Total Protein Production Research Articles

Forsythiaside attenuates Escherichia coli lipopolysaccharide-induced liver acute inflammatory response in chicken

This study investigated the effects of forsythiaside on the acute inflammatory response induced by Escherichia coli lipopolysaccharide (LPS) in liver of broiler chickens. Fifteen-day-old chickens were randomly assigned to three groups (n = 20 for each group, orally treated with 0, 30, or 60 mg/kg BW of forsythiaside) for 7 days. At 21 days of age, the chickens were intravenously injected with either LPS (200 μg/kg BW) or sterile saline (200 μg/kg BW, control group). All the chickens were humanely euthanized by cervical dislocation 2 h after the LPS injection. The results showed that the injection of LPS induced some indexes, including total proteins, nitric oxide (NO), interleukin-1beta (IL-1β), interleukin-6 (IL-6), and interleukin-17 (IL-17) production ( P < 0.05) and increased the mRNA expression of LPS-induced tumor necrosis factor-alpha (LITAF), IL-1β, IL-17, IL-6, and inducible nitric oxide synthase (iNOS) ( P < 0.05). Forsythiaside supplementation alleviated the LPS-induced inflammatory response by inhibiting the production of total proteins, NO, LITAF, IL-1β, IL-17, and IL-6 and down-regulating the mRNA expression of pro-inflammatory cytokines and iNOS. In conclusion, forsythiaside is a potential treatment for LPS-induced liver acute inflammation in chicken.

Pharmacological stimulation of NQO1 decreases NADPH levels and ameliorates acute pancreatitis in mice

Reactive oxygen species (ROS) regulates the activation of inflammatory cascades and tissue damage in acute pancreatitis. NADPH oxidase (NOX) is upregulated in pancreatitis and is one of the major enzymes involved in ROS production using NADPH as a general rate-limiting substrate. Dunnione, a well-known substrate of NAD(P)H:quinone oxidoreductase 1 (NQO1), reduces the ratio of cellular NADPH/NADP+ through the enzymatic action of NQO1. This study assessed whether a reduction in cellular NADPH/NADP+ ratio can be used to regulate caerulein-induced pancreatic damage associated with NOX-induced ROS production in animal models. Dunnione treatment significantly reduced the cellular NADPH/NADP+ ratio and NOX activity through the enzymatic action of NQO1 in the pancreas of the caerulein-injection group. Similar to these results, total ROS production and expressions of mRNA and protein for NOX subunits Nox1, p27phox, p47phox, and p67phox also decreased in the dunnione-treated group. In addition, caerulein-induced pancreatic inflammation and acinar cell injury were significantly reduced by dunnione treatment. This study is the first to demonstrate that modulation of the cellular NADPH:NADP+ ratio by enzymatic action of NQO1 protects acute pancreatitis through the regulation of NOX activity. Furthermore, these results suggest that modulation of the NADPH:NADP+ ratio in cells by NQO1 may be a novel therapeutic strategy for acute pancreatitis.

Transdentinal cytotoxicity and macrophage phenotype of a novel quaternary ammonium silane cavity disinfectant

ObjectiveTo evaluate the transdentinal cytotoxicity and macrophage phenotype response to a novel quaternary ammonium silane (QAS) cavity disinfectant. MethodsNIH 3T3 mouse fibroblasts were cultured in Dulbecco’s Modified Eagle's Medium and incubated for 3 days. The cells (3×104) were seeded on the pulpal side of dentine discs and the occlusal side of the discs were treated with different cavity disinfectants: Group 1: de-ionized water (control); Group 2: 2% chlorhexidine (CHX); Group 3: 2% QAS; Group 4: 5% QAS, and Group 5: 10% QAS. Cell morphology of NIH 3T3 cells was examined using scanning electron microscopy (SEM) and cell viability was assessed using Trypan blue assay. The eluates were collected and applied on cells seeded in 24-well plates. The total protein production, alkaline phosphatase activity and deposition of mineralized nodules were evaluated after 7 and 14 days. Immunofluorescence staining was performed on the samples with primary antibodies of CD68+, CD80+, and CD163+ assessing the macrophage M1/M2 phenotypes. The macrophages were imaged using a confocal scanning light microscope with an excitation wavelength of 488nm. ResultsNo significant difference in cell viability (p<0.0001), total protein production (p<0.01) and mineralized nodule production (p<0.05) was found between 2% QAS and the control, which was significantly higher than 2% CHX, 5% and 10% QAS after 14 days. Alkaline phosphatase production of 2% QAS was significantly lower than the control (p<0.001), but higher than 2% CHX at 14 days. The M1/M2 macrophage ratio was also significantly lower in the 2% and 10% QAS groups (p<0.05) compared to the control and 2% CHX groups. SignificanceThe 2% QAS cavity disinfectant does not have cytotoxic effects on 3T3 NIH mouse fibroblast cells and the predominance of the anti-inflammatory phenotype after its application may stimulate healing and tissue repair.

