Relationship between recombinant activated protein C secretion rates and mRNA levels in baby hamster kidney cells

Analysis of 12 baby hamster kidney (BHK) clones in exponential growth revealed a linear relationship between cell-specific recombinant activated protein C (APC) production rates and APC mRNA levels. This correlation indicated that mRNA levels limited APC productivity. Two strategies were employed to increase APC mRNA levels and APC productivity. First, sodium butyrate was added to increase mRNA levels by two- to sixfold in five APC-producing clones to obtain up to 2.7-fold increase in APC production rate. The second strategy was to retransfect an APC-producing BHK cell line with a vector containing additional APC cDNA and a mutant DHFR. This mutant DHFR gene allowed the selection of retransfected clones in higher MTX concentrations. Over two-fold higher mRNA levels were obtained in these retransfected clones and the cell-specific APC production rate increased twofold. At the highest level of APC secretion, increases in mRNA levels did not result in higher rates of APC production. Analysis of the intracellular APC content revealed a possible saturation in the secretory pathway at high mRNA levels. The relation between mRNA level and APC secretion rate was also investigated in batch culture. The levels of total cellular RNA, APC mRNA, and beta-actin mRNA were relatively stable while cells were in the exponential growth phase, but rapidly decreased when cells reached the stationary phase. The decline of cell-specific APC mRNA levels correlated with a decline in APC secretion rates, which indicated that the mRNA levels continued to limit the rates beyond the exponential phase and into the declining growth and stationary phases of batch APC production.     

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.     

Impact of depth filtration on disulfide bond reduction during downstream processing of monoclonal antibodies from CHO cell cultures

Monoclonal antibody interchain disulfide bond reduction was observed in a Chinese Hamster Ovary manufacturing process that used single-use technologies. A similar reduction has been reported for processes that involved high mechanical shear recovery unit operations, such as continuous flow centrifugation and when the clarified harvest was stored under low dissolved oxygen (DO) conditions (Trexler-Schmidt et al., 2010. Biotechnology and Bioengineering, 106(3), 452–461). The work described here identifies disposable depth filtration used during cell culture harvest operations as a shear-inducing unit operation causing cell lysis. As a result, reduction of antibody interchain disulfide bonds was observed through the same mechanisms described for continuous flow centrifugation. Small-scale depth-filtration models were developed, and the differential pressure (Δ P) of the primary depth filter was identified as the key factor contributing to cell lysis. Strong correlations of Δ P and cell lysis were generated by measuring the levels of lactate dehydrogenase and thiol in the filtered harvest material. A simple risk mitigation strategy was implemented during manufacturing by providing an air overlay to the headspace of a single-use storage bag to maintain sufficient DO in the clarified harvest. In addition, enzymatic characterization studies determined that thioredoxin reductase and glucose-6-phosphate dehydrogenase are critical enzymes involved in antibody reduction in a nicotinamide adenine dinucleotide phosphate (NADP ⁺)/NADPH-dependent manner.     

Identifying rare and common disease associated variants in genomic data using Parkinson’s disease as a model

Background

Genome-wide association studies have been successful in identifying common genetic variants for human diseases. However, much of the heritable variation associated with diseases such as Parkinson’s disease remains unknown suggesting that many more risk loci are yet to be identified. Rare variants have become important in disease association studies for explaining missing heritability. Methods for detecting this type of association require prior knowledge on candidate genes and combining variants within the region. These methods may suffer from power loss in situations with many neutral variants or causal variants with opposite effects.

Results

We propose a method capable of scanning genetic variants to identify the region most likely harbouring disease gene with rare and/or common causal variants. Our method assigns a score at each individual variant based on our scoring system. It uses aggregate scores to identify the region with disease association. We evaluate performance by simulation based on 1000 Genomes sequencing data and compare with three commonly used methods. We use a Parkinson’s disease case–control dataset as a model to demonstrate the application of our method.Our method has better power than CMC and WSS and similar power to SKAT-O with well-controlled type I error under simulation based on 1000 Genomes sequencing data. In real data analysis, we confirm the association of α-synuclein gene (SNCA) with Parkinson’s disease (p = 0.005). We further identify association with hyaluronan synthase 2 (HAS2, p = 0.028) and kringle containing transmembrane protein 1 (KREMEN1, p = 0.006). KREMEN1 is associated with Wnt signalling pathway which has been shown to play an important role for neurodegeneration in Parkinson’s disease.

