Rural Revolt in Mexico: U.S. Intervention and the Domain of Subaltern Politics

Rural Revolt in Mexico: U.S. Intervention and the Domain of Subaltern Politics. Daniel Nugent. ed. Durham, NC: Duke University Press, 1998. 384 pp.     

Evaluation of a novel Wave Bioreactor® cellbag for aerobic yeast cultivation

The Wave Bioreactor is widely used in cell culture due to the benefits of disposable technology and ease of use. A novel cellbag was developed featuring a frit sparger to increase the system's oxygen transfer. The purpose of this work was to evaluate the sparged cellbag for yeast cultivation. Oxygen mass transfer studies were conducted in simulated culture medium and the sparged system's maximum oxygen mass transfer coefficient (kLa) was 38 h(-1). These measurements revealed that the sparger was ineffective in increasing the oxygen transfer capacity. Cultures of Saccharomyces cerevisiae were successfully grown in oxygen-blended sparged and oxygen-blended standard cellbags. Under steady state conditions for both cellbag designs, kLa values as high as 60 h(-1) were obtained with no difference in growth characteristics. This is the first report of a successful cultivation of a microbe in a Wave Bioreactor comparing conventional seed expansion in shake flasks and stirred tank bioreactors.     

Modeling the influence of body size on V(O2) peak: effects of model choice and body composition.

This study examined the bivariate relationship between peak oxygen uptake (V(O2) peak); l/min) and body size in adult men (n = 1,314, age 17-66 yr), using both "simple" and "full" iterative nonlinear allometric models. The simple model was described by V(O2) peak = M(b) (or FFM(b)) exp(c SR-PA) exp(a + d age) epsilon (where M is body mass in kg; FFM is fat-free mass in kg; SR-PA is self-reported physical activity; epsilon is a multiplicative error term; and exp indicates natural antilogarithms). The full model was described by V(O2) peak = M(b) (or FFM(b)) exp(c SR-PA) exp(a + d age) + e (epsilon), where e is a permitted Y-intercept term. The M exponent obtained from simple allometry was 0.65 [95% confidence interval (CI), 0.59-0.71], suggestive of a curvilinear relationship constrained to pass through the origin. This "zero Y-intercept" assumption was examined via the full allometric model, which revealed an M exponent of 1.00 (95% CI, 0.7-1.31), together with a positive Y-intercept term (e) of 1.13 (95% CI, 0.54-1.73). The FFM exponents were not significantly different from unity in either the simple or full allometric models. It appears that the curvilinearity of the simple allometric model (using total M) is fictitious and is due to the inappropriate forcing of the regression line through the origin. Utilizing FFM as the body-size variable revealed a linear relationship between body size and V(O2) peak, irrespective of model choice. We conclude that the population mass exponent for V(O2) peak is close to unity.     

Dependence of morphology on agitation intensity in fed-batch cultures of Aspergillus oryzae and its implications for recombinant protein production

We previously reported that, although agitation conditions strongly affected mycelial morphology, such changes did not lead to different levels of recombinant protein production in chemostat cultures of Aspergillus oryzae (Amanullah et al., 1999). To extend this finding to another set of operating conditions, fed-batch fermentations of A. oryzae were conducted at biomass concentrations up to 34 g dry cell weight/L and three agitation speeds (525, 675, and 825 rpm) to give specific power inputs between 1 and 5 kWm(-3). Gas blending was used to control the dissolved oxygen level at 50% of air saturation except at the lowest speed where it fell below 40% after 60-65 h. The effects of agitation intensity on growth, mycelial morphology, hyphal tip activity, and recombinant protein (amyloglucosidase) production in fed-batch cultures were investigated. In the batch phase of the fermentations, biomass concentration, and AMG secretion increased with increasing agitation intensity. If in a run, dissolved oxygen fell below approximately 40% because of inadequate oxygen transfer associated with enhanced viscosity, AMG production ceased. As with the chemostat cultures, even though mycelial morphology was significantly affected by changes in agitation intensity, enzyme titers (AGU/L) under conditions of substrate limited growth and controlled dissolved oxygen of >50% did not follow these changes. Although the measurement of active tips within mycelial clumps was not considered, a dependency of the specific AMG productivity (AGU/g biomass/h) on the percentage of extending tips was found, suggesting that protein secretion may be a bottle-neck in this strain during fed-batch fermentations.     

Understanding Market Size and Reporting Gaps for Paediatric TB in Indonesia,Nigeria and Pakistan: Supporting Improved Treatment of Childhood TB in the Advent of New Medicines

MethodologyExploratory assessments were carried out in Indonesia, Nigeria and Pakistan, reaching a range of facility types in two selected areas of each country. Record reviews and interviews of healthcare providers were carried out to assess numbers of unreported paediatric TB cases, diagnostic pathways followed and treatment regimens prescribed.

Main Findings

A total of 985 unreported diagnosed paediatric TB cases were identified over a three month period in 2013 in Indonesia from 64 facilities, 463 in Pakistan from 35 facilities and 24 in Nigeria from 20 facilities. These represent an absolute additional annualised yield to 2013 notifications reported to WHO of 15% for Indonesia, 2% for Nigeria and 7% for Pakistan. Only 12% of all facilities provided age and sex-disaggregated data. Findings highlight the challenges of confirming childhood TB. Diagnosis patterns in Nigeria highlight a very low suspicion for childhood TB. Providers note the need for paediatric medicines aligned to WHO recommendations.

Conclusion: How Market Data Can Support Better Public Health Interventions

This study emphasises the impact of incomplete reporting on the estimation of disease burden and potential market size of paediatric TB medicines. Further studies on “hubs” (facilities treating large numbers of childhood TB cases) will improve our understanding of the epidemic, support introduction efforts for new treatments and better measure markets for new paediatric medicines.     

