Predicting industrial-scale cell culture seed trains-A Bayesian framework for model fitting and parameter estimation, dealing with uncertainty in measurements and model parameters, applied to a nonlinear kinetic cell culture model, using an MCMC method.

For production of biopharmaceuticals in suspension cell culture, seed trains are required to increase cell number from cell thawing up to production scale. Because cultivation conditions during the seed train have a significant impact on cell performance in production scale, seed train design, monitoring, and development of optimization strategies is important. This can be facilitated by model-assisted prediction methods, whereby the performance depends on the prediction accuracy, which can be improved by inclusion of prior process knowledge, especially when only few high-quality data is available, and description of inference uncertainty, providing, apart from a "best fit"-prediction, information about the probable deviation in form of a prediction interval. This contribution illustrates the application of Bayesian parameter estimation and Bayesian updating for seed train prediction to an industrial Chinese hamster ovarian cell culture process, coppled with a mechanistic model. It is shown in which way prior knowledge as well as input uncertainty (e.g., concerning measurements) can be included and be propagated to predictive uncertainty. The impact of available information on prediction accuracy was investigated. It has been shown that through integration of new data by the Bayesian updating method, process variability (i.e., batch-to-batch) could be considered. The implementation was realized using a Markov chain Monte Carlo method.

Identification of a DMBT1 polymorphism associated with increased breast cancer risk and decreased promoter activity.

According to present estimations, the unfavorable combination of alleles with low penetrance but high prevalence in the population might account for the major part of hereditary breast cancer risk. Deleted in Malignant Brain Tumors 1 (DMBT1) has been proposed as a tumor suppressor for breast cancer and other cancer types. Genomewide mapping in mice further identified Dmbt1 as a potential modulator of breast cancer risk. Here, we report the association of two frequent and linked single-nucleotide polymorphisms (SNPs) with increased breast cancer risk in women above the age of 60 years: DMBT1 c.-93C>T, rs2981745, located in the DMBT1 promoter; and DMBT1 c.124A>C, p.Thr42Pro, rs11523871(odds ratio [OR]=1.66, 95% confidence interval [CI]=1.21-2.29, P=0.0017; and OR=1.66; 95% CI=1.21-2.28, P=0.0016, respectively), based on 1,195 BRCA1/2 mutation-negative German breast cancer families and 1,466 unrelated German controls. Promoter studies in breast cancer cells demonstrate that the risk-increasing DMBT1 -93T allele displays significantly decreased promoter activity compared to the DMBT1 -93C allele, resulting in a loss of promoter activity. The data suggest that DMBT1 polymorphisms in the 5'-region are associated with increased breast cancer risk. In accordance with previous results, these data link decreased DMBT1 levels to breast cancer risk.

The functional genetic variant Ile646Val located in the kinase binding domain of the A-kinase anchoring protein 10 is associated with familial breast cancer.

Overexpression of cAMP-dependent protein kinase A (PKA) is a hallmark of the great majority of human cancers including breast cancer. A-kinase anchoring proteins (AKAPs) coordinate the specificity of PKA signalling by localizing the kinase to its subcellular sites. We tested the hypothesis whether the functional amino acid exchange Ile646Val, located in the kinase-binding domain of AKAP10, is a low-penetrance familial breast cancer risk factor. Ile646Val alters the binding of AKAP10 to PKA and is associated with morbidity. The analysis of 787 BRCA1/2 mutation-negative familial breast cancer patients and 993 controls revealed an association of the AKAP10 Ile646Val polymorphism with increased familial breast cancer risk [odds ratio (OR)=1.25, 95% confidence interval (CI) 1.03-1.51, P=0.024]. Our previous study has shown that AKAP13 Lys526Gln is associated with familial breast cancer (OR=1.58). Here, we discovered that carriers of both variants, AKAP10 Ile646Val and AKAP13 Lys526Gln, are at a further enhanced breast cancer risk (OR=2.41, 95% CI 1.30-4.46, P=0.005). PKA is a major target of therapeutic anticancer strategies. Phosphorylation of the estrogen receptor (ER) alpha by PKA induces resistance against the anti-estrogen tamoxifen. Our results indicate for the first time the importance of AKAP10 Ile646Val for familial breast cancer susceptibility. Due to the impact of Ile646Val on the subcellular localization of PKA, it will be interesting to investigate whether this polymorphism influences the effectiveness of PKA and tamoxifen based therapeutic anticancer concepts.

Associations of genetic variants in the estrogen receptor coactivators PPARGC1A, PPARGC1B and EP300 with familial breast cancer.

