A map of the rubisco biochemical landscape.

Rubisco is the primary CO2 fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineered E. coli where enzyme function is coupled to growth. By assaying >99% of single amino acid mutants across CO2 concentrations, we inferred enzyme velocity and CO2 affinity for thousands of substitutions. We identified many highly conserved positions that tolerate mutation and rare mutations that improve CO2 affinity. These data suggest that non-trivial kinetic improvements are readily accessible and provide a comprehensive sequence-to-function mapping for enzyme engineering efforts.

The CbbQO-type rubisco activases encoded in carboxysome gene clusters can activate carboxysomal form IA rubiscos.

The CO2-fixing enzyme rubisco is responsible for almost all carbon fixation. This process frequently requires rubisco activase (Rca) machinery, which couples ATP hydrolysis to the removal of inhibitory sugar phosphates, including the rubisco substrate ribulose 1,5-bisphosphate (RuBP). Rubisco is sometimes compartmentalized in carboxysomes, bacterial microcompartments that enable a carbon dioxide concentrating mechanism (CCM). Characterized carboxysomal rubiscos, however, are not prone to inhibition, and often no activase machinery is associated with these enzymes. Here, we characterize two carboxysomal rubiscos of the form IAC clade that are associated with CbbQO-type Rcas. These enzymes release RuBP at a much lower rate than the canonical carboxysomal rubisco from Synechococcus PCC6301. We found that CbbQO-type Rcas encoded in carboxysome gene clusters can remove RuBP and the tight-binding transition state analog carboxy-arabinitol 1,5-bisphosphate from cognate rubiscos. The Acidithiobacillus ferrooxidans genome encodes two form IA rubiscos associated with two sets of cbbQ and cbbO genes. We show that the two CbbQO activase systems display specificity for the rubisco enzyme encoded in the same gene cluster, and this property can be switched by substituting the C-terminal three residues of the large subunit. Our findings indicate that the kinetic and inhibitory properties of proteobacterial form IA rubiscos are diverse and predict that Rcas may be necessary for some α-carboxysomal CCMs. These findings will have implications for efforts aiming to introduce biophysical CCMs into plants and other hosts for improvement of carbon fixation of crops.

Insights into the mechanism and regulation of the CbbQO-type Rubisco activase, a MoxR AAA+ ATPase.

The vast majority of biological carbon dioxide fixation relies on the function of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). In most cases the enzyme exhibits a tendency to become inhibited by its substrate RuBP and other sugar phosphates. The inhibition is counteracted by diverse molecular chaperones known as Rubisco activases (Rcas). In some chemoautotrophic bacteria, the CbbQO-type Rca Q2O2 repairs inhibited active sites of hexameric form II Rubisco. The 2.2-Å crystal structure of the MoxR AAA+ protein CbbQ2 from Acidithiobacillus ferrooxidans reveals the helix 2 insert (H2I) that is critical for Rca function and forms the axial pore of the CbbQ hexamer. Negative-stain electron microscopy shows that the essential CbbO adaptor protein binds to the conserved, concave side of the CbbQ2 hexamer. Site-directed mutagenesis supports a model in which adenosine 5'-triphosphate (ATP)-powered movements of the H2I are transmitted to CbbO via the concave residue L85. The basal ATPase activity of Q2O2 Rca is repressed but strongly stimulated by inhibited Rubisco. The characterization of multiple variants where this repression is released indicates that binding of inhibited Rubisco to the C-terminal CbbO VWA domain initiates a signal toward the CbbQ active site that is propagated via elements that include the CbbQ α4-ß4 loop, pore loop 1, and the presensor 1-ß hairpin (PS1-ßH). Detailed mechanistic insights into the enzyme repair chaperones of the highly diverse CO2 fixation machinery of Proteobacteria will facilitate their successful implementation in synthetic biology ventures.

Probing the rice Rubisco-Rubisco activase interaction via subunit heterooligomerization.

