Smart process development: Application of machine-learning and integrated process modeling for inclusion body purification processes

The development of a biopharmaceutical production process usually occurs sequentially, and tedious optimization of each individual unit operation is very time-consuming. Here, the conditions established as optimal for one-step serve as input for the following step. Yet, this strategy does not consider potential interactions between a priori distant process steps and therefore cannot guarantee for optimal overall process performance. To overcome these limitations, we established a smart approach to develop and utilize integrated process models using machine learning techniques and genetic algorithms. We evaluated the application of the data-driven models to explore potential efficiency increases and compared them to a conventional development approach for one of our development products. First, we developed a data-driven integrated process model using gradient boosting machines and Gaussian processes as machine learning techniques and a genetic algorithm as recommendation engine for two downstream unit operations, namely solubilization and refolding. Through projection of the results into our large-scale facility, we predicted a twofold increase in productivity. Second, we extended the model to a three-step model by including the capture chromatography. Here, depending on the selected baseline-process chosen for comparison, we obtained between 50% and 100% increase in productivity. These data show the successful application of machine learning techniques and optimization algorithms for downstream process development. Finally, our results highlight the importance of considering integrated process models for the whole process chain, including all unit operations.     

Five batches representative of Ontario-grown American ginseng root produce comparable reductions of postprandial glycemia in healthy individuals

Evidence indicates that the glycemia-lowering effect of American ginseng root may be batch dependent. We therefore evaluated the effect of 5 root batches, representative of Ontario-grown American ginseng, on postprandial glucose and insulin indices. Twelve healthy subjects (5 male, 7 female), mean +/- SE age 26.5 +/- 2 years, body mass index 23.96 +/- 3.41 kg/m2, fasting blood glucose 4.77 +/- 0.04 mmol/L, were assigned to consume 9 g of American ginseng from 5 farms (A-E), administered in randomized sequence on 5 separate visits, and a water-control during the 6th and last visit. Treatments were consumed 40 min before a 2-hour 75-gram oral glucose tolerance test. Plasma glucose and insulin were measured at baseline, before, and during the test. Compared with control, batches A and C reduced glucose incremental area under the curve (IAUC) by 35.2% (156 vs. 240 mmol.min/L) and 32.6% (162 vs. 240 mmol.min/L), respectively. Batches A, C, and E reduced incremental peak glucose by 1.3, 1.2, and 1.1 mmol/L, respectively. Batch C reduced the insulin IAUC by 27.7% (15.8 vs. 21.8 nmol.min/L). Effects on glucose and insulin parameters were not different across ginseng treatments. The mean of the 5 ginseng treatments reduced peak postprandial glucose by 1.0 mmol/L, glucose IAUC by 27.7% (173 vs. 240 mmol.min/L), and insulin IAUC by 23.8% (16.6 vs. 21.8 nmol.min/L) relative to control. (All results statistically significant at p < 0.05.) American ginseng decreased postprandial glycemia and insulinemia; however, 40% of the batches did not reduce glycemia with the anticipated magnitude, irrespective of their saponin composition.     

The relative importance of different direct benefits in the mate choices of a field cricket

Discussions about the evolution of female mating preferences have often suggested that females should express multiple strong preferences when different male traits are correlated with different mating benefits, yet few studies have directly tested this hypothesis by comparing the strength of female preferences for male traits known to be correlated with different benefits. In the variable field cricket, Gryllus lineaticeps, females receive fecundity and fertility benefits from mating with males with higher chirp rates and life-span benefits from mating with males with longer chirp durations. Although females prefer higher chirp rates and longer chirp durations when the other trait is held constant, it is possible that they give priority to one of these song traits when both vary. In this study, we examined the relative importance of chirp rate and chirp duration in female mate choice using single-stimulus presentations of songs that varied in both chirp rate and chirp duration. Females expressed both directional and stabilizing preferences based on chirp rate, responding most strongly to a chirp rate approximately one standard deviation above the population mean. Females did not express preferences based on chirp duration, and did not express correlational preferences. These results suggest that females may give priority to the reproductive benefits provided by males that produce higher chirp rates.     

