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Decidual remodelling of midluteal endometrium leads to a short implantation window after which the uterine mucosa either breaks down or is transformed into a robust matrix that accommodates the placenta throughout pregnancy. To gain insights into the underlying mechanisms, we established and characterised endometrial assembloids, consisting of gland organoids and primary stromal cells. Single-cell transcriptomics revealed that decidualized assembloids closely resemble midluteal endometrium, harbouring differentiated and senescent subpopulations in both glands and stroma. We show that acute senescence in glandular epithelium drives secretion of multiple canonical implantation factors, whereas in the stroma it calibrates the emergence of anti-inflammatory decidual cells and pro-inflammatory senescent decidual cells. Pharmacological inhibition of stress responses in pre-decidual cells accelerated decidualization by inhibiting senescence and mesenchymal-epithelial transition, processes involved in endometrial breakdown and regeneration, respectively. Accelerated decidualization resulted in entrapment of co-cultured human blastocysts in a largely static decidual matrix. By contrast, the presence of senescent decidual cells created a dynamic implantation environment, enabling embryo expansion and attachment, although their persistence led to gradual disintegration of assembloids. Our findings demonstrate that senescence controls endometrial fate decisions at implantation and highlight how endometrial assembloids may accelerate the discovery of new treatments to prevent reproductive failure.

STUDY QUESTIONCan the accuracy of timing of luteal phase endometrial biopsies based on urinary ovulation testing be improved by measuring the expression of a small number of genes and a continuous, non-categorical modelling approach?SUMMARY ANSWERMeasuring the expression levels of six genes (IL2RB, IGFBP1, CXCL14, DPP4, GPX3, and SLC15A2) is sufficient to obtain substantially more accurate timing estimates and assess the reliability of timing estimates for each sample.WHAT IS KNOWN ALREADYCommercially available endometrial timing approaches based on gene expression require much larger gene sets and use a categorical approach that classifies samples as pre-receptive, receptive, or post-receptive.STUDY DESIGN, SIZE, DURATIONGene expression was measured by RT-qPCR in 260 endometrial biopsies obtained 4 to 12 days after a self-reported positive home ovulation test. A further 36 endometrial samples were profiled by RT-qPCR as well as RNA-sequencing.PARTICIPANTS/MATERIALS, SETTING, METHODSA computational procedure, named ‘EndoTime’, was established that models the temporal profile of each gene and estimates the timing of each sample. Iterating these steps, temporal profiles are gradually refined as sample timings are being updated, and confidence in timing estimates is increased. After convergence, the method reports updated timing estimates for each sample while preserving the overall distribution of time points.MAIN RESULTS AND THE ROLE OF CHANCEThe Wilcoxon Rank Sum Test was used to confirm that ordering samples by EndoTime estimates yields sharper temporal expression profiles for held-out genes (not used when determining sample timings) than ordering the same expression values by patient-reported times (GPX3: p < 0.005; CXCL14: p < 2.7e-6; DPP4: p < 3.7e-13). Pearson correlation between EndoTime estimates for the same sample set but based on RT-qPCR or RNA-sequencing data showed high degree of congruency between the two (p = 8.6e-10, R2 = 0.687).LIMITATIONS, REASONS FOR CAUTIONTiming estimates are predominantly informed by glandular gene expression and will only represent the temporal state of other endometrial cell types if in synchrony with the epithelium. Methods that estimate the day of ovulation are still required as these data are essential inputs in our method. Our approach - in its current iteration – performs batch correction such that larger sample batches impart greater accuracy to timing estimations. In theory, our method requires endometrial samples obtained at different days in the luteal phase. In practice, however, this is not a concern as timings based on urinary ovulation testing are associated with a sufficient level of noise to ensure that a variety of time points will be sampled.WIDER IMPLICATIONS OF THE FINDINGSOur method is the first to assay the temporal state of luteal-phase endometrial samples on a continuous domain. It is freely available with fully shared data and open source software. EndoTime enables accurate temporal profiling of any gene in luteal endometrial samples for a wide range of research applications and, potentially, clinical use.STUDY FUNDING/COMPETING INTEREST(S)This study was supported by a Wellcome Trust Investigator Award (Grant/Award Number: 212233/Z/18/Z) and the Tommy’s National Miscarriage Research Centre. None of the authors have any competing interests. JL was funded by the Biotechnology and Biological Sciences Research Council (UK) through the Midlands Integrative Biology Training Partnership (MIBTP).TRIAL REGISTRATION NUMBERN/A.

Pregnancy depends on the wholesale transformation of the endometrium, a process driven by differentiation of endometrial stromal cells (EnSC) into specialist decidual cells. Upon embryo implantation, decidual cells impart the tissue plasticity needed to accommodate a rapidly growing conceptus and invading placenta, although the underlying mechanisms are unclear. Here we characterize a discrete population of highly proliferative mesenchymal cells (hPMC) in midluteal human endometrium, coinciding with the window of embryo implantation. Single-cell transcriptomics demonstrated that hPMC express genes involved in chemotaxis and vascular transmigration. Although distinct from resident EnSC, hPMC also express genes encoding pivotal decidual transcription factors and markers, most prominently prolactin. We further show that hPMC are enriched around spiral arterioles, scattered throughout the stroma, and occasionally present in glandular and luminal epithelium. The abundance of hPMC correlated with thein vitrocolony-forming unit activity of midluteal endometrium and, conversely, clonogenic cells in culture express a gene signature partially conserved in hPMC. Cross-referencing of single-cell RNA-sequencing data sets indicated that hPMC differentiate into a recently discovered decidual subpopulation in early pregnancy. Finally, we demonstrate that recurrent pregnancy loss is associated with hPMC depletion. Collectively, our findings characterize midluteal hPMC as novel decidual precursors that are likely derived from circulating bone marrow-derived mesenchymal stem/stromal cells and integral to decidual plasticity in pregnancy.Significance statementTransformation of cycling endometrium into the decidua of pregnancy requires extensive tissue remodeling. Perturbations in this process lead to breakdown of the maternal-fetal interface and miscarriage. Here we report the characterization of decidual precursor cells during the window of implantation. We demonstrate that decidual precursors are clonogenic and primed for exponential growth. They likely originate from bone marrow-derived MSC and give rise to a distinct decidual subpopulation in pregnancy. Recurrent pregnancy loss is associated with loss of decidual precursor cells prior to conception, raising the possibility that they can be harnessed for the prevention of pregnancy disorders, including miscarriages and preterm labor.

Objective: To determine the value of uterine CD138+ cells, as a marker of chronic endometritis, in predicting subsequent reproductive outcome in women with history of recurrent pregnancy loss.\ud Design: A prospective longitudinal study.\ud Setting: Tertiary specialised clinic.\ud Patients: Women with history of recurrent pregnancy loss or implantation failure over a 12-months follow-up period. \ud Intervention: We quantified the CD138+ cells/high powered field (hpf) using immunohistochemistry and image analysis of endometrial biopsies obtained during the secretory stage post ovulation. \ud Main Outcome Measures: live birth and subsequent pregnancy loss. We calculated the receiver operator curve for predicting subsequent pregnancy loss and reported using relative risk (RR) and 95% confidence intervals (CI).\ud Results: We enrolled 344 women of whom eighty-eight became pregnant (88/344, 25.5%). Half of them had a subsequent live birth (47/88, 53%) and the rest lost their pregnancy (41/88, 46%). The median CD138+ score was significantly lower in the live birth group (p

Background: Recurrent pregnancy loss (RPL) is associated with the loss of endometrial mesenchymal stem-like progenitor cells (eMSC). DPP4 inhibitors may increase homing and engraftment of bone marrow-derived cells to sites of tissue injury by preventing inactivation of stromal cell-derived factor-1α, a potent chemoattractant. Aims: To assess the effect of the DPP4 inhibitor sitagliptin on eMSC in women with RPL, to evaluate the impact on endometrial decidualization, and to assess the feasibility of a full-scale clinical trial. Methods: A double-blind, randomised, placebo-controlled feasibility trial on women aged 18 to 42 years with a history of 3 or more consecutive pregnancy losses, regular ovulatory menstrual cycles, and no contraindications to sitagliptin. Thirty-eighty subjects were randomised to either 100 mg sitagliptin daily for 3 consecutive cycles or identical placebo capsules. Computer generated, permuted block randomisation was used to allocate treatment packs. The primary outcome measure was the posttreatment eMSC count, assessed by colony forming unit (CFU) assays, in midluteal biopsies. Secondary outcome measures were change in CFU count before and after placebo or sitagliptin treatment, endometrial thickness, study acceptability, and subsequent pregnancy outcome within 12 months following the study. Tissue samples were subjected to explorative investigations. Findings: CFU counts following sitagliptin were higher compared to placebo when adjusted for baseline CFU counts and age (RR:1.52, 95%CI:1.32-1.75, P

