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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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

A novel proteinaceous molecule produced by Lysinibacillus sp. OF-1 depends on the Ami oligopeptide transporter to kill Streptococcus pneumoniae.

acceptedVersio

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

Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules

BACKGROUND: Affibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to identify potential improvements in terms of yield, ease of production and purification advantages. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. RESULTS: We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, was identified to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both of the two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind their target,\ua0human HER3, as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530\ua0mg Affibody molecule/L. CONCLUSION: This study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching a high titer, and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli

Strain-level bacterial typing directly from patient samples using optical DNA mapping

For bacterial infections, it is important to rapidly and accurately identify and characterize the type of bacteria involved so that optimal antibiotic treatment can be given quickly to the patient. However, current diagnostic methods are sometimes slow and cannot be used for mixtures of bacteria. We have, therefore, developed a method to identify bacteria directly from patient samples. The method was tested on two common species of disease-causing bacteria - Escherichia coli and Klebsiella pneumoniae - and it could correctly identify the bacterial strain or subtype in both urine samples and mixtures. Hence, the method has the potential to provide fast diagnostic information for choosing the most suited antibiotic, thereby reducing the risk of death and suffering. Nyblom, Johnning et al. develop an optical DNA mapping approach for bacterial strain typing of patient samples. They demonstrate rapid identification of clinically relevant E. coli and K. pneumoniae strains, without the need for cultivation. BackgroundIdentification of pathogens is crucial to efficiently treat and prevent bacterial infections. However, existing diagnostic techniques are slow or have a too low resolution for well-informed clinical decisions.MethodsIn this study, we have developed an optical DNA mapping-based method for strain-level bacterial typing and simultaneous plasmid characterisation. For the typing, different taxonomical resolutions were examined and cultivated pure Escherichia coli and Klebsiella pneumoniae samples were used for parameter optimization. Finally, the method was applied to mixed bacterial samples and uncultured urine samples from patients with urinary tract infections.ResultsWe demonstrate that optical DNA mapping of single DNA molecules can identify Escherichia coli and Klebsiella pneumoniae at the strain level directly from patient samples. At a taxonomic resolution corresponding to E. coli sequence type 131 and K. pneumoniae clonal complex 258 forming distinct groups, the average true positive prediction rates are 94% and 89%, respectively. The single-molecule aspect of the method enables us to identify multiple E. coli strains in polymicrobial samples. Furthermore, by targeting plasmid-borne antibiotic resistance genes with Cas9 restriction, we simultaneously identify the strain or subtype and characterize the corresponding plasmids.ConclusionThe optical DNA mapping method is accurate and directly applicable to polymicrobial and clinical samples without cultivation. Hence, it has the potential to rapidly provide comprehensive diagnostics information, thereby optimizing early antibiotic treatment and opening up for future precision medicine management