Identification of a regeneration-organizing cell in the Xenopus tail.

Unlike mammals, Xenopus laevis tadpoles have a high regenerative potential. To characterize this regenerative response, we performed single-cell RNA sequencing after tail amputation. By comparing naturally occurring regeneration-competent and -incompetent tadpoles, we identified a previously unrecognized cell type, which we term the regeneration-organizing cell (ROC). ROCs are present in the epidermis during normal tail development and specifically relocalize to the amputation plane of regeneration-competent tadpoles, forming the wound epidermis. Genetic ablation or manual removal of ROCs blocks regeneration, whereas transplantation of ROC-containing grafts induces ectopic outgrowths in early embryos. Transcriptional profiling revealed that ROCs secrete ligands associated with key regenerative pathways, signaling to progenitors to reconstitute lost tissue. These findings reveal the cellular mechanism through which ROCs form the wound epidermis and ensure successful regeneration.

Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes - a method for quantitative transcriptional reprogramming.

Full-grown Xenopus oocytes in first meiotic prophase contain an immensely enlarged nucleus, the Germinal Vesicle (GV), that can be injected with several hundred somatic cell nuclei. When the nuclei of mammalian somatic cells or cultured cell lines are injected into a GV, a wide range of genes that are not transcribed in the donor cells, including pluripotency genes, start to be transcriptionally activated, and synthesize primary transcripts continuously for several days. Because of the large size and abundance of Xenopus laevis oocytes, this experimental system offers an opportunity to understand the mechanisms by which somatic cell nuclei can be reprogrammed to transcribe genes characteristic of oocytes and early embryos. The use of mammalian nuclei ensures that there is no background of endogenous maternal transcripts of the kind that are induced. The induced gene transcription takes place in the absence of cell division or DNA synthesis and does not require protein synthesis. Here we summarize new as well as established results that characterize this experimental system. In particular, we describe optimal conditions for transplanting somatic nuclei to oocytes and for the efficient activation of transcription by transplanted nuclei. We make a quantitative determination of transcript numbers for pluripotency and housekeeping genes, comparing cultured somatic cell nuclei with those of embryonic stem cells. Surprisingly we find that the transcriptional activation of somatic nuclei differs substantially from one donor cell-type to another and in respect of different pluripotency genes. We also determine the efficiency of an injected mRNA translation into protein.

The factorial decomposition of the Mahalanobis distances in habitat selection studies.

The Mahalanobis distances have been introduced in habitat selection studies for the estimation of environmental suitability maps (ESMs). The pixels of raster maps of a given area correspond to points in the multidimensional space defined by the mapped environmental variables (ecological space). The Mahalanobis distances measure the distances in this space between these points and the mean of the ecological niche (i.e., the hypothesized optimum for the species) regarding the structure of the niche. The map of these distances over the area of interest is an estimated ESM. Several authors recently noted that the use of a single optimum for the niche of a species may lead to biased predictions of animal occurrence. They proposed to use instead a minimum set of basic habitat requirements, found by partitioning the Mahalanobis distances into a restricted set of biologically meaningful axes. However, the statistical approach they proposed does not take into account the environmental conditions on the area where the niche was sampled (i.e., the environmental availability), and we show that including this availability is necessary. We used their approach as a basis to develop a new exploratory tool, the Mahalanobis distance factor analysis (MADIFA), which performs an additive partitioning of the Mahalanobis distances taking into account this availability. The basic habitat requirements of a species can be derived from the axes of the MADIFA. This method can also be used to compute ESMs using only this small number of basic requirements, therefore including only the biologically relevant information. We also prove that the MADIFA is complementary to the commonly used ecological-niche factor analysis (ENFA). We used the MADIFA method to analyze the niche of the chamois Rupicapra rupicapra in a mountainous area. This method adds to the existing set of tools for the description of the niche.

Two-dimensional morphogen gradient in Xenopus: boundary formation and real-time transduction response.

Morphogen gradients play an important role in pattern formation in embryo. However, the interpretation of position in a morphogen gradient is not well understood. Because it is hard to analyze morphogen gradients especially in opaque embryos such as those of Xenopus, it is necessary to fix and section the embryo, thereby eliminating the possibility of real-time observation, and making more difficult the interpretation of events that take place in three dimensions. We describe here a two-dimensional preparation of cells from a Xenopus blastula animal cap, in which an activin concentration gradient appears to be formed and interpreted at the same rate and in the same way as in normal embryos. We use two-dimensional preparations of this kind to contribute the following new information about gradient formation and interpretation in embryo. We determine the dynamics of formation of an activin activity gradient in real time. We demonstrate that this gradient is established by diffusion of activin through intercellular space and does not require internalization of receptor or ligand. We also show that the generation of a boundary of gene expression depends on the interpretation, rather than a change of composition, of the concentration gradient.

