Long chain capsaicin analogues synthetized by CALB-CLEAs show cytotoxicity on glioblastoma cell lines.

Glioblastoma is one of the most lethal tumors, displaying striking cellular heterogeneity and drug resistance. The prognosis of patients suffering from glioblastoma after 5 years is only 5%. In the present work, capsaicin analogues bearing modifications on the acyl chain with long-chain fatty acids showed promising anti-tumoral activity by its cytotoxicity on U-87 and U-138 glioblastoma multiforme cells. The capsaicin analogues were enzymatically synthetized with cross-linked enzyme aggregates of lipase B from Candida antarctica (CALB). The catalytic performance of recombinant CALB-CLEAs was compared to their immobilized form on a hydrophobic support. After 72 h of reaction, the synthesis of capsaicin analogues from linoleic acid, docosahexaenoic acid, and punicic acid achieved a maximum conversion of 69.7, 8.3 and 30.3% with CALB-CLEAs, respectively. Similar values were obtained with commercial CALB, with conversion yields of 58.3, 24.2 and 22% for capsaicin analogues from linoleic acid, DHA and punicic acid, respectively. Olvanil and dohevanil had a significant cytotoxic effect on both U-87 and U-138 glioblastoma cells. Irrespective of the immobilization form, CALB is an efficient biocatalyst for the synthesis of anti-tumoral capsaicin derivatives. KEY POINTS: • This is the first report concerning the enzymatic synthesis of capsaicin analogues from docosahexaenoic acid and punicic acid with CALB-CLEAs. • The viability U-87 and U-138 glioblastoma cells was significantly affected after incubation with olvanil and dohevanil. • Capsaicin analogues from fatty acids obtained by CALB-CLEAs are promising candidates for therapeutic use as cytotoxic agents in glioblastoma cancer cells.

Combined effect of copper sulfate and water temperature on key freshwater trophic levels - Approaching potential climatic change scenarios.

This work relied on the use microcosms to evaluate the individual and the combined effects of different levels of copper sulfate (0.0, 0.013, 0.064 and 0.318mg Cu L-1) - a fungicide commonly exceeding allowable thresholds in agricultural areas - and a range of water temperature increase scenarios (15, 20 and 25°C) on freshwater species belonging to different functional groups. Hence, the growth inhibition of primary producers (the microalgae Raphidocelis subcapitata and the macrophyte Lemna minor), as well as the survival and feeding behavior of a shredder species (the Trichoptera Schizopelex sp.) were evaluated. The results revealed that copper was toxic to primary producers growth, as well as shredders growth and survival, being the growth of L. minor particularly affected. Higher water temperatures had generally enhanced the growth of primary producers under non-contaminated (microalgae and macrophytes) or low-contaminated (macrophytes) conditions. Despite the tendency for a more pronounced toxicity of copper under increasing water temperatures, a significant interaction between the two factors was only observed for microalgae. Since the test organisms represent relevant functional groups for sustaining freshwater systems functions, the present results may raise some concerns on the impacts caused by possible future climate change scenarios in aquatic habitats chronically exposed to the frequent or intensive use of the fungicide copper sulfate.

Ecotoxicological assessment of the herbicide Winner Top and its active substances-are the other formulants truly inert?

Formulants used in Plant Protection Products (PPPs) to promote their efficiency are normally undisclosed in the PPP documentation, unless they bear a human health or environmental hazardous potential per se. PPP regulation also demands the assessment of putative interactions among formulants within each product recipe and consequent effects, but these results are often unavailable. Such a case is that of the herbicide Winner Top (Selectis®, Portugal), which we selected as a model commercial formulation in the present study specifically aiming at (i) characterising its aquatic toxicity towards sensitive eco-receptors (Raphidocelis subcapitata, Chlorella vulgaris, Lemna minor and Lemna gibba), as well as that of its active substances (a.s.) nicosulfuron and terbuthylazine; (ii) comparing the ecotoxicity among the commercial formulation, the corresponding mixture of its a.s. and this a.s.'s mixture increasingly enriched with the formulants. Single chemical testing revealed that terbuthylazine was the strongest microalgae growth inhibitor and nicosulfuron was the strongest macrophyte growth inhibitor. On the other hand, the commercial formulation was consistently less toxic than the corresponding mixture of the a.s., suggesting that Winner Top formulants (72.9% of the commercial formulation) interact with the a.s., promoting less than additive effects in the selected non-target species. Importantly, this environmentally protective effect of the formulation can be apparent. Because macrophytes share most physiological features with the weeds targeted by the studied herbicide, it is likely that increased application doses are required to reach desired efficacy levels with the consequent detrimental increase of PPP residues load in edge-of-field freshwater ecosystems.

