The production of graceful skeletal movements requires coordinated activation of multiple muscles that produce torques around multiple joints. The work described herein is focused on one such movement, stance, that requires coordinated activation of extensor muscles acting around the hip, knee and ankle joints. The forces evoked in these muscles by external stimulation all have a complex dependence on muscle length and shortening velocities, and some of these muscles are bi-articular. In order to recreate sit-to-stand maneuvers in the anesthetized feline, we excited the hind limb musculature using intrafascicular multielectrode stimulation (IFMS) of the muscular branch of the sciatic nerve, the femoral nerve, and the main branch of the sciatic nerve. Stimulation was achieved with either acutely or chronically implanted Utah Slanted Electrode Arrays (USEAs) via subsets of electrodes 1) that activated motor units in the extensor muscles of the hip, knee, and ankle joints, 2) that were able to evoke large extension forces, and 3) that manifested minimal coactivation of the targeted motor units. Three hind limb force-generation strategies were investigated, including sequential activation of independent motor units to increase force, and interleaved or simultaneous IFMS of three sets of six or more USEA electrodes that excited the hip, knee, and ankle extensors. All force-generation strategies evoked stance, but the interleaved IFMS strategy also reduced muscle fatigue produced by repeated sit-to-stand maneuvers compared with fatigue produced by simultaneous activation of different motor neuron pools. These results demonstrate the use of interleaved IFMS as a means to recreate coordinated, fatigue-resistant multi-joint muscle forces in the unilateral hind limb. This muscle activation paradigm could provide a promising neuroprosthetic approach for the restoration of sit-to-stand transitions in individuals who are paralyzed by spinal cord injury, stroke, or disease.
Neural prosthetics; intrafascicular stimulation; motor system; stance
This paper characterizes the Utah Slant Optrode Array (USOA) as a means to deliver infrared light deep into tissue. An undoped crystalline silicon (100) substrate was used to fabricate 10 × 10 arrays of optrodes with rows of varying lengths from 0.5 mm to 1.5 mm on a 400-μm pitch. Light delivery from optical fibers and loss mechanisms through these Si optrodes were characterized, with the primary loss mechanisms being Fresnel reflection, coupling, radiation losses from the tapered shank and total internal reflection in the tips. Transmission at the optrode tips with different optical fiber core diameters and light in-coupling interfaces was investigated. At λ = 1.55μm, the highest optrode transmittance of 34.7%, relative to the optical fiber output power, was obtained with a 50-μm multi-mode fiber butt-coupled to the optrode through an intervening medium of index n = 1.66. Maximum power is directed into the optrodes when using fibers with core diameters of 200 μm or less. In addition, the output power varied with the optrode length/taper such that longer and less tapered optrodes exhibited higher light transmission efficiency. Output beam profiles and potential impacts on physiological tests were also examined. Future work is expected to improve USOA efficiency to greater than 64%.
(170.3890) Medical optics instrumentation; (220.4610) Optical fabrication; (230.7380) Waveguides, channeled; (260.3060) Infrared
Endosymbiotic bacteria were identified in the parasitic ciliate Ichthyophthirius multifiliis, a common pathogen of freshwater fish. PCR amplification of DNA prepared from two isolates of I. multifiliis, using primers that bind conserved sequences in bacterial 16S rRNA genes, generated an ∼1,460-bp DNA product, which was cloned and sequenced. Sequence analysis demonstrated that 16S rRNA gene sequences from three classes of bacteria were present in the PCR product. These included Alphaproteobacteria (Rickettsiales), Sphingobacteria, and Flavobacterium columnare. DAPI (4′,6-diamidino-2-phenylindole) staining showed endosymbionts dispersed throughout the cytoplasm of trophonts and, in most, but not all theronts. Endosymbionts were observed by transmission electron microscopy in the cytoplasm, surrounded by a prominent, electron-translucent halo characteristic of Rickettsia. Fluorescence in situ hybridization demonstrated that bacteria from the Rickettsiales and Sphingobacteriales classes are endosymbionts of I. multifiliis, found in the cytoplasm, but not in the macronucleus or micronucleus. In contrast, F. columnare was not detected by fluorescence in situ hybridization. It likely adheres to I. multifiliis through association with cilia. The role that endosymbiotic bacteria play in the life history of I. multifiliis is not known.
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 vivax4, 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.
