Search

Sleep is one of the most ubiquitous but also complex animal behaviors. It is regulated at the global, systems level scale by circadian and homeostatic processes. Across the 24-h day, distribution of sleep/wake activity differs between species, with global sleep states characterized by defined patterns of brain electric activity and electromyography. Sleep patterns have been most intensely investigated in mammalian species. The present review begins with a brief overview on current understandings on the regulation of sleep, and its interaction with aging. An overview on age-related variations in the sleep states and associated electrophysiology and oscillatory events in humans as well as in the most common laboratory rodents follows. We present findings observed in different studies and meta-analyses, indicating links to putative physiological changes in the aged brain. Concepts requiring a more integrative view on the role of circadian and homeostatic sleep regulatory mechanisms to explain aging in sleep are emerging.

With the advent of personalized medicine, there is a movement to develop “smaller” and “smarter” microdevices that are able to distinguish similar cancer subtypes. Tumor cells display major differences when compared to their natural counterparts, due to alterations in fundamental cellular processes such as glycosylation. Glycans are involved in tumor cell biology and they have been considered to be suitable cancer biomarkers. Thus, more selective cancer screening assays can be developed through the detection of specific altered glycans on the surface of circulating cancer cells. Currently, this is only possible through time-consuming assays. In this work, we propose the “intelligent” Lab on Fiber (iLoF) device, that has a high-resolution, and which is a fast and portable method for tumor single-cell type identification and isolation. We apply an Artificial Intelligence approach to the back-scattered signal arising from a trapped cell by a micro-lensed optical fiber. As a proof of concept, we show that iLoF is able to discriminate two human cancer cell models sharing the same genetic background but displaying a different surface glycosylation profile with an accuracy above 90% and a speed rate of 2.3 seconds. We envision the incorporation of the iLoF in an easy-to-operate microchip for cancer identification, which would allow further biological characterization of the captured circulating live cells. This work was partially funded by the projects NanoSTIMA and NORTE-01-0145-FEDER-000029, both supported by the North Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, and through the European Regional Development Fund (ERDF); and by the Portuguese Foundation for Science and Technology, within the scope of the PhD grant PD/BD/135023/2017 and the projects: PTDC/BBB-EBI/0567/2014 (to CAR) and UID/BIM/04293/2013. It was also funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE (POCI-01-0145-FEDER-016585; POCI-01-0145-FEDER-007274; PPBI-POCI-01-0145-FEDER-022122). MB acknowledges the Marie Sklodowska-Curie grant agreement No. 748880.

This review provides insights on the impact of glycosylation in cancer biology and its influence in the current approaches of targeted cancer therapies in the clinical setting. The roles of glycosylation in cancer signaling, tumor progression, and metastasis are reviewed as well as glycans and glycan-binding proteins in tumor immunomodulation. Moreover, the latest reports on glycans influencing targeted therapeutic approaches in cancer are summarized. Finally, we discuss the future challenges of the field, outlining potential applications of glycan-based biomarkers for patient stratification and strategies for improving personalized cancer treatment.

Organic UV-filters are emergent contaminants continuously released into the aquatic ecosystems. These compounds are persistent showing potential for bioaccumulation. Partial life-cycle tests may underestimate the toxicity of UV-filters especially since these compounds have shown to act as endocrine disruptors. In the present study, the benthic aquatic insect Chironomus riparius was exposed to a gradient of Benzophenone-3 (BP3) concentrations over two generations to assess effects over a full life cycle from the first-instar larvae in the parental (P) generation (emergence, fecundity and fertility) until emergence in the subsequent generation (filial - F1). Recovery from exposure was also assessed after one generational exposure. Our results showed that concentrations of up to 8mg BP3/kg, elicited no effects regarding emergence rate and development time of C. riparius in the P generation. Our results also showed that C. riparius fecundity was not affected by BP3 exposure, but a strong dose-response relationship was observed for fertility with none of the egg ropes hatching at 8mg BP3/kg. Regarding effects observed in the F1 generation, emergence and development time were impaired by continuous exposure to BP3. Moreover, reduced emergence and changes in development time were observed in the F1 generation maintained in control/clean conditions but whose parents were exposed to BP3. Results found in this two-generational study clearly show reproductive effects of BP3 on C. riparius that would not be detected using standard tests. Full life cycle and multigenerational assays are critical to properly evaluate the population level effects of endocrine disrupting compounds such as organic UV-filters. published

Increasing salinity levels in freshwaters due to natural and anthropogenic sources pose risk to exposed aquatic organisms. However, there is a paucity of information on how salinity may influence the effects of other chemical stressors especially psychiatric pharmaceuticals. Freshwater planarians which have been suggested as bioindicator species in aquatic habitats were used in this study to evaluate toxic effects of sodium chloride (NaCl) used here as a surrogate for increasing salinity, and its influence on the effects of the antidepressant, fluoxetine. Effects of NaCl on Schmidtea mediterranea were evaluated using survival, regeneration, locomotion, feeding, and reproduction as endpoints. Subsequently, combined effects of NaCl and fluoxetine on planarians’ locomotion and reproduction were also evaluated. Result showed that exposure to increased NaCl concentrations is toxic to planarians with 48 and 96 h LC50 of 9.15 and 7.55 g NaCl L−1 respectively and exposure to sub-lethal concentrations led to reductions in feeding (LOEC of 0.75 g NaCl L−1 or 1906 μS cm−1 at 20 °C) and reproduction (LOEC 3.0 g NaCl L−1 or 5530 μS cm−1 at 20 °C), delayed head regeneration (LOEC of 1.5 g NaCl L−1 or 3210 μS cm−1 at 20 °C), and also slight decreases in locomotor activity. Moreover, some developmental malformations were observed in regenerating planarians, as well as delayed or inhibition of wound healing and degeneration after fissioning and during head regeneration. A significant interaction between fluoxetine and NaCl was observed for locomotor activity and unlike planarians exposed to fluoxetine alone, fissioned planarians and their pieces from the combined exposure treatments were also unable to regenerate missing portions. Results show that S. mediterranea can be highly sensitive to low NaCl concentrations and that this stressor can alter the effects of fluoxetine. The implication of these effects for planarian populations in the natural habitat is discussed as well as the need for more research on the effects of neuroactive pharmaceuticals under relevant exposure scenarios.

