Fluorogenic and Affinity Derivatization Methods to Enable Proteomic Study of 3-Nitrotyrosine and 3,4-Dihydroxyphenylalanine as Markers of Oxidative Stress

Purpose: Oxidative post-translational modification of protein-bound tyrosine residues can have a significant impact on protein structure and function and thus may be important to physiological and pathological processes. Oxidative stress has been correlated with biological aging and many disease states, including diabetes, atherosclerosis, and neurodegeneration. Proteomic methods targeted to these modifications are important tools for determining which specific modifications may be significant in these conditions. Toward this end, a method designed to fluorogenically label the protein oxidation products 3-nitrotyrosine (3NY) and 3,4-dihydroxyphenylalanine (DOPA) using benzylamine-dependent chemistry is applied to model peptides and proteins, as well as cardiac tissue samples from a rat model for aging. Methods: Peptides or proteins are reacted with excess benzylamine (or a derivative thereof), in the presence of potassium ferricyanide, to fluorescently label DOPA residues by forming 2-phenylbenzoxazole derivatives. To label 3NY residues, the peptides or proteins are first reduced with sodium dithionite to give 3-aminotyrosine, which can undergo a similar reaction with benzylamine and oxidant to give the same products. Products are characterized by fluorescence spectroscopy, high-performance liquid chromatography (HPLC) with UV and fluorescence detection, mass spectrometry, and amino acid analysis. For enrichment by boronate-affinity HPLC, the benzylamine derivative (3R, 4S)-1-(4-(aminomethyl)phenylsulfonyl) pyrrolidine-3,4-diol (APPD) is used as the tagging reagent. Results: Cardiac proteins have been fluorescently labeled and separated, and some putative identifications have been made. A model protein, glycogen phosphorylase b (Ph-b), has been nitrated in vitro and labeled within a matrix of cardiac homogenate, and the products exhibit concentration-dependent fluorescence. The loss of 3NY from nitrated Ph-b upon mixing with cardiac homogenate has been observed and examined. Five model peptides have been labeled with APPD to determine the effect of primary structure on labeling efficiency, fluorescence quantum yield, and molar absorptivity. Conclusions: This method has great potential to aid identification of the protein oxidation products DOPA and 3NY in proteomic studies of tissue samples and can also be adapted for affinity enrichment and relative quantification of these low-abundance species

Tyrosine Modifications in Aging

This is the publisher's version, also available electronically from http://online.liebertpub.com/doi/abs/10.1089/ars.2012.4595Significance: The understanding of physiological and pathological processes involving protein oxidation, particularly under conditions of aging and oxidative stress, can be aided by proteomic identification of proteins that accumulate oxidative post-translational modifications only if these detected modifications are connected to functional consequences. The modification of tyrosine (Tyr) residues can elicit significant changes in protein structure and function, which, in some cases, may contribute to biological aging and age-related pathologies, such as atherosclerosis, neurodegeneration, and cataracts. Recent Advances: Studies characterizing proteins in which Tyr has been modified to 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, 3,3′-dityrosine and other cross-links, or 3-chlorotyrosine are reviewed, with an emphasis on structural and functional consequences. Critical Issues: Distinguishing between inconsequential modifications and functionally significant ones requires careful biochemical and biophysical analysis of target proteins, as well as innovative methods for isolating the effects of the multiple modifications that often occur under oxidizing conditions. Future Directions: The labor-intensive task of isolating and characterizing individual modified proteins must continue, especially given the expanding list of known modifications. Emerging approaches, such as genetic and metabolic incorporation of unnatural amino acids, hold promise for additional focused studies of this kind. Antioxid. Redox Signal. 17, 1571–1579

Phase I Study of Ipilimumab Combined with Whole Brain Radiation Therapy or Radiosurgery for Melanoma Patients with Brain Metastases

Purpose: We performed a phase I study to determine the maximum tolerable dose (MTD) and safety of ipilimumab with stereotactic radiosurgery (SRS) or whole brain radiotherapy (WBRT) in patients with brain metastases (BM) from melanoma. Methods: Based on intracranial (IC) disease burden, patients were treated with WBRT (Arm A) or SRS (Arm B). Ipilimumab starting dose was 3 mg/kg (every 3 weeks, starting on day 3 of WBRT or 2 days after SRS). Ipilimumab was escalated to 10 mg/kg using a two-stage, 3+3 design. The primary endpoint was to determine the MTD of ipilimumab combined with radiotherapy. Secondary endpoints were overall survival (OS), IC and extracranial (EC) control, progression free survival (PFS), and toxicity. This trial is regis- tered with ClinicalTrials.gov, number NCT01703507. Results: Characteristics of the 16 patients enrolled between 2011 and 2014 were: mean age, 60; median BM, 2 (1 to \u3e10); number with EC disease, 13 (81%). Treatment included WBRT (n=5), SRS (n=11), ipilimumab 3mg/kg (n=7), 10 mg/kg (n=9). Median follow-up was 8 months (Arm A) and 10.5 months (Arm B). There were 21 grade 1-2 neuro- toxic effects with no dose-limiting toxicities (DLTs). One patient experienced grade 3 neurotoxicity prior to ipilimumab administration. Ten additional grade 3 toxicities were reported with gastrointestinal (n=5, 31%) as the most common. There were no grade 4/5 toxicities. Median PFS and OS, respectively, in Arm A were 2.5 months and 8 months, and in Arm B were 2.1 months and not reached. Conclusion: Concurrent ipilimumab 10 mg/kg with SRS is safe. The WBRT arm was closed early due to slow accrual, but demonstrated safety with ipilimumab 3 mg/kg. No patient experienced DLT. Larger studies with ipilimumab 10 mg/kg and SRS are warranted