Although guidance exists for the use of many laboratory tests in a wide range of clinical situations, this guidance is spread among a range of literature sources, and is often directed at laboratory specialists rather than test users. Individual general practices display large variations in standardised test requesting, yet much of their testing activity involves a relatively small range of tests. This paper describes a methodological approach to review the available evidence and guidance and to extract relevant primary research work to examine a range of testing scenarios in general practice, with the aim of formulating guidance based on the best available evidence or consensus opinions.
appropriateness; best practice; evidence based pathology; multidisciplinary
Apolipoproteins (apo) A-I and C-III are components of high-density lipoprotein-cholesterol (HDL-C), a quantitative trait negatively correlated with risk of cardiovascular disease (CVD). We analyzed the contribution of individual and pairwise combinations of single nucleotide polymorphisms (SNPs) in the APOA1/APOC3 genes to HDL-C variability to evaluate (1) consistency of published single-SNP studies with our single-SNP analyses; (2) consistency of single-SNP and two-SNP phenotype–genotype relationships across race-, gender-, and geographical location-dependent contexts; and (3) the contribution of single SNPs and pairs of SNPs to variability beyond that explained by plasma apo A-I concentration. We analyzed 45 SNPs in 3,831 young African–American (N=1,858) and European–American (N=1,973) females and males ascertained by the Coronary Artery Risk Development in Young Adults (CARDIA) study. We found three SNPs that significantly impact HDL-C variability in both the literature and the CARDIA sample. Single-SNP analyses identified only one of five significant HDL-C SNP genotype relationships in the CARDIA study that was consistent across all race-, gender-, and geographical location-dependent contexts. The other four were consistent across geographical locations for a particular race–gender context. The portion of total phenotypic variance explained by single-SNP genotypes and genotypes defined by pairs of SNPs was less than 3%, an amount that is miniscule compared to the contribution explained by variability in plasma apo A-I concentration. Our findings illustrate the impact of context-dependence on SNP selection for prediction of CVD risk factor variability.
APOA1 gene; APOC3 gene; High-density lipoprotein-cholesterol; Cardiovascular disease
Aims: To compare differences in microbiology testing activity between general practices within and between five hospitals in two National Health Service (NHS) regions in England.
Methods: Retrospective capture of standardised microbiology testing activity from the laboratory computer databases. Six equivalent tests were identified and compared. Data were obtained for 174 general practices in eight primary care groups, served by two NHS hospital trusts and three public health laboratories. The total catchment population was 1 180 000 people. Comparative test activities were displayed graphically and differences in median test activity and the hospital activity distributions were examined by the Wilcoxon signed rank test.
Results: Median testing activity differed by 200% (urine) to 800% (wound swabs) between the trusts that performed the highest and the lowest number of tests, and from 300% to 1900% between the top and bottom 10% activity bands of general practices. Large and significant differences were found between the hospitals, irrespective of whether they belonged to the same trust, and irrespective of their geographical location.
Conclusions: Large differences in microbiology testing exist within individual trust catchment areas in primary care, and there are also considerable differences between trusts. These inequalities may also introduce a selection bias into epidemiological and antibiotic resistance surveillance. This indicates a widespread need to examine and deal with the reasons responsible for these differences.
Appropriateness; evidence based medicine; microbiology; primary care
Despite years of research and hundreds of reports on tumour markers in oncology, the number of markers that have emerged as clinically useful is pitifully small. Often initially reported studies of a marker show great promise, but subsequent studies on the same or related markers yield inconsistent conclusions or stand in direct contradiction to the promising results. It is imperative that we attempt to understand the reasons that multiple studies of the same marker lead to differing conclusions. A variety of methodological problems have been cited to explain these discrepancies. Unfortunately, many tumour marker studies have not been reported in a rigorous fashion, and published articles often lack sufficient information to allow adequate assessment of the quality of the study or the generalisability of the study results. The development of guidelines for the reporting of tumour marker studies was a major recommendation of the US National Cancer Institute and the European Organisation for Research and Treatment of Cancer (NCI-EORTC) First International Meeting on Cancer Diagnostics in 2000. Similar to the successful CONSORT initiative for randomised trials and the STARD statement for diagnostic studies, we suggest guidelines to provide relevant information about the study design, preplanned hypotheses, patient and specimen characteristics, assay methods, and statistical analysis methods. In addition, the guidelines suggest helpful presentations of data and important elements to include in discussions. The goal of these guidelines is to encourage transparent and complete reporting so that the relevant information will be available to others to help them to judge the usefulness of the data and understand the context in which the conclusions apply.
tumour marker; guidelines; REMARK; NCI; EORTC; prognostic
Aims: To determine the value of squamous mucosal histology in the assessment of patients with gastro-oesophageal reflux symptoms.
