A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery

Background: Several human pathologies, including neoplasia and ischemic cardiovascular diseases, course with an unbalance between oxygen supply and demand ( hypoxia). Cells within hypoxic regions respond with the induction of a specific genetic program, under the control of the Hypoxia Inducible Factor (HIF), that mediates their adaptation to the lack of oxygen. The activity of HIF is mainly regulated by the EGL-nine homolog (EGLN) enzymes that hydroxylate the alpha subunit of this transcription factor in an oxygen-dependent reaction. Hydroxylated HIF is then recognized and ubiquitinilated by the product of the tumor suppressor gene, pVHL, leading to its proteosomal degradation. Under hypoxia, the hydroxylation of HIF by the EGLNs is compromised due to the lack of oxygen, which is a reaction cosubstrate. Thus, HIF escapes degradation and drives the transcription of its target genes. Since the progression of the aforementioned pathologies might be influenced by activation of HIF-target genes, development of small molecules with the ability to interfere with the HIF-regulatory machinery is of great interest.Results: Herein we describe a yeast three-hybrid system that reconstitutes mammalian HIF regulation by the EGLNs and VHL. In this system, yeast growth, under specific nutrient restrictions, is driven by the interaction between the beta domain of VHL and a hydroxyproline-containing HIF alpha peptide. In turn, this interaction is strictly dependent on EGLN activity that hydroxylates the HIFa peptide. Importantly, this system accurately preserves the specificity of the hydroxylation reaction toward specific substrates. We propose that this system, in combination with a matched control, can be used as a simple and inexpensive assay to identify molecules that specifically modulate EGLN activity. As a proof of principle we show that two known EGLN inhibitors, dimethyloxaloylglycine (DMOG) and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamide (S956711), have a profound and specific effect on the yeast HIF/EGLN/VHL system.Conclusion: The system described in this work accurately reconstitutes HIF regulation while preserving EGLN substrate specificity. Thus, it is a valuable tool to study HIF regulation, and particularly EGLN biochemistry, in a cellular context. In addition, we demonstrate that this system can be used to identify specific inhibitors of the EGLN enzymes

Successful Transplantation of Human Kidneys Deemed Untransplantable but Resuscitated by Ex Vivo Normothermic Machine Perfusion

We report the successful transplantation of a pair of human kidneys that were declined for transplantation due to inadequate in situ perfusion but subsequently transplanted after perfusion and assessment using ex vivo normothermic perfusion (EVNP). The kidneys were from a 35-year-old man, a donation after circulatory death donor. Both kidneys were declined by all UK transplant centers. On arrival, the kidneys had significant areas of incomplete clearance of blood from the microcirculation that did not clear after a further attempt to flush them. Kidneys underwent 60 min of EVNP with an oxygenated packed red blood cell–based solution warmed to 35.2°C. During EVNP, the patchy areas cleared in both kidneys. The mean renal blood flow and total urine output were 68.0 mL/min/100 g and 560 mL in the left kidney and 59.9 mL/min/100 g, 430 mL in the right, respectively. Based on the EVNP perfusion parameters, both kidneys were deemed suitable for transplantation. They were transplanted without any complications, and both recipients had initial graft function. The serum creatinine levels at 3 months were 1.2 mg/dl in the recipient of the left kidney and 1.62 mg/dl in the recipient of the right kidney. EVNP technology can be used to assess and rescue kidneys previously deemed unsuitable for transplantation.This study was supported by Kidney Research UK. The research was also funded by the National Institute for Health Research Blood and Transplant Research Unit (NIHR BTRU) in Organ Donation and Transplantation at the University of Cambridge in collaboration with Newcastle University and in partnership with NHS Blood and Transplant (NHSBT). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, the Department of Health, or NHSBT

Monitoreo neurofisiológico intraoperatorio en la cirugía de la médula anclada

Presentamos nuestra experiencia en cirugía de médula anclada utilizando el monitoreo neurofisiológico como método de asistencia intraoperatoria realizada en el Hospital de Niños "Superiora Sor María Ludovica" de La Plata entre los años 1998 y 2006. Se intervinieron quirúrgicamente 40 pacientes con una edad promedio de 5.2 años. Las causas de anclaje medular más frecuentes fueron el re-anclaje secundario a cirugía de mielomeningocele (n: 14) y el filum terminale hipertrófico (n: 9). Las motivos de consulta principales fueron trastornos motores (n: 19) y estigmas cutáneos (n: 15). Utilizando el registro electromiográfico intraoperatorio, se logró en todos los casos preservar la función vesical, mejorar significativamente la espasticidad (n: 15) y evitar la progresividad de la signosintomatología. El monitoreo neurofisiológico resulta entonces un método indispensable para evitar déficit postoperatorios definitivos, guiando la cirugía a través de áreas carentes de inervación.We present our experience in the surgical treatment of tethered cord using the assistance of neurophysiological monitoring from 1998 to 2006. The procedure was done in 40 patients (mean age 5.2 years). The most frequent causes of tethered cord were that secondary to surgical treatment of myelomeningocele (n: 14), and hypertrophic phylum terminale (n: 9). Main presenting manifestations included muscle weakness (n: 19) and cutaneous stigmata (n: 15). By using the intraoperative electromyographic monitoring it was possible to preserve bladder function in all the cases, improve significatively the development of spasticity (n: 15) and avoid progression of clinical symptoms and signs. Thus, this procedure becomes absolutely necessary to avoid postoperative sequela guiding the surgical practice through anatomic areas devoid of nerves

