Antisense oligodeoxynucleotide inhibition as a potent diagnostic tool for gene function in plant biology

Antisense oligodeoxynucleotide (ODN) inhibition emerges as an effective means for probing gene function in plant cells. Employing this method we have established the importance of the SUSIBA2 transcription factor for regulation of starch synthesis in barley endosperm, and arrived at a model for the role of the SUSIBAs in sugar signaling and source-sink commutation during cereal endosperm development. In this addendum we provide additional data demonstrating the suitability of the antisense ODN technology in studies on starch branching enzyme activities in barley leaves. We also comment on the mechanism for ODN uptake in plant cells. Antisense ODNs are short (12-25 nt-long) stretches of single-stranded ODNs that hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. They are naturally occurring in both prokaryotes and eukaryotes where they partake in gene regulation and defense against viral infection. The mechanisms for antisense ODN inhibition are not fully understood but it is generally considered that the ODN either sterically interferes with translation or promotes transcript degradation by RNase H activation. The earliest indication of the usefulness of antisense ODN technology for the purposes of molecular biology and medical therapy was the demonstration in 1978 that synthetic ODNs complementary to Raos sarcoma virus could inhibit virus replication in tissue cultures of chick embryo fibroblasts. Since then the antisense ODN technology has been widely used in animal sciences and as an important emerging therapeutic approach in clinical medicine. However, antisense ODN inhibition has been an under-exploited strategy for plant tissues, although the prospects for plant cells in suspension cultures to take up single-stranded ODNs was reported over a decade ago. In 2001, two reports from Malho and coworker demonstrated the use of cationic-complexed antisense ODNs to suppress expression of genes encoding pollen-signaling proteins in pollen tubes from the lilly Agapanthus umbellatus. For the uptake of DNA pollen tubes represent a unique system since the growing tip is surrounded by a loose matrix of hemicellulose and pectins, exposing the plasma membrane7 and the first uptake of ODNs by pollen tubes was reported as early as 1994. A breakthrough in the employment of antisense ODN inhibition as a powerful approach in plant biology was recently presented through our work on intact barley leaves. As was illustrated by confocal microscopy and fluorescently labeled ODNs, naked ODNs were taken up through the leaf petiole and efficiently imported into the plant cell and the nucleus. The work portrayed in that study demonstrate the applicability of antisense ODN inhibition in plant biology, e.g. as a rapid antecedent to time-consuming transgenic studies, and that it operates through RNase H degradation. We employed the antisense ODN strategy to demonstrate the importance of the SUSIBA2 transcription factor in regulation of starch synthesis, and to depict a possible mechanism for sugar signaling in plants and how it might confer endosperm-specific gene expression during seed development. We also described the employment of the antisense ODN strategy for studies on in vitro spike cultures of barley. Here we present further evidence as to the value of the antisense ODN approach in plant biology by following the effects on starch branching enzyme (SBE) accumulation in barley leaves after suppression of individual SBE genes. In agreement with transcript analyses of SBE expression in barley leaves, a zymogram assay (Fig. 1) revealed that sucrose treatment of barley leaves increased the number of SBE activity bands as compared to sorbitol treatment. In the presence of antisense SBEI or SBEIIA ODNs, zymograms of sucrose-treated leaves displayed only a subset of these activities with bands in the top portion of the zymogram gel missing or diminished. With antisense SBEIIB ODN, all activity bands in the top portion of the gel as well as the lowest band were absent. Based on these data we provide a tentative annotation for the various SBE activity bands. In animal experiments, naked ODNs are usually not taken up by the cells since both the ODNs and the outside of the plasma membrane carry a net negative charge. Thus the uptake of naked ODNs into barley leaf cells was surprising and called for an explanation. As demonstrated in our subsequent paper, the answer seems to be that the ODNs slip into the cells through sugar translocators as they are activated in the presence of the appropriate sugar (Fig. 2). Whether it is the structural resemblance between the sugar (deoxyribose) backbone of the ODNs and the transported sugars that allows for the ODNs to be transferred, or if other mechanisms are involved, remains to be elucidated

A Systematic Study of Gene Mutations in Urothelial Carcinoma; Inactivating Mutations in TSC2 and PIK3R1