During the implantation window, the endometrium becomes poised to transition to a pregnant state, a process driven by differentiation of stromal cells into decidual cells (DC). Perturbations in this process, termed decidualization, leads to breakdown of the feto-maternal interface and miscarriage, but the underlying mechanisms are poorly understood. Here, we reconstructed the decidual pathway at single-cell level in vitro and demonstrate that stromal cells first mount an acute stress response before emerging as DC or senescent DC (snDC). In the absence of immune cell-mediated clearance of snDC, secondary senescence transforms DC into progesterone-resistant cells that abundantly express extracellular matrix remodelling factors. Additional single-cell analysis of midluteal endometrium identified DIO2 and SCARA5 as marker genes of a diverging decidual response in vivo. Finally, we report a conspicuous link between a pro-senescent decidual response in peri-implantation endometrium and recurrent pregnancy loss, suggesting that pre-pregnancy screening and intervention may reduce the burden of miscarriage., Lucas, Vrljicak et al characterise how the cycling endometrium transitions to a pregnant state. Through single-cell analysis of endometrial stromal cells and timed endometrial biopsies they identify DIO2 and SCARA5 as markers of a divergent decidual response.

In cycling human endometrium, menstruation is followed by rapid estrogen-dependent growth. Upon ovulation, progesterone and rising cellular cAMP levels activate the transcription factor Forkhead box O1 (FOXO1) in endometrial stromal cells (EnSCs), leading to cell cycle exit and differentiation into decidual cells that control embryo implantation. Here we show that FOXO1 also causes acute senescence of a subpopulation of decidualizing EnSCs in an IL-8 dependent manner. Selective depletion or enrichment of this subpopulation revealed that decidual senescence drives the transient inflammatory response associated with endometrial receptivity. Further, senescent cells prevent differentiation of endometrial mesenchymal stem cells in decidualizing cultures. As the cycle progresses, IL-15 activated uterine natural killer (uNK) cells selectively target and clear senescent decidual cells through granule exocytosis. Our findings reveal that acute decidual senescence governs endometrial rejuvenation and remodeling at embryo implantation, and suggest a critical role for uNK cells in maintaining homeostasis in cycling endometrium.

SummaryIn cycling human endometrium, menstruation is followed by rapid estrogen-dependent growth. Upon ovulation, progesterone and rising cellular cAMP levels activate the transcription factor Forkhead box O1 (FOXO1) in endometrial stromal cells (EnSCs), leading to cell cycle exit and differentiation into decidual cells that control embryo implantation. Here we show that FOXO1 also causes acute senescence of a subpopulation of decidualizing EnSCs in an IL-8 dependent manner. Selective depletion or enrichment of this subpopulation revealed that decidual senescence drives the transient inflammatory response associated with endometrial receptivity. Further, senescent cells prevent differentiation of endometrial mesenchymal stem cells in decidualizing cultures. As the cycle progresses, IL-15 activated uterine natural killer (uNK) cells selectively target and clear senescent decidual cells through granule exocytosis. Our findings reveal that acute decidual senescence governs endometrial rejuvenation and remodeling at embryo implantation, and suggest a critical role for uNK cells in maintaining homeostasis in cycling endometrium.

SummaryThe transcription factor SOX2 is widely known to play a critical role in the central nervous system; however, its role in peripheral neurogenesis remains poorly understood. We recently developed an hESC-based model in which migratory cells undergo epithelial to mesenchymal transition (EMT) to acquire properties of neural crest (NC) cells. In this model, we found that migratory NC progenitors downregulate SOX2, but then start re-expressing SOX2 as they differentiate to form neurogenic dorsal root ganglion (DRG)-like clusters. SOX2 downregulation was sufficient to induce EMT and resulted in massive apoptosis when neuronal differentiation was induced. In vivo, downregulation of SOX2 in chick and mouse NC cells significantly reduced the numbers of neurons within DRG. We found that SOX2 binds directly to NGN1 and MASH1 promoters and is required for their expression. Our data suggest that SOX2 plays a key role for NGN1-dependent acquisition of neuronal fates in sensory ganglia.

Regulated neuron production within the vertebrate nervous system relies on input from multiple signalling pathways. Work in the Drosophila retina has demonstrated that PI3-kinase and downstream TOR signalling regulate the timing of photoreceptor differentiation; however, the function of such signals during vertebrate neurogenesis is not well understood. Here we show that mutant mice lacking PKB activity downstream of PDK1, the master kinase of the PI3-kinase pathway, exhibit deficient neuron production. We further demonstrate expression of PI3-kinase signalling components and active PKB and TOR signalling in the chick spinal cord, an early site of neurogenesis. Neuron production was also attenuated in the chick neural tube following exposure to small molecule inhibitors of PI3-kinase (LY294002) or TOR (Rapamycin) activity. Furthermore, Rapamycin repressed expression of early neuronal differentiation genes, such as Ngn2, but did not inhibit expression of Sox1B genes characteristic of proliferating neural progenitors. In addition, some cells expressing an early neuronal marker were mis-localised at the ventricular surface in the presence of Rapamycin and remained aberrantly within the cell cycle. These findings suggest that TOR signalling is necessary to initiate neuronal differentiation and that it may facilitate coordination of cell cycle and differentiation programmes. In contrast, stimulating PI3-kinase signalling did not increase neuron production, suggesting that such activity is simply permissive for vertebrate neurogenesis.

During early vertebrate development Fibroblast Growth Factor (FGF) signalling is required for multiple activities including specification of mesodermal, neural and heart tissue, as well as gastrulation movements and regulation of differentiation and pattern onset in the extending body axis. A current challenge is to understand how FGF signalling generates such diverse outcomes. A key FGF downstream pathway is the Ras-MAPK/Erk1/2 cascade, which culminates in the phosphorylation of target proteins, such as the Ets family of transcription factors. To begin to assess specificity downstream of FGF in the chick embryo we have characterised the patterns of Fgfr1–4 expression and Erk1/2 activation, as well as expression of the Erk1/2 specific phosphatase, Mkp3 and of three Ets factor genes (Erm, Pea3 and Er81) from early blastula to the 10 somite stage. We identify new sites of Fgfr expression and show that nearly all regions of Erk1/2 activity are within Fgfr expression domains and require FGF signalling. Differences in intensity, duration, distribution and sub-cellular localisation of activated Erk1/2 are observed in distinct cell populations within the embryo and during wound healing. With few exceptions, a tight correspondence between Erk1/2 activation and Mkp3 expression is found, while specific combinations of Ets factors are associated with distinct regions of Erk1/2 activation. These findings provide a comprehensive spatial and temporal map of FGF/Erk1/2 activity during early chick development and identify region and tissue specific differences in expression of Fgfrs as well as Erk1/2 phosphorylation and transcriptional targets which help to define response specificity.

Studies in humans and animal models show that neuronal insulin resistance increases the risk of developing Alzheimer's Disease (AD), and that insulin treatment may promote memory function. Cholinergic neurons play a critical role in cognitive and attentional processing and their dysfunction early in AD pathology may promote the progression of AD pathology. Synthesis and release of the neurotransmitter acetylcholine (ACh) is closely linked to the activity of the high-affinity choline transporter protein (CHT), but the impact of insulin receptor signaling and neuronal insulin resistance on these aspects of cholinergic function are unknown. In this study, we used differentiated SH-SY5Y cells stably-expressing CHT proteins to study the effect of insulin signaling on CHT activity and function. We find that choline uptake activity measured after acute addition of 20 nM insulin is significantly lower in cells that were grown for 24 h in media containing insulin compared to cells grown in the absence of insulin. This coincides with loss of ability to increase phospho-Protein Kinase B (PKB)/Akt levels in response to acute insulin stimulation in the chronic insulin-treated cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase) in cells significantly lowers phospho-PKB/Akt levels and decreases choline uptake activity. We show total internal reflection microscopy (TIRF) imaging of the dynamic movement of CHT proteins in live cells in response to depolarization and drug treatments. These data show that acute exposure of depolarized cells to insulin is coupled to transiently increased levels of CHT proteins at the cell surface, and that this is attenuated by chronic insulin exposure. Moreover, prolonged inhibition of PI3-kinase results in enhanced levels of CHT proteins at the cell surface by decreasing their rate of internalization.

A novel method of initialising and applying genetic algorithms to the limit analysis problem of masonry arch collapse mode identification is described. The method of encoding an individual uses half the resources (with no accompanying loss of information) in comparison with a simple genetic algorithm (SGA) implementation. The new genetic algorithm (GA) uses a set of heuristic rules to seed the initial population and control subsequent genetic operations (mutation and reproduction). The heuristics developed are seen to be applicable to different sized problems. The heuristic GA (HGA) results are compared with simple GA results and are shown to be significantly more efficient. The heuristic GA finds better solutions than the simple GA after far less iteration and with smaller populations. Several conventionally challenging problems illustrate the solving efficiency of the heuristically initialised genetic algorithm formulation.

