港西地区河流相砂岩油藏剩余油分布研究

The Gangxi oil field has reached a stage of high water production. The reservoir parameters, such as reservoir physical characteristics, pore structure, fluid, have obviously changed. This thesis therefore carries out a study of these parameters that control reservoir characteristics, physical and chemical actions that have taken place within the reservoirs due to fluid injection, subsequent variations of reservoir macroscopic physical features, microscopic pore structures, seepages, and formation fluid properties. This study rebuilds a geologic model for this oil field, establishes a log-interpreting model, proposes a methodology for dealing with large pore channels and remnant oil distribution, and offers a basis for effective excavation of potential oil, recovery planning, and improvement of water-injection techniques. To resolve some concurrent key problems in the process of exploration of the Gangxi area, this thesis carries out a multidisciplinary research into reservoir geology, physical geography, reservoir engineering, and oil-water well testing. Taking sandstone and flow unit as objects, this study establishes a fine geologic model by a quantificational or semi-quantificational approach in order to understand the remnant oil distribution and the reservoir potential, and accordingly proposes a plan for further exploration. By rebuilding a geological model and applying reservoir-engineering methods, such as numerical simulation, this thesis studies the oil-water movement patterns and remnant-oil distribution, and further advances a deployment plan for the necessary adjustments and increase of recoverable reserves. Main achievements of this study are as follows: 1. The Minghazhen Formation in the Gangxi area is featured by medium-sinuosity river deposits, manifesting themselves as a transitional type between typical meandering and braided rivers. The main microfacies are products of main and branch channels, levee, inter-channel overflows and crevasse-splay floodplains. The Guantao Group is dominantly braided river deposit, and microfacies are mainly formed in channel bar, braided channel and overbank. Main lithofacies include conglomerate, sandstone, siltstone and shale, with sandstone facies being the principal type of the reservoir. 2. The reservoir flow unit of the Gangxi area can be divided into three types: Type I is a high-quality heterogeneous seepage unit, mainly distributed in main channel; Type II is a moderate-quality semi-heterogeneous seepage unit, mainly distributed in both main and branch channels, and partly seen within inter-channel overflow microfacies; Type III is a low-quality, relatively strong heterogeneous seepage unit, mainly distributed in inter-channel overflow microfacies and channel flanks. 3. Flow units and sedimentary microfacies have exerted relatively strong controls on the flowing of underground oil-water: (1) injection-production is often effective in the float units of Type I and II, whilst in the same group of injection-production wells, impellent velocity depends on flow unit types and injection-production spacing; (2) The injection-production of Type III flow unit between the injection-production wells of Type I and II flow units, however, are little effective; (3) there can form a seepage shield in composite channels between channels, leading to inefficient injection and production. 4. Mainly types of large-scale remnant-oil distribution are as follows: (1) remnant oil reservoir of Type III flow unit; (2) injection-production well group of remnant oil area of Type III flow unit; (3) remnant oil reservoirs that cannot be controlled by well network, including reservoir featured by injection without production, reservoir characterized by production without injection, and oil reservoir at which no well can arrive; (4) remnant oil area where injection-production system is not complete. 5. Utilizing different methods to deal with different sedimentary types, sub-dividing the columns of up to 900 wells into 76 chronostratigraphic units. Four transitional sandstone types are recognized, and contrast modes of different sandstone facies are summarized Analyzing in details the reservoirs of different quality by deciphering densely spaced well patterns, dividing microscopic facies and flow units, analyzing remnant oil distribution and its effect on injection-production pattern, and the heterogeneity. Theory foundation is therefore provided for further excavation of remnant oil. Re-evaluating well-log data. The understanding of water-flood layers and conductive formations in the Gangxi area have been considerably improved, and the original interpretations of 233 wells have changed by means of double checking. Variations of the reservoirs and the fluid and formation pressures after water injection are analyzed and summarized Studies are carried out of close elements of the reservoirs, fine reservoir types, oil-water distribution patterns, as well as factors controlling oil-gas enrichment. A static geological model and a prediction model of important tracts are established. Remaining recoverable reserves are calculated of all the oil wells and oil-sandstones. It is proposed that injection-production patterns of 348 oil-sandstones should be adjusted according to the analysis of adaptability of all kinds of sandstones in the injection-production wells.

