Research Note: Phenotypic trends for the multigenerational advanced intercross of the Virginia body weight lines of chickens.

Random samples from generation S41 of the Virginia high and low 8-week body weight lines formed the base population for producing a multigenerational reciprocal intercross population. Although genetic mapping from this intercross has been reported, lacking are phenotypic trends across multiple generations. Here, we provide phenotypic information for the parental base population, the F1 reciprocal cross, and subsequent segregating recombinant generations F2 to F17. Heterosis for the selected trait in the F1 was negative for both reciprocal crosses. Phenotypic correlations for the selected trait in the recombinant generations were essentially nil for both males and females as was percent sexual dimorphism and coefficients of variation.

Low-coverage sequencing in a deep intercross of the Virginia body weight lines provides insight to the polygenic genetic architecture of growth: novel loci revealed by increased power and improved genome-coverage.

Genetic dissection of highly polygenic traits is a challenge, in part due to the power necessary to confidently identify loci with minor effects. Experimental crosses are valuable resources for mapping such traits. Traditionally, genome-wide analyses of experimental crosses have targeted major loci using data from a single generation (often the F2) with individuals from later generations being generated for replication and fine-mapping. Here, we aim to confidently identify minor-effect loci contributing to the highly polygenic basis of the long-term, bi-directional selection responses for 56-d body weight in the Virginia body weight chicken lines. To achieve this, a strategy was developed to make use of data from all generations (F2-F18) of the advanced intercross line, developed by crossing the low and high selected lines after 40 generations of selection. A cost-efficient low-coverage sequencing based approach was used to obtain high-confidence genotypes in 1Mb bins across 99.3% of the chicken genome for >3,300 intercross individuals. In total, 12 genome-wide significant, and 30 additional suggestive QTL reaching a 10% FDR threshold, were mapped for 56-d body weight. Only 2 of these QTL reached genome-wide significance in earlier analyses of the F2 generation. The minor-effect QTL mapped here were generally due to an overall increase in power by integrating data across generations, with contributions from increased genome-coverage and improved marker information content. The 12 significant QTL explain >37% of the difference between the parental lines, three times more than 2 previously reported significant QTL. The 42 significant and suggestive QTL together explain >80%. Making integrated use of all available samples from multiple generations in experimental crosses are economically feasible using the low-cost, sequencing-based genotyping strategies outlined here. Our empirical results illustrate the value of this strategy for mapping novel minor-effect loci contributing to complex traits to provide a more confident, comprehensive view of the individual loci that form the genetic basis of the highly polygenic, long-term selection responses for 56-d body weight in the Virginia body weight chicken lines.

Population dynamics of a long-term selection experiment in White Plymouth Rock chickens selected for low or high body weight.

The population dynamics of 2 lines of chickens from a long-term (59 generations) selection experiment were assessed based on pedigree data. These lines were propagated from phenotypic selection for low and high 8-wk BW in White Plymouth Rock chickens. Our objective was to determine whether the 2 lines maintained similar population structures over the selection horizon to allow meaningful comparisons of their performance data. A complete pedigree of 31,909 individuals, consisting of 102 founders, 1,064 from the parental generation, and 16,245 low weight (LWS) and 14,498 high weight (HWS) select chickens, was available. Inbreeding (F) and average relatedness (AR) coefficients were computed. Average F per generation and AR coefficients were 1.3 (SD 0.8) % and 0.53 (SD 0.001) for LWS, and 1.5 (SD 1.1) % and 0.66 (SD 0.001) for HWS. Mean F for the entire pedigree was 0.26 (0.16) and 0.33 (0.19), and maximum F was 0.64 and 0.63, in LWS and HWS, respectively. Based on Wright's fixation index, at generation 59, substantial genetic differences were established between lines. The effective population size was 39 in LWS and 33 in HWS. The effective number of founders was 17 and 15, effective number of ancestors were 12 and 8, and genome equivalents were 2.5 and 1.9 in LWS and HWS, respectively. About 30 founders explained the marginal contribution to both lines. By generation 59, only 7 male and 6 female founders contributed to both lines. Moderately high levels of inbreeding and low effective population sizes were inevitable, as this was a closed population. However, effects on the fitness of the population were expected to be less substantial because founders were a combination of 7 lines. The effective numbers of founders and ancestors were relatively low compared to the actual number of founders, as few ancestors contributed to descendants. Based on these evaluations, it can be inferred that LWS and HWS had similar population structures. Comparisons of selection responses in the 2 lines therefore should be reliable.

