Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst. As compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 x 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of >or= 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration. Stepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.

Stress effects caused by the expression of a mutant cellobiohydrolase I and proteasome inhibition in Trichoderma reesei Rut-C30

Trichoderma reesei Rut-C30 is used widely as an expression host for various gene products. We have explored cellular effects caused by the expression of a mutant form of cellobiohydrolase I (CBHI), the major secreted protein of T. reesei using biochemical and transcriptomic analyses and confocal laser scanning microscopy. The mutated CBHI was tagged fluorescently with Venus to establish the subcellular location of the fusion protein and its potential association with the proteasome, an organelle assigned for the disposal of misfolded proteins. Expression of the mutant CBHI in the high protein-secreting host Rut-C30 caused physiological changes in the fungal hyphae, affected protein secretion and elicited ER stress. A massive upregulation of UPR- and ERAD-related genes sec61, der1, uba1, bip1, pdi1, prp1, cxl1 and lhs1 was observed by qRT-PCR in the CBHIΔ4-Venus strain with four mutations introduced in the DNA encoding the core domain of CBHI. Further stress was applied to this strain by inhibiting function of the proteasome with MG132 (N-benzoylcarbonyl(Cbz)-Leu-Leu-leucinal). The effect of MG132 was found to be specific to the proteasome-associated genes. There are no earlier reports on the effect of proteasome inhibition on protein quality control in filamentous fungi. Confocal fluorescence microscopy studies suggested that the mutant CBHI accumulated in the ER and colocalized with the fungal proteasome. These results provide an indication that there is a limit to how far T. reesei Rut-C30, already under secretion stress, can be pressed to produce higher protein yields.

Characterization and novel activation of 72-kDa metalloproteinase in retinal interphotoreceptor matrix and Y-79 cell culture medium

Analysis of bovine interphotoreceptor matrix and conditioned medium from human Y-79 retinoblastoma cells by gelatin SDS-PAGE zymography reveals abundant activity of a 72-kDa M(r) gelatinase. The 72-kDa gelatinase from either source is inhibited by EDTA but not aprotinin or NEM, indicating that it is a metalloproteinase (MMP). The 72-kDa MMP is converted to a 62-kDa species with APMA treatment after gelatin sepharose affinity purification typical of previously described gelatinase MMP-2. The latent 72-kDa gelatinase from either bovine IPM or Y-79 media autoactivates without APMA in the presence of calcium and zinc after 72 hr at 37°C, producing a fully active mixture of proteinase species, 50 (48 in Y-79 medium), 38 and 35 kDa in size. The presence of inhibitory activity was examined in both whole bovine IPM and IPM fractions separated by SDS-PAGE. Whole IPM inhibited gelatinolytic activity of autoactivated Y-79-derived MMP in a dose-dependent manner. Inhibitory activities are observed in two protein fractions of 27-42 and 20-25 kDa. Western blots using antibodies to human tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and -2) reveal the presence of two TIMP-1-like proteins at 21 and 29 kDa in inhibitory fractions of the bovine IPM. TIMP-2 was not detected in the inhibitory IPM fractions, consistent with the observed autoactivation of bovine IPM 72-kDa gelatinase. Potential roles for this IPM MMP-TIMP system include physiologic remodelling of the neural retina-RPE cell interface and digestion of shed rod outer segment as well as pathological processes such as retinal detachment, PE cell migration, neovascularization and tumor progression. Cultured Y-79 cells appear to be a good model for studying the production and regulation of this proteinase system.

Converse results to the Wiener attack on RSA

A well-known attack on RSA with low secret-exponent d was given by Wiener about 15 years ago. Wiener showed that using continued fractions, one can efficiently recover the secret-exponent d from the public key (N,e) as long as d < N 1/4. Interestingly, Wiener stated that his attack may sometimes also work when d is slightly larger than N 1/4. This raises the question of how much larger d can be: could the attack work with non-negligible probability for d=N 1/4 + ρ for some constant ρ > 0? We answer this question in the negative by proving a converse to Wiener’s result. Our result shows that, for any fixed ε > 0 and all sufficiently large modulus lengths, Wiener’s attack succeeds with negligible probability over a random choice of d < N δ (in an interval of size Ω(N δ )) as soon as δ > 1/4 + ε. Thus Wiener’s success bound d 1/4. The known attacks in this class (by Verheul and Van Tilborg and Dujella) run in exponential time, so it is natural to ask whether there exists an attack in this class with subexponential run-time. Our second converse result answers this question also in the negative.

