River ecosystem metabolism and carbon biogeochemistry in a changing world.

River networks represent the largest biogeochemical nexus between the continents, ocean and atmosphere. Our current understanding of the role of rivers in the global carbon cycle remains limited, which makes it difficult to predict how global change may alter the timing and spatial distribution of riverine carbon sequestration and greenhouse gas emissions. Here we review the state of river ecosystem metabolism research and synthesize the current best available estimates of river ecosystem metabolism. We quantify the organic and inorganic carbon flux from land to global rivers and show that their net ecosystem production and carbon dioxide emissions shift the organic to inorganic carbon balance en route from land to the coastal ocean. Furthermore, we discuss how global change may affect river ecosystem metabolism and related carbon fluxes and identify research directions that can help to develop better predictions of the effects of global change on riverine ecosystem processes. We argue that a global river observing system will play a key role in understanding river networks and their future evolution in the context of the global carbon budget.

Can Common Pool Resource Theory Catalyze Stakeholder-Driven Solutions to the Freshwater Salinization Syndrome?

Freshwater salinity is rising across many regions of the United States as well as globally, a phenomenon called the freshwater salinization syndrome (FSS). The FSS mobilizes organic carbon, nutrients, heavy metals, and other contaminants sequestered in soils and freshwater sediments, alters the structures and functions of soils, streams, and riparian ecosystems, threatens drinking water supplies, and undermines progress toward many of the United Nations Sustainable Development Goals. There is an urgent need to leverage the current understanding of salinization's causes and consequences─in partnership with engineers, social scientists, policymakers, and other stakeholders─into locally tailored approaches for balancing our nation's salt budget. In this feature, we propose that the FSS can be understood as a common pool resource problem and explore Nobel Laureate Elinor Ostrom's social-ecological systems framework as an approach for identifying the conditions under which local actors may work collectively to manage the FSS in the absence of top-down regulatory controls. We adopt as a case study rising sodium concentrations in the Occoquan Reservoir, a critical water supply for up to one million residents in Northern Virginia (USA), to illustrate emerging impacts, underlying causes, possible solutions, and critical research needs.

Global change-driven effects on dissolved organic matter composition: Implications for food webs of northern lakes.

Northern ecosystems are experiencing some of the most dramatic impacts of global change on Earth. Rising temperatures, hydrological intensification, changes in atmospheric acid deposition and associated acidification recovery, and changes in vegetative cover are resulting in fundamental changes in terrestrial-aquatic biogeochemical linkages. The effects of global change are readily observed in alterations in the supply of dissolved organic matter (DOM)-the messenger between terrestrial and lake ecosystems-with potentially profound effects on the structure and function of lakes. Northern terrestrial ecosystems contain substantial stores of organic matter and filter or funnel DOM, affecting the timing and magnitude of DOM delivery to surface waters. This terrestrial DOM is processed in streams, rivers, and lakes, ultimately shifting its composition, stoichiometry, and bioavailability. Here, we explore the potential consequences of these global change-driven effects for lake food webs at northern latitudes. Notably, we provide evidence that increased allochthonous DOM supply to lakes is overwhelming increased autochthonous DOM supply that potentially results from earlier ice-out and a longer growing season. Furthermore, we assess the potential implications of this shift for the nutritional quality of autotrophs in terms of their stoichiometry, fatty acid composition, toxin production, and methylmercury concentration, and therefore, contaminant transfer through the food web. We conclude that global change in northern regions leads not only to reduced primary productivity but also to nutritionally poorer lake food webs, with discernible consequences for the trophic web to fish and humans.

Whole-stream 13C tracer addition reveals distinct fates of newly fixed carbon.

