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Amino groups on the antibody surface (amino terminus and Lys) are very interesting conjugation targets due to their substantial quantities and selectivity toward various reactive groups. Oriented immobilization of antibodies via amino moieties on the Fc region instead of the antigen-binding fragment (Fab) is highly appreciated to conserve antigen-binding capacity. In this paper, targeting amino moieties on distinct regions, three antibody immobilization strategies were compared with the recognition ability of corresponding adsorbents. Our results demonstrate that oriented immobilization of antibodies onto heterofunctional chelate-epoxy support selectively involving Lys residues placed at the bottom of the Fc region, thus preserved the highest antigen recognition capacity (over 75% functionality). For homofunctional aldehyde support, immobilization at pH 10 demonstrates 50% remaining functionality due to the random orientation of tethered antibodies; while only 10% functionality remained when N-terminus were specifically conjugated at pH 8.5. With the rationalization of moieties density onto heterofunctional support, 2-fold recognition capacity was exhibited over randomly immobilization for antigens with higher size (β-galactosidase, 425 kDa vs. horseradish peroxidase, 40 kDa). Meanwhile, at least 97% of antigens with a varied concentration in diluted human serum were efficiently captured by the optimized chelate-epoxy support. Therefore, our antibody immobilization protocol proved the potential to be utilized as a promising candidate to capture voluminous antigens (large proteins and cells) in real samples.

To protect native biodiversity and habitats from the negative impacts of biological invasions, comprehensive studies and measures to anticipate invasions are required, especially across countries in a transfrontier context. Species distribution models (SDMs) can be particularly useful to integrate different types of data and predict the distribution of invasive species across borders, both for current conditions and under scenarios of future environmental changes. We used SDMs to test whether predicting invasions and potential spatial conflicts with protected areas in a transfrontier context, under current and future climatic conditions, would provide additional insights on the patterns and drivers of invasion when compared to models obtained from predictions for individual regions/countries (different modelling strategies). The framework was tested with the invasive alien plant Acacia dealbata in North of Portugal/NW Spain Euro-region, where the species is predicted to increase its distribution under future climatic conditions. While SDMs fitted in a transfrontier context and using “the national strategy (with Portugal calibration data) presented similar patterns, the distribution of the invasive species was higher in the former. The transfrontier strategy expectedly allowed to capture a more complete and accurate representation of the species’ niche. Predictions obtained in a transfrontier context are therefore more suitable to support resource prioritisation for anticipation and monitoring impacts of biological invasions, while also providing additional support for international cooperation when tackling issues of global change. Our proposed framework provided useful information on the potential patterns of invasion by A. dealbata in a transfrontier context, with an emphasis on protected areas. This information is crucial for decision-makers focusing on the prevention of invasions by alien species inside protected areas in a transfrontier context, opening a new way for collaborative management of invasions.

Different immobilized biocatalysts of Thermomyces lanuginosus lipase (TLL) exhibited different properties for the ethanolysis of high oleic sunflower oil in solvent-free systems. TLL immobilized by interfacial adsorption on octadecyl (C-18) supports lost its 1,3-regioselectivity and produced more than 99% of ethyl esters. This reaction was influenced by mass-transfer limitations. TLL adsorbed on macroporous C-18 supports (616 Å of pore diameter) was 10-fold more active than TLL adsorbed on mesoporous supports (100–200 Å of pore diameter) in solvent-free systems. Both derivatives exhibited similar activity when working in hexane in the absence of diffusional limitations. In addition, TLL adsorbed on macroporous Purolite C-18 was 5-fold more stable than TLL adsorbed on mesoporous Sepabeads C-18. The stability of the best biocatalyst was 20-fold lower in anhydrous oil than in anhydrous hexane. Mild PEGylation of immobilized TLL greatly increased its stability in anhydrous hexane at 40 °C, fully preserving the activity after 20 days. In anhydrous oil at 40 °C, PEGylated TLL-Purolite C-18 retained 65% of its initial activity after six days compared to 10% of the activity retained by the unmodified biocatalyst. Macroporous and highly hydrophobic supports (e.g., Purolite C-18) seem to be very useful to prepare optimal immobilized biocatalysts for ethanolysis of oils by TLL in solvent-free systems. Spanish Ministry of Economy, Industry and Competitiveness (BIO2012-36861; CTQ2015-70348; IJCI-2014-19260) Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) (2016/10636-8; 2015/10530-2; 2013/20826-0) 3.503 JCR (2019) Q2, 47/156 Biotechnology & Aplied Microbiology 0.992 SJR (2019) Q1, 60/359 Biotechnology No data IDR 2019 UEM

