Combinatorial impact of Sec signal peptides from Bacillus subtilis and bioprocess conditions on heterologous cutinase secretion by Corynebacterium glutamicum

The impact of Sec signal peptides (SPs) from Bacillus subtilis in combination with isopropyl-β- d -1-thiogalactopyranoside concentration and feeding profile was investigated for heterologous protein secretion performance by Corynebacterium glutamicum using cutinase as model enzyme. Based on a comprehensive data set of about 150 bench-scale bioreactor cultivations in fed-batch mode and choosing the cutinase yield as objective, it was shown that relative secretion performance for bioprocesses remains very similar, irrespective of the applied SP enabling Sec-mediated cutinase secretion. However, to achieve the maximal absolute cutinase yield, careful adjustment of bioprocess conditions was found to be necessary. A model-based, two-step multiple regression approach resembled the collected data in a comprehensive way. The corresponding results suggest that the choice of the heterologous Sec SP and its interaction with the adjusted exponential feeding profile is highly relevant to maximize absolute cutinase yield in this study. For example, the impact of Sec SP is high at low growth rates and low at high growth rates. However, promising Sec SPs could be inferred from less complex batch cultivations. The extensive data were also evaluated in terms of cutinase productivity, highlighting the well-known trade-off between yield and productivity in bioprocess development in detail. Conclusively, only the right combination of target protein, Sec SP, and bioprocess conditions is the key to success.     

Potential pitfalls of the PTV concept in dose-to-medium planning optimization

In typical treatment planning of 3D IMRT, the incident energy fluence is optimized to achieve a homogeneous dose distribution to the PTV. The PTV includes the tumour but also healthy tissues that may have a different dose response for the same incident energy fluence, like bony structures included in the PTV (mandibles in head and neck tumours or femoral bones in sarcomas). Dose to medium optimization compensates for this heterogeneous response, leading to a non-homogeneous energy fluence in the PTV and a non-homogeneous dose in the CTV in the presence of geometric errors. We illustrate qualitatively this statement in a cylindrical geometry where the PTV includes a CTV (7 cm diameter) made of water surrounded by ICRU compact bone (1.2 cm thickness); such configuration was chosen to exaggerate the aforementioned effect. Optimization was performed assuming dose equals photon energy fluence times mass energy absorption coefficient. Bone has a 4% lower dose response in a 6 MV flattening filter free spectrum. After optimization either in medium or assuming everything as water composition, the geometry was shifted by 1.2 cm and dose recomputed. As expected, compensating for the under-response of the bone material during optimization in medium leads to an overdosage of the CTV when patient geometric errors are taken into account. Optimization in dose assuming everything as water composition leads to a uniform coverage. Robust optimization or forcing a uniform atomic composition in the PTV margin may resolve this incompatibility between the PTV concept and dose to medium optimization.     

Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52: production and some properties

AIMS: An investigation was carried out on an oxidative and SDS-stable alkaline protease secreted by Bacillus clausii of industrial significance. METHODS AND RESULTS: Maximum enzyme activity was produced when the bacterium was grown in the medium containing (g l-1): soyabean meal, 15; wheat flour, 10; liquid maltose, 25; K2HPO4, 4; Na2HPO4, 1; MgSO4.7H2O, 0.1; Na2CO3, 6. The enzyme has an optimum pH of around 11 and optimum temperature of 60 degrees C. The alkaline protease showed extreme stability towards SDS and oxidizing agents, which retained its activity above 75 and 110% on treatment for 72 h with 5% SDS and 10% H2O2, respectively. Inhibition profile exhibited by phenylmethylsulphonyl fluoride suggested that the protease from B. clausii belongs to the family of serine proteases. CONCLUSIONS: Bacillus clausii produced high levels of an extracellular protease having high stability towards SDS and H2O2. SIGNIFICANCE AND IMPACT OF THE STUDY: The alkaline protease from B. clausii I-52 is significant for an industrial perspective because of its ability to function in broad pH and temperature ranges in addition to its tolerance and stability in presence of an anionic surfactant, like SDS and oxidants like peroxides and perborates. The enzymatic properties of the protease also suggest its suitable application as additive in detergent formulations.     

