Estimation of the mass density of biological matter from refractive index measurements.

The quantification of physical properties of biological matter gives rise to novel ways of understanding functional mechanisms. One of the basic biophysical properties is the mass density (MD). It affects the dynamics in sub-cellular compartments and plays a major role in defining the opto-acoustical properties of cells and tissues. As such, the MD can be connected to the refractive index (RI) via the well known Lorentz-Lorenz relation, which takes into account the polarizability of matter. However, computing the MD based on RI measurements poses a challenge, as it requires detailed knowledge of the biochemical composition of the sample. Here we propose a methodology on how to account for assumptions about the biochemical composition of the sample and respective RI measurements. To this aim, we employ the Biot mixing rule of RIs alongside the assumption of volume additivity to find an approximate relation of MD and RI. We use Monte-Carlo simulations and Gaussian propagation of uncertainty to obtain approximate analytical solutions for the respective uncertainties of MD and RI. We validate this approach by applying it to a set of well-characterized complex mixtures given by bovine milk and intralipid emulsion and employ it to estimate the MD of living zebrafish (Danio rerio) larvae trunk tissue. Our results illustrate the importance of implementing this methodology not only for MD estimations but for many other related biophysical problems, such as mechanical measurements using Brillouin microscopy and transient optical coherence elastography.

Influence of Feeding Substrates on the Presence of Toxic Metals (Cd, Pb, Ni, As, Hg) in Larvae of Tenebrio molitor: Risk Assessment for Human Consumption.

Larvae of Tenebrio molitor L. (Coleoptera: Tenebrionidae) are particularly suitable as novel food for the human consumption; nevertheless, there are some potential safety risks linked with insect consumption. In this study we investigated the presence of Cd, Pb, Ni, As, Hg in new feeding substrates coming from solid residues generated by olive fruits processing, called olive-pomace, and their influence on the metal content in larvae of T. molitor. Morover, bioaccumulation factor and the mercury-selenium balance were evaluated. Analyses were carried out via graphite furnace atomic absorption spectrophotometry for Cd, Pb, Ni, As and Se, and via Direct Mercury Analyzer for Hg. All metal concentrations found in feeding substrates were below the legal limit of undesirable substances in animal feed (2002/32/EC). Concentrations in larvae were in the range (mg kg-1 wet weight): Cd 0.008-0.016, Pb 0.063-0.079, Ni 0.03-0.63, As 0.021-0.023, Hg 0.12 × 10-3-0.49 × 10-3, and Se 0.057-0.085. Statistically significant correlation between metal content in feeding substrates and in larvae was evidenced only for Hg, which bioaccumulates. Se protects from mercury toxicity, with a Selenium Health Benefit Value (HVBSe) of > 0. Overall, our results indicate that the risk of exposure to metals from consumption of mealworm larvae is relatively low and in compliance with European Union regulations.

Standardization of sample homogenization for mosquito identification using an innovative proteomic tool based on protein profiling.

The rapid spread of vector-borne diseases demands the development of an innovative strategy for arthropod monitoring. The emergence of MALDI-TOF MS as a rapid, low-cost, and accurate tool for arthropod identification is revolutionizing medical entomology. However, as MS spectra from an arthropod can vary according to the body part selected, the sample homogenization method used and the mode and duration of sample storage, standardization of protocols is indispensable prior to the creation and sharing of an MS reference spectra database. In the present study, manual grinding of Anopheles gambiae Giles and Aedes albopictus mosquitoes at the adult and larval (L3) developmental stages was compared to automated homogenization. Settings for each homogenizer were optimized, and glass powder was found to be the best sample disruptor based on its ability to create reproducible and intense MS spectra. In addition, the suitability of common arthropod storage conditions for further MALDI-TOF MS analysis was kinetically evaluated. The conditions that best preserved samples for accurate species identification by MALDI-TOF MS were freezing at -20°C or in liquid nitrogen for up to 6 months. The optimized conditions were objectified based on the reproducibility and stability of species-specific MS profiles. The automation and standardization of mosquito sample preparation methods for MALDI-TOF MS analyses will popularize the use of this innovative tool for the rapid identification of arthropods with medical interest.

Assessment of water quality in urban streams based on larvae of Hydropsyche angustipennis (Insecta, Trichoptera).

