The role of stormwater infrastructure in regional methane emissions.

Stormwater infrastructure has been recently indicated as a potential hotspot for methane (CH4) emissions. Although local assessments based on direct CH4 measurements are increasingly available, there is currently no standardized approach for evaluating CH4 emissions from different types of stormwater infrastructure, including permanently impounded or fast-draining structures in Urban Drainage Systems (UDS). Therefore, a comparative analysis with wastewater infrastructure systems, such as wastewater treatment plants (WWTPs), is not yet possible. Here, we present a conceptual framework for the first-order quantification and upscaling of CH4 emissions from stormwater infrastructure at local and national scales. We combined in-situ and ex-situ measurements of CH4 emissions with purposely acquired data from selected stormwater facilities to provide initial estimates of CH4 emissions and emission factors for stormwater infrastructure in Germany. The results show that while stormwater infrastructure might emit comparable amounts of CH4 per area as natural and anthropogenically impacted inland waters, it may exhibit higher mean emission factors (up to 7 times) than conventional WWTPs, indicating less efficiency in limiting CH4 emissions than WWTPs. This is particularly true for permanently impounded facilities, which showed substantially higher mean surface CH4 emissions (up to 632 mg m-2 d-1) than fast-draining infrastructure (0.5-1.28 mg m-2 d-1). Permanently impounded sedimentation basins for stormwater management alone may reach up to 60% of the total CH4 emissions originating from WWTPs in Germany. These results are in conflict with the ongoing trend towards increasing implementation of impounded stormwater infrastructure systems, highlighting the urgent need for more extensive assessments of their impact on CH4 dynamics.

How does greywater separation impact the operation of conventional wastewater treatment plants?

Source separation has thus far been addressed mainly within the context of decentralization in new development areas; centralized approaches for resource-oriented sanitation remained, however, largely disregarded. By means of inhabitant-specific load and volume flow balances, based on typical reference values for municipal wastewater in Germany, a stepwise transition towards on-site greywater recycling was investigated for a model wastewater treatment plant (WWTP). Up to 17% transition (separation of greywater from 17% of the total inhabitants), greywater separation was proven to benefit plant operation by reducing energy consumption for aeration. From 17% transition onwards, however, unfavorable carbon to nitrogen ratios (C/N) were reported, as less biodegradable carbon reaches denitrification, thus shifting C/N ratios negatively. Therefore, nitrogen recovery/removal from N-rich sludge sidestreams would be required. At 35% transition, a 50% N recovery from sludge liquor was proven to be sufficient in order to ensure full denitrification; combined with greywater separation, nutrient recovery yielded 14% reduction in power demand for aeration (on the actual state). Additionally, extensive mainstream process changeovers could be avoided by separating N-rich urine alongside greywater from the main wastewater stream. Urine separation was proven to maintain denitrification stability as well as reduce power demand for aeration. The calculations show that, under consideration of specific boundary conditions, existing WWTP can be successfully integrated in transition concepts for resource-oriented sanitation.

Aging: a portrait from gene expression profile in blood cells.

The availability of reliable biomarkers of aging is important not only to monitor the effect of interventions and predict the timing of pathologies associated with aging but also to understand the mechanisms and devise appropriate countermeasures. Blood cells provide an easily available tissue and gene expression profiles from whole blood samples appear to mirror disease states and some aspects of the aging process itself. We report here a microarray analysis of whole blood samples from two cohorts of healthy adult and elderly subjects, aged 43±3 and 68±4 years, respectively, to monitor gene expression changes in the initial phase of the senescence process. A number of significant changes were found in the elderly compared to the adult group, including decreased levels of transcripts coding for components of the mitochondrial respiratory chain, which correlate with a parallel decline in the maximum rate of oxygen consumption (VO2max), as monitored in the same subjects. In addition, blood cells show age-related changes in the expression of several markers of immunosenescence, inflammation and oxidative stress. These findings support the notion that the immune system has a major role in tissue homeostasis and repair, which appears to be impaired since early stages of the aging process.

Transcriptional regulation of the human Raver2 ribonucleoprotein gene.

Raver2 is a putative modulator of the activity of the polypyrimidine-tract binding protein (PTB), one of the most intensively studied splicing repressors. Little is known about Raver2 expression, and all current data is from mice where it shows tissue specificity. In the present study, by comparing Raver2 transcript expression in human and mouse tissues, we found that human Raver2 is ubiquitously expressed in adult tissues. In order to investigate human Raver2 transcription regulation, we identified and characterized a putative promoter region in a 1000bp region upstream of the transcription starting site of the gene. Dual luciferase reporter assays demonstrated that this region had promoter activity conferred by the first 160bp. By mutagenic analyses of putative cis-acting regulatory sequences, we identified an individual site that decreased the promoter activity by up to 40% when mutated. Together, our results suggest that regulation of human Raver2 expression involves TATA-less transcriptional activity.

