Muonic atom spectroscopy with microgram target material.

Muonic atom spectroscopy-the measurement of the x rays emitted during the formation process of a muonic atom-has a long standing history in probing the shape and size of nuclei. In fact, almost all stable elements have been subject to muonic atom spectroscopy measurements and the absolute charge radii extracted from these measurements typically offer the highest accuracy available. However, so far only targets of at least a few hundred milligram could be used as it required to stop a muon beam directly in the target to form the muonic atom. We have developed a new method relying on repeated transfer reactions taking place inside a 100 bar hydrogen gas cell with an admixture of 0.25% deuterium that allows us to drastically reduce the amount of target material needed while still offering an adequate efficiency. Detailed simulations of the transfer reactions match the measured data, suggesting good understanding of the processes taking place inside the gas mixture. As a proof of principle we demonstrate the method with a measurement of the 2p-1s muonic x rays from a 5  µ g gold target.

Demonstration of Muon-Beam Transverse Phase-Space Compression.

We demonstrate efficient transverse compression of a 12.5 MeV/c muon beam stopped in a helium gas target featuring a vertical density gradient and crossed electric and magnetic fields. The muon stop distribution extending vertically over 14 mm was reduced to a 0.25 mm size (rms) within 3.5 µs. The simulation including cross sections for low-energy µ^{+}-He elastic and charge exchange (µ^{+}↔ muonium) collisions describes the measurements well. By combining the transverse compression stage with a previously demonstrated longitudinal compression stage, we can improve the phase space density of a µ^{+} beam by a factor of 10^{10} with 10^{-3} efficiency.

Experimental adaptation of an RNA virus mimics natural evolution.

Identification of virulence determinants of viruses is of critical importance in virology. In search of such determinants, virologists traditionally utilize comparative genomics between a virulent and an avirulent virus strain and construct chimeras to map their locations. Subsequent comparison reveals sequence differences, and through analyses of site-directed mutants, key residues are identified. In the absence of a naturally occurring virulent strain, an avirulent strain can be functionally converted to a virulent variant via an experimental evolutionary approach. However, the concern remains whether experimentally evolved virulence determinants mimic those that have evolved naturally. To provide a direct comparison, we exploited a plant RNA virus, soybean mosaic virus (SMV), and its natural host, soybean. Through a serial in vivo passage experiment, the molecularly cloned genome of an avirulent SMV strain was converted to virulent variants on functionally immune soybean genotypes harboring resistance factor(s) from the complex Rsv1 locus. Several of the experimentally evolved virulence determinants were identical to those discovered through a comparative genomic approach with a naturally evolved virulent strain. Thus, our observations validate an experimental evolutionary approach to identify relevant virulence determinants of an RNA virus.

Gain of virulence on Rsv1-genotype soybean by an avirulent Soybean mosaic virus requires concurrent mutations in both P3 and HC-Pro.

In soybean, Rsv1, a single dominant resistance gene, invokes extreme resistance (ER) against most Soybean mosaic virus (SMV) strains, including SMV-N, but not SMV-G7, which provokes a virulent lethal systemic hypersensitive response (LSHR). The elicitor functions of the two viruses provoking Rsv1-mediated ER and LSHR have been mapped to the N-terminal 271 amino acids of P3 from SMV-N and SMV-G7, respectively, which differ by nine residues between the two strains. To identify amino acids of P3 from SMV-N provoking Rsv1-mediated ER, the unique residues of SMV-G7 were substituted with those of SMV-N. Of the mutants tested on Rsv1-genotype soybean, only SMV-G7(I788R) and SMV-G7(T948A) lost virulence. However, substitution of amino acids of SMV-N, individually or in combination, with the reciprocal residues from SMV-G7 at these two positions failed to confer virulence to SMV-N. In the search for additional virulence determinants, a series of SMV-N chimeras was generated in which fragments within a region from near the middle of the helper-component proteinase (HC-Pro) cistron to the 5' end of the cytoplasmic inclusion cistron, nucleotides 1,605 to 3,787, were replaced with those of SMV-G7. Only SMV-N-derived chimeras harboring the 3' region of HC-Pro, at least from nucleotide 2,013, and the entire 5' end of P3 (nucleotides 2,430 to 3,237) from SMV-G7 were virulent whereas reciprocal exchanges resulted in loss of SMV-G7 virulence. This region of HC-Pro differs by three amino acids between SMV-N and SMV-G7. Analyses of SMV-G7-derived HC-Pro site-directed mutants showed that only SMV-G7(M683R) lost virulence on Rsv1-genotype soybean; however, SMV-N(R682M) failed to gain virulence. Nevertheless, an SMV-N derived mutant with three concurrent substitutions, R682M+R787I+A947T, gained virulence. The data indicate that both P3 and HC-Pro are involved in virulence of SMV on Rsv1-genotype soybean.

