Reducing donor-site complications in DIEP flap breast reconstruction with closed incisional negative pressure therapy: A cost-benefit analysis.

INTRODUCTION: Deep inferior epigastric perforator (DIEP) flaps are considered the gold standard for autologous breast reconstruction but create large abdominal incisions that risk donor-site morbidity during harvest. Closed incision negative pressure therapy (ciNPT) is emerging as an effective alternative to standard postoperative dressings, but there is a paucity of data in DIEP flap donor sites. METHODS: We conducted a retrospective case-control study investigating the use of ciNPT in DIEP flap donor sites at a single institution between March 2017 and September 2021. Patients who underwent microsurgical autologous breast reconstruction with DIEP flaps were included. Patients were divided into those with donor incision sites managed with ciNPT (n = 24) and those with conventional postoperative wound dressings (n = 20). We compared patient demographics, wound drainage volumes and postoperative outcomes between the two groups. A cost-benefit analysis was employed to compare the overall costs associated with each complication and differences in length of stay between the two groups. RESULTS: There was no statistically significant difference in age, body mass index (BMI), comorbidity burden or smoking status between the two groups. Both groups had similar lengths of stay and wound drainage volumes with no readmissions or reoperations in either group. There was a statistically significant reduction in donor-site complications (p = 0.018), surgical site infections (p = 0.014) and seroma formation (p = 0.016) in those with ciNPT. Upon cost-benefit analysis, the ciNPT group had a mean reduction in cost-per-patient associated with postoperative complications of £420.77 (p = 0.031) and £446.47 (p = 0.049) when also accounting for postoperative length of stay CONCLUSION: ciNPT appears to be an effective alternative incision management system with the potential to improve complication rates and postoperative morbidity in DIEP flap donor sites. Our analysis demonstrates improved cost-benefit outweighing the increase in costs associated with ciNPT. We recommend a multicentre prospective trial with formal cost-utility analysis to strengthen these findings.

Role of Adjuvant Radiotherapy in Acinic Cell Carcinoma of the Salivary Glands: A Systematic Review.

A systematic review was carried out to evaluate if adjuvant radiotherapy for acinic cell carcinomas (ACCs) of salivary glands improves survival. Twelve retrospective studies published between 2000 and 2020 that analysed the effect of radiotherapy on salivary gland neoplasms and ACCs of salivary glands and met the inclusion criteria were included in the review. The overall quality of the studies was moderate to low. There was no high-quality evidence for improved survival with radiotherapy for ACCs of the salivary gland. Some evidence suggests that there may be an advantage for patients with high-grade tumours, but these data should be interpreted with caution due to the small number of patients and low-quality evidence. Good quality of evidence is lacking. Recommendation for adjuvant radiotherapy for tumours with poor prognostic factors will require discussion and shared decision-making with the patients.

Epigenetic regulation of endometrium during the menstrual cycle.

The endometrium undergoes morphological and functional changes during the menstrual cycle which are essential for uterine receptivity. These changes are driven by estrogen and progesterone and involve the fine control of many different genes-several of which have been identified as being epigenetically regulated. Epigenetic modification may therefore influence the functional changes in the endometrium required for successful implantation. There is, however, only limited information on epigenetic regulation in endometrium. We review the potential role of epigenetic regulation of key processes during the menstrual cycle and present our own findings following a preliminary study into global acetylation levels in the human endometrium. A changing epigenetic state is associated with the differentiation of stem cells into different lineages and thus may be involved in endometrial regeneration. Histone acetylation is implicated in the vascular endothelial growth factor pathway during angiogenesis, and studies using histone deacetylase inhibitors suggest an involvement in endometrial proliferation and differentiation. The processes of decidualization and implantation are also associated with epigenetic change and epigenetic modulators show variable expression across the menstrual cycle. Our own studies found that endometrial global histone acetylation, as determined by western blotting, changed throughout the menstrual cycle and correlated well with expected transcription activity during the different phases. This suggests that epigenetics may be involved in the regulation of endometrial gene expression during the menstrual cycle and that abnormal epigenetic modifications may therefore be associated with implantation failure and early pregnancy loss as well as with other endometrial pathologies.

Bleeding gastric ulcer from a pulmonary artery 20 years post-oesophagectomy: a common presentation with a rare cause.

