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ABSTRACTOver the past two decades, there has been a growing interest in the role of universities in developing generic skills, in addition to disciplinary ones, to help students adapt to a changing workplace. The need to develop these skills is particularly critical for healthcare students who must face challenges in health systems. This scoping study, following Arksey and O'Malley's ([2005]. Scoping studies: Towards a methodological framework. International Journal of Social Research Methodology, 8(1), 19–32. https://doi.org/10.1080/1364557032000119616) method provides an overview of the scientific literature on generic skills in health studies. The definitions, categorizations, and measurement tools from 43 articles were mapped and summarized and gaps were identified. Definitions and terms were varied but overall, generic skills are considered cross-disciplinary and cross-professional. Nevertheless, the term skill itself was not defined. The lack of consensus on categorization and assessment led us to propose a typology to group all the skills mentioned into four categories: personal, ideas and object-related, interpersonal and community-related skills. One conclusion is that generic skills are valued foremost in relation to labor market demands with an emphasis on skills such as knowledge, communication, and planning over ethical and citizenship skills that are essential for future healthcare professionals. It is imperative to consider generic skills that are necessary to face the complex challenges of today’s world.

This study reports that inhibition of Leishmania Topo-I with the minor groove-binding ligands (MGBLs) Hoechst 33342 (Ho342) blocks parasite growth in culture by mechanisms involving DNA breakage. While Ho342 inhibited the replication of several species of Leishmania in a dose- and time-dependent manner, Ho258 was not effective. Cytofluorometric analysis suggested that superior effectiveness of Ho342 over Ho258 was attributed to Leishmania parasites being more permeable toward Ho342. This observation was supported by the ability of both Ho342 and Ho258 to block the relaxation of supercoiled pBR322 DNA by Leishmania Topo-I. The Ho342 specificity toward L. donovani Topo-I was reinforced by the observation that increased Topo-I gene expression and Topo-I activity in Leishmania was paralleled by augmented resistance for this compound. Furthermore, the capacity of NaCl treatment to reverse MGBL-mediated DNA break suggests that Ho342 targetted Topo-I. Moreover, we observed that Ho342-inducible arrest of Leishmania growth was accompanied by G1 arrest and induction of cell death that closely resembles apoptosis. Taken together, our results suggest that MGBL compounds show promise as Topo-I inhibitors against Leishmania infection.

Three, genetically identical, Nigerian Dwarf bucks produced by somatic cell nuclear transfer (NT) of fetal fibroblasts were monitored for sexual maturation and fertility. Starting at four months of age, these male clones were trained to serve an artificial vagina (AV). Average age of the NT-derived bucks at first semen collection was 20 weeks, which was not different from that of other young bucks of this breed (average age at first collection = 20 weeks). Average sperm production at 5 months of age for the NT-derived bucks was 5.0 × 108 spermatozoa, which was comparable to that of dwarf bucks of similar age (3.4 × 108 spermatozoa). At seven months of age, semen collected from two NT-derived bucks was used to artificially inseminate six females (three does per buck). Five does were confirmed pregnant by ultrasound at day 42. Nine healthy kids, four males and five females, were born in March and April 2000. Viable spermatozoa were collected from one of the F1 males at 28 weeks of age. These results demonstrated that NT-derived bucks and one of their male offspring developed sexually within the normal timeframe for their breed and that the clones were fertile.

It has been suggested that the major function of DNA topoisomerase I in Escherichia coli is to suppress the formation of R-loops, which could inhibit growth. Although the currently available data suggest that the inhibitory effect of R-loops is exerted at the level of gene expression, this has never been demonstrated. In the present report, we show that rRNA synthesis is significantly impaired at the level of transcription elongation in a bacterial strain lacking DNA topoisomerase I. We found that this inhibition is due to transcriptional blocks. RNase H overproduction is also shown to considerably reduce the extent of such transcriptional blocks during rRNA synthesis. Moreover, one of these transcriptional blockage sites is located within a region where extensive R-loop formation was previously shown to occur on a plasmid DNA in the absence of DNA topoisomerase I. Together, these results allow us to propose that an important function of DNA topoisomerase I is to inhibit the formation of R-loops, which may otherwise translate into roadblocks for RNA polymerases. Our results also highlight the potential regulatory role of DNA supercoiling at the level of transcription elongation.

