Hybrid Stem Intervention as New Post-Pandemic Approach to Motivate Students to Stem

There has long been a struggle over how to increase student interest in careers in STEM and meet the labour market’s need for specialised knowledge and skills. The long-standing debate at the EU level about the role of formal, informal, and non-formal education in meeting these challenges has not yet reached a clear conclusion. In the last decade, there has been a significant increase in the number of STEM programmes offered by various non-governmental organisations in Croatia. These interventions are often localised and have limited social impact, but there is a strong willingness to create an environment for their greater inclusion in the formal education system, triggered by comprehensive curriculum reform in Croatia. Motivation, especially intrinsic motivation, is a crucial driving force in our lives. In our pilot study, conducted with 6th grade elementary students, we aimed to explore the extent to which STEM interventions encourage students to learn more about the topic and whether it is possible to incorporate lessons learned from the pandemic into the design of future interventions. Our results show that there is no significant difference in student motivation after a 45-minute whole-class interactive intervention between face-to-face and virtual delivery. Although the intervention was entertaining, students perceived the science as interesting and useful rather than entertaining. Considering that students have positive attitudes toward Nature as a school subject, an early intervention with students at this age could be useful in maintaining their interest and preventing a decline in interest later in life. This finding is particularly important in the context of the transformation of the Croatian elementary school system into a “whole-day school”, which provides room for incorporating this type of intervention into a regular school system

Human Adenovirus Type 26 Infection Mediated by αvβ3 Integrin Is Caveolin-1-Dependent

Human adenovirus type 26 (HAdV26) has been recognized as a promising platform for vaccine vector development, and very recently vaccine against COVID-19 based on HAdV26 was authorized for emergency use. Nevertheless, basic biology of this virus, namely, pathway which HAdV26 uses to enter the cell, is still insufficiently known. We have shown here that HAdV26 infection of human epithelial cells expressing low amount of αvβ3 integrin involves clathrin and is caveolin-1- independent, while HAdV26 infection of cells with high amount of αvβ3 integrin does not involve clathrin but is caveolin-1-dependent. Thus, this study demonstrates that caveolin-1 is limiting factor in αvβ3 integrin-mediated HAdV26 infection. Regardless of αvβ3 integrin expression, HAdV26 infection involves dynamin-2. Our data provide for the first-time description of HAdV26 cell entry pathway, hence increase our knowledge of HAdV26 infection. Knowing that functionality of adenovirus vector is influenced by its cell entry pathway and intracellular trafficking, our results will contribute to better understanding of HAdV26 immunogenicity and antigen presentation when used as vaccine vector

Nanoparticle-Based Adjuvants and Delivery Systems for Modern Vaccines

Ever since the development of the first vaccine, vaccination has had the great impact on global health, leading to the decrease in the burden of numerous infectious diseases. However, there is a constant need to improve existing vaccines and develop new vaccination strategies and vaccine platforms that induce a broader immune response compared to traditional vaccines. Modern vaccines tend to rely on certain nanotechnology platforms but are still expected to be readily available and easy for large-scale manufacturing and to induce a durable immune response. In this review, we present an overview of the most promising nanoadjuvants and nanoparticulate delivery systems and discuss their benefits from tehchnological and immunological standpoints as well as their objective drawbacks and possible side effects. The presented nano alums, silica and clay nanoparticles, nanoemulsions, adenoviral-vectored systems, adeno-associated viral vectors, vesicular stomatitis viral vectors, lentiviral vectors, virus-like particles (including bacteriophage-based ones) and virosomes indicate that vaccine developers can now choose different adjuvants and/or delivery systems as per the requirement, specific to combatting different infectious diseases

Interactions of variously coated gold and silver nanoparticles with a bis(triarylborane) photodyanmic therapy (PDT)-dye; their cellular uptake, cytotoxicity and photo-activity

Background and purpose: Diethynylarene-linked bis(triarylborane) tetracations can be used as probes for fluorimetric and Raman sensing of biomacromolecules, as well as promising theragnostic agents. Among them, bis(triarylborane) fluorophore (TAB3), when bonded to Ag nanoparticles (NP), stood out with specific properties such as Raman signal enhancemen of the TAB3 dye in a cuvette. However, TAB3 dye - nanoparticle composites have not been studied in biological systems. For this reason, questions arose as to whether different types of metal nanoparticles (Au or Ag-based) with different coatings (negatively charged citrate or neutral PVP) could be efficiently stained with the TAB3 dye in a cuvette. The aim of this research was to examine Au and Ag nanoparticles of similar size (20-25 nm) with different stabilizers for their cellular uptake, cytotoxicity in the dark and under visible light radiation, to characterize the interactions of nanoparticles with the TAB3 fluorophore, and to study NP-TAB3 composites in cells, evaluate their intracellular staining, as well as possible photoinduced release and biological activity. Materials and methods: The binding constants of Au- and Ag- based nanoparticles with TAB3 were determined by fluorimetric titrations. The cytotoxic effect of NPs was determined by the survival of A549 cells (MTT assay). Cellular uptake of both NP and NP-TAB3 composites were performed by live cell imaging experiments. Results: The Au- or Ag-based NPs with different coatings bind to the TAB3 with high affinity. These NPs, as well as TAB3-NP complexes, efficiently enter living human cells, accumulating in cytoplasm with no apparent selectivity for a particular organelle. Even prolonged 3-day treatment with the NPs studied did not show any toxic effect on the cells. Bioimaging studies in cells revealed that the TAB3-NP complex does not intracellularly dissociate; the previously reported photo-bioactivity of TAB3 is completely inhibited by binding to NPs. Conclusion: Au- and Ag NPs were non-covalently stained by TAB3, irrespective of the different coatings, with similar binding affinities. Emission from TAB3 is strongly quenched by the NPs, but not completely. Experiments on living human cells revealed that neither free NPs, nor their composites with TAB3, were toxic. Bioimaging studies by confocal microscopy revealed that all NPs efficiently enter living cells within 90 min. Colocalization experiment with simultaneous collection of data in the reflection and fluorescence modes demonstrated that the TAB3 dye remained bound to NPs inside cells. Strong irradiation of TAB3-NP inside cells with a 457 nm laser did not yield any damage to the cells, at variance with our previously shown very strong photo-bioactivity of the TAB3 dye alone. Thus, binding of a chromophore to a nanoparticle can inhibit the chromophore’s ability to undergo photo-induced singlet oxygen production, consequently blocking its photo-bioactivity

