Outer membrane vesicles and the outer membrane protein OmpU govern Vibrio cholerae biofilm matrix assembly.

Biofilms are matrix-encased microbial communities that increase the environmental fitness and infectivity of many human pathogens including Vibrio cholerae. Biofilm matrix assembly is essential for biofilm formation and function. Known components of the V. cholerae biofilm matrix are the polysaccharide Vibrio polysaccharide (VPS), matrix proteins RbmA, RbmC, Bap1, and extracellular DNA, but the majority of the protein composition is uncharacterized. This study comprehensively analyzed the biofilm matrix proteome and revealed the presence of outer membrane proteins (OMPs). Outer membrane vesicles (OMVs) were also present in the V. cholerae biofilm matrix and were associated with OMPs and many biofilm matrix proteins suggesting that they participate in biofilm matrix assembly. Consistent with this, OMVs had the capability to alter biofilm structural properties depending on their composition. OmpU was the most prevalent OMP in the matrix, and its absence altered biofilm architecture by increasing VPS production. Single-cell force spectroscopy revealed that proteins critical for biofilm formation, OmpU, the matrix proteins RbmA, RbmC, Bap1, and VPS contribute to cell-surface adhesion forces at differing efficiency, with VPS showing the highest efficiency whereas Bap1 showing the lowest efficiency. Our findings provide new insights into the molecular mechanisms underlying biofilm matrix assembly in V. cholerae, which may provide new opportunities to develop inhibitors that specifically alter biofilm matrix properties and, thus, affect either the environmental survival or pathogenesis of V. cholerae.IMPORTANCECholera remains a major public health concern. Vibrio cholerae, the causative agent of cholera, forms biofilms, which are critical for its transmission, infectivity, and environmental persistence. While we know that the V. cholerae biofilm matrix contains exopolysaccharide, matrix proteins, and extracellular DNA, we do not have a comprehensive understanding of the majority of biofilm matrix components. Here, we discover outer membrane vesicles (OMVs) within the biofilm matrix of V. cholerae. Proteomic analysis of the matrix and matrix-associated OMVs showed that OMVs carry key matrix proteins and Vibrio polysaccharide (VPS) to help build biofilms. We also characterize the role of the highly abundant outer membrane protein OmpU in biofilm formation and show that it impacts biofilm architecture in a VPS-dependent manner. Understanding V. cholerae biofilm formation is important for developing a better prevention and treatment strategy framework.

The role of spatial structure in at-risk metapopulation recoveries.

Metapopulations are often managed as a single contiguous population despite the spatial structure underlying their local and regional dynamics. Disturbances from human activities can also be spatially structured with mortality impacts concentrated to just a few local populations among the aggregate. Scale transitions between local and regional processes can generate emergent properties whereby the whole system can fail to recover as quickly as expected for an equivalent single population. Here, we draw on theory and empirical case studies to ask: what is the consequence of spatially structured ecological and disturbance processes on metapopulation recoveries? We suggest that exploring this question could help address knowledge gaps for managing metapopulations including: Why do some metapopulations recover quickly while others remain collapsed? And, what risks are unaccounted for when metapopulations are managed at aggregate scales? First, we used model simulations to examine how scale transitions among ecological and disturbance conditions interact to generate emergent metapopulation recovery outcomes. In general, we found that the spatial structure of disturbance was a strong determinant of recovery outcomes. Specifically, disturbances that unevenly impacted local populations consistently generated the slowest recoveries and highest conservation risks. Ecological conditions that dampened metapopulation recoveries included low dispersal, variable local demography, sparsely connected habitat networks, and spatially and temporally correlated stochastic processes. Second, we illustrate the unexpected challenges of managing metapopulations by examining the recoveries of three USA federally listed endangered species: Florida Everglade snail kites, California and Alaska sea otters, and Snake River Chinook salmon. Overall, our results show the pivotal role of spatial structure in metapopulation recoveries whereby the interplay between local and regional processes shapes the resilience of the whole system. With this understanding, we provide guidelines for resource managers tasked with conserving and managing metapopulations and identify opportunities for research to support the application of metapopulation theory to real-world challenges.

3-deazaadenosine (3DA) alleviates senescence to promote cellular fitness and cell therapy efficiency in mice.

