Deep-sequencing of Solanum commersonii small RNA libraries reveals riboregulators involved in cold stress response.

Among wild species used in potato breeding, Solanum commersonii displays the highest tolerance to low temperatures under both acclimated (ACC) and non-acclimated (NACC) conditions. It is also the first wild potato relative with a known whole genome sequence. Recent studies have shown that abiotic stresses induce changes in the expression of many small non-coding RNA (sncRNA). We determined the small non-coding RNA (sncRNAome) of two clones of S. commersonii contrasting in their cold response phenotypes via smRNAseq. Differential analysis provided evidence that expression of several miRNAs changed in response to cold stress conditions. Conserved miR408a and miR408b changed their expression under NACC conditions, whereas miR156 and miR169 were differentially expressed only under ACC conditions. We also report changes in tasiRNA and secondary siRNA expression under both stress conditions. Our results reveal possible roles of sncRNA in the regulatory networks associated with tolerance to low temperatures and provide useful information for a more strategic use of genomic resources in potato breeding.

Larval zebrafish as a model for glucose metabolism: expression of phosphoenolpyruvate carboxykinase as a marker for exposure to anti-diabetic compounds.

The zebrafish model system is one of the most widely used animal models for developmental research and it is now becoming an attractive model for drug discovery and toxicological screening. The completion of sequencing the zebrafish genome and the availability of full-length cDNAs and DNA microarrays for expression analysis, in addition to techniques for generating transgenic lines and targeted mutations, have made the zebrafish model even more attractive to researchers. Recent data indicate that the regulation of glucose metabolism in zebrafish, through the production of insulin, is similar to mammalian models, and many of the genes involved in regulating blood glucose levels have been identified in zebrafish. The data presented here show that adult zebrafish respond to anti-diabetic drugs similarly to mammalian models, by reducing blood glucose levels. Furthermore, we show that the expression of phosphoenolpyruvate carboxykinase (PEPCK), which catalyzes a rate-limiting step in gluconeogenesis and is transcriptionally regulated by glucagon and insulin, is regulated in larval zebrafish similarly to that seen in mammalian systems, and changes in PEPCK expression can be obtained through real-time PCR analysis of whole larval RNA. Taken together, these data suggest that larval zebrafish may be an appropriate model for the examination of glucose metabolism, using PEPCK as an indicator of blood glucose levels.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure of normal human dermal fibroblasts results in AhR-dependent and -independent changes in gene expression.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in a variety of lesions in mammals including severe skin lesions. The majority of TCDD's biological effects are mediated through activation of the aryl hydrocarbon receptor (AhR). We have chosen to examine the effect of TCDD and the AhR pathway on dermal fibroblasts because this cell type plays an integral role in skin homeostasis through the production of cytokines and other factors that regulate epidermal proliferation and differentiation. Our data show that normal human dermal fibroblasts (NHDFs) are responsive to TCDD, as demonstrated by induction of cytochrome p450 1B1 (CYP1B1) expression. Further, our data demonstrate that TCDD treatment of NHDFs results in significant (75-90%) decrease in expression of Id-1 and Id-3, proteins that are involved in regulation of cell proliferation and differentiation. The Id (Inhibitor of DNA binding) proteins are transcriptional inhibitors that function by forming inactive heterodimers with other HLH proteins. TCDD-repression of Id-1 and -3 is independent of de novo protein synthesis; co-treatment with cycloheximide has no effect on TCDD inhibition of Id-1 and Id-3. Co-treatment with the AhR antagonist alpha-naphthoflavone also does not block inhibition of Id-1 and Id-3 by TCDD, suggesting that TCDD inhibition of Id-1 and Id-3 is, at least in part, mediated independently of the AhR pathway. Our data also show that TCDD inhibits expression of the cell cycle regulatory gene p16(ink4a), which is often linked to Id expression. TCDD-induced reduction of p16(ink4a) expression is also independent of protein synthesis and the AhR pathway.

Expression of the helix-loop-helix protein inhibitor of DNA binding-1 (ID-1) is activated by all-trans retinoic acid in normal human keratinocytes.

The ID (inhibitor of differentiation or DNA binding) helix-loop-helix proteins are important mediators of cellular differentiation and proliferation in a variety of cell types through regulation of gene expression. Overexpression of the ID proteins in normal human keratinocytes results in extension of culture lifespan, indicating that these proteins are important for epidermal differentiation. Our hypothesis is that the ID proteins are targets of the retinoic acid signaling pathway in keratinocytes. Retinoids, vitamin A analogues, are powerful regulators of cell growth and differentiation and are widely used in the prevention and treatment of a variety of cancers in humans. Furthermore, retinoic acid is necessary for the maintenance of epithelial differentiation and demonstrates an inhibitory action on skin carcinogenesis. We examined the effect of all-trans retinoic acid on expression of ID-1, -2, -3, and -4 in normal human keratinocytes and found that exposure of these cells to all-trans retinoic acid causes an increase in both ID-1 and ID-3 gene expression. Furthermore, our data show that this increase is mediated by increased transcription involving several cis-acting elements in the distal portion of the promoter, including a CREB-binding site, an Egr1 element, and an YY1 site. These data demonstrate that the ID proteins are direct targets of the retinoic acid signaling pathway. Given the importance of the ID proteins to epidermal differentiation, these results suggest that IDs may be mediating some of the effects of all-trans retinoic acid in normal human keratinocytes.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces matrix metalloproteinase (MMP) expression and invasion in A2058 melanoma cells.

There has been a 34% increase in melanoma related mortality in the United States from 1973 to 1992. Although few successful treatments for malignant melanoma exist, it is known that genetic susceptibility and environmental factors contribute to the initiation and progression of melanoma. Excessive UV exposure is considered the main etiological factor in melanoma initiation, however, epidemiological and experimental evidence suggests that exposure to environmental carcinogens contribute to melanoma. We propose that exposure to environmental chemicals that activate the aryl hydrocarbon receptor pathway contribute to melanoma progression, specifically through stimulation of the expression and activity of the matrix metalloproteinases (MMPs). Therefore, we investigated the effect of AhR activation on normal human melanocytes and several melanoma cell lines. The data presented here demonstrate that normal melanocytes and melanoma cells express the AhR and Arnt and are responsive to activation by TCDD. Furthermore, activation of this pathway in transformed melanoma cells (A2058) results in increased expression and activity of MMP-1, MMP-2 and MMP-9, as well as increased invasion using in vitro invasion assays. Furthermore, TCDD-induced expression of the MMP-1 promoter in melanoma cells appears to require different elements than those required in untransformed cells, indicating that this pathway may have multiple mechanisms for activation of MMP expression.

Lymphoscintigraphy with intraoperative gamma probe sentinel node detection: clinical impact in patients with head and neck melanomas.

AIM: The aims of this paper were to evaluate the clinical relevance of lymphoscintigraphy with intraoperative gamma-probe detection in identifying sentinel nodes (SNs) and to study the prognostic value of SN biopsy in head and neck melanoma patients. METHODS: Sixty-one patients had lymphoscintigraphy with intradermal injections of 99mTc-Nanocoll (40 MBq), 24 h before surgery. Tumor-positive SNs patients underwent total lymph node dissection. Postoperative histological examination was performed. Patients were followed up for 1 to 5 years (median 3 years). The tumor relapses and the overall survival were evaluated by means of statistical methods. RESULTS: Lymphoscintigraphy showed lymphatic distribution to more than one basin in 45 patients (74%), in 15 patients one basin was visualized and no basin in 1 patient. In 41 patients the SN was negative for metastases, while in 20 was positive. In a high percentage of patients (30%), metastatic involvement occurred in more than one lymph node basin. During follow-up in the negative SN group, 40 patients remained disease free and 1 relapsed. In the positive SN group, 10 patients remained disease free and 10 relapsed. Recurrence time ranged from 6 to 11 months. The overall survival of the SNs negative group was significantly higher than the positive SN group. CONCLUSIONS: This approach was able to distinguish: a) patients with tumor-negative SNs with a favorable clinical course (98% did not relapse, P<0.001); b) patients with tumor-positive SNs with a high rate of tumor relapse (50%, P<0.001). Therefore SN biopsy may give information about prognosis in head and neck melanoma patients.

Comparative evaluation of two methods to assay thyroglobulin serum concentrations in patients with differentiated thyroid carcinomas.

AIM: This study was aimed at the comparative assessment of the analytical and clinical performances of 2 tests for thyroglobulin (Tg) assays: the Dynotest Tg-Plus immunoradiometric assay (IRMA), a new method that might be of interest for its claimed superior sensitivity compared to other methods, and the HTGK-2 IRMA, one of the test currently used in clinical routine. METHODS: The study was performed in serum samples from 157 patients with differentiated thyroid carcinoma (DTC). The clinical sensitivity of the test was evaluated in patients with and without thyroid stimulating hormone (TSH) suppression. RESULTS: The lowest detectable Tg concentration values and the within-assay coefficient of variation (CV) were 0.4 and 0.8 microg/L and 5% and 3% for the Dynotest Tg-Plus assay and the HTGK-2 assay, respectively; the between-assay CV was 6% for both assays. The clinical results of the Dynotest Tg-Plus and those of the HTGK-2 kit were similar in both DTC patient populations, either under or off the TSH suppressive treatment. In spite of the manufacturer's statement that the calibrators of both assays had been standardized against the same common reference (standard CRM 457 of the Community Bureau of References), the Dynotest Tg-Plus test underrated by a factor of 0.5 the Tg values measured by means of HTGK-2 IRMA. CONCLUSION: The sensitivity of the Dynotest Tg-Plus IRMA appears to be similar to that of the HTGK-2 assay.

Position of nuclear medicine techniques in the diagnostic work-up of neuroendocrine tumors.