Toxicity testing with the benthic diatom Navicula libonensis (Schoeman 1970): procedure optimisation and assessment of the species sensitivity to reference chemicals.

Periphytic communities are good indicators of river quality due to their general sensitivity to several pollutants. The primary objective of this study was to develop and optimize an ecotoxicological testing methodology using the freshwater benthic diatom Navicula libonensis. This species was selected due to its ubiquity and suitability for use under laboratory conditions. In the most suitable test medium (Chu10) the diatom demonstrated comparable sensitivity to potassium dichromate and 3,5-dichlorophenol using growth rate as the reference parameter, with median effect concentrations (ErC50) in the same order of magnitude (0.119 and 0.799 mg L(-1)) respectively. Yield-based estimates did not confirm this pattern and potassium dichromate was one order of magnitude more toxic than 3,5-dichlorophenol. The sensitivity of N. libonensis to the reference chemicals was higher than that published in the literature for several standard planktonic microalgae. This advantage, as well as the ability to grow the species in the laboratory, supports further efforts towards the standardisation of a toxicity testing protocol. In addition, the functional role of benthic diatoms in lotic ecosystems justifies their inclusion in risk assessment test batteries to better cover an environmental compartment that has so far been neglected.

Potential effects of the discharge of wastewater treatment plant (WWTP) effluents in benthic communities: evidence from three distinct WWTP systems.

Wastewater treatment plant (WWTP) effluents can be sources of environmental contamination. In this study, we aimed to understand whether effluents of three different WWTPs may have ecological effects in riverine recipient ecosystems. To achieve this, we assessed benthic phytobenthos and macroinvertebrate communities at three different locations relative to the effluent discharge: immediately upstream, immediately downstream and 500-m downstream the effluent discharge. Two approaches were employed: the ecological status classification as defined in the Water Framework Directive (WFD) based on biological indicators; constrained multivariate analysis to disentangle the environmental drivers (physicochemical variables and contaminants, namely metals, polycyclic aromatic hydrocarbons, pharmaceuticals, and personal care products) of ecological changes across the study sites. The results showed inconsistencies between the WFD approach and the multivariate approach, as well as between the responses of macroinvertebrates and diatoms. The WWTP effluents impacted benthic communities in a single case: macroinvertebrates were negatively affected by one of the WWTP effluents, likely by the transported pharmaceuticals (other stressors are essentially homogeneous among sites). Given the findings and the scarcity of consistent evidence on ecological impacts that WWTP effluents may have in recipient ecosystems, further research is needed towards more sustainable regulation and linked environmental protection measures.

A contribution to lipid digestion of Odobenidae family: Computational analysis of gastric and pancreatic lipases from walrus (Odobenus rosmarus divergens).

Triacylglycerols (TAGs) are a primary energy source for marine mammals during lipid digestion. Walruses (Odobenus rosmarus divergens) consume prey with a high content of long-chain polyunsaturated fatty acids; however, their digestive physiology and lipid digestion remain poorly studied. The present study aims to model and characterize the gastric (PWGL) and pancreatic (PWPL) lipases of Pacific walruses using an in-silico approach. The confident 3D models of PWGL and PWPL were obtained via homology modeling and protein threading and displayed the structural features of lipases. Molecular docking analysis demonstrated substrate selectivity for long-chain TAG (Trieicosapentaenoin; TC20:5n-3) in PWGL and short-chain TAG (Trioctanoin; TC8:0) in PWPL. Molecular dynamics simulations demonstrate that PWGL bound to tridocosahexaenoin (TC22:6n-3), the protein is considerably stable at all three salinity conditions, but fluctuations are observed in the regions associated with catalytic sites and the lid, indicating the potential hydrolysis of the substrate. This is the first study to report on the digestion of TAGs in walruses, including modeling and lipases characterization and proposing a digestive tract for pinnipeds.

