Feasibility and acceptability of a whole-school social-marketing intervention to prevent unintended teenage pregnancies and promote sexual health: evidence for progression from a pilot to a phase III randomised trial in English secondary schools.

BACKGROUND: Reducing unintended teenage pregnancy and promoting adolescent sexual health remains a priority in England. Both whole-school and social-marketing interventions are promising approaches to addressing these aims. However, such interventions have not been rigorously trialled in the UK and it is unclear if they are appropriate for delivery in English secondary schools. We developed and pilot trialled Positive Choices, a new whole-school social marketing intervention to address unintended teenage pregnancy and promote sexual health. Our aim was to assess the feasibility and acceptability of the intervention and trial methods in English secondary schools against pre-defined progression criteria (relating to randomisation, survey follow-up, intervention fidelity and acceptability and linkage to birth/abortion records) prior to carrying out a phase III trial of effectiveness and cost-effectiveness. METHODS: Pilot RCT with integral process evaluation involving four intervention and two control schools in south-east England. The intervention comprised a student needs survey; a student/staff-led school health promotion council; a classroom curriculum for year-9 students (aged 13-14); whole-school student-led social-marketing activities; parent information; and a review of local and school-based sexual health services. Baseline surveys were conducted with year 8 (aged 12-13) in June 2018. Follow-up surveys were completed 12 months later. Process evaluation data included audio recording of staff training, surveys of trained staff, staff log books and researcher observations of intervention activities. Survey data from female students were linked to records of births and abortions to assess the feasibility of these constituting a phase III primary outcome. RESULTS: All six schools were successfully randomised and retained in the trial. Response rates to the survey were above 80% in both arms at both baseline and follow-up. With the exception of the parent materials, the fidelity target for implementation of essential elements in three out of four schools was achieved. Student surveys indicated 80% acceptability among those who reported awareness of the programme and interviews with staff suggested strong acceptability. Linkage to birth/abortion records was feasible although none occurred among participants. CONCLUSIONS: The criteria for progression to a phase III trial were met. Our data suggest that a whole-school social-marketing approach may be appropriate for topics that are clearly prioritised by schools. A phase III trial of this intervention is now warranted to establish effectiveness and cost-effectiveness. Births and terminations are not an appropriate primary outcome measure for such a trial. TRIAL REGISTRATION: ISRCTN65324176.

ATP-dependent proteases controlling mitochondrial function in the yeast Saccharomyces cerevisiae.

Regulated protein degradation by ATP-dependent proteases plays a fundamental role in the biogenesis of mitochondria. Membrane-bound and soluble ATP-dependent proteases have been identified in various subcompartments of this organelle. Subunits composing these proteases are evolutionarily conserved from yeast to humans and, in support of an endosymbiotic origin of mitochondria, evolved from prokaryotic ancestors: the PIM1/Lon protease is active in the matrix of mitochondria, while the i-AAA protease and the m-AAA protease mediate the turnover of inner membrane proteins. Most of the knowledge concerning the biogenesis and the physiological role of ATP-dependent proteases comes from studies in the yeast Saccharomyces cerevisiae. Proteases were found to be required for mitochondrial stasis, for the maintenance of the morphology of the organelle and for mitochondrial genome integrity. ATP-dependent proteolysis is crucial for the expression of mitochondrially encoded subunits of respiratory chain complexes and for the assembly of these complexes. Hence, mitochondrial ATP-dependent proteases exert multiple roles which are essential for the maintenance of cellular respiratory competence.

Mcx1p, a ClpX homologue in mitochondria of Saccharomyces cerevisiae.

Members of the Hsp100/Clp-family of molecular chaperones form regulatory subunits of ATP-dependent Clp proteases and fulfill crucial roles for cellular thermotolerance. We have identified a Clp-like protein in Saccharomyces cerevisiae, Mcx1p, which shares approximately 30% sequence identity with ClpX-proteins in bacteria, plants and nematodes. Mcx1p localizes to the matrix space of mitochondria and is peripherally associated with the inner membrane. A homologue of E. coli ClpP protease was not identified when screening the yeast genome. We therefore propose that Mcx1p represents a novel molecular chaperone of mitochondria with non-proteolytic function.

The ATP-dependent PIM1 protease is required for the expression of intron-containing genes in mitochondria.

