Estimating the potential refolding yield of recombinant proteins expressed as inclusion bodies

Recombinant protein production in bacteria is efficient except that insoluble inclusion bodies form when some gene sequences are expressed. Such proteins must undergo renaturation, which is an inefficient process due to protein aggregation on dilution from concentrated denaturant. In this study, the protein-protein interactions of eight distinct inclusion-body proteins are quantified, in different solution conditions, by measurement of protein second virial coefficients (SVCs). Protein solubility is shown to decrease as the SVC is reduced (i.e., as protein interactions become more attractive). Plots of SVC versus denaturant concentration demonstrate two clear groupings of proteins: a more aggregative group and a group having higher SVC and better solubility. A correlation of the measured SVC with protein molecular weight and hydropathicity, that is able to predict which group each of the eight proteins falls into, is presented. The inclusion of additives known to inhibit aggregation during renaturation improves solubility and increases the SVC of both protein groups. Furthermore, an estimate of maximum refolding yield (or solubility) using high-performance liquid chromatography was obtained for each protein tested, under different environmental conditions, enabling a relationship between yield and SVC to be demonstrated. Combined, the results enable an approximate estimation of the maximum refolding yield that is attainable for each of the eight proteins examined, under a selected chemical environment. Although the correlations must be tested with a far larger set of protein sequences, this work represents a significant move beyond empirical approaches for optimizing renaturation conditions. The approach moves toward the ideal of predicting maximum refolding yield using simple bioinformatic metrics that can be estimated from the gene sequence. Such a capability could potentially screen, in silico, those sequences suitable for expression in bacteria from those that must be expressed in more complex hosts. (C) 2004 Wiley Periodicals, Inc.

Serum triglycerides as a risk factor for cardiovascular diseases in the Asia-Pacific Region

Background-The importance of serum triglyceride levels as a risk factor for cardiovascular diseases is uncertain. Methods and Results-We performed an individual participant data meta-analysis of prospective studies conducted in the Asia-Pacific region. Cox models were applied to the combined data from 26 studies to estimate the overall and region-, sex-, and age-specific hazard ratios for major cardiovascular diseases by fifths of triglyceride values. During 796 671 person-years of follow-up among 96 224 individuals, 670 and 667 deaths as a result of coronary heart disease (CHD) and stroke, respectively, were recorded. After adjustment for major cardiovascular risk factors, participants grouped in the highest fifth of triglyceride levels had a 70% (95% CI, 47 to 96) greater risk of CHD death, an 80% (95% CI, 49 to 119) higher risk of fatal or nonfatal CHD, and a 50% (95% CI, 29% to 76%) increased risk of fatal or nonfatal stroke compared with those belonging to the lowest fifth. The association between triglycerides and CHD death was similar across subgroups defined by ethnicity, age, and sex. Conclusions-Serum triglycerides are an important and independent predictor of CHD and stroke risk in the Asia-Pacific region. These results may have clinical implications for cardiovascular risk prediction and the use of lipid-lowering therapy.

Operational regimes for a simplified one-step artificial chaperone refolding method

The artificial chaperone method for protein refolding developed by Rozema et al. (Rozema, D.; Gellman, S. H. J. Am. Chem. Soc. 1995, 117 (8), 2373-2374) involves the sequential dilution of denatured protein into a buffer containing detergent (cetyltrimethylammonium bromide, CTAB) and then into a refolding buffer containing cyclodextrin WD). In this paper a simplified one-step artificial chaperone method is reported, whereby CTAB is added directly to the denatured solution, which is then diluted directly into a refolding buffer containing P-cyclodextrin (P-CD). This new method can be applied at high protein concentrations, resulting in smaller processing volumes and a more concentrated protein solution following refolding. The increase in achievable protein concentration results from the enhanced solubility of CTAB at elevated temperatures in concentrated denaturant. The refolding yields obtained for the new method were significantly higher than for control experiments lacking additives and were comparable to the yields obtained with the classical two-step approach. A study of the effect of beta-CD and CTAB concentrations on refolding yield suggested two operational regimes: slow stripping ( beta-CDXTABsimilar to1), most suited for higher protein concentrations, and fast stripping (beta-CD/CTABsimilar to2.7), best suited for lower protein concentrations. An increased chaotrope concentration resulted in higher refolding yields and an enlarged operational regime.

