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Urban slums are proliferating in the developing countries. A corollary of this structural transformation is the increasing recognition of an urban penalty wherein slum populations exhibit notable inequalities in health relative to non-slum urban residents and even rural populations. The built urban environment, in turn, is a crucial context within which the social production of disproportionate morbidity and mortality is enacted. The authors develop this assertion and use bivariate and partial correlation analysis to highlight the association of urban slum prevalence, or proportion of the total population living in urban slum conditions, with indicators of mortality and gender parity, measured at the national level. Data for 99 developing countries show that greater urban slum prevalence is strongly correlated with higher levels of infant, child, and maternal mortality. Further, urban slum prevalence exhibits strong, deleterious correlations with gender parity (measured by the gender development index) and fertility rate, factors that have a crucial direct impact in shaping variant mortality levels. Future research is warranted on the social inequalities in health and illness derived from the expansion of urban slum conditions in the developing countries. [ABSTRACT FROM AUTHOR]

This case study describes the innovative and unique U.S. Army Wounded Warrior Program (AW2), which provides nonmedical case management to the most severely wounded, injured, and ill soldiers and their families. The study describes the program and identifies the features for a successful nonmedical case management program of an identified population who has complex medical needs. Although the article focuses primarily on the role of the AW2 advocate, key components of the program are discussed, including successful initiatives as well as areas that required adjustment. The lessons learned are identified as well as recommendations for future nonmedical case management initiatives. [ABSTRACT FROM AUTHOR]

DNA- based vaccines have demonstrated the potential as a safe and effective modality. PlaCCine, a DNA-based vaccine approach described subsequently relies on a synthetic DNA delivery system and is independent of virus or device. The synthetic functionalized polymer combined with DNA demonstrated stability over 12 months at 4C and for one month at 25C. Transfection efficiency compared to naked DNA increased by 5-15-fold in murine skeletal muscle. Studies of DNA vaccines expressing spike proteins from variants D614G (pVAC15), Delta (pVAC16), or a D614G + Delta combination (pVAC17) were conducted. Mice immunized intramuscular injection (IM) with pVAC15, pVAC16 or pVAC17 formulated with functionalized polymer and adjuvant resulted in induction of spike-specific humoral and cellular responses. Antibody responses were observed after one immunization. And endpoint IgG titers increased to greater than 1x 10 5 two weeks after the second injection. Neutralizing antibodies as determined by a pseudovirus competition assay were observed following vaccination with pVAC15, pVAC16 or pVAC17. Spike specific T cell immune responses were also observed following vaccination and flow cytometry analysis demonstrated the cellular immune responses included both CD4 and CD8 spike specific T cells. The immune responses in vaccinated mice were maintained for up to 14 months after vaccination. In an immunization and challenge study of K18 hACE2 transgenic mice pVAC15, pVAC16 and pVAC17 induced immune responses lead to decreased lung viral loads by greater than 90 % along with improved clinical score. These findings suggest that PlaCCine DNA vaccines are effective and stable and further development against emerging SARS-CoV-2 variants is warranted., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Subeena Sood, Majed M. Matar, Jessica Kim, Meredyth Kinsella, Kempaiah Rayavara, Olivia Signer, John Henderson, Joseph Rogers, Austin Arnold, Jennifer S. Rice, Alanna M. Smith, Daishui Sui, Jeff Sparks, Corinne Le Goff, Jean D. Boyer and Khursheed Anwer report other support from Imunon Corporation during the conduct of the study. No disclosures were reported by the other authors.]., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Climate change, energy system transitions, and socioeconomic change are compounding influences affecting the growth of electricity demand. While energy efficiency initiatives and distributed resources can address a significant amount of this demand, the United States will likely still need new utility-scale generation resources. The energy sector uses capacity expansion planning models to determine the aggregate need for new generation, but these models are typically at the state or regional scale and are not equipped to address the wide range of location- and technology-specific issues that are increasingly a factor in power plant siting. To help address these challenges, we have developed the Geospatial Raster Input Data for Capacity Expansion Regional Feasibility (GRIDCERF) data package, a high-resolution product to evaluate siting suitability for renewable and non-renewable power plants in the conterminous United States. GRIDCERF offers 264 suitability layers for use with 56 power plant technologies in a harmonized format that can be easily ingested by geospatially-enabled modeling software allowing for customization to robustly address science objectives when evaluating varying future conditions., (© 2023. The Author(s).)

