Comparative proteomics profiling of a phospholamban mutant mouse model of dilated cardiomyopathy reveals progressive intracellular stress responses

Defective mobilization of Ca2+ by cardiomyocytes can lead to cardiac insufficiency, but the causative mechanisms leading to congestive heart failure (HF) remain unclear. In the present study we performed exhaustive global proteomics surveys of cardiac ventricle isolated from a mouse model of cardiomyopathy overexpressing a phospholamban mutant, R9C (PLN-R9C), and exhibiting impaired Ca2+ handling and death at 24 weeks and compared them with normal control littermates. The relative expression patterns of 6190 high confidence proteins were monitored by shotgun tandem mass spectrometry at 8, 16, and 24 weeks of disease progression. Significant differential abundance of 593 proteins was detected. These proteins mapped to select biological pathways such as endoplasmic reticulum stress response, cytoskeletal remodeling, and apoptosis and included known biomarkers of HF (e.g. brain natriuretic peptide/atrial natriuretic factor and angiotensin-converting enzyme) and other indicators of presymptomatic functional impairment. These altered proteomic profiles were concordant with cognate mRNA patterns recorded in parallel using high density mRNA microarrays, and top candidates were validated by RT-PCR and Western blotting. Mapping of our highest ranked proteins against a human diseased explant and to available data sets indicated that many of these proteins could serve as markers of disease. Indeed we showed that several of these proteins are detectable in mouse and human plasma and display differential abundance in the plasma of diseased mice and affected patients. These results offer a systems-wide perspective of the dynamic maladaptions associated with impaired Ca2+ homeostasis that perturb myocyte function and ultimately converge to cause HF.     

Estimating the complete ground reaction forces with pressure insoles in walking

This study presented a method to estimate the complete ground reaction forces from pressure insoles in walking. Five male subjects performed 10 walking trials in a laboratory. The complete ground reaction forces were collected during a right foot stride by a force plate at 1000Hz. Simultaneous plantar pressure data were collected at 100Hz by a pressure insole system with 99 sensors covering the whole plantar area. Stepwise linear regressions were performed to individually reconstruct the complete ground reaction forces in three directions from the 99 individual pressure data until redundancy among the predictors occurred. An additional linear regression was performed to reconstruct the vertical ground reaction force by the sum of the value of the 99 pressure sensors. Five other subjects performed the same walking test for validation. Estimated ground reaction forces in three directions were calculated with the developed regression models, and were compared with the real data recorded from force plate. Accuracy was represented by the correlation coefficient and the root mean square error. Results showed very good correlation in anterior-posterior (0.928) and vertical (0.989) directions, and reasonable correlation in medial-lateral direction (0.719). The root mean square error was about 12%, 5% and 28% of the peak recorded value. Future studies should aim to generalize the methods or to establish specific methods to other subjects, patients, motions, footwear and floor conditions. The method gives an extra option to study an estimation of the complete ground reaction forces in any environment without the constraints from the number and location of force plates.     

The acidic domain of GPIHBP1 is important for the binding of lipoprotein lipase and chylomicrons

GPIHBP1, a glycosylphosphatidylinositol-anchored endothelial cell protein of the lymphocyte antigen 6 (Ly6) family, plays a key role in the lipolysis of triglyceride-rich lipoproteins (e.g. chylomicrons). GPIHBP1 is expressed along the luminal surface of endothelial cells of heart, skeletal muscle, and adipose tissue, and GPIHBP1-expressing cells bind lipoprotein lipase (LPL) and chylomicrons avidly. GPIHBP1 contains an amino-terminal acidic domain (amino acids 24-48) that is enriched in aspartate and glutamate residues, and we previously speculated that this domain might be important in binding ligands. To explore the functional importance of the acidic domain, we tested the ability of polyaspartate or polyglutamate peptides to block the binding of ligands to pgsA-745 Chinese hamster ovary cells that overexpress GPIHBP1. Both polyaspartate and polyglutamate blocked LPL and chylomicron binding to GPIHBP1. Also, a rabbit antiserum against the acidic domain of GPIHBP1 blocked LPL and chylomicron binding to GPIHBP1-expressing cells. Replacing the acidic amino acids within GPIHBP1 residues 38-48 with alanine eliminated the ability of GPIHBP1 to bind LPL and chylomicrons. Finally, mutation of the positively charged heparin-binding domains within LPL and apolipoprotein AV abolished the ability of these proteins to bind to GPIHBP1. These studies indicate that the acidic domain of GPIHBP1 is important and that electrostatic interactions play a key role in ligand binding.     

A mechanical supination sprain simulator for studying ankle supination sprain kinematics

This study presents a free-fall mechanical supination sprain simulator for evaluating the ankle joint kinematics during a simulated ankle supination sprain injury. The device allows the foot to be in an anatomical position before the sudden motion, and also allows different degrees of supination, or a combination of inversion and plantarflexion. Five subjects performed simulated supination sprain trials in five different supination angles. Ankle motion was captured by a motion analysis system, and the ankle kinematics were reported in plantarflexion/dorsiflexion, inversion/eversion and internal/external rotation planes. Results showed that all sprain motions were not pure single-plane motions but were accompanied by motion in other two planes, therefore, different degrees of supination were achieved. The presented sprain simulator allows a more comprehensive study of the kinematics of ankle sprain when compared with some previous laboratory research designs.     

Abnormal patterns of lipoprotein lipase release into the plasma in GPIHBP1-deficient mice

GPIHBP1-deficient mice (Gpihbp1(-/-)) exhibit severe chylomicronemia. GPIHBP1 is located within capillaries of muscle and adipose tissue, and expression of GPIHBP1 in Chinese hamster ovary cells confers upon those cells the ability to bind lipoprotein lipase (LPL). However, there has been absolutely no evidence that GPIHBP1 actually interacts with LPL in vivo. Heparin is known to release LPL from its in vivo binding sites, allowing it to enter the plasma. After an injection of heparin, we reasoned that LPL bound to GPIHBP1 in capillaries would be released very quickly, and we hypothesized that the kinetics of LPL entry into the plasma would differ in Gpihbp1(-/-) and control mice. Indeed, plasma LPL levels peaked very rapidly (within 1 min) after heparin in control mice. In contrast, plasma LPL levels in Gpihbp1(-/-) mice were much lower 1 min after heparin and increased slowly over 15 min. In keeping with that result, plasma triglycerides fell sharply within 10 min after heparin in wild-type mice, but were negligibly altered in the first 15 min after heparin in Gpihbp1(-/-) mice. Also, an injection of Intralipid released LPL into the plasma of wild-type mice but was ineffective in releasing LPL in Gpihbp1(-/-) mice. The observed differences in LPL release cannot be ascribed to different tissue stores of LPL, as LPL mass levels in tissues were similar in Gpihbp1(-/-) and control mice. The differences in LPL release after intravenous heparin and Intralipid strongly suggest that GPIHBP1 represents an important binding site for LPL in vivo.     

A propeptide toolbox for secretion optimization of <Emphasis Type="Italic">Flavobacterium meningosepticum</Emphasis> endopeptidase in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Background

Lactic acid bacteria are a family of “generally regarded as safe” organisms traditionally used for food fermentation. In recent years, they have started to emerge as potential chassis for heterologous protein production. And more recently, due to their beneficial properties in the gut, they have been examined as potential candidates for mucosal delivery vectors, especially for acid-sensitive enzymes. One such application would be the delivery of gluten-digesting endopeptidases for the treatment of celiac disease. To facilitate these applications, an efficient recombinant protein expression toolbox is required, especially for recombinant protein secretion. While current tools for enhancing protein secretion consist mainly of signal peptides, secretion propeptides have also been observed to play a crucial role for protein secretion and improved yields.

