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Progress in proteomics research has led to a demand for powerful analytical tools with high separation efficiency and sensitivity for confident identification and quantification of proteins, posttranslational modifications, and protein complexes expressed in cells and tissues. This demand has significantly increased interest in capillary electrophoresis‐mass spectrometry (CE‐MS) in the past few years. This review provides highlights of recent advances in CE‐MS for proteomics research, including a short introduction to top‐down mass spectrometry and native mass spectrometry (native MS), as well as a detailed overview of CE methods. Both the potential and limitations of these methods for the analysis of proteins and peptides in synthetic and biological samples and the challenges of CE methods are discussed, along with perspectives about the future direction of CE‐MS. @ 2019 Wiley Periodicals, Inc. Mass Spec Rev 00:1–16, 2019. [ABSTRACT FROM AUTHOR]

Additive manufacturing of metallic components is regarded as one of the more exciting developments in engineering. The combined attractions of near net shape, tailored composition, and geometry optimisation have led to much interest in the various processes used and a drive to improve the mechanical properties to match those of wrought parts. In this paper, we reflect on the apparent lack of ambition in optimising the structural integrity of parts made using these new manufacturing processes. The current research focus seems to be either on largely irrelevant static properties, or on quantifying the fatigue response in a way that would be familiar to engineers in the 19th Century. Given the work on the role of microstructure and fatigue, which dates back to Ewing and Humphrey in 1903 reaching its zenith in the 1980s and 90s with Keith Miller in the vanguard, and recent developments in both imaging technologies and sophisticated numerical modelling, all the elements are in place for a much more rigorous, and ultimately more fruitful, approach to understand the structural integrity of additive manufactured components. [ABSTRACT FROM AUTHOR]

Highlights: [•] Spatial proteomics was used to monitor protein localization following MMS treatment. [•] MMS treatment affected the levels of many proteins in the chromatin-enriched fraction. [•] Proteins involved in chromatin organization were responsive to MMS treatment. [•] MMS treatment also affected components of the ubiquitin proteasome system. [•] Decreased phosphorylation of many nuclear proteins was observed after MMS treatment. [Copyright &y& Elsevier]

Abstract: A modelling strategy is proposed to link the meso-scale mechanical response of a solid material to the macroscopic material behaviour. The model is based on a regular lattice of truncated octahedral cells, with sites at the cell centres linked by two sets of bonds. The relationship between the macroscopic elastic behaviour of the model and the elastic properties of the bonds is studied numerically. The results demonstrate that, in contrast to previously proposed lattice arrangements, any elastic properties of metallic or cementitious materials can be obtained by appropriate selection of the axial and the shear stiffness of the bonds. Discussion of the modelling approach includes the potential of the site-bond model to simulate the evolution of damage driven not only by mechanical deformation but also by processes that involve the interaction of different mechanisms. [Copyright &y& Elsevier]

In this article, the author discusses how mass spectrometry has developed from the atomic structure study, and characterizing molecules' incarnation as a technique in proteomic analysis. He states that the birth of mass spectrometry was attributed to the physicist J. J. and was used to examine the atomic structure's fundamental aspects, and in the soft ionization method of Michael Barber and colleagues. Moreover, the rise of proteomics has improved peptide and protein sequencing.

Mass spectrometry (MS) is the most comprehensive and versatile tool in large-scale proteomics. In this review, we dissect the overall framework of the MS experiment into its key components. We discuss the fundamentals of proteomic analyses as well as recent developments in the areas of separation methods, instrumentation, and overall experimental design. We highlight both the inherent strengths and limitations of protein MS and offer a rough guide for selecting an experimental design based on the goals of the analysis. We emphasize the versatility of the Orbitrap, a novel mass analyzer that features high resolution (up to 150,000), high mass accuracy (2-5 ppm), a mass-to-charge range of 6000, and a dynamic range greater than 10³. High mass accuracy of the Orbitrap expands the arsenal of the data acquisition and analysis approaches compared with a low-resolution instrument. We discuss various chromatographic techniques, including multidimensional separation and ultra-performance liquid chromatography. Multidimensional protein identification technology (MudPIT) involves a continuum sample preparation, orthogonal separations, and MS and software solutions. We discuss several aspects of MudPIT applications to quantitative phosphoproteomics. MudPIT application to large-scale analysis of phosphoproteins includes (a) a fractionation procedure for motif-specific enrichment of phosphopeptides, (b) development of informatics tools for interrogation and validation of shotgun phosphopeptide data, and (c) in-depth data analysis for simultaneous determination of protein expression and phosphorylation levels, analog to western blot measurements. We illustrate MudPIT application to quantitative phosphoproteomics of the beta adrenergic pathway. We discuss several biological discoveries made via mass spectrometry pipelines with a focus on cell signaling proteomics. [ABSTRACT FROM AUTHOR]