During photosynthesis the AAA+ protein and essential molecular chaperone Rubisco activase (Rca) constantly remodels inhibited active sites of the CO2-fixing enzyme Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) to release tightly bound sugar phosphates. Higher plant Rca is a crop improvement target, but its mechanism remains poorly understood. Here we used structure-guided mutagenesis to probe the Rubisco-interacting surface of rice Rca. Mutations in Ser-23, Lys-148, and Arg-321 uncoupled adenosine triphosphatase and Rca activity, implicating them in the Rubisco interaction. Mutant doping experiments were used to evaluate a suite of known Rubisco-interacting residues for relative importance in the context of the functional hexamer. Hexamers containing some subunits that lack the Rubisco-interacting N-terminal domain displayed a ∼2-fold increase in Rca function. Overall Rubisco-interacting residues located toward the rim of the hexamer were found to be less critical to Rca function than those positioned toward the axial pore. Rca is a key regulator of the rate-limiting CO2-fixing reactions of photosynthesis. A detailed functional understanding will assist the ongoing endeavors to enhance crop CO2 assimilation rate, growth, and yield.

Dirofilaria immitis exposure status in client-owned cats with or without lower airway/lung-associated signs: case-control study in a canine heartworm-endemic area.

Objectives Heartworm-associated respiratory disease (HARD) is a recently recognised pathological manifestation in cats caused by Dirofilaria immitis exposure. This study aimed to estimate the percentage of cats at risk of developing HARD in a heartworm-endemic area (Taipei, Taiwan), and to test the correlation of heartworm exposure and the presence of lower airway/lung clinical signs (LA/L signs). Methods This was a prospective case-control study. The study design called for the enrolment of at least 80 cats with LA/L signs and at least 80 cats without such clinical signs in a 1 year period. The D immitis antibody seroprevalence of the two cohorts was compared. Results From February 2014 to January 2015, 187 client-owned cats were prospectively enrolled: 83 clinical cases with LA/L signs and 104 cats without such signs. Antibody seropositivity was approximately twice as frequent in cats with LA/L signs (13.3%) than in cats without signs (7.8%) (odds ratio [OR] 1.814); nevertheless, no statistically significant difference between the two cohorts ( P = 0.22) was found. We used 41 frozen samples from free-roaming cats to examine the possibility of different exposure rates to mosquito bites between client-owned cats and stray cats, finding the seroprevalence to be 7.5% in free-roaming cats - a result not statistically different to that in client-owned cats ( P = 0.60). Outdoor access was a significant risk factor for heartworm exposure in client-owned cats (OR 3.748; P = 0.03); however, living entirely indoors did not provide complete protection from exposure/infection. Conclusions and relevance Our results did not show statistically significant differences in antibody seroprevalence between cats with and without LA/L signs. LA/L signs were not always present under conditions of natural exposure. However, exposure to D immitis is not rare among client-owned cats, suggesting that heartworm prophylactics should be a part of routine care in all cats living in areas endemic for canine heartworm.

Characterization of the heterooligomeric red-type rubisco activase from red algae.

The photosynthetic CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) is inhibited by nonproductive binding of its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. Reactivation requires ATP-hydrolysis-powered remodeling of the inhibited complexes by diverse molecular chaperones known as rubisco activases (Rcas). Eukaryotic phytoplankton of the red plastid lineage contain so-called red-type rubiscos, some of which have been shown to possess superior kinetic properties to green-type rubiscos found in higher plants. These organisms are known to encode multiple homologs of CbbX, the α-proteobacterial red-type activase. Here we show that the gene products of two cbbX genes encoded by the nuclear and plastid genomes of the red algae Cyanidioschyzon merolae are nonfunctional in isolation, but together form a thermostable heterooligomeric Rca that can use both α-proteobacterial and red algal-inhibited rubisco complexes as a substrate. The mechanism of rubisco activation appears conserved between the bacterial and the algal systems and involves threading of the rubisco large subunit C terminus. Whereas binding of the allosteric regulator RuBP induces oligomeric transitions to the bacterial activase, it merely enhances the kinetics of ATP hydrolysis in the algal enzyme. Mutational analysis of nuclear and plastid isoforms demonstrates strong coordination between the subunits and implicates the nuclear-encoded subunit as being functionally dominant. The plastid-encoded subunit may be catalytically inert. Efforts to enhance crop photosynthesis by transplanting red algal rubiscos with enhanced kinetics will need to take into account the requirement for a compatible Rca.