In this study, Penicillium sp. section Gracilenta CBMAI 1583 was used to produce lipase under submerged conditions. The enzyme was purified and the biochemical properties of both the crude and purified enzymes were evaluated. Maximum lipase production (1.62 U mL−1) was obtained using olive oil 0.5% (w v−1) after 72 h of cultivation, representing a 90% increase in the lipase initially produced. The enzyme was purified using hydrophobic interaction chromatography (phenyl Sepharose) under conditions which allowed its interfacial activation. The partially purified sample showed an enzyme with esterase activity (65.4 kDa) on α- and β-naphthyl acetate and other with lipase activity (52.9 kDa) on octyl oleate. Optimum activity of crude and purified lipase was observed at pH 4.0 and 70 °C. The purified lipase was activated by NaCl, BaCl2, NH4Cl, MnSO4 and MgSO4; it also presented high stability in organic solvents such as hexane, 2.2.4-trimethylpentane, acetone, DMSO and toluene. Maximum enzyme activity was observed with p-nitrophenyl decanoate as substrate; and the enzyme kinetics showed to be directly affected by Triton X-100. The enzyme shows potential application in processes that operate in acid pH, such as treatment of dairy and industry effluents, resolution of esters in the pharmaceutical industry or in the food industry, as well in synthesis reaction under non-aqueous conditions., The authors gratefully acknowledge to São Paulo Research Foundation - FAPESP, Brazil, for the scholarship granted to the first author and Spanish Ministry of Science and Innovation - MICINN, Spain (Project BIO-2012–36861).

This paper reports the development of a novel strategy for the easy immobilization of low molecular weight haptens to microarray platforms by coating with modified lipase molecules. The chimers consist of a dextran network covalently coupled to bacterial thermoalkalophilic lipase (BTL2). The relative high surface hydrophobicity of lipase allows easy immobilization of the conjugates onto glass planar waveguides previously modified with 1,1,1,3,3,3-hexamethyldisilazane, via nonspecific hydrophobic interactions, while the dextran layer provides a three-dimensional network that can be easily modified with different functional groups for further hapten immobilization on the glass substrate. The conjugates have been applied to the development of microarray biosensors for the simultaneous detection of three water pollutants namely, atrazine (ATR), a triazine pesticide, enrofloxacin (ENRO), a fluoroquinolone antimicrobial, and the hepatotoxin microcystin LR (MCLR). In an alternative approach, the chimeric hapten-dextran-BTL2 constructs were synthesized, purified and further immobilized on the hydrophobic transducer surface through their hydrophobic faces (Janus character). Detection of the three model targets was based in a competitive immunoassay format. A combination of detection and tracer antibodies was optimized to avoid significant cross-reactivity. Evanescent wave excitation was used to excite the fluorescent immunocomplexes formed on the planar waveguide and the identity of the target analyte was encoded by its location in the detection platform. The developed microarray allows the simultaneous detection of the target pollutants with IC 50 values, of 4.4 ± 0.7, 75 ± 9, 18 ± 4 ng L −1 for ATR, ENRO and MCLR, respectively and relative standard deviations (RSDs) lower than 15%. Waveguide functionalization using the dextran-BTL2 chimers is rapid, simple and allows the development of highly sensitive microarrays that can be easily applied to the multiplexed detection of pollutants in environmental samples.