Abstract: A major scientific challenge at the present time for cancer research is the determination of the underlying biological basis for cancer development. It is further complicated by the heterogeneity of cancer''s origin. Understanding the molecular basis of cancer requires studying the dynamic and spatial interactions among proteins in cells, signaling events among cancer cells, and interactions between the cancer cells and the tumor microenvironment. Recently, it has been proposed that large-scale protein expression analysis of cancer cell proteomes promises to be valuable for investigating mechanisms of cancer transformation. Advances in mass spectrometry technologies and bioinformatics tools provide a tremendous opportunity to qualitatively and quantitatively interrogate dynamic protein–protein interactions and differential regulation of cellular signaling pathways associated with tumor development. In this review, progress in shotgun proteomics technologies for examining the molecular basis of cancer development is presented and discussed. [Copyright &y& Elsevier]

Tuberous sclerosis is a serious inherited disease which poses major challenges for affected families and those caring for them. Identification of the genes causing the condition and study of their protein products has shed light on the pathogenesis of the disease and provided valuable new information about signalling pathways regulating protein synthesis and cell growth. There is now the exciting possibility of drug therapy for some of the manifestations of the disease.European Journal of Human Genetics (2006) 14, 1065–1073. doi:10.1038/sj.ejhg.5201625; published online 26 July 2006 [ABSTRACT FROM AUTHOR]

Proteomic analysis of digested complex protein mixtures has become a useful strategy to identify proteins involved in biological processes. We have evaluated the use of a new mass spectrometer that combines a linear ion trap and an Orbitrap to create a hybrid tandem mass spectrometer. A digested submandibular/sublingual saliva sample was used for the analysis. We find the instrument is capable of mass resolution in excess of 40 000 and mass measurement accuracies of less than 2 ppm for the analysis of complex peptide mixtures. Such high mass accuracy allowed the elimination of virtually any false positive peptide identifications, suggesting that peptides that do not match the specificity of the protease used in the digestion of the sample should not automatically be considered as false positives. Tandem mass spectra from the linear ion trap and from the Orbitrap have very similar ion abundance ratios. We conclude this instrument will be well suited for shotgun proteomic types of analyses. [ABSTRACT FROM AUTHOR]

Abstract: The global identification of post-translationally modified proteins is a difficult challenge that is currently being addressed by many researchers in the field of mass spectrometry (MS)-based proteomics. The ability to identify thousands of proteins by shotgun-based strategies has made the mere idea of a global analysis of a particular protein modification seem reasonable. There has been much progress in the development of methods that make use of shotgun-based protein identification in the analysis of a wide variety of protein modifications, some of which will be discussed here. [Copyright &y& Elsevier]

Presents a probability based method for protein identification using tandem mass spectra and protein databases. Use of the method for generating a score characterizing the randomness of a database sequence match to a tandem mass spectrum; Hypergeometric distribution to model frequencies of matches between fragment ion employed in the method; Test used to check the validity of the Hypergeometric model in describing fragment ion matches.

Membrane proteins perform some of the most important functions in the cell, including the regulation of cell signaling through surface receptors, cell-cell interactions, and the intracellular compartmentalization of organelles. Recent developments in proteomic strategies have focused on the inclusion of membrane proteins in highthroughput analyses. While slow and steady progress continues to be made in gel-based technologies, significant advances have been reported in non-gel shotgun methods using liquid chromatography coupled to mass spectrometry (LC/MS). These latter strategies facilitate the identification of large numbers of membrane proteins and modifications, and have the potential to provide insights into protein topology and orientation in membranes. [ABSTRACT FROM AUTHOR]

