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.     

Relationship between ventilatory threshold and muscle fiber conduction velocity responses in trained cyclists

The relationship between surface electromyography (SEMG) amplitude and the ventilatory threshold has been extensively studied. However, previous studies of muscle fiber conduction velocity (MFCV) are scarce and present insufficient evidence concerning the relationship between MFCV and metabolic responses during cycling. Based on that fact, the purpose of this study is twofold: (1) to investigate the existence of a MFCV threshold (MFCVT) during cycling and (2) to verify if this possible breakpoint is correlated with the ventilatory threshold (VT) and the SEMG threshold (SEMGT). Eight trained male cyclists (age 36.0 ± 9.7 years) performed an incremental cycling test with initial workload of 150 W gradually incremented by 20 W min^?1 until the exhaustion. Gas analyses were conducted using a breath-by-breath open-circuit spirometry and SEMG were registered from vastus lateralis in each pedaling cycle with a linear array of electrodes. A bi-segmental linear regression computer algorithm was used to estimate VT, MFCVT and SEMGT respectively in the carbon dioxide production (VCO₂), MFCV and electromyography root mean square (EMG RMS) curves. The one way ANOVA for repeated measures did not reveal any significant difference among VT (77.1 ± 7.5% of VO₂max), MFCVT (80.3 ± 10.4% of VO₂max) and SEMGT (81.9 ± 11.7% of VO₂max). The Bland and Altman procedure confirmed a good concordance between SEMGT and VT (Bias = 5.5 of %VO₂max) as well as MFCVT and VT (Bias = 5.2 of %VO₂max). The present findings suggest that muscle fiber conduction velocity threshold is a valid and reliable non-invasive tool to obtain information about ventilatory threshold in trained cyclists.     

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     

Floral and vegetative morphometrics of three Platonia insignis Mart. (Clusiaceae) populations,a native tree from the Brazilian Amazon

Platonia insignis Mart. (Clusiaceae), the bacurizeiro, is a native tree species from the Brazilian Amazon forests. Three populations of P. insignis have been observed in the north-east region of the state of Maranhão that differ in flower color: the red population that produces dark pink flowers, the pink population that produces light pink flowers, and the white population with yellowish-white flowers. From multivariate statistical analysis, we aimed at characterizing such populations using morpho-anatomical leaf and flower morphology parameters. A total of 40 P. insignis individuals have been sampled in the cities of São Luís and Chapadinha. The morphological traits varied more than the anatomical traits. Area, fresh mass, and dry mass were the leaf parameters that show more variations. Platonia insignis have hypostomatic or amphihypostomatic leaves. The length of the gynoecium+the length of the nectary, the total length and the length of gynoecium were the principal components considering flower analysis. The three populations did not show significant differences nor did they group using Ward's method. Individuals from the Chapadinha and São Luís red population have been separated according to leaf and flower morphological traits, and the morphological difference between individuals may represent early stages of geographical speciation.     

Characterization and use of the total head soluble cholinesterases from mosquitofish (Gambusia holbrooki) for screening of anticholinesterase activity

Inhibition of cholinesterases (ChE) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. Different ChE isoforms may be present in the same tissue and may present distinct sensitivities towards environmental contaminants. The present work characterises the soluble ChE present in mosquitofish (Gambusia holbrooki) total head homogenates, through the use of different substrates and selective inhibitors of cholinesterasic activity. Furthermore, the effects of sodium dodecylsulphate (SDS) on the enzymatic activity were investigated, both in vivo and in vitro. These results showed that acetylcholinesterase (AChE) seemed to be the predominant form present in head homogenates of G. holbrooki, despite the inhibition by tetraisopropylpyrophosphoramide (iso-OMPA) found at high concentrations. SDS was responsible for in vitro, but not in vivo, inhibitory effects. The in vitro AChE inhibitory effects of SDS was partially prevented by the use of increasing amounts of ethanol, suggesting that the inhibition was induced by an emulsion effect, which may explain the lack of effect in vivo.     

Adenosine Inhibition of 45Ca Uptake by Synaptosomes as Function of Time of Electrical Stimulation

Abstract

The effect of adenosine on ⁴⁵Ca uptake by rat brain synaptosomes electrically stimulated was studied as function of time of stimulation (10, 30, 120 s). Inhibition of ⁴⁵Ca uptake was more evident for 120 s.     

A new genus and species of Coenosiini from Costa Rica (Diptera,Muscidae, Coenosiinae)

Palpilongus gen. n. is herein described for one species – Palpilongus bifurcus sp. n., from Costa Rica, based on male and females. The striking morphological characters of the species – palpus very long, about as long as prementum; upper calypter truncate and very short and setae of male sternite 5 bifurcated, confirm that this new species is also a new genus in the tribe Coenosiini. Male and female terminalia were dissected and illustrated.