Mycorrhization and phosphorus may be an alternative for increasing the production of metabolites in Myracrodruon urundeuva

‘Aroeira-do-sertao’ (Myracrodruon urundeuva Allemao; Anacardiaceae) is one of the most-used medicinal species by the population of northeastern Brazil and is on the list of plants threatened with extinction. In this context, alternatives that could contribute to reduced extractive use must be designed to establish crops of this species, which have high concentrations of metabolites. Inoculation with arbuscular mycorrhizal fungi (AMF) and the addition of phosphorus (P) to soil increases the production of primary and secondary metabolites in some plants. We tested the hypothesis that the effects of mycorrhization in ‘aroeira’ can be maximized by the application of P. The dry matter and the concentration of metabolites were evaluated. Mycorrhization increased the accumulation of phytomass, which was also favored at the highest dosage of P tested. The production of phenols and total proteins was maximized by mycorrhization only at an intermediate dosage of P. The inoculation did not favor the synthesis of flavonoids and soluble carbohydrates. The production of proanthocyanidins was influenced only by phosphate supplementation. It is concluded that the mycorrhization of seedlings of ‘aroeira-do-sertao’ associated with P fertilization maximizes the production of total phenols and total foliar proteins, making the phytomass more attractive to the herbal industry and resulting in the use of P fertilization.

Does adopting legume-based cropping practices improve the food security of small-scale farm households? Panel survey evidence from Zambia

This study provides empirical evidence on whether and how integrating legumes into production systems affects measures of small-scale farm households’ food availability and access. We used nationally representative household panel survey data from Zambia to estimate the differential effects on cereal-growing households of incorporating grain legumes into their farms via cereal-legume intercropping, cereal-legume rotation, and other means (such as legume monocropping). Specifically, we tested the hypotheses that with all else equal, cereal-growing small-scale farm households that integrate grain legumes into their production systems have: (1) more availability of food as measured by total production of calories and protein; (2) more income from crop production or sales; and (3) increased food access. Results suggest that cereal-legume rotation was associated with statistically significant increases in production of calories and protein by a household as well as their gross value of crop sales; it may also improve their food access. In contrast, we found little evidence of statistically significant effects of cereal-legume intercropping and other forms of legume production on household food availability and access in Zambia.

Whole synthetic pathway engineering of recombinant protein production.

The output from protein biomanufacturing systems is a function of total host cell biomass synthetic capacity and recombinant protein production per unit cell biomass. In this study, we describe how these two properties can be simultaneously optimized via design of a product-specific combination of synthetic DNA parts to maximize flux through the protein synthetic pathway and the use of a host cell chassis with an increased capability to synthesize both cell and product biomass. Using secreted alkaline phosphatase (SEAP) production in Chinese hamster ovary cells as our example, we demonstrate how an optimal composition of input components can be assembled from a minimal toolbox containing rationally designed promoters, untranslated regions, signal peptides, product coding sequences, cell chassis, and genetic effectors. Product titer was increased 10-fold, compared with a standard reference system by (a) identifying genetic components that acted in concert to maximize the rates of SEAP transcription, translation, and translocation, (b) selection of a cell chassis with increased biomass synthetic capacity, and (c) engineering the host cell factory's capacity for protein folding and secretion. This whole synthetic pathway engineering process to design optimal expression cassette-chassis combinations should be applicable to diverse recombinant protein and host cell-type contexts.