东营凹陷沙三中亚段湖相浊积岩储层特征及油气成藏规律研究

Turbidity sandstone reservoirs have been an important field of hydrocarbon exploration and development in the basins all over the world, as well as in China. Lithologic pools are composed of turbidity sandstones and other sandstones are frequently found in the Jiyang Depression that is a Mesozoic-Cenozoic non-marine oil-bearing basin. The Dongying Sag lies in the sedimentary center of the basin. The subtle traps with turbidity reservoirs are generally difficult to be predicted and described by using current techniques. The studies on turbidity reservoirs plays thus an important theoretical and theoretical practical role in exploration and development in the Jiyang Depression. The attention is, in this thesis, focused on the petrologic properties and oil accumulating behaviors in lake turbidity sedimentary systems in the middle part of the third section of Shahejie Formation in the Dongying Sag, especially in Dongxin area, which lies on the central uplift of the Sag. The paper has disclosed the origin types of turbidity sandstones, distribution pattern and controlling factors of turbidity sandstones, and set up hydrocarbon accumulation patterns of the middle part of the third section of Shahejie Formation in Dongxin, based on nonmarine high resolution sequence stratigraphy, event sedimentology and new theories of hydrocarbon forming. By studying prediction method and technology of turbidity sandstone reservoirs, using precise geological model developing, new techniques of high resolution seismic inversion constrained by logging, the paper has forecast low permeability turbidity sandstone reservoirs and pointed out advantage exploration aims to progressive exploration and development. The paper has obtained mainly many productions and acknowledges as follows: 1.Turbidity sandstone reservoirs of the third section of Shahejie Formationin Dongying Sag are formed in such specifical geological background as rift and extension of basin. The inherited Dongying delta and transgression make up many turbidity distribution areas by overlaying and joining together. The hydrocarbon migrates from depression area to adjacent turbidity sandstone continuously. Accumulation area which is sufficient in oil is formed. 2.The paper has confirmed distinguishable sign of sequence boundary , established stratigraphic framework of Dongying Sag and realized isotime stratigraphic correlation. Es3 of Dongying delta is divided into eleven stages. Among them, the second period of the lower section in Es3, the sixth period of the middle section in Es3, the third period of the upper section in Es3 correspond to eleven sedimentary isotime surface in seismic profile, namely Es3 is classified into eleven Formations. 3.According to such the features of turbidity sandstone as deep in burial, small in area, strong in subtle property, overlaying and joining together and occurring in groups, management through fault and space variations of restriction quantum are realized and the forecast precision of turbidity sandstone by using precise geological model developing, new techniques of high resolution seismic inversion constrained by logging, based on the analysis of all kinds of interwell seismic inversion techniques. 4.According to the features of low permeable turbidity sandstone reservoirs, new method of log interpretation model is put forward. At the same time, distinguish technology of familiar low resistivity oil layer in the turbidity sandstone reservoirs is studied based on petrophysical laboratory work and "four properties" interrelationship between lithological physical Jogging and bearing hydrocarbon properties. Log interpretation model and reservoir index interpretation model of low resistivity oil layer are set up. So the log interpretation precision is improved. 5.The evolution law and its difference of the turbidity sandstone are embodies as follows: the source of sediments come from the south and east of the study area in the middle period of Es3. East source of sediments is pushed from west to east. However, the south source supply of sediments in the early and middle period of Es3 is in full, especially in Es3. subsequently, the supply is decreased gradually. Turbidity fan moves back toward the south and the size of fan is minished accordingly. The characteristic of turbidity sandstone in Dongying Sag is different in different structural positions. Dongxin in the middle-east of the central lift and Niuzhuang Sag He in Dongying delta front and prodelta deep lake subfacies. Although the turbidity sandstone of the two areas root in the Dongying delta sedimentary system, the sand body has different remarkably characteristic. 6.The sedimentary model of the turbiditys in study area have three types as follows: (1) collapse turbidity fan in respect of delta; (2) fault trench turbidity fan; (3) other types of microturbidity sandstone. Middle fan and outer fan, can be found mainly in sublacustrine fan. Middle fan includes braided channel microfacies, central microfacies and braided interchannel microfacies, which is main prospecting oil-bearing subfacies. The middle section of the third section of Shahejie Formation in study area (for example the central lift) can be divided into middle-lower and upper part. The middle-lower part is characteristic of turbidity fan. The upper part is sedimented mainly by delta-collapse fan. 7.The turbidity reservoirs of the middle part of the third section of Shahejie Formation in study area characterize by low maturity both in component and texture, strong in diagenesis and low in permeability. The reservoir can be classified into four types. Type III is the body of reservoir and comprises two types of H a and HI b. M a belongs to middle porosity - low permeability reservoir and distributes in the central lift. Hlb belongs to low porosity - low permeability and distributes in Haojia region. 8.A11 single sand body of lens turbidity reservoir of the middle part of the third section of Shahejie Formation in study area are surrounded by thick dark source rocks. The oil-water system is complex and behaves that every sandstone is single seal unit. The water body is 1/3-1-5 of the sand body. The edge water is not active. The gas exists in the top of reservoir in the form of mixed gas. For far-range turbidity fan with big scale channel, the area and volume of sand body is large and the gap is big in oil packing degree. There are lots of edge water and bottom water, and the latter increases rapidly during the course of development. 9.By exerting the modern hydrocarbon forming theories, the third section of Shahejie Formation in study area belongs to abnormally pressured fluid compartment. The lithological reservoir of the third section of Shahejie Formation is formed in the compartment. The reservoir-formed dynamic system belongs to lower self-source enclosed type. The result and the practice indicate that the form and accumulation of lithological oil reservoirs are controlled by the temperature and pressure of stratum, microfacies, thickness of sand body, fault and reservoir heterogeneity. 10. Based on studies above, the emphases focus on in south and north part of Dongying structure, west Dongxin region and south part Xinzhen structure in the application of production. The practice proves that the turbidity sandstone reservoirs in Ying 11 block and the fault-lithological reservoirs in Xin 133 block have been obtained significant breakthrough. The next target is still sandstone groups of the third section of Shahejie Formation in the bordering areas of Dongxin region for instance Xin 149 area, He 89 area, Ying 8 area etc.