Regional climate models can be used to examine how past weather events might unfold under different climate conditions by simulating analogue versions of those events with modified thermodynamic conditions (i.e., warming signals). Here, we apply this approach by dynamically downscaling a 40-year sequence of past weather from 1980-2019 driven by atmospheric re-analysis, and then repeating this 40-year sequence a total of 8 times using a range of time-evolving thermodynamic warming signals that follow 4 80-year future warming trajectories from 2020-2099. Warming signals follow two emission scenarios (SSP585 and SSP245) and are derived from two groups of global climate models based on whether they exhibit relatively high or low climate sensitivity. The resulting dataset, which contains 25 hourly and over 200 3-hourly variables at 12 km spatial resolution, can be used to examine a plausible range of future climate conditions in direct reference to previously observed weather and enables a systematic exploration of the ways in which thermodynamic change influences the characteristics of historical extreme events., (© 2023. Springer Nature Limited.)

Land surface models such as the Community Land Model Version 5 (CLM5) are essential tools for simulating the behavior of the terrestrial system. Despite the extensive application of CLM5, limited attention has been paid to the underlying uncertainties associated with its hydrological parameters and how these uncertainties affect water resource applications. To address this long-standing issue, we use five meteorological datasets to conduct a comprehensive hydrological parameter uncertainty characterization of CLM5 over the hydroclimatic gradients of the conterminous United States. Key datasets produced from the uncertainty characterization experiment include: a benchmark dataset of CLM5 default hydrological performance, parameter sensitivities for 28 hydrological metrics, and large-ensemble outputs for CLM5 hydrological predictions. The presented datasets will assist CLM5 calibration and support broad applications, such as evaluating drought and flood vulnerabilities. The datasets can be used to identify the hydroclimatological conditions under which parametric uncertainties demonstrate substantial effects on hydrological predictions and clarify where further investigations are needed to understand how hydrological prediction uncertainties interact with other Earth system processes., (© 2023. Battelle Memorial Institute.)

Shelter-in-place orders and business closures related to COVID-19 changed the hourly profile of electricity demand and created an unprecedented source of uncertainty for the grid. The potential for continued shifts in electricity profiles has implications for electricity sector investment and operating decisions that maintain reserve margins and provide grid reliability. This study reveals that understanding this uncertainty requires an understanding of the underlying drivers at the customer-class scale. This paper utilizes three datasets to compare the impacts of COVID-19 on electricity consumption across a range of spatiotemporal and customer scales. At the utility/customer-class scale, COVID-19-induced shutdowns in the spring of 2020 shifted weekday residential load profiles to resemble weekend profiles from previous years. Total commercial loads declined, but the commercial diurnal load profile was unchanged. With only total loads available at the balancing authority scale, the apparent impact of COVID-19 was smaller during the summer due in part to phased re-opening and spatial variability in re-opening, but there were still clear variations once total loads were broken down zonally. Monthly data at the state scale showed an increase in state-level residential electricity sales, a decrease in commercial sales, and a small net decrease in total sales in most states from April-August 2020. Analyses that focus on total load or a single scale may miss important changes that become apparent when the load is broken down regionally or by customer class., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Elsevier Ltd.)

Drinking water supplies of cities are exposed to potential contamination arising from land use and other anthropogenic activities in local and distal source watersheds. Because water quality sampling surveys are often piecemeal, regionally inconsistent, and incomplete with respect to unregulated contaminants, the United States lacks a detailed comparison of potential source water contamination across all of its large cities. Here we combine national-scale geospatial datasets with hydrologic simulations to compute two metrics representing potential contamination of water supplies from point and nonpoint sources for over a hundred U.S. cities. We reveal enormous diversity in anthropogenic activities across watersheds with corresponding disparities in the potential contamination of drinking water supplies to cities. Approximately 5% of large cities rely on water that is composed primarily of runoff from non-pristine lands (e.g., agriculture, residential, industrial), while four-fifths of all large cities that withdraw surface water are exposed to treated wastewater in their supplies., (© 2021. Battelle Memorial Institute.)

There is evidence that warming leads to greater evapotranspiration and surface drying, thus contributing to increasing intensity and duration of drought and implying that mitigation would reduce water stresses. However, understanding the overall impact of climate change mitigation on water resources requires accounting for the second part of the equation, i.e., the impact of mitigation-induced changes in water demands from human activities. By using integrated, high-resolution models of human and natural system processes to understand potential synergies and/or constraints within the climate-energy-water nexus, we show that in the United States, over the course of the 21st century and under one set of consistent socioeconomics, the reductions in water stress from slower rates of climate change resulting from emission mitigation are overwhelmed by the increased water stress from the emissions mitigation itself. The finding that the human dimension outpaces the benefits from mitigating climate change is contradictory to the general perception that climate change mitigation improves water conditions. This research shows the potential for unintended and negative consequences of climate change mitigation.

