Charakterisierung der Latenz des murinen Cytomegalovirus in vivo

Die primäre, produktive Cytomegalovirus (CMV)-Infektion wird im immunkompetenten Patienten effizient durch antivirale CD8+ T-Zellen kontrolliert. Das virale Genom besitzt jedoch die Fähigkeit, in einem nicht replikativen, Latenz genannten Zustand, in gewissen Zelltypen zu persistieren, ohne dass infektiöse Nachkommenviren produziert werden. Die molekularen Mechanismen, welche der Etablierung und Aufrechterhaltung der Latenz zugrundeliegen, sind noch weitestgehend unbekannt. Es gibt Hinweise darauf, dass zelluläre Verteidigungsmechanismen die Zirkularisierung und Chromatinisierung viraler Genome hervorrufen und dadurch die virale Genexpression größtenteils verhindert wird (Marks & Spector, 1984; Reeves et al., 2006).rnAllerdings liegen die Genome nicht in einem komplett inaktiven Zustand vor. Vielmehr konnte für das murine CMV (mCMV) bereits die sporadische Transkription der Gene ie1 und ie2 während der Latenz nachgewiesen werden (Kurz et al., 1999; Grzimek et al., 2001).rnIn der vorliegenden Arbeit wurde zum ersten Mal eine umfassende in vivo Latenz-Analyse zur Charakterisierung der viralen Transkription in einer Kinetik anhand der alle drei kinetischen Klassen repräsentierenden Transkripte IE1, IE3, E1, m164, M105 und M86 vorgenommen.rnNach Latenz-Etablierung, verifiziert durch Abwesenheit von infektiösem Virus, konnten alle getesteten Transkripte in der Lunge quantifiziert werden. Interessanterweise war die transkriptionelle Aktivität zu keinem Analyse-Zeitpunkt mit der klassischen IE-E-L-Kinetik der produktiven Infektion kompatibel. Stattdessen lag eine stochastische Transkript-Expression vor, deren Aktivität mit voranschreitender Zeit immer weiter abnahm.rnWährend der Latenz exprimierte Transkripte, die für antigene Peptide kodieren, können infizierte Zellen für das Immunsystem sichtbar machen, was zu einer fortwährenden Restimulation des memory T-Zell-pools führen würde. Durch zeitgleiche Analyse der Transkript-Expression, sowie der Frequenzen Epitop-spezifischer CD8+ T-Zellen während der Latenz (IE1, m164, M105), wurde eine möglicher Zusammenhang zwischen der transkriptionellen Aktivität und der Expansion des memory T-Zell-pools untersucht. Die weitere Charakterisierung von Subpopulationen der Epitop-spezifischen CD8+ T-Zellen identifizierte die SLECs (short-lived-effector cells; CD127low CD62Llow KLRG1high) als die dominante Population in Lunge und Milz während der mCMV-Latenz.rnIn einem weiteren Teil der Arbeit sollte untersucht werden, ob IE-Genexpression zur Etablierung von Latenz notwendig ist. Mit Hilfe der Rekombinanten mCMV-Δie2-DTR, die die Gensequenz des Diphtherietoxin-Rezeptors (DTR) anstelle des Gens ie2 trägt, konnten infizierte, DTR exprimierende Zellen durch eine DT-Applikation konditional depletiert werden.rnIm latent infizierbaren Zelltyp der Leber, den LSECs (liver sinusoidal endothelial cells) wurde die virale Load durch 90-stündige DT–Applikation nach mCMV-Δie2-DTR Infektion auf das Level latent infizierter LSECs reduziert. Diese Daten sprechen für die Hypothese eines von Beginn an inaktiven Genoms, das keine IE-Genexpression zur Latenz-Etablierung benötigt. Zusätzlich stellt dieser Ansatz ein neues Tier-Modell zur Latenz-Etablierung dar. Verringerte Wartezeiten bis zur vollständigen Latenz-Etablierung, im Vergleich zum bisherigen Knochenmarktransplantations-Modell, könnten anfallende Tierhaltungskosten erheblich reduzieren und das Voranschreiten der Forschung beschleunigen.

