Sample preparation and fractionation techniques for intact proteins for mass spectrometric analysis.

The analysis of proteins in biological samples is highly desirable, given their connection to myriad biological functions and disease states, as well as the growing interest in the development of protein-based pharmaceuticals. The introduction and maturation of "soft" ionization methods, such as electrospray ionization and matrix-assisted laser desorption/ionization, have made mass spectrometry an indispensable tool for the analysis of proteins. Despite the availability of powerful instrumentation, sample preparation and fractionation remain among the most challenging aspects of protein analysis.

Physiological demands and signaling associated with snake venom production and storage illustrated by transcriptional analyses of venom glands.

Despite the extensive body of research on snake venom, many facets of snake venom systems, such as the physiology and regulation of the venom gland itself, remain virtually unstudied. Here, we use time series gene expression analyses of the rattlesnake venom gland in comparison with several non-venom tissues to characterize physiological and cellular processes associated with venom production and to highlight key distinctions of venom gland cellular and physiological function. We find consistent evidence for activation of stress response pathways in the venom gland, suggesting that mitigation of cellular stress is a crucial component of venom production.

1,2-Disubstituted Benzimidazoles by the Iron Catalyzed Cross-Dehydrogenative Coupling of Isomeric -Phenylenediamine Substrates.

Benzimidazoles are common in nature, medicines, and materials. Numerous strategies for preparing 2-arylbenzimidazoles exist. In this work, 1,2-disubstituted benzimidazoles were prepared from various mono- and disubstituted -phenylenediamines (OPD) by iron-catalyzed oxidative coupling.

Estimation of cerebral blood flow velocity during breath-hold challenge using artificial neural networks.

Unlabelled: The effect of untreated Obstructive Sleep Apnoea (OSA) on cerebral haemodynamics and CA impairment is an active field of research interest. A breath-hold challenge is usually used in clinical and research settings to simulate cardiovascular and cerebrovascular changes that mimic OSA events. This work utilises temporal arterial oxygen saturation (SpO) and photoplethysmography (PPG) signals to estimate the temporal cerebral blood flow velocity (CBFv) waveform.

Quantification of lung water in heart failure using cardiovascular magnetic resonance imaging.

Background: Pulmonary edema is a cardinal feature of heart failure but no quantitative tests are available in clinical practice. The goals of this study were to develop a simple cardiovascular magnetic resonance (CMR) approach for lung water quantification, to correlate CMR derived lung water with intra-cardiac pressures and to determine its prognostic significance.

Methods: Lung water density (LWD, %) was measured using a widely available single-shot fast spin-echo acquisition in two study cohorts.

Multi-Staged Regulation of Lipid Signaling Mediators during Myogenesis by COX-1/2 Pathways.

Cyclooxygenases (COXs), including COX-1 and -2, are enzymes essential for lipid mediator (LMs) syntheses from arachidonic acid (AA), such as prostaglandins (PGs). Furthermore, COXs could interplay with other enzymes such as lipoxygenases (LOXs) and cytochrome P450s (CYPs) to regulate the signaling of LMs. In this study, to comprehensively analyze the function of COX-1 and -2 in regulating the signaling of bioactive LMs in skeletal muscle, mouse primary myoblasts and C2C12 cells were transfected with specific COX-1 and -2 siRNAs, followed by targeted lipidomic analysis and customized quantitative PCR gene array analysis.

Online Comprehensive High pH Reversed Phase × Low pH Reversed Phase Approach for Two-Dimensional Separations of Intact Proteins in Top-Down Proteomics.

A proof-of-concept study is presented on the use of comprehensive two-dimensional liquid chromatography mass spectrometry (LC × LC-MS) for the separation of intact protein mixtures using a different mobile phase pH in each dimension. This system utilizes mass spectrometry (MS) friendly pH modifiers for the online coupling of high pH reversed phase liquid chromatography (HPH-RPLC) in the first dimension (D) followed by low pH reversed phase liquid chromatography (LPH-RPLC) in the second dimension (D). Owing to the ionic nature of proteins, the use of a different mobile phase pH was successful to provide altered selectivity between the two dimensions, even for closely related protein variants, such as bovine cytochrome and equine cytochrome , which differ by only three amino acids.

Multi-species comparisons of snakes identify coordinated signalling networks underlying post-feeding intestinal regeneration.

