of other retinyl-ester isomers. This enzymatic activity implies the existence of an 11-cis-specific retinyl-ester synthase in Müller cells. Here, we evaluated multifunctional O-acyltransferase (MFAT) as a candidate for this 11-cis-RE-synthase. MFAT exhibited much higher catalytic efficiency as a synthase of 11-cis-REs versus other retinyl-ester isomers. Further, we show that MFAT is expressed in Müller cells. Finally, homogenates of cells coexpressing DES1 and MFAT catalyzed the conversion of all-trans-ROL to 11-cis-RP, similar to what we observed with chicken-retina homogenates. MFAT is therefore an excellent candidate for the retinyl-ester synthase that cooperates with DES1 to drive synthesis of 11-cis-retinoids by mass action.Light perception begins with the absorption of a photon by an opsin pigment in the membranous outer segment (OS) of a rod or cone photoreceptor cell. The light-absorbing chromophore in most vertebrate opsins is 11-cis-retinaldehyde (11-cis-RAL). Photon capture isomerizes the 11-cis-RAL to all-trans-retinaldehyde (all-trans-RAL), inducing conformational changes in the protein that lead to its active meta-II state. After a brief period of signaling through the transduction cascade, meta II decays to yield apo-opsin and free all-trans-RAL. Light sensitivity is restored to the apo-opsin when it combines with 11-cis-RAL to regenerate the pigment. Conversion of all-trans-RAL to 11-cis-RAL is carried out by a multistep enzyme pathway called the visual cycle, located in cells of the retinal pigment epithelium (RPE) (1, 2). The retinoid isomerase in this pathway is Rpe65, which converts an all-trans-retinyl ester (all-trans-RE), such as all-trans-retinyl palmitate (all-trans-RP), to 11-cis-retinol (11-cis-ROL) and a free fatty acid (3–5). Retinyl esters are synthesized in RPE cells by lecithin:retinol acyl transferase (LRAT), which transfers a fatty acid from phosphatidylcholine to retinol (6, 7). LRAT converts both all-trans-ROL and 11-cis-ROL to their cognate esters with similar catalytic efficiency (8).A second visual cycle is present in Müller cells of the retina, providing 11-cis-ROL to cones (9–11). Cones, but not rods, can use 11-cis-ROL as a chromophore precursor to regenerate bleached opsin pigments (10, 12, 13). The isomerase in the noncanonical pathway is dihydroceramide desaturase (DES1) (11). DES1 catalyzes rapid equilibrium isomerization of retinol (11). At equilibrium, 11-cis-ROL is much less abundant than all-trans-ROL, due to the 4.1 kcal/mole difference in free energy between these isomers (14). Accordingly, a secondary source of energy is required to drive the conversion of all-trans-ROL to 11-cis-ROL by DES1. Retinas from cone-dominant species contain 11-cis-retinyl esters (11-cis-REs), whereas retinyl esters are much less abundant in retinas from rod-dominant species (11, 13, 15). Homogenates from cone-dominant chicken and ground-squirrel retinas convert all-trans-ROL predominantly to 11-cis-REs in the presence of palmitoyl CoA (palm CoA) (13, 16, 17). These observations suggest that selective esterification of 11-cis-ROL may be the driving force for 11-cis-retinoid formation. In the current work, we sought to identify the protein responsible for the 11-cis-RE-synthase activity in Müller cells. We evaluated multifunctional O-acyltransferase (MFAT) as a candidate for this synthase. MFAT, also called acyl-CoA wax-alcohol acyltransferase-2 (AWAT2), catalyzes palm CoA-dependent synthesis of triglycerides, wax monoesters, and retinyl esters (18). It is present in the endoplasmic reticulum and predominantly expressed in skin (18). The retinol-isomer specificity of MFAT, and its expression in ocular tissues, has not been studied.     

Impending paradoxical embolus: Clinical imaging and successful surgical treatment

In a patient with pulmonary emboli, transesophageal echocardiography showed a thrombus straddling the foramen ovale (impending paradoxical embolism). Proximal pulmonary emboli were visualized by spiral computed tomography and subsequent surgical treatment, consisting of removal of intracardiac clot, closure of the open foramen ovale and pulmonary embolectomy, was successful.     

A combined method for the determination of myocardial perfusion in experimental animals using microspheres and CMR.

A convenient method is introduced for myocardial perfusion research by combining multislice short-axis cine cardiovascular magnetic resonance (MSA-CMR) and colored microspheres (CM). In canine control-ischemia-reperfusion (n = 11), Cardiac output (CO) was measured using MSA-CMR (COMSA), Phase Contrast CMR (PC-CMR, COPC) and CM (from reference blood samples, COmicro) in 3 experimental periods per animal. COmicro significantly and systematically overestimated COMSA (median deviation: 291 mL/min, p < 0.01), while there was excellent agreement between PC-CMR and MSA-CMR without significant over-or underestimation (median deviation: -14 ml/min, p = NS). In quantitative myocardial perfusion assay by CM, CMR should be used instead of reference blood sampling. This should improve the accuracy of absolute regional perfusion measurement.     

