Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing ³¹P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, ³¹P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.     

酵母双杂交技术应用进展

酵母双杂交技术是鉴定蛋白互作最有效和最广泛的分子生物学技术。该技术能直接作用于活细胞,检测细胞内蛋白质互作,具有成本低、易操作、可达到全基因组水平、能进行品种间的互作鉴定等诸多优点。较之传统的检测方法有明显优势,已在越来越多的领域得到应用。对酵母双杂交的技术原理以及应用进行了综述,介绍了该技术在发现新蛋白质、探究蛋白质功能、建立基因组蛋白连锁图、研究人类DNA文库和筛选药物作用位点等方面的重要应用,以期为该技术的广泛应用提供参考。     

In vitro-engineered non-antibody protein therapeutics

Antibodies have proved to be a valuable mode of therapy for numerous diseases, mainly owing to their high target binding affinity and specificity. Unfortunately, antibodies are also limited in several respects, chief amongst those being the extremely high cost of manufacture. Therefore, non-antibody binding proteins have long been sought after as alternative therapies. New binding protein scaffolds are constantly being designed or discovered with some already approved for human use by the FDA. This review focuses on protein scaffolds that are either already being used in humans or are currently being evaluated in clinical trials. Although not all are expected to be approved, the significant benefits ensure that these molecules will continue to be investigated and developed as therapeutic alternatives to antibodies. Based on the location of the amino acids that mediate ligand binding, we place all the protein scaffolds under clinical development into two general categories: scaffolds with ligand-binding residues located in exposed flexible loops, and those with the binding residues located in protein secondary structures, such as α-helices. Scaffolds that fall under the first category include adnectins, anticalins, avimers, Fynomers, Kunitz domains, and knottins, while those belonging to the second category include affibodies, β-hairpin mimetics, and designed ankyrin repeat proteins (DARPins). Most of these scaffolds are thermostable and can be easily produced in microorganisms or completely synthesized chemically. In addition, many of these scaffolds derive from human proteins and thus possess very low immunogenic potential. Additional advantages and limitations of these protein scaffolds as therapeutics compared to antibodies will be discussed.     

γ-decalactone production by Yarrowia lipolytica and Lindnera saturnus in crude glycerol

Flavor compounds are commonly obtained from chemical synthesis or extracted from plants. These sources have disadvantages, such as racemic mixture generation, more steps to yield the final product, low yield, and high cost, making the microbial fermentation an alternative and potential way to obtain flavor compounds. The most important lactone for flavor application is γ-decalactone, which has an aroma of peach and can be obtained by ricinoleic acid biotransformation through yeast peroxisomal β-oxidation. The aim of this work was to use crude glycerol, a residual biodiesel industry, for the production of bioaroma from two different yeasts. Yarrowia lipolytica CCMA 0357 and Lindnera saturnus CCMA 0243 were grown at different concentrations (10, 20, and 30% w/v) of substrates (castor oil and crude glycerol) for γ-decalactone production. L. saturnus CCMA 0243 produced higher concentration of y-decalactone (5.8?g/L) in crude glycerol, whereas Y. lipolytica CCMA 0357 showed a maximum production in castor oil (3.5?g/L). Crude glycerol showed better results for γ-decalactone production when compared to castor oil. L. saturnus CCMA 0243 has been shown to have a high potential for γ-decalactone production from crude glycerol.     

Lumping kinetics of ABE fermentation wastewater treatment by oleaginous yeast Trichosporon cutaneum

Lumping kinetics models were built for the biological treatment of acetone–butanol–ethanol (ABE) fermentation wastewater by oleaginous yeast Trichosporon cutaneum with different fermentation temperatures. Compared with high temperature (33°C, 306?K) and low temperature (23°C, 296?K), medium temperature (28°C, 301?K) was beneficial for the cell growth and chemical oxygen demand (COD) degradation during the early stage of fermentation but the final yeast biomass and COD removal were influenced little. By lumping method, the materials in the bioconversion network were divided into five lumps (COD, lipid, polysaccharide, other intracellular products, other extracellular products), and the nine rate constants (k₁–k₉) for the models can well explain the bioconversion laws. The Gibbs free energy (G) for this bioconversion was positive, showing that it cannot happen spontaneous, but the existence of yeast can after the chemical equilibrium and make the bioconversion to be possible. Overall, the possibility of using lumping kinetics for elucidating the laws of materials conversion in the biological treatment of ABE fermentation wastewater by T. cutaneum has been initially proved and this method has great potential for further application.     

Diffusible signal factor‐dependent quorum sensing in pathogenic bacteria and its exploitation for disease control

Cell‐to‐cell signals of the diffusible signal factor (DSF) family are cis‐2‐unsaturated fatty acids of differing chain length and branching pattern. DSF signalling has been described in diverse bacteria to include plant and human pathogens where it acts to regulate functions such as biofilm formation, antibiotic tolerance and the production of virulence factors. DSF family signals can also participate in interspecies signalling with other bacteria and interkingdom signalling such as with the yeast Candida albicans. Interference with DSF signalling may afford new opportunities for the control of bacterial disease. Such strategies will depend in part on detailed knowledge of the molecular mechanisms underlying the processes of signal synthesis, perception and turnover. Here, I review both recent progress in understanding DSF signalling at the molecular level and prospects for translating this knowledge into approaches for disease control.     

Effects of salinity variations on the rates of photosynthesis and respiration of the juvenile giant clam (Tridacna gigas,Bivalvia, Cardiidae)

Giant clams are in symbiotic relationship with the photosynthesizing zooxanthellae and dwell in the shallow coral reefs of the Indo-Pacific region where they may be exposed to fluctuating salinities during high levels of precipitation. This study evaluated the effects of reduced salinities (18, 25, and 35‰ as control) on the rates of photosynthesis and respiration in juvenile Tridacna gigas for 14 days. At 18‰, total mortality was recorded after day 4, so no photosynthetic measurements were subsequently conducted for this treatment. However, results showed no significant differences in the photosynthetic rates among treatments. Respiration rates were significantly increased at both low salinities resulting in low P_g/R ratios. After 14 days of exposure, photosynthetic parameters at 25‰ were not significantly different from the control which suggests that juvenile T. gigas may be able to acclimate to osmotic stress and adjust its photophysiology following reductions in salinity of up to 25‰.