An integrated practical implementation of continuous aqueous two‐phase systems for the recovery of human IgG: From the microdevice to a multistage bench‐scale mixer‐settler device

Aqueous two‐phase systems (ATPS) are a liquid‐liquid extraction technology with clear process benefits; however, its lack of industrial embracement is still a challenge to overcome. Antibodies are a potential product to be recovered by ATPS in a commercial context. The objective of this work is to present a more integral approach of the different isolated strategies that have arisen in order to enable a practical, generic implementation of ATPS, using human immunoglobulin G (IgG) as experimental model. A microfluidic device is used for ATPS parameters preselection for product recovery. ATPS were continuously operated in a mixer‐settler device in one stage, multistage and multistage with recirculation configuration. Single‐stage pure IgG extraction with a polyethylene glycol (PEG) 3350‐phophates ATPS within continuous operation allowed a 65% recovery. Further implementation of a multistage platform promoted a higher particle partitioning reaching a 90% recovery. The processing of IgG from a cell supernatant culture harvest in a multistage system with top phase recirculation resulted in 78% IgG recovery in bottom phase. This work conjugates three not widely spread methodologies for ATPS: microfluidics, continuous and multistage operation.     

Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form.     

Maternal separation and early stress cause long‐lasting effects on dopaminergic and endocannabinergic systems and alters dendritic morphology in the nucleus accumbens and frontal cortex in rats

A considerable amount experimental studies have shown that maternal separation (MS) is associated with adult offspring abnormal behavior and cognition disorder. Accordingly, this experimental procedure has been proposed as a predictor for alcohol and drug dependence based on the neurodevelopmental soon after birth. Endocannabinoid system (eCBs) has been implicated in reward processes, including drug abuse and dependence. MS and associated stress causes changes in the eCBs that seem to facilitate alcohol consumption. In this study, we seek to evaluate potential morphological changes in neurons of the frontal cortex (FCx) and nucleus accumbens (NAcc), in the expression of receptors and enzymes of the endocannabinoid and dopamine systems and in second messengers, such as Akt, in adult rats subjected to MS and early stress (MS + ES; 2 × 180 min daily) vs. nonseparated rats (NMS). Results showed that MS + ES induces higher D2R expression and lower D3R, FAAH, and MAGL expression compared with NMS rats. Alterations in total dendritic length were also detected and were characterized by increases in the NAcc while there were decreases in the FCx. We believe MS + ES‐induced changes in the dopaminergic and endocannabinergic systems and in the neuronal microstructure might be contributing to alcohol seeking behavior and, potential vulnerability to other drugs in rats. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 819–831, 2016     

Effects of the Infusion of 4% or 20% Human Serum Albumin on the Skeletal Muscle Microcirculation in Endotoxemic Rats

Background

Sepsis-induced microcirculatory alterations contribute to tissue hypoxia and organ dysfunction. In addition to its plasma volume expanding activity, human serum albumin (HSA) has anti-oxidant and anti-inflammatory properties and may have a protective role in the microcirculation during sepsis. The concentration of HSA infused may influence these effects. We compared the microcirculatory effects of the infusion of 4% and 20% HSA in an experimental model of sepsis.

Methods

Adult male Wistar rats were equipped with arterial and venous catheters and received an intravenous infusion of lipopolysaccharide (LPS, serotype O127:B8, 10 mg/kg over 30 minutes) or vehicle (SHAM, n = 6). Two hours later, endotoxemic animals were randomized to receive 10 mL/kg of either 4% HSA (LPS+4%HSA, n = 6), 20% HSA (LPS+20%HSA, n = 6) or 0.9% NaCl (LPS+0.9%NaCl, n = 6). No fluids were given to an additional 6 animals (LPS). Vessel density and perfusion were assessed in the skeletal muscle microcirculation with sidestream dark field videomicroscopy at baseline (t0), 2 hours after LPS injection (t1), after HSA infusion (t2) and 1 hour later (t3). The mean arterial pressure (MAP) and heart rate were recorded. Serum endothelin-1 was measured at t2.

Results

MAP was stable over time in all groups. The microcirculatory parameters were significantly altered in endotoxemic animals at t1. The infusion of both 4% and 20% HSA similarly increased the perfused vessel density and blood flow velocity and decreased the flow heterogeneity to control values. Microvascular perfusion was preserved in the LPS+20%HSA group at t3, whereas alterations reappeared in the LPS+4%HSA group.

Conclusions

In a rat model of normotensive endotoxemia, the infusion of 4% or 20% HSA produced a similar acute improvement in the microvascular perfusion in otherwise unresuscitated animals.     

