Nonlinear state estimation as tool for online monitoring and adaptive feed in high throughput cultivations

Robotic facilities that can perform advanced cultivations (e.g., fed-batch or continuous) in high throughput have drastically increased the speed and reliability of the bioprocess development pipeline. Still, developing reliable analytical technologies, that can cope with the throughput of the cultivation system, has proven to be very challenging. On the one hand, the analytical accuracy suffers from the low sampling volumes, and on the other hand, the number of samples that must be treated rapidly is very large. These issues have been a major limitation for the implementation of feedback control methods in miniaturized bioreactor systems, where observations of the process states are typically obtained after the experiment has finished. In this work, we implement a Sigma-Point Kalman Filter in a high throughput platform with 24 parallel experiments at the mL-scale to demonstrate its viability and added value in high throughput experiments. The filter exploits the information generated by the ammonia-based pH control to enable the continuous estimation of the biomass concentration, a critical state to monitor the specific rates of production and consumption in the process. The objective in the selected case study is to ensure that the selected specific substrate consumption rate is tightly controlled throughout the complete Escherichia coli cultivations for recombinant production of an antibody fragment.     

Ventral medullary pH and ventilatory responses to hyperperfusion and hypoxia

The role of a sudden increase in brain perfusion on ventral medullary surface pH (Vm pH) and minute ventilation (VI) was assessed in anesthetized peripherally chemo denervated cats. Acute hypertension (AH), produced by rapid inflation of an aortic balloon, and hypoxemia, produced with either inhalation of 1% CO (COHx) or inhalation of a hypoxic gas (HHx), were used to increase brain blood flow. In the AH group, increasing arterial blood pressure (from 122 +/- 3 to 180 +/- 5 mmHg) caused a rapid (less than 5 s) increase in Vm pH in every trial (n = 18). Associated with the mean peak increases in Vm pH (0.003 +/- 0.0004 pH units) were significant decrease in tidal volume (7-9%). In the COHx group, 17% HbCO caused a significant increase in Vm pH (0.003 +/- 0.0005 pH unit) and diminution of VI (9%). Further increases in HbCO caused a progressive ventral medullary acidosis and greater reductions in VI. The results from the HHX group were qualitatively similar to the COHx group; there was a biphasic response of Vm pH, i.e., an initial increase in Vm pH (0.008 +/- 0.001) followed by a steady decrease in Vm pH, with reductions in VI associated with both phases. We conclude that hyperperfusion, per se, produces an increase in Vm pH and a reduction in VI equivalent in magnitude to that predicted from the CO2 stimulus-response curve; the alkalotic shift in Vm pH and concomitant diminution in VI associated with mild hypoxia is probably related to an increase in ventral medullary perfusion; and the ventilatory depression associated with the medullary acidosis of moderate brain hypoxia must be attributed to another mechanism.     

A model-based framework for parallel scale-down fed-batch cultivations in mini-bioreactors for accelerated phenotyping

Concentration gradients that occur in large industrial-scale bioreactors due to mass transfer limitations have significant effects on process efficiency. Hence, it is desirable to investigate the response of strains to such heterogeneities to reduce the risk of failure during process scale-up. Although there are various scale-down techniques to study these effects, scale-down strategies are rarely applied in the early developmental phases of a bioprocess, as they have not yet been implemented on small-scale parallel cultivation devices. In this study, we combine mechanistic growth models with a parallel mini-bioreactor system to create a high-throughput platform for studying the response of Escherichia coli strains to concentration gradients. As a scaled-down approach, a model-based glucose pulse feeding scheme is applied and compared with a continuous feed profile to study the influence of glucose and dissolved oxygen gradients on both cell physiology and incorporation of noncanonical amino acids into recombinant proinsulin. The results show a significant increase in the incorporation of the noncanonical amino acid norvaline in the soluble intracellular extract and in the recombinant product in cultures with glucose/oxygen oscillations. Interestingly, the amount of norvaline depends on the pulse frequency and is negligible with continuous feeding, confirming observations from large-scale cultivations. Most importantly, the results also show that a larger number of the model parameters are significantly affected by the scale-down scheme, compared with the reference cultivations. In this example, it was possible to describe the effects of oscillations in a single parallel experiment. The platform offers the opportunity to combine strain screening with scale-down studies to select the most robust strains for bioprocess scale-up.     

Hydrocarbon Stapled Antimicrobial Peptides

Antimicrobial peptides are promising candidates for anti-infective pharmaceuticals. Unfortunately, because of their low proteolytic and chemical stability, their usage is generally narrowed down to topical formulations. Until now, numerous approaches to increase peptide stability have been proposed. One of them, peptide hydrocarbon stapling, a modification based on stabilizing peptide secondary structure with a side-chain covalent hydrocarbon bridge, have been successfully applied to many peptides. Moreover, constraining secondary structure of peptides have also been proven to increase their biological activity. This review article describes studies on hydrocarbon stapled antimicrobial peptides with respect to improved drug-like properties.     