Differential regulation of NGF receptors in primary sensory neurons by adjuvant-induced arthritis in the rat.

In the adult brain, neurotrophins play a key role in adaptive processes linked to increased neuronal activity. A growing body of evidence suggests that chronic pain results from long-term plasticity of central pathways involved in nociception. We have investigated the involvement of nerve growth factor (NGF) in adaptive responses of primary sensory neurons during the course of a long-lasting inflammatory pain model. The amount and distribution of the NGF receptors p75(NTR) and TrkA were measured in the dorsal horn and dorsal root ganglia (DRG) of animals subjected to Freund's adjuvant-induced arthritis (AIA). We observed an increased immunoreactivity of both receptors in the central terminals of primary sensory neurons in the arthritic state. The increases were seen in the same population of afferent terminals in deep dorsal horn laminae. These changes paralleled the variations of clinical and behavioral parameters that characterize the course of the disease. They occurred in NGF-sensitive, but not GDNF-sensitive, nerve terminals. However, p75(NTR) and TrkA protein levels in the DRG (in the cell body of these neurons) showed different response patterns. An immediate rise of p75(NTR) was seen in parallel with the initial inflammation that developed after administration of Freund's adjuvant in hindpaws. In contrast, increases of the mature (gp140(trk)) form of TrkA occurred later and seemed to be linked to the development of the long-lasting inflammatory response. The changes in receptor expression were observed exclusively at lumbar levels, L3-L5, somatotopically appropriate for the inflammation. Together, these results implicate NGF in long-term mechanisms accompanying chronic inflammatory pain, via the up-regulation of its high affinity receptor, and offer additional evidence for differential processes underlying short- versus long-lasting inflammatory pain.

An interim determination of health gain from oral cancer and precancer screening: 3. Preselecting high risk individuals.

OBJECTIVE: To perform a sensitivity analysis using a decision model for simulating opportunistic screening for oral cancer and precancer in order to obtain an estimate of health gain from screening high risk dental patients, identified with the aid of artificial intelligence. DESIGN: A hypothetical opportunistic screening programme based on preselection of high risk patients was compared with screening all eligible patients and with a 'do nothing' scenario. The basic assumptions and data used in the model have been described previously. SETTING: Screening would be carried out in dental practices. PARTICIPANTS: A notional population of 100,000 adults of average age 55 years and 20 years life expectancy. INTERVENTIONS: Systematic clinical examination of the oral mucosa of high risk individuals identified by artificial intelligence on the basis of personal characteristics and life style. OUTCOME MEASURES: QALYs and equivalent lives saved RESULTS: The following estimates of health gain were obtained: the preselected screened group (n = 25,000) would achieve 1,993,294 QALYs. Without preselection, screening an unselected population of 100,000 eligible adults would result in 1,993,094 QALYs. Under the 'do nothing' scenario, 100,000 unscreened individuals would achieve 1,992,982 QALYs. Selective screening could avoid the equivalent of 15 deaths per 100,000 subjects examined. Screening preselected subjects at heightened risk would save the equivalent of two to three times the number of healthy lives compared with non-selective screening. Only one quarter of the population would need to be examined. CONCLUSIONS: Opportunistic screening of preselected dental patients for oral cancer and precancer appears to be a promising health promotion strategy and should be subject to formal economic appraisal.

Cell cycle phase-specific surface expression of nerve growth factor receptors TrkA and p75(NTR).

Expression of the nerve growth factor (NGF) receptors TrkA and p75(NTR) was found to vary at the surface of PC12 cells in a cell cycle phase-specific manner. This was evidenced by using flow cytometric and microscopic analysis of cell populations labeled with antibodies to the extracellular domains of both receptors. Differential expression of these receptors also was evidenced by biotinylation of surface proteins and Western analysis, using antibodies specific for the extracellular domains of TrkA and p75(NTR). TrkA is expressed most strongly at the cell surface in M and early G1 phases, whereas p75(NTR) is expressed mainly in late G1, S, and G2 phases. This expression reflects the molecular and cellular responses to NGF in specific phases of the cell cycle; in the G1 phase NGF elicits both the anti-mitogenic effect, i.e., inhibition of the G1 to S transition, and the differentiation response whereas a survival effect is provoked elsewhere in the cell cycle. A model is proposed relating these responses to the surface expression of the two receptors. These observations open the way for novel approaches to the investigation of the mechanism of NGF signal transduction.

An interim determination of health gain from oral cancer and precancer screening: 1. Obtaining health state utilities.

OBJECTIVE: To obtain preliminary health state utility values for oral precancer, and stage 1 and stage 2 (or greater) oral cancer, for use in a study involving quality of life measurement. DESIGN: Members of the general public were personally interviewed and asked to complete a standard gamble questionnaire in which each individual had to choose between having oral cancer or precancer (a description of each stage and its treatment was given), and being perfectly healthy but with a probability (p) from 0 to 1, of immediate death. There were three questions, the first related to a state of oral precancer, the second to a small cancer (< 2 cm) and the third to a large oral cancer (> 2 cm). The utility of each state was equal to the probability that the subject would opt for having the disease (1-p). The pooled responses of all subjects were analysed using a Wilcoxon matched-pairs signed-ranks test. SETTING: The premises of a commercial company. PARTICIPANTS: A convenience, quota sample of 100 employees aged 40 years or over. RESULTS: The mean utility values obtained were: oral precancer = 0.92; small oral cancer = 0.88; large oral cancer = 0.68. There was a significant difference between all three states (P < 0.05). CONCLUSIONS: The study relied on a convenience sample to elicit the public's perceptions of different oral cancer states. Nevertheless, despite this limitation, it was considered that the utility values derived were suitable for incorporation in measurements of quality adjusted life years.

An interim determination of health gain from oral cancer and precancer screening: 2. Developing a model of population screening.

OBJECTIVE: To design a simulation model of population screening for oral cancer and precancer and to obtain estimates of quality adjusted life years (QALYs) and lives gained from screening, using decision analysis. DESIGN: A hypothetical opportunistic screening programme was compared with the status quo (no screening). Variables included in the model were attendance (50 per cent of the eligible population); prevalence of oral cancer (0.098 per cent) and precancer (2.57 per cent), positive (0.67) and negative (0.99) predictive values of screening, stage distribution of cancer (40 per cent stage 1, 60 per cent stage 2+ without screening; 60 per cent stage 1, 40 per cent stage 2+ with screening); average survival (precancer, 19.2 years; stage 1, 14.6 years; stage 2+, 10.8 years); and the public's perceived utilities of the various outcomes (health, 1.00; precancer, 0.92; stage 1 cancer, 0.88; stage 2+ cancer, 0.68). One cycle of the programme was modelled. SETTING: Screening would be carried out in dental practices. PARTICIPANTS: A notional population of 100,000 adults of average age 55 years and 20 years life expectancy entered each decision pathway (screening or status quo). INTERVENTIONS: Systematic clinical examination of the oral soft tissues. OUTCOME MEASURES: QALYs and equivalent lives saved. RESULTS: Assuming 50 per cent attended, the population offered screening achieved 1,993,094 QALYs. With no screening the corresponding end point was 1,992,982 QALYs. The gain from screening was therefore 56 QALYs representing an equivalent of 2.8 lives saved. CONCLUSIONS: Though a simplification, this model is likely to be more reliable than intuitive prediction and is amenable to sensitivity analysis of different screening strategies. A modest health gain from screening was predicted by the model.

Evaluation of health care workers' accuracy in recognising oral cancer and pre-cancer.

The aim of the study was to evaluate dental health care workers' ability to recognise oral cancer and pre-cancer from colour photographic slides. A set of 80 slides was prepared, 40 were negative (normal mucosa or lesions with no malignant potential) and 40 were positive (cancer or potentially malignant lesions). The slides were arranged in random order and projected in a standard format. The performance was evaluated in terms of mean sensitivity, specificity, mean correct score and likelihood ratio. There were no significant differences between the three groups of dentists (consultants, juniors hospital dentists and general dentists), all performed better than the auxiliaries. The chances of a consultant making a correct decision were 5.5 times better than for an auxiliary and 2.7 times better than for a junior hospital dentist. The slide show enables a comparison of health care workers to be made and may be useful for the evaluation, training and calibration of examiners for an oral cancer screening programme.

Isolation and Characterization of Biotin Carboxylase from Pea Chloroplasts.

Pea (Pisum sativum L.) leaf acetyl-coenzyme A carboxylase (ACCase) exists as two structurally different forms: a major, chloroplastic, dissociable form and a minor, multifunctional enzyme form located in the leaf epidermis. The dissociable form is able to carboxylate free D-biotin as an alternate substrate in place of the natural substrate, biotin carboxyl carrier protein. Here we report the purification of the biotin carboxylase component of the chloroplastic pea leaf ACCase. The purified enzyme, free from carboxyltransferase activity, is composed of two firmly bound polypeptides, one of which (38 kD) is biotinylated. In contrast to bacterial biotin carboxylase, which retains full activity upon removal of the biotin carboxyl carrier component, attempts to dissociate the two subunits of the plant complex led to a complete loss of biotin carboxylase activity. Steady-state kinetic studies of the biotin carboxylase reaction reveal that addition of all substrates on the enzyme is sequential and that no product release is possible until all three substrates (MgATP, D-biotin, bicarbonate) are bound to the enzyme and all chemical processes at the active site are completed. In agreement with this mechanism, bicarbonate-dependent ATP hydrolysis by the enzyme is found to be strictly dependent on the presence of exogenous D-biotin in the reaction medium.

The use of artificial intelligence to identify people at risk of oral cancer and precancer.

Artificial intelligence is being used increasingly as an aid to diagnosis in medicine. The purpose of this study was to evaluate the ability of a neural network to predict the likelihood of an individual having a malignant or potentially malignant oral lesion based on knowledge of their risk habits. Performance of the network was compared with a group of dental screeners in a screening programme involving 2027 adults. The screening performance was measured in terms of sensitivity, specificity and likelihood ratios. All subjects were examined independently by a dental screener and a specialist, who provided a definitive diagnosis, or 'gold standard', for each individual. All subjects also completed an interview questionnaire regarding personal details, dental attendance and smoking and drinking habits. The neural network was trained on 1662 of the screened population using ten input variables derived from the questionnaire along with the outcome of the specialist's diagnosis. Following training, the network was asked to classify the remaining unseen proportion (365 individuals) of the screened population as positive or negative for the presence of cancer or precancer. The overall sensitivity and specificity of the dentists were 0.74 [95% confidence interval (CI), 0.62-0.86] and 0.99 (95% CI, 0.985-0.994) respectively compared with 0.80 (99% CI, 0.55-1.00) and 0.77 (95% CI, 0.73-0.81) for the neural network. In view of the potential costs involved in implementing a screening programme, this neural network may be of value for the identification of individuals with a high risk of oral cancer or precancer for further clinical examination or health education.

Case-control study of oral dysplasia and risk habits among patients of a dental hospital.

Several studies have investigated risk factors for oral cancer but few have considered precancer. Records accumulated from 1975 to 1993 of dental hospital patients with histologically confirmed oral dysplasia provided the opportunity for a retrospective case-control study of the association between oral precancer and smoking tobacco and drinking alcohol. Seventy sets of case notes were available and each case was matched with records of a control subject, known to be free from dysplasia from another study, for birth date, gender and presumed ethnicity. The relative risk (OR) of having a dysplastic lesion for smokers compared with non-smokers, or ex-smokers for > 10 years, was 7.00. Logistic multiple regression revealed a dose-response relationship for tobacco dependent upon the level of cigarette consumption. Also subjects with moderate or severe dysplasia included a higher proportion of smokers than those with mild dysplasia. No overall increased risk from alcohol was found. However, the proportion of subjects who drank spirits was significantly higher among cases than controls. The study reaffirms the role of dental practitioners in identifying individuals at risk of mucosal disease, the importance of public education about the risk factors, and the necessity for counselling patients with precancerous lesions on avoiding further risk.

Attendance and compliance at an oral cancer screening programme in a general medical practice.

The purpose of this study was to measure the attendance and compliance rates in a demonstration invitational screening programme for oral cancer. 4348 subjects aged 40 years or over registered at an inner city medical practice in north London were invited for screening by post. The socioeconomic profile of the group was determined by analysis of residential areas. Screening was conducted by one of several dentists and a referral pathway was established for patients requiring follow-up. Attendance rates for screening and referral for follow-up were measured. The response rate was 985/3826 (25.7%) after removing 522 subjects whose invitations could not be delivered or who refused appointments. No reply was obtained for 2841 patients. Attendance for referral of lesions considered to have malignant potential was 67% (8/12), compared to 92% (11/12) for patients requiring referral for incidental benign lesions. The low compliance suggests that oral cancer screening may not be able to achieve the desired benefits of reducing morbidity and mortality, and establishment of such a programme may not, therefore, be cost-effective. Further research is required into how to identify people in high risk groups and motivate them to present themselves for screening.

Evaluation of screening for oral cancer and precancer in a company headquarters.