How well does B-type natriuretic peptide predict death and cardiac events in patients with heart failure: systematic review

Objective To assess how well B-type natriuretic peptide (BNP) predicts prognosis in patients with heart failure. Design Systematic review of studies assessing BNP for prognosis m patients with heart failure or asymptomatic patients. Data sources Electronic searches of Medline and Embase from January 1994 to March 2004 and reference lists of included studies. Study selection and data extraction We included all studies that estimated the relation between BNP measurement and the risk of death, cardiac death, sudden death, or cardiovascular event in patients with heart failure or asymptomatic patients, including initial values and changes in values in response to treatment. Multivariable models that included both BNP and left ventricular ejection fraction as predictors were used to compare the prognostic value of each variable. Two reviewers independently selected studies and extracted data. Data synthesis 19 studies used BNP to estimate the relative risk of death or cardiovascular events in heart failure patients and five studies in asymptomatic patients. In heart failure patients, each 100 pg/ml increase was associated with a 35% increase in the relative risk of death. BNP was used in 35 multivariable models of prognosis. In nine of the models, it was the only variable to reach significance-that is, other variables contained no prognostic information beyond that of BNP. Even allowing for the scale of the variables, it seems to be a strong indicator of risk. Conclusion Although systematic reviews of prognostic studies have inherent difficulties, including die possibility of publication bias, the results of the studies in this review show that BNP is a strong prognostic indicator for both asymptomatic patients mid for patients with heart failure at all stages of disease.

A comparison of lipid variables as predictors of cardiovascular disease in the Asia Pacific Region

PURPOSE: Many guidelines advocate measurement of total or low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL), and triglycerides (TG) to determine treatment recommendations for preventing coronary heart disease (CHD) and cardiovascular disease (CVD). This analysis is a comparison of lipid variables as predictors of cardiovascular disease. METHODS: Hazard ratios for coronary and cardiovascular deaths by fourths of total cholesterol (TC), LDL, HDL, TG, non-HDL, TC/HDL, and TG/HDL values, and for a one standard deviation change in these variables, were derived in an individual participant data meta-analysis of 32 cohort studies conducted in the Asia-Pacific region. The predictive value of each lipid variable was assessed using the likelihood ratio statistic. RESULTS: Adjusting for confounders and regression dilution, each lipid variable had a positive (negative for HDL) log-linear association with fatal CHD and CVD. Individuals in the highest fourth of each lipid variable had approximately twice the risk of CHD compared with those with lowest levels. TG and HDL were each better predictors of CHD and CVD risk compared with TC alone, with test statistics similar to TC/HDL and TG/HDL ratios. Calculated LDL was a relatively poor predictor. CONCLUSIONS: While LDL reduction remains the main target of intervention for lipid-lowering, these data support the potential use of TG or lipid ratios for CHD risk prediction. (c) 2005 Elsevier Inc. All rights reserved.

Targeting Antigen-loaded Liposomes to Myeloid Antigen Presenting Cells

Objective: To target antigen-loaded liposomes to myeloid APC in vivo for immunotherapy and to manipulate immune function through liposome composition. Method: Liposomes were loaded with ovalbumin, the lipophilic red fluorescent marker, DiI, with or without QuilA adjuvant then injected either i.v. or s.c. to naı¨ ve C57Bl/6 mice. Spleen, liver and draining LN were stained with MHC class II and various myeloid markers to determine the uptake of liposomes. Frozen sections of spleen and draining LN were stained with FITC-labeled mAb to determine which cells take up the liposomes. To determine the effect on OVA-specific T cell responses, liposomes were administered to Balb/c mice which received DO11.10 OVAspecific TCR transgenic T cells labelled with CFSE. Results: The DiI fluorescence was visualized in MHC class II+ macrophages and DC in draining lymph nodes after s.c. injection and in spleen and liver after i.v injection. Immunofluorescence microscopy shows liposome uptake in marginal zone macrophages and some DC in the T cell areas of the spleen after i.v. injection. Administration of ova-liposomes with or without QuilA stimulated a specific T cell response as measured by CFSE dilution. Conclusion: APC of liver, spleen and LN, and subsequent antigen presentation to T cells can be targeted for immunotherapy by the administration of liposomes encapsulating antigen and adjuvant. Varying the composition and routes of liposome administration is expected to alter the function of the targeted APC and the T cell response.

Modelling of species in hood fires by conditional moment closure

Carbon monoxide, the chief killer in fires, and other species are modelled for a series of enclosure fires. The conditions emulate building fires where CO is formed in the rich, turbulent, nonpremixed flame and is transported frozen to lean mixtures by the ceiling jet which is cooled by radiation and dilution. Conditional moment closure modelling is used and computational domain minimisation criteria are developed which reduce the computational cost of this method. The predictions give good agreement for CO and other species in the lean, quenched-gas stream, holding promise that this method may provide a practical means of modelling real, three-dimensional fire situations. (c) 2005 The Combustion Institute. Published by Elsevier Inc. All rights reserved.

Problems associated with spray drying of sugar-rich foods

Stickiness is a major reason that limits the spray drying of various sugar-rich food products. Higher hygroscopicity of amorphous powder, increase in solubility of sugars with temperature, and lower melting point and glass transition temperature, contribute to the stickiness problem. So far, the glass transition temperature has been widely accepted as a best indicator for stickiness. There are various manoeuvres that have been applied to spray dry such products. Some of them are the addition of drying aids, modification of drier design and use of mild drying temperature conditions. This review paper highlights the major research works that deal with the stickiness property of sugar-rich foods.