Bis-Pyrene Photo-Switch Open- and Closed-Form Differently Bind to ds-DNA, ds-RNA and Serum Albumin and Reveal Light-Induced Bioactivity

Newly designed and synthesized diarylethene (DAE) derivatives with aliphatic amine sidearms and one with two pyrenes, revealed excellent photo-switching property of central DAE core in MeOH and water. The only exception was bis-pyrene analogue, its DAE core very readily photochemically closed, but reversible opening completely hampered by aromatic stacking interaction of pyrene(s) with cyclic DAE. In this process, pyrene fluorescence showed to be a reliable monitoring method, an open form characterized by strong emission at 480 nm (typical for pyrene-aggregate), while closed form emitted weakly at 400 nm (typical for pyrene-DAE quenching). Only open DAE-bis-pyrene form interacted measurably with ds-DNA/RNA by flexible insertion in polynucleotide grooves, while self-stacked closed form did not bind to DNA/RNA. For the same steric reasons, flexible open DAE-bis-pyrene form was bound to at least three different binding sites at bovine serum albumin (BSA), while rigid, self-stacked closed form interacted dominantly with only one BSA site. Preliminary screening of antiproliferative activity against human lung carcinoma cell line A549 revealed that all DAE-derivatives are non-toxic. However, bis-pyrene analogue efficiently entered cells and located in the cytoplasm, whereby irradiation by light (315–400 nm) resulted in a strong, photo-induced cytotoxic effect, typical for pyrene-related singlet oxygen species production

Increased Adenovirus Type 5 Mediated Transgene Expression Due to RhoB Down-regulation

Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using a study model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5- mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We show that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking

Impact of the Histidine‐Triazole and Tryptophan‐Pyrene Exchange in the WHW Peptide: Cu(II) Binding, DNA/RNA Interactions and Bioactivity

n three novel peptidoids based on the tryptophan—histidine— tryptophan (WHW) pep‐ tide, the central histidine was replaced by Ala‐(triazole), and two derivatives also had one trypto‐ phan replaced with pyrene‐alkyls of different lengths and flexibility. Pyrene analogues show strong fluorescence at 480–500 nm, attributed to intramolecular exciplex formation with trypto‐ phan. All three peptidoids bind Cu2+ cation in water with strong affinity, with Trp‐ Ala‐(triazole)‐Trp binding comparably to the parent WHW, and the pyrene analogues even stronger, demonstrating that replacement of histidine with triazole in peptides does not hamper Cu2+ coordination. The studied peptidoids strongly bind to ds‐DNA and ds‐RNA, whereby their complexes with Cu2+ exhibit distinctively different interactions in comparison to metal‐free ana‐ logues, particularly in the stabilization of ds‐DNA against thermal denaturation. The pyrene pep‐ tidoids efficiently enter living cells with no apparent cytotoxic effect, whereby their red‐shifted emission compared to the parent pyrene allows intracellular confocal microscopy imaging, show‐ ing accumulation in cytoplasmic organelles. However, irradiation with 350 nm light resulted in evident antiproliferative effect on cells treated with micromolar concentrations of the pyrene ana‐ logues, presumably attributed to pyrene‐induced production of singlet oxygen and consecutive cellular damage

The Revolving Door of Adenovirus Cell Entry: Not All Pathways Are Equal

Adenoviruses represent exceptional candidates for wide-ranging therapeutic applications, from vectors for gene therapy to oncolytics for cancer treatments. The first ever commercial gene therapy medicine was based on a recombinant adenovirus vector, while most recently, adenoviral vectors have proven critical as vaccine platforms in effectively controlling the global coronavirus pandemic. Here, we discuss factors involved in adenovirus cell binding, entry, and trafficking ; how they influence efficiency of adenovirus-based vectors ; and how they can be manipulated to enhance efficacy of genetically modified adenoviral variants. We focus particularly on endocytosis and how different adenovirus serotypes employ different endocytic pathways to gain cell entry, and thus, have different intracellular trafficking pathways that subsequently trigger different host antiviral responses. In the context of gene therapy, the final goal of the adenovirus vector is to efficiently deliver therapeutic transgenes into the target cell nucleus, thus allowing its functional expression. Aberrant or inefficient endocytosis can impede this goal, therefore, it should be considered when designing and constructing adenovirus-based vectors

Increased Adenovirus Type 5 Mediated Transgene Expression Due to RhoB Down-Regulation

Abstract Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using an in vitro model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5-mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We propose that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking