Techno-economic evaluation of an inclusion body solubilization and recombinant protein refolding process

Expression of recombinant proteins in Escherichia coli is normally accompanied by the formation of inclusion bodies (IBs). To obtain the protein product in an active (native) soluble form, the IBs must be first solubilized, and thereafter, the soluble, often denatured and reduced protein must be refolded. Several technically feasible alternatives to conduct IBs solubilization and on-column refolding have been proposed in recent years. However, rarely these on-column refolding alternatives have been evaluated from an economical point of view, questioning the feasibility of their implementation at a preparative scale. The presented study assesses the economic performance of four distinct process alternatives that include pH induced IBs solubilization and protein refolding (pH_IndSR); IBs solubilization using urea, dithiothreitol (DTT), and alkaline pH followed by batch size-exclusion protein refolding; inclusion bodies (IBs) solubilization using urea, DTT, and alkaline pH followed by simulated moving bed (SMB) size-exclusion protein refolding, and IBs solubilization using urea, DTT and alkaline pH followed by batch dilution protein refolding. The economic performance was judged on the basis of the direct fixed capital, and the production cost per unit of product (P(C)). This work shows that (1) pH_IndSR system is a relatively economical process, because of the low IBs solubilization cost; (2) substituting β-mercaptoethanol for dithiothreithol is an attractive alternative, as it significantly decreases the product cost contribution from the IBs solubilization; and (3) protein refolding by size-exclusion chromatography becomes economically attractive by changing the mode of operation of the chromatographic reactor from batch to continuous using SMB technology.     

Relationships among fire frequency, rainfall and vegetation patterns in the wet–dry tropics of northern Australia: an analysis based on NOAA-AVHRR data

Aim To quantify the regional‐scale spatio‐temporal relationships among rainfall, vegetation and fire frequency in the Australian wet–dry tropics (AWDT). Location Northern Australia: Cape York Peninsula, central Arnhem, central Kimberly, Einasleigh Uplands, Gulf Fall Uplands and northern Kimberley. Methods Monthly ‘fraction of photosynthetic active radiation absorbed by green vegetation’ (fAPAR) was decomposed into monthly evergreen (EG) and monthly raingreen (RG) components using time‐series techniques applied to monthly normalized difference vegetation index (NDVI) data from Advanced Very High Resolution Radiometer (AVHRR) imagery. Fire affected areas were independently mapped at the same spatio‐temporal resolution from AVHRR imagery. Weather station records were spatially interpolated to create monthly rainfall surfaces. Vegetation structural classes were derived from a digitized map of northern Australian vegetation communities (1 : 1,000,000). Generalized linear models were used to quantify relationships among the fAPAR, EG and RG signals, vegetation structure, rainfall and fire frequency, for the period November 1996–December 2001. Results The fAPAR and EG signals are positively correlated with annual rainfall and canopy cover, notably: EG_{closed forest} > EG_{open heathland} > EG_{open forest} > EG_woodland > EG_{open woodland} > EG_{low woodland} > EG_{low open woodland} > EG_{open grassland}. Vegetation height and fAPAR are positively correlated, excluding the special case of open heathland. The RG signal is highest where intermediate annual rainfall and strong seasonality in rainfall coincide, and is associated with vegetation structure as follows: RG_{open grassland} > RG_woodland > RG_{open forest} > RG_{open heathland} > RG_{low woodland} > RG_{open woodland} > RG_{low open woodland} > RG_{closed forest}. Monthly RG tracks monthly rainfall. Annual proportion of area burnt (PB) is maximal where high RG coincides with low EG (open grassland, several woodland communities). PB is minimal in vegetation where both RG and EG are low (low open woodland); and in vegetation where EG is high (closed forest, open heathland). Conclusions The RG–EG scheme successfully reflects digitally mapped tree and grass covers in relation to rainfall. RG–EG patterns are strongly associated with fire frequency patterns. PB is maximal in areas of high RG, where high biomass production during the wet season supports abundant fine fuel during the dry season. PB is minimal in areas with high EG, where relatively moist fuel limits fire ignition; and in areas with low EG and RG, where a relative short supply of fuel limits fire spread.     

