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The market expansion due to globalization generated more anthropogenic contaminants, enhancing their negative impact on the environment. In order to prevent and balanced their negative effect, more rapid, sensitive and eco-friendly ecotoxicological studies as risk assessments were developed. Each country developed national programs to ensure the research infrastructure for implementation and development of new testing methodologies, generating new experimental data transferable to governmental authorities, economic agents and academia. In this review, we intend to highlighted the progress of Romanian researchers in the last 10 years in the field of ecotoxicology and aquatic risk assessment. The studies focused on aquatic systems as a non-targeted system to the anthropogenic pollutants impact. The implemented methodologies according with international requirements for chemical control like REACH Regulation, Detergents Regulation, Biocide Products Regulation or the Aquatic Risk Assessment Guidelines were presented. The ecotoxicological effects of various contaminants such as detergents, pharmaceuticals, pesticides, biocides or heavy metals and environmental polluted samples (surface water, sediment or sludge) were provided. Overall, the studies presented in our studies have a nationally novelty through the variety of test substances, the complexity of laboratory experiments and data processing. The research work has a continue applicability in industry and for control authorities. New accredited testing services that providing useful information about the chemicals and their impact on the environmental were introduced. In addition, the ecotoxicology studies and their challenges were a great source for professional training like PhD and master studies and for impact published papers.

The worldwide population rise corroborated with the raise of the health-care standards have generated an escalation of the antibiotic production and uncontrolled usage. The subsequent effects of this escalation have led to an increase of the antibiotic resistance rates, Romania is in the top of the EU countries regarding the antibiotic resistance rates, and to a continuous presence in the environment, including the aquatic environment. Unfortunately, the present design of the classical WWTPs is not optimized for the efficient removal of antibiotics since these compounds may have highly soluble and polar molecular structures. Instead, antibiotics removal using microorganisms could be an ecofriendly solution to this environmental issue, as long as their antibiotic degradation structures are not more toxic than the antibiotic itself. In the present review, we focus on the environmental presence and biodegradation of the most commonly used antibiotics as well as on their biodegradation, based on bacterial model, monitored by mass-spectrometric methods.

Three new samarium(III) complex compounds with chlorhexidine as ligand and mixed ligands chlorhexidine/o-phenanthroline have been prepared and characterized by elemental and thermogravimetrical analyses, infrared, electronic and luminescence spectra. The complexes corresponded to the formulas: [Sm(CHX)(NO3)2]� NO3, [Sm(CHX)(o-phen)2]� (NO3)3 and [Sm2(CHX)(o-phen)2(NO3)4]�(NO3)2, where CHX was the chlorhexidine. Chlorhexidine acted as neutral tetradentate NNNN donor, coordinating through the four imine nitrogen atoms. The two mixed ligands complexes showed a strong luminescent emission in solid state, characteristic of samarium(III) ion. The metal complexes and the chlorhexidine diacetate were in vitro evaluated for their antimicrobial activity against two Gram negative bacteria. The results revealed that all compounds were very effective in reducing the bacterial growth rate, even at low concentration.

The aim of this paper was to assess de ecotoxicity effects of four biocides, used as chemical disinfectants for swimming pool water treatment, on non-target organisms such as Daphnia magna and Heterocypris incongruens crustaceans. The tests showed a significant difference of sensitivity between species. The biocides, at the recommended disinfection doses, induced a very toxic or toxic effect on Daphnia magna, based on their mortality percentage. On the contrary, the observed ostracods lethal effects were generally less than 50% mortality and no significant growth inhibitions were observed. Overall, the ostracods had a higher tolerance to subacute doses, including disinfection doses of 20 mg/L (potassium mono persulfate), 5 mg/L (sodium dichloroisocyanurate), 8 mg/L (trichloroisocyanuric acid 90%) and 7 mg/L (trichloroisocyanuric acid 87-88%). In the context of a continuous biocides market expansion, the paper highlighted that our research proposed testing methods to obtain fast and accurate ecotoxicity data for a sustainable biocide production / authorization and also for an environmental hazard assessment.