An investigation of the anti-pronation effect of two taping methods after application and exercise

This study investigated the effect of two anti-pronation taping techniques on vertical navicular height, an indicator of foot pronation, after its application and 20 min of exercise. The taping techniques were: the low dye (LD) and low dye with the addition of calcaneal slings and reverse sixes (LDCR). A repeated measures study was used. It found that LDCR was superior to LD and control immediately after application and exercise. LD was better than control immediately after application but not after exercise. These findings provide practical directions to clinicians regularly using anti-pronation taping techniques.

Intracellular free Ca2+ and basal Mn2+ influx in cultured aortic smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats.

Numerous studies investigating the possible role of altered Ca2+ homeostasis in hypertension have compared resting and agonist-stimulated intracellular free Ca2+ ([Ca2+](i)) in cultured aortic smooth muscle cells from spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. However, such studies have not given consistent results. Differences in the method used to load cells with the Ca2+-sensitive indicator fura-2 have been investigated here as a possible source of variability between studies. We also describe the adaptation of a fluorescence technique for the assessment of basal Ca2+ permeability in SHR and WKY through the measurement of Mn2+ influx. The results are consistent with the hypothesis that basal Ca2+ influx is elevated in cultured aortic smooth muscle cells from SHR compared to those from WKY. However, this was not reflected as a significant difference between the two strains in basal or angiotensin II (200 nmol/L)stimulated [Ca2+](i). Furthermore, this result was not dependent on the protocol used to load cells with fura-2. Hence, measurement of bulk [Ca2+](i) does not appear to be the most sensitive parameter for altered Ca2+ homeostasis in SHR. Other compartments of the cell may better reflect altered Ca2+ fluxes in hypertension and are discussed in this work.

The family attitude scale: Reliability and validity of a new scale for measuring the emotional climate of families

Research on outcomes from psychiatric disorders has highlighted the importance of expressed emotion (EE), but its cost-effective measurement remains a challenge. This article describes development of the Family Attitude Scale (FAS), a 30-item instrument that can be completed by any informant. Its psychometric characteristics are reported in parents of undergraduate students and in 70 families with a schizophrenic member. The total FAS had high internal consistency in all samples, and reports of angry behaviour in FAS items showed acceptable inter-rater agreement. The FAS was associated with the reported anger, anger expression and anxiety of respondents. Substantial associations between the parents' FAS and the anger and anger expression of students was also observed. Parents of schizophrenic patients had higher FAS scores than parents of students, and the FAS was higher if disorder duration was longer or patient functioning was poorer. Hostility, high criticism and low warmth on the Camberwell Family Interview (CFI) were associated with a more negative FAS. The highest FAS in the family was a good predictor of a highly critical environment on the CFI. The FAS is a reliable and valid indicator of relationship stress and expressed anger that has wide applicability. (C) 1997 Elsevier Science Ireland Ltd.

Human settlements: Australia state of the environment report, 2001

Australia’s transition to the 21st century has been marked by an extended period of economic prosperity unmatched for several decades, but one in which a series of question marks are being raised in three principal areas: in relation to the environment, the social well-being of the population, and the future path of economic development. The first concern, which is of primary interest in this report, relates to the physical environment of cities and their surrounding regions, and the range of pressures exerted by population and human activity. The report begins by noting the increasing divergence of the prime indicator of national economic performance—gross domestic product (GDP)—from the Genuine Progress Indicator (GPI). GPI is a new experimental measure of sustainable development that accommodates factors currently unaccounted for in GDP, such as income distribution, value of household work, cost of unemployment, and various other social and environmental costs. The divergence of these two indicators in recent decades suggests that Australia’s growth has been heavily dependent on the draw-down of the nation’s stocks of capital assets (its infrastructure), its human and social capital, and its natural capital (Hamilton 1997).

Economics of wildlife tourism

After introducing the importance of the topic, we examine the economic impacts of wildlife tourism on income and employment as an indicator of the importance of this form of tourism. While such indicators can be important politically and to particular interest groups, they are shown to be an inadequate guide to the economic use and conservation of resources, including wildlife used in tourism. One reason for this (amongst others) is that total economic value must be taken into account in determining economic resource use and this is shown to be quite important in the case of wildlife species. Empirical procedures, such as use of the travel cost method and stated preference methods (for example, contingent valuation) are outlined and the way in which they can be used for determining the optimal economic allocation of land and other resources for wildlife tourism is explained. Economic implications and limitations of some empirical estimates of the importance of wildlife tourism are discussed. This leads on to a consideration of the purpose and usefulness of using economic instruments to manage wildlife tourism.

Conclusions

The researchers focussed on the impact of the heroin shortage that occurred in early 2001 on three Australian jurisdictions, namely NSW, Vic and SA. They interviewed 82 heroin dependent heroin users and 172 key informants from health and law enforcement agencies. They also examined a range of indicator data such as drug seizures, drug-related arrests, deaths related to drug use, emergency department admissions for overdose or drug induced psychosis , calls to telephone help lines about drug use, needles distributed for drug use, and notifications about blood borne diseases. In addition a range of documentation, in particular from the law enforcement sector, was examined.