Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

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Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Conducting Polymers in the Fields of Energy, Environmental Remediation, and Chemical-Chiral Sensors.

Conducting polymers (CPs), thanks to their unique properties, structures made on-demand, new composite mixtures, and possibility of deposit on a surface by chemical, physical, or electrochemical methodologies, have shown in the last years a renaissance and have been widely used in important fields of chemistry and materials science. Due to the extent of the literature on CPs, this review, after a concise introduction about the interrelationship between electrochemistry and conducting polymers, is focused exclusively on the following applications: energy (energy storage devices and solar cells), use in environmental remediation (anion and cation trapping, electrocatalytic reduction/oxidation of pollutants on CP based electrodes, and adsorption of pollutants) and finally electroanalysis as chemical sensors in solution, gas phase, and chiral molecules. This review is expected to be comprehensive, authoritative, and useful to the chemical community interested in CPs and their applications.

PGC-1α-Dependent Mitochondrial Adaptation Is Necessary to Sustain IL-2-Induced Activities in Human NK Cells.

Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in antitumor defense reactions. NK cell effector functions are critically dependent on cytokines and metabolic activity. Among various cytokines modulating NK cell function, interleukin-2 (IL-2) can induce a more potent cytotoxic activity defined as lymphokine activated killer activity (LAK). Our aim was to determine if IL-2 induces changes at the mitochondrial level in NK cells to support the bioenergetic demand for performing this enhanced cytotoxic activity more efficiently. Purified human NK cells were cultured with high IL-2 concentrations to develop LAK activity, which was assessed by the ability of NK cells to lyse NK-resistant Daudi cells. Here we show that, after 72 h of culture of purified human NK cells with enough IL-2 to induce LAK activity, both the mitochondrial mass and the mitochondrial membrane potential increased in a PGC-1α-dependent manner. In addition, oligomycin, an inhibitor of ATP synthase, inhibited IL-2-induced LAK activity at 48 and 72 h of culture. Moreover, the secretion of IFN-γ from NK cells with LAK activity was also partially dependent on PGC-1α expression. These results indicate that PGC-1α plays a crucial role in regulating mitochondrial function involved in the maintenance of LAK activity in human NK cells stimulated with IL-2.

Aldosterone increases oxygen consumption of rectal epithelia of normal, sodium-deprived and sodiumloaded rats.

INTRODUCTION: The colonic epithelium is a classical aldosterone target, but the effect of the hormone on the oxygen consumption rate (QO2 ) of this tissue is unknown. Objectives. We aimed at assessing, in the rectal epithelium of rats fed with diets of different sodium content, the effect of epithelial sodium channel (ENaC) blockade on short-circuit current (ISC ) and QO2 , and the acute effect of aldosterone incubation on ISC and QO2 . METHODS: Adult male rats were fed with normal, low or high-sodium diets for 8 days. Plasma sodium and serum aldosterone were measured. Isolated mucosa preparations from the rectal portion of the colon were mounted in Ussing chambers modified to measure ISC and QO2. RESULTS: Baseline ISC and QO2 were highest in sodium-deprived rats. Both were proportionally reduced by amiloride (0.1 mM) in this group and in the normal sodium group, but not in sodium-loaded rats. In separate experiments, incubation with aldosterone (10 nM) for 7 h increased ISC and QO2 in all groups; increases were larger in the normal and sodium-loaded groups. Amiloride decreased both ISC and QO2 , abolishing the differences between groups. Linear regression of the decrease in QO2 and ISC after amiloride showed the steepest slope for the sodium-deprived group and the flattest one for the sodium-loaded group. CONCLUSIONS: Baseline epithelial QO2 of sodium-deprived and control rats is reduced by ENaC blockade. Aldosterone increased QO2 proportionally to ISC augmentation in all groups, but the coupling between aerobic metabolism and electrogenic transport seems more efficient in sodium-deprived animals.

Downscaling the chemical oxygen demand test.