Several snake species that feed infrequently in nature have evolved the ability to massively upregulate intestinal form and function with each meal. While fasting, these snakes downregulate intestinal form and function, and upon feeding restore intestinal structure and function through major increases in cell growth and proliferation, metabolism and upregulation of digestive function. Previous studies have identified changes in gene expression that underlie this regenerative growth of the python intestine, but the unique features that differentiate this extreme regenerative growth from non-regenerative post-feeding responses exhibited by snakes that feed more frequently remain unclear.

Qualitative evaluation of high pH mass spectrometry-compatible reversed phase liquid chromatography for altered selectivity in separations of intact proteins.

Intact proteins are increasingly being recognized as potential biomarkers and biotherapeutic agents for cancer and other serious diseases. Low pH reversed phase plays an important role in both single and multidimensional protein separations for resolving complex protein samples prior to mass spectrometric detection. In this work, we evaluated the use of high pH reversed phase liquid chromatography as an alternative chromatographic separation to gain different selectivity while maintaining the high resolving power and MS compatibility of reversed phase separations.

Supercharging and multiple reaction monitoring of high-molecular-weight intact proteins using triple quadrupole mass spectrometry.

Rationale: Different supercharging agents were tested to study their effect on the intensity and charge state distributions of high-molecular-weight intact proteins. The goal of this work was to increase chargeability and ionization efficiency for proteins ranging from 66 to 150 kDa, to enable subsequent optimization of multiple reaction monitoring (MRM) mode transitions with a triple quadrupole mass spectrometer for potential top-down quantitative analysis.

Methods: Supercharging agents, such as meta-nitrobenzyl alcohol (m-NBA), dimethylsulfoxide, trifluoroethanol (TFE), and sulfolane were tested in different concentrations in 50/50 acetonitrile/water with 0.

Exercise in cirrhosis: Translating evidence and experience to practice.

Physical inactivity, sarcopenia, and frailty are highly prevalent, independent predictors of morbidity and mortality in patients with cirrhosis. Across a range of chronic diseases, exercise training is a key recommendation supported by guidelines and, for some conditions, even by governmental funding of exercise programmes. Consistent with the broader chronic disease literature, the evidence for a benefit of exercise in cirrhosis is promising.

Perfluorinated alcohol induced coacervates as extraction media for proteomic analysis.

We describe a novel method for using hexafluoroisopropanol (HFIP) induced coacervates with a variety of surfactants to extract proteins from a yeast whole cell lysate and conduct a global proteomic investigation on the extracted proteins. Yeast whole cell lysates were prepared and proteins were extracted using two workflows: 1) Proteins were extracted into the coacervate generated from the mixture of HFIP, surfactant, and cell lysate. 2) Proteins were extracted from cell lysate using a surfactant solution, and HFIP was added to the supernatant to generate a coacervate phase with concentrated proteins.

Growth and stress response mechanisms underlying post-feeding regenerative organ growth in the Burmese python.

Background: Previous studies examining post-feeding organ regeneration in the Burmese python (Python molurus bivittatus) have identified thousands of genes that are significantly differentially regulated during this process. However, substantial gaps remain in our understanding of coherent mechanisms and specific growth pathways that underlie these rapid and extensive shifts in organ form and function. Here we addressed these gaps by comparing gene expression in the Burmese python heart, liver, kidney, and small intestine across pre- and post-feeding time points (fasted, one day post-feeding, and four days post-feeding), and by conducting detailed analyses of molecular pathways and predictions of upstream regulatory molecules across these organ systems.

Obesity and the built environment at different urban scales: examining the literature.

The majority of people now live in an urban (or suburban) environment. The built (material) environment, its vehicular and pedestrian infrastructure, buildings, and public realm places, are the places used for working, living, and recreating. The environment currently favors and facilitates motorized vehicles generally, and private automobiles especially.

Understanding quality-of-life while living with late-stage lung cancer: an exploratory study.

U.S. Veterans have a higher prevalence of advanced lung cancer and poorer survival outcomes compared to the general population; yet, no studies exist which specifically explore the psychosocial and existential quality-of-life (QOL) of late-stage lung cancer among this population.

Integrative molecular profiling reveals asparagine synthetase is a target in castration-resistant prostate cancer.

The identification of new and effective therapeutic targets for the lethal, castration-resistant stage of prostate cancer (CRPC) has been challenging because of both the paucity of adequate frozen tissues and a lack of integrated molecular analysis. Therefore, in this study, we performed a genome-wide analysis of DNA copy number alterations from 34 unique surgical CRPC specimens and 5 xenografts, with matched transcriptomic profiling of 25 specimens. An integrated analysis of these data revealed that the asparagine synthetase (ASNS) gene showed a gain in copy number and was overexpressed at the transcript level.

