Use of polyethyleneimine polymer in cell culture as attachment factor and lipofection enhancer

BACKGROUND: Several cell lines and primary cultures benefit from the use of positively charged extracellular matrix proteins or polymers that enhance their ability to attach to culture plates. Polyethyleneimine is a positively charged polymer that has gained recent attention as a transfection reagent. A less known use of this cationic polymer as an attachment factor was explored with several cell lines. RESULTS: Polyethyleneimine compared favorably to traditional attachment factors such as collagen and polylysine. PC-12 and HEK-293 cells plated on dishes coated with polyethyleneimine showed a homogeneous distribution of cells in the plate, demonstrating strong cell adhesion that survived washing procedures. The polymer could also be used to enhance the adherence and allow axonal outgrowth from zebrafish retinal explants. The effects of this coating agent on the transfection of loosely attaching cell lines were studied. Pre-coating with polyethyleneimine had the effect of enhancing the transfection yield in procedures using lipofection reagents. CONCLUSION: Polyethyleneimine is an effective attachment factor for weakly anchoring cell lines and primary cells. Its use in lipofection protocols makes the procedures more reliable and increases the yield of expressed products with commonly used cell lines such as PC-12 and HEK-293 cells

Mangrove diversity is more than fringe deep

AbstractMangroves form coastal tropical forests in the intertidal zone and are an important component of shoreline protection. In comparison to other tropical forests, mangrove stands are thought to have relatively low genetic diversity with population genetic structure gradually increasing with distance along a coastline. We conducted genetic analyses of mangrove forests across a range of spatial scales; within a 400 m2 parcel comprising 181 Rhizophora mangle (red mangrove) trees, and across four sites ranging from 6–115 km apart in Honduras. In total, we successfully genotyped 269 R. mangle trees, using a panel of 677 SNPs developed with 2b-RAD methodology. Within the 400 m2 parcel, we found two distinct clusters with high levels of genetic differentiation (FST = 0.355), corresponding to trees primarily located on the seaward fringe and trees growing deeper into the forest. In contrast, there was limited genetic differentiation (FST = 0.027–0.105) across the sites at a larger scale, which had been predominantly sampled along the seaward fringe. Within the 400 m2 parcel, the cluster closest to the seaward fringe exhibited low genetic differentiation (FST = 0.014–0.043) with the other Honduran sites, but the cluster further into the forest was highly differentiated from them (FST = 0.326–0.414). These findings contradict the perception that genetic structure within mangroves forests occurs mainly along a coastline and highlights that there is greater genetic structure at fine spatial scales.</jats:p

How do markets manage water resources? An experiment

We experimentally test how a private monopoly, a duopoly and a public utility allocate water of differing qualities to households and farmers. Most of our results are in line with the theoretical predictions. Overexploitation of the resources is observed independently of the market structure. Stock depletion for the public utility is the fastest, followed by the private duopoly and private monopoly. On the positive aspects of centralized public management, we find that the average quality to price ratio offered by the public monopoly is substantially higher than that offered by the private monopoly or duopoly

The effect of seasoning with herbs on the nutritional, safety and sensory properties of reduced-sodium fermented Cobrançosa cv. table olives

This study aimed at evaluating the effectiveness of seasoning Cobrancosa table olives in a brine with aromatic ingredients, in order to mask the bitter taste given by KCl when added to reduced-sodium fermentation brines. Olives were fermented in two different salt combinations: Brine A, containing 8% NaCl and, Brine B, a reduced-sodium brine, containing 4% NaCl + 4% KCl. After the fermentation the olives were immersed in seasoning brines with NaCl (2%) and the aromatic herbs (thyme, oregano and calamintha), garlic and lemon. At the end of the fermentation and two weeks after seasoning, the physicochemical, nutritional, organoleptic, and microbiological parameters, were determined. The olives fermented in the reduced-sodium brines had half the sodium concentration, higher potassium and calcium content, a lower caloric level, but were considered, by a sensorial panel, more bitter than olives fermented in NaCl brine. Seasoned table olives, previously fermented in Brine A and Brine B, had no significant differences in the amounts of protein (1.23% or 1.11%), carbohydrates (1.0% or 0.66%), fat (20.0% or 20.5%) and dietary fiber (3.4% or 3.6%). Regarding mineral contents, the sodium-reduced fermented olives, presented one third of sodium, seven times more potassium and three times more calcium than the traditional olives fermented in 8% NaCl. Additionally, according to the panelists' evaluation, seasoning the olives fermented in 4% NaCl + 4% KCl, resulted in a decrease in bitterness and an improvement in the overall evaluation and flavor. Escherichia coli and Salmonella were not found in the olives produced.info:eu-repo/semantics/publishedVersio