Abstract BACKGROUND: Urothelial carcinoma (UC) is characterized by frequent gene mutations of which activating mutations in FGFR3 are the most frequent. Several downstream targets of FGFR3 are also mutated in UC, e.g., PIK3CA, AKT1, and RAS. Most mutation studies of UCs have been focused on single or a few genes at the time or been performed on small sample series. This has limited the possibility to investigate co-occurrence of mutations. METHODOLOGY/PRINCIPAL FINDINGS: We performed mutation analyses of 16 genes, FGFR3, PIK3CA, PIK3R1 PTEN, AKT1, KRAS, HRAS, NRAS, BRAF, ARAF, RAF1, TSC1, TSC2, APC, CTNNB1, and TP53, in 145 cases of UC. We show that FGFR3 and PIK3CA mutations are positively associated. In addition, we identified PIK3R1 as a target for mutations. We demonstrate a negative association at borderline significance between FGFR3 and RAS mutations, and show that these mutations are not strictly mutually exclusive. We show that mutations in BRAF, ARAF, RAF1 rarely occurs in UC. Our data emphasize the possible importance of APC signaling as 6% of the investigated tumors either showed inactivating APC or activating CTNNB1 mutations. TSC1, as well as TSC2, that constitute the mTOR regulatory tuberous sclerosis complex were found to be mutated at a combined frequency of 15%. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate a significant association between FGFR3 and PIK3CA mutations in UC. Moreover, the identification of mutations in PIK3R1 further emphasizes the importance of the PI3-kinase pathway in UC. The presence of TSC2 mutations, in addition to TSC1 mutations, underlines the involvement of mTOR signaling in UC

High Temperature Triggers Latent Variation among Individuals: Oviposition Rate and Probability for Outbreaks

It is anticipated that extreme population events, such as extinctions and outbreaks, will become more frequent as a consequence of climate change. To evaluate the increased probability of such events, it is crucial to understand the mechanisms involved. Variation between individuals in their response to climatic factors is an important consideration, especially if microevolution is expected to change the composition of populations.Here we present data of a willow leaf beetle species, showing high variation among individuals in oviposition rate at a high temperature (20 °C). It is particularly noteworthy that not all individuals responded to changes in temperature; individuals laying few eggs at 20 °C continued to do so when transferred to 12 °C, whereas individuals that laid many eggs at 20 °C reduced their oviposition and laid the same number of eggs as the others when transferred to 12 °C. When transferred back to 20 °C most individuals reverted to their original oviposition rate. Thus, high variation among individuals was only observed at the higher temperature. Using a simple population model and based on regional climate change scenarios we show that the probability of outbreaks increases if there is a realistic increase in the number of warm summers. The probability of outbreaks also increased with increasing heritability of the ability to respond to increased temperature.If climate becomes warmer and there is latent variation among individuals in their temperature response, the probability for outbreaks may increase. However, the likelihood for microevolution to play a role may be low. This conclusion is based on the fact that it has been difficult to show that microevolution affect the probability for extinctions. Our results highlight the urge for cautiousness when predicting the future concerning probabilities for extreme population events

Mäns våld mot kvinnor : Våldsutövarens bakgrund och behandling

Detta arbete handlar om mäns våld mot kvinnor i hemmet, och vilken bakgrund de män som utövar våldet kan ha, samt vilken behandling som finns att få. Vi ville skriva detta för att både vi och andra ska lära oss något mer om detta problem som vi ofta kommer att stöta på i vårat framtida yrke. Vad är det som gör att männen slår, är det något i deras uppväxt eller är det genetiskt eller kanske till och med samhällets fel med alla normer som finns idag? Vi fick genom böcker och Internet samt intervjuer med manscentran fram ett antal förklaringar som kan användas för att få våldet begripligt, men i slutändan så är det ändå mannens ansvar. Det finns inget som gör våldet försvarbart. Både böcker, information via nätet och de som jobbar med behandling på manscentran är inne på samma linje när det gäller orsaker till våldet och vad som bör göras. Det handlar om att lyfta fram vem som är ansvarig för våldet, det vill säga männen, och få dem att hitta ett annat sätt än att med våld hantera konflikter, och att kunna kontrollera sina egna känslor. Männen som frivilligt söker behandling är dock inte de som mest skulle behöva den. De män som man verkligen vill nå är de som inte kommit så långt att de insett att de behöver hjälp. Att arbetet på de manscentran som finns ger resultat ser de som jobbar med detta. Många män är kvar i det gamla förhållandet och kan med hjälp av stöd och råd fungera ”normalt”. Ett fåtal avbryter behandlingen och någon enstaka får återfall. I övrigt verkar det fungera bra. Förhoppningsvis så kommer det att satsas mer ekonomiskt på denna form av behandling för män, för ekonomin är det största hindret för manscentrans överlevnad

Paper properties of TMP and CTMP handsheets from spruce, as affected by the press load and temperature during sheet drying

The objective of this investigation was to evaluate how physical properties of laboratory sheets, made from different thermomechanical and chemithermomechanical pulps, depend on the pressure and temperature levels during pressing and drying. Sheets were formed from long fibre fractions, defined as the pulp retained on a 50 mesh wire in a Britt Dynamic Drainage Jar, and pressed and dried according to a number of different pressure and temperature settings. The results showed that there were large differences in the sheet properties depending on what type of method was used for pressing and drying. High freeness pulps were more sensitive than low freeness pulps to the methods used for pressing and drying. The results indicate that the difference in sheet properties between ISO and Rapid Kothen sheets may be explained by the fact that ISO sheets are wet pressed only at room temperature, the fibres are then allowed to spring back when pressure is released whereas the fibres in Rapid Kothen sheets are locked in their positions as the sheets are pressed and dried at high temperature until dry

Paper properties of TMP and CTMP handsheets from spruce, as affected by the solids content from pressing

This paper discusses how the properties of dry handsheets from two different mechanical pulps and one chemithermomechanical pulp and their respective long fibre fractions depend on the level of dryness to which the sheets are pressed at high temperature. Sheets were pressed and dried to different dryness levels at 400 kPa and 93 degrees C in a modified Rapid Kothen dryer. Prior to testing of the physical properties, the sheets were dried without pressure at, room temperature to full dryness and kept in a conditioning room. This was performed with the aim of evaluating the consolidation and development of final strength properties with respect to dryness after a press section in a paper machine.The most interesting finding from these trials was that the increase in density and strength were far from linear with increased press dryness, especially for the long fibre fractions when looking at the Z-strength development. There was a distinct inflection of the curve when the sheets reached a press dryness level of some 50%. The most important dryness interval for internal strength development was found between 50 and 80%.The fact that most paper and board machines press the sheet to only some 50% dryness, before feeding it into the drying section, implies that much of the inherent strength potential of mechanical pulps may be unexploited. Further research on pressing to higher dryness levels will in the future be continued at FSCN at Mid Sweden University

Mortality from peptic ulcer bleeding: the impact of comorbidity and the use of drugs that promote bleeding.

Summary Background Use of drugs promoting peptic ulcer bleed has increased several folds. Aim To make a time-trend analysis of peptic ulcer bleed patients and evaluate the impact of age, gender, comorbidity and use of drugs promoting peptic ulcer bleed on outcome. Methods Retrospective review of hospitalizations for peptic ulcer bleed at Lund University Hospital during 1984, 1994 and 2004. Univariate analyses between years and multivariable logistic regression for risk factors of fatal outcome. Results Incidence decreased from 62.0 to 32.1 per 100 000 inhabitants between 1984 and 2004. Mortality rates were stable. Median age (70-77 years; P = 0.001), number of comorbidities (mean +/- s.d.: 0.88 +/- 0.96 to 1.16 +/- 0.77; P = 0.021), use of aspirin (16-57%; P < 0.001) and warfarin (5-17%; P = 0.02) increased. Pharmacological and endoscopic therapy improved. Age above 65 years (OR: 1.11, 95% CI: 1.02-1.23) and number of comorbidities (OR: 6.00, 95% CI: 2.56-17.4) were independent risk factors for in-hospital mortality. Bleeding promoting drugs did not influence outcome negatively. Aspirin decreased the risk of fatal outcome (OR: 0.12, 95% CI: 0.012-0.67). Conclusions Incidence of peptic ulcer bleed decreased despite higher prescription rates of bleeding promoting drugs. The in-hospital mortality remained unchanged. The effect of improved therapy against peptic ulcer bleed is probably outweighed by older and more comorbid patients. The decreased risk of fatal outcome in aspirin users warrants further investigations