The persistent pollutant azo dye, Acid Orange 10, was removed from the environment by an Amberlite IRA 400 anion-exchange resin and then biodegraded by two bacterial strains. Moreover, the pollutant ecotoxicity was studied by a bacterial model. The objective of this study was to offer a new synergetic/compatible physico-chemical and biological method to remove and biodegrade the azo dye, Acid Orange 10, from the environment. Bach method was applied. The Acid Orange 10 sorption on the resin (Acid Orange 10 concentration, its contact time and stability) was characterized based on the various parameters such as pH and temperature. The biodegradation and the ecotoxicity effect of Acid Orange 10 have been monitorized on two bacterial strains such as Salmonella enterica (gram-negative bacteria) and Enterococcus faecalis (gram-positive bacteria). Experimental data detected from ion-exchange studies showed that the strongly basic anion-exchange resin Amberlite IRA 400, can removed efficiently up to 96.8% at 10−2 M of Acid Orange 10 concentration. Moreover, 10−2 M Acid Orange 10 induced in 1 h a significant 50% growth inhibition on S. enterica, but not on the E. faecalis. This result was linked to E. faecalis ability to degrade more than 60% of Acid Orange 10 compared to S. enterica (30% Acid Orange 10 degradation). The methods tested in this study can be used for removed/biodegradation of Acid Orange 10 from different polluted waters.

In this present paper the behavior of two important pharmaceutical compounds, namely: nicotinic acid (vitamin PP/B3) and para-aminobenzoic acid (vitamin B10) to the transport through a liquid membrane of chloroform containing Aliquat 336 have been studied. The influence of operational parameters such as: solute concentration in the feed phase, stripping agent concentration in the stripping phase, transport time was monitored. The assessment of the obtained results allowed establishing of optimal transport conditions as well as identification of a kinetic transport model for nicotinic acid corresponding to consecutive irreversible 1st order reactions. The maximum transport efficiency was 96% and it was obtained at a concentration of 10-4 mol/L nicotinic acid in the feed phase and a concentration of 1 mol/L sodium hydroxide in the stripping phase. The transport time necessary to achieve this efficiency was 6 h. The results obtained led to important conclusions regarding the possibility of separating these two compounds. Analytical control of the process was done spectrophotometrically. The maximum absorbance was obtained at their characteristic wavelengths, namely 262 nm for nicotinic acid and respectively 267 nm for para-aminobenzoic acid.

Organic UV-filters, including 4-hydroxybenzophenone (4-HBP) and 2,4-dihydroxybenzophenone (BP-1), are persistent emerging contaminants whose presence in the environment poses a threat to aquatic organisms due to their endocrine disruptor’s properties. For this reason, finding suitable technological processes for their safety and efficient removal from the environment represent a priority for the scientific community. To the author’s knowledge, until now, there are no studies reporting the biodegradation of 4-HBP and BP-1 by a single bacteria strain. In this paper, there were tested the 4-HBP and BP-1 biodegradation potential of two Gram-positive (Staphylococcus aureus and Enterococcus faecalis) and two Gram-negative (Salmonella typhimurium and Serratia rubidae). The 4-HPB biodegradation process was observed only in the presence of Gram-negative bacterial strains. Thus, the biodegradation rates of 4-HBP reached up to 12.7% after 24h of incubation in presence of Salmonella thyphimurium and up to 24.0% after 24h of incubation with Serratia rubidae. Staphylococcus aureus was able to biodegrade 26.7% of BP-1, while Salmonella thiphymurium was able to biodegrade 14.7% of BP-1 after 24h of incubation. Their biodegradation products generated during the 4-HBP biodegradation process by Serratia rubidae were analyzed through LC-MS/MS analysis. The (bio)degradation products were benzophenone and a multi-hydroxylated derivative of 4-HBP and the degradation pathways were proposed. The data obtained in this study gave important information regarding the 4-HBP and BP-1 potential biodegradation by single bacterial strains.