Computational insights of the molecular recognition between volatile molecules and odorant binding proteins from the red palm weevil Rhynchophorus ferrugineus.

The red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae) is one of the most harmful pests for palm trees, causing serious economic damage worldwide. The present work aims to model and study the 3D structures of highly expressed odorant binding proteins from R. ferrugineus (RferOBPs) and identify possible binding modes and ligand release mechanism by docking and molecular dynamics. Highly confident 3D structures of a total of 11 odorant binding proteins (OBPs) were obtained with AlphaFold2. All 3D RferOBPs modeled structures displayed six characteristic α-helices, except for RfeOBP7 and RfeOBP10, which had an extra terminal α-helix. Among the eleven modeled RferOBPs, RferOBP4 was highly expressed in the antennae and subsequently selected for further analyses. Molecular docking analyses demonstrated that ferruginol, α-pinene, DEET, and picaridin can favorably bind the RferOBP4 cavity with low affinity energies. Molecular dynamic simulations of RferOBP4 bound to ferruginol at different pH values showed that low pH environments dictate a structural change into an apo-state that modifies the number of tunnels where the ligand can coexist, further triggering ligand release by a pH-dependent mechanism. This is the first report concerning the modelling and study of ligand binding modes and release mechanism of R. ferrugineus OBPs.Communicated by Ramaswamy H. Sarma.

Structural Behavior of Amphiphilic Triblock Copolymer P104/Water System.

A detailed study of the different structural transitions of the triblock copolymer PEO27-PPO61-PEO27 (P104) in water, in the dilute and semi-dilute regions, is addressed here as a function of temperature and P104 concentration (CP104) by mean of complimentary methods: viscosimetry, densimetry, dynamic light scattering, turbidimetry, polarized microscopy, and rheometry. The hydration profile was calculated through density and sound velocity measurements. It was possible to identify the regions where monomers exist, spherical micelle formation, elongated cylindrical micelles formation, clouding points, and liquid crystalline behavior. We report a partial phase diagram including information for P104 concentrations from 1 × 10-4 to 90 wt.% and temperatures from 20 to 75 °C that will be helpful for further interaction studies with hydrophobic molecules or active principles for drug delivery.

Thermoplastic Starch Biocomposite Films Reinforced with Nanocellulose from Agave tequilana Weber var. Azul Bagasse.

In this work, cellulose nanocrystals (CNCs), bleached cellulose nanofibers (bCNFs), and unbleached cellulose nanofibers (ubCNFs) isolated by acid hydrolysis from Agave tequilana Weber var. Azul bagasse, an agro-waste from the tequila industry, were used as reinforcements in a thermoplastic starch matrix to obtain environmentally friendly materials that can substitute contaminant polymers. A robust characterization of starting materials and biocomposites was carried out. Biocomposite mechanical, thermal, and antibacterial properties were evaluated, as well as color, crystallinity, morphology, rugosity, lateral texture, electrical conductivity, chemical identity, solubility, and water vapor permeability. Pulp fibers and nanocelluloses were analyzed via SEM, TEM, and AFM. The water vapor permeability (WVP) decreased by up to 20.69% with the presence of CNCs. The solubility decreases with the presence of CNFs and CNCs. The addition of CNCs and CNFs increased the tensile strength and Young's modulus and decreased the elongation at break. Biocomposites prepared with ubCNF showed the best tensile mechanical properties due to a better adhesion with the matrix. Images of bCNF-based biocomposites demonstrated that bCNFs are good reinforcing agents as the fibers were dispersed within the starch film and embedded within the matrix. Roughness increased with CNF content and decreased with CNC content. Films with CNCs did not show bacterial growth for Staphylococcus aureus and Escherichia coli. This study offers a new theoretical basis since it demonstrates that different proportions of bleached or unbleached nanofibers and nanocrystals can improve the properties of starch films.

Acute and chronic toxicity of Betanal(®)Expert and its active ingredients on nontarget aquatic organisms from different trophic levels.