Background: Changes in glycosylation are known to play critical roles during gastric carcinogenesis. Expression of truncated O-glycans, such as the Sialyl-Tn (STn) antigen, is a common feature shared by many cancers and is associated with cancer aggressiveness and poor-prognosis. Methods: Glycoengineered cell lines were used to evaluate the impact of truncated O-glycans in cancer cell biology using in vitro functional assays, transcriptomic analysis and in vivo models. Tumor patients ‘samples and datasets were used for clinical translational significance evaluation. Findings: In the present study, we demonstrated that gastric cancer cells expressing truncated O-glycans display major phenotypic alterations associated with higher cell motility and cell invasion. Noteworthy, the glycoengineered cancer cells overexpressing STn resulted in tumor xenografts with less cohesive features which had a critical impact on mice survival. Furthermore, truncation of O-glycans induced activation of EGFR and ErbB2 receptors and a transcriptomic signature switch of gastric cancer cells. The disclosed top activated genes were further validated in gastric tumors, revealing that SRPX2 and RUNX1 are concomitantly overexpressed in gastric carcinomas and its expression is associated with patients’ poor-survival, highlighting their prognosis potential in clinical practice. Interpretation: This study discloses novel molecular links between O-glycans truncation frequently observed in cancer and key cellular regulators with major impact in tumor progression and patients’ clinical outcome. This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE ( POCI-01-0145-FEDER-016585 ; POCI-01-0145-FEDER-007274 ; POCI-01-0145-FEDER-028489 ) and National Funds through the Foundation for Science and Technology (FCT) , under the projects: PTDC/BBB-EBI/0567/2014 (to CAR), PTDC/MED-ONC/28489/2017 (to AM) and UID/BIM/04293/2013 ; and the project NORTE-01-0145-FEDER-000029 , supported by Norte Portugal Regional Programme ( NORTE 2020 ), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). The authors acknowledge the support by Gastric Glyco Explorer Initial Training Network (European Union Seventh Framework Programme GastricGlycoExplorer project, grant number 316929 ). DF acknowledges the FCT PhD Programmes and Programa Operacional Potencial Humano (POPH), specifically the BiotechHealth Programe (Doctoral Programme on Cellular and Molecular Biotechnology Applied to Health Sciences), with the reference PD/0016/2012 funded by FCT . Grants were received from FCT, POPH and FSE (Fundo Social Europeu): SFRH/BD/110636/2015 to DF and SFRH/BPD/115730/2016 to FP. We thank Catharina Steentoft and Henrik Clausen for the cell line models and helpful scientific discussions. We are grateful for Nuno Mendes's technical support. The authors acknowledge the support of José Luis Costa and Mafalda Rocha from the i3S Genomics Platform (GenCore), and Maria G Lazaro from the Bioimaging I3S Scientific Platform , member of the PPBI ( PPBI-POCI-01-0145-FEDER-022122 ).

Herein, sixteen priority sixteen priority polycyclic aromatic hydrocarbons (PAHs) and seven polychlorinated biphenyls (PCBs), considered seawater quality indicators, were quantified in four beaches of the Iberian (Portuguese) northwest coastline, including one ornithological reserve. These seashores are close to an important industrial region and a harbour. Water samples were extracted for PAHs and PCBs in their aqueous phase (DAP) and suspended particulate matter (SPM) and analysed by GC-MS/MS. Annual average concentrations reached ∼8 ng/L (DAP) and ∼100 ng/g dw (SPM) for PAHs and ∼3 ng/L (DAP) and ∼19 ng/g dw (SPM) for PCBs. PAHs and PCBs had fluctuations suggestive of seasonality. The possible origins of PAHs point to pyrogenic (forest fires) and petrogenic activities linked to maritime transports. The source(s) of PCBs appear to be related with leaks from waste sorting centres located in the vicinity of the sampling sites, together with the burning of contaminated materials. Toxic equivalents (TEQs) suggested a low carcinogenic potential for PAHs in seawater samples (∼0.3 ng/L and ∼4 ng/g dw, for DAP and SPM). Evaluation of risk coefficients for Σ16 PAHs revealed "Low-risk" of both matrices in all sampling areas. PCBs thyroid toxicity equivalents (TEQs-TH), suggested a low impact on biota (∼7.0E-05 ng/L and ∼5.2E-04 ng/g dw, for DAP and SPM). However, in vivo acute assays with Artemia salina exposed to environmental concentrations of PAHs, PCBs, and mixtures, confirmed the theoretical approach, showing that this coastline is not "Risk-free". This fact calls for further toxicological approaches to fully understand the risks posed by these compounds locally.

Freshwater sediments are a repository of microplastics (MPs) resulting from inland anthropogenic activities. Benthic invertebrates, particularly endobenthic sediment–ingesting species such as the annelid Lumbriculus variegatus (blackworm), are commonly found in contaminated sediments where they likely find and ingest MPs. In the present study, L. variegatus was exposed to concentrations between 0.51 and 20 g kg−1 dry sediment of four size-classes of irregularly-shaped polyethylene MPs (PE-MPs; size-class A: 32–63, B: 63–125, C: 125–250 and D: 250–500 μm) for 48 h to assess their sub-cellular responses to particles ingested, and for 28 days to determine chronic effects on worm's reproduction and biomass. After the short-term exposure (48 h), number of PE-MPs in blackworms' gut were related to MPs concentration in the sediment. In general, PE-MPs ingestion by blackworms induced depletion of their energy reserves (e.g., sugars in all size classes and lipids in the size-classes of PE-MPs > 125 μm), concomitant with the activation of antioxidant and detoxification mechanisms (increased level of total glutathione in all size-classes, and increased glutathione-S-transferase activity in PE-MPs > 250 μm), preventing lipid peroxidation. In addition, it was observed a reduction of aerobic energy production (decreased activity of the electron transport system) and a slight increase in neurotransmission (cholinesterase activity). After a long-term exposure (28 d), the presence and ingestion of PE-MPs did not affect reproduction and biomass of L. variegatus. The activation and efficiency of the antioxidant and detoxification mechanisms allied with the anatomy and physiology of L. variegatus, its feeding strategy and potentially dynamic ingestion/egestion capacity seem to be key features preventing MP deleterious effects under short- and chronic-exposures. Considering the MPs levels reported for freshwater sediments, and despite evidence of MPs ingestion and some sub-organismal effects, our results suggest no adverse impacts of PE-MPs contamination on L. variegatus populations fitness. This study applies an integrative approach in which data concerning the ingestion of different sized MPs and subsequent sub-cellular and apical responses are delivered, raising knowledge on endobenthic invertebrates’ strategies to potentially overcome MP toxicity in field contaminated sites.

Bioinsecticides based on the bacterium Bacillus thuringiensis subsp. israelensis (Bti) are increasingly being applied directly into aquatic compartments to control nuisance mosquitoes and blackflies and are generally considered environmentally friendly alternatives to synthetic insecticides. Bti-based insecticides are considered highly selective, being Diptera-specific, and supposedly decompose rapidly in the environment. Nevertheless, their safety to non-target species and freshwater ecosystems has been questioned by recent studies, which in fact document possible indirect effects in aquatic food webs such as the decrease of prey availability to predators. This work aimed to evaluate the potential effects of a Bti-based insecticide (VectoBac® 12AS) on a freshwater macroinvertebrate community and on stream ecological functions by using artificial microcosm streams. Artificial microcosm streams were colonized with a macroinvertebrate community plus periphyton collected in a stream together with Alnus glutinosa leaf packs. They were exposed for 7 days to different Bti treatments (0, 12, 120, 1200 μg/L), which are within the recommended concentrations of application in aquatic compartments for blackfly and mosquito control. Besides invertebrate community structure and abundance, effects were evaluated regarding leaf decomposition and primary production as measures of ecosystem functioning. Community structure was significantly altered in all Bti treatments after 7 days of exposure, mostly due to a decline in chironomids, followed by oligochaetes, which both belong to the deposit-feeders’ functional group. Direct effects on oligochaetes are surprising and require further research. Also, reductions of leaf decomposition due to Bti-induced sublethal effects on shredders (reduced feeding) or mortality of chironomids (that can also feed on coarse organic matter) observed in our study, represent potential indirect effects of Bti in aquatic ecosystems. Our short-exposure experiment evidenced some negative effects on stream benthic invertebrate communities and on ecosystem functioning that must be considered whenever Bti is used in water bodies for blackfly or mosquito control programs.

