DNA methylation as a transcriptional regulator of the immune system.

DNA methylation is a dynamic epigenetic modification with a prominent role in determining mammalian cell development, lineage identity, and transcriptional regulation. Primarily linked to gene silencing, novel technologies have expanded the ability to measure DNA methylation on a genome-wide scale and uncover context-dependent regulatory roles. The immune system is a prototypic model for studying how DNA methylation patterning modulates cell type- and stimulus-specific transcriptional programs. Preservation of host defense and organ homeostasis depends on fine-tuned epigenetic mechanisms controlling myeloid and lymphoid cell differentiation and function, which shape innate and adaptive immune responses. Dysregulation of these processes can lead to human immune system pathology as seen in blood malignancies, infections, and autoimmune diseases. Identification of distinct epigenotypes linked to pathogenesis carries the potential to validate therapeutic targets in disease prevention and management.

Predictors of improved functional outcome following inpatient rehabilitation for patients with traumatic brain injury.

OBJECTIVE: To determine factors associated with response to inpatient rehabilitation treatment among TBI patients. SETTING: Inpatient rehabilitation service at a Level I trauma center. PARTICIPANTS: Moderate-severe TBI patients ages ≥ 18years old admitted between January 1, 2002 and December 31, 2012. MAIN MEASURES: Response to inpatient rehabilitation, measured by the Functional Independence Measure (FIM) score. DESIGN: Retrospective cohort study. RESULTS: Of 1,984 patients treated for TBI, 184 (10.8%) underwent inpatient rehabilitation. The largest proportion of patients improved in mobility (98.9%), followed by self-care (93.7%), communication/social cognition (84.0%), and sphincter control (65.7%). Of these, 99 (53.8%) improved by 2 or more levels of functional independence and were considered rehabilitation responders. Responders were younger (53.1 years vs. 63.8, p < 0.01), had longer average rehabilitation stays (15.4 days vs. 12.2, p < 0.01), and were less likely to have an admission SBP <100 mmHg (7.1% vs. 17.1%, p = 0.01). On multivariate analysis, normotension at admission (AOR 0.06, p = 0.01) and longer rehabilitation LOS (AOR 1.11, p < 0.01) were associated with a response to inpatient rehabilitation. CONCLUSION: Of the TBI patients who qualified for same-center inpatient rehabilitation, approximately half responded to treatment. Longer rehabilitation time and normotension at admission predicted response to rehabilitation. Further efforts are necessary to identify and optimize TBI patients for inpatient rehabilitation.

Charge site mass spectra: conformation-sensitive components of the electron capture dissociation spectrum of a protein.

A conventional electron capture dissociation (ECD) spectrum of a protein is uniquely characteristic of the first dimension of its linear structure. This sequence information is indicated by summing the primary c (m+) and z (m+•) products of cleavage at each of its molecular ion's inter-residue bonds. For example, the ECD spectra of ubiquitin (M + nH)(n+) ions, n = 7-13, provide sequence characterization of 72 of its 75 cleavage sites from 1843 ions in seven c ((1-7)+) and eight z ((1-8)+•) spectra and their respective complements. Now we find that each of these c/z spectra is itself composed of "charge site (CS)" spectra, the c (m+) or z (m+•) products of electron capture at a specific protonated basic residue. This charge site has been H-bonded to multiple other residues, producing multiple precursor ion forms; ECD at these residues yields the multiple products of that CS spectrum. Closely similar CS spectra are often formed from a range of charge states of ubiquitin and KIX ions; this indicates a common secondary conformation, but not the conventional α-helicity postulated previously. CS spectra should provide new capabilities for comparing regional conformations of gaseous protein ions and delineating ECD fragmentation pathways.

Thiamin biosynthesis: still yielding fascinating biological chemistry.

The present paper describes the biosynthesis of the thiamin thiazole in Bacillus subtilis and Saccharomyces cerevisiae. The two pathways are quite different: in B. subtilis, the thiazole is formed by an oxidative condensation of glycine, deoxy-D-xylulose 5-phosphate and a protein thiocarboxylate, whereas, in S. cerevisiae, the thiazole is assembled from glycine, NAD and Cys205 of the thiazole synthase.

How ubiquitin unfolds after transfer into the gas phase.

The structural evolution of ubiquitin after transfer into the gas phase was studied by electron capture dissociation. Site-specific fragment yields show that ubiquitin's solution fold is overall unstable in the gas phase, but unfolding caused by loss of solvent is slowest in regions stabilized by salt bridges.

A century of progress in molecular mass spectrometry.

The first mass spectrum of a molecule was measured by J.J. Thomson in 1910. Mass spectrometry (MS) soon became crucial to the study of isotopes and atomic weights and to the development of atomic weapons for World War II. Its notable applications to molecules began with the quantitative analysis of light hydrocarbons during World War II. When I joined the Dow Chemical Company in 1950, MS was not favored by organic chemists. This situation improved only with an increased understanding of gaseous ion chemistry, which was obtained through the use of extensive reference data. Gas chromatography-MS was developed in 1956, and tandem MS was first used a decade later. In neutralization-reionization MS, an unusual, unstable species is prepared by ion-beam neutralization and characterized by reionization. Electrospray ionization of a protein mixture produces its corresponding ionized molecules. In top-down proteomics, ions from an individual component can be mass separated and subjected to collision-activated and electron-capture dissociation to provide extensive sequence information.

