Author Correction: Microbial spatial footprint as a driver of soil carbon stabilization.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Microbial spatial footprint as a driver of soil carbon stabilization.

Increasing the potential of soil to store carbon (C) is an acknowledged and emphasized strategy for capturing atmospheric CO2. Well-recognized approaches for soil C accretion include reducing soil disturbance, increasing plant biomass inputs, and enhancing plant diversity. Yet experimental evidence often fails to support anticipated C gains, suggesting that our integrated understanding of soil C accretion remains insufficient. Here we use a unique combination of X-ray micro-tomography and micro-scale enzyme mapping to demonstrate for the first time that plant-stimulated soil pore formation appears to be a major, hitherto unrecognized, determinant of whether new C inputs are stored or lost to the atmosphere. Unlike monocultures, diverse plant communities favor the development of 30-150 µm pores. Such pores are the micro-environments associated with higher enzyme activities, and greater abundance of such pores translates into a greater spatial footprint that microorganisms make on the soil and consequently soil C storage capacity.

Advancing the Sustainability of US Agriculture through Long-Term Research.

Agriculture in the United States must respond to escalating demands for productivity and efficiency, as well as pressures to improve its stewardship of natural resources. Growing global population and changing diets, combined with a greater societal awareness of agriculture's role in delivering ecosystem services beyond food, feed, fiber, and energy production, require a comprehensive perspective on where and how US agriculture can be sustainably intensified, that is, made more productive without exacerbating local and off-site environmental concerns. The USDA's Long-Term Agroecosystem Research (LTAR) network is composed of 18 locations distributed across the contiguous United States working together to integrate national and local agricultural priorities and advance the sustainable intensification of US agriculture. We explore here the concept of sustainable intensification as a framework for defining strategies to enhance production, environmental, and rural prosperity outcomes from agricultural systems. We also elucidate the diversity of factors that have shaped the past and present conditions of cropland, rangeland, and pastureland agroecosystems represented by the LTAR network and identify priorities for research in the areas of production, resource conservation and environmental quality, and rural prosperity. Ultimately, integrated long-term research on sustainable intensification at the national scale is critical to developing practices and programs that can anticipate and address challenges before they become crises.

Correction: eEF-2 kinase is a critical regulator of Warbrug effect through controlling PP2A-A synthesis.

In this article, the authors recently noticed that the tubulin blots in Figs. 2a and 6a were inadvertently misplaced during the preparation of these figures due to their similarity. The amended versions of the figures are now shown below. The conclusions of this paper are not affected. The authors sincerely apologize for these errors.

eEF-2 kinase is a critical regulator of Warburg effect through controlling PP2A-A synthesis.

Cancer cells predominantly metabolize glucose by glycolysis to produce energy in order to meet their metabolic requirement, a phenomenon known as Warburg effect. Although Warburg effect is considered a peculiarity critical for survival and proliferation of cancer cells, the regulatory mechanisms behind this phenomenon remain incompletely understood. We report here that eukaryotic elongation factor-2 kinase (eEF-2K), a negative regulator of protein synthesis, has a critical role in promoting glycolysis in cancer cells. We showed that deficiency in eEF-2K significantly reduced the uptake of glucose and decreased the productions of lactate and adenosine triphosphate in tumor cells and in the Ras-transformed mouse embryonic fibroblasts. We further demonstrated that the promotive effect of eEF-2K on glycolysis resulted from the kinase-mediated restriction of synthesis of the protein phosphatase 2A-A (PP2A-A), a key factor that facilitates the ubiquitin-proteasomal degradation of c-Myc protein, as knockdown of eEF-2K expression led to a significant increase in PP2A-A protein synthesis and remarkable downregulation of c-Myc and pyruvate kinase M2 isoform, the key glycolytic enzyme transcriptionally activated by c-Myc. In addition, depletion of eEF-2K reduced the ability of the transformed cells to proliferate and enhanced the sensitivity of tumor cells to chemotherapy both in vitro and in vivo. These results, which uncover a role of the eEF-2K-mediated control of PP2A-A in tumor cell glycolysis, provide new insights into the regulation of the Warburg effect.

CD82/KAI expression prevents IL-8-mediated endothelial gap formation in late-stage melanomas.

Melanoma cells facilitate endothelial gap formation, the first step during tumor transendothelial migration, which is mediated by both adhesion and endogenously produced chemokines (in particular, interleukin-8 (IL-8)). Tetraspanins are localized to the cell surface in cancer and participate in various functions including invasion of tissues mediated by secretion of cytokines and matrix metalloproteinases. However, little is known about the role of CD82 tetraspanins in malignant melanomas during cancer cell invasion. In this study, we investigated the functional importance of CD82 expression in melanoma-mediated gap formation by using cDNAs to induce CD82 expression in highly invasive melanoma cell lines. Results showed that CD82 expression inhibited melanoma cell-induced gap formation, melanoma cell extravasation in vitro and subsequent lung metastasis development in vivo. Mechanistic studies showed that inducible expression of CD82 in highly metastatic melanoma cells significantly increased p21 expression upon binding of Duffy antigen receptor group (DARC), inducing tumor cell senescence and interrupting IL-8-mediated vascular endothelial (VE)-cadherin disassembly. Taken together, these studies provide a rationale for using drug therapies that restore CD82 expression and inhibit IL-8 production to inhibit late-stage melanoma cell extravasation and subsequent metastasis development.

