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.

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.

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.

Reduction of mortality in specific-pathogen-free layer chickens by a caprine serum fraction after infection with Pasteurella multocida.

Caprine serum was fractionated by size, and its proteinaceous material <8,000 Da [caprine serum fraction immunomodulator 2 (CSF-I2)] was evaluated for its ability to impart immunoresistance to specific-pathogen-free (SPF) layer chickens. The SPF layers were challenged with 18 to 30 cfu of Pasteurella multocida X-73 (serotype 1) at 5 wk of age. A high degree of mortality was apparent 24 and 48 h later (62+/-14% and 88+/-7%, respectively). Mortality observed after 48 h was minimal. Noting the rapid onset of mortality, we administered CSF-I2 (material that expressed no direct antimicrobial activity but was believed to be an immunostimulant) 1 d before challenge and coincident to time of challenge. The group of birds that received CSF-I2 (either 5 or 10 mg per administration) expressed significant reduction in mortality throughout the 1-wk study period. Reduction in mortality appeared to be dose dependent. Birds that received two administrations of 10 mg CSF-I2 had significantly fewer deaths than did the group of birds that received half that amount. No deaths were recorded through 24 h, whereas, at 48 h, the percentage mortality was 13 in CSF-I2-treated birds. This study demonstrates that one or more small molecular weight compounds isolated from caprine serum were able to reduce mortality in SPF layers infected with Pasteurella multocida.

Pep3p/Pep5p complex: a putative docking factor at multiple steps of vesicular transport to the vacuole of Saccharomyces cerevisiae.

Pep3p and Pep5p are known to be necessary for trafficking of hydrolase precursors to the vacuole and for vacuolar biogenesis. These proteins are present in a hetero-oligomeric complex that mediates transport at the vacuolar membrane. PEP5 interacts genetically with VPS8, implicating Pep5p in the earlier Golgi to endosome step and/or in recycling from the endosome to the Golgi. To understand further the cellular roles of Pep3p and Pep5p, we isolated and characterized a set of pep3 conditional mutants. Characterization of mutants revealed that pep3(ts) mutants are defective in the endosomal and nonendosomal Golgi to vacuole transport pathways, in the cytoplasm to vacuole targeting pathway, in recycling from the endosome back to the late Golgi, and in endocytosis. PEP3 interacts genetically with two members of the endosomal SNARE complex, PEP12 (t-SNARE) and PEP7 (homologue of mammalian EEA1); Pep3p and Pep5p associate physically with Pep7p as revealed by two-hybrid analysis. Our results suggest that a core Pep3p/Pep5p complex promotes vesicular docking/fusion reactions in conjunction with SNARE proteins at multiple steps in transport routes to the vacuole. We propose that this complex may be responsible for tethering transport vesicles on target membranes.

Malignant spinal neurofibrosarcoma.

STUDY DESIGN: A report of a case of metastatic spinal neurofibrosarcoma. OBJECTIVE: To document metastatic neurofibrosarcoma as a cause of spinal cord compression and to review the literature. SUMMARY OF BACKGROUND DATA: Three previously reported cases of metastatic neurofibrosarcoma of the spine were reviewed. METHODS: The patient's clinical record and radiologic investigations as well as the result of a search of the English literature are reported. Magnetic resonance images, computed tomographic scans, and histology photomicrographs are displayed. RESULTS: Paraparesis developed in this patient, due to a posterior extradural thoracic spinal cord compression by a neurofibrosarcoma believed to be metastatic from a neurofibrosarcoma of the femoral nerve. CONCLUSIONS: Malignant spinal metastasis remains a rare complication of neurofibromatosis, with a very poor prognosis.

Orbital metastasis due to interval lobular carcinoma of the breast: a potential mimic of lymphoma.

A 53-year-old woman had an orbital mass composed of a neoplastic small round cell infiltrate and no apparent extraorbital primary tumor. Although the initial diagnosis was primary orbital lymphoma, a combination of mucin histochemistry and immunohistochemical staining for cytokeratin and estrogen receptors led to the discovery of an impalpable lobular carcinoma of the breast. We discuss how detailed histopathological assessment can lead to beneficial therapy.