Benzenthonium chloride (BEC, Hyamine 1622) is a quaternary ammonium surfactant with cationic properties widely used in cleaning, sanitation, and medical products that can become harmful to humans and also to the environment. This study aimed to evaluate its acute effects on Cyprinus carpio fish in terms of oxidative stress and morphological changes on hepatic tissue in order to show the sub-lethal toxicity of BEC. Fish were exposed to 1 mg/L BEC for 24, 48, and 96 h, and the liver samples were collected. The most significant changes were noticed after 96 h of exposure when the entire antioxidant enzyme system was affected. The activities of catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase decreased by 44%, 31%, 30%, and 45%, respectively, compared to control. Glucose-6-phosphate dehydrogenase activity decreased by 29% after 96 h of control, inducing a reduction of NADPH formation which decreased by half the level of reduced glutathione, the main non-enzymatic antioxidant. These effects correlated with the raised value of lipid peroxidation after 96 h and the morphology changes on hepatic tissue, such as cytoplasmic vacuolization and nuclear hypertrophy that could affect the normal function of the liver. All of these results showed acute toxicity of BEC on C. carpio after 96 h of exposure, causing oxidative stress response at the hepatic level.

This study investigates the synergistic effects of ion exchange and biodegradation methods to remove the Acid Blue 193 also called Gryfalan Navy Blue RL (GNB) dye from wastewater. Ion exchange studies were performed using a strongly basic anion exchange resin Amberlite IRA 400. The equilibrium was characterized by a kinetic and thermodynamic points of view, establishing that the sorption of the GNB dye was subject to the Freundlich isotherm model with R2 = 0.8710. Experimental results showed that the activated resin can removed up to 93.4% when the concentration of dye solution is 5.62�10-2 mM. The biodegradation of the GNB was induced by laccase, an enzyme isolated from white-rot fungus. It was also analyzed the role of pH and dye concentration on GNB biodegradation, so 5�10-2 mM dye had a maximum discoloration efficiency of 82.9% at pH of 4. The laccase showed a very fast and robust activity reaching in a few minutes a Km value of 2.2�10-1mM. In addition, increasing the GNB concentration up to 8�10-1 mM did not triggered a substrat inhibition effect on the laccase activity. Overall, in this study we proposed a mixt physicochemical and biological approach to enhance the GNB removal and biodegradability from the wastewaters and subsequently the environment.

The spread of a growing number of antibiotic-resistant bacteria (ARB) outside the clinical setting into the environment has been observed. The surface water plays an important role in ARB dissemination by being both habitats and transport systems for microorganisms. The ecological and touristic importance of the Danube Delta make it a European priority for close monitoring of its freshwater system. The main goal of this paper was to analyze how the St. Gheorghe branch of the Danube Delta microbiological contamination and their antibiotic-resistant profile were influenced by climate change, especially the global warming from 2013 up to 2019. In the surface water from all sampling points, total and fecal coliform bacteria showed a constant colony forming units (CFU) increase tendency during the years, with a sharp rise from 1500 CFU/mL in 2015 to more than 20,000 CFU/mL in 2019. The bacterial population’s analyses revealed an indirect proportionality between coliform bacteria density in water and sediment during the years in accordance with global warming. The most commonly identified bacterial strains such as Escherichia coli, Klebsiella oxytoca, Citrobacter freundii and Proteus mirabilis have been shown a resistance rate of approximatively 70% to beta-lactam antibiotics, especially to ampicillin and amoxicillin-clavulanate.

Metallic elements copper (Cu), zinc (Zn), nickel (Ni) and manganese (Mn) are some of the most commonly found in water and sediment samples collected from the Danube - Danube Delta. These elements are important as essential micronutrients, being normally present at low concentrations in biological organisms, but in high concentrations they become toxic with immediate and delayed effects. The role of this metals is still controversial, that�s why bioconcentration potential is so important. In this non-clinical study, we tested in vitro effect of heavy metals on carp, Cyprinus carpio, reproducing in vivo presence of Cu, Zn, Ni and Mn in the Romanian�s surface water. The toxicity tests were performed according to OECD 203 by detecting the average (50%) lethal concentration - LC50 on aquatic organisms (freshwater fish) at 96h. The results pointed out that, copper value for LC 50 at 96h was estimated as 3.4 mg/L (concentrations tested in the range of 0.1 - 4.75 mg/L). Zinc value for LC 50 at 96h was estimated as 20.8 mg/L (concentrations tested in the range of 0.028 � 29.6 mg/L). Nickel value for LC 50 at 96h was estimated as 40.1 mg/L (concentrations tested in the range of 0.008 - 84.5 mg/L). For manganese the mortality effects has recorded at LC 50 at 96h at estimated value higher than 53 mg/L (concentrations tested in the range of 0.04 - 53.9 mg/L). The accuracy of the testing metals concentration was insured by the screening of the dilution water, as well as food and control fish, acclimated in laboratory conditions.

