In recent years, the use of biochemical markers, especially in the assessment of toxic effects and modes of action, under controlled laboratory conditions has increased. However, transposing their ...use to in situ monitoring or risk assessment evaluations has encountered barriers, mainly related to the difficulty in interpreting the meaning of biochemical variation. In this work, we aimed at understanding if biochemical marker activities (cholinesterase, glutathione S-transferase and lactate dehydrogenase) can be used to monitor the health status of natural populations of fish (
Gambusia affinis
) and daphnids (
Daphnia magna
). For that, two ponds with different water properties were chosen as study sites, and organisms collected at four sampling periods along the year. The pattern of biochemical marker responses was not the same in the two species, showing higher integrated biochemical marker response values in the winter for
G. affinis
and in the autumn for
D. magna
, suggesting specificities that must be taken into account in biomonitoring programmes by including representative species of several trophic levels. In the case of
G. affinis
, the differences in key physicochemical parameters between the two ponds (especially dissolved oxygen levels) did not seem to affect biochemical marker levels as if organisms were already perfectly adapted to their environment. In general, seasonal variation of water quality seems to have an important role on biochemical marker responses. Several parameters above Environmental Quality Standards were identified such as dissolved oxygen (DO), ammonia, nitrites, sulphides and metals, but eventual responses to these stressors could not be discriminated from natural variation except for particular cases.
Free-ranging animals are often used as bioindicators of both short- and long-term changes in ecosystem health, mainly to detect the presence and effects of contaminants. Birds, and gulls in ...particular, have been used as bioindicators over a broad range of marine and terrestrial ecosystems. In this study, we standardise the conditions for the use of a suite of biochemical markers in non-destructive matrices of Lesser Black-backed Gull (Larus fuscus) to facilitate future biomonitoring of marine and terrestrial contaminants. We characterized cholinesterase (ChE) in plasma and optimized assay conditions for ChE activity as a marker of neurotoxic damage. Moreover, we quantified variation in activity of ChE, lactate dehydrogenase (LDH), glutathione-S-transferase (GST) and catalase (CAT) as well as variation ranges of lipid peroxidation (LPO), in free-ranging adults and captive chicks. The main ChE form present in plasma of both adults and chicks was butyrylcholinesterase (BChE) followed by acetylcholinesterase (AChE), whose relative proportion in plasma tended to decrease with increased chick age. LPO levels and GST activity in blood cells (BCs) decreased significantly with increasing chick age, while BChE and LDH activity in plasma were not age-dependent. CAT in BCs tended to decline non-significantly in older chicks. Results of this study underscore the importance of standardising assay conditions and assessing intrinsic baseline variation in biochemical markers, before biochemical quantification. Data presented here provide a foundation for future use of BChE and LDH activity in plasma, as well as oxidative stress markers (LPO, CAT and GST) in BCs, to monitor environmental stress effects in Lesser Black-backed gulls.
•BChE and AChE were both detected in plasma of adults and chicks.•Main ChE form in plasma of both adults and chicks was BChE.•AChE proportion in plasma decreased with increasing age.•Age-dependent differences in LPO and GST levels were detected in blood cells.
Generalist species can potentially exploit a wide variety of resources, but at the individual level they often show a certain degree of foraging specialization. Specific foraging strategies, however, ...may increase exposure to environmental contaminants that can alter the cost-benefit balance of consuming particular food items. The Lesser Black-backed Gull (Larus fuscus) is known to opportunistically feed on a wide range of marine and terrestrial prey that differ in contaminant load, such as mercury (Hg) that strongly biomagnifies through the aquatic food web. The hypothesis tested in this study were: i) a predominant use of marine prey by females during egg-formation and by both parents during chick rearing increases the exposure to Hg during embryonic development and chick growth, and ii) this affects parental investment in clutch volume, chick growth and body condition. Total Hg burden and isotopic signatures of carbon (δ13C) and nitrogen (δ15N) were determined for eggs, down feathers, and primary feathers of L. fuscus chicks collected at a coastal colony in Belgium. As expected, eggs and feathers of chicks from parents with a stable isotope signature that suggested a predominantly marine diet had higher levels of Hg. The use of marine resources by females during the egg-formation period positively correlated to maternal investment in egg size, though entailing the cost of increased Hg-concentrations which in turn negatively affected clutch volume. Furthermore, it is shown that the use of chick down feathers is a suitable matrix to non-lethally estimate Hg concentrations in eggs. Contrary to our expectations, no relationship between Hg exposure and chick growth or chick body condition was found, which may be due the low concentrations found. We conclude that currently Hg contamination does not constitute a risk for development and condition of L. fuscus offspring at the levels currently observed at the Belgian coast.