AmpG inactivation restores susceptibility of pan-beta-lactam-resistant Pseudomonas aeruginosa clinical strains

Constitutive AmpC hyperproduction is the most frequent mechanism of resistance to the weak AmpC inducers antipseudomonal penicillins and cephalosporins. Previously, we demonstrated that inhibition of the β-N-acetylglucosaminidase NagZ prevents and reverts this mechanism of resistance, which is caused by ampD and/or dacB (PBP4) mutations in Pseudomonas aeruginosa. In this work, we compared NagZ with a second candidate target, the AmpG permease for GlcNAc-1,6-anhydromuropeptides, for their ability to block AmpC expression pathways. Inactivation of nagZ or ampG fully restored the susceptibility and basal ampC expression of ampD or dacB laboratory mutants and impaired the emergence of one-step ceftazidime-resistant mutants in population analysis experiments. Nevertheless, only ampG inactivation fully blocked ampC induction, reducing the MICs of the potent AmpC inducer imipenem from 2 to 0.38 μg/ml. Moreover, through population analysis and characterization of laboratory mutants, we showed that ampG inactivation minimized the impact on resistance of the carbapenem porin OprD, reducing the MIC of imipenem for a PAO1 OprD mutant from >32 to 0.5 μg/ml. AmpG and NagZ targets were additionally evaluated in three clinical isolates that are pan-β-lactam resistant due to AmpC hyperproduction, OprD inactivation, and overexpression of several efflux pumps. A marked increase in susceptibility to ceftazidime and piperacillin-tazobactam was observed in both cases, while only ampG inactivation fully restored wild-type imipenem susceptibility. Susceptibility to meropenem, cefepime, and aztreonam was also enhanced, although to a lower extent due to the high impact of efflux pumps on the activity of these antibiotics. Thus, our results suggest that development of small-molecule inhibitors of AmpG could provide an excellent strategy to overcome the relevant mechanisms of resistance (OprD inactivation plus AmpC induction) to imipenem, the only currently available β-lactam not significantly affected by P. aeruginosa major efflux pumps.     

Associação entre síndrome de burnout e ansiedade em residentes e anestesiologistas do Distrito Federal

Background and objective

There is a shortage of studies addressing the association between burnout syndrome and anxiety among anesthesiologists. Identifying the relationship between these two conditions is of fundamental importance for the prevention, follow‐up, and treatment of the professionals. Thus, we evaluated the association between burnout syndrome and anxiety in anesthesiologists and residents of anesthesiology in the Federal District.

Laparoscopic Treatment of Median Arcuate Ligament Syndrome: Analysis of Long-Term Outcomes and Predictive Factors

Background

Laparoscopic arcuate ligament release has been demonstrated a valid therapeutic option for arcuate ligament syndrome. Nevertheless, long-term follow-up and predictive factors have not been described for this treatment.

Methods

Clinical and surgical data and short- and long-term outcomes together with the impact of the degree of stenosis of the celiac trunk were analyzed in 13 consecutive patients who underwent laparoscopic arcuate ligament release between 2001 and 2013.

Results

Thirteen patients (12 F/1 M) underwent surgery. The median age was 32 years old, and their mean body mass index was 20.7 (range 14.7–25). The 13 patients presented with intense postprandial abdominal pain. Ten cases were associated with weight loss. The median duration of symptoms was 24 months (range 2–240). Three patients presented symptoms associated with superior mesenteric artery syndrome. Median operative time was 120 min (range 90–240), and there were no conversions to open surgery. Median hospital stay was 3 days (range 2–14). Over a median follow-up of 117 months (range 45–185), nine patients had excellent results although two required endovascular procedures at 70 and 24 months after surgery. Four patients (30.7%) experienced poor outcomes. When we analyzed the impact of the degree of occlusion of the celiac trunk, we observed that in patients with severe occlusion (>?70%), better results were obtained, with complete resolution of symptoms in 71% of cases.

Conclusion

Laparoscopic arcuate ligament release constitutes an excellent treatment for arcuate ligament syndrome. The degree of occlusion of the celiac trunk may be a factor predictive of long-term outcomes.

     

Axonal fusion via conduit-based delivery of hydrophilic polymers

Background

Hydrophilic polymers have been shown to improve physiologic recovery following repair of transected nerves with microsutures. Our study was designed to combine hydrophilic polymer therapy with nerve tubes (NT) to enhance polymer delivery to the site of nerve injury.

Methods

Using a rat sciatic nerve injury model, a single transection injury was repaired in an end-to-end fashion with NT + polyethylene glycol (PEG) to NT alone. Compound action potentials (CAPs) were recorded before nerve transection and after repair. Behavioral testing was performed for 5 weeks.

Results

PEG therapy restored CAPS in all, but one, animals, while no CAPS were recorded in animals not receiving PEG. Behavioral nerve function was measured using the standardized functional assessment technique and foot fault asymmetry scores (FF). FF scores were improved for the PEG therapy groups on postoperative days 7, 14, and 21. However, after expected eventual axonal outgrowth, the benefit was less noticeable at days 28 and 35. Immunohistochemistry of the distal axon segments showed an increase number of sensory and motor axons in the NT + PEG group as compared to NT alone.

Conclusion

These data suggest that PEG delivery via a conduit may provide a simple, effective way to fuse severed axons and regain early nerve function. For proximal nerve injuries in large animals, recovery of axonal continuity could dramatically improve outcomes, even if fusion only occurs in a small percentage of axons.