Elevação do seio maxilar versus colocação de implantes curtos

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária

Hydrocarbon Potential of the Infra-salt Strata

This schematic geological cross-section of Angola offshore is representative of the majority of the Atlantic-type divergent margins. It illustrates the main geological features allowing to understand the different petroleum systems occurring, particularly, in South Atlantic divergent margins : (i) Pre-Pangea rocks (Precambrian granite-gneiss basement, volcanic rocks an/ or Paleozoic sediments, more or less, metamorphosed), which lie underneath the pre-rifting unconformity (PRU), in blue in the cross-section ; (ii) The rift-type basins developed during the lengthening of the Pangea supercontinent ; (iii) The breakup unconformity (BUU), which highlight the upper limit of the rift-type basins, in which organic rich lacustrine shales with a parallel internal configuration are potential source-rocks (organic matter type I) ; (iv) The SDRs (seaward dipping reflectors), which, generally, do not have any generating hydrocarbon potential (just 5 m of lacustrine shales are known in Austral basin) ; (v) The BUU is fossilized by SDRs (subaerial volcanism) or by margin infra-salt sediments (forming the mistakenly called by some American geoscientists "sag basin") ; (vi) The Loeme salt basin, which is a twin of the Brazilian salt basin, that is to say, that both basins have always been individualized ; (vii) The transgressive (backstepping) and regressive (forestepping) phases of the post-Pangea continental encroachment cycle ; (v) The interface between these sedimentary phases, correspond to the emplacement of potential marine source-rocks (organic matter type-II) ; (vi) Potential dispersive source rocks (organic matter type III) are possible in the regressive sedimentary interval.

