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Extracellular vesicles (EVs) play important roles in cell-to-cell communications and carry high potential as markers targeted in disease diagnosis, prognosis, and therapeutic development. The main obstacles to EV study are their high heterogeneity; low amounts present in samples; and physical similarity to the abundant, interfering matrix components. Multiple rounds of separation and purification are often needed prior to EV characterization and function assessment. Herein, we report the offline coupling of asymmetrical flow field-flow fractionation (AF4) and capillary electrophoresis (CE) for EV analysis. While AF4 provides gentle and fast EV separation by size, CE resolves EVs from contaminants with similar sizes but different surface charges. Employing Western Blotting, ELISA, and SEM, we confirmed that intact EVs were eluted within a stable time window under the optimal AF4 and CE conditions. We also proved that EVs could be resolved from free proteins and high-density lipoproteins by AF4 and be further separated from the low-density lipoproteins co-eluted in AF4. The effectiveness of the coupled AF4-CE system in EV analysis was demonstrated by monitoring the changes in EV secretion from cells and by direct injection of human serum and detection of serum EVs. We believe that coupling AF4 and CE can provide rapid EV quantification in biological samples with much reduced matrix interference and be valuable for the study of total EVs and EV subpopulations produced by cells or present in clinical samples.

Acetylcholinesterase (EC 3.1.1.7), a key acetylcholine-hydrolyzing enzyme in cholinergic neurotransmission, is present in a variety of states in situ, including monomers, C-terminally disulfide-linked homodimers, homotetramers, and up to three tetramers covalently attached to structural subunits. Could oligomerization that ensures high local concentrations of catalytic sites necessary for efficient neurotransmission be affected by environmental factors? Using small-angle X-ray scattering (SAXS) and cryo-EM, we demonstrate that homodimerization of recombinant monomeric human acetylcholinesterase (hAChE) in solution occurs through a C-terminal four-helix bundle at micromolar concentrations. We show that diethylphosphorylation of the active serine in the catalytic gorge or isopropylmethylphosphonylation by the RP enantiomer of sarin promotes a 10-fold increase in homodimer dissociation. We also demonstrate the dissociation of organophosphate (OP)-conjugated dimers is reversed by structurally diverse oximes 2PAM, HI6, or RS194B, as demonstrated by SAXS of diethylphosphoryl-hAChE. However, binding of oximes to the native ligand-free hAChE, binding of high-affinity reversible ligands, or formation of an SP-sarin-hAChE conjugate had no effect on homodimerization. Dissociation monitored by time-resolved SAXS occurs in milliseconds, consistent with rates of hAChE covalent inhibition. OP-induced dissociation was not observed in the SAXS profiles of the double-mutant Y337A/F338A, where the active center gorge volume is larger than in wildtype hAChE. These observations suggest a key role of the tightly packed acyl pocket in allosterically triggered OP-induced dimer dissociation, with the potential for local reduction of acetylcholine-hydrolytic power in situ. Computational models predict allosteric correlated motions extending from the acyl pocket toward the four-helix bundle dimerization interface 25 Å away.

High-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.

Immunoassays and mass spectrometry are powerful single-cell protein analysis tools; however, interfacing and throughput bottlenecks remain. Here, we introduce three-dimensional single-cell immunoblots to detect both cytosolic and nuclear proteins. The 3D microfluidic device is a photoactive polyacrylamide gel with a microwell array-patterned face (xy) for cell isolation and lysis. Single-cell lysate in each microwell is "electrophoretically projected" into the 3rd dimension (z-axis), separated by size, and photo-captured in the gel for immunoprobing and confocal/light-sheet imaging. Design and analysis are informed by the physics of 3D diffusion. Electrophoresis throughput is > 2.5 cells/s (70× faster than published serial sampling), with 25 immunoblots/mm2 device area (>10× increase over previous immunoblots). The 3D microdevice design synchronizes analyses of hundreds of cells, compared to status quo serial analyses that impart hours-long delay between the first and last cells. Here, we introduce projection electrophoresis to augment the heavily genomic and transcriptomic single-cell atlases with protein-level profiling.

Doxorubicin (DOX) is one of the most widely used anticancer agents. DOX is known for inducing cardiotoxicity, resulting in the long-term development of heart failure. Intravascular delivery of DOX may benefit from the carriage by red blood cells (RBCs), as they can limit the systemic toxicity while delivering the DOX to the tumor. This study proposes a methodology for the synthesis of electrophoretically DOX-loaded red blood cells (RBC-DOX), as well as the assessment of its antitumorigenic effects in human colon cancer cells (HT-29), and in colon cancer xenograft models. In addition, healthy mice without tumors were dosed with RBC-DOX to assess cardiotoxicity via assessment of indexes of cardiac function after multiple doses of RBC-DOX. The HT-29 IC50 was found to be lower for RBC-DOX compared to free DOX. Tumor volume for the RBC-DOX group was smaller than the free DOX groups in HT-29 xenografts models. Statistically higher concentrations of DOX were found in the liver, spleen, and lungs for the RBC-DOX group compared to the free DOX group. However, the heart and the skin had statistically lower DOX concentrations for the RBC-DOX group compared to the free DOX group, with no significant differences in tumor biodistribution. All hemodynamic and cardiac function parameters were closer to control parameters for the RBC-DOX treated compared to for the free DOX-treated mice. These results suggest that RBC-DOX can be an alternative to prolong treatments with DOX, with superior antitumorigenic effects, decreased myelosuppression, and limited cardiac toxicity compared to equivalent doses of free DOX.