A Modified Valley Restrained Monte Carlo Method to Efficiently Search the Low Energy Structures of Peptides

Abstract

A new Monte Carlo sampling scheme, namely the Modified Valley Restrained Monte Carlo procedure, is used to obtain the global energy minimum conformations for polypeptides, such as Met-enkephalin and Melittin. For each peptide, we found close agreement with previous results from both theoretical and experimental studies. The simple idea for controlling the step size according to the Valley Function, provides useful suggestions in searching the global energy minimum structures, and furthermore helps solve the multiple minima problem.     

Overcoming difficulties on synthesis of cardiac troponin-I

Human cardiac troponin-I (cTnI) is one of the most sensitive and specific indicators, used in the diagnosis of myocardial infarction. To produce the protein efficiently, Escherichia coli and Pichia pastoris systems were used. Initial trials for the expression in E. coli were not successful, although different expression vectors with different promoters were tested. This led us to use P. pastoris for the expression. After several trials with two different expression strains of P. pastoris, it was concluded that P. pastoris was also not an optimal expression host for cTnI. Comprehensive analysis of the expression systems indicated that an efficient expression is only possible when the gene is optimized for expression in E. coli. For this purpose, the gene was optimized in-silico, but edited manually afterwards. It was synthesized and cloned into pQE-2 vector. Expression was performed using routine experimental conditions. Thus, cTnI could be efficiently expressed from the optimized gene in E. coli. The expression and purification were practical and may be used for commercial purposes since a total yield of 25µg highly pure protein per milliliter of culture could be obtained. The protein was in its ready-to-use form for many biological applications, including as a standard in diagnostic tests and an antigen for antibody production.     

纤维素降解菌L-06的筛选、鉴定及其产酶条件的分析

从用于堆肥的水稻秸秆中初筛出一株高效纤维素降解菌L-06, 根据18S rRNA基因序列和菌株形态分析, 初步鉴定该菌为斜卧青霉(Penicillium decumbens)。研究了液体发酵培养基中氮源、碳源、表面活性剂、培养温度、起始pH以及接种量对该菌株各纤维素酶活力的影响。在最适条件下, 该菌的b-葡萄糖苷酶(BGL)、外切纤维素酶(CBH)于培养第3天酶活力分别达到1662 u/mL和2770 u/mL; 内切纤维素酶(EG)、滤纸糖化力(FPase)于培养第4天酶活力分别达到18064 u/mL和4035 u/mL。在产酶优化实验中, 该菌的EG和CBH在pH10的培养条件下分别保持了70%和43%的酶活性; 在50oC培养条件下EG和CBH分别保持了68%和59%的酶活性。表现出了耐热, 耐碱的特性。     

Dietary Enrichment with Medium Chain Triglycerides (AC-1203) Elevates Polyunsaturated Fatty Acids in the Parietal Cortex of Aged Dogs: Implications for Treating Age-Related Cognitive Decline

Dogs demonstrate an age-related cognitive decline, which may be related to a decrease in the concentration of omega-3 polyunsaturated fatty acids (n-3 PUFA) in the brain. Medium chain triglycerides (MCT) increase fatty acid oxidation, and it has been suggested that this may raise brain n-3 PUFA levels by increasing mobilization of n-3 PUFA from adipose tissue to the brain. The goal of the present study was to determine whether dietary MCT would raise n-3 PUFA concentrations in the brains of aged dogs. Eight Beagle dogs were randomized to a control diet (n = 4) or an MCT (AC-1203) enriched diet (n = 4) for 2 months. The animals were then euthanized and the parietal cortex was removed for phospholipid, cholesterol and fatty acid determinations by gas-chromatography. Dietary enrichment with MCT (AC-1203) resulted in a significant increase in brain phospholipid and total lipid concentrations (P < 0.05). In particular, n-3 PUFA within the phospholipid, unesterified fatty acid, and total lipid fractions were elevated in AC-1203 treated subjects as compared to controls (P < 0.05). Brain cholesterol concentrations did not differ significantly between the groups (P > 0.05). These results indicate that dietary enrichment with MCT, raises n-3 PUFA concentrations in the parietal cortex of aged dogs.     