As a way to improve the efficacy to target organisms, new pesticide generation is based on technologically advanced coformulations of two or more active ingredients. One example is Betanal(®)Expert, a postemergence herbicide composed of an Advanced Micro Droplet coformulation of phenmedipham, desmedipham, and ethofumesate. Although its composed formulation brings an increase in the pesticide performance, it can also enhance its toxicity to nontarget species. Therefore, the present study intends to contribute with relevant information on ecotoxicological effects of Betanal(®)Expert and its active ingredients on a battery of bioassays using aquatic species from different trophic levels: bacteria (Vibrio fischeri), microalgae (Pseudokirchneriella subcapitata, Chlorella vulgaris, and Chlamydomonas pseudocostata), macrophyte (Lemna minor), and cladocerans (Daphnia magna and Daphnia longispina) species. Across the organisms tested and endpoints measured, different responses concerning the toxicity of the active ingredients were found: (i) phenmedipham was the most toxic to V. fischeri and L. minor; (ii) desmedipham was the most toxic to P. subcapitata, D. magna, and D. longispina; (iii) and ethofumesate was the most toxic to C. pseudocostata and C. vulgaris. Furthermore, for C. pseudocostata and daphnids, the toxicity observed for some active ingredients was higher than the toxicity of the commercial formulation. In fact, in an attempt to evaluate the contribution of each active ingredient to the overall toxicity of Betanal(®)Expert, it was observed that, in general, the toxicity values obtained for desmedipham and phenmedipham were close or even lower to the values determined for Betanal(®)Expert, indicating that the ethofumesate can act as an antagonist in the three-way coformulation. In spite of the most impaired species being the photosynthetic ones, this study also showed pernicious effects on nonphotosynthetic organisms with distinct target sites. Therefore, our results underline the importance of clarifying the mode of action and metabolic pathways of these compounds on nonphotosynthetic species.

Characterization, comparative, and functional analysis of arylacetamide deacetylase from Gnathostomata organisms.

BACKGROUND: Arylacetamide deacetylase (AADAC) is a lipolytic enzyme involved in xenobiotic metabolism. The characterization in terms of activity and substrate preference has been limited to a few mammalian species. The potential role and catalytic activities of AADAC from other organisms are still poorly understood. Therefore, in this work, the physicochemical properties, proteomic analysis, and protein-protein interactions from Gnathostomata organisms were investigated. RESULTS: The analysis were performed with 142 orthologue sequences with ~ 48-100% identity with human AADAC. The catalytic motif HGG[A/G] tetrapeptide block was conserved through all AADAC orthologues. Four variations were found in the consensus pentapeptide GXSXG sequence (GDSAG, GESAG, GDSSG, and GSSSG), and a novel motif YXLXP was found. The prediction of N-glycosylation sites projected 4, 1, 6, and 4 different patterns for amphibians, birds, mammals, and reptiles, respectively. The transmembrane regions of AADAC orthologues were not conserved among groups, and variations in the number and orientation of the active site and C-terminal carboxyl were observed among the sequences studied. The protein-protein interaction of AADAC orthologues were related to cancer, lipid, and xenobiotic metabolism genes. CONCLUSION: The findings from this computational analysis offer new insight into one of the main enzymes involved in xenobiotic metabolism from mammals, reptiles, amphibians, and birds and its potential use in medical and veterinarian biotechnological approaches.

Texture Analysis in MRI of the Knee for an Early Diagnosis of Osteoarthritis.

Osteoarthritis is one of the most disabling diseases in developed countries. Its etiology is not firmly established, and the diagnosis is made by observing radiographs, assigning a degree of severity based on the information displayed. For this reason, the diagnosis is usually late and determined by the subjectivity of the doctor, which implies a restriction of the treatment. Magnetic resonance imaging (MRI) has allowed us to see in greater detail the alterations produced in soft joint structures. In this work, biomarkers for an early diagnosis of knee osteoarthritis have been developed by means of textures analysis on MRI. For this purpose, 50 subjects underwent T1-weighted MR image acquisitions: 25 controls and 25 diagnosed with knee osteoarthritis between grades I and III. Six regions were segmented on these images, corresponding to the femorotibial cartilage, femoral condyles, and tibial plateau. 43 textures were extracted for each region of interest (ROI) employing 5 statistical methods and 5 different predictive models were trained and compared. In addition, a study of the thickness of the cartilage was carried out to make a comparison with the texture analysis. The best result has been obtained using a K-nearest neighbor model with the combination of 33 textures (maximum value of AUC = 0.7684). Furthermore, in the analysis of the cartilage thickness, no statistically significant differences were found. Finally, it is concluded that the texture analysis has great potential for the diagnosis of knee osteoarthritis. Clinical Relevance - The current study establishes a methodology for an early diagnosis of knee osteoarthritis by means of MRI-based texture analysis, in a fast and objective manner.