Cellular senescence is a stable type of cell cycle arrest triggered by different stresses. As such, senescence drives age-related diseases and curbs cellular replicative potential. Here, we show that 3-deazaadenosine (3DA), an S-adenosyl homocysteinase (AHCY) inhibitor, alleviates replicative and oncogene-induced senescence. 3DA-treated senescent cells showed reduced global Histone H3 Lysine 36 trimethylation (H3K36me3), an epigenetic modification that marks the bodies of actively transcribed genes. By integrating transcriptome and epigenome data, we demonstrate that 3DA treatment affects key factors of the senescence transcriptional program. Remarkably, 3DA treatment alleviated senescence and increased the proliferative and regenerative potential of muscle stem cells from very old mice in vitro and in vivo. Moreover, ex vivo 3DA treatment was sufficient to enhance the engraftment of human umbilical cord blood (UCB) cells in immunocompromised mice. Together, our results identify 3DA as a promising drug enhancing the efficiency of cellular therapies by restraining senescence.

Toxicological impacts of roadway deicers on aquatic resources and human health: A review.

During winter, snow and ice on roads in regions with cold weather can increase traffic crashes and casualties, resulting in travel delays and financial burdens to society. Anti-icing or deicing the roads can serve a cost-effective method to significantly reduce such risks. Although traditionally the main priorities of winter road maintenance (WRM) have been level of service, cost-effectiveness, and corrosion reduction, it is increasingly clear that understanding the environmental impacts of deicers is vital. One of the most important problems in this regard is environmental contamination caused by cumulative use of deicers, which has many detrimental effects on the aquatic systems. Among the deicers, the chloride-based ones raise the most toxicological concerns because they are highly soluble, can migrate quickly in the environment and have cumulative effects over time. In this review, we summarize and organize existing data, including the latest findings about the adverse effects of deicers on surface water and groundwater, aquatic species, and human health, and identify future research priorities. In addition, the data provided can be used to develop a framework for quantifying some of the variables that stakeholders and agencies use when preparing guidelines and standards for WRM programs. PRACTITIONER POINTS: Pollution from the increasing use of roadway deicers may have detrimental effects on the environment. Of particular concern are the acute and cumulative risks that chloride salts pose to aquatic species. Chloride salts are water-soluble, very difficult to remove, highly mobile, and non-degradable. Deicers cause water stratification, change the chemicophysical properties of water, and affect aquatic species and human health. Current guidelines may not be appropriate for environmental protection and need to be revised.

The small GTPase MglA together with the TPR domain protein SgmX stimulates type IV pili formation in M. xanthus.

Bacteria can move across surfaces using type IV pili (T4P), which undergo cycles of extension, adhesion, and retraction. The T4P localization pattern varies between species; however, the underlying mechanisms are largely unknown. In the rod-shaped Myxococcus xanthus cells, T4P localize at the leading cell pole. As cells reverse their direction of movement, T4P are disassembled at the old leading pole and then form at the new leading pole. Thus, cells can form T4P at both poles but engage only one pole at a time in T4P formation. Here, we address how this T4P unipolarity is realized. We demonstrate that the small Ras-like GTPase MglA stimulates T4P formation in its GTP-bound state by direct interaction with the tetratricopeptide repeat (TPR) domain-containing protein SgmX. SgmX, in turn, is important for polar localization of the T4P extension ATPase PilB. The cognate MglA GTPase activating protein (GAP) MglB, which localizes mainly to the lagging cell pole, indirectly blocks T4P formation at this pole by stimulating the conversion of MglA-GTP to MglA-GDP. Based on these findings, we propose a model whereby T4P unipolarity is accomplished by stimulation of T4P formation at the leading pole by MglA-GTP and SgmX and indirect inhibition of T4P formation at the lagging pole by MglB due to its MglA GAP activity. During reversals, MglA, SgmX, and MglB switch polarity, thus laying the foundation for T4P formation at the new leading pole and inhibition of T4P formation at the new lagging pole.

Application of an atmospheric tracer ratio method to estimation of PM2.5 emission rates from wheat conveying operations at a wheat pile storage facility.

Particulate matter (PM) pollution is associated with adverse effects on human health and the environment. There is no designated PM2.5 emission factor for horizontal grain conveyors. Instead, in Washington state, the air permitting agency uses an emission factor for headhouse and grain handling operations to issue permits. There is concern that this factor does not accurately represent the conveyor operations and limits the size and operation of wheat pile facilities. The primary goal of this work was to estimate the PM2.5 emission rate (which can further be converted to an emission factor) from wheat conveying operations at a large wheat pile storage facility in eastern Washington using an atmospheric tracer ratio method, with CO2 gas as the tracer. The field study results yield an emission rate of 5.2[Formula: see text]1.7 grams of PM2.5 per hour and these emissions are due to the transfer point from an upper belt to a lower belt. This rate is approximately 320 times lower than the emission rate for headhouse operations which has been used previously to represent conveyor operations. The emission rate was in relatively good agreement with results of an inverse Gaussian plume model calculation of emissions using measured ambient PM2.5 levels at a very short distance downwind of the transfer point. A consistent PM2.5 to tracer gas ratio over the tests showed that PM2.5 and CO2 disperse in a similar manner and confirmed that the CO2 tracer release was a reliable simulation of the PM2.5 pollutant source over distances involved in the study (less than 10 meters). The results also indicate a need for the Environmental Protection Agency to develop a designated PM2.5 emission factor for wheat conveyance. IMPLICATIONS: There are presently no emission factors available for large wheat pile storage facilities where wheat is transferred via long horizontal conveyor belts. As a result, local and state permitting agencies use emission factors for other types of grain handling systems. In this paper, we report the first measurements of PM2.5 emission rates (that can further be converted to emission factors using a known grain rate on the conveyor) for horizontal grain conveyors used at wheat pile storage facilities. The measured emission rate is much less than the emission rate derived from the surrogate emission factor currently used for permit purposes. This has implications for the size and operation of wheat pile storage facilities.

Spatial control of the GTPase MglA by localized RomR-RomX GEF and MglB GAP activities enables Myxococcus xanthus motility.

The rod-shaped Myxococcus xanthus cells move with defined front-rear polarity using polarized motility systems. A polarity module consisting of the small GTPase MglA, its cognate GTPase activating protein (GAP) MglB and RomR establishes this polarity. Agl-Glt gliding motility complexes assemble and disassemble at the leading and lagging pole, respectively. These processes are stimulated by MglA-GTP at the leading and MglB at the lagging pole. Here, we identify RomX as an integral component of the polarity module. RomX and RomR form a complex that has MglA guanine nucleotide exchange factor (GEF) activity and also binds MglA-GTP. In vivo RomR recruits RomX to the leading pole forming the RomR-RomX complex that stimulates MglA-GTP formation and binding, resulting in a high local concentration of MglA-GTP. The spatially separated and opposing activities of the RomR-RomX GEF at the leading and the MglB GAP at the lagging cell pole establish front-rear polarity by allowing the spatially separated assembly and disassembly of Agl-Glt motility complexes. Our findings uncover a regulatory system for bacterial cell polarity that incorporates a nucleotide exchange factor as well as an NTPase activating protein for regulation of a nucleotide-dependent molecular switch and demonstrate a spatial organization that is conserved in eukaryotes.

A new baseline for countrywide α-diversity and species distributions: illustration using >6,000 plant species in Panama.