Pavel Siffel (1954–2003) or Life full of chlorophyll

Life and research results of Pavel Siffel, a talented but untimely deceased Czech scientist in photosynthesis, are reviewed. He studied biophysics and physiology of chlorophyll, its complexes with proteins, their absorption and fluorescence spectra, activities in mutants and transformants, dealt with chlorophyll biosynthesis and protochlorophyllide photoreduction, pigments in plants grown at CO₂ deficiency and under simulated acid rain, with changes accompanying leaf and plant development, photobleaching, etc. He participated in construction of specialised spectrofluorometers, finally he built the kinetic spectrophotometer SpeKin.     

Non-linear cancer classification using a modified radial basis function classification algorithm

Summary This paper proposes a modified radial basis function classification algorithm for non-linear cancer classification. In the algorithm, a modified simulated annealing method is developed and combined with the linear least square and gradient paradigms to optimize the structure of the radial basis function (RBF) classifier. The proposed algorithm can be adopted to perform non-linear cancer classification based on gene expression profiles and applied to two microarray data sets involving various human tumor classes: (1) Normal versus colon tumor; (2) acute myeloid leukemia (AML) versus acute lymphoblastic leukemia (ALL). Finally, accuracy and stability for the proposed algorithm are further demonstrated by comparing with the other cancer classification algorithms.     

Ecogeographic size variations in Sifakas: a test of the resource seasonality and resource quality hypotheses

Ecogeographic size variations have been documented in some but not all sifakas. Few morphometric or body weight data have been available for two critically endangered subspecies of diademed sifakas: Perrier's sifakas (Propithecus diadema perrieri) and silky sifakas (Propithecus diadema candidus). The objectives of our study were to determine size variations in sifakas and if these variations are related to resource quality and/or resource seasonality. P. d. perrieri and P. d. candidus were captured, weighed, and measured in northern Madagascar. Body weights and morphometrics were compared with other subspecies of diademed sifakas and indris (Indri indri). Differences in body weights and morphometrics between taxa are particularly pronounced for P. d. perrieri compared to P. d. diadema, P. d. edwardsi, and I. indri. Most morphometrics varied in comparisons between P. d. candidus and the other Indriidae (P. d. diadema, P. d. edwardsi, and I. indri). Average body size in sifakas is positively correlated with annual rainfall and negatively correlated with length of dry season. Sifaka body size is not correlated with protein-to-fiber ratios. Thus, size variations in sifakas are related to resource seasonality rather than resource quality. The relationships between the temporal availability of food resources and sifaka body size reflect complex and regionally varying causalities. Detailed, longitudinal information on the ecological factors underlying food selection and nutrient requirements in sifakas are needed to determine the relationship between ecogeographic variables and body size in sifakas.     

钙蛋白酶介导模拟失重大鼠心肌肌钙蛋白抑制亚基的降解

本文旨在观察尾部悬吊模拟失重大鼠心肌钙蛋白酶(calpain)与钙蛋白酶抑素(calpastatin)表达的变化,以探讨心肌肌钙蛋白抑制亚基(cardiac troponin I,cTnI)降解的可能机制。采用尾部悬吊模拟失重大鼠模型,Western blotting技术观测心肌calpain-1、calpain-2与calpastatin的表达;PD150606抑制calpain活性,分析cTnI降解程度的变化。结果显示:与同步对照组相比,悬吊2周与4周组大鼠心肌calpastatin表达呈显著性降低(P0.05),calpain-1表达未改变,calpain-2表达略有降低;但是,心肌calpain-1/calpastatin及calpain-2/calpastatin的比值在悬吊2周与4周组明显增高(P0.05,P0.01)。悬吊4周组cTnI降解显著高于对照组(P0.01);然而,用calpain非特异性抑制剂PD150606处理后,对照组及悬吊组cTnI的降解均被显著抑制(P0.01)。这些结果提示模拟失重大鼠心肌calpain活性增高可能增加cTnI的降解。     