Criminal narrative experience: relating emotions to offence narrative roles during crime commission

A neglected area of research within criminality has been that of the experience of the offence for the offender. The present study investigates the emotions and narrative roles that are experienced by an offender while committing a broad range of crimes and proposes a model of Criminal Narrative Experience (CNE). Hypotheses were derived from the Circumplex of Emotions (Russell, 1997), Frye (1957), Narrative Theory (McAdams, 1988) and its link with Investigative Psychology (Canter, 1994). The analysis was based on 120 cases. Convicted for a variety of crimes, incarcerated criminals were interviewed and the data were subjected to Smallest Space Analysis (SSA). Four themes of Criminal Narrative Experience (CNE) were identified: Elated Hero, Calm Professional, Distressed Revenger and Depressed Victim in line with the recent theoretical framework posited for Narrative Offence Roles (Youngs & Canter, 2012). The theoretical implications for understanding crime on the basis of the Criminal Narrative Experience (CNE) as well as practical implications are discussed

ZTF 18aaqeasu (SN 2018byg): A Massive Helium-shell Double Detonation on a Sub-Chandrasekhar Mass White Dwarf

The detonation of a helium shell on a white dwarf has been proposed as a possible explosion triggering mechanism for Type Ia supernovae. Here, we report ZTF 18aaqeasu (SN 2018byg/ATLAS 18pqq), a peculiar Type I supernova, consistent with being a helium-shell double-detonation. With a rise time of $\approx 18$ days from explosion, the transient reached a peak absolute magnitude of $M_R \approx -18.2$ mag, exhibiting a light curve akin to sub-luminous SN 1991bg-like Type Ia supernovae, albeit with an unusually steep increase in brightness within a week from explosion. Spectra taken near peak light exhibit prominent Si absorption features together with an unusually red color ($g-r \approx 2$ mag) arising from nearly complete line blanketing of flux blue-wards of 5000 \AA. This behavior is unlike any previously observed thermonuclear transient. Nebular phase spectra taken at and after $\approx 30$ days from peak light reveal evidence of a thermonuclear detonation event dominated by Fe-group nucleosynthesis. We show that the peculiar properties of ZTF 18aaqeasu are consistent with the detonation of a massive ($\approx 0.15$ M$_\odot$) helium shell on a sub-Chandrasekhar mass ($\approx 0.75$ M$_\odot$) white dwarf after including mixing of $\approx 0.2$ M$_\odot$ of material in the outer ejecta. These observations provide evidence of a likely rare class of thermonuclear supernovae arising from detonations of massive helium shells.Comment: 10 pages, 6 figures. Submitted to ApJ

Discovery of functionally distinct anti-C7 monoclonal antibodies and stratification of anti-nicotinic AChR positive Myasthenia Gravis patients

Myasthenia Gravis (MG) is mediated by autoantibodies against acetylcholine receptors that cause loss of the receptors in the neuromuscular junction. Eculizumab, a C5-inhibitor, is the only approved treatment for MG that mechanistically addresses complement-mediated loss of nicotinic acetylcholine receptors. It is an expensive drug and was approved despite missing the primary efficacy endpoint in the Phase 3 REGAIN study. There are two observations to highlight. Firstly, further C5 inhibitors are in clinical development, but other terminal pathway proteins, such as C7, have been relatively understudied as therapeutic targets, despite the potential for lower and less frequent dosing. Secondly, given the known heterogenous mechanisms of action of autoantibodies in MG, effective patient stratification in the REGAIN trial may have provided more favorable efficacy readouts. We investigated C7 as a target and assessed the in vitro function, binding epitopes and mechanism of action of three mAbs against C7. We found the mAbs were human, cynomolgus monkey and/or rat cross-reactive and each had a distinct, novel mechanism of C7 inhibition. TPP1820 was effective in preventing experimental MG in rats in both prophylactic and therapeutic dosing regimens. To enable identification of MG patients that are likely to respond to C7 inhibition, we developed a patient stratification assay and showed in a small cohort of MG patients (n=19) that 63% had significant complement activation and C7-dependent loss of AChRs in this in vitro set up. This study provides validation of C7 as a target for treatment of MG and provides a means of identifying patients likely to respond to anti-C7 therapy based on complement-activating properties of patient autoantibodies