Epigenetic modifiers identified as regulators of food intake in a unique hypophagic chicken model.

DNA methylation is an epigenetic modification that influences gene transcription; however, the effects of methylation-influencing chemicals on appetite are unknown. We evaluated the effects of single administration of a methyl donor, S-Adenosylmethionine (SAM), or methylation inhibitor, 5-Azacytidine (AZA), on immediate and later-age food intake in an anorexic chick model. The doses of intracerebroventricularly-injected SAM were 0 (vehicle), 0.1, 1, and 10 µg, and of AZA were 0 (vehicle), 1, 5, and 25 µg. When injected on day 5 posthatch, there was no effect of SAM on food intake in either fed or fasted chicks, whereas AZA increased food consumption in the fasted state but decreased it in fed chicks. We then performed a single injection (same doses) at hatch and measured food intake on day 5 in response to neuropeptide Y (NPY; 0.2 µg) injection. Irrespective of NPY, chicks injected with 1 µg of SAM ate more than others on day 5. In contrast, chicks injected with AZA (5 and 25 µg doses) consumed less on day 5. In conclusion, we identified DNA methylation-regulating chemicals as regulators of food intake. AZA but not SAM affected food intake in the short-term, feeding state dependently. Later, both chemicals injected on the day of hatch were associated with food intake changes at a later age, suggesting that feeding pathways might be altered through changes in methylation.

A survey of health care professionals and oncology patients at the McGill University Health Centre reveals enthusiasm for establishing a postmortem rapid tissue donation program.

Background: In the early developmental phase of a postmortem rapid tissue donation (rtd) program for patients with metastatic cancer, we surveyed health care professionals (hcps) and oncology patients at the McGill University Health Centre (muhc) to assess their knowledge and attitudes pertaining to rtd from metastatic cancer patients for research purposes. Methods: A 23-item survey was developed and distributed to hcps at tumour board meetings, and a related 26-item survey was developed and distributed to oncology patients at the muhc Cedars Cancer Centre. Results: The survey attracted participation from 73 hcps, including 37 attending physicians, and 102 oncology patients. Despite the fact that 88% of hcps rated their knowledge of rtd as none or limited, 42% indicated that they would feel comfortable discussing rtd with their cancer patients. Of the responding hcps, 67% indicated that their current knowledge of rtd would affect their decision to discuss such a program with patients, which implies the importance of education for hcps to facilitate enrolment of patients into a rtd program. Of responding patients, 78% indicated that they would not be uncomfortable if their doctor discussed rtd with them, and 61% indicated that they would like it if their doctor were to discuss rtd with them. The hcps and patients felt that the best time for patients to be approached about consenting to a rtd program would be at the transition to palliative care when no treatment options remain. Conclusions: At the muhc, hcps and patients are generally enthusiastic about adopting a rtd program for patients with metastatic cancer. Education of hcps and patients will be an important determinant of the program's success.

A genomic inference of the White Plymouth Rock genealogy.

Crossing of populations has been, and still is, a central component in domestication and breed and variety formation. It is a way for breeders to utilize heterosis and to introduce new genetic variation into existing plant and livestock populations. During the mid-19th century, several chicken breeds that had been introduced to America from Europe and Asia became the founders for those formed in the USA. Historical records about the genealogy of these populations are often unclear and inconsistent. Here, we used genomics in an attempt to describe the ancestry of the White Plymouth Rock (WPR) chicken. In total, 150 chickens from the WPR and 8 other stocks that historical records suggested contributed to its formation were whole-genome re-sequenced. The admixture analyses of the autosomal and sex chromosomes showed that the WPR was likely founded as a cross between a paternal lineage that was primarily Dominique, and a maternal lineage where Black Java and Cochin contributed in essentially equal proportions. These results were consistent and provided quantification with the historical records that they were the main contributors to the WPR. The genomic analyses also revealed genome-wide contributions (<10% each) by Brahma, Langshan, and Black Minorca. When viewed on an individual chromosomal basis, contributions varied considerably among stocks.