It has recently been shown that RNase H overproduction can partially compensate for the growth defect due to the absence of DNA topoisomerase I in Escherichia coli (Drolet, M., Phoenix, P., Menzel, R., Massé, E., Liu, L. F., and Crouch, R. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3526-3530). This result has suggested a model in which inhibitory R-loops occur during transcription in topA mutants. Results presented in this report further support this notion and demonstrate that transcription-induced supercoiling is involved in R-loop formation. First, we show that stable R-loop formation during in vitro transcription with E. coli RNA polymerase only occurs in the presence of DNA gyrase. Second, extensive R-loop formation in vivo, revealed by the production of RNase H-sensitive hypernegatively supercoiled plasmid DNAs, is observed under conditions where topA mutants fail to grow. Furthermore, we have demonstrated that the coupling of transcription and translation in bacteria is an efficient way of preventing R-loop formation.

It has been suggested that the essential function of DNA topoisomerase I in Escherichia coli is to prevent chromosomal DNA from reaching an unacceptably high level of global negative supercoiling. However, other in vivo studies have shown that DNA topoisomerase I is very effective in removing local negative supercoiling generated during transcription elongation. To determine whether topoisomerase I is essential for controlling global or local DNA supercoiling, we have prepared a set of topA null mutant strains in combination with different plasmid DNAs. Although we found a correlation between the severity of the growth defect with both transcription-induced and global supercoiling, near to complete growth inhibition correlated only with transcription-induced supercoiling. This result strongly suggests that the major function of DNA topoisomerase I is to relax local negative supercoiling generated during transcription elongation.

In order to clone the gene encoding a type I DNA topoisomerase from Leishmania donovani, a PCR-amplified DNA fragment obtained with degenerate oligodeoxyribonucleotides was used to screen a genomic library from this parasite. An open reading frame of 1905 bases encoding a putative protein of 635 amino acid residues was isolated. A substantial part of the protein shares a significant degree of homology with the sequence of other known members of the IB topoisomerase family, in a highly conserved region of these enzymes termed the core domain. However, homology is completely lost after this conserved central core. Moreover, no conventional active tyrosine site could be identified. In fact, the protein expressed in Escherichia coli did not show any relaxation activity in vitro and was unable to complement a mutant deficient in topoisomerase I activity. The results of Southern blot experiments strongly suggested that the cloned gene was not a pseudogene. Northern analysis revealed that the gene was transcribed in its full length and also excluded the possibility that some form of splicing is necessary to produce a mature messenger. Furthermore, our results indicate that the gene is preferentially expressed in actively growing L.donovani promastigotes and that it is also expressed in other kinetoplastid parasites.

The hemA gene of Agrobacterium radiobacter ATCC4718 was identified by hybridization with a hemA probe from Rhizobium meliloti and cloned by complementation of a hemA mutant of Escherichia coli K12. E. coli hemA transformants carrying the hemA gene of Agrobacterium showed d-aminolevulinic acid synthetase (d-ALAS) activity in vitro. The hemA gene was carried on a 4.4 kb EcoRI fragment which could be reduced to a 2.6 kb EcoRI-SstI fragment without affecting its complementing or d-ALAS activity. The sequence of the hemA gene showed an open reading frame of 1215 nucleotides, which could code for a protein of 44361 Da. This is very close to the molecular weight of the HemA protein obtained using an in vitro coupled transcription-translation system (45000 Da). Comparison of amino acid sequences of the d-ALAS of A. radiobacter and Bradyrhizobium japonicum showed strong homology between the two enzymes; less, but still significant, homology was observed when A. radiobacter and human d-ALAS were compared. Primer extension experiments enabled us to identify two promoters for the hemA gene of A. radiobacter. One of these promoters shows some similarity to the first promoter of the hemA gene of R. meliloti.

Previous biochemical studies have suggested a role for bacterial DNA topoisomerase (TOPO) I in the suppression of R-loop formation during transcription. In this report, we present several pieces of genetic evidence to support a model in which R-loop formation is dynamically regulated during transcription by activities of multiple DNA TOPOs and RNase H. In addition, our results suggest that events leading to the serious growth problems in the absence of DNA TOPO I are linked to R-loop formation. We show that the overexpression of RNase H, an enzyme that degrades the RNA moiety of an R loop, can partially compensate for the absence of DNA TOPO I. We also note that a defect in DNA gyrase can correct several phenotypes associated with a mutation in the rnhA gene, which encodes the major RNase H activity. In addition, we found that a combination of topA and rnhA mutations is lethal.

