Inline-tandem purification of viruses from cell lysate by agarose-based chromatography

An efficient chromatography-based virus purification method has been developed and validated for the nonpathogenic infectious virus PRD1. Compared to the conventional method that consists of relatively time-consuming and labour-intensive precipitation and density gradient ultracentrifugation steps, the method developed here is performed in a single flow using tandem-coupled anion exchange and size exclusion chromatography (AIEX-SEC) columns. This inline approach helps to minimize the loss of virus in the process and streamlines time consumption, since no physical transfer of the sample is required between purification steps. In the development process, sample feed composition, dynamic binding capacity and elution conditions for the AIEX resin as well as different exclusion limits for SEC resins were optimized to achieve maximal yield of pure infectious viruses. Utilizing this new approach, a high-quality virus sample was produced from a lysate feed in 320 min with a total yield of 13 mg purified particles per litre of cell lysate, constituting a 3.5-fold yield increase as compared to the conventional method, without compromising the high specific infectivity of the product (6 x 1012 to 7 x 10(12) pfu/mg of protein). The yield of infectious viruses of the lysate feed was 54%. The easy scalability of chromatography-based methods provide a direct route to industrial usage without any significant changes needed to be made to the purification regime. This is especially interesting as the method has high potential to be used for purification of various viruses and nanoparticles, including adenovirus.Peer reviewe

An unstructured 5′-coding region of the prfA mRNA is required for efficient translation

Expression of virulence factors in the human bacterial pathogen Listeria monocytogenes is almost exclusively regulated by the transcriptional activator PrfA. The translation of prfA is controlled by a thermosensor located in the 5′-untranslated RNA (UTR), and is high at 37°C and low at temperatures <30°C. In order to develop a thermoregulated translational expression system, the 5′-UTR and different lengths of the prfA-coding sequences were placed in front of lacZ. When expressed in Escherichia coli, the β-galactosidase expression was directly correlated to the length of the prfA-coding mRNA lying in front of lacZ. A similar effect was detected with gfp as a reporter gene in both L. monocytogenes and E. coli, emphasizing the requirement of the prfA-coding RNA for maximal expression. In vitro transcription/translation and mutational analysis suggests a role for the first 20 codons of the native prfA-mRNA for maximal expression. By toe-print and RNA-probing analysis, a flexible hairpin-loop located immediately downstream of the start-codon was shown to be important for ribosomal binding. The present work determines the importance of an unstructured part of the 5′-coding region of the prfA-mRNA for efficient translation

Characterization and Transcriptome Analysis of Mycobacterium tuberculosis Persisters

Tuberculosis continues to be a major public health problem in many parts of the world. Significant obstacles in controlling the epidemic are the length of treatment and the large reservoir of latently infected people. Bacteria form dormant, drug-tolerant persister cells, which may be responsible for the difficulty in treating both acute and latent infections. We find that in Mycobacterium  tuberculosis, low numbers of drug-tolerant persisters are present in lag and early exponential phases, increasing sharply at late exponential and stationary phases to make up ~1% of the population. This suggests that persister formation is governed by both stochastic and deterministic mechanisms. In order to isolate persisters, an exponentially growing population was treated with d-cycloserine, and cells surviving lysis were collected by centrifugation. A transcriptome of persisters was obtained by using hybridization to an Affymetrix array. The transcriptome shows downregulation of metabolic and biosynthetic pathways, consistent with a certain degree of dormancy. A set of genes was upregulated in persisters, and these are likely involved in persister formation and maintenance. A comparison of the persister transcriptome with transcriptomes obtained for several in vitro dormancy models identified a small number of genes upregulated in all cases, which may represent a core dormancy response

IntaRNA: efficient prediction of bacterial sRNA targets incorporating target site accessibility and seed regions