Combination antiretroviral therapy can suppress human immunodeficiency virus (HIV) infection but cannot completely eradicate the virus. A major obstacle in the quest for a cure is the difficulty in targeting and measuring latently infected cells. To date, a single person seems to have been cured of HIV. Hematopoietic stem cell transplantation (HSCT) preceded this cancer patient's long-term sustained HIV remission, but researchers have been unable to replicate this cure, and the mechanisms that led to HIV remission remain to be established. In February 2014, the National Institute of Allergy and Infectious Diseases sponsored a workshop that provided a venue for in-depth discussion of whether HSCT could be exploited to cure HIV in cancer patients requiring such procedures. Participants also discussed how HSCT might be applied to a broader community of HIV-infected persons in whom the risks of HSCT currently outweigh the likelihood and benefits of HIV cure., (Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Accurate assessment of shark population status is essential for conservation but is often constrained by limited and unreliable data. To provide a basis for improved management of shark resources, we analyzed a long-term record of species-specific catches, sizes, and sexes of sharks collected by onboard observers in the western and central Pacific Ocean from 1995 to 2010. Using generalized linear models, we estimated population-status indicators on the basis of catch rate and biological indicators of fishing pressure on the basis of median size to identify trends for blue (Prionace glauca), mako (Isurus spp.), oceanic whitetip (Carcharhinus longimanus), and silky (Carcharhinus falciformis) sharks. Standardized catch rates of longline fleets declined significantly for blue sharks in the North Pacific (by 5% per year [CI 2% to 8%]), for mako sharks in the North Pacific (by 7% per year [CI 3% to 11%]), and for oceanic whitetip sharks in tropical waters (by 17% per year [CI 14% to 20%]). Median lengths of silky and oceanic whitetip sharks decreased significantly in their core habitat, and almost all sampled silky sharks were immature. Our results are consistent with results of analyses of similar data sets. Combined, these results and evidence of targeted fishing for sharks in some regional fisheries heighten concerns for sustainable utilization, particularly for oceanic whitetip and North Pacific blue sharks. Regional regulations that prohibit shark finning (removal of fins and discarding of the carcass) were enacted in 2007 and are in many cases the only form of control on shark catches. However, there is little evidence of a reduction of finning in longline fisheries. In addition, silky and oceanic whitetip sharks are more frequently retained than finned, which suggests that even full implementation of and adherence to a finning prohibition may not substantially reduce mortality rates for these species. We argue that finning prohibitions divert attention from assessing whether catch levels are sustainable and that the need for management of sharks should not be addressed by measures that are simple to implement but complex to enforce and evaluate., (© 2012 Society for Conservation Biology.)

How self tolerance is maintained during B cell development in the bone marrow has been a focal area of study in immunology. Receptor editing, anergy and clonal deletion all play important roles in the regulation of autoimmunity in the immature population. The mechanisms of tolerance induction in the periphery, however, are less well characterized. Overexpression of the apoptosis inhibitor Bcl-2 rescues autoreactive B cells from deletion and can contribute to the development of autoimmune disease in certain genetic backgrounds. Using a peptide-induced autoimmunity model, we recently identified a peripheral tolerance checkpoint in antigen-activated B cells that have undergone class switching and somatic hypermutation. At this checkpoint, receptor editing, induced by antigen engagement, dampened the autoantibody response. In this study, we show that receptor editing fails to be induced in antigen-activated DNA-reactive B cells that overexpress Bcl-2 (Bcl-2 Tg). The failure to induce RAG and receptor editing is likely due, at least partially, to the lack of self antigen. First, the levels of circulating DNA and of apoptotic bodies in the spleen of Bcl-2 Tg mice are significantly lower than in control mice. Second, in Bcl-2 Tg mice, RAG can be induced in a population of antigen-activated B cells by providing exogenous soluble antigen. These data suggest that, in addition to its anti-apoptotic activity, Bcl-2 may indirectly inhibit tolerance induction in B cells acquiring anti-nuclear antigen reactivity after peripheral activation by limiting the availability of self antigen., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Biotoxin activity assays typically involve multistep sample preparation, multicomponent reactions, multistep analysis, or a combination thereof. We report a single-step, real-time ricin activity assay that requires little or no sample preparation and employs direct analysis in real time mass spectrometry. The release of adenine from the inhomogeneous substrate herring sperm DNA by ricin was determined to be 53 +/- 2 pmol adenine per picomole of ricin per hour. This procedure can be readily adapted to any enzyme for which a reactant or product of low molecular weight (up to approximately 600) can be identified.

