Carbon concentration and oxygen availability affect lipid and carotenoid production by carob pulp syrup‐grown Rhodosporidium toruloides NCYC 921

The simultaneous effect of oxygen availability and carbon source concentration on yeast lipid and carotenoid production has never been studied before. In this work, a Doehlert distribution design was used to study the simultaneous effect of carbon concentration and oxygen availability on Rhodosporidium toruloides NCYC 921 carotenoid and lipid production. A cheap industrial byproduct was used as carbon source (carob pulp syrup). A total sugar concentration of 106.3 g/L and a medium volume of 0.120 L induced the highest total carotenoid and total fatty acid productivities (4.60 μg/Lh and 0.029 g/Lh, respectively). Flow cytometry was used to assess yeast stress response under different cultivation conditions. The highest proportion of cells with permeabilised membrane (>20%) was induced when the cultivations were carried out at the highest sugar concentration studied (130.0 g/L) or when the culture reached the minimum final medium pH (4.60). The results showed that the total sugar concentration had a positive influence on the yeast biomass and carotenoid content, while the oxygen availability had little influence on the biomass concentration, but had a slight positive influence on the carotenoid content. Regarding the fatty acids, the two factors had a negative impact on the synthesis of these compounds.     

Toxocara canis: Potential activity of natural products against second-stage larvae in vitro and in vivo

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox® was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox® was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox®, the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo.     

Modulation of bone marrow mesenchymal stem cell secretome by ECM-like hydrogels

It has been demonstrated that bone marrow mesenchymal stem cell (BM-MSCs) transplantation has beneficial effects on several central nervous system (CNS) debilitating conditions. Growing evidence indicate that trophic factors secreted by these cells are the key mechanism by which they are acting. These cells are frequently used in combination with 3D artificial matrices, for instance hydrogels, in tissue engineering-based approaches. However, so far, no study has been reported on the influence of such matrices, namely the presence or absence of extracellular matrix motifs, on BM-MSCs secretome and its effects in neuronal cell populations. In this sense, we herein studied the impact of a hydrogel, gellan gum, on the behavior and secretome of BM-MSCs, both in its commercial available form (commonly used in tissue engineering) and in a fibronectin peptide-modified form. The results showed that in the presence of a peptide in the gellan gum hydrogel, BM-MSCs presented higher proliferation and metabolic activity than in the regular hydrogel. Moreover, the typical spindle shape morphology of BM-MSCs was only observed in the modified hydrogel. The effects of the secretome of BM-MSCs were also affected by the chemical nature of the extracellular matrix. BM-MSCs cultured in the modified hydrogel were able to secrete factors that induced higher metabolic viabilities and neuronal cell densities, when compared to those of the unmodified hydrogel. Thus adding a peptide sequence to the gellan gum had a significant effect on the morphology, activity, proliferation and secretome of BM-MSCs. These results highlight the importance of mimicking the extracellular matrix when BM-MSCs are cultured in hydrogels for CNS applications.     

“Protein aggregates” contain RNA and DNA,entrapped by misfolded proteins but largely rescued by slowing translational elongation

All neurodegenerative diseases feature aggregates, which usually contain disease‐specific diagnostic proteins; non‐protein constituents, however, have rarely been explored. Aggregates from SY5Y‐APP_Sw neuroblastoma, a cell model of familial Alzheimer''s disease, were crosslinked and sequences of linked peptides identified. We constructed a normalized “contactome” comprising 11 subnetworks, centered on 24 high‐connectivity hubs. Remarkably, all 24 are nucleic acid‐binding proteins. This led us to isolate and sequence RNA and DNA from Alzheimer''s and control aggregates. RNA fragments were mapped to the human genome by RNA‐seq and DNA by ChIP‐seq. Nearly all aggregate RNA sequences mapped to specific genes, whereas DNA fragments were predominantly intergenic. These nucleic acid mappings are all significantly nonrandom, making an artifactual origin extremely unlikely. RNA (mostly cytoplasmic) exceeded DNA (chiefly nuclear) by twofold to fivefold. RNA fragments recovered from AD tissue were ~1.5‐to 2.5‐fold more abundant than those recovered from control tissue, similar to the increase in protein. Aggregate abundances of specific RNA sequences were strikingly differential between cultured SY5Y‐APP_Sw glioblastoma cells expressing APOE3 vs. APOE4, consistent with APOE4 competition for E‐box/CLEAR motifs. We identified many G‐quadruplex and viral sequences within RNA and DNA of aggregates, suggesting that sequestration of viral genomes may have driven the evolution of disordered nucleic acid‐binding proteins. After RNA‐interference knockdown of the translational‐procession factor EEF2 to suppress translation in SY5Y‐APP_Sw cells, the RNA content of aggregates declined by >90%, while reducing protein content by only 30% and altering DNA content by ≤10%. This implies that cotranslational misfolding of nascent proteins may ensnare polysomes into aggregates, accounting for most of their RNA content.     