The ATP-dependent PIM1 protease, a Lon-like protease localized in the mitochondrial matrix, is required for mitochondrial genome integrity in yeast. Cells lacking PIM1 accumulate lesions in the mitochondrial DNA (mtDNA) and therefore lose respiratory competence. The identification of a multicopy suppressor, which stabilizes mtDNA in the absence of PIM1, enabled us to characterize novel functions of PIM1 protease during mitochondrial biogenesis. The synthesis of mitochondrially encoded cytochrome c oxidase subunit I (CoxI) and cytochrome b (Cob) is impaired in pim1 mutants containing mtDNA. PIM1-mediated proteolysis is required for the translation of mature COXI mRNA. Moreover, deficiencies in the splicing of COXI and COB transcripts, which appear to be restricted to introns encoding mRNA maturases, were observed in cells lacking the PIM1 gene. Transcripts of COXI and COB genes harboring multiple introns are degraded in the absence of PIM1. These results establish multiple, essential functions of the ATP-dependent PIM1 protease during mitochondrial gene expression.

An 18.3 kb DNA fragment from yeast chromosome VII carries four unknown open reading frames, the gene for an Asn synthase, remnants of Ty and three tRNA genes.

An 18.3 kb DNA segment from yeast Saccharomyces cerevisiae VII encompasses the previously characterized MEP1, NUP57 and PPT1 genes as well as seven new open reading frames (ORFs) of at least 100 residues. G6358 is an ubiquitous glutamine-dependent asparagine synthase. G6362 is membrane protein highly homologous to a protein of unknown function in the yeast Schizosaccharomyces pombe. Three ORFs (G6324, G6335 and G6365) have no significant homology with previously reported proteins of characteristic motifs. G6321 and G6359, enclosed in longer ORFs, are not likely to be coding. The segment also contains tRNA genes for Asn, Arg and Ile as well as sigma element and two solo deltas. ORFs and genetic elements are named according to a preliminary working nomenclature.

Autocatalytic processing of the ATP-dependent PIM1 protease: crucial function of a pro-region for sorting to mitochondria.

The biogenesis of the ATP-dependent PIM1 protease of mitochondria was studied by mutational analysis. The ATPase and proteolytic activities of PIM1 were shown to be essential for mitochondrial function. A proteolytically inactive mutant form of PIM1 protease accumulated as a pro-form in mitochondria, revealing a two-step processing of PIM1: the matrix targeting signal is removed by the mitochondrial processing peptidase and then a pro-region of 61 amino acids is cleaved off in an autocatalytic reaction. This latter process depended on the ATP-dependent assembly of PIM1 protease subunits and can occur by an intermolecular and, most probably, also an intramolecular pathway. The respiratory competence of cells harboring mutant PIM1 protease lacking the pro-region was strongly impaired. Subcellular fractionation revealed a cytosolic localization of mutant PIM1 protease. This demonstrates the requirement for the propeptide for efficient sorting of PIM1 protease to mitochondria.

Substitution of PIM1 protease in mitochondria by Escherichia coli Lon protease.

PIM1 protease in mitochondria belongs to a conserved family of ATP-dependent proteases, which includes the Escherichia coli Lon protease. Yeast cells lacking PIM1 are largely defective in degrading misfolded proteins in the mitochondrial matrix, are respiratory deficient, and lose integrity of mitochondrial DNA. In order to analyze whether E. coli Lon protease is functionally equivalent to mitochondrial PIM1 protease, yeast cells lacking the PIM1 gene were transformed with a construct consisting of a mitochondrial targeting sequence fused onto the Lon protease. In these cells, the fusion protein was expressed and imported into mitochondria, and the targeting sequence was removed. In the absence of PIM1 protease, the E. coli Lon protease mediated the degradation of misfolded proteins in the matrix space in cooperation with the mitochondrial hsp70 system. These cells maintained the integrity of the mitochondrial genome and the respiratory function at 30 degrees C but not at 37 degrees C. Stabilization of mitochondrial DNA in Deltapim1 cells depended on protein degradation by the E. coli Lon protease, as a proteolytically inactive Lon variant was not capable of substituting for a loss of PIM1 protease. These results demonstrate functional conservation of Lon-like proteases from prokaryotes to eukaryotes and shed new light on the role of Lon-like proteases in mitochondrial biogenesis.

An 8.2 kb DNA segment from chromosome XIV carries the RPD3 and PAS8 genes as well as the Saccharomyces cerevisiae homologue of the thiamine-repressed nmt1 gene and a chromosome III-duplicated gene for a putative aryl-alcohol dehydrogenase.