This case presents an unusually late complication of oesophagectomy 20-years post-surgery, with upper gastrointestinal bleeding. Further investigation revealed a gastric conduit ulcer eroding into the lower lobe of the right lung, forming a fistula with a basal branch of the right pulmonary artery. Upon successful embolisation, the HydroCoil® was visible on endoscopy. This case highlights the need for lifetime proton pump inhibitor cover post-oesophagectomy and demonstrates that when approaching uncommon presentations of common problems, careful consideration to treatment technique is essential.

The Sec34/35 Golgi transport complex is related to the exocyst, defining a family of complexes involved in multiple steps of membrane traffic.

The specificity of intracellular vesicle transport is mediated in part by tethering factors that attach the vesicle to the destination organelle prior to fusion. We have identified a protein, Dor1p, that is involved in vesicle targeting to the yeast Golgi apparatus and found it to be associated with seven further proteins. Identification of these revealed that they include Sec34p and Sec35p, the two known components of the Sec34/35 complex previously proposed to tether vesicles to the Golgi. Of the six previously uncharacterized components, four have homologs in higher eukaryotes, including a subunit of a mammalian Golgi transport complex. Furthermore, several of the proteins show distant homology to components of two other putative tethering complexes, the exocyst and the Vps52/53/54 complex, revealing that tethering factors involved in different membrane traffic steps are structurally related.

Localization of proteins to the Golgi apparatus.

For the Golgi apparatus to perform its various unique roles it must maintain a population of resident proteins. These residents include the enzymes that modify the proteins and lipids passing through the Golgi, as well as the proteins involved in vesicle formation and protein sorting. For several of these residents, it has been possible to identify regions that are crucial for specifying a Golgi localization. Consideration of how these targeting domains could function has provided insights into the organization of the Golgi and its protein and lipid content.

Accumulation of caveolin in the endoplasmic reticulum redirects the protein to lipid storage droplets.

Caveolin-1 is normally localized in plasma membrane caveolae and the Golgi apparatus in mammalian cells. We found three treatments that redirected the protein to lipid storage droplets, identified by staining with the lipophilic dye Nile red and the marker protein ADRP. Caveolin-1 was targeted to the droplets when linked to the ER-retrieval sequence, KKSL, generating Cav-KKSL. Cav-DeltaN2, an internal deletion mutant, also accumulated in the droplets, as well as in a Golgi-like structure. Third, incubation of cells with brefeldin A caused caveolin-1 to accumulate in the droplets. This localization persisted after drug washout, showing that caveolin-1 was transported out of the droplets slowly or not at all. Some overexpressed caveolin-2 was also present in lipid droplets. Experimental reduction of cellular cholesteryl ester by 80% did not prevent targeting of Cav-KKSL to the droplets. Cav-KKSL expression did not grossly alter cellular triacylglyceride or cholesteryl levels, although droplet morphology was affected in some cells. These data suggest that accumulation of caveolin-1 to unusually high levels in the ER causes targeting to lipid droplets, and that mechanisms must exist to ensure the rapid exit of newly synthesized caveolin-1 from the ER to avoid this fate.

A functionally distinct member of the DP family of E2F subunits.

E2F transcription factors regulate genes involved in cell-cycle progression. In mammalian cells, physiological E2F exists as an E2F/DP heterodimer. Currently, eight E2F and two DP subunits have been characterized. We report here the characterization of a new member of the DP family, DP-4. While DP-4 exhibits certain similarities with members of the DP family, it also possesses a number of significant differences. Thus, DP-4 forms a heterodimer with E2F subunits, binds to the E2F site and associates with pocket proteins including pRb. In contrast to DP-1, however, DP-4/E2F-1 complexes exhibit reduced DNA binding activity. Furthermore, DP-4 interferes with E2F-1-dependent transcription and delays cell-cycle progression. These results highlight an emerging complexity in the DP family of E2F subunits, and suggest that DP-4 may endow E2F heterodimers with distinct transcription properties.

The notch signalling regulator fringe acts in the Golgi apparatus and requires the glycosyltransferase signature motif DXD.

