US oil and gas system emissions from nearly one million aerial site measurements.

As airborne methane surveys of oil and gas systems continue to discover large emissions that are missing from official estimates1-4, the true scope of methane emissions from energy production has yet to be quantified. We integrate approximately one million aerial site measurements into regional emissions inventories for six regions in the USA, comprising 52% of onshore oil and 29% of gas production over 15 aerial campaigns. We construct complete emissions distributions for each, employing empirically grounded simulations to estimate small emissions. Total estimated emissions range from 0.75% (95% confidence interval (CI) 0.65%, 0.84%) of covered natural gas production in a high-productivity, gas-rich region to 9.63% (95% CI 9.04%, 10.39%) in a rapidly expanding, oil-focused region. The six-region weighted average is 2.95% (95% CI 2.79%, 3.14%), or roughly three times the national government inventory estimate5. Only 0.05-1.66% of well sites contribute the majority (50-79%) of well site emissions in 11 out of 15 surveys. Ancillary midstream facilities, including pipelines, contribute 18-57% of estimated regional emissions, similarly concentrated in a small number of point sources. Together, the emissions quantified here represent an annual loss of roughly US$1 billion in commercial gas value and a US$9.3 billion annual social cost6. Repeated, comprehensive, regional remote-sensing surveys offer a path to detect these low-frequency, high-consequence emissions for rapid mitigation, incorporation into official emissions inventories and a clear-eyed assessment of the most effective emission-finding technologies for a given region.

Preferential redox regulation of cysteine-based protein tyrosine phosphatases: structural and biochemical diversity.

Protein phosphorylation is a major post-translational modification involved in cell signalling that regulates many physiological and pathological processes. Despite their biological importance, protein phosphatases are less studied than protein kinases. Importantly, the activity of Cys-based protein tyrosine phosphatases (PTPs) can be regulated by reversible oxidation. The initial two-electron oxidation product of the active site Cys is a sulfenic acid (Cys-SOH) that can then undergo distinct outcomes, such as the disulfide bond or a sulfenyl amide formation. Here, we review the biochemical and structural features of PTPs to find patterns that might specify their oxidation products, aiming to get insights into redox regulatory mechanisms. Initially, the structure and biochemistry of PTP1B is presented. Then, we describe structural aspects that are relevant for substrate recognition and catalysis. Notably, all PTPs contain critical Cys residues for the catalysis of dephosphorylation that is prone to oxidative inactivation, which are frequently found oxidized in cells under physiological conditions, such as upon growth factor stimuli. However, direct oxidations of Cys residues in PTPs by H2 O2 are rather slow. Therefore, we discuss possible mechanisms that may account for this apparent contradiction between biological and chemical redox aspects of PTPs. Furthermore, we performed a systematic analysis of the distance between active site cysteine and its backdoor cysteine with the attempt to analyse the preference between disulfide bond formation or sulfenyl amide interaction upon oxidation. In summary, PTPs have been showing many possibilities to auto-protect from irreversible oxidation, which is important for cell signalling regulation.

Fusion of genomic, proteomic and phenotypic data: the case of potyviruses.

Data fusion has been widely applied to analyse different sources of information, combining all of them in a single multivariate model. This methodology is mandatory when different omic data sets must be integrated to fully understand an organism using a systems biology approach. Here, a data fusion procedure is presented to combine genomic, proteomic and phenotypic data sets gathered for Tobacco etch virus (TEV). The genomic data correspond to random mutations inserted in most viral genes. The proteomic data represent both the effect of these mutations on the encoded proteins and the perturbation induced by the mutated proteins to their neighbours in the protein-protein interaction network (PPIN). Finally, the phenotypic trait evaluated for each mutant virus is replicative fitness. To analyse these three sources of information a Partial Least Squares (PLS) regression model is fitted in order to extract the latent variables from data that explain (and relate) the significant variables to the fitness of TEV. The final output of this methodology is a set of functional modules of the PPIN relating topology and mutations with fitness. Throughout the re-analysis of these diverse TEV data, we generated valuable information on the mechanism of action of certain mutations and how they translate into organismal fitness. Results show that the effect of some mutations goes beyond the protein they directly affect and spreads on the PPIN to neighbour proteins, thus defining functional modules.

The impact of high-order epistasis in the within-host fitness of a positive-sense plant RNA virus.

