At elevated salt concentrations, the structure of chromatin is destabilized. This paper is concerned with the processes by which DNA is released from nucleosome core particles in free, uncomplexed form. Our experiments indicate that the DNA release reaction has distinctly different characteristics below and above approximately 0.75 M NaCl. Below this concentration of salt, release of the histones from the DNA is highly cooperative, so that no dissociation intermediates are even seen. Above this salt concentration, histone release is not so cooperative; H2A and H2B are released from the DNA more readily than are H3 and H4. This results in an apparently heterogeneous population of (H2A, H2B)-depleted intermediate species sedimenting at rates between that of free DNA and that of intact core particles. Dissociation of core particles at NaCl concentrations below 0.75 M is readily reversible. Reassociation of DNA and histones from higher salt concentrations is nearly quantitative if carried out by gradual decrease of salt concentration, but rapid dilution to low salt results in the formation of a fraction of metastable nucleosome multimers. To help organize our description of the DNA release process, we introduce a stability diagram for the core particle, defined with respect to the independent variables of salt concentration and particle concentration. We draw upon our own experimental work and also upon the work of several other laboratories. We distinguish five major regions in this diagram.

Chemical constituents of an organic film collected from an impervious urban surface, namely the exterior surface of windows, have been characterized. The organic portion of this film, that ranged in thickness from 11 to 100 nm, constitutes a previously unrecognized site for exchange of semivolatile organic contaminants in urban environments. The concentrations of total n-alkanes, total polycyclic aromatic compounds (PAH), and total polychlorinated biphenyls (PCB) ranged from 1010 to 22 500, 900 to 62 100, and 8 to 5820 ng/m2 of window surface, respectively. Whereas concentrations varied, the patterns of chemical and congener abundance were similar among samples. Alkanes were derived from mainly biogenic sources, while the PAH showed a weathered pattern and PCB congener patterns generally indicated an enrichment in higher chlorinated congeners. Film-to-air partition ratios, K FA, were calculated for selected PAH and PCB congeners and were comparable to reported values of octanol−air partition coefficients, K OA, suggesting that gas-phase compounds partition into this organic film. Potential biological effects of an organic film extract were evaluated using zebrafish embryo assays. Incubation at a concentration of 270 ppb ΣPAH plus 420 ppb ΣPCB showed 100% lethality, while concentrations 3−10 times lower produced a dose-dependent syndrome of abnormalities including cardiovascular, hematopoietic, neural crest-related, and behavioral defects.

BackgroundSystemic inflammation is a whole body reaction having an infection-positive (i.e., sepsis) or infection-negative origin. It is important to distinguish between these two etiologies early and accurately because this has significant therapeutic implications for critically ill patients. We hypothesized that a molecular classifier based on peripheral blood RNAs could be discovered that would (1) determine which patients with systemic inflammation had sepsis, (2) be robust across independent patient cohorts, (3) be insensitive to disease severity, and (4) provide diagnostic utility. The goal of this study was to identify and validate such a molecular classifier.Methods and FindingsWe conducted an observational, non-interventional study of adult patients recruited from tertiary intensive care units (ICUs). Biomarker discovery utilized an Australian cohort (n = 105) consisting of 74 cases (sepsis patients) and 31 controls (post-surgical patients with infection-negative systemic inflammation) recruited at five tertiary care settings in Brisbane, Australia, from June 3, 2008, to December 22, 2011. A four-gene classifier combining CEACAM4, LAMP1, PLA2G7, and PLAC8 RNA biomarkers was identified. This classifier, designated SeptiCyte Lab, was validated using reverse transcription quantitative PCR and receiver operating characteristic (ROC) curve analysis in five cohorts (n = 345) from the Netherlands. Patients for validation were selected from the Molecular Diagnosis and Risk Stratification of Sepsis study (ClinicalTrials.gov, NCT01905033), which recruited ICU patients from the Academic Medical Center in Amsterdam and the University Medical Center Utrecht. Patients recruited from November 30, 2012, to August 5, 2013, were eligible for inclusion in the present study. Validation cohort 1 (n = 59) consisted entirely of unambiguous cases and controls; SeptiCyte Lab gave an area under curve (AUC) of 0.95 (95% CI 0.91–1.00) in this cohort. ROC curve analysis of an independent, more heterogeneous group of patients (validation cohorts 2–5; 249 patients after excluding 37 patients with an infection likelihood of “possible”) gave an AUC of 0.89 (95% CI 0.85–0.93). Disease severity, as measured by Sequential Organ Failure Assessment (SOFA) score or Acute Physiology and Chronic Health Evaluation (APACHE) IV score, was not a significant confounding variable. The diagnostic utility of SeptiCyte Lab was evaluated by comparison to various clinical and laboratory parameters available to a clinician within 24 h of ICU admission. SeptiCyte Lab was significantly better at differentiating cases from controls than all tested parameters, both singly and in various logistic combinations, and more than halved the diagnostic error rate compared to procalcitonin in all tested cohorts and cohort combinations. Limitations of this study relate to (1) cohort compositions that do not perfectly reflect the composition of the intended use population, (2) potential biases that could be introduced as a result of the current lack of a gold standard fo...

The bone morphogenetic proteins, BMP-2 and OP-1, are candidates for growth factors that control renal branching morphogenesis. We examined their effects in embryonic kidney explants and in the mIMCD-3 cell model of collecting duct morphogenesis (mIMCD-3 cells are derived from the terminal inner medullary collecting duct of the SV40 mouse). Osteogenic protein-1 (OP-1), at a dose of 0.25 nM, increased explant growth by 30% ( P = 0.001). In contrast, 100-fold greater concentrations of OP-1 (28 nM) decreased explant growth by 10% ( P < 0.001). BMP-2 was entirely inhibitory (maximum inhibition of 7% at 5 nM, P < 0.0004). In an in vitro model for branching morphogenesis utilizing the kidney epithelial cell line, mIMCD-3, low doses of OP-1 (<0.5 nM) increased the number of tubular structures formed by 28 ± 5% ( P = 0.01), whereas concentrations >0.5 nM decreased that number by 22 ± 8% ( P = 0.02). All concentrations of BMP-2 (0.05–10 nM) were inhibitory (maximum inhibition at 10 nM of 88 ± 3%, P < 0.0001). Stimulatory doses of OP-1 increased tubular length ( P = 0.003) and the number of branch points/structure (3.2-fold increase, P= 0.0005) compared with BMP-2. To determine the molecular basis for these effects, we demonstrated that BMP-2 is bound to mIMCD-3 cells by the type I serine/threonine kinase receptor, ALK-3, and that OP-1 bound to an ∼80-kDa protein using ligand-receptor affinity assays. To demonstrate that OP-1 can exert both stimulatory and inhibitory effects within a developing kidney, embryonic explants were treated with agarose beads saturated with 2 μM OP-1. OP-1 decreased the number of ureteric bud/collecting duct branches adjacent to the beads by 58 ± 1% ( P < 0.0001). In contrast, the number of branches in tissue distal to the OP-1 beads was enhanced, suggesting a stimulatory effect at lower doses of OP-1. We conclude that OP-1 and BMP-2 directly control branching morphogenesis and that the effects of OP-1 are dependent on its local concentration within developing kidney tissue.

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