Direct and transdentinal biostimulatory effects of grape seed extract rich in proanthocyanidin on pulp cells.

To evaluate the potential biostimulatory effects of grape seed extract (GSE) on a primary culture of human pulp cells. Human molars were used to obtain the primary pulp cell culture and 0.5-mm dentine discs. For GSE direct exposure, dose-response (0.0065-6.5%) and time response (1-60min of contact) were examined. For transdentinal exposure, 0.65% of GSE was tested for 24h. Cellular metabolism, nitric oxide and collagen production, and cell morphology alterations were assessed at periods of 24 and 72h. After cell differentiation and direct exposure to GSE, the total protein production (TP), alkaline phosphatase activity (ALP) and formation of mineralization nodules (MN) were assessed. The results were analysed by parametric tests or non-parametric tests (α=0.05). The lower concentration of GSE tested (0.0065%) was associated with an increase in cellular metabolism, a reduction in the production of nitric oxide and an increase in extracellular matrix synthesis (collagen). Distinct behaviours were observed for the different concentrations, without a reduction of cellular metabolism >10% compared with the control, either when applied directly or transdentinally. SEM revealed no significant change in cell morphology, except for the positive control (H2 O2 ). There was no difference in TP, ALP or MN between the control group and the group exposed to GSE. Treatment with grape seed extract, even at the highest concentration and longest period, caused neither direct nor transdentinal cytotoxic effects on human pulp cells. Grape seed extract components may play a biostimulatory role and protect dental pulp cells when in direct contact.

A combined treatment using ethylmethane sulfonate and ultraviolet light to compare amylase production by three Bacillus sp. isolates

In this study, three Bacillus sp.-producing amylase enzymes were isolated from soil samples and identified using 16S rDNA sequence analysis. Amylase production and total protein productions were spectrophotometrically measured. The following media were tested to increase enzyme production: LB medium and molasses. Three Bacillus sp. were identified as follows: Bacillus subtilis subtilis, Bacillus thuringiensis, and Bacillus cereus. Amylase production levels were in the range of 10 U/mL, whereas total protein production levels were at 15 mg/mL. Higher amylase activity was found in the Bacillus subtilis isolate. Ethylmethane sulfonate (EMS) and ultraviolet (UV) mutagenesis in combination were applied to compare amylase production. Amylase activity was increased to around 58% in the treatment with 0.03 mL of EMS and UV when compared to the control group. A pilot scale bioreactor with a total working volume of 10 liters was used to produce amylase by B. subtilis subtilis. In conclusion, B. subtilis subtilis can be used to produce amylase enzyme for various industrial purposes, and, for the first time, the amylase activities of B. subtilis can be enhanced with EMS and UV treatment.

Effects of short-term fasting on in vivo rumen microbiota and in vitro rumen fermentation characteristics.

ObjectiveFasting may lead to changes in the microbiota and activity in the rumen. In the present study, the effects of fasting on rumen microbiota and the impact of fasting on in vitro rumen fermentation were evaluated using molecular culture-independent methods.MethodsThree ruminally cannulated Holstein steers were fed rice straw and concentrates. The ruminal fluids were obtained from the same steers 2 h after the morning feeding (control) and 24 h after fasting (fasting). The ruminal fluid was filtrated through four layers of muslin, collected for a culture-independent microbial analysis, and used to determine the in vitro rumen fermentation characteristics. Total DNA was extracted from both control and fasting ruminal fluids. The rumen microbiota was assessed using denaturing gradient gel electrophoresis (DGGE) and quantitative polymerase chain reaction. Microbial activity was evaluated in control and fasting steers at various intervals using in vitro batch culture with rice straw and concentrate at a ratio of 60:40.ResultsFasting for 24 h slightly affected the microbiota structure in the rumen as determined by DGGE. Additionally, several microorganisms, including Anaerovibrio lipolytica, Eubacterium ruminantium, Prevotella albensis, Prevotella ruminicola, and Ruminobacter amylophilus, decreased in number after fasting. In addition, using the ruminal fluid as the inoculum after 24 h of fasting, the fermentation characteristics differed from those obtained using non-fasted ruminal fluid. Compared with the control, the fasting showed higher total gas production, ammonia, and microbial protein production (p<0.05). No significant differences, however, was observed in pH and dry matter digestibility.ConclusionWhen in vitro techniques are used to evaluate feed, the use of the ruminal fluid from fasted animals should be used with caution.