LKB1 deficiency in T cells promotes the development of gastrointestinal polyposis.

Germline mutations in STK11, which encodes the tumor suppressor liver kinase B1 (LKB1), promote Peutz-Jeghers syndrome (PJS), a cancer predisposition syndrome characterized by the development of gastrointestinal (GI) polyps. Here, we report that heterozygous deletion of Stk11 in T cells (LThet mice) is sufficient to promote GI polyposis. Polyps from LThet mice, Stk11+/- mice, and human PJS patients display hallmarks of chronic inflammation, marked by inflammatory immune-cell infiltration, signal transducer and activator of transcription 3 (STAT3) activation, and increased expression of inflammatory factors associated with cancer progression [interleukin 6 (IL-6), IL-11, and CXCL2]. Targeting either T cells, IL-6, or STAT3 signaling reduced polyp growth in Stk11+/- animals. Our results identify LKB1-mediated inflammation as a tissue-extrinsic regulator of intestinal polyposis in PJS, suggesting possible therapeutic approaches by targeting deregulated inflammation in this disease.

Long-term selection of chickens for body weight alters muscle satellite cell behaviors.

Muscle satellite cells (SC) are resident stem-like cells that play an integral role in skeletal muscle growth and repair. Understanding how SC maintain their identities and dynamic properties is critical to animal growth. However, the genetic and environmental factors governing SC behaviors and the underpinning mechanisms remain unknown. To explore whether genetic selection influences SC behaviors, we used 2 lines of chickens selected for over 50 generations with over a 10-fold difference in body weight at 56 d of age-the Virginia high weight selection (HWS) and low weight selection (LWS) lines. To study these 2 lines, we performed both in vivo and in vitro experiments. In vivo, we studied the abundance of SC in normal physiological settings and tested their functional roles in muscle regeneration using a muscle injury model. In vitro, we isolated SC from chicken skeletal muscle and assayed their ability to proliferate and differentiate under cultured conditions. Immunohistochemical staining of breast muscle (pectoralis major) revealed that muscle fibers from HWS chickens possessed more SC than those from LWS. Further analysis showed that the SC pool from HWS muscles contained a higher percentage of activated SC compared to that of LWS. When isolated SC from HWS and LWS muscles were cultured, HWS SC exhibited greater abilities to proliferate and differentiate than those SC from LWS. To test whether the observed in vitro differences in SC properties could be confirmed in vivo, we subjected chicken breast muscle to barium chloride to induce muscle injury and regeneration. Consistent with in vitro data, breast muscle in HWS chicken experienced a faster and more robust recovery than that of LWS, as evidenced by quicker regeneration and larger muscle fiber size. Taken together, these findings suggest divergent selection for body weight not only results in correlated responses in SC number, but also changes SC growth kinetics. Further dissection of the molecular mechanism will aid the identification of the target molecules for growth intervention in chickens.

Asymmetries, heterosis, and phenotypic profiles of red junglefowl, White Plymouth Rocks, and F1 and F2 reciprocal crosses.

During the domestication of farm animals, humans have manipulated genetic variation for growth and reproduction through artificial selection. Here, data are presented for growth, reproductive, and behavior traits for the red junglefowl, a line of White Plymouth Rock chickens, and their F1 and F2 reciprocal crosses. Intra- and intergenerational comparisons for growth related traits reflected considerable additive genetic variation. In contrast, those traits associated with reproduction exhibited heterosis. The role of sexual selection was seen in the evolution of prominent secondary sexual ornaments that lend to female choice and male-male competition. The large differences between parental lines in fearfulness to humans were only mitigated slightly in the intercross generations. Whereas, overall F1 generation heterosis was not transferred to the F2, there was developmental stability in the F2, as measured by relative asymmetry of bilateral traits. Through multigenerational analyses between the red junglefowl and the domestic White Plymouth Rocks, we observed plasticity and considerable residual genetic variation. These factors likely facilitated the adaptability of the chicken to a broad range of husbandry practices throughout the world.

Genomic signatures of 60 years of bidirectional selection for 8-week body weight in chickens.

Sixty years, constituting 60 generations, have passed since the founding of the Virginia body weight lines, an experimental population of White Plymouth Rock chickens. Using a stringent breeding scheme for divergent 8-week body weight, the lines, which originated from a common founder population, have responded to bidirectional selection with an approximate 15-fold difference in the selected trait. They provide a model system to study the genetics of complex traits in general and the influences of artificial selection on quantitative genetic architectures in particular. As we reflect on the 60th anniversary of the initiation of the Virginia body weight lines, there is opportunity to discuss the findings obtained using different analytical and experimental genetic and genomic strategies and integrate them with a recent pooled genome resequencing dataset. Hundreds of regions across the genome show differentiation between the 2 lines, reinforcing previous findings that response to selection relied on standing variation across many genes and giving insights into the haplotype complexity underlying regions associated with body weight.

Stress-induced suppression of neuropeptide Y-induced hunger in anorexic chicks involves corticotrophin-releasing factor signalling and the paraventricular nucleus of the hypothalamus.

The Virginia lines of chickens have been selected for low (LWS) or high (HWS) juvenile body weight and have different severities of anorexia and obesity, respectively. The LWS that are exposed to stressors at hatch are refractory to neuropeptide Y (NPY)-induced food intake and the objective of the present study was to determine the underlying mechanisms. Chicks were exposed to a stressor (-20°C for 6 minutes and 22°C and delayed access to food for 24 hours) after hatching and the hypothalamic nuclei, including the lateral hypothalamus (LH), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH) and arcuate nucleus (ARC), were collected 5 days later. In LWS but not HWS, stress exposure up-regulated corticotrophin-releasing factor (CRF), CRF receptor subtypes 1 and 2 (CRFR1 and CRFR2, respectively), melanocortin receptor 4 and urocortin 3 in the PVN, as well as CRFR2 mRNA in the VMH and ARC. In LWS, stress exposure was also associated with greater NPY and NPY receptor subtype 5 mRNA in the ARC and PVN, respectively, as well as decreased agouti-related peptide mRNA in the ARC. In HWS, stress exposure was associated with increased CRFR1 and decreased cocaine- and amphetamine-regulated transcript in the ARC and PVN, respectively. Refractoriness of the food intake response to NPY in LWS may thus result from the over-riding anorexigenic tone in the PVN associated with CRF signalling. Indeed, the orexigenic effect of NPY was restored when LWS were injected with a CRF receptor antagonist, astressin, before stress exposure. The results of the present study provide insights into the molecular basis of eating disorders and suggest that CRF signalling in the PVN may exacerbate the anorexic phenotype in the presence of environmental stressors.

Correlated responses to long-term divergent selection for 8-week body weight in female White Plymouth Rock chickens: Sexual maturity.

Reported here are correlated responses for reproductive traits to long-term divergent selection (54 generations) for 8-week body weight (BW8). Comparisons involved both selected and relaxed lines. Traits measured were age at first egg (AFE), body weight at first egg (WFE), and ratio of body weight and age at first egg (WAFE). Although sexual maturity was delayed in the selected lines, the effect was more pronounced in the low than high selected and relaxed lines. Selection for low BW resulted in decreases in WFE and WAFE. Correlated responses to selection for high BW were increased WFE and WAFE. Minimum AFE, WFE, and WAFE in relation to sexual maturity were line specific and influenced by selection for BW8. WAFE provided a "yardstick" for target body weights that were optimum for successful attainment of sexual maturity and higher reproductive rates. Such may be line specific. There was opposition between relaxed and artificial selection, resulting in a higher reproductive performance and fitness for the former.

Dual functioning ovaries and atresia in chickens. Is it a coincidence?

During the course of a QTL study involving an intercross between White Plymouth Rock chickens and Red Jungle Fowl, certain reproductive anomalies such as atresia and double ovary-oviduct became evident. Observed in reciprocal pedigreed F1 crosses were 2 full-sib pullets with dual functioning ovaries. One also had complete functioning left and right oviducts. The other had asymmetrical reproductive tracts with a typical left oviduct and a rudimentary right oviduct. In addition, there were 3 females with atresia. Although they had different sires and dams, their grandparents were related, and all 5 traced to a common ancestor. Pedigrees, photos, and biological aspects of these conditions are presented and discussed.

Differential expression of appetite-regulating genes in avian models of anorexia and obesity.

Chickens from lines that have been selected for low (LWS) or high (HWS) juvenile body weight for more than 57 generations provide a unique model by which to research appetite regulation. The LWS display different severities of anorexia, whereas all HWS become obese. In the present study, we measured mRNA abundance of various factors in appetite-associated nuclei in the hypothalamus. The lateral hypothalamus (LHA), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), dorsomedial nucleus (DMN) and arcuate nucleus (ARC) were collected from 5 day-old chicks that were fasted for 180 minutes or provided with continuous access to food. Fasting increased neuropeptide Y receptor subtype 1 (NPYR1) mRNA in the LHA and c-Fos in the VMH, at the same time as decreasing c-Fos in the LHA, neuropeptide Y receptor subtype 5 and ghrelin in the PVN, and neuropeptide Y receptor subtype 2 in the ARC. Fasting increased melanocortin receptor subtype 3 (MC3R) expression in the DMN and NPY in the ARC of LWS but not HWS chicks. Expression of NPY was greater in LWS than HWS in the DMN. neuropeptide Y receptor subtype 5 mRNA was greater in LWS than HWS in the LHA, PVN and ARC. Expression of orexin was greater in LWS than HWS in the LHA. There was greater expression of NPYR1, melanocortin receptor subtype 4 and cocaine- and amphetamine-regulated transcript in HWS than LWS and mesotocin in LWS than HWS in the PVN. In the ARC, agouti-related peptide and MC3R were greater in LWS than HWS and, in the VMH, orexin receptor 2 and leptin receptor were greater in LWS than HWS. Greater mesotocin in the PVN, orexin in the LHA and ORXR2 in the VMH of LWS may contribute to their increased sympathetic tone and anorexic phenotype. The results of the present study also suggest that an increased hypothalamic anorexigenic tone in the LWS over-rides orexigenic factors such as NPY and AgRP that were more highly expressed in LWS than HWS in several nuclei.

Selection for juvenile body weight in chickens: Standardizing for scaling.

The influence of scaling on phenotypic responses to long-term selection was evaluated in lines of chickens selected for high (HWS) and low (LWS) 8-week body weight. The focus of the experiment was on phenotypic correlations between means and standard deviations, and unadjusted vs. standardized responses. Traits measured were body weight at 4 (BW4) and 8 wk (BW8). Correlations between means and standard deviations for BW8 (the selected trait) were positive and greater for BW4 in LWS than HWS. In both lines, scaling masked the degree more than the pattern of response; however, after standardization, it was greater in LWS than HWS. Selection changed the standard deviation units of juvenile BW with a 2-fold difference in BW8 for both HWS and LWS in which the latter reached a physiological limit. Standardization is a robust method to adjust for scaling, thus enhancing the interpretability of responses in long-term selection experiments. Because of changes in variances associated with those in phenotypic means, this method to correct for part-whole correlations was useful.

Long-term results of ultrasound guided fine needle aspiration cytology in conjunction with sentinel node biopsy support step-wise approach in melanoma.