Methods: Sixty six patients with reflux symptoms underwent endoscopy with oesophageal biopsy, manometry, and 24 hour oesophageal pH testing. The following histological features were assessed in squamous mucosa: the degree of basal cell hyperplasia, the degree of papillary zone elongation, and the density of neutrophil and eosinophil infiltration. Comparisons were made between the histological findings and the oesophageal function tests.
Results: The correlation between the traditionally accepted histological markers of gastro-oesophageal reflux disease in squamous mucosa and 24 hour pH testing was predominantly negative, with the exception of neutrophil inflammation in the squamous mucosa of patients with complicated reflux disease.
Conclusions: This study was unable to confirm the value of the Ismail-Beigi criteria as histological markers of acid reflux. By inference, biopsy of the oesophageal squamous mucosa is of limited value in the assessment of patients with reflux symptoms.
ambulatory pH monitoring; gastro-oesphageal reflux disease; histology; oesophagitis
survival analysis; missing data; validation; repeated events
survival analysis; Cox model; AFT model; model checking; choice of coavriates; goodness of fit
survival analysis; Cox model; AFT model; model selection
survival analysis; statistical methods; Kaplan-Meier
BACKGROUND—The cause of inflammation in cardiac mucosa at the gastro-oesophageal junction (GOJ) is unclear, both gastro-oesophageal reflux disease (GORD) and Helicobacter pylori having been implicated.
AIMS—To describe patterns of gastritis in patients with symptomatic GORD.
METHODS—In 150 patients (126 normally located Z-line, 24 Barrett's oesophagus) with symptoms of GORD, biopsies were taken of the GOJ, corpus, and antrum. Inflammation was assessed using the updated Sydney System.
RESULTS—For the 126 patients with a normally located Z-line, biopsies of the GOJ revealed cardiac mucosa in 96, fundic mucosa in 29, and squamous mucosa in one. Inflammation in glandular mucosa at the GOJ was present in 99/125 specimens (79%), including 87/96 (91%) with cardiac mucosa and 12/29 (41%) with fundic mucosa. Inflammation in fundic mucosa was closely related to H pylori and active inflammation was only seen in its presence. Inflammation in cardiac mucosa was less closely linked to H pylori. When H pylori was present in cardiac mucosa (28/96, 29%) active inflammation was usually present (25/28, 89%). However, active inflammation was also found in 34/68 (50%) cardiac mucosa specimens without H pylori. Overall, 28/87 (32%) biopsies with carditis were colonised with H pylori and 59/87 (68%) were not. In H pylori colonised patients, inflammation was seen throughout the stomach, while in non-colonised patients, it was confined to cardiac mucosa.
CONCLUSIONS—Patients with symptomatic GORD had a high prevalence of carditis. This was of two types, H pylori associated and unassociated. Except on Giemsa staining, the two were morphologically identical, suggesting mediation by a similar immunological mechanism.
Keywords: cardiac mucosa; carditis; gastro- oesophageal junction; gastro-oesophageal reflux; Helicobacter pylori; inflammation
About 6000 women in the United Kingdom develop ovarian cancer each year and about two-thirds of the women will die from the disease. Establishing the prognosis of a woman with ovarian cancer is an important part of her evaluation and treatment. Prognostic models and indices in ovarian cancer should be developed using large databases and, ideally, with complete information on both prognostic indicators and long-term outcome. We developed a prognostic model using Cox regression and multiple imputation from 1189 primary cases of epithelial ovarian cancer (with median follow-up of 4.6 years). We found that the significant (P≤ 0.05) prognostic factors for overall survival were age at diagnosis, FIGO stage, grade of tumour, histology (mixed mesodermal, clear cell and endometrioid versus serous papillary), the presence or absence of ascites, albumin, alkaline phosphatase, performance status on the ZUBROD-ECOG-WHO scale, and debulking of the tumour. This model is consistent with other models in the ovarian cancer literature; it has better predictive ability and, after simplification and validation, could be used in clinical practice. http://www.bjcancer.com © 2001 Cancer Research Campaignhttp://www.bjcancer.com
ovarian cancer; prognostic model; overall survival
In Drosophila, male accessory gland fluid (seminal fluid) has multiple effects on the female's reproductive efficiency. Here, we show the effect of seminal fluid on rate of egg hatch immediately following mating. Singly mated females were remated to two classes of sterile males, one with seminal fluid and one without seminal fluid. Transfer of seminal fluid results in a strong reduction in egg hatch shortly after the mating. Also, it is shown that remating with normal males causes an immediate reduction of egg hatch followed by recovery to normal egg hatch. In all cases, unhatched eggs contained no sperm. These results are consistent with a role for seminal fluid in sperm competition, mediated by incapacitation or inefficient use of resident sperm.