Antifungal, Acute Toxicity and Mutagenicity Activity of Extracts from Datura stramonium, Jacquinia macrocarpa and Krameria erecta on Fusarium verticillioides

The effect of Baccharis glutinosa, Jacquinia macrocarpa, and Krameria erecta extracts was investigated on the growth and the spore germination of Fusarium verticillioides (ATCC 52539). Brine shrimp (Artemia salina) was used to evaluate the potential acute toxicity of the fractions obtained from plant extracts. The butanol fraction of J. macrocarpa totally inhibited the radial growth for 144 h and up to 95% after 168 h. The ethyl acetate fraction of B. glutinosa caused 100% of radial growth inhibition for 96 h. The ethyl acetate fractions of B. glutinosa and K. erecta caused the higher inhibitory effect on F. verticillioides spore germination, 100 and 95%, respectively. All plant fractions tested at a concentration of 5.0 mg mL-1 caused 100% brine shrimp lethality after 24 h. The Ames test did not reveal the presence of an evident mutagenic activity.Keywords: Antifungal Activity, Plant Extracts, Brine Shrimp Bioassay, Mutagenicity Assay, Fusarium verticillioide

Is the even distribution of insecticide-treated cattle essential for tsetse control? Modelling the impact of baits in heterogeneous environments

Background: Eliminating Rhodesian sleeping sickness, the zoonotic form of Human African Trypanosomiasis, can be achieved only through interventions against the vectors, species of tsetse (Glossina). The use of insecticide-treated cattle is the most cost-effective method of controlling tsetse but its impact might be compromised by the patchy distribution of livestock. A deterministic simulation model was used to analyse the effects of spatial heterogeneities in habitat and baits (insecticide-treated cattle and targets) on the distribution and abundance of tsetse. Methodology/Principal Findings: The simulated area comprised an operational block extending 32 km from an area of good habitat from which tsetse might invade. Within the operational block, habitat comprised good areas mixed with poor ones where survival probabilities and population densities were lower. In good habitat, the natural daily mortalities of adults averaged 6.14% for males and 3.07% for females; the population grew 8.46in a year following a 90% reduction in densities of adults and pupae, but expired when the population density of males was reduced to <0.1/km2; daily movement of adults averaged 249 m for males and 367 m for females. Baits were placed throughout the operational area, or patchily to simulate uneven distributions of cattle and targets. Gaps of 2–3 km between baits were inconsequential provided the average imposed mortality per km2 across the entire operational area was maintained. Leaving gaps 5–7 km wide inside an area where baits killed 10% per day delayed effective control by 4–11 years. Corrective measures that put a few baits within the gaps were more effective than deploying extra baits on the edges. Conclusions/Significance: The uneven distribution of cattle within settled areas is unlikely to compromise the impact of insecticide-treated cattle on tsetse. However, where areas of >3 km wide are cattle-free then insecticide-treated targets should be deployed to compensate for the lack of cattle

Ex vivo normothermic perfusion of isolated segmental porcine bowel: a novel functional model of the small intestine.

BACKGROUND: There is an unmet need for suitable ex vivo large animal models in experimental gastroenterology and intestinal transplantation. This study details a reliable and effective technique for ex vivo normothermic perfusion (EVNP) of segmental porcine small intestine. METHODS: Segments of small intestine, 1.5-3.0 m in length, were retrieved from terminally anaesthetized pigs. After a period of cold ischaemia, EVNP was performed for 2 h at 37°C with a mean pressure of 80 mmHg using oxygenated autologous blood diluted with Ringer's solution. The duration of EVNP was extended to 4 h for a second set of experiments in which two segments of proximal to mid-ileum (1.5-3.0 m) were retrieved from each animal and reperfused with whole blood (control) or leucocyte-depleted blood to examine the impact of leucocyte depletion on reperfusion injury. RESULTS: After a mean cold ischaemia time of 5 h and 20 min, EVNP was performed in an initial group of four pigs. In the second set of experiments, five pigs were used in each group. In all experiments bowel segments were well perfused and exhibited peristalsis during EVNP. Venous glucose levels significantly increased following luminal glucose stimulation (mean(s.e.m.) basal level 1.8(0.6) mmol/l versus peak 15.5(5.8) mmol/l; P < 0.001) and glucagon-like peptide 1 (GLP-1) levels increased in all experiments, demonstrating intact absorptive and secretory intestinal functions. There were no significant differences between control and leucocyte-depleted animals regarding blood flow, venous glucose, GLP-1 levels or histopathology at the end of 4 h of EVNP. CONCLUSIONS: This novel model is suitable for the investigation of gastrointestinal physiology, pathology and ischaemia reperfusion injury, along with evaluation of potential therapeutic interventions