Impact of OqxR loss of function on the envelope proteome of Klebsiella pneumoniae and susceptibility to antimicrobials.

In Klebsiella pneumoniae, loss-of-function mutations in the transcriptional repressors RamR and OqxR both have an impact on the production of efflux pumps and porins relevant to antimicrobial efflux/entry. To define, in an otherwise isogenic background, the relative effects of OqxR and RamR loss-of-function mutations on envelope protein production, envelope permeability and antimicrobial susceptibility. We also investigated the clinical relevance of an OqxR loss-of-function mutation, particularly in the context of β-lactam susceptibility. Envelope permeability was estimated using a fluorescent dye accumulation assay. Antimicrobial susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and quantitative RT-PCR was used to measure transcript levels. Loss of RamR or OqxR reduced envelope permeability in K. pneumoniae by 45%-55% relative to the WT. RamR loss activated AcrAB efflux pump production ∼5-fold and this reduced β-lactam susceptibility, conferring ertapenem non-susceptibility even in the absence of a carbapenemase. In contrast, OqxR loss specifically activated OqxAB efflux pump production >10 000-fold. This reduced fluoroquinolone susceptibility but had little impact on β-lactam susceptibility even in the presence of a β-lactamase. Whilst OqxR loss and RamR loss are both seen in K. pneumoniae clinical isolates, only RamR loss significantly stimulates AcrAB efflux pump production. This means that only RamR mutants have significantly reduced β-lactamase-mediated β-lactam susceptibility and therefore represent a greater clinical threat.

Influence of alternative soil amendments on mycorrhizal fungi and cowpea production

Alternative soil amendments (worm compost, pyrolyzed carbon [biochar]) and crop symbioses with arbuscular mycorrhizal (AM) fungi have the potential to reduce food production costs while promoting sustainable agriculture by improving soil quality and reducing commercial (N and P) fertilizer use. Our greenhouse studies investigated the influence of alternative soil amendments on AM fungi associated with cowpea (Vigna unguiculata [L.] Walp.) and common bean (Phaseolus vulgaris L.) by examining productivity and plant nutrition. We conducted an experiment to select a cowpea or common bean genotype based on AM fungal colonization, seed production, and seed nutritional content. We then grew the selected cowpea genotype (Resina) in low-fertility soil with 10 different soil amendments (combinations of biochar, worm compost, and/or commercial fertilizers) plus a non-amended control. There were no significant differences in AM fungal colonization of cowpea plants grow with different soil amendments. However, an amendment blend containing worm compost, biochar, and 50% of the typically recommended commercial fertilizer rate produced plants with similar aboveground biomass, protein concentration, and total protein production, with increased tissue K, P, and Zn concentration and total content, compared to plants receiving only the recommended (100%) rate of commercial fertilizer. As previous research links uptake of P and Zn with plant-mycorrhizal symbioses, our results indicate cowpea nutritional benefits may be derived from AM partnership and alternative soil amendments. These synergies between alternative soil amendments and AM fungi may help reduce farm costs while maintaining or improving crop yield and nutrition, thus increasing global food and nutrition security.

