Synthetic studies on the bioactive tetramic acid JBIR-22 using a late stage Diels-Alder reaction.

A late stage Diels-Alder reaction is used to prepare a mixture of JBIR-22, a natural product from the Equisetin family of tetramic acids, and one of its diastereomers. This is achieved in just 8 steps from pyruvate. The success of the late stage DA approach is discussed in the context of the biosynthesis of JBIR-22 (and perhaps related natural products).

A comprehensive qualitative investigation of the factors that affect surgical site infection prevention in cardiac surgery in England using observations and interviews.

BACKGROUND: Interview and questionnaire studies have identified barriers and challenges to preventing surgical site infections (SSIs) by focusing on compliance with recommendations and care bundles using interviews, questionnaires and expert panels. This study proposes a more comprehensive investigation by using observations of clinical practice plus interviews which will enable a wider focus. AIM: To comprehensively identify the factors which affect SSI prevention using cardiac surgery as an exemplar. METHODS: The study consisted of 130 h of observed clinical practice followed by individual semi-structured interviews with 16 surgeons, anaesthetists, theatre staff, and nurses at four cardiac centres in England. Data were analysed thematically. FINDINGS: The factors were complex and existed at the level of the intervention, the individual, the team, the organization, and even the wider society. Factors included: the attributes of the intervention; the relationship between evidence, personal beliefs, and perceived risk; power and hierarchy; leadership and culture; resources; infrastructure; supplies; organization and planning; patient engagement and power; hospital administration; workforce shortages; COVID-19 pandemic; 'Brexit'; and the war in Ukraine. CONCLUSION: This is one of the first studies to provide a comprehensive overview of the factors affecting SSI prevention. The factors are complex and need to be fully understood when trying to reduce SSIs. A strong evidence base was insufficient to ensure implementation of an intervention.

p53 and cell cycle independent dysregulation of autophagy in chronic lymphocytic leukaemia.

BACKGROUND: Activation of wild-type p53 with the small molecule sirtuin inhibitor Tenovin-6 (Tnv-6) induces p53-dependent apoptosis in many malignant cells. In contrast, Tnv-6 reduces chronic lymphocytic leukaemia (CLL) cell viability with dysregulation of autophagy, without increasing p53-pathway activity. METHODS: Here, we have investigated whether a quiescent phenotype (unique to CLL) determines the Tnv-6 response, by comparing the effects of Tnv-6 on activated and proliferating CLL. We further studied if these responses are p53-dependent. RESULTS: Unlike quiescent cells, cell death in activated cultures treated with Tnv-6 was consistently associated with p53 upregulation. However, p53 acetylation remained unchanged, without caspase-3 cleavage or apoptosis on electron microscopy. Instead, cellular ultrastructure and protein profiles indicated autophagy inhibition, with reduced ubiquitin-proteasome activity. In specimens with mutant TP53 cultured with Tnv-6, changes in the autophagy-associated protein LC3 occurred independently of p53. Cells treated with Tnv-6 analogues lacking sirtuin inhibitory activity had attenuated LC3 lipidation compared with Tnv-6 (P0.01), suggesting that autophagy dysregulation occurs predominantly through an effect on sirtuins. CONCLUSION: These cell cycle and p53-independent anti-leukaemic mechanisms potentially offer novel therapeutic approaches to target leukaemia-sustaining cells in CLL, including in disease with p53-pathway dysfunction. Whether targets in addition to sirtuins contribute to autophagy dysregulation by Tnv-6, requires further investigation.

Barriers and facilitators for surgical site infection surveillance for adult cardiac surgery in a high-income setting: an in-depth exploration.

BACKGROUND: Surgical site infection (SSI) surveillance aims to facilitate a reduction in SSIs through identifying infection rates, benchmarking, triggering clinical review and instituting infection control measures. Participation in surveillance is, however, variable suggesting opportunities to improve wider adoption. AIM: To gain an in-depth understanding of the barriers and facilitators for SSI surveillance in a high-income European setting. METHODS: Key informant interviews with 16 surveillance staff, infection prevention staff, nurses and surgeons from nine cardiac hospitals in England. Data were analysed thematically. FINDINGS: SSI surveillance was reported to be resource intensive. Barriers to surveillance included challenges associated with data collection: data being located in numerous places, multiple SSI data reporting schemes, difficulty in finding denominator data, lack of interface between computerized systems, 'labour intensive' or 'antiquated' methods to collect data (e.g., using postal systems for patient questionnaires). Additional reported concerns included: relevance of definitions, perceived variability in data reporting, lack of surgeon engagement, unsupportive managers, low priority of SSIs among staff, and a 'blame culture' around high SSI rates. Facilitators were increased resources, better use of digital technologies (e.g., remote digital wound monitoring), integrating surveillance within routine clinical work, having champions, mandating surveillance, ensuring a closer relationship between surveillance and improved patient outcomes, increasing the focus on post-discharge surveillance, and integration with primary care data. CONCLUSION: Using novel interviews with 'front-line' staff, identified opportunities for improving participation in SSI surveillance. Translating these findings into action will increase surveillance activity and bring patient safety benefits to a larger pool of surgical patients.

A High-throughput Screening of a Chemical Compound Library in Ovarian Cancer Stem Cells.

BACKGROUND: Epithelial ovarian cancer has a poor prognosis, mostly due to its late diagnosis and the development of drug resistance after a first platinum-based regimen. The presence of a specific population of "cancer stem cells" could be responsible of the relapse of the tumor and the development of resistance to therapy. For this reason, it would be important to specifically target this subpopulation of tumor cells in order to increase the response to therapy. METHOD: We screened a chemical compound library assembled during the COST CM1106 action to search for compound classes active in targeting ovarian stem cells. We here report the results of the high-throughput screening assay in two ovarian cancer stem cells and the differentiated cells derived from them. RESULTS AND CONCLUSION: Interestingly, there were compounds active only on stem cells, only on differentiated cells, and compounds active on both cell populations. Even if these data need to be validated in ad hoc dose response cytotoxic experiments, the ongoing analysis of the compound structures will open up to mechanistic drug studies to select compounds able to improve the prognosis of ovarian cancer patients.

Bypassing the proline/thiazoline requirement of the macrocyclase PatG.

Biocatalysis is a fast developing field in which an enzyme's natural capabilities are harnessed or engineered for synthetic chemistry. The enzyme PatG is an extremely promiscuous macrocyclase enzyme tolerating both non-natural amino acids and non-amino acids within the substrate. It does, however, require a proline or thiazoline at the C-terminal position of the core peptide which means the final product must contain this group. Here, we show guided by structural insight we have identified two synthetic routes, triazole and a double cysteine, that circumvent this requirement. With the triazole, we show PatGmac can macrocyclise substrates that do not contain any amino acids in the final product.

Differentially expressed genes in adult familial myelodysplastic syndromes.

The precise genetic events leading to myelodysplastic syndromes (MDSs) and leukemic transformation remain poorly defined. Even less is known about adult familial MDS. We report an adult MDS family in whom enriched tissue-specific transcripts were derived by subtractive hybridization of cDNA from the mononuclear and CD34+ cells of affected and unaffected family members. These expression libraries were then hybridized to Genome Discovery arrays containing 18 404 genes and expressed sequence tags, and several clusters of differentially expressed genes were identified. A group of 21 genes was underexpressed (>5-fold) in affected vs unaffected family members, and among these were transcription factors and genes involved in myeloid differentiation, such as ZNF140 and myeloid nuclear differentiation antigen (MNDA). Another group of 36 genes was overexpressed (>5-fold), and these encoded proteins belonging to signaling pathways, such as Ras- and Fos-related genes. The top two genes downregulated in this MDS family, ZNF140 and MNDA, were similarly altered in another MDS family, and in some cases of sporadic MDS. Our data suggest that we have identified genes differentially expressed in adult familial MDS, and that alteration of some of these genes may also be important for the evolution of different stages or severity of sporadic MDS.

The importance of growth hormone in the regulation of erythropoiesis, red cell mass, and plasma volume in adults with growth hormone deficiency.

Total body water (TBW) is reduced in adult GH deficiency (GHD) largely due to a reduction of extracellular water. It is unknown whether total blood volume (TBV) contributes to the reduced extracellular water in GHD. GH and insulin-like growth factor I (IGF-I) have been demonstrated to stimulate erythropoiesis in vitro, in animal models, and in growing children. Whether GH has a regulatory effect on red cell mass (RCM) in adults is not known. We analyzed body composition by bioelectrical impedance and used standard radionuclide dilution methods to measure RCM and plasma volume (PV) along with measuring full blood count, ferritin, vitamin B12, red cell folate, IGF-I, IGF-binding protein-3, and erythropoietin in 13 adult patients with GHD as part of a 3-month, double blind, placebo-controlled trial of GH (0.036 U/kg.day). TBW and lean body mass significantly increased by 2.5 +/- 0.53 kg (mean +/- SEM; P < 0.004) and 3.4 +/- 0.73 kg (P < 0.004), respectively, and fat mass significantly decreased by 2.4 +/- 0.32 kg (P < 0.001) in the GH-treated group. The baseline RCM of all patients with GHD was lower than the predicted normal values (1635 +/- 108 vs. 1850 +/- 104 mL; P < 0.002). GH significantly increased RCM, PV, and TBV by 183 +/- 43 (P < 0.006), 350 +/- 117 (P < 0.03), and 515 +/- 109 (P < 0.004) mL, respectively. The red cell count increased by 0.36 +/- 0.116 x 10(12)/L (P < 0.03) with a decrease in ferritin levels by 39.1 +/- 4.84 micrograms/L (P < 0.001) after GH treatment. Serum IGF-I and IGF-binding protein-3 concentrations increased by 3.0 +/- 0.43 (P < 0.001) and 1.3 +/- 0.15 (P < 0.001) SD, respectively, but the erythropoietin concentration was unchanged after GH treatment. No significant changes in body composition or blood volume were recorded in the placebo group. Significant positive correlations could be established between changes in TBW and TBV, lean body mass and TBV (r = 0.78; P < 0.04 and r = 0.77; P < 0.04, respectively), and a significant negative correlation existed between changes in fat mass and changes in TBV in the GH-treated group (r = -0.95; P < 0.02). We conclude that 1) erythropoiesis is impaired in GHD; 2) GH stimulates erythropoiesis in adult GHD; and 3) GH increases PV and TBV, which may contribute to the increased exercise performance seen in these patients.

Activated phenotype in neutrophils and monocytes from patients with primary proliferative polycythaemia.

AIM: To investigate whether monocytes and neutrophils from patients with primary proliferative polycythaemia (PPP) exhibit increased expression of markers of cell activation and, if so, whether they are associated with the phagocytic activity of these cells and concentrations of circulating cytokines. METHODS: Expression of CD11b, CD14, CD18, and CD64 on monocytes and neutrophils was assessed by flow cytometry. Phagocytosis was analysed using immunoglobulin opsonised Escherichia coli. Serum concentrations of granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) were determined by bioassays, and interferon-gamma (IFN-gamma) by enzyme linked immunosorbent assay (ELISA). RESULTS: Patients with PPP (n = 18), when compared with normal subjects (n = 10), had increased percentages of CD64+ monocytes (52% v 36%) and neutrophils (42% v 11%) and of CD14+ neutrophils (36% v 18%). Monocytes from patients with PPP exhibited increased expression of CD64 (47 v 26) and of CD11b (65 v 36). These abnormalities were not found in patients with secondary (n = 8) or apparent (n = 13) polycythaemia. The percentage of neutrophils undergoing phagocytosis was higher in patients with PPP (mean 64%; n = 6) than in normal subjects (mean 42%; n = 5). G-CSF, GM-CSF and IFN-gamma concentrations in patients' serum samples were comparable with normal; M-CSF was not detected in any of the samples. There was no correlation between cytokine concentrations and the expression of CD11b, CD14, CD18, and CD64 on patients' phagocytes. CONCLUSIONS: Increased expression of CD11b and CD64 by monocytes, increased percentages of CD14+ and CD64+ neutrophils and the high phagocytic activity of neutrophils suggests that these cells are activated in vivo in patients with PPP. The phenotypic changes of PPP phagocytes were not associated with increased concentrations of circulating cytokines and probably reflect intrinsic abnormalities within the neoplastic PPP clone.

Diagnostic applications of haemopoietic progenitor culture techniques in polycythaemias and thrombocythaemias.