Immunoediting of leukocyte functions within the tumor microenvironment promotes cancer metastasis development.

Attachment of tumor cells to the endothelium (EC) under flow conditions is critical for migration of tumor cells out of the vascular system to establish metastases. We found that neutrophils (PMN) increased melanoma cell extravasation. Endogenous IL-8 liberated from melanoma cells or from PMN induced by melanoma cells contributed to PMN-facilitated melanoma cell arrest on the EC in the microcirculation. Functional blocking of IL-8 receptors on PMN or neutralizing soluble IL-8 in the tumor circulation decreased the level of CD11b/CD18 up-regulation on PMN and subsequently reduced melanoma cell extravasation. We also found that targeting mutant V600EB-Raf interrupted melanoma cell extravasation in vitro and subsequent lung metastasis development in vivo. B-Raf encodes a RAS-regulated kinase that mediates cell growth and malignant transformation kinase pathway activation. Results showed that inhibition of V600EB-Raf reduced IL-8 secretion from melanoma cells and reduced the capacity of IL-8 production from the tumor microenvironment involving PMN. Furthermore, reduction in intercellular adhesion molecule-1 (ICAM-1) expression on melanoma cells was found after V600EB-Raf knockdown. These results provide new evidence for the complex role of secreted chemokine and PMN-melanoma adhesion in the recruitment of metastatic cancer cells to the EC, which are significant in fostering new approaches to cancer treatment through anti-inflammatory therapeutics.

Measuring simultaneous fluxes from soil of N2O and N2 in the field Using the 15N-gas "nonequilibium" technique.

Our purpose was to measure simultaneous fluxes from soil of both N2O and N2 from the same plot in the field using the 15N-gas "nonequilibrium" technique (i.e., the "Hauck" technique) as used previously for N2. We accommodated analysis of N2O by modifying the head amplifier of our mass spectrometer. Our system accurately measured the 15N enrichments of labeled soil slurries for both N2 and N2O. In the field, we measured flux of N2 and N2O during soil denitrification from a 15N-labeled plot of winter wheat. Nine chamber incubations were conducted over 4 days. N2 flux ranged from below detection limit (<0.022 g x m(-2) x d(-1)) to 0.055 g x m(-2) x d(-1). N2O flux ranged from 0.0002 to 0.0027 g N2O-N x m(-2) x d(-1), with a detection limit of 1.0 x 10(-6) g N2O-N x m(-2) x d(-1). For N2O flux, the 15N-gas technique and gas chromatography technique agreed well (r = 0.98). The 15N enrichment of the soil mineral pool undergoing denitrification, measured nondestructively using the N2O data, dropped from about 0.82 to 0.72 atom fraction 15N over 4 days. Applying the 15N-gas nonequilibrium technique to N2O complements its use for 15N-N2 analysis when studying the relative production of N2O and N2 during denitrification.

Aberrant CpG-island methylation has non-random and tumour-type-specific patterns.

CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

Identification and validation of tumor suppressor genes.

Cancers are associated with frequent deletions of genetic material that select for the loss of genes regulating normal cellular physiology. Although several cancer suppressor genes have been identified from these areas of deletion, the identities of the vast majority remain unknown, making approaches leading to their localization, identification, and validation an important continuing endeavor. Those currently characterized cancer suppressors include regulators of aspects of the cell cycle, growth and transcriptional regulators, DNA repair enzymes, differentiation factors, cell motility elements, and regulators of signal transduction. Several inherited cancer predisposition genes have been mapped and cloned using meiotic genetic linkage mapping but less success has been achieved identifying those genes involved in nonfamilial cancer. The future localization, identification, and validation of these genes are likely to involve a combination of complementary position-oriented and function-driven approaches, some of which are detailed in this article.

The chromosome 10 monosomy common in human melanomas results from loss of two separate tumor suppressor loci.

Alteration of chromosome 10 is common in human melanomas and usually entails the loss of an entire chromosome homologue. Although the reasons for monosomy in cancer has remained obscure, one possibility is that multiple tumor suppressor genes residing on this chromosome must be lost in unison during tumor progression, and this is easier to accomplish by chromosome segregation rather than by multiple mutational and/or deletion events. The localization and identification of these genes has been hampered by the monosomy itself, which has resulted in a paucity of small defining deletions in tumors. Here, we have addressed the issue of monosomy in tumor development by using functional complementation mapping to localize and demonstrate the existence of different melanoma suppressor genes on chromosome 10 and assigned each locus a distinct tumorigenic phenotype. We report that a locus on 10q distal to 10q23.1, likely involving the PTEN tumor suppressor, causes a severe reduction in the kinetics of melanoma tumor formation in animals. In contrast, a previously unrecognized region at 10p15.3 has a distinct, but lesser, effect on in vivo melanoma growth. Thus, the loss of both of these regions, which is accomplished by tumor-associated monosomy, provides a significant growth advantage over the individual loss of either region, thereby explaining the monosomy observed in sporadic melanomas.