During the past few decades, the anthropogenic activities induced worldwide changes in the ecological systems, including the aquatic systems. This work analysed the contamination level of groundwater resources from a rural agglomeration (Central-Western part of Prahova County) by biological and physico-chemical approaches. The study was performed during the autumn of 2016 on several sampling sites (four drilling wells, depth higher than 100 m supplying three villages; two wells lower than 10 m depth and one spring). The water quality was evaluated by comparison with the limit values of the drinking water quality legislation (Law no.458/2002) and the Order 621/2014 (applicable to all groundwater bodies of Romania). The results showed that phenols and metals (iron and manganese) exceeded the threshold values in all sampling sites. Moreover, the anthropogenic factors including agriculture, use of fertilizers, manures, animal husbandry led to an increase of the bacterial load, particularly at wells sites.

Nowadays, environmental pollution is constantly increasing, and depollution is the most important challenge of the 21st century. This paper is a review of organic pollutants in wastewater that can be removed by the photocatalysis process. Also are presented specialized studies that demonstrate the removal of organic pollutants from wastewater and the experimental conditions necessary to obtain high treatment efficiencies. The important classes of pollutants in wastewater are hydrocarbons, halo compounds, carbonyl derivatives, alcohols, phenols, pesticides, surfactants, nitrogen containing compounds and dyes, and represent a major source of water pollution. These organic pollutants are photodegradable and can be easily removed by the photocatalysis process.

The infectious hematopoietic necrosis virus (IHNV; Rhabdoviridae, Novirhabdovirus) infects teleost fish, such as salmon and trout, and is responsible for significant losses in the aquaculture industry and in wild fish populations. Although IHNV enters the host through the skin at the base of the fins, the viral adhesion and entry mechanisms are not fully understood. In recent years, evidence has accumulated in support of the key roles played by protein-carbohydrate interactions between host lectins secreted to the extracellular space and virion envelope glycoproteins in modulating viral adhesion and infectivity. In this study, we assessed in vitro the potential role(s) of zebrafish (Danio rerio) proto type galectin-1 (Drgal1-L2) and a chimera galectin-3 (Drgal3-L1) in IHNV adhesion to epithelial cells. Our results suggest that the extracellular Drgal1-L2 and Drgal3-L1 interact directly and in a carbohydrate-dependent manner with the IHNV glycosylated envelope and glycans on the epithelial cell surface, significantly reducing viral adhesion.

International Symposium "The Environment and the Industry", 26-27 September 2019, Bucharest, Romania, pp. 18-19

International Symposium "The Environment and the Industry", 26-27 September 2019, Bucharest, Romania, pp. 82-83

The economic factor of the microalgae harvesting step acts as a barrier to scaling up microalgae-based technology designed for wastewater treatment. In view of that, this study presents an alternative microalgae-bacteria system, which is proposed for eliminating the economic obstacle. Instead of the microalgae-bacteria (activated algae) flocs, the study aimed to develop activated algae granules comprising the microalgae Chlorella sp. as a target species. The presence of the filamentous microalgae (Phormidium sp.) was necessary for the occurrence of the granulation processes. A progressive decrease in frequency of the free Chlorella sp. cells was achieved once with the development of the activated algae granules as a result of the target microalgae being captured in the dense and tangled network of filaments. The mature activated algae granules ranged between 600 and 2,000 µm, and were characterized by a compact structure and significant settling ability (21.6 ± 0.9 m/h). In relation to the main aim of this study, a microalgae recovery efficiency of higher than 99% was achieved only by fast sedimentation of the granules; this performance highlighted the viability of the granular activated algae system for sustaining a microalgae harvesting procedure with neither cost nor energy inputs.

The study aims to assess the trophic state of Danube River and the main tributaries in the Southern part of Romania through chlorophyll „a” (Chl a) dynamics during 2015. The samples were collected with a seasonally frequency. Several environmental variables such as water temperature, pH, salinity, dissolved oxygen, nutrients were considered. In addition, the qualitative analysis of phytoplankton community was performed. The integrative approach, which included both in-situ and laboratory analysis, highlighted positive correlation between abiotic and biotic parameters. The chlorophyll „a” dynamics showed a close dependence with the sampling sites characteristics, the point sources of pollution, phytoplankton community and specific changes of chemical parameters.