Microplastics (1 μm–5 mm), a ubiquitous and persistent marine pollutant, pose a severe threat to coral reefs when recently associated with physiological distress and increased diseases on corals. Studies conducted so far have only reported effects on scleractinian species. Knowledge about its effects on other corals (e.g. Order Zoantharia) remains uncovered, and responses at biochemical levels remain poorly documented. This study aimed to assess the potential effects induced by the presence of microplastics (1 and 10 mg L−1 low-density polyethylene, LDPE MP, or polyvinyl chloride, PVC MP) in the tropical and subtropical cosmopolitan species Zoanthus sociatus (order Zoantharia. Anthozoa: Hexacorallia), at organism level (survival and behaviour), endosymbionts (photosynthetic efficiency) and the cellular level (oxidative stress, detoxification capacity and energy metabolism). In a short-term exposure (96 h), this species was more sensitive to PVC MP. The presence of this polymer at a concentration of 10 mg L−1 caused a ten-fold higher adhesion to the coral epidermis, increased photosynthetic efficiency, lipid peroxidation, and antioxidant defences; without, however, inducing energetic costs. Although the observed physiological and biochemical effects did not compromise Z. sociatus survival in the short term, it does not rule out potential long-term (cumulative) effects that could endanger this and other physiologically similar species that underlie coral reefs.

CD44 variant isoforms are often upregulated in cancer and associated with increased aggressive tumor phenotypes. The CD44v9 is one of the major protein splice variant isoforms expressed in human gastrointestinal cancer cells. Immunodetection of CD44 isoforms like CD44v9 in tumor tissue is almost exclusively performed by using specific monoclonal antibodies. However, the structural variability conferred by both the alternative splicing and CD44 protein glycosylation is disregarded. In the present work, we have evaluated the role of O-glycosylation using glycoengineered gastric cancer models in the detection of CD44v9 by monoclonal antibodies. We demonstrated, using different technical approaches, that the presence of immature O-glycan structures, such as Tn and STn, enhance CD44v9 protein detection. These findings can have significant implications in clinical applications mainly at the detection and targeting of this cancer-related CD44v9 isoform and highlight the utmost importance of considering glycan structures in cancer biomarker detection and in therapy targeting. This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE, grant numbers POCI-01-0145-FEDER-016585; POCI-01-0145-FEDER-007274; OCI-01-0145-FEDER-031028; and national funds through the Foundation for Science and Technology (FCT), grant numbers PTDC/BBB-EBI/0567/2014 (to CAR), UID/BIM/04293/2013, and PTDC/MED-QUI/29780/2017; and the project NORTE-01-0145-FEDER-000029, supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). F. Pinto received a fellowship from FCT (SFRH/BPD/115730/2016).

Ubiquitination is an essential posttranslational modification that has emerged as a key regulator of multiple cellular processes in multiple manners. Different enzymes are involved in the control of ubiquitin homeostasis, being deubiquitinases (DUBs) responsible for cleaving the linkage between ubiquitin and target proteins. DUBs can be classified into seven families, among which ubiquitin-specific proteases (USPs) stand out due to their wide functional diversity. USPs are involved in a plethora of cellular processes, several of which are frequently altered in cancer. Consistent with this, altered USP function has been related to different human tumor types, and in recent years, the knowledge about pro- and antitumorigenic roles played by USPs has grown considerably. This fact has turned USPs into attractive targets for anticancer drug therapies. In this chapter we attempt to summarize the involvement of USPs in a variety of cellular processes and highlight their critical implications in different types of human cancer. Finally, we discuss the growing relevance of using USPs as targets in cancer therapy and the current status of small inhibitory molecules against USP functions.

The high levels of microplastics (MPs) found in freshwaters, particularly in riverine sediments, may impose a threat to the macroinvertebrate communities with possible consequences at ecosystem-level. The present study aimed to assess the effects of a mixture of different sizes of polyethylene microplastics (PE-MPs) on the composition and structure of macroinvertebrate communities and key-functions, such as primary production and leaf litter decomposition. MPs were mixed in the sediment at three different concentrations (0.1, 1, and 10 g kg-1) already found in freshwater sediments to enhance the relevance of the work. After eight days of exposure to PE-MPs, the observed changes in macroinvertebrate community structure were mostly due to the reduction in the abundance of deposit-feeders and grazers that were reduced by ca 31-50% and 34-39%, in the two highest MPs concentrations respectively, in comparison with the control treatment after 8 days of exposure. MPs internal concentrations were detected only in organisms exposed to plastic particles within artificial streams with chironomids and mayflies presenting higher MPs internal levels (average of 115 particles/individual found in chironomids, 166/individual for Baetis sp. and 415 particles/individual for Ephemerella sp.) suggesting higher ingestion of plastic microparticles. Nevertheless, the alterations in the community structure did not translate into impairments in the functional endpoints analysed, leaf litter decomposition and primary production, that were expected due to possible sub-lethal effects (e.g., feeding inhibition) on detritivores and grazers. This study represents one of the few assessments of MPs effects on freshwater benthic macroinvertebrate community structure and the first that simultaneously considered ecosystem-level functional endpoints. Further research combining different microplastics and longer exposure periods are needed to raise knowledge on potential ecological consequences of MPs to freshwaters.