Water transport across yeast vacuolar and plasma membrane-targeted secretory vesicles occurs by passive diffusion.

To determine whether solute transport across yeast membranes was facilitated, we measured the water and solute permeations of vacuole-derived and late secretory vesicles in Saccharomyces cerevisiae; all permeations were consistent with passive diffusive flow. We also overexpressed Fps1p, the putative glycerol facilitator in S. cerevisiae, in secretory vesicles but observed no effect on water, glycerol, formamide, or urea permeations. However, spheroplasts prepared from the strain overexpressing Fps1p showed enhanced glycerol uptake, suggesting that Fps1p becomes active only upon insertion in the plasma membrane.

Spontaneous intralesional haemorrhage in dysembryoplastic neuroepithelial tumours: a series of five cases.

Five patients with dysembryoplastic neuroepithelial tumour (DNT) showing extensive secondary haemorrhage, a finding not previously associated with these neoplasms, are described. The clinical presentations, neuroimaging findings, and histopathological features of these patients are reviewed. One patient, a previously asymptomatic 12 year old girl, presented with an acute intracerebral haemorrhage into a DNT. A further four young adults with histories of intractable partial and generalised seizures dating from childhood showed significant chronic haemorrhages within DNT, the MRI appearances in one patient giving a false impression of a cavernoma. Histopathology disclosed vascular abnormalities within these tumours which, together with other factors discussed, may have predisposed these tumours to haemorrhage.

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.

Genetic interaction with vps8-200 allows partial suppression of the vestigial vacuole phenotype caused by a pep5 mutation in Saccharomyces cerevisiae.

pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases. In addition, they show a vestigial vacuole morphology and a sensitivity to growth on media containing excess divalent cations. This pleiotropic phenotype observed for pep5::TRP1 mutants is partially suppressed by the vps8-200 allele. pep5::TRP1 vps8-200 mutants show near wild-type levels of mature-sized soluble vacuolar hydrolases, growth on zinc-containing medium, and a more "wild-type" vacuolar morphology; however, aminopeptidase I and alkaline phosphatase accumulate as precursors. These data suggest that Pep5p is a bifunctional protein and that the TRP1 insertion does not eliminate function, but results in a shorter peptide that can interact with Vps8-200p, allowing for partial function. vps8 deletion/disruption mutants contain a single enlarged vacuole. This genetic interaction was unexpected, since Pep5p was thought to interact more directly with the vacuole, and Vps8p is thought to play a role in transport between the Golgi complex and the prevacuolar compartment. The data are consistent with Pep5p functioning both at the site of Vps8p function and more closely proximal to the vacuole. They also provide evidence that the three transport pathways to the vacuole either converge or share gene products at late step(s) in the pathway(s).

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.

Genetic interactions between a pep7 mutation and the PEP12 and VPS45 genes: evidence for a novel SNARE component in transport between the Saccharomyces cerevisiae Golgi complex and endosome.

The PEP7 gene from Saccharomyces cerevisiae encodes a 59-kD hydrophilic polypeptide that is required for transport of soluble vacuolar hydrolase precursors from the TGN to the endosome. This study presents the results of a high-copy suppression analysis of pep7-20 mutant phenotypes. This analysis demonstrated that both VPS45 and PEP12 are allele-specific high-copy suppressors of pep7-20 mutant phenotypes. Overexpression of VPS45 was able to completely suppress the Zn2+ sensitivity and partially suppress the carboxypeptidase Y deficiency. Overexpression of PEP12 was able to do the same, but to a lesser extent. Vps45p and Pep12p are Sec1p and syntaxin (t-SNARE) homologues, respectively, and are also thought to function in transport between the TGN and endosome. Two additional vacuole pathway SNARE complex homologues, Vps33p (Sec1p) and Pth1p (syntaxin), when overexpressed, were unable to suppress pep7-20 or any other pep7 allele, further supporting the specificity of the interactions of pep7-20 with PEP12 and VPS45. Because several other vesicle docking/fusion reactions take place in the cell without discernible participation of Pep7p homologues, we suggest that Pep7p is a step-specific regulator of docking and/or fusion of TGN-derived transport vesicles onto 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.