Upper limits on the extent of seafloor anoxia during the PETM from uranium isotopes

This is the final version. Available on open access from Nature Research via the DOI in this recordData availability: All data generated or analyzed during this study are included in this published article (and its Supplementary information files). Source data are provided with this paper.The Paleocene Eocene Thermal Maximum (PETM) represents a major carbon cycle and climate perturbation that was associated with ocean de-oxygenation, in a qualitatively similar manner to the more extensive Mesozoic Oceanic Anoxic Events. Although indicators of ocean de-oxygenation are common for the PETM, and linked to biotic turnover, the global extent and temporal progression of de-oxygenation is poorly constrained. Here we present carbonate associated uranium isotope data for the PETM. A lack of resolvable perturbation to the U-cycle during the event suggests a limited expansion of seafloor anoxia on a global scale. We use this result, in conjunction with a biogeochemical model, to set an upper limit on the extent of global seafloor de-oxygenation. The model suggests that the new U isotope data, whilst also being consistent with plausible carbon emission scenarios and observations of carbon cycle recovery, permit a maximum ~10-fold expansion of anoxia, covering <2% of seafloor area.European Union Horizon 2020Natural Environment Research Council (NERC

Daily mycoprotein consumption for one week does not affect insulin sensitivity or glycaemic control but modulates the plasma lipidome in healthy adults: a randomised controlled trial

This is the author accepted manuscript. The final version is available from Cambridge University Press via the DOI in this record.Mycoprotein consumption has been shown to improve acute postprandial glycaemic control and decrease circulating cholesterol concentrations. We investigated the impact of incorporating mycoprotein into the diet on insulin sensitivity (IS), glycaemic control and plasma lipoprotein composition. Twenty healthy adults participated in a randomised, parallel-group trial in which they consumed a 7 d fully-controlled diet where lunch and dinner contained either meat/fish (CON) or mycoprotein (MYC) as the primary source of dietary protein. Oral glucose tolerance tests were performed pre- and post- intervention, and 24h continuous blood glucose monitoring was applied throughout. Fasting plasma samples were obtained pre- and post- intervention and were analysed using quantitative, targeted NMR-based metabonomics. There were no changes within or between groups in blood glucose or serum insulin responses, nor in IS (Cederholm; 51±3 to 51±3 and 54±3 to 53±3 mg.L2/mmol.mU.min in CON and MYC, respectively; P<0.05) or 24 h glycaemic profiles. No differences between groups were found for 171 of the 224 metabonomic targets. Forty five lipid concentrations of different lipoprotein fractions (VLDL, LDL, IDL and HDL) remained unchanged in CON but showed a coordinated decrease (7-27 %; all P<0.05) in MYC. Total plasma cholesterol, free-C, LDL-C, HDL2-C, DHA and omega-3 fatty acids decreased to a larger degree in MYC (14-19 %) compared with CON (3-11 %; P<0.05). Substituting meat/fish for mycoprotein twice-daily for one week did not modulate whole-body IS or glycaemic control but resulted in changes to plasma lipid composition; the latter primarily consisting of a coordinated reduction in circulating cholesterol containing lipoproteins.QuornMarlow Foods Lt

Use of Ex Vivo Normothermic Perfusion for Quality Assessment of Discarded Human Donor Pancreases.

A significant number of pancreases procured for transplantation are deemed unsuitable due to concerns about graft quality and the associated risk of complications. However, this decision is subjective and some declined grafts may be suitable for transplantation. Ex vivo normothermic perfusion (EVNP) prior to transplantation may allow a more objective assessment of graft quality and reduce discard rates. We report ex vivo normothermic perfusion of human pancreases procured but declined for transplantation, with ABO-compatible warm oxygenated packed red blood cells for 1-2 h. Five declined human pancreases were assessed using this technique after a median cold ischemia time of 13 h 19 min. One pancreas, with cold ischemia over 30 h, did not appear viable and was excluded. In the remaining pancreases, blood flow and pH were maintained throughout perfusion. Insulin secretion was observed in all four pancreases, but was lowest in an older donation after cardiac death pancreas. Amylase levels were highest in a gland with significant fat infiltration. This is the first study to assess the perfusion, injury, as measured by amylase, and exocrine function of human pancreases using EVNP and demonstrates the feasibility of the approach, although further refinements are required.This study was financially supported by a grant from the Mason Medical Research Foundation.This is the author accepted manuscript. The final version is available via Wiley at http://onlinelibrary.wiley.com/doi/10.1111/ajt.13303/abstract