A 8.2 kb DNA segment from the left arm of Saccharomyces cerevisiae chromosome XIV (GenBank/EMBL accession number: X83226) encompasses four open reading frames (ORFs) longer than 100 residues. The ORF N0295 is highly similar to the Aspergillus parasiticus and Schizosaccharomyces pombe nmt1 gene products, which are involved in thiamine biosynthesis and are strongly repressed by thiamine. N0300 is 76% identical to YCR107w, a hypothetical protein of yeast chromosome III, and 55% identical to a ligninolytic aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. In addition, this fragment encodes Rpd3, a pleiotropic transcription factor (Vidal and Gaber, 1991), and part of Pas8, a protein essential for the biogenesis of peroxisomes (Voorn-Brouwer et al., 1993).

The sequence of a 13.5 kb DNA segment from the left arm of yeast chromosome XIV reveals MER1; RAP1; a new putative member of the DNA replication complex and a new putative serine/threonine phosphatase gene.

The nucleotide sequence of two adjacent ClaI fragments from the left arm of Saccharomyces cerevisiae chromosome XIV has been determined. Analysis of the 13,520 bp DNA segment reveals nine open reading frames (ORFs) longer than 300 bp. N1302 contains the consensus sequence for a phosphate-binding loop common to ATP- and GTP-binding proteins and a strictly conserved 'SRC' sequence of unknown function present in all accessory proteins of replicative polymerases. N1306 shares homologies with serine/threonine phosphatases. N1310 encodes RAP1 (TUF or SBF-E), a transcription regulator. N1330 is the MER1 gene required for chromosome pairing and genetic recombination. Two ORFs show no homology with proteins in the databases and no particular features. N1311 is not likely to be expressed as it is located on the complementary strand of N1310.

Analysis of a 17.4 kb DNA segment of yeast chromosome II encompassing the ribosomal protein L19 as well as proteins with homologies to components of the hnRNP and snRNP complexes and to the human proliferation-associated p120 antigen.

We report the nucleotide sequence of a 17.4 kb DNA segment from the left arm of Saccharomyces cerevisiae chromosome II. This sequence contains 12 open reading frames (ORFs) longer than 300 bp and a putative autonomously replicating sequence (ARS). The ORF YBL0418 contains the KH motif present in several nucleic acid-binding proteins and shares homologies with the mouse X protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complexes involved in pre-mRNA processing. YBL0424 is the yeast member of the ribosomal protein L19 (YL14) family. YBL0425 is related to the D1 core polypeptide of the small nuclear ribonucleoprotein (snRNP) particles involved in the splicing of introns. YBL0437 is a putative homologue of the human protein p120, one of the major antigens associated with malignant tumours. Mcm2, a protein important for ARS activity, as well as Aac2, one of the three isoforms of the mitochondrial ATP/ADP carrier, were previously described (Yan et al., 1991; Lawson and Douglas, 1988). Four ORFs show no homology or particular features that could help to assess their functions. The last ORFs are not likely to be expressed for they are localized on the complementary strand of longer ORFs.

Molecular chaperones cooperate with PIM1 protease in the degradation of misfolded proteins in mitochondria.

ATP dependent proteolytic degradation of misfolded proteins in the mitochondrial matrix is mediated by the PIM1 protease and depends on the molecular chaperone proteins mt-hsp70 and Mdj1p. Chaperone function is essential to maintain misfolded proteins in a soluble state, a prerequisite for their degradation by PIM1 protease. In the absence of functional mt-hsp70 or Mdj1p misfolded proteins either remain associated with mt-hsp70 or form aggregates and thereby are no longer substrates for PIM1 protease. Mdj1p is shown to regulate the ATP dependent association of an unfolded polypeptide chain with mt-hsp70 affecting binding to as well as release from mt-hsp70. These findings establish a central role of molecular chaperone proteins in the degradation of misfolded proteins by PIM1 protease and thereby demonstrate a functional interrelation between components of the folding machinery and the proteolytic system within mitochondria.

The sequence of an 8.8 kb segment on the left arm of chromosome II from Saccharomyces cerevisiae reveals four new open reading frames including homologs of animal DNA polymerase alpha-primases and bacterial GTP cyclohydrolase II.

The DNA sequence of two contiguous 7648 bp and 1194 bp BamHI fragments from the cosmid alpha 1201 located about 60 kb from the centromere on the left arm of chromosome II from Saccharomyces cerevisiae has been determined. Sequence analysis reveals four new open reading frames longer than 300 bp: YBL0415 (309 bp), YBL0416 (4539 bp), YBL0417 (1035 bp) and YBL0414 (2115 bp), which extends into the neighbouring 5.2 kb BamHI fragment. The YBL0414 shows homologies to the mouse 68 kDa and Drosophila melanogaster 76 kDa subunits of the DNA polymerase alpha-primase complex. The YBL0417 is homologous to bacterial GTP cyclohydrolase II (EC 3.5.4.25).