Immunoassays such as ELISAs and Western blotting have been the common choice for protein validation studies for the past several decades. Technical advancements and modifications are continuously being developed to enhance the detection sensitivity of these procedures. Among them, Streptavidin-containing poly-horseradish peroxidase (PolyHRP) based detection strategies have been shown to improve signals in ELISA. The use of commercially available Streptavidin and antibodies conjugated with many HRPs (PolyHRPs) to potentially enhance the detection sensitivity in Western blotting has not been previously investigated in a comprehensive manner. The use of PolyHRP-secondary antibody instead of HRP-secondary antibody increased the Western blotting sensitivity up to 85% depending on the primary antibody used. The use of a biotinylated secondary antibody and commercially available Streptavidin-conjugated with HRP or PolyHRP all resulted in increased sensitivity with respect to antigen detection. Utilizing a biotinylated secondary antibody and Streptavidin-conjugated PolyHRP resulted in as much as a 110-fold increase in Western blotting sensitivity over traditional Western blotting methods. Quantification of troponin I in rat heart lysates showed that the traditional Western blotting method only detected troponin I in ≥2 μg of lysate while Streptavidin-conjugated PolyHRP20 detected troponin I in ≥50 ng of lysate. A modified blocking procedure is also described that eliminated the interference caused by the endogenous biotinylated proteins. These results suggest that Streptavidin-conjugated PolyHRP and PolyHRP secondary antibodies are likely to be commonly utilized for Western blots in the future.

While profiling of cell surface receptors grants valuable insight on cell phenotype, surface receptors alone cannot fully describe activated downstream signaling pathways, detect internalized receptor activity, or indicate constitutively active signaling in subcellular compartments. To measure surface-bound and intracellular targets in the same cell, we introduce a tandem single-cell assay that combines immunofluorescence of surface-bound epithelial cellular adhesion molecule (EpCAM) with subsequent protein polyacrylamide gel electrophoresis (PAGE) of unfixed MCF7 breast cancer cells. After surface staining and cell lysis, surface EpCAM is analyzed by single-cell PAGE, concurrent with immunoprobing of intracellular targets. Consequently, the single-cell electrophoresis step reports localization of both surface and intracellular targets. Unbound intracellular EpCAM is readily resolved from surface EpCAM immunocomplex owing to a ∼30% mobility shift. Flow cytometry and immunofluorescence are in concordance with single-cell PAGE. Lastly, we challenged the stability of the EpCAM immunocomplexes by varying ionic and non-ionic component concentrations in the lysis buffer, the lysis time, and electrophoresis duration. As expected, the harsher conditions proved most disruptive to the immunocomplexes. The compatibility of live-cell immunostaining with single-cell PAGE eliminates the need to perform single-cell imaging by condensing read-out of both surface-bound proteins (as low mobility immune complexes) and intracellular targets to a single immunoblot, thus linking cell type and state.

We utilized pulsed-field gel electrophoresis (PFGE) to purify high-molecular-weight DNA from HIV-infected cells. This purification, in combination with our previously described droplet digital PCR (ddPCR) assay, was used to develop a method to quantify proviral integrated HIV DNA free of lower-molecular-weight species of HIV DNA. Episomal 2-long-terminal-repeat (2-LTR) circles were completely cleared from HIV DNA samples. Technical replicates of the complete assay, starting with the same specimens, resulted in no statistical differences in quantification of integrated HIV gag sequences in cellular DNA from cells from HIV-infected subjects after prolonged treatment with antiretroviral therapy (ART). The PFGE ddPCR assay was compared to the Alu-gag quantitative PCR (qPCR) assay, the most widely used assay to measure proviral integrated HIV DNA. Spearman's rho nonparametric correlation determined PFGE ddPCR results to be positively correlated with Alu-gag qPCR results (r = 0.7052; P = 0.0273). In summary, PFGE ddPCR is a sensitive, reproducible, and robust method to measure proviral integrated HIV DNA and is theoretically more accurate than previously described assays, because it is a direct measure of integrated HIV DNA.

The c-MYC (MYC) oncoprotein is deregulated in over 50% of cancers, yet regulatory mechanisms controlling MYC remain unclear. To this end, we interrogated the MYC interactome using BioID mass spectrometry (MS) and identified PP1 (protein phosphatase 1) and its regulatory subunit PNUTS (protein phosphatase-1 nuclear-targeting subunit) as MYC interactors. We demonstrate that endogenous MYC and PNUTS interact across multiple cell types and that they co-occupy MYC target gene promoters. Inhibiting PP1 by RNAi or pharmacological inhibition results in MYC hyperphosphorylation at multiple serine and threonine residues, leading to a decrease in MYC protein levels due to proteasomal degradation through the canonical SCFFBXW7 pathway. MYC hyperphosphorylation can be rescued specifically with exogenous PP1, but not other phosphatases. Hyperphosphorylated MYC retained interaction with its transcriptional partner MAX, but binding to chromatin is significantly compromised. Our work demonstrates that PP1/PNUTS stabilizes chromatin-bound MYC in proliferating cells.

