Proteome analysis using capillary electrophoresis coupled to mass spectrometry (CE‐MS) has been used in a number of clinical applications in the past years. The main focus of CE‐MS‐based studies has ...been on the investigation of urine, due to the stability of the urinary proteome, ease of collection, and also the low molecular weight range of the urinary proteome, mostly peptides below 30 kDa. The reproducibility of this approach has enabled analysis of over 20 000 samples in a comparable way, giving enormous statistical power to any additional study involving this methodological setup. In this article, we review the major technological issues associated with the application of CE‐MS in the routine investigation of the urinary proteome for clinical applications. We pinpoint recent developments that may have a chance to improve on the currently used approach, and highlight obstacles that need to be solved. In the second part of the article, we review the recent clinical applications, aiming to highlight relevant issues, and possible future routine applications in clinical diagnosis. In the end, we provide a short outlook, and indicate future developments to be expected, as well as problems that need to be solved to enable routine application of CE‐MS in a clinical setting.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Iron is an essential micronutrient for most bacteria and is obtained from iron-chelating siderophores or directly from iron-containing host proteins. For Gram-negative bacteria, classical iron ...transport systems consist of an outer membrane receptor, a periplasmic binding protein, and an inner membrane ABC transporter, which work in concert to deliver iron from the cell surface to the cytoplasm. We recently showed that Pectobacterium spp. are able to acquire iron from ferredoxin, a small and stable 2Fe-2S iron sulfur cluster containing protein and identified the ferredoxin receptor, FusA, a TonB-dependent receptor that binds ferredoxin on the cell surface. The genetic context of fusA suggests an atypical iron acquisition system, lacking a periplasmic binding protein, although the mechanism through which iron is extracted from the captured ferredoxin has remained unknown. Here we show that FusC, an M16 family protease, displays a highly targeted proteolytic activity against plant ferredoxin, and that growth enhancement of Pectobacterium due to iron acquisition from ferredoxin is FusC-dependent. The periplasmic location of FusC indicates a mechanism in which ferredoxin is imported into the periplasm via FusA before cleavage by FusC, as confirmed by the uptake and accumulation of ferredoxin in the periplasm in a strain lacking fusC. The existence of homologous uptake systems in a range of pathogenic bacteria suggests that protein uptake for nutrient acquisition may be widespread in bacteria and shows that, similar to their endosymbiotic descendants mitochondria and chloroplasts, bacteria produce dedicated protein import systems.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Increasing attention is being paid to the welfare of decapod crustaceans. Legislation exists for their humane slaughter in several countries and this is being debated in others. Electrical stunning ...may have potential for humane slaughter of crustaceans in some circumstances, although scientific data on the effectiveness of electrical stunning when applied to various species are limited. Assessment criteria for effective stunning have so far been based mainly on behavioural assessments, but these do not always reflect neural insensibility. In this study direct recordings of neural activity, both centrally and peripherally, have been used to provide more direct measures of the state of sensibility. We have also examined whether electrical stunning acts as a physiological stressor, using measures of haemolymph L-lactate. Experiments were performed on a commercially important decapod species, the brown crab Cancer pagurus L. Spontaneous activity within the CNS was arrested by electrical stunning, which is an indication of loss of sensibility. There were also specific effects on the peripheral nervous system, with loss of responsiveness to sensory stimulation, rendering the animals unresponsive to external stimuli, and a failure of motor activation. All these effects were apparent immediately after a 10s stun, and persisted for as long as tested (4h) indicating that the animals were also killed by the procedure. No autotomy of limbs occurred. Haemolymph L-lactate was found to be no greater following electrical stunning than after handling and sampling alone, and both were significantly lower than values reached in a range of environmental and commercial situations. For all these reasons we find that electrical stunning may meet criteria for humane slaughter of C. pagurus.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
4.
Mycotoxins in aquaculture: feed and food Gonçalves, Rui A.; Schatzmayr, Dian; Albalat, Amaya ...