Uniformly convergent finite element and finite difference methods for singularly perturbed ordinary differential equations

This thesis is concerned with uniformly convergent finite element and finite difference methods for numerically solving singularly perturbed two-point boundary value problems. We examine the following four problems: (i) high order problem of reaction-diffusion type; (ii) high order problem of convection-diffusion type; (iii) second order interior turning point problem; (iv) semilinear reaction-diffusion problem. Firstly, we consider high order problems of reaction-diffusion type and convection-diffusion type. Under suitable hypotheses, the coercivity of the associated bilinear forms is proved and representation results for the solutions of such problems are given. It is shown that, on an equidistant mesh, polynomial schemes cannot achieve a high order of convergence which is uniform in the perturbation parameter. Piecewise polynomial Galerkin finite element methods are then constructed on a Shishkin mesh. High order convergence results, which are uniform in the perturbation parameter, are obtained in various norms. Secondly, we investigate linear second order problems with interior turning points. Piecewise linear Galerkin finite element methods are generated on various piecewise equidistant meshes designed for such problems. These methods are shown to be convergent, uniformly in the singular perturbation parameter, in a weighted energy norm and the usual L2 norm. Finally, we deal with a semilinear reaction-diffusion problem. Asymptotic properties of solutions to this problem are discussed and analysed. Two simple finite difference schemes on Shishkin meshes are applied to the problem. They are proved to be uniformly convergent of second order and fourth order respectively. Existence and uniqueness of a solution to both schemes are investigated. Numerical results for the above methods are presented.

The role of metabolism in the pathogenesis of adherent invasive Escherichia coli (AIEC)

Crohn’s disease (CD) is a chronic, relapsing inflammatory condition affecting the gastrointestinal tract of humans, of which there is currently no cure. The precise etiology of CD is unknown, although it has become widely accepted that it is a multifactorial disease which occurs as a result of an abnormal immune response to commensal enteric bacteria in a genetically susceptible host. Recent studies have shown that a new group of Escherichia coli, called Adherent Invasive Escherichia coli (AIEC) are present in the guts of CD patients at a higher frequency than in healthy subjects, suggesting that they may play a role in the initiation and/or maintenance of the inflammation associated with CD. Two phenotypes define an AIEC and differentiate them from other groups of E. coli. Firstly, AIEC can adhere to and invade epithelial cells; and secondly, they can replicate in macrophages. In this study, we use a strain of AIEC which has been isolated from the colonic mucosa of a CD patient, called HM605, to examine the relationship between AIEC and the macrophage. We show, using a systematic mutational approach, that while the Tricarboxylic acid (TCA) cycle, the glyoxylate pathway, the Entner-Doudoroff (ED) pathway, the Pentose Phosphate (PP) pathway and gluconeogenesis are dispensable for the intramacrophagic growth of HM605, glycolysis is an absolute requirement for the ability of this organism to replicate intracellularly. We also show that HM605 activates the inflammasome, a major driver of inflammation in mammals. Specifically, we show that macrophages infected with HM605 produce significantly higher levels of the pro-inflammatory cytokine IL-1β than macrophages infected with a non-AIEC strain, and we show by immunoblotting that this is due to cleavage of caspase-1. We also show that macrophages infected with HM605 exhibit significantly higher levels of pyroptosis, a form of inflammatory cell death, than macrophages infected with a non-AIEC strain. Therefore, AIEC strains are more pro-inflammatory than non-AIEC strains and this may have important consequences in terms of CD pathology. Moreover, we show that while inflammasome activation by HM605 is independent of intracellular bacterial replication, it is dependent on an active bacterial metabolism. Through the establishment of a genetic screen aimed at identifying mutants which activate the inflammasome to lower levels than the wild-type, we confirm our observation that bacterial metabolism is essential for successful inflammasome activation by HM605 and we also uncover new systems/structures that may be important for inflammasome activation, such as the BasS/BasR two-component system, a type VI secretion system and a K1 capsule. Finally, in this study, we also identify a putative small RNA in AIEC strain LF82, which may be involved in modulating the motility of this strain. Overall this works shows that, in the absence of specialised virulence factors, the ability of AIEC to metabolise within the host cell may be a key determinant of its pathogenesis.