This paper presents a new way to assess the environmental impact on historical artifacts using binary logistic regression. The prediction of the impact on the exhibits during certain pollution scenarios (environmental impact) was calculated by a mathematical model based on the binary logistic regression; it allows the identification of those environmental parameters from a multitude of possible parameters with a significant impact on exhibitions and ranks them according to their severity effect. Air quality (NO2, SO2, O3 and PM2.5) and microclimate parameters (temperature, humidity) monitoring data from a case study conducted within exhibition and storage spaces of the Romanian National Aviation Museum Bucharest have been used for developing and validating the binary logistic regression method and the mathematical model. The logistic regression analysis was used on 794 data combinations (715 to develop of the model and 79 to validate it) by a Statistical Package for Social Sciences (SPSS 20.0). Th...

The aquatic environment is under increased pressure by pharmaceutically active compounds (PhACs) due to anthropogenic activities. In spite of being found at very low concentrations (ng/L to μg/L) in the environment, PhACs represent a real danger to aquatic ecosystems due to their bioaccumulation and long-term effects. In this study, the presence in the aquatic environment of six non-steroidal anti-inflammatory drugs (ibuprofen, diclofenac, acetaminophen, naproxen, indomethacin, and ketoprofen), caffeine, and carbamazepine were monitored. Moreover, their aquatic risk and ecotoxicity by three biological models were evaluated. The monitoring studies performed in Romania showed that all studied PhACs were naturally present at concentrations >0.01 μg/L, pointing out the necessity to perform further toxicity tests for environmental risk assessment. The toxicity studies were carried out on aquatic organisms or bacteria and they indicated, for most of the tested PhACs, an insignificant or low toxicity effects: lethal concentrations (LC50) on fish Cyprinus carpio ranged from 42.60 mg/L to more than 100 mg/L; effective concentrations (EC50) on planktonic crustacean Daphnia magna ranged from 11.02 mg/L to more than 100 mg/L; inhibitory concentrations (IC50)/microbial toxic concentrations (MTC) on Vibrio fischeri and other bacterial strains ranged from 7.02 mg/L to more than 100 mg/L. The PhAC aquatic risk was assessed by using the ratio between measured environmental concentration (MEC) and predicted no effect concentration (PNEC) calculated for each type of organism. The average of quotient risks (RQs) revealed that the presence of these compounds in Romania's aquatic environment induced a lower or moderate aquatic risk.

Oral cancer is one of the most aggressive epithelial malignancies, whose incidence is on the rise. Previous studies have shown that in a subset of human oral squamous cell carcinoma (OSCC) tumor specimens, overexpression of the DPAGT1 gene, encoding the dolichol-P-dependent N-acetylglucoseamine-1-phosphate transferase, a key regulator of the metabolic pathway of protein N-glycosylation, drives tumor cell discohesion by inhibiting E-cadherin adhesive function. Recently, we reported that DPAGT1 was a target of the canonical Wnt signaling pathway. Here, we link overexpression of DPAGT1 in human OSCC tumor specimens to aberrant activation of canonical Wnt signaling. We report dramatic increases in β- and γ-catenins at the DPAGT1 promoter and correlate them with reduced expression of a Wnt inhibitor, Dickkopf-1 (Dkk-1). Using human squamous carcinoma cell lines of the head and neck, we show that partial inhibition of DPAGT1 reduces canonical Wnt signaling, indicating that DPAGT1 and canonical Wnt signaling function in a positive feedback loop. We provide evidence that E-cadherin inhibits DPAGT1, canonical Wnt signaling and the OSCC cancer phenotype by depleting nuclear β- and γ-catenins, with hypoglycosylated E-cadherin being the most effective. This suggests that in human OSCC, extensive N-glycosylation of E-cadherin compromises its ability to inhibit canonical Wnt signaling and DPAGT1 expression. Our studies reveal a novel interplay between DPAGT1/N-glycosylation and canonical Wnt signaling and suggest that dysregulation of this crosstalk is a key mechanism underlying OSCC. They also suggest that partial inhibition of DPAGT1 may represent an effective way to restore normal interactions among these essential pathways in oral cancer.