Organellar compartments involved in secretion are expanded during the transition from late pregnancy (basal secretory state) to lactation (maximal secretory state) to accommodate for the increased secretory function required for copious milk production in mammary epithelial cells. The Golgi complex is a major organelle of the secretory pathway and functions to sort, package, distribute, and post-translationally modify newly synthesized proteins and membrane lipids. These complex functions of the Golgi are reflected in the protein complement of the organelle. Therefore, using proteomics, the protein complements of Golgi fractions isolated at two functional states (basal and maximal) were compared to identify some of the molecular changes that occur during this transition. This global analysis has revealed that only a subset of the total proteins is up-regulated from steady state during the transition. Identification of these proteins by tandem mass spectrometry has revealed several classes of proteins involved in the regulation of membrane fusion and secretion. This first installment of the functional proteomic analysis of the Golgi complex begins to define the molecular basis for the transition from basal to maximal secretion. [ABSTRACT FROM AUTHOR]

Standard single‐cell (sc) proteomics of disease states inferred from multicellular organs or organoids cannot currently be related to single‐cell physiology. Here, a scPatch‐Clamp/Proteomics platform is developed on single neurons generated from hiPSCs bearing an Alzheimer's disease (AD) genetic mutation and compares them to isogenic wild‐type controls. This approach provides both current and voltage electrophysiological data plus detailed proteomics information on single‐cells. With this new method, the authors are able to observe hyperelectrical activity in the AD hiPSC‐neurons, similar to that observed in the human AD brain, and correlate it to ≈1400 proteins detected at the single neuron level. Using linear regression and mediation analyses to explore the relationship between the abundance of individual proteins and the neuron's mutational and electrophysiological status, this approach yields new information on therapeutic targets in excitatory neurons not attainable by traditional methods. This combined patch‐proteomics technique creates a new proteogenetic‐therapeutic strategy to correlate genotypic alterations to physiology with protein expression in single‐cells. [ABSTRACT FROM AUTHOR]

Describes a method to correlate uninterpreted tandem mass spectra of modified peptides produced under low-energy collision conditions with amino acid sequences in a protein database. Algorithm of modified searches; Fragmentation patterns observed in the tandem mass spectra of peptides containing covalent modifications.

Summary: We present an updated version of the TFold software for pinpointing differentially expressed proteins in shotgun proteomics experiments. Given an FDR bound, the updated approach uses a theoretical FDR estimator to maximize the number of identifications that satisfy both a fold-change cutoff that varies with the t-test P-value as a power law and a stringency criterion that aims to detect lowly abundant proteins. The new version has yielded significant improvements in sensitivity over the previous one.Availability: Freely available for academic use at http://pcarvalho.com/patternlab.Contact: paulo@pcarvalho.com [ABSTRACT FROM PUBLISHER]

A new combined experimental and modelling approach has been developed in order to understand the physical mechanisms that lead to crack nucleation in a polycrystalline aluminium alloy AA2024 undergoing cyclic loading. Four‐point bending low‐cycle fatigue tests were performed inside the chamber of a scanning electron microscope on specimens with a through‐thickness central hole, introduced to localize stresses and strains in a small region on the top surface of the sample. Fatigue crack initiation and small crack growth mechanisms were analyzed through high‐resolution scanning electron microscope images, local orientation measurements using electron‐back‐scattered‐diffraction, and local strain measurements using digital image correlation. A crystal plasticity finite element model was developed to simulate the cyclic deformation behaviour of AA2024. Two‐dimensional Voronoi‐based microstructures were generated, and the material parameters for the constitutive equations (including both isotropic and kinematic hardening) were identified using monotonic and fully reversed cyclic tests. A commonly used fatigue crack initiation criterion found in the literature, the maximum accumulated plastic slip, was evaluated in the crystal plasticity finite element model but could not predict the formation of cracks away from the edge of the hole in the deformed specimens. A new criterion combining 2 parameters: The maximum accumulated slip over each individual (critical) slip system and the maximum accumulated slip over all slip systems were formulated to reproduce the experimental locations of crack nucleation in the microstructure. [ABSTRACT FROM AUTHOR]

In this article, the authors comment on the potential of informatics to drive mass-spectroscopy-based protein analysis to make large-scale methods for proteomics. They say that the developers of computer software must provide a positive control with a standard data set and must offer results together with the parameter file in order for the new users to become confident that they make the analyses correctly. They also note that software tools evolve with newer versions and capacities.

Presents information on levels and the location of production of the protein Waldo in the yeast Saccharomyces cerevisiae. Use of the yeast in the analysis of cellular processes; Techniques and methods used to reveal the identities of various proteins; Use of homologous recombination to integrate DNA sequence; Findings on several open reading frames that did not encode authentic gene products.