Transcatheter aortic valve implantation (TAVI) versus sutureless aortic valve replacement (SUAVR) for aortic stenosis: a systematic review and meta-analysis of matched studies.

BACKGROUND: With improving technologies and an increasingly elderly populations, there have been an increasing number of therapeutic options available for patients requiring aortic valve replacement. Recent evidence suggests that transcatheter aortic valve implantation (TAVI) is one suitable option for high risk inoperable patients, as well as high risk operable patients. Sutureless valve technology has also been developed concurrently, with facilitates surgical aortic valve replacement (SUAVR) by allow resection and replacement of the native aortic valve with minimal sutures and prosthesis anchoring required. For patients amenable for both TAVI and SUAVR, the evidence is unclear with regards to the benefits and risks of either approach. The objectives are to compare the perioperative outcomes and intermediate-term survival rates of TAVI and SUAVR in matched or propensity score matched studies. METHODS: A systematic literature search was performed to include all matched or propensity score matched studies comparing SUAVR versus TAVI for severe aortic stenosis. A meta-analysis with odds ratios (OR) and mean differences were performed to compare key outcomes including paravalvular regurgitation and short and intermediate term mortality. RESULTS: Six studies met our inclusion criteria giving a total of 741 patients in both the SUAVR and TAVI arm of the study. Compared to TAVI, SUAVR had a lower incidence of paravalvular leak (OR =0.06; 95% CI: 0.03-0.12, P<0.01). There was no difference in perioperative mortality, however SUAVR patients had significantly better survival rates at 1 (OR =2.40; 95% CI: 1.40-4.11, P<0.01) and 2 years (OR =4.62; 95% CI: 2.62-8.12, P<0.01). CONCLUSIONS: The present study supports the use of minimally invasive SUAVR as an alternative to TAVI in high risk patients requiring aortic replacement. The presented results require further validation in prospective, randomized controlled studies.

Association between post-sternotomy tracheostomy and deep sternal wound infection: a retrospective analysis.

BACKGROUND: Tracheostomy has traditionally been used as a means of facilitated mechanical ventilation in patients requiring respiratory management following cardiac surgery. However in the clinical setting, the advantages of tracheostomy has been questioned by concerns surrounding evidence of its association with increased risk of deep sternal wound infections (DSWI). The present study sought to evaluate retrospectively our experience with post-sternotomy tracheostomy among cardiac surgery patients and association with DSWI. METHODS: Between July 2003 and June 2013, 11,795 patients underwent open cardiac surgery via sternotomy in our department. Among these, 225 underwent post-sternotomy tracheostomy. Data were obtained by reviewing and analyzing the Cardiac Surgical and Cardiac Intensive Care Unit (ICU) databases for adult cardiac patients. RESULTS: Out of the 11,795 sternotomy patients analyzed, 225 (1.9%) underwent tracheostomy. The overall mortality rate for post-sternotomy tracheostomy patients was 21.3%. DSWI developed in 23 patients (10.2%) of the tracheostomy group. Seven of these 23 patients had DSWI after insertion of tracheostomy. DSWI was significantly higher in tracheostomy versus no-tracheostomy patients (10.2% vs. 0.48%; P<0.001). DSWI was also associated with higher mortality rates compared to non-DSWI patients (11.4% vs. 2.3%; P<0.001). CONCLUSIONS: The present study demonstrated that tracheostomy was an independent risk factor for post-sternotomy DSWI, and that DSWI was a predictor of mortality. For tracheostomy patients, coronary artery bypass grafting (CABG) procedures and longer durations of tracheostomy were strong predictors of DSWI. Across all sternotomy patients, tracheostomy, diabetes, urgency status and blood transfusions were significant risk factors for DSWI. As such, the decision for tracheostomy post-sternotomy should be carefully considered on a case by case basis.