BACKGROUND: Signalling via the Notch receptor is a key regulator of many developmental processes. The differential responsiveness of Notch-expressing cells to the ligands Delta and Serrate is controlled by Fringe, itself essential for normal patterning in Drosophila and vertebrates. The mechanism of Fringe action, however, is not known. The protein has an amino-terminal hydrophobic stretch resembling a cleaved signal peptide, which has led to the widespread assumption that it is a secreted signalling molecule. It also has distant homology to bacterial glycosyltransferases, although it is not clear if this reflects a shared enzymatic activity, or merely a related structure. RESULTS: We report that a functional epitope-tagged form of Drosophila Fringe was localised in the Golgi apparatus. When the putative signal peptide was replaced by a confirmed one, Fringe no longer accumulated in the Golgi, but was instead efficiently secreted. This change in localisation dramatically reduced its biological activity, implying that the wild-type protein normally acts inside the cell. We show that Fringe specifically binds the nucleoside diphosphate UDP, a feature of many glycosyltransferases. Furthermore, specific mutation of a DxD motif (in the single-letter amino acid code where x is any amino acid), a hallmark of most glycosyltransferases that use nucleoside diphosphate sugars, did not affect the Golgi localisation of the protein but completely eliminated in vivo activity. CONCLUSIONS: These results indicate that Fringe does not exert its effects outside of the cell, but rather acts in the Golgi apparatus, apparently as a glycosyltransferase. They suggest that alteration in receptor glycosylation can regulate the relative efficiency of different ligands.

A yeast homolog of the mammalian mannose 6-phosphate receptors contributes to the sorting of vacuolar hydrolases.

The soluble hydrolases of the mammalian lysosome are marked for delivery to this organelle by the addition of mannose 6-phosphate to their N-glycans. Two related mannose 6-phosphate receptors (MPRs) recognize this feature in the trans Golgi network (TGN) and deliver the hydrolases to the late endosome. In contrast, the vacuolar hydrolases of the yeast Saccharomyces cerevisiae do not contain 6-phosphate monoesters on their N-glycans, and the only sorting receptor so far identified in this organism is the product of the VPS10 gene. This protein also cycles between the Golgi and the late endosome, but is unrelated to the vertebrate MPRs, and recognizes a specific amino acid sequence of carboxypeptidase Y (CPY). This has led to the notion that although yeast and mammals share many components in Golgi to endosome traffic, they use unrelated receptor systems to sort their abundant soluble hydrolases. In this paper, we report that the yeast genome does in fact contain an uncharacterized ORF (YPR079w) that encodes a membrane protein that is distantly related to mammalian MPRs. The protein encoded by this gene (which we term MRL1) cycles through the late endosome. Moreover, there is a strong synergistic effect on the maturation of proteinases A and B when both MRL1 and VPS10 are deleted, which suggests that Mrl1p may serve as a sorting receptor in the delivery of vacuolar hydrolases.

The GRIP domain - a novel Golgi-targeting domain found in several coiled-coil proteins.

Many large coiled-coil proteins are being found associated peripherally with the cytoplasmic face of the organelles of the secretory pathway. Various roles have been proposed for these proteins, including the docking of donor vesicles or organelles to an acceptor organelle prior to fusion, and, in the case of the Golgi apparatus, the stacking of the cisternae [1] [2] [3] [4] [5]. Such critical roles require accurate recruitment to the correct organelle. For the endosomal coiled-coil protein EEA1, targeting requires a carboxy-terminal FYVE domain, which interacts with Rab5 and phosphatidylinositol 3-phosphate (PI(3)P), whereas the Golgi protein GM130 interacts with Golgi membranes via the protein GRASP65 [3] [6] [7]. In this paper, we show that two other mammalian Golgi coiled-coil proteins, golgin-245/p230 and golgin-97, have a conserved domain of about 50 amino acids at their carboxyl termini. This 'GRIP' domain is also found at the carboxyl terminus of several other large coiled-coiled proteins of unknown function, including two human proteins and proteins in the genomes of Caenorhabditis elegans and yeasts. The GRIP domains from several of these proteins, including that from the yeast protein Imh1p, were sufficient to specify Golgi targeting in mammalian cells when fused to green fluorescent protein (GFP). This result suggests that this small domain functions to recruit specific coiled-coil proteins to the Golgi by recognising a determinant that has been well conserved in eukaryotic evolution.

The pleckstrin homology domain of oxysterol-binding protein recognises a determinant specific to Golgi membranes.