This study covers a modified semi-quantitative approach to detecting signature peptides for body fluid identification. A liquid chromatography tandem mass spectrometer normally used for toxicology was adapted to detect target ion transitions for five semen or saliva specific peptides. Peptide concentrations were measured based on a mixture of synthetic peptide standards. Samples were processed using a three-hour trypsin digestion and Microcon membrane filtration. This method generates PCR compatible DNA and peptide fractions that can be typed without any further treatment. Preliminary validation tests covered stains on different substrates, semen/saliva mixtures, limit of detection, and repeatability. All signature peptides were present at different concentrations, varied amongst donors, and were tissue specific. Saliva peptides were detected at lower concentrations and had a higher limit of detection (LOD). Semen peptides had higher concentrations and were detected even as a minor component in a mixture. All semen peptides and all, but one, saliva peptides were detected on the various substrates. Semen peptide concentrations had relative standard deviations (RSDs) lower than 20%, indicating high repeatability, different from saliva where higher RSDs were observed. DNA fractions did not show signs of degradation or PCR inhibition.

Improving biodiversity predictions is essential if we are to meet the challenges posed by global change. As knowledge is key to feed models, we need to evaluate how debated theory can affect models. An important ongoing debate is whether environmental constraints limit the number of species that can coexist in a community (saturation), with recent findings suggesting that species richness in many communities might be unsaturated. Here, we propose that biodiversity models could address this issue by accounting for a duality: considering communities as unsaturated but where species composition is constrained by different scale-dependent biodiversity drivers. We identify a variety of promising advances for incorporating this duality into commonly applied biodiversity modelling approaches and improving their spatial predictions., Trends The majority of biodiversity modelling approaches do not explicitly address the question of saturation. Theoretical and methodological implications of saturation or unsaturation in biodiversity modelling. Addressing saturation or unsaturation is vital to produce more reliable conservation strategies. Integrative community modelling frameworks may be the way forward.

Background: Anosmia/hyposomia have been described as early signs of COVID-19 infection in adults, including young asymptomatic patients who commonly refer olfactory disfunction as their only clinical manifestation. Very few studies involving paediatric age patients have been published until now. This study is aimed to determine the presence of olfactory dysfunction in children with COVID-19 infection through the use of a self-reported questionnaire and a new olfactory screening tool. Methods: Nested case-control study. All paediatric patients screened by reverse transcription polymerase chain reaction (RT-PCR) and Anti-SARS-CoV-2 antibodies for COVID-19 infection, during the study period (March-May 2020), were asked to respond to a questionnaire about olfactory disfunction symptoms. Patients above six years of age also performed an odor identification test based on seven odorants (Kradeo®). This test was designed based on our cultural context and eating habits. Findings: 126 patients were recruited, including 33 with COVID-19 infection. 15% of the infected children referred anosmia and/or dysgeusia on the questionnaire, all of them were older than eleven years. The results of the odor test (69 patients) revealed subtle disturbances in the infected group (mostly misrecognition of odorants). Median odorant recognition was 3 odors [Interquartile range (ICR) 2-4] in case group and 4 [ICR 3-5] in controls. Male patients showed significantly bigger disturbances than girls in both groups (p=0·03). Interpretation: Self-referred prevalence of olfactory disfunction in our sample of infected children is lower than that described in adults, especially among the youngest ones, maybe due to immature development of angiotensin-converting enzyme 2 (ACE2) receptors expressed in nasal mucosa. Nevertheless, one month after infection, subtle disturbances (misrecognition of odors) were recognised among the infected children. Our screening olfactory test seems to be a hygienic, easy to perform tool, suitable for children older than six in our population. Funding Statement: This work received no specific funding. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The study was approved by the Regional Ethics Committee.

This work evaluated the immobilization of Candida antarctica lipase B (CALB) on a new heterofunctional support, prepared by silanization of silica particles with triethoxy(octyl)silane and trietoxy(3-aminopropyl)silane, followed by activation with glutaraldehyde. The new support (octyl-silica-amino-glutaraldehyde, OSGlu) exhibits features of an anionic exchanger and hydrophobic adsorbent capable of adsorbing and covalently linking the lipase under mild conditions in a single step. As comparison, CALB was also immobilized on silica coated with octyl, amino-glutaraldehyde, octyl and glyoxyl, and octyl and epoxy groups. Among these supports, CALB immobilized on OSGlu (namely CALB-OSGlu), showed the highest recovered activity (71 ± 2%) and thermal stability in tert -butyl alcohol (full activity was recovered after 100 h at 65 °C). CALB immobilized on silica coated with octyl or amino-glutaraldehyde yielded biocatalysts with lower recovered activities, compared to CALB-OSGlu, with values of 43% and 32%, respectively. CALB-OSGlu was chosen to be evaluated in the synthesis of fructose oleate in tert -butyl alcohol medium and at high temperature, achieving conversions above 70% in nine 6 h-cycles at 55 °C. CALB-OSGlu showed better operational stability than benchmark immobilized CALB (Novozym 435) in the same reaction conditions, with the conversion of the later decreasing from 84% (first cycle) to 53% (ninth cycle).