BACKGROUND: US-FNAC is a common diagnostic tool in the work-up of many cancers. Results in melanoma were initially poor (sensitivity 20-40%). Introduction of the Berlin Morphology criteria has shown potential improvement up to 65-80% in selected patients. AIM: This cohort study evaluates the long-term survival outcome of melanoma patients undergoing Ultrasound (US) guided Fine Needle Aspiration Cytology (FNAC) prior to sentinel node biopsy (SNB) or direct lymphadenectomy. METHODS: Between 2001 and 2010 over 1000 consecutive melanoma patients prospectively underwent targeted US-FNAC prior to SNB. The Berlin US morphology criteria: peripheral perfusion (PP), loss of central echoes (LCE) and balloon shape (BS) were registered. FNAC was performed if any factor was present. All patients underwent SNB or lymphadenectomy in case of positive FNAC. RESULTS: Median follow-up was 61 months (IQR 40-95). SN positivity rate was 21%. Survival analyses demonstrated that patients with positive US-FNAC had poor survival. After adjustment for SN status and other known prognostic features, patients with positive US-FNAC (hazard ratio (HR) 1.80, 95% CI 1.10-2.96) had worse survival than patients with normal US (reference). Patients with suspicious US and negative FNAC (HR 1.13, 95% CI 0.71-1.78) had survival comparable to patients with normal US. CONCLUSIONS: The long-term US-FNAC results support this step-wise approach to melanoma patients. Patients with positive US-FNAC have a poor survival and can be spared a SNB. Patients with suspicious US and negative FNAC should undergo SNB to detect microscopic occult disease. Completely US-FNAC negative patients might only require follow-up and no SN staging at all.

Timing of completion lymphadenectomy after positive sentinel node biopsy in patients with melanoma.

BACKGROUND: Nodal staging with sentinel node biopsy (SNB) and completion lymph node dissection (CLND) provides prognostic information to patients with melanoma and their physicians. It is not known whether the timing of CLND is associated with survival outcome and/or CLND tumour load. This study investigated whether CLND timing is associated with CLND tumour load, disease-free survival (DFS) and/or melanoma-specific survival (MSS). METHODS: A retrospective cohort of patients with SNB-positive melanoma from nine European Organisation for Research and Treatment of Cancer (EORTC) Melanoma Group centres undergoing surgery between 1993 and 2009 were examined. Patients were selected based on availability of CLND and follow-up data. The CLND interval was defined as the number of days between diagnosis and CLND. Patient and tumour characteristics were collected. Five-year DFS and MSS rates were calculated. Cox and logistic regression analysis were performed, adjusting for known prognostic/predictive indicators. RESULTS: A total of 784 patients were included in the study. Their median age was 51 (i.q.r. 40-62) years, and 418 patients (53·3 per cent) were men. Median Breslow thickness was 3·0 (i.q.r. 2·0-5·0) mm, and 148 patients (18·9 per cent) had a residual tumour load. Median CLND interval was 84 (i.q.r. 65-105) days. Five-year DFS and MSS rates were not significantly different for patients operated on with a median CLND interval of less than 84 days and those with an interval of at least 84 days (DFS: 54·2 versus 53·3 per cent respectively; MSS: 66·9 versus 65·1 per cent). In a multivariable Cox model, CLND interval was not a significant prognostic indicator. CLND interval was negatively correlated with identification of positive non-sentinel nodes, but following adjustment for known risk factors this effect was no longer found. CONCLUSION: The time interval between diagnosis of melanoma and CLND did not influence CLND tumour load, DFS or MSS.

The Cdc42/Rac1 regulator CdGAP is a novel E-cadherin transcriptional co-repressor with Zeb2 in breast cancer.