RNA viruses are the main source of emerging infectious diseases because of the evolutionary potential bestowed by their fast replication, large population sizes and high mutation and recombination rates. However, an equally important property, which is usually neglected, is the topography of the fitness landscape. How many fitness maxima exist and how well they are connected is especially interesting, as this determines the number of accessible evolutionary pathways. To address this question, we have reconstructed a region of the fitness landscape of tobacco etch potyvirus constituted by mutations observed during the experimental adaptation of the virus to the novel host Arabidopsis thaliana. Fitness was measured for many genotypes and showed the existence of multiple peaks and holes in the landscape. We found prevailing epistatic effects between mutations, with cases of reciprocal sign epistasis being common among pairs of mutations. We also found that high-order epistasis was as important as pairwise epistasis in their contribution to fitness. Therefore, results suggest that the landscape was rugged due to the existence of holes caused by lethal genotypes, that a very limited number of potential neutral paths exist and that it contained a single adaptive peak.

Magnitude and sign epistasis among deleterious mutations in a positive-sense plant RNA virus.

How epistatic interactions between mutations determine the genetic architecture of fitness is of central importance in evolution. The study of epistasis is particularly interesting for RNA viruses because of their genomic compactness, lack of genetic redundancy, and apparent low complexity. Moreover, interactions between mutations in viral genomes determine traits such as resistance to antiviral drugs, virulence and host range. In this study we generated 53 Tobacco etch potyvirus genotypes carrying pairs of single-nucleotide substitutions and measured their separated and combined deleterious fitness effects. We found that up to 38% of pairs had significant epistasis for fitness, including both positive and negative deviations from the null hypothesis of multiplicative effects. Interestingly, the sign of epistasis was correlated with viral protein-protein interactions in a model network, being predominantly positive between linked pairs of proteins and negative between unlinked ones. Furthermore, 55% of significant interactions were cases of reciprocal sign epistasis (RSE), indicating that adaptive landscapes for RNA viruses maybe highly rugged. Finally, we found that the magnitude of epistasis correlated negatively with the average effect of mutations. Overall, our results are in good agreement to those previously reported for other viruses and further consolidate the view that positive epistasis is the norm for small and compact genomes that lack genetic robustness.

Preparation of single cells for imaging mass spectrometry.

Characterizing the molecular contents of individual cells is critical for understanding fundamental mechanisms of biological processes. Imaging mass spectrometry (IMS) of biological systems has been steadily gaining popularity for its ability to create precise chemical images of biological samples, thereby revealing new biological insights and improving understanding of disease. In order to acquire mass spectral images from single cells that contain relevant molecular information, samples must be prepared such that cell-culture components, especially salts, are eliminated from the cell surface and that the cell contents are accessible to the mass spectrometer. We have demonstrated a cellular preparation technique for IMS that preserves the basic morphology of cultured cells, allows mass spectrometric chemical profiling of cytosol, and removes the majority of the interfering species derived from the cellular growth medium. Using this protocol, we achieve high-quality, reproducible IMS images from three diverse cell types: MCF7 human breast cancer cells, Madin-Darby canine kidney (MDCK) cells, and NIH/3T3 mouse fibroblasts. This preparation method allows rapid and routine IMS analysis of cultured cells, making possible a wide variety of experiments to further scientific understanding of molecular processes within individual cells.

Preparation of single cells for imaging/profiling mass spectrometry.

Characterizing chemical changes within individual cells is important for determining fundamental mechanisms of biological processes that will lead to new biological insights and improved disease understanding. Analyzing biological systems with imaging and profiling mass spectrometry (MS) has gained popularity in recent years as a method for creating chemical maps of biological samples. To obtain mass spectra that provide relevant molecular information about individual cells, samples must be prepared so that salts and other cell culture components are removed from the cell surface and that the cell contents are rendered accessible to the desorption beam. We have designed a cellular preparation protocol for imaging/profiling MS that removes the majority of the interfering species derived from the cellular growth medium, preserves the basic morphology of the cells, and allows chemical profiling of the diffusible elements of the cytosol. Using this method, we are able to reproducibly analyze cells from three diverse cell types: MCF7 human breast cancer cells, Madin-Darby canine kidney (MDCK) cells, and NIH/3T3 mouse fibroblasts. This preparation technique makes possible routine imaging/profiling MS analysis of individual cultured cells, allowing for understanding of molecular processes within individual cells.

Proteomic comparison of nasopharyngeal cancer cell lines C666-1 and NP69 identifies down-regulation of annexin II and beta2-tubulin for nasopharyngeal carcinoma.