Identification of target genes of the T-box proteins in Drosophila melanogaster

In a prior bioinformatic analysis by Hüyseyin Binbas, potential Tbx targets sequences in wing-related genes have been identified. Guided by this information, enhancer trap/reporter lacZ insertions were characterized by X-gal staining first in wildtype and then in l(1)omb imaginal discs.rnIn several lines I observed an increase in reporter expression in a l(1)omb mutant background. Since Omb is assumed to function predominantly as a transcriptional repressor, this may indicate direct regulation. Repression by Omb was observed e.g. for brk and tkv. These genes are negatively regulated by Dpp, while omb is induced by Dpp. Omb which mediates the effects of Dpp on proliferation could, thus, also mediate the Dpp effect on patterning of the wing disc. However, brk and tkv were not completely derepressed in l(1)omb indicating that Dpp represses these genes also by an Omb-independent mechanism.rnMore frequently I observed loss of reporter expression in an l(1)omb mutant background. In these cases, regulation by Omb presumably is indirect. For example, STAT92E-lacZ expression in the wildtype eye was symmetrically expressed at the dorsal and ventral margins. In l(1)omb, ventral expression was selectively lost. Loss of omb is known to cause ventral overproliferation of the eye by activation of the Jak/STAT pathway. STAT92E expression is negatively regulated by Jak/STAT signaling suggesting that loss of omb activates Jak/STAT further upstream in the pathway.rnRegional overproliferation of eye and wing in the l(1)omb mutant background proved a complicating issue in the search for Omb targets. This effect made it difficult to decide whether an expanded reporter expression pattern was due to tissue expansion or reporter gene derepression. For instance hth-lacZ appeared to expand along the ventral eye disc margin in l(1)omb. Without addtional experiments it cannot be concluded whether this is due to de-repression or to activation in association with the proliferative state. Parallel to my experiments, evidence accumulated in our laboratory that loss of omb may attenuate Wg and Hegehog signaling. Since these diffusible proteins are the main patterning molecules in the wing imaginal disc, with dpp being downstream of Hh, many of the observed effects could be secondary to reduced Wg and Hh activity. Examples are ab-lacZ, Dll-lacZ and vgBE-lacZ (reduced expression on the dorso-ventral boundary) and inv-lacZ (late larval expression in the anterior wing disc compartment is lost) or sal-lacZ. Epistasis experiment will be required to clarifiy these issues.rnFurthermore, loss of omb appeared to induce cell fate changes. It was reported previously that in an omb null mutant, the dorsal determinant apterous (ap) is ectopically expressed in the ventral compartment (an effect I did not observe with the strongly hypomorphic l(1)omb15, indicating strong dose dependence). Ventral repression of ap is maintained by epigenetic mechanisms. The patchy and variable nature of ectopic expression of ap or grn-1.1-lacZ points to an effect of omb on epigenetic stability.rnIn the second part of my thesis, an analysis of Omb expression in the Drosophila embryonic ventral nervous system was performed. Omb was found co-expressed with Eve in the medial aCC and RP2 motorneurons as well as the fpCC interneuron and the mediolateral CQ neurons. Additionally, Omb was detected in the Eg positive NB7-3 GW serotonergic motoneuron and the N2-4 neurons. Omb was not found in Repo positive glial cells. During embryonic stage 14, Omb showed some coepression with Dpn or Pros. At the embryonic stage 16, Omb was expressed in minor subset of Mid and Wg positive cells.

