Ion Specificity and Nonmonotonic Protein Solubility from Salt Entropy

The addition of salt to protein solutions can either increase or decrease the protein solubility, and the magnitude of this effect depends on the salt used. We show that these effects can be captured using a theory that includes attractive and repulsive electrostatic interactions, nonelectrostatic protein-ion interactions, and ion-solvent interactions via an effective solvated ion radius. We find that the ion radius has significant effects on the translational entropy of the salt, which leads to salt specificity in the protein solubility. At low salt, the dominant effect comes from the entropic cost of confining ions within the aggregate, whereas at high concentrations, the salt drives a depletion attraction that favors aggregation. Our theory explains the reversal in the Hofmeister series observed in lysozyme cloud point measurements and semi-quantitatively describes the solubility of lysozyme and chymosin crystals. We present a comparison of the contributions to the free energy and give guidelines for when salting in or salting out should be expected.     

Therapeutic efficacy of TBC3711 in monocrotaline-induced pulmonary hypertension

Background

Modified function of immune cells in nasal secretions may play a role in the enhanced susceptibility to respiratory viruses that is seen in smokers. Innate immune cells in nasal secretions have largely been characterized by cellular differentials using morphologic criteria alone, which have successfully identified neutrophils as a significant cell population within nasal lavage fluid (NLF) cells. However, flow cytometry may be a superior method to fully characterize NLF immune cells. We therefore characterized immune cells in NLF by flow cytometry, determined the effects of live attenuated influenza virus (LAIV) on NLF and peripheral blood immune cells, and compared responses in samples obtained from smokers and nonsmokers.

Methods

In a prospective observational study, we characterized immune cells in NLF of nonsmokers at baseline using flow cytometry and immunohistochemistry. Nonsmokers and smokers were inoculated with LAIV on day 0 and serial nasal lavages were collected on days 1-4 and day 9 post-LAIV. LAIV-induced changes of NLF cells were characterized using flow cytometry. Cell-free NLF was analyzed for immune mediators by bioassay. Peripheral blood natural killer (NK) cells from nonsmokers and smokers at baseline were stimulated in vitro with LAIV followed by flow cytometric and mediator analyses.

Results

CD45(+)CD56(-)CD16(+) neutrophils and CD45(+)CD56(+) NK cells comprised median 4.62% (range 0.33-14.52) and 23.27% (18.29-33.97), respectively, of non-squamous NLF cells in nonsmokers at baseline. LAIV did not induce changes in total NK cell or neutrophil percentages in either nonsmokers or smokers. Following LAIV inoculation, CD16(+) NK cell percentages and granzyme B levels increased in nonsmokers, and these effects were suppressed in smokers. LAIV inoculation enhanced expression of activating receptor NKG2D and chemokine receptor CXCR3 on peripheral blood NK cells from both nonsmokers and smokers in vitro but did not induce changes in CD16(+) NK cells or granzyme B activity in either group.

Conclusions

These data are the first to identify NK cells as a major immune cell type in the NLF cell population and demonstrate that mucosal NK cell cytotoxic function is suppressed in smokers following LAIV. Altered NK cell function in smokers suggests a potential mechanism that may enhance susceptibility to respiratory viruses.     

Expression of a GALACTINOL SYNTHASE gene in tomato seeds is up-regulated before maturation desiccation and again after imbibition whenever radicle protrusion is prevented

Raffinose family oligosaccharides (RFOs) have been implicated in mitigating the effects of environmental stresses on plants. In seeds, proposed roles for RFOs include protecting cellular integrity during desiccation and/or imbibition, extending longevity in the dehydrated state, and providing substrates for energy generation during germination. A gene encoding galactinol synthase (GOLS), the first committed enzyme in the biosynthesis of RFOs, was cloned from tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds, and its expression was characterized in tomato seeds and seedlings. GOLS (LeGOLS-1) mRNA accumulated in developing tomato seeds concomitant with maximum dry weight deposition and the acquisition of desiccation tolerance. LeGOLS-1 mRNA was present in mature, desiccated seeds but declined within 8 h of imbibition in wild-type seeds. However, LeGOLS-1 mRNA accumulated again in imbibed seeds prevented from completing germination by dormancy or water deficit. Gibberellin-deficient (gib-1) seeds maintained LeGOLS-1 mRNA amounts after imbibition unless supplied with gibberellin, whereas abscisic acid (ABA) did not prevent the loss of LeGOLS-1 mRNA from wild-type seeds. The presence of LeGOLS-1 mRNA in ABA-deficient (sitiens) tomato seeds indicated that wild-type amounts of ABA are not necessary for its accumulation during seed development. In all cases, LeGOLS-1 mRNA was most prevalent in the radicle tip. LeGOLS-1 mRNA accumulation was induced by dehydration but not by cold in germinating seeds, whereas both stresses induced LeGOLS-1 mRNA accumulation in seedling leaves. The physiological implications of LeGOLS-1 expression patterns in seeds and leaves are discussed in light of the hypothesized role of RFOs in plant stress tolerance.     