On the parametrization of lateral dose profiles in proton radiation therapy

PurposeThe accurate evaluation of the lateral dose profile is an important issue in the field of proton radiation therapy. The beam spread, due to Multiple Coulomb Scattering (MCS), is described by the Molière's theory. To take into account also the contribution of nuclear interactions, modern Treatment Planning Systems (TPSs) generally approximate the dose profiles by a sum of Gaussian functions. In this paper we have compared different parametrizations for the lateral dose profile of protons in water for therapeutical energies: the goal is to improve the performances of the actual treatment planning.MethodsWe have simulated typical dose profiles at the CNAO (Centro Nazionale di Adroterapia Oncologica) beamline with the FLUKA code and validated them with data taken at CNAO considering different energies and depths. We then performed best fits of the lateral dose profiles for different functions using ROOT and MINUIT.ResultsThe accuracy of the best fits was analyzed by evaluating the reduced χ², the number of free parameters of the functions and the calculation time. The best results were obtained with the triple Gaussian and double Gaussian Lorentz–Cauchy functions which have 6 parameters, but good results were also obtained with the so called Gauss–Rutherford function which has only 4 parameters.ConclusionsThe comparison of the studied functions with accurate and validated Monte Carlo calculations and with experimental data from CNAO lead us to propose an original parametrization, the Gauss–Rutherford function, to describe the lateral dose profiles of proton beams.     

华中五味子ISSR-PCR反应体系优化及引物筛选

系统研究了华中五味子ISSR PCR反应体系中的主要影响因子,确立华中五味子ISSR-PCR最适反应体系,并筛选出12条有效引物。单因子实验结果显示,华中五味子ISSR-PCR反应体系中各主要成分的适宜浓度范围为,Mg²⁺ 1.50~3.50 mmol·L^-1,dNTPs 0.10~0.35 mmol·L^-1,引物0.25~0.60 μmol·L^-1,Taq酶0.50~1.50 U。4因子3水平正交实验确立了最适反应体系,即20 μL体系中包含2.50 mmol·L^-1 Mg²⁺、0.20 mmol·L^-1 dNTPs、0.25 μmol·L^-1引物、1.50 U Taq酶、60 ng DNA模板、2.50 μL 10×PCR Buffer。本研究为华中五味子种质资源的评估及遗传多样性分析奠定基础。     

甲型流感病毒核蛋白在大肠杆菌中的可溶性高效表达与纯化

为了在大肠杆菌中高效表达甲型流感病毒A/京科/30/95(H3N2)核蛋白NP,以便对原核表达的NP蛋白进行免疫原性研究,本研究通过密码子优化及全基因合成等方法,将3种形式的NP基因:与6×His标签融合的NP基因NP(His)、非融合的野生型NP基因NPwt及非融合的按大肠杆菌优势密码子改造的基因NP(O)分别插入原核表达载体pET-30a,构建了表达3种形式NP基因的3种原核表达质粒并研究不同质粒中NP蛋白的表达形式、条件、纯化工艺及抗原性。限制性酶切反应与测序表明,三种形式的NP基因均正确插入原核表达质粒pET-30a;SDS-PAGE凝胶电泳显示,三种形式的NP基因均能在大肠杆菌中表达,NP(O)基因的表达量最高;在不同温度诱导条件下,NP蛋白呈现可溶性表达,NP(O)基因可溶性高效表达的条件为:T=25℃,t=10 h;经阴离子交换和凝胶过滤层析两步纯化,可溶性表达的NP蛋白纯度可达90%;Western blot检测显示,纯化的NP能与流感病毒A/PR/8/34(H1N1)株感染小鼠的血清发生特异性结合。这些结果表明,非融合表达的密码子优化甲型流感病毒NP蛋白能在大肠杆菌中高效表达和纯化,同时保持良好的免疫反应活性。