Background: The poor prognosis associated with ovarian cancer is primarily the result of delayed diagnosis and the lack of an effective treatment for advanced disease. Use of novel immunotherapy strategies are being evaluated that work to enhance local and systemic immune responses against cancer cells and can possibly work together with traditional cytotoxic chemotherapy regimens to produce more effective treatment options., Methods: In the present study, we describe a gene-based therapy whereby the anticancer cytokine interleukin-12 gene (pmIL-12) is formulated with a synthetic polymeric delivery vehicle (PPC) and administered intraperitoneally into a mouse model of disseminated ovarian cancer., Results: The administration of pmIL-12/PPC in tumor-bearing mice was associated with a shift towards a Th1 immune state, including significant increases in murine IL-12 (mIL-12) and interferon (IFN)-gamma (mIFN-gamma) in ascites fluid, with little change in systemic levels of these proteins. The mIL-12 protein was detectable for several days and could be reintroduced with subsequent injections. We show that treatment delayed the onset of ascites formation and improved survival in a dose-dependent manner. A significant decrease in vascular endothelial growth factor was associated with pmIL-12/PPC delivery and believed to play a predominant role in inhibiting ascites accumulation. Administration of pmIL-12/PPC was associated with minimal toxicity and, when combined with standard chemotherapies, resulted in additive improvement in survival., Conclusions: Taken together, these results suggest that pmIL-12/PPC may be an effective strategy for inhibiting progression of disseminated ovarian cancer and may offer a new option for treatment of advanced disease that can be used to complement standard therapies.

Numerous autoimmune and inflammatory disorders stem from the dysregulation of hematopoietic cell activation. The activity of inositol lipid and protein tyrosine phosphatases, and the receptors that recruit them, is critical for prevention of these disorders. Balanced signaling by inhibitory and activating receptors is now recognized to be an important factor in tuning cell function and inflammatory potential. In this review, we provide an overview of current knowledge of membrane proximal events in signaling by inhibitory/regulatory receptors focusing on structural and functional characteristics of receptors and their effectors Src homology 2 (SH2) domain-containing tyrosine phosphatase 1 and SH2 domain-containing inositol 5-phosphatase-1. We review use of new strategies to identify novel regulatory receptors and effectors. Finally, we discuss complementary actions of paired inhibitory and activating receptors, using Fc gammaRIIA and Fc gammaRIIB regulation human basophil activation as a prototype.

B cells and autoimmunity: cells of the immune system have the capacity to recognize/neutralize a myriad array of disease-causing pathogens, while simultaneously minimizing damage to self tissue. Obvious breakdowns in this ability to distinguish between self and non-self are evident in multiple forms of autoimmune disease, where B and T cells mount damaging attacks on cells and organs. B cells may directly damage tissue by producing pathogenic antibodies that bind self antigen, fix complement or form immune complexes. Recent evidence also suggests B cells indirectly induce autoimmunity by concentrating low avidity self antigen through the B cell receptor and presenting self-peptides to autoreactive T cells. B cells may also initiate autoimmunity when provided sufficient help from autoreactive T cells that have escaped deletion in the thymus. Here, we will review the role of anergy in maintenance of tolerance and how alterations in the normal balance of positive and negative signals may contribute to the development of autoimmune disease in mouse models and humans.

The products formed from the degradation of the blister agent sulfur mustard [bis(2-chloroethyl) sulfide] on concrete were identified using gas chromatography with mass spectrometry detection (GC/MSD), (1)H NMR, 2D (1)H-(13)C NMR and (13)C solid state magic angle spinning (SSMAS) NMR. In situ and extraction experiments were performed. Sulfur mustard was detected in the in situ (13)C SSMAS samples for 12 weeks, whereas less than 5% of the sulfur mustard was detected in extracts from the concrete monoliths after 8 days. Sulfonium ions and (2-chloroethylthio)ethyl ether (T) were observed on the in situ samples after a period of 12 weeks, whereas vinyl species and bis(2-chloroethyl) sulfoxide were observed in the extracts of the concrete monoliths within 24h. The differences between the extraction and the SSMAS data indicated that the sulfur mustard existed in the concrete in a non-extractable form prior to its degradation. Extraction methods alone were not sufficient to identify the products; methods to identify the presence of non-extractable degradation products were also required.

