A label-free mass spectrometry method for the quantification of protein isotypes

Successful quantitative mass spectrometry (MS) requires strategies to link the mass spectrometer response to the analyte abundance, with the response being dependent on more factors than just analyte abundance. Label-dependent strategies rely on the incorporation of an isotopically labeled internal standard into the sample. Current label-free strategies (performed without internal standards) are useful for analyzing samples that are unsuitable for isotopic labeling but are less accurate. Here we describe a label-free technique applicable to analysis of products from related genes (isotypes). This approach enables the invariant tryptic peptide sequences within the family to serve as “built-in” internal standards and the isotype-specific peptide sequences to report the amount of the various isotypes. A process of elimination segregates reliably trypsin-released standard and reporter peptides from unreliably released peptides. The specific MS response factors for these reporter and standard peptides can be determined using synthetic peptides. Analysis of HeLa tubulin digests revealed peptides from βI-, βII-, βIII-, βIVb-, and βV-tubulin, eight of which were suitable; along with five standard peptides for quantification of the β-tubulin isotypes. To show the utility of this method, we determined that βI-tubulin represented 77% and βIIItubulin represented 3.2% of the total HeLa β-tubulin

Interpellations : Three Essays on Kent Monkman = Trois essais sur Kent Monkman

"In Interpellations. Three Essays on Kent Monkman the art historians Jonathan D. Katz, Richard W. Hill and Todd Porterfield offer perspectives and analyses on Monkman's work that address history and genre painting, the queered Romantic landscape, the shifting and unfixed subject, race, sexuality conquest and soverignty, and modern versus discontinuous temporality." -- p. [4] of cover

Tension Pneumocephalus Related to Spontaneous Skull Base Dehiscence in a Patient on BiPAP

Spontaneous pneumocephalus is an uncommon phenomenon that may develop in patients with occult skull base defects. There have been reports of pneumocephalus occurring spontaneously in the setting of continuous positive airway pressure (CPAP) use (1). Tension pneumocephalus represents a neurosurgical emergency where intracranial air is trapped with increasing pressures resulting in neurological deterioration (2). Previous literature has also documented the growing understanding of how obesity, elevated intracranial pressure (ICP), obstructive sleep apnea (OSA), and cortical skull thinning are associated with spontaneous tegmen dehiscence and cerebrospinal fluid (CSF) leakage (3). Other mechanisms for spontaneous CSF leak include aberrant arachnoid granulation, congenital skull base dehiscences, increased abdominal and thoracic pressure resulting in reduced cerebral venous drainage, and age-related cortical thinning. In this report, we present the case of a bilevel positive airway pressure (BiPAP) user with an undiagnosed spontaneous tegmen dehiscence who developed spontaneous tension pneumocephalus

Kinetic and Spectroscopic Characterization of the H178A Methionyl Aminopeptidase from \u3cem\u3eEscherichia coli\u3c/em\u3e

To gain insight into the role of the strictly conserved histidine residue, H178, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H178A mutant enzyme was prepared. Metal-reconstituted H178A binds only one equivalent of Co(II) or Fe(II) tightly with affinities that are identical to the WT enzyme based on kinetic and isothermal titration calorimetry (ITC) data. Electronic absorption spectra of Co(II)-loaded H178A EcMetAP-I indicate that the active site divalent metal ion is pentacoordinate, identical to the WT enzyme. These data indicate that the metal binding site has not been affected by altering H178. The effect of altering H178 on activity is, in general, due to a decrease in kcat. The kcat value for Co(II)-loaded H178A decreased 70-fold toward MGMM and 290-fold toward MP-p-NA compared to the WT enzyme, while kcat decreased 50-fold toward MGMM for the Fe(II)-loaded H178A enzyme and 140-fold toward MP-p-NA. The Km values for MGMM remained unaffected, while those for MP-p-NA increased approximately 2-fold for Co(II)- and Fe(II)-loaded H178A. The kcat/Km values for both Co(II)- and Fe(II)-loaded H178A toward both substrates ranged from ∼50- to 580-fold reduction. The pH dependence of log Km, log kcat, and log(kcat/Km) of both WT and H178A EcMetAP-I were also obtained and are identical, within error, for H178A and WT EcMetAP-I. Therefore, H178A is catalytically important but is not required for catalysis. Assignment of one of the observed pKa values at 8.1 for WT EcMetAP-I was obtained from plots of molar absorptivity at λmax(640) vs pH for both WT and H178A EcMetAP-I. Apparent pKa values of 8.1 and 7.6 were obtained for WT and H178A EcMetAP-I, respectively, and were assigned to the deprotonation of a metal-bound water molecule. The data reported herein provide support for the key elements of the previously proposed mechanism and suggest that a similar mechanism can apply to the enzyme with a single metal in the active site

Paper Session II-A - Biomedical Applications from Microgravity Experiments Flown on the CMIX Commercial Shuttle Flights

NASA\u27s initiatives to encourage the US private sector to invest in space hardware, products, and services are approximately 10 years old. These initiatives have worked and have encouraged the private sector to invest in commercial space projects. 1be Office of Advanced Concepts and Technology (formally NASA\u27s Office of Commercial Programs) has over the years initiated several innovative programs to provide access to space for commercial entities having developed their own hardware with private sector resources. These innovative agreements range from direct pay to fly agreements to barter arrangements with a commercial entity. The purp:lse of this paper is to present an overview of the QJmmercial ,MDA ff A E.EJerimems (CMIX) Program, which has flown two Space Shuttle missions during the past 16 months. The paper will show typical data results of new biomedical applications that can be obtained from space processing operations that can be a benefit to the US

Microscopy with ultraviolet surface excitation for rapid slide-free histology.

Histologic examination of tissues is central to the diagnosis and management of neoplasms and many other diseases, and is a foundational technique for preclinical and basic research. However, commonly used bright-field microscopy requires prior preparation of micrometre-thick tissue sections mounted on glass slides, a process that can require hours or days, that contributes to cost, and that delays access to critical information. Here, we introduce a simple, non-destructive slide-free technique that within minutes provides high-resolution diagnostic histological images resembling those obtained from conventional haematoxylin-and-eosin-histology. The approach, which we named microscopy with ultraviolet surface excitation (MUSE), can also generate shape and colour-contrast information. MUSE relies on ~280-nm ultraviolet light to restrict the excitation of conventional fluorescent stains to tissue surfaces, and it has no significant effects on downstream molecular assays (including fluorescence in situ hybridization and RNA-seq). MUSE promises to improve the speed and efficiency of patient care in both state-of-the-art and low-resource settings, and to provide opportunities for rapid histology in research

Pilot Safety Evaluation of Varenicline for the Treatment of Methamphetamine Dependence.

Despite the worldwide extent of methamphetamine dependence, no medication has been shown to effectively treat afflicted individuals. One relatively unexplored approach is modulation of cholinergic system function. Animal research suggests that enhancement of central cholinergic activity, possibly at nicotinic acetylcholine receptors (nAChRs), can reduce methamphetamine-related behaviors. Further, preliminary findings indicate that rivastigmine, a cholinesterase inhibitor, may reduce craving for methamphetamine after administration of the drug in human subjects. We therefore performed a double-blind, placebo-controlled, crossover pilot study of the safety and tolerability of varenicline in eight methamphetamine-dependent research subjects. Varenicline is used clinically to aid smoking cessation, and acts as a partial agonist at α4β2 nAChRs with full agonist properties at α7 nAChRs. Oral varenicline dose was titrated over 1 week to reach 1 mg bid, and then was co-administered with 30 mg methamphetamine, delivered in ten intravenous infusions of 3 mg each. Varenicline was found to be safe in combination with IV methamphetamine, producing no cardiac rhythm disturbances or alterations in vital sign parameters. No adverse neuropsychiatric sequelae were detected either during varenicline titration or following administration of methamphetamine. The results suggest that varenicline warrants further investigation as a potential treatment for methamphetamine dependence