N-glycosylation of E-cadherin has been shown to inhibit cell-cell adhesion. Specifically, our recent studies have provided evidence that the reduction of E-cadherin N-glycosylation promoted the recruitment of stabilizing components, vinculin and serine/threonine protein phosphatase 2A (PP2A), to adherens junctions (AJs) and enhanced the association of AJs with the actin cytoskeleton. Here, we examined the details of how N-glycosylation of E-cadherin affected the molecular organization of AJs and their cytoskeletal interactions. Using the hypoglycosylated E-cadherin variant, V13, we show that V13/β-catenin complexes preferentially interacted with PP2A and with the microtubule motor protein dynein. This correlated with dephosphorylation of the microtubule-associated protein tau, suggesting that increased association of PP2A with V13-containing AJs promoted their tethering to microtubules. On the other hand V13/γ-catenin complexes associated more with vinculin, suggesting that they mediated the interaction of AJs with the actin cytoskeleton. N-glycosylation driven changes in the molecular organization of AJs were physiologically significant because transfection of V13 into A253 cancer cells, lacking both mature AJs and tight junctions (TJs), promoted the formation of stable AJs and enhanced the function of TJs to a greater extent than wild-type E-cadherin. These studies provide the first mechanistic insights into how N-glycosylation of E-cadherin drives changes in AJ composition through the assembly of distinct β-catenin- and γ-catenin-containing scaffolds that impact the interaction with different cytoskeletal components.

Basem T Jamal1,3, Mihai Nita-Lazar1,3, Zhennan Gao1, Bakr Amin1, Janice Walker2, Maria A Kukuruzinska11Department of Molecular and Cell Biology, Boston University Medical Center, Boston, MA, USA; 2Department of Anatomy, Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA; 3These authors contributed equally to this workAbstract: N-glycosylation of E-cadherin has been shown to inhibit cell–cell adhesion. Specifically, our recent studies have provided evidence that the reduction of E-cadherin N-glycosylation promoted the recruitment of stabilizing components, vinculin and serine/threonine protein phosphatase 2A (PP2A), to adherens junctions (AJs) and enhanced the association of AJs with the actin cytoskeleton. Here, we examined the details of how N-glycosylation of E-cadherin affected the molecular organization of AJs and their cytoskeletal interactions. Using the hypoglycosylated E-cadherin variant, V13, we show that V13/β-catenin complexes preferentially interacted with PP2A and with the microtubule motor protein dynein. This correlated with dephosphorylation of the microtubule-associated protein tau, suggesting that increased association of PP2A with V13-containing AJs promoted their tethering to microtubules. On the other hand, V13/γ-catenin complexes associated more with vinculin, suggesting that they mediated the interaction of AJs with the actin cytoskeleton. N-glycosylation driven changes in the molecular organization of AJs were physiologically significant because transfection of V13 into A253 cancer cells, lacking both mature AJs and tight junctions (TJs), promoted the formation of stable AJs and enhanced the function of TJs to a greater extent than wild-type E-cadherin. These studies provide the first mechanistic insights into how N-glycosylation of E-cadherin drives changes in AJ composition through the assembly of distinct β-catenin- and γ-catenin-containing scaffolds that impact the interaction with different cytoskeletal components.Keywords: E-cadherin, N-glycosylation, PP2A, microtubules, actin cytoskeleton

Influenza patients frequently display increased susceptibility to Streptococcus pneumoniae co-infection and sepsis, the prevalent cause of mortality during influenza pandemics. However, the detailed mechanisms by which an influenza infection predisposes patients to suffer pneumococcal pneumonia are not fully understood. A murine model for influenza infection closely reflects the observations in human patients, since if the animals that have recovered from influenza A virus (IAV) sublethal infection are challenged with S. pneumoniae, they undergo a usually fatal uncontrolled cytokine response. We have previously demonstrated both in vitro and in vivo that the expression and secretion of galectin-1 (Gal1) and galectin-3 (Gal3) are modulated during IAV infection, and that the viral neuraminidase unmasks galactosyl moieties in the airway epithelia. In this study we demonstrate in vitro that the binding of secreted Gal1 and Gal3 to the epithelial cell surface modulates the expression of SOCS1 and RIG1, and activation of ERK, AKT or JAK/STAT1 signaling pathways, leading to a disregulated expression and release of pro-inflammatory cytokines. Our results suggest that the activity of the viral and pneumococcal neuraminidases on the surface of the airway epithelial cells function as a "danger signal" that leads to rapid upregulation of SOCS1 expression to prevent an uncontrolled inflammatory response. The binding of extracellular Gal1 or Gal3 to the galactosyl moieties unmasked on the surface of airway epithelial cells can either "fine-tune" or severely disregulate this process, respectively, the latter potentially leading to hypercytokinemia.

