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2.  PspK of Streptococcus pneumoniae Increases Adherence to Epithelial Cells and Enhances Nasopharyngeal Colonization 
Infection and Immunity  2013;81(1):173-181.
Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and can cause invasive disease aided by the pneumococcal capsule. Group II nontypeable S. pneumoniae (NTSp) lacks a polysaccharide capsule, and a subgroup of NTSp carriage isolates has been found to have a novel gene, pneumococcal surface protein K (pspK), which replaces the capsule locus. A recent rise in the number of NTSp isolates colonizing the human nasopharynx has been observed, but the colonization factors of NTSp have not been well studied. PspK has been shown to play a role in mouse colonization. We therefore examined PspK-mediated immune evasion along with adherence to host cells and colonization. PspK bound human secretory immunoglobulin A (sIgA) but not the complement regulator factor H and did not decrease C3b deposition on the pneumococcal surface. PspK increased binding of pneumococci to epithelial cells and enhanced pneumococcal colonization independently of the genetic background. Understanding how NTSp colonizes and survives within the nasopharynx is important due to the increase in NTSp carriage. Our data suggest that PspK may aid in the persistence of NTSp within the nasopharynx but is not involved in invasion.
PMCID: PMC3536125  PMID: 23115034
3.  Biomarker Discovery Using New Metabolomics Software for Automated Processing of High Resolution LC-MS Data 
Robust biomarkers of target engagement and efficacy are required in different stages of drug discovery. Liquid chromatography coupled to high resolution mass spectrometry provides sensitivity, accuracy and wide dynamic range required for identification of endogenous metabolites in biological matrices. LCMS is widely-used tool for biomarker identification and validation. Typical high resolution LCMS profiles from biological samples may contain greater than a million mass spectral peaks corresponding to several thousand endogenous metabolites. Reduction of the total number of peaks, component identification and statistical comparison across sample groups remains to be a difficult and time consuming challenge. Blood samples from four groups of rats (male vs. female, fully satiated and food deprived) were analyzed using high resolution accurate mass (HRAM) LCMS. All samples were separated using a 15 minute reversed-phase C18 LC gradient and analyzed in both positive and negative ion modes. Data was acquired using 15K resolution and 5ppm mass measurement accuracy. The entire data set was analyzed using software developed in collaboration between Bristol Meyers Squibb and Thermo Fisher Scientific to determine the metabolic effects of food deprivation on rats. Metabolomic LC-MS data files are extraordinarily complex and appropriate reduction of the number of spectral peaks via identification of related peaks and background removal is essential. A single component such as hippuric acid generates more than 20 related peaks including isotopic clusters, adducts and dimers. Plasma and urine may contain 500-1500 unique quantifiable metabolites. Noise filtering approaches including blank subtraction were used to reduce the number of irrelevant peaks. By grouping related signals such as isotopic peaks and alkali adducts, data processing was greatly simplified by reducing the total number of components by 10-fold. The software processes 48 samples in under 60minutes. Principle Component Analysis showed substantial differences in endogenous metabolites levels between the animal groups. Annotation of components was accomplished via searching the ChemSpider database. Tentative assignments made using accurate mass need further verification by comparison with the retention time of authentic standards.
PMCID: PMC3186631
4.  Culture of Impure Human Islet Fractions in the Presence of Alpha-1-Antitrypsin Prevents Insulin Cleavage and Improves Islet Recovery 
Transplantation proceedings  2010;42(6):2055-2057.
Exocrine tissue is commonly cotransplanted with islets in autografting and allotransplantation of impure preparations. Proteases and insulin are released by acinar cells and islets, respectively, during pretransplantation culture and also systemically after transplantation. We hypothesized that released proteases could cleave insulin molecules and that addition of alpha 1 antitrypsin (A1AT) to impure islet cultures would block this cleavage, improving islet recovery and function.
Trypsin, chymotrypsin, and elastase (TCE) activity and insulin levels were measured in culture supernates of pure (n = 5) and impure (n = 5) islet fractions, which were isolated from deceased donors. SDS-PAGE was used to detect insulin after incubation with proteases. We assessed the effects of A1AT supplementation (0.5 mg/mL; n = 4] on TCE activity, insulin levels, culture recovery, and islet quality. The ultrastructure of islets exposed to TCE versus control medium was examined using electron microscopy (EM).