The mitogen effect of the ovarian steroid estrogen is a strong risk factor for breast cancer development. This effect is mainly mediated by the estrogen receptor alpha, a hormone inducible transcription factor, which activates gene expression through recruiting multiple coactivators, such as PPARGC1A, PPARGC1B and EP300. We tested the hypothesis that non-conservative, putative functional amino acid exchanges in PPARGC1A, PPARGC1B and EP300 act as low-penetrance familial breast cancer risk factors. The analysis of 816 BRCA1/2 mutation-negative familial breast cancer patients and 1012 controls revealed an association of the PPARGC1A Thr612Met polymorphism with familial breast cancer (OR = 1.35, 95% CI 1.00-1.81, P = 0.049), high-risk familial breast cancer (OR = 1.51, 95% CI 1.08-2.12, P = 0.017) and bilateral familial breast cancer (OR = 2.30, 95% CI 1.24-4.28, P = 0.009). Logistic regression analyses of the PPARGC1B Ala203Pro variant showed an increased familial breast cancer risk of heterozygous and homozygous variant allele carriers (OR = 1.48, 95% CI 1.15-1.91, P = 0.002). The genotype-combination analysis of the associated PPARGC1A Thr612Met variant and the associated PPARGC1B Ala203Pro variant suggests an allele dose-dependent breast cancer risk (P(trend) = 0.0004). Our results indicate for the first time the importance of inherited variants in the estrogen receptor coactivator genes PPARGC1A and PPARGC1B for familial breast cancer susceptibility. Owing to their impact on estrogen signaling, these polymorphisms might also influence adjuvant anti-estrogen therapy, using agents such as tamoxifen and raloxifen, and outcome of breast cancer patients.

Detection of herpes simplex virus type 1, herpes simplex virus type 2 and varicella-zoster virus in skin lesions. Comparison of real-time PCR, nested PCR and virus isolation.

BACKGROUND: Herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2) and varicella-zoster virus (VZV) cause a wide range of signs and symptoms, varying from trivial mucocutaneous lesions to life-threatening infections, especially in immuno-suppressed patients. Since antiviral drugs are available, rapid and sensitive laboratory diagnosis of these virus infections is important. OBJECTIVE: To set up and evaluate HSV-1, HSV-2 and VZV qualitative real-time PCR on the Lightcycler system and to compare the results with those of the 'in-house' nested PCR and virus isolation. STUDY DESIGN: 110 consecutive samples from dermal or genital lesions from patients suspected of having HSV infections and another 110 samples from patients with suspected VZV infections were tested with real-time PCR, nested PCR and virus isolation. RESULTS: 24 samples (22%) were positive for HSV-1 by virus isolation and nested PCR, whereas 26 (24%) were positive by real-time PCR. HSV-2 was detected in 28 samples (25%) by virus isolation, in 41 (37%) by nested PCR and in 40 (36%) by real-time PCR. VZV was isolated in 15 samples (14%) and VZV DNA was detected in 51 samples (46%) by nested PCR as well as by real-time PCR. Nucleic acid amplification increased the detection rate of HSV-2 and VZV DNA in particular compared to virus isolation. No significant difference in sensitivity was found between real-time PCR and nested PCR. CONCLUSION: Real-time PCR has the advantage of rapid amplification, a reduced risk for contamination and it is a suitable method for diagnosis of VZV and HSV in specimens from skin lesions.

Population dynamics of a heterogeneous Borrelia burgdorferi B31 strain in an experimental mouse-tick infectious cycle.

We have recently shown that low-passage, infectious Borrelia burgdorferi strain B31 MI is a heterogeneous mixture of clones varying in colony morphology, growth rate, protein profiles, plasmid content and infectivity. In this study, we asked whether there is a selection for certain clonal populations during the infectious cycle when uncloned B31 MI is used as the starting strain. B31 MI derivatives were reisolated from various tissues of two mice after completion of a mouse-tick-mouse infectious cycle and their protein and plasmid profiles were analyzed. Both mice developed ostensibly clonal infections despite the fact that the infectious cycle was started with a heterogeneous strain. Moreover, the mice became infected with two different clones varying in protein profile and growth phenotype. Comparison of the mouse reisolates to uncloned B31 MI and clonal variants derived from B31 MI before mouse-tick-mouse passage suggests that they were derived from clonal populations present in the uncloned B31 MI. Our results indicate the presence of at least two distinct populations within B31 MI that are competent to complete an experimental mouse-tick infectious cycle. The study provides insight into infectivity profiles and infection dynamics of different clonal populations present in a low-passage, infectious B. burgdorferi strain.