The survival of young patients (⩽ 50 years of age) with carcinoma of the oesophagus or stomach has been reported to be poorer than that of their older counterparts. The aim of the current study was to review the outcome of such young patients with oesophagogastric cancer and to compare the outcome in patients with carcinoma of the oesophagus/cardia with patients with carcinoma of the more distal stomach. The study population was 50 patients. Tumour location was oesophagus/cardia (n=33) and gastric body/antrum (n=17). The most common presenting symptoms were weight loss (66%), epigastric pain (54%), dysphagia (50%), and heartburn (40%). Seventeen patients had experienced foregut symptoms for a period of ⩾6 months. These patients were more likely to have symptoms of gastro-oesophageal reflux disease and to have received acid suppression therapy than patients with shorter symptom durations. Only 20 patients underwent a potentially curative resection, while 10 underwent open and close laparotomy. The overall median survival was 7 months and the 5-year survival was 8%. Multivariate analysis revealed that surgical resection and UICC stage were the only factors that significantly influenced survival. There was no difference in the survival of patients with proximally situated tumours compared to those with distally located tumours. Wide variations in clinical practice were seen between different surgeons. Consequently, a multidisciplinary team designed to manage all patients with oesophagogastric cancer according to nationally agreed protocols has been established in our hospital. Earlier diagnosis of these tumours is to be encouraged, even if this necessitates the more liberal use of endoscopy in the evaluation of young patients with persistent foregut symptoms.
Keywords: oesophagus; stomach; carcinoma; surgery; age-factors
Yersinia pestis, the causative agent of plague, and the enteropathogen Yersinia pseudotuberculosis have nearly identical nucleotide similarity yet cause markedly different diseases. To investigate this conundrum and to study Yersinia pathogenicity, we developed a high-density oligonucleotide array-based modification of signature-tagged mutagenesis (STM). Y. pseudotuberculosis YPIII mutants constructed with the tagged transposons were evaluated in the murine yersiniosis infection model. The DNA tags were amplified using biotinylated primers and hybridized to high-density oligonucleotide arrays containing DNA complementary to the tags. Comparison of the hybridization signals from input pools and output pools identified a mutant whose relative abundance was significantly reduced in the output pool. Sequence data from 31 transposon insertion regions was compared to the complete Y. pestis CO92 genome sequence. The 26 genes present in both species were found to be almost identical, but five Y. pseudotuberculosis genes identified through STM did not have counterparts in the Y. pestis genome and may contribute to the different tropisms in these closely related pathogens. Potential virulence genes identified include those involved in lipopolysaccharide biosynthesis, adhesion, phospholipase activity, iron assimilation, and gene regulation. The phospholipase A (PldA) mutant exhibited reduced phospholipase activity compared to the wild-type strain and in vivo attenuation of the mutant was confirmed. The combination of optimized double tag sequences and high-density array hybridization technology offers improved performance, efficiency, and reliability over classical STM and permits quantitative analysis of data.
