Pertinent chemical reactions of AA, hemoglobin, and NO.

<p>A) Ascorbic acid (AA) can lose an electron to become monodehydroascorbate (MDHA) radical, which can lose an electron to become dehydroascorbate (DHA). B) The nitric oxide dioxygenase (NOD) reaction; oxy hemoglobins (Hb) will react with NO to make nitrate and ferric Hb. If the ferric Hb is reduced (in this case by AA), it will bind oxygen (if present) to start the reaction again. C) The AA/glutathione cycle in plants is associated with scavenging of peroxide, superoxide, and hydroxyl radical, and is hypothesized to reduce nsHbs in support of NOD.</p

nsHb reduction by AA, DHA, and MDHAR.

<p>A) ferric rice nsHb (5 µM) was mixed with 3 mM AA in air-saturated buffer. The absorbance changes in the visible and B) Soret regions are associated with nsHb reduction and oxygen binding. C) Time courses for nsHb reduction by various concentrations of AA. D) The rate constants for AA reduction of nsHb and Mb (5 µM) are plotted as a function of AA concentration, which have slopes equal to the bimolecular rate constants for reduction of nsHb.</p

Production of NO from nitrite and AA.

<p>NO production from solutions of nitrite (10 mM unless otherwise indicated) were stimulated by addition of AA at pH values ranging from 6 to 7. A) NO production is measurable at pH 6.75 and increases with decreasing pH. When 5 µM nsHb is included (red traces) there is a decrease in the level NO proportional to the Hb concentration. B) The amount of NO produced is directly related to nitrite concentration, as is evident from the high levels produced even at pH 7 when nitrite concentration is very high.</p

EPR measurements of MDHA radical in solutions of AA (A), and produced from AA reduction of nsHb (B).

<p>A) Freshly dissolved 3 mM AA solutions contain MDHA, which decays to a lower equilibrium level over several hours. B) Addition of ferric nsHb to AA increases the EPR signal associated with MDHA.</p

The effect of MDHAR on AA reduction of nsHb.

<p>A) Recombinant rice MDHAR was purified and quantified based on its flavin absorbance spectrum. B) The EPR signal associated with MDHA was used to demonstrate the activity of AAOx (which increases the MDHA signal in the presence of AA), and MDHAR (which decreases MDHA in the presence of NADH). These data also show that NO reacts directly with MDHA. C) MDHAR oxidizes NADH in the presence of MDHA (produced from AA and AAOx, as in (B)). D) Time courses for nsHb reduction by AA in the absence and present of MDHAR and NADH are nearly identical. E) When nsHb concentration is greater than AA, reduction is very slow, and slightly faster in the presence of MDHAR and NADH.</p

A Practical Guide to the Preparation of Liquid Crystal-Templated Microparticles

We provide a practical guide to methods and protocols that use polymer networks templated from droplets of liquid crystal (LC) to synthesize micrometer-sized polymeric particles that are chemically patchy, are anisometric in shape, possess anisotropic optical properties, and/or are mesoporous. We describe a range of methods that permit the preparation of LC droplets (containing reactive monomers) as templates for polymerization, including formation of LC-in-water emulsions by mechanical methods (e.g., vortexing), encapsulation in polymeric shells, or microfluidics. The relative merits of the methods, including ease of use and potential pitfalls, and the resulting droplet size distributions, are described. We also report a menu of approaches that can be used to control the internal configurations of the LC droplets, including changes in composition of the continuous solvent phases (e.g., addition of glycerol) and adsorption of surfactants or colloids at the interfaces of the LC droplets. Photopolymerization of the LC droplets in bipolar, radial, axial, or preradial configurations and subsequent extraction of the nonreactive mesogens generates polymeric particles that have spindle, spherical, spherocylindrical, or tear shapes, respectively. Finally, we describe how to characterize these polymeric particles, including their shape, internal structure, optical properties, and porosity. The methods described in this paper, which provide access to complex microparticles with properties relevant to separation processes, drug delivery, and optical devices, are general and versatile and can be readily developed further (e.g., by changing the choice of LC) to create an even greater diversity of microparticles

Th17 responses in SrtA/SCPA immunized mice were resolved promptly following bacterial clearance.

<p>Mice were immunized and challenged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168861#pone.0168861.g001" target="_blank">Fig 1A</a> and sacrificed on the indicated days after challenge. (A) CFUs in NALT were determined on blood agar plate. Data are means ± SEM (n = 4). (B) Frequencies of IL-17⁺ cells in CD4⁺ NALT cells were determined by flow cytometry. Data are presented as the means ± SEM (n = 6) from two independent experiments. * <i>P</i> ≤ 0.05, ** <i>P</i> ≤ 0.01, *** <i>P</i> ≤ 0.001, Unpaired t-test (A) and Kruskal-Wallis test (B).</p

SrtA/SCPA co-immunization induced more efficient protection against GAS infection.

<p>(A) Mice were i.n immunized three times at 1-week intervals with indicated antigens (SrtA, 10 μg and SCPA, 20 μg) and CTB or infected with GAS (Materials and Methods). 10 days after the last immunization or infection, mice were challenged with a sublethal dose of GAS M1 (Spec^r). NALTs were taken and homogenized for CFU counting 24 hr after challenge. Data are geometric means of two independent experiments. (B) Mice were co-immunized with SrtA/SCPA and CTB or infected with GAS M49 (5 x 10⁷) for three times at 1-week intervals and challenged with M49 (2 x 10⁸) 14 days after the last immunization or infection. CFUs in NALT were determined 24 hr after challenge. * <i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.01, Kruskal-Wallis test.</p

SrtA/SCPA immunization induced rapid neutrophil recruitment to NALT.

<p>Mice were immunized and challenged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168861#pone.0168861.g001" target="_blank">Fig 1A</a>. (A) Four hr after GAS challenge mice were sacrificed. Recruitment of neutrophils to NALT was determined by flow cytometry and summarized in the bar graph. Data are presented as the means ± SEM (n = 12) from at least two independent experiments. (B) Mice were euthanized four hr after challenge, and MPO activity in NALT cells was measured. Data are presented as the means ± SEM (n = 14) from three independent experiments. (C and D) Mice were euthanized at indicated time points, and neutrophil influx (C) was analyzed by flow cytometry on NALT cells and CFUs (D) in NALT were determined on blood agar plates. Data are presented as the means ± SEM (n = 6) from two independent experiments. * <i>P</i> ≤ 0.05, ** <i>P</i> ≤ 0.01, *** <i>P</i> ≤ 0.001, Kruskal-Wallis test.</p

SrtA/SCPA co-immunization induced high levels of SCPA specific antibodies.

<p>Mice were immunized and challenged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168861#pone.0168861.g001" target="_blank">Fig 1A</a>. Serum and saliva samples were taken 24 hr after challenge. (A) Titers of SCPA-specific serum IgG. (B) Titers of SCPA-specific saliva IgA. All of the data are presented as the means ± SEM (n = 10) from at least two independent experiments. * <i>P</i> ≤ 0.05, ** <i>P</i> ≤ 0.01, *** <i>P</i> ≤ 0.001, Kruskal-Wallis test.</p