Novel porphyrin amino acids as building blocks for artificial photosynthetic reaction centers : photoinduced energy and electron transfer

This thesis reports on the synthesis and characterisation of trans-(M)AB2C meso-substituted porphyrin amino acid esters (PAr) (M = 2H or Zn) with tunable electron donating and electron withdrawing Ar substituents at B positions (Ar = 4-C6H4OnBu, 4-C6H4OMe, 2,4,6-C6H2Me3, 4-C6H4Me, C6H5, 4-C6H4F, 4-C6H4CF3, C6F5). These porphyrins were used as key building blocks for photosynthetic LHC (LHC = light-harvesting antenna complex) and RC (RC = reaction center) model compounds.rnBased on free-base or zinc(II) porphyrin amino acid esters and porphyrin acids several amide linked free-base bis(porphyrins) PAr1-PAr2 (Ar1 = 2,4,6-C6H2Me3, C6F5 and Ar2 = 2,4,6-C6H2Me3, 4-C6H4F, 4-C6H4CF3, C6F5), mono metallated bis(porphyrin) PAr1-(Zn)PAr2 (Ar1 = 2,4,6-C6H2Me3 and Ar2 =4-C6H4F) and its doubly zincated complexes (Zn)PAr1-(Zn)PAr2 were prepared. In the fluorescence spectra of free-base bis(porphyrins) the porphyrin with the strongest electron donating power of Ar substituents at B positions is the light emitting unity. The emission of mono metallated bis(porphyrin) occurs only from the free-base porphyrin building block. This phenomenon is caused by an efficient energy transfer likely via the Dexter through-bond mechanism.rnLinking of anthraquinone (Q) as electron acceptor (A) to the N-terminus of porphyrin amino acid esters ((M)PAr) and aminoferrocene (Fc) as electron donor (D) to the C-terminus of the porphyrin resulting in Q-(M)PAr-Fc triads (M = 2H or Zn, Ar = 4-C6H4OnBu, 4-C6H4OMe, 2,4,6-C6H2Me3, 4-C6H4Me, C6H5, 4-C6H4F, 4-C6H4CF3, C6F5) with tunable electron density at the porphyrin chromophore. In these triads initial oxidative PET (Q←(M)PAr) and reductive PET ((M)PAr→Fc) (PET = photoinduced electron transfer) are possible. Both processes leads to an emission quenching of (M)PAr. The efficiency of the PET pathways occurring in the Marcus normal region is controlled by the specific porphyrin electron density.rnAmide-linked conjugates PAr-Fc (Ar = 2,4,6-C6H2Me3, C6F5) and Fmoc-Fc-PAr1 (N-Fmoc-Fc = N-Fmoc protected 1,1’-ferrocene amino acid; Ar1 = C6H5, 4-C6H4F, 4-C6H4CF3, C6F5) as well as hinges PAr2-Fc-PAr1 (Ar1 = C6H5, 4-C6H4F and Ar2 = 2,4,6-C6H2Me3) were studied with respect to the reductive PET. The PET driving force (−GET) in dyads increases with the increasing electron withdrawing character of Ar substituents. Additionally, intramolecular energy transfer between porphyrins PAr1 and PAr2 is feasible in the hinges via the Förster mechanism.rn

Excitability properties of mouse motor axons in the mutant SOD1(G93A) model of amyotrophic lateral sclerosis

Non-invasive excitability studies of motor axons in patients with amyotrophic lateral sclerosis (ALS) have revealed a changing pattern of abnormal membrane properties with disease progression, but the heterogeneity of the changes has made it difficult to relate them to pathophysiology. The SOD1(G93A) mouse model of ALS displays more synchronous motoneuron pathology. Multiple excitability measures of caudal and sciatic nerves in mutant and wild-type mice were compared before onset of signs and during disease progression (4-19 weeks), and they were related to changes in muscle fiber histochemistry. Excitability differences indicated a modest membrane depolarization in SOD1(G93A) axons at about the time of symptom onset (8 weeks), possibly due to deficient energy supply. Previously described excitability changes in ALS patients, suggesting altered sodium and potassium conductances, were not seen in the mice. This suggests that those changes relate to features of the human disease that are not well represented in the animal model.

Patterns of morphological changes and hybridization between sympatric whitefish morphs (Coregonus spp.) in a Swiss lake: a role for eutrophication?