Derivatized-agarose supports are suitable for enzyme immobilization by different methods, taking advantage of different physical, chemical and biological conditions of the protein and the support. In this study, agarose particles were modified with MANAE, PEI and glyoxyl groups and evaluated to stabilize polygalacturonase from Streptomyces halstedii ATCC 10897. A new immobilized biocatalyst was developed using glyoxyl-agarose as support; it exhibited high performance in degrading polygalacturonic acid and releasing oligogalacturonides. Maximal enzyme activity was detected at 5 h of reaction using 0.05 g/mL of immobilized biocatalyst, which released 3 mg/mL of reducing sugars and allowed the highest product yield conversion and increased stability. These results are very favorable for pectin degradation with reusability up to 18 successive reactions (90 h) and application in juice clarification. Plum (4.7 °Bx) and grape (10.6 °Bx) juices were successfully clarified, increasing reducing sugars content and markedly decreasing turbidity and viscosity. Fil: Ramírez Tapias, Yuly Andrea. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Biotecnología Sustentable; Argentina Fil: Rivero, Cintia Wanda. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Biotecnología Sustentable; Argentina Fil: Gallego, Fernando López. Heterogeneous Biocatalysis Group; España. Ikerbasque; España Fil: Guisán, José M.. Institute of Catalysis and Petrochemistry; España Fil: Trelles, Jorge Abel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Biotecnología Sustentable; Argentina

l - lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to l -lactate using NADH as a cofactor. In this work, we have optimized the immobilization of LDH from rabbit muscle in glyoxyl-agarose to have an active and stable preparation which is able to synthesize l -lactic acid. Optimization of various parameters during immobilization allowed the preparation of an active and highly stable immobilized derivative of LDH. A 90.1% immobilization and 72.0% yield was achieved using 300 mM trehalose during the immobilization process. Thermal stabilization factors attained for the immobilized LDH were 1600 times greater as compared to its soluble counterpart. The immobilized preparation was also stabilized against ethanol where it recovered 75% of its initial activity after 48 h while the soluble enzyme was completely inactivated after only 10 min under the same conditions. The enzyme activity profile versus pH and temperature showed a wider range for the immobilized preparation as compared to the soluble enzyme, whilst maintaining optimal values. Production of l -lactic acid was achieved in a batch reactor with the immobilized LDH and this preparation resisted 15 reuses without the loss of activity. We believe that the immobilization strategy optimized here broadened the possibilities of utilizing LDH for biocatalytic applications.

The enantioselectivity of carrier-bound and carrier-free immobilized lipase from Candida rugosa (CRL) was studied. CRL was immobilized in six agarose-based carriers functionalized with different reactive groups and in two different CRL cross-linked aggregates. Both, activity and enantioselectivity of all the immobilized lipase preparations were evaluated with different racemic esters under different reaction conditions (temperature, pH and solvent polarity). A strong effect of reaction media and immobilization protocol on enzyme activity and selectivity was found. Enzyme immobilization and reaction engineering allowed us obtaining the best immobilization protocol and reaction conditions to achieve high activity and enantioselectivty of CRL as heterogeneous catalyst. CRL immobilized on an agarose-based carrier activated with primary amino groups preferentially hydrolyzed (S)-phenylethyl acetate with E > 200 under pH 7, 4 °C and 30% of acetonitrile. On the other hand, CRL aggregated and cross-linked through their carboxylic groups preferentially hydrolyzed the (S)-isomer of ethyl 2-hydroxy-4-phenylbutyrate with an E = 39 under pH 5, 4 °C and 30% of acetonitrile. This work demonstrates the success of the combinatorial enzyme engineering for the production of highly enantioselective heterogeneous biocatalysts by screening different immobilization protocols and reaction media conditions.