The longitudinal changes of BOLD response and cerebral hemodynamics from acute to subacute stroke. A fMRI and TCD study

<p>Abstract</p> <p>Background</p> <p>By mapping the dynamics of brain reorganization, functional magnetic resonance imaging MRI (fMRI) has allowed for significant progress in understanding cerebral plasticity phenomena after a stroke. However, cerebro-vascular diseases can affect blood oxygen level dependent (BOLD) signal. Cerebral autoregulation is a primary function of cerebral hemodynamics, which allows to maintain a relatively constant blood flow despite changes in arterial blood pressure and perfusion pressure. Cerebral autoregulation is reported to become less effective in the early phases post-stroke.</p> <p>This study investigated whether any impairment of cerebral hemodynamics that occurs during the acute and the subacute phases of ischemic stroke is related to changes in BOLD response.</p> <p>We enrolled six aphasic patients affected by acute stroke. All patients underwent a Transcranial Doppler to assess cerebral autoregulation (Mx index) and fMRI to evaluate the amplitude and the peak latency (time to peak-TTP) of BOLD response in the acute (i.e., within four days of stroke occurrence) and the subacute (i.e., between five and twelve days after stroke onset) stroke phases.</p> <p>Results</p> <p>As patients advanced from the acute to subacute stroke phase, the affected hemisphere presented a BOLD TTP increase (p = 0.04) and a deterioration of cerebral autoregulation (Mx index increase, p = 0.046). A similar but not significant trend was observed also in the unaffected hemisphere. When the two hemispheres were grouped together, BOLD TTP delay was significantly related to worsening cerebral autoregulation (Mx index increase) (Spearman's rho = 0.734; p = 0.01).</p> <p>Conclusions</p> <p>The hemodynamic response function subtending BOLD signal may present a delay in peak latency that arises as patients advance from the acute to the subacute stroke phase. This delay is related to the deterioration of cerebral hemodynamics. These findings suggest that remodeling the fMRI hemodynamic response function in the different phases of stroke may optimize the detection of BOLD signal changes.</p

The Stakes in Bayh-Dole: Public Values Beyond the Pace of Innovation

Evaluation studies of the Bayh-Dole Act are generally concerned with the pace of innovation or the transgressions to the independence of research. While these concerns are important, I propose here to expand the range of public values considered in assessing Bayh-Dole and formulating future reforms. To this end, I first examine the changes in the terms of the Bayh-Dole debate and the drift in its design. Neoliberal ideas have had a definitive influence on U.S. innovation policy for the last thirty years, including legislation to strengthen patent protection. Moreover, the neoliberal policy agenda is articulated and justified in the interest of “competitiveness.” Rhetorically, this agenda equates competitiveness with economic growth and this with the public interest. Against that backdrop, I use Public Value Failure criteria to show that values such as political equality, transparency, and fairness in the distribution of the benefits of innovation, are worth considering to counter the “policy drift” of Bayh-Dole

ZTF 18aaqeasu (SN2018byg): A Massive Helium-shell Double Detonation on a Sub-Chandrasekhar-mass White Dwarf

The detonation of a helium shell on a white dwarf (WD) has been proposed as a possible explosion triggering mechanism for SNe Ia. Here, we report ZTF 18aaqeasu (SN 2018byg/ATLAS 18pqq), a peculiar Type I supernova, consistent with being a helium-shell double-detonation. With a rise time of ≈18 days from explosion, the transient reached a peak absolute magnitude of M_R ≈ −18.2 mag, exhibiting a light curve akin to sub-luminous SN 1991bg-like SNe Ia, albeit with an unusually steep increase in brightness within a week from explosion. Spectra taken near peak light exhibit prominent Si absorption features together with an unusually red color (g − r ≈ 2 mag) arising from nearly complete line blanketing of flux blueward of 5000 Å. This behavior is unlike any previously observed thermonuclear transient. Nebular phase spectra taken at and after ≈30 days from peak light reveal evidence of a thermonuclear detonation event dominated by Fe-group nucleosynthesis. We show that the peculiar properties of ZTF 18aaqeasu are consistent with the detonation of a massive (≈0.15 M⊙) helium shell on a sub-Chandrasekhar mass (≈0.75 M⊙) WD after including mixing of ≈0.2 M⊙ of material in the outer ejecta. These observations provide evidence of a likely rare class of thermonuclear supernovae arising from detonations of massive helium shells