Search

Metals and metalloids are integral to biological processes and play key roles in physiology and metabolism.Nonetheless, overexposure to some metals or lack of others can lead to serious health consequences. In this study,eight zebrafish facilities collaborated to generate a multielement analysis of their centralized recirculating watersystems. We report a first set of average concentrations for 46 elements detected in zebrafish facilities. Our resultshelp to establish an initial baseline for trouble-shooting purposes, and in general for safe ranges of metalconcentrations in recirculating water systems, supporting reproducible scientific research outcomes with zebrafish.

IntroductionRecommendations have been issued to prevent workplace-related SARS-CoV-2 outbreaks. To discuss their impact, we assessed non-healthcare workers of a company which implemented such recommendations from early on. .Material and methodsWe performed molecular and serological tests to SARS-CoV-2 in workers of a Portuguese electrical company, in June 2020. Workers were also subject to an epidemiological survey. .ResultsA total of 1359 workers (out of 5850) underwent SARS-CoV-2 testing and answered the survey. Twenty-five participants (1.8%) had positive testing results.ConclusionsWe observed low frequency of SARS-CoV-2 infection in a company that early on implemented policies to decrease COVID-19 risk.

Many flowering plants contain stylar S-RNases that are involved in self-incompatibility and S-like RNases of which the biological function is uncertain. This paper reports the deduced amino acid sequence of an S-like RNase gene (PD1) from the self-incompatible plant Prunus dulcis (almond). The amino acid sequence of PD1, which was derived from cDNA and genomic DNA clones, showed 34-86% identity to acidic plant S-like RNases reported so far, with the highest degree of similarity being to an S-like RNase from Japanese pear (Pyrus pyrifolia). Based on RNA hybridisation experiments it appears that, like for many other S-like RNases, the expression of PD1 is not pistil-specific. Analysis of the genomic structure revealed the presence of three introns, of which one is similar in location to that of the related S-RNase gene from Solanaceae and Rosaceae. At least four bands hybridising to PD1 were found upon Southern hybridisation, suggesting the presence of a multigene family of S-like RNase genes in almond. The putative biological function of PD1 is discussed.

This boom in research has led its aquaculture to grow exponentially worldwide without sufficient accompanying studies on novel methods for husbandry and larval rearing which would optimize an intensive production of fish for research with adequate standardization and fish welfare. With new transgenic tools being rapidly developed, the zebrafish mutation project as well as its current role as a powerful tool in drug screens, it became essential to find better protocols and methods to breed and raise zebrafish faster, more reliably and with better welfare. However, little is known on the nutritional requirements of zebrafish [29, 30], being mostly reared with information available for Cyprinaformes [30, 31]. This is becoming a major concern within the research community as it imposes a difficulty to standardize a husbandry protocol in different facilities [32, 33]. Proper nutrition is not only important for individual growth and survival [29] but also to the reproductive success [34] which directly affects offspring fitness [35].

A husbandry workshop on July 3, 2017, at the 10th European Zebrafish Meeting in Budapest, Hungary (July 3–July 7, 2017), focused on the standardization, optimization, and streamlining of fish facility procedures. Standardization can be achieved for example by developing novel software and hardware tools, such as a fish facility database for husbandry and environmental facility management (Zebrabase, Oltova), or a hand-held, air-pressurized fish feeder for consistent food distribution (Blowfish, Argenton). Streamlining is achieved when work hours are reduced, as with the standardized fish feeder, or by limiting the number and types of fish diets and observing the effect on animal welfare and performance (Barton). Testing the characteristics of new fish diets and observing whether they produce better experimental outcomes (Certal) optimizes diets and improves fish productivity. Collectively, the workshop presentations emphasized how consistency and harmonization of husbandry procedures within and across aquatic facilities yield reproducible scientific outcomes.

In this project we propose a computer vision method, based on background subtraction, to estimate the number of zebrafish inside a tank. We addressed questions related to the best choice of parameters to run the algorithm, namely the threshold blob area for fish detection and the reference area from which a blob area in a threshed frame may be considered as one or multiple fish. Empirical results obtained after several tests show that the method can successfully estimate, within a margin of error, the number of zebrafish (fries or adults) inside fish tanks proving that adaptive background subtraction is extremely effective for blob isolation and fish counting.

A multi-approach was used to study different aspects of self-incompatibility (SI) in almond (Prunus dulcis). First, a population of almond cultivars was characterised as to their individual S-allele combination using separation of stylar protein extracts (non-equilibrium pH gradient electrofocusing) followed by staining for RNase activity, which led to the identification of one putative new allele and several new S-allele combinations. Second, a field pollination scheme was designed to study pollen tube progression and to obtain a spatial and temporal characterisation of this reproductive stage in both incompatible and compatible crosses. In addition, an anti-serum was raised against a synthetic peptide designed from an almond S-protein (S8) and used for immunological in situ detection in pistil cryosections. S-RNases were found to accumulate intercellularly in the stylar transmitting tissue as previously reported for other rosaceous species. The results are discussed in view of the evolution of the gametophytic SI system and the models proposed for its mechanism. Gametophyte selection is also proposed as an important intraspecific barrier to fertilisation in this species.

The molecular bases of self-incompatibility have been intensively studied in a restricted number of model species, but for most families the expression and distribution of S-proteins is unknown. In this work, pistil cryosections from apple were used for in situ detection of S-proteins. Two specific antibodies, one against the S3-protein and another against all apple S-proteins were used. S-proteins were shown to be localised in the intercellular space of the transmitting tissue, both in the stigma and style, which agrees with the proposed mechanism of action for S-RNases in gametophytic self-incompatibility. Some intracellular labelling was also observed in all ovary sections, confined to one layer of the nucellus surrounding the embryo sac, but this labelling was found to be non-S-allele-specific. Nevertheless, the signal in the ovary was tissue-specific, which may indicate that some component not encoded by the S-locus but similar to S-proteins was detected. To the best of our knowledge this is the first report on the precise distribution of S-RNases in a rosaceous species.

