Biogeosciences Perspectives on Integrated, Coordinated, Open, Networked (ICON) Science.

This article is composed of three independent commentaries about the state of Integrated, Coordinated, Open, Networked (ICON) principles in the American Geophysical Union Biogeosciences section, and discussion on the opportunities and challenges of adopting them. Each commentary focuses on a different topic: (a) Global collaboration, technology transfer, and application (Section 2), (b) Community engagement, community science, education, and stakeholder involvement (Section 3), and (c) Field, experimental, remote sensing, and real-time data research and application (Section 4). We discuss needs and strategies for implementing ICON and outline short- and long-term goals. The inclusion of global data and international community engagement are key to tackling grand challenges in biogeosciences. Although recent technological advances and growing open-access information across the world have enabled global collaborations to some extent, several barriers, ranging from technical to organizational to cultural, have remained in advancing interoperability and tangible scientific progress in biogeosciences. Overcoming these hurdles is necessary to address pressing large-scale research questions and applications in the biogeosciences, where ICON principles are essential. Here, we list several opportunities for ICON, including coordinated experimentation and field observations across global sites, that are ripe for implementation in biogeosciences as a means to scientific advancements and social progress.

ASPM and microcephalin expression in epithelial ovarian cancer correlates with tumour grade and survival.

BACKGROUND: The clinico-pathological and molecular heterogeneity of epithelial ovarian cancer (EOC) complicates its early diagnosis and successful treatment. Highly aneuploid tumours and the presence of ascitic fluids are hallmarks of EOC. Two microcephaly-associated proteins, abnormal spindle-like microcephaly-associated protein (ASPM) and microcephalin, are involved in mitosis and DNA damage repair. Their expression is deregulated at the RNA level in EOC. Here, ASPM and microcephalin protein expression in primary cultures established from the ascites of patients with EOC was determined and correlated with clinical data to assess their suitability as biomarkers. METHODS: Five established ovarian cancer cell lines, cells derived from two benign ovarian ascites samples and 40 primary cultures of EOC derived from ovarian ascites samples were analysed by protein slot blotting and/or immunofluorescence to determine ASPM and microcephalin protein levels and their cellular localisation. Results were correlated with clinico-pathological data. RESULTS: A statistically significant correlation was identified for ASPM localisation and tumour grade, with high levels of cytoplasmic ASPM correlating with grade 1 tumours. Conversely, cytoplasmic microcephalin was only identified in high-grade tumours. Furthermore, low levels of nuclear microcephalin correlated with reduced patient survival. CONCLUSION: Our results suggest that ASPM and microcephalin have the potential to be biomarkers in ovarian cancer.

Loss of expression of oestrogen receptor beta in colon cancer and its association with Dukes' staging.

Gender differences in the incidence and behaviour of colon cancer suggest a hormonal influence and epidemiological data suggest a protective effect for hormone replacement therapy. Recently, it has been shown that oestrogen receptor (ER) beta is the predominant ER in colon tissue. The aim of this study was to examine the expression and distribution of ERbeta in normal and colorectal cancer samples, using immunohistochemistry and (in a subset of patients) real-time quantitative reverse transcriptase polymerase chain reaction in a well-defined patient cohort and to correlate this with clinico-pathological outcome. Immunohistochemical analyses of normal colon revealed strong specific nuclear immuno-reactivity in all epithelial cells lining the colonic crypts. In colon cancer, ERbeta expression was lost in 21% of samples irrespective of patient age or gender. Interestingly loss of ERbeta expression was higher in left colon and rectal cancers (27%) compared to right colon cancers (8%). A correlation between loss of ERbeta expression and advanced Dukes stage was observed. Loss of ERbeta with increased Dukes' stage suggests that it may be affording a protective effect against colon carcinogenesis. Its presence may be a favourable prognostic marker in this disease and could explain the protective effect of oestrogens against colon cancer development.

Association between family history and mismatch repair in colorectal cancer.