Protease (TCE) activity in culture supernates was directly proportional to the percentage purity of islets: pure, impure, or highly impure. Increasingly lower levels of insulin were detected in culture supernates with higher protease activity levels. Insulin levels measured in supernates of 2000 IE aliquots of impure and highly impure islet preparations were 61 ± 23.7% and 34 ± 33% of that in pure preparations, respectively. Incubation with commercially available proteases (TCE) or exocrine acinar cell supernates cleaved insulin molecules as assessed using SDS-PAGE. Addition of A1AT to impure islet preparations reduced protease activity and restored normal insulin levels as detected using enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE of culture supernates. A1AT improved insulin levels to 98% ± 1.3% in impure and 78% ± 34.2% in highly impure fractions compared with pure islet fractions. A1AT supplementation improved postculture recovery of islets in impure preparations compared with nontreated controls (72% ± 9% vs 47% ± 15%). Islet viability as measured using membrane integrity assays was similar in both the control (98% ± 2%) and the A1AT-treated groups (99% ± 1%). EM results revealed reduction or absence of secretory granules after exposure to proteases (TCE).
Culture of impure human islet fractions in the presence of A1AT prevented insulin cleavage and improved islet recovery. A1AT supplementation of islet culture media, therefore, may increase the proportion of human islet products that meet release criteria for transplantation.
PMCID: PMC2924667  PMID: 20692406
5.  The genome of the simian and human malaria parasite Plasmodium knowlesi 
Nature  2008;455(7214):799-803.
Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the ‘kra’ monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia1,2. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated3, and it has a close phylogenetic relationship to Plasmodium vivax​4, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or ‘hypnozoite’ in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone5) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome4 and other sequenced Plasmodium genomes6-8. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs9, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.
PMCID: PMC2656934  PMID: 18843368
6.  The genome of the social amoeba Dictyostelium discoideum 
Eichinger, L. | Pachebat, J.A. | Glöckner, G. | Rajandream, M.-A. | Sucgang, R. | Berriman, M. | Song, J. | Olsen, R. | Szafranski, K. | Xu, Q. | Tunggal, B. | Kummerfeld, S. | Madera, M. | Konfortov, B. A. | Rivero, F. | Bankier, A. T. | Lehmann, R. | Hamlin, N. | Davies, R. | Gaudet, P. | Fey, P. | Pilcher, K. | Chen, G. | Saunders, D. | Sodergren, E. | Davis, P. | Kerhornou, A. | Nie, X. | Hall, N. | Anjard, C. | Hemphill, L. | Bason, N. | Farbrother, P. | Desany, B. | Just, E. | Morio, T. | Rost, R. | Churcher, C. | Cooper, J. | Haydock, S. | van Driessche, N. | Cronin, A. | Goodhead, I. | Muzny, D. | Mourier, T. | Pain, A. | Lu, M. | Harper, D. | Lindsay, R. | Hauser, H. | James, K. | Quiles, M. | Babu, M. Madan | Saito, T. | Buchrieser, C. | Wardroper, A. | Felder, M. | Thangavelu, M. | Johnson, D. | Knights, A. | Loulseged, H. | Mungall, K. | Oliver, K. | Price, C. | Quail, M.A. | Urushihara, H. | Hernandez, J. | Rabbinowitsch, E. | Steffen, D. | Sanders, M. | Ma, J. | Kohara, Y. | Sharp, S. | Simmonds, M. | Spiegler, S. | Tivey, A. | Sugano, S. | White, B. | Walker, D. | Woodward, J. | Winckler, T. | Tanaka, Y. | Shaulsky, G. | Schleicher, M. | Weinstock, G. | Rosenthal, A. | Cox, E.C. | Chisholm, R. L. | Gibbs, R. | Loomis, W. F. | Platzer, M. | Kay, R. R. | Williams, J. | Dear, P. H. | Noegel, A. A. | Barrell, B. | Kuspa, A.
Nature  2005;435(7038):43-57.
The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes encode ~12,500 predicted proteins, a high proportion of which have long repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal rDNA element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal/fungal lineage after the plant/animal split, but Dictyostelium appears to have retained more of the diversity of the ancestral genome than either of these two groups.