Receptor editing or secondary Ig gene rearrangement occurs in immature, autoreactive B cells to maintain self-tolerance. Here we show that nonspontaneously autoimmune mice immunized with a peptide mimetope of DNA develop peptide- and DNA-reactive antibodies. Antigen-specific B cells display a follicular B cell phenotype. As these cells move into the memory compartment, many express RAG protein and acquire expression of both kappa and lambda light chains. Thus, this study provides evidence for receptor editing occurring in a mature, antigen-activated B cell population. Because the receptor editing observed here occurred in an autoreactive response to antigen, it may function to maintain peripheral tolerance.

Although cells of the innate immune response have a variety of pattern recognition receptors that are triggered by blood classes of markers, a critical feature of the adaptive immune response is antigenic specificity. Yet it is becoming increasingly clear that the specificity of lymphocyte receptors admits of some laxity. Cross-reactivity may, in fact, be necessary for lymphocyte survival as antigen receptor signaling maintains cellular viability in the absence of antigen activation. Studies of molecular mimicry have revealed many instances in which antibodies to microbial antigens bind also to self-antigens; in some cases, this cross-reactivity has pathogenic potential. In this chapter, we describe cross-reactivity between two self-antigens, DNA and NMDA receptors, and how antibodies with specificity for DNA in patients with splenic lupus may cause central nervous system damage by virtue of binding also to neuronal receptors. This example serves as a reminder that cross-reactivity may exist among self-antigens as well as between foreign and self-antigens.

The solution structure of an 11-mer DNA duplex, d(CGGTCA*CGAGG) x d(CCTCGTGACCG), containing a 10R adduct at dA* that corresponds to the cis addition of the N(6)-amino group of dA(6) to (+)-(9S,10R)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene was studied by 2D NMR methods. The NOESY cross-peak patterns indicate that the hydrocarbon is intercalated on the 5'-side of the modified base. This observation is the same as that observed for other oligonucleotides containing (10R)-dA adducts but opposite to that observed for the corresponding (10S)-dA adducts which are intercalated on the 3'-side of the modified base. The hydrocarbon is intercalated from the major groove without significant disruption of either the anti glycosidic torsion angle of the modified residue or the base pairing of the modified residue with the complementary residue on the opposite strand. The ensemble of 10 structures determined exhibits relatively small variations (6-15 degrees) in the characteristic hydrocarbon-base dihedral angles (alpha' and beta') as well as the glycosidic torsion angle chi. These angles are similar to those in a previously determined cis-opened benzo[a]pyrene diol epoxide-(10R)-dA adduct structure. Comparison of the present structure with the cis-opened diol epoxide adduct suggests that the absence of the 7- and 8-hydroxyl groups results in more efficient stacking of the aromatic moiety with the flanking base pairs and deeper insertion of the hydrocarbon into the helix. Relative to normal B-DNA, the duplex containing the present tetrahydroepoxide adduct is unwound at the lesion site, whereas the diol epoxide adduct structure is more tightly wound than normal B-DNA. Buckling of the adducted base pair as well as the C(5)-G(18) base pair that lies immediately above the hydrocarbon is much less severe in the present adducted structure than its cis-opened diol epoxide counterpart.

The difficulty in characterizing antigen-specific B cells that arise in the native B cell repertoire has been a formidable obstacle to understanding both protective and pathogenic antibody responses. We have developed a tetramer-based technique for identifying antigen-specific B cells. Biotin-labeled antigen is made tetrameric by interaction with streptavidin. The enhanced avidity of this antigenic compound for the B cell membrane permits the visualization, characterization and isolation of antigen-specific B cells.

2D NMR has been used to examine the structure and dynamics of a 12-mer DNA duplex, d(T(1)A(2)G(3)T(4)C(5)A(6)A(7)G(8)G(9)G(10)C(11)A(12))-d(T(13)G(14)C( 15)C(16)C(17)T(18)T(19)G(20)A(21)C(22)T(23)A(24)), containing a 10R adduct at dA(7) that corresponds to trans addition of the N(6)-amino group of dA(7) to (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-(S,R,R,S)-BP DE-2]. This DNA duplex contains the base sequence for the major dA mutational hot spot in the HPRT gene when Chinese hamster V79 cells are given low doses of the highly carcinogenic (+)-(R,S,S,R)-BP DE-2 enantiomer. NOE data indicate that the hydrocarbon is intercalated on the 5'-side of the modified base as has been seen previously for other oligonucleotides containing BP DE-2 (10R)-dA adducts. 2D chemical exchange-only experiments indicate dynamic behavior near the intercalation site especially at the 10R adducted dA, such that this base interconverts between the normal anti conformation and a less populated syn conformation. Ab initio molecular orbital chemical shift calculations of nucleotide and dinucleotide fragments in the syn and anti conformations support these conclusions. Although this DNA duplex containing a 10R dA adduct exhibits conformational flexibility as described, it is nevertheless more conformationally stable than the corresponding 10S adducted duplex corresponding to trans opening of the carcinogenic isomer (+)-(R,S,S, R)-BP DE-2, which was too dynamic to permit NMR structure determination. UV and imino proton NMR spectral observations indicated pronounced differences between these two diastereomeric 12-mer duplexes, consistent with conformational disorder at the adduct site and/or an equilibrium with a nonintercalated orientation of the hydrocarbon in the duplex containing the 10S adduct. The existence of conformational flexibility around adducts may be related to the occurrence of multiple mutagenic outcomes resulting from a single DE adduct.