In recent years nuclear medicine has contributed to the impressive development of the knowledge of neuroendocrine tumors in terms of biology (receptor scintigraphy), pharmacology (development of new tracers), and therapy (radiometabolic therapy). At present, it is impossible to plan the management of a patient affected by a neuroendocrine tumor without performing nuclear medicine examinations. The contribution of nuclear medicine had affected and improved the management of these patients by offering various important options that are part of the modern diagnosis and treatment protocols. The clinical experience and the literature confirm that, among the wide variety of tracers and nuclear medicine modalities available today, metaiodobenzylguanidine (MIBG) and DTPA-D-Phe-octreotide (pentetreotide) are the radiopharmaceuticals of current clinical use. Several new somatostatin analogues are under investigation. Positron emission tomography (PET) supplies a range of labelled compounds to be used for the visualization of tumor biochemistry. In addition to the first routinely used PET tracer in oncology, 18F-labelled deoxyglucose (FDG), a number of radiopharmaceuticals based on different precursors such as fluorodopamine and 5-hydroxytryptophan (5-HTP) are going to gain a clinical role. Of course, the diagnosis of neuroendocrine tumors has to be based on integrated information derived from different examinations including nuclear medicine studies. The clinical presentation of neuroendocrine tumors is highly variable: sometimes they manifest typical or atypical symptoms but they may also be detected by chance during an X-ray or ultrasound examination carried out for other reasons. At disease presentation nuclear medicine modalities are sometimes able to direct physicians towards the clinical diagnosis thanks to the specificity of their imaging mechanisms. They also play a role in disease staging and restaging, patient follow-up and treatment monitoring. In addition, the biological characterisation of neuroendocrine tissues (receptor status, glucose metabolism, differentiation, etc.) allows the interpretation of radiopharmaceutical uptake as a prognostic parameter and sometimes as a predictor of the response to treatment.

Gallium scan in adolescents and children with Hodgkin's disease (HD). Treatment response assessment and prognostic value.

AIM: The aim of the present paper is to describe the accuracy of gallium ((67)Ga) scintigraphy in adolescents and children with Hodgkin's disease (HD). We have studied the diagnostic value of this nuclear imaging technique at disease presentation (staging) and its prognostic value based on changes in (67)Ga uptake observed after treatment (response assessment). METHODS: From April 1985 to July 1999 74 consecutive untreated patients with a median age of 13 y underwent (67)Ga scans 48-72 h after injection of 37-111 MBq of (67)Ga-citrate. Planar whole-body scintigraphy was performed, supplemented with single photon emission tomography (SPET) of the mediastinum from 1996 onwards. Three patients did not undergo further scintigraphic examination because they were treated with radical surgery. After the 1st examination 71 of the 74 patients were monitored by 1-3 (67)Ga scans during the course of their disease. All of them had at least one (67)Ga scintigraphy at the end of the induction phase of chemotherapy, before any other therapeutic regimens were planned. RESULTS: At disease presentation (67)Ga scintigraphy was positive in all patients, detecting 285 of 335 (85.0%) lymph nodal sites of disease. The best sensitivity was observed in the mediastinum (100%; 63/63) and the laterocervical supraclavicular region (85.6%; 125/146); it was lower for axillary (72.7%; 16/22) and retroperitoneal (68.7%; 11/16) lymph node masses. In detecting visceral involvement the sensitivity of (67)Ga scintigraphy was 66.6% (8/12) for lung and 80% (4/5) for bone involvement. Among 71 patients in follow-up, 2 showed rapid progression of disease during induction therapy while 69 patients were monitored for a long period. The response to therapy has been classified according to the changes observed on nuclear medicine or radiological images as complete response (CR) or partial response (PR). On the basis of (67)Ga scans 55 patients (72.4%) were considered as having a CR, while with radiological modalities (chest X-ray, CT, MRI) CR was observed in only 29 patients (40.8%). PR or progression was found with (67)Ga scintigraphy in 16 patients (22.5%) and with radiological modalities in 42 patients (59.1%). (67)Ga scan was concordant with clinical outcome in 97% (28/29). The diagnostic effectiveness of this imaging technique has been analysed by comparing the scintigraphic or radiological changes at the 1st scintigraphic/radiological follow-up examination after induction therapy with the clinical outcome. In this population the relapse rate was 50% (8/16) in the group that did not achieve a CR according to post-treatment (67)Ga scintigraphy, while it was only 10.9% (6/55) in the group that achieved a CR on the basis of scintigraphy findings. The overall survival (OS) and disease-free survival (DFS) were calculated by means of Kaplan-Meier cumulative survival plotting. When the 2 groups of patients with complete (CR) or incomplete normalisation (PR or progression) of (67)Ga scintigraphy were compared, both OS and DFS were found to be statistically different (p=0.0001 and p=0.0004, respectively). By contrast, no statistical difference was found when the radiological findings were considered as the criterion for assessment of tumour response. On the basis of X-ray results the relapse rate was 13.7% in patients with negative post-therapy findings and 23.8% in patients with positive radiological imaging. CONCLUSION: Our data demonstrate the high value of (67)Ga scintigraphy in HD staging in paediatric patients. In addition, evaluation of the (67)Ga uptake is very useful as a prognostic parameter; changes in (67)Ga uptake after therapy indicate a favourable prognosis, whereas children still positive on post-treatment (67)Ga scintigrams should be given more aggressive treatment.