Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping.

Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples

Design and numerical analysis of floating photovoltaic array for fjord conditions

To reduce anthropogenic climate change and its negative consequences, renewable energy is key. Solar energy is one of the cheapest forms of renewable energy. However, it demands large land areas, which may cause conflicts. Therefore, floating photovoltaic (FPV) systems have been developed. Existing FPV systems are mainly found on calm water bodies with minimal environmental loads. These areas may also introduce conflicts, and in some regions calm water bodies are a scarcity. Therefore, developing FPV systems that can withstand harsher marine environmental conditions could give the possibility of harvesting solar power in much larger quantities, and with less conflicts related to the use of area. As this is a rather new area of innovation, only few projects exist. This thesis aims to design and analyse an innovative FPV array that can withstand waves based on environmental loads from Norwegian fjords, with a significant wave height of 2,1 m. These conditions are much harsher than what is seen for most existing FPVs and may also serve as a step towards developing fully offshore FPV systems. The thesis analyses four different connection and mooring configurations to identify the effect on the response of the array. The suggested design consists of modules with a 2,5 x 2,5 m platform constructed of a grid of aluminium beams, with two 2 x 1 m solar panels and a 0,5 m wide maintenance walkway that allows access to all solar panels and connections, on top of four upright cylindric floaters. The modules are meant to be connected in arrays. The hydrostatic and hydrodynamic properties of a single module is calculated in the frequency domain using potential theory. The properties are implemented in Sima – Simo - Riflex, where a 3x3 array of modules is assembled and the behaviour is studied in a time domain analysis with irregular waves. The results show that the design is not suitable for the given environmental conditions without improvements. The connector and mooring stiffness impact the array behaviour, and the results may be used as an inspiration for future designs. The results also indicate that small lightweight FPV systems in large waves may be prone to large motions that are outside the limits of the applied method of analysis

Breeding And Maternal Behavior Of The Steller Sea Lion (Eumetopias Jubata) In Alaska

Thesis (M.S.) University of Alaska Fairbanks, 197

SegH and Hef: two novel homing endonucleases whose genes replace the mobC and mobE genes in several T4-related phages

T4 contains two groups of genes with similarity to homing endonucleases, the seg-genes (similarity to endonucleases encoded by group I introns) containing GIY-YIG motifs and the mob-genes (similarity to mobile endonucleases) containing H-N-H motifs. The four seg-genes characterized to date encode homing endonucleases with cleavage sites close to their respective gene loci while none of the mob-genes have been shown to cleave DNA. Of 18 phages screened, only T4 was found to have mobC while mobE genes were found in five additional phages. Interestingly, three phages encoded a seg-like gene (hereby called segH) with a GIY-YIG motif in place of mobC. An additional phage has an unrelated gene called hef (homing endonuclease-like function) in place of the mobE gene. The gene products of both novel genes displayed homing endonuclease activity with cleavage site specificity close to their respective genes. In contrast to intron encoded homing endonucleases, both SegH and Hef can cleave their own DNA as well as DNA from phages without the genes. Both segH and mobE (and most likely hef) can home between phages in mixed infections. We discuss why it might be a selective advantage for phage freestanding homing endonucleases to cleave both HEG-containing and HEG-less genomes

Characterization of Fusiformicin : a novel antimicrobial molecule with activity against Streptococcus pneumoniae