Techniques for disrupting gene expression are invaluable tools for the analysis of the biological role(s) of a gene product. Because of its genetic tractability and multiple advantages over conventional mammalian models, the zebrafish (Danio rerio) is recognized as a powerful system for gaining new insight into diverse aspects of human health and disease. Among the multiple mammalian gene families for which the zebrafish has shown promise as an invaluable model for functional studies, the galectins have attracted great interest due to their participation in early development, regulation of immune homeostasis, and recognition of microbial pathogens. Galectins are β-galactosyl-binding lectins with a characteristic sequence motif in their carbohydrate recognition domains (CRDs), which comprise an evolutionary conserved family ubiquitous in eukaryotic taxa. Galectins are emerging as key players in the modulation of many important pathological processes, which include acute and chronic inflammatory diseases, autoimmunity and cancer, thus making them potential molecular targets for innovative drug discovery. Here, we provide a review of the current methods available for the manipulation of gene expression in the zebrafish, with a focus on gene knockdown [morpholino (MO)-derived antisense oligonucleotides] and knockout (CRISPR-Cas) technologies.

The process of endoplasmic reticulum-associated degradation (ERAD) involved in the degradation of misfolded N-linked glycoproteins utilizes Cdc48p which extracts misfolded glycoproteins from the lumen to the cytosol to present them for deglycosylation and degradation. Pkc1p has been identified as a component of the ERAD pathway, because deletion of the pkc1 gene impairs ERAD and causes accumulation of CPY* in the lumen of the ER, most probably because of the mislocalization of Cdc48p. In addition, we show that Cdc48p interacts in the cytosol with the deglycosylation enzyme, PNGase, only when Cdc48p is associated with a misfolded glycoprotein.

In the Gram-negative bacterium Campylobacter jejuni there is a pgl (protein glycosylation) locus-dependent general N-glycosylation system of proteins. One of the proteins encoded by pgl locus, PglB, a homolog of the eukaryotic oligosaccharyltransferase component Stt3p, is proposed to function as an oligosaccharyltransferase in this prokaryotic system. The sequence requirements of the acceptor polypeptide for N-glycosylation were analyzed by reverse genetics using the reconstituted glycosylation of the model protein AcrA in Escherichia coli. As in eukaryotes, the N-X-S/T sequon is an essential but not a sufficient determinant for N-linked protein glycosylation. This conclusion was supported by the analysis of a novel C. jejuni glycoprotein, HisJ. Export of the polypeptide to the periplasm was required for glycosylation. Our data support the hypothesis that eukaryotic and bacterial N-linked protein glycosylation are homologous processes.

Journal of Biotechnology, vol. 231, pp. S88-S89, 2016; European Biotechnology Congress, May 2016, Latvia

N-linked protein glycosylation is the most abundant posttranslation modification of secretory proteins in eukaryotes. A wide range of functions are attributed to glycan structures covalently linked to asparagine residues within the asparagine-X-serine/threonine consensus sequence (Asn-Xaa-Ser/Thr). We found an N-linked glycosylation system in the bacterium Campylobacter jejuni and demonstrate that a functional N-linked glycosylation pathway could be transferred into Escherichia coli . Although the bacterial N-glycan differs structurally from its eukaryotic counterparts, the cloning of a universal N-linked glycosylation cassette in E. coli opens up the possibility of engineering permutations of recombinant glycan structures for research and industrial applications.