Both dimerization and interdomain processing are essential for caspase-4 activation

A subgroup of caspase family of inflammatory caspases (-1, -4, -5, -11, and -12) play important role during cytokine maturation and inflammation but their regulation is not well understood as much as the initiator and effector caspases. Here, the biochemical mechanism of caspase-4 activation is elucidated. With citrate, a well-known kosmotrope to enhance the monomer-dimer transition, caspase-4 was activated approximately 40 times that was comparable with that of caspase-9 ( approximately 75-fold increments). The activation reaction was mainly bimolecular (n=1.67+/-0.04) for monomeric caspase-4. In addition, the interdomain cleavage was also responsible to activate caspase-4 more than 100-fold, again comparable with that of effector caspases where the proteolytic processing is considered as the sole activation mechanism. Thus, caspase-4 shows a novel activation mechanism of the synergism between dimerization and proteolysis that sharply differs from the established activation mechanism of dimerization for initiators and interdomain cleavage for effector caspases.     

Tricarboxylic Acid Cycle and One-Carbon Metabolism Pathways Are Important in Edwardsiella ictaluri Virulence

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of channel catfish (ESC). The disease causes considerable economic losses in the commercial catfish industry in the United States. Although antibiotics are used as feed additive, vaccination is a better alternative for prevention of the disease. Here we report the development and characterization of novel live attenuated E. ictaluri mutants. To accomplish this, several tricarboxylic acid cycle (sdhC, mdh, and frdA) and one-carbon metabolism genes (gcvP and glyA) were deleted in wild type E. ictaluri strain 93-146 by allelic exchange. Following bioluminescence tagging of the E. ictaluri ΔsdhC, Δmdh, ΔfrdA, ΔgcvP, and ΔglyA mutants, their dissemination, attenuation, and vaccine efficacy were determined in catfish fingerlings by in vivo imaging technology. Immunogenicity of each mutant was also determined in catfish fingerlings. Results indicated that all of the E. ictaluri mutants were attenuated significantly in catfish compared to the parent strain as evidenced by 2,265-fold average reduction in bioluminescence signal from all the mutants at 144 h post-infection. Catfish immunized with the E. ictaluri ΔsdhC, Δmdh, ΔfrdA, and ΔglyA mutants had 100% relative percent survival (RPS), while E. ictaluri ΔgcvP vaccinated catfish had 31.23% RPS after re-challenge with the wild type E. ictaluri.     

The kinetic mechanism for cytochrome P450 metabolism of type II binding compounds: evidence supporting direct reduction

The metabolic stability of a drug is an important property that should be optimized during drug design and development. Nitrogen incorporation is hypothesized to increase the stability by coordination of nitrogen to the heme iron of cytochrome P450, a binding mode that is referred to as type II binding. However, we noticed that the type II binding compound 1 has less metabolic stability at sub-saturating conditions than a closely related type I binding compound 3. Three kinetic models will be presented for type II binder metabolism; (1) Dead-end type II binding, (2) a rapid equilibrium between type I and II binding modes before reduction, and (3) a direct reduction of the type II coordinated heme. Data will be presented on reduction rates of iron, the off rates of substrate (using surface plasmon resonance) and the catalytic rate constants. These data argue against the dead-end, and rapid equilibrium models, leaving the direct reduction kinetic mechanism for metabolism of the type II binding compound 1.     

Comparative Spatial Dynamics of Japanese Encephalitis and Acute Encephalitis Syndrome in Nepal

Japanese Encephalitis (JE) is a vector-borne disease of major importance in Asia. Recent increases in cases have spawned the development of more stringent JE surveillance. Due to the difficulty of making a clinical diagnosis, increased tracking of common symptoms associated with JE—generally classified as the umbrella term, acute encephalitis syndrome (AES) has been developed in many countries. In Nepal, there is some debate as to what AES cases are, and how JE risk factors relate to AES risk. Three parts of this analysis included investigating the temporal pattern of cases, examining the age and vaccination status patterns among AES surveillance data, and then focusing on spatial patterns of risk factors. AES and JE cases from 2007–2011 reported at a district level (n = 75) were examined in relation to landscape risk factors. Landscape pattern indices were used to quantify landscape patterns associated with JE risk. The relative spatial distribution of landscape risk factors were compared using geographically weighted regression. Pattern indices describing the amount of irrigated land edge density and the degree of landscape mixing for irrigated areas were positively associated with JE and AES, while fragmented forest measured by the number of forest patches were negatively associated with AES and JE. For both JE and AES, the local GWR models outperformed global models, indicating spatial heterogeneity in risks. Temporally, the patterns of JE and AES risk were almost identical; suggesting the relative higher caseload of AES compared to JE could provide a valuable early-warning signal for JE surveillance and reduce diagnostic testing costs. Overall, the landscape variables associated with a high degree of landscape mixing and small scale irrigated agriculture were positively linked to JE and AES risk, highlighting the importance of integrating land management policies, disease prevention strategies and promoting healthy sustainable livelihoods in both rural and urban-fringe developing areas.     

Computational and Biochemical Docking of the Irreversible Cocaine Analog RTI 82 Directly Demonstrates Ligand Positioning in the Dopamine Transporter Central Substrate-binding Site

The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3β-(p-chlorophenyl)tropane-2β-carboxylic acid, 4′-azido-3′-iodophenylethyl ester ([¹²⁵I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [¹²⁵I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors.