Summary Care Records in urgent and emergency care in England.

Summary Care Records (SCRs) have been created for more than 50% of the population of England. The number is increasing at about 100,000 records a week. Fewer than 1.5% of people have elected not to have a SCR. SCRs contain updated details of patient medication, allergies and adverse reactions, electronically extracted from the GP record. A patient and their GP can also agree to have additional information included. SCRs are being viewed by authorised healthcare staff in urgent and emergency care settings all over England. Benefits are being reported in relation to increased patient safety, improved clinical decision making, improved efficiency and improved quality of care. NHS England strongly supports the uptake and adoption of SCRs by Trusts in England.

Detection of trace sub-micron (nano) plastics in water samples using pyrolysis-gas chromatography time of flight mass spectrometry (PY-GCToF).

The identification and quantification of micro and nanoplastics (MPs and NPs respectively) requires the development of standardised analytical methods. Thermal analysis methods are generally not considered a method of choice for MPs analysis, especially in aqueous samples due to limited sample size introduction to the instrument, decreasing the detection levels. In this article, pyrolysis - Gas chromatography time of flight mass spectrometry (Py-GCToF) is used as a method of choice for detection of MPs and NPs due to its unprecedented detection capabilities, in combination with PTFE membranes as sample support, allow for smaller particle sizes (>0.1 µm) in water samples to be identified. The utilisation of these widely used membranes and the identification of several and specific (marker) ions for the three plastics in study (polypropylene (PP), polystyrene (PS) and polyvinyl chloride (PVC)), allows for the extraction of individual plastics from complex signals at trace levels. The method was validated against a number of standards, containing known quantities of MPs. Detection levels were then determined for PVC and PS and were found to be below <50 µg/L, with repeatable data showing good precision (%RSD <20%). Further verification of this new method was achieved by the analysis of a complex sample, sourced from a river. The results were positive for the presence of PS with a semi-quantifiable result of 241.8 µg/L. Therefore PY-GCToF seems to be a fit for purpose method for the identification of MPs and NPs from complex mixtures and matrices which have been deposited on PTFE membranes.

Apparent endocytosis of fluorescein isothiocyanate-conjugated dextran by Saccharomyces cerevisiae reflects uptake of low molecular weight impurities, not dextran.

Concurrent with Riezman's report (Riezman, H. 1985, Cell. 40:1001-1009) that fluid-phase endocytosis of the small molecule Lucifer yellow occurs in the yeast Saccharomyces cerevisiae, Makarow (Makarow, M. 1985. EMBO [Eur. Mol. Biol. Organ.] J. 4:1861-1866) reported the endocytotic uptake of 70-kD FITC-dextran (FD) and its subsequent compartmentation into the yeast vacuole. Samples of FD synthesized and purified here failed to label yeast vacuoles under conditions that allowed labeling using commercial FD. Chromatography revealed that the commercial FD was heavily contaminated with at least three low molecular weight fluorescent compounds. Dialysis was ineffective for removing the contaminants. After purification (Sephadex G25, ethanol extraction), commercial FD was incapable of labeling vacuoles. Extracts of cells labeled with partially purified FD contained FITC, not FD, based on Sephadex and thin layer chromatography. In either the presence or absence of unlabeled 70-kD dextran, authentic FITC (10 micrograms/ml) was an effective labeling agent for vacuoles. The rapid kinetics (0.28 pmol/min per 10(6) cells at pH 5.5) and the pH dependence of FITC uptake suggest that the mechanism of FITC uptake involves diffusion rather than endocytosis. In view of these results, labeling experiments that use unpurified commercial FD should be interpreted with caution.

Processing pathway for protease B of Saccharomyces cerevisiae.

The vacuolar protease B of Saccharomyces cerevisiae is a subtilisin-like protease encoded by the PRB1 gene. Antibodies raised against a synthetic peptide and an Escherichia coli-derived PRB1 open reading frame (ORF) protein cross-react with authentic protease B from yeast. By using these antibodies, the posttranslational biosynthetic pathway of protease B has been elucidated. Preproprotease B is a 76-kD unglycosylated precursor that enters the endoplasmic reticulum (ER), where it receives one asparagine-linked (Asn-linked) and an undetermined number of non-Asn-linked carbohydrate side chains. The large glycosylated intermediate is proteolytically processed to a 39-kD form before exiting the ER. In the Golgi complex, the 39-kD form becomes 40 kD, due to elaboration of the Asn-linked side chain. The carboxyterminal end of the 40-kD proprotease B undergoes protease A-mediated processing to a 37-kD intermediate, which in turn is quickly processed to 31-kD mature protease B. The ultimate processing step removes a peptide containing the Asn-linked chain; mature PrB has only non-Asn-linked carbohydrate.

Clathrin requirement for normal growth of yeast.

Clathrin-coated membranes and coated vesicles take part in the selective transfer of proteins between different subcellular compartments of eukaryotic cells. To allow assessment of the role of clathrin in vesicular transport, genetic analysis of the clathrin heavy chain gene (CHC1) in Saccharomyces cerevisiae was initiated. The complete heavy chain gene was cloned, and the effects of deletion of this gene were studied. The null mutation (chc1-delta) is lethal unless a suppressor of clathrin deficiency (scd1) is present. Even in the presence of the suppressor gene, mutants lacking the clathrin heavy chain grow slowly, are genetically unstable, are morphologically abnormal, and show loss of or reduction in several yeast functions. These results indicate that clathrin is required for normal growth of yeast, and, therefore, most likely, for growth of all eukaryotic cells.

Do faculty and resident physicians discuss their medical errors?

BACKGROUND: Discussions about medical errors facilitate professional learning for physicians and may provide emotional support after an error, but little is known about physicians' attitudes and practices regarding error discussions with colleagues. METHODS: Survey of faculty and resident physicians in generalist specialties in Midwest, Mid-Atlantic and Northeast regions of the US to investigate attitudes and practices regarding error discussions, likelihood of discussing hypothetical errors, experience role-modelling error discussions and demographic variables. RESULTS: Responses were received from 338 participants (response rate = 74%). In all, 73% of respondents indicated they usually discuss their mistakes with colleagues, 70% believed discussing mistakes strengthens professional relationships and 89% knew at least one colleague who would be a supportive listener. Motivations for error discussions included wanting to learn whether a colleague would have made the same decision (91%), wanting colleagues to learn from the mistake (80%) and wanting to receive support (79%). Given hypothetical scenarios, most respondents indicated they would likely discuss an error resulting in no harm (77%), minor harm (87%) or major harm (94%). Fifty-seven percent of physicians had tried to serve as a role model by discussing an error and role-modelling was more likely among those who had previously observed an error discussion (OR 4.17, CI 2.34 to 7.42). CONCLUSIONS: Most generalist physicians in teaching hospitals report that they usually discuss their errors with colleagues, and more than half have tried to role-model discussions. However, a significant number of these physicians report that they do not usually discuss their errors and some do not know colleagues who would be supportive listeners.

Protease B of the lysosomelike vacuole of the yeast Saccharomyces cerevisiae is homologous to the subtilisin family of serine proteases.

The PRB1 gene of Saccharomyces cerevisiae encodes the vacuolar endoprotease protease B. We have determined the DNA sequence of the PRB1 gene and the amino acid sequence of the amino terminus of mature protease B. The deduced amino acid sequence of this serine protease shares extensive homology with those of subtilisin, proteinase K, and related proteases. The open reading frame of PRB1 consists of 635 codons and, therefore, encodes a very large protein (molecular weight, greater than 69,000) relative to the observed size of mature protease B (molecular weight, 33,000). Examination of the gene sequence, the determined amino-terminal sequence, and empirical molecular weight determinations suggests that the preproenzyme must be processed at both amino and carboxy termini and that asparagine-linked glycosylation occurs at an unusual tripeptide acceptor sequence.

Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae PEP3 gene was cloned from a wild-type genomic library by complementation of the carboxypeptidase Y deficiency in a pep3-12 strain. Subclone complementation results localized the PEP3 gene to a 3.8-kb DNA fragment. The DNA sequence of the fragment was determined; a 2,754-bp open reading frame predicts that the PEP3 gene product is a hydrophilic, 107-kDa protein that has no significant similarity to any known protein. The PEP3 predicted protein has a zinc finger (CX2CX13CX2C) near its C terminus that has spacing and slight sequence similarity to the adenovirus E1a zinc finger. A radiolabeled PEP3 DNA probe hybridized to an RNA transcript of 3.1 kb in extracts of log-phase and diauxic lag-phase cells. Cells bearing pep3 deletion/disruption alleles were viable, had decreased levels of protease A, protease B, and carboxypeptidase Y antigens, had decreased repressible alkaline phosphatase activity, and contained very few normal vacuolelike organelles by fluorescence microscopy and electron microscopy but had an abundance of extremely small vesicles that stained with carboxyfluorescein diacetate, were severely inhibited for growth at 37 degrees C, and were incapable of sporulating (as homozygotes). Fractionation of cells expressing a bifunctional PEP3::SUC2 fusion protein indicated that the PEP3 gene product is present at low abundance in both log-phase and stationary cells and is a vacuolar peripheral membrane protein. Sequence identity established that PEP3 and VPS18 (J. S. Robinson, T. R. Graham, and S. D. Emr, Mol. Cell. Biol. 11:5813-5824, 1991) are the same gene.

The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases.

pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases. The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation. Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment. This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide. Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts. Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI. Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family. A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986). Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.

Novel syntaxin homologue, Pep12p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast.

pep12/vps6 mutants of Saccharomyces cerevisiae are defective in delivery of soluble vacuolar hydrolases to the vacuole. Morphological analysis by electron microscopy revealed that pep12 cells accumulate 40- to 50-nm vesicles. Furthermore, pep12 cells have enlarged vacuoles characteristic of class D pep/vps mutants. PEP12 encodes a protein of 288 amino acids that has a C-terminal hydrophobic region and shares significant sequence similarity with members of the syntaxin protein family. These proteins appear to participate in the docking and fusion of intracellular transport vesicles. Pep12p is the first member of the syntaxin family to be implicated in transport between the Golgi and the vacuole/lysosome. Pep12p-specific polyclonal antisera detected a 35-kDa protein that fractionated as an integral membrane protein. Subcellular fractionation experiments revealed that Pep12p was associated with membrane fractions of two different densities; the major pool (approximately 90%) of pep12p may associate with the endosome, while a minor pool (approximately 10%) cofractionated with the late Golgi marker Kex2p. These observations suggest that Pep12p may mediate the docking of Golgi-derived transport vesicles at the endosome.

Pep7p provides a novel protein that functions in vesicle-mediated transport between the yeast Golgi and endosome.

Saccharomyces cerevisiae pep7 mutants are defective in transport of soluble vacuolar hydrolases to the lysosome-like vacuole. PEP7 is a nonessential gene that encodes a hydrophilic protein of 515 amino acids. A cysteine-rich tripartite motif in the N-terminal half of the polypeptide shows striking similarity to sequences found in many other eukaryotic proteins. Several of these proteins are thought to function in the vacuolar/lysosomal pathway. Mutations that change highly conserved cysteine residues in this motif lead to a loss of Pep7p function. Kinetic studies demonstrate that Pep7p function is required for the transport of the Golgi-precursors of the soluble hydrolases carboxypeptidase Y, proteinase A, and proteinase B to the endosome. Integral membrane hydrolase alkaline phosphatase is transported to the vacuole by a parallel intracellular pathway that does not require Pep7p function. pep7 mutants accumulate a 40-60-nm vesicle population, suggesting that Pep7p functions in a vesicle consumption step in vesicle-mediated transport of soluble hydrolases to the endosome. Whereas pep7 mutants demonstrate no defects in endocytic uptake at the plasma membrane, the mutants demonstrate defects in transport of receptor-mediated macromolecules through the endocytic pathway. Localization studies indicate that Pep7p is found both as a soluble cytoplasmic protein and associated with particulate fractions. We conclude that Pep7p functions as a novel regulator of vesicle docking and/or fusion at the endosome.

Synthesis and maturation of the yeast vacuolar enzymes carboxypeptidase Y and aminopeptidase I.

