50,225 research outputs found
Like-a-PRO: WP2:T2.1: Samples (Olypep/Krill hydrolysate powder) provided by Rimfrost to Like-a-PRO partners
No full text<p>The dataset provides an overview of dates, quantites and sample types sent from Rimfrost to partners in the Like-a-PRO project. All samples are specifically form the krill species: Antarctic krill (Euphausia superba). In addition, a brief explination of intial and futher processing along with simplified goals for the samples is added in the overview. </p>
<p>Contact the data collectors (contact persons) for more information regarding specifics on the processing/development and/or goals for each sample.</p>
Elucidating antibiotic mode of action through multi-omics analysis of bacterial stress responses
No full textIn 2023, the World Health Organization stated that antimicrobial resistance (AMR) remains one of the top ten threats to global health. In 2019, an estimated 4.95 million deaths were associated with antibiotic-resistant infections. Furthermore, AMR could cost the global economy up to USD 100 billion by 2050 if we fail to reduce the spread of resistance. Therefore, the development of new antibiotics to treat AMR is crucial to avoid a global health crisis.
β-clamps are ring-shaped proteins found in bacteria that act as a platform for a number of important proteins involved in DNA replication. These proteins bind to the β-clamp using special peptide sequences, known as binding motifs. β-clamp targeting peptides (BTP) are a new class of antibiotic drug candidates that have broad-spectrum activity, prevent biofilm formation, inhibit mutagenesis, and yield rapid bacterial death. BTPs bind to the β-clamp at the same site as replicative proteins using a binding motif known as APIM, thereby blocking replication.
BTPs exhibit a rapid bactericidal effect, even in stationary phase bacteria where the replication rate is greatly reduced. This observation suggests that BTPs possess multiple modes of action (MoA). In papers I and III, we explored other possible MoAs for BTPs by using transcriptomics, proteomics, and metabolomics to study a BTP variant called BTP-001. Both papers demonstrate that BTP-001 induces the production of reactive oxygen species (ROS) in Staphylococcus aureus (paper I) and Escherichia coli (paper III). Furthermore, paper III revealed that BTP-001 induces a cell envelope stress response. To investigate this further, we examined the impact of BTP-001 on E. coli mutants with a compromised cell envelope stress response. These mutants displayed reduced susceptibility to the rapid bactericidal effect of BTP-001, and they also produced lower levels of ROS. This suggests that ROS induced by cell envelope stress plays a significant role in the rapid bactericidal effect of BTP-001.
Two antibiotics with a synergistic or additive combination effect offer several potential benefits, including broad-spectrum activity, lower resistance development, and reduced toxicity. BTP-001 has a strong additive effect in combination with halogenated pyrrolopyrimidines against S. aureus. In paper I, we showed that this effect is due to the activation of several stress responses that were unique to the combination treatment. To investigate the MoA of these two antibiotic drug candidates, we developed a proteomics-based method to analyze changes in signaling proteins after treatment. By focusing on this subset of proteins, rather than all proteins within a bacterial cell, we could determine whether these proteins were activated or deactivated, thereby significantly enhancing our understanding of the MoA.
The SOS response is a DNA damage stress response that increases the mutation rate and, consequently, the development of resistance in bacteria. Previously, the SOS response was thought to be activated gradually, with SOS genes expressed at different times after DNA damage. In paper II, we used transcriptomics and metabolomics to show that most SOS genes are expressed shortly after the SOS response is induced and that elevated pyrimidine pools in the cell may play an important role in increased mutagenesis. Knowing exactly how DNA repair and mutation processes take place is crucial for fighting AMR and developing new antibiotics that target bacteria in more effective ways.