Probing of C-terminal lysine variation in a recombinant monoclonal antibody production using Chinese hamster ovary cells with chemically defined media

C-terminal lysine (C-K) variants are commonly observed in therapeutic monoclonal antibodies and recombinant proteins. Heterogeneity of C-K residues is believed to result from varying degree of proteolysis by endogenous carboxypeptidase(s) during cell culture production. The achievement of batch-to-batch culture performance and product quality reproducibility is a key cell culture development criterion. Understanding the operational parameters affecting C-K levels provides valuable insight into the cell culture process. A CHO cell line X expressing a recombinant antibody was selected as the model cell line due to the exhibited sensitivity of its C-K level to the process conditions. A weak cation exchange chromatography (WCX) method with or without carboxypeptidase B (CpB) treatment was developed to monitor the C-K level for in-process samples. The effects of operating conditions (i.e., temperature and culture duration) and media trace elements (copper and zinc) on C-K variants were studied. The dominant effect on C-K level was identified as the trace elements concentration. Specifically, increased C-K levels were observed with increase of copper concentration and decrease of zinc concentration in chemically defined medium. Further, a hypothesis for C-K processing with intracellular and extracellular carboxypeptidase activity was proposed, based on preliminary intracellular carboxypeptidase Western blot results and the extracellular HCCF holding study.     

Comparative metabolite analysis to understand lactate metabolism shift in Chinese hamster ovary cell culture process

A metabolic shift from lactate production (LP) to net lactate consumption (LC) phenotype was observed in certain Chinese hamster ovary (CHO) cell lines during the implementation of a new chemically defined medium (CDM) formulation for antibody production. In addition, this metabolic shift typically leads to process performance improvements in cell growth, productivity, process robustness, and scalability. In our previous studies, a correlation between a key media component, copper, and this lactate metabolism shift was observed. To further investigate this phenomenon, two complementary studies were conducted. In the first study, a single cell line was cultivated in two media that only differed in their copper concentrations, yet were known to generate an LP or LC phenotype with that cell line. In the second study, two different cell lines, which were known to possess inherently different lactate metabolic characteristics, were cultivated in the same medium with a high level of copper; one cell line produced lactate throughout the duration of the culture, and the other consumed lactate after an initial period of LP. Cell pellet and supernatant samples from both studies were collected at regular time intervals, and their metabolite profiles were investigated. The primary finding from the metabolic analysis was that the cells in LP conditions exhibited a less efficient energy metabolism, with glucose primarily being converted into pyruvate, sorbitol, lactate, and other glycolytic intermediates. This decrease in energy efficiency may be due to an inability of pyruvate and acetyl-CoA to progress into the TCA cycle. The lack of progression into the TCA cycle or overflow metabolism in the LP phenotype resulted in the inadequate supply of ATP for the cells. As a consequence, the glycolysis pathway remained the major source of ATP, which in turn, resulted in continuous LP throughout the culture. In addition, the accumulation of free fatty acids was observed; this was thought to be a result of phospholipid catabolism that was being used to supplement the energy produced through glycolysis in order to meet the needs of LP cells. A thorough review of the metabolic profiles indicated that the lactate metabolic shift could be related to the oxidative metabolic capacity of cells.     

Multivariate classification analysis of metabolomic data for candidate biomarker discovery in type 2 diabetes mellitus

Recent advances in genomics, metabolomics and proteomics have made it possible to interrogate disease pathophysiology and drug response on a systems level. The analysis and interpretation of the complex data obtained using these techniques is potentially fertile but equally challenging. We conducted a small clinical trial to explore the application of metabolomics data in candidate biomarker discovery. Specifically, serum and urine samples from patients with type 2 diabetes mellitus (T2DM) were profiled on metabolomics platforms before and after 8 weeks of treatment with one of three commonly used oral antidiabetic agents, the sulfonyurea glyburide, the biguanide metformin, or the thiazolidinedione rosiglitazone. Multivariate classification techniques were used to detect serum or urine analytes, obtained at baseline (pre-treatment) that could predict a significant treatment response after 8 weeks. Using this approach, we identified three analytes, measured at baseline, that were associated with response to a thiazolidinedione after 8 weeks of treatment. Although larger and longer-term studies are required to validate any of the candidate biomarkers, pharmacometabolomic profiling, in combination with multivariate classification, is worthy of further exploration as an adjunct to clinical decision making regarding treatment selection and for patient stratification within clinical trials. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cu(II) organizes beta-2-microglobulin oligomers but is released upon amyloid formation

beta-2-Microglobulin (beta2m) is deposited as amyloid fibrils in the bones and joints of patients undergoing long-term dialysis treatment as a result of kidney failure. Previous work has shown that biologically relevant amounts of Cu(II) can cause beta2m to be converted to amyloid fibrils under physiological conditions in vitro. In this work, dynamic light scattering, mass spectrometry, and size-exclusion chromatography are used to characterize the role that Cu plays in the formation of oligomeric intermediates that precede fibril formation. Cu(II) is found to be necessary for the stability of the dimer and an initial form of the tetramer. The initially formed tetramer then undergoes a structural change to a state that no longer binds Cu(II) before progressing to a hexameric state. Based on these results, we propose that the lag phase associated with beta2m fibril formation is partially accounted for by the structural transition of the tetramer that results in Cu(II) loss. Consistent with this observation is the determination that the mature beta2m amyloid fibrils do not contain Cu. Thus, Cu(II) appears to play a catalytic role by enabling the organization of the necessary oligomeric intermediates that precede beta2m amyloid formation.