The usefulness of the standard chemical oxygen demand (COD) test for water characterization is offset to some extent by its requirement for highly toxic or expensive Cr, Ag, and Hg species. In addition, oxidation of the target samples by chromate requires a 2-3 h heating step. We have downscaled this method to obtain a reduction of up to ca. 80% in the use and generation of toxic residues and a time reduction of up to ca. 67%. This also translates into considerable energy savings by reducing the time required for heating as well as costly labour time. Such reductions can be especially important for analytical laboratories with heavy loads of COD analyses. Numerical results obtained with the standard COD method for laboratory KHP samples (potassium hydrogen phthalate) show an average relative error of 1.41% vs. an average of 2.14% obtained with the downsized or small-scale version. The average % standard deviation when using the former is 2.16% vs. 3.24% obtained with the latter. When analysing municipal wastewater samples, the relative error is smaller for the proposed small-scale method than for the standard method (0.05 vs. 0.58, respectively), and the % std. dev. is 1.25% vs. 1.06%. The results obtained with various industrial wastewaters show good agreement with those obtained using the standard method. Chloride ions do not interfere at concentrations below 2000 mg Nacl/L. This highly encouraging proof-of-concept offers a potentially alternative greener approach to COD analysis.

The effect of acute hypoxia on short-circuit current and epithelial resistivity in biopsies from human colon.

BACKGROUND AND AIMS: In isolated colonic mucosa, decreases in short-circuit current (ISC) and transepithelial resistivity (RTE) occur when hypoxia is either induced at both sides or only at the serosal side of the epithelium. We assessed in human colon biopsies the sensitivity to serosal-only hypoxia and mucosal-only hypoxia and whether Na, K-ATPase blockade with ouabain interacts with hypoxia. MATERIALS AND METHODS: Biopsy material from patients undergoing colonoscopy was mounted in an Ussing chamber for small samples (1-mm2 window). In a series of experiments we assessed viability and the electrical response to the mucolytic, dithiothreitol (1 mmol/l). In a second series, we explored the effect of hypoxia without and with ouabain. In a third series, we evaluated the response to a cycle of hypoxia and reoxygenation induced at the serosal or mucosal side while keeping the oxygenation of the opposite side. RESULTS: 1st series: Dithiothreitol significantly decreased the unstirred layer and ISC but increased RTE. 2nd series: Both hypoxia and ouabain decreased ISC, but ouabain increased RTE and this effect on RTE prevailed even during hypoxia. 3rd series: Mucosal hypoxia caused lesser decreases of ISC and RTE than serosal hypoxia; in the former, but not in the latter, recovery was complete upon reoxygenation. CONCLUSIONS: In mucolytic concentration, dithiothreitol modifies ISC and RTE. Oxygen supply from the serosal side is more important to sustain ISC and RTE in biopsy samples. The different effect of hypoxia and Na, K-ATPase blockade on RTE suggests that their depressing effect on ISC involves different mechanisms.

Extra-virgin olive oil ameliorates high-fat diet-induced seminal and testicular disorders by modulating the cholesterol pathway.

BACKGROUND: Rabbits are sensitive to dietary cholesterol and rapidly develop hypercholesterolemia, leading to prominent subfertility. Sterol regulatory element-binding protein isoform 2 drives the intracellular cholesterol pathway in many tissues, including the testicles. Its abnormal regulation could be the mainly responsible for the failure of suppressing cholesterol synthesis in a cholesterol-enriched environment, ultimately leading to testicular and seminal alterations. However, extra-virgin olive oil consumption has beneficial properties that promote lowering of cholesterol levels, including the recovery of seminal parameters altered under a high-fat diet. OBJECTIVES: Our goal was to investigate the effects of high-fat diet supplementation with extra-virgin olive oil at the molecular level on rabbit testes, by analyzing sterol regulatory element-binding protein isoform 2 protein and its corresponding downstream effectors. MATERIALS AND METHODS: During 12 months, male rabbits were fed a control diet, high-fat diet, or 6-month high-fat diet followed by 6-month high-fat diet plus extra-virgin olive oil. Serum lipids, testosterone levels, bodyweight, and seminal parameters were tested. The mRNA and protein levels of sterol regulatory element-binding protein isoform 2, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, and low-density lipoprotein receptor were determined by semi-quantitative polymerase chain reaction and Western blotting techniques. The expression pattern of sterol regulatory element-binding protein isoform 2 protein in the rabbit testicles was studied by indirect immunofluorescence. In addition, testicular cholesterol was detected and quantified by filipin staining and gas chromatography. RESULTS: The data showed that the addition of extra-virgin olive oil to high-fat diet reduced testicular cholesterol levels and recovered the expression of sterol regulatory element-binding protein isoform 2, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, and low-density lipoprotein receptor initially altered by the high-fat diet. DISCUSSION AND CONCLUSIONS: The combination of high-fat diet with extra-virgin olive oil encourages testicular recovery by modifying the expression of the enzymes related to intracellular cholesterol management.