Purpose:Loss of retinal capillary endothelial cells and pericytes through apoptosis is an early event in diabetic retinopathy (DR). Inflammatory pathways play a role in early DR, yet the biochemical mechanisms are poorly understood. In this study, we investigated the role of indoleamine 2,3-dioxygenase (IDO), an inflammatory cytokine-inducible enzyme, on retinal endothelial apoptosis and capillary degeneration in the diabetic retina. Methods:IDO was detected in human and mouse retinas by immunohistochemistry or Western blotting. Interferon-γ (IFN-γ) levels were measured by ELISA. IDO levels were measured in human retinal capillary endothelial cells (HREC) cultured in the presence of IFN-γ ± 25 mM D-glucose. Reactive oxygen species (ROS) were measured using CM-H2DCFDA dye and apoptosis was measured by cleaved caspase-3. The role of IDO in DR was determined in IDO knockout (IDO-/-) mice with streptozotocin-induced diabetes. Results:The IDO and IFN-γ levels were higher in human diabetic retinas with retinopathy relative to nondiabetic retinas. Immunohistochemical data showed that IDO is present in capillary endothelial cells. IFN-γ upregulated the IDO and ROS levels in HREC. The blockade of either IDO or kynurenine monooxygenase led to inhibition of ROS in HREC. Apoptosis through this pathway was inhibited by an ROS scavenger, TEMPOL. Capillary degeneration was significantly reduced in diabetic IDO-/- mice compared to diabetic wild-type mice. Conclusions:The results suggest that the kynurenine pathway plays an important role in the inflammatory damage in the diabetic retina and could be a new therapeutic target for the treatment of DR.

Tools for measuring affinities and stoichiometries of protein-protein complexes are valuable for elucidating the role of protein-protein interactions (PPIs) in governing cell functions and screening for PPI modulators. Such measurements can be challenging because PPIs can span a wide range of affinities and include stoichiometries from dimers to high order oligomers. Also, most techniques require large amounts of protein which can hamper research for difficult to obtain proteins. Protein cross-linking capillary electrophoresis (PXCE) has the potential to directly measure PPIs and even resolve multiple PPIs while consuming attomole quantities. Previously PXCE has only been used for high affinity, 1 : 1 complexes; here we expand the utility of PXCE to access a wide range of PPIs including weak and multimeric oligomers. Use of glutaraldehyde as the cross-linking agent was key to advancing the method because of its rapid reaction kinetics. A 10 s reaction time was found to be sufficient for cross-linking and quantification of seven different PPIs with Kd values ranging from low μM to low nM including heat shock protein 70 (Hsp70) interacting with heat shock organizing protein (3.8 ± 0.7 μM) and bcl2 associated anthanogene (26 ± 6 nM). Non-specific cross-linking of protein aggregates was found to be minimal at protein concentrations

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

BackgroundBiochemical analysis of mutant huntingtin (mHTT) aggregation species in HD mice is a common measure to track disease. A longitudinal and systematic study of how tissue processing affects detection of conformers has not yet been reported. Understanding the homeostatic flux of mHTT over time and under different processing conditions would aid in interpretation of pre-clinical assessments of disease interventions.ObjectiveProvide a systematic evaluation of tissue lysis methods and molecular and biochemical assays in parallel with behavioral readouts in R6/2 mice to establish a baseline for HTT exon1 protein accumulation.MethodsEstablished biochemical methods were used to process tissue from R6/2 mice of specific ages following behavior tasks. Aggregation states and accumulation of mHTT exon 1 protein were evaluated using multiple break and assay methods to determine potential conformational flux assay specificity in detection of mHTT species, and tissue specificity of conformers.ResultsDetection of mHTT exon 1 protein species varied based on biochemical processing and analysis providing a baseline for subsequent studies in R6/2 mice. Insoluble, high molecular weight species of mHTT exon 1 protein increased and tracked with onset of behavioral impairments in R6/2 mice using multiple assay methods.ConclusionsConformational flux from soluble monomer to high molecular weight, insoluble species of mHTT exon 1 protein was generally consistent for multiple assay methods throughout R6/2 disease progression; however, the results support the use of multiple biochemical techniques to detect mHTT exon 1 protein species for preclinical assessments in HD mouse models expressing mHTT exon 1 protein.

Protein and peptide oligomers are thought to play important roles in the pathogenesis of a number of neurodegenerative diseases. For this reason, considerable effort has been devoted to understanding the oligomerization process and to determining structure-activity relationships among the many types of oligomers that have been described. We discuss here a method for producing pure populations of amyloid β-protein (Aβ) of specific sizes using the most pathologic form of the peptide, Aβ42. This work was necessitated because Aβ oligomerization produces oligomers of many different sizes that are non-covalently associated, which means that dissociation or further assembly may occur. These characteristics preclude rigorous structure-activity determinations. In studies of Aβ40, we have used the method of photo-induced cross-linking of unmodified proteins (PICUP) to produce zero-length carbon-carbon bonds among the monomers comprising each oligomer, thus stabilizing the oligomers. We then isolated pure populations of oligomers by fractionating the oligomers by size using SDS-PAGE and then extracting each population from the stained gel bands. Although this procedure worked well with the shorter Aβ40 peptide, we found that a significant percentage of Aβ42 oligomers had not been stabilized. Here, we discuss a new method capable of yielding stable Aβ42 oligomers of sizes from dimer through dodecamer.