Clonogenic cultures support the proliferation of haemopoietic cells into colonies of differentiated progeny. Using these techniques it has been established that normal haemopoietic progenitors are dependent upon growth factors for their survival, proliferation and differentiation in vitro. However, progenitors from patients with polycythaemia vera (PV) generate erythroid colonies in cultures deprived of exogenous erythropoietin. These have been termed endogenous erythroid colonies (EEC). Recently, endogenous megakaryocytic colonies (EMC), those arising in assays without megakaryocyte growth factors, have been reported, particularly in patients with primary thrombocythaemia (PT). Many investigators have found an EEC positive culture to have 100% diagnostic specificity and sensitivity for PV. Similarly, EMC have been shown by some to be an unequivocal marker of PT. Accordingly, clonogenic assays for EEC and EMC have been advocated as diagnostic markers of PV and PT. However, the specificity of these assays is not universally attested to as there are some reports of EEC in patients with secondary polycythaemia and of EEC and EMC in normal subjects. Thus, for diagnostic use, EEC and EMC assays must be exhaustively validated for specificity using clinically appropriate controls. Furthermore, clonogenic culture techniques are not amenable to external quality assurance, are technically demanding and are unlikely to be available in most haematology laboratories. These considerations must be taken into account when assigning weighting to EEC and EMC assays as diagnostic criteria in myeloproliferative disorders.

T lymphocyte subpopulations in patients with B-cell chronic lymphocyte leukaemia: relationship to infective episodes.

Patients with B-cell chronic lymphocytic leukaemia (B-CLL) have an increased susceptibility to infection. Quantitative abnormalities of T-cells have been previously reported in B-CLL, although the relationship between such abnormalities and the incidence of infection still remains to be established. We therefore enumerated lymphocyte subpopulations in 22 patients with B-CLL grouped according to the number of infective episodes in the previous three years. No significant differences were found between the patient groups and the mean number of T-cells subsets (helper, suppressor, suppressor-inducer and suppressor effector) or NK cells, but patients with frequent infections were found to have significantly higher CD5+ B-cell counts. Thus, we confirm that T-cell subpopulations are numerically altered in patients with B-CLL, but found that such changes are not predictive of susceptibility to infection. Our results however suggest that the malignant B-cells may exhibit immunosuppressive activity.

IgG subclass levels in patients with B cell chronic lymphocytic leukaemia.

Patients with B-cell chronic lymphocytic leukaemia (BCLL) have low levels of serum IgG. In order to determine if this is a pan IgG deficiency or a selective suppression of one or more IgG subclasses, levels of IgG 1, 2, 3 and 4 in nine BCLL patients were determined and compared to those of nine age and sex matched controls. No significant differences were found in the levels of IgG1 and IgG2, but the patients were found to have significantly lower levels of IgG3 (p < 0.05) and IgG4 (p < 0.05). Selective deficiencies of these isotypes may explain the particular pattern of infection seen in BCLL patients.

Serum erythropoietin and circulating BFU-E in patients with multiple myeloma and anaemia but without renal failure.

In 30 patients with multiple myeloma (MM) and mild to moderate anaemia (mean Hb 107 g/l, 95% confidence limit (CL) 102-113) but no evidence of renal failure (serum creatinine < 110 mumol/l), serum erythropoietin (EPO) showed significant inverse logarithmic correlation with the haemoglobin level (r = -0.57, p = 0.001). The observed/expected ratio of log-EPO in patients with MM (mean 0.96, CL 0.89-1.04) was similar to that of 119 subjects (mean 1.01, CL 0.96-1.05) with or without anaemia (mean Hb 116 g/L, CL 110-121) but without renal failure. The concentration of circulating erythroid progenitors (BFU-E) in 10 MM patients in plateau phase was significantly reduced (mean 0.70 x 10(5)/l of blood, CL 0.34-1.06) compared to that of 8 normal controls (mean 3.57, CL 1.60-5.55, p = 0.011) In vitro sensitivity of the BFU-E to EPO in the patients with MM was comparable to that of the normal controls. It appears that in MM there is an appropriate EPO response to anaemia but even in the plateau phase the number of circulating BFU-E is reduced, reflecting a degree of marrow failure. However, the progenitors are normally sensitive to EPO in such patients, and therapeutic doses of EPO may correct the anaemia by a pharmacological rather than a physiological effect.

Vascular uptake of catecholamines in perfused lungs of the rat occurs by the same process as Uptake1 in noradrenergic neurones.

