Determination of protein concentration in downstream biomanufacturing processes by in-line index of refraction

Protein concentration determination is a necessary in-process control for the downstream operations within biomanufacturing. As production transitions from batch mode to an integrated continuous bioprocess paradigm, there is a growing need to move protein concentration quantitation from off-line to in-line analysis. One solution to fulfill this process analytical technology need is an in-line index of refraction (IoR) sensor to measure protein concentration in real time. Here the performance of an IoR sensor is evaluated through a series of experiments to assess linear response, buffer matrix effects, dynamic range, sensor-to-sensor variability, and the limits of detection and quantitation. The performance of the sensor was also tested in two bioprocessing scenarios, ultrafiltration and capture chromatography. The implementation of this in-line IoR sensor for real-time protein concentration analysis and monitoring has the potential to improve continuous bioprocess manufacturing.     

The citrus plant pathogen Xanthomonas citri has a dual polyamine-binding protein

ATP-Binding Cassette transporters (ABC transporters) are protein complexes involved in the import and export of different molecules, including ions, sugars, peptides, drugs, and others. Due to the diversity of substrates, they have large relevance in physiological processes such as virulence, pathogenesis, and antimicrobial resistance. In Xanthomonas citri subsp. citri, the phytopathogen responsible for the citrus canker disease, 20% of ABC transporters components are expressed under infection conditions, including the putative putrescine/polyamine ABC transporter, PotFGHI. Polyamines are ubiquitous molecules that mediate cell growth and proliferation and play important role in bacterial infections. In this work, we characterized the X. citri periplasmic-binding protein PotF (XAC2476) using bioinformatics, biophysical and structural methods. PotF is highly conserved in Xanthomonas sp. genus, and we showed it is part of a set of proteins related to the import and assimilation of polyamines in X. citri. The interaction of PotF with putrescine and spermidine was direct and indirectly shown through fluorescence spectroscopy analyses, and experiments of circular dichroism (CD) and small-angle X-ray scattering (SAXS), respectively. The protein showed higher affinity for spermidine than putrescine, but both ligands induced structural changes that coincided with the closing of the domains and increasing of thermal stability.     

Reduced impact of ocean acidification on growth and swimming performance of newly hatched tropical sharks (Chiloscyllium plagiosum)

ABSTRACT

Sharks have been facing unprecedented pressure over the last decades, and ocean acidification may represent an additional threat, particularly during their most susceptible life stages. Hence, the present study aimed to investigate the effects of ocean acidification (control pCO₂ ~ 400 μatm; high pCO₂ ~ 900 μatm) on the growth, swimming performance and cholinergic system of juvenile white-spotted bamboo sharks (Chiloscyllium plagiosum). After 45 days of exposure, we observed that high CO₂ did not affect most of the end-points studied. However, somatic growth rate and the percentage of time that sharks spent swimming was significantly reduced under high CO₂ conditions. Moreover, AChE activity decreased in two of the seven brain macroareas analyzed, the telencephalon and optic lobes. As this near-threatened shark species showed small sub-lethal effects to high CO₂ levels, we argue that within a longer time-frame they can potentially reduce individual performance with cascading consequences to shark population dynamics.     

Iron mobilization by succinylacetone methyl ester in rats. A model study for hereditary tyrosinemia and porphyrias characterized by 5-Aminolevulinic acid overload

Accumulation of 5-aminolevulinic acid (ALA) is an event characteristic of porphyrias that may contribute to their pathological manifestations. To investigate effects of ALA independent of porphyrin accumulation we treated rats with the methyl ester of succinylacetone, an inhibitor of 5-aminolevulinic acid dehydratase that accumulates in the porphyric-like syndrome hereditary tyrosinemia. Acute 2-day treatment of fasted rats with succinylacetone methyl ester (SAME) promoted a 27% increase in plasma ALA. This increase in plasma ALA was accompanied by augmentation of the level of total nonheme iron in liver (37%) and brain (20%). Mobilization of iron was also indicated by 49% increase in plasma iron and a 77% increase in plasma transferrin saturation. Liver responded with a mild (12%) increase in ferritin. Under these acute conditions, some indications of oxidative stress were evident: a 15% increase in liver reactive protein carbonyls, and a 42% increase in brain subcellular membrane TBARS. Brain also showed a 44% increase in CuZnSOD activity, consistent with observations in treatment with ALA. Overall, the data indicate that SAME promotes ALA-driven changes in iron metabolism that could lead to increased production of free radicals. The findings support other evidence that accumulation of ALA in porphyrias and hereditary tyrosinemia may induce iron-dependent biological damage that contributes to neuropathy and hepatoma.     

The Cytotoxic and Proapoptotic Activities of Hypnophilin are Associated with Calcium Signaling in UACC‐62 Cells

Hypnophilin (HNP) is a sesquiterpene that is isolated from Lentinus cf. strigosus and has cytotoxic activities. Here, we studied the calcium signaling and cytotoxic effects of HNP in UACC‐62 cells, a human skin melanoma cell line. HNP was able to increase the intracellular calcium concentration in UACC‐62 cells, which was blocked in cells stimulated in Ca²⁺‐free media. HNP treatment with BAPTA‐AM, an intracellular Ca²⁺ chelator, caused an increase in calcium signals. HNP showed cytotoxicity against UACC‐62 cells in which it induced DNA fragmentation and morphological alterations, including changes in the nuclear chromatin profile and increased cytoplasmatic vacuolization, but it had no effect on the plasma membrane integrity. These data suggest that cytotoxicity in UACC‐62 cells, after treatment with HNP, is associated with Ca²⁺ influx. Together, these findings suggest that HNP is a relevant tool for the further investigation of new anticancer approaches. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:479‐485, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21507     