Although Metarhizium anisopliae is one of the most studied fungal biocontrol agents, its infection mechanism is far from being completely understood. Using multidimensional protein identification technology (MudPIT), we evaluated the differential secretome of M. anisopliae E6 induced by the host Rhipicephalus microplus cuticle. The proteomic result showed changes in the expression of 194 proteins after exposure to host cuticle, such as proteins involved in adhesion, penetration, stress and fungal defense. Further, we performed a comparative genomic distribution of differentially expressed proteins of the M. anisopliae secretome against another arthropod pathogen, using the Beauveria bassiana ARSEF2860 protein repertory. Among 47 analyzed protein families, thirty were overexpressed in the M. anisopliae E6 predicted genome compared to B. bassiana. An in vivo toxicity assay using a Galleria mellonella model confirmed that the M. anisopliae E6 secretome was more toxic in cattle tick infections compared to other secretomes, including B. bassiana with cattle ticks and M. anisopliae E6 with the insect Dysdereus peruvianus , which our proteomic results had also suggested. These results help explain molecular aspects associated with host infection specificity due to genetic differences and gene expression control at the protein level in arthropod-pathogenic fungi. [ABSTRACT FROM AUTHOR]

Background: Clostridioides difficile (C. difficile) is a leading cause of healthcare-associated infections. Using opioids while infected with C. difficile may hypothetically lead to reduced clearance of the organism and higher risk of progressing to severe or fulminant infection. Objective: The objective of this study was to determine if opioid use leads to worsening of C. difficile infection. Methods: This was a single-center, retrospective cohort study of patients with C. difficile infection. The primary endpoint was progression to severe or fulminant disease, defined as serum creatinine greater than 1.5 mg/dL or over 50% of baseline, white blood cells above 15,000 cells/mm3, shock requiring vasopressors, ileus, toxic megacolon, or vancomycin dose increase. Secondary outcomes included hospital length of stay and time to resolution of diarrhea. The groups were stratified based on average morphine milligram equivalents received during the treatment. Results: A total of 73 patients were included in the non-opioid group and 93 patients in the opioid group. The composite outcome occurred in 16 patients (21.9%) without opioids vs 26 patients (28.0%) with opioids; (P = 0.37). The average length of stay was 7.2 days without opioids and 9.3 days with opioids (P = 0.11) and the average time to resolution of diarrhea was 3.5 days without opioids and 5.5 days with opioids (P = 0.40). Conclusion: There was no significant difference in the rate of progression to severe or fulminant disease. There was a numerical trend towards increase in progression in patients who had opioids, primarily driven by those who had higher dosages of opioids used. [ABSTRACT FROM AUTHOR]

Background: Post-operative ileus and delayed return of gastrointestinal function are complications seen frequently in patients undergoing colorectal surgery. Many enhanced recovery after surgery protocols include alvimopan to inhibit the effects of opiates in the gastrointestinal tract and lidocaine to augment analgesics. Limited data exist regarding alvimopan's efficacy in opiate-sparing regimens. Methods: This single-center, retrospective cohort analysis was conducted in a randomly selected population of adult patients undergoing colorectal resection between February 2018 and October 2019. Patients meeting inclusion criteria were divided into four groups dependent upon whether or not they received alvimopan (A or a) and/or lidocaine (L or l). The primary endpoint in this study was median time to first bowel movement or discharge, whichever came first. Our secondary endpoint was length of stay. Results: Of the 430 patients evaluated, a total of 192 patients were included in the final evaluation in the following groups: AL (n = 93), Al (n = 34), aL (n = 44), and al (n = 21). A significant difference was found among the groups for the primary outcome of median time to bowel movement or discharge (p = 0.001). Three subsequent pair-wise comparisons resulted in a significant difference in the primary outcome: group AL 39.4 h vs. group aL 54.0 h (p = 0.003), group AL 39.4 h vs. group al 55.4 h (p = 0.001), and group Al 44.9 h vs. group al 55.4 h (p = 0.01). Length of stay was significantly reduced by 1.8 days in groups AL and Al compared to group aL (p < 0.001). Conclusion: Treatment with alvimopan resulted in a significant improvement in time to GI recovery and decreased length of stay in an established ERAS program. While lidocaine's reduction in opiates was minimal, the group receiving both alvimopan and lidocaine had the greatest reduction in time to GI recovery and length of stay. [ABSTRACT FROM AUTHOR]

