[Electives during preclinical medical curriculum: the Geneva experience ]. / Modules à option au cours des études de médecine: l'expérience genevoise.

Electives have come of age in Medical Schools throughout Europe since the Bologna Guidelines were issued. At the Faculty of Medicine of Geneva its importance was recognized early to satisfy the students' curiosity, enlarge their visions, or deepen their knowledge in certain aspects of their curriculum. It was therefore decided to develop a great number of different electives ranging from basic biomedical research to emblematic clinical syndromes and humanitarian medicine for 2nd and 3rd year Bachelor students. The Electives taken have to be validated through specific examinations corresponding to 10% of the yearly ECTS credits. The experience has so far been a success, with high satisfaction as well of the student as of the teacher body. A major challenge for the future will be opening further these Electives to other Faculties and other professions.

Impact of the maximum ring difference on image quality and noise characteristics of a total-body PET/CT scanner.

The sensitivity of a PET system highly depends on the axial acceptance angle or maximum ring difference (MRD), which can be particularly high for total-body scanners due to their larger axial field of views (aFOVs). This study aims to evaluate the impact on image quality (IQ) and noise performance when MRD85 (18°), the current standard for clinical use, is increased to MRD322 (52°) for the Biograph Vision Quadra (Siemens Healthineers). METHODS: Studies with a cylindrical phantom covering the 106 cm aFOV and an IEC phantom filled with 18F, 68Ga and 89Zr were performed for acquisition times from 60 to 1800 s and activity concentrations from 0.4 to 3 kBq/ml to assess uniformity, contrast recovery coefficients (CRCs) and to characterize noise by coefficient of variation (CV). Spatial resolution was compared for both MRDs by sampling a quadrant of the FOV with a point source. Further IQ, CV, liver SUVmean and SUVmax were compared for a cohort of 5 patients scanned with [18F]FDG (3 MBq/kg, 1 h p.i.) from 30 to 300 s. RESULTS: CV was improved by a factor of up to 1.49 and is highest for short acquisition times, peaks at the center field of view and mitigates parabolic in axial direction with no difference to MRD85 beyond the central 80 cm. No substantial differences between the two evaluated MRDs in regards to uniformity, SUVmean or CRC for the different isotopes were observed. A degradation of the average spatial resolution of 0.9 ±â€¯0.2 mm in the central 40 cm FOV was determined with MRD322. Depending on the acquisition time MRD322 resulted in a decrease of SUVmax between 23.8% (30 s) and 9.0% (300 s). CONCLUSION: Patient and phantom studies revealed that scan time could be lowered by approximately a factor of two with MRD322 while maintaining similar noise performance. The moderate degradation in spatial resolution for MRD322 is worth to exploit the full potential of the Quadra by either shorten scan times or leverage noise performance in particular for low count scenarios such as ultra-late imaging or dynamic studies with high temporal resolution.

Automatic electrode scalar location assessment after cochlear implantation using a novel imaging software.

As of today, image-based assessment of cochlear implant electrode array location is not part of the clinical routine. Low resolution and contrast of computer tomography (CT) imaging, as well as electrode array artefacts, prevent visibility of intracochlear structures and result in low accuracy in determining location of the electrode array. Further, trauma assessment based on clinical-CT images requires a uniform image-based trauma scaling. Goal of this study was to evaluate the accuracy of a novel imaging software to detect electrode scalar location. Six cadaveric temporal bones were implanted with Advanced Bionics SlimJ and Mid-Scala electrode arrays. Clinical-CT scans were taken pre- and postoperatively. In addition, micro-CTs were taken post-operatively for validation. The electrode scalar location rating done by the software was compared to the rating of two experienced otosurgeons and the micro-CT images. A 3-step electrode scalar location grading scale (0 = electrode in scala tympani, 1 = interaction of electrode with basilar membrane/osseous spiral lamina, 2 = translocation of electrode into scala vestibuli) was introduced for the assessment. The software showed a high sensitivity of 100% and a specificity of 98.7% for rating the electrode location. The correlation between rating methods was strong (kappa > 0.890). The software gives a fast and reliable method of evaluating electrode scalar location for cone beam CT scans. The introduced electrode location grading scale was adapted for assessing clinical CT images.

From RNA helicases to RNPases.

In eukaryotic cells, all aspects of cellular RNA metabolism require putative RNA helicases of the DEAD and DExH protein families (collectively known as DExD/H families). Based on data from biochemical studies of a few of these RNA helicases, they are generally considered to be involved in the unwinding of duplex RNA molecules. However, recent reports provide evidence indicating that these proteins might also be involved in the active disruption of RNA-protein interactions.

Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families.

Members of the RNA-helicase family are defined by several evolutionary conserved motifs. They are found in all organisms - from bacteria to humans - and many viruses. The minimum number of RNA helicases present within a eukaryotic cell can be predicted from the complete sequence of the Saccharomyces cerevisiae genome. Recent progress in the functional analysis of various family members has given new insights into, and confirmed the significance of these proteins for, most cellular RNA metabolic processes.

mRNA export: travelling with DEAD box proteins.

Recent studies have shown that the putative RNA helicase protein UAP56 and its yeast homologue Sub2p are not only involved in pre-mRNA splicing but also required for the export of mRNA out of the nucleus, even if the mRNA is encoded by an intron-less gene.

A yeast homolog of chromatin assembly factor 1 is involved in early ribosome assembly.

Cells have a recurrent need for the correct assembly of protein-nucleic acid complexes. We have studied a yeast homolog of the smallest subunit of chromatin assembly factor 1 (CAF1), encoded by YMR131c and termed "RRB1". Unlike other yeast homologs, Msi1p, and Hat2p, Rrb1p is essential for cell viability. Impairment of Rrb1p function results in decreased levels of free 60S ribosomal subunits and the appearance of half-mer polysomes, suggesting its involvement in ribosome biogenesis. Using tandem affinity purification (TAP ) combined with mass spectrometry, we show that Rrb1p is associated with ribosomal protein L3. A fraction of Rrb1p is also found in a protein-precursor rRNA complex containing at least ten other early-assembling ribosomal proteins. We propose that Rrb1p is required for proper assembly of preribosomal particles during early ribosome biogenesis, presumably by targeting L3 onto the 35S precursor rRNA. This action may resemble the mechanism by which CAF1 assembles histones H3/H4 onto newly replicated DNA.

Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases.

The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA. This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B. These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation. To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae. We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function. Only in rare cases could we make a conservative substitution without affecting cell growth. The mutants show a clear correlation between their growth and in vivo translation rates. One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature. These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products.

Synthetic lethality with conditional dbp6 alleles identifies rsa1p, a nucleoplasmic protein involved in the assembly of 60S ribosomal subunits.

Dbp6p is an essential putative ATP-dependent RNA helicase that is required for 60S-ribosomal-subunit assembly in the yeast Saccharomyces cerevisiae (D. Kressler, J. de la Cruz, M. Rojo, and P. Linder, Mol. Cell. Biol. 18:1855-1865, 1998). To identify factors that are functionally interacting with Dbp6p, we have performed a synthetic lethal screen with conditional dbp6 mutants. Here, we describe the cloning and the phenotypic analysis of the previously uncharacterized open reading frame YPL193W, which we renamed RSA1 (ribosome assembly 1). Rsa1p is not essential for cell viability; however, rsa1 null mutant strains display a slow-growth phenotype, which is exacerbated at elevated temperatures. The rsa1 null allele synthetically enhances the mild growth defect of weak dbp6 alleles and confers synthetic lethality when combined with stronger dbp6 alleles. Polysome profile analysis shows that the absence of Rsa1p results in the accumulation of half-mer polysomes. However, the pool of free 60S ribosomal subunits is only moderately decreased; this is reminiscent of polysome profiles from mutants defective in 60S-to-40S subunit joining. Pulse-chase labeling of pre-rRNA in the rsa1 null mutant strain indicates that formation of the mature 25S rRNA is decreased at the nonpermissive temperature. Interestingly, free 60S ribosomal subunits of a rsa1 null mutant strain that was grown for two generations at 37 degrees C are practically devoid of the 60S-ribosomal-subunit protein Qsr1p/Rpl10p, which is required for joining of 60S and 40S subunits (D. P. Eisinger, F. A. Dick, and B. L. Trumpower, Mol. Cell. Biol. 17:5136-5145, 1997). Moreover, the combination of the Deltarsa1 and qsr1-1 mutations leads to a strong synthetic growth inhibition. Finally, a hemagglutinin epitope-tagged Rsa1p localizes predominantly to the nucleoplasm. Together, these results point towards a function for Rsa1p in a late nucleoplasmic step of 60S-ribosomal-subunit assembly.

Dbp6p is an essential putative ATP-dependent RNA helicase required for 60S-ribosomal-subunit assembly in Saccharomyces cerevisiae.