Adaptation of Soybean mosaic virus avirulent chimeras containing P3 sequences from virulent strains to Rsv1-genotype soybeans is mediated by mutations in HC-Pro.

In Rsv1-genotype soybean, Soybean mosaic virus (SMV)-N (an avirulent isolate of strain G2) elicits extreme resistance (ER) whereas strain SMV-G7 provokes a lethal systemic hypersensitive response (LSHR). SMV-G7d, an experimentally evolved variant of SMV-G7, induces systemic mosaic. Thus, for Rsv1-genotype soybean, SMV-N is avirulent whereas SMV-G7 and SMV-G7d are both virulent. Exploiting these differential interactions, we recently mapped the elicitor functions of SMV provoking Rsv1-mediated ER and LSHR to the N-terminal 271 amino acids of P3 from SMV-N and SMV-G7, respectively. The phenotype of both SMV-G7 and SMV-G7d were rendered avirulent on Rsv1-genotype soybean when the part of the genome encoding the N-terminus or the entire P3 cistron was replaced with that from SMV-N; however, reciprocal exchanges did not confer virulence to SMV-N-derived P3 chimeras. Here, we describe virulent SMV-N-derived P3 chimeras containing the full-length or the N-terminal P3 from SMV-G7 or SMV-G7d, with or without additional mutations in P3, that were selected on Rsv1-genotype soybean by sequential transfers on rsv1 and Rsv1-genotype soybean. Sequence analyses of the P3 and helper-component proteinase (HC-Pro) cistrons of progeny recovered from Rsv1-genotype soybean consistently revealed the presence of mutations in HC-Pro. Interestingly, the precise mutations in HC-Pro required for the adaptation varied among the chimeras. No mutation was detected in the HC-Pro of progeny passaged continuously in rsv1-genotype soybean, suggesting that selection is a consequence of pressure imposed by Rsv1. Mutations in HC-Pro alone failed to confer virulence to SMV-N; however, reconstruction of mutations in HC-Pro of the SMV-N-derived P3 chimeras resulted in virulence. Taken together, the data suggest that HC-Pro complementation of P3 is essential for SMV virulence on Rsv1-genotype soybean.

Strain-specific P3 of Soybean mosaic virus elicits Rsv1-mediated extreme resistance, but absence of P3 elicitor function alone is insufficient for virulence on Rsv1-genotype soybean.

