A cDNA microarray assessment of gene expression in the liver of rainbow trout (Oncorhynchus mykiss) in response to a handling and confinement stressor.

A purpose-designed microarray platform (Stressgenes, Phase 1) was utilised to investigate the changes in gene expression within the liver of rainbow trout during exposure to a prolonged period of confinement. Tissue and blood samples were collected from trout at intervals up to 648 h after transfer to a standardised confinement stressor, together with matched samples from undisturbed control fish. Plasma ACTH, cortisol, glucose and lactate were analysed to confirm that the neuroendocrine response to confinement was consistent with previous findings and to provide a phenotypic context to assist interpretation of gene expression data. Liver samples for suppression subtractive hybridisation (SSH) library construction were selected from within the experimental groups comprising "early" stress (2-48 h) and "late" stress (96-504 h). In order to reduce redundancy within the four SSH libraries and yield a higher number of unique clones an additional subtraction was carried out. After printing of the arrays a series of 55 hybridisations were executed to cover 6 time points. At 2 h, 6 h, 24 h, 168 h and 504 h 5 individual confined fish and 5 individual control fish were used with control fish only at 0 h. A preliminary list of 314 clones considered differentially regulated over the complete time course was generated by a combination of data analysis approaches and the most significant gene expression changes were found to occur during the 24 h to 168 h time period with a general approach to control levels by 504 h. Few changes in expression were apparent over the first 6 h. The list of genes whose expression was significantly altered comprised predominantly genes belonging to the biological process category (response to stimulus) and one cellular component category (extracellular region) and were dominated by so-called acute phase proteins. Analysis of the gene expression profile in liver tissue during confinement revealed a number of significant clusters. The major patterns comprised genes that were up-regulated at 24 h and beyond, the primary examples being haptoglobin, beta-fibrinogen and EST10729. Two representative genes from each of the six k-means clusters were validated by qPCR. Correlations between microarray and qPCR expression patterns were significant for most of the genes tested. qPCR analysis revealed that haptoglobin expression was up-regulated approximately 8-fold at 24 h and over 13-fold by 168 h.

Molecular profiling techniques and bioinformatics in cancer research.

AIMS: Our aim was to describe the commonly used molecular profiling techniques in cancer research, to examine their limitations and to discuss the challenges of bioinformatics. METHODS: A literature search was performed using the PubMed database to identify publications relevant to this review. Citations from these articles were also examined to yield further relevant publications. RESULTS: We describe the use of DNA microarrays, comparative genomic hybridisation, tissue microarrays and digital differential display. The limitations of these technologies, their contribution to cancer research and the challenges of bioinformatics are also discussed. CONCLUSIONS: Although these high throughput technologies each have their own limitations they are rapidly developing and contributing significantly to our understanding of cancer genetics. They have also led to the emergence of bioinformatics as a rapidly developing and vital field.

Effect of systemic progesterone concentration on the expression of progesterone-responsive genes in the bovine endometrium during the early luteal phase.

Increasing evidence indicates an association between the concentration of systemic progesterone during the early luteal phase of the oestrous cycle and embryo survival rate in cattle. We examined the relationship between the concentration of systemic progesterone on Days 4 to 8 post-ovulation and expression of progesterone receptor (PGR), oestrogen receptor +/- (ESR1) and retinol-binding protein (RBP) mRNA in the bovine endometrium. Heifers were blood sampled from the day of ovulation (Day 0) to Day 8 post-ovulation. On Day 4, animals were divided into low progesterone control (LC) and high progesterone control (HC) groups based on their plasma progesterone concentrations. Half of each group was supplemented with exogenous progesterone resulting in two further groups, low progesterone supplemented (LS) and high progesterone supplemented (HS). Endometrial tissues were recovered from all groups on Day 6 or Day 8 and gene expression was analysed following Northern blotting. Increasing progesterone concentrations were associated with decreased PGR and ESR1 expression. Duration-dependent effects of progesterone supplementation on ESR1 were evident and there was an effect of systemic progesterone concentrations between Day 0 and Day 4 on the expression of RBP at Days 6 and 8. Such progesterone-responsive changes in uterine gene expression are likely to affect embryo development.

Associations between milk progesterone concentration on different days and with embryo survival during the early luteal phase in dairy cows.

The relationships between the concentration of milk progesterone and early embryo survival on Days 4-8 inclusive and between the concentration of progesterone on different days from Days 0-8 inclusive following ovulation and insemination were examined in dairy cows. The relationships were examined following 77 randomly chosen artificial inseminations to cows in standing oestrus. There was a significant (P < 0.05) linear and quadratic relationship between the concentration of milk progesterone on each of Days 4-6 after ovulation and the probability of embryo survival. There was no association (P > 0.05) between milk progesterone concentration and probability of embryo survival on Days 7 and 8 after ovulation. There were no associations between milk progesterone concentration on Days 0-2 and the concentrations on Days 4-7, however, progesterone concentrations on Days 4 and 5 were highly predictive of the concentration on Days 6 and 7, respectively. Overall, the results indicate that suboptimal progesterone support during the early luteal phase is likely to deleteriously affect embryo viability and in addition, that it is possible to predict milk progesterone concentrations during the early luteal phase based on earlier stage concentrations and thus identify cows at risk of early embryo loss.

Scottish attitudes to blood donation and AIDS.

OBJECTIVE: To see whether the issue of AIDS has influenced the observed decline in blood donation in Scotland. DESIGN: Two methods: a quantitative survey using personal interviews based on a questionnaire and a qualitative survey based on group discussions. SETTING: Interviews based on the questionnaire were conducted in the respondents' homes. The group discussions were held in the homes of professional market research interviewers. PARTICIPANTS: For the quantitative survey a representative sample of 976 Scottish adults was selected by multistage sampling. In the qualitative survey 16 groups of five to eight respondents assigned according to donating experience and sociodemographic criteria took part. MAIN RESULTS: AIDS was not mentioned as a discouraging factor in donation, and off putting aspects identified before AIDS became a public issue remained salient--for example, fear of needles. Many (75%) thought it unlikely that donation entailed a risk of developing AIDS. Nevertheless, respondents were reluctant to consider the AIDS issue personally. Being asked to do so, as in the routine screening of donors, aroused fears and resentment. CONCLUSIONS: The issue of AIDS, including fear of infection, has not directly influenced the declining numbers of donors, but the unpleasant associations of AIDS have had an indirect effect, particularly in undermining the emotional benefits of giving blood. For example, the screening process, which requires potential donors to consider their personal risk from AIDS, had had the effect of discouraging donors in general. Redressing the balance is difficult as screening must be universally applied. Rather than minimising the issue of AIDS, publicity needs urgently to reassert the positive benefits of and rewards from giving blood.

Cytologic evaluation of Papanicolaou-stained bone marrow aspirates.

Aspirates obtained from bone marrow were prepared for routine Papanicolaou staining and screened by a cytologist. As a preliminary study, 100 consecutive bone marrow aspirations were examined. It was shown that metastatic carcinoma and primary bone marrow disorders (including leukemia, myelodysplastic syndromes, and multiple myeloma) can be recognized on Papanicolaou-stained marrow aspirates using the same cytologic criteria employed in the evaluation of cells from any site. Evaluation of bone marrow aspirates in the cytology laboratory is feasible and can be used to augment parallel services employing air-dried Wright-Giemsa-stained specimens in the hematology laboratory.

Cirrhosis with atypia. A potential pitfall in the interpretation of liver aspirates.

Needle aspirations of the liver yielding highly atypical hepatocytes present a diagnostic challenge, with the differential diagnosis lying between hepatocellular carcinoma and benign reactive atypia. A case of a healing liver abscess in a patient with cirrhosis, mistakenly diagnosed as an hepatocellular carcinoma, is presented. Criteria for the avoidance of false-positive diagnoses of hepatocellular carcinoma on needle aspirates are presented, and the concept of "liver cell dysplasia" as a cytodiagnostic entity is discussed.

Amebae resembling Entamoeba gingivalis in the genital tracts of IUD users.

A total of 2,252 vaginopancervical (Fast) smears were studied as a continuation of an ongoing program to further investigate the occurrence of amebae in the genital tracts of women using intrauterine devices (IUDs). Of the 947 IUD users, 1% harbored trophic amebae. No amebae, however, were found in any of the 1,164 smears from non-IUD wearers. In all instances the amebae were associated with Actinomyces. Both organisms disappeared promptly after removal of the IUD. In preparations stained according to the Papanicolaou, iron-hematoxylin and Gomori-Wheatley trichrome methods, the amebae could be diagnosed as belonging to the genus Entamoeba. These trophozoites failed to stain with fluorescein-labeled anti-Entamoeba-histolytica serum. Although on the basis of the presently available evidence these trophozoites cannot be assigned with certainty to any known species, they appear to share many morphologic characteristics with Entamoeba gingivalis.