A previously uncharacterized Saccharomyces cerevisiae open reading frame, YNR038W, was analyzed in the context of the European Functional Analysis Network. YNR038W encodes a putative ATP-dependent RNA helicase of the DEAD-box protein family and was therefore named DBP6 (DEAD-box protein 6). Dbp6p is essential for cell viability. In vivo depletion of Dbp6p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase labeling of pre-rRNA and steady-state analysis of pre-rRNA and mature rRNA by Northern hybridization and primer extension show that Dbp6p depletion leads to decreased production of the 27S and 7S precursors, resulting in a depletion of the mature 25S and 5.8S rRNAs. Furthermore, hemagglutinin epitope-tagged Dbp6p is detected exclusively within the nucleolus. We propose that Dbp6p is required for the proper assembly of preribosomal particles during the biogenesis of 60S ribosomal subunits, probably by acting as an rRNA helicase.

Fal1p is an essential DEAD-box protein involved in 40S-ribosomal-subunit biogenesis in Saccharomyces cerevisiae.

A previously uncharacterized Saccharomyces cerevisiae gene, FAL1, was found by sequence comparison as a homolog of the eukaryotic translation initiation factor 4A (eIF4A). Fal1p has 55% identity and 73% similarity on the amino acid level to yeast eIF4A, the prototype of ATP-dependent RNA helicases of the DEAD-box protein family. Although clearly grouped in the eIF4A subfamily, the essential Fal1p displays a different subcellular function and localization. An HA epitope-tagged Fal1p is localized predominantly in the nucleolus. Polysome analyses in a temperature-sensitive fal1-1 mutant and a Fal1p-depleted strain reveal a decrease in the number of 40S ribosomal subunits. Furthermore, these strains are hypersensitive to the aminoglycoside antibiotics paromomycin and neomycin. Pulse-chase labeling of pre-rRNA and steady-state-level analysis of pre-rRNAs and mature rRNAs by Northern hybridization and primer extension in the Fal1p-depleted strain show that Fal1p is required for pre-rRNA processing at sites A0, A1, and A2. Consequently, depletion of Fal1p leads to decreased 18S rRNA levels and to an overall deficit in 40S ribosomal subunits. Together, these results implicate Fal1p in the 18S rRNA maturation pathway rather than in translation initiation.

Characterization and mutational analysis of yeast Dbp8p, a putative RNA helicase involved in ribosome biogenesis.

RNA helicases of the DEAD box family are involved in almost all cellular processes involving RNA molecules. Here we describe functional characterization of the yeast RNA helicase Dbp8p (YHR169w). Our results show that Dbp8p is an essential nucleolar protein required for biogenesis of the small ribosomal subunit. In vivo depletion of Dbp8p resulted in a ribosomal subunit imbalance due to a deficit in 40S ribosomal subunits. Subsequent analyses of pre-rRNA processing by pulse-chase labeling, northern hybridization and primer extension revealed that the early steps of cleavage of the 35S precursor at sites A(1) and A(2) are inhibited and delayed at site A(0). Synthesis of 18S rRNA, the RNA moiety of the 40S subunit, is thereby blocked in the absence of Dbp8p. The involvement of Dbp8p as a bona fide RNA helicase in ribosome biogenesis is strongly supported by the loss of Dbp8p in vivo function obtained by site-directed mutagenesis of some conserved motifs carrying the enzymatic properties of the protein family.

Dbp10p, a putative RNA helicase from Saccharomyces cerevisiae, is required for ribosome biogenesis.

Ribosome biogenesis requires, in addition to rRNA molecules and ribosomal proteins, a multitude of trans-acting factors. Recently it has become clear that in the yeast Saccharomyces cerevisiae many RNA helicases of the DEAD-box and related families are involved in ribosome biogenesis. Here we show that the previously uncharacterised open reading frame YDL031w (renamed DBP10 for DEAD-box protein 10) encodes an essential putative RNA helicase that is required for accurate ribosome biogenesis. Genetic depletion of Dbp10p results in a deficit in 60S ribosomal subunits and an accumulation of half-mer polysomes. Furthermore, pulse-chase analyses of pre-rRNA processing reveal a strong delay in the maturation of 27SB pre-rRNA intermediates into 25S rRNA and 7S pre-rRNA. Northern blot analyses indicate that this delay leads to higher steady-state levels of 27SB species and reduced steady-state levels of 7S pre-rRNA and 25S/5.8S mature rRNAs, thus explaining the final deficit in 60S subunit and the formation of half-mer polysomes. Consistent with a direct role in ribosome biogenesis, Dbp10p was found to be located predominantly in the nucleolus.

Expression of translation initiation factor 4A from yeast and mouse in Saccharomyces cerevisiae.