The papers in this thesis demonstrate that "omics" methods provide new insights into the MoA of antibiotics and how they affect stress-related processes. Using these methods, we show that BTP-001 has at least two MoAs. This makes BTPs promising antibiotic drug candidates in the fight against AMR
Genotypic and Phenotypic Characterization of Norwegian Farmhouse Ale Yeast Cultures: A Domestication-Driven Evolution
No full textThere is an immense gap in our knowledge regarding biodiversity of yeasts and the available untapped yeasts that can be found in nature. Studying both domesticated and wild species is important as it can enable us to better understand the natural history, and potentially reveal how selective pressures have affected and shaped its evolution. The yeast Saccharomyces cerevisiae has been used for thousands of years by mankind to produce the most consumed fermented beverage in the world; beer. Recently, the domestication and divergence of S. cerevisiae beer yeasts has been thoroughly studied. However, very little is known about the evolution and phenotypic behaviour of the Norwegian farmhouse ale yeast (NFAY) cultures that were used in traditional home-brewed beer.
In this work, 14 different NFAY cultures with a total of 24 yeast isolates, were characterized and compared with S. cerevisiae laboratory strains and commercially available beer yeasts. The yeasts were taxonomically classified at genus and species levels by sequencing of rRNA gene regions; internal transcribed spacer (ITS) region and partially sequencing of the large subunit (LSU), referred to as LSU1 and LSU2. Since incomplete fermentation of maltotriose is a common problem in the brewing industry and maltotriose utilization is highly associated with domestication, the presence and distribution of a gene encoding an α-glucoside transporter, AGT1, was investigated. This transporter has a wide substrate specificity for common sugars in the wort, including maltotriose. Moreover, the ploidy was investigated with flow cytometry, and a small-scale fermentation of the samples was carried out at 22 °C and 35 °C. The fermentation performance, as well as its flavor and aroma profile, was determined with chromatography and mass spectrometry instrumentation (headspace GC-MS, HPLC and LC-MS).
The species differentiation based on the ITS and LSU regions suggested that the NFAY cultures consist of at least three different species; Saccharomyces cerevisiae, Saccharomyces bayanus and the less familiar non-Saccharomyces yeast Meyerozyma caribbica. The Saccharomyces species seems to be closely related, as it was generally observed low DNA sequence divergence among them. The AGT1 allele was present in all Saccharomyces species except two of the the laboratory strains. It was discovered that a particular AGT1 gene mutation producing a premature stop codon may not be limited to lager strains, as presumed before. Interestingly, the NFAY samples carried a variant of the AGT1 gene that is more similar to the domesticated ones rather than wild S. cerevisiae variants. This raises the suspicion of them originating from domesticated ancestors. Phenotypic variants among the NFAY samples were also observed, and were different from the performance of commercial yeasts. This diversity is believed to have emerged from the farmhouse brewing traditions, through high fermentation temperatures, unique storage techniques and repitching. However, the phenotypic traits were not conserved within the three species, nor did they carry geographical structure. This can indicate that the phenotypic characteristics are mainly strain dependent. This, together with high maltotriose utilization, low sequence divergence and wide geographical dispersal, further supports the theory of a domestication-driven evolution in the NFAY cultures
Characterization of genotype and beer fermentation properties of Norwegian Farmhouse Ale Yeasts
No full textEight different Norwegian Farmhouse Ale Yeasts from Voss, Stranda, Lærdal, Hornindal and Olden were donated to IBT for analysis. The yeast were phenotypically characterized and two beer styles were produced under laboratory conditions, a pale ale and a traditional Honndalsøl. The sensory properties were assessed on an untrained panel as a Duo-trio test and the composition of volatile compounds produced during fermentation and after bottling at different fermentation temperatures was determined using dynamic Head space GC-MS. A genetic analysis of the yeasts internally transcribed spacer 1 and 2 region was performed to assess their species and genetic relationship between the strains. The Kveik strains were compared to reference yeast. All Kveik except one was classified as the genus Saccharomyces, two were classified as S. boulardii, two were classified as S. cerevisiae, one was classified as S. bayanus / S. pastorianus, two were unclassified specie of the Saccharomyces genus. Phenotypical characterization showed a variation of maximum growth rate with over 20 \% of selected Kveik strains. Flocculation varies between fully flocculating and non-flocculating. As fermentation temperature increase, the concentration of esters and alcohols increase except longer chained esters. During the fermentation, the concentration of alcohols decrease, esters decrease over time as well except ethyl acetate, ethyl octanoate and ethyl decanoate. A sensory panel could distinguish between 3 of the 5 tested yeast strains
Isolation and genotypic characterization of different yeast strains from Nepalese mixed culture (Murcha)
No full text14 different yeast strains were isolated from the Nepalese yeast cakes (murcha) of six different places varying in geographical condition. The isolation was entirely random based on the morphologies of the colonies, were genotypically characterized using the ITS and LSU region. The fermentation was carried out with modified synthetic must and concentrations of sugars, acid and ethanol was analyzed using HPLC and composition of volatiles were determined using headspace GC-MS. A genetic analysis was made using the internal transcribed spacer (ITS) sequencing to identify the species and establish genetic relationships between the strains. The isolated strains were classified as Vanderwaltozyma, Wickerhamomyces and Saccharomyces after LSU sequencing.
