Mutagenesis of Trichoderma reesei endoglucanase I: impact of expression host on activity and stability at elevated temperatures.

BackgroundTrichoderma reesei is a key cellulase source for economically saccharifying cellulosic biomass for the production of biofuels. Lignocellulose hydrolysis at temperatures above the optimum temperature of T. reesei cellulases (~50°C) could provide many significant advantages, including reduced viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature biomass pretreatment, and faster rates of hydrolysis. These potential advantages motivate efforts to engineer T. reesei cellulases that can hydrolyze lignocellulose at temperatures ranging from 60-70°C.ResultsA B-factor guided approach for improving thermostability was used to engineer variants of endoglucanase I (Cel7B) from T. reesei (TrEGI) that are able to hydrolyze cellulosic substrates more rapidly than the recombinant wild-type TrEGI at temperatures ranging from 50-70°C. When expressed in T. reesei, TrEGI variant G230A/D113S/D115T (G230A/D113S/D115T Tr_TrEGI) had a higher apparent melting temperature (3°C increase in Tm) and improved half-life at 60°C (t1/2 = 161 hr) than the recombinant (T. reesei host) wild-type TrEGI (t1/2 = 74 hr at 60°C, Tr_TrEGI). Furthermore, G230A/D113S/D115T Tr_TrEGI showed 2-fold improved activity compared to Tr_TrEGI at 65°C on solid cellulosic substrates, and was as efficient in hydrolyzing cellulose at 60°C as Tr_TrEGI was at 50°C. The activities and stabilities of the recombinant TrEGI enzymes followed similar trends but differed significantly in magnitude depending on the expression host (Escherichia coli cell-free, Saccharomyces cerevisiae, Neurospora crassa, or T. reesei). Compared to N.crassa-expressed TrEGI, S. cerevisiae-expressed TrEGI showed inferior activity and stability, which was attributed to the lack of cyclization of the N-terminal glutamine in Sc_TrEGI and not to differences in glycosylation. N-terminal pyroglutamate formation in TrEGI expressed in S. cerevisiae was found to be essential in elevating its activity and stability to levels similar to the T. reesei or N. crassa-expressed enzyme, highlighting the importance of this ubiquitous modification in GH7 enzymes.ConclusionStructure-guided evolution of T. reesei EGI was used to engineer enzymes with increased thermal stability and activity on solid cellulosic substrates. Production of TrEGI enzymes in four hosts highlighted the impact of the expression host and the role of N-terminal pyroglutamate formation on the activity and stability of TrEGI enzymes

Enhanced nicotinic receptor mediated relaxations in gastroesophageal muscle fibers from Barrett\u27s esophagus patients

BACKGROUND: Increased nicotinic receptor mediated relaxation in the gastroesophageal antireflux barrier may be involved in the pathophysiology of reflux. This study is designed to determine whether the defects we previously identified in GERD patients in-vivo are due to abnormalities of the gastric sling, gastric clasp or lower esophageal circular (LEC) muscle fibers. METHODS: Muscle strips from whole stomachs and esophagi were obtained from 16 normal donors and 15 donors with histologically proven Barrett's esophagus. Contractile and relaxant responses of gastric sling, gastric clasp or LEC fibers were determined to increasing concentrations of carbachol and to nicotine after inducing maximal contraction to bethanechol. Muscarinic receptor density was measured using subtype selective immunoprecipitation. KEY RESULTS: Barrett's esophagus gastric sling and LEC fibers have decreased carbachol induced contractions. Barrett's esophagus sling fibers have decreased M(2) muscarinic receptors and LEC fibers have decreased M(3) receptors. Relaxations of all 3 fiber types are greater in Barrett's esophagus specimens to both high carbachol concentrations and to nicotine following bethanechol pre-contraction. The maximal response to bethanechol is greater in Barrett esophagus sling and LEC fibers. CONCLUSIONS & INFERENCES: The increased contractile response to bethanechol in Barrett's specimens indicates that the defect is likely not due to the smooth muscle itself. The enhanced nicotinic receptor mediated response may be involved in greater relaxation of the muscles within the high pressure zone of the gastroesophageal junction during transient lower esophageal sphincter relaxations and during deglutitive inhibition and may be involved in the pathophysiology of gastro esophageal reflux disease

Did a physician-targeted intervention that reduced potentially inappropriate prescribing to elderly patients also reduce related hospitalizations?