Pengaruh Pemberian Ekstrak Daun Pepaya (Carica papaya Linn) dan Kunyit (Curcuma domestica) Terhadap Fermentabilitas Rumen Sapi Perah Secara In vitro

This research was conducted to determine the effect of using papaya leaves extract, turmeric extract and the combination on ruminal pH, the concentrations of amonia (NH 3 ), Volatile Fatty Acids (VFA), Acetate, Propionate, Butyrate, Methane (CH 4 ), CO 2 and Total Protein. The material was used rumen fluid of dairy cows, papaya leaves extract and turmeric extract. The study was arranged in a completely randomized design with 4 treatments and 4 replications. The treatment was control diet (T1); control diet supplemented with 0.005 ml papaya leaves extract (T2); control diet supplemented with 0.005 ml turmeric extract (T3) and control diet supplemented with 0.0025 ml papaya leaves extract and 0.0025 ml turmeric extract (T4). The rumen fluid was analyzed after 3 hours fermentation. Data were analyzed using ANOVA and continued by Duncan test. The result showed that single supplementation of either papaya leaves or turmeric extract alone and supplementation of both herbal did not affect ruminal pH, NH 3 , CO 2 and total protein production. Moreover, there were an increase in concentrations of total VFA, acetate, propionate, butyrate and methane. The combination of papaya leaves and turmeric extract supplementation resulting in the highest concentrations of VFA (445 mMol/l), acetate (13.5 mMol/l), propionate (4.24 mMol/l), butyrate (1.87 mMol/l) and CH 4 (17.17%) in the rumen. In conclusion, supplementation of papaya leaves and turmeric alone, and the combination of both herbal could enhance rumen fermentability of dairy cow.

The Effects of Plant Growth Regulators on Cell Growth, Protein, Carotenoid, PUFAs and Lipid Production of Chlorella pyrenoidosa ZF Strain

In the present study, eight kinds plant growth regulators—salicylic acid (SA), 1-naphthaleneacetic acid (NAA), gibberellic acid (GA3), 6-benzylaminopurine (6-BA), 2, 4-epi-brassinolide (EBR), abscisic acid (ABA), ethephon (ETH), and spermidine (SPD)—were used to investigate the impact on microalgal biomass, lipid, total soluble protein, carotenoids, and polyunsaturated fatty acids (PUFAS) production of Chlorella pyrenoidosa ZF strain. The results showed the quickest biomass enhancement was induced by 50 mg·L−1 NAA, with a 6.3-fold increase over the control; the highest protein content was increased by 0.005 mg·L−1 ETH, which produced 3.5-fold over the control; total carotenoids content was induced most effectively by 1 mg·L−1 NAA with 3.6-fold higher production than the control; the most efficient elicitor for lipid production was 5 mg·L−1 GA3 at 1.9-fold of the control; 0.2 mg·L−1 ETH induced the abundant production of 1.82 ± 0.23% linoleic acid; 0.65 ± 0.01% linolenic acid was induced by 1 mg·L−1 NAA; 2.53 ± 0.15% arachidonic acid and 0.44 ± 0.05% docosahexaenoic acid were induced by 5 mg·L−1 GA3. Transcriptional expression levels of seven lipid-related genes, including ACP, BC, FAD, FATA, KAS, MCTK, and SAD, were studied by real-time RT-q-PCR. 5 mg·L−1 GA3 was the most effective regulator for transcriptional expressions of these seven genes, producing 23-fold ACP, 31-fold BC, 25-fold FAD, 6-fold KAS, 12-fold MCTK compared with the controls, respectively.

Envelope proteome changes driven by RamA overproduction in Klebsiella pneumoniae that enhance acquired β-lactam resistance.

In Klebsiella pneumoniae, overproduction of RamA results in reduced envelope permeability and reduced antimicrobial susceptibility but clinically relevant resistance is rarely observed. Here we have tested whether RamA overproduction can enhance acquired β-lactam resistance mechanisms in K. pneumoniae and have defined the envelope protein abundance changes upon RamA overproduction during growth in low and high osmolarity media. Envelope permeability was estimated using a fluorescent dye accumulation assay. β-Lactam susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and transcript levels were quantified using real-time RT-PCR. RamA overproduction enhanced β-lactamase-mediated β-lactam resistance, in some cases dramatically, without altering β-lactamase production. It increased production of efflux pumps and decreased OmpK35 porin production, though micF overexpression showed that OmpK35 reduction has little impact on envelope permeability. A survey of K. pneumoniae bloodstream isolates revealed ramA hyperexpression in 3 of 4 carbapenemase producers, 1 of 21 CTX-M producers and 2 of 19 strains not carrying CTX-M or carbapenemases. Whilst RamA is not a key mediator of antibiotic resistance in K. pneumoniae on its own, it is potentially important for enhancing the spectrum of acquired β-lactamase-mediated β-lactam resistance. LC-MS/MS proteomics analysis has revealed that this enhancement is achieved predominantly through activation of efflux pump production.

