In situ hybridization to mRNA: from black art to guiding light

In situ hybridization to mRNA in embryo sections or wholemount embryos is one of the most powerful analytical tools available to the molecular developmental biologist. For many workers, this procedure provides the first insights into the function of newly isolated genes, allowing the formulation of hypotheses and setting the course for further research. This paper presents a personal historical perspective of the development of in situ hybridization, looks at the theory and practice of the technique, summarizes the current state of the art, and speculates on possible directions for the future as a tool in functional genomics.

The influence of calcium on granular sludge in a full-scale UASB treating paper mill wastewater

Calcium precipitation can have a number of effects on the performance of high-rate anaerobic performance including cementing of the sludge bed, limiting diffusion, and diluting the active biomass. The aim of this study was to observe the influence of precipitation in a stable full-scale system fed with high-calcium paper factory wastewater. Granules were examined from an upflow anaerobic sludge blanket reactor (volume 1,805 m(3)) at a recycled paper mill with a loading rate of 5.7-6.6 kgCOD.m(-3).d(-1) and influent calcium concentration of 400-700 gCa(.)m(-3). The granules were relatively small (1 mm), with a 200-400 mum core of calcium precipitate as observed with energy dispersive X-ray spectroscopy. Compared to other granules, Methanomicrobiales not Methanobacteriales were the dominant hydrogen or formate utilisers, and putative acidogens were filamentous. The strength of the paper mill fed granules was very high when compared to granules from other full-scale reactors, and a partial linear correlation between granule strength and calcium concentration was identified.

Hyaline tissue of thermally unaltered conodont elements and the enamel of vertebrates

Comparison of the ultrastructure of the hyaline tissue of conodont elements and the enamel of vertebrates provides little support for a close phylogenetic relationship between conodonts and vertebrates. Transmission and scanning electron microscopy shows that the mineralised component of the hyaline tissue of Panderodus and of Cordylodus elements consists of large, flat, oblong crystals, arranged in layers that run parallel to the long axis of the conodont. Enamel in the dentition of a living vertebrate, the lungfish Neoceratodus forsteri, has crystals of calcium hydroxyapatite, arranged in layers, and extending in groups from the dentine-enamel junction; the crystals are slender, elongate spicules perpendicular to the surface of the tooth plate, Similar crystal arrangements to those of lungfish are found in other vertebrates, but none resembles the organisation of the hyaline tissue of conodont elements, The crystals of hydroxyapatite in conodont hyaline tissue are exceptionally large, perpendicular or parallel to the surface of the element, with no trace of prisms, unlike the protoprismatic radial crystallite enamel of fish teeth and scales, or the highly organised prismatic enamel of mammals.

Growth and hard tissue remodelling in the dentition of the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi)

The extant lungfish, including three genera, the Australian, South American and African lungfishes, retain a dentition that appeared first in the Devonian, in some of the oldest members of this group. The dentition consists of permanent tooth plates with persistent cusps that appear early in development of the fish. The cusps, separate early in development, form ridges that are arranged in a radiating pattern, and fusion of the cusps to each other and to the underlying jaw bone produces a tooth plate. The lungfish dentition is based on a template of mantle dentine that surrounds bone trabeculae enclosed in the tooth plate. The mantle layer is covered by enamel. In most derived dipnoans, this framework encloses two further forms of dentine, known as interdenteonal and circumdenteonal dentines. The tooth plates grow in area and in depth without evidence of macroscopic resorption of dentines or of enamel. Increase in size and changes in shape of lungfish tooth plates is actually achieved by a process involving microscopic remodelling of the bone contained within the margin of each tooth plate, and the later addition of new dentines and enamel within and around the bone. This is accomplished without creating weakness in the structural integrity of the tooth plate and bone complex, and proceeds in line with growth and remodelling of the jaw bones attached to the tooth plates.