Worldwide, millions of people are exposed to arsenic in drinking water that exceeds the World Health Organization standard of 10 microg/liter by as much as 50-300-fold, yet little is known about the molecular basis for arsenic excretion. Here we show that transport of arsenic into bile depends on the MRP2/cMOAT transporter and that glutathione is obligatory for such transport. Using reversed phase liquid chromatography/mass spectrometry, we demonstrate that two arsenic-glutathione complexes not previously identified in vivo, arsenic triglutathione and methylarsenic diglutathione, account for most of the arsenic in the bile. The structure of the compounds was also confirmed by nuclear magnetic resonance spectroscopy. Our findings may help explain the increased susceptibility of malnourished human populations to arsenic.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants whose metabolism in mammals results in deleterious cell transformation. Covalent modification of DNA by diol epoxides metabolically formed from PAHs such a benzo[a]pyrene (BaP) provides a mechanism for the genotoxicity, mutagenicity, and carcinogenicity of PAHs. We had previously reported NMR evidence for a minor conformer of the duplex d(G1G2T3C4A5*C6G7A8G9).d(C10T11C12G13G14G15A16C17C18) containing a dG14 mismatch opposite a dA5* residue modified at the exocyclic amino group by trans addition to (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene [Yeh, H.J.C., Sayer, J.M., Liu, X., Altieri, A.S., Byrd, R.A., Lashman, M.K., Yagi, H., Schurer, E.J., Gorenstein, D.G., & Jerina, D.M. (1995) Biochemistry 34, 13570-13581]. In the present work, we describe the structure of this minor conformer (ca. 17% of the total conformer population). This represents the first structural determination of a minor conformer of a carcinogen-lesion DNA adduct. Two-dimensional NOESY, ROESY, TOCSY, and exchange-only spectra at 750 MHz allowed nearly complete sequential assignment of both conformers. In the minor conformer, the adducted base assumes an anti-glycosidic torsion angle whereas in the major conformer it assumes an unusual syn-glycosidic torsion angle. The aromatic hydrocarbon in the minor conformer is intercalated between dG13 and dG14, preserving the energetically favorable stacking interactions found in the major conformer. The major structural differences between the two conformers appear to be near the lesion site as evidenced by the large chemical shift differences between major and minor conformer protons near the lesion site; away from this site, the chemical shifts of the major and minor conformer protons are nearly identical. Because any of the conformations of benzo[a]pyrene diol epoxide-modified DNA may contribute to tumorigenic activity, structural determination of all conformations is essential for the elucidation of the mechanism of cell transformation initiated by covalent modification of DNA by PAHs.

Neutral and achiral backbone linkers are promising replacements for the phosphodiester linkages of antisense oligonucleotides that target mRNA sequences. Results are presented here for the solution structure elucidation by NMR of an RNA x DNA hybrid duplex, r(GCGCAAAACGCG) x d(CGCGTT-SCH2O-TTGCGC) (designated RIII), containing a 3'-thioformacetal (3'-TFMA) backbone substitution in the DNA strand. The 3'-thioformacetal linker can be accommodated in the hybrid duplex in a conformation that is drastically different from its form in a DNA x DNA duplex but close to that of the canonical A-form helix, reflecting the sequence requirement for hybridization. While the global features of RIII are similar to what are described in the literature, the 3'-TFMA modification drives sugar puckers of the adjacent residues to more C3'-endo-like conformations and causes distortions in related twist angles and helical rises. The helical conformation analyses of each of the two strands and the hybrid duplex enable a clear account of the conformational variability of both the DNA and RNA strands. The A-tract in the RNA strand features an overall straight helix and a more prominent bend at the 3'-end CG step. The structure of RIII provides a structural basis for the improved thermal stability of RIII compared to the corresponding DNA x DNA duplex and insights into the factors that are important concerns for the design of new, effective antisense oligonucleotides.

