ProbeMaker: an extensible framework for design of sets of oligonucleotide probes

BACKGROUND: Procedures for genetic analyses based on oligonucleotide probes are powerful tools that can allow highly parallel investigations of genetic material. Such procedures require the design of large sets of probes using application-specific design constraints. RESULTS: ProbeMaker is a software framework for computer-assisted design and analysis of sets of oligonucleotide probe sequences. The tool assists in the design of probes for sets of target sequences, incorporating sequence motifs for purposes such as amplification, visualization, or identification. An extension system allows the framework to be equipped with application-specific components for evaluation of probe sequences, and provides the possibility to include support for importing sequence data from a variety of file formats. CONCLUSION: ProbeMaker is a suitable tool for many different oligonucleotide design and analysis tasks, including the design of probe sets for various types of parallel genetic analyses, experimental validation of design parameters, and in silico testing of probe sequence evaluation algorithms

Flera om samma brott – en kritisk analys av medgärningsmannaskap

I svensk rätt kan kretsen för vem som kan dömas som gärningsman utvidgas genom att döma flera personer i medgärningsmannaskap. När flera personer tillsammans uppfyller brottsrekvisiten i en straffbelagd gärning, kan domstolen döma flera gärningsmän. Hur begreppet har tillämpats i praktiken av både åklagare och domare, har fått stor kritik. Praxis på området spretar åt olika håll, vilket ger knapphändig vägledning för hur begreppet ska tolkas och tillämpas. Syftet med uppsatsens är att problematisera och belysa diskussionen kring begreppet medgärningsmannaskap ur ett kritiskt perspektiv. Om domstolarna har en benägenget att döma flera personer kollektivt, och om detta är förenligt med rättssäkerheten är frågeställningar som uppsatsen ämnar att besvara. Hur begreppet ska tillämpas kom att förändras efter Lindomefallet, där två personer frikändes på grund av att det inte gick att fastställa vem som hade utfört vilken gärning. Domen upprörde allmänheten och krav ställdes på lagstiftarna. Fallet fick effekter för efterkommande fall, däribland Pinnstolsfallet, där omständigheterna liknade dem i Lindomefallet men utgången var den motsatta. Efter diskoteksbranden i Göteborg dömdes fyra individer i medgärningsmannaskap med otydlig motivering, vilket visade på utvecklingen av det utvidgade medgärningsmannaskapet. I uppsatsen behandlas vidare mordet på Björn Söderberg, morden på Sturecompagniet och Pirate Bay-målet. I analysen argumenterar jag för att det går att se tendenser på att domarna dömer hela kollektivet hellre än att göra individuella prövningar. Vidare diskuteras den moralfilosofiska frågeställningen om rättssäkerhet, där vikten att kunna säkerställa rättssäkerheten för både gärningsman och offer understryks. Uppsatsen avslutas med att fastslå att mer forskning behövs för att rättvist kunna tillämpa begreppet medgärningsmannaskap

PieceMaker: selection of DNA fragments for selector-guided multiplex amplification

We describe PieceMaker, a software tool for the design of applications of selector probes–oligonucleotide probes that direct circularization of target nucleic acid molecules. Such probes can be combined in parallel to circularize a selection of fragments from restriction digested total genomic DNA. These fragments can then be amplified in a single PCR using a common primer pair, yielding substrates for subsequent analyses, such as parallel genotyping or sequencing. However, designing multiplex selector assays is a laborious task. The PieceMaker program alleviates this problem by selecting restriction enzymes to generate suitable fragments for selection, and generating the output data required to design the selector probes

Tullåtgärder vid privatinförsel av kopierade och förfalskade varor - en analys av förordning (EU) nr 608/2013

