Identification of Salmonella Typhimurium Deubiquitinase SseL Substrates by Immunoaffinity Enrichment and Quantitative Proteomic Analysis.

Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here, we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification, and global quantitative proteomic analysis. As a model system to identify substrates, we used a virulence-related deubiquitinase, SseL, secreted by Salmonella enterica serovar Typhimurium into host cells. Using this approach, two SseL substrates were identified in the RAW 264.7 murine macrophage-like cell line, S100A6 and heterogeneous nuclear ribonuclear protein K, in addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. This method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.

Multi-omic data integration links deleted in breast cancer 1 (DBC1) degradation to chromatin remodeling in inflammatory response.

This study investigated the dynamics of ubiquitinated proteins after the inflammatory stimulation of RAW 264.7 macrophage-like cells with bacterial lipopolysaccharide. Ubiquitination is a common protein post-translational modification that regulates many key cellular functions. We demonstrated that levels of global ubiquitination and K48 and K63 polyubiquitin chains change after lipopolysaccharide stimulation. Quantitative proteomic analysis identified 1199 ubiquitinated proteins, 78 of which exhibited significant changes in ubiquitination levels following stimulation. Integrating the ubiquitinome data with global proteomic and transcriptomic results allowed us to identify a subset of 88 proteins that were targeted for degradation after lipopolysaccharide stimulation. Using cellular assays and Western blot analyses, we biochemically validated DBC1 (a histone deacetylase inhibitor) as a degradation substrate that is targeted via an orchestrated mechanism utilizing caspases and the proteasome. The degradation of DBC1 releases histone deacetylase activity, linking lipopolysaccharide activation to chromatin remodeling in caspase- and proteasome-mediated signaling.

Management of gastroesophageal reflux disease.

Gastroesophageal reflux disease (GERD) continues to be one of the most prevalent gastrointestinal tract disorders. Management of GERD is individualized for each patient depending on severity of symptoms, complications of GERD and patient/physician preference. The different management options include life style modification, pharmacological therapy, minimally invasive procedures and surgery. The final decision regarding management should be made based on an individualized patient centered approach on a case-by-case basis in consultation with a multidisciplinary team including primary care physician, gastroenterologist and surgeon. We provide a comprehensive review for the management of GERD.

Accuracy of endoscopic ultrasound in esophageal cancer staging.

Since its advent in the 1980s endoscopic ultrasound (EUS) has played an important role in the diagnosis, staging, and therapeutic management of various gastrointestinal malignancies. EUS has emerged as a vital tool in the evaluation of esophageal cancer as it provides a detailed view of the layers of the esophageal wall and surrounding tissues. This permits determination of tumor invasion depth and local lymph node metastases. It is the most sensitive and specific method available for locoregional staging of esophageal cancer. The information obtained via EUS is vital in determining the appropriate diagnosis, prognosis, and treatment options. Thus, this article aims to present a review of the accuracy and utilization of EUS in the staging of esophageal cancer.

Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter.

Many pathogenic bacteria of the family Enterobacteriaceae use type III secretion systems to inject virulence proteins, termed "effectors," into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from the Enterobacteriaceae intracellular pathogens Salmonella enterica serovar Typhimurium and Citrobacter rodentium. We identified 54 high-confidence host interactors for the Salmonella effectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for the Citrobacter effectors EspT, NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfH Salmonella protein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction. IMPORTANCE During infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets of Salmonella and Citrobacter effectors, which will help elucidate their mechanisms of action.

Improved proteomic analysis following trichloroacetic acid extraction of Bacillus anthracis spore proteins.

Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Analysis of cellular proteins is dependent upon efficient extraction from bacterial samples, which can be challenging with increasing complexity and refractory characteristics. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrichment for certain classes of proteins. The method presented here is technically simple, does not require specialized equipment such as a mechanical disrupter, and is effective for protein extraction of the particularly challenging sample type of Bacillus anthracis Sterne spores. The ability of Trichloroacetic acid (TCA) extraction to isolate proteins from spores and enrich for spore-specific proteins was compared to the traditional mechanical disruption method of bead beating. TCA extraction improved the total average number of proteins identified within a sample as compared to bead beating (547 vs 495, respectively). Further, TCA extraction enriched for 270 spore proteins, including those typically identified by first isolating the spore coat and exosporium layers. Bead beating enriched for 156 spore proteins more typically identified from whole spore proteome analyses. The total average number of proteins identified was equal using TCA or bead beating for easily lysed samples, such as B. anthracis vegetative cells. As with all assays, supplemental methods such as implementation of an alternative preparation method may simplify sample preparation and provide additional insight to the protein biology of the organism being studied.

A method to determine lysine acetylation stoichiometries.

Lysine acetylation is a common protein posttranslational modification that regulates a variety of biological processes. A major bottleneck to fully understanding the functional aspects of lysine acetylation is the difficulty in measuring the proportion of lysine residues that are acetylated. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of protein lysine acetylation. Using this technique, we determined the modification occupancy for ~750 acetylated peptides from mammalian cell lysates. Furthermore, the acetylation on N-terminal tail of histone H4 was cross-validated by treating cells with sodium butyrate, a potent deacetylase inhibitor, and comparing changes in stoichiometry levels measured by our method with immunoblotting measurements. Of note we observe that acetylation stoichiometry is high in nuclear proteins, but very low in mitochondrial and cytosolic proteins. In summary, our method opens new opportunities to study in detail the relationship of lysine acetylation levels of proteins with their biological functions.

Optical scattering and backscattering by organic and inorganic particulates in U.S. coastal waters.

We present the results of a study of optical scattering and backscattering of particulates for three coastal sites that represent a wide range of optical properties that are found in U.S. near-shore waters. The 6000 scattering and backscattering spectra collected for this study can be well approximated by a power-law function of wavelength. The power-law exponent for particulate scattering changes dramatically from site to site (and within each site) compared with particulate backscattering where all the spectra, except possibly the very clearest waters, cluster around a single wavelength power-law exponent of -0.94. The particulate backscattering-to-scattering ratio (the backscattering ratio) displays a wide range in wavelength dependence. This result is not consistent with scattering models that describe the bulk composition of water as a uniform mix of homogeneous spherical particles with a Junge-like power-law distribution over all particle sizes. Simultaneous particulate organic matter (POM) and particulate inorganic matter (PIM) measurements are available for some of our optical measurements, and site-averaged POM and PIM mass-specific cross sections for scattering and backscattering can be derived. Cross sections for organic and inorganic material differ at each site, and the relative contribution of organic and inorganic material to scattering and backscattering depends differently at each site on the relative amount of material that is present.

Relationship between mercury accumulation in young-of-the-year yellow perch and water-level fluctuations.

A three-year (2001-2003) monitoring effort of 14 northeastern Minnesota lakes was conducted to document relationships between water-level fluctuations and mercury bioaccumulation in young-of-the-year (YOY) yellow perch (Perca flavescens) collected in the fall of each year at fixed locations. Six of those lakes are located within or adjacent to Voyageurs National Park and are influenced by dams on the outlets of Rainy and Namakan lakes. One site on Sand Point Lake coincides with a location that has nine years of previous monitoring suitable for addressing the same issue over a longer time frame. Mean mercury concentrations in YOY yellow perch at each sampling location varied significantly from year to year. For the 12-year monitoring site on Sand Point Lake, values ranged from 38 ng gww(-1) in 1998 to 200 ng gww(-1) in 2001. For the 14-lake study, annual mean concentrations ranged by nearly a factor of 2, on average, for each lake over the three years of record. One likely factor responsible for these wide variations is that annual water-level fluctuations are strongly correlated with mercury levels in YOY perch for both data sets.

Uniqueness in remote sensing of the inherent optical properties of ocean water.

We examine the problem of uniqueness in the relationship between the remote-sensing reflectance (Rrs) and the inherent optical properties (IOPs) of ocean water. The results point to the fact that diffuse reflectance of plane irradiance from ocean water is inherently ambiguous. Furthermore, in the 400 < lambda < 750 nm region of the spectrum, Rrs(lambda) also suffers from ambiguity caused by the similarity in wavelength dependence of the coefficients of absorption by particulate matter and of absorption by colored dissolved organic matter. The absorption coefficients have overlapping exponential responses, which lead to the fact that more than one combination of IOPs can produce nearly the same Rrs spectrum. This ambiguity in absorption parameters demands that we identify the regions of the Rrs spectrum where we can isolate the effects that are due only to scattering by particulates and to absorption by pure water. The results indicate that the spectral shape of the absorption coefficient of phytoplankton, a(ph)(lambda), cannot be derived from a multiparameter fit to Rrs(lambda). However, the magnitude and the spectral dependence of the absorption coefficient can be estimated from the difference between the measured Rrs(lambda) and the best fit to Rrs(lambda) in terms of IOPs that exclude a(ph)(lambda).