Multi-step evolution of protein conformation on electrospray into the gas phase.

In the gas phase, some properties of native versus denatured protein conformations correspond to those in solution, such as affinity for protons and physical cross section. However, the capacity for hydrogen/deutrerium exchange is the opposite, with ubiquitin 7+ and 13+ ions exchanging >-60 D and approximately 15 D atoms, respectively. A variety of experimental methods now delineate a series of conformational perturbations that can occur in the 10(-12) s to 10(+2) s following electrospray, including side-chain collapse, hydrophobic and electrostatic non-covalent bond unfolding and refolding into a variety of non-native structures.

Chemokine CXC receptor 4--mediated glioma tumor tracking by bone marrow--derived neural progenitor/stem cells.

Malignant gliomas manifest frequent tumor recurrence after surgical resection and/or other treatment because of their nature of invasiveness and dissemination. The recognized brain tumor-tracking property of neural progenitor/stem cells opened the possibility of targeting malignant brain tumors using neural progenitor/stem cells. We and others have previously shown that fetal neural progenitor/stem cells can be used to deliver therapeutic molecules to brain tumors. Our recent work has further shown that gene delivery by bone marrow-derived neural progenitor/stem cells achieves therapeutic effects in a glioma model. In this study, we isolate and characterize bone marrow-derived neural progenitor/stem cells, which also express the chemokine receptor chemokine CXC receptor 4 (CXCR4). We show that CXCR4 is required for their chemotaxis and extracellular matrix invasion against a gradient of glioma soluble factors. Furthermore, beta-galactosidase-labeled bone marrow-derived neural progenitor/stem cells implanted in the contralateral side of the brain were shown to track gliomas as early as day 1 and increased through days 3 and 7. Intracranial glioma tracking by bone marrow-derived neural progenitor/stem cells is significantly inhibited by preincubation of bone marrow-derived neural progenitor/stem cells with a blocking anti-CXCR4 antibody, suggesting a CXCR4-dependent tracking mechanism. Glioma tracking bone marrow-derived neural progenitor/stem cells were found to express progenitor/stem cell markers, as well as CXCR4. Although bromodeoxyuridine incorporation assays and proliferating antigen staining indicated that tumor tracking bone marrow-derived neural progenitor/stem cells were mostly nonproliferating, these cells survive in the local tumor environment with little apoptosis. Elucidating the molecular mechanism of brain tumor tracking by adult source stem cells may provide basis for the development of future targeted therapy for malignant brain tumors.

IgE receptor-mediated alteration of membrane-cytoskeleton interactions revealed by mass spectrometric analysis of detergent-resistant membranes.

We use electrospray ionization mass spectrometry to quantify >100 phospholipid (PL) components in detergent-resistant membrane (DRM) domains that are related to ordered membrane compartments commonly known as lipid rafts. We previously compared PL compositions of DRMs with plasma membrane vesicles and whole cell lipid extracts from RBL mast cells, and we made the initial observation that antigen stimulation of IgE receptors (FcepsilonRI) causes a significant change in the PL composition of DRMs [Fridriksson, E. K., et al. (1999) Biochemistry 38, 8056-8063]. We now characterize the signaling requirements and time course for this change, which is manifested as an increase in the recovery of polyunsaturated PL in DRM, particularly in phosphatidylinositol species. We find that this change is largely independent of tyrosine phosphorylation, stimulated by engagement of FcepsilonRI, and can be activated by Ca(2+) ionophore in a manner independent of antigen stimulation. Unexpectedly, we found that inhibitors of actin polymerization (cytochalasin D and latrunculin A) cause a similar, but more rapid, change in the PL composition of DRMs in the absence of FcepsilonRI activation, indicating that perturbations in the actin cytoskeleton affect the organization of plasma membrane domains. Consistent with this interpretation, a membrane-permeable stabilizer of F-actin, jasplakinolide, prevents antigen-stimulated changes in DRM PL composition. These results are confirmed by a detailed analysis of multiple experiments, showing that receptor and cytochalasin D-stimulated changes in DRM lipid composition follow first-order kinetics. Analysis in terms of the number of double bonds in the fatty acid chains is valid for total PL of the major headgroups and for headgroups individually. In this manner, we show that, on average, concentrations of saturated or monounsaturated PL decrease in the DRM, whereas concentrations of PL with two or more double bonds (polyunsaturated PL) increase due to cytoskeletal perturbation. We find that these changes are independent of fatty acid chain length. Our mass spectrometric analyses provide a detailed accounting of receptor-activated alterations in the plasma membrane that are regulated by the actin cytoskeleton.

Preparation and characterization of antioxidant nanospheres from multiple alpha-lipoic acid-containing compounds.

