Tandem Mass Spectrometry de novo Sequencing of the Skin Defense Peptides of the Central Slovenian Agile Frog Rana dalmatina.

Peptides released on frogs' skin in a stress situation represent their only weapon against micro-organisms and predators. Every species and even population of frog possesses its own peptidome being appropriate for their habitat. Skin peptides are considered potential pharmaceuticals, while the whole peptidome may be treated as a taxonomic characteristic of each particular population. Continuing the studies on frog peptides, here we report the peptidome composition of the Central Slovenian agile frog Rana dalmatina population. The detection and top-down de novo sequencing of the corresponding peptides was conducted exclusively by tandem mass spectrometry without using any chemical derivatization procedures. Collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), electron transfer dissociation (ETD) and combined MS3 method EThcD with stepwise increase of HCD energy were used for that purpose. MS/MS revealed the whole sequence of the detected peptides including differentiation between isomeric Leu/Ile, and the sequence portion hidden in the disulfide cycle. The array of the discovered peptide families (brevinins 1 and 2, melittin-related peptides (MRPs), temporins and bradykinin-related peptides (BRPs)) is quite similar to that of R. temporaria. Since the genome of this frog remains unknown, the obtained results were compared with the recently published transcriptome of R. dalmatina.

Mass Spectrometry Differentiation between Rana arvalis Populations Based on Their Skin Peptidome Composition.

Skin secretion of amphibians often represents the only weapon of these species against pathogens and predators. Peptides constitute the major portion of active molecules of that weapon and may be treated as potential pharmaceuticals for future generations. The first step of their efficient use involves establishing of their primary structure, i.e., sequencing. De novo sequencing by means of mass spectrometry was applied to Rana arvalis species, collected in the spring 2021 in Central Slovenia (vicinity of Ljubljana). HPLC-ESI-HRMS/MS with Orbitrap instruments was used to establish the skin peptidome of these species and compare it with the earlier identified skin peptidome of the Moscow population of Rana arvalis. Application of CID, HCD, ETD, and EThcD enabled detecting and sequencing 18 peptides; five of them were novel and may be treated as possible biomarkers of the Ljubljana population of Rana arvalis. Interestingly, representatives of two peptide families (temporins and brevinins 2) were not found in the Moscow population. MS3 modes, first of all EThcD, demonstrated their great potential in the de novo sequencing, including extraction of the sequence information from the intact peptides with disulfide cycle (rana box) in their structure and differentiation of isomeric Leu/Ile residues. Thus, all six isomeric residues were reliably distinguished in the novel melittin-related peptide AK-23-1. In addition, another post-translational modification dealing with carbonylation of the N-terminal Gly of novel temporin AVa was established using the MS3 mode. The obtained results demonstrate the efficiency of the use of MS3 tools in proteomics/peptidomics.

Using the structure-behavior-function model in conjunction with augmented reality helps students understand the complexity of the circulatory system.

This study examines how lower secondary school students understand the circulatory system, using the structure-behavior-function (SBF) framework for conceptual representation. It evaluates the progress of students' understanding after interventions with two different teaching approaches, one using a biology textbook supported by augmented reality (AR) technology and the other using only a textbook as a main source of information. The data analysis is based on the assumption that systemic understanding demands the perception of three system dimensions: the components forming the system at all levels of organization (its structures), the interactions and mechanisms between them (its behavior), and the function as a whole outcome (its phenomena). The results indicate that both learning approaches contribute to a higher level of understanding the circulatory system. The group using AR-supported educational materials showed statistically significant better improvements in their knowledge of the circulatory system, including all three components of the SBF framework.NEW & NOTEWORTHY This study examines how lower secondary school students understand the circulatory system, using the structure-behavior-function (SBF) framework for conceptual representation. It confirms that the use of augmented reality (AR) can help lower secondary school students understand the complexity of the circulatory system. The group using AR-supported instructional materials showed statistically significant better improvements in their knowledge of the circulatory system, including all three components of the SBF framework.

Do tick-borne diseases affect outdoor teaching in Slovenian preschools?

Childhood experiences with nature are decisive for children's wellbeing. The need for outdoor activities has been duly recognized in the preschool curriculum in Slovenia. However, there are some obstacles to this approach-specifically, tick-borne diseases. This study examines the views and experiences of Slovenian preschool teachers (n = 158) with ticks and tick-borne diseases in order to create a safer environment for the further development of outdoor education. The majority of the respondents engage in outdoor teaching once or several times a week. They have very frequent contact with ticks and tick-borne diseases in their professional lives. More than four-fifths of the respondents carry out some tick-prevention measures in preschools, but room for improvement remains. The respondents report rare cases in which parents or guardians have tried to prohibit them from visiting outdoor areas because of tick-borne diseases, and they explain how they dealt with the situation. The attitude of preschool teachers toward ticks and tick-borne diseases did not correlate significantly with the frequency with which they went outdoors in preschools. This study therefore concludes that, despite the objective risks associated with tick-borne diseases, there is still broad consensus among educators and parents on the importance of outdoor education in the early years.

Development and validation of a framework for the assessment of school curricula on the presence of evolutionary concepts (FACE).