Candida inconspicua from Baglung showed lesser gravity fall during fermentation. Maximum utilization of sugars was shown by Saccharomyces while strains of Saccharomycopsis utilized less sugars. Thus, establishing themselves with high and low ethanol producers respectively. The production of ester compounds is found to be very less except for ethyl acetate. Ethyl acetate production was observed very high in most of the strains
Analysis of DNA Repair Proteins and Chromatin Remodeling Factors Using iPOND
No full textMost chemotherapeutic treatments rely on induction of severe DNA damage to kill the cancer cells. However, DNA repair pathways can repair the induced lesions, leading to survival of the tumor cells. Thus, inhibition of DNA repair pathways may increase the effect of chemotherapeutic drugs. Proliferating cell nuclear antigen (PCNA) functions as a binding platform for many proteins and has an essential role in co-ordination of DNA replication, DNA repair and other crucial processes for cell survival. In 2009, Gilljam and co-workers identified the peptide sequence AlkB homologue 2 PCNA-interacting motif (APIM), important for binding of proteins to PCNA. APIM is found in proteins involved in epigenetics, genome maintenance and cell cycle control, many of which are important after DNA damage. Studies have shown that APIM peptides sensitize cells to DNA damaging agents; hence APIM has a potential in cancer therapy. One hypothesis is that overexpressed APIM blocks the binding sites on PCNA and impairs the binding of APIM-containing proteins to PCNA, thus prevent optimal response to DNA damage.
Isolation of proteins on nascent DNA (iPOND) is a newly developed method for analysis of proteins involved in replication-related processes. The method relies on incorporation of the thymidine analog 5-ethynyl-2?-deoxyuridine (EdU) to nascent DNA. Crosslinking of proteins to DNA and biotin-conjugation to EdU results in biotin-tagged fragments of nascent DNA with bound proteins. The DNA-protein complexes are purified by exploiting the strong binding of biotin to streptavidin, before the proteins are eluted and analyzed by Western blotting.
The aim of this Master thesis has been to optimize iPOND to function with the Flp-INTM T-RexTM-293 APIM-YFP cell line and for the detection of APIM-containing proteins with low abundance close to replication forks. Furthermore, the purpose of this study was to analyze how close proteins involved in epigenetics and DNA repair are to the replisome, and to evaluate if overexpression of APIM affects the presence of these proteins on nascent DNA, both before and after inducing DNA damage.