Handeln med piratkopierade och varumärkesförfalskade varor, det vill säga varor som gör intrång i immateriella rättigheter, har ökat kraftigt. Utvecklingen har gått från småskalig verksamhet till en global industri. De intrångsgörande varorna orsakar omfattande skador för rättighetshavare, bland annat genom förlorade intäkter och dåligt rykte. Vidare kan varorna medföra risker för konsumenternas säkerhet och hälsa. Handeln med intrångsgörande varor resulterar även i färre arbetstillfällen, minskad drivkraft för forskning och innovation och en ny bransch för den organiserade brottsligheten. I syfte att hålla sådana varor borta från unionsmarknaden påbörjades under 1980-talet arbetet med ett gemensamt system för tullkontroll av misstänkta intrångsgörande produkter, vilket ledde till att den första tullförordningen trädde i kraft år 1986. Förordningen har därefter varit föremål för granskning och revidering ett flertal gånger. Den 1 januari 2014 trädde förordning (EU) nr 608/2013 i kraft och ersatte tidigare bestämmelser. Syftet med uppsatsen är att analysera den nya tullförordningens utökade tillämpningsområde med särskilt avseende på den enskilde konsumentens införsel av varor som misstänks göra intrång i immateriella rättigheter till EU. När tullåtgärder kan vidtas mot konsumenter och om tullförordningen är rättssäker för konsumenten är frågeställningar som uppsatsen ämnar att besvara. I analysen konstateras att tullförordningens utökade tillämpningsområde tangerar skyddet i de immaterialrättsliga reglerna, vilket ger rättighetshavaren ett effektivt verktyg för att skydda sina rättigheter. Vidare diskuteras de huvudsakliga förändringarna i den nya tullförordningen. Därefter diskuteras den moralfilosofiska frågeställningen om rättssäkerhet, där vikten av enhetlig lagstiftning, likartad tillämpning och klargörande praxis understryks. Uppsatsen avslutas med att fastslå att säkerställandet av skyddet för immateriella rättigheter är ett ständigt aktuellt ämne. EU strävar idag efter ett fördjupat samarbete mellan tullmyndigheter i EU och tredjeländer, och vill med gemensamma insatser bekämpa intrång i immateriella rättigheter vid EU:s yttre gränser. Bestämmelser gällande privatimport förefaller inte få fäste i det nya varumärkespaketet, varför EU-domstolen än en gång måste klargöra förhållandet mellan tullförordningen och konsumenters införsel av intrångsgörande varor.Trade with pirated and counterfeited goods has increased considerably. The development has transited from small scale production towards becoming a global industry. Infringed goods cause large damage for right holders, not only do they lose revenue, but also the companies’ image and market value is under fire. Furthermore, infringing goods cause damage not only to right holders, but also affect consumer health and security. Additionally the trade also affects the market for labour, a loss in incitement for research and development, and constitutes a new market for organized criminality. In order to keep such items away from the European Union’s market, collaboration began in the 1980s, in which a joint system for custom controls of suspected infringed goods were made. This led to the first Regulation (1986). The Regulation has since been reviewed and revised several times. On 1 January 2014, the Regulation (EU) No 608/2013 was taken into force and replaced the previous. This thesis analyses the expanded scope of the new Customs Regulations. Particular focus is given regarded the individual, and how he or she might import goods to the European Union which infringe intellectual property rights. Further, this thesis analyses when customs actions can be taken against consumers and how these affect the Rule of Law. It is concluded that the Customs Regulation’s expanded scope follows the laws of intellectual property rights, which provides the right holders with a proper and effective tool in protecting their rights. Further, the main changes in the Regulation are also discussed, as well as a section of moral philosophical thoughts regarding the importance of a joint legislation, a uniform application, and the possible effects on the Rule of Law. The thesis concludes that the enforcement of intellectual property rights is a recurring and ever present topic, and that the European Union strives after a unified collaboration in-between custom authorizations and third part countries, regarding the pursuit of infringement. Conditions regarding private import do not appear to firmly adhere in the new Regulation. Hence, the European Union court once again may need to clarify the relationship between the Custom Regulation and consumers’ importation of counterfeited goods

Selectivity in the Reinnervation of the Lateral Gastrocnemius Muscle after Nerve Repair with Ethyl Cyanoacrylate in the Rat