One-pot multi-step conversions are desirable to achieve more efficient and sustainable chemical processes. In this context, the immobilization of multi-enzyme systems allows the reusability of several stabilized biocatalysts working in cascade and orthogonal reactions to access more complex synthetic schemes. Herein, we have shown the one-pot tandem hydrolysis and oxidation of racemic esters (1-phenylethyl acetate) to yield quantitative conversion of prochiral ketones (acetophenone) catalyzed by a consortium of immobilized enzymes. Eukaryotic lipase and catalase, and microbial thermophilic alcohol dehydrogenase and NADH oxidase are covalently, irreversibly and individually immobilized onto four different carriers, achieving high immobilization yields (>95%) for all the enzymes, and residual activities >50% for both thermophilic alcohol dehydrogenase and NADH oxidase, 18% for the catalase and 10% for the lipase. This heterogeneous system efficiently recycles NAD+ with a maximum turnover frequency (TOF) of 294 h−1 and can be reused for up to 10 operational cycles, retaining more than 80% of its initial activity.

Limited conservation resources mean that management decisions are often made on the basis of scarce biological information. Species distribution models (SDMs) are increasingly proposed as a way to improve the representation of biodiversity features in conservation planning, but the extent to which SDMs are used in conservation planning is unclear. We reviewed the peer-reviewed and grey conservation planning literature to explore if and how SDMs are used in conservation prioritisations. We use text mining to analyse 641 peer-reviewed conservation prioritisation articles published between 2006 and 2012 and find that only 10% of articles specifically mention SDMs in the abstract, title, and/or keywords. We use topic modelling of all peer-reviewed articles plus a detailed review of a random sample of 40 peer-reviewed and grey literature plans to evaluate factors that might influence whether decision-makers use SDMs to inform prioritisations. Our results reveal that habitat maps, expert-elicited species distributions, or metrics representing landscape processes (e.g. connectivity surfaces) are used more often than SDMs as biodiversity surrogates in prioritisations. We find four main reasons for using such alternatives in place of SDMs: (i) insufficient species occurrence data (particularly for threatened species); (ii) lack of biologically-meaningful predictor data relevant to the spatial scale of planning; (iii) low concern about uncertainty in biodiversity data; and (iv) a focus on accounting for ecological, evolutionary, and cumulative threatening processes that requires alternative data to be collected. Our results suggest that SDMs are perceived as best-suited to dealing with traditional reserve selection objectives and accounting for uncertainties such as future climate change or mapping accuracy. The majority of planners in both the grey and peer-reviewed literature appear to trade off the benefits of using SDMs for the benefits of including information on multiple threats and processes. We suggest that increasing the complexity of species distribution modelling methods might have little impact on their use in conservation planning without a corresponding increase in research aiming at better incorporation of a range of ecological, evolutionary, and threatening processes.

Differently activated agarose-based supports were evaluated for co-immobilization of a crude extract from Penicillium janczewskii containing xylanase, β-xylosidase and α-l-arabinofuranosidase activities. Adequately selecting support and immobilization conditions (8 h, using agarose with 10% crosslinking) increased enzyme levels substantially, mainly in relation to the xylanase (2-fold). A coating with dextran aldehyde MW 6000 Da, partially oxidized, covalently attached the enzymes to the support. Optimum activity was verified in the pH range 2-4, and at 50, 65 and 80 °C for the xylanase, α-l-arabinofuranosidase and β-xylosidase, respectively. The xylanase was highly thermostable retaining more than 70% of activity even after 24 h incubation at 60 and 70 °C; and at 80 °C its half-life was 1.7 h. The half-lives of the β-xylosidase and α-l-arabinofuranosidase at 50 °C were 2.3 and 3.8 h, respectively. The co-immobilization of the enzymes on a single support give raise to a functional multi-enzymatic biocatalyst acting in the complete hydrolysis of different and complex substrates such as oat spelt and wheat arabinoxylans, with xylose yield higher than 40%. The xylanase and the α-l-arabinofuranosidase presented high stability retaining 86.6 and 88.0% of activity after 10 reuse cycles.