Many estimates of freshwater carbon (C) fluxes focus on inputs, processing, and storage of terrestrial C; yet inland waters have high rates of internally fixed (autochthonous) C production. Some fraction of newly fixed C may be released as biologically available, dissolved organic C (DOC) and stimulate microbial-driven biogeochemical cycles soon after fixation, but the fate of autochthonous C is difficult to measure directly. Tracing newly fixed C can increase our understanding of fluxes and fate of autochthonous C in the context of freshwater food webs and C cycling. We traced autochthonous C fixation and fate using a dissolved inorganic C stable isotope addition (13C(DIC)). We added 13C(DIC) to North Fork French Creek, Wyoming, USA during two days in August. We monitored changes in 13C pools, fluxes, and storage for 44 d after the addition. Two-compartment flux models were used to quantify net release of newly fixed 13C(DOC) and 13C(DIC) into the water column. We compared net 13C fixation with tracer 13C(DIC) removal and gross primary production (GPP) to account for the mass of tracer fixed, released, lost to the atmosphere, and exported downstream. Much of the fixed C turned over rapidly and did not enter longer-term storage pools. Net C fixed was 70% of GPP measured with O2. Algae likely released the remaining 30% via 13C(DOC) exudation and respiration of newly fixed C. Primary producers released 13C(DOC) at rates of up to 16% per day during the 13C addition, but exudation of new labile C declined to near zero by day 6. DIC production from newly fixed C accounted for 21% of ecosystem respiration the day after the 13C addition. All measured organic C (OC) pools were enriched with 13C 1 d after the tracer addition. 20% of fixed 13C remained in benthic OC by day 44, and average residence time of autochthonous C in benthic OC was 62 d. Newly fixed C had two distinct fates: short-term (< 1 week) exudation and respiration or longer-term storage and downstream export. Autochthonous C in streams likely fuels short-term microbial production and biogeochemical cycling, in addition to providing a longer-term resource for consumers.

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) triggers MSH2 and Cdt2 protein-dependent degradation of the cell cycle and mismatch repair (MMR) inhibitor protein p21Waf1/Cip1.

p21(Waf1/Cip1) protein levels respond to DNA damage; p21 is induced after ionizing radiation, but degraded after UV. p21 degradation after UV is necessary for optimal DNA repair, presumably because p21 inhibits nucleotide excision repair by blocking proliferating cell nuclear antigen (PCNA). Because p21 also inhibits DNA mismatch repair (MMR), we investigated how p21 levels respond to DNA alkylation by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which triggers the MMR system. We show that MNNG caused rapid degradation of p21, and this involved the ubiquitin ligase Cdt2 and the proteasome. p21 degradation further required MSH2 but not MLH1. p21 mutants that cannot bind PCNA or cannot be ubiquitinated were resistant to MNNG. MNNG induced the formation of PCNA complexes with MSH6 and Cdt2. Finally, when p21 degradation was blocked, MNNG treatment resulted in reduced recruitment of MMR proteins to chromatin. This study describes a novel pathway that removes p21 to allow cells to efficiently activate the MMR system.

De novo constitutional MLH1 epimutations confer early-onset colorectal cancer in two new sporadic Lynch syndrome cases, with derivation of the epimutation on the paternal allele in one.

Lynch syndrome is an autosomal dominant cancer predisposition syndrome classically caused by germline mutations of the mismatch repair genes, MLH1, MSH2, MSH6 and PMS2. Constitutional epimutations of the MLH1 gene, characterized by soma-wide methylation of a single allele of the promoter and allelic transcriptional silencing, have been identified in a subset of Lynch syndrome cases lacking a sequence mutation in MLH1. We report two individuals with no family history of colorectal cancer who developed that disease at age 18 and 20 years. In both cases, cancer had arisen because of the de novo occurrence of a constitutional MLH1 epimutation and somatic loss-of-heterozygosity of the functional allele in the tumors. We show for the first time that the epimutation in one case arose on the paternally inherited allele. Analysis of 13 tumors from seven individuals with constitutional MLH1 epimutations showed eight tumors had lost the second MLH1 allele, two tumors had a novel pathogenic missense mutation and three had retained heterozygosity. Only 1 of 12 tumors demonstrated the BRAF V600E mutation and 3 of 11 tumors harbored a mutation in KRAS. The finding that epimutations can originate on the paternal allele provides important new insights into the mechanism of origin of epimutations. It is clear that the second hit in MLH1 epimutation-associated tumors typically has a genetic not epigenetic basis. Individuals with mismatch repair-deficient cancers without the BRAF V600E mutation are candidates for germline screening for sequence or methylation changes in MLH1.

Linking calcification by exotic snails to stream inorganic carbon cycling.