PMCID: PMC1352341  PMID: 15875012
7.  Sleep · 7: Positive airway pressure therapy for obstructive sleep apnoea/hypopnoea syndrome 
Thorax  2005;60(1):68-75.
The use of continuous positive airway pressure (CPAP) in treating symptoms associated with OSAHS is reviewed. Although it is an imperfect intervention, it continues to evolve and improve in such a way that patients who would not have been able to use this treatment even in the recent past can benefit from it today.
PMCID: PMC1747175  PMID: 15618587
8.  Regional implementation of the NWC guideline on ST-elevation myocardial infarction 
Netherlands Heart Journal  2005;13(11):401-407.
The NVVC guideline on ST-elevation myocardial infarction forms the basis for the regional prehospital triage (PHT) project in Zuidoost Brabant. In this project diagnosis and treatment strategies are determined in the ambulance.
To summarise quality assessment and clinical results after one year.
We evaluated the protocol and patient record form, the patient's call, assignment of tasks, diagnosis, treatment, time intervals, information to hospitals, cooperation and data transmission. Time delays were compared with time delays in a regional dry run before the start of the project and with time delays reported in the literature.
Patients still wait over one hour before seeking medical attention. The GP received the majority (65%) of patient calls. In half of all cases (51%), GPs call the ambulance centre only after they have seen the patient. When the patient calls the ambulance centre (35%), information to the GP is either prompt or absent. In 77% of calls to 112 it remains unclear whether the GP was informed at all. The treatment strategy was correct in 97% of cases. Time between symptoms and call decreased in comparison with our local preliminary investigation. Quality assessment after one year shows protocol deviations that are either logical procedural improvements or correctable flaws with no substantial negative influence.
Short-term clinical results are good, but structured follow-up is needed to reduce mortality in the long term, especially after thrombolysis. A guideline is a snapshot of a dynamic process. The PHT project allows rapid adaptations to be made to new paradigms.
PMCID: PMC2497361
STEMI; prehospital thrombolysis; primary PCI
9.  Choroidal folds and papilloedema 
The British Journal of Ophthalmology  1999;83(10):1139-1143.
AIMS—To assess the clinical and fluorescein angiographic features of choroidal folds seen in association with papilloedema.
METHODS—In a retrospective study, the clinical data from a database on patients with choroidal folds (1963-97), including fundus photography and fluorescein angiography, from 32 patients (64 eyes) with choroidal folds in association with papilloedema were reviewed. The clinical and fluorescein angiographic features and the clinical course of choroidal folds in these patients are described.
RESULTS—32 patients had choroidal folds associated with papilloedema. Folds of two distinct categories were observed, either coarse folds or wrinkles. The folds persisted in all cases, even after resolution of papilloedema. Follow up ranged from 1 month to 20 years. Only one patient suffered permanent visual impairment as a result of a choroidal fold.
CONCLUSIONS—Choroidal folds exist in two forms, coarse folds and wrinkles. They persist even after papilloedema has resolved. Final visual acuity did not appear to be affected by the presence of choroidal folds in the majority of patients.

PMCID: PMC1722841  PMID: 10502574
12.  Papilloedema, a complication of progressive diaphyseal dysplasia: a series of three case reports 
The British Journal of Ophthalmology  1998;82(9):1042-1048.
BACKGROUND/AIMS—Progressive diaphyseal dysplasia (PDD) is a rare, autosomal dominant, osteosclerotic dysplasia affecting both endochondrally and intramembranously derived bones. Severely affected patients can develop progressive stenosis of the optic canals and compressive optic neuropathy. Although raised intracranial pressure (ICP) has been described in patients with PDD in whom visual loss has occurred, the elevation of ICP in those patients has been thought to be either non-contributory or only partially responsible for the accompanying visual loss.
METHODS—Three cases were reviewed and the clinical and radiological characteristics are described here.
RESULTS—All three patients had bilateral optic disc swelling with no radiological evidence of either compressive optic neuropathy or thrombosis of the intracranial venous sinuses. The aetiology of the disc swelling was proved to be papilloedema in the first two cases and was probably the dominant cause in the third case.