Oral cancer and precancer appear to fulfil many of the criteria for a disease suitable for mass screening. Several commercial organisations in the UK have introduced screening for their employees. One program has been formally evaluated over the course of 1 yr. Of 553 company headquarters staff aged > or = 40 yr, 292 (53%) responded to the well-publicised screening invitation and received a simple clinical examination of the oral mucosa from one of two company dentists. In addition, 17 staff were screened from a separate company work-site. After screening, subjects were examined independently by an oral medicine specialist with access to the relevant diagnostic aids. The dentists' screening decisions were validated against the specialist's definitive diagnoses (the 'gold standard'). The true prevalence of subjects with lesions diagnosed as positive (white patch, red patch or ulcer of greater than 2 weeks' duration) was 17 (5.5%). Overall, sensitivity was 0.71 and specificity, 0.99. The compliance rate to screening among headquarters subjects in seven occupational categories did not differ significantly from the occupational profile for all headquarters personnel. Estimates of relative risk of a positive diagnosis were calculated by logistic regression for five independent variables; gender, age, moderate smoking, heavy smoking, and smoking combined with greater than low risk alcohol consumption. Only heavy smoking (> or = 20 cigarettes per day) produced a significant odds ratio (3.43, P < 0.05).

Evaluation of a screening test for the early detection of oral cancer and precancer.

The purpose of this study was to establish the sensitivity and specificity of a clinical examination for the detection of early oral cancer and precancer. A screening programme was conducted over a period of one year and 2027 subjects aged 40 years and over were examined. Screening took place at two sites; opportunistically in outpatient departments at a dental hospital and by postal invitation at an inner city medical practice. The screening procedure included a questionnaire on habits and an oral examination by two independent dentists. The first examining dentist (the screener) was either a general dental practitioner, a community dental officer or a junior hospital dentist. There were 24 dentists in the screener group. A second single examining dentist (a specialist) provided the definitive diagnosis, or 'gold standard', with which the screeners' results were compared. A screen was defined as positive if a white patch, a red patch, or an ulcer of longer than two weeks duration was detected. Each subject was categorised as either positive or negative by both the screener and the specialist. The screener and specialist were unaware of each other's findings. The prevalence of disease according to the specialist was 2.7 per cent. The results for all 24 screeners were pooled and gave an overall sensitivity of 0.74 (95 per cent CI, 0.62 to 0.86), specificity of 0.99 (95 per cent CI, 0.985-0.994) and positive and negative predictive values of 0.67 and 0.99 respectively.

Effects of cohort, sex, and birth date on body development of roe deer (Capreolus capreolus) fawns.

We investigated the effects of the year of birth (cohort effect), sex and birth date on the variations observed in birth weight and postnatal growth rate in 209 newborn fawns marked during eight consecutive fawning seasons from a highly productive roe deer population under good nutritional conditions. Roe deer exhibited a fast body development with a birth weight of 1628 g and a postnatal growth rate of 139 g/day constant over the first 20 days of their life. As previously reported for dimorphic and polygynous ungulates, there was a marked cohort effect on the rate of body development. This variability was partly explained by climatic conditions during late gestation. When fewer than 5 days with rainfall over 5 mm occurred in April, fawns were heavier. High temperatures during April and during the winter could also be involved in fast body development of roe deer fawns. Sex of fawn did not affect roe deer growth pattern. This was expected on the basis of low sexual dimorphism in size and low polygyny level characteristic of roe deer. Lastly, the date of birth did not affect the body development of roe deer fawns. High constancy of mean birth dates and high synchrony of births observed in this population could account for this result.

Sex differentiation in postnatal growth rate: a test in a wild boar population.

In wild boar individual growth rate is linear between 0.5 and 6 months after birth, based on successive body weight measurements. Contrary to expectation for a dimorphic and polygynous mammal like wild boar, no sexual dimorphism in growth rate could be detected between 0.5 and 6 months. We argue that high total maternal invesment in offspring due to large litter size and/or strong selection for early reproduction in this population with a short generation time could explain this absence of early differentiation in postnatal growth rate according to offspring sex.

Control of Culex pipiens by Bacillus sphaericus and role of nontarget arthropods in its recycling.

Bacillus sphaericus was used to control Culex pipiens breeding in a water treatment settling basin near Montpellier, France. Four treatments with 4 liters/ha (3.6 lb/acre) of commercially available B. sphaericus formulation (Vectolex) reduced the larval population of Cx. pipiens with 50-600 spores of B. sphaericus/ml recorded in the treated portions of the water plant 14 days following the last treatment. Natural recycling of the bacteria was shown to take place at the water surface where germination of spores was evidenced. The appearance of vegetative forms of B. sphaericus may be linked to the passage through larval guts of several filter-feeding arthropods.