The limit analysis of a segmental arch is considered. A collapse work rate minimisation problem is formulated, with constraints on the minimisation that incorporate a mesh description of geometric compatibility based on Kirchhoff’s network laws. A single quadratic objective function and a set of linear constraint equations are used to perform a limit analysis that considers both rotational and frictional failures in the arch. For comparison with a tested linear optimisation technique for arch analysis, the quadratic objective function is reduced to a linear function, and a Genetic Algorithm (GA) is adopted for the solution method. Ordinal ranking is based on the geometric suitability and energy requirements of a collapse mechanism. The solution set is then driven towards efficiency by the GA. The GA uses a roulette wheel selection mechanism based on the fitness of an individual to select parents for subsequent generations. Goals are dynamically redefined as the optimal solutions in a population improve. A conventionally challenging problem shows the solving efficiency of the GA formulation.

The antimalarial drug artemisinin and a number of its derivatives were tested for their effects on proliferation of undifferentiated neuroblastoma Nb2a cells and glioma C6 cells in culture as well as their ability to inhibit neurite outgrowth from Nb2a cells differentiated by removal of serum and addition of dibutyryl cyclic AMP. In the Nb2a and C6 cell cultures, all drugs except desoxyartemisinin significantly inhibited cell proliferation in a dose-related manner with the lowest effective concentration being that of artemisinin at 0.1 microM. Artemether, arteether, artemisinin and dihydroartemisinin also produced a dose-related decrease in the number of neurites/extensions formed by differentiating Nb2a cells, with an effect of dihydroartemisinin at a concentration as low as 1 nM. Desoxyartemisinin had no effect on extension/neurite formation. We propose a potential mechanism for neurotoxicity of artemisinin and its derivatives that involves the endoperoxide bridge which is also known to be necessary for their antimalarial action.

The neural crest is a population of migratory cells that follows specific pathways during development, eventually differentiating to form parts of the face, heart, and peripheral nervous system, the latter of which includes contributions from placodal cells derived from the ectoderm. Stationary, premigratory neural crest cells acquire the capacity to migrate by undergoing an epithelial-to-mesenchymal transition that facilitates their emigration from the dorsal neural tube. This emigration involves, in part, the dismantling of cell-cell junctions, including apically localized tight junctions in the neuroepithelium. In this study, we have characterized the role of the transmembrane tight junction protein claudin-1 during neural crest and placode ontogeny. Our data indicate that claudin-1 is highly expressed in the developing neuroepithelium but is down-regulated in migratory neural crest cells, although expression persists in the ectoderm from which the placode cells arise. Depletion or overexpression of claudin-1 augments or reduces neural crest cell emigration, respectively, but does not impact the development of several cranial placodes. Taken together, our results reveal a novel function for a tight junction protein in the formation of migratory cranial neural crest cells in the developing vertebrate embryo.

Background: The neural crest is a multipotent cell type unique to the vertebrate lineage and capable of differentiating into a large number of varied cell types, including ganglia of the peripheral nervous system, cartilage, and glia. An early step in neural crest specification occurs at the neural plate border, a region defined by the overlap of transcription factors of the Zic, Msx, and Pax families. Results: Here we identify a novel chick gene with close homology to double-stranded RNA-binding protein Interleukin enhancer binding factor 3 (ILF-3) in other species. Our results show that chick Ilf-3 is required for proper expression of the transcription factor, Zic-1, at the neural plate border. Conclusion: We have identified a novel chick gene and show it has a role in the correct specification of Zic-1 at the neural plate border.

12 pages.-- PMID: 19487820 [PubMed]., The events that convert adherent epithelial cells into individual migratory cells that can invade the extracellular matrix are known collectively as epithelial-mesenchymal transition (EMT). Throughout evolution, the capacity of cells to switch between these two cellular states has been fundamental in the generation of complex body patterns. Here, we review the EMT events that build the embryo and further discuss two prototypical processes governed by EMT in amniotes: gastrulation and neural crest formation. Cells undergo EMT to migrate and colonize distant territories. Not surprisingly, this is also the mechanism used by cancer cells to disperse throughout the body.

The successful management of faecal incontinence can dramatically improve the quality of life of affected children. The introduction of the non-refluxing, catheterisable appendico-caecostomy provides the opportunity to treat previously resistant patients. Over a 6-year period, 29 children had a Malone antegrade continent stoma for enema administration (MACE). Incontinence was related to spina bifida in 12 children, ano-rectal anomaly in 12, Hirschprung's disease in 2, followed excision of a pelvic tumour in 2, and was secondary to intractable chronic constipation in 1. The conduit was fashioned from the appendix (20), a caecal tube (8), or a gastric tube (1). Surgical complications were stomal stenosis (11), wound infection (1), anastomotic leak (1), MACE stoma prolapse (1), and a pressure sore (1). Colonic irrigation was achieved with washouts of saline (24), saline plus phosphate (4), and saline plus Picolax (1). Twenty-three patients have complete control of bowel function, but 4 still soil. Two remain incontinent, 1 of whom is still being instructed. One child subsequently had a colostomy, but still uses the MACE stoma. Successful bowel management requires motivation, dedication, commitment, and the input of a clinical nurse specialist. The MACE is a relatively straightforward operative procedure that provides an effective washout technique that is acceptable to both parents and children.