The Ewing Sarcoma protein (EWS) is a multifaceted RNA binding protein (RBP) with established roles in transcription, pre-mRNA processing and DNA damage response. By generating high quality EWS-RNA interactome, we uncovered its specific and prevalent interaction with a large subset of primary microRNAs (pri-miRNAs) in mammalian cells. Knockdown of EWS reduced, whereas overexpression enhanced, the expression of its target miRNAs. Biochemical analysis revealed that multiple elements in target pri-miRNAs, including the sequences flanking the stem-loop region, contributed to high affinity EWS binding and sequence swap experiments between target and non-target demonstrated that the flanking sequences provided the specificity for enhanced pri-miRNA processing by the Microprocessor Drosha/DGCR8. Interestingly, while repressing Drosha expression, as reported earlier, we found that EWS was able to enhance the recruitment of Drosha to chromatin. Together, these findings suggest that EWS may positively and negatively regulate miRNA biogenesis via distinct mechanisms, thus providing a new foundation to understand the function of EWS in development and disease.

IntroductionWestern blotting is one of the most commonly used techniques in molecular biology and proteomics. Since western blotting is a multistep protocol, variations and errors can occur at any step reducing the reliability and reproducibility of this technique. Recent reports suggest that a few key steps, such as the sample preparation method, the amount and source of primary antibody used, as well as the normalization method utilized, are critical for reproducible western blot results. Areas covered: In this review, improvements in different areas of western blotting, including protein transfer and antibody validation, are summarized. The review discusses the most advanced western blotting techniques available and highlights the relationship between next generation western blotting techniques and its clinical relevance. Expert commentary: Over the last decade significant improvements have been made in creating more sensitive, automated, and advanced techniques by optimizing various aspects of the western blot protocol. New methods such as single cell-resolution western blot, capillary electrophoresis, DigiWest, automated microfluid western blotting and microchip electrophoresis have all been developed to reduce potential problems associated with the western blotting technique. Innovative developments in instrumentation and increased sensitivity for western blots offer novel possibilities for increasing the clinical implications of western blot.

Despite years of fully suppressive antiretroviral therapy (ART), HIV persists in its hosts and is never eradicated. One major barrier to eradication is that the virus infects multiple cell types that may individually contribute to HIV persistence. Tissue macrophages are critical contributors to HIV pathogenesis; however, their specific role in HIV persistence during long-term suppressive ART has not been established. Using humanized myeloid-only mice (MoM), we demonstrate that HIV infection of tissue macrophages is rapidly suppressed by ART, as reflected by a rapid drop in plasma viral load and a dramatic decrease in the levels of cell-associated viral RNA and DNA. No viral rebound was observed in the plasma of 67% of the ART-treated animals at 7 weeks after ART interruption, and no replication-competent virus was rescued from the tissue macrophages obtained from these animals. In contrast, in a subset of animals (∼33%), a delayed viral rebound was observed that is consistent with the establishment of persistent infection in tissue macrophages. These observations represent the first direct evidence, to our knowledge, of HIV persistence in tissue macrophages in vivo.

AimExtended-spectrum β-lactamase-producing (ESBL) Escherichia coli are a public threat worldwide. This study aimed at analyzing the genomic and functional attributes of nine ESBLs taken from rectal swabs.Materials & methodsSamples were isolated from patients admitted for gastrointestinal and urological procedures at the University Medical Center-Rizk Hospital (UMCRH) in Lebanon. Illumina paired-end libraries were prepared and sequenced.ResultsThe isolates were distributed into five lineages: ST131, ST648, ST405, ST73 and ST38, and harbored bla OXA-1, bla TEM-1B, bla TEM-1C and aac(6')Ib-cr. ST131 isolates were carriers of stx2 converting I phage.ConclusionThis is the first comprehensive genomic analysis performed on ESBLs in Lebanon.

AimExtended-spectrum β-lactamase-producing (ESBL) Escherichia coli are a public threat worldwide. This study aimed at analyzing the genomic and functional attributes of nine ESBLs taken from rectal swabs.Materials & methodsSamples were isolated from patients admitted for gastrointestinal and urological procedures at the University Medical Center-Rizk Hospital (UMCRH) in Lebanon. Illumina paired-end libraries were prepared and sequenced.ResultsThe isolates were distributed into five lineages: ST131, ST648, ST405, ST73 and ST38, and harbored bla OXA-1, bla TEM-1B, bla TEM-1C and aac(6')Ib-cr. ST131 isolates were carriers of stx2 converting I phage.ConclusionThis is the first comprehensive genomic analysis performed on ESBLs in Lebanon.

