The Clean Water Act: Financing Combined Sewer Overflow Projects

In 1987 Congress expanded the scope of the Clean Water Act to include combined sewer overflows (CSOs) despite continuing to reduce federal assistance for water-pollution abatement and despite the fact that CSO abatement is far more costly than previous water-quality mandates. As a result, many low-income deindustrializing cities are now subject to an additional federal mandate that many of them cannot afford without extensive federal or state assistance. The authors conclude that, in lieu of increased federal funding for CSO abatement, U.S. Environmental Protection Agency regulatory guidelines and the Clean Water Act be amended to include an assessment of the fiscal and economic impact of CSO mandates. Such action would provide a basis for targeting the available resources where needs are greatest and the effect of CSO abatement is likely to result in tangible beneficial uses

Crystal structure of Bacillus anthracis dihydrofolate reductase with the dihydrophthalazine-based trimethoprim derivative RAB1 provides a structural explanation of potency and selectivity

Bacillus anthracis possesses an innate resistance to the antibiotic trimethoprim due to poor binding to dihydrofolate reductase (DHFR); currently, there are no commercial antibacterials that target this enzyme in B. anthracis. We have previously reported a series of dihydrophthalazine-based trimethoprim derivatives that are inhibitors for this target. In the present work, we have synthesized one compound (RAB1) displaying favorable 50% inhibitory concentration (54 nM) and MIC (</=12.8 ug/ml) values. RAB1 was cocrystallized with the B. anthracis DHFR in the space group P212121, and X-ray diffraction data were collected to a 2.3-A resolution. Binding of RAB1 causes a conformational change of the side chain of Arg58 and Met37 to accommodate the dihydrophthalazine moiety. Unlike the natural substrate or trimethoprim, the dihydrophthalazine group provides a large hydrophobic anchor that embeds within the DHFR active site and accounts for its selective inhibitory activity against B. anthracis.Peer reviewedVeterinary PathobiologyChemistr

Building programmable multicompartment artificial cells incorporating remotely activated protein channels using microfluidics and acoustic levitation

Abstract: Intracellular compartments are functional units that support the metabolism within living cells, through spatiotemporal regulation of chemical reactions and biological processes. Consequently, as a step forward in the bottom-up creation of artificial cells, building analogous intracellular architectures is essential for the expansion of cell-mimicking functionality. Herein, we report the development of a droplet laboratory platform to engineer complex emulsion-based, multicompartment artificial cells, using microfluidics and acoustic levitation. Such levitated models provide free-standing, dynamic, definable droplet networks for the compartmentalisation of chemical species. Equally, they can be remotely operated with pneumatic, heating, and magnetic elements for post-processing, including the incorporation of membrane proteins; alpha-hemolysin; and mechanosensitive channel of large-conductance. The assembly of droplet networks is three-dimensionally patterned with fluidic input configurations determining droplet contents and connectivity, whilst acoustic manipulation can be harnessed to reconfigure the droplet network in situ. The mechanosensitive channel can be repeatedly activated and deactivated in the levitated artificial cell by the application of acoustic and magnetic fields to modulate membrane tension on demand. This offers possibilities beyond one-time chemically mediated activation to provide repeated, non-contact, control of membrane protein function. Collectively, this expands our growing capability to program and operate increasingly sophisticated artificial cells as life-like materials

An Analysis of Private School Closings

We add to the small literature on private school supply by exploring exits of K-12 private schools. We find that the closure of private schools is not an infrequent event, and use national survey data from the National Center for Education Statistics to study closures of private schools. We assume that the probability of an exit is a function of excess supply of private schools over the demand, as well as the school's characteristics such as age, size, and religious affiliation. Our empirical results generally support the implications of the model. Working Paper 07-0

Determination of the Deep Inelastic Contribution to the Generalised Gerasimov-Drell-Hearn Integral for the Proton and Neutron

The virtual photon absorption cross section differences [sigma_1/2-sigma_3/2] for the proton and neutron have been determined from measurements of polarised cross section asymmetries in deep inelastic scattering of 27.5 GeV longitudinally polarised positrons from polarised 1H and 3He internal gas targets. The data were collected in the region above the nucleon resonances in the kinematic range nu < 23.5 GeV and 0.8 GeV**2 < Q**2 < 12 GeV**2. For the proton the contribution to the generalised Gerasimov-Drell-Hearn integral was found to be substantial and must be included for an accurate determination of the full integral. Furthermore the data are consistent with a QCD next-to-leading order fit based on previous deep inelastic scattering data. Therefore higher twist effects do not appear significant.Comment: 6 pages, 3 figures, 1 table, revte

Effect of HLA DR epitope de-immunization of Factor VIII \u3ci\u3ein vitro\u3c/i\u3e and \u3ci\u3ein vivo\u3c/i\u3e

T cell-dependent development of anti-Factor VIII (FVIII) antibodies that neutralize FVIII activity is a major obstacle to replacement therapy in hemophilia A. To create a less immunogenic therapeutic protein, recombinant FVIII can be modified to reduce HLA binding of epitopes based on predicted anchoring residues. Here, we used immunoinformatic tools to identify C2 domain HLA DR epitopes and predict site-specific mutations that reduce immunogenicity. Epitope peptides corresponding to original and modified sequences were validated in HLA binding assays and in immunizations of hemophilic E16 mice, DR3 and DR4 mice and DR3 × E16 mice. Consistent with immunoinformatic predictions, original epitopes are immunogenic. Immunization with selected modified sequences lowered immunogenicity for particular peptides and revealed residual immunogenicity of incompletely de-immunized modified peptides. The stepwise approach to reduce protein immunogenicity by epitope modification illustrated here is being used to design and produce a functional full-length modified FVIII for clinical use

Bromodomain protein BRD4 is a transcriptional repressor of autophagy and lysosomal function

Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation

Intraspecies Variation in the Emergence of Hyperinfectious Bacterial Strains in Nature

Salmonella is a principal health concern because of its endemic prevalence in food and water supplies, the rise in incidence of multi-drug resistant strains, and the emergence of new strains associated with increased disease severity. Insights into pathogen emergence have come from animal-passage studies wherein virulence is often increased during infection. However, these studies did not address the prospect that a select subset of strains undergo a pronounced increase in virulence during the infective process- a prospect that has significant implications for human and animal health. Our findings indicate that the capacity to become hypervirulent (100-fold decreased LD50) was much more evident in certain S. enterica strains than others. Hyperinfectious salmonellae were among the most virulent of this species; restricted to certain serotypes; and more capable of killing vaccinated animals. Such strains exhibited rapid (and rapidly reversible) switching to a less-virulent state accompanied by more competitive growth ex vivo that may contribute to maintenance in nature. The hypervirulent phenotype was associated with increased microbial pathogenicity (colonization; cytotoxin production; cytocidal activity), coupled with an altered innate immune cytokine response within infected cells (IFN-β; IL-1β; IL-6; IL-10). Gene expression analysis revealed that hyperinfectious strains display altered transcription of genes within the PhoP/PhoQ, PhoR/PhoB and ArgR regulons, conferring changes in the expression of classical virulence functions (e.g., SPI-1; SPI-2 effectors) and those involved in cellular physiology/metabolism (nutrient/acid stress). As hyperinfectious strains pose a potential risk to human and animal health, efforts toward mitigation of these potential food-borne contaminants may avert negative public health impacts and industry-associated losses