The role of HRS phosphorylation in endosomal trafficking and signalling.

Hepatocyte growth factor receptor substrate (HRS) is a component of the ESCRT-0 complex. It is involved in the sorting of ubiquitylated receptors into the lumen of multivesicular bodies (MVBs), thus targeting them for lysosomal degradation. HRS is highly phosphorylated in response to stimulation by various growth factors, but despite this, the functional role of its phosphorylation is still contentious. Preliminary data from our laboratory suggested that HRS may also act as a signalling adaptor, involved in the crosstalk between receptor tyrosine kinases (RTKs) and G protein couple receptors (GPCRs). In order to investigate this hypothesis, I first examined the EGF dependent interactors of HRS by mass spectrometry. I then developed tools for the measurement of endosomal cAMP production and PKA signalling. My data suggests that HRS interacts with the E3 ligase deltex3-like (DTX3L) in an EGF dependent manner. Although an association between these proteins has previously been made, its EGF dependence is novel. DTX3L has previously been shown to modulate the recruitment of HRS to endosomes in response to stimulation of the G-protein coupled receptor CXCR4. This new finding offers a theoretical means through which different receptor classes could crosstalk and elicit an integrated signalling response. I further show that GFP-HRS is recruited directly to newly formed vesicles containing EGF and its receptor. Recruitment of HRS to endosomes/vesicles in response to TGFα, but not EGF, is partly dependent on phospho-tyrosine residues Y329 and Y334. Furthermore, HRS has recently been implicated in the recruitment of the recycling complex WASH to endosomes. This, combined with the previous observation, suggests that phosphorylation of these two sites may be involved in receptor recycling. Overall, my thesis generates new tools for the study of HRS function and for the analysis of endosomal signalling. I provide evidence for the role of HRS in RTK and GPCR crosstalk alongside providing insights into the mechanisms governing recruitment of HRS

Super-resolution microscopy as a potential approach to platelet granule disorder diagnosis

BACKGROUND: Many platelet functions are dependent on bioactive molecules released from their granules. Deficiencies of these granules in number, shape or content are associated with bleeding. The small size of these granules is such that imaging them for diagnosis has traditionally required electron microscopy. However, recently developed super-resolution microscopes provide sufficient spatial resolution to effectively image platelet granules. When combined with automated image analysis, these methods provide a quantitative, unbiased, rapidly acquired dataset that can readily and reliably reveal differences in platelet granules between individuals. OBJECTIVE: To demonstrate the ability of structured illumination microscopy (SIM) to efficiently differentiate between healthy volunteers and three patients with Hermansky-Pudlak syndrome PATIENT METHODS: Blood samples were taken from three patients with Hermansky Pudlak syndrome and seven controls. Patients 1-3 have gene defects in HPS1, HPS6 and HPS5 respectively; all controls were healthy volunteers. Platelet-rich plasma was isolated from blood and the platelets fixed, stained for CD63, and processed for analysis by immunofluorescence microscopy, using a custom-built SIM microscope. RESULTS: SIM can successfully resolve CD63-positive structures in fixed platelets. A determination of the number of CD63-positive structures per platelet allowed us to conclude that each patient was significantly different from all of the controls with 99% confidence. CONCLUSIONS: A super-resolution imaging approach is effective and rapid in objectively differentiating between patients with a platelet bleeding disorder and healthy volunteers. CD63 is a useful marker for predicting Hermansky-Pudlak Syndrome and could be used in the diagnosis of patients suspected of other Platelet granule disorders. This article is protected by copyright. All rights reserved

A generalized framework unifying image registration and respiratory motion models and incorporating image reconstruction, for partial image data or full images.

Surrogate-driven respiratory motion models relate the motion of the internal anatomy to easily acquired respiratory surrogate signals, such as the motion of the skin surface. They are usually built by first using image registration to determine the motion from a number of dynamic images, and then fitting a correspondence model relating the motion to the surrogate signals. In this paper we present a generalized framework that unifies the image registration and correspondence model fitting into a single optimization. This allows the use of 'partial' imaging data, such as individual slices, projections, or k-space data, where it would not be possible to determine the motion from an individual frame of data. Motion compensated image reconstruction can also be incorporated using an iterative approach, so that both the motion and a motion-free image can be estimated from the partial image data. The framework has been applied to real 4DCT, Cine CT, multi-slice CT, and multi-slice MR data, as well as simulated datasets from a computer phantom. This includes the use of a super-resolution reconstruction method for the multi-slice MR data. Good results were obtained for all datasets, including quantitative results for the 4DCT and phantom datasets where the ground truth motion was known or could be estimated