Made available in DSpace on 2022-04-29T08:33:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-15 European Commission Fundação para a Ciência e a Tecnologia Ministério da Ciência, Tecnologia e Ensino Superior Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) European Regional Development Fund Centro de Investigação em Materiais Cerâmicos e Compósitos Centro de Estudos Ambientais e Marinhos, Universidade de Aveiro Microplastics and nanomaterials are applied in a myriad of commercial and industrial applications. When leaked to natural environments, such small particles might threaten living organisms' health, particularly when considering their potential combination that remains poorly investigated. This study investigated the physiological and biochemical effects of polyethylene (PE; 64-125 μm in size, 0.1, 1.0, and 10.0 mg·L−1) single and combined with an engineered nanomaterial applied in antifouling coatings, the copper-aluminium layered double hydroxides (Cu-Al LDH; 0.33, 1.0, and 3.33 mg·L−1) in the flatfish Solea senegalensis larvae (8 dph) after 3 h exposure, in a full factorial design. Particles ingestion, histopathology, and biochemical biomarkers were assessed. Fish larvae presented

Freshwater riverbeds are a major repository of microplastics (MPs) from inland activities. Benthic macroinvertebrates that live in close contact with sediments seem to ingest a considerable amount of such plastic particles. The effects of MPs on life-history traits are relatively well-known, but the suborganismal mechanisms underlying such effects remain unclear. This study addressed the potential effects of low-density polyethylene (LDPE) MPs on Chironomus riparius larvae at cellular and molecular levels. Fourth instar C. riparius larvae were exposed to 0.025 and 2.5 g/kg LDPE of dry sediment (sizes:32 and 32-45 μm; with irregular shape) under laboratory conditions for 48 h. These short-term exposures to environmental concentrations of LDPE MPs induced changes in the energy reserves (mostly by decreasing carbohydrates and increasing lipids), increased antioxidant and detoxification responses (tGSH, CAT, and GST), and induced increases in the activity of AChE (related to neurotransmission). In addition, at the gene level, exposure to MPs modified mRNA levels of InR, Dis, EcR, Dronc, Met (endocrine system), Def (immune system), PARP, ATM, NLK, and Decay (DNA repair), generating important alterations in the C. riparius development and response to unfavorable situations. This study provides new evidence of the effects of LDPE MPs at the suborganismal level, filling the gap in knowledge regarding the mechanisms underlying the toxicity of MPs and spotlighting gene expression analyses as early indicators of MP toxicity in C. riparius which were confirmed by Integrated biomarker response analyses highlighting the gene expression as sensible and useful endpoints for LPDE pollution in freshwaters. These results, coupled with previous investigations on responses at the organismal level, emphasizes the potential adverse effects of LDPE MPs on C. riparius, which may compromise freshwater benthic communities, considering its ecological role within these habitats.

Despite the frequent detection of organic ultraviolet-filters (UV-filters) in freshwater sediments, there is a lack of ecotoxicological data undermining a correct risk assessment for these emerging contaminants. The present study assessed the effects of three of the most commonly used UV-filters (benzophenone-3 - BP3; 3-(4-methylbenzylidene)camphor - 4-MBC and octocrylene - OC) on Chironomus riparius life history and biochemical responses. Standard ecotoxicological assays confirmed that all compounds impaired growth of C. riparius larvae and induced developmental effects such as delayed emergence and a reduction of imagoes weight. Concerning the biochemical responses analysed no evidences of oxidative damage in lipids or neurotoxicity (tested assessing acetylcholinesterase activity) were observed for any of the tested compounds. However, 4-MBC exposure induced a decrease in catalase activity and an increase in glutathione-S-transferase activity at 14.13mg/Kg while OC exposure caused an increase in total glutathione levels at 0.23 and 18.23mg/Kg. Exposure to all UV-filters tested, increased energy consumption on C. riparius with significant differences above 1.00mg/Kg for BP3, 0.09mg/Kg for 4-MBC and 2.13mg/Kg for OC. These results suggest that environmental relevant concentrations of UV-filters can cause deleterious effects to aquatic benthic species, such as C. riparius, and call for further research concerning effects of organic UV-filters on natural invertebrate communities and ecosystem functioning.

MUC16 is a large transmembrane mucin expressed on the apical surfaces of the epithelium covering the ocular surface, respiratory system and female reproductive tract. The transmembrane mucin is overexpressed by ovarian carcinomas, it is one of the most frequently used diagnostic markers for the disease and it is considered a promising target for immunotherapeutic intervention. Immunodetection of the mucin has to date been through antibodies that recognize its exceptionally large ectodomain. Similar to other membrane anchored mucins, MUC16 has a short cytoplasmic tail (CT), but studies of the biological relevance of the C-terminal domain of MUC16 has been limited by lack of availability of monoclonal antibodies that recognize the native CT. Here, we report the development of a novel monoclonal antibody to the CT region of the molecule that recognizes native MUC16 and its enzymatically released CT region. The antibody is useful for immunoprecipitation of the released CT domain as demonstrated with the OVCAR3 ovarian cancer cell line and can be used for detailed cytolocalization in cells as well as in frozen sections of ocular surface and uterine epithelium.

Physiological responses allow populations to cope with metal contamination and can be involved in the evolution of tolerance under historical metal contamination scenarios. Here we investigate physiological aspects that might be underlying the heritable high tolerance to cadmium (Cd) in two Chironomus riparius populations collected from historically metal contaminated sites in comparison to two populations from reference sites. To evaluate differences in the physiological response to short-term Cd exposure, protein expression profiles, metallothioneins [MTs] and several antioxidant defences such as total glutathione (GSHt), catalase (CAT) and glutathione-S-transferases [GSTs], were measured in all four populations reared for at least 8 generations under laboratory clean conditions. Cd-induced oxidative damage in lipids and energy related parameters (energy consumption and energy reserves) were also assessed. Results showed two major gradients of protein profiles according to Cd concentration and population tolerance. Furthermore, Cd-tolerant populations showed higher baseline levels of MTs and GSHt while Cd-sensitive populations, collected from reference sites, showed significant induction of GSHt levels with Cd exposure that were nonetheless insufficient to avoid increased oxidative damage to lipids. Cd exposure had no clear effects on the antioxidant enzymes or energy reserves but triggered a general increase in energy consumption. Finally, energy consumption was higher in Cd-tolerant populations across experimental conditions. Altogether, results demonstrate that inherited Cd-tolerance in these midge populations is related, at least in part, with different constitutive levels and plasticity of different defence mechanisms confirming the validity of using multiple physiological traits when studying evolution of tolerance.

The insecticide thiamethoxam (TMX) is a systemic neonicotinoid widely used for pest control in several agricultural crops. TMX mimics the action of acetylcholine causing uncontrolled muscular contraction eventually leading to insect death. TMX is being found in freshwater ecosystems at concentrations of up to 225µg/L. Still, chronic toxicity data for freshwater invertebrates is limited. Therefore, the aim of this study was to evaluate the acute and chronic effects (at organismal and biochemical levels) of TMX on the freshwater insect Chironomus riparius. C. riparius life history responses were significantly affected by TMX exposure, namely with a decrease in growth and delay in emergence. Concerning the biochemical responses, after a short exposure (48h) to TMX, our results showed that low concentrations of TMX significantly reduced CAT activity and LPO levels of C. riparius. No effects were observed in AChE, GST and ETS activities. Effects in terms of survival, development rates and biochemical responses of C. riparius exposed to low concentrations of TMX observed in this study suggest potential deleterious effects of this neonicotinoid on aquatic insects inhabiting freshwaters environments near agricultural areas.

Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter-cellular communication and mediating a broad spectrum of biological functions. EVs cargo iscomposed of a large repertoire of molecules, including glycoconjugates. Herein, we report the firststudy on the impact of the isolation strategy on the EV populations’glycosylation profile. The use ofdifferent state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isola-tion (TEI), OptiPrepTMdensity gradient (ODG) and size exclusion chromatography (SEC) resulted in EVpopulations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODGand SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated themost distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EVglycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn(STn) were identified as packaged cargo into EVs independently of the isolation methodology. STncarrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-relatedproteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of usingdifferent isolation methodologies in the populations of EVs that are obtained, with consequences inthe glycosylation profile of the isolated population. Furthermore, our results highlight the importanceof selecting adequate EV isolation protocols and cell culture conditions to determine the structuraland functional complexity of the EV glycoconjugates. This work was funded by FEDER funds through theOperational Programme for Competitiveness Factors-COMPETE (POCI-01-0145-FEDER-016585; POCI-01-0145-FEDER-007274; POCI-01-0145-FEDER-028489) and NationalFunds through the Foundation for Science and Technology(FCT), under the projects: PTDC/BBB-EBI/0567/2014 (toCAR), PTDC/MED-ONC/28489/2017 (to AM) and UID/BIM/04293/2013; and the project NORTE-01-0145-FEDER-000029, supported by Norte Portugal Regional Programme(NORTE 2020), under the PORTUGAL 2020 PartnershipAgreement, through the European Regional DevelopmentFund (ERDF). DF acknowledges the FCT PhD Programmesand Programa Operacional Potencial Humano (POPH), speci-fically the Biotech Health Programme (Doctoral Programme onCellular and Molecular Biotechnology Applied to HealthSciences), with the reference PD/0016/2012 funded by FCTand the grant SFRH/BD/110636/2015 from FCT, POPH andFSE (Fundo Social Europeu); MB acknowledges the EuropeanUnion’s Horizon 2020 research and innovation programmeunder the Marie Sklodowska-Curie grant agreement No.748880; and JP acknowledges FCT (SFRH/BD/137319/2018).The authors acknowledge Rede Nacional de Espectrometria deMassa, ROTEIRO/0028/2013, ref. LISBOA-01-0145-FEDER-022125, supported by COMPETE and North PortugalRegional Operational Programme (Norte2020), under thePORTUGAL 2020 Partnership Agreement, through theEuropean Regional Development Fund (ERDF). SV acknowl-edges the Danish National Research Foundation (DNRF107).

Giant tortoises are among the longest-lived vertebrate animals and, as such, provide an excellent model to study traits like longevity and age-related diseases. However, genomic and molecular evolutionary information on giant tortoises is scarce. Here, we describe a global analysis of the genomes of Lonesome George—the iconic last member of Chelonoidis abingdonii—and the Aldabra giant tortoise (Aldabrachelys gigantea). Comparison of these genomes with those of related species, using both unsupervised and supervised analyses, led us to detect lineage-specific variants affecting DNA repair genes, inflammatory mediators and genes related to cancer development. Our study also hints at specific evolutionary strategies linked to increased lifespan, and expands our understanding of the genomic determinants of ageing. These new genome sequences also provide important resources to help the efforts for restoration of giant tortoise populations., We thank Banco Santander for funding a short stay of S.F.-R. and D.C.-I. at Yale University. V.Q. is supported by grants from the Principado de Asturias and Ministerio de Economía y Competitividad, including FEDER funding. L.F.K.K. is supported by an FPI fellowship associated with BFU2014-55090-P (FEDER). T.M.-B. is supported by MINECO BFU2017-86471-P (MINECO/FEDER, UE), an NIH U01 MH106874 grant, the Howard Hughes International Early Career programme, Obra Social ‘La Caixa’ and Secretaria d’Universitats i Recerca, and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya. C.L.-O. is supported by grants from the European Research Council (DeAge; ERC Advanced Grant), Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (RTICC) and Progeria Research Foundation. The Instituto Universitario de Oncología is supported by Fundación Bancaria Caja de Ahorros de Asturias. We also thank staff at the Galapagos National Park and Galapagos Conservancy for logistic and financial support.

Leukemia cell lines have been widely used in the hematology field to unravel mechanistic insights and to test new therapeutic strategies. Myelodysplastic syndromes (MDS) comprise a heterogeneous group of diseases that are characterized by ineffective hematopoiesis and frequent progress to acute myeloid leukemia (AML). A few cell lines have been established from MDS patients after progression to AML but their characterization is incomplete. Here we provide a detailed description of the immunophenotypic profile of the MDS-derived cell lines SKK-1, SKM-1, F-36P; and MOLM-13. Specifically, we analyzed a comprehensive panel of markers that are currently applied in the diagnostic routine for myeloid disorders. To provide high-resolution genetic data comprising copy number alterations and losses of heterozygosity we performed whole genome single nucleotide polymorphism-based arrays and included the cell line OHN-GM that harbors the frequent chromosome arm 5q deletion. Furthermore, we assessed the mutational status of 83 disease-relevant genes. Our results provide a resource to the MDS and AML field that allows researchers to choose the best-matching cell line for their functional studies. © 2016 Wiley Periodicals, Inc.

// Julia M. Fraile 1 , Diana Campos-Iglesias 1 , Francisco Rodriguez 1 , Yaiza Espanol 1 and Jose M.P. Freije 1 1 Departamento de Bioquimica y Biologia Molecular, Facultad de Medicina, Instituto Universitario de Oncologia, Universidad de Oviedo, Oviedo, Spain Correspondence to: Jose M.P. Freije, email: // Keywords : degradome, CSCs, ubiquitin-specific proteases, gene targeting Received : August 04, 2016 Accepted : October 13, 2016 Published : October 19, 2016 Abstract Ubiquitin-Specific Proteases (USPs) are deubiquitinating enzymes frequently deregulated in human malignancies. Here, we show that USP54 is overexpressed in intestinal stem cells and demonstrate that its downregulation in colorectal carcinoma cells impedes tumorigenesis. We have generated mutant mice deficient for this deubiquitinase, which are viable and fertile, and protected against chemically-induced colorectal carcinoma. Furthermore, we show that USP54 is upregulated in human colon cancer and associates with poor prognosis. In agreement with these results, Usp54 downregulation in mouse melanoma cells inhibits lung metastasis formation. Collectively, this work has uncovered the pro-tumorigenic properties of USP54, highlighting the importance of deubiquitinating enzymes as promising targets for the development of specific anti-cancer therapies.

Stream ecosystems face ever-increasing pressures by the presence of emergent contaminants, such as, personal care products. N, N-diethyl-3-methylbenzamide (DEET) is a synthetic insect repellent that is being found in surface waters environments in concentrations up to 33.4 μg/L. Information concerning DEET's toxicity in the aquatic environment is still limited and focused only on its acute effects on model species. Our main objective was to assess the effects of DEET exposure to a caddisfly non-target species using sub-lethal endpoints. For that, we chose Sericostoma vittatum, an important shredder in Portuguese freshwaters that has been already used in different ecotoxicological assays. Besides acute tests, S. vittatum were exposed during 6 days to a gradient of DEET concentrations (8, 18 and 40.5 mg/L) to assess effects on feeding behaviour and biochemical responses, such as, lipid peroxidation levels (LPO), catalase and acetylcholinesterase (AChE) activities, and also assess effects on energy reserves and consumption. Acute tests revealed a 48 h-LC50 of 80.12 mg/L and DEET exposure caused feeding inhibition with a LOEC of 36.80 mg/L. Concerning the biochemical responses, DEET caused no effects in LPO nor on catalase activity. A non-significant decrease in AChE activity was observed. Regarding energetic reserves, exposure to DEET caused a significant reduction in S. vittatum carbohydrates levels. These results add important information for the risk assessment of insect repellents in the aquatic environment and suggest that reported environmental concentrations of DEET are not toxic to non-target freshwater insects.

Paraquat (PQ) is still used in several countries worldwide as an herbicide for weed control in agricultural production, ponds, reservoirs and irrigation canals. Thus, PQ is frequently found in surface water systems and is potentially toxic to aquatic organisms, since it can cause mitochondrial dysfunction altering in the redox state of cells. This study aimed to investigate the chronic effects of PQ to Chironomus riparius and Girardia tigrina, and compare their physiological strategies to cope with environmental stress. The mean emergence time was the most sensitive endpoint for Chironomids, with the lowest observed effect concentrations (LOEC) being 0.02 for males and 0.1 mg PQ L−1 for females. Moreover, PQ reduced the body weight of male and female imagoes, with LOECs of 0.5 and 2.5 mg PQ L−1, respectively. Paraquat also decreased the respiration rate (LOEC = 2.5 mg PQ L−1) and total glutathione (tGSH) content (LOEC = 0.5 mg PQ L−1). Thus, the aerobic production of energy was not affected and allowed chironomids to cope with oxidative stress induced by PQ, but with consequent physiological costs in terms of development rates and weight of adults. In planarians, PQ decreased the locomotion and feeding activity, and delayed photoreceptor regeneration (LOECs = 2.5 mg PQ L−1 for all endpoints). Despite increased aerobic energy production (LOEC = 0.5 mg PQ L−1), planarians were not able to cope with oxidative stress induced by the highest PQ concentrations, since lipid peroxidation levels were significantly increased (LOEC = 2.5 mg PQ L−1) concomitantly with a significant decrease of tGSH (LOEC = 2.5 mg PQ L−1). These results showed that planarians were unable to cope with oxidative stress induced by PQ with consequent impairments of behavior and regeneration despite an increased aerobic energy production.

Despite being considered environmentally safe, a deeper environmental risk assessment is needed for microbial insecticides; special attention should be devoted to their sublethal toxicity to non-target species. This study evaluated effects of VectoBac® 12AS - VB (based on the bacterium Bacillus thurigiensis var. israelensis) and Naturalis®-L - NL (based on the fungus Beauveria bassiana) on the aquatic insect Chironomus riparius life- history and biochemical responses. Acute tests estimated a 48 h-LC50 (median lethal concentration) of 1.85 μg/L (VB) and 34.7 mg/L (NL). Under sub- lethal exposure, VB decreased adults' emergence (LOEC – lowest observed effect concentration of 80 ng/L) while NL impaired larval growth (LOEC of 0.32 mg/L) and delayed emergence (LOEC of 2 mg/L for males and 0.8 mg/L for females). Despite not being monotonic, phenoloxidase activity increased (LOEC of 20 ng/L (VB) and 2 mg/L (NL)), suggesting activation of the immune system. There were no indications of oxidative damage nor neurotox- icity. Catalase activity was stimulated with all VB treatments, possibly associated with detoxification of immune response products. Under NL exposure, glutathione-S-transferase activity increased but did not show a dose- dependent response and, total glutathione decreased in the highest concentration. Exposure to both formulations caused the increase in protein content, while carbohydrate and lipids were not altered. This study revealed the susceptibility of C. riparius to VB and NL at concentrations below the ones recommended for field application, with potential population-level effects. These results add important information for the risk assessment of these microbial insecticides in aquatic ecosystems, considering relevant sublethal endpoints and raising concern about the adverse effects on non-target aquatic organisms. published

Successful application of potent antibody-based T-cell engaging immunotherapeutic strategies is currently limited mainly to hematological cancers. One major reason is the lack of well-characterized antigens on solid tumors with sufficient cancer specific expression. Aberrantly O-glycosylated proteins contain promising cancer-specific O-glycopeptide epitopes suitable for immunotherapeutic applications, but currently only few examples of such antibody epitopes have been identified. We previously showed that chimeric antigen receptor T-cells directed towards aberrantly O-glycosylated MUC1 can control malignant growth in a mouse model. Here, we present a discovery platform for the generation of cancer-specific monoclonal antibodies targeting aberrant O-glycoproteins. The strategy is based on cancer cell lines engineered to homogeneously express the truncated Tn O-glycoform, the so-called SimpleCells. We used SimpleCells of different cancer origin to elicit monoclonal antibodies with selectivity for aberrant O-glycoproteins. For validation we selected and characterized one monoclonal antibody (6C5) directed to a Tn-glycopeptide in dysadherin (FXYD5), known to be upregulated in cancer and promote metastasis. While dysadherin is widely expressed also in normal cells, we demonstrated that the 6C5 epitope is specifically expressed in cancer.

Proteases play key roles in the execution and regulation of most if not all biological functions, and alterations in their activity, expression, or location are associated with multiple pathological conditions, including cancer and aging. In this regard, the use of RNA interference-based approaches to specifically target the expression of individual proteases constitutes an invaluable tool to identify enzymes involved in central aspects of these processes and to explore their potential as targets of therapeutic interventions. Here we describe simple protocols to optimize and monitor the specific silencing of cancer- and aging-related proteases.

Circulating O-glycoproteins shed from cancer cells represent important serum biomarkers for diagnostic and prognostic purposes. We have recently shown that selective detection of cancer-associated aberrant glycoforms of circulating O-glycoprotein biomarkers can increase specificity of cancer biomarker assays. However, the current knowledge of secreted and circulating O-glycoproteins is limited. Here, we used the COSMC KO "Simple- Cell" (SC) strategy to characterize the O-glycoproteome of two gastric cancer SimpleCell lines (AGS, MKN45) as well as a gastric cell line (KATO III) which naturally expresses at least partially truncated O-glycans. Overall, we identified 499 O-glycoproteins and 1236 O-glycosites in gastric cancer SimpleCells, and a total 47 O-glycoproteins and 73 O-glycosites in the KATO III cell line. We next modified the glycoproteomic strategy to apply it to pools of sera from gastric cancer and healthy individuals to identify circulating O-glycoproteins with the STn glycoform. We identified 37 O-glycoproteins in the pool of cancer sera, and only nine of these were also found in sera from healthy individuals. Two identified candidate O-glycoprotein biomarkers (CD44 and GalNAc-T5) circulating with the STn glycoform were further validated as being expressed in gastric cancer tissue. A proximity ligation assay was used to show that CD44 was expressed with the STn glycoform in gastric cancer tissues. The study provides a discovery strategy for aberrantly glycosylated O-glycoproteins and a set of O-glycoprotein candidates with biomarker potential in gastric cancer. This work was supported by The Danish Research Councils, The Mizutani Foundation, The Danish National Research Foundation (DNRF107) and Fundacão para a Ciência e a Tecnologia (FCT) and COMPETE (Programa Operacional Temático Factores de Competitividade, comparticipado pelo fundo comunitário europeu FEDER) in the framework of the projects: PTDC/BBB-EBI/0786/2012; EXPL/CTM-BIO/0762/2013. Grants were received from FCT (SFRH/BD/73717/2010 to DC), (SFRH/BPD/75871/2011 to AM), (SFRH/BPD/96510/2013 to CG) and (SFRH/BPD/66288/2009 to JAF). IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education, and is partially supported by FCT.