Recent in vivo and in vitro studies have suggested an important role for DNA topoisomerases in regulating R-loop formation during transcription in Escherichia coli. In the present report we present genetic and biochemical evidence strongly suggesting that R-loop formation can occur during transcription of a portion of the rrnB operon and that it is regulated by DNA topoisomerase activity. We found that a multicopy plasmid (pBR322) carrying an heavily transcribed portion of the rrnB operon cannot be transformed in topA mutants unless RNase H is overproduced. Transcription of the 567-base pair HindIII fragment from the rrnB operon allows the extraction of large amount of R-looped plasmid DNAs from a topA mutant, in a manner that depends on the intracellular level of RNase H activity. When DNA gyrase is sufficiently active, hypernegatively supercoiled plasmid DNA is produced if the same DNA fragment is transcribed in a topA mutant. The formation of such topoisomers most likely reflect the presence of extensive R-loops since it is sensitive to the intracellular level of RNase H activity. Finally, the formation of R-looped plasmid DNAs in an in vitro transcription system using phage RNA polymerases is also detected when the 567-base pair HindIII fragment is transcribed on a negatively supercoiled DNA template.

We have recently found that stable R-loop formation occurs in vivo and in vitro when a portion of the Escherichia coli rrnB operon is transcribed preferentially in its physiological orientation. Our results also suggested that the formation of such structures was more frequent in topA mutants and was sensitive to the template DNA supercoiling level. In the present report we investigated in greater detail the involvement of DNA topoisomerases in this process. By using an in vitro transcription system with phage RNA polymerases, we found that hypernegative supercoiling of plasmid DNAs in the presence of DNA gyrase is totally abolished by RNase H, suggesting that extensive R-looping occurs during transcription in the presence of DNA gyrase. When RNase A is present, significant hypernegative supercoiling occurs only when the 567-base pair rrnB HindIII fragment is transcribed in its physiological orientation. This result suggests that more stable R-loops are being produced in this orientation. Our results also suggest that DNA gyrase can participate in the process of R-loop elongation. The strong transcription-induced relaxing activity of E. coli DNA topoisomerase I is shown to efficiently counteract the effect of DNA gyrase and thus inhibit extensive R-looping. In addition, we found that an R-looped plasmid DNA is a better substrate for relaxation by E. coli DNA topoisomerase I as compared with a non-R-looped substrate.

All combinations of cooling versus warming and finger versus temporal artery were used in the present study, the aim of which was to identify an optimal biofeedback training site and to assess the specific effects of skin temperature biofeedback upon migraine. After an initial 4-week baseline phase, during which daily records of headache activity and medication were kept, 24 migraine patients were randomly assigned to one of the four experimental conditions. Training sessions for all patients were of 50-min duration and occurred once per week for 8 weeks. The headache charts were completed for another 4 weeks after treatment was terminated and again at 6-month follow-up. The results showed significant reductions in migraine activity and drug usage. However, self-regulation of skin temperature in different directions at different sites did not in fact result in significantly different magnitudes of change in migraine activity across groups. The role of nonspecific factors is discussed and a physiological model is proposed to explain how warming and cooling may produce clinical improvement in migraine.

DNA of plasmid pSAS1002TH (F' ilv+ hemD+ hemC+ cya+) was used to clone the hemD gene of Escherichia coli K-12. Due to poor transformability of the heme-deficient mutants, the restriction fragments of the F' plasmid were first cloned into a mobilizable derivative of pBR322, pSAS1211LP, which was then mobilized into a hemD recA mutant (E. coli SASX419AN). One recombinant plasmid, carrying a HindIII fragment of about 5 kilobases (kb), was shown to complement the hemD mutant and also a cya mutant of E. coli K-12, as well as a hemC mutant of Salmonella typhimurium LT2. Further subcloning of the insert enabled us to locate the hemD gene to a BamHI-PstI fragment (approximately 2.3 kb) which also carried the hemC gene. The hemD gene occupies a region close to the PstI end, since the deletion of a 0.6-kb fragment from this end resulted in loss of the ability to complement the hemD mutation. The use of the promoter-probe vector pK01 and the results of complementation showed that the hemD gene was transcribed under physiological conditions from the same promoter as the hemC gene, the direction of transcription being hemC-hemD. This allows us to define a new polycistronic operon of E. coli K-12, for which we propose the designation Uro operon. Sequencing of the hemD gene showed the presence of an open reading frame (ORF) of 738 nucleotides which could code for a protein with a molecular weight of 27,766, which should correspond to the hemD protein; the ORF starts with the last nucleotide of the hemC gene, the two genes having different reading frames. An ORF of at least 480 base pairs follows the hemD gene after a few nucleotides. The corresponding gene X, the function of which is unknown, might represent a third member of the Uro operon.

It is obvious, to anyone who has worked in motion picture sounds that is is important for the heads of a magnetic recorder or reproducer to be aligned to the correct azimuth. The precise implication of operating with an incorrect azimuth though, is something that is less well understood. This article will explain why proper azimuth adjustments are desirable, define the magnitude of the consequences of azimuth error and outline a method for determining the accuracy of head alignment on tape and magnetic film recording and reproducing equipment. [ABSTRACT FROM AUTHOR]