Naloxone reduces ventilatory depression of brain hypoxia

To assess whether endogenous opioids participate in respiratory depression due to brain hypoxia, we determined the ventilatory response to progressive carboxyhemoglobinemia (1% CO, 40% O2) before and after administration of naloxone (NLX, 0.1 mg/kg iv). Minute ventilation (VI) and ventral medullary surface pH (Vm pH) were measured in six anesthetized, peripherally chemodenervated cats. NLX consistently increased base-line hyperoxic VI from 618 +/- 99 to 729 +/- 126 ml/min (P less than 0.05). Although NLX did not alter the Vm pH response to CO [initial alkalosis, Vm pH +0.011 +/- 0.003 pH units, followed by acidosis, Vm pH -0.082 +/- 0.036 at carboxyhemoglobin (HbCO) 55%], NLX attenuated the amount of ventilatory depression; increasing HbCO to 55% decreased VI to 66 +/- 6% of base line before NLX and to 81 +/- 9% of base line after NLX (P less than 0.05). The difference in response after NLX was primarily the result of a linear increase in tidal volume (VT) with decreasing Vm pH (delta VT = 60.3 ml/-pH unit) which was absent before NLX. To assess whether the site of action of the endogenous opioid effect was the central chemosensors, the ventilatory and Vm pH response to progressive HbCO was determined in three additional cats before and after topical application of NLX (3 X 10(-4) M) to the ventral medullary surface. The effect of topical NLX was similar to systemic NLX; significant attenuation of the reduction in VI with increasing HbCO. We conclude that 1) endogenous opioids mediate a portion of the depression of ventilation due to acute brain hypoxia, and 2) this effect is probably at the central chemosensitive regions.     

Correction to: Moving Beyond Global Warming Potentials to Quantify the Climatic Role of Ecosystems

Ecosystems - In our 2015 paper, we dropped the e(− dt/τ) term from Equations (1) and (2) when an ecosystem removed CH4 or N2O from the atmosphere, reasoning that greenhouse gases...     

Neutral salt effects on the velocity and activation volume of the lactate dehydrogenase reaction: Evidence for enzyme hydration changes during catalysis

The effects of different neutral salts on the maximal velocity (V) and activation volume ($\text{Δ}\text{V3}$) of the M₄-lactate dehydrogenase reaction were studied to determine the mechanistic basis of the inhibitory effects of these salts. For salting-in salts (which increase protein group solubility), increasing salt concentrations led to reductions in V and increases in $\text{Δ}\text{V3}$, with the order of salt effectiveness following the Hofmeister (lyotropic) series: KSCN > KI > KBr. A 50% reduction in V was associated with an approximately 17 cm³ mol^?1 increase in $\text{Δ}\text{V3}$ for different concentrations of the same salt and for equal concentrations of different salting-in salts. Salting-out salts were also inhibitory, but no uniform correlation between changes in V and $\text{Δ}\text{V3}$ was observed. The strongly salting-out salt KF decreased $\text{Δ}\text{V3}$ at all concentrations. The weaker salting-out salt K₂SO₄ increased $\text{Δ}\text{V3}$ at concentrations below 0.1 m and decreased $\text{Δ}\text{V3}$ at higher concentrations. KCl increased $\text{Δ}\text{V3}$ as the salt concentration was raised to approximately 0.2 m; further increases in KCl concentration were without effect on $\text{Δ}\text{V3}$. The rate and volume effects of these neutral salts, especially the highly regular covariation in V and $\text{Δ}\text{V3}$ found for salting-in salts, seem difficult to explain in terms of salt-induced changes in the geometry of the active site. We propose instead that these salt effects can all be explained in terms of the energy and volume changes which accompany transfers of protein groups (amino acid side chains and peptide backbone linkages) between the hydrophobic interior of the enzyme and the enzyme-water interface during catalytic conformational changes.     

Changes in creatine transporter function during cardiac maturation in the rat

Background  

It is well established that the immature myocardium preferentially utilises non-oxidative energy-generating pathways. It exhibits low energy-transfer capacity via the creatine kinase (CK) shuttle, reflected in phosphocreatine (PCr), total creatine and CK levels that are much lower than those of adult myocardium. The mechanisms leading to gradually increasing energy transfer capacity during maturation are poorly understood. Creatine is not synthesised in the heart, but taken up exclusively by the action of the creatine transporter protein (CrT). To determine whether this transporter is ontogenically regulated, the present study serially examined CrT gene expression pattern, together with creatine uptake kinetics and resulting myocardial creatine levels, in rats over the first 80 days of age.     

Role of the Guanine Nucleotide Exchange Factor Rom2 in Cell Wall Integrity Maintenance of Aspergillus fumigatus

Aspergillus fumigatus is a mold and the causal agent of invasive aspergillosis, a systemic disease with high lethality. Recently, we identified and functionally characterized three stress sensors implicated in the cell wall integrity (CWI) signaling of this pathogen, namely, Wsc1, Wsc3, and MidA. Here, we functionally characterize Rom2, a guanine nucleotide exchange factor with essential function for the cell wall integrity of A. fumigatus. A conditional rom2 mutant has severe growth defects under repressive conditions and incorporates all phenotypes of the three cell wall integrity sensor mutants, e.g., the echinocandin sensitivity of the Δwsc1 mutant and the Congo red, calcofluor white, and heat sensitivity of the ΔmidA mutant. Rom2 interacts with Rho1 and shows a similar intracellular distribution focused at the hyphal tips. Our results place Rom2 between the cell surface stress sensors Wsc1, Wsc3, MidA, and Rho1 and their downstream effector mitogen-activated protein (MAP) kinase module Bck1-Mkk2-MpkA.