BACKGROUND AND AIMS: Germline mutations in mismatch repair (MMR) genes cause a greatly increased risk of cancer of the gastrointestinal and female reproductive tracts (hereditary non-polyposis colorectal cancer (HNPCC)). Loss of MMR expression is common in colorectal cancer (CRC) overall. Such loss is assumed to be acquired predominantly, although a population of CRC cases will include individuals with unrecognised MMR mutations. This study examines the association between MMR gene expression and family history of cancer among the CRC population. METHODS: Individuals with CRC were identified from two well characterised populations: (1) consecutive hospital patients (n = 644) and (2) a population based cases series (n = 249). CRC was examined for expression of hMLH1 and hMSH2 using immunohistochemistry, and expression was related to family history using logistic regression. RESULTS: hMLH1 and hMSH2 expression was assessed in 732 CRCs with 8% showing loss of expression. No association was seen overall for hMLH1 or hMSH2 expression and family history of CRC. Loss of hMSH2 was predicted by family history of extracolonic cancer (odds ratio (OR) 5.78 (95% confidence interval (CI) 0.95-35.18)) and family history suggestive of HNPCC (OR 27.84 (95% CI 4.37-177.56)). Loss of hMLH1 was not predicted by family history of extracolonic cancer or a family history suggestive of HNPCC but was for a family history of at least two affected relatives (OR 4.88 (95% CI 1.25-19.03)). CONCLUSIONS: Individuals with hMSH2 deficient CRC in the general population exhibit a family history and other characteristics suggestive of HNPCC, and may carry germline MMR mutations. Loss of hMLH1 is only associated with a strong family history of extracolonic cancer at older ages, suggesting a novel mechanism of susceptibility.

Cytogenetic alterations in ovarian clear cell carcinoma detected by comparative genomic hybridisation.

Ovarian clear cell carcinoma (OCCC) accounts for a small but significant proportion of all ovarian cancers and is a distinct clinical and pathological entity. It tends to be associated with poorer response rates to chemotherapy and with a worse prognosis. Little is known about possible underlying genetic changes. DNA extracted from paraffin-embedded samples of 18 pure OCCC cases was analysed for genetic imbalances using comparative genomic hybridisation (CGH). All of the 18 cases showed genomic alterations. The mean number of alterations detected by CGH was 6 (range 1-15) indicating a moderate level of genetic instability. Chromosome deletions were more common than amplifications. The most prominent change involved chromosome 9 deletions in 10 cases (55%). This correlates with changes seen in other epithelial ovarian cancers. This deletion was confirmed using microsatellite markers to assess loss of heterozygosity (LOH) at four separate loci on chromosome 9. The most distinct region of loss detected was around the IFNA marker at 9p21 with 41% (11 out of 27 cases) LOH. Other frequent deletions involved 1p (five out of 18; 28%); 11q (four out of 18; 22%) and 16 (five out of 18; 28%). Amplification was most common at chromosome 3 (six out of 18; 33%); 13q (four out of 18; 22%) and 15 (three out of 18; 17%). No high-level amplifications were identified. These features may serve as useful prognostic indicators in the management of OCCC.

BCL10 in malignant lymphomas--an evaluation using fluorescence in situ hybridization.

BCL10 is a tumour suppressor gene originally cloned from a t(1;14)(p22;q32) breakpoint in a case of mucosa-associated lymphoid tissue (MALT) lymphoma. Translocations involving this gene, though uncommon, are sometimes encountered in MALT lymphomas. This gene is thought to play an important role in the development of malignant lymphomas. Fluorescence in situ hybridization (FISH) was therefore undertaken on 22 cases of malignant lymphoma of varying histology to establish the incidence of rearrangements involving the BCL10 gene. Initially, one case with a novel t(1;2)(p22;p12) translocation involving the BCL10 gene was identified, in a marginal zone lymphoma of the MALT type, and was reported elsewhere. Seven other cases were subsequently identified with abnormalities in the 1p region, including a translocation with a breakpoint in the 1p22 region in a case of lymphoblastic lymphoma. However, none of these involved the BCL10 gene. Mutation analysis of BCL10 was then performed on 57 cases of malignant lymphoma, including 17 MALT lymphomas, by single-strand conformational polymorphism (SSCP) analysis of tumour DNA. Tissue was obtained for mutation analysis for 12 of the 22 cases analysed by FISH. Selected cases with SSCP band shifts were further studied by direct sequencing. Polymorphisms were identified in eight cases, but no mutations of pathogenic significance were identified. Further RT-PCR and mutation analysis was performed on cDNAs from 12 cases (four MALT, seven diffuse large B-cell lymphoma, one Hodgkin's disease) in which DNA analysis had already been completed. This included the MALT lymphoma with the t(1;2)(p22;p12) rearrangement. Again, no mutations were identified in the coding sequence. This study confirms that rearrangements of the BCL10 gene are uncommon in lymphoma (1/22) and may be limited tothe MALT subtype of non-Hodgkin's lymphomas. It was also found that breakpoints or rearrangements in the 1p22 region do not necessarily involve the BCL10 gene. Moreover, the absence of mutations at both the DNA (0/60) and the mRNA (0/12) level indicates that this gene is not frequently inactivated by mutation, in those tumours in which it is not involved in translocations. Our findings suggest that the BCL10 gene is unlikely to have a frequent or key role in general lymphomagenesis.