A region of tissue plasminogen activator that affects plasminogen activation differentially with various fibrin(ogen)-related stimulators.

The dissolution of blood clots by plasmin is normally initiated in vivo by the activation of plasminogen to plasmin through the activity of tissue plasminogen activator (t-PA). The rate of plasminogen activation can be stimulated several orders of magnitude by the presence of fibrin-related proteins. Here we describe the kinetic analysis of both recombinant human t-PA (wild-type) and a t-PA variant produced by site-directed mutagenesis in which the original sequence from amino acids 296 to 299, KHRR, has been altered to AAAA. This tetra-alanine variant form of t-PA, K296A/H297A/R298A/R299A t-PA, we refer to as "KHRR" t-PA here. The plasminogen activating kinetics of wild-type t-PA (Activase alteplase) showed a catalytic efficiency which changed over 100-fold dependent on the stimulator in the assay. The lowest rate was in the absence of a stimulator. The following stimulators showed increasing ability to accelerate the catalytic efficiency of the reaction: fibrinogen, fragments of fibrinogen obtained by digestion with plasmin, fibrin, and slightly degraded fibrin. This increase in efficiency was driven primarily by decreases in the Michaelis constant (KM) of the reaction, whereas the catalytic rate constant (kcat) of the reaction did not change significantly. The "KHRR" variant of t-PA displayed novel kinetics with all stimulators tested. In the absence of a stimulator or with the poorer stimulators (fibrinogen and fibrinogen fragments), the KM values of the reaction with Activase alteplase and "KHRR" t-PA were similar. The kcat however, was lower with "KHRR" t-PA than with wild-type t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ulva lactuca and U. flexuosa (Chlorophyta,Ulvophyceae) cultivation in Brazilian tropical waters: recruitment,growth, and ulvan yield

Ulva spp. are used in a wide range of commercial applications, including bioremediation, food, bioenergy, pharmaceuticals, and agriculture. The sulfated polysaccharide ulvan obtained from Ulva spp. is of interest for triggering plant defenses against disease. However, the cultivation of Ulva spp. is still in its infancy. This study verified the feasibility of cultivating Ulva lactuca and Ulva flexuosa at two sites on the tropical Brazilian coast. We investigated the following: (a) methods to induce sporulation, (b) comparison of seeding ropes inoculated in vitro versus seeding at sea over 40 days, (c) production and harvest cycles at 15 and 30 days, (d) growth productivity of U. flexuosa at sea and in outdoor tanks, and (e) comparison of ulvan yields from biomass cultivated in tanks and the sea. High nutrient treatment was the most efficient method to induce sporulation (7,540?±?3,133 spores m^?1). Sea-based cultivation of U. flexuosa was only successful at one site. Seeding of ropes in vitro was more efficient than seeding at sea (0.31?±?0.20 g m^?2 day^?1), and 15-day harvest cycles were most efficient (20.1?±?1.8 % day^?1; 0.46?±?0.11 g m^?2 day^?1). Despite differences in plant growth in tanks (27.9?±?4.4 % day^?1) and at sea (20.1?±?1.8 % day^?1), the dry biomass and ulvan yields (17.7?±?5.0 %) did not differ between these systems. Cultivation of U. flexuosa was feasible at sea using in vitro seeding with a production cycle of 15 days in Brazilian tropical waters and tanks with high irradiance and enriched seawater.     

Internal virus polarization model for virus retention by the Ultipor® VF Grade DV20 membrane

Several recent studies have reported a decline in virus retention during virus challenge filtration experiments, although the mechanism(s) governing this phenomenon for different filters remains uncertain. Experiments were performed to evaluate the retention of PP7 and PR772 bacteriophage through Ultipor VF Grade DV20 virus filters during constant pressure filtration. While the larger PR772 phage was fully retained under all conditions, a 2‐log decline in retention of the small PP7 phage was observed at high throughputs, even under conditions where there was no decline in filtrate flux. In addition, prefouling the membrane with an immunoglobulin G solution had no effect on phage retention. An internal polarization model was developed to describe the decline in phage retention arising from the accumulation of phage in the upper (reservoir) layer within the filter which increases the challenge to the lower (rejection) layer. Independent support for this internal polarization phenomenon was provided by confocal microscopy of fluorescently labeled phage within the membrane. The model was in good agreement with phage retention data over a wide range of phage titers, confirming that virus retention is throughput dependent and supporting current recommendations for virus retention validation studies. These results provide important insights into the factors governing virus retention by membrane filters and their dependence on the underlying structure of the virus filter membrane. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:856–863, 2014     

Latitudinal Variation in Termite Species Richness and Abundance along the Brazilian Atlantic Forest Hotspot