Pancreatic islet β-cells are particularly susceptible to endoplasmic reticulum (ER) stress, which is implicated in β-cell dysfunction and loss during the pathogenesis of type 1 diabetes (T1D). The peripheral membrane protein GAD65 is an autoantigen in human T1D. GAD65 synthesizes γ-aminobutyric acid, an important autocrine and paracrine signaling molecule and a survival factor in islets. We show that ER stress in primary β-cells perturbs the palmitoylation cycle controlling GAD65 endomembrane distribution, resulting in aberrant accumulation of the palmitoylated form in trans-Golgi membranes. The palmitoylated form has heightened immunogenicity, exhibiting increased uptake by antigen-presenting cells and T-cell stimulation compared with the nonpalmitoylated form. Similar accumulation of GAD65 in Golgi membranes is observed in human β-cells in pancreatic sections from GAD65 autoantibody-positive individuals who have not yet progressed to clinical onset of T1D and from patients with T1D with residual β-cell mass and ongoing T-cell infiltration of islets. We propose that aberrant accumulation of immunogenic GAD65 in Golgi membranes facilitates inappropriate presentation to the immune system after release from stressed and/or damaged β-cells, triggering autoimmunity.

Many gene expression factors contain repetitive phosphorylation sites for single kinases, but the functional significance is poorly understood. Here we present evidence for hyperphosphorylation as a mechanism allowing UPF1, the central factor in nonsense-mediated decay (NMD), to increasingly attract downstream machinery with time of residence on target mRNAs. Indeed, slowing NMD by inhibiting late-acting factors triggers UPF1 hyperphosphorylation, which in turn enhances affinity for factors linking UPF1 to decay machinery. Mutational analyses reveal multiple phosphorylation sites contributing to different extents to UPF1 activity with no single site being essential. Moreover, the ability of UPF1 to undergo hyperphosphorylation becomes increasingly important for NMD when downstream factors are depleted. This hyperphosphorylation-dependent feedback mechanism may serve as a molecular clock ensuring timely degradation of target mRNAs while preventing degradation of non-targets, which, given the prevalence of repetitive phosphorylation among central gene regulatory factors, may represent an important general principle in gene expression.

The 1966 GENETICS papers by John Hubby and Richard Lewontin were a landmark in the study of genome-wide levels of variability. They used the technique of gel electrophoresis of enzymes and proteins to study variation in natural populations of Drosophila pseudoobscura, at a set of loci that had been chosen purely for technical convenience, without prior knowledge of their levels of variability. Together with the independent study of human populations by Harry Harris, this seminal study provided the first relatively unbiased picture of the extent of genetic variability in protein sequences within populations, revealing that many genes had surprisingly high levels of diversity. These papers stimulated a large research program that found similarly high electrophoretic variability in many different species and led to statistical tools for interpreting the data in terms of population genetics processes such as genetic drift, balancing and purifying selection, and the effects of selection on linked variants. The current use of whole-genome sequences in studies of variation is the direct descendant of this pioneering work.

The virtual two-dimensional gel electrophoresis/mass spectrometry (virtual 2D gel/MS) technology combines the premier, high-resolution capabilities of 2D gel electrophoresis with the sensitivity and high mass accuracy of mass spectrometry (MS). Intact proteins separated by isoelectric focusing (IEF) gel electrophoresis are imaged from immobilized pH gradient (IPG) polyacrylamide gels (the first dimension of classic 2D-PAGE) by matrix-assisted laser desorption/ionization (MALDI) MS. Obtaining accurate intact masses from sub-picomole-level proteins embedded in 2D-PAGE gels or in IPG strips is desirable to elucidate how the protein of one spot identified as protein 'A' on a 2D gel differs from the protein of another spot identified as the same protein, whenever tryptic peptide maps fail to resolve the issue. This task, however, has been extremely challenging. Virtual 2D gel/MS provides access to these intact masses. Modifications to our matrix deposition procedure improve the reliability with which IPG gels can be prepared; the new procedure is described. Development of this MALDI MS imaging (MSI) method for high-throughput MS with integrated 'top-down' MS to elucidate protein isoforms from complex biological samples is described and it is demonstrated that a 4-cm IPG gel segment can now be imaged in approximately 5min. Gel-wide chemical and enzymatic methods with further interrogation by MALDI MS/MS provide identifications, sequence-related information, and post-translational/transcriptional modification information. The MSI-based virtual 2D gel/MS platform may potentially link the benefits of 'top-down' and 'bottom-up' proteomics.

Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (Δ247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The Δ247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type Δ247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of Δ247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases Δ247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the Δ247-Brr2 RP mutants compared with the wild type Δ247-Brr2. When stimulated by Prp8, wild type Δ247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices.

The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false-positive rates of

Whole genome sequencing (WGS) was compared to pulse-field gel electrophoresis (PFGE) of XbaI-digested genomic DNA, as methods by which to evaluate a potential transmission of carbapenem-resistant Klebsiella pneumoniae between 2 hospital inpatients. PFGE result demonstrated only 1-band difference between the isolates, suggesting probable relatedness. In contrast, while WGS data demonstrated the same sequence type and very similar chromosomal sequences, over 20 single nucleotide variants were identified between the isolates, bringing into question whether there was a transmission event. WGS also identified an additional plasmid, with an XbaI restriction site in the isolates of the second patient that was not identified by PFGE. While WGS provided additional information that was not available by PFGE, in this study, neither method could definitively conclude the relatedness between the isolates.