This study focuses on the characterization of the stability and conformation of the antisense oligodeoxyribonucleotides, d[CGCGTT x TTGCGC] [x = phosphodiester (-O-P(O)2-O-), formacetal (FMA, -O-CH2-O-), or 3'-thioformacetal linkage (TFMA, -S-CH2-O-)], in DNA.DNA and RNA.DNA duplexes (designated DI-III and RI-III, respectively). NMR analysis of two RNA.DNA hybrid duplexes containing a single FMA (the RII duplex) or 3'-TFMA (the RIII duplex) modification has been carried out. The conformations of these duplexes are compared with that of the unmodified hybrid duplex RI and with those of the DI-III duplexes. These analyses and comparisons indicate that the residue containing a 3'-FMA linker has a preference for the C2'-endo sugar pucker and adopts a canonical backbone conformation. In contrast, the residue containing a 3'-TFMA linker has a much increased preference for the C3'-endo sugar pucker and adopts different backbone conformations in the DNA.DNA and RNA.DNA duplexes. UV and NMR melting studies of the six duplexes demonstrate that the DNA.DNA duplexes are more stable than the corresponding RNA.DNA hybrid duplexes and that both FMA and 3'-TFMA destabilize the duplex. The 3'-TFMA modified duplex is less stable than the FMA duplex in the context of DNA.DNA recognition and is slightly more stable than the FMA hybrid duplex in the context of RNA.DNA recognition. These results suggest a correlation between the conformational preference of backbone modifications and the stability of antisense duplexes. The implications of these studies for optimized incorporation of FMA and 3'-TFMA linkers into oligonucleotides and for better design of antisense oligonucleotide analogs are discussed.

Neocarzinostatin chromophore (NCS chrom) belongs to a family of highly potent enediyne antitumor antibiotics which bind to specific DNA sequences and cause single- and/or double-strand lesions. NCS chrom-DNA complexes have eluded structural studies since the native form of the drug is extremely labile in aqueous conditions. We report the three-dimensional structure of the stable glutathione post-activated NCS chrom (NCSi-glu)-DNA complex [NCSi-glu-d(GGAGCGC).d(GCGCTCC)] using NMR and distance geometry-molecular dynamics simulation methods. NCSi-glu interacts with the GCTC tetranucleotide on one strand and with the AGC trinucleotide on the other strand through the unique intercalation at the 5'-CT/5'-AG step and minor groove binding. The DNA-drug complex exhibits an extended, unwound V-shaped intercalation site and wider and shallower grooves than the free DNA duplex. The structure of the complex manifests specific van der Waals interactions and H-bond formation between the carbohydrate moiety and a specific DNA sugar/phosphate. Prominent among those are the contacts of the NCSi-glu residues with the functional groups in the minor groove that are characteristic of individual DNA bases. These results provide a structural model for understanding the sequence specificity of the single- and double-strand cleavage at the AGC and related sites by the enediyne NCS chrom.

The present studies demonstrate that matrix Gla protein (MGP), a 10-kDa vitamin K-dependent protein, is phosphorylated at 3 serine residues near its N-terminus. Phosphoserine was identified at residues 3, 6, and 9 of bovine, human, rat, and lamb MGP by N-terminal protein sequencing. All 3 modified serines are in tandemly repeated Ser-X-Glu sequences. Two of the serines phosphorylated in shark MGP, residues 2 and 5, also have glutamate residues in the n + 2 position in tandemly repeated Ser-X-Glu sequences, whereas the third, shark residue 3, would acquire an acidic phosphoserine in the n + 2 position upon phosphorylation of serine 5. The recognition motif found for MGP phosphorylation, Ser-X-Glu/Ser(P), has been seen previously in milk caseins, salivary proteins, and a number of regulatory peptides. A review of the literature has revealed an intriguing dichotomy in the extent of serine phosphorylation among secreted proteins that are phosphorylated at Ser-X-Glu/Ser(P) sequences. Those phosphoproteins secreted into milk or saliva are fully phosphorylated at each target serine, whereas phosphoproteins secreted into the extracellular environment of cells are partially phosphorylated at target serine residues, as we show here for MGP and others have shown for regulatory peptides and the insulin-like growth factor binding protein 1. We propose that the extent of serine phosphorylation regulates the activity of proteins secreted into the extracellular environment of cells, and that partial phosphorylation can therefore be explained by the need to ensure that the phosphoprotein be poised to gain or lose activity with regulated changes in phosphorylation status.(ABSTRACT TRUNCATED AT 250 WORDS)