Populations inhabiting metal-impacted freshwater systems located nearby industrial and urban areas may be under intense selection. The present study aims to address two fundamental microevolutionary aspects of metal contamination in the midge Chironomus riparius (Meigen): Are populations inhabiting historically metal contaminated sites genetically adapted to metals? And, are populations from these sites genetically eroded? To answer these questions, C. riparius populations were sampled from three sites with well-known histories of metal contamination and three nearby-located references. Genetic adaptation to metals was investigated through acute and chronic exposures to cadmium (Cd), after rearing all populations for at least six generations under laboratory clean conditions. Genetic diversity was estimated based on the allelic variation of seven microsatellite markers. Results showed higher acute tolerance to Cd in populations originating from metal contaminated sites compared to their respective references and significant differences in two out of three pairwise comparisons. However, there was a mismatch between acute and chronic tolerance to Cd with results of the partial life-cycle tests suggesting fitness costs under control clean conditions in two metal-adapted populations. Despite no evidences of genetic erosion in populations sampled from metal contaminated sites, our results suggest genetically inherited tolerance to Cd in populations inhabiting historically contaminated sites. These findings lend support to the use of C. riparius as a model organism in evolutionary toxicology and highlight the importance of coupling measures of neutral genetic diversity with assessments of chemical tolerance of populations for a better understanding of contaminant-induced adaptation and evolutionary processes.

KRAS is the most frequently mutated oncogene in human cancer, but its therapeutic targeting remains challenging. Here, we report a synthetic lethal screen with a library of deubiquitinases and identify USP39, which encodes an essential splicing factor, as a critical gene for the viability of KRAS-dependent cells. We show that splicing fidelity inhibitors decrease preferentially the proliferation rate of KRAS-active cells. Moreover, depletion of DHX38, encoding an USP39-interacting splicing factor, also reduces the viability of these cells. In agreement with these results, USP39 depletion caused a significant reduction in pre-mRNA splicing efficiency, as demonstrated through RNA-seq experiments. Furthermore, we show that USP39 is up-regulated in lung and colon carcinomas and its expression correlates with KRAS levels and poor clinical outcome. Accordingly, our work provides critical information for the development of splicing-directed antitumor treatments and supports the potential of USP39-targeting strategies as the basis of new anticancer therapies.

In Portugal, Eucalyptus plantations replaced native species and presently occupy at least 26% of the total forest area. These changes in the biodiversity affect riparian corridors and eucalyptus leaves are nowadays common primary sources of energy and carbon for aquatic benthic communities. The diverse and complex chemical composition of eucalyptus leaf litter and the resulting leachates may alter the homeostatic balance of exposed aquatic organisms. The present study focused on the evaluation of ecotoxicological responses of Chironomus riparius to Eucalyptus globulus leaf leachates at different levels of biological organization, including larval survival, growth and emergence, and biochemical endpoints, namely biomarkers related to cholinergic neurotransmission (acetylcholinesterase, AChE, activity), oxidative stress (catalase, CAT, and glutathione- S -transferases, GSTs, activities), total glutathione contents (tGSH), and peroxidative damage (lipid peroxidation, LPO) and also to oxygen consumption rate (electron transport system, ETS). The here-obtained results showed that E. globulus leaf leachates, in comparison with leaf leachates of the autochthonous species Alnus glutinosa , impaired C. riparius growth and development rates, altering their emergence patterns, and also changed key biochemical pathways, namely with putative effects on energetic balance, and neurophysiology.

Acute myeloid leukemia (AML) is a heterogeneous group of hematological neoplasms with distinct clinical and genetic features. Chromosomal abnormalities are detectable by conventional cytogenetic an...

Summary Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, plays a key role in the pathogenesis of gas gangrene. CpPLC may lead to cell lysis at concentrations that cause extensive degradation of plasma membrane phospholipids. However, at sublytic concentrations it induces cytotoxicity without inducing evident membrane damage. The results of this work demonstrate that CpPLC becomes internalized in cells by a dynamin-dependent mechanism and in a time progressive process: first, CpPLC colocalizes with caveolin both at the plasma membrane and in vesicles, and later it colocalizes with early and late endosomes and lysosomes. Lysosomal damage in the target cells is evident 9 h after CpPLC exposure. Our previous work demonstrated that CpPLCinduces ERK1/2 activation, which is involved in its cytotoxic effect. In this work we found that cholesterol sequestration, dynamin inhibition, as well as inhibition of actin polymerization, prevent CpPLC internalization and ERK1/2 activation, involving endocytosis in the signalling events required for CpPLC cytotoxic effect at sublytic concentrations. These results provide new insights about the mode of action of this bacterial phospholipase C, previously considered to act only locally on cell membrane.

Organic ultraviolet filters (UV-filters) used for protection against radiation in personal care products and other materials (e.g. textiles, plastic products) are considered emerging contaminants of aquatic ecosystem. Benzophenone-3 (BP3) and 3-(4-methylbenzylidene)camphor (4-MBC) are the most commonly used organic UV-filters and have been reported in freshwater environments due to contamination through discharges from wastewater treatment plants and swimming pools or by direct contamination from recreational activities. Our aim was to evaluate the ecotoxicological effects of these UV-filters using the freshwater caddisfly Sericostoma vittatum’ biochemical biomarkers and energy processing related endpoints (feeding behaviour, energy reserves and cellular metabolism). In laboratory trials, both compounds induced feeding inhibition of S. vittatum at 3.55 mg/kg of BP3 and at concentrations ≥2.57 mg/kg of 4-MBC, decreased carbohydrates content at 3.55 and 6.95 mg/kg of BP3 and 4-MBC respectively, and increased total glutathione levels at concentrations ≥1.45 and 1.35 mg/kg of BP3 and 4-MBC respectively. No significant effects were observed on endpoints associated with oxidative stress, antioxidant defences, phase II biotransformation or neurotoxicity after exposure to the two UV-filters. Our results show that environmental relevant concentrations of BP3 and 4-MBC, can negatively impact freshwater insects and demonstrate the importance of monitoring the ecological effects of organic UV-filters using non-model invertebrate species.