Motivation: During the last few years, several new small regulatory RNAs (sRNAs) have been discovered in bacteria. Most of them act as post-transcriptional regulators by base pairing to a target mRNA, causing translational repression or activation, or mRNA degradation. Numerous sRNAs have already been identified, but the number of experimentally verified targets is considerably lower. Consequently, computational target prediction is in great demand. Many existing target prediction programs neglect the accessibility of target sites and the existence of a seed, while other approaches are either specialized to certain types of RNAs or too slow for genome-wide searches

The ζ Toxin Induces a Set of Protective Responses and Dormancy

The ζε module consists of a labile antitoxin protein, ε, which in dimer form (ε2) interferes with the action of the long-living monomeric ζ phosphotransferase toxin through protein complex formation. Toxin ζ, which inhibits cell wall biosynthesis and may be bactericide in nature, at or near physiological concentrations induces reversible cessation of Bacillus subtilis proliferation (protective dormancy) by targeting essential metabolic functions followed by propidium iodide (PI) staining in a fraction (20–30%) of the population and selects a subpopulation of cells that exhibit non-inheritable tolerance (1–5×10−5). Early after induction ζ toxin alters the expression of ∼78 genes, with the up-regulation of relA among them. RelA contributes to enforce toxin-induced dormancy. At later times, free active ζ decreases synthesis of macromolecules and releases intracellular K+. We propose that ζ toxin induces reversible protective dormancy and permeation to PI, and expression of ε2 antitoxin reverses these effects. At later times, toxin expression is followed by death of a small fraction (∼10%) of PI stained cells that exited earlier or did not enter into the dormant state. Recovery from stress leads to de novo synthesis of ε2 antitoxin, which blocks ATP binding by ζ toxin, thereby inhibiting its phosphotransferase activity

Ciprofloxacin Causes Persister Formation by Inducing the TisB toxin in Escherichia coli

Persisters are specialized survivor cells that arise in populations of E. coli after antibiotic-mediated DNA damage induces the production of a small membrane-acting peptide TisB, which causes reversible dormancy. The TisB-dependent persisters are tolerant to multiple antibiotics

Small RNA-mediated Regulation of Gene Expression in Escherichia coli

Non-coding RNAs are highly abundant regulators of gene expression in all kingdoms of life that often play important roles in vital cellular functions. In bacteria, small regulatory RNAs (sRNAs) usually act post-transcriptionally by regulating mRNAs through base pairing within ribosome binding sites (RBS), thereby inhibiting translation initiation. tisB encodes a toxin, TisB, whose synthesis is controlled by the sRNA IstR-1. Intriguingly, IstR-1 base pairs far upstream of the RBS but nevertheless inhibits translation initiation. The tisB mRNA is unusual in that ribosomes cannot access the RBS directly, but instead need an unstructured upstream region. This is precisely where IstR-1 exerts its inhibitory effect. We propose this region to serve as a ribosome loading site (standby site) which permits ribosomes to overcome the obstacle of inhibitory RBS-containing structures. Sequence-independent ribosome binding to the standby site allows for efficient relocation to the RBS structure when it is transiently open. Thus, standby sites are translation enhancer elements. I also characterized TisB-mediated toxicity. The hydrophobic protein TisB is targeted to the inner membrane and causes damage. This decreases the intracellular ATP concentration and entails decreased  replication, transcription and translation rates. It is likely that this toxin is involved in multidrug tolerance under certain conditions. We identified the sRNA MicF as a negative regulator of lrp expression. Lrp is a global transcription factor that controls genes involved in amino acid metabolism and transport of small molecules. Interestingly, Lrp also downregulates MicF. Thus, this study established that the mutual downregulation of MicF/Lrp creates a positive feedback loop which gives a switch-like behavior important for fast adaptations

Transfer pricing –The influences from accounting traditions in the interpretation of OECD:s guidelines