Objectives: To determine whether a general practitioner focused intervention aimed at decreasing PIM prescribing in the elderly can decrease the risk of PIM-related hospitalizations. Poster presented at 2016 ISPOR conference in Washington DC.https://jdc.jefferson.edu/jcphposters/1005/thumbnail.jp

Padded Helmet Shell Covers in American Football: A Comprehensive Laboratory Evaluation with Preliminary On-Field Findings

Protective headgear effects measured in the laboratory may not always translate to the field. In this study, we evaluated the impact attenuation capabilities of a commercially available padded helmet shell cover in the laboratory and field. In the laboratory, we evaluated the efficacy of the padded helmet shell cover in attenuating impact magnitude across six impact locations and three impact velocities when equipped to three different helmet models. In a preliminary on-field investigation, we used instrumented mouthguards to monitor head impact magnitude in collegiate linebackers during practice sessions while not wearing the padded helmet shell covers (i.e., bare helmets) for one season and whilst wearing the padded helmet shell covers for another season. The addition of the padded helmet shell cover was effective in attenuating the magnitude of angular head accelerations and two brain injury risk metrics (DAMAGE, HARM) across most laboratory impact conditions, but did not significantly attenuate linear head accelerations for all helmets. Overall, HARM values were reduced in laboratory impact tests by an average of 25% at 3.5 m/s (range: 9.7 - 39.6%), 18% at 5.5 m/s (range: -5.5 - 40.5%), and 10% at 7.4 m/s (range: -6.0 - 31.0%). However, on the field, no significant differences in any measure of head impact magnitude were observed between the bare helmet impacts and padded helmet impacts. Further laboratory tests were conducted to evaluate the ability of the padded helmet shell cover to maintain its performance after exposure to repeated, successive impacts and across a range of temperatures. This research provides a detailed assessment of padded helmet shell covers and supports the continuation of in vivo helmet research to validate laboratory testing results.Comment: 49 references, 8 figure

Peripartum Pulmonary Embolism

Pregnancy and peripartum increase the risk of venous thromboembolism (VTE) by many folds. Interestingly, the VTE is more common during the pregnancy, whereas the pulmonary embolism is more frequent in postpartum period. There are various risk factors for the VTE and pulmonary embolism in these patients. The important risks are improper thromboprophylaxis, obesity, and multigravida. The clinical parameters and the d-dimer are not used for diagnosis of thromboembolism during pregnancy and in the postpartum period. The compression ultrasonography (CUSG) is commonly used for VTE diagnosis; for the pulmonary embolism diagnosis, one has to consider the radiation hazard to the fetus as well as to the mothers. Ventilation/perfusion scan is the imaging of choice for patient who has respiratory signs with normal chest radiograph. If chest X-ray is abnormal with suspicion of peripartum pulmonary embolism (PPE), the choice should be computed tomographic angiography. Heparin and its derivatives remained the anticoagulation of choice for the treatment of VTE as well as the PPE, as it is a shorter acting, easy to reverse with protamine sulfate. Proper thromboprophylaxis is the key for prevention of VTE and peripartum pulmonary embolism

Peripartum Cardiomyopathy: Facts and Figures

Peripartum cardiomyopathy (PPCM) is a rare clinical entity during pregnancy. PPCM is a diagnosis of exclusion. These patients do not have prior history of heart disease, and there are no other known possible causes of heart failure. It is more common in African countries, may be related to the consumption of kanwa, in the postpartum period. The multiparity, African descent and pregnancy-induced hypertension are a few risk factors for PPCM. The exact etiology of PPCM is not known; possible theories range from myocarditis to the maladaptation to the changes of pregnancy. The clinical manifestation varies from shortness of breath to thromboembolic phenomenon. Echocardiography is essential for diagnosis as well as differential diagnosis of PPCM. These patients preferably are managed in tertiary healthcare facilities. Anticoagulation and antiarrhythmic medications are pillars for the management of PPCM patients. If required, mechanical devices should be used temporarily. PPCM patients may need heart transplant. The beneficial role of bromocriptine and immunosuppression is not clear in PPCM patients. Subsequent pregnancies should be avoided to prevent the PPCM occurrence