The purpose of this study was to prepare and characterize antioxidant nanospheres composed of multiple alpha-lipoic acid-containing compounds (mALAs). It was found that the nanospheres were remarkably stable under physiologic conditions, maintained the antioxidant property of alpha-lipoic acid, and could be destabilized oxidatively and enzymatically. The preparations were simple and highly reproducible providing a new strategy for the development of nanometer-sized antioxidant biomaterials. The nanospheres may find applications as antioxidant therapeutics and oxidation-responsive antioxidant nanocontainers in drug delivery for pathological conditions characterized by oxidative stress including cancer and neurodegenerative diseases.

Stimuli-responsive antioxidant nanoprodrugs of NSAIDs.

A novel group of alpha-lipoic acid-containing hydrophobic prodrugs of non-steroidal anti-inflammatory drugs (NSAIDs) was synthesized and transformed into nanometer-sized prodrugs (nanoprodrugs). Three NSAIDs, indomethacin, ibuprofen and naproxen were linked to alpha-lipoic acid via tetraethylene glycol through hydrolytically degradable ester bonds. The three bifunctional derivatives were dissolved in organic solvents and capable of forming stable nanoprodrugs upon addition of the organic solutions into aqueous phase through the spontaneous emulsification mechanism. Antioxidant property and stimuli-responsiveness of the nanoprodrugs were demonstrated by hypochlorous acid (HOCl) scavenging followed by oxidative destabilization of the nanoprodrugs. The effect of varying NSAIDs on the in vitro hydrolytic prodrug activation catalyzed by porcine liver esterase was investigated by monitoring the rates of NSAIDs hydrolysis from the nanoprodrugs. The remarkable feature of these nanoprodrugs is that despite the highly hydrophobic nature of the derivatives NSAIDs were readily hydrolyzed enzymatically from the nanoprodrugs. Furthermore, the rate of hydrolysis was higher when the nanoprodrugs were oxidized and destabilized upon HOCl scavenging suggesting an enhanced activation of the nanoprodrugs in the oxidative environment.

Stepwise evolution of protein native structure with electrospray into the gas phase, 10(-12) to 10(2) s.

Mass spectrometry (MS) has been revolutionized by electrospray ionization (ESI), which is sufficiently "gentle" to introduce nonvolatile biomolecules such as proteins and nucleic acids (RNA or DNA) into the gas phase without breaking covalent bonds. Although in some cases noncovalent bonding can be maintained sufficiently for ESI/MS characterization of the solution structure of large protein complexes and native enzyme/substrate binding, the new gaseous environment can ultimately cause dramatic structural alterations. The temporal (picoseconds to minutes) evolution of native protein structure during and after transfer into the gas phase, as proposed here based on a variety of studies, can involve side-chain collapse, unfolding, and refolding into new, non-native structures. Control of individual experimental factors allows optimization for specific research objectives.

Early structural evolution of native cytochrome c after solvent removal.

Electrospray ionization transfers thermally labile biomolecules, such as proteins, from solution into the gas phase, where they can be studied by mass spectrometry. Covalent bonds are generally preserved during and after the phase transition, but it is less clear to what extent noncovalent interactions are affected by the new gaseous environment. Here, we present atomic-level computational data on the structural rearrangement of native cytochrome c immediately after solvent removal. The first structural changes after desolvation occur surprisingly early, on a timescale of picoseconds. For the time segment of up to 4.2 ns investigated here, we observed no significant breaking of native noncovalent bonds; instead, we found formation of new noncovalent bonds. This generally involves charged residues on the protein surface, resulting in transiently stabilized intermediate structures with a global fold that is essentially the same as that in solution. Comparison with data from native electron capture dissociation experiments corroborates both its mechanistic postulations and our computational predictions, and suggests that global structural changes take place on a millisecond timescale not covered by our simulations.

Top-down identification and characterization of biomolecules by mass spectrometry.

The most widely used modern mass spectrometers face severe performance limitations with molecules larger than a few kDa. For far larger biomolecules, a common practice has been to break these up chemically or enzymatically into fragments that are sufficiently small for the instrumentation available. With its many sophisticated recent enhancements, this "bottom-up" approach has proved highly valuable, such as for the rapid, routine identification and quantitation of DNA-predicted proteins in complex mixtures. Characterization of smaller molecules, however, has always measured the mass of the molecule and then that of its fragments. This "top-down" approach has been made possible for direct analysis of large biomolecules by the uniquely high (>10(5)) mass resolving power and accuracy ( approximately 1 ppm) of the Fourier-transform mass spectrometer. For complex mixtures, isolation of a single component's molecular ions for MS/MS not only gives biomolecule identifications of far higher reliability, but directly characterizes sequence errors and post-translational modifications. Protein sizes amenable for current MS/MS instrumentation are increased by a "middle-down" approach in which limited proteolysis forms large (e.g., 10 kDa) polypeptides that are then subjected to the top-down approach, or by "prefolding dissociation." The latter, which extends characterization to proteins >200 kDa, was made possible by greater understanding of how molecular ion tertiary structure evolves in the gas phase.