Identification and characterization of multiple rubisco activases in chemoautotrophic bacteria.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is responsible for almost all biological CO2 assimilation, but forms inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. The distantly related AAA+ proteins rubisco activase and CbbX remodel inhibited rubisco complexes to effect inhibitor release in plants and α-proteobacteria, respectively. Here we characterize a third class of rubisco activase in the chemolithoautotroph Acidithiobacillus ferrooxidans. Two sets of isoforms of CbbQ and CbbO form hetero-oligomers that function as specific activases for two structurally diverse rubisco forms. Mutational analysis supports a model wherein the AAA+ protein CbbQ functions as motor and CbbO is a substrate adaptor that binds rubisco via a von Willebrand factor A domain. Understanding the mechanisms employed by nature to overcome rubisco's shortcomings will increase our toolbox for engineering photosynthetic carbon dioxide fixation.

Effect of Grape Seed Proanthocyanidin-Gelatin Colloidal Complexes on Stability and in Vitro Digestion of Fish Oil Emulsions.

The colloidal complexes composed of grape seed proanthocyanidin (GSP) and gelatin (GLT), as natural antioxidants to improve stability and inhibit lipid oxidation in menhaden fish oil emulsions, were evaluated. The interactions between GSP and GLT, and the chemical structures of GSP/GLT self-assembled colloidal complexes, were characterized by isothermal titration calorimetry (ITC), circular dichroism (CD), and Fourier transform infrared spectroscopic (FTIR) studies. Fish oil was emulsified with GLT to obtain an oil-in-water (o/w) emulsion. After formation of the emulsion, GLT was fixed by GSP to obtain the GSP/GLT colloidal complexes stabilized fish oil emulsion. Menhaden oil emulsified by GSP/GLT(0.4 wt %) colloidal complexes yielded an emulsion with smaller particles and higher emulsion stability as compared to its GLT emulsified counterpart. The GSP/GLT colloidal complexes inhibited the lipid oxidation in fish oil emulsions more effectively than free GLT because the emulsified fish oil was surrounded by the antioxidant GSP/GLT colloidal complexes. The digestion rate of the fish oil emulsified with the GSP/GLT colloidal complexes was reduced as compared to that emulsified with free GLT. The extent of free fatty acids released from the GSP/GLT complexes stabilized fish oil emulsions was 63.3% under simulated digestion condition, indicating that the fish oil emulsion was considerably hydrolyzed with lipase.

Sutureless aortic valve replacement: a systematic review and meta-analysis.

BACKGROUND: Sutureless aortic valve replacement (SU-AVR) has emerged as an innovative alternative for treatment of aortic stenosis. By avoiding the placement of sutures, this approach aims to reduce cross-clamp and cardiopulmonary bypass (CPB) duration and thereby improve surgical outcomes and facilitate a minimally invasive approach suitable for higher risk patients. The present systematic review and meta-analysis aims to assess the safety and efficacy of SU-AVR approach in the current literature. METHODS: Electronic searches were performed using six databases from their inception to January 2014. Relevant studies utilizing sutureless valves for aortic valve implantation were identified. Data were extracted and analyzed according to predefined clinical endpoints. RESULTS: Twelve studies were identified for inclusion of qualitative and quantitative analyses, all of which were observational reports. The minimally invasive approach was used in 40.4% of included patients, while 22.8% underwent concomitant coronary bypass surgery. Pooled cross-clamp and CPB duration for isolated AVR was 56.7 and 46.5 minutes, respectively. Pooled 30-day and 1-year mortality rates were 2.1% and 4.9%, respectively, while the incidences of strokes (1.5%), valve degenerations (0.4%) and paravalvular leaks (PVL) (3.0%) were acceptable. CONCLUSIONS: The evaluation of current observational evidence suggests that sutureless aortic valve implantation is a safe procedure associated with shorter cross-clamp and CPB duration, and comparable complication rates to the conventional approach in the short-term.

Ministernotomy or minithoracotomy for minimally invasive aortic valve replacement: a Bayesian network meta-analysis.