Evolution is a key concept of biology, fundamental to understand the world and address important societal problems, but research studies show that it is still not widely understood and accepted. Several factors are known to influence evolution acceptance and understanding, but little information is available regarding the impacts of the curriculum on these aspects. Very few curricula have been examined to assess the coverage of biological evolution. The available studies do not allow comparative analyses, due to the different methodologies employed by the authors. However, such an analysis would be useful for research purposes and for the development of appropriate educational policies to address the problem of a lack of evolution acceptance in some countries. In this paper we describe the steps through which we developed a valid and reliable instrument for curricula analysis known as FACE: "Framework to Assess the Coverage of biological Evolution by school curricula." This framework was developed based on the "Understanding Evolution Conceptual Framework" (UECF). After an initial pilot study, our framework was reformulated based on identified issues and experts' opinions. To generate validity and reliability evidence in support of the framework, it was applied to four European countries' curricula. For each country, a team of a minimum of two national and two foreign coders worked independently to assess the curriculum using this framework for content analysis. Reliability evidence was estimated using Krippendorf's alpha and resulted in appropriate values for coding the examined curricula. Some issues that coders faced during the analysis were discussed and, to ensure better reliability for future researchers, additional guidelines and one extra category were included in the framework. The final version of the framework includes six categories and 34 subcategories. FACE is a useful tool for the analysis and the comparison of curricula and school textbooks regarding the coverage of evolution, and such results can guide curricula development.

EThcD Discrimination of Isomeric Leucine/Isoleucine Residues in Sequencing of the Intact Skin Frog Peptides with Intramolecular Disulfide Bond.

Our scientific interests involve de novo sequencing of non-tryptic natural amphibian skin peptides including those with intramolecular S-S bond by means of exclusively mass spectrometry. Reliable discrimination of the isomeric leucine/isoleucine residues during peptide sequencing by means of mass spectrometry represents a bottleneck in the workflow for complete automation of the primary structure elucidation of these compounds. MS3 is capable of solving the problem. Earlier we demonstrated the advanced efficiency of ETD-HCD method to discriminate Leu/Ile in individual peptides by consecutive application of ETD to the polyprotonated peptides followed by HCD applied to the manually selected primary z-ions with the targeted isomeric residues at their N-termini and registration of the characteristic w-ions. Later this approach was extended to deal with several (4-7) broad band mass ranges, without special isolation of the primary z-ions. The present paper demonstrates an advanced version of this method when EThcD is applied in the whole mass range to a complex mixture of natural non-tryptic peptides without their separation and intermediate isolation of the targeted z-ions. The proposed EThcD method showed over 81% efficiency for the large natural peptides with intact disulfide ring, while the interfering process of radical site migration is suppressed. Due to higher speed and sensitivity, the proposed EThcD approach facilitates the analytical procedure and allows for the automation of the entire experiment and data processing. Moreover, in some cases it gives a chance to establish the nature of the residues in the intact intramolecular disulfide loops. Graphical Abstract ᅟ.

Differentiation of frogs from two populations belonging to the Pelophylax esculentus complex by LC-MS/MS comparison of their skin peptidomes.

LC-MS/MS was applied to establish the composition of the skin peptidome of a Slovenian green frog belonging to the Pelophylax esculentus complex. As this was similar to the peptidome of the Moscow population of Pelophylax ridibundus, it allowed us to identify the Slovenian frog from the Pelophylax esculentus complex as Pelophylax ridibundus. The sequences of six new peptides from the brevinin 2 family are reported for the first time on the basis of manual interpretation of their tandem mass spectra. The structural similarity of the brevinin 2 peptides from the Moscow and Slovenian populations of Pelophylax ridibundus enables peptides from this family to be utilized as biomarkers for Pelophylax ridibundus inter- and intraspecies differentiation, and the proposed approach can be used as an analytical tool for differentiating the corresponding species and populations. The potential biological activities of the novel peptides were estimated by 2D mass mapping. The results allowed us to classify all of the available peptides belonging to the brevinin 2 family. Graphical Abstract Intraspecies identification within the green frog complex.

Beyond the use of an Explanatory Key Accompanying Submicroscopic Representations.

When learning chemistry, students encounter various visualizations introducing scientific concepts and processes unobservable to the naked eye. It is often assumed that these visualizations support students' understanding and enable them to solve problems more efficiently. The research presented here investigates students' use of an explanatory key while solving tasks based on submicroscopic representations. The following features of the explanatory key were examined: 1) colored versus black-and-white, and 2) pictorial versus textual. Eye-fixation patterns and students' verbal explanations indicated that the presence of color in the key does not influence students' task solving. However, significant differences were observed with regard to a textual versus pictorial key used in the tasks: students spent more time and fixated more frequently on the key while solving tasks using a textual key in comparison to a pictorial key. The results indicate that the type of explanatory key might play an important role in revealing students' representational competence with regard to submicroscopic representations.

LTQ Orbitrap Velos in routine de novo sequencing of non-tryptic skin peptides from the frog Rana latastei with traditional and reliable manual spectra interpretation.

RATIONALE: Mass spectrometry has shown itself to be the most efficient tool for the sequencing of peptides. However, de novo sequencing of novel natural peptides is significantly more challenging in comparison with the same procedure applied for the tryptic peptides. To reach the goal in this case it is essential to select the most efficient methods of triggering fragmentation and combine all the possible complementary techniques. METHODS: Collision-induced dissociation (CID), high-energy collision dissociation (HCD), and electron-transfer dissociation (ETD) tandem mass spectra recorded with a LTQ Orbitrap Velos instrument were used for the elucidation of the sequence of the natural non-tryptic peptides from the skin secretion of Rana latastei. Manual interpretation of the spectra was applied. RESULTS: The combined approach using CID, HCD, and ETD tandem mass spectra of the multiprotonated peptides in various charge states, as well as of their proteolytic fragments, allowed the sequences of seven novel peptides from the skin secretion of Rana latastei to be established. CONCLUSIONS: Manual mass spectrometry sequencing of natural non-tryptic peptides from the skin secretion of Rana latastei provided the opportunity to work successfully with these species and demonstrated once again its advantage over automatic approaches.