Optoelectronic tweezers (OET) has advanced within the past decade to become a promising tool for cell and microparticle manipulation. Its incompatibility with high conductivity media and limited throughput remain two major technical challenges. Here a novel manipulation concept and corresponding platform called Self-Locking Optoelectronic Tweezers (SLOT) are proposed and demonstrated to tackle these challenges concurrently. The SLOT platform comprises a periodic array of optically tunable phototransistor traps above which randomly dispersed single cells and microparticles are self-aligned to and retained without light illumination. Light beam illumination on a phototransistor turns off the trap and releases the trapped cell, which is then transported downstream via a background flow. The cell trapping and releasing functions in SLOT are decoupled, which is a unique feature that enables SLOT's stepper-mode function to overcome the small field-of-view issue that all prior OET technologies encountered in manipulation with single-cell resolution across a large area. Massively parallel trapping of more than 100,000 microparticles has been demonstrated in high conductivity media. Even larger scale trapping and manipulation can be achieved by linearly scaling up the number of phototransistors and device area. Cells after manipulation on the SLOT platform maintain high cell viability and normal multi-day divisibility.

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2'-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Pore-gradient microgel arrays enable thousands of parallel high-resolution single-cell protein electrophoresis separations for targets accross a wide molecular mass (25-289 kDa), yet within 1 mm separation distances. Dual crosslinked hydrogels facilitate gel-pore expansion after electrophoresis for efficient and uniform immunoprobing. The photopatterned, light-activated, and acid-expandable hydrogel underpins single-cell protein analysis, here for oncoprotein-related signaling in human breast biopsy.

Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein.

ObjectivesThe aim of this study was to identify the genetic relatedness of multiple drug-resistant (MDR) Acinetobacter baumannii clinical isolates recovered from a hospital in Los Angeles.MethodsTwenty-one MDR A. baumannii isolates were collected and their antibiotic susceptibilities determined according to Clinical and Laboratory Standards Institute guidelines. Genes coding for antibiotic resistance were identified by PCR, and their identities were confirmed by DNA sequencing. Clonal relationships were studied by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).ResultsMDR consistently correlated with the presence of oxacillinases, mostly in the form of the plasmid-mediated OXA-23 enzyme, which was detected in 12 (57.1%) isolates. GES-type carbapenemases were found in 20 (95.2%) strains, AAC in all 21 (100%) strains, and PER in seven (33.3%) strains, and ISAba1 was detected in 16 (76.2%) isolates. The association between ISAba1 and resistance genes confirms insertion elements as a source of β-lactamase production. Of the 21 clinical isolates, five were found to be related to sequence type 1 (ST1) and 16 to ST2, as analyzed by MLST. PFGE demonstrated that the majority of clinical isolates were highly related (>85%).ConclusionsThis study supports a more complete understanding of genotyping of antibiotic resistance for better assessment of MDR strain transmission.

Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a "lateral e-flow assay" and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to selectively immobilize unmodified capture antigen along the microchannel in a barcode-like pattern. The channel-filling polyacrylamide hydrogel incorporates a photoactive moiety (benzophenone) to immobilize capture antigen to the hydrogel without a priori antigen modification. We report a heterogeneous sandwich assay using low-power electrophoresis to drive biospecimen through the capture antigen barcode. Fluorescence barcode readout is collected via a low-resource appropriate imaging system (CellScope). We characterize lateral e-flow assay performance and demonstrate a serum assay for antibodies to the hepatitis C virus (HCV). In a pilot study, the lateral e-flow assay positively identifies HCV+ human sera in 60 min. The lateral e-flow assay provides a flexible format for conducting multiplexed immunoassays relevant to confirmatory diagnosis in near-patient settings.

Autophagy, the process for recycling cytoplasm in the lysosome, depends on membrane trafficking. We previously identified Drosophila Sbf as a Rab21 guanine nucleotide exchange factor (GEF) that acts with Rab21 in endosomal trafficking. Here, we show that Sbf/MTMR13 and Rab21 have conserved functions required for starvation-induced autophagy. Depletion of Sbf/MTMR13 or Rab21 blocked endolysosomal trafficking of VAMP8, a SNARE required for autophagosome-lysosome fusion. We show that starvation induces Sbf/MTMR13 GEF and RAB21 activity, as well as their induced binding to VAMP8 (or closest Drosophila homolog, Vamp7). MTMR13 is required for RAB21 activation, VAMP8 interaction and VAMP8 endolysosomal trafficking, defining a novel GEF-Rab-effector pathway. These results identify starvation-responsive endosomal regulators and trafficking that tunes membrane demands with changing autophagy status.