Whitefish, genus Coregonus, show exceptional levels of phenotypic diversity with sympatric morphs occurring in numerous postglacial lakes in the northern hemisphere. Here, we studied the effects of human-induced eutrophication on sympatric whitefish morphs in the Swiss lake, Lake Thun. In particular, we addressed the questions whether eutrophication (i) induced hybridization between two ecologically divergent summer-spawning morphs through a loss of environmental heterogeneity, and (ii) induced rapid adaptive morphological changes through changes in the food web structure. Genetic analysis based on 11 microsatellite loci of 282 spawners revealed that the pelagic and the benthic morph represent highly distinct gene pools occurring at different relative proportions on all seven known spawning sites. Gill raker counts, a highly heritable trait, showed nearly discrete distributions for the two morphs. Multilocus genotypes characteristic of the pelagic morph had more gill rakers than genotypes characteristic of benthic morph. Using Bayesian methods, we found indications of recent but limited introgressive hybridization. Comparisons with historical gill raker data yielded median evolutionary rates of 0.24 haldanes and median selection intensities of 0.27 for this trait in both morphs for 1948-2004 suggesting rapid evolution through directional selection at this trait. However, phenotypic plasticity as an alternative explanation for this phenotypic change cannot be discarded. We hypothesize that both the temporal shifts in mean gill raker counts and the recent hybridization reflect responses to changes in the trophic state of the lake induced by pollution in the 1960s, which created novel selection pressures with respect to feeding niches and spawning site preferences.

Excitability and recruitment patterns of spinal motoneurons in human sleep as assessed by F-wave recordings

This study examines the excitability and recruitment of spinal motoneurons in human sleep. The main objective was to assess whether supraspinal inhibition affects the different subpopulations of the compound spinal motoneuron pool in the same way or rather in a selective fashion in the various sleep stages. To this end, we studied F-conduction velocities (FCV) and F-tacheodispersion alongside F-amplitudes and F-persistence in 22 healthy subjects in sleep stages N2, N3 (slow-wave sleep), REM and in wakefulness. Stimuli were delivered on the ulnar nerve, and F-waves were recorded from the first dorsal interosseus muscle. Repeated sets of stimuli were stored to obtain at least 15 F-waves for each state of vigilance. F-tacheodispersion was calculated based on FCVs using the modified Kimura formula. Confirming the only previous study, excitability of spinal motoneurons was generally decreased in all sleep stages compared with wakefulness as indicated by significantly reduced F-persistence and F-amplitudes. More importantly, F-tacheodispersion showed a narrowed range of FCV in all sleep stages, most prominently in REM. In non-REM, this narrowed range was associated with a shift towards significantly decreased maximal FCV and mean FCV as well as with a trend towards lower minimal FCV. In REM, the lowering of mean FCV was even more pronounced, but contrary to non-REM sleep without a shift of minimal and maximal FCV. Variations in F-tacheodispersion between sleep stages suggest that different supraspinal inhibitory neuronal circuits acting on the spinal motoneuron pool may contribute to muscle hypotonia in human non-REM sleep and to atonia in REM sleep.

Glycogen-rich pleomorphic xanthoastrocytoma with clear-cell features: confirmatory report of a rare variant with implications for differential diagnosis

Central nervous system space-occupying lesions with clear-cell features encompass a nosologically heterogeneous array, ranging from reactive histiocytic proliferations to neuroepithelial or meningothelial neoplasms of various grades and to metastases. In the face of such differential diagnostic breadth, recognizing cytoplasmic lucency as part of the morphological spectrum of some low grade gliomas will directly have an impact on patient care. We describe a prevailing clear-cell change in an epileptogenic left temporal pleomorphic xanthoastrocytoma surgically resected from a 36-year-old man. Mostly subarachnoid and focally calcified, the tumor was composed of fascicles of moderately atypical spindle cells with optically lucent cytoplasm that tended to intermingle with a desmoplastic mesh of reticulin fibers. Immunohistochemically, coexpression of S100 protein, vimentin, GFAP, and CD34 was noted. Conversely, neither punctate staining for EMA nor positivity for CD68 was seen. Mitotic activity was absent, and the MIB1 labeling index was 2-3% on average. Diastase-sensitive PAS-positive granula indicated clear-cell change to proceed from glycogen storage. Electron microscopy showed tumor cell cytoplasm to be largely obliterated by non-lysosomal-bound pools of glycogen, while hardly any fat vacuole was encountered. Neither ependymal-derived organelles nor annular lamellae suggesting oligodendroglial differentiation were detected. The latter differential diagnosis was further invalidated by lack of codeletion of chromosomal regions 1p36 and 19q13 on molecular genetic testing. By significantly interfering with pattern recognition as an implicit approach in histopathology, clear-cell change in pleomorphic xanthoastrocytoma is likely to suspend its status as a "classic", and to prompt more deductive differential diagnostic strategies to exclude look-alikes, especially clear-cell ependymoma and oligodendroglioma.