CONCLUSION—The visual loss documented in at least two of the three patients reported appears to be solely attributable to raised ICP. Normalisation of the ICP has led to an improvement and stabilisation of the visual function in all three patients. Patients with PDD probably require periodic ophthalmic assessments.

 Keywords: progressive diaphyseal dysplasia; papilloedema
PMCID: PMC1722750  PMID: 9893596
13.  Ocular neuromyotonia. 
AIMS/BACKGROUND: Ocular neuromyotonia is characterised by spontaneous spasm of extraocular muscles and has been described in only 14 patients. Three further cases, two with unique features, are described, and the underlying mechanism reviewed in the light of recent experimental evidence implicating extracellular potassium concentration in causing spontaneous firing in normal and demyelinated axons. METHODS: Two patients had third nerve neuromyotonia, one due to compression by an internal carotid artery aneurysm, which has not been reported previously, while the other followed irradiation of a pituitary tumour, a common association in the published reports. Selective activation occurred in both, where neuromyotonic activity was triggered by prolonged voluntary activation of specific extraocular muscles with or without spread of activity to other third nerve muscles. The other patient had fourth nerve involvement, where spasms of the superior oblique muscle were induced only by alcohol, a phenomenon which has not been described. RESULTS: The two patients with third nerve involvement responded to carbamazepine and in one, an improvement in a chronic partial third nerve paresis occurred. The other has not required treatment and remains asymptomatic by refraining from alcohol. CONCLUSIONS: A careful examination, including the effects of prolonged voluntary muscle action is required to initiate episodes and to demonstrate selective activation. Imaging is mandatory to exclude compressive intracranial lesions, particularly where there is no history of pituitary fossa irradiation. A trial of anticonvulsants should be considered in all patients. Extracellular potassium may play a role in spontaneous firing and ephatic transmission in ocular neuromyotonia.
PMCID: PMC505464  PMID: 8703889
14.  After the strike: using facilitation in a residency training program 
Methods of alternative dispute resolution, including facilitation, can be used to identify and resolve areas of conflict. Facilitation was used by the University of Saskatchewan's Department of Family Medicine (Saskatoon division) after the strike by residents in July and August 1995 so as to allow optimal use of the remaining educational time. Through facilitation, experiences of the strike and areas of potential conflict were explored. Participants had a broad range of responses to the strike. Specific coping strategies were developed to deal with identified concerns. Although outcomes were not measured formally, levels of trust improved and collegial relationships were restored. Because so many changes occur in health care and medical education, conflict inevitably arises. Facilitation offers one way of dealing with change constructively, thereby making possible the optimal use of educational time.
PMCID: PMC1229008  PMID: 9526479
18.  Regulation of the chicken ovalbumin gene by estrogen and corticosterone requires a novel DNA element that binds a labile protein, Chirp-1. 
Molecular and Cellular Biology  1996;16(5):2015-2024.
Because induction of the chicken ovalbumin (Ov) gene by steroid hormones requires concomitant protein synthesis, efforts have focused on defining the binding site in the Ov gene for a labile transcription factor. Previous gel mobility shift studies identified one such site in the steroid-dependent regulatory element (SDRE) between -900 and -853. To ascertain whether estrogen and glucocorticoid affect the binding of this labile protein, genomic footprinting of the Ov gene was done by treating primary oviduct cell cultures with dimethyl sulfate. Several alterations that include steroid-dependent protection of guanine residues -889 and -885 and hypersensitivity of adenine residues -892 and -865 were observed. Of particular importance, the in vivo footprinting data are corroborated by two functional studies, one with linker-scanning mutations and the other with point mutations. Ten-base-pair linker-scanning mutations between -900 and -878 severely reduced the induction by estrogen and glucocorticoid. Likewise, point mutations of the protected guanine residues profoundly attenuated the response to these steroid hormones. In addition, in vitro binding activity correlated with in vivo functional activity. For example, mutant A4e shows no transcriptional activity in response to steroid hormones, and a corresponding oligomer does not bind protein in vitro. In contrast, mutant A4c is fully active in both contexts. These data support the contention that the ovalbumin gene is regulated by a steroid hormone-induced transcriptional cascade that culminates in the binding of chicken ovalbumin induced regulatory protein or protein complex (Chirp-I) to a DNA element from -891 to -878 in the SDRE.