GRP78-CAR T cell effector function against solid and brain tumors is controlled by GRP78 expression on T cells.

Lack of targetable antigens is a key limitation for developing successful T cell-based immunotherapies. Members of the unfolded protein response (UPR) represent ideal immunotherapy targets because the UPR regulates the ability of cancer cells to resist cell death, sustain proliferation, and metastasize. Glucose-regulated protein 78 (GRP78) is a key UPR regulator that is overexpressed and translocated to the cell surface of a wide variety of cancers in response to elevated endoplasmic reticulum (ER) stress. We show that GRP78 is highly expressed on the cell surface of multiple solid and brain tumors, making cell surface GRP78 a promising chimeric antigen receptor (CAR) T cell target. We demonstrate that GRP78-CAR T cells can recognize and kill GRP78+ brain and solid tumors in vitro and in vivo. Additionally, our findings demonstrate that GRP78 is upregulated on CAR T cells upon T cell activation; however, this expression is tumor-cell-line specific and results in heterogeneous GRP78-CAR T cell therapeutic response.

Modular chimeric cytokine receptors with leucine zippers enhance the antitumour activity of CAR T cells via JAK/STAT signalling.

The limited availability of cytokines in solid tumours hinders maintenance of the antitumour activity of chimeric antigen receptor (CAR) T cells. Cytokine receptor signalling pathways in CAR T cells can be activated by transgenic expression or injection of cytokines in the tumour, or by engineering the activation of cognate cytokine receptors. However, these strategies are constrained by toxicity arising from the activation of bystander cells, by the suboptimal biodistribution of the cytokines and by downregulation of the cognate receptor. Here we show that replacement of the extracellular domains of heterodimeric cytokine receptors in T cells with two leucine zipper motifs provides optimal Janus kinase/signal transducer and activator of transcription signalling. Such chimeric cytokine receptors, which can be generated for common γ-chain receptors, interleukin-10 and -12 receptors, enabled T cells to survive cytokine starvation without induction of autonomous cell growth, and augmented the effector function of CAR T cells in vitro in the setting of chronic antigen exposure and in human tumour xenografts in mice. As a modular design, leucine zippers can be used to generate constitutively active cytokine receptors in effector immune cells.

IPNV modulation of pro and anti-inflammatory cytokine expression in Atlantic salmon might help the establishment of infection and persistence.

IPNV is the agent of a well-characterized acute disease that produces a systemic infection and high mortality in farmed fish species and persistent infection in surviving fish after outbreaks. Because modulation of the host expression of pro and anti-inflammatory cytokines can help establish persistence, in this study, we examined the expression of IL-1ß, IL-8, IFNα1 and IL-10 during acute and persistent IPNV infection of Atlantic salmon. Results showed that IPNV infection induces an increase of the IFNα1 and IL-10 mRNA levels in the spleen and head kidney (HK) of fish after acute experimental infection. Levels of the pro-inflammatory cytokines IL-1ß and IL-8 did not rise in the spleen although an increase of IL-1ß, but not of IL-8, was observed in head kidney. In carrier asymptomatic salmon, cytokine gene expression of IFNα1 in the spleen and IL-10 in head kidney were also significantly higher than expression in non-carrier fish. Interestingly, a decrease of IL-8 expression was also observed. IPNV infection of SHK-1, which is a macrophage-like cell line of salmon, also induced an increase of expression of the anti-inflammatory cytokine IL-10 with no effects on the expression of IL-1ß and IL-8. The effects are induced by an unknown mechanism during viral infection because poly I:C and the viral genomic dsRNA showed the opposite effects on cytokine expression in SHK-1 cells. In summary, IPNV always induces up-regulation of the anti-inflammatory cytokine IL-10 in Atlantic salmon. As this is accompanied by a lack of induction of the pro-inflammatory cytokines IL-1ß and IL-8, the anti-inflammatory milieu may explain the high frequency, prevalence and persistence of IPNV in salmon. Effects might be part of the viral mechanisms of immune evasion.