Lantgården som resurs för samhället och organisationen av dess välfärdsinsatser

Målet med denna rapport är att presentera hur kommuner och regioner kan ta hjälp av lantgårdar i utformningen av välfärdsinsatser, exempelvis inom social omsorg, utbildning, sjukvård, alternativa arbetsuppgifter och integration. Ett ytterligare mål är att belysa hur dessa verksamheter bör organiseras för att få ett långsiktigt hållbart samarbete mellan de svenska lantgårdarna och samhället. Historiskt har lantgårdar används som social insats med vinster för många individer och samhället, men detta minskade succesivt under urbaniseringen. Flera europeiska länder har återinfört lantgården som en resurs för olika utsatta målgrupper i samhället och har återuppbyggt en nationell struktur för dess genomförande. Utländsk forskning har visat att lantgårdar kan ge ökad motivation, social kontakt, känsla av att känna sig behövd, upplevelse av att kunna klara av uppgifter, ökad rörelse, ökat psykiskt välmående, och flera andra funktioner viktiga för god mänsklig välfärd. Svensk forskning har visat att lantgårdar kan ge stöd inom social omsorg och för skolelever med olika utmaningar, men många fler målgrupper skulle kunna gynnas av en lantgårdsbaserad insats (LBI). I Sverige finns ett 80-tal godkända Grön Arena gårdar, samt flera andra gårdar som kommuner och regioner kan upphandla för olika insatser. En utbyggnad av LBI i Sverige skulle gynna landsbygdsföretagande, och särskilt kvinnligt företagande. Det saknas dock en stödjande struktur och ett nationellt politiskt ställningstagande inom detta område. Rapporten utmynnar i flera rekommendationer. Tillsätt en statlig utredning med uppdrag till förslagsvis Socialstyrelsen, Tillväxtverket och Jordbruksverket med uppdrag att föreslå hur offentlig förvaltning kan underlätta för och stödja LBI. Ge uppdrag till Sveriges Kommuner och Regioner (SKR) att skapa en samordning från nationell, till regional, till kommunal nivå mellan myndigheter och företag för utbyggnad av LBI. Ta fram nationella riktlinjer och kvalitetssystem för användning av lantgårdar för sociala insatser. Skapa ett dokument gällande kvalitetssäkring för LBI på nationell nivå i samverkan mellan olika aktörer, t ex Hushållningssällskapet och Socialstyrelsen. Flerårig upphandling med rimlig ersättning måste tillämpas. Ställ krav på att alla som får kontrakt genom offentlig upphandling har dokumentation på att de har genomgått en godkänd kurs, att deras gård bedömts för risker och att de är lämpliga för övrigt. Bygg upp en stödjande struktur som är flexibel för användning av LBI för olika målgrupper inom kommun och region. Forskningsmedel behöver ges för tvärvetenskaplig forskning utifrån så väl brukarperspektivet som verksamhetsperspektivet. Avsätt forskningsmedel till samhällsekonomisk analys där kostnaderna för lantgårdsbaserad insats jämförs med annan insats. Slutsatsen är att lantgårdsbaserade välfärdsinsatser kan utgöra en värdefull resurs och ett komplement till traditionella insatser för flera olika målgrupper

Mental disorder is a cause of crime: The cornerstone of forensic psychiatry

The assumption that mental disorder is a cause of crime is the foundation of forensic psychiatry, but conceptual. epistemological. and empirical analyses show that neither mental nor crime, or the causation implied, are clear-cut concepts. "Mental" denotes heterogeneous aspects of a per-son such as inner experiences. cognitive abilities, and behaviour patterns described in a non-physical vocabulary. In psychology and psychiatry, mental describes law-bound, caused aspects of human functioning that are predictable and generalizable. Problems defined as mental disorders are end-points of dimensional inter-individual differences rather than natural categories. Deficits in cognitive faculties, such as attention, verbal understanding, impulse control, and reality assessment, may be susceptibility factors that relate to behaviours (Such as crimes) by increasing the probability (risk) for a negative behaviour or constitute causes in the sense of INUS conditions (insufficient but Non-redundant parts of Unnecessary but Sufficient conditions). Attributing causes to complex behaviours such as crimes is not an unbiased process, and mental disorders will attract disproportionate attention when it comes to explanations of behaviours that we wish to distance ourselves from. Only by rigorous interpretation of what psychiatry actually can inform us about, using empirical analyses of quantified aggressive antisocial behaviours and their possible explanatory factors, can we gain a clearer notion of the relationship between mental disorder and crime. (C) 2009 Elsevier Ltd. All rights reserved