A simple defined medium for growth and maintenance of the mixotrophic protist Ochromonas danica.

A simple-defined medium was formulated that allows robust axenic-growth of the model mixotrophic protist Ochromonas danica at a neutral pH. This new defined medium, with a minimum number of constituents, facilitates more highly controlled studies of mixotrophic metabolism and nutrient regeneration than have previously been possible.

Working together, nurses can make a difference in resuscitation outcomes: an update on the American Heart Association's 2010 guidelines for cardiopulmonary resuscitation and emergency cardiovascular care.

The International Liaison Committee on Resuscitation (ILCOR) was formed in 1992 to provide a forum for resuscitation organizations worldwide. According to its mission, ILCOR provides a mechanism by which international science and knowledge that are relevant to cardiopulmonary resuscitation (CPR) and emergency cardiovascular care (ECC) can be identified, reviewed, and evaluated. Every 5 years, based on a rigorous review of the published, peer-reviewed science, ILCOR develops a consensus on resuscitation science and carries that forward into internationally agreed-on treatment recommendations.

Characterization of in vivo anti-rotavirus activities of saponin extracts from Quillaja saponaria Molina.

Rotavirus is the leading cause of severe diarrhea disease in newborns and young children worldwide with approximately 300,000 pre-adolescent deaths each year. Quillaja saponins are a natural aqueous extract obtained from the Chilean soapbark tree. The extract is approved for use in humans by the FDA for use in beverages as a food addictive.

Enhanced functional brain imaging by using adaptive filtering and a depth compensation algorithm in diffuse optical tomography.

Reflectance diffuse optical tomography (rDOT) of brain function is limited by its high sensitivity to the superficial tissues (i.e., the scalp and skull) and by its severe decrease in measurement sensitivity with increased depth.

Evolution of the Schlafen genes, a gene family associated with embryonic lethality, meiotic drive, immune processes and orthopoxvirus virulence.

Genes of the Schlafen family, first discovered in mouse, are expressed in hematopoietic cells and are involved in immune processes. Previous results showed that they are candidate genes for two major phenomena: meiotic drive and embryonic lethality (DDK syndrome). However, these genes remain poorly understood, mostly due to the limitations imposed by their similarity, close location and the potential functional redundancy of the gene family members.

Epigenetic, transposon and small RNA determinants of hybrid dysfunctions.

Accumulating evidence suggests that hybrid genetic dysfunctions accrue not only because of sequence divergence of incompatible alleles but also result from a broad variety of mechanisms related to the maintenance of chromatin integrity. For example, it has been observed that hybridization in plants and mammals disrupts patterns of DNA methylation and imprinting. These epigenetic changes can be associated with transcriptional activation and mobilization of transposable elements in hybrids.

Gene expression analysis of the ovary of hybrid females of Xenopus laevis and X. muelleri.

Background: Interspecific hybrids of frogs of the genus Xenopus result in sterile hybrid males and fertile hybrid females. Previous work has demonstrated a dramatic asymmetrical pattern of misexpression in hybrid males compared to the two parental species with relatively few genes misexpressed in comparisons of hybrids and the maternal species (X. laevis) and dramatically more genes misexpressed in hybrids compared to the paternal species (X.

Features of the mammalian orthoreovirus 3 Dearing l1 single-stranded RNA that direct packaging and serotype restriction.

A series of recombinant mammalian orthoreoviruses (mammalian orthoreovirus 3 Dearing, MRV-3De) were generated that express an MRV-3De lambda3-CAT fusion protein. Individual viruses contain L1CAT double-stranded (ds) RNAs that range in length from a minimum of 1020 bp to 4616 bp. The engineered dsRNAs were generated from in vitro-transcribed single-stranded (ss) RNAs and incorporated into infectious virus particles by using reverse genetics.

Antiviral activity obtained from aqueous extracts of the Chilean soapbark tree (Quillaja saponaria Molina).

Natural, aqueous extracts of Quillaja saponaria, the Chilean soapbark tree, contain several physiologically active triterpenoid saponins that display strong adjuvant activity when used in either human or animal vaccines. In this paper, we describe studies that demonstrate a novel antiviral activity of Quillaja extracts against six viruses: vaccinia virus, herpes simplex virus type 1, varicella zoster virus, human immunodeficiency viruses 1 and 2 (HIV-1, HIV-2) and reovirus. We demonstrate that microgram amounts of extract, while exhibiting no cell cytotoxicity or direct virucidal activity, prevent each of the six viruses tested from infecting their host cells.