When challenged by mechanical inoculation, the Rsv1 gene of soybean invokes extreme resistance (ER) against Soybean mosaic virus (SMV) strain N, but not SMV-G7 and its experimentally evolved variant, SMV-G7d. SMV-G7 provokes a lethal systemic hypersensitive response (LSHR), whereas SMV-G7d induces systemic mosaic. Thus, for Rsv1-genotype soybean, SMV-G7 and SMV-G7d are both virulent virus strains. The elicitor function of SMV-G7 provoking Rsv1-mediated LSHR was recently mapped to P3, and the influence of amino acids 823, 953, and 1112 of the precursor polypeptide of SMV-G7d on evasion of Rsv1-mediated recognition provoking LSHR was demonstrated. We have now extended this study to SMV-N. Initially, amino acids corresponding to those of SMV-G7d at these positions were substituted, individually or in combinations. All the mutants remained replication competent on rsv1-genotype soybean; however, none lost the elicitor function provoking Rsv1-mediated ER. Subsequently, P3 of SMV-N was precisely replaced with P3 of SMV-G7 or SMV-G7d and vice versa. All the chimeras were replication competent on rsv1-genotype soybean, but surprisingly SMV-N/G7P3 and SMV-N/G7dP3 failed to gain virulence on Rsv1-genotype soybeans. However, SMV-G7/NP3 and SMV-G7d/NP3 lost virulence, and this loss of virulence function was mapped to the N-terminus domain of SMV-N P3. The data indicate that SMV strain-specific P3 provokes Rsv1-mediated ER; however, virulence on Rsv1-genotype soybean is not solely a consequence of the absence of the P3 elicitor functions provoking Rsv1-mediated ER and LSHR.

Loss and gain of elicitor function of soybean mosaic virus G7 provoking Rsv1-mediated lethal systemic hypersensitive response maps to P3.

Rsv1, a single dominant resistance gene in soybean PI 96983 (Rsv1), confers extreme resistance against all known American strains of Soybean mosaic virus (SMV), except G7 and G7d. SMV-G7 provokes a lethal systemic hypersensitive response (LSHR), whereas SMV-G7d, an experimentally evolved variant of SMV-G7, induces systemic mosaic. To identify the elicitor of Rsv1-mediated LSHR, chimeras were constructed by exchanging fragments between the molecularly cloned SMV-G7 (pSMV-G7) and SMV-G7d (pSMV-G7d), and their elicitor functions were assessed on PI 96983 (Rsv1). pSMV-G7-derived chimeras containing only P3 of SMV-G7d lost the elicitor function, while the reciprocal chimera of pSMV-G7d gained the function. The P3 regions of the two viruses differ by six nucleotides, of which two are translationally silent. The four amino acid differences are located at positions 823, 915, 953, and 1112 of the precursor polypeptide. Analyses of the site-directed point mutants of both the viruses revealed that nucleotide substitutions leading to translationally silent mutations as well as reciprocal amino acid substitution at position 915 did not influence the loss or gain of the elicitor function. pSMV-G7-derived mutants with amino acid substitutions at any of the other three positions lost the ability to provoke LSHR but induced SHR instead. Two concomitant amino acid substitutions at positions 823 (V to M) and 953 (K to E) abolished pSMV-G7 elicitor function, provoking Rsv1-mediated SHR. Conversely, pSMV-G7d gained the elicitor function of Rsv1-mediated LSHR by a single amino acid substitution at position 823 (M to V), and mutants with amino acid substitutions at position 953 or 1112 induced SHR instead of mosaic. Taken together, the data suggest that strain-specific P3 of SMV is the elicitor of Rsv1-mediated LSHR.

Evolution of Soybean mosaic virus-G7 molecularly cloned genome in Rsv1-genotype soybean results in emergence of a mutant capable of evading Rsv1-mediated recognition.

Plant resistance (R) genes direct recognition of pathogens harboring matching avirluent signals leading to activation of defense responses. It has long been hypothesized that under selection pressure the infidelity of RNA virus replication together with large population size and short generation times results in emergence of mutants capable of evading R-mediated recognition. In this study, the Rsv1/Soybean mosaic virus (SMV) pathosystem was used to investigate this hypothesis. In soybean line PI 96983 (Rsv1), the progeny of molecularly cloned SMV strain G7 (pSMV-G7) provokes a lethal systemic hypersensitive response (LSHR) with up regulation of a defense-associated gene transcript (PR-1). Serial passages of a large population of the progeny in PI 96983 resulted in emergence of a mutant population (vSMV-G7d), incapable of provoking either Rsv1-mediated LSHR or PR-1 protein gene transcript up regulation. An infectious clone of the mutant (pSMV-G7d) was synthesized whose sequences were very similar but not identical to the vSMV-G7d population; however, it displayed a similar phenotype. The genome of pSMV-G7d differs from parental pSMV-G7 by 17 substitutions, of which 10 are translationally silent. The seven amino acid substitutions in deduced sequences of pSMV-G7d differ from that of pSMV-G7 by one each in P1 proteinase, helper component-proteinase, and coat protein, respectively, and by four in P3. To the best of our knowledge, this is the first demonstration in which experimental evolution of a molecularly cloned plant RNA virus resulted in emergence of a mutant capable of evading an R-mediated recognition.