CONTEXT: Nasopharyngeal carcinoma (NPC), common in southern China and North Africa, has a complex etiology involving interplay between viral, environmental, and hereditary factors and is almost constantly associated with the Epstein-Barr virus. Since the prognosis of locally advanced and metastatic diseases is poor, increased understanding of the pathogenesis of NPC would be important for discovering novel markers for patients' management. OBJECTIVES: To compare the proteomic expression profile between an Epstein-Barr virus-associated NPC cell line (C666-1) and a normal NP cell line (NP69). The proteins with differential expression were analyzed in 40 undifferentiated NPC paraffin-embedded specimens. DESIGN: Differentially expressed proteins discovered between the two cell lines were identified by mass spectrometry. After confirmation by immunocytochemical staining, their expression in patient samples was measured using 40 pairs of undifferentiated NPCs together with their adjacent normal epithelia. RESULTS: Proteomic findings indicated that adenosine triphosphate synthase alpha chain was up-regulated, whereas annexin II, annexin V, beta(2)-tubulin, and profilin 1 were down-regulated. After confirming the results in agar-processed cell lines, annexin II and beta(2)-tubulin expression were found to be lower in tumor cells than in adjacent normal epithelial cells in 100% and 90% of the patients' specimens, respectively. Finally, annexin II down-regulation was positively associated with lymph node metastasis, suggesting that it may be a prognostic factor in NPC. CONCLUSIONS: The results suggest that annexin II and beta(2)-tubulin down-regulation is important in NPC formation and may represent potential targets for further investigations.

Low genetic variability in the coat and movement proteins of American plum line pattern virus isolates from different geographic origins.

Until very recently isolates of American plum line pattern virus (APLPV) had not been reported from outside North America. The nucleotide sequences corresponding to the movement (MP) and coat (CP) proteins of eight APLPV isolates from five Mediterranean countries were determined. Sequence analysis showed that both MP and CP genes are highly conserved irrespective of geographic origin. The study of the distribution of synonymous and nonsynonymous changes along both open reading frames revealed that these proteins are under the effect of negative purifying selection. The MP and CP of APLPV possess most of the functional motifs described for other members of the genus Ilarvirus.

The contribution of bifunctional SkipDewax pretreatment solution, rabbit monoclonal antibodies, and polymer detection systems in immunohistochemistry.

CONTEXT: In immunohistochemistry, nonstandardized antigen retrieval protocols and fluids, poor-quality antibodies, and the presence of endogenous biotin frequently lead to incorrect results. Recently, advanced reagents including bifunctional SkipDewax pretreatment solution (BSPS), rabbit monoclonal (RM) antibodies, and biotin-free polymer detection systems (PDSs) have been developed, which, it is claimed, resolve these problems. OBJECTIVES: To determine whether BSPS, RM antibodies, and biotin-free PDSs improve the accuracy of immunohistochemistry; to optimize a new protocol consisting of a combination of BSPS, RM antibodies, and PDSs; and to compare it with a conventional protocol. DESIGN: The efficacies of BSPS, RM antibodies, and PDSs were compared with those of their respective conventional reagents using multitissue spring-roll sections. The new protocol was compared with a conventional protocol using Ki-67 immunostaining of 49 colorectal carcinoma specimens. RESULTS: For antigen retrieval, BSPS resulted in similar or better tissue staining than an EDTA solution, but the efficacy of BSPS decreased when it was reused. Most RM antibodies resulted in a greater proportion of positive cells than the corresponding non-RM antibodies, which did not produce satisfactory results in the absence of antigen retrieval. The PDSs Bond, ChemMate, and SuperPicture resulted in a high percentage of positive cells, good staining intensities, and low backgrounds. Other PDSs, except that from Ventana, resulted in high backgrounds and false positivity. The new combined protocol resulted in better Ki-67 staining than the conventional assay. CONCLUSIONS: Bifunctional SkipDewax pretreatment solution, RM antibodies, and PDSs improve staining quality and diagnostic accuracy of immunohistochemistry assays and provide a foundation for standardization.

A real-time RT-PCR assay for quantifying the fitness of tobacco etch virus in competition experiments.

Relative fitness determination has become a standard tool in experimental virus evolution studies. In this type of studies, the tested strain is mixed with a reference strain, which differs in an easy-to-score and genetically stable marker, and allowed to compete for a limited common pool of resources during a given number of generations. In this report, a TaqMan real-time PCR methodology is proposed for quantifying the relative fitness of tobacco etch potyvirus strains (TEV) in in planta mixed infections with a reference TEV strain. Two different forward primers along with a common reverse one are used into separated reactions mixes from the same RNA preparation. The reference strain, named TEV-PC1, was genetically engineered to carry a neutral marker in a highly conserved region of the RNA polymerase NIb gene. This marker allows tracking the frequency of both competitors during competition experiments by real-time quantitative PCR using specific primers. Both the reproducibility and sensitivity of the method have been explored. Reproducibility was assessed by running multiple competition experiments for the same genotype. Sensitivity was assessed by comparing the results of competition experiments against TEV-PC1 of 24 single-nucleotide substitutions mutants.

Chemical and biological differentiation of three human breast cancer cell types using time-of-flight secondary ion mass spectrometry.