Introduction Glycosylation alterations are frequently found in cancer and specific aberrant glycan structures are associated with tumour development and progression.1 The characterisation of glycosylation modifications occurring in cancer is of high interest and represents a source of putative new biomarkers for cancer detection, therapeutic intervention and patient stratification. Material and methods Glycomics, glycoproteomics, glycoprofiling, and in situ Proximity Ligation Assays of glycoengineered cancer cell models and tissue samples from gastrointestinal cancer patients were performed. Results and discussions This presentation reports the recent discoveries applying several novel approaches for: (A) the characterisation of glycosylation changes in the cancer cells; (B) the identification of the aberrant expression of specific glycan structures in cancer, like terminal sialylated glycans, which lead to the activation of tyrosine kinase receptors, such as HER2, MET, and RON;2,3,4,5 (C) the identification of altered glycosylated proteins, carrying simple mucin-type carbohydrate structures, in engineered cancer cell models and in sera of cancer patients.5,6 Conclusion These results demonstrate aberrant glycan structures as key functional players in tumour biology and highlight their potential as novel biomarkers and as therapeutic targets in the clinical management of cancer patients.1,5,6

N,N-diethyl-3-methylbenzamide (DEET) is the active ingredient of many commercial insect repellents. Despite being detected worldwide in effluents, surface water and groundwater, there is still limited information on DEET's toxicity toward non-target aquatic invertebrates. Thus, our main objective was to assess the effects of DEET in the life cycle of Chironomus riparius and assess its biochemical effects. Laboratory assays showed that DEET reduced developmental rates (reduced larval growth, delayed emergence) of C. riparius larvae and also caused a decrease in the size of adult midges. Concerning the biochemical responses, a short exposure to DEET caused no effects in lipid peroxidation, despite the significant inhibition of catalase and glutathione-S-transferase activities and of total glutathione contents. Moreover, inhibition of acetylcholinesterase activity was also observed showing neurotoxic effects. Environmental risk assessment of insect repellents is needed. Our results showed moderate toxicity of DEET toward C. riparius, however, due to their mode of action, indirect ecological effects of DEET and of other insect repellents cannot be excluded and should be evaluated.

The most used cancer serum biomarker is the CA125 immunoassay for ovarian cancer that detects the mucin glycoprotein MUC16. Several monoclonal antibodies (mAbs) including OC125 and M11 are used in CA125 assays. However, despite considerable efforts, our knowledge of the molecular characteristics of the recognized epitopes and the role played by glycosylation has remained elusive. Here a comprehensive set of recombinant MUC16 tandem repeats (TRs) expressed in glycoengineered mammalian cells and E. coli, together with overlapping peptides, was used to probe antigen-binding epitopes. We present a complete analysis of N- and O-glycosylation sites of a MUC16 TR expressed in CHO cells and demonstrate that neither N- nor O-glycosylation appear to substantially influence binding of OC125 and M11 mAbs. A series of successive N- and C-terminal truncations of a MUC16 TR construct expressed in E. coli narrowed down the epitopes for OC125 and M11 to a segment containing parts of two consecutive SEA domains with a linker. Thus, a complete SEA domain is not required. These findings suggest that binding epitopes of mAbs OC125 and M11 are dependent on conformation but not on glycosylation. The availability of recombinant TR constructs with and without aberrant glycosylation now opens the way for vaccine studies.

Detritus processing is vital for freshwater ecosystems that depend on the leaf litter from riparian vegetation and is mediated by microorganisms and aquatic invertebrates. Shredder invertebrates transform coarse particulate organic matter into fine particulate organic matter used as food by collector species. Direct and indirect effects of contaminants can impair detritus processing and thus affect the functioning of these ecosystems. Here, we assessed the combined effects of a toxic metal (cadmium) and resource quality (leaf species) on detritus processing and shredder-collector interactions. We considered two types of leaves, alder and eucalyptus that were microbially conditioned under different Cd concentrations in the laboratory. The microbial communities present on leaves were analyzed by Denaturing Gradient Gel Electrophoresis (DGGE), and we also measured microbial respiration rates. Sericostoma vittatum (a caddisfly shredder) and Chironomus riparius (a midge collector) were also exposed to Cd and allowed to consume the corresponding alder or eucalyptus leaves. We evaluated C. riparius growth and leaf mass loss in multispecies microcosms. Cadmium exposure affected leaf conditioning and fungal diversity on both leaf species, as assessed by DGGE. Cadmium exposure also affected the mass loss of alder leaves by reductions in detritivore feeding, and impaired C. riparius growth. Chironomus riparius consumed alder leaf discs in the absence of shredders, but S. vittatum appear to promote C. riparius growth in treatments containing eucalyptus. These results show that indirect effects of contaminants along detritus-processing chains can occur through effects on shredder-collector interactions such as facilitation but they also depend on the nutritional quality of detritus and on sensitivity and feeding plasticity of detritivore species.

Evaluation of: Pedersen JW, Blixt O, Bennett EP et al. Seromic profiling of colorectal cancer patients with novel glycopeptide microarray. Int. J. Cancer 128(8), 1860-1871 (2011). Autoantibodies to cancer-associated antigens hold promise as sensitive biomarkers for cancer detection. Based on this hypothesis, and knowing that O-glycans on proteins constitute a source of possible epitopes recognized by autoantibodies, Pedersen and colleagues have generated a glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from human mucins. The profiling of sera immunoreactivity of colon cancer patients allowed the identification of cancer-associated autoantibodies to various mucin (MUC)1 and MUC4 glycopeptides carrying aberrant glycosylation. This article provides evidence for the value of glycopeptides displaying cancer-associated glycans in diagnostic applications, and opens new avenues for the expansion to other protein glycoforms, as well as to further applications of such a microarray strategy for other post-translational modifications of proteins in the search for cancer biomarker.

Sialyl-Lewis X (SLe(x)) is a sialylated glycan antigen expressed on the cell surface during malignant cell transformation and is associated with cancer progression and poor prognosis. The increased expression of sialylated glycans is associated with alterations in the expression of sialyltransferases (STs). In this study we determined the capacity of ST3GAL3 and ST3GAL4 sialyltransferases to synthesize the SLe(x) antigen in MKN45 gastric carcinoma cells and evaluated the effect of SLe(x) overexpression in cancer cell behavior both in vitro and in vivo using the chicken chorioallantoic membrane (CAM) model. The activation of tyrosine kinase receptors and their downstream molecular targets was also addressed. Our results showed that the expression of ST3GAL4 in MKN45 gastric cancer cells leads to the synthesis of SLe(x) antigens and to an increased invasive phenotype both in vitro and in the in vivo CAM model. Analysis of phosphorylation of tyrosine kinase receptors showed a specific increase in c-Met activation. The characterization of downstream molecular targets of c-Met activation, involved in the invasive phenotype, revealed increased phosphorylation of FAK and Src proteins and activation of Cdc42, Rac1 and RhoA GTPases. Inhibition of c-Met and Src activation abolished the observed increased cell invasive phenotype. In conclusion, the expression of ST3GAL4 leads to SLe(x) antigen expression in gastric cancer cells which in turn induces an increased invasive phenotype through the activation of c-Met, in association with Src, FAK and Cdc42, Rac1 and RhoA GTPases activation.