Estimating α-diversity and species distributions provides baseline information to understand factors such as biodiversity loss and erosion of ecosystem services. Yet, species surveys typically cover a small portion of any country's landmass. Public, global databases could help, but contain biases. Thus, the magnitude of bias should be identified and ameliorated, the value of integration determined, and application to current policy issues illustrated. The ideal integrative approach should be powerful, flexible, efficient, and conceptually straightforward. We estimated distributions for >6,000 species, integrating species sightings (S) from the Global Biodiversity Information Facility (GBIF), systematic survey data (S2 ), and "bias-adjustment kernels" (BaK) using spatial and species trait databases (S2 BaK). We validated our approach using both locational and species holdout sets, and then applied our predictive model to Panama. Using sightings alone (the most common approach) discriminated relative probabilities of occurrences well (area under the curve [AUC] = 0.88), but underestimated actual probabilities by ~4,000%, while using survey data alone omitted over three-quarters of the >6,000 species. Comparatively, S2 BaK had no systematic underestimation, and substantially stronger discrimination (AUC = 0.96) and predictive power (deviance explained = 47%). Our model suggested high diversity (~200% countrywide mean) where urban development is projected to occur (the Panama Canal watershed) and also suggested this is not due to higher sampling intensity. However, portions of the Caribbean coast and eastern Panama (the Darién Gap) were even higher, both for total plant biodiversity (~250% countrywide mean), and CITES listed species. Finally, indigenous territories appeared half as diverse as other regions, based on survey observations. However, our model suggested this was largely due to site selection, and that richness in and out of indigenous territories was roughly equal. In brief, we provide arguably the best estimate of countrywide plant α-diversity and species distributions in the Neotropics, and make >6,000 species distributions available. We identify regions of overlap between development and high biodiversity, and improve interpretation of biodiversity patterns, including for policy-relevant CITES species, and locations with limited access (i.e., indigenous territories). We derive a powerful, flexible, efficient and simple estimation approach for biodiversity science.

Control of hexuronate metabolism in Escherichia coli by the two interdependent regulators, ExuR and UxuR: derepression by heterodimer formation.

Two homologous proteins, UxuR and ExuR, were previously predicted to repress synthesis of enzymes required for hexuronic acid metabolism, but little is known about the relative roles of these proteins in gene regulation. We confirmed the previous report that UxuR is essential for rapid growth with d-glucuronate as the primary source of carbon and energy. In contrast, an exuR mutant grew more rapidly on d-glucuronate than the parent. Transcription of exuR is initiated at a σ70-dependent promoter predicted in silico. Purified ExuR bound to the exuR regulatory region in the presence, but not in the absence, of d-glucuronate. Apparently weaker UxuR binding in the presence of glucuronate was also detected, and its addition decreased ExuR binding by forming ExuR-UxuR heterodimers. Glucuronate induced exuR transcription in the parental strain, but not in the exuR mutant. No evidence was obtained for cAMP-dependent regulation of exuR by the catabolite repressor protein (CRP). A previous study reported that the divergent yjjM and yjjN genes, essential for l-galactonate metabolism, are repressed by UxuR. We showed that ExuR binds to the yjjM-yjjN regulatory region, and that the binding is also glucuronate-dependent. As for the exuR promoter, UxuR appeared to decrease ExuR binding. ExuR is required for glucuronate induction of yjjM and yjjN, and CRP is required for their transcription. The combined data established that UxuR and ExuR fulfil contrasting roles in regulating hexuronic acid metabolism and indicate that ExuR can function as a transcription activator, possibly by inactivating the repressor function of UxuR by heterodimer formation.

Structural modeling of the ExuR and UxuR transcription factors of E. coli: search for the ligands affecting their regulatory properties.

Gammaproteobacteria get energy for their growth from different carbon sources using either glycolysis or alternative metabolic pathways induced in stress conditions. These metabolic switches are coordinated by complex interplay of regulatory proteins sensing concentrations of available metabolites by mechanisms yet to be understood. Here, we use two transcriptional regulators, ExuR and UxuR, controlling d-galacturonate (d-gal) and d-glucuronate metabolism in Escherichia coli, as the targets for computational search of low-molecular compounds capable to bind their ligand-binding domains. Using a flexible molecular docking, we modeled the interactions of these proteins with substrates and intermediates of glycolysis, Ashwell and Entner-Doudoroff pathways. For UxuR, the two preferred sites of ligand binding were found: one is located within the C-terminal domain, while another occupies the interdomain space. For ExuR, the only one preferred site was detected in the interdomain area. Availability of this area to different ligands suggests that, similar to the Lac repressor, the DNA-binding properties of UxuR and ExuR may be changed by repositioning of their domains. Experimental assays confirmed the ability of ligands with highest affinities to bind the regulatory proteins and affect their interaction with DNA. d-gal that is carried into the cell by the ExuT transporter appeared to be the best ligand for repressor of the exuT transcription, ExuR. For UxuR, the highest affinity was found for d-fructuronate transported by GntP, which biosynthesis is repressed by UxuR. Providing a feedback loop to balance the concentrations of different nutrients, such ligand-mediated modulation can also coordinate switching between different metabolic pathways in bacteria.

A Direct in vivo RNAi screen identifies MKK4 as a key regulator of liver regeneration.

The liver harbors a distinct capacity for endogenous regeneration; however, liver regeneration is often impaired in disease and therefore insufficient to compensate for the loss of hepatocytes and organ function. Here we describe a functional genetic approach for the identification of gene targets that can be exploited to increase the regenerative capacity of hepatocytes. Pools of small hairpin RNAs (shRNAs) were directly and stably delivered into mouse livers to screen for genes modulating liver regeneration. Our studies identify the dual-specific kinase MKK4 as a master regulator of liver regeneration. MKK4 silencing robustly increased the regenerative capacity of hepatocytes in mouse models of liver regeneration and acute and chronic liver failure. Mechanistically, induction of MKK7 and a JNK1-dependent activation of the AP1 transcription factor ATF2 and the Ets factor ELK1 are crucial for increased regeneration of hepatocytes with MKK4 silencing.

Senescence surveillance of pre-malignant hepatocytes limits liver cancer development.

Upon the aberrant activation of oncogenes, normal cells can enter the cellular senescence program, a state of stable cell-cycle arrest, which represents an important barrier against tumour development in vivo. Senescent cells communicate with their environment by secreting various cytokines and growth factors, and it was reported that this 'secretory phenotype' can have pro- as well as anti-tumorigenic effects. Here we show that oncogene-induced senescence occurs in otherwise normal murine hepatocytes in vivo. Pre-malignant senescent hepatocytes secrete chemo- and cytokines and are subject to immune-mediated clearance (designated as 'senescence surveillance'), which depends on an intact CD4(+) T-cell-mediated adaptive immune response. Impaired immune surveillance of pre-malignant senescent hepatocytes results in the development of murine hepatocellular carcinomas (HCCs), thus showing that senescence surveillance is important for tumour suppression in vivo. In accordance with these observations, ras-specific Th1 lymphocytes could be detected in mice, in which oncogene-induced senescence had been triggered by hepatic expression of Nras(G12V). We also found that CD4(+) T cells require monocytes/macrophages to execute the clearance of senescent hepatocytes. Our study indicates that senescence surveillance represents an important extrinsic component of the senescence anti-tumour barrier, and illustrates how the cellular senescence program is involved in tumour immune surveillance by mounting specific immune responses against antigens expressed in pre-malignant senescent cells.

Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution.

BACKGROUND: New high-throughput sequencing technologies promise a very sensitive and high-resolution analysis of DNA methylation patterns in quantitative terms. However, a detailed and comprehensive comparison with existing validated DNA methylation analysis methods is not yet available. Therefore, a systematic cross-validation of 454 sequencing and conventional pyrosequencing, both of which offer exact quantification of methylation levels with a single CpG dinucleotide resolution, was performed. RESULTS: To this end the methylation patterns of 12 loci (GSTπ1, p16INK4a, RASSF1A, SOCS1, MAL, hsa-mir-1-1, hsa-mir-9-3, hsa-mir-34a, hsa-mir-596, hsa-mir-663, MINT31, and LINE-1) were analyzed in ten primary hepatocellular carcinoma specimens. After applying stringent quality control criteria, 35749 sequences entered further analysis. The methylation level of individual CpG dinucleotides obtained by 454 sequencing was systematically compared with the corresponding values obtained by conventional pyrosequencing. Statistical analyses revealed an excellent concordance of methylation levels for all individual CpG dinucleotides under study (r2 = 0.927). CONCLUSIONS: Our results confirm that 454 sequencing of bisulfite treated genomic DNA provides reliable high quality quantitative methylation data and identify MAL, hsa-mir-9-3, hsa-mir-596, and hsa-mir-663 as new targets of aberrant DNA methylation in human hepatocellular carcinoma. In addition, the single molecule resolution of 454 sequencing provides unprecedented information about the details of DNA methylation pattern heterogeneity in clinical samples.

Global reactivation of epigenetically silenced genes in prostate cancer.