During optimization of iPOND, it was found that 3x107 cells/dish gives optimal EdU-incorporation and that 2x108 cells/sample is necessary for detection of proteins with low abundance close to replication forks. To maintain the proliferation rate, the cells need to be passaged the day before adding tetracycline, at a concentration of 0,02 µg/mL to induce and sustain APIM-expression. Finally, it was found that 0.5-1 mM methyl methanesulphonate (MMS) introduces DNA damage without excessive stalling of the replication machinery.
iPOND detected proteins involved in nucleotide excision repair (NER) (XPA and XPF)) and direct repair (hABH2) at newly replicated DNA, suggesting a function of these proteins in post-replicative repair. iPOND also verified the chromatin remodeling factors UHRF1 and hSNF5 as replisome proteins and trimethylated H3K9 (H3K9me3) and acetylated H4K16 (H4K16ac) as chromatin-bound proteins. Furthermore, a slightly reduced presence of XPA, hABH2 and hSNF5 (APIM-containing proteins) and of H3K9me3 and H4K16ac on nascent DNA was observed in MMS-treated APIM-expressing cells compared to cells not expressing APIM. The APIM-containing proteins EHMT1 and MRG15 are found in protein complexes that participate in trimethylation of H3K9 and acetylation of H4K16, respectively. Thus, overexpressed APIM seems to perturb the binding of APIM-containing proteins to nascent DNA, and to affect the function of APIM-containing protein complexes responsible for certain histone modifications
A translational medicine approach to hypertensive nephropathy: Prevalence, diagnosis, and pathophysiology
No full textNORSK SAMMENDRAG
Translasjonsmedisinske studier av hypertensiv nefropati: Prevalens, diagnose og patofysiologi
Kronisk nyresjukdom defineres som nedsatt nyrefunksjon (estimert glomerulær filtrasjonsrate (eGFR) <60 mL/min/1,73m2), og/eller avvik i urinfunn (proteinuri, hematuri) eller bildediagnostikk av nyrene, som varer i mer enn 3 måneder. Kronisk nyresjukdom finnes hos rundt én av ti voksne i industrialiserte land. Tilstanden utgjør en byrde på samfunn og helsevesen, spesielt på grunn av den økte risikoen for hjerte-/karsjukdom og slag som følger med kronisk nyresjukdom. Tidlig diagnose og behandling, med vekt på blant annet blodtrykk, proteinlekkasje i urin og kolesterol, bremser forverringa av nyrefunksjonen. Dagens diagnoseverktøy med blod- og urinprøver gir utslag først når nyrefunksjonen er moderat redusert, og nyresjukdommen er etablert. Vi trenger bedre diagnoseredskaper for å finne de som har tidlig nyresjukdom, og skille ut de som gradvis forverres til endestadium nyresvikt. Vi trenger også mer kunnskap om hvilke sjukdomsmekanismer som er viktigst i utviklinga av nyresvikt, for å kunne finne nye behandlinger.
Den vanligste årsaken til endestadium nyresvikt i Norge er nyresjukdom på grunn av høgt blodtrykk, såkalt hypertensiv nefropati. Etter sukkersyke er det den vanligste årsaken til endestadium nyresvikt i industrialiserte land. Sjøl om tilstanden er godt kjent, er det paradoksalt nok debatt rundt både definisjonen, utbredelsen, årsakene og de grunnleggende sjukdomsmekanismene bak sjukdommen.
Hypertensiv nefropati har ofte vært antatt årsak til kronisk nyresjukdom hos individer med langvarig høgt blodtrykk, kun sparsomme urinfunn (lite blod eller protein i urinen), og som ikke har tegn til andre sjukdommer som kan gi nyreskade (for eksempel sukkersyke, cystenyrer eller kronisk nyrebetennelse/glomerulonefritt). Hos disse har man tidligere ofte antatt diagnosen hypertensiv nefropati uten å ta vevsprøve fra nyrene (nyrebiopsi) for bekreftelse. Det har vært omdiskutert hvor presise disse kliniske kriteriene er. Dette har vært undersøkt hos afrikansk-amerikanere og i enkelte andre etniske grupper, men ikke så godt hos hvite europeere. I artikkel 2 gjennomgikk vi 4920 nyrebiopserte pasienter fra Norsk nyreregister, hvorav 918 hadde biopsi-bekreftet hypertensiv nefropati. Blant de 918 hadde mange urinfunn, hvorav 34 % hadde blod i urinen og 57 % hadde protein i urinen. Vi fant at de tradisjonelle kliniske diagnosekriteriene var relativt upresise (sensitivitet 0,12, spesifisitet 0,96), og at mens fravær av disse kriteriene utelukket diagnosen relativt presist (negativ prediktiv verdi 0,83), klarte ikke disse kriteriene, de gangene de var tilstede, å forutsi diagnosen presist (positiv prediktiv verdi 0,41). Kriteriene som var sterkest assosiert med biopsi-bekreftet hypertensive nefropati var høg alder, høgt diastolisk blodtrykk, lav proteinuri, fravær av blod i urin, hankjønn og fravær av sukkersyke. Vi fant at de kliniske kriteriene var mest presise hvis man brukte alder >50 år, proteinuri 90 mmHg, og ingen blod i urinen.
I artikkel 1 beskreiv vi forekomsten av kronisk nyresjukdom i Norge på to tidspunkter med ti års mellomrom ved hjelp av tall fra tverrsnittsundersøkelsene HUNT2 i 1995-97 og HUNT3 i 2006-08. Hovedfunnet var at forekomsten av kronisk nyresjukdom holdt seg stabil på rundt 11 %. I denne perioden fant det sted en klar reduksjon av høgt blodtrykk, noe reduksjon av kolesterol og økt fysisk aktivitet, samt moderate økninger i både sukkersyke og overvekt. Vi tror at blodtrykksreduksjonen har bidratt til at nyresjukdom-tallene har holdt seg stabile, på tross av økt sukkersyke og overvekt.
I artikkel 3 undersøkte vi proteiner og peptider i urinen hos pasienter med langtkommen nyresjukdom av forskjellige typer, og fant at et sett av 273 forskjellige urin-proteiner og peptider skiller mellom nyresjuke og –friske. Urin-proteinene som var mest forskjellige, peker i retning mot forstyrrelser i bindevev- og arrvevsproduksjonen hos de nyresjuke.
I artikkel 4 viste kombinerte gen- og urin-analyser at individer med hypertensiv nefropati har endringer innen flere områder av metabolismen eller stoffomsetninga, med spesielt redusert utskillelse i urin av flere typer aminosyrer sammenliknet med friske. Disse forskjellene peker mot forstyrrelser av blodtrykksregulering, åreforkalkning, arrdannelse/fibrose og oksidativt stress, som er kjente mekanismer i dannelsen av hypertensiv nefropati og nyresjukdom generelt
Isolation and genotypic characterization of different yeast strains from Nepalese mixed culture (Murcha)
No full text14 different yeast strains were isolated from the Nepalese yeast cakes (murcha) of six different places varying in geographical condition. The isolation was entirely random based on the morphologies of the colonies, were genotypically characterized using the ITS and LSU region. The fermentation was carried out with modified synthetic must and concentrations of sugars, acid and ethanol was analyzed using HPLC and composition of volatiles were determined using headspace GC-MS. A genetic analysis was made using the internal transcribed spacer (ITS) sequencing to identify the species and establish genetic relationships between the strains. The isolated strains were classified as Vanderwaltozyma, Wickerhamomyces and Saccharomyces after LSU sequencing.
Candida inconspicua from Baglung showed lesser gravity fall during fermentation. Maximum utilization of sugars was shown by Saccharomyces while strains of Saccharomycopsis utilized less sugars. Thus, establishing themselves with high and low ethanol producers respectively. The production of ester compounds is found to be very less except for ethyl acetate. Ethyl acetate production was observed very high in most of the strains
Genotypic and Phenotypic Characterization of Norwegian Farmhouse Ale Yeast Cultures: A Domestication-Driven Evolution
No full textThere is an immense gap in our knowledge regarding biodiversity of yeasts and the available untapped yeasts that can be found in nature. Studying both domesticated and wild species is important as it can enable us to better understand the natural history, and potentially reveal how selective pressures have affected and shaped its evolution. The yeast Saccharomyces cerevisiae has been used for thousands of years by mankind to produce the most consumed fermented beverage in the world; beer. Recently, the domestication and divergence of S. cerevisiae beer yeasts has been thoroughly studied. However, very little is known about the evolution and phenotypic behaviour of the Norwegian farmhouse ale yeast (NFAY) cultures that were used in traditional home-brewed beer.
In this work, 14 different NFAY cultures with a total of 24 yeast isolates, were characterized and compared with S. cerevisiae laboratory strains and commercially available beer yeasts. The yeasts were taxonomically classified at genus and species levels by sequencing of rRNA gene regions; internal transcribed spacer (ITS) region and partially sequencing of the large subunit (LSU), referred to as LSU1 and LSU2. Since incomplete fermentation of maltotriose is a common problem in the brewing industry and maltotriose utilization is highly associated with domestication, the presence and distribution of a gene encoding an α-glucoside transporter, AGT1, was investigated. This transporter has a wide substrate specificity for common sugars in the wort, including maltotriose. Moreover, the ploidy was investigated with flow cytometry, and a small-scale fermentation of the samples was carried out at 22 °C and 35 °C. The fermentation performance, as well as its flavor and aroma profile, was determined with chromatography and mass spectrometry instrumentation (headspace GC-MS, HPLC and LC-MS).
The species differentiation based on the ITS and LSU regions suggested that the NFAY cultures consist of at least three different species; Saccharomyces cerevisiae, Saccharomyces bayanus and the less familiar non-Saccharomyces yeast Meyerozyma caribbica. The Saccharomyces species seems to be closely related, as it was generally observed low DNA sequence divergence among them. The AGT1 allele was present in all Saccharomyces species except two of the the laboratory strains. It was discovered that a particular AGT1 gene mutation producing a premature stop codon may not be limited to lager strains, as presumed before. Interestingly, the NFAY samples carried a variant of the AGT1 gene that is more similar to the domesticated ones rather than wild S. cerevisiae variants. This raises the suspicion of them originating from domesticated ancestors. Phenotypic variants among the NFAY samples were also observed, and were different from the performance of commercial yeasts. This diversity is believed to have emerged from the farmhouse brewing traditions, through high fermentation temperatures, unique storage techniques and repitching. However, the phenotypic traits were not conserved within the three species, nor did they carry geographical structure. This can indicate that the phenotypic characteristics are mainly strain dependent. This, together with high maltotriose utilization, low sequence divergence and wide geographical dispersal, further supports the theory of a domestication-driven evolution in the NFAY cultures
Characterization of genotype and beer fermentation properties of Norwegian Farmhouse Ale Yeasts
No full textEight different Norwegian Farmhouse Ale Yeasts from Voss, Stranda, Lærdal, Hornindal and Olden were donated to IBT for analysis. The yeast were phenotypically characterized and two beer styles were produced under laboratory conditions, a pale ale and a traditional Honndalsøl. The sensory properties were assessed on an untrained panel as a Duo-trio test and the composition of volatile compounds produced during fermentation and after bottling at different fermentation temperatures was determined using dynamic Head space GC-MS. A genetic analysis of the yeasts internally transcribed spacer 1 and 2 region was performed to assess their species and genetic relationship between the strains. The Kveik strains were compared to reference yeast. All Kveik except one was classified as the genus Saccharomyces, two were classified as S. boulardii, two were classified as S. cerevisiae, one was classified as S. bayanus / S. pastorianus, two were unclassified specie of the Saccharomyces genus. Phenotypical characterization showed a variation of maximum growth rate with over 20 \% of selected Kveik strains. Flocculation varies between fully flocculating and non-flocculating. As fermentation temperature increase, the concentration of esters and alcohols increase except longer chained esters. During the fermentation, the concentration of alcohols decrease, esters decrease over time as well except ethyl acetate, ethyl octanoate and ethyl decanoate. A sensory panel could distinguish between 3 of the 5 tested yeast strains
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