Characterization of a monoclonal antibody recognizing a DAG-containing epitope conserved in aphid transmissible potyviruses: evidence that the DAG motif is in a defined conformation.

Two viral proteins, the helper component-protease and the coat protein, are required for the non-persistent aphid transmission of potyviruses. In the potyvirus coat protein, the tripeptide aspartate-alanine-glycine (DAG) has often been shown to be involved. A monoclonal antibody, raised against a synthetic decapeptide containing the DAG tripeptide, reacted with the peptide as well as with isolates of soybean mosaic, tobacco etch and tobacco vein mottling potyviruses. Experiments indicate that the monoclonal antibody recognizes a conformational rather than a sequential epitope. The data support the suggestion that the DAG region plays a structural role to determine a coat protein-helper component-protease conformation that influences aphid transmission.

Transfer of the movement protein gene between two tobamoviruses: influence on local lesion development.

The effects of transfer of the movement gene between the tobamoviruses tobacco mosaic virus (TMV) and tobacco mild green mosaic virus (TMGMV) were studied. The movement protein (MP) gene of TMGMV was cloned into an infectious cDNA of TMV to build the recombinant virus V23. V23, like TMV and TMGMV, caused systemic infection in Nicotiana tabacum Xanthi. In N. sylvestris V23 and TMV spread systemically although TMGMV produces necrotic local lesions on this host. V23 and TMV cause systemic infection on tomato plants while TMGMV does not infect tomato. In Xanthi nc plants, V23 produced necrotic local lesions similar in size to those produced by TMGMV. On the other hand in transgenic Xanthi nc tobacco plants that express a gene encoding the MP of TMV the necrotic lesions produced by V23 and TMGMV were similar in size to those produced by TMV. These results indicate that the size of necrotic lesions produced by TMGMV and TMV on Xanthi nc plants is influenced by the MP gene.

The nucleotide sequence of a soybean mosaic virus coat protein-coding region and its expression in Escherichia coli, Agrobacterium tumefaciens and tobacco callus.

A DNA complementary to the 3'-terminal 1168 nucleotides of the genome of the N strain of soybean mosaic virus (SMV) has been cloned and sequenced. cDNA sequence and coat protein analyses indicate that the SMV coat protein-coding region is at the 3' end of the genome, and that the coat protein is processed from a larger protein. The coat protein-coding sequence is predicted to be 795 nucleotides in length, encoding a protein of 265 amino acids with a calculated Mr of 29,857. The 3' untranslated region is 259 nucleotides in length and is followed by a polyadenylate tract. The SMV coat protein-coding region, along with a small amount of upstream sequence, has been expressed in Escherichia coli as a beta-galactosidase fusion protein. The size of the protein was less than predicted for the fusion protein, suggesting processing in E. coli. The coat protein-coding region has also been expressed in Agrobacterium tumefaciens and transgenic tobacco callus as an unfused protein under the control of the cauliflower mosaic virus 35S promoter. The coat protein produced in transgenic tobacco callus had an electrophoretic mobility identical to that of SMV coat protein and constituted approximately 0.05% (w/w) of the total extracted protein.

[Unusual ophthalmic findings in a case of Niemann-Pick disease (author's transl)]. / Aussergewöhnlicher Augenbefund bei Niemann-Pick-Krankheit.

Case report on a woman patient who, although not Jewish, is suffering from Niemann-Pick disease. At the time of publication she is 55 years old, although this disease usually proves lethal in early childhood. Related atypical ocular changes were diagnosed, such as tortuosity of the retinal vessels and a conjunctival affection, which was demonstrated histologically.