Den ökade globalisering som råder gynnar internationell handel på många plan, vilket även har bidragit till att internprissättning blivit en allt mer central fråga. Däremot ställs det högre krav på att adressera de problem som systemet resulterar i. Detta med hänsyn till att ett internpris är av påverkande karaktär på organisationens resultat på grund av skattemässiga motiv och incitament från ledningen. Även om OECD:s riktlinjer TPG är utformade av det politiskt styrande organet i respektive medlemsland är dessa riktlinjer inte tvingande. Systemet möjliggör subjektivitet i internprissättning. Problematiken belyses i synnerhet när redovisningstraditioner och deras eventuella påverkan på internprissättningen i Sverige och Storbritannien tas i beaktning.Studien har som syfte att genom en komparativ analys undersöka hur Sveriges och Storbritanniens redovisningstraditioner påverkar respektive lands tolkning av TPG. Ländernas skattemyndigheter, Skatteverket och HMRC, har gett ut en handledning respektive en intern manual rörande internprissättning där myndighetens tolkning av TPG framgår.Studien är en innehållsanalys av kvalitativ karaktär och datamaterialet som har använts är textutdrag från myndigheternas handledning och manual samt respektive lands lagstiftning på vissa områden. Studien har som ambition att undersöka fyra områden avseende internprissättning, myndigheternas referering till gällande rätt, armlängdsprincipen, metoder för prissättning och bevisförfarande. Tolkningsmodellen som har använts i studien har utvecklats fram ifrån Johanssons (2010) modell avseende substance over form. Tolkningsmodellen tillsammans med den teoretiska referensramen har fungerat som ett verktyg i analysen kring hur redovisningstraditionerna i respektive land påverkar tolkningen av TPG.Studiens slutsats indikerar att det finns inslag av redovisningstraditionerna i respektive lands tolkning av TPG. Både Storbritannien och Sverige refererar till OECD kontinuerligt men studien har resulterat i vissa skillnader som belyser det faktum att länderna har olika redovisningstraditioner. I Skatteverkets handledning identifierar resultatet vissa inslag av den kontinentala redovisningstradition som Sverige präglas av, det påträffas även inslag av det regelbaserade synsättet samt spår av det rättssystem som karaktäriserar Sverige, code law. Resultatet visar även att Storbritanniens redovisningstraditioner framträder i HMRC:s tolkning av TPG. Det återfinns inslag av den anglosaxiska traditionen, det principbaserade synsättet och även här syns spår av deras rättssystem, common law. Studien bidrar med en ökad förståelse kring hur nationella redovisningstraditioner påverkar tolkningen av internationella riktlinjer. Den bidrar även med en praktisk kunskap för företag, myndigheter och internationella verkställande organ.Increased globalization stimulates international trade in many ways. It has led to increased importance of transfer pricing. However, there is a need to address several issues that have arisen since a transfer price affects the organization's profit or loss due to tax consequences and incentives from management. Although the guidelines known as TPG are drafted by the governments in members’ states, these guidelines are not mandatory. The system allows for subjectivity in transfer pricing. The problem is can be demonstrated by comparing the accounting traditions and their possible impact on transfer pricing in Sweden and the UK.The purpose of the study is to investigate how Sweden's and Britain's accounting traditions affect the countries' interpretation of the TPG. The tax authorities of these countries, the Swedish Tax Agency and HMRC, have each issued a guide and an internal manual on transfer pricing, which explains the authorities’ interpretation of the TPG. The aim of the study is to make a comparative analysis of these guides and internal manuals to see if and how the countries’ interpretation of the TPG differ due to the countries’ accounting traditions. Further, the study is a content analysis of qualitative nature and the data used has been extracted from the authorities' guide and internal manual, as well as the legislation of each country in certain areas. The aim of the study is to investigate four areas of transfer pricing, the authorities' reference to current law, the arm's length principle, pricing methods and evidence procedures. The research model used in this study has been developed from Johanssons (2010) model regarding substance over form. The resarch model together with the theoretical reference framework have served as a tool in the analysis of how accounting traditions in each country affect the interpretation of TPG.The study’s conclusion indicates that there are elements of accounting traditions in each country’s interpretation of TPG. Both the UK and Sweden refer to the OECD repeatedly but the study has resulted in some differences highlighting the fact that the countries have different accounting traditions. In the Swedish Tax Agency’s guidance, the result identifies certain elements of the continental accounting tradition that Sweden is characterized by, it also finds elements of the rule-based approach as well as traces of the legal system code law that characterizes Sweden. Likewise, the result also shows that Britain's accounting traditions appear in HMRC's interpretation of TPG. There are elements of Anglo-Saxon tradition, the principle-based approach, and also traces of their legal system common law. The study contributes to an increased understanding of how national accounting traditions affect the interpretation of international guidelines. It also provides useful information for companies, authorities and international executive agencies.This paper is hereinafter written in Swedish