BACKGROUND: Peripheral membrane proteins are targeted to the cytoplasmic face of specific intracellular membranes. The organelle-specific ligands recognised by peripheral proteins include other proteins and lipids. Oxysterol-binding protein (OSBP) translocates from the cytoplasm to the Golgi apparatus on binding oxygenated derivatives of cholesterol. The mechanism by which OSBP recognises the Golgi is unknown. It does, however, contain a pleckstrin homology (PH) domain, which in other proteins has been found to mediate regulated membrane binding, although in all previously studied examples the binding is to the plasma membrane. RESULTS: The PH domain of OSBP and of a yeast homologue, Osh1p, were sufficient to target proteins specifically to mammalian Golgi membranes. In addition, high level expression disrupted Golgi architecture and prevented forward traffic of cargo protein. In vitro, the OSBP PH domain bound to Golgi membranes in a manner apparently dependent on phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) or a related phosphatidylinositide. The OSBP PH domain bound to PI(4,5)P2 in liposomes with a submicromolar dissociation constant. CONCLUSIONS: The PH domains of OSBP and its yeast homologue recognise a determinant which is specific to Golgi membranes and important for Golgi function. The determinant appears to be a combination or a phosphatidylinositol polyphosphate and a second, Golgi-specific feature.

Dual targeting of Osh1p, a yeast homologue of oxysterol-binding protein, to both the Golgi and the nucleus-vacuole junction.

Oxysterol binding protein (OSBP) is the only protein known to bind specifically to the group of oxysterols with potent effects on cholesterol homeostasis. Although the function of OSBP is currently unknown, an important role is implicated by the existence of multiple homologues in all eukaryotes so far examined. OSBP and a subset of homologues contain pleckstrin homology (PH) domains. Such domains are responsible for the targeting of a wide range of proteins to the plasma membrane. In contrast, OSBP is a peripheral protein of Golgi membranes, and its PH domain targets to the trans-Golgi network of mammalian cells. In this article, we have characterized Osh1p, Osh2p, and Osh3p, the three homologues of OSBP in Saccharomyces cerevisiae that contain PH domains. Examination of a green fluorescent protein (GFP) fusion to Osh1p revealed a striking dual localization with the protein present on both the late Golgi, and in the recently described nucleus-vacuole (NV) junction. Deletion mapping revealed that the PH domain of Osh1p specified targeting to the late Golgi, and an ankyrin repeat domain targeting to the NV junction, the first such targeting domain identified for this structure. GFP fusions to Osh2p and Osh3p showed intracellular distributions distinct from that of Osh1p, and their PH domains appear to contribute to their differing localizations.

Inositol phosphorylceramide synthase is located in the Golgi apparatus of Saccharomyces cerevisiae.

The plasma membrane of eukaryotic cells differs in lipid composition from most of the internal organelles, presumably reflecting differences in many of its functions. In particular, the plasma membrane is rich in sphingolipids and sterols, one property of which is to decrease the permeability and increase the thickness of lipid bilayers. In this paper, we examine the length of transmembrane domains throughout the yeast secretory pathway. Although the transmembrane domains of cis and medial Golgi residents are similar to those of endoplasmic reticulum proteins, these domains lengthen substantially beyond the medial Golgi, suggesting a thickening of the bilayer. Yeast sphingolipids have particularly long acyl chains, and Aur1p, the inositol phosphorylceramide synthase that initiates yeast sphingolipid synthesis, was found to be located in the Golgi apparatus by both immunofluorescence and membrane fractionation, with its active site apparently in the Golgi lumen. Thus, it appears that sphingolipid synthesis in yeast takes place in the Golgi, separated from glycerophospholipid synthesis in the endoplasmic reticulum. A similar separation has been found in mammalian cells, and this conservation suggests that such an arrangement of enzymes within the secretory pathway could be important for the creation of bilayers of different thickness within the cell.

Surveying the attitudes of acute mental health nurses.

Recent UK policy and guidance indicates the importance of positive attitudes towards mental health service users. This is especially true in acute inpatient care, where service users are often at their most vulnerable and have higher levels of contact with mental health staff. The following paper details secondary analysis of data collected for the validation of an attitude measurement scale with a sample of 140 nursing staff in acute settings. The results demonstrate that a wide range of attitudes are held by mental health nurses towards acute mental health care. Overall, the results indicate generally positive attitudes. Significant differences were found between qualified and unqualified staff, and males and females for some questions. Recommendations are made for future attitudinal research of mental health staff.

Drug competition for thyroxine binding to transthyretin (prealbumin): comparison with effects on thyroxine-binding globulin.