Biostimulant activity of humic substances extracted from leonardites

Biostimulants are natural compounds that enhance plant growth and plant nutrient use efficiency. In this study, biostimulant effects of humic substances (HS) extracted from leonardites were analysed on the metabolism of maize plants grown in hydroponic conditions. HS extracted from four leonardites were tested for their auxin-like and gibberellin-like activities. Then, 11 day old maize seedlings were treated for 48 h with five concentrations (0, 0.1, 0.5, 1, and 10 mg C L−1) of HS. After sampling, root growth and morphology, glutamine synthetase (GS) activity, glutamate synthase (GOGAT) activity, total protein content, soluble sugars content, phenylalanine ammonia-lyase (PAL) activity, soluble phenols, and free phenolic acids were analysed. HS from leonardites had similar spectroscopic pattern, with small differences. The HS from the South Dakota lignite (HS_USA) had more carboxylic groups, whereas the three from Turkish mines had more aromatic and aliphatic structures. HS_USA best enhanced total root growth, root surface area, and proliferation of secondary roots. Plant nutrient use efficiency was enhanced by HS_4, HS_USA and HS_B, with increment of GS and GOGAT enzymes activity and total protein production. HS stimulated also PAL enzyme activity, followed by a higher production of total soluble phenols, p-hydroxybenzoic acid, p-coumarilic acid, and chlorogenic acid. This study found that, although the activity of the HS depended on the origin of the leonardite, these compounds can be attributed to the biostimulant products, eliciting plant growth, nitrogen metabolism, and accumulation of phenolic substances.

“Metabolism of Odontoblast-like cells submitted to transdentinal irradiation with blue and red LED”

ObjectivesThe present study evaluated the trans-dentinal effect of light emitting diodes (LEDs) irradiation on the metabolism of odontoblast-like cells. MethodsSeventy-two dentin discs (0.2mm thick) were obtained from human molar teeth. MDPC-23 cells (20,000 cells/disc) were seeded on the pulpal side of the discs using DMEM, supplemented with 10% fetal bovine serum (FBS). After 12h, the culture medium was replaced with DMEM containing 0.5% FBS. After additional 12h, blue (455±10nm) or red (630±10nm) LEDs were used at irradiances of 80 and 40mW/cm2, respectively, to irradiate the occlusal side of the discs. The energy doses were fixed at 2 or 4J/cm2. Cell viability, alkaline phosphatase activity (ALP), total protein production and collagen synthesis were evaluated 72h after irradiation. Data were submitted to Kruskal-Wallis and Mann-Whitney tests (α=0.05). ResultsRed light promoted proliferative effects at the energy dose of 4J/cm2. Conversely, cell cultures irradiated with 2J/cm2 emitted by the blue light showed reduced viability. ALP production was stimulated by red light in comparison with blue light at 4J/cm2. Total protein production was reduced after exposure to blue light at 4J/cm2, while no effect was observed on collagen production. ConclusionsIrradiation with red LED at 4J/cm2 bio-stimulated the viability of odontoblast-like cells, whilst blue light had unfavorable effects on the cellular metabolism.

Synthesis and Biological Evaluation of Ionizable Lipid Materials for the In Vivo Delivery of Messenger RNA to B Lymphocytes.