We have studied the two vacuolar enzymes carboxypeptidase Y and aminopeptidase I from Saccharomyces cerevisiae with respect to biosynthesis, maturation and transfer from their site of synthesis into the organelle. The levels of translatable mRNA for these two proteins increase more than 10-fold at the end of the exponential growth period on glucose as carbon source and decrease again in the stationary phase. Two precursors of carboxypeptidase Y have been identified by in vivo pulse-labelling with [35S]methionine. These differ in their amount of carbohydrate as shown by inhibition of N-linked glycosylation with tunicamycin. The first is a protein with an apparent molecular weight of 67 kDa, which can be converted into the mature 60-kDa protein via an intermediate of 69 kDa. In the pep4-3 mutant, which is disturbed in the maturation of several vacuolar enzymes (Hemmings, B.A., Zubenko, G.S., Hasilik, A. and Jones, E.W. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 435-439), the 69-kDa precursor accumulates in the vacuole. This suggests that the final proteolytic cleavage of carboxypeptidase Y can occur in the vacuole.

Nonsense mutations in the ade3 locus of Saccharomyces cerevisiae.

Fifty seven mutations at the ade3 locus have been crossed to ochre, amber and ochre-amber suppressors. 70% (39/56) of the mutations at this locus are nonsense mutations; 61% (34/56) are ochre mutations and 9% (5/56) are amber mutations. The frequency of nonsense mutations among ade3 alleles recovered is very high and raises the interesting possibility that only polar mutations at this locus are recovered. An hypothesis to explain these genetical findings as well as physiological properties of these mutations is proposed.

Fine Structure Analysis of the ade3 Locus in SACCHAROMYCES CEREVISIAE.

Twenty-six spontaneous mutants at the ade3 locus of Saccharomyces cerevisiae have been mapped and characterized with respect to revertibility, osmotic remediability and temperature sensitivity. Twelve of the twenty-six are temperature sensitive, 25 of 26 are osmotic remedial and 21 of 26 revert. Two of the mutants map as deletions. At least five of the 26 are nonsense mutations but are also, unexpectedly, osmotic remedial. Three nonsense mutations are also temperature sensitive, again an unexpected result. The two multisite mutations are both temperature sensitive and osmotic remedial. For mutants at this locus osmotic remediability and temperature sensitivity cannot be considered diagnostic criteria for missense mutations.

Proteinase mutants of Saccharomyces cerevisiae.

Fifty-nine mutants with reduced ability to cleave the chymotrypsin substrate N-acetyl-DL-phenylalanine beta-naphthyl ester have been isolated in S. cerevisiae. All have reduced levels of one or more of the three well-characterized proteinases in yeast. All have reduced levels of proteinase C (carboxy-peptidase Y). These mutations define 16 complementation groups.

Bipartite structure of the ade3 locus of Saccharomyces cerevisiae.

Forty ade3 mutants were examined with respect to their growth requirements, levels of the tetrahydrofolate interconversion enzymes, and/or map positions. Four deletions were detected. Mutations that result in a requirement for adenine and histidine map in one region of the locus; those which result in a requirement for adenine only map in a quite separate region of the locus, a region not disclosed in previous studies. No correlation was observed between growth properties of the strains and enzyme levels.

Pth1/Vam3p is the syntaxin homolog at the vacuolar membrane of Saccharomyces cerevisiae required for the delivery of vacuolar hydrolases.

The PEP12 homolog Pth1p (Pep twelve homolog 1) is predicted to be similar in size to Pep12p, the endosomal syntaxin homolog that mediates docking of Golgi-derived transport vesicles and, like other members of the syntaxin family, is predicted to be a cytoplasmically oriented, integral membrane protein with a C-terminal transmembrane domain. Kinetic analyses indicate that deltapth1/vam3 mutants fail to process the soluble vacuolar hydrolase precursors and that PrA, PrB and most of CpY accumulate within the cell in their Golgi-modified P2 precursor forms. This is in contrast to a pep12 mutant in which P2CpY is secreted from the cell. Furthermore, pep12 is epistatic to pth1/vam3 with respect to the CpY secretion phenotype. Alkaline phosphatase, a vacuolar membrane hydrolase, accumulates in its precursor form in the deltapth1/vam3 mutant. Maturation of pro-aminopeptidase I, a hydrolase precursor delivered directly to the vacuole from the cytoplasm, is also blocked in the deltapth1/vam3 mutant. Subcellular fractionation localizes Pth1/Vam3p to vacuolar membranes. Based on these data, we propose that Pth1/Vam3p is the vacuolar syntaxin/t-SNARE homolog that participates in docking of transport vesicles at the vacuolar membrane and that the function of Pth1/Vam3p impinges on at least three routes of protein delivery to the yeast vacuole.