Conclusions

Our method is time efficient and less sensitive to inclusion of neutral variants and direction effect of causal variants. It can narrow down a genomic region or a chromosome to a disease associated region. Using Parkinson’s disease as a model, our method not only confirms association for a known gene but also identifies two genes previously found by other studies. In spite of many existing methods, we conclude that our method serves as an efficient alternative for exploring genomic data containing both rare and common variants.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0088-9) contains supplementary material, which is available to authorized users.     

Kernel-based association test

Association mapping (i.e., linkage disequilibrium mapping) is a powerful tool for positional cloning of disease genes. We propose a kernel-based association test (KBAT), which is a composite function of "P-values of single-locus association tests" and "kernel weights related to intermarker distances and/or linkage disequilibria." The KBAT is a general form of some current test statistics. This method can be applied to the study of candidate genes and can scan each chromosome using a moving average procedure. We evaluated the performance of the KBAT through simulation studies that considered evolutionary parameters, disease models, sample sizes, kernel functions, test statistics, window attributes, empirical P-value estimations, and genetic/physical maps. The results showed that the KBAT had a well-controlled false positive rate and high power compared to existing methods. In addition, the KBAT was also applied to analyze a genomewide data set from the Collaborative Study on the Genetics of Alcoholism. Important genes associated with alcoholism dependence were identified. In summary, the merits of the KBAT are multifold: the KBAT is robust against the inclusion of nuisance markers, is invariant to the map scale, and accommodates different types of genomic data, study designs, and study purposes. The proposed methods are packaged in the user-friendly software, KBAT, available at http://www.stat.sinica.edu.tw/hsinchou/genetics/association/KBAT.htm.     

Summarizing techniques that combine three non-parametric scores to detect disease-associated 2-way SNP-SNP interactions

Identifying susceptibility genes that influence complex diseases is extremely difficult because loci often influence the disease state through genetic interactions. Numerous approaches to detect disease-associated SNP-SNP interactions have been developed, but none consistently generates high-quality results under different disease scenarios. Using summarizing techniques to combine a number of existing methods may provide a solution to this problem. Here we used three popular non-parametric methods—Gini, absolute probability difference (APD), and entropy—to develop two novel summary scores, namely principle component score (PCS) and Z-sum score (ZSS), with which to predict disease-associated genetic interactions. We used a simulation study to compare performance of the non-parametric scores, the summary scores, the scaled-sum score (SSS; used in polymorphism interaction analysis (PIA)), and the multifactor dimensionality reduction (MDR). The non-parametric methods achieved high power, but no non-parametric method outperformed all others under a variety of epistatic scenarios. PCS and ZSS, however, outperformed MDR. PCS, ZSS and SSS displayed controlled type-I-errors (< 0.05) compared to GS, APDS, ES (> 0.05). A real data study using the genetic-analysis-workshop 16 (GAW 16) rheumatoid arthritis dataset identified a number of interesting SNP-SNP interactions.     

Diagnostic Validity of the Generalized Anxiety Disorder - 7 (GAD-7) among Pregnant Women

Objective

Generalized anxiety disorder (GAD) during pregnancy is associated with several adverse maternal and perinatal outcomes. A reliable and valid screening tool for GAD should lead to earlier detection and treatment. Among pregnant Peruvian women, a brief screening tool, the GAD-7, has not been validated. This study aims to evaluate the reliability and validity of the GAD-7.