Multiclass Classification of Prostate Tumors Following an MR Image Analysis-Based Radiomics Approach.

Prostate cancer is one of the most common cancers in men, with symptoms that may be confused with those caused by benign prostatic hyperplasia. One of the key aspects of treating prostate cancer is its early detection, increasing life expectancy and improving the quality of life of those patients. However, the tests performed are often invasive, resulting in a biopsy. A non-invasive alternative is the magnetic resonance imaging (MRI)-based PI-RADS v2 classification. The aim of this work was to find objective biomarkers that allow the PI-RADS classification of prostate lesions using a radiomics approach on Multiparametric MRI. A total of 90 subjects were analyzed. From each segmented lesion, 609 different texture features were extracted using five different statistical methods. Two feature selection methods and eight multiclass predictive models were evaluated. This was a multiclass study in which the best AUC result was 0.7442 ± 0.0880, achieved with the Naïve Bayes model using a subset of 120 features. Valuable results were also obtained using the Random Forests model, obtaining an AUC of 0.7394 ± 0.0965 with a lower number of features (52). Clinical Relevance- The current study establishes a methodology for classifying prostate cancer and supporting clinical decision-making in a fast and efficient manner and avoiding additional invasive procedures using MRI.

Testing the response of benthic diatom assemblages to common riverine contaminants.

Benthic diatoms constitute keystone assemblages in riverine ecosystems, and their structure is used to support regulatory water quality assessment. However, no standard ecotoxicological tests exist using integrated responses of the benthic diatom assemblages. This work aimed to assess whether benthic diatom assemblages are responsive to different riverine contaminants through a previously developed rapid toxicity test, supporting future attempts towards its standardization and integration in both prospective and retrospective Environmental Risk Assessment (ERA) schemes. We selected two benthic diatoms assemblages likely responding similarly to pollution (similar IPS diatom index score), collected from two rivers in Northern-Central Portugal (sites: Palhal and Cabreia). Fresh whole diatom assemblages were exposed for 48 h to five model contaminants (glyphosate, imidacloprid, SDS, CuSO4, and Pb). At the end of the test, changes induced by the exposures in overall yield and in the yield of each diatom genus were assessed. The assemblage collected at Palhal was invariably more responsive and sensitive than that collected at Cabreia, both considering overall and genus-specific yields, regardless of the tested contaminant. Achnanthes, Fragilaria and Navicula were the most responsive genus, regardless of the tested contaminant or assemblage. The distinct response profiles observed for the two assemblages to the same contaminants at the same concentration ranges suggest that using this test method to support prospective ERA is inadequate. However, the method can be an asset supporting retrospective ERA, as the responses seem to be shaped by the interplay of resilience drivers promoted by the local conditions, e.g. adaptive changes in assemblage structure.

Responses of benthic diatoms to waters affected by post-fire contamination.

Wildfire effects go beyond direct impact in terrestrial ecosystems. Specifically, the periphytic communities of aquatic ecosystems standing within and downstream the burnt areas are relevant ecological receptors of post-fire runoff contamination. Nevertheless, the off-site impacts of wildfires in these communities are limitedly studied so far. The present study aimed to assess the effects of river water contaminated with ash-loaded runoff in the growth benthic diatom Navicula libonensis (Schoeman 1970). Four surface water samples were collected approximately one year after the wildfire for laboratory testing with the diatom: one was collected from a site upstream the burnt area, within the Unhais river (UU); three were collected from sites standing within the burnt area, one in the Unhais river (UB) and two in the Zêzere river (Z1 and Z2), reflecting different hydrological regimes. N. libonensis was proven able to discriminate among river sites affected and unaffected by wildfire runoff, reflecting, in general, the expected trends considering the physico-chemical characterization of the water samples. The water samples from the sites standing within the burnt area inhibited the biomass yield and growth rate of the tested diatom, ranking the samples regarding toxicity as follows: Z1 > UB > Z2 > UU. However, UB rather than Z1 presented the highest contaminant burden, namely metal elements, and some were found above widely accepted safety benchmarks (polycyclic aromatic hydrocarbons were not detected). This inconsistency can be linked to unknown interactions among metals within each water sample, to differential nutrient enrichment of samples, as well as hydrological factors. Overall, our results suggest that monospecific laboratory assays with sensitive diatoms can be valuable as cost-effective screening tools to prioritize sites affected by wildfires runoff requiring in-depth monitoring of negative effects in benthic producer communities.