Zebrafish is already one of the most used model organisms in biomedical sciences and other research fields. It is therefore becoming increasingly important to assure that zebrafish maintained in laboratory aquaculture conditions are raised and housed under rigorous standards that promote health and welfare to guarantee the required quality and reproducibility of research data. Specifying the programs each facility is adopting would be the first step to achieve this by allowing other facilities to compare, improve, and discuss their protocols and fish performance. We provide in this article a detailed description of an integrated facility health management program, with protocols and readouts, fully designed and aimed at maximizing fish health, welfare, and performance for research.

Interdigital tissue regression during embryonic development is one of the most representative model systems of morphogenetic cell death, but the degenerative cascade accounting for this process awaits clarification. Although the canonical apoptotic caspase pathway appears to be activated in the interdigital mesenchyme committed to die, neither genetic nor chemical blockage of caspases or their downstream effectors, is sufficient to prevent cell death. Hence, alternative and/or complementary dying pathways must also be responsible for this degenerative process. In this work we have chosen to study the endonucleases during the regression of the interdigital tissue of avian embryos to gain insights into the molecular mechanisms accounting for programmed cell death in this system. We show that caspase activated DNase, which is a neutral DNase associated with the caspase apoptotic pathway, appears to be the main endonuclease only at an initial phase of interdigit regression. However at peak stages of the degenerative process, the acidic DNases L-DNase II and lysosomal DNase IIB become predominant in the system and markers for cell autophagy become moderately up-regulated. Consistent with the activation of acidic endonucleases we observed that microenvironmental pH value in the interdigits decreased to levels only appropriate for acidic enzymes. Furthermore, we found that overexpression of lysosomal DNase IIB in embryonic limb mesoderm promoted cell death, which was also accompanied by up-regulation and activation of L-DNase II. Up-regulation of acidic DNases was maintained in interdigits explanted to culture dishes, where the participation of exogenous professional phagocytes of hematopoietic origin is avoided. Finally, and consistent with all our findings, up-regulation of acidic DNases was much reduced in the webbed interdigits of duck embryos, characterized by a rudimentary interdigital degenerative process. We conclude that the regression of the interdigital tissue involves a coordinated and sequential activation of the caspase and lysosomal degenerative molecular cascades.

Polarized growth in pollen tubes results from exocytosis at the tip and is associated with conspicuous polarization of Ca2+, H+, K+, and Cl− -fluxes. Here, we show that cell polarity in Nicotiana tabacum pollen is associated with the exclusion of a novel pollen-specific H+-ATPase, Nt AHA, from the growing apex. Nt AHA colocalizes with extracellular H+ effluxes, which revert to influxes where Nt AHA is absent. Fluorescence recovery after photobleaching analysis showed that Nt AHA moves toward the apex of growing pollen tubes, suggesting that the major mechanism of insertion is not through apical exocytosis. Nt AHA mRNA is also excluded from the tip, suggesting a mechanism of polarization acting at the level of translation. Localized applications of the cation ionophore gramicidin A had no effect where Nt AHA was present but acidified the cytosol and induced reorientation of the pollen tube where Nt AHA was absent. Transgenic pollen overexpressing Nt AHA-GFP developed abnormal callose plugs accompanied by abnormal H+ flux profiles. Furthermore, there is no net flux of H+ in defined patches of membrane where callose plugs are to be formed. Taken together, our results suggest that proton dynamics may underlie basic mechanisms of polarity and spatial regulation in growing pollen tubes.

JJM was supported by a fellowship (SFRH/BD/45131/2008) from Fundacao para a Ciencia e Tecnologia (FCT), Portugal. ACC was supported by a postdocoral fellowship (SFRH/BPD/29957/2006) from Fundacao para a Ciencia e Tecnologia (FCT), Portugal. This work was supported by the following grants: european FP6-Cells into Organs, FCT (Grant PTDC/BIA-BCM/100867/2008 and POCTI-ISFL-4-664) and Ministerio de Ciencia e Innovacion, Spain (Subprograma Ramon y Cajal, reference RYC-2008-02753 and grant BFU2009-12279). JRL is supported by the Subprogram Ramon y Cajal from Ministerio de Ciencia e Innovacion, Spain (RYC-2008-02753). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H+ ef'flux. The onset and intensity of both V-ATPase expression and H+ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H+ pump is necessary for aldh1a2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration. published

Research in sexual plant reproduction has been boosted by the concentration of means and minds around central issues, such as incompatibility, microspore development or, to a less extent, the progamic phase of fertilisation. These areas are now pivotal since they involve basic biological questions still unanswered and which became recently accessible due to powerful genetic and molecular techniques. While these combined efforts resulted in the outcome of major findings that projected the field to a place not easy to by-pass even in the general context of Biology, this was only possible because of the existence of a few model species, particularly adapted to the experimental needs of the molecular weaponry. Most notably, Arabidopsis now plays a central role, since it possesses most of the development paths that characterise the majority of Angiosperms (with the exception of secondary growth). While this approach is not questionable, since it permits a faster advance in models and concepts, the reductionism underlying the focus on a few model species may create a void of knowledge on the natural variability and diversity of the cellular and molecular mechanisms. Most likely, the majority of the flowering species will never find their place as models species since they cannot compete as regards easiness to grow, maintain and analyse. Yet, an historical perspective shows beyond doubt that some of these species may bear variations to the basic models which may open new avenues and question established paradigms. Such was the case of Arabidopsis, for instance, often neglected and rejected for funding because of its condition of weed (Goldberg 1996).