The toxicity caused by the artemisinin derivative dihydroartemisinin in differentiated NB2a neuroblastoma cells was studied by transmission and scanning electron microscopy, Western blotting and immunocytochemistry. Western blotting with monoclonal antibodies failed to detect any specific changes in the cell cytoskeleton, nor were any changes detected by immunocytochemistry. This was consistent with electron microscopy of surviving cell neurites. Transmission electron microscopy revealed that dihydroartemisinin damaged NB2a cell mitochondrial cristae and endoplasmic reticulum. Scanning electron microscopy revealed that dihydroartemisinin depleted the filopodia-like processes projecting from the surface of the cell body and neurites. Some, or all, of these drug-induced changes in differentiating NB2a cells may have a role in the neurotoxicity of artemisinin derivatives.

The binding characteristics of dihydroartemisinin to neural tissues and cells in culture were studied by incubating differentiating C6 and NB2a cells and rat cerebral cortex homogenate with 1 to 200 microM 14C-labelled dihydroartemisinin with or without 2 microM haemin for 24 h. The role of protein thiol and amine groups in dihydroartemisinin binding was assessed by pre-incubation of the cortex homogenate with sodium cyanate and/or iodoacetamide. Rosenthal plots of binding data demonstrated that there were two discrete phases of dihydroartemisinin binding. Haemin increased the total binding of dihydroartemisinin to both cortex and cell proteins. In each preparation, haemin increased Bmax of the high affinity binding sites and also increased kD of NB2a high affinity binding sites and decreased kD of cortex low affinity binding sites. The effects of haemin on the binding parameters of cortex resembled its effects on C6 cells rather than NB2a cells. NB2a high and low affinity binding sites had a greater affinity for dihydroartemisinin than those of either rat cortex or C6 cells. Iodoacetamide and sodium cyanate reduced binding to cortex proteins by approximately 70%. Co-incubation of 14C-dihydroartemisinin with arteether reduced binding of dihydroartemisinin but co-incubation with desoxyartemisinin did not. These studies demonstrate that the known haemin-induced increase in toxicity of dihydroartemisinin to differentiating neuroblastoma cells is accompanied by a corresponding increase in dihydroartemisinin binding to cell proteins, that protein thiol and amine groups react with the products of the reaction between dihydroartemisinin and haemin and point to a relationship between the toxicity of artemisinin derivatives and protein binding.

Objective assessment of surgical technique is currently impossible. A virtual reality simulator for laparoscopic surgery (MIST VR) models the movements needed to perform minimally invasive surgery and can generate a score for various aspects of psychomotor skill. Two studies were performed using the simulator: first to assess surgeons of different surgical experience to validate the scoring system; second to assess in a randomised controlled way, the effect of a standard laparoscopic surgery training course. Experienced surgeons (100 laparoscopic cholecystectomies) were significantly more efficient, made less correctional submovements and completed the virtual reality tasks faster than trainee surgeons or non-surgeons. The training course caused an improvement in efficiency and a reduction in errors, without a significant increase in speed when compared with the control group. The MIST VR simulator can objectively assess a number of desirable qualities in laparoscopic surgery, and can distinguish between experienced and novice surgeons. We have also quantified the beneficial effect of a structured training course on psychomotor skill acquisition.

The accuracy of an automated compounder in preparing parenteral nutrient solutions was evaluated. A total of 480 1-, 2-, and 3--L solutions representing 68 different formulations, some containing electrolytes, were prepared with two Automix devices. The actual weight of the final product was compared with the calculated weight of all ingredients plus the weight of the empty final container. The weight of each ingredient was calculated by multiplying the specific gravity by the volume. A solution was considered acceptable if it weight 95% to 105% of the estimated weight. Only two bags were outside the acceptable range. The mean weight as a percentage of estimated weight for all bags was 100.07 +/- 1.26%. Of the 480 nutrient solutions prepared by an automated compounder, 478 were within 5% of their expected final weight.

The role of haem in the neurotoxicity of artemisinin derivatives has been studied in vitro by examining neurite outgrowth measured by image analysis and cellular metabolism of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) measured spectrophotometrically in the neuroblastoma cell line NB2a, and by examining binding of radiolabelled dihydroartemisinin to NB2a cell and rat brain proteins. In the cases of artemether, dihydroartemisinin, and arteether, haemin (ferriprotoporphyrin IX) significantly increased the dose-related inhibition of neurite outgrowth from differentiating NB2a cells and significantly increased the dose-dependent inhibition of MTT metabolism. Inhibition of neurite outgrowth and metabolism of MTT in the presence or absence of haemin ranged from 72% to 93% and from 27% to 49% at a drug concentration of 300 nM. Haemin also significantly increased the dose-related binding of radiolabelled dihydroartemisinin to proteins from NB2a cells approximately twofold and to rat brain between three- and sixfold. Haemin did not enhance the neurotoxicity of desoxyarteether, a structural analogue of arteether with an ether linkage in the place of the endoperoxide bridge. It is suggested that haemin may catalyse the transformation of these derivatives via an interaction with the endoperoxide bridge of the artemisinin derivative to produce free radicals or electrophilic intermediates that are toxic to neuronal cells.

The ability of a number of different solvent systems to extract lipid from a range of plant tissues was compared by measurement of phospholipid, glycolipid, sterol lipid and total acyl lipid content. A chloroform-methanol extraction method based upon the principles of Bligh and Dyer was considered to be the most efficient system for use with the majority of plant tissues. Cereal seeds were anomalous in that water saturated n - butanol was the preferred solvent system due to its superior ability to extract bound lysophospholipids present in large amounts in the endosperm portion of the tissue.

A procedure has been developed for measuring the extent of oxidative deterioration of polyunsaturated fatty acids. The method is based on the conversion of fatty acid hydroperoxides and compounds derived from them, oxodienes and hydroxydienes, to conjugated fatty acids with strongly chromophoric triene and tetraene structures. This is done in two steps in which reduction with sodium borohydride is followed by dehydration under acid conditions; the chromophores produced are measured by absorbance in the ultraviolet. The method has been investigated using methyl linoleate and methyl linolenate as model compounds. Dienoic fatty acid oxidation products give rise to a conjugated triene chromophore whilst the more polyunsaturated fatty acids form a conjugated tetraene chromophore. The sum of absorbance changes at 268 and 301 nm measure the degree of oxidation. The ratio of absorbance change at 301 nm divided by that at 268 nm varies from zero for a pure triene to 2.8 for a pure tetraene and is a measure of the type of polyunsaturated fatty acid being oxidised. The concentration of oxodiene component present in the original mixture may be estimated separately by the decrease in the 275 nm region during the reduction step.

Amyloplasts were separated from other subcellular organelles by sedimentation through a discontinuous sucrose density gradient. Purified amyloplast envelope membranes were similar in most characteristics to those from other types of plastids. A substantial proportion of the carotenoid content of these membranes was present in the esterified form. In contrast to published work on chloroplasts of photosynthetic tissue, our results showed that the acylase (acyl-CoA:sn-glycerol 3-phosphate acyltransferase) is firmly bound to the envelope membranes. Evidence was obtained to indicate that digalactosyldiacylglycerol, phosphatidylglycerol and sulpholipid, but not monogalactosyldiacylglycerol, are exclusively found in the cell as amyloplast lipids.

Cucumber flesh contains lipids which are qualitatively similar to those of other non-photosynthetic plant tissues. Phospholipids account for nearly half the weight of lipids. The neutral lipids are characterised by a high proportion of 1,2- and 1,3-diacylglycerols relative to triacylglycerol. The sterol composition is similar to that reported for cucumber seedlings; β-sitosterol together with C29 unsaturated sterols being the major components. Linolenic acid is the major fatty acid component of the neutral, glycolipid and phospholipid fractions and this is reflected in the high levels (> 40%) present in all major lipid classes except phosphatidylethanolamine (25%).

Homogenization of fresh tissue from cucumber fruits results in a loss of endogenous lipid catalysed by acyl hydrolase enzymes. Deacylation of lipids is not accompanied by accumulation of free fatty acids. The levels of both saturated (mainly palmitic) and polyunsaturated (linoleic and linolenic) fatty acids in the lipids are reduced. Losses of the major acyl lipid constituents of cucumber (triacylglycerols and phospholipids) are mainly responsible for the observed hydrolysis. Triacylglycerol acyl hydrolase (lipase), phospholipase D and polar lipid acyl hydrolase enzyme activities were demonstrated. It is suggested that hydrolytic attack on endogenous lipids is the initial event on disruption of cucumber tissue, in the formation of lipid degradation products, amongst which are the volatile carbonyl compounds responsible for the characteristic flavour of cucumber.