Genetic events during the transformation of a tamoxifen-sensitive human breast cancer cell line into a drug-resistant clone.

Tamoxifen resistance is a serious clinical problem commonly encountered in the management of patients with breast cancer. The mechanisms leading to its development are unclear. Tamoxifen acts via multiple pathways and has diverse effects. Hence transformation from a tamoxifen-sensitive to a resistant phenotype could involve multiple genetic events. Knowledge of the genetic pathways leading to resistance may facilitate the development of novel therapeutic strategies. In this study, a variation of conventional comparative genomic hybridization (CGH) has been employed to detect genetic alterations associated with tamoxifen resistance. MCF-7, a tamoxifen-sensitive human breast cancer cells line, and its tamoxifen-resistant clone, CL-9 were used. Both cell lines showed extensive areas of concordance but consistent differences were seen with the acquisition of tamoxifen resistance. These differences included the amplification of 2p16.3 approximately p23.2, 2q21 approximately q34, 3p12.3 approximately p14.1, 3p22 approximately p26, 3q, 12q13.2 approximately q22, 13q12 approximately q14, 17q21.3 approximately q23, 20q11.2 approximately q13.1 and 21q11.2 approximately q21 as well as the deletion of 6p21.1, 6p23 approximately p25, 7q11.1 approximately q31, 7q35 approximately q36, 11p15, 11q24, 13q33, 17p, 18q12 approximately q21.1, 19p, 19q13.3, 22q13.1 approximately q13.2. These findings were supported by conventional cytogenetics and chromosome painting. The regions identified by CGH potentially harbor genes that could be important in the development of tamoxifen resistance.

Methylation and colorectal cancer.

Statistics rate colorectal adenocarcinoma as the most common cause of cancer death on exclusion of smoking-related neoplasia. However, the reported accumulation of genetic lesions over the adenoma to adenocarcinoma sequence cannot wholly account for the neoplastic phenotype. Recently, heritable, epigenetic changes in DNA methylation, in association with a repressive chromatin structure, have been identified as critical determinants of tumour progression. Indeed, the transcriptional silencing of both established and novel tumour suppressor genes has been attributed to the aberrant cytosine methylation of promoter-region CpG islands. This review aims to set these epigenetic changes within the context of the colorectal adenoma to adenocarcinoma sequence. The role of cytosine methylation in physiological and pathological gene silencing is discussed and the events behind aberrant cytosine methylation in ageing and cancer are appraised. Emphasis is placed on the interrelationships between epigenetic and genetic lesions and the manner in which they cooperate to define a CpG island methylator phenotype at an early stage in tumourigenesis. Finally, the applications of epigenetics to molecular pathology and patient diagnosis and treatment are reviewed.

Endogenous electric current is associated with normal development of the vertebrate limb.

A steady ionic current is driven out of both developing and regenerating amphibian limbs. In the developing limbs of anurans and urodeles, focal outwardly directed current (0.5-2 microA/cm(2)) predicts the location of mesenchyme accumulations producing the early bud. Here, we report measurements of a similar outwardly directed ionic current associated with the development of the limb bud in the mouse and chick embryo by using a noninvasive, self-referencing electrode for the measurement of extracellular current. In both the mouse and chick embryo, flank currents were usually inwardly directed - the direction of Na(+) uptake by ectoderm. Outward currents associated with the mouse limb bud ranged from 0.04-10.8 microA/cm(2). Mouse limb bud and flank currents were similar to those measured in amphibian larvae, because they were reversibly collapsed and/or reversed by application of 30 microM amiloride, a Na(+) channel blocker. Unlike the amphibian embryos, flank ectoderm adjacent to the mouse limb bud in the anterior/posterior axis was usually associated with outwardly directed ionic current. This raises the possibility of a different, or changing, gradient of extracellular voltage experienced by mesenchyme cells in this plane of development than that observed in other regions of the limb bud. In the chick flank caudal to the somites, a striking reversal of the inwardly directed flank currents to very large ( approximately 100 microA/cm(2)) outwardly directed currents occurred three developmental stages before limb bud formation. We tested the relevance of this outwardly directed ionic current to limb formation in the chick embryo by reversing it by using an artificially applied "countercurrent" pulled through a microelectrode inserted just beneath the caudal ectoderm of the embryo. This application was performed for approximately 6 hr 2.5-3 developmental stages before hindlimb bud formation. This method resulted in abnormal limb formation by the tenth day of gestation in some embryos, whereas all control embryos developed normally. These data suggest an early physiological control of limb development.