Results

To expand the propeptide library for secretion optimization, we have mined and characterized three naturally occurring propeptides from the sequenced genomes of 109 Lactococcus species. These newly-mined propeptides were introduced after the N-terminal USP45 secretion signal to characterize and compare their effects on the secretion of Escherichia coli thioredoxin (TRX) and Flavobacterium meningosepticum prolyl endopeptidase (Fm PEP) in Lactococcus lactis NZ9000. All three propeptides, along with the positive control LEISSTCDA, improved volumetric secretion yields by 1.4–2.3-folds. However, enhancement of secretion yield is dependent on protein of interest. For TRX, the optimal combination of USP45 signal peptide and LEISSTCDA produced a 2.3-fold increase in secretion yields. Whilst for Fm PEP, propeptide 1 with USP45 signal peptide improved volumetric secretion yields by 2.2-fold compared to a 1.4-fold increase by LEISSTCDA. Similar trends in Fm PEP activity and protein yield also demonstrated minimal effect of the negative charged propeptides on PEP activity and thus folding.

Conclusions

Overall, we have characterized three new propeptides for use in L. lactis secretion optimization. From success of these propeptides for improvement of secretion yields, we anticipate this collection to be valuable to heterologous protein secretion optimisation in lactic acid bacteria. We have also demonstrated for the first time, secretion of Fm PEP in L. lactis for potential use as a therapy agent in celiac disease.

     

Rubidium Multication Perovskite with Optimized Bandgap for Perovskite‐Silicon Tandem with over 26% Efficiency

Rubidium (Rb) is explored as an alternative cation to use in a novel multication method with the formamidinium/methylammonium/cesium (Cs) system to obtain 1.73 eV bangap perovskite cells with negligible hysteresis and steady state efficiency as high as 17.4%. The study shows the beneficial effect of Rb in improving the crystallinity and suppressing defect migration in the perovskite material. The light stability of the cells examined under continuous illumination of 12 h is improved upon the addition of Cs and Rb. After several cycles of 12 h light–dark, the cell retains 90% of its initial efficiency. In parallel, sputtered transparent conducting oxide thin films are developed to be used as both rear and front transparent contacts on quartz substrate with less than 5% parasitic absorption of near infrared wavelengths. Using these developments, semi‐transparent perovskite cells are fabricated with steady state efficiency of up to 16.0% and excellent average transparency of ≈84% between 720 and 1100 nm. In a tandem configuration using a 23.9% silicon cell, 26.4% efficiency (10.4% from the silicon cell) in a mechanically stacked tandem configuration is demonstrated which is very close to the current record for a single junction silicon cell of 26.6%.     

A comparison of HIV-1 integrase inhibition by aqueous and methanol extracts of Chinese medicinal herbs

The aqueous and methanol extracts of twenty herbs traditionally used in Chinese medicine were screened for anti-HIV-1 integrase activity in a non-radioactive ELISA-based HIV-1 integrase assay. The screening was performed at an herb extract concentration of 50 microg/ml. It was found that most of the aqueous and methanol herb extracts could elicit strong inhibition of HIV-I integrase activity. The inhibition was most likely due to tannins or polyphenolics in the herb extracts. In most of the herb extracts, 40-80% of the anti-HIV-1 integrase activity could be removed after passing through a minicolumn of polyamide resin. After removal of polyphenolic compounds, the methanol extract of Paeonia suffruticosa still exerted potent inhibition of HIV-1 integrase (EC50 = 15 microg/ml) and the aqueous extract of Prunella vulgaris caused moderate inhibition (EC50 = 45 microg/ml). The results support the view that herbs represent a rich source of anti-HIV compounds.     

Taxane diterpenoids from the bark of Taxus yunnanensis

Further investigation on the alcohol extract of the barks of Taxus yunnanensis led to the isolation of four taxoids, namely, 7beta-xylosyl-taxol D, taxuyunnanines P, Q and R, along with the known taxuyuntin G (2). Four are rearranged taxoids with an 11(15-->1)-abeotaxoid skeleton and an opened oxetane ring moiety. Structures were determined by spectroscopic and chemical means.