The eukaryotic translation initiation factor 4A (eIF-4A) plays an important role in regulating initiation. To analyze its function in yeast, we carried out a mutational analysis of the TIF1 and TIF2 genes, which encode eIF-4A. Expression of these two yeast genes has also been investigated at the transcriptional level and it has been found that both are expressed in wild-type yeast cells. Analysis of the expression of eIF-4A-beta-galactosidase fusion proteins reveals that the TIF2 gene is more highly expressed than the TIF1 gene. Interestingly, the yeast eIF-4A protein shows a high degree of amino acid sequence similarity to the mouse homologue. However, we find that the mammalian factor does not support protein synthesis in yeast either in vivo or in vitro.

Plasmid pSC101 replication mutants generated by insertion of the transposon Tn1000.

A derivative of pSC101, pLC709, was constructed by ligation of the HincII-A fragment of pSC101 to the mini-colEI plasmid pVH51 and to a DNA fragment encoding resistance to the antibiotics streptomycin and spectinomycin. Insertions of the transposon Tn1000 (gamma-delta) into the pSC101 replication region of pLC709 were isolated following cotransfer of the plasmid with the sex factor F. The sites of insertion of the transposon were determined by restriction enzyme analysis and the replication and incompatibility properties of the insertion plasmids and DNA fragments cloned from them were analysed. The insertion mutations defined a locus, inc, of approximately 200 base-pairs that is responsible for pSC101-specific incompatibility. Two mutations adjacent to this region inactivate pSC101 replication but can be complemented in trans by a wild-type pSC101 plasmid, and thus define a trans-acting replication function, rep. The inc locus is within a larger region of some 450 base-pairs that is essential for pSC101 replication and that includes the origin of replication. This 450 base-pair segment can replicate in the presence of a helper plasmid that supplies the rep function in trans.

An essential replication gene, repA, of plasmid pSC101 is autoregulated.

Measurements of the rate of replication of a mutant pSC101 plasmid, cloned into a ColE1 vector, showed that insertions of the transposon Tn1000 into the repA gene of pSC101 abolished replication activity, but could be complemented in trans, albeit at a low level. The promoter of the repA gene was mapped by the construction of repA-lacZ gene fusions, and one of the fusions was used to demonstrate that repA protein, provided in trans, could repress expression of beta-galactosidase activity. This repression was primarily due to reduction of transcription of the repA-lacZ fusion. The sequence analysis of mutants of the repA-lacZ fusion gene which were no longer sensitive to the presence of repA protein showed that the site of action of repA was a 22 base-pair sequence, present as an inverted repeat, overlapping the repA promoter. The repA gene is thus autoregulated.

NMR in vitro effects on proliferation, apoptosis, and viability of human chondrocytes and osteoblasts.

This study presents findings on the proliferation rate, cellular apoptosis, and viability of human chondrocyte and osteoblast cultures before and after treatment with NMR pulse sequences. A commercially available nuclear magnetic resonance machine (MBST(R)-Nuclear Magnetic Resonance Therapy) was used for treatment. The study was carried out for 19 days, including 9 days of NMR exposure in a controlled, double-blind, randomized manner, using commercially available human cell lines. The study revealed that NMR treatment did not induce apoptosis or inhibit cell viability, but revealed a tendency of an elevated cell proliferation rate as observed by cell count.

The ADE2 gene from Saccharomyces cerevisiae: sequence and new vectors.

We have determined the sequence of a DNA fragment encoding the ADE2 gene from Saccharomyces cerevisiae. A DNA fragment of 2241 bp capable of complementing ade2 mutations was modified so it is available as a single BglII fragment for use in yeast vectors or for gene disruptions. The minimal fragment codes for a putative protein which is highly similar to the protein encoded by the ADE6 gene from Schizosaccharomyces pombe and to the proteins encoded by the purEK operon of Escherichia coli.

Formation constants for the complexes of orthophosphate with magnesium and hydrogen ions.

The system of orthophosphate, magnesium and hydrogen ions in aqueous solution at 25 degrees and I = 0.2M chloride has been characterized by means of glass-electrode potentiometry. Protonation constants for orthophosphate species and formation constants for the complexes MgH(2)PO(+)(4), MgHPO(4), MgPO(4)(-) and MgOH(+) are reported.

Correction of formation constants for ionic strength, from only one or two data points: An examination of the use of the extended Debye-Hückel equation.

The results of an extensive examination show the extended Debye-Hückel equation to be efficacious for the use of as few as one or two formation constant values, for a given metal-complex species, for calculation of the value at a lower ionic strength. Approaches to choosing values for the constants in the extended Debye-Hückel equation are described. A statistical analysis shows the chief source of error in the resulting predicted formation constants to be uncertainties in the literature formation-constant values used as data.