Defective fluid secretion and NaCl absorption in the parotid glands of Na+/H+ exchanger-deficient mice.

Multiple Na(+)/H(+) exchangers (NHEs) are expressed in salivary gland cells; however, their functions in the secretion of saliva by acinar cells and the subsequent modification of the ionic composition of this fluid by the ducts are unclear. Mice with targeted disruptions of the Nhe1, Nhe2, and Nhe3 genes were used to study the in vivo functions of these exchangers in parotid glands. Immunohistochemistry indicated that NHE1 was localized to the basolateral and NHE2 to apical membranes of both acinar and duct cells, whereas NHE3 was restricted to the apical region of duct cells. Na(+)/H(+) exchange was reduced more than 95% in acinar cells and greater than 80% in duct cells of NHE1-deficient mice (Nhe1(-/-)). Salivation in response to pilocarpine stimulation was reduced significantly in both Nhe1(-/-) and Nhe2(-/-) mice, particularly during prolonged stimulation, whereas the loss of NHE3 had no effect on secretion. Expression of Na(+)/K(+)/2Cl(-) cotransporter mRNA increased dramatically in Nhe1(-/-) parotid glands but not in those of Nhe2(-/-) or Nhe3(-/-) mice, suggesting that compensation occurs for the loss of NHE1. The sodium content, chloride activity and osmolality of saliva in Nhe2(-/-) or Nhe3(-/-) mice were comparable with those of wild-type mice. In contrast, Nhe1(-/-) mice displayed impaired NaCl absorption. These results suggest that in parotid duct cells apical NHE2 and NHE3 do not play a major role in Na(+) absorption. These results also demonstrate that basolateral NHE1 and apical NHE2 modulate saliva secretion in vivo, especially during sustained stimulation when secretion depends less on Na(+)/K(+)/2Cl(-) cotransporter activity.

The beta-lactamases and beta-lactam antibiotic susceptibility of Yersinia enterocolitica.

One hundred and forty-five isolates of Yersinia enterocolitica of different serotypes and biotypes, including atypical biotypes, collected from various parts of the world, were examined for their susceptibility to beta-lactam antibiotics and expression of intracellular beta-lactamases. The reasons for the specificity of patterns of susceptibility to beta-lactams for each biotype or subtype of Y. enterocolitica were elucidated by examining their ss-lactamase activity. Whilst the biotypes and subtypes were uniformly susceptible to the newer beta-lactam antibiotics, the susceptibility pattern observed with other beta-lactams was specific to each biotype or subtype, because of the characteristics of beta-lactamase expression by strains within these groups. The susceptibility to these beta-lactam agents depended entirely on the extent of elaboration or the absence of one of the two beta-lactamases, enzyme A and enzyme B, found in the species. Detection of enzyme B by a disc diffusion test yielded inconsistent results, but detection of enzyme A by disc diffusion was highly reliable. This test clearly distinguished strains of biotype 2, serotype O:5,27 from those of biotype 2, serotype O:9 and biotype 3, serotypes O:1, 2a-3, O:3 and O:5.

Novel translocation of the BCL10 gene in a case of mucosa associated lymphoid tissue lymphoma.

Interest has focused on a recently identified gene, BCL10, thought to play an important role in the genesis of extranodal, marginal zone (MALT) lymphomas. This gene belongs to a family containing caspase recruitment domains (CARD), that are involved in the apoptotic pathway. Translocations of the BCL10 gene to the immunoglobulin heavy chain locus at 14q32 have been described. We report herein a case of MALT lymphoma showing t(1; 2)(p22; p12). The translocation was shown to involve the BCL10 gene and the immunoglobulin kappa light chain locus by fluorescence in situ hybridization.

Intraventricular insulin suppresses the acoustic startle response in rats.

We and others have previously reported that the hormone insulin alters brain noradrenergic function at the synaptic and molecular levels. In the present study, we examined the in vivo effect of insulin (administered chronically via osmotic minipumps at a dose of 5 mU/day into the third cerebral ventricle) on the acoustic startle response. Rats receiving chronic intraventricular insulin had a significantly reduced startle response relative to vehicle-treated controls (i.e., 47 +/- 21% of baseline control startle response). Because our previous findings suggest that on an acute basis, insulin may enhance endogenous noradrenergic activity by inhibiting norepinephrine reuptake, we speculate here that the chronic effect of insulin is similar to that of the noradrenergic reuptake blocker, desipramine, which has been reported to decrease baseline startle performance.

Genetic modification of cultured skin substitutes by transduction of human keratinocytes and fibroblasts with platelet-derived growth factor-A.