Using honey to heal diabetic foot ulcers

Diabetic ulcers seem to be arrested in the inflammatory/proliferative stage of the healing process, allowing infection and inflammation to preclude healing. Antibiotic-resistant bacteria have become a major cause of infections, including diabetic foot infections. It is proposed here that the modern developments of an ancient and traditional treatment for wounds, dressing them with honey, provide the solution to the problem of getting diabetic ulcers to move on from the arrested state of healing. Honeys selected to have a high level of antibacterial activity have been shown to be very effective against antibiotic-resistant strains of bacteria in laboratory and clinical studies. The potent anti-inflammatory action of honey is also likely to play an important part in overcoming the impediment to healing that inflammation causes in diabetic ulcers, as is the antioxidant activity of honey. The action of honey in promotion of tissue regeneration through stimulation of angiogenesis and the growth of fibroblasts and epithelial cells, and its insulin-mimetic effect, would also be of benefit in stimulating the healing of diabetic ulcers. The availability of honey-impregnated dressings which conveniently hold honey in place on ulcers has provided a means of rapidly debriding ulcers and removing the bacterial burden so that good healing rates can be achieved with neuropathic ulcers. With ischemic ulcers, where healing cannot occur because of lack of tissue viability, these honey dressings keep the ulcers clean and prevent infection occurring

Interpretation of evidence in data by untrained medical students: a scenario-based study

<p>Abstract</p> <p>Background</p> <p>To determine which approach to assessment of evidence in data - statistical tests or likelihood ratios - comes closest to the interpretation of evidence by untrained medical students.</p> <p>Methods</p> <p>Empirical study of medical students (N = 842), untrained in statistical inference or in the interpretation of diagnostic tests. They were asked to interpret a hypothetical diagnostic test, presented in four versions that differed in the distributions of test scores in diseased and non-diseased populations. Each student received only one version. The intuitive application of the statistical test approach would lead to rejecting the null hypothesis of no disease in version A, and to accepting the null in version B. Application of the likelihood ratio approach led to opposite conclusions - against the disease in A, and in favour of disease in B. Version C tested the importance of the p-value (A: 0.04 versus C: 0.08) and version D the importance of the likelihood ratio (C: 1/4 versus D: 1/8).</p> <p>Results</p> <p>In version A, 7.5% concluded that the result was in favour of disease (compatible with p value), 43.6% ruled against the disease (compatible with likelihood ratio), and 48.9% were undecided. In version B, 69.0% were in favour of disease (compatible with likelihood ratio), 4.5% against (compatible with p value), and 26.5% undecided. Increasing the p value from 0.04 to 0.08 did not change the results. The change in the likelihood ratio from 1/4 to 1/8 increased the proportion of non-committed responses.</p> <p>Conclusions</p> <p>Most untrained medical students appear to interpret evidence from data in a manner that is compatible with the use of likelihood ratios.</p

The extraordinary evolutionary history of the reticuloendotheliosis viruses

The reticuloendotheliosis viruses (REVs) comprise several closely related amphotropic retroviruses isolated from birds. These viruses exhibit several highly unusual characteristics that have not so far been adequately explained, including their extremely close relationship to mammalian retroviruses, and their presence as endogenous sequences within the genomes of certain large DNA viruses. We present evidence for an iatrogenic origin of REVs that accounts for these phenomena. Firstly, we identify endogenous retroviral fossils in mammalian genomes that share a unique recombinant structure with REVs—unequivocally demonstrating that REVs derive directly from mammalian retroviruses. Secondly, through sequencing of archived REV isolates, we confirm that contaminated Plasmodium lophurae stocks have been the source of multiple REV outbreaks in experimentally infected birds. Finally, we show that both phylogenetic and historical evidence support a scenario wherein REVs originated as mammalian retroviruses that were accidentally introduced into avian hosts in the late 1930s, during experimental studies of P. lophurae, and subsequently integrated into the fowlpox virus (FWPV) and gallid herpesvirus type 2 (GHV-2) genomes, generating recombinant DNA viruses that now circulate in wild birds and poultry. Our findings provide a novel perspective on the origin and evolution of REV, and indicate that horizontal gene transfer between virus families can expand the impact of iatrogenic transmission events

Attitude toward contraception and abortion among Curaçao women. Ineffective contraception due to limited sexual education?

Background In Curaçao is a high incidence of unintended pregnancies and induced abortions. Most of the induced abortions in Curaçao are on request of the woman and performed by general practitioners. In Curaçao, induced abortion is strictly prohibited, but since 1999 there has been a policy of connivance. We present data on the relevance of economic and socio-cultural factors for the high abortion-rates and the ineffective use of contraception. Methods Structured interviews to investigate knowledge and attitudes toward sexuality, contraception and abortion and reasons for ineffective use of contraceptives among women, visiting general practitioners. Results Of 158 women, 146 (92%) participated and 82% reported that their education on sexuality and about contraception was of good quality. However 'knowledge of reliable contraceptive methods' appeared to be - in almost 50% of the cases - false information, misjudgements or erroneous views on the chance of getting pregnant using coitus interruptus and about the reliability and health effects of oral contraceptive pills. Almost half of the interviewed women had incorrect or no knowledge about reliability of condom use and IUD. 42% of the respondents risked by their behavior an unplanned pregnancy. Most respondents considered abortion as an emergency procedure, not as contraception. Almost two third experienced emotional, physical or social problems after the abortion. Conclusions Respondents had a negative attitude toward reliable contraceptives due to socio-cultural determined ideas about health consequences and limited sexual education. Main economic factors were costs of contraceptive methods, because most health insurances in Curaçao do not cover contraceptives. To improve the effective use of reliable contraceptives, more adequate information should be given, targeting the wrong beliefs and false information. The government should encourage health insurance companies to reimburse contraceptives. Furthermore, improvement of counseling during the abortion procedure is important

