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The extremely radiation and desiccation resistant bacterium Deinococcus radiodurans possesses three genes encoding Endonuclease III-like enzymes (DrEndoIII1, DrEndoIII2, DrEndoIII3). In vitro enzymatic activity measurements revealed that DrEndoIII2 is the main Endonuclease III in this organism, while DrEndoIII1 and 3 possess unusual and, so far, no detectable EndoIII activity, respectively. In order to understand the role of these enzymes at a cellular level, DrEndoIII knockout mutants were constructed and subjected to various oxidative stress related conditions. The results showed that the mutants are as resistant to ionizing and UV-C radiation as well as H2O2 exposure as the wild type. However, upon exposure to oxidative stress induced by methyl viologen, the knockout strains were more resistant than the wild type. The difference in resistance may be attributed to the observed upregulation of the EndoIII homologs gene expression upon addition of methyl viologen. In conclusion, our data suggest that all three EndoIII homologs are crucial for cell survival in stress conditions, since the knockout of one of the genes tend to be compensated for by overexpression of the genes encoding the other two.

Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H2O2 detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H2O2 substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP.

Abstract Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.

The development of a lipid nano-delivery system was attempted for three specific poly (ADP-ribose) polymerase 1 (PARP1) inhibitors: Veliparib, Rucaparib, and Niraparib. Simple lipid and dual lipid formulations with 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1′-glycerol) sodium salt (DPPG) and 1,2-dipalmitoyl-sn-glycero-3-phosphocoline (DPPC) were developed and tested following the thin-film method. DPPG-encapsulating inhibitors presented the best fit in terms of encapsulation efficiency (>40%, translates into concentrations as high as 100 µM), zeta potential values (below −30 mV), and population distribution (single population profile). The particle size of the main population of interest was ~130 nm in diameter. Kinetic release studies showed that DPPG-encapsulating PARP1 inhibitors present slower drug release rates than liposome control samples, and complex drug release mechanisms were identified. DPPG + Veliparib/Niraparib presented a combination of diffusion-controlled and non-Fickian diffusion, while anomalous and super case II transport was verified for DPPG + Rucaparib. Spectroscopic analysis revealed that PARP1 inhibitors interact with the DPPG lipid membrane, promoting membrane water displacement from hydration centers. A preferential membrane interaction with lipid carbonyl groups was observed through hydrogen bonding, where the inhibitors’ protonated amine groups may be the major players in the PARP1 inhibitor encapsulation mode.

Endonuclease III (EndoIII) is a bifunctional DNA glycosylase with specificity for a broad range of oxidized DNA lesions. The genome of an extremely radiation- and desiccation-resistant bacterium, Deinococcus radiodurans, possesses three genes encoding for EndoIII-like enzymes (DrEndoIII1, DrEndoIII2 and DrEndoIII3), which reveal different types of catalytic activities. DrEndoIII2 acts as the main EndoIII in this organism, while DrEndoIII1 and 3 demonstrate unusual and no EndoIII activity, respectively. In order to understand the role of DrEndoIII1 and DrEndoIII3 in D. radiodurans, we have generated mutants which target non-conserved residues in positions considered essential for classic EndoIII activity. In parallel, we have substituted residues coordinating the iron atoms in the [4Fe-4S] cluster in DrEndoIII2, aiming at elucidating the role of the cluster in these enzymes. Our results demonstrate that the amino acid substitutions in DrEndoIII1 reduce the enzyme activity without altering the overall structure, revealing that the residues found in the wild-type enzyme are essential for its unusual activity. The attempt to generate catalytic activity of DrEndoIII3 by re-designing its catalytic pocket was unsuccessful. A mutation of the iron-coordinating cysteine 199 in DrEndoIII2 appears to compromise the structural integrity and induce the formation of a [3Fe-4S] cluster, but apparently without affecting the activity. Taken together, we provide important structural and mechanistic insights into the three EndoIIIs, which will help us disentangle the open questions related to their presence in D. radiodurans and their particularities.

Extracellular electron transfer pathways allow bacteria to transfer electrons from the cell metabolism to extracellular substrates, such as metal oxides in natural environments and electrodes in microbial electrochemical technologies (MET). Studies of electroactive microorganisms and mainly of Shewanella oneidensis MR-1 have demonstrated that extracellular electron transfer pathways relies on several multiheme c-type cytochromes. The small tetraheme cytochrome c (STC) is highly conserved among Shewanella species and is one of the most abundant cytochromes in the periplasmic space. It transfers electrons from the cell metabolism delivered by the inner-membrane tetraheme cytochrome CymA, to the porin-cytochrome complex MtrCAB in the outer-membrane, to reduce solid electron acceptors outside the cell, or electrodes in the case of MET. In this work knock-out strains of STC of S. oneidensis MR-1, expressing STC from distinct Shewanella species were tested for their ability to perform extracellular electron transfer, allowing to explore the effect of protein mutations in living organisms. These studies, complemented by a biochemical evaluation of the electron transfer properties of the individual proteins, revealed a considerable plasticity in the molecular components involved in extracellular electron transfer. The results of this work are pioneering and of significant relevance for future rational design of cytochromes in order to enhance extracellular electron transfer and thus contribute to the practical implementation of MET.

The Deinococcus radiodurans bacterium is extremely resistant to ionising radiation and desiccation and can withstand a 200-fold higher radiation dose than most other bacteria with no loss of viability. The mechanisms behind this extreme resistance are not fully understood, but it is clear that several factors contribute to this phenotype. Efficient scavenging of reactive oxygen species and repair of damaged DNA are two of these. In this review, we summarise the results from a decade of structural and functional studies of the DNA repair machinery of Deinococcus radiodurans and discuss how these studies have contributed to an improved understanding of the molecular mechanisms underlying DNA repair and to the outstanding resistance of Deinococcus radiodurans to DNA damaging agents.

Upon infection by pathogenic bacteria, production of reactive oxygen species (ROS) is part of the host organism's first line of defence. ROS damage a number of macromolecules, and in order to withstand such a harsh environment, the bacteria need to have well‐functioning ROS scavenging and repair systems. Herein, MutT is an important nucleotide‐pool sanitization enzyme, which degrades 8‐oxo‐dGTP and thus prevents it from being incorporated into DNA. In this context, we have performed a comparative biochemical and structural analysis of MutT from the fish pathogenAliivibrio salmonicida (AsMutT) and the human pathogenVibrio cholerae (VcMutT), in order to analyse their function as nucleotide sanitization enzymes and also determine possible cold‐adapted properties ofAsMutT. The biochemical characterisation revealed that both enzymes possess activity towards the 8‐oxo‐dGTP substrate, and thatAsMutT has a higher catalytic efficiency thanVcMutT at all temperatures studied. Calculations based on the biochemical data also revealed a lower activation energy (Ea) forAsMutT compared toVcMutT, and differential scanning calorimetry experiments showed thatAsMutT displayed an unexpected higher melting temperature (Tm) value thanVcMutT. A comparative analysis of the crystal structure ofVcMutT, determined to 2.42 Å resolution, and homology models ofAsMutT indicate that three unique Gly residues in loops ofVcMutT, and additional long range ion‐pairs inAsMutT could explain the difference in temperature stability of the two enzymes. We conclude thatAsMutT is a stable, cold‐active enzyme with high catalytic efficiency and reducedEa, compared to the mesophilicVcMutT.

I denne studien undersøkes betydningen av den koronarelaterte nedstengningen av norsk arbeidsliv for opplevelsen av jobbfellesskap. Dette gjøres ved å undersøke 1) om det har vært noen endring i opplevd jobbfellesskap fra tiden like før nedstengningen 12. mars 2020 til slutten av juni 2021 og 2) betydningen av koronarelaterte endringer i arbeidssituasjonen. I analysene av endring over tid benyttes data fra spørreskjemaundersøkelsene Medbestemmelsesbarometeret 2020 (N=3575) og Pandemiundersøkelsen 2021 (N=3067). Endringene i arbeidssituasjonen studeres i pandemiutvalget, og vi ser mer konkret på betydningen av hjemmekontor, permitteringer, endringer i arbeidsoppgaver og arbeidstid. Resultatene viste ingen endring i opplevelsen av jobbfellesskap fra før pandemien til juni 2021. Utviklingen over tid var imidlertid forskjellig på Sør- og Vestlandet sammenliknet med i Oslo og omegn. Når det gjelder endringene i arbeidssituasjonen, fant vi ingen sammenheng for hjemmekontor eller permitteringer. Sammenhengen mellom endring i arbeidsoppgaver og opplevd jobbfellesskap kunne heller ikke påvises med sikkerhet. Analysen viste videre at både reduksjon og økning i arbeidstid var negativt korrelert med opplevelsen av jobbfellesskap. Funnene diskuteres opp imot ulike perspektiver på fellesskapsdannelse og organisatoriske endringer. Studien bidrar til å belyse hvordan opplevelsen av jobbfellesskap kan være på det samme nivået til tross for store endringer i sosiale og materielle strukturer.

Little research has been done in Norway about what motivates students to choose a career in teaching. This is a qualitative study aimed at examining students’ motivation for becoming teachers, as well as examining if the Teacher Education program of study promoted their motivation and development as teachers. The study is anchored in motivation theory about mastery experiences and values. In addition, we choose to make use of theoretical perspectives involving Bildung, engagement and connectedness to school, since these revealed themselves to be important in the students’ motivation, both before and during their studies. The empirical basis for this study is 40 student logs, all written halfway through the first year of study. The texts are studied using qualitative analysis. The study’s findings are presented in six categories: For the Wellbeing of School Children; Social Commitment; Inspiring Influences; Previous Accomplishments; Fundamental Importance of the Student Practicum period; and But, What About the Teacher Training College?