Restricted or severely hindered diffusion of intramyocellular lipids in human skeletal muscle shown by in vivo proton MR spectroscopy

Although magnetic resonance spectroscopy can be used as a unique tool to study molecular diffusion, it is rarely used to measure the diffusion properties of intramyocellular and extramyocellular lipids. Lipids have very low apparent diffusion coefficients (ADCs), which make these measurements difficult and necessitate strong diffusion gradients and long diffusion times. Consequence is that these measurements have inherently low signal-to-noise ratio and are prone to artifacts. The addition of physiological triggering and individual storage and processing of the spectra is seen to be a possible approach to maximize signal intensity and achieve high reproducibility of the experiments. Thus, the optimized measurement protocol was used to investigate the diffusion properties of lipids in human skeletal muscle in vivo. At a diffusion time of about 110 ms, intramyocellular lipids show a significantly lower ADC (2.0 × 10(-6) mm(2)/s, 95% confidence interval 1.10 × 10(-6) to 2.94 × 10(-6) mm(2)/s) than extramyocellular lipids (1.58 × 10(-5) mm(2)/s, 95% confidence interval 1.41 × 10(-5) to 1.75 × 10(-5) mm(2)/s). Because the chemical properties of both lipid pools can be assumed to be similar, the difference can only be attributed to restricted or severely hindered diffusion in the intramyocellular droplets.

Spatial and temporal variation in population genetic structure of wild Nile tilapia (Oreochromis niloticus) across Africa

Background: Reconstructing the evolutionary history of a species is challenging. It often depends not only on the past biogeographic and climatic events but also the contemporary and ecological factors, such as current connectivity and habitat heterogeneity. In fact, these factors might interact with each other and shape the current species distribution. However, to what extent the current population genetic structure reflects the past and the contemporary factors is largely unknown. Here we investigated spatio-temporal genetic structures of Nile tilapia (Oreochromis niloticus) populations, across their natural distribution in Africa. While its large biogeographic distribution can cause genetic differentiation at the paleo-biogeographic scales, its restricted dispersal capacity might induce a strong genetic structure at micro-geographic scales. Results: Using nine microsatellite loci and 350 samples from ten natural populations, we found the highest genetic differentiation among the three ichthyofaunal provinces and regions (Ethiopian, Nilotic and Sudano-Sahelian) (R(ST) = 0.38 - 0.69). This result suggests the predominant effect of paleo-geographic events at macro-geographic scale. In addition, intermediate divergences were found between rivers and lakes within the regions, presumably reflecting relatively recent interruptions of gene flow between hydrographic basins (R(ST) = 0.24 - 0.32). The lowest differentiations were observed among connected populations within a basin (R(ST) = 0.015 in the Volta basin). Comparison of temporal sample series revealed subtle changes in the gene pools in a few generations (F = 0 - 0.053). The estimated effective population sizes were 23 - 143 and the estimated migration rate was moderate (m similar to 0.094 - 0.097) in the Volta populations. Conclusions: This study revealed clear hierarchical patterns of the population genetic structuring of O. niloticus in Africa. The effects of paleo-geographic and climatic events were predominant at macro-geographic scale, and the significant effect of geographic connectivity was detected at micro-geographic scale. The estimated effective population size, the moderate level of dispersal and the rapid temporal change in genetic composition might reflect a potential effect of life history strategy on population dynamics. This hypothesis deserves further investigation. The dynamic pattern revealed at micro-geographic and temporal scales appears important from a genetic resource management as well as from a biodiversity conservation point of view.

Screening of Swiss hot spring resorts for potentially pathogenic free-living amoebae

Free-living amoebae (FLA) belonging to Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, and Sappinia pedata are known to cause infections in humans and animals leading to severe brain pathologies. Worldwide, warm aquatic environments have been found to be suitable habitats for pathogenic FLA. The present study reports on screening for potentially pathogenic FLA in four hot spring resorts in Switzerland. Water samples were taken from water filtration units and from the pools, respectively. Amoebae isolated from samples taken during, or before, the filtration process were demonstrated to be morphologically and phylogenetically related to Stenoamoeba sp., Hartmannella vermiformis, Echinamoeba exundans, and Acanthamoeba healyi. With regard to the swimming pools, FLA were isolated only in one resort, and the isolate was identified as non-pathogenic and as related to E. exundans. Further investigations showed that the isolates morphologically and phylogenetically related to A. healyi displayed a pronounced thermotolerance, and exhibited a marked in vitro cytotoxicity upon 5-day exposure to murine L929 fibroblasts. Experimental intranasal infection of Rag2-immunodeficient mice with these isolates led to severe brain pathologies, and viable trophozoites were isolated from the nasal mucosa, brain tissue, and lungs post mortem. In summary, isolates related to A. healyi were suggestive of being potentially pathogenic to immunocompromised persons. However, the presence of these isolates was limited to the filtration units, and an effective threat for health can therefore be excluded.

Cardiac sodium channel NaV1.5 distribution in myocytes via interacting proteins: the multiple pool model

The cardiac sodium current (INa) is responsible for the rapid depolarization of cardiac cells, thus allowing for their contraction. It is also involved in regulating the duration of the cardiac action potential (AP) and propagation of the impulse throughout the myocardium. Cardiac INa is generated by the voltage-gated Na(+) channel, NaV1.5, a 2016-residue protein which forms the pore of the channel. Over the past years, hundreds of mutations in SCN5A, the human gene coding for NaV1.5, have been linked to many cardiac electrical disorders, including the congenital and acquired long QT syndrome, Brugada syndrome, conduction slowing, sick sinus syndrome, atrial fibrillation, and dilated cardiomyopathy. Similar to many membrane proteins, NaV1.5 has been found to be regulated by several interacting proteins. In some cases, these different proteins, which reside in distinct membrane compartments (i.e. lateral membrane vs. intercalated disks), have been shown to interact with the same regulatory domain of NaV1.5, thus suggesting that several pools of NaV1.5 channels may co-exist in cardiac cells. The aim of this review article is to summarize the recent works that demonstrate its interaction with regulatory proteins and illustrate the model that the sodium channel NaV1.5 resides in distinct and different pools in cardiac cells. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Effects of in situ freezing on soil net nitrogen mineralization and net nitrification in fertilized grassland of northern China

Effects of soil freezing on nitrogen (N) mineralization have been the subject of increased attention in the ecological literature, though fewer studies have examined N mineralization responses to successive mild freezing, severe freezing and cyclic freeze–thaw events. Even less is known about relationships of responses to soil N status. This study measured soil N mineralization and nitrification in the field along an experimental N gradient in a grassland of northern China during the dormant season (October 2005–April 2006), a period in which freezing naturally occurs. Net N mineralization exhibited great temporal variability, with nitrification being the predominant N transformation process. Soil microbial biomass C and N and extractable NH4 + pools declined by 40, 52, and 56%, respectively, in April 2006, compared with their initial concentrations in October 2005; soil NO3– pools increased by 84%. Temporal patterns of N mineralization were correlated with soil microbial biomass C and N. N mineralization and nitrification increased linearly with added N. Microbial biomass C in treated soils increased by 10% relative to controls, whereas microbial N declined by 9%. Results further suggest that freezing events greatly alter soil N dynamics in the dormant season at this site, with considerable available N accumulating during this period.

Virus isolation vs RT-PCR: which method is more successful in detecting VHSV and IHNV in fish tissue sampled under field conditions?