Regulation of the proteinase B structural gene PRB1 in Saccharomyces cerevisiae.

The expression of PRB1, the gene that encodes the precursor to the soluble vacuolar proteinase B (PrB) in Saccharomyces cerevisiae, is regulated by carbon and nitrogen sources and by growth phase. Little or no PRB1 mRNA is detectable during exponential growth on glucose as the carbon source; it begins to accumulate as cells exhaust the glucose. Previous work has shown that glucose repression of PRB1 transcription is not mediated by HXK2 or by the SNF1, SNF4, and SNF6 genes (C. M. Moehle and E. W. Jones, Genetics 124:39-55, 1990). We analyzed the effects of mutations in the MIG1, TUP1, and GRR1 genes on glucose repression of PRB1 and found that mutations in each partially alleviate glucose repression. tup1 and mig1 mutants fail to translocate all of the Prb1p into the lumen of the endoplasmic reticulum. A screen for new mutants revealed mutations in MIG1 and REG1, genes already known to regulate glucose repression, as well as in three new genes that we have named PBD1 to PBD3; all cause derepressed expression. Mutations that result in failure to completely derepress PRB1 were also identified in two new genes, named PND1 and PND2. Good nitrogen sources, like ammonia, repress PRB1 transcription; mutations in URE2 do not affect this response. Derepression upon transfer to a poor nitrogen source is dependent upon GLN3.

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.

An immunocytochemical assessment of 19 cases of cutaneous angiosarcoma.

Four endothelial cell markers, two selective cytokeratin markers and a monoclonal smooth muscle antibody (SMA) were employed in the assessment of 19 cases of cutaneous angiosarcoma classified according to their degree of tumour differentiation. No labelling was seen for SMA or with cytokeratin markers MNF116 and CBL170. Expression of factor VIII-related antigen was seen in two tumours and positivity for CD34 (QBend 10 antibody) was found in four tumours. By contrast the pan-endothelial cell marker Ulex europeaus agglutinin 1 (UEA-1) and the CD31 marker JC70A labelled all cases of cutaneous angiosarcoma with the exception of one poorly differentiated tumour. These data confirm the endothelial cell origin of angiosarcoma, they demonstrate that CD31 and UEA1 are reliable markers in routinely processed tissue, and they suggest a lymphatic derivation for the tumour. This finding is in marked contrast to Kaposi's sarcoma where CD34 is the most reliable marker.

vph6 mutants of Saccharomyces cerevisiae require calcineurin for growth and are defective in vacuolar H(+)-ATPase assembly.

We have characterized a Saccharomyces cerevisiae mutant strain that is hypersensitive to cyclosporin A (CsA) and FK506, immunosuppressants that inhibit calcineurin, a serine-threonine-specific phosphatase (PP2B). A single nuclear mutation, designated cev1 for calcineurin essential for viability, is responsible for the CsA-FK506-sensitive phenotype. The peptidyl-prolyl cis-trans isomerases cyclophilin A and FKBP12, respectively, mediate CsA and FK506 toxicity in the cev1 mutant strain. We demonstrate that cev1 is an allele of the VPH6 gene and that vph6 mutant strains fail to assemble the vacuolar H(+)-ATPase (V-ATPase). The VPH6 gene was mapped on chromosome VIII and is predicted to encode a 181-amino acid (21 kD) protein with no identity to other known proteins. We find that calcineurin is essential for viability in many mutant strains with defects in V-ATPase function or vacuolar acidification. In addition, we find that calcineurin modulates extracellular acidification in response to glucose, which we propose occurs via calcineurin regulation of the plasma membrane H(+)-ATPase PMA1. Taken together, our findings suggest calcineurin plays a general role in the regulation of cation transport and homeostasis.