Antibiotikaresistens regnes som et av de største problemene som verden står ovenfor i dag i forhold til global helse, matsikkerhet og utvikling. Det arbeides med å finne nye antibiotika med aktivitet mot bakterier som bidrar til resistensproblemet. Dette inkluderer Streptococcus pneumoniae, en viktig humanpatogen bakterie med iboende evne til å tilegne seg gener for resistens. I 2020 ble det antimikrobielle stoffet Fusiformicin oppdaget. Fusiformicin produseres av Lysinibacillus fusiformis og har smalspektret aktivitet hovedsakelig mot streptokokker. Stoffets aktivitet har vist direkte sammenheng med peptidtransportsystemet Ami. I denne oppgaven ble det gjort arbeid for å karakterisere stoffet og finne innledende svar på stoffets virkningsmekanisme på S. pneumoniae. Med proteinase K-behandling og S. pneumoniae-overlay, ble det funnet at Fusiformicin er et peptid, trolig et klasse I- eller II-bakteriosin. Ved å bekrefte uttrykk av AmiE(D184A) og AmiF(D190A) (punktmutasjon i Walker B-motivet) i S. pneumoniae med et immunoblott, ble det basert på tidligere resultater vist at Fusiformicin trenger aktiv form av AmiCDEF for å fungere. Med mutasjon av aminosyrer (S503 og W504) viktige for peptidbinding i AmiA ble det vist at AmiA ikke er nødvendig for Fusiformicins virkning på S. pneumoniae. Likevel viste ektopisk uttrykk av villtype AmiA at proteinet kan bistå i økning av Fusiformicins aktivitet. Motsigende resultater i forhold til Fusiformicins virkningsmekanisme viser til et behov for flere tester for å få svar på om stoffet virker ekstra- eller intracellulært. Dog, dersom virkningsmekanismen er intracellulær ble det tydelig at den ikke er lik virkningsmekanismen til ampicillin eller ciprofloksasin, siden celler behandlet med disse antibiotikumene hadde signifikante endringer i morfologi sammenliknet med Fusiformicin-behandlede celler. Predikasjon av AmiA 3D-struktur viste at proteinet har en peptidbindende kløft med konserverte aminosyrer viktig for peptidbinding lik det homologe proteinet OppA fra L. lactis. Til slutt ble det funnet at AmiACDEF virket toksisk på E. coli, som gjorde at det ikke var mulig å introdusere Ami i andre Gram-positive bakterier slik det var tenkt. Videre arbeid bør inkludere rensing av Fusiformicin til >95% renhet slik at aminosyresekvensen til peptidet (og tilhørende gensekvens) kan avdekkes.Antibiotic resistance is considered one of the biggest problems that the world faces today related to global health, food security and development. Efforts are being made to find new antibiotics with activity against bacteria that contribute to the resistance problem. This includes Streptococcus pneumoniae, an important human pathogenic bacterium with an inherent ability to acquire resistance genes. In 2020, the antimicrobial compound Fusiformicin was discovered. Fusiformicin is produced by Lysinibacillus fusiformis and has narrow-spectrum activity mainly against streptococci. The activity of the substance has shown a direct connection with the peptide transport system Ami. In this thesis, work was done to characterize the substance and find an initial answer to the substance's mechanism of action on S. pneumoniae. With proteinase K treatment and an S. pneumoniae overlay, it was found that Fusiformicin is a peptide, probably a class I or II bacteriocin. By confirming expression of AmiE(D184A) and AmiF(D190A) (point mutation in the Walker B motif) in S. pneumoniae with an immunoblot, it was shown based on previous results that Fusiformicin requires the active form of AmiCDEF to have antimicrobial activity. With mutation of amino acids (S503 and W504) important for peptide binding in AmiA, it was shown that AmiA is not necessary for the action of Fusiformicin on S. pneumoniae. Nevertheless, ectopic expression of wild-type AmiA showed that the protein may assist in increasing the activity of Fusiformicin. Due to contradictory results in relation to the mechanism of action of Fusiformicin, several tests must be performed to obtain an answer as to whether the substance acts extra- or intracellularly. However, if the mechanism of action is intracellular, it became clear that it is not similar to the mechanism of action of ampicillin or ciprofloxacin, since cells treated with these antibiotics showed significant different morphological changes compared with Fusiformicin treated cells. Prediction of AmiA 3D structure showed that the protein has a peptide binding cleft with conserved amino acids important for peptide binding similar to the homologous protein OppA from L. lactis. Finally, AmiACDEF was found to be toxic to E. coli, making it difficult to introduce Ami into other Gram-positive bacteria as intended. Further work should include purification of Fusiformicin to >95% purity so that the amino acid sequence of the peptide (and associated gene sequence) can be detected.M-BIOTE

Den kvartärgeologiska forskningen i Sverige under de senaste 25 åren

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Målsättningar och motivation kopplade till fysisk aktivitet: En tvärsnittsstudie baserat i självbestämmandeteorin

Syftet med föreliggande studie var att med utgångspunkt i självbestämmandeteorin, undersöka sambanden mellan målsättningar och motivation kopplat till fysisk aktivitet. Vidare undersöktes om målsättningar kunde predicera fysisk aktivitet via typ av motivation. En enkätundersökning med 97 deltagare genomfördes för att undersöka sambanden mellan enskilda målsättningar i förhållande till motivation och fysisk aktivitet. En medieringsanalys genomfördes för att även se hur sambandet mellan målsättningar och fysisk aktivitet medieras av typ av motivation. Resultatet visade på blandade korrelationer mellan fysisk aktivitet och typ av målsättningar. Samtliga målsättningar korrelerade med självbestämmande motivation, men kontrollerad motivation uppvisade bara signifikanta samband med de två yttre målsättningarna. Vidare hade fysisk aktivitet en positiv korrelation med självbestämmande motivation, men inte till kontrollerad motivation. Resultaten av medieringsanalysen visade att det fanns en signifikant indirekt effekt från samtliga målsättningar till fysisk aktivitet via självbestämmande motivation. Däremot fanns det ingen signifikant indirekt effekt via kontrollerad motivation. De samlade resultaten följer till stor del tidigare forskning med vissa skillnader, som diskuteras

Parallel within-host evolution alters virulence factors in an opportunistic Klebsiella pneumoniae during a hospital outbreak

Bacterial pathogens adapt to host niches because of selective within-host pressures, an evolutionary process that offers invaluable insights into host-pathogen interactions. Here, we retrospectively track the evolution of a single multiresistant Klebsiella pneumoniae clone in 110 patients over a 5-year nosocomial outbreak. We combine comparative genomics with phenotypic characterization of mucoviscosity, serum survival, iron utilization, biofilm formation, and infection potential in Galleria mellonella for all isolates. Strong positive selection within patients targeted key virulence factors. Notably, convergent evolutionary trajectories were dominated by reduced acute virulence and recurrent changes in iron uptake regulation, capsule and lipopolysaccharide, and enhanced biofilm formation. These phenotypic changes likely represent clinical niche adaptations, with some resulting in trade-offs during gastrointestinal colonization. This study underscores the dynamic nature of within-host evolution and its role in shaping virulence in opportunistic pathogens, even on short time scales

Экономика образования: нравственный аспект

В связи с обострением социально-экономических противоречий современного общества особую актуальность приобретают вопросы изучения нравственного аспекта экономической деятельности. В статье рассматриваются предпосылки изучения нравственных основ экономики, изучается мнение ученых-экономистов о влиянии нравственности на экономику. А также делается вывод о непосредственном участии сферы высшего образования в формировании личности, разделяющей традиционные нравственные ценности общества