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•Chick down feathers are a suitable matrix to estimate Hg concentrations in eggs.•Marine diet increases maternal investment on egg size during egg-formation.•Positive effects of marine diet on egg size are restrained at higher Hg exposure.•Levels of Hg observed did not affect chick development or condition.
In this study, the electropolymerization of 4-hydroxyphenylacetic acid (4-HPA) over graphite electrodes (GE) was optimized, aiming its application as a functionalized electrochemical platform for ...oligonucleotides immobilization. It was investigated for the number of potential cycles and the scan rate influence on the monomer electropolymerization by using cyclic voltammetry technique. It was observed that the polymeric film showed a redox response in the region of +0.53/+0.38 V and the increase in the number of cycles produces more electroactive platforms because of the better electrode coverage. On the other hand, the decrease of scan rate produces more electroactive platforms because of the occurrence of more organized coupling. Scanning electron microscopy (SEM) images showed that the number of potential cycles influences the coverage and morphology of the electrodeposited polymeric film. However, the images also showed that at different scan rates a more organized material was produced. The influence of these optimized polymerization parameters was evaluated both in the immobilization of specific oligonucleotides and in the detection of hybridization with complementary target. Poly(4-HPA)/GE platform has shown efficient and sensitive for oligonucleotides immobilization, as well as for a hybridization event with the complementary oligonucleotide in all investigated cases. The electrode was modified with 100 cycles at 75 mV/s presented the best responses in function of the amplitude at the monitored peak current values for the Methylene Blue and Ethidium Bromide intercalators. The construction of the genosensor to detect a specific oligonucleotide sequence for the
bacillus confirmed the results regarding the poly(4-HPA)/GE platform efficiency since it showed excellent sensitivity. The limit of detection and the limit of quantification was found to be 0.56 (±0.05) μM and 8.6 (±0.7) μM, respectively operating with very low solution volumes (15 µL of probe and 10 µL target). The biosensor development was possible with optimization of the probe adsorption parameters and target hybridization, which led to an improvement in the decrease of the Methylene Blue (MB) reduction signal from 14% to 34%. In addition, interference studies showed that the genosensor has satisfactory selectivity since the hybridization with a non-specific probe resulted in a signal decrease (46% lower) when compared to the specific target.
Electropolymerization of 4-aminobenzoic acid (4-ABA) on graphite electrodes (GEs) was investigated for the development of electrochemically functionalized platforms applied to the immobilization of ...biomolecules. The electrogeneration of 4-ABA was carried out in perchloric acid solutions using cyclic voltammetry (CV) and chronoamperometry (CA) techniques. In the case of CV studies, the GEs were modified by applying 100 consecutive potential cycles, while, in the case of CA studies, the electrodes were modified at different potentials (
E
/V vs. Ag/AgCl): 0.95, 1.05, and 1.15. The modified GEs were characterized in HClO
4
solutions in the presence and absence of the ferricyanide/ferrocyanide redox couple (redox probe) using the CV and electrochemical impedance spectroscopy techniques. Scanning electron microscopy was used for morphological characterization. In the case of CA, the best electrochemical activities for the electropolymerization reaction are in the following order of performance: 1.05 > 1.15 > 0.95 V. The poly(4-ABA) platforms were investigated for the immobilization and direct detection of purine bases (adenine and guanine), where higher values of the anodic peak current (
I
p,a
) were observed for the transducers electroformed using CV. In the case of immobilization of poly(GA) oligonucleotides, as well as for the recognition of the hybridization event with the complementary target poly(CT), methylene blue (MB) and ethidium bromide (EB) were used as the indicator and intercalator, respectively. MB was reduced at −0.26 V resulting in the cathodic peak current (
I
p,c
) for the
ss
DNA, while EB was oxidized at +0.58 V yielding the higher anodic peak current (
I
p,a
) for the
ds
DNA. The platforms were also evaluated for immobilization of the DD K peptide, with the antibacterial activity and biological recognition being verified using the complementary (phospholipid 1-palmitoyl-2-oleoyl phosphatidylcholine—POPC) and noncomplementary (phospholipid POPC + cholesterol) targets. The recognition mechanism was monitored from impedance measurements, with a good interaction of the DD K peptide with the POPC mimetic membrane being verified. In addition, the interaction was affected by the presence of cholesterol, revealing that the use of poly(4-ABA) platforms is very promising for the development of biosensors.