The impact of first pregnancy and delivery on pelvic floor dysfunction

Background: The first childbirth has the greatest impact on a woman’s pelvic floor when major changes occur. The aim of this study was to comprehensively describe pelvic floor dysfunction (PFD) in young nulliparous women, and its correlation with postnatal pathology. Methods: A prospective study was performed at Cork University Maternity Hospital, Ireland. Initially 1484 nulliparous women completed the validated Australian Pelvic Floor Questionnaire at 15 weeks’ gestation and repeatedly at one year postnatally (N=872). In the second phase, at least one year postnatally, 202 participants without subsequent pregnancies attended the clinical follow up which included: pelvic organ prolapse quantification, a 3D-Transperineal ultrasound scan and collagen level assessment. Results: A high pre-pregnancy prevalence of various types of PFD was detected, which in the majority of cases persisted postnatally and included multiple types of PFD. The first birth had a negative impact on severity of pre-pregnancy symptoms in <15% of cases. Apart from prolapse, vaginal delivery, including instrumental delivery did not increase the risk of PFD symptoms, where as Caesarean section was protective for all types of PFD. The first birth had a bigger impact on pre-existing symptoms of overactive bladder compared to stress urinary incontinence. Pelvic organ prolapse is extremely prevalent in young primiparous women, however usually it is low grade and asymptomatic. Congenital factors and high collagen type III levels play an important role in the aetiology of pelvic organs prolapse. Levator ani trauma is present in one in three women after the first pregnancy and delivery. Conclusion: The main damage to the pelvic floor most likely occurs due to an undiagnosed congenital intrinsic weakness of the pelvic floor structures. PFD is highly associated with first childbirth, however it seems that pregnancy and delivery are contributing factors only which unmask the congenital intrinsic weakness of the pelvic floor support.

Irisin and FNDC5 in retrospect: An exercise hormone or a transmembrane receptor?

FNDC5 (fibronectin domain-containing [protein] 5) was initially discovered and characterized by two groups in 2002. In 2011 FNDC5 burst into prominence as the parent of irisin, a small protein containing the fibronectin type III domain. Irisin was proposed to be secreted by skeletal muscle cells in response to exercise, and to circulate to fat tissue where it induced a transition to brown fat. Since brown fat results in dissipation of energy, this pathway is of considerable interest for metabolism and obesity. Here I review the original discoveries of FNDC5 and the more recent discovery of irisin. I note in particular three problems in the characterization of irisin: the antibodies used to detect irisin in plasma lack validity; the recombinant protein used to demonstrate activity in cell culture was severely truncated; and the degree of shedding of soluble irisin from the cell surface has not been quantitated. The original discovery proposing that FNDC5 may be a transmembrane receptor may deserve a new look.

Irisin - a myth rather than an exercise-inducible myokine.

The myokine irisin is supposed to be cleaved from a transmembrane precursor, FNDC5 (fibronectin type III domain containing 5), and to mediate beneficial effects of exercise on human metabolism. However, evidence for irisin circulating in blood is largely based on commercial ELISA kits which are based on polyclonal antibodies (pAbs) not previously tested for cross-reacting serum proteins. We have analyzed four commercial pAbs by Western blotting, which revealed prominent cross-reactivity with non-specific proteins in human and animal sera. Using recombinant glycosylated and non-glycosylated irisin as positive controls, we found no immune-reactive bands of the expected size in any biological samples. A FNDC5 signature was identified at ~20 kDa by mass spectrometry in human serum but was not detected by the commercial pAbs tested. Our results call into question all previous data obtained with commercial ELISA kits for irisin, and provide evidence against a physiological role for irisin in humans and other species.

Protocol for the "Implementation, adoption, and utility of family history in diverse care settings" study.

BACKGROUND: Risk assessment with a thorough family health history is recommended by numerous organizations and is now a required component of the annual physical for Medicare beneficiaries under the Affordable Care Act. However, there are several barriers to incorporating robust risk assessments into routine care. MeTree, a web-based patient-facing health risk assessment tool, was developed with the aim of overcoming these barriers. In order to better understand what factors will be instrumental for broader adoption of risk assessment programs like MeTree in clinical settings, we obtained funding to perform a type III hybrid implementation-effectiveness study in primary care clinics at five diverse healthcare systems. Here, we describe the study's protocol. METHODS/DESIGN: MeTree collects personal medical information and a three-generation family health history from patients on 98 conditions. Using algorithms built entirely from current clinical guidelines, it provides clinical decision support to providers and patients on 30 conditions. All adult patients with an upcoming well-visit appointment at one of the 20 intervention clinics are eligible to participate. Patient-oriented risk reports are provided in real time. Provider-oriented risk reports are uploaded to the electronic medical record for review at the time of the appointment. Implementation outcomes are enrollment rate of clinics, providers, and patients (enrolled vs approached) and their representativeness compared to the underlying population. Primary effectiveness outcomes are the percent of participants newly identified as being at increased risk for one of the clinical decision support conditions and the percent with appropriate risk-based screening. Secondary outcomes include percent change in those meeting goals for a healthy lifestyle (diet, exercise, and smoking). Outcomes are measured through electronic medical record data abstraction, patient surveys, and surveys/qualitative interviews of clinical staff. DISCUSSION: This study evaluates factors that are critical to successful implementation of a web-based risk assessment tool into routine clinical care in a variety of healthcare settings. The result will identify resource needs and potential barriers and solutions to implementation in each setting as well as an understanding potential effectiveness. TRIAL REGISTRATION: NCT01956773.