Mechanical and Physicochemical Properties of 3D-Printed Agave Fibers/Poly(lactic) Acid Biocomposites.

In order to provide a second economic life to agave fibers, an important waste material from the production of tequila, filaments based on polylactic acid (PLA) were filled with agave fibers (0, 3, 5, 10 wt%), and further utilized to produce biocomposites by fused deposition modeling (FDM)-based 3D printing at two raster angles (-45°/45° and 0°/90°). Differential scanning calorimetry, water uptake, density variation, morphology, and composting of the biocomposites were studied. The mechanical properties of the biocomposites (tensile, flexural, and Charpy impact properties) were determined following ASTM international norms. The addition of agave fibers to the filaments increased the crystallinity value from 23.7 to 44.1%. However, the fibers generated porous structures with a higher content of open cells and lower apparent densities than neat PLA pieces. The printing angle had a low significant effect on flexural and tensile properties, but directly affected the morphology of the printed biocomposites, positively influenced the impact strength, and slightly improved the absorption values for biocomposites printed at -45°/45°. Overall, increasing the concentrations of agave fibers had a detrimental effect on the mechanical properties of the biocomposites. The disintegration of the biocomposites under simulated composting conditions was slowed 1.6-fold with the addition of agave fibers, compared to neat PLA.

Optimization of the Obtaining of Cellulose Nanocrystals from Agave tequilana Weber Var. Azul Bagasse by Acid Hydrolysis.

A multilevel factorial design of 23 with 12 experiments was developed for the preparation of cellulose nanocrystals (CNC) from Agave tequilana Weber var. Azul bagasse, an agro-industrial waste from tequila production. The studied parameters were acid type (H2SO4 and HCl), acid concentration (60 and 65 wt% for H2SO4, 2 and 8N for HCl) temperature (40 and 60 °C for H2SO4, 50 and 90 °C for HCl), and hydrolysis time (40, 55 and 70 min for H2SO4; and 30, 115 and 200 min for HCl). The obtained CNC were physical and chemically characterized using dynamic light scattering (DLS), atomic force microscopy (AFM), Fourier-transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XDR) techniques. The maximum CNC yield was 90 and 96% for HCL and H2SO4, respectively, and the crystallinity values ranged from 88-91%. The size and morphology of A. tequilana CNC strongly depends on the acid type and hydrolysis time. The shortest CNC obtained with H2SO4 (65 wt%, 40 °C, and 70 min) had a length of 137 ± 68 nm, width 33 ± 7 nm, and height 9.1 nm, whereas the shortest CNC obtained with HCl (2 N, 50 °C and 30 min) had a length of 216 ± 73 nm, width 69 ± 17 nm, and height 8.9 nm. In general, the obtained CNC had an ellipsoidal shape, whereas CNC prepared from H2SO4 were shorter and thinner than those obtained with HCl. The total sulfate group content of CNC obtained with H2SO4 increased with time, temperature, and acid concentration, exhibiting an exponential behavior of CSG=aebt.

Using flow cytometry for bacterioplankton community analysis as a complementary tool to Water Framework Directive to signal putatively impacted sites.