Vie di ossidazione della frazione extramitocondriale dei coenzimi piridinici: I. - Sull'Efficienza In Vivo del sistema ascorbato/deidroascorbato

Abstract

OXIDATION PATHWAYS OF EXTRAMITOCHONDRIAL PIRIDINE COENZYMES. I. - ON THE « IN VIVO » EFFICIENCY OF THE ASCORBATE-DEYHDROASCORBATE SYSTEM. — An evaluation of the efficiency in vivo of the AA-DHA couple as an electron carrier system has been attempted, by measuring after short time of anaerobiosis the rate of the increase of AA and of the dicrease of DHA in etiolated pea internode segments and in potato tuber disks. The changes of reduced glutathione (GSH) contents as induced by anaerobiosis or by the addition of DHA to the incubation medium were also followed.

In the pea segments anaerobiosis induced a significant increase of AA and a corresponding decrease of DHA. These changes were almost completed after 10 minutes from starting anaerobiosis. The value (extrapolated to 0 time) of the initial rate of DHA desappearance under anaerobiosis was taken as representing the rate of DHA reduction to AA « in vivo », under aerobic conditions. As this rate — in a steady state situation — corresponds to that of the inverse process of oxidation of AA to DHA, this value should give and indication on the « in vivo » efficiency of the AA-DHA system as an electron carrier in respiration. As some AA was probably reoxidized to DHA in the very short period required to kill the tissue, the value of the AA DHA turnover thus calculated is probably somewhat lower than the real one.

According to the present work, the oxidative turnover of the AA-DHA system would results of 0,7 micromoles/g. fr. weght/h. for the pea internode tissues and of 0,9 micromoles/g. fr. weght^?h for the potato tuber (aged disks). These values would account for 5% of total oxygen uptake, in the former, and for 3% in the latter material.

The very high AA/DHA ratio usually prevailing in living cells suggests that the contents in DHA (and thus the activity of the AA oxidizing systems) is a limiting factor for the efficiency of the AA-DHA system as an electron carrier. This view is supported also by experiments in which DHA (at pH 5) was fed to pea internode segments and to potato tuber disks : as the presence of DHA into the medium induced — under anaerobiotic conditions — a rapid increase of the level of AA in both types of materials. In aerobiosis uptake and reduction of DHA to AA was evident in the potato tuber tissue, while it appeared very scarce in the pea internodes. As an interpretation of this behaviour it is suggested that, in aerobiosis, the very active and probably surface localized ascorbic acid oxidase of the pea tissue re-oxidises the AA formed from reduction of the DHA fed; an accumulation of DHA into the cells would follow, and this excess of DHA would inhibit the enzyme GSH-DHA reductase. This enzyme, in fact, appears, from « in vitro » experiments, to be strongly inhibited by DHA when the DHA/GSH ratio becomes higher than 1. On the other hand, the same hypothesis is also supported by the finding that the addition of DHA to the medium induces a significant drop in the GSH level (probably due to its oxidation to GSSG) only under those conditions in which DHA is absorbed and reduced to AA; that is, in the pea internodes, under anaerobiosis, and in the potato disks, under both anaerobiosis and aerobiosis. These results are also taken as confirming the indication from the enzymatic data that GSH is acting, in vivo as a reducing agent for DHA. The results of this investigation are thus interpred as showing that a comparatively small, but by no means negligeable fraction of respiration is mediated, in higher plant tissues such as those of the pea stem and the potato tuber, by and electron transfer system including glutathione and the ascorbate-dehydroascorbate couple. The efficiency of this system in the materials investigated appears to account for 3–5% of the total 0₂ uptake (minimum value). As enzyme systems transferring electrons from TPNH to ox. glutathione are widely distributed and generally very active in higher plant tissues, it is suggested that the sequence TPNH-GSH-AA/DHA - O₂ is probably of considerable importance in mediating the reoxidation of extramitochondrial trophosphoridine nucleotide and thus in permitting the operation of the TPN requiring pentose phosphate pathway of respiration.     

Peptide inhibitors against herpes simplex virus infections

Herpes simplex virus (HSV) is a significant human pathogen causing mucocutaneous lesions primarily in the oral or genital mucosa. Although acyclovir (ACV) and related nucleoside analogs provide successful treatment, HSV remains highly prevalent worldwide and is a major cofactor for the spread of human immunodeficiency virus. Encephalitis, meningitis, and blinding keratitis are among the most severe diseases caused by HSV. ACV resistance poses an important problem for immunocompromised patients and highlights the need for new safe and effective agents; therefore, the development of novel strategies to eradicate HSV is a global public health priority. Despite the continued global epidemic of HSV and extensive research, there have been few major breakthroughs in the treatment or prevention of the virus since the introduction of ACV in the 1980s. A therapeutic strategy at the moment not fully addressed is the use of small peptide molecules. These can be either modeled on viral proteins or derived from antimicrobial peptides. Any peptide that interrupts protein–protein or viral protein–host cell membrane interactions is potentially a novel antiviral drug and may be a useful tool for elucidating the mechanisms of viral entry. This review summarizes current knowledge and strategies in the development of synthetic and natural peptides to inhibit HSV infectivity. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.