High levels of the vitamin K-dependent matrix Gla protein (MGP) have been found in the calcified costal cartilage of the cow and the calcified vertebral cartilage of the soupfin shark (Galeorhinus galeus). In both species, MGP accounts for 35-40% of the total protein in the acid demineralization extract of calcified cartilage, and the mineral content of calcified cartilage is comparable to that of bovine cortical bone. Shark and bovine MGP are both nearly insoluble in neutral buffers, a conserved property that indicates that self-aggregation could be important to the as yet unknown function of MGP. The complete amino acid sequence of shark MGP was determined to compare the structure of the elasmobranch protein to the several currently known mammalian MGP sequences. Shark MGP contains 4 residues of the vitamin K-dependent amino acid gamma-carboxyglutamic acid in its 102 residue sequence and has a calculated molecular weight = 12,770 daltons. The first 76 residues of shark MGP are homologous in sequence to mammalian MGPs, with 37% sequence identity, but the C-terminal 23 residues of the shark protein have no counterpart in the mammalian MGPs. This C-terminal segment of shark MGP contains 8 basic residues and no acidic residues. Among the features conserved in shark MGP, in all mammalian MGPs, and in all other currently known vitamin K-dependent mammalian proteins are a 15-residue region of sequence homology that has been shown to function as the gamma-carboxylase recognition sequence and an invariant sequence of unknown function, Gla-Xaa-Xaa-Xaa-Gla-Xaa-Cys.

Acremonium coenophialum, a fungal endosymbiont in tall fescue, is responsible for the production of alkaloid toxins. Animals grazing endophyte-infected tall fescue often show toxicosis. In marginal environments, the endophyte is important for long-term survival of tall fescue. Few differences in endophyte isolates from individual tall fescue plants have been reported. To aid development of a toxicosis-free tall fescue, it is important to identify differences in endophyte isolates. This report describes variation in nitrogen utilization in a defined culture medium by endophyte isolates from Kentucky-31 tall fescue. Overall, the best nitrogen sources for dry weight production of mycelium were proline and potassium nitrate. Thirty-four isolates grown on agar-solidified defined media with single nitrogen sources showed variation in nitrogen utilization. Fifty percent of the isolates were unable to utilize two or more amino acids. Manipulation of endophyte variation could lead to development of a toxicosis-free tall fescue cultivar.

To better understand the biochemistry of matrix-forming cells, we developed a simple and reproducible procedure for the isolation and identification by N-terminal sequencing of proteins secreted by cells into culture medium and applied this procedure to the analysis of the major Coomassie blue-staining proteins under 100 kD that are secreted from three different human osteoblastic cell cultures. The major proteins secreted by normal human osteoblasts from adult trabecular bone were identified by N-terminal sequencing to be gelatinase, osteonectin, the C-terminal propeptides of the alpha 1 and alpha 2 chains of type I collagen, tissue inhibitor of metalloproteinase 1 (TIMP-1), and beta 2-microglobulin. The amounts of each of these proteins secreted into medium over a 24 h interval did not change over the 7 consecutive days of culture under serum-free conditions, which indicates that this pattern of protein secretion is not significantly affected by the serum-free conditions needed for protein identification by this method. In addition, radioimmunoassay for bone gla protein (BGP), a marker for osteoblast phenotype, revealed that BGP secretion remained high over 7 days of culture under serum-free conditions and was comparable to the rate of BGP secretion in control cultures with 10% serum. The major proteins secreted by MG-63 cells were identified by N-terminal sequencing to be gelatinase, a novel 40 kD human bone protein we termed YKL-40, TIMP-1, the recently discovered TIMP-2, and beta 2-microglobulin. Further studies revealed that YKL-40 is the only protein detectable by Coomassie staining of SDS gels of MG-63 media proteins that is induced by extended time at confluence or by treatment with 1,25-(OH)2D3. The apparent absence of detectable Coomassie-stained bands corresponding to the C-terminal propeptides of collagen in the medium of MG-63 cells suggests that these transformed cells may not be a good model for bone matrix formation. The major proteins secreted by normal fetal osteoblastic cells were identified by N-terminal sequencing to be osteonectin and the C-terminal propeptides of the alpha 1 and alpha 2 chains of type I collagen. Gelatinase and TIMP could not be detected among the conditioned medium proteins by these methods. These observations indicate that fetal osteoblasts primarily express proteins that are matrix constituents and adult human osteoblasts secrete, in addition to these, proteins that could function in matrix turnover.