Expression of liver-specific member of the 3β-hydroxysteroid dehydrogenase family, an isoform possessing an almost exclusive 3-ketosteroid reductase activity

We have recently characterized two types of rat 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) isoenzymes expressed in adrenals and gonads. In addition, we have cloned a third type of cDNA encoding a predicted type III 3β-HSD protein specifically expressed in the male rat liver which shares 80% similarity with the two other isoenzymes. Transient expression in human HeLa cells of the cDNAs reveals that the type III 3β-HSD protein does not display oxidative activity for the classical substrates of 3β-HSD, in contrast to the type I 3β-HSD isoenzyme. However, in the presence of NADH, type III isoenzyme, in common with the type I isoform, converts 5α-androstane-3,17-dione (A-dione) and 5α-dihydrotestosterone (DHT) to the corresponding 3β-hydroxysteroids. In fact, the type I and the type III isoenzymes have the same affinity for DHT with K(m) values of 5.05 and 6.16 μM, respectively. When NADPH is used as cofactor, the affinity for DHT of the type III isoform becomes higher than that of the type I isoform with K(m) values of 0.12 and 1.18 μM, respectively. The type III isoform is thus a 3-ketoreductase using NADPH as preferred cofactor which is responsible for the conversion of 3-keto-saturated steroids such as DHT and A-dione into less active steroids.

Ice-binding proteins adsorb to their ligand via anchored clathrate waters

The main success of my thesis has been to establish the mechanism by which antifreeze proteins (AFPs) bind irreversibly to ice crystals, and hence prevent their growth. AFPs organize ice-like water on their ice-binding site, which then merges and freezes with the quasi-liquid layer of ice. This was revealed from studying the exceptionally large (ca. 1.5-MDa) Ca 2+-dependent AFP from the Antarctic bacterium Marinomonas primoryensis (MpAFP). The 34-kDa antifreeze- active region of MpAFP was predicted to fold as a novel Ca 2+-binding β-helix. Site-directed mutagenesis confirmed the model and demonstrated that its ice-binding site (IBS) consisted of solvent-exposed Thr and Asx parallel arrays on the Ca 2+-binding turns. The X-ray crystal structure of the antifreeze region was solved to a resolution of 1.7 Å. Two of the four molecules within the unit cell of the crystal had portions of their IBSs freely exposed to solvent. Identical clathrate-like cages of water molecules were present on each IBS. These waters were organized by the hydrophobic effect and anchored to the protein via hydrogen bonds. They matched the spacing of water molecules in an ice lattice, demonstrating that anchored clathrate waters bind AFPs to ice. This mechanism was extended to other AFPs including the globular type III AFP from fishes. Site-directed mutagenesis and a modified ice-etching technique demonstrated this protein uses a compound ice-binding site, comprised of two flat and relatively hydrophobic surfaces, to bind at least two planes of ice. Reinvestigation of several crystal structures of type III AFP identified anchored clathrate waters on the solvent-exposed portion of its compound IBS that matched the spacing of waters on the primary prism plane of ice. Ice nucleation proteins (INPs), which can raise the temperature at which ice forms in solution to just slightly below 0oC, have the opposite effect to AFPs. A novel dimeric β-helical model was proposed for the INP produced by the bacterium Pseudomonas borealis. Molecular dynamics simulations showed that INPs are also capable of ordering water molecules into an ice- like lattice. However, their multimerization brings together sufficient ordered waters to form an ice nucleus and initiate freezing.