Gene therapy promises the potential for improved treatment of cutaneous wounds. This study evaluated whether genetically modified cultured skin substitutes can act as vehicles for gene therapy in an athymic mouse model of wound healing. Human keratinocytes and fibroblasts were genetically engineered by retroviral transduction to overexpress human platelet-derived growth factor-A chain. Three types of skin substitutes were prepared from collagen-glycosaminoglycan substrates populated with fibroblasts and keratinocytes: HF-/HK-, containing both unmodified fibroblasts and keratinocytes; HF-/HK+, containing unmodified fibroblasts and modified keratinocytes; and HF+/HK-, containing modified fibroblasts and unmodified keratinocytes. Skin substitutes were cultured for two weeks before grafting to full-thickness wounds on athymic mice. The modified skin substitutes secreted significantly elevated levels of platelet-derived growth factor throughout the culture period. Expression of retroviral platelet-derived growth factor-A mRNA was maintained after grafting to mice, and was detected in all HF-/HK+ grafts and one HF+/HK- graft at two weeks after surgery. Although no differences were seen between control and modified grafts, the results suggest that genetically modified cultured skin substitutes can be a feasible mechanism for cutaneous gene therapy. The cultured skin model used for these studies has advantages over other skin analogs containing only epidermal cells; because it contains both fibroblasts and keratinocytes, it therefore offers greater opportunities for genetic modification and potential modulation of wound healing.

Enhanced vascularization of cultured skin substitutes genetically modified to overexpress vascular endothelial growth factor.

Cultured skin substitutes have been used as adjunctive therapies in the treatment of burns and chronic wounds, but they are limited by lack of a vascular plexus. This deficiency leads to greater time for vascularization compared with native skin autografts and contributes to graft failure. Genetic modification of cultured skin substitutes to enhance vascularization could hypothetically lead to improved wound healing. To address this hypothesis, human keratinocytes were genetically modified by transduction with a replication incompetent retrovirus to overexpress vascular endothelial growth factor, a specific and potent mitogen for endothelial cells. Cultured skin substitutes consisting of collagen-glycosaminoglycan substrates inoculated with human fibroblasts and either vascular endothelial growth factor-modified or control keratinocytes were prepared, and were cultured in vitro for 21 d. Northern blot analysis demonstrated enhanced expression of vascular endothelial growth factor mRNA in genetically modified keratinocytes and in cultured skin substitutes prepared with modified cells. Furthermore, the vascular endothelial growth factor-modified cultured skin substitutes secreted greatly elevated levels of vascular endothelial growth factor protein throughout the entire culture period. The bioactivity of vascular endothelial growth factor protein secreted by the genetically modified cultured skin substitutes was demonstrated using a microvascular endothelial cell growth assay. Vascular endothelial growth factor-modified and control cultured skin substitutes were grafted to full-thickness wounds on athymic mice, and elevated vascular endothelial growth factor mRNA expression was detected in the modified grafts for at least 2 wk after surgery. Vascular endothelial growth factor-modified grafts exhibited increased numbers of dermal blood vessels and decreased time to vascularization compared with controls. These results indicate that genetic modification of keratinocytes in cultured skin substitutes can lead to increased vascular endothelial growth factor expression, which could prospectively improve vascularization of cultured skin substitutes for wound healing applications.

Targeted disruption of the Nhe1 gene prevents muscarinic agonist-induced up-regulation of Na(+)/H(+) exchange in mouse parotid acinar cells.

The onset of salivary gland fluid secretion in response to muscarinic stimulation is accompanied by up-regulation of Na(+)/H(+) exchanger (NHE) activity. Although multiple NHE isoforms (NHE1, NHE2, and NHE3) have been identified in salivary glands, little is known about their specific function(s) in resting and secreting acinar cells. Mice with targeted disruptions of the Nhe1, Nhe2, and Nhe3 genes were used to investigate the contribution of these proteins to the stimulation-induced up-regulation of NHE activity in mouse parotid acinar cells. The lack of NHE1, but not NHE2 or NHE3, prevented intracellular pH recovery from an acid load in resting acinar cells, in acini stimulated to secrete with the muscarinic agonist carbachol, and in acini shrunken by hypertonic addition of sucrose. In HCO(3)(-)-containing solution, the rate of intracellular pH recovery from a muscarinic agonist-stimulated acid load was significantly inhibited in acinar cells from mice lacking NHE1, but not in cells from NHE2- or NHE3-deficient mice. These data demonstrate that NHE1 is the major regulator of intracellular pH in both resting and muscarinic agonist-stimulated acinar cells and suggest that up-regulation of NHE1 activity has an important role in modulating saliva production in vivo.