Biotic calcification is rarely considered in freshwater C budgets, despite calculations suggesting that calcifying animals can alter inorganic C cycling. Most studies that have quantified biocalcification in aquatic ecosystems have not directly linked CO(2) fluxes from biocalcification with whole-ecosystem rates of inorganic C cycling. The freshwater snail, Melanoides tuberculata, has achieved a high abundance and 37.4 g biomass m(-2) after invading Kelly Warm Springs in Grand Teton National Park. This high biomass suggests that introduced populations of Melanoides may alter ecosystem processes. We measured Melanoides growth rates and biomass to calculate the production of biomass, shell mass, and CO(2). We compared Melanoides biomass and inorganic C production with ecosystem C pools and fluxes, as well as with published rates of CO(2) production by other calcifying organisms. Melanoides calcification in Kelly Warm Springs produced 12.1 mmol CO(2) m(-2) day(-1) during summer months. We measured high rates of gross primary productivity and respiration in Kelly Warm Springs (-378 and 533 mmol CO(2) m(-2) day(-1), respectively); CO(2) produced from biocalcification increased net CO(2) production in Kelly Warm Springs from 155 to 167 mmol CO(2) m(-2) day(-1). This rate of CO(2) production via biocalcification is within the published range of calcification by animals. But these CO(2) fluxes are small when compared to ecosystem C fluxes from stream metabolism. The influence of animals is relative to ecosystem processes, and should always be compared with ecosystem fluxes to quantify the importance of a specific animal in its environment.

Author Correction: Carbon dioxide stimulates lake primary production.

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

Chemopreventive properties of pinoresinol-rich olive oil involve a selective activation of the ATM-p53 cascade in colon cancer cell lines.

UNLABELLED: The Mediterranean diet is rich in extra virgin olive oil (EVOO) and associated with a lower incidence of colorectal cancer. EVOO contains phenolic extracts with potential anticarcinogenic activity. AIM: To assess the anticancer properties of EVOO phenolic extracts using in vitro models. METHODS: Phenolic profiles of two different EVOOs (A and B) were determined. RKO and HCT116 (both p53 proficient), SW480 (p53 mutant) and HCT116(p53-/-) (p53 knocked out) cell lines were treated with EVOO extracts and assessed for cell viability. Apoptosis was determined by terminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay and changes in Bax transcript levels. Cell cycle analysis was determined by flow cytometry and western blots. To confirm the data, analysis of cell viability and cell cycle was performed with purified pinoresinol. RESULTS: Chemical characterization showed that pinoresinol is the main phenol in EVOO-A, and oleocanthal predominates in EVOO-B. Only EVOO-A affected cell viability, which was significantly more pronounced in p53-proficient cells. At a concentration of 200 nM, p53-proficient cells showed increased apoptosis and G(2)/M arrest. In p53-proficient cells, ataxia telangiectasia mutated (ATM) and its downstream-controlled proteins were upregulated after treatment, with a parallel decrease of cyclin B/cdc2. Identical results on cell viability and cell cycle were obtained with purified pinoresinol, but this required a higher concentration than in EVOO-A. CONCLUSION: Our results demonstrate that pinoresinol-rich EVOO extracts have potent chemopreventive properties and specifically upregulate the ATM-p53 cascade. This result was achieved at substantially lower concentrations in EVOO than with purified pinoresinol, indicating a possible synergic effect between the various polyphenols in olive oil.

Carbon dioxide stimulates lake primary production.

Gross primary production (GPP) is a fundamental ecosystem process that sequesters carbon dioxide (CO2) and forms the resource base for higher trophic levels. Still, the relative contribution of different controls on GPP at the whole-ecosystem scale is far from resolved. Here we show, by manipulating CO2 concentrations in large-scale experimental pond ecosystems, that CO2 availability is a key driver of whole-ecosystem GPP. This result suggests we need to reformulate past conceptual models describing controls of lake ecosystem productivity and include our findings when developing models used to predict future lake ecosystem responses to environmental change.

Annurca apple polyphenols have potent demethylating activity and can reactivate silenced tumor suppressor genes in colorectal cancer cells.

The CpG island methylator phenotype is characterized by DNA hypermethylation in the promoters of tumor suppressor genes with silencing of transcription. Hypermethylation of the promoter of hMLH1 and subsequent microsatellite instability occurs in approximately 12% of sporadic colorectal cancers (CRC). Annurca apple, a variety of southern Italy, is rich in polyphenols that are associated with anticancer properties. Populations in southern Italy have lower incidences of CRC than elsewhere in the western world. We evaluated the mechanisms of putative anticancer effects of Annurca polyphenol extract (APE) in in vitro models of CRC. We extracted polyphenols from Annurca apples and treated RKO, SW48, and SW480 cells with APE and assessed the cell viability, apoptosis, and cell cycle. DNA methylation of selected tumor suppressor genes was evaluated after treatment with APE and was compared with the synthetic demethylating agent 5-aza-2'deoxycytidine (5-aza-2dC). DNA methyltransferase (DNMT)-1 and -3b levels were evaluated. Decreased cell viability and induction of apoptosis was evident after treatment. We found no significant changes in cell cycle dynamics. We observed significant increases of p53 protein expression in RKO after treatment. APE treatment strongly reduced DNA methylation in the promoters of hMLH1, p14(ARF), and p16(INK4a) with consequent restoration of normal expression. These effects were qualitatively comparable with those obtained with 5-aza-2dC. We observed a significant reduction in expression of DNMT proteins after treatment without changes in messenger RNA. In conclusion, APE have potent demethylating activity through the inhibition of DNMT proteins. The lack of toxicity in Annurca extracts makes them excellent candidates for the chemoprevention of CRC.