B lymphocytes regulate several aspects of immunity including antibody production, cytokine secretion, and T-cell activation; moreover, B cell misregulation is implicated in autoimmune disorders and cancers such as multiple sclerosis and non-Hodgkin's lymphomas. The delivery of messenger RNA (mRNA) into B cells can be used to modulate and study these biological functions by means of inducing functional protein expression in a dose-dependent and time-controlled manner. However, current in vivo mRNA delivery systems fail to transfect B lymphocytes and instead primarily target hepatocytes and dendritic cells. Here, the design, synthesis, and biological evaluation of a lipid nanoparticle (LNP) system that can encapsulate mRNA, navigate to the spleen, transfect B lymphocytes, and induce more than 60 pg of protein expression per million B cells within the spleen is described. Importantly, this LNP induces more than 85% of total protein production in the spleen, despite LNPs being observed transiently in the liver and other organs. These results demonstrate that LNP composition alone can be used to modulate the site of protein induction in vivo, highlighting the critical importance of designing and synthesizing new nanomaterials for nucleic acid delivery.

Stable and Transient Expression of Human Coagulation Factor IX in Mammalian Expression Systems; CHO Versus HEK Cells

Background: Selecting a host system for the expression of recombinant proteins should be carefully evaluated prior to the initiation of any bio therapeutic development programs. Since different hosts express proteins with various efficiencies and with different posttranslational modifications, changing hosts may impact the expected activity of the protein. The main expression systems have members of the mammalian cell family, however, there are remarkable differences between the species. The most generally used mammalian hosts for the production of recombinant proteins are CHO and HEK293 cells. Objectives: In order to compare the differences between HEK versus CHO cells, the human coagulation factor IX in a transient and stable expression was examined. Methods: After transfection of CHO and HEK cells with an hFIX-expressing mammalian plasmid, pcDNA-hFIX, transient expression was analyzed on the culture supernatant during 72 hours. The stable clones were also recovered after 10 weeks of geneticin selection. The expression and activity of the hFIX was evaluated by performing enzyme-linked immunosorbent assay (ELISA) as well as a coagulation test, respectively. The γ-carboxylation of the hFIX was confirmed by evaluation of the expressed protein, after being precipitated with barium citrate. Results: Although the stable CHO clones secreted hFIX protein 30% higher than HEK cells, the transient HEK cell line proved to be superior in the production of total hFIX protein (%42 increased) and functional hFIX (%29 higher) relative to the CHO cell line. Moreover, specific activity and the fully γ-carboxylated hFIX are almost constant in transient and stable expression, indicating that γ-carboxylation efficiency in both CHO and HEK cell lines is almost equal. Conclusions: In conclusion, transient transfection studies with HEK cells provide a simple and strong method for a high production of recombinant proteins in weeks, a process which is more time consuming in the CHO cell line.

Evaluating biochemical response of filamentous algae integrated with different water bodies

The present study prompted the second attempts to evaluate biochemical responses of filamentous algae under different physico-chemical variables in various water bodies in Turkey. These variables were investigated by use of multivariate approach in the years of 2013 (May and November) and 2014 (May and October). Studied ecoregions had the different geographic position, climate, land-use, and anthropogenic activities, could strongly affect physico-chemical variables of water bodies, which caused to change or regulate in algal biomass composition due to the different response of filamentous species. Besides, biochemical responses of species changed at different sampling times and stations. Multivariate analyses indicated that temperature, heavy metals, and nutrient contents of aquatic systems were found to be major variables driving the spatial and temporal occurrence and biochemical contents of filamentous species. Total protein and pigment production by filamentous algae were high in water bodies having high nutrients, whereas they were low in high heavy metal contents. Amount of malondialdehyde (MDA), H2O2, total thiol group, total phenolic compounds, proline, total carbohydrate, and bioaccumulation of metals by filamentous algae were closely related with heavy metal contents of water bodies, indicated by the multivariate approach. Significant increase in aforementioned biochemical compounds with a distinct range of habitats and sensitive-tolerance to environmental conditions could make them highly valuable indicators.