The formation of titan cells in Cryptococcus neoformans depends on the mouse strain and correlates with induction of Th2‐type responses

Cryptococcus neoformans is a pathogenic yeast that can form titan cells in the lungs, which are fungal cells of abnormal enlarged size. Little is known about the factors that trigger titan cells. In particular, it is not known how the host environment influences this transition. In this work, we describe the formation of titan cells in two mouse strains, CD1 and C57BL/6J. We found that the proportion of C. neoformans titan cells was significantly higher in C57BL/6J mice than in CD1. This higher proportion of titan cells was associated with a higher dissemination of the yeasts to the brain. Histology sections demonstrated eosinophilia in infected animals, although it was significantly lower in the CD1 mice which presented infiltration of lymphocytes. Both mouse strains presented infiltration of granulocytes, but the amount of eosinophils was higher in C57BL/6J. CD1 mice showed a significant accumulation of IFN‐γ, TNF‐α and IL17, while C57BL/BL mice had an increase in the anti‐inflammatory cytokine IL‐4. IgM antibodies to the polysaccharide capsule and total IgE were more abundant in the sera from C57BL/6J, confirming that these animals present a Th2‐type response. We conclude that titan cell formation in C. neoformans depends, not only on microbe factors, but also on the host environment.     

Engineering high α‐amylase levels in wheat grain lowers Falling Number but improves baking properties

Late maturity α‐amylase (LMA) and preharvest sprouting (PHS) are genetic defects in wheat. They are both characterized by the expression of specific isoforms of α‐amylase in particular genotypes in the grain prior to harvest. The enhanced expression of α‐amylase in both LMA and PHS results in a reduction in Falling Number (FN), a test of gel viscosity, and subsequent downgrading of the grain, along with a reduced price for growers. The FN test is unable to distinguish between LMA and PHS; thus, both defects are treated similarly when grain is traded. However, in PHS‐affected grains, proteases and other degradative process are activated, and this has been shown to have a negative impact on end product quality. No studies have been conducted to determine whether LMA is detrimental to end product quality. This work demonstrated that wheat in which an isoform α‐amylase (TaAmy3) was overexpressed in the endosperm of developing grain to levels of up to 100‐fold higher than the wild‐type resulted in low FN similar to those seen in LMA‐ or PHS‐affected grains. This increase had no detrimental effect on starch structure, flour composition and enhanced baking quality, in small‐scale 10‐g baking tests. In these small‐scale tests, overexpression of TaAmy3 led to increased loaf volume and Maillard‐related browning to levels higher than those in control flours when baking improver was added. These findings raise questions as to the validity of the assumption that (i) LMA is detrimental to end product quality and (ii) a low FN is always indicative of a reduction in quality. This work suggests the need for a better understanding of the impact of elevated expression of specific α‐amylase on end product quality.     

Small interfering RNA targeting NF-κB attenuates lipopolysaccharide-induced acute lung injury in rats

Background

Veterinary cardiology, especially electrocardiography, has shown major advancements for all animal species. Consequently, the number of ovine species used as experimental animals has increased to date. Few studies have been published on ovine systematic electrocardiography, particularly with respect to lamb physiology and neonatology. This study aimed to standardize the values of normal waves, complexes, and intervals of the electrocardiogram (ECG) in clinically Bergamasca healthy neonatal lambs, used as experimental animals. Serial computerized electrocardiography was performed in 10 male and 12 female neonates on the 1st, 7th, 14th, 21st, 28th, and 35th days of age. The following parameters were analyzed: heart rate and rhythm, duration and amplitude of waves, duration of intervals, and heart electrical axis.

Results

During the first 35 days of life, (1) the sinusal heart rhythm was predominant, (2) there was a progressive decrease in the heart rate and R and T wave amplitude, and (3) a progressive increase in the PR, QT, and RR intervals. Finally, we confirmed that various components of neonatal evolution were more discernible in the augmented unipolar leads (aVF), which we recommend should be preferentially used in future studies. No significant statistical alterations were observed between males and females in relation to the analyzed parameters.

Conclusions

The information assimilated in this study is anticipated to enhance the diagnosis of multiple congenital heart defects in Bergamasca lambs and could be implemented in studies that use ovine species as experimental models.

     

Model-based strategy for cell culture seed train layout verified at lab scale

Cell culture seed trains—the generation of a sufficient viable cell number for the inoculation of the production scale bioreactor, starting from incubator scale—are time- and cost-intensive. Accordingly, a seed train offers potential for optimization regarding its layout and the corresponding proceedings. A tool has been developed to determine the optimal points in time for cell passaging from one scale into the next and it has been applied to two different cell lines at lab scale, AGE1.HN_AAT and CHO-K1. For evaluation, experimental seed train realization has been evaluated in comparison to its layout. In case of the AGE1.HN_AAT cell line, the results have also been compared to the formerly manually designed seed train. The tool provides the same seed train layout based on the data of only two batches.