Galectins are characterized by their binding affinity for ß-galactosides, a unique binding site sequence motif, and wide taxonomic distribution and structural conservation in vertebrates, invertebrates, protista, and fungi. Since their initial description, galectins were considered to bind endogenous (self) glycans and mediate developmental processes and cancer. In the past few years, however, numerous studies have described the diverse effects of galectins on cells involved in both innate and adaptive immune responses, and the mechanistic aspects of their regulatory roles in immune homeostasis. More recently, however, evidence has accumulated to suggest that galectins also bind exogenous (non-self) glycans on the surface of potentially pathogenic microbes, parasites, and fungi, suggesting that galectins can function as pattern recognition receptors (PRRs) in innate immunity. Thus, a perplexing paradox arises by the fact that galectins also recognize lactosamine-containing glycans on the host cell surface during developmental processes and regulation of immune responses. According to the currently accepted model for non-self recognition, PRRs recognize pathogens via highly conserved microbial surface molecules of wide distribution such as LPS or peptidoglycan (pathogen-associated molecular patterns; PAMPs), which are absent in the host. Hence, this would not apply to galectins, which apparently bind similar self/non-self molecular patterns on host and microbial cells. This paradox underscores first, an oversimplification in the use of the PRR/PAMP terminology. Second, and most importantly, it reveals significant gaps in our knowledge about the diversity of the host galectin repertoire, and the subcellular targeting, localization, and secretion. Furthermore, our knowledge about the structural and biophysical aspects of their interactions with the host and microbial carbohydrate moieties is fragmentary, and warrants further investigation.

Protein–carbohydrate interactions mediated by lectins have been recognized as key components of innate immunity in vertebrates and invertebrates, not only for recognition of potential pathogens, but also for participating in downstream effector functions, such as their agglutination, immobilization, and complement-mediated opsonization and killing. More recently, lectins have been identified as critical regulators of mammalian adaptive immune responses. Fish are endowed with virtually all components of the mammalian adaptive immunity, and are equipped with a complex lectin repertoire. In this review, we discuss evidence suggesting that: (a) lectin repertoires in teleost fish are highly diversified, and include not only representatives of the lectin families described in mammals, but also members of lectin families described for the first time in fish species; (b) the tissue-specific expression and localization of the diverse lectin repertoires and their molecular partners is consistent with their distinct biological roles in innate and adaptive immunity; (c) although some lectins may bind endogenous ligands, others bind sugars on the surface of potential pathogens; (d) in addition to pathogen recognition and opsonization, some lectins display additional effector roles, such as complement activation and regulation of immune functions; (e) some lectins that recognize exogenous ligands mediate processes unrelated to immunity: they may act as anti-freeze proteins or prevent polyspermia during fertilization.

Epithelial cell-cell adhesion is controlled by multiprotein complexes that include E-cadherin-mediated adherens junctions (AJs) and ZO-1-containing tight junctions (TJs). Previously, we reported that reduction of E-cadherin N-glycosylation in normal and cancer cells promoted stabilization of AJs through changes in the composition and cytoskeletal association of E-cadherin scaffolds. Here, we show that enhanced interaction of hypoglycosylated E-cadherin-containing AJs with protein phosphatase 2A (PP2A) represents a mechanism for promoting TJ assembly. In MDCK cells, attenuation of cellular N-glycosylation with siRNA to DPAGT1, the first gene in the N-glycosylation pathway, reduced N-glycosylation of surface E-cadherin and resulted in increased recruitment of stabilizing proteins gamma-catenin, alpha-catenin, vinculin and PP2A to AJs. Greater association of PP2A with AJs correlated with diminished binding of PP2A to ZO-1 and claudin-1 and with increased pools of serine-phosphorylated ZO-1 and claudin-1. More ZO-1 was found in complexes with occludin and claudin-1, and this corresponded to enhanced transepithelial resistance (TER), indicating physiological assembly of TJs. Similar maturation of AJs and TJs was detected after transfection of MDCK cells with the hypoglycosylated E-cadherin variant, V13. Our data indicate that E-cadherin N-glycans coordinate the maturity of AJs with the assembly of TJs by affecting the association of PP2A with these junctional complexes.

Metastasis, the spread of cancer cells throughout the body, claims 90% of solid tumor deaths. During metastasis, cancer cells undergo discohesion. An understanding of how to control and strengthen cell adhesion through junction formation could lead to methods for decreasing metastatic tendencies.Copyright © 2010 by ASME