Analysis of an 11.7 kb DNA fragment of chromosome XI reveals a new tRNA gene and four new open reading frames including a leucine zipper protein and a homologue to the yeast mitochondrial regulator ABF2.

We report the nucleotide sequence of an 11.7 kb fragment from the left arm of Saccharomyces cerevisiae chromosome XI. Analysis reveals a new tRNA for valine and four unknown open reading frames among which YKL245 shows homology with a yeast mitochondrial regulatory protein and YKL244, YKL246 and YKL247 are unknown.

PIM1 encodes a mitochondrial ATP-dependent protease that is required for mitochondrial function in the yeast Saccharomyces cerevisiae.

The PIM1 nuclear gene in the yeast Saccharomyces cerevisiae encodes a mitochondrial ATP-dependent protease that exhibits over 30% identity with ATP-dependent protease La from Escherichia coli, Lon from Bacillus brevis, and one from Myxococcus xanthus. In addition, Pim1 is 1133 amino acids long and has a putative mitochondrial import signal in the N-terminal region. Enzymatic comparisons of normal PIM1+ and deficient pim1-delta strains revealed that the ATP-dependent protease is located within the mitochondrial matrix. The pim1-delta strains are unable to utilize nonfermentable substrates as the sole carbon source and are unable to maintain functional mitochondrial DNA, indicating that the Pim1 protease is required for mitochondrial function. PIM1 mRNA is constitutively expressed but is increased after thermal stress, suggesting that Pim1 may play a role in the heat shock response.

PDR5, a novel yeast multidrug resistance conferring transporter controlled by the transcription regulator PDR1.

The complete sequence of the pleiotropic drug resistance gene PDR5 from Saccharomyces cerevisiae is reported and analyzed. PDR5 encodes a 160-kDa protein with a predicted duplicated six membrane-span domain and a repeated putative ATP-binding domain. PDR5 shares this structural feature with the mammalian multidrug resistance pumps as well as the functional capacity of conferring resistance to various inhibitors upon amplification (Leppert, G., McDevitt, R., Falco, S. C., Van Dyk, T. K., Ficke, M. B., and Golin, J. (1990) Genetics 125, 13-20). The yeast PDR5 is thus a new member of the ABC (ATP-binding cassette) protein superfamily. Mutations in another yeast pleiotropic drug resistance gene, PDR1, encoding a putative transcription regulator (Balzi, E., Chen, W., Ulaszewski, S., Capieaux, E., and Goffeau, A. (1987) J. Biol. Chem. 262, 16871-16879), increase markedly the mRNA levels of the PDR5 and STE6 genes. The multidrug resistance mutations pdr1-3 and pdr1-6 also lead to considerable overexpression of the PDR5 plasma membrane protein.

Regulation of the expression of the H(+)-ATPase genes PMA1 and PMA2 during growth and effects of octanoic acid in Saccharomyces cerevisiae.

A peak of plasma membrane H(+)-ATPase activity during exponential growth is correlated with the expression of the PMA1 gene as monitored by measurements of the beta-galactosidase activity from a PMA1-lacZ fusion. This peak of activity is also correlated to the content of the H(+)-ATPase protein in yeast plasma membrane as shown by quantitative immunodetection. The PMA2-lacZ fusion assay indicates that the expression of the PMA2 gene is activated somewhat later during exponential phase but under all circumstances its activity remains at least 500-fold lower than that of the PMA1-lacZ fusion. A slight but significant stimulation of ATPase activity by low concentrations of octanoic acid coincides with a decrease in the PMA1 gene expression. It is concluded that octanoic acid stimulates de PMA1 ATPase activity by posttranslational mechanisms.

The sequence of a 17.5 kb DNA fragment on the left arm of yeast chromosome XI identifies the protein kinase gene ELM1, the DNA primase gene PRI2, a new gene encoding a putative histone and seven new open reading frames.

A 17.5 kb DNA fragment of chromosome XI, located between the genetic loci mif2 and mak11 was sequenced and analysed. Ten open reading frames were identified. Two of them are the previously sequenced genes ELM1 and PRI2, two (YKL253 and YKL256) show homologies to proteins from other organisms and one (YKL262) to yeast and mouse histone.