Made-to-order nanocarbons through deterministic plasma nanotechnology

Through a combinatorial approach involving experimental measurement and plasma modelling, it is shown that a high degree of control over diamond-like nanocarbon film sp3/sp2 ratio (and hence film properties) may be exercised, starting at the level of electrons (through modification of the plasma electron energy distribution function). Hydrogenated amorphous carbon nanoparticle films with high percentages of diamond-like bonds are grown using a middle-frequency (2 MHz) inductively coupled Ar + CH4 plasma. The sp3 fractions measured by X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy in the thin films are explained qualitatively using sp3/sp2 ratios 1) derived from calculated sp3 and sp2 hybridized precursor species densities in a global plasma discharge model and 2) measured experimentally. It is shown that at high discharge power and lower CH4 concentrations, the sp3/sp2 fraction is higher. Our results suggest that a combination of predictive modeling and experimental studies is instrumental to achieve deterministically grown made-to-order diamond-like nanocarbons suitable for a variety of applications spanning from nano-magnetic resonance imaging to spin-flip quantum information devices. This deterministic approach can be extended to graphene, carbon nanotips, nanodiamond and other nanocarbon materials for a variety of applications

Centralspindlin and α-catenin regulate Rho signalling at the epithelial zonula adherens

The biological impact of Rho depends critically on the precise subcellular localization of its active, GTP-loaded form. This can potentially be determined by the balance between molecules that promote nucleotide exchange or GTP hydrolysis. However, how these activities may be coordinated is poorly understood. We now report a molecular pathway that achieves exactly this coordination at the epithelial zonula adherens. We identify an extramitotic activity of the centralspindlin complex, better understood as a cytokinetic regulator, which localizes to the interphase zonula adherens by interacting with the cadherin-associated protein, α-catenin. Centralspindlin recruits the RhoGEF, ECT2, to activate Rho and support junctional integrity through myosin IIA. Centralspindlin also inhibits the junctional localization of p190 B RhoGAP, which can inactivate Rho. Thus, a conserved molecular ensemble that governs Rho activation during cytokinesis is used in interphase cells to control the Rho GTPase cycle at the zonula adherens

The burden attributable to mental and substance use disorders as risk factors for suicide : findings from the Global Burden of Disease Study 2010

Background The Global Burden of Disease Study 2010 (GBD 2010) identified mental and substance use disorders as the 5th leading contributor of burden in 2010, measured by disability adjusted life years (DALYs). This estimate was incomplete as it excluded burden resulting from the increased risk of suicide captured elsewhere in GBD 2010's mutually exclusive list of diseases and injuries. Here, we estimate suicide DALYs attributable to mental and substance use disorders. Methods Relative-risk estimates of suicide due to mental and substance use disorders and the global prevalence of each disorder were used to estimate population attributable fractions. These were adjusted for global differences in the proportion of suicide due to mental and substance use disorders compared to other causes then multiplied by suicide DALYs reported in GBD 2010 to estimate attributable DALYs (with 95% uncertainty). Results Mental and substance use disorders were responsible for 22.5 million (14.8-29.8 million) of the 36.2 million (26.5-44.3 million) DALYs allocated to suicide in 2010. Depression was responsible for the largest proportion of suicide DALYs (46.1% (28.0%-60.8%)) and anorexia nervosa the lowest (0.2% (0.02%-0.5%)). DALYs occurred throughout the lifespan, with the largest proportion found in Eastern Europe and Asia, and males aged 20-30 years. The inclusion of attributable suicide DALYs would have increased the overall burden of mental and substance use disorders (assigned to them in GBD 2010 as a direct cause) from 7.4% (6.2%-8.6%) to 8.3% (7.1%-9.6%) of global DALYs, and would have changed the global ranking from 5th to 3rd leading cause of burden. Conclusions Capturing the suicide burden attributable to mental and substance use disorders allows for more accurate estimates of burden. More consideration needs to be given to interventions targeted to populations with, or at risk for, mental and substance use disorders as an effective strategy for suicide prevention.

Estimating the burden of disease attributable to smoking in South Africa in 2000

Objectives To quantify the burden of disease attributable to smoking in South Africa for 2000. Design The absolute difference between observed lung cancer death rate and the level in non-smokers, adjusted for occupational and indoor exposure to lung carcinogens, was used to estimate the proportion of lung cancer deaths attributable to smoking and the smoking impact ratio (SIR). The SIR was substituted for smoking prevalence in the attributable fraction formula for chronic obstructive pulmonary disease (COPD) and cancers to allow for the long lag between exposure and outcome. Assuming a shorter lag between exposure and disease, the current prevalence of smoking was used to estimate the population-attributable fractions (PAF) for the other outcomes. Relative risks (RR) from the American Cancer Society cancer prevention study (CPS-II) were used to calculate PAF. Setting South Africa. Outcome measures Deaths and disability-adjusted life years (DALYs) due to lung and other cancers, COPD, cardiovascular conditions, respiratory tuberculosis, and other respiratory and medical conditions. Results Smoking caused between 41 632 and 46 656 deaths in South Africa, accounting for 8.0 - 9.0% of deaths and 3.7 - 4.3% of DALYs in 2000. Smoking ranked third (after unsafe sex/sexually transmitted disease and high blood pressure) in terms of mortality among 17 risk factors evaluated. Three times as many males as females died from smoking. Lung cancer had the largest attributable fraction due to smoking. However, cardiovascular diseases accounted for the largest proportion of deaths attributed to smoking. Conclusion Cigarette smoking accounts for a large burden of preventable disease in South Africa. While the government has taken bold legislative action to discourage tobacco use since 1994, it still remains a major public health priority.

Estimating the burden of disease attributable to excess body weight in South Africa in 2000

Objective. To estimate the burden of disease attributable to excess body weight using the body mass index (BMI), by age and sex, in South Africa in 2000. Design. World Health Organization comparative risk assessment (CRA) methodology was followed. Re-analysis of the 1998 South Africa Demographic and Health Survey data provided mean BMI estimates by age and sex. Populationattributable fractions were calculated and applied to revised burden of disease estimates. Monte Carlo simulation-modelling techniques were used for the uncertainty analysis. Setting. South Africa. Subjects. Adults 30 years of age. Outcome measures. Deaths and disability-adjusted life years (DALYs) from ischaemic heart disease, ischaemic stroke, hypertensive disease, osteoarthritis, type 2 diabetes mellitus, and selected cancers. Results. Overall, 87% of type 2 diabetes, 68% of hypertensive disease, 61% of endometrial cancer, 45% of ischaemic stroke, 38% of ischaemic heart disease, 31% of kidney cancer, 24% of osteoarthritis, 17% of colon cancer, and 13% of postmenopausal breast cancer were attributable to a BMI 21 kg/m2. Excess body weight is estimated to have caused 36 504 deaths (95% uncertainty interval 31 018 - 38 637) or 7% (95% uncertainty interval 6.0 - 7.4%) of all deaths in 2000, and 462 338 DALYs (95% uncertainty interval 396 512 - 478 847) or 2.9% of all DALYs (95% uncertainty interval 2.4 - 3.0%). The burden in females was approximately double that in males. Conclusions. This study shows the importance of recognising excess body weight as a major risk to health, particularly among females, highlighting the need to develop, implement and evaluate comprehensive interventions to achieve lasting change in the determinants and impact of excess body weight.