Mitomycin C (MC) and Decarbamoylmitomycin C (DMC) - a derivative of MC lacking the carbamate on C10 - are DNA alkylating agents. Their cytotoxicity is attributed to their ability to generate DNA monoadducts as well as intrastrand and interstrand cross-links (ICLs). The major monoadducts generated by MC and DMC in tumor cells have opposite stereochemistry at carbon one of the guanine-mitosene bond: trans (or alpha) for MC and cis (or beta) for DMC. We hypothesize that local disruptions of DNA structure from trans or cis adducts are responsible for the different biochemical responses produced by MC and DMC. Access to DNA substrates bearing cis and trans MC/DMC lesions is essential to verify this hypothesis. Synthetic oligonucleotides bearing trans lesions can be obtained by bio-mimetic methods. However, this approach does not yield cis adducts. This report presents the first chemical synthesis of a cis mitosene DNA adduct. We also examined the stereopreference exhibited by the two drugs at the mononucleotide level by analyzing the formation of cis and trans adducts in the reaction of deoxyguanosine with MC or DMC using a variety of activation conditions. In addition, we performed Density Functional Theory calculations to evaluate the energies of these reactions. Direct alkylation under autocatalytic or bifunctional conditions yielded preferentially alpha adducts with both MC and DMC. DFT calculations showed that under bifunctional activation, the thermodynamically favored adducts are alpha, trans, for MC and beta, cis, for DMC. This suggests that the duplex DNA structure may stabilize/oriente the activated pro-drugs so that, with DMC, formation of the thermodynamically favored beta products are possible in a cellular environment.

As anthropogenic degradation of biodiversity and ecosystems increases, so does the potential threat to the supply of ecosystem services, a key contribution of nature to people. Biodiversity has often been used in spatial conservation planning and has been regarded as one among multiple services delivered by ecosystems. Hence, biodiversity conservation planning should be integrated in a framework of prioritizing services in order to inform decision-making. Here, we propose a prioritization approach based on scenarios maximising both the provision of ecosystem services and the conservation of biodiversity hotspots. Different weighting scenarios for the α-diversity in four taxonomic groups and 10 mapped ecosystem services were used to simulate varying priorities of policymakers in a mountain region. Our results illustrate how increasing priorities to ecosystem services can be disadvantageous to biodiversity. Moreover, the analysis to identify priority areas that best compromise the conservation of α-diversity and ecosystem services are predominantly not located within the current protected area network. Our analyses stress the need for an appropriate weighting of biodiversity within decision making that seek to integrate multiple ecosystem services. Our study paves the way toward further integration of multiple biodiversity groups and components, ecosystem services and various socio-economic scenarios, ultimately fuelling the development of more informed, evidence-based spatial planning decisions for conservation.

Molecular technologies, such as metabarcoding, have become powerful tools for conservation purposes. Here, we present a non-invasive study analyzing the diet of one population of European pond turtle (Emys orbicularis) during its whole activity period and of four other populations during the same period, based on faecal sample, and using for the first time on this species, a long metabarcoding approach. Emys orbicularis is an emblematic freshwater species of wetlands in Europe. In several countries, this species is endangered and, in Switzerland, Emys orbicularis is ranked as critically endangered on the Swiss Red List. A national conservation program was created to reintroduce this species and raised the question if this reintroduced species could be a threat for other endangered species. We developed a new method of long metabarcoding analysis, using universal PCR primers to determine prey species occurrence in the faeces. The analysis conducted on 174 faeces collected on 142 individuals revealed 1153 preys from 270 species. Emys orbicularis consumed plants throughout the year with a more diverse diet during the reproduction period (April–June). This study therefore not only determines precisely the omnivorous and opportunistic diet of the Emys orbicularis, but also shows that this species is not a threat to its environment, as 85.5% of the consumed species were not list on the Swiss Red List. Moreover, it also demonstrated that the genetic analyses of faeces could be an efficient tool to determine trophic interaction with a high level of precision, yielding promising perspectives for food web ecology.