Discovery of mouse spleen signaling responses to anthrax using label-free quantitative phosphoproteomics via mass spectrometry.

Inhalational anthrax is caused by spores of the bacterium Bacillus anthracis (B. anthracis), and is an extremely dangerous disease that can kill unvaccinated victims within 2 weeks. Modern antibiotic-based therapy can increase the survival rate to ∼50%, but only if administered presymptomatically (within 24-48 h of exposure). To discover host signaling responses to presymptomatic anthrax, label-free quantitative phosphoproteomics via liquid chromatography coupled to mass spectrometry was used to compare spleens from uninfected and spore-challenged mice over a 72 h time-course. Spleen proteins were denatured using urea, reduced using dithiothreitol, alkylated using iodoacetamide, and digested into peptides using trypsin, and the resulting phosphopeptides were enriched using titanium dioxide solid-phase extraction and analyzed by nano-liquid chromatography-Linear Trap Quadrupole-Orbitrap-MS(/MS). The fragment ion spectra were processed using DeconMSn and searched using both Mascot and SEQUEST resulting in 252,626 confident identifications of 6248 phosphopeptides (corresponding to 5782 phosphorylation sites). The precursor ion spectra were deisotoped using Decon2LS and aligned using MultiAlign resulting in the confident quantitation of 3265 of the identified phosphopeptides. ANOVAs were used to produce a q-value ranked list of host signaling responses. Late-stage (48-72 h postchallenge) Sterne strain (lethal) infections resulted in global alterations to the spleen phosphoproteome. In contrast, ΔSterne strain (asymptomatic; missing the anthrax toxin) infections resulted in 188 (5.8%) significantly altered (q<0.05) phosphopeptides. Twenty-six highly tentative phosphorylation responses to early-stage (24 h postchallenge) anthrax were discovered (q<0.5), and ten of these originated from eight proteins that have known roles in the host immune response. These tentative early-anthrax host response signaling events within mouse spleens may translate into presymptomatic diagnostic biomarkers of human anthrax detectable within circulating immune cells, and could aid in the identification of pathogenic mechanisms and therapeutic targets.

Expression of CD105 and CD34 receptors controls BMP-induced in vitro mineralization of mouse adipose-derived stem cells but does not predict their in vivo bone-forming potential.

Adipose-derived stem cells (ADSCs) can be excellent alternative to bone marrow derived stem cells for enhancing fracture repair since ADSCs can be isolated comparatively in large numbers from discarded lipoaspirates. However, osteogenic potential of ADSCs in vivo is very controversial. We hypothesized that adipose-derived stem cells (ADSCs) that respond maximally to bone morphogenetic proteins (BMPs) in vitro would possess maximum bone-forming potential. Four purified populations of mouse ADSCs: CD105(+) CD34(+), CD105(-) CD34(-), CD105(+) CD34(-) and CD105(-) CD34(+) were obtained using fluorescence-activated cell sorting (FACS) and their BMP-responsiveness was determined in vitro. CD105(+) CD34(-) population showed the strongest response to BMPs in terms of robust increase in mineralization. Expression of CD105 correlated with high BMP-responsive phenotype and larger cell size while expression of CD34 correlated with low BMP-responsive phenotype and smaller cell size. CD105(+) CD34(-) population displayed higher gene expression of Alk1 or Alk6 receptors in comparison with other populations. However, CD105(+) CD34(-) ADSCs failed to induce ectopic bone formation in vivo after they were transplanted into syngeneic mice, indicating that in vitro BMP-responsiveness is not a good indicator to predict in vivo bone forming potential of ADSCs. Therefore greater precautions should be executed during selection of competent ADSCs for bone repair.