Transcriptional silencing associated with aberrant promoter hypermethylation is a common mechanism of inactivation of tumor suppressor genes in cancer cells. To globally profile the genes silenced by hypermethylation in prostate cancer, we screened a whole genome expression microarray for genes reactivated in the LNCaP, DU-145, PC-3, and MDA2b prostate tumor cell lines after treatment with the demethylating drug 5-aza-2-deoxycytidine and the histone deacetylation-inhibiting drug trichostatin A. A total of 2,997 genes showed at least 2-fold upregulation of expression after drug treatment in at least one prostate tumor cell line. For validation, we examined the first 45 genes, ranked by upregulation of expression, which had a typical CpG island and were known to be expressed in the normal cell counterpart. Two important findings were, first, that several genes known to be frequently hypermethylated in prostate cancer were apparent, and, second, that validation studies revealed eight novel genes hypermethylated in the prostate tumor cell lines, four of which were unmethylated in normal prostate cells and hypermethylated in primary prostate tumors (SLC15A3, 66%; KRT7, 54%; TACSTD2, 17%; GADD45b, 3%). Thus, we established the utility of our screen for genes hypermethylated in prostate cancer cells. One of the novel genes was TACSTD2/TROP2, a marker of human prostate basal cells with stem cell characteristics. TACSTD2 was unmethylated in prostatic intraepithelial neoplasia and may have utility in emerging methylation-based prostate cancer tests. Further study of the hypermethylome will provide insight into the biology of the disease and facilitate translational studies in prostate cancer.

Epigenetic inactivation of tumour suppressor gene KLF11 in myelodysplastic syndromes*.

The identification of aberrantly hypermethylated genes may lead to the development of new diagnostic markers and the identification of novel targets of epigenetic therapy in myelodysplastic syndromes (MDS). We therefore investigated the methylation status of transcription factor genes KLF5, KLF11, and MAFB, shown to be aberrantly methylated in myelogeneous leukaemia cells, in a series of 115 MDS patient as well as in 25 control subjects. Using quantitative high-resolution pyrosequencing methodology, KLF11, MAFB, and KLF5 were shown for the first time to be hypermethylated in 17 (15%), 8 (7%), and 2 (1.7%) cases, respectively, but not in any of the patients with an isolated 5q-deletion. Patient samples harbouring KLF11 methylation displayed reduced KLF11 mRNA expression and KLF11 hypermethylation correlated with a high International Prognostic Scoring System score (P < 0.05). In conclusion, epigenetic inactivation and subsequent transcriptional repression of the KLF11 gene is quite frequent in MDS. Patients with an isolated 5q-deletion seem to harbour a distinct epigenetic profile.

Promoter hypermethylation of the PALB2 susceptibility gene in inherited and sporadic breast and ovarian cancer.

The partner and localizer of BRCA2 (PALB2) gene was recently identified as a BRCA2-interacting protein and subsequently shown to be a Fanconi anemia gene (FANCN). Disease-associated point mutations resulting in protein truncation have been found in BRCA1/2 mutation-negative breast cancer families identifying PALB2 as a susceptibility gene for breast cancer. Aberrant promoter hypermethylation is a mechanism of inactivation of many tumor suppressor genes, including BRCA1 and p16(INK4a), in breast and ovarian cancer. We therefore investigated the methylation status of a 1512 bp typical CpG island located in the promoter and exon 1 region of the PALB2 gene in 130 sporadic and familial breast and ovarian primary tumors, 9 cell lines, and 10 normal cell specimens. We found two primary breast tumors from BRCA2 mutation carriers, four sporadic primary breast tumors, and four sporadic primary ovarian tumors showed hypermethylation of the core promoter region of PALB2. All 10 normal tissue DNA had an unmethylated PALB2 promoter region. Quantitative real-time reverse transcription-PCR showed PALB2 expression to be reduced 28-fold in primary breast tumor with PALB2 promoter hypermethylation compared with matched normal breast tissue RNA. Aberrant promoter hypermethylation of PALB2 is more frequent than the reported level of PALB2 point mutations in breast tumors from BRCA1/2-negative families and is similar to the frequency of BRCA1 hypermethylation in inherited and sporadic breast and ovarian cancers.