In this study, we investigated the termites of the Brazilian Atlantic Forest, one of the most threatened biodiversity hotspots in the world, in regularly spaced sites from 7° S to 27° S latitude. To our knowledge, this is the only report of a latitudinal survey of termites at species level performed with a standardized sampling protocol. We evaluate termite diversity and abundance, and describe patterns of species composition based on feeding groups along the latitudinal gradient. We also describe the relative contribution of environmental variables to explain diversity patterns. Termite assemblages were investigated by standardized surveys at 15 Atlantic Forest sites, on six transects divided into five sections of 10 m², with 30 sections per site (or 300 m²/site), which were investigated by one trained person for one hour. Observed species richness and abundance were negatively correlated with latitude. The influence of latitude was explained mainly by variables related to temperature, precipitation and ambient energy (potential evapotranspiration). Our results also suggest that temperature exerts a greater constraint on Atlantic Forest termites than productivity, because ambient productivity increases with latitude in this forest but termite diversity decreases. Termite species richness in the Atlantic Forest showed a different pattern than those described for other organisms, increasing in diversity where the coastal‐forest strip narrows. Overall, our results indicate comparatively high termite species richness at northeastern sites and a significant impoverishment of termite assemblages in the southeastern and southern regions of the Atlantic Forest.     

Expression profiles of growth-related genes in two Nile tilapia strains and their crossbred provide insights into introgressive breeding effects

The Nile tilapia (Oreochromis niloticus) is a prominent farmed fish in aquaculture worldwide. Crossbreeding has recently been carried out between the Red-Stirling and the wt Chitralada strains of Nile tilapia, producing a heterotic hybrid (7/8 Chitralada and 1/8 Red-Stirling) that combines the superior growth performance of the Chitralada with the reddish coloration of the Red-Stirling strain. While classical selective breeding and crossbreeding strategies are well known, the molecular mechanisms underlying the phenotypic expression of economically advantageous traits in tilapia remain largely unknown. Molecular investigations have shown that variable expression of growth hormone (gh), insulin-like growth factors (igf1 and 2) and somatolactin (smtla) – components of the growth hormone/insulin-like growth factor (GH/IGF) axis – and myostatin (mstn) genes can affect traits of economic relevance in farmed animals. The aim of this study was to assess and compare the gene expression signature among Chitralada, Red-Stirling and their backcross hybrid in order to gain insights into the effects of introgressive breeding in modulation of the GH/IGF axis. Gene expression analyses in distinct tissues showed that most genes of the GH/IGF axis were up-regulated and mstn was down-regulated in backcross animals in comparison with Red-Stirling and Chitralada animals. These gene expression profiles revealed that backcross animals displayed a distinctive expression signature, which attests to the effectiveness of the introgressive breeding technique. Our findings also suggest that the GH/IGF axis and mstn genes might be candidate markers for fish performance and prove useful within genetic improvement programs aimed at the production of superior-quality tilapia strains using introgressive breeding.     

Metabolic pathway for propionate utilization by phosphorus-accumulating organisms in activated sludge: 13C labeling and in vivo nuclear magnetic resonance

In vivo 13C and 31P nuclear magnetic resonance techniques were used to study propionate metabolism by activated sludge in enhanced biological phosphorus removal systems. The fate of label supplied in [3-13C]propionate was monitored in living cells subjected to anaerobic/aerobic cycles. During the anaerobic phase, propionate was converted to polyhydroxyalkanoates (PHA) with the following monomer composition: hydroxyvalerate, 74.2%; hydroxymethylvalerate, 16.9%; hydroxymethylbutyrate, 8.6%; and hydroxybutyrate, 0.3%. The isotopic enrichment in the different carbon atoms of hydroxyvalerate (HV) produced during the first anaerobic stage was determined: HV5, 59%; HV4, 5.0%; HV3, 1.1%; HV2, 3.5%; and HV1, 2.8%. A large proportion of the supplied label ended up on carbon C-5 of HV, directly derived from the pool of propionyl-coenzyme A (CoA), which is primarily labeled on C-3; useful information on the nature of operating metabolic pathways was provided by the extent of labeling on C-1, C-2, and C-4. The labeling pattern on C-1 and C-2 was explained by the conversion of propionyl-CoA to acetyl-CoA via succinyl-CoA and the left branch of the tricarboxylic acid cycle, which involves scrambling of label between the inner carbons of succinate. This constitutes solid evidence for the operation of succinate dehydrogenase under anaerobic conditions. The labeling in HV4 is explained by backflux from succinate to propionyl-CoA. The involvement of glycogen in the metabolism of propionate was also demonstrated; moreover, it was shown that the acetyl moiety to the synthesis of PHA was derived preferentially from glycogen. According to the proposed metabolic scheme, the decarboxylation of pyruvate is coupled to the production of hydrogen, and the missing reducing equivalents should be derived from a source other than glycogen metabolism.