Replacing dichromate with hydrogen peroxide in the chemical oxygen demand (COD) test.

The widely used standard method for chemical oxygen demand (COD) involves hazardous chromium species, and its two-hour heating protocol entails a substantial amount of energy expenditure. In the present work we report a proof of concept for a major modification of this method in the range 10-800 mgCOD/L, whereby H2O2 is proposed as a replacement oxidizer. This modification not only reduces the use of unsafe chromium species but also allows for the use of milder conditions that decrease the total energy outlay. The results are comparable with those obtained either with the standard method or with a commercial Hach® kit.

A novel combined electrochemical-magnetic method for water treatment.

Electrocoagulation (EC) is a wastewater treatment process in which aqueous pollutants can be removed by adsorption, entrapment, precipitation or coalescence during a coagulation step produced by electrochemically generated metallic species. When using Fe as the sacrificial electrode, Fe(2+) and Fe(3+) ions are formed. As Fe(3+) species are paramagnetic, this property can in principle be used to facilitate their removal through the application of a magnetic field. In the present work we present a proof-of-concept for a combined electrochemical-magnetic method for pollutant removal. For this approach, the amounts of Fe(2+) and Fe(3+) produced in an EC cell at various voltages were measured by spectroscopic methods to confirm that Fe(3+) species predominate (up to 84%). The effectiveness of the presence of a magnetic field in the precipitation of coagulants from a suspension was confirmed by monitoring the turbidity change versus time with and without exposure to a magnetic field, up to a 30% improvement.

Electrogenic transport, oxygen consumption, and sensitivity to acute hypoxia of human colonic epithelium.

INTRODUCTION: It is recognized that epithelial ion transport depends on oxygen supply, but this dependence has not been characterized in the human colon in vitro despite its surgical and clinical implications. PURPOSES: The aim of this study is to measure the oxygen consumption of colonic epithelium under conditions which preserve vectorial ion transport and to assess the sensitivity of the human colonic epithelium short-circuit current (I (sc)) to acute hypoxia induced in vitro. METHODS: Isolated mucosa preparations from human sigmoid colon were placed in a modified Ussing chamber which allows simultaneous measurement of short-circuit current (I (sc)) and oxygen consumption (QO(2)). In separate experiments, the sensitivity to acute hypoxia induced in a conventional Ussing chamber was assessed. RESULTS: Basal mean ± SEM values (n = 8) were I (sc) = 3.3 ± 0.5 µEq h(-1) cm(-2) and QO(2) = 8.09 ± 0.55 µmol h(-1) cm(-2). The contribution of the serosal side to the oxygen supply was higher than that of the mucosal side (p = 0.0023). Ouabain reduced I (sc) by 70% (P < 0.0001) and QO(2) by 26% (n = 8; P = 0.0009), suggesting that a large fraction of QO(2) is needed to support ouabain-sensitive electrogenic transport. Induction of hypoxia at both sides of the Ussing chamber caused a rapid decrease in I (sc) after 2 min. I (sc) was also significantly depressed when hypoxia was induced by 5 min in the serosal side (n = 6, P < 0.0001), but was unaffected by hypoxia induced in the mucosal side. CONCLUSION: The present results allow a better understanding of the clinical consequences of acute hypoxia on intestinal ion transport.

The actin filament network associated to Sertoli cell ectoplasmic specializations.

Junctional devices in Sertoli cells conform the blood-testis barrier and play a key role in maturation and differentiation of germ cells. The spacial distribution of ectoplasmic specializations of Sertoli cells was studied by beta-actin immunolabelling, using laser confocal and transmission electron microscopy. For confocal microscopy, beta-actin immunolabelling of ectoplasmic specializations was studied over the background of either prosaposin or glutaredoxin immunolabelling of the Sertoli cytoplasm. Labelling was found near the basal lamina, surrounding early spermatocytes (presumably in leptotene-zygotene) or at one of two levels in the seminiferous epithelium: (1) around deep infoldings of the Sertoli cell cytoplasm, in tubular stages before spermiation, and (2) in the superficial part of the seminiferous epithelium, in tubular stages after or during spermiation. For transmission electron microscopy, beta-actin immunolabelling of ectoplasmic specializations was also used. Ectoplasmic specializations were found at two different levels of the seminiferous epithelium. We also used freeze fracture to analyze the characteristics of tubulo-bulbar complexes, a known component of apical ectoplasmic specializations. Also, these different approaches allowed us to study the complex arrangement of the actin cytoskeleton of Sertoli cells branches, which surround germ cells in different stages of the spermatogenic cycle. Our results show a consistent labelling for beta-actin before, during and after the release of spermatozoa in the tubular lumen (spermiation) suggesting a significant role of the actin network in spermatic cell differentiation. In conclusion, significant interrelations among the beta-actin network, the junctional complexes of the blood-testis barrier and the ectoplasmic specializations were detected at different stages of the seminiferous cycle.