Protein degradation, meiosis and sporulation in proteinase-deficient mutants of Saccharomyces cerevisiae.

During the process of sporulation, a/alpha diploids degrade about 50% of their vegetative proteins. This degradation is not sporulation specific, for asporogenous diploids of a/a mating type degrade their vegetative proteins in a fashion similar to that of their a/alpha counterparts. Diploids lacking carboxypeptidase Y activity, prc1/prc1, show about 80% of wild-type levels of protein degradation, but are unimpaired in the production of normal asci. Diploids lacking proteinase B activity, prb1/prb1, show about 50% of wild-type levels of protein degradation. The effect on degradation of the proteinase B deficiency is epistatic to the degradation deficit attributable to the carboxypeptidase Y deficiency. The prb1 homozygotes undergo meiosis and produce spores, but the asci and, possibly, the spores are abnormal. Diploids homozygous for the pleiotropic pep4-3 mutation show only 30% of the wild-type levels of degradation when exposed to a sporulation regimen, and do not undergo meiosis or sporulation. Neither proteinase B nor carboxypeptidase Y is necessary for germination of spores. Approximately half of the colonies arising from a/a or alpha/alpha diploids exposed to the sporulation regimen that express an initially heterozygous drug-resistance marker (can1) appear to arise from mating-type switches followed by meiosis and sporulation.

The PBN1 gene of Saccharomyces cerevisiae: an essential gene that is required for the post-translational processing of the protease B precursor.

The vacuolar hydrolase protease B in Saccharomyces cerevisiae is synthesized as an inactive precursor (Prb1p). The precursor undergoes post-translational modifications while transiting the secretory pathway. In addition to N- and O-linked glycosylations, four proteolytic cleavages occur during the maturation of Prb1p. Removal of the signal peptide by signal peptidase and the autocatalytic cleavage of the large amino-terminal propeptide occur in the endoplasmic reticulum (ER). Two carboxy-terminal cleavages of the post regions occur in the vacuole: the first cleavage is catalyzed by protease A and the second results from autocatalysis. We have isolated a mutant, pbn1-1, that exhibits a defect in the ER processing of Prb1p. The autocatalytic cleavage of the propeptide from Prb1p does not occur and Prb1p is rapidly degraded in the cytosol. PBN1 was cloned and is identical to YCL052c on chromosome III. PBN1 is an essential gene that encodes a novel protein. Pbn1p is predicted to contain a sub-C-terminal transmembrane domain but no signal sequence. A functional HA epitope-tagged Pbn1p fusion localizes to the ER. Pbn1p is N-glycosylated in its amino-terminal domain, indicating a lumenal orientation despite the lack of a signal sequence. Based on these results, we propose that one of the functions of Pbn1p is to aid in the autocatalytic processing of Prb1p.

Consequences of growth media, gene copy number, and regulatory mutations on the expression of the PRB1 gene of Saccharomyces cerevisiae.

Glucose represses PRB1 expression at the level of transcription. However, release from glucose repression initially does not result in accumulation of protease B (PrB) activity despite transcriptional derepression. PrB activity accumulates only upon a second transcriptional derepression as the cells approach stationary phase. Increasing the PRB1 gene dosage on 2 mu-based plasmids does not overcome glucose repression. Glucose-mediated repression of PRB1 is not subject to the same genetic controls as SUC2. Mutation of the HXK2 gene, which confers glucose-insensitive expression of secreted invertase, had no effect on PRB1 expression at the level of PrB activity. Strains bearing a mutation in any of the SNF1-SNF6 genes cannot derepress secreted invertase synthesis, but did derepress PrB synthesis when grown in the absence of glucose. Mutation of the SNF2 or SNF5 gene led to accumulation of PrB activity to levels ten times that of wild type. Polymorphism for a suppressor gene was observed: in snf5-bearing strains, one allele of this suppressor gene resulted in elevated levels of PrB and the other allele resulted in wild-type levels of PrB; neither allele suppressed the Suc- phenotype of the snf5 mutant. Re-examination of published data on SUC2 expression in snf2 and snf5 mutants and examination of PRB1 expression in these mutants paradoxically suggest that the SNF2 and SNF5 gene products might act as negative regulators of gene expression.