A structural basis for different antifreeze protein roles

Antifreeze proteins (AFPs) are produced by a variety of organisms to either protect them from freezing or help them tolerate being frozen. Recent structural work has shown that AFPs bind to ice using ordered surface waters on a particular surface of the protein called the ice-binding site (IBS). These 'anchored clathrate' waters fuse to particular planes of an ice crystal and hence irreversibly bind the AFP to its ligand. An AFP isolated from the perennial ryegrass, Lolium perenne (LpAFP) was previously modelled as a right-handed beta helix with two proposed IBSs. Steric mutagenesis, where small side chains were replaced with larger ones, determined that only one of the putative IBSs was responsible for binding ice. The mutagenesis work also partly validated the fold of the computer-generated model of this AFP. In order to determine the structure of the protein, LpAFP was crystallized and solved to 1.4 Å resolution. The protein folds as an untwisted left-handed beta-helix, of opposite handedness to the model. The IBS identified by mutagenesis is remarkably flat, but less regular than the IBS of most other AFPs. Furthermore, several of the residues constituting the IBS are in multiple conformations. This irregularity may explain why LpAFP causes less thermal hysteresis than many other AFPs. Its imperfect IBS is also argued to be responsible for LpAFP's heightened ice-recrystallization inhibition activity. The structure of LpAFP is the first for a plant AFP and for a protein responsible for providing freeze tolerance rather than freeze resistance. To help understand what constitutes an IBS, a non-ice-binding homologue of type III AFP, sialic acid synthase (SAS), was engineered for ice binding. Point mutations were made to the germinal IBS of SAS to mimic key features seen in type III AFP. The crystal structures of some of the mutant proteins showed that the potential IBS became less charged and flatter as the mutations progressed, and ice affinity was gained. This proof-of-principle study highlights some of the difficulties in AFP engineering.

Plasma biomarker profiling in the detection of growth promoter use in calves

The detection of illicit growth promoter use during meat production within the European Union is reliant on residue testing which is a limiting factor on the number of animals which can be tested and consequently compromises the efficacy of testing procedures. The present study examined a novel detection strategy based on the profiling of plasma component concentrations in response to growth promoter administrations. Calves subjected to nortestosterone decanoate, 17 beta-oestradiol benzoate and dexamethasone were found to have altered urea, aminoterminal propeptide of type III procollagen and sex hormone binding globulin profiles in response to treatments. These findings demonstrate the potential of using the identification of perturbed profiles within a panel of biomarkers which cover a spectrum of biological activity to reveal growth promoter abuse.

Histological and ultrastructural investigation of retinal microaneurysm development in diabetic patients

BACKGROUND: Although microaneurysms are a clinicopathological hallmark of diabetic retinopathy, there have been few ultrastructural studies of these important lesions. As a result, knowledge of the mechanisms involved in the pathogenesis of microaneurysms remains fragmentary. This study provides histological and ultrastructural evidence of various stages in microaneurysm formation within the retinal vasculature. METHODS: The eyes of three type II diabetic patients, obtained within 24 hours of death, were studied by the trypsin digest technique. Eyes from two further type II diabetics were fixed in 2.5% glutaraldehyde within 12 hours of death and processed for electron microscopy. RESULTS: In the trypsin digest preparations, small saccular and fusiform microaneurysms were observed in the peripheral retinal. In the central retina, the microaneurysms ranged in morphology from thin walled, cellular forms to dense, acellular, hyalinised forms. Ultrastructurally, four distinct groups of microaneurysm were observed. Type I showed an extensive accumulation of polymorphonuclear cells into the lumen. The endothelium remained intact, although pericytes were invariably absent. Type II microaneurysms were typified by large numbers of red blood cells (RBCs) in the lumen. Endothelial cells and pericytes were completely absent. The type III microaneurysm was also non-perfused and contained aggregates of irregularly shaped RBC profiles and RBC breakdown products. Recanalisation by new vessels into the occluded lumen was observed in one microaneurysm. Type IV microaneurysms were almost or completely sclerosed, with extensive fibrosis and lipid infiltration into the lumen and basement membrane wall. CONCLUSION: This investigation describes several distinctive stages in the formation of microaneurysms during diabetic retinopathy. With reference to the pathogenesis of retinal microaneurysms, the interaction of various cell types is discussed and the significance of vascular cell death and localised hypertensive events highlighted.