Selenomethionine induces p53 mediated cell cycle arrest and apoptosis in human colon cancer cells.

While there is an increasing interest in selenium chemoprevention against human colon polyp recurrence and other cancers, the mechanism(s) by which these agents inhibit carcinogenesis are uncertain. Some of the proposed mechanisms include the inhibition of cytosine methyltransferases, carcinogen bioactivation, and inhibition of cyclooxygenase (COX). More recently, it has been suggested that selenium may exert growth inhibitory effects by activating p53. However, the molecular mechanisms of action of selenomethionine, an organoselenium compound present in selenized yeast and currently being investigated in human clinical trials for colon polyp prevention, are unclear. In the present study we tested the hypothesis that selenomethionine might affect colon cancer cell growth by p53 mediated apoptosis and/or cell cycle regulation. Four human colon cancer cell lines including HCT116 and RKO (wild type p53), HCT116-p53KO (isogenic control of HCT116 cells with p53 knocked out) and Caco-2 (mutant p53) were treated with 0-100 microM of selenomethionine for 24, 48 and 72 h. Cell viability rates were determined by the MTT assay. Cell cycle analysis was performed by flow cytometry and apoptosis measured by Annexin V-Cy5 staining. Expression of p53 protein was determined by Western blotting and immunofluorescence assays. All cell lines showed concentration and time dependent growth inhibition with selenomethionine, although HCT116 and RKO cells were the most sensitive to such treatments. Interestingly, although HCT116 and HCT116-p53KO are isogenic cell lines, selenomethionine caused a G2/M cell cycle arrest in HCT116 and RKO cells, but not in HCT116-p53KO cells. Similarly, both HCT116 and RKO demonstrated a significant increase in apoptosis (100-170%; p < 0.01) with 50-100 microM selenomethionine. Cell cycle arrest and apoptosis observed in HCT116 and RKO cell lines were accompanied by a marked increase in p53 protein expression following selenium treatment. These results clearly suggest that selenomethionine exerts p53 dependent growth inhibitory effects in colon cancer cells by inducing G2/M cell cycle arrest as well as apoptosis.

Novel Mutations in MLH1 and MSH2 Genes in Mexican Patients with Lynch Syndrome.

Background. Lynch Syndrome (LS) is characterized by germline mutations in the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2. This syndrome is inherited in an autosomal dominant pattern and is characterized by early onset colorectal cancer (CRC) and extracolonic tumors. The aim of this study was to identify mutations in MMR genes in three Mexican patients with LS. Methods. Immunohistochemical analysis was performed as a prescreening method to identify absent protein expression. PCR, Denaturing High Performance Liquid Chromatography (dHPLC), and Sanger sequencing complemented the analysis. Results. Two samples showed the absence of nuclear staining for MLH1 and one sample showed loss of nuclear staining for MSH2. The mutations found in MLH1 gene were c.2103+1G>C in intron 18 and compound heterozygous mutants c.1852_1854delAAG (p.K618del) and c.1852_1853delinsGC (p.K618A) in exon 16. In the MSH2 gene, we identified mutation c.638dupT (p.L213fs) in exon 3. Conclusions. This is the first report of mutations in MMR genes in Mexican patients with LS and these appear to be novel.

Transient stress lymphocytosis: an immunophenotypic characterization of the most common cause of newly identified adult lymphocytosis in a tertiary hospital.