Metal contamination, as well as pesticides, organic matter and nutrient input are main factors leading to freshwater ecosystems degradation. The Water Framework Directive (WFD) was implemented within the European Union with the ultimate goal of promoting a good ecological status in all European waterbodies. However, the broad implementation of the bioassessment behind WFD is costly and time-consuming and the search for complementary methodologies has been given significant attention. In this context, the main goal of this study was to evaluate whether flow cytometry (FCM) and denaturing gradient gel electrophoresis (DGGE) can be used as cellular/molecular tools to efficiently assess riverine bacterioplankton communities and relevantly inform on the ecological quality of these ecosystems. Caima river was chosen as case study using three sampling sites reflecting different levels and types of contamination (point-source organic and metal input). Both bacterioplankton community assessment approaches (DGGE and FCM), as well as macroinvertebrate and periphyton communities were consistent in signaling organic contamination. The putatively metal-loaded site bears some contradictory results depending on the community focused, possibly due to the overall low levels of metals actually found and seasonality. When comparing the two bacterioplankton community analysis tools, DGGE and FCM, the results obtained were essentially coherent, with FCM being simpler, faster and still accurate for screening bacteria communities via quantification of bacteria of high and low DNA content. This highlights the suitability of the FCM approach for prioritization of contaminated sampling sites and reinforces the suitability of using bacterioplankton communities as the focus of rapid tools to complement bioassessment sensu the WFD methodology, e.g. assisting the prioritization of potentially impacted areas.

Cross-linked enzyme aggregates of recombinant Candida antarctica lipase B for the efficient synthesis of olvanil, a nonpungent capsaicin analogue.

Despite the proven therapeutic role of capsaicin in human health, its usage is still hampered by its high pungency. In this sense, nonpungent capsaicin analogues as olvanil are a feasible alternative to the unpleasant sensations produced by capsaicin while maintaining a similar pharmacological profile. Olvanil can be obtained by a lipase-catalyzed chemoenzymatic process. In the present work, recombinant Candida antarctica lipase B (CALB) was expressed in Pichia pastoris and subsequently immobilized by cross-linked enzyme aggregate (CLEA) methodology for the synthesis of olvanil. The CALB-CLEAs were obtained directly from the fermentation broth of P. pastoris without any purification step in order to assess the role of the contaminant proteins of the crude extract as co-feeders. The CALB-CLEAs were also bioimprinted to enhance the catalytic performance in olvanil synthesis. When CALB was precipitated with isopropanol, the obtained CALB-CLEAs exhibited the highest activity in the synthesis of olvanil, regardless of the glutaraldehyde concentration. The maximum product synthesis was found at 72 hr obtaining 6.8 g L-1 of olvanil with a reaction yield of 16%. When CALB was bioimprinted with olvanil, the synthesis was enhanced 1.3 times, reaching 10.7 g L-1 of olvanil at 72 hr of reaction with a reaction yield of 25%. Scanning electron microscopy images indicated different morphologies of the CLEAs depending on the precipitating agent and the template used for bioimprinting. Recombinant CALB-CLEAs obtained directly from the fermentation broth are a suitable alternative to commercial enzymatic preparations for the synthesis of olvanil in organic medium.

Is the aquatic toxicity of cationic polyelectrolytes predictable from selected physical properties?

Cationic acrylamide-based polyelectrolytes (cPAM) are widely used in industry. They can be designed for optimal performance in a specific application, but this opportunity means the environmental safety of all different alternatives needs to be addressed. Both the inclusion of environmental toxicity as a design variable and the establishment of relationships between structure and ecotoxicity are thus current challenges. The aim of this study was to assess whether structural variables such as molecular weight, charge density and the integrative intrinsic viscosity parameter can be used to predict the environmental safety of cPAMs, as well as if these relationships are stable when the biological models change. Five cPAMs comprising molecular weight and charge density gradients were tested against bacteria, microalgae, macrophytes and daphnids. While correlations were found between physical properties of cPAMs as expected, no clear ecotoxicity patterns could be identified. All cPAMs can be classified as harmful to aquatic life on the basis of the responses elicited in the most sensitive organisms, microalgae and daphnids. Unicellular bacteria were the least sensitive eco-receptors possibly due to cell wall structure or the protective effect of the ionic strength of the test medium. The macrophytes were also tolerant to cPAMs exposure, which may be related to exposure avoidance mechanisms. The order of toxicity of cPAMs depended on the test organism, preventing the establishment of stable structure-ecotoxicity relationships. Therefore, the study leads to the overall generalist recommendation of relying on the most sensitively responding test organisms when developing new (eco)safe-by-design cPAMs.