The loss of E-cadherin causes dysfunction of the cell-cell junction machinery, which is an initial step in epithelial-to-mesenchymal transition (EMT), facilitating cancer cell invasion and the formation of metastases. A set of transcriptional repressors of E-cadherin (CDH1) gene expression, including Snail1, Snail2 and Zeb2 mediate E-cadherin downregulation in breast cancer. However, the molecular mechanisms underlying the control of E-cadherin expression in breast cancer progression remain largely unknown. Here, by using global gene expression approaches, we uncover a novel function for Cdc42 GTPase-activating protein (CdGAP) in the regulation of expression of genes involved in EMT. We found that CdGAP used its proline-rich domain to form a functional complex with Zeb2 to mediate the repression of E-cadherin expression in ErbB2-transformed breast cancer cells. Conversely, knockdown of CdGAP expression led to a decrease of the transcriptional repressors Snail1 and Zeb2, and this correlated with an increase in E-cadherin levels, restoration of cell-cell junctions, and epithelial-like morphological changes. In vivo, loss of CdGAP in ErbB2-transformed breast cancer cells impaired tumor growth and suppressed metastasis to lungs. Finally, CdGAP was highly expressed in basal-type breast cancer cells, and its strong expression correlated with poor prognosis in breast cancer patients. Together, these data support a previously unknown nuclear function for CdGAP where it cooperates in a GAP-independent manner with transcriptional repressors to function as a critical modulator of breast cancer through repression of E-cadherin transcription. Targeting Zeb2-CdGAP interactions may represent novel therapeutic opportunities for breast cancer treatment.

The role of claudins in cancer metastasis.

TJs are large intercellular adhesion complexes that maintain cell polarity in normal epithelia and endothelia. During the metastatic process, TJs must be 'loosened' or dismantled in cancer cells to enable migration and dissemination. Diminished TJ integrity must also occur within endothelial cells to allow intravasation and extravasation of cancer cells across endothelial barriers. Claudins are critical components of TJs, forming homo- and heteromeric interactions between the adjacent cells, which have been implicated as key modulators of carcinogenesis and metastasis. Numerous epithelial-derived cancers display altered claudin expression patterns and certain claudins can now be used as biomarkers to predict patient prognosis. Moreover, claudins have been functionally implicated in numerous steps of the metastatic cascade. The distinct roles played by claudins during the cancer progression to metastatic disease are just starting to be elucidated. A more complete understanding of the mechanisms through which claudins augment cancer metastasis is required to develop new therapeutic agents against this family of proteins. In this review, we will summarize the relationship between the claudin expression and clinical outcomes in diverse cancers, discuss tumor intrinisic roles through which claudins regulate metastasis and explore claudin-mediated functions within stromal cells that influence the metastatic process. Finally, we will consider possible strategies for targeting claudins that have the potential to improve the management of metastatic cancer.

Anorexia is Associated with Stress-Dependent Orexigenic Responses to Exogenous Neuropeptide Y.

Chicken lines that have been divergently selected for either low (LWS) or high (HWS) body weight at 56 days of age for more than 57 generations have different feeding behaviours in response to a range of i.c.v. injected neurotransmitters. The LWS have different severities of anorexia, whereas the HWS become obese. Previously, we demonstrated that LWS chicks did not respond, whereas HWS chicks increased food intake, after central injection of neuropeptide Y (NPY). The present study aimed to determine the molecular mechanisms underlying the loss of orexigenic function of NPY in LWS. Chicks were divided into four groups: stressed LWS and HWS on day of hatch, and control LWS and HWS. The stressor was a combination of food deprivation and cold exposure. On day 5 post-hatch, each chick received an i.c.v. injection of vehicle or 0.2 nmol of NPY. Only the LWS stressed group did not increase food intake in response to i.c.v. NPY. Hypothalamic mRNA abundance of appetite-associated factors was measured at 1 h post-injection. Interactions of genetic line, stress and NPY treatment were observed for the mRNA abundance of agouti-related peptide (AgRP) and synaptotagmin 1 (SYT1). Intracerebroventricular injection of NPY decreased and increased AgRP and SYT1 mRNA, respectively, in the stressed LWS and increased AgRP mRNA in stressed HWS chicks. Stress was associated with increased NPY, orexin receptor 2, corticotrophin-releasing factor receptor 1, melanocortin receptor 3 (MC3R) and growth hormone secretagogue receptor expression. In conclusion, the loss of responsiveness to exogenous NPY in stressed LWS chicks may be a result of the decreased and increased hypothalamic expression of AgRP and MC3R, respectively. This may induce an intensification of anorexigenic melanocortin signalling pathways in LWS chicks that block the orexigenic effect of exogenous NPY. These results provide insights onto the anorexic condition across species, and especially for forms of inducible anorexia such as human anorexia nervosa.