Estimating the burden of disease attributable to physical inactivity in South Africa in 2000

Objectives. To quantify the burden of disease attributable to physical inactivity in persons 15 years or older, by age group and sex, in South Africa for 2000. Design. The global comparative risk assessment (CRA) methodology of the World Health Organization was followed to estimate the disease burden attributable to physical inactivity. Levels of physical activity for South Africa were obtained from the World Health Survey 2003. A theoretical minimum risk exposure of zero, associated outcomes, relative risks, and revised burden of disease estimates were used to calculate population-attributable fractions and the burden attributed to physical inactivity. Monte Carlo simulation-modelling techniques were used for the uncertainty analysis. Setting. South Africa. Subjects. Adults ≥ 15 years. Outcome measures. Deaths and disability-adjusted life years (DALYs) from ischaemic heart disease, ischaemic stroke, breast cancer, colon cancer, and type 2 diabetes mellitus. Results. Overall in adults ≥ 15 years in 2000, 30% of ischaemic heart disease, 27% of colon cancer, 22% of ischaemic stroke, 20% of type 2 diabetes, and 17% of breast cancer were attributable to physical inactivity. Physical inactivity was estimated to have caused 17 037 (95% uncertainty interval 11 394 - 20 407), or 3.3% (95% uncertainty interval 2.2 - 3.9%) of all deaths in 2000, and 176 252 (95% uncertainty interval 133 733 - 203 628) DALYs, or 1.1% (95% uncertainty interval 0.8 - 1.3%) of all DALYs in 2000. Conclusions. Compared with other regions and the global average, South African adults have a particularly high prevalence of physical inactivity. In terms of attributable deaths, physical inactivity ranked 9th compared with other risk factors, and 12th in terms of DALYs. There is a clear need to assess why South Africans are particularly inactive, and to ensure that physical activity/inactivity is addressed as a national health priority.

Estimating the burden of disease attributable to four selected environmental risk factors in South Africa

INTRODUCTION: The first South African National Burden of Disease study quantified the underlying causes of premature mortality and morbidity experienced in South Africa in the year 2000. This was followed by a Comparative Risk Assessment to estimate the contributions of 17 selected risk factors to burden of disease in South Africa. This paper describes the health impact of exposure to four selected environmental risk factors: unsafe water, sanitation and hygiene; indoor air pollution from household use of solid fuels; urban outdoor air pollution and lead exposure. METHODS: The study followed World Health Organization comparative risk assessment methodology. Population-attributable fractions were calculated and applied to revised burden of disease estimates (deaths and disability adjusted life years, [DALYs]) from the South African Burden of Disease study to obtain the attributable burden for each selected risk factor. The burden attributable to the joint effect of the four environmental risk factors was also estimated taking into account competing risks and common pathways. Monte Carlo simulation-modeling techniques were used to quantify sampling, uncertainty. RESULTS: Almost 24 000 deaths were attributable to the joint effect of these four environmental risk factors, accounting for 4.6% (95% uncertainty interval 3.8-5.3%) of all deaths in South Africa in 2000. Overall the burden due to these environmental risks was equivalent to 3.7% (95% uncertainty interval 3.4-4.0%) of the total disease burden for South Africa, with unsafe water sanitation and hygiene the main contributor to joint burden. The joint attributable burden was especially high in children under 5 years of age, accounting for 10.8% of total deaths in this age group and 9.7% of burden of disease. CONCLUSION: This study highlights the public health impact of exposure to environmental risks and the significant burden of preventable disease attributable to exposure to these four major environmental risk factors in South Africa. Evidence-based policies and programs must be developed and implemented to address these risk factors at individual, household, and community levels.

Estimating the burden of disease attributable to high cholesterol in South Africa in 2000

Objectives To estimate the burden of disease attributable to high cholesterol in adults aged 30 years and older in South Africa in 2000. Design World Health Organization comparative risk assessment (CRA) methodology was followed. Small community studies were used to derive the prevalence by population group. Population-attributable fractions were calculated and applied to revised burden of disease estimates for the relevant disease categories for each population group. The total attributable burden for South Africa in 2000 was obtained by adding the burden attributed to high cholesterol for the four population groups. Monte Carlo simulation-modelling techniques were used for uncertainty analysis. Setting South Africa. Subjects Black African, coloured, white and Indian adults aged 30 years and older. Outcome measures Mortality and disability-adjusted life years (DALYs) from ischaemic heart disease (IHD) and ischaemic stroke. Results Overall, about 59% of IHD and 29% of ischaemic stroke burden in adult males and females (30+ years) were attributable to high cholesterol (≥ 3.8 mmol/l), with marked variation by population group. High cholesterol was estimated to have caused 24 144 deaths (95% uncertainty interval 22 404 - 25 286) or 4.6% (95% uncertainty interval 4.3 - 4.9%) of all deaths in South Africa in 2000. Since most cholesterol-related cardiovascular disease events occurred in middle or old age, the loss of life years comprised a smaller proportion of the total: 222 923 DALYs (95% uncertainty interval 206 712 - 233 460) or 1.4% of all DALYs (95% uncertainty interval 1.3 - 1.4%) in South Africa in 2000. Conclusions High cholesterol is an important cardiovascular risk factor in all population groups in South Africa.