Early energy deficit in Huntington disease: identification of a plasma biomarker traceable during disease progression

Huntington disease (HD) is a fatal neurodegenerative disorder, with no effective treatment. The pathogenic mechanisms underlying HD has not been elucidated, but weight loss, associated with chorea and cognitive decline, is a characteristic feature of the disease that is accessible to investigation. We, therefore, performed a multiparametric study exploring body weight and the mechanisms of its loss in 32 presymptomatic carriers and HD patients in the early stages of the disease, compared to 21 controls. We combined this study with a multivariate statistical analysis of plasma components quantified by proton nuclear magnetic resonance ((1)H NMR) spectroscopy. We report evidence of an early hypermetabolic state in HD. Weight loss was observed in the HD group even in presymptomatic carriers, although their caloric intake was higher than that of controls. Inflammatory processes and primary hormonal dysfunction were excluded. (1)H NMR spectroscopy on plasma did, however, distinguish HD patients at different stages of the disease and presymptomatic carriers from controls. This distinction was attributable to low levels of the branched chain amino acids (BCAA), valine, leucine and isoleucine. BCAA levels were correlated with weight loss and, importantly, with disease progression and abnormal triplet repeat expansion size in the HD1 gene. Levels of IGF1, which is regulated by BCAA, were also significantly lower in the HD group. Therefore, early weight loss in HD is associated with a systemic metabolic defect, and BCAA levels may be used as a biomarker, indicative of disease onset and early progression. The decreased plasma levels of BCAA may correspond to a critical need for Krebs cycle energy substrates in the brain that increased metabolism in the periphery is trying to provide.     

False promises: females spurn cheating males in a field cricket

Females commonly prefer to mate with males that provide greater material benefits, which they often select using correlated male signals. When females select higher-benefit males based on correlated signals, however, males can potentially deceive females by producing exaggerated signals of benefit quality. The handicap mechanism can prevent lower-quality males from producing exaggerated signals, but cannot prevent cheating by higher-quality males that choose to withhold the benefit, and this poses a major problem for the evolution of female choice based on direct benefits. In a field cricket, Gryllus lineaticeps, females receive seminal fluid products from males with preferred songs that increase their fecundity and lifespan. We tested the hypothesis that female behaviour penalizes males that provide lower-quality benefits. When females were paired with males that varied in benefit quality but had experimentally imposed average songs, they were less likely to re-mate with males that provided lower-quality benefits in the initial mating. This type of conditional female re-mating may be a widespread mechanism that penalizes males that cheat on direct benefits.     

Purification and analysis of a 5kDa component of enamel matrix derivative

High performance liquid chromatography (HPLC) methods were used to analyse a 5kDa component purified from enamel matrix derivative (EMD), the active ingredient in Emdogain, a commercial product for periodontal tissue regeneration. After initial purification by size-exclusion chromatography (SEC) on a 100 cm x 5 cm column (Bio-Gel P-30 Fine, 280 nm), collected fractions were analysed by size-exclusion HPLC (SE HPLC; TSK-Gel Super SW2000, 220 nm). The fractions containing only the 5kDa component were analysed by reversed-phase high-pressure chromatography (RP HPLC; YMC-Pack ODS-A, 200 nm), revealing four peaks of the 5kDa component. From 1200 mg of EMD (of which 9% is the 5kDa component), approximately 65 mg of lyophilised 5kDa component were obtained, corresponding to a recovery of 60%. The SE HPLC method was mainly suitable for qualitative analysis, whereas the RP HPLC method was appropriate for both qualitative and quantitative analysis.     

Identification of DNA motifs implicated in maintenance of bacterial core genomes by predictive modeling

Bacterial biodiversity at the species level, in terms of gene acquisition or loss, is so immense that it raises the question of how essential chromosomal regions are spared from uncontrolled rearrangements. Protection of the genome likely depends on specific DNA motifs that impose limits on the regions that undergo recombination. Although most such motifs remain unidentified, they are theoretically predictable based on their genomic distribution properties. We examined the distribution of the “crossover hotspot instigator,” or Chi, in Escherichia coli, and found that its exceptional distribution is restricted to the core genome common to three strains. We then formulated a set of criteria that were incorporated in a statistical model to search core genomes for motifs potentially involved in genome stability in other species. Our strategy led us to identify and biologically validate two distinct heptamers that possess Chi properties, one in Staphylococcus aureus, and the other in several streptococci. This strategy paves the way for wide-scale discovery of other important functional noncoding motifs that distinguish core genomes from the strain-variable regions.