BACKGROUND: The magnitude and scope of Clostridium difficile infection in the United States continue to evolve. METHODS: In 2011, we performed active population- and laboratory-based surveillance across 10 geographic areas in the United States to identify cases of C. difficile infection (stool specimens positive for C. difficile on either toxin or molecular assay in residents ≥ 1 year of age). Cases were classified as community-associated or health care-associated. In a sample of cases of C. difficile infection, specimens were cultured and isolates underwent molecular typing. We used regression models to calculate estimates of national incidence and total number of infections, first recurrences, and deaths within 30 days after the diagnosis of C. difficile infection. RESULTS: A total of 15,461 cases of C. difficile infection were identified in the 10 geographic areas; 65.8% were health care-associated, but only 24.2% had onset during hospitalization. After adjustment for predictors of disease incidence, the estimated number of incident C. difficile infections in the United States was 453,000 (95% confidence interval [CI], 397,100 to 508,500). The incidence was estimated to be higher among females (rate ratio, 1.26; 95% CI, 1.25 to 1.27), whites (rate ratio, 1.72; 95% CI, 1.56 to 2.0), and persons 65 years of age or older (rate ratio, 8.65; 95% CI, 8.16 to 9.31). The estimated number of first recurrences of C. difficile infection was 83,000 (95% CI, 57,000 to 108,900), and the estimated number of deaths was 29,300 (95% CI, 16,500 to 42,100). The North American pulsed-field gel electrophoresis type 1 (NAP1) strain was more prevalent among health care-associated infections than among community-associated infections (30.7% vs. 18.8%, P

A considerable body of evidence supports the role of mitochondrial dysfunction in psychiatric disorders and mitochondrial DNA (mtDNA) mutations are known to alter brain energy metabolism, neurotransmission, and cause neurodegenerative disorders. Genetic studies focusing on common nuclear genome variants associated with these disorders have produced genome wide significant results but those studies have not directly studied mtDNA variants. The purpose of this study is to investigate, using next generation sequencing, the involvement of mtDNA variation in bipolar disorder, schizophrenia, major depressive disorder, and methamphetamine use. MtDNA extracted from multiple brain regions and blood were sequenced (121 mtDNA samples with an average of 8,800x coverage) and compared to an electronic database containing 26,850 mtDNA genomes. We confirmed novel and rare variants, and confirmed next generation sequencing error hotspots by traditional sequencing and genotyping methods. We observed a significant increase of non-synonymous mutations found in individuals with schizophrenia. Novel and rare non-synonymous mutations were found in psychiatric cases in mtDNA genes: ND6, ATP6, CYTB, and ND2. We also observed mtDNA heteroplasmy in brain at a locus previously associated with schizophrenia (T16519C). Large differences in heteroplasmy levels across brain regions within subjects suggest that somatic mutations accumulate differentially in brain regions. Finally, multiplasmy, a heteroplasmic measure of repeat length, was observed in brain from selective cases at a higher frequency than controls. These results offer support for increased rates of mtDNA substitutions in schizophrenia shown in our prior results. The variable levels of heteroplasmic/multiplasmic somatic mutations that occur in brain may be indicators of genetic instability in mtDNA.

Expression of hTS (human thymidylate synthase), a key enzyme in thymidine biosynthesis, is regulated on the translational level through a feedback mechanism that is rarely found in eukaryotes. At low substrate concentrations, the ligand-free enzyme binds to its own mRNA and stabilizes a hairpin structure that sequesters the start codon. When in complex with dUMP (2'-deoxyuridine-5'-monophosphate) and a THF (tetrahydrofolate) cofactor, the enzyme adopts a conformation that is unable to bind and repress expression of mRNA. Here, we have used a combination of X-ray crystallography, RNA mutagenesis and site-specific cross-linking studies to investigate the molecular recognition of TS mRNA by the hTS enzyme. The interacting mRNA region was narrowed to the start codon and immediately flanking sequences. In the hTS enzyme, a helix-loop-helix domain on the protein surface was identified as the putative RNA-binding site.

Down syndrome (DS) is one of the most common causes of intellectual disability, owing to trisomy of all or part of chromosome 21. DS is also associated with the development of Alzheimer disease (AD) neuropathology after the age of 40 years. To better clarify the cellular and metabolic pathways that could contribute to the differences in DS brain, in particular those involved in the onset of neurodegeneration, we analyzed the frontal cortex of DS subjects with or without significant AD pathology in comparison with age-matched controls, using a proteomics approach. Proteomics represents an advantageous tool to investigate the molecular mechanisms underlying the disease. From these analyses, we investigated the effects that age, DS, and AD neuropathology could have on protein expression levels. Our results show overlapping and independent molecular pathways (including energy metabolism, oxidative damage, protein synthesis, and autophagy) contributing to DS, to aging, and to the presence of AD pathology in DS. Investigation of pathomechanisms involved in DS with AD may provide putative targets for therapeutic approaches to slow the development of AD.