This paper describes a new methodology for characterising the plastic zone ahead of a fatigue crack. This methodology is applied to a set of experimental data obtained by synchrotron X‐ray diffraction on a bainitic steel compact tension specimen. The methodology is based on generating the equivalent Von Mises strain field from the X‐ray experimental elastic strain maps. Based on the material response, a threshold is then applied on the equivalent strain maps to identify the size and shape of the plastic zone. The experimental plastic zone lies between the plane strain and plane stress Westergaard's bounds but closer to the plane strain theoretical prediction confirming that the volume analyzed is predominantly subjected to plane strain conditions. However, the observed plastic zone has a somewhat flatter shape, extending further from the crack plane but less extended in the crack growing direction. Highlights: This paper describes a new methodology to infer the plastic zone in the bulk of materials.Methodology was tested successfully on a CT specimen made of bainitic steel.Results are consistent with data collected at the midplane through the thickness. [ABSTRACT FROM AUTHOR]

Summary: The centrosome consists of a pair of centrioles and surrounding pericentriolar material (PCM). Many vertebrate cells also have an array of granules, termed centriolar satellites, that localize around the centrosome and are associated with centrosome and cilium function. Centriole duplication occurs once per cell cycle and is effected by a set of proteins including PLK4, CEP192, CEP152, CEP63, and CPAP. Information on the relationships between these components is limited due to the difficulty in assaying interactions in the context of the centrosome. Here, we used proximity-dependent biotin identification (BioID) to identify proximity interactions among centriole duplication proteins. PLK4, CEP192, and CEP152 BioID identified known physically interacting proteins and a new interaction between CEP152 and CDK5RAP2 consistent with a function of CEP152 in PCM recruitment. BioID for CEP63 and its paralog CCDC67 revealed extensive proximity interactions with centriolar satellite proteins. Focusing on these satellite proteins identified two new regulators of centriole duplication, CCDC14 and KIAA0753. Both proteins colocalize with CEP63 to satellites, bind to CEP63, and identify other satellite proteins by BioID. KIAA0753 positively regulates centriole duplication and CEP63 centrosome localization, whereas CCDC14 negatively regulates both processes. These results suggest that centriolar satellites have a previously unappreciated function in regulating centriole duplication. [ABSTRACT FROM AUTHOR]

Highlights: [•] Pore space-informed model for meso-scale diffusion with limited experimental data. [•] Sorption effects and up-scaling via film coefficients incorporated. [•] Pore space structure and solute size control topology of diffusion network. [•] Macroscopic validation for radionuclide diffusion in bentonite. [•] Transient diffusion descriptors for experimental meso-scale validation proposed. [ABSTRACT FROM AUTHOR]

Many neurons in the adult central nervous system, including retinal ganglion cells (RGCs), degenerate and die after injury. Early axon protein and organelle trafficking failure is a key component in many neurodegenerative disorders yet changes to axoplasmic transport in disease models have not been quantified. We analyzed early changes in the protein 'transportome' from RGC somas to their axons after optic nerve injury and identified transport failure of an anterograde motor protein Kif5a early in RGC degeneration. We demonstrated that manipulating Kif5a expression affects anterograde mitochondrial trafficking in RGCs and characterized axon transport in Kif5a knockout mice to identify proteins whose axon localization was Kif5a-dependent. Finally, we found that knockout of Kif5a in RGCs resulted in progressive RGC degeneration in the absence of injury. Together with expression data localizing Kif5a to human RGCs, these data identify Kif5a transport failure as a cause of RGC neurodegeneration and point to a mechanism for future therapeutics. [ABSTRACT FROM AUTHOR]

In recent years, the advent of staring array detectors has made Thermoelastic Stress Analysis (TSA) a technique with considerable potential for fatigue and fracture mechanics applications. The technique is noncontacting and provides full field stress maps from the surface of cyclically loaded components. In addition, the technique appears to have a great potential in the evaluation of the effective stress intensity factor range during fatigue since fracture mechanics parameters are derived directly from the temperature changes in the vicinity of the crack tip rather than from remote data. In the current work TSA is presented as a novel methodology for measuring the effective stress intensity factor from the analysis of thermoelastic images. ΔK values inferred using TSA have been employed to estimate an equivalent opening/closing load at different R-ratios in a cracked aluminium 2024 CT specimen. Results have been compared with those obtained using the strain-offset technique showing a good level of agreement. [ABSTRACT FROM AUTHOR