Functional localization of a melanoma tumor suppressor gene to a small (< or = 2 Mb) region on 11q23.

We have previously demonstrated the existence of a melanoma tumor suppressor gene(s) on the long arm of chromosome 11 through suppression of tumorigenicity assays. Although loss of heterozygosity studies also support this finding, only a large critical region (44 cM) has been identified to date on 11q22-25. To further localize a tumor suppressor gene(s) within this region, we have now generated and characterized nine melanoma microcell hybrids, each retaining an introduced fragment of 11q. Of the nine hybrids, four were suppressed for tumor formation in nude mice, while five formed tumors at the same rate as the parental melanoma cell line (UACC 903). Molecular analysis of the hybrids with 118 microsatellite markers narrowed the location of a putative suppressor gene to a small (< or =2 Mb) candidate region on 11q23 between the markers D11S1786 and D11S2077 and within the larger region frequently deleted in melanoma tumors and cell lines. While multiple tumor suppressor genes are likely to reside on 11q22-25, the presence of this region in all four suppressed hybrids supports the simplest model that a single locus is responsible for the suppressed phenotype observed in UACC 903.

In vitro loss of heterozygosity targets the PTEN/MMAC1 gene in melanoma.

Gross genetic lesions of chromosome 10 occur in 30-50% of sporadic human melanomas. To test the functional significance of this observation, we have developed an in vitro loss of heterozygosity approach in which a wild-type chromosome 10 was transferred into melanoma cells, where there was selection for its breakage and regional deletion to relieve its growth suppressive effects. The overlap of these events was at band 10q23, the site of the recently isolated PTEN/MMAC1 tumor suppressor gene, suggesting it as a potential target. Although the gene was expressed in the parental cells, both of its chromosomal alleles contained truncating mutations. In vitro loss of heterozygosity resulted in loss of the chromosomally introduced wild-type PTEN/MMAC1, and ectopic expression of the gene caused cell growth suppression. Thus, this approach identified PTEN/MMAC1 as a target in malignant melanoma and may provide an alternative means to localizing tumor suppressor genes.

A panel of transferable fragments of human chromosome 11q.

Cytogenetic and molecular studies have implicated one or more tumor suppressor genes on the long arm of human chromosome 11 in the malignant progression of several human solid tumors, including malignant melanoma and carcinomas of the breast, cervix, ovary, and lung. Microcell-mediated chromosome transfer of an intact copy of chromosome 11 into tumor cell lines has provided additional evidence of tumor suppressor gene function in melanoma, breast cancer, and cervical cancer. However, sublocalization of the region(s) conferring the tumor suppressive effect has been difficult. To facilitate mapping of tumor suppressor gene(s) on chromosome 11, we have generated a panel of 25 mouse donor cell lines containing neo-tagged fragments of human chromosome 11q which can be transferred into cell lines to test for tumor suppressor activity. The chromosome fragments in these cell lines have been characterized by fluorescence in situ hybridization with probes to human DNA and to the centromere of chromosome 11, and also by analysis of microsatellite markers spanning chromosome 11. Finally, to demonstrate the usefulness of these cell lines as donors for microcell-mediated chromosome transfer, two fragments were transferred into the human melanoma cell line UACC 903. This panel of selectable subchromosomal fragments, derived from the long arm of human chromosome 11, will be useful for the regional localization of tumor suppressors and other genes by means of functional assays.

Mechanisms of human melanoma cell growth and tumor suppression by chromosome 6.

Breaks and deletions of chromosome 6 are among the most frequent karyotypic abnormalities that appear in human malignant melanoma cells, and chromosome transfer experiments have provided functional evidence for the presence of a melanoma tumor suppressor locus on chromosome 6[J.M. Trent et al., Science (Washington DC), 247: 568-571, 1990]. We have investigated the genetic mechanism of this suppression. We have found that the suppression of tumorigenicity that follows the introduction of a normal copy of chromosome 6 into the UACC 903 human melanoma cell line is correlated with increased chromosome 6 dosage, rather than with the presence of the transferred normal copy of the chromosome. Transfer of chromosome 6 into another human melanoma cell line, MelJuSo, does not result in suppression of primary tumor formation, although the additional copy of chromosome 6 does reduce the cell growth rate in vitro. Finally, we have identified a substantial portion of the chromosome that is evidently not involved in tumor suppression. We have observed that a copy of chromosome 6 derived from a normal cell but with a deletion involving chromosome bands 6q22-6q24 suppresses primary tumor formation in UACC 903 cells as effectively as the intact chromosome. We have thus provided additional information about the chromosomal location of this tumor suppressor and about its mode of action.