BACKGROUND: Establishing the relative merits of ministernotomy (MS) and minithoracotomy (MT) approaches to minimally invasive aortic valve replacement (MIAVR) is difficult given the limited available direct evidence. Network meta-analysis is a Bayesian approach that can combine direct and indirect evidence to better define the benefits and risks of MS and MT. METHODS: Electronic searches were performed using six databases from their inception to June 2014. Relevant studies utilizing a minimally invasive approach for aortic valve replacement were identified. Data were extracted and analyzed according to predefined clinical endpoints. Both traditional and Bayesian meta-analysis approaches were conducted. RESULTS: Compared to full sternotomy, MT was associated with longer cardiopulmonary bypass (CPB) duration (WMD, 9.99; 95% CI, 3.91, 16.07; I(2)=55%; P=0.001) and cross-clamp duration (WMD, 7.64; 95% CI, 2.86, 12.42; P=0.002; I(2)=74%). When compared to MS using network meta-analysis, no significant difference in duration was detected. Postoperative outcomes including 30-day mortality, stroke, and reoperation for bleeding and wound infection were comparable between MS and MT using both traditional and Bayesian meta-analysis techniques. CONCLUSIONS: The current evidence demonstrates that MIAVR via MS or MT is a safe and efficacious alternative to conventional median sternotomy. MT may be associated with longer CPB and cross-clamp durations, but has similar post-operative outcomes compared to MS. An individualized approach tailored to both the patient and surgical team is likely to provide optimal outcomes.

Surgical left atrial appendage occlusion during cardiac surgery for patients with atrial fibrillation: a meta-analysis.

OBJECTIVES: Concomitant left atrial appendage occlusion (LAAO) during surgical ablation has emerged as a potential treatment strategy to reduce stroke and perioperative mortality in patients with atrial fibrillation (AF). The present meta-analysis aims to assess current evidence on the efficacy and safety between LAAO and LAA preservation cohorts for patients undergoing cardiac surgery. METHODS: Electronic searches were performed using six electronic databases from their inception to November 2013, identifying all relevant comparative randomized and observational studies comparing LAAO with non-LAAO in AF patients undergoing cardiac surgery. Data were extracted and analysed according to predefined endpoints including mortality, stroke, postoperative AF and reoperation for bleeding. RESULTS: Seven relevant studies identified for qualitative and quantitative analyses, including 3653 patients undergoing LAAO (n = 1716) versus non-LAAO (n = 1937). Stroke incidence was significantly reduced in the LAAO occlusion group at the 30-day follow-up [0.95 vs 1.9%; odds ratio (OR) 0.46; P = 0.005] and the latest follow-up (1.4 vs 4.1%; OR 0.48; P = 0.01), compared with the non-LAAO group. Incidence of all-cause mortality was significantly decreased with LAAO (1.9 vs 5%; OR 0.38; P = 0.0003), while postoperative AF and reoperation for bleeding was comparable. CONCLUSIONS: While acknowledging the limitations and inadequate statistical power of the available evidence, this study suggests LAAO as a promising strategy for stroke reduction perioperatively and at the short-term follow-up without a significant increase in complications. Larger randomized studies in the future are required, with clearer surgical and anticoagulation protocols and adequate long-term follow-up, to validate the clinical efficacy of LAAO versus non-LAAO groups.

Biatrial ablation vs. left atrial concomitant surgical ablation for treatment of atrial fibrillation: a meta-analysis.

AIMS: Surgical ablation performed concomitantly with cardiac surgery has emerged as an effective curative strategy for atrial fibrillation (AF). Left atrial (LA) lesion sets for ablation have been suggested to reduce procedural times and post-surgical bradycardia compared with biatrial (BA) lesions. Given the inconclusive literature regarding BA vs. LA ablation, the present meta-analysis sought to assess the current evidence. METHODS AND RESULTS: Electronic searches were performed using six databases from their inception to December 2013, identifying all relevant randomized trials and observational studies comparing BA vs. LA surgical ablation AF patients undertaking cardiac surgery. In 10 included studies, 2225 patient results were available for analysis to compare BA (n = 888) vs. LA (n = 1337) ablation. Sinus rhythm prevalence was higher in the BA cohort compared with the LA cohort at 6-month and 12-month follow-up, but similar beyond 1 year. Permanent pacemaker implantations were higher in the BA cohort, but 30-day and late mortality, neurological events, and reoperation for bleeding were similar between BA and LA groups. CONCLUSIONS: Biatrial and LA ablations produced comparable 30-day and late mortality but LA was associated with significantly reduced permanent pacemaker implantation rates. Biatrial ablation appeared to be more efficacious than LA ablation in achieving SR at 1 year, but this difference was not maintained beyond 1 year. Trends appear to be driven by the preferential selection of long-standing and persistent AF patients for the BA approach. Future randomized studies of adequate follow-up are required to validate risks and benefits of BA vs. LA surgical ablation.