Expression of toll-like receptors 2 and 4 is upregulated during hospital admission in traumatic patients: lack of correlation with blunted innate immune responses.

BACKGROUND: There are reports with conflicting results on the expression of toll-like receptors (TLRs) in trauma patients. In addition, these studies analyzed TLR expression only at patients' hospital admission but not later when complications usually arise. OBJECTIVES: To analyze the surface expression of TLR2 and TLR4 on circulating monocytes from trauma patients during the hospitalization period and to correlate this with cytokine production after stimulation with TLR2 and TLR4 agonists. The phagocytic capacity of monocytes was analyzed at the same time points of TLR expression analysis; to correlate these molecular findings with the presence or absence of infections. METHODS: Prospective and observational study from June 2005 to June 2007. In all analysis, a control group composed of healthy subjects was included. RESULTS: We studied 70 trauma patients admitted to the intensive care unit (ICU) of a tertiary hospital, and 30 healthy volunteers. Blood samples were collected at hospital admission, on day 7 and 14. Forty-four patients (63%) developed at least one episode of infection. Monocytes from trauma patients expressed higher levels of TLR2 and TLR4 than monocytes from control subjects at all time points. Expression of TLR2 and TLR4 in monocytes from those patients who developed any infection was significantly lower than in those patients without infection but still significantly higher than in control subjects. Cellular responses to TLR4 agonist were impaired. Monocytes from traumatic patients phagocytosized less efficiently than monocytes from control subjects. CONCLUSIONS: These results indicate that trauma patients present a dysregulation of the innate immune system that persists during the first 14 days after hospital admission.

The exocyst controls lysosome secretion and antigen extraction at the immune synapse of B cells.

B lymphocytes capture antigens from the surface of presenting cells by forming an immune synapse. Local secretion of lysosomes, which are guided to the synaptic membrane by centrosome repositioning, can facilitate the extraction of immobilized antigens. However, the molecular basis underlying their delivery to precise domains of the plasma membrane remains elusive. Here we show that microtubule stabilization, triggered by engagement of the B cell receptor, acts as a cue to release centrosome-associated Exo70, which is redistributed to the immune synapse. This process is coupled to the recruitment and activation of GEF-H1, which is required for assembly of the exocyst complex, used to promote tethering and fusion of lysosomes at the immune synapse. B cells silenced for GEF-H1 or Exo70 display defective lysosome secretion, which results in impaired antigen extraction and presentation. Thus, centrosome repositioning coupled to changes in microtubule stability orchestrates the spatial-temporal distribution of the exocyst complex to promote polarized lysosome secretion at the immune synapse.

Efficient degradation of selected polluting dyes using the tetrahydroxoargentate ion, Ag(OH)4-, in alkaline media.

The use of soluble and highly oxidizing Ag(III) in the form of the tetrahydroxoargentate ion Ag(OH)4- is reported for the oxidation of surrogate organic recalcitrant dyes (i.e., rhodamine 6G (Rh6G) and fluorescein (Fl)). The possible use of Ag(OH)4- for the treatment of these and other refractory compounds is assessed. Such dyes were selected due to their common occurrence, stability, refractory nature, the relatively high toxicity of Rh6G, and their structural similarity to Fl. Several reaction intermediates/products were identified. The results showed that the highly oxidizing tetrahydroxoargentate anion was capable of degrading these recalcitrant dyes. Furthermore, the final degradation products do not represent a higher environmental risk than the original surrogates themselves. In addition, the partial mineralization of the dyes was proven.