Protein arginine methyltransferase 7 (PRMT7) methylates arginine residues on various protein substrates and is involved in DNA transcription, RNA splicing, DNA repair, cell differentiation, and metastasis. The substrate sequences it recognizes in vivo and the enzymatic mechanism behind it, however, remain to be explored. Here we characterize methylation catalyzed by a bacterially expressed GST-tagged human PRMT7 fusion protein with a broad range of peptide and protein substrates. After confirming its type III activity generating only ω-N(G)-monomethylarginine and its distinct substrate specificity for RXR motifs surrounded by basic residues, we performed site-directed mutagenesis studies on this enzyme, revealing that two acidic residues within the double E loop, Asp-147 and Glu-149, modulate the substrate preference. Furthermore, altering a single acidic residue, Glu-478, on the C-terminal domain to glutamine nearly abolished the activity of the enzyme. Additionally, we demonstrate that PRMT7 has unusual temperature dependence and salt tolerance. These results provide a biochemical foundation to understanding the broad biological functions of PRMT7 in health and disease.

Papain has long been known to cause the gelation of mammalian fibrinogens. It has also been reported that papain-fibrin is insoluble in dispersing solvents like strong urea or sodium bromide solutions, similar to what is observed with thrombin-generated clots in the presence of factor XIIIa and calcium. In those old studies, both the gelation and subsequent clot stabilization were attributed to papain, although the possibility that the second step might be due to contaminating factor XIII in fibrinogen preparations was considered. I have revisited this problem in light of knowledge acquired over the past half-century about thiol proteases like papain, which mostly cleave peptide bonds, and transglutaminases like factor XIIIa that catalyze the formation of ε-lysyl-γ-glutamyl cross-links. Recombinant fibrinogen, inherently free of factor XIII and other plasma proteins, formed a stable gel when treated with papain alone. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the intermolecular cross-linking in papain-fibrin leads to γ-chain dimers, trimers, and tetramers, just as is the case with thrombin-factor XIIIa-stabilized fibrin. Mass spectrometry of bands excised from gels showed that the cross-linked material is quite different from what occurs with factor XIIIa, however. With papain, the cross-linking occurs between γ chains in neighboring protofibrils becoming covalently linked in a "head-to-tail" fashion by a transpeptidation reaction involving the α-amino group of γ-Tyr1 and a papain cleavage site at γ-Gly403 near the carboxy terminus, rather than by the (reciprocal) "tail-to-tail" manner that occurs with factor XIIIa and that depends on cross-links between γ-Lys406 and γ-Gln398.

Intratumor heterogeneity remains a major obstacle to effective cancer therapy and personalized medicine. Current understanding points to differential therapeutic response among subpopulations of tumor cells as a key challenge to successful treatment. To advance our understanding of how this heterogeneity is reflected in cell-to-cell variations in chemosensitivity and expression of drug-resistance proteins, we optimize and apply a new targeted proteomics modality, single-cell western blotting (scWestern), to a human glioblastoma cell line. To acquire both phenotypic and proteomic data on the same, single glioblastoma cells, we integrate high-content imaging prior to the scWestern assays. The scWestern technique supports thousands of concurrent single-cell western blots, with each assay comprised of chemical lysis of single cells seated in microwells, protein electrophoresis from those microwells into a supporting polyacrylamide (PA) gel layer, and in-gel antibody probing. We systematically optimize chemical lysis and subsequent polyacrylamide gel electrophoresis (PAGE) of the single-cell lysate. The scWestern slides are stored for months then reprobed, thus allowing archiving and later analysis as relevant to sparingly limited, longitudinal cell specimens. Imaging and scWestern analysis of single glioblastoma cells dosed with the chemotherapeutic daunomycin showed both apoptotic (cleaved caspase 8- and annexin V-positive) and living cells. Intriguingly, living glioblastoma subpopulations show up-regulation of a multidrug resistant protein, P-glycoprotein (P-gp), suggesting an active drug efflux pump as a potential mechanism of drug resistance. Accordingly, linking of phenotype with targeted protein analysis with single-cell resolution may advance our understanding of drug response in inherently heterogeneous cell populations, such as those anticipated in tumors.

Human TPH2 (hTPH2) catalyzes the rate-limiting step in CNS serotonin biosynthesis. We characterized a single-nucleotide polymorphism (C2755A) in the hTPH2 gene that substitutes tyrosine for serine at position 41 in the regulatory domain of the enzyme. This polymorphism is associated with bipolar disorder and peripartum depression in a Chinese population. Recombinant h TPH2 human proteins were expressed in bacteria and also stably expressed in PC12 cells. Following bacterial expression and purification, the tyrosine for serine substitution at position 41 (S41Y) polymorphic enzyme displayed increased Vmax with unchanged Km values. By contrast, enzyme stability was decreased in vitro from 32 min to 4 min (37°C) for the S41Y enzyme (as compared to the wild-type enzyme). The S41Y polymorphism decreased cyclic AMP-dependent protein kinase A-mediated phosphorylation ~ 50% relative to wild-type hTPH2, suggesting that the S41Y mutation may disrupt the post-translational regulation of this enzyme. Transfected PC12 cells expressed hTPH2 mRNA, active protein, and synthesized and released serotonin. Paradoxically, while S41Y-transfected PC12 cells expressed higher levels of hTPH2 than wild type, they synthesized less serotonin. These findings suggest a modified regulation of the S41Y gene variant leading to altered regulation and reduced neurotransmitter synthesis that may contribute to association of the polymorphism with bipolar disorder and depression. © 2014 International Society for Neurochemistry.