Role of small subunit in mediating assembly of red-type form I Rubisco.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO2 to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into green- and red-type. Some red-type enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the green-type RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone-independent manner. We find that the C-terminal ß-hairpin extension of red-type RbcS, which is absent in green-type RbcS, is critical for efficient assembly. The ß-hairpins of four RbcS subunits form an eight-stranded ß-barrel that protrudes into the central solvent channel of the RbcL core complex. The two ß-barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal ß-hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing red-type Rubisco.

Venoarterial extracorporeal membrane oxygenation for cardiogenic shock and cardiac arrest: a meta-analysis.

OBJECTIVE: To evaluate the effect of extracorporeal membrane oxygenation (ECMO) on survival and complication rates in adults with refractory cardiogenic shock or cardiac arrest. DESIGN: Meta-analysis. SETTING: University hospitals. PARTICIPANTS: One thousand one hundred ninety-nine patients from 22 observational studies. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Observational studies published from the year 2000 onwards, examining at least 10 adult patients who received ECMO for refractory cardiogenic shock or cardiac arrest were included. Pooled estimates with 95% confidence intervals were calculated based on the Freeman-Tukey double-arcsine transformation and DerSimonian-Laird random-effect model. Survival to discharge was 40.2% (95% confidence intervals [CI], 33.9-46.7), while survival at 3, 6, and 12 months was 55.9% (95% CI, 41.5-69.8), 47.6% (95% CI, 25.4-70.2), and 54.4% (95% CI, 36.6-71.7), respectively. Survival up to 30 days was higher in cardiogenic shock patients (52.5%, 95% CI, 43.7%-61.2%) compared to cardiac arrest (36.2%, 95% CI, 23.1%-50.4%). Concurrently, complication rates were particularly substantial for neurologic deficits (13.3%, 95% CI, 8.3-19.3), infection (25.1%, 95%CI, 15.9-35.5), and renal impairment (47.4%, 95% CI, 30.2-64.9). Significant heterogeneity was detected, although its levels were similar to previous meta-analyses that only examined short-term survival to discharge. CONCLUSIONS: Venoarterial ECMO can improve short-term survival in adults with refractory cardiogenic shock or cardiac arrest. It also may provide favorable long-term survival at up to 3 years postdischarge. However, ECMO also is associated with significant complication rates, which must be incorporated into the risk-benefit analysis when considering treatment. These findings require confirmation by large, adequately controlled and standardized trials with long-term follow-up.

Hydrogel microspheres for stabilization of an antioxidant enzyme: effect of emulsion cross-linking of a dual polysaccharide system on the protection of enzyme activity.

Catalase is an antioxidant enzyme abundant in natural resources. However, the enzyme is usually inactivated by gastric acid and digestive enzymes after oral ingestion. In this study, carboxymethyl chitosan (CM-chitosan) and hyaluronic acid (HA) conjugate hydrogel microspheres have been prepared by an emulsion cross-linking technique to retain the activity of catalase in simulated gastrointestinal (GI) fluids. Cross-linking reduced the swelling capability and increased the resistance toward hyaluronidase digestion of prepared HA-CM-chitosan hydrogel microspheres. Catalase entrapped in the hydrogel microspheres exhibited superior stability over a wide pH range (pH 2.0 and 6.0-8.0) as compared to the native enzyme. The entrapped catalase was also protected against degradation by digestive enzymes. Following the treatments, the catalase-loaded microspheres, in contrast to native catalase, could effectively decrease the intracellular H2O2 level and protect HT-29 colonic epithelial cells against H2O2-induced oxidative damage to preserve cell viability. These results suggested that the HA-CM-chitosan hydrogel microspheres can be used for entrapment, protection and intestinal delivery of catalase for H2O2 scavenging.