We prospectively evaluated 52 consecutive cases of newly identified absolute lymphocytosis to determine the hematologic and immunophenotypic features of transient stress lymphocytosis (TSL). The lymphocytosis in all cases was associated with an acute stressful event and ranged from 4,000 to 10,400/microL (4.0-10.4 x 10(9)/L). Compared with healthy individuals, patients with TSL showed an increase in the total WBC, absolute lymphocyte (ALC), absolute neutrophil (ANC), and platelet counts with no difference in hemoglobin levels. Immunophenotypic analyses of 38 cases revealed increases in absolute numbers of T B, and natural killer cells. Both CD4+ and CD8+ T cells were increased, predominantly accountedfor by an increase in memory cell subsets, with no change in gamma/delta T cells. Follow-up studies showed a significant reduction in the ALC with a concurrent increase in the ANC and reduction in hemoglobin values. The reduction in lymphocytes at resolution was accompanied by reduction in all broad Iymphocyte subsets. However, naive and memory subsets showed different patterns of alteration within the CD4+ and CD8+ populations, suggesting that acute stress differentially affects the in vivo distribution of these subsets.

Prohibitin attenuates colitis-associated tumorigenesis in mice by modulating p53 and STAT3 apoptotic responses.

Although inflammatory bowel disease is associated with higher risk of colorectal cancer, the precise pathogenic mechanisms underlying this association are not completely understood. Prohibitin 1 (PHB), a protein implicated in the regulation of proliferation, apoptosis, and transcription, is decreased in intestinal inflammation. In this study, we have established a key function for PHB in mediating colitis-associated cancer. Wild-type and transgenic (Tg) mice specifically overexpressing PHB in intestinal epithelial cells were subjected to a classical two-stage protocol of colitis-associated carcinogenesis. In addition, wild-type and p53 null human cell models were used to assess PHB interaction with STAT3 and p53. Wild-type mice exhibited decreased mucosal PHB protein expression during colitis-associated carcinogenesis. Tg mice exhibited decreased susceptibility in a manner associated with increased apoptosis, p53, Bax, and Bad expression plus decreased Bcl-xL and Bcl-2 expression. PHB overexpression in wild-type but not p53 null human cells increased expression of Bax, Bad, and caspase-3 cleavage. In wild-type p53 cells, PHB overexpression decreased basal and interleukin-6-induced STAT3 activation and expression of the STAT3 responsive genes Bcl-xL and Bcl-2. PHB coimmunoprecipitated with phospho-STAT3 in addition to p53 in cultured cell lysates and colon mucosa. This is the first study to show interaction between PHB and STAT3 in vivo. In summary, our findings suggest that PHB protects against colitis-associated cancer by modulating p53- and STAT3-mediated apoptosis. Modulation of PHB expression in intestinal epithelial cells may offer a potential therapeutic approach to prevent colitis-associated carcinogenesis.

Oncogenic T-antigen of JC virus is present frequently in human gastric cancers.

BACKGROUND: JC virus (JCV) is a polyomavirus that commonly infects humans and is the causative agent of progressive multifocal leukoencephalopathy in immune-compromised patients. An association between JCV and human cancers long has been suspected, because this virus induces brain tumors in several animal models. The oncogenic potential of JCV is mediated by a transforming protein, the T-antigen (T-Ag), which is a multifunctional protein that transforms cells through interactions with various growth-regulatory genes, including p53 and pRb, and by stabilizing beta-catenin. Previously, the laboratory at the authors' institution demonstrated that JCV is present frequently in the human gastrointestinal tract and may play a role in colorectal carcinogenesis. However, to date, no studies have determined whether JCV sequences are present specifically in gastric cancers. The current study was designed to investigate whether JCV sequences and expression are found in human gastric cancers. METHODS: DNA was extracted from 23 paraffin embedded and 14 frozen gastric cancer specimens. For the detection of JCV gene sequences, polymerase chain reaction amplifications were performed using gene-specific primers for T-Ag, VP-1 (a JCV capsid gene), and the viral regulatory region (or transcriptional control region). Immunohistochemical staining was performed with an anti-T-Ag monoclonal antibody to detect protein expression. RESULTS: Twenty-one of 37 gastric cancers (57%) harbored JCV T-Ag sequences, and 13 of 37 gastric cancers (30%) contained VP-1 sequences. T-Ag sequences also were found in adjacent nonneoplastic mucosa. In addition, JCV regulatory region sequences were present frequently in gastric cancers and adjacent nonneoplastic mucosa. T-Ag protein expression was found in 9 of 23 gastric cancers (39%), whereas no expression was observed in any of the nonneoplastic tissues. CONCLUSIONS: To the authors' knowledge, this is the first demonstration of the presence of JCV T-Ag expression in human gastric cancers. These findings suggest a possible role for this polyomavirus in gastric carcinogenesis.