p53 gene mutation is the most common genetic alteration in neoplastic diseases, including breast cancer, for which p53 alteration may indicate poor prognosis. Recent clinical evidence suggests that prostate-specific antigen (PSA) expression may identify breast cancer patients with favourable outcome. Assessment of p53 and PSA in combination, potentially offering improved prediction, has not yet been performed. Extracts from 952 primary breast carcinomas were assayed for PSA and p53 by quantitative enzyme-linked immunosorbent assays (ELISAs) developed by the authors. Concentrations of each marker were classified as negative or positive on the basis of median and 30th percentile cut-off points for p53 and PSA respectively. Patients followed for a median of 6 years having different combinations of negative or positive status for PSA and p53 were compared with respect to the relative risks (RRs) for relapse and death by Cox proportional hazards regression analysis, in which an interaction term was also evaluated, and with respect to disease-free survival (DFS) and overall survival (OS) probabilities by Kaplan–Meier plots and log-rank tests. Multivariate models were adjusted for oestrogen and progesterone receptor status, nodal status, patient age, tumour size, DNA ploidy, S phase fraction and receipt of chemotherapy. Interactions were not found between the status of PSA and p53 in the Cox models, in which PSA-negativity (RR = 1.47, P = 0.020 for DFS, and RR = 1.49, P = 0.023 for OS) and p53-positivity (RR = 1.46, P = 0.017 for DFS, and RR = 1.41, P = 0.033 for OS) were individually associated with prognosis. Evaluation of a combined three-level variable revealed that PSA(–)/p53(+) patients had significantly higher risks for relapse (RR = 2.13, P < 0.001) and death (RR = 2.08, P = 0.001) than PSA(+)/p53(–) patients, and that patients positive or negative for both markers had intermediate risks for the outcome events in the same multivariate analysis (RR = 1.45 for both DFS and OS). The results of our study demonstrate that the assessment of combined PSA and p53 expression status by ELISAs, easily applicable to breast tumour extracts prepared for steroid hormone receptor analyses, may stratify breast cancer patients into groups differing by relapse and death risks of greater magnitude than offered by the assessment of either p53 or PSA alone. © 1999 Cancer Research Campaign
prostate-specific antigen; protein, p53; ELISA; breast neoplasms
Recent dengue outbreaks in the Caribbean and Central and South America and the presence of competent mosquito vectors increase the likelihood of future autochthonous transmission in Florida. During April 1997 to March 1998, a laboratory-based active surveillance program detected 18 cases of dengue involving all four dengue serotypes. All patients reported recent travel to countries with indigenous dengue transmission. These results demonstrate that dengue infections are imported into Florida at a much higher rate than reflected by previous passive surveillance; therefore, the risk for local dengue transmission may be increasing.
BACKGROUND: The aim of this study was to determine whether significant differences exist in lung bioavailability between generic (Salamol, Salbulin) and innovator (Ventolin) formulations of inhaled salbutamol given by metered dose inhalers. METHODS: Ten healthy volunteers of mean age 20.5 years with a forced expiratory volume in one second (FEV1) of 112.1% predicted were studied in a randomised double blind single dosing crossover study. Salbutamol, 1200 micrograms, was given with mouth rinsing and lung bioavailability was assessed by measuring plasma salbutamol levels and urine excretion of salbutamol. RESULTS: No differences were seen between innovator and generic salbutamol metered dose inhalers in plasma salbutamol levels, urinary salbutamol excretion, or extrapulmonary beta 2 activity. The maximum plasma salbutamol concentration (mean Cmax) and time to maximum concentration (median Tmax) were: Ventolin (2.93 ng/ml, 10 minutes), Salbulin (3.01 ng/ml, 10 minutes), Salamol (3.33 ng/ml, 10 minutes). The geometric mean ratio and 95% confidence interval for Cmax were: Salbulin/Ventolin 1.02 (0.69 to 1.41) and Salamol/Ventolin 1.13 (0.94 to 1.35). CONCLUSIONS: No differences are apparent between the two generic and innovator formulations of salbutamol metered dose inhaler in terms of lung bioavailability.
The X-ray crystal structure of the complex between the synthetic antitumour and antiviral DNA binding ligand SN 7167 and the DNA oligonucleotide d(CGCGAATTCGCG)2 has been determined to an R factor of 18.3% at 2.6 A resolution. The ligand is located within the minor groove and covers almost 6 bp with the 1-methylpyridinium ring extending as far as the C9-G16 base pair and the 1-methylquinolinium ring lying between the G4-C21 and A5-T20 base pairs. The ligand interacts only weakly with the DNA, as evidenced by long range contacts and shallow penetration into the groove. This structure is compared with that of the complex between the parent compound SN 6999 and the alkylated DNA sequence d(CGC[e6G]AATTCGCG)2. There are significant differences between the two structures in the extent of DNA bending, ligand conformation and groove binding.
The hydrolysis of DL-alanine-beta-naphthylamide and D-alanine-p-nitroanilide for identification of Listeria spp. has been studied with 227 cultures. All species of Listeria, except L. monocytogenes, hydrolyzed these substrates. The reactions were detected by simple chromogenic reactions and could substitute for the CAMP test.
The crystal structure is reported of a complex between the dodecanucleotide sequence d(CGCGAATTCGCG)2and an analogue of the DNA binding drug Hoechst 33258, in which the piperazine ring has been replaced by an amidinium group and the phenol ring by a phenylamidinium group. The structure has been refined to an R factor of 19.5% at 2.2 A resolution. The drug is held in the minor groove by five strong hydrogen bonds, together with bridging water molecules at both ends. There are few other contacts with the floor of the groove, indicating a lack of isohelicity with the groove and suggesting (i) that the observed high DNA affinity of this drug is primarily due to the array of hydrogen bonds and (ii) that these more than compensate for its poor isohelicity.