There is a need for complementary surgical techniques that enable rapid and reliable primary repair of transected nerves. Previous studies after peripheral nerve transection and repair with synthetic adhesives have demonstrated regeneration to an extent comparable to that of conventional techniques. The aim of this study was to compare two different repair techniques on the selectivity of muscle reinnervation after repair and completed regeneration. We used the cholera toxin B technique of retrograde axonal tracing to evaluate the morphology, the number, and the three-dimensional location of α-motoneurons innervating the lateral gastrocnemius muscle and compared the results after repair with either ethyl cyanoacrylate (ECA) or epineural sutures of the transected parent sciatic nerve. In addition, we recorded the wet weight of the muscle. Six months after transection and repair of the sciatic nerve, the redistribution of the motoneuron pool was markedly disorganized, the motoneurons had apparently increased in number, and they were scattered throughout a larger volume of the spinal cord gray matter with a decrease in the synaptic coverage compared to controls. A reduction in muscle weight was observed as well. No difference in morphometric variables or muscle weight between the two repair methods could be detected. We conclude that the selectivity of motor reinnervation following sciatic nerve transection and subsequent repair with ECA is comparable to that following conventional micro suturing

Diagnostic application of padlock probes—multiplex detection of plant pathogens using universal microarrays

Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection. Since target recognition is separated from downstream processing, PLPs enable the development of flexible and extendable diagnostic systems, targeting diverse organisms. To adapt padlock technology for diagnostic purposes, we optimized PLP design to ensure high specificity and eliminating ligation on non-target sequences under real-world assay conditions. We designed and tested 11 PLPs to target various plant pathogens at the genus, species and subspecies levels, and developed a prototype PLP-based plant health chip. Excellent specificity was demonstrated toward the target organisms. Assay background was determined for each hybridization using a no-target reference sample, which provided reliable and sensitive identification of positive samples. A sensitivity of 5 pg genomic DNA and a dynamic range of detection of 100 were observed. The developed multiplex diagnostic system was validated using genomic DNAs of characterized isolates and artificial mixtures thereof. The demonstrated system is adaptable to a wide variety of applications ranging from pest management to environmental microbiology

High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification

BACKGROUND: Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation. RESULTS: SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe. CONCLUSIONS: Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring

CK1δ restrains lipin-1 induction, lipid droplet formation and cell proliferation under hypoxia by reducing HIF-1α/ARNT complex formation.

Proliferation of cells under hypoxia is facilitated by metabolic adaptation, mediated by the transcriptional activator Hypoxia Inducible Factor-1 (HIF-1). HIF-1α, the inducible subunit of HIF-1 is regulated by oxygen as well as by oxygen-independent mechanisms involving phosphorylation. We have previously shown that CK1δ phosphorylates HIF-1α in its N-terminus and reduces its affinity for its heterodimerization partner ARNT. To investigate the importance of this mechanism for cell proliferation under hypoxia, we visually monitored HIF-1α interactions within the cell nucleus using the in situ proximity ligation assay (PLA) and fluorescence recovery after photobleaching (FRAP). Both methods show that CK1δ-dependent modification of HIF-1α impairs the formation of a chromatin binding HIF-1 complex. This is confirmed by analyzing expression of lipin-1, a direct target of HIF-1 that mediates hypoxic neutral lipid accumulation. Inhibition of CK1δ increases lipid droplet formation and proliferation of both cancer and normal cells specifically under hypoxia and in an HIF-1α- and lipin-1-dependent manner. These data reveal a novel role for CK1δ in regulating lipid metabolism and, through it, cell adaptation to low oxygen conditions.This work was supported by the “ARISTEIA ΙΙ” Action of the “OPERATIONAL PROGRAMME EDUCATION AND LIFELONG LEARNING” and was co-funded by the European Social Fund (ESF) and National Resources. Partial support was provided by the Proof of Concept Studies for the ESFRI project Euro-BioImaging (Greek BioImaging Facility, PCS facility Nr. 9, Unit 2). N.-N.G., M.A.R. and Z.L. were supported by a grant from the European Research Council and S.S. was supported by a Medical Research Council Senior Fellowship (grant number G0701446).This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S0898656815000637