This study compared the results of reverse transcription-polymerase chain reaction (RT-PCR) and traditional virus isolation on cell culture in detection of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). RT-PCR was used for 172 tissue sample pools (total of 859 fish) originating from a field survey on the occurrence of VHSV and IHNV in farmed and wild salmonids in Switzerland. These samples represented all sites with fish that were either identified as virus-positive by means of virus isolation (three sites, four positive tissue sample pools) and/or demonstrated positive anti-VHSV-antibody titres (83 sites, 121 positive blood samples) in a serum plaque neutralization test (SPNT). The RT-PCR technique confirmed the four VHSV-positive tissue sample pools detected by virus isolation and additionally identified one VHSV-positive sample that showed positive anti-VHSV-AB titres, but was negative in virus isolation. With IHNV, RT-PCR detected two positive samples not identified by virus isolation while in these fish the SPNT result had been questionable. One of the IHNV-positive samples represents the first detection of IHNV-RNA in wild brown trout in Switzerland. Compared to SPNT, the RT-PCR method detected, as with virus isolation, a much lower number of positive cases; reasons for this discrepancy are discussed. Our results indicate that RT-PCR can not only be successfully applied in field surveys, but may also be slightly more sensitive than virus isolation. However, in a titration experiment under laboratory conditions, the sensitivity of RT-PCR was not significantly higher when compared with virus isolation.

Spinal muscular atrophy: SMN2 pre-mRNA splicing corrected by a U7 snRNA derivative carrying a splicing enhancer sequence

Spinal muscular atrophy (SMA) is a lethal hereditary disease caused by homozygous deletion/inactivation of the survival of motoneuron 1 (SMN1) gene. The nearby SMN2 gene, despite its identical coding capacity, is only an incomplete substitute, because a single nucleotide difference impairs the inclusion of its seventh exon in the messenger RNA (mRNA). This splicing defect can be corrected (transiently) by specially designed oligonucleotides. Here we have developed a more permanent correction strategy based on bifunctional U7 small nuclear RNAs (snRNAs). These carry both an antisense sequence that allows specific binding to exon 7 and a splicing enhancer sequence that will improve the recognition of the targeted exon. When expression cassettes for these RNAs are stably introduced into cells, the U7 snRNAs become incorporated into small nuclear ribonucleoprotein (snRNP) particles that will induce a durable splicing correction. We have optimized this strategy to the point that virtually all SMN2 pre-mRNA becomes correctly spliced. In fibroblasts from an SMA patient, this approach induces a prolonged restoration of SMN protein and ensures its correct localization to discrete nuclear foci (gems).

Will Whole-Tree Harvest of Jack Pine (Pinus banksiana) deplete soil nutrients in low-productivity sand soils?

In 2009 and 2010 a study was conducted on the Hiawatha National Forest (HNF) to determine if whole-tree harvest (WTH) of jack pine would deplete the soil nutrients in the very coarse-textured Rubicon soil. WTH is restricted on Rubicon sand in order to preserve the soil fertility, but the increasing construction of biomass-fueled power plants is expected to increase the demand for forest biomass. The specific objectives of this study were to estimate biomass and nutrient content of above- and below-ground tree components in mature jack pine (Pinus banksiana) stands growing on a coarse-textured, low-productivity soil, determine pools of total C and N and exchangeable soil cations in Rubicon sand, and to compare the possible impacts of conventional stem-only harvest (CH) and WTH on soil nutrient pools and the implications for productivity of subsequent rotations. Four even-aged jack pine stands on Rubicon soil were studied. Allometric equations were used to estimate above-ground biomass and nutrients, and soil samples from each stand were taken for physical and chemical analysis. Results indicate that WTH will result in cation deficits in all stands, with exceptionally large Ca deficits occurring in two stands. Where a deficit does not occur, the cation surplus is small and, chemical weathering and atmospheric deposition is not anticipated to replace the removed cations. CH will result in a surplus of cations, and will likely not result in productivity declines during the first rotation. However even under CH, the surplus is small, and chemical weathering and atmospheric deposition will not supply enough cations for the second rotation.