Murine Dendritic Cells Transcriptional Modulation upon Paracoccidioides brasiliensis Infection

Limited information is available regarding the modulation of genes involved in the innate host response to Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis. Therefore, we sought to characterize, for the first time, the transcriptional profile of murine bone marrow-derived dendritic cells (DCs) at an early stage following their initial interaction with P. brasiliensis. DCs connect innate and adaptive immunity by recognizing invading pathogens and determining the type of effector T-cell that mediates an immune response. Gene expression profiles were analyzed using microarray and validated using real-time RT-PCR and protein secretion studies. A total of 299 genes were differentially expressed, many of which are involved in immunity, signal transduction, transcription and apoptosis. Genes encoding the cytokines IL-12 and TNF-α, along with the chemokines CCL22, CCL27 and CXCL10, were up-regulated, suggesting that P. brasiliensis induces a potent proinflammatory response in DCs. In contrast, pattern recognition receptor (PRR)-encoding genes, particularly those related to Toll-like receptors, were down-regulated or unchanged. This result prompted us to evaluate the expression profiles of dectin-1 and mannose receptor, two other important fungal PRRs that were not included in the microarray target cDNA sequences. Unlike the mannose receptor, the dectin-1 receptor gene was significantly induced, suggesting that this β-glucan receptor participates in the recognition of P. brasiliensis. We also used a receptor inhibition assay to evaluate the roles of these receptors in coordinating the expression of several immune-related genes in DCs upon fungal exposure. Altogether, our results provide an initial characterization of early host responses to P. brasiliensis and a basis for better understanding the infectious process of this important neglected pathogen

Contamination level and spatial distribution of heavy metals in water and sediments of El Guájaro Reservoir, Colombia

Heavy metals have become a subject of special concern worldwide, mainly due to high persistence in the environment, toxicity, biogeochemical recycling and ecological risk. Therefore, the objective of this investigation was to analyze the spatial–temporal distribution of heavy metals in water and sediments to determine the environmental status of El Guájaro Reservoir, where such studies have not been developed. Two measurement campaigns (dry and wet period) were carried out and eight sampling stations were selected. A comparison of water and sediment quality parameters with existing national and international regulations was done. Also, heavy metal distribution maps were generated, and the geoaccumulation index was calculated to identify sources and sediments contamination level. Based on the obtained results, agriculture and mining activities are the main causes of the reservoir contamination. This metals levels could be a potential risk for the aquatic life and the populations that are supplied from this water body

Endogenous Signaling by Omega-3 Docosahexaenoic Acid-derived Mediators Sustains Homeostatic Synaptic and Circuitry Integrity

The harmony and function of the complex brain circuits and synapses are sustained mainly by excitatory and inhibitory neurotransmission, neurotrophins, gene regulation, and factors, many of which are incompletely understood. A common feature of brain circuit components, such as dendrites, synaptic membranes, and other membranes of the nervous system, is that they are richly endowed in docosahexaenoic acid (DHA), the main member of the omega-3 essential fatty acid family. DHA is avidly retained and concentrated in the nervous system and known to play a role in neuroprotection, memory, and vision. Only recently has it become apparent why the surprisingly rapid increases in free (unesterified) DHA pool size take place at the onset of seizures or brain injury. This phenomenon began to be clarified by the discovery of neuroprotectin D1 (NPD1), the first-uncovered bioactive docosanoid formed from free DHA through 15-lipoxygenase-1 (15-LOX-1). NPD1 synthesis includes, as agonists, oxidative stress and neurotrophins. The evolving concept is that DHA-derived docosanoids set in motion endogenous signaling to sustain homeostatic synaptic and circuit integrity. NPD1 is anti-inflammatory, displays inflammatory resolving activities, and induces cell survival, which is in contrast to the pro-inflammatory actions of the many of omega-6 fatty acid family members. We highlight here studies relevant to the ability of DHA to sustain neuronal function and protect synapses and circuits in the context of DHA signalolipidomics. DHA signalolipidomics comprises the integration of the cellular/tissue mechanism of DHA uptake, its distribution among cellular compartments, the organization and function of membrane domains containing DHA phospholipids, and the precise cellular and molecular events revealed by the uncovering of signaling pathways regulated by docosanoids endowed with prohomeostatic and cell survival bioactivity. Therefore, this approach offers emerging targets for prevention, pharmaceutical intervention, and clinical translation involving DHA-mediated signaling

Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering. [Methodology/Principal Findings]: We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome. [Conclusions/Significance]: The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.This work was supported by grant BIO2008-00133 from the Spanish Ministry of Science and Innovation to ML. CGP was a recipient of a predoctoral fellowship from the University of Cantabria, Spain.Peer reviewe

Quality changes and shelf-life prediction of a fresh fruit and vegetables purple smoothie

The sensory, microbial and bioactive quality changes of untreated (CTRL) and mild heat−treated (HT; 90 ºC/45 s) smoothies were studied and modelled throughout storage (5, 15 and 25 ºC). The overall acceptability was better preserved in HT samples being highly correlated (hierarchical clustering) with the flavour. The sensory quality data estimated smoothie shelf−life (CTRL/HT) of 18/55 (at 5 ºC), 4.5/12 (at 15 ºC), 2.4/5.8 (at 25 ºC) days. The yeast and moulds growth rate was lower in HT compared to CTRL while a lag phase for mesophiles/psychrophiles was observed in HT−5/15 ºC. HT and 5 ºC−storage stabilized the phenolics content. FRAP reported the best correlation (R2=0.94) with the studied bioactive compounds, followed by ABTS (R2=0.81) while DPPH was the total antioxidant capacity method with the lowest adjustment (R2=0.49). Conclusively, modelling was used to estimate the shelf−life of a smoothie based on quality retention after a short time−high temperature heat treatment that better preserved microbial and nutritional quality during storage.The financial support of this research was provided by the Ministerio Español de Economía y Competitividad MINECO (Projects AGL2013−48830−C2−1−R and AGL2013−48993−C2−1−R) and by FEDER funds. G.A. González−Tejedor thanks to Panamá Government for the scholarship to carry out his PhD Thesis. A. Garre (BES−2014−070946) is grateful to the MINECO for awarding him a pre−doctoral grant. We are also grateful to E. Esposito and N. Castillejo for their skilful technical assistance

Optical and near-infrared observations of SN 2013dx associated with GRB 130702A

We present optical and near-infrared light curves and optical spectra of SN 2013dx, associated with the nearby (redshift 0.145) gamma-ray burst GRB 130702A. The prompt isotropic gamma-ray energy released from GRB 130702A is measured to be $E_{\gamma,\mathrm{iso}} = 6.4_{-1.0}^{+1.3} \times 10^{50}$erg (1keV to 10MeV in the rest frame), placing it intermediate between low-luminosity GRBs like GRB 980425/SN 1998bw and the broader cosmological population. We compare the observed $g^{\prime}r^{\prime}i^{\prime}z^{\prime}$ light curves of SN 2013dx to a SN 1998bw template, finding that SN 2013dx evolves $\sim20$% faster (steeper rise time), with a comparable peak luminosity. Spectroscopically, SN 2013dx resembles other broad-lined Type Ic supernovae, both associated with (SN 2006aj and SN 1998bw) and lacking (SN 1997ef, SN 2007I, and SN 2010ah) gamma-ray emission, with photospheric velocities around peak of $\sim$21,000 km s$^{-1}$. We construct a quasi-bolometric ($g^{\prime}r^{\prime}i^{\prime}z^{\prime}yJH$) light curve for SN 2013dx, and, together with the photospheric velocity, we derive basic explosion parameters using simple analytic models. We infer a $^{56}$Ni mass of $M_{\mathrm{Ni}} = 0.38\pm 0.01$M$_{\odot}$, an ejecta mass of $M_{\mathrm{ej}} = 3.0 \pm 0.1$ M$_{\odot}$, and a kinetic energy of $E_{\mathrm{K}} = (8.2 \pm 0.40) \times 10^{51}$erg (statistical uncertainties only), consistent with previous GRB-associated SNe. When considering the ensemble population of GRB-associated SNe, we find no correlation between the mass of synthesized $^{56}$Ni and high-energy properties, despite clear predictions from numerical simulations that $M_{\mathrm{Ni}}$ should correlate with the degree of asymmetry. On the other hand, $M_{\mathrm{Ni}}$ clearly correlates with the kinetic energy of the supernova ejecta across a wide range of core-collapse events