Reviews in aquaculture,
February 2020, Volume:
12, Issue:
1
Journal Article
Peer reviewed
Open access
Mycotoxins, secondary metabolites produced by moulds, are responsible for causing significant economic losses due to spoilage of agricultural products but also due to direct or indirect health impact ...on livestock upon ingestion of mycotoxin contaminated feedstuffs. Aquaculture farmed species are not an exception and studies reporting mycotoxin‐related issues in the aquaculture industry have been increasing. However, our understanding on the prevalence and impact of mycotoxins in the aquaculture sector is still lower compared to the terrestrial livestock sector. Consequently, regulatory limits and guidance values have been defined based on the studies on terrestrial farm animals. The aim of this review is to compile and critically assess mycotoxin occurrence and co‐occurrence in aquaculture finished feeds, and understand the risk of mycotoxin carry‐over in aquaculture seafood products. Furthermore, we aim with this review to raise awareness to the scientific community, the regulatory authorities and the aquaculture industry to the need for specific aquaculture mycotoxin maximum concentration levels for both aquaculture feeds and foods.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Decapod crustaceans provide a valuable food source worldwide, both through wild capture and captive rearing. They are also used extensively in scientific research, both as subjects for the ...investigation of basic biological processes and as model organisms for the detection of environmental changes. There is now an increasing acceptance that decapod crustaceans are sentient, and legislation is being introduced in numerous countries to ensure crustacean welfare when they are harvested or held captive. Moreover, methods for the humane slaughter of these animals are being developed, and of these electrical stunning is a prime candidate. Optimisation of electrical stunning is underway for a range of commercially-exploited or scientifically-important species, and the present study contributes to that process by examining further crab ( Carcinus maenas ) and lobster ( Homarus gammarus ) species using a rigorous neurophysiological approach. By recording nerve activity in both the central and peripheral nervous systems, we have found that electrical stunning with a standard commercial instrument arrests nerve activity in both these species at all levels: sensory, motor and central, rendering the animals neurologically insensible. This methodology is the most direct and effective way to establish if insensibility is successfully achieved. However, ultimately the routine monitoring of effective stunning in commercial and laboratory settings will have to depend on simple, yet reliable physiological or behavioural indicators, following their calibration against neurological methods. Monitoring heartbeat is used widely to establish metabolic activity, locomotory performance, agonistic interactions and responses to environmental conditions. We have therefore assessed the potential to use cardiac activity as an indicator for the state of sensibility. We recorded the heartbeat in both C. maenas and H. gammarus before and after electrical stunning, but find that in many cases even though nerve activity has ceased both centrally and peripherally, cardiac activity actually continues (though at a reduced rate) for a substantial time (>1h). The heartbeat is therefore not a reliable indicator of the state of sensibility, making it an unsuitable indicator of effective stunning. Possible reasons for these findings, and ways to validate behavioural measures that may be more appropriate for routinely establishing effective electrical stunning, are discussed.
The farming of decapod crustaceans is a key economic driver in many countries, with production reaching around 9.4 million tonnes (USD 69.3 billion) in 2018. These efforts are currently dominated by ...the farming of Pacific whiteleg shrimp,
Penaeus vannamei
, which translates into approximately 167 billion farmed
P. vannamei
being harvested annually. Further production growth is expected in the future and hence the need for more research into its health and welfare is required. Herein, from an extensive survey of the available literature, we scrutinise farming practices and the challenges associated with the production of
P. vannamei
from an animal-centric welfare perspective (1), we propose potential welfare indicators (2) and we critically review current scientific evidence of sentience in penaeid shrimp among other commercially important decapods (3), since it is plausible that in the near future not only the largest, but in fact all decapod crustaceans will receive welfare protection. This review highlights that despite the wide knowledge on crustacean stress physiology and immunology as well as disease control, still little is known about some key parameters related to the five welfare dimensions. We recommend that further research should focus on developing a systematic integrated welfare assessment encompassing all the different aspects of the crustaceans farming and life cycle up to slaughter. Furthermore, direct and indirect species-specific operational welfare indicators should be developed for all decapod crustaceans currently farmed, similar to the ones suggested in this review for
P. vannamei
.
The external mucosal surfaces of the fish harbor complex microbial communities, which may play pivotal roles in the physiological, metabolic, and immunological status of the host. Currently, little ...is known about the composition and role of these communities, whether they are species and/or tissue specific and whether they reflect their surrounding environment. Co-culture of fish, a common practice in semi-intensive aquaculture, where different fish species cohabit in the same contained environment, is an easily accessible and informative model toward understanding such interactions. This study provides the first in-depth characterization of gill and skin microbiomes in co-cultured Nile tilapia (
) and grey mullet (
) in semi-intensive pond systems in Egypt using 16S rRNA gene-based amplicon sequencing. Results showed that the microbiome composition of the external surfaces of both species and pond water was dominated by the following bacterial phyla:
, and
. However, water microbial communities had the highest abundance and richness and significantly diverged from the external microbiome of both species; thus, the external autochthonous communities are not a passive reflection of their allochthonous communities. The autochthonous bacterial communities of the skin were distinct from those of the gill in both species, indicating that the external microbiome is likely organ specific. However, gill autochthonous communities were clearly species specific, whereas skin communities showed higher commonalities between both species. Core microbiome analysis identified the presence of shared core taxa between both species and pond water in addition to organ-specific taxa within and between the core community of each species. These core taxa included possibly beneficial genera such as
,
, and
and opportunistic potential pathogens such as
,
, and
. This study provides the first in-depth mapping of bacterial communities in this semi-intensive system that in turn provides a foundation for further studies toward enhancing the health and welfare of these cultured fish and ensuring sustainability.
Photoperiod is thought to be the main zeitgeber that induces smoltification in salmonids. However, its effects on the smoltification of rainbow trout (Oncorhynchus mykiss) are not fully understood ...and no published data documents the effects of the photoperiod regime currently used commercially, continuous light (LL). The present study compared the effect of four different photoperiod regimes (i.e. advanced phase photoperiod (APP), delayed phase photoperiod (DPP), LL and simulated natural photoperiod (SNP)) on the smoltification and growth of juvenile rainbow trout during their freshwater phase of winter-spring and the following summer post smolt phase. Smoltification was evaluated by monitoring gill Na+,K+–ATPase (NKA) activity and transcription of NKA α-subunit isoforms 1a and 1b, and Na+,K+,2Cl‐cotransporter 1a. Growth was measured as specific growth rate of both length and weight, and through molecular growth proxies such as the levels of circulating insulin-like growth factor 1 (IGF-I) in plasma and transcription of igf-I, igf binding protein 1b (igfbp1b), growth hormone receptor 1 (ghr1) and cathepsin L (ctsl) in the liver. Results indicate that APP induces a longer smolt window and higher levels of plasma IGF-I in both freshwater and seawater (two months post transfer), while DPP led to a shorter smolt window, lower plasma IGF-I levels in freshwater and seawater, an earlier decrease in liver igf-I and ctsl transcription in freshwater (as seen by modelling over time) and lower specific growth rate in freshwater. The transcription analysis of osmoregulatory genes complemented NKA activity and allowed for the detection of a transient response to light and of differences between the osmoregulatory capacity of parr and desmolted fish. Furthermore, an upregulation of the liver transcription of igf-I, ghr1 and ctsl was found in all treatments during the smolt window, which corresponded to the periods with highest growth. Finally, both plasma IGF-I and liver igf-I in seawater were found to be significantly correlated to fish growth in seawater. However, our data did not show that plasma IGF-I prior to seawater transfer could be used as a reliable predictor of growth in seawater. Overall, and especially when compared with other salmonid species, photoperiod seems to be a weaker inducer of smoltification in rainbow trout, according to the parameters that were tested, suggesting that other environmental cues might be more important drivers of this process.
•nkaα1a, nkaα1b and nkcc1a provide relevant information that the NKA activity does not reflect.•The liver transcription of igf-I, ghr1 and ctsl is directly related to fish SGRL.•While the best photoperiod in terms of duration of the smolt window and abundance of plasma IGF-I seemed to be APP, DPP was clearly the worst one.•While plasma IGF-I and liver igf-I in seawater was correlated to growth, plasma IGF-I prior seawater transfer was not a predictor of growth.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Rainbow trout (Oncorhynchus mykiss) is a salmonid species with a complex life-history. Wild populations are naturally divided into freshwater residents and sea-run migrants. Migrants undergo an ...energy-demanding adaptation for life in seawater, known as smoltification, while freshwater residents display these changes in an attenuated magnitude and rate. Despite this, in seawater rainbow trout farming all fish are transferred to seawater. Under these circumstances, weeks after seawater transfer, a significant portion of the fish die (around 10%) or experience growth stunting (GS; around 10%), which represents an important profitability and welfare issue. The underlying causes leading to GS in seawater-transferred rainbow trout remain unknown. In this study, we aimed at characterising the GS phenotype in seawater-transferred rainbow trout using untargeted and targeted approaches. To this end, the liver proteome (LC-MS/MS) and lipidome (LC-MS) of GS and fast-growing phenotypes were profiled to identify molecules and processes that are characteristic of the GS phenotype. Moreover, the transcription, abundance or activity of key proteins and hormones related to osmoregulation (Gill Na+, K + -ATPase activity), growth (plasma IGF-I, and liver igf1, igfbp1b, ghr1 and ctsl) and stress (plasma cortisol) were measured using targeted approaches. No differences in Gill Na+, K + -ATPase activity and plasma cortisol were detected between the two groups. However, a significant downregulation in plasma IGF-I and liver igf1 transcription pointed at this growth factor as an important pathomechanism for GS. Changes in the liver proteome revealed reactive-oxygen-species-mediated endoplasmic reticulum stress as a key mechanism underlying the GS phenotype. From the lipidomic analysis, key observations include a reduction in triacylglycerols and elevated amounts of cardiolipins, a characteristic lipid class associated with oxidative stress, in GS phenotype. While the triggers to the activation of endoplasmic reticulum stress are still unknown, data from this study point towards a nutritional deficiency as an underlying driver of this phenotype.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The sea-run phenotype of rainbow trout (Oncorhynchus mykiss), like other anadromous salmonids, present a juvenile stage fully adapted to life in freshwater known as parr. Development in freshwater is ...followed by the smolt stage, where preadaptations needed for seawater life are developed making fish ready to migrate to the ocean, after which event they become post-smolts. While these three life stages have been studied using a variety of approaches, proteomics has never been used for such purpose. The present study characterised the blood plasma proteome of parr, smolt and post-smolt rainbow trout using a gel electrophoresis liquid chromatography tandem mass spectrometry approach alone or in combination with low-abundant protein enrichment technology (combinatorial peptide ligand library). In total, 1,822 proteins were quantified, 17.95% of them being detected only in plasma post enrichment. Across all life stages, the most abundant proteins were ankyrin-2, DNA primase large subunit, actin, serum albumin, apolipoproteins, hemoglobin subunits, hemopexin-like proteins and complement C3. When comparing the different life stages, 17 proteins involved in mechanisms to cope with hyperosmotic stress and retinal changes, as well as the downregulation of nonessential processes in smolts, were significantly different between parr and smolt samples. On the other hand, 11 proteins related to increased growth in post-smolts, and also related to coping with hyperosmotic stress and to retinal changes, were significantly different between smolt and post-smolt samples. Overall, this study presents a series of proteins with the potential to complement current seawater-readiness assessment tests in rainbow trout, which can be measured non-lethally in an easily accessible biofluid. Furthermore, this study represents a first in-depth characterisation of the rainbow trout blood plasma proteome, having considered three life stages of the fish and used both fractionation alone or in combination with enrichment methods to increase protein detection.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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