PMCID: PMC231188  PMID: 8628267
20.  Vision. 
PMCID: PMC1073593  PMID: 7608708
22.  High resolution magnetic resonance imaging of the anterior visual pathway in patients with optic neuropathies using fast spin echo and phased array local coils. 
High resolution MRI of the anterior visual pathways was evaluated using frequency selective fat suppressed fast spin echo (FSE) sequences in conjunction with phased array local coils in patients with optic neuropathies. Fifteen normal controls and 57 patients were examined. Coronal T2 weighted fat suppressed FSE images were obtained in 11 minutes with an in plane resolution of 0.39 x 0.39 mm. The optic nerve and its sheath containing CSF were clearly differentiated. Central retinal vessels were often visible. In demyelinating optic neuritis and in anterior ischaemic optic neuropathy high signal within the nerve was readily delineated. Meningiomas and gliomas involving the optic nerve were precisely visualised both in the orbit and intracranially. Extrinsic compression of the optic nerves was readily visualised in carotid artery ectasia and dysthyroid eye disease. Enlarged subarachnoid spaces around the optic nerves were demonstrated in benign intracranial hypertension. High resolution MRI of the anterior visual pathway represents an advance in the diagnosis and management of patients presenting with optic neuropathy.
PMCID: PMC1073486  PMID: 7745403
24.  Incidence of acute symptomatic toxoplasma retinochoroiditis in south London according to country of birth. 
BMJ : British Medical Journal  1995;310(6986):1037-1040.
OBJECTIVE--To determine the incidence of acute symptomatic toxoplasma retinochoroiditis presenting to ophthalmologists for patients born in Britain and elsewhere. DESIGN--Population based, cross sectional study. SETTING--11 districts in south Greater London. SUBJECTS--All patients presenting to NHS ophthalmologists with symptoms due to acute toxoplasma retinochoroiditis in 1992-3. MAIN OUTCOME MEASURE--Intraocular inflammation in association with a retinochoroidal scar, active adjoining retinitis, and IgG serum antibodies to toxoplasma. RESULTS--The estimated incidence of acute symptomatic retinochoroiditis for all people born in Britain was 0.4/100,000/year. If a mean of two symptomatic episodes per lifetime is assumed, 100 people born in Britain may be affected each year, about a fifth of the estimated 500-600 congenitally infected people born each year. CONCLUSIONS--A substantial proportion of people with acute symptomatic toxoplasma retinochoroiditis were born outside the country, and the number born in Britain was smaller than the number previously estimated to develop retinochoroidal lesions due to congenital toxoplasmosis. These findings suggest that prenatal screening for toxoplasmosis in Britain may be of limited benefit.
PMCID: PMC2549430  PMID: 7728057
25.  Optic nerve sheath decompression for the treatment of visual failure in chronic raised intracranial pressure. 
The records of all patients undergoing optic nerve sheath decompression for visual failure in chronic raised intracranial pressure performed over a 15 year period have been reviewed. The aim was to study the visual outcome and relation to any shunting procedures. Fourteen patients (20 eyes) were identified in whom follow up information of at least one year was available. Eleven patients had benign intracranial hypertension (idiopathic intracranial hypertension) and three had dural venous sinus occlusive disease. Eight patients had unilateral surgery and six had bilateral surgery. Visual acuity and fields either improved or stabilised in 17 out of 20 eyes and three deteriorated. Of the eight patients undergoing unilateral surgery, the other eye remained stable in seven and deteriorated in one. Four patients required optic nerve sheath decompression despite previous shunting or subtemporal decompression. Five patients required shunts or subtemporal decompression after optic nerve sheath decompression because of persistent headache in three cases and for uncontrolled visual failure in two cases. No patients lost vision as a direct consequence of surgery. It is concluded that optic nerve sheath decompression is a safe and important therapeutic option in the management of chronic raised intracranial pressure complicated by visual loss. Vision can be saved after shunt failure, and in other cases may be maintained without the need for a shunt. Shunts may still be required, however, after optic nerve sheath decompression, especially for persistent headache.
PMCID: PMC1073203  PMID: 7964827

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