We examined the effect of 26 drugs on T4 binding to transthyretin (TTR; prealbumin) and T4-binding globulin (TBG) by determining their ability to inhibit [125I]T4 binding to TTR isolated from normal human plasma and to serum diluted 1:10,000, respectively. The hierarchies for drug inhibition of T4 binding differed greatly for these two proteins. Relative to T4, the drugs were much more potent inhibitors of [125I]T4 binding to TTR than to TBG. Compounds of the anthranilic acid class, such as flufenamic, meclofenamic, and mefenamic acids, interacted particularly strongly with TTR. Flufenamic acid was more potent than T4 itself in inhibiting [125I]T4 binding [175 +/- 17% (+/- SD); cf. T4; n = 3; P less than 0.001], while mefenamic acid, diflunisal, and meclofenamic acid were 20-26% as potent as T4 in their interaction with TTR. The reactivity of diclofenac, fenclofenac, indomethacin, sulindac, and the diuretic ethacrynic acid was 0.8-2.1% relative to that of T4. In contrast, furosemide, the drug most highly reactive with TBG, was only 0.11 +/- 0.03% (n = 7) as potent as T4, followed by meclofenamic acid greater than mefenamic acid greater than fenclofenac greater than flufenamic acid greater than diflunisal greater than milrinone. Aspirin and sodium salicylate were, respectively, 0.05% and 0.20% as active as unlabeled T4 as inhibitors of [125I]T4 binding to TTR, but these compounds had only 3-4 x 10(-6)% of the activity of T4 for TBG binding. Diphenylhydantoin had no detectable effect on T4 binding to TTR and was 2.9 x 10(-4)% as reactive as T4 with TBG. Amiodarone did not interact with either binding site. Drug interactions with TTR may be important when this protein becomes a major circulating T4-binding protein, as in patients with complete or partial TBG deficiency, or when serum T4 is markedly elevated. Such interactions may also be important where TTR is the dominant tissue T4-binding protein, as in the choroid plexus. In addition, the drug competitors described here may be useful as probes to further define the structural basis for specific ligand interactions with different classes of T4-binding sites.

What can yeast tell us about N-linked glycosylation in the Golgi apparatus?

The N-glycans found on eukaryotic glycoproteins occur in a vast range of different structures. A universal N-glycan core is attached to proteins during synthesis in the endoplasmic reticulum, and then diversity is generated as the proteins pass through the Golgi apparatus. Many of the Golgi-localised glycosyltransferases have now been identified in both yeast and mammalian cells, but it is still unclear how these enzymes are integrated into the Golgi and the rest of the cell so as to ensure efficient and specific processing of passing substrates. This review discusses the potential of the yeast system to address these issues.

Solution conformation of endothelin, a potent vaso-constricting bicyclic peptide. A combined use of 1H NMR spectroscopy and distance geometry calculations.

The solution structure of endothelin-1, a newly discovered potent bicyclic peptide vaso-constrictor agent, has been investigated using 1H NMR conformational constraints and distance geometry calculations. The conformation is constrained by two disulphide bridges between Cys1-Cys15 and Cys3-Cys11 but the NMR data and computed conformers show additional helical structure between residues Leu6 and Cys11. Our results are compared with previous conflicting reports on the solution conformation of this peptide.

The solution structure of a monocyclic analogue of endothelin [1,15 Aba]-ET-1, determined by 1H NMR spectroscopy.

The structure of [1,15 Aba]-ET-1 has been determined in aqueous acetonitrile solution (10% acetonitrile, 1.5% acetic acid). [1,15 Aba]-ET-1 is an analogue of endothelin (ET-1) in which the disulfide bridge linking residues 1 and 15 has been removed by replacement of the cysteine residues with the mimicking group alpha-aminobutyric acid (Aba). The structure has been determined by 1H NMR spectroscopy and simulated annealing calculations based on NOE constraints, 3JHN-H alpha scalar coupling constants, and amide-proton-exchange rates. Distance information was extracted from 2D NOESY spectra using full-relaxation matrix techniques (utilizing the program DISCON). The structure can be described in terms of three defined segments: a type I beta-turn over residues 5-8, a helix over residues 9-16, and a structured C-terminus over residues 17-21. The data indicate that some conformational averaging occurs throughout the peptide, particularly for residues 1-4 in the N-terminus, where no preferred conformation is present. The structure is compared with those previously reported for native ET-1. In general, removal of the disulfide bridge does not cause a major structural change in the helical and turn regions of the sequence, but increased structural disorder is noted at the N-terminus. The implications of the monocyclic analogue's conformation for the pharmacological activity and the ETA/ETB selectivity of the endothelin family of peptides and analogues are described. The N-terminus is proposed to be a key structural region for differentiation of binding activity at the ETA and ETB receptor sites.