The aim of the study was to determine whether the uptake process for catecholamines in rat lungs is Uptake1, Uptake2 or a distinct process with some properties of both Uptake1 and Uptake2. The initial rate of uptake of noradrenaline was measured in isolated lungs of rats perfused with 2 nmol/l 3H-(-)-noradrenaline for 2 min with monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) inhibited, in the absence or presence of drugs that are substrates or inhibitors of Uptake1 or Uptake2 or of alterations in the ionic composition of the Krebs solution. The rank order of the IC50 values for inhibition of uptake of noradrenaline in the lungs by drugs that are substrates or inhibitors of Uptake1 or Uptake2 is compatible with the conclusion that uptake of catecholamines in rat lungs occurs by Uptake1, and not by a process with the properties of Uptake2. Additional evidence was provided by the marked inhibition of uptake in the lungs when the Na+ concentration in the Krebs solution was decreased from 143 to 25 mmol/l and by the lack of inhibition when the K+ concentration was increased from 5.9 mmol/l to either 10.9 or 20.9 mmol/l. Further experiments were included in the study to obtain data additional to histological evidence (Hughes et al. 1969; Nicholas et al. 1974) regarding the site of Uptake1 in rat lungs. Pretreatment of rats with either 6-hydroxydopamine (to destroy noradrenergic neurones) or reserpine (to inhibit synaptic vesicle uptake) had no effect on the deamination or accumulation of noradrenaline in lungs perfused with 3H-noradrenaline (COMT inhibited).(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for uptake1-mediated efflux of catecholamines from pulmonary endothelial cells of perfused lungs of rats.

Previous pharmacological studies have demonstrated that pulmonary endothelial cells and noradrenergic neurones possess the same transporter for inward transport of catecholamines, uptake1. In noradrenergic neurones, it has been shown that uptake1 is also involved in the carrier-mediated outward transport, or efflux, of noradrenaline and dopamine. The aim of the present study was to examine the efflux of noradrenaline and dopamine from perfused lungs of rats to determine whether uptake1, in addition to diffusion, mediates efflux of catecholamines from pulmonary vascular endothelial cells. The effects of reducing the cellular sodium gradient and of substrates and inhibitors of uptake1 on the efflux of 3H-noradrenaline and 3H-dopamine from rat lungs were measured. Isolated perfused lungs of rats (monoamine oxidase and catechol-O-methyltransferase inhibited) were loaded with 3H-(-)-noradrenaline or 3H-dopamine for 10 min followed by perfusion with either (1) a low sodium, amine-free Krebs solution, in which NaCl was replaced by either Tris.HCl or LiCl, for 15 or 10 min, respectively or (2) amine-free Krebs solution for 30 min in the absence or presence of a substrate or inhibitor of uptake1 for the last 15 min. The rate constants for spontaneous efflux of noradrenaline and dopamine from the lungs were 0.0163 min-1 and 0.0466 min-1, respectively. When NaCl was replaced by Tris.HCl during efflux, the rate constants for efflux of noradrenaline and dopamine were increased 2.5-fold and 3-fold, respectively, whereas, when NaCl was replaced by LiCl, the rate constants were increased 8-fold and 4-fold, respectively. The uptake1 substrates, dopamine (1 and 3 mumol/l) and adrenaline (40 mumol/l), both caused a rapid and marked increase in the efflux of noradrenaline, while noradrenaline (4 mumol/l) had a similar effect on the efflux of dopamine. The uptake1 inhibitors, imipramine (3 and 10 mumol/l) and nisoxetine (50 nmol/l), caused small and gradual increases in the efflux of noradrenaline and dopamine from rat lungs. These results demonstrate that efflux of noradrenaline and dopamine from rat lungs is affected by alterations in the normal sodium gradient across the cell and by drugs that interact with the uptake1 transporter. Thus, it can be concluded that the spontaneous efflux of catecholamines from pulmonary vascular endothelial cells is mediated predominantly by uptake1. In addition, efflux of catecholamines from the lungs has a diffusional component, which, combined with inhibition of reuptake, accounts for the small increase in amine efflux by inhibitors of uptake1.

Screening and synthesis: high throughput technologies applied to parasitology.

High throughput technologies continue to develop in response to the challenges set by the genome projects. This article discusses how the techniques of both high throughput screening (HTS) and synthesis can influence research in parasitology. Examples of the use of targeted and phenotype-based HTS using unbiased compound collections are provided. The important issue of identifying the protein target(s) of bioactive compounds is discussed from the synthetic chemist's perspective. This article concludes by reviewing recent examples of successful target identification studies in parasitology.