Optimal synthesis of chromatographic trains for downstream protein processing

Downstream bioprocessing and especially chromatographic steps, commonly used for the purification of multicomponent systems, are significant cost drivers in the production of therapeutic proteins. There has been an increased interest in the development of systematic methods for the design of such processes, and the appropriate selection of a series of chromatographic steps is still a major challenge to be addressed. Several models have been developed previously but have assumed that 100% recovery of the desired product is obtained at each chromatographic step. In this work, a mathematical framework is proposed, based on mixed integer optimisation techniques, that removes this assumption and allows full flexibility on the position of retention time cut-points, between which the desired product fraction is collected. The proposed model is demonstrated on three example protein mixtures, each containing up to 13 contaminants and selecting from a set of up to 21 candidate steps. The proposed model results in a reduction of one to three chromatographic steps over solutions that no losses are allowed.     

Impact of peroxisome proliferator-activated receptors gamma and delta on adiposity in toddlers and preschoolers in the GENESIS Study

Peroxisome proliferator-activated receptor gamma (PPAR gamma) and peroxisome proliferator-activated receptor delta (PPAR delta) are promising candidate genes for obesity. Associations between adiposity-related phenotypes and genetic variation in PPAR gamma (Pro12Ala and C1431T), as well as PPAR delta (T+294C) were assessed in 2,102 Greek children aged 1-6 years, as part of a large-scale epidemiological study (Growth, Exercise and Nutrition Epidemiological Study In preSchoolers). In girls aged 3-4 years, the Ala12 allele was associated with higher mid-upper arm (P = 0.010) and hip (P = 0.005) circumferences, as well as subscapular (P = 0.008) and total skinfolds (P = 0.011) that explained 2.0, 3.7, 2.1, and 1.9% of the phenotypic variance, respectively, while the T1431 allele was associated with higher mean values for waist circumference (P = 0.018) and suprailiac skinfold (P = 0.017), genotype accounting for 1.6% of the variance in both phenotypes. No significant effects of PPAR delta T+294C polymorphism or the interaction of the PPAR delta and PPAR gamma variants on adiposity-related phenotypes were observed in any age group or gender. Haplotype-based analysis including both PPAR gamma polymorphisms revealed that in girls aged 3-4 years, the Ala-T haplotype was associated with higher waist (P = 0.014) and hip (P = 0.007) circumferences compared to the common Pro-C haplotype. The PPAR gamma Pro12Ala and C1431T polymorphisms are associated with increased adiposity during early childhood in a gender- and age-specific manner and independently of the PPAR delta T+294C polymorphism.     

Impact of KRAS,BRAF, PIK3CA Mutations,PTEN, AREG,EREG Expression and Skin Rash in ≥2nd Line Cetuximab-Based Therapy of Colorectal Cancer Patients

Background

To investigate the predictive significance of KRAS, BRAF, PIK3CA mutational status, AREG- EREG mRNA expression, PTEN protein expression and skin rash in metastatic colorectal cancer (mCRC) patients treated with cetuximab containing salvage chemotherapy.

Methods

Primary tumors from 112 mCRC patients were analyzed. The worst skin toxicity during treatment was recorded.

Results

KRAS, BRAF and PIK3CA mutations were present in 37 (33%), 8 (7.2%) and 11 (9.8%) cases, respectively, PTEN was lost in 21 (19.8%) cases, AREG and EREG were overexpressed in 48 (45%) and 51 (49%) cases. In the whole study population, time to tumor progression (TTP) and overall survival (OS) was significantly lower in patients with KRAS (p = 0.001 and p = 0.026, respectively) or BRAF (p = 0.001 and p<0.0001, respectively) mutant tumors, downregulation of AREG (p = 0.018 and p = 0.013, respectively) or EREG (p = 0.002 and p = 0.004, respectively) and grade 0-1 skin rash (p<0.0001 and p<0.0001, respectively). In KRAS wt patients TTP and OS was significantly lower in patients with BRAF (p = 0.0001 and p<0.0001, respectively) mutant tumors, downregulation of AREG (p = 0.021 and p = 0.004, respectively) or EREG (p = 0.0001 and p<0.0001, respectively) and grade 0-1 skin rash (p<0.0001 and p<0.0001, respectively). TTP was significantly lower in patients with PIK3CA mutations (p = 0.01) or lost PTEN (p = 0.002). Multivariate analysis revealed KRAS (Hazard Ratio [HR] 4.3, p<0.0001), BRAF mutation (HR: 5.1, p<0.0001), EREG low expression (HR: 1.6, p = 0.021) and absence of severe/moderate skin rash (HR: 4.0, p<0.0001) as independent prognostic factors for decreased TTP. Similarly, KRAS (HR 2.9, p = 0.01), BRAF mutation (HR: 3.0, p = 0.001), EREG low expression (HR: 1.7, p = 0.021), absecence of severe/moderate skin rash (HR: 3.7, p<0.0001) and the presence of undifferantited tumours (HR: 2.2, p = 0.001) were revealed as independent prognostic factors for decreased OS.

Conclusions

These results underscore that KRAS-BRAF mutations and EREG expression can be used as biomarkers to further select patients undergoing anti-EGFR treatment.     

Encoding of spatio-temporal input characteristics by a CA1 pyramidal neuron model

The in vivo activity of CA1 pyramidal neurons alternates between regular spiking and bursting, but how these changes affect information processing remains unclear. Using a detailed CA1 pyramidal neuron model, we investigate how timing and spatial arrangement variations in synaptic inputs to the distal and proximal dendritic layers influence the information content of model responses. We find that the temporal delay between activation of the two layers acts as a switch between excitability modes: short delays induce bursting while long delays decrease firing. For long delays, the average firing frequency of the model response discriminates spatially clustered from diffused inputs to the distal dendritic tree. For short delays, the onset latency and inter-spike-interval succession of model responses can accurately classify input signals as temporally close or distant and spatially clustered or diffused across different stimulation protocols. These findings suggest that a CA1 pyramidal neuron may be capable of encoding and transmitting presynaptic spatiotemporal information about the activity of the entorhinal cortex-hippocampal network to higher brain regions via the selective use of either a temporal or a rate code.     

Mild stretch activates cPLA2 in alveolar type II epithelial cells independently through the MEK/ERK and PI3K pathways

Alveolar epithelial type II cells (AT II) in which lung surfactant synthesis and secretion take place, are subjected to low magnitude stretch during normal breathing. The aim of the study was to explore the effect of mild stretch on phospholipase A(2) (PLA(2)) activation, an enzyme known to be involved in surfactant secretion. In A549 cells (a model of AT II cells), we showed, using a fluorometric assay, that stretch triggers an increase of total PLA(2) activity. Western blot experiments revealed that the cytosolic isoform cPLA(2) is rapidly phosphorylated under stretch, in addition to a modest increase in cPLA(2) mRNA levels. Treatment of A549 cells with selective inhibitors of the MEK/ERK pathway significantly attenuated the stretch-induced cPLA(2) phosphorylation. A strong interaction of cPLA(2) and pERK enzymes was demonstrated by immunoprecipitation. We also found that inhibition of PI3K pathway attenuated cPLA(2) activation after stretch, without affecting pERK levels. Our results suggest that low magnitude stretch can induce cPLA(2) phosphorylation through the MEK/ERK and PI3K-Akt pathways, independently.