A Bayesian method for calculating real-time quantitative PCR calibration curves using absolute plasmid DNA standards

<p>Abstract</p> <p>Background</p> <p>In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignored in calibration calculations and in some cases impossible to characterize. A flexible statistical method that can account for uncertainty between plasmid and genomic DNA targets, replicate testing, and experiment-to-experiment variability is needed to estimate calibration curve parameters such as intercept and slope. Here we report the use of a Bayesian approach to generate calibration curves for the enumeration of target DNA from genomic DNA samples using absolute plasmid DNA standards.</p> <p>Results</p> <p>Instead of the two traditional methods (classical and inverse), a Monte Carlo Markov Chain (MCMC) estimation was used to generate single, master, and modified calibration curves. The mean and the percentiles of the posterior distribution were used as point and interval estimates of unknown parameters such as intercepts, slopes and DNA concentrations. The software WinBUGS was used to perform all simulations and to generate the posterior distributions of all the unknown parameters of interest.</p> <p>Conclusion</p> <p>The Bayesian approach defined in this study allowed for the estimation of DNA concentrations from environmental samples using absolute standard curves generated by real-time qPCR. The approach accounted for uncertainty from multiple sources such as experiment-to-experiment variation, variability between replicate measurements, as well as uncertainty introduced when employing calibration curves generated from absolute plasmid DNA standards.</p

Appropriate criteria for identification of near-miss maternal morbidity in tertiary care facilities: A cross sectional study

<p>Abstract</p> <p>Background</p> <p>The study of severe maternal morbidity survivors (near miss) may be an alternative or a complement to the study of maternal death events as a health care indicator. However, there is still controversy regarding the criteria for identification of near-miss maternal morbidity. This study aimed to characterize the near miss maternal morbidity according to different sets of criteria.</p> <p>Methods</p> <p>A descriptive study in a tertiary center including 2,929 women who delivered there between July 2003 and June 2004. Possible cases of near miss were daily screened by checking different sets of criteria proposed elsewhere. The main outcome measures were: rate of near miss and its primary determinant factors, criteria for its identification, total hospital stay, ICU stay, and number and kind of special procedures performed.</p> <p>Results</p> <p>There were two maternal deaths and 124 cases of near miss were identified, with 102 of them admitted to the ICU (80.9%). Among the 126 special procedures performed, the most frequent were central venous access, echocardiography and invasive mechanical ventilation. The mean hospital stay was 10.3 (± 13.24) days. Hospital stay and the number of special procedures performed were significantly higher when the organ dysfunction based criteria were applied.</p> <p>Conclusion</p> <p>The adoption of a two level screening strategy may lead to the development of a consistent severe maternal morbidity surveillance system but further research is needed before worldwide near miss criteria can be assumed.</p

Women and postfertilization effects of birth control: consistency of beliefs, intentions and reported use

BACKGROUND: This study assesses the consistency of responses among women regarding their beliefs about the mechanisms of actions of birth control methods, beliefs about when human life begins, the intention to use or not use birth control methods that they believe may act after fertilization or implantation, and their reported use of specific methods. METHODS: A questionnaire was administered in family practice and obstetrics and gynecology clinics in Salt Lake City, Utah, and Tulsa, Oklahoma. Participants included women ages 18–50 presenting for any reason and women under age 18 presenting for family planning or pregnancy care. Analyses were based on key questions addressing beliefs about whether specific birth control methods may act after fertilization, beliefs about when human life begins, intention to use a method that may act after fertilization, and reported use of specific methods. The questionnaire contained no information about the mechanism of action of any method of birth control. Responses were considered inconsistent if actual use contradicted intentions, if one intention contradicted another, or if intentions contradicted beliefs. RESULTS: Of all respondents, 38% gave consistent responses about intention to not use or to stop use of any birth control method that acted after fertilization, while 4% gave inconsistent responses. The corresponding percentages for birth control methods that work after implantation were 64% consistent and 2% inconsistent. Of all respondents, 34% reported they believed that life begins at fertilization and would not use any birth control method that acts after fertilization (a consistent response), while 3% reported they believed that life begins at fertilization but would use a birth control method that acts after fertilization (inconsistent). For specific methods of birth control, less than 1% of women gave inconsistent responses. A majority of women (68% or greater) responded accurately about the mechanism of action of condoms, abstinence, sterilization, and abortion, but a substantial percentage of women (between 19% and 57%) were uncertain about the mechanisms of action of oral contraceptives, intrauterine devices (IUDs), Depo-Provera, or natural family planning. CONCLUSION: Women who believe that life begins at fertilization may not intend to use a birth control method that could have postfertilization effects. More research is needed to understand the relative importance of postfertilization effects for women in other populations, and in relation to other properties of and priorities for birth control methods. However, many women were uncertain about the mechanisms of action of specific methods. To respect the principles of informed consent, some women may need more education about what is known and not known about the mechanisms of action of birth control methods