En studie av anbudsskedets process vid införande av nytt kvalitetssystem

Almroth Byggnads AB är mitt i processen att revidera sina olika ledningssystem. Entré, som nyligen började användas som ekonomiledningssystem, i ett steg på vägen för att kunna utvecklas till ett verksamhetsledningssystem. Dit ska man komma genom att samarbeta med Povel, ett branschanpassat ledningssystem för alla typer av byggföretag. Detta kräver mycket justering av dokument för att passa Almroth, då Povels dokument är väldigt generella, och gäller både stora och små företag. Det finns styrande dokument för anbudsskedet i Povel, de är sådana att de måste justeras för att passa Almroth. Denna studie tar reda på vad som bör ingå i Almroths dokument, vad Almroth bör gå igenom från det att man får in en anbudsförfrågan tills arbetet med anbudet är färdigt och om ledningssystemet kommer medföra att Almroths arbete med anbud blir effektivare. De styrande dokumenten jag tar fram ska vara utformade så att de når kraven i ISO 9001, ISO 14001 och AFS2001:1. Jag har undersökt vilken typ av arbeten man har och hur man bör gå till väga vid olika typer av entreprenader och upphandlingar. Mina resultat av teori och empiri om anbudsskedets process är presenterade i kapitel 4, där gör jag även en analys över hur ledningssystemen kommer påverka anbudsprocessen och snuddar även lite bredare runt dessa system. Vidare finns mina checklistor och rutinbeskrivning som bilagor, med grund i kapitel 4 och 5, analysen samt mina slutsatser

Small RNA-mediated Regulation of Gene Expression in Escherichia coli

Non-coding RNAs are highly abundant regulators of gene expression in all kingdoms of life that often play important roles in vital cellular functions. In bacteria, small regulatory RNAs (sRNAs) usually act post-transcriptionally by regulating mRNAs through base pairing within ribosome binding sites (RBS), thereby inhibiting translation initiation. tisB encodes a toxin, TisB, whose synthesis is controlled by the sRNA IstR-1. Intriguingly, IstR-1 base pairs far upstream of the RBS but nevertheless inhibits translation initiation. The tisB mRNA is unusual in that ribosomes cannot access the RBS directly, but instead need an unstructured upstream region. This is precisely where IstR-1 exerts its inhibitory effect. We propose this region to serve as a ribosome loading site (standby site) which permits ribosomes to overcome the obstacle of inhibitory RBS-containing structures. Sequence-independent ribosome binding to the standby site allows for efficient relocation to the RBS structure when it is transiently open. Thus, standby sites are translation enhancer elements. I also characterized TisB-mediated toxicity. The hydrophobic protein TisB is targeted to the inner membrane and causes damage. This decreases the intracellular ATP concentration and entails decreased  replication, transcription and translation rates. It is likely that this toxin is involved in multidrug tolerance under certain conditions. We identified the sRNA MicF as a negative regulator of lrp expression. Lrp is a global transcription factor that controls genes involved in amino acid metabolism and transport of small molecules. Interestingly, Lrp also downregulates MicF. Thus, this study established that the mutual downregulation of MicF/Lrp creates a positive feedback loop which gives a switch-like behavior important for fast adaptations