Simulated microgravity‐induced epigenetic changes in human lymphocytes

Real space flight and modeled microgravity conditions result in changes in the expression of genes that control important cellular functions. However, the mechanisms for microgravity‐induced gene expression changes are not clear. The epigenetic changes of DNA methylation and chromatin histones modifications are known to regulate gene expression. The objectives of this study were to investigate whether simulated microgravity alters (a) the DNA methylation and histone acetylation, and (b) the expression of DNMT1, DNMT3a, DNMT3b, and HDAC1 genes that regulate epigenetic events. To achieve these objectives, human T‐lymphocyte cells were grown in a rotary cell culture system (RCCS) that simulates microgravity, and in parallel under normal gravitational conditions as control. The microgravity‐induced DNA methylation changes were detected by methylation sensitive‐random amplified polymorphic DNA (MS‐RAPD) analysis of genomic DNA. The gene expression was measured by Quantitative Real‐time PCR. The expression of DNMT1, DNMT3a, and DNMT3b was found to be increased at 72 h, and decreased at 7 days in microgravity exposed cells. The MS‐RAPD analysis revealed that simulated microgravity exposure results in DNA hypomethylation and mutational changes. Gene expression analysis revealed microgravity exposure time‐dependent decreased expression of HDAC1. Decreased expression of HDAC1 should result in increased level of acetylated histone H3, however a decreased level of acetylated H3 was observed in microgravity condition, indicating thereby that other HDACs may be involved in regulation of H3 deacetylation. The findings of this study suggest that epigenetic events could be one of the mechanistic bases for microgravity‐induced gene expression changes and associated adverse health effects. J. Cell. Biochem. 111: 123–129, 2010. © 2010 Wiley‐Liss, Inc.     

Use of Two Industrial Wastes as Soil Amendments: Effect on Dissolved Reactive Phosphorus in Runoff

To control the dissolved reactive phosphorus (DRP) concentration in a soil solution, a number of soil amendments were tested. In the current study, Blast Furnace Slag (BFS) and Water Treatment Residues (WTR) were tested on bare soil under two rainfall intensities and two soil roughness levels. The soil was fertilized with P (KH₂PO₄) at a rate of 400 kg ha^?1 while BFS and WTR were applied at a rate of 5 g per 100 g of soil. Two soil roughness levels were exposed to artificial rainfall intensities of 30 and 65 mm h^?1. Three rainfall events were performed on each treatment. The runoff water generated over an area of 0.5 m² with a slope of 8% was collected at different time intervals and analyzed for DRP, Al, Fe, and K concentrations. The results showed that, regardless of rainfall intensity and soil roughness, the concentration of DRP in the runoff water increased with increasing runoff time from the unamended plots. However, in the BFS- and WTR-amended soils, the DRP concentration decreased with runoff time. Dissolved reactive P and DRP loads were the lowest from the WTR-amended plots, followed by the control and the BFS treatment plots. Water treatment residues reduced the mean DRP concentration by 27.3% and the DRP load by 32% compared to unamended plots. The two rainfall intensities significantly affected the DRP concentration and load. Under the low rainfall intensity, the DRP concentration and load were higher compared to the high rainfall intensity. The overall DRP concentration was not affected by changes in soil roughness. However, the DRP loads were higher from the plots with low soil roughness levels, especially during the first and second runs. Both the BFS and WTR were also effective in reducing the DRP concentrations in the drain water collected during the runoff events. The concentrations of Al, Fe, and K in the runoff water were not affected by the soil amendments. However, the electrical conductivity and pH readings were higher from the BFS-amended plots.