Hydropsyche angustipennis (Insecta, Trichoptera) larvae were used as indicators of stream contamination in the city of Lódz, Poland. The larvae of H. angustipennis were present at 9 sampling sites established for this study. Significant differences between the sampling sites were noted for environmental parameters as well as concentration of chemicals in water and biodiversity of aquatic invertebrates. Statistical analyses showed significant correlations between quantity and quality of water pollutants and density of H. angustipennis larvae, concentration of metals in larval bodies, and the appearance of morphological anomalies in tracheal gills and anal papillae. In comparison to literature data, the level of contaminants in water, including heavy metals, for each of the studied streams of Lódz was surprisingly low while concentration of these metals in the whole bodies of H. angustipennis larvae was very high. Some of the heavy metals present in the water might be identified only after conducting analyses of their concentration in the larval bodies. Therefore, long life cycle of H. angustipennis and heavy metal tolerance with a possibility of their accumulation in the larval bodies may constitute a support to traditional chemical assessment of water quality or traditional biomonitoring.

Purification and identification of angiotensin I-converting enzyme-inhibitory peptides from apalbumin 2 during simulated gastrointestinal digestion.

BACKGROUND: Bee larvae are considered to be an important reservoir for proteins. However, little attention has been paid to the release of potential bioactive peptides from bee larva proteins. In this study the major protein in bee larvae was hydrolyzed in vitro by gastrointestinal enzymes. The peptide profile of the hydrolysis was characterized by gel filtration chromatography and tricine-SDS-PAGE. Furthermore, the bioactive peptide was isolated and identified by Q-TOF-MS/MS. RESULTS: The major bee larva protein was identified as apalbumin 2 and was more digestible into peptides with molecular weights lower than 3 kDa. The hydrolysate obtained after 3 h of digestion exhibited angiotensin I-converting enzyme (ACE)-inhibitory activity and was purified sequentially by gel filtration and RP-HPLC. The molecular weights of peptide fractions with ACE-inhibitory activity were distributed between 0.5 and 1.5 kDa. A novel peptide with highest ACE-inhibitory activity (IC50 54.9 µmol L(-1) ) was purified by further RP-HPLC. The amino acid sequence of this peptide was identified as LLKPY (632.40 Da). CONCLUSION: ACE-inhibitory peptides could be formed from bee larvae through gastrointestinal digestion. The most active peptide (LLKPY) is potentially useful as a therapeutic agent in treating hypertension.

Assessment and partial purification of serine protease inhibitors from Rhipicephalus (Boophilus) annulatus larvae.

Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatus is an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.

Assessment and partial purification of serine protease inhibitors from Rhipicephalus (Boophilus) annulatus larvae / Avaliação e purificação parcial dos inibidores da serina protease de larvas do Rhipicephalus (Boophilus) annulatus

Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatus is an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.

Carrapatos são uma rica fonte de inibidores da serina protease, particularmente aqueles que previnem coagulação e respostas inflamatórias durante a alimentação com sangue. O carrapato Rhipicephalus (B.) annulatus é um ectoparasita importante de bovinos. O objetivo deste estudo foi caracterizar e purificar os inibidores da serina protease presentes no extrato de larva do R. (B.) annulatus. Os inibidores foram caracterizados através de zimografia reversa uni e bidimensional e purificados com cromatografia de afinidade em uma coluna de sepharose-tripsina. A análise do extrato de larva pela zimografia reversa uni e bidimensional mostrou atividade inibitória de tripsina entre 13 e 40 kDa. Através de SDS-PAGE e zimografia reversa para proteínas purificadas pela cromatografia por sepharose-tripsina, algumas bandas de proteínas com pesos moleculares entre 13 e 34 kDa foram detectadas. Western blotting mostrou que cinco bandas de proteínas a 48, 70, 110, 130 e 250 kDa reagiram positivamente com o soro imune, enquanto não houve reação positiva nas bandas 13-40 kDa. Inibidores da serina protease do R. (B.) annulatus têm atividade antitripsina semelhante àquelas dos inibidores de outras espécies de carrapatos duros, sugerindo, assim, que essas proteínas podem ser úteis como alvo de vacinas contra carrapatos.

An assessment of the antibacterial activity in larval excretion/secretion of four species of insects recorded in association with corpses, using Lucilia sericata Meigen as the marker species.