Nuclear RelB(+) cells are found in normal lymphoid organs and in peripheral tissue in the context of inflammation, but not under normal resting conditions

Differentiated dendritic cells (DC) have been identified by the presence of nuclear RelB (nRelB) and HLA-DR, and the absence of CD20 or high levels of CD68, in lymph nodes and active rheumatoid arthritis synovial tissue. The current studies aimed to identify conditions in which nRelB is expressed in human tissues, by single and double immunohistochemistry of formalin-fixed peripheral and lymphoid tissue. Normal peripheral tissue did not contain nRelB(+) cells. nRelB(+) DC were located only in T- or B-cell areas of lymphoid tissue associated with normal organs or peripheral tissues, including tonsil, colon, spleen and thymus, or in association with T cells in inflamed peripheral tissue. Inflamed sites included skin delayed-type hypersensitivity reaction, and a wide range of tissues affected by autoimmune disease. Nuclear RelB(+) -HLA-DR- follicular DC were located in B-cell follicles in lymphoid organs and in lymphoid-like follicles of some tissues affected by autoimmune disease. Lymphoid tissue T-cell areas also contained nRelB(-) -HLA-DR+ cells, some of which expressed CD123 and/or CD68. Nuclear RelB(+) cells are found in normal lymphoid organs and in peripheral tissue in the context of inflammation, but not under normal resting conditions.

A mixture model-based approach to the clustering of microarray expression data

Motivation: This paper introduces the software EMMIX-GENE that has been developed for the specific purpose of a model-based approach to the clustering of microarray expression data, in particular, of tissue samples on a very large number of genes. The latter is a nonstandard problem in parametric cluster analysis because the dimension of the feature space (the number of genes) is typically much greater than the number of tissues. A feasible approach is provided by first selecting a subset of the genes relevant for the clustering of the tissue samples by fitting mixtures of t distributions to rank the genes in order of increasing size of the likelihood ratio statistic for the test of one versus two components in the mixture model. The imposition of a threshold on the likelihood ratio statistic used in conjunction with a threshold on the size of a cluster allows the selection of a relevant set of genes. However, even this reduced set of genes will usually be too large for a normal mixture model to be fitted directly to the tissues, and so the use of mixtures of factor analyzers is exploited to reduce effectively the dimension of the feature space of genes. Results: The usefulness of the EMMIX-GENE approach for the clustering of tissue samples is demonstrated on two well-known data sets on colon and leukaemia tissues. For both data sets, relevant subsets of the genes are able to be selected that reveal interesting clusterings of the tissues that are either consistent with the external classification of the tissues or with background and biological knowledge of these sets.

Adjusting for fat-free mass in metabolic studies

in a recent publication, Eriksson et al. [1] explored the relationship between size at birth and resting metabolic rate and body composition in adulthood in a cohort of over 300 men and women. They reported an unexpected finding that people of both sexes who had a low birth weight also had a higher metabolic activity per unit muscle tissue. This conclusion was drawn from an analysis where resting metabolic rate (expressed as kcal/kg fat-free mass) in adulthood was examined relative to the birth weight of the subject. One explanation that they suggested was that the apparent increased activity of muscle tissue resulted from an increased sympathetic drive associated with low birth weight. There may be a less physiological reason for the findings of Eriksson et al. Whilst the data are not given specifically in the text, it can be seen clearly from Fig. 1 in the paper that the mean fat-free mass measured in adulthood increased, in both sexes, from the lightest birth weight group to the heaviest birth weight group when the cohort were divided into tertiles based on birth weight. The crux of the issue is that in many - indeed most - cases, expressing resting energy expenditure as kcal/kg fat-free mass does not totally adjust for fat-free mass [2 - 5], and a bias is introduced so that those who have a higher fat-free mass will tend to have a lower resting energy expenditure when expressed per kg fat-free mass. This bias found when expressing many physiological parameters relative to body size, body weight or body composition has long been known [6], and should be carefully considered by appropriate adjustment and hence analysis.

Specific orofacial problems experienced by musicians

Background: Patients who play musical instruments (especially wind and stringed instruments) and vocalists are prone to particular types of orofacial problems. Some problems are caused by playing and some are the result of dental treatment. This paper proposes to give an insight into these problems and practical guidance to general practice dentists. Method: Information in this paper is gathered from studies published in dental, music and occupational health journals, and from discussions with career musicians and music teachers. Results: Orthodontic problems, soft tissue trauma, focal dystonia, denture retention, herpes labialis, dry mouth and temporomandibular joint (TMJ) disorders were identified as orofacial problems of career musicians. Options available for prevention and palliative treatment as well as instrument selection are suggested to overcome these problems. Conclusions: Career musicians express reluctance to attend dentists who are not sensitive to their specific needs. General practitioner dentists who understand how the instruments impact on the orofacial structures and are aware of potential problems faced by musicians are able to offer preventive advice and supportive treatment to these patients, especially those in the early stages of their career.

Selective loss of expression of glutamate GluR2/R3 receptor subunits in cerebellar tissue from a patient with olivopontocerebellar atrophy

Expression of the mRNAs encoding the astrocytic (EAAT1, EAAT2) and neuronal (EAAT3, EAAT4) excitatory amino acid transporters and the AMPA-type glutamate receptor subunits GluR2 and GluR3 was investigated in postmortem cerebellar extracts from a patient with olivopontocerebellar atrophy (OPCA) and in material from three age-matched controls. Decreased expression in the steady state level of EAAT4 mRNA in the OPCA sample was correlated with the selective loss of Purkinje cells. Neuropathological evaluation revealed reactive gliosis and concomitantly increased expression of the mRNA encoding astrocytic glial fibrillary acidic protein (GFAP). Expression of the mRNAs encoding the AMPA receptor subunits GluR2 and GluR3 subunits was found to be decreased in OPCA suggesting that excitotoxic mechanism could play a role in the pathogenesis of the selective neuronal cell death in this disorder.

Apoptosis and cell proliferation in the development of gastric carcinomas: Associations with c-myc and p53 protein expression

Background and Aim: Patients with gastric carcinomas have a poor prognosis and low survival rates. The aim of the present paper was to characterize cellular and molecular properties to provide insight into aspects of tumor progression in early compared with advanced gastric cancers. Methods: One hundred and nine graded gastric carcinomas (early or advanced stage, undifferentiated or differentiated type) with paired non-cancer tissue were studied to define the correlation between apoptosis (morphology, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling), cell proliferation (Ki-67 expression, morphology) and expression and localization of two proteins frequently having altered expression in cancers, namely p53 and c-myc. Results: Overall, apoptosis was lower in early stage, differentiated and undifferentiated gastric carcinomas compared with advanced-stage cancers. Cell proliferation was comparatively high in all stages. There was a high level of p53 positivity in all stages. Only the early- and advanced-stage undifferentiated cancers that were p53 positive had a significantly higher level of apoptosis (P< 0.05). Cell proliferation was significantly greater (P < 0.05) only in the early undifferentiated cancers that had either c-myc or p53-positivity. Conclusions: The results indicate that low apoptosis and high cell proliferation combine to drive gastric cancer development. The molecular controls for high cell proliferation of the early stage undifferentiated gastric cancers involve overexpression of both p53 and c-myc. Overexpression of p53 may also control cancer development in that its expression is associated with higher levels of apoptosis in early and late-stage undifferentiated, cancers. (C) 2002 Blackwell Publishing Asia Pty Ltd.

The role of Doppler left ventricular filling indexes and Doppler tissue echocardiography in the assessment of cardiac involvement in hereditary hemochromatosis

Although cardiac dysfunction in hereditary hemochromatosis (HHC) can be evaluated by conventional echocardiography, findings are often not specific. To test the hypothesis that the assessment of (1) conventional Doppler left ventricular filling indexes and (2) intrinsic elastic properties of the myocardium by Doppler tissue echocardiography can both enhance the accuracy of echocardiographic diagnosis of cardiac involvement in HHC, a group of 18 patients with HHC (mean age 50+/-7 years) and 22 age-matched healthy subjects were studied. The following indexes were characteristic for HHC: (1) the duration of atrial reversal measured from pulmonary venous flow (ms) was longer(118+/-20 vs 90+/-16; P

Responses to a clinical test of mechanical provocation of nerve tissue in whiplash associated disorder

Involvement of nerve tissue may contribute to the persistence of pain following a whiplash injury. This study aimed to investigate responses to the brachial plexus provocation test (BPPT) in 156 subjects with chronic whiplash associated disorder (WAD) with and without associated arm pain and 95 asymptomatic control subjects. The range of elbow extension (ROM) and visual analogue scale (VAS) pain scores were measured. Subjects with chronic WAD demonstrated significantly less ROM and higher VAS scores with the BPPT than the asymptomatic subjects (P

The effect of soft tissue deloading tape on thoracic spine pressure pain thresholds in asymptomatic subjects

The application of tape to deload soft tissue is used in the management of thoracic spine pain. A reported clinical feature of this treatment is reduced tenderness of the spine during postero-anterior mobilizations. A randomized, single blind, placebo controlled, repeated measures design study was employed to investigate the effects of deloading tape on pressure pain threshold measurements at the level of the T7 spinous process in an asymptomatic group of 24 subjects. Pressure pain thresholds were assessed prior to and following the application of deloading tape, placebo sham tape and no-tape control conditions. All subjects received all three conditions in a randomized order on three separate days. Differences between the pre- and post-measurements were used as indicators of change in a subject's pressure pain threshold. No significant change in pressure pain threshold measurements was found between conditions. In summary, this study demonstrated that deloading tape applied to the level of the T7 spinous process did not significantly change pressure pain threshold measurements in asymptomatic subjects, raising the possibility that any pain relieving effect may well be conditional upon pain being present. (C) 2002 Elsevier Science Ltd. All rights reserved.

Determinants of tissue Doppler measures of regional diastolic function during dobutamine stress echocardiography

Background Diastolic dysfunction induced by ischemia may alter transmitral blood flow, but this reflects global ventricular function, and pseudonormalization may occur with increased preload. Tissue Doppler may assess regional diastolic function and is relatively load-independent, but limited data exist regarding its application to stress testing. We sought to examine the stress response of regional diastolic parameters to dobutomine echocardiography (DbE). Methods Sixty-three patients underwent study with DbE: 20 with low probability of coronary artery disease (CAD) and 43 with CAD who underwent angiography. A standard DbE protocol was used, and segments were categorized as ischemic, scar, or normal. Color tissue Doppler was acquired at baseline and peak stress, and waveforms in the basal and mid segments were used to measure early filling (Em), late filling (Am), and E deceleration time. Significant CAD was defined by stenoses >50% vessel diameter. Results Diastolic parameters had limited feasibility because of merging of Em and Am waves at high heart rates and limited reproducibility. Nonetheless, compared with normal segments, segments subtended with significant stenoses showed a lower Em velocity at rest (6.2 +/- 2.6 cm/s vs 4.8 +/- 2.2 cm/s, P < .0001) and peak (7.5 +/- 4.2 cm/s vs 5.1 +/- 3.6 cm/s, P < .0001), Abnormal segments also showed a shorter E deceleration time (51 +/- 27 ms vs 41 +/- 27 ms, P = .0001) at base and peak. No changes were documented in Am. The same pattern was seen with segments identified as ischemic with wall motion score. However, in the absence of ischemia, segments of patients with left ventricular hypertrophy showed a lower Em velocity, with blunted Em responses to stress. Conclusion Regional diastolic function is sensitive to ischemia. However, a number of practical limitations limit the applicability of diastolic parameters for the quantification of stress echocardiography.