Degradation of ascorbic acid in ethanolic solutions.

Ascorbic acid occurs naturally in many wine-making fruits. The industry also uses ascorbic acid as an antioxidant and color stabilizer in the making of alcoholic beverages including white wine, wine cooler, alcopop, and fruit liqueur. However, the degradation of ascorbic acid itself may cause browning and the deterioration of color quality. This study was aimed to monitor the degradation of ascorbic acid, the formation of degradation products, and the browning in storage of ascorbic acid containing 0-40% (v/v) ethanolic solutions buffered at pH 3.2 as models of alcoholic beverages. The results show that ascorbic acid degradation in the ethanolic solutions during storage follows first-order reaction, that the degradation and browning rates increase with the increase of ethanol concentration, that the activation energy for the degradation of ascorbic acid is in the range 10.35-23.10 (kcal/mol), that 3-hydroxy-2-pyrone is an indicator and a major product of ascorbic acid degradation, and that aerobic degradation pathway dominants over anaerobic pathway in ascorbic acid degradation in ethanolic solutions.

Chaperonin cofactors, Cpn10 and Cpn20, of green algae and plants function as hetero-oligomeric ring complexes.

The chloroplast chaperonin system of plants and green algae is a curiosity as both the chaperonin cage and its lid are encoded by multiple genes, in contrast to the single genes encoding the two components of the bacterial and mitochondrial systems. In the green alga Chlamydomonas reinhardtii (Cr), three genes encode chaperonin cofactors, with cpn10 encoding a single ∼10-kDa domain and cpn20 and cpn23 encoding tandem cpn10 domains. Here, we characterized the functional interaction of these proteins with the Escherichia coli chaperonin, GroEL, which normally cooperates with GroES, a heptamer of ∼10-kDa subunits. The C. reinhardtii cofactor proteins alone were all unable to assist GroEL-mediated refolding of bacterial ribulose-bisphosphate carboxylase/oxygenase but gained this ability when CrCpn20 and/or CrCpn23 was combined with CrCpn10. Native mass spectrometry indicated the formation of hetero-oligomeric species, consisting of seven ∼10-kDa domains. The cofactor "heptamers" interacted with GroEL and encapsulated substrate protein in a nucleotide-dependent manner. Different hetero-oligomer arrangements, generated by constructing cofactor concatamers, indicated a preferential heptamer configuration for the functional CrCpn10-CrCpn23 complex. Formation of heptamer Cpn10/Cpn20 hetero-oligomers was also observed with the Arabidopsis thaliana (At) cofactors, which functioned with the chloroplast chaperonin, AtCpn60α(7)ß(7). It appears that hetero-oligomer formation occurs more generally for chloroplast chaperonin cofactors, perhaps adapting the chaperonin system for the folding of specific client proteins.

Clinical comparison of recovery from total intravenous anesthesia with propofol and inhalation anesthesia with isoflurane in dogs.

The characteristics of recovery from total intravenous anesthesia (TIVA) with propofol and inhalation anesthesia with isoflurane was clinically compared in 149 client-owned dogs that anesthetized for surgical or diagnostic procedures. In all dogs, anesthesia was induced with an intravenous injection of propofol following premedication with acepromazine or diazepam. As a result, 58 dogs anesthetized with propofol-TIVA showed slower but smoother recovery than 91 dogs anesthetized with isoflurane anesthesia. The dogs stood at 34.5 +/- 19.3 and 27.7 +/- 17.2 min after propofol-TIVA and isoflurane anesthesia, respectively. Adverse effects, including hypersalivation, neurologic excitement (paddling, muscle tremor/twitching, opisthotonos) and vomiting/retching, were observed in similar infrequent incidences during the recovery from both anesthetic protocols. Propofol-TIVA is suggested to be an alternative anesthetic protocol for canine practice.