Methods

Of 2,978 women who attended their first perinatal care visit and had the GAD-7 screening, 946 had a Composite International Diagnostic Interview (CIDI). The Cronbach’s alpha was calculated to examine the reliability. We assessed the criterion validity by calculating operating characteristics. The construct validity was evaluated using factor analysis and association with health status on the CIDI. The cross-cultural validity was explored using the Rasch Rating Scale Model (RSM).

Results

The reliability of the GAD-7 was good (Cronbach’s alpha = 0.89). A cutoff score of 7 or higher, maximizing the Youden Index, yielded a sensitivity of 73.3% and a specificity of 67.3%. One-factor structure of the GAD-7 was confirmed by exploratory and confirmatory factor analysis. Concurrent validity was supported by the evidence that higher GAD-7 scores were associated with poor self-rated physical and mental health. The Rasch RSM further confirmed the cross-cultural validity of the GAD-7.

Conclusion

The results suggest that the Spanish-language version of the GAD-7 may be used as a screening tool for pregnant Peruvian women. The GAD-7 has good reliability, factorial validity, and concurrent validity. The optimal cutoff score obtained by maximizing the Youden Index should be considered cautiously; women who screened positive may require further investigation to confirm GAD diagnosis.     

Effect of D57N mutation on membrane activity and molecular unfolding of cobra cardiotoxin.

Cobra cardiotoxins (CTXs) are able to adopt a three-fingered beta-strand structure with continuous hydrophobic patch that is capable of interacting with zwitterionic phospholipid bilayer. In addition to the four disulfide bonds that form the rigid core of CTXs, Asp57 near the C-terminus interacts electrostatically with Lys2 near the N-terminus (Chiang et al. 1996. Biochemistry. 35:9177-9186). We indicate herein, using circular dichroism and the time-resolved polarized tryptophan fluorescence measurement, that Asp57 to Asn57 (D57N) mutation perturbs the structure of CTX molecules at neutral pH. The structural stability of the D57N mutant was found to be lower, as evidenced by the reduced effective concentration of the 2,2,2-trifluoethanol (TFE)-induced beta-sheet to alpha-helix transition. Interestingly, the single mutation also allows a greater degree of molecular unfolding, because the rotational correlation time of the TFE-induced unfolding intermediate is larger for the D57N mutant. It is suggested that the electrostatic interaction between N- and C-termini also contributes to the formation of the functionally important continuous hydrophobic stretch on the distant end of CTX molecules, because both the binding to anilinonaphthalene fluorescent probe and the interaction with phospholipid bilayer were also reduced for D57N mutant. The result emphasizes the importance of the hydrophobic amino acid residues near the tip of loop 3 as a continuous part of the three-fingered beta-strand CTX molecule and indicates how a distant electrostatic interaction might be involved. It is also implicated that electrostatic interaction plays a role in expanding the radius of gyration of the folding/unfolding intermediate of proteins.     

Ascorbic acid promotes prostanoid release in human lung parenchyma

Ascorbic acid reduces airway reactivity to inhaled bronchoconstrictor agents in man and guinea pigs. The precise mechanism(s) responsible for this effect are unknown, but in both species an acute indomethacin treatment reverses the action of the ascorbic acid. To determine if ascorbic acid promotes prostanoid synthesis and/or inhibits degradation, human lung parenchymal slices (100–200mg) were incubated for 60 minutes in oxygenated Tyrode's solution alone or with sodium ascorbate (0.001M–1M) and/or methacholine (1μM–100μM) and/or indomethacin (0.17μM–17μM). Aliquots of the incubation medium were assayed by radioimmunoassay for PGE₂, PGF_2α, thromboxane B₂ and 6-keto-PGF_1α. Ascorbic acid increased the accumulation of all four prostanoids in the incubation medium, especially thromboxane B₂ and 6-keto-PGF_1α. This stimulatory effect of ascorbic acid was concentration-dependent and was inhibited by indomethacin. We conclude that ascorbic acid can alter prostanoid generation by human lung tissue and this effect may, in part, explain its antibronchoconstrictor activity in man.