Estimating the burden of disease attributable to urban outdoor air pollution in South Africa in 2000

Objectives To quantify the mortality burden attributed to urban outdoor air pollution in South Africa in 2000. Design The study followed comparative risk assessment (CRA) methodology developed by the World Heath Organization (WHO). In most urban areas, annual mean concentrations of particulate matter (PM) with diameters less than 10 μum (PM10) from monitoring network data and PM with diameters less than 2.5 μm (PM2.5) derived using a ratio method were weighted according to population size. PM10 and PM2.5 data from air-quality assessment studies in areas not covered by the network were also included. Population-attributable fractions calculated using risk coefficients presented in the WHO study were weighted by the proportion of the total population (33%) in urban environments, and applied to revised estimates of deaths and years of life lost (YLLs) for South Africa in 2000. Setting South Africa. Subjects Children under 5 years and adults 30 years and older. Outcome measures Mortality and YLLs from lung cancer and cardiopulmonary disease in adults (30 years and older), and from acute respiratory infections (ARIs) in children aged 0 - 4 years. Results Outdoor air pollution in urban areas in South Africa was estimated to cause 3.7% of the national mortality from cardiopulmonary disease and 5.1% of mortality attributable to cancers of the trachea, bronchus and lung in adults aged 30 years and older, and 1.1% of mortality from ARIs in children under 5 years of age. This amounts to 4 637 or 0.9% (95% uncertainty interval 0.3 - 1.5%) of all deaths and about 42 000 YLLs, or 0.4% (95% uncertainty interval 0.1 - 0.7%) of all YLLs in persons in South Africa in 2000. Conclusion Urban air pollution has under-recognised public health impacts in South Africa. Fossil fuel combustion emissions and traffic-related air pollution remain key targets for public health in South Africa.

Estimating the burden of disease attributable to high blood pressure in South Africa in 2000

Objectives To estimate the burden of disease attributable to high blood pressure (BP) in adults aged 30 years and older in South Africa in 2000. Design World Health Organization comparative risk assessment (CRA) methodology was followed. Mean systolic BP (SBP) estimates by age and sex were obtained from the 1998 South African Demographic and Health Survey adult data. Population-attributable fractions were calculated and applied to revised burden of disease estimates for the relevant disease categories for South Africa in 2000. Monte Carlo simulation modelling techniques were used for uncertainty analysis. Setting South Africa Subjects Adults aged 30 years and older. Outcome measures Mortality and disability-adjusted life years (DALYs) from ischaemic heart disease (IHD), stroke, hypertensive disease and other cardiovascular disease (CVD). Results High BP was estimated to have caused 46 888 deaths (95% uncertainty interval 44 878 - 48 566) or 9% (95% uncertainty interval 8.6 - 9.3%) of all deaths in South Africa in 2000, and 390 860 DALYs (95% uncertainty interval 377 955 - 402 256) or 2.4% of all DALYs (95% uncertainty interval 2.3 - 2.5%) in South Africa in 2000. Overall, 50% of stroke, 42% of IHD, 72% of hypertensive disease and 22% of other CVD burden in adult males and females (30+ years) were attributable to high BP (systolic BP ≥ 115 mmHg). Conclusions High BP contributes to a considerable burden of CVD in South Africa and results indicate that there is considerable potential for health gain from implementing BP-lowering interventions that are known to be highly costeffective.