Functional characterization of the ribonucleoprotein, PTB-binding 1/Raver1 promoter region.

Raver1 is a ribonucleoprotein, evolutionarily conserved in mammals, which acts as a polypyrimidine tract-binding protein (PTB/hnRNPI) co-repressor in regulating alternative splicing events. The mouse homologue has been identified as a dual compartment protein that interacts with PTB within perinucleolar structures, and localizes at microfilament plasma membrane attachment sites in fibroblasts, epithelial and muscle cells. Human Raver1 gene is localized on chromosome 19p13.2 and encodes for an inferred 756 amino acid protein sharing 87% similarity with the mouse orthologue. The human Raver1 gene expression has not been previously investigated. Here we report the mRNA expression profile of human Raver1 gene and the molecular characterization of its promoter region. From the in silico analysis of 1542 bp of the Raver1 5'-flanking region (GC content=61%), no canonical TATA or CAAT boxes can be highlighted, whereas several consensus Sp1 putative binding sequences can be predicted within 1 kb from the transcription start site (TSS) that we determined by 5'-RACE. Functional analyses established a minimal region involved in the regulation of the human Raver1 promoter activity. Mutational analyses and transfection studies indicated that a GGGAGCTCCC sequence at -531 represents a putative cis signal acting as a negative regulator element of the promoter function. Altogether, our results indicate that human Raver1 gene promoter region shares common features of ubiquitously expressed gene with the interacting splicing regulator PTB.

Identification and analysis of the human neural polypyrimidine tract binding protein (nPTB) gene promoter region.

Neural polypyrimidine tract-binding protein nPTB, originally identified as the neuronal counterpart of the hnRNPI/PTB protein, is an RNA binding protein involved into tissue-specific alternative splicing regulation. Here we describe the functional characterization of the promoter sequence of nPTB in HeLa and neuroblastoma cells. By means of genomic sequence analysis we have isolated and cloned a 1587-base pair region upstream the human nPTB coding region. The nPTB proximal promoter, although rich in G+C content and presenting putative binding sites for the transcription factors Sp1, NF-1, NF-kB and Oct-1, lacks a typical TATA box. Luciferase transient expression assays using deletion mutants have identified the proximal promoter region at -125 relative to the transcription start site. Alignment of human, murine and chimpanzee genomic sequences upstream the nPTB exon 1 has provided evidences for the evolutionary conservation of specific transcription factor binding sites.

Nuclear localization domains in human thyroid transcription factor 2.

Thyroid transcription factor-2 (TTF2) is a nuclear protein involved in morphogenesis and gene expression in thyroid gland, belonging to the family of the forkhead/winged-helix transcription factors. In the present study we have investigated the sequence determinants for transport and accumulation into the nucleus of the TTF2 protein. By transient expression of fusion proteins constructed by joining different parts of TTF2 to the reporter gene of the jellyfish green fluorescent protein (GFP) and, in a separate set of deleted constructs, the glutathione S-transferase (GST) coding sequence, we have demonstrated that a basic amino acid stretch present at both ends of the DNA-binding domain is a bona fide nuclear localization signal (NLS). We have analyzed the subcellular localization of deleted GFP-GST-TTF2 fusion proteins and have shown that residues inside the forkhead domain (FHD) contributed to the complete nuclear TTF2 protein accumulation. Furthermore, by means of GST binding assays we have shown that distinct TTF2 fragments, containing the NLS, were able to bind the nuclear import receptor importin alpha. Taken together, our results provide the first documentation about nuclear targeting of a forkhead protein containing two identical NLS signal flanking the DNA-binding domain.

Importin alpha binds to an unusual bipartite nuclear localization signal in the heterogeneous ribonucleoprotein type I.

The heterogeneous nuclear ribonucleoprotein (hnRNP) type I, a modulator of alternative splicing, localizes in the nucleoplasm of mammalian cells and in a discrete perinucleolar structure. HnRNP I contains a novel type of bipartite nuclear localization signal (NLS) at the N-terminus of the protein that we have previously named nuclear determinant localization type I (NLD-I). Recently, a neural counterpart of hnRNP I has been identified that contains a putative NLS with two strings of basic amino acids separated by a spacer of 30 residues. In the present study we show that the neural hnRNP I NLS is necessary and sufficient for nuclear localization and represents a variant of the novel bipartite NLS present in the NLD-I domain. Furthermore, we demonstrate that the NLD-I is transported into the nucleus by cytoplasmic factor(s) with active transport modality. Binding assays using recombinant importin alpha show an interaction with NLD-I similar to that of SV40 large T antigen NLS. Deletion analysis indicates that both stretches of basic residues are necessary for binding to importin alpha. The above experimental results lead to the conclusion that importin alpha acts as cytoplasmic receptor for proteins characterized by a bipartite NLS signal that extends up to 37 residues.