We use time-of-flight secondary ion mass spectrometry (TOF-SIMS) to image and classify individual cells on the basis of their characteristic mass spectra. Using statistical data reduction on the large data sets generated during TOF-SIMS analysis, similar biological materials can be differentiated on the basis of a combination of small changes in protein expression, metabolic activity and cell structure. We apply this powerful technique to image and differentiate three carcinoma-derived human breast cancer cell lines (MCF-7, T47D, and MDA-MB-231). In homogenized cells, we show the ability to differentiate the cell types as well as cellular compartments (cytosol, nuclear, and membrane). These studies illustrate the capacity of TOF-SIMS to characterize individual cells by chemical composition, which could ultimately be applied to detect and identify single aberrant cells within a normal cell population. Ultimately, we anticipate characterizing rare chemical changes that may provide clues to single cell progression within carcinogenic and metastatic pathways.

Atypical epithelial proliferations in acquired renal cystic disease harbor cytogenetic aberrations.

Acquired renal cystic disease (ARCD) complicating end-stage renal failure confers an increased risk for renal cell carcinoma, and atypical epithelial proliferation in the cysts may represent the precursor lesion. In this report we used an interphase cytogenetic technique to analyze the karyotypic features of various forms of atypical epithelial proliferations in a patient with ARCD. Both kidneys harbored numerous simple and atypical cysts. In addition, papillary tufts and a hitherto undescribed cribriform epithelial proliferation were found in the right kidney. The left kidney contained a 10-mm renal cell carcinoma with features indeterminate between clear cell and papillary types. There was gain of chromosome 7 in the papillary tufts; gain of chromosomes 7 and 17 in the cribriform lesion; gain of chromosomes 7, 12, 17, 20, and Y in the atypical cysts; and gain of chromosomes 7, 12, 17, and 20 in the renal cell carcinoma. These chromosomal aberrations suggest that atypical epithelial proliferations in ARCD represent early neoplastic lesions.

Presence of filterable and nonfilterable mRNA in the plasma of cancer patients and healthy individuals.

BACKGROUND: As RNA is labile, we investigated whether circulating RNA in human plasma may be present in a particle-associated form. METHODS: Blood was collected from 27 healthy individuals and 16 hepatocellular carcinoma (HCC) patients. The plasma from each individual was processed by two means: filtration through filters with different pore sizes (from 5 microm to 0.22 microm) and ultracentrifugation. We assessed plasma RNA content by a real-time quantitative reverse transcription-PCR assay for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts and plasma DNA by a real-time quantitative PCR assay for the beta-globin gene. RESULTS: The plasma GAPDH mRNA concentrations in the healthy individuals were significantly different in every pair of these filter sizes (P <0.05 for each pair). Overall, the plasma GAPDH mRNA concentration was higher by a median of 15-fold (interquartile range, 10- to 24-fold) in the paired unfiltered sample than in the sample filtered through a 0.22 microm filter. In contrast, no significant difference was seen in beta-globin DNA concentrations among different pore-size-filtered plasma samples (P = 0.455). Similarly, a significant difference was observed for RNA, but not DNA, between unfiltered plasma and ultracentrifuged plasma (P <0.05). No significant difference in GAPDH mRNA concentrations was seen between the 0.22-microm-filtered plasma samples and the ultracentrifuged plasma samples (P >0.05). In HCC patients, filtration with a 0.22 microm filter produced a median 9.3-fold (interquartile range, 6.9- to 311-fold) reduction in GAPDH mRNA concentration in plasma. Plasma GAPDH mRNA concentrations in HCC patients were significantly higher than those in healthy individuals, both with or without filtration (P <0.0 5 for filtered plasma samples; P <0.005 for unfiltered plasma samples). CONCLUSIONS: A substantial proportion of plasma mRNA species is particle-associated. In HCC patients, both circulating particle- and non-particle-associated plasma RNA are increased.

Cost of host radiation in an RNA virus.

Although host radiation allows a parasite to expand its ecological niche, traits governing the infection of multiple host types can decrease fitness in the original or alternate host environments. Reasons for this reduction in fitness include slower replication due to added genetic material or modifications, fitness trade-offs across host environments, and weaker selection resulting from simultaneous adaptation to multiple habitats. We examined the consequences of host radiation using vesicular stomatitis virus (VSV) and mammalian host cells in tissue culture. Replicate populations of VSV were allowed to evolve for 100 generations on the original host (BHK cells), on either of two novel hosts (HeLa and MDCK cells), or in environments where the availability of novel hosts fluctuated in a predictable or random way. As expected, each experimental population showed a substantial fitness gain in its own environment, but those evolved on new hosts (constant or fluctuating) suffered reduced competitiveness on the original host. However, whereas evolution on one novel host negatively correlated with performance on the unselected novel host, adaptation in fluctuating environments led to fitness improvements in both novel habitats.