Environmental stressors have the potential to induce perturbations in the development of young individuals, leading to aberrant and unstable development. This may manifest as fluctuating asymmetry ...(FA; small, non-directional changes in the bilateral symmetry of morphological traits). Although widely regarded as a proxy for stress effects, the use of FA as a biomarker is still a topic of much debate. We investigated the applicability of FA as an indicator of nutritional stress (brought about by energetic constraints) by experimental manipulation of the diet composition and quantity during the growth of Lesser Black-backed Gull (Larus fuscus) chicks. FA as an endpoint was measured across the tarsus, wing and 10th primary feather when chicks reached 30 days of age. Although levels of asymmetry were found to increase with stress in the feather, relationships with tarsus and wing FA were mixed and mostly non-significant. Furthermore, we did not find any correlations in unsigned FA between traits, indicating the absence of organism-wide asymmetry. Our study was therefore unable to find unequivocal evidence in support of the application of FA as a reliable estimator of nutritional stress.
Tryptophyllins constitute a heterogeneous group of peptides that are one of the first classes of peptides identified from amphibian’s skin secretions. Here, we report the structural characterization ...and antioxidant properties of a novel tryptophyllin‐like peptide, named PpT‐2, isolated from the Iberian green frog Pelophylax perezi. The skin secretion of P. perezi was obtained by electrical stimulation and fractionated using RP‐HPLC. De novo peptide sequencing was conducted using MALDI MS/MS. The primary structure of PpT‐2 (FPWLLS‐NH2) was confirmed by Edman degradation and subsequently investigated using in silico tools. PpT‐2 shared physicochemical properties with other well‐known antioxidants. To test PpT‐2 for antioxidant activity in vitro, the peptide was synthesized by solid phase and assessed in the chemical‐based ABTS and DPPH scavenging assays. Then, a flow cytometry experiment was conducted to assess PpT‐2 antioxidant activity in oxidatively challenged murine microglial cells. As predicted by the in silico analyses, PpT‐2 scavenged free radicals in vitro and suppressed the generation of reactive species in PMA‐stimulated BV‐2 microglia cells. We further explored possible bioactivities of PpT‐2 against prostate cancer cells and bacteria, against which the peptide exerted a moderate antiproliferative effect and negligible antimicrobial activity. The biocompatibility of PpT‐2 was evaluated in cytotoxicity assays and in vivo toxicity with Galleria mellonella. No toxicity was detected in cells treated with up to 512 µg/ml and in G. mellonella treated with up to 40 mg/kg PpT‐2. This novel peptide, PpT‐2, stands as a promising peptide with potential therapeutic and biotechnological applications, mainly for the treatment/prevention of neurodegenerative disorders.
SLE has a relapsing-remitting course with disease activity flares over time. This study aims to identify clinical predictors of SLE flares.
This prospective cohort study over 24 months included all ...SLE patients on follow-up at one academic lupus clinic. Flare was defined as an increase in SLEDAI-2K score ≥4 points. Baseline clinical and demographic parameters were compared using survival analysis for time-to-flare outcome with univariate log-rank tests. Variables with significant differences were further evaluated as predictors with multivariate Cox regression models adjusting for potential confounding or contributing factors and hazard ratio (HR) calculation.
A total of 202 SLE patients were included. Over the follow-up period, 1083 visits were documented and 16.8% of patients presented with flares. In multivariate analysis, the following parameters emerged as flare predictors: SLE diagnosis up to 25 years of age (HR = 2.14, P = 0.03), lupus nephritis previous to baseline visit (HR = 4.78, P < 0.0001) and immunosuppressor treatment for severe SLE (HR = 3.22, P < 0.001). Baseline disease activity, disease duration and treatment with prednisone or HCQ were not predictive factors.
Patients with an SLE diagnosis before age 25 years, lupus nephritis or immunosuppressor treatment for severe SLE present greater HRs for flares, suggesting the need for tighter clinical monitoring. Current immunosuppressive strategies seem to be inefficient in providing flare prevention.
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