The relative antibacterial activities of excretion/secretion (ES) from two carrion-feeding insects, Calliphora vicina Robineau-Desvoidy and Dermestes maculatus DeGeer, and a detritivore, Tenebrio molitor Linnaeus, were compared to that of Lucilia sericata Meigen, a species with ES of known antibacterial capacity, in order to explore the antimicrobial potential of other carrion and detritivore species. Viable counts were used to assess time-kill of ES against five bacterial species, Staphylococcus aureus, Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa and Proteus mirabilis. Antibacterial activity was recorded in all four insect species although T. molitor and D. maculatus were the most effective in controlling growth of P. mirabilis. The blowflies were more effective in controlling a wider range of both Gram-positive and Gram-negative bacteria. The larval ES from all species was shown to reduce bacterial growth rate although differences in antibacterial spectrum were noted and the degree of potency varied between the four species. These differences may be explained ecologically by the different colonisation times of each insect species on the corpse. Overall, this study demonstrates that research into other carrion-feeding insect species has potential to provide an increased source of antimicrobial chemicals to broaden the range of bacterial species beyond that currently controlled using L. sericata.

Polychlorinated biphenyls and Hudson River white perch: implications for population-level ecological risk assessment and risk management.

Risk assessments and risk management decisions concerning risks to wild fish populations resulting from exposures to polychlorinated biphenyls (PCBs) and related chemicals have been based primarily on observations of effects of chemicals on individual organisms. Although the development and application of population-level ecological risk-assessment methods is proceeding at a rapid pace, the organism-level approach is still being justified by arguments that population-level ecological risk assessment is in an early stage of development and has not been shown to be reliable. This article highlights the importance of including population-level effects in risk-management decision-making, by examining the effects of exposures to PCBs on fish populations inhabiting the Hudson River, New York, USA, a system in which data have been collected for approximately 30 y concerning both concentrations of PCBs in sediment and fish tissue and the abundance and reproduction of exposed fish populations. We previously tested hypotheses concerning the effects of PCBs on the striped bass population of the Hudson River, and found that the available data conflicted with all of these hypotheses. Here, we report results of similar analyses of effects of historic PCB exposures on the Hudson River white perch population, using an extended data set that recently became available. As with striped bass, we found no correlation between maternal PCB tissue concentrations and any measure of reproductive success in Hudson River white perch during the 30-y period covered by the data set. Together with results of studies performed on fish populations exposed to PCBs at other sites, our results clearly demonstrate that physiological and genetic adaptation, biological compensation, and other ecological processes influence responses of fish populations to PCB exposures and should be considered in risk management decision-making.

Cane toad toxicity: an assessment of extracts from early developmental stages and adult tissues using MDCK cell culture.

Extracts of the cane toad (Bufo [Chaunus] marinus) adversely affected the growth of Mardin-Darby canine kidney (MDCK) cells during culture. In a similar manner to ouabain treatment, application of toad extracts over a 24 h period resulted in high levels of cytotoxicity, as indicated by cell detachment, increased membrane permeability and loss of mitochondrial function. Cell viability and growth were unchanged for controls (PBS) and increased with the application of Limnodynastes peronii tadpole and adult frog extracts. We investigated the general cytotoxicity of cane toad developmental stages (e.g., eggs, embryonic hatchlings, tadpoles and post-metamorphic toadlets) as well as selected adult tissues (e.g. skin, gut, liver). Our results showed that pre-metamorphic cane toad aqueous extracts used at 1 mg/ml on MDCK cells generated cytotoxicity levels comparable to ouabain treatment (3 microM). After normalisation, extracts from 2-3-month-old toadlets appeared less toxic than pre- and early metamorphic stages. Adult tissues revealed a gradient of cytotoxicity levels ranging from non-toxic brain to highly toxic dorsal skin extracts.

Markov chain Monte Carlo methods for assigning larvae to natal sites using natural geochemical tags.

Geochemical signatures deposited in otoliths are a potentially powerful means of identifying the origin and dispersal history of fish. However, current analytical methods for assigning natal origins of fish in mixed-stock analyses require knowledge of the number of potential sources and their characteristic geochemical signatures. Such baseline data are difficult or impossible to obtain for many species. A new approach to this problem can be found in iterative Markov Chain Monte Carlo (MCMC) algorithms that simultaneously estimate population parameters and assign individuals to groups. MCMC procedures only require an estimate of the number of source populations, and post hoc model selection based on the deviance information criterion can be used to infer the correct number of chemically distinct sources. We describe the basics of the MCMC approach and outline the specific decisions required when implementing the technique with otolith geochemical data. We also illustrate the use of the MCMC approach on simulated data and empirical geochemical signatures in otoliths from young-of-the-year and adult weakfish, Cynoscion regalis, from the U.S. Atlantic coast. While we describe how investigators can use MCMC to complement existing analytical tools for use with otolith geochemical data, the MCMC approach is suitable for any mixed-stock problem with a continuous, multivariate data.