Changes in proteins and lipids were followed during the storage of freeze-dried turkey breast muscle in air and in nitrogen. Oxidation of sulphydryl groups accounted for part of the oxygen uptake of air-stored samples, and was accompanied by a decrease in soluble nitrogen greater than in controls stored in nitrogen. The major deteriorative process at low moisture content was a type of ‘lipid’ browning reaction which was dependent on oxygen and caused discoloration and objectionable odours. Autoxidation of lipids catalysed by haem pigments and resulting in rancid flavours was not a major deteriorative process in freeze-dried turkey muscle. Lipid hydrolysis occurred at water contents of 8·2 and 5·4% but not at 0·8%.

A study by reflectance spectroscopy of the haem compounds of turkey breast and leg muscle showed that the structure of the main component, myoglobin, changed during the freeze-drying process to form a low-spin complex, ferromyochromagen. Ferrimyoglobin, either present in fresh muscle or introduced as an oxidation product during dehydration, forms the corresponding ferrimyochromagen. During storage in the freeze-dried state the iron complexes do not catalyse the autoxidation of unsaturated lipids. When freeze-dried muscle is rehydrated to a water content not less than 50% of that of fresh muscle, a proportion of the haem pigment is present in the form of a high-spin complex, metmyoglobin, which catalyses autoxidation of unsaturated lipids in the rehydrated muscle.

Dėhydrated carrot develops an unpleasant “off”-flavour when stored in the presence of oxygen. The off-flavour has been described as “having an odour similar to violets” and it was suggested to be due to the formation of β-ionone formed by oxidation of β-carotene. This suggestion is supported by the fact that loss of colour, which is mainly due to the presence of β-carotene, accompanies the development of off-flavour. If the above suggestion is true it should be possible to establish a direct relationship between loss of β-carotene and off-flavour, and this has now been done in a series of storage tests in which both carotene analyses and taste panel assessment for natural and off-flavour were carried out.

Diced turkey leg and breast muscle stored for 22 days at 0° and —3°, and for up to one year at —10°, —20° and —60° was examined at intervals for lipid composition. Free fatty acids increased at all temperatures except —60°, with a Q10 of 3-4 between 0° and —20° 90% of the fatty acids liberated were unsaturated, matching in composition the unsaturated acids of the muscle glycerophospholipids. Demonstration of linear relationships between increase in free fatty acids and decrease in phosphatidylethanolamine or increase in lysophosphatidylcholine confirmed phospholipase A2 as the enzyme mainly responsible. The composition of the fatty acids liberated during storage at 0° and —3° showed that both lipase and phospholipase were active. A slight decrease in extractability, resulting in an apparent loss of phospholipid-P, was observed after storage at low temperatures.

About 85% of the fatty acids liberated during the manufacture of black tea can be attributed to autolysis of 4 major polar lipid classes in tea leaf tissue, phosphatidylcholine, monogalactosyldiglyceride, digalactosyldiglyceride and phosphatidylethanolamine. Linolenic, linoleic and palmitic acids are the principal fatty acids released from these lipids and they all undergo further degradation. Linolenic acid (60% of fatty acids released) is derived mainly from galactolipids and thus the upper limit of release is dependent on the chloroplast maturity and content of the leaf tissues. Lipid breakdown is complete after 2 hr fermentation and, as there appears to be no accumulation of long chain fatty acid intermediates, it is probable that volatile production has also ceased at this time.

Integrated optimization of structural topology and control system is considered. The problem is formulated as mixed discrete-continuous multi-objective programming with a linear quadratic regulator cost index, and measures of robustness and controllability as objectives. The Genetic Algorithm (GA), a guided random search technique, is adopted for the problem-solving. A member elimination strategy that allows deleted members to be recovered is suggested in the search procedure. As verification for the proposed method, optimum layout and actuator placement for a 45-bar truss is illustrated. Numerical results indicate that the genetic algorithm can converge to optimum solutions by searching only a minor fraction of the solution space. Discussions on the algorithm are presented. © 1998 John Wiley & Sons, Ltd.

WHEN dehydrated carrot is stored in the presence of oxygen its colour, due to β-carotene, is destroyed1 and simultaneously an off-flavour develops which has been described as having an odour similar to that of violets2. Although it was earlier suggested that the off-flavour was due to the formation of β-ionone2, no unequivocal proof of the nature of the product or products responsible has yet been put forward.