A New Nβ‐alkanoyl‐5‐hydroxytryptamide Induces Anti‐inflammatory Activity in a Mice Subcutaneous Air Pouch Model of Inflammation

The C18‐5HT, a new Nβ‐alkanoyl‐5‐hydroxytryptamide is naturally found in the surface wax of coffee beans (Speer et al., 2006). Some amides of the serotonin class demonstrated an anti‐inflammatory effect by inhibiting the expression of caspases participants in inflammatory process (Meijerink et al., 2013). The aim of this study was to evaluate the anti‐inflammatory activity of C18‐5HT, a new fatty acid amide of serotonin.C18‐5HT (0.1–10 mg/kg, p.o.) effects were assessed in the carrageenan‐induced cell migration into the subcutaneous air pouch (SAP) with quantification of total leukocyte number, total proteins, nitric oxide (NO), tumor necrosis factor α (TNF‐α), interleukin 1β (IL‐1β), interferon γ (IFN‐γ) and interleukin 10 (IL‐10). A possible inhibitory effect in reactive oxygen species (ROS) production was evaluated ex vivo, after activation of leukocytes collected from the SAP with phorbolmyristate acetate (PMA). Cells were pretreated ex vivo with C18‐5HT (1–10 μM). The fluorescence was captured in the FL‐1 channel of a flow cytometer and expressed as fluorescence intensity. The protocol for the use of animals was approved by CAUAP/UFRJ and received the number DFBCICB015‐04/16. Statistical analysis was performed by ANOVA and Bonferroni's test (*p&lt;0.05).Pretreatment of animals with different doses of C18‐5HT significantly reduced cell migration to the SAP: PBS in SAP=3.5±1.4×106cells/mL; carrageenan in SAP=69.2±9.9×106cells/mL; 0.1 mg/kg= 51.9±8.4×106cells/mL; 1 mg/kg= 41.7±6.7×106*cells/mL; 10 mg/kg=37.1±3.1×106*cells/mL. C18‐5HT also significantly decrease the amount of total proteins and NO (Table 1) With respect to quantification of TNF‐α, IFN‐γ and IL‐1β, C18‐5HT were able to reduce the values when compared with carrageenan group in SAP (Table 1). Even though C18‐5HT was able to significantly increase the release of IL‐10 only at the dose of 10 mg/kg (table 1).C18‐5HT also reduced ROS production (control=0.19×105±0.12×104, PMA stimulated=1.54×105±0.62×104; 1 μM=0.24×105±0.80×104*; 3 μM=0.21×105±0.30×104*; 10 μM=0.11×105±0.20×104*.Thereby we can conclude that C18‐5HT inhibited leukocyte migration, production of total proteins, NO, TNF‐α, IFN‐γ and IL‐1β. In addition, increase the release of IL‐10 and decreased ROS production. Data suggest that this substance could be used as new prototype forthe development of new anti‐inflammatory drugs.Support or Funding InformationAlan Minho for technical assistance, Instituto Vital Brazil (Niterói, Brazil) for donation of mice. Financial support: CAPES, CNPq and FAPERJ Effect of C18‐5HT on inflammatory mediators accumulated in the subcutaneous air pouch after carrageenan injection. Injection in SAP Treatment mg/kg Total protein (μg/ml) NO (μM) TNF‐α (pg/ml) IFN‐γ (pg/ml) IL‐1β (pg/ml) IL‐10 (pg/ml) PBS ‐ ‐ 24.5±13.3 34.6±14.6 55.6±9.0 62.5±25.6 768.5±149.4 905.6±59.3 Carrageenan Vehicle ‐ 244.5±36.0 330.0±32.4 599.7±83.1 1,301.5±132.6 1,584.0±350.7 2,525.3±215.7 C18‐5HT 0.1 206.3±52.2 164.1±30.7* 386.1±40.8* 1,212.2±185.6 1,322.9±192.3 2,274.4±462.3 1 186.4±13.0* 118.8±39.5* 317.5±58.0* 1,272.5±151.1 733.8±69.1* 2,580.8±268.8 10 158.5±31.9* 82.3±14.0* 271.5±36.1* 808.0±128.2* 622.9±53.2* 4,213.8±453.8* Statistical significance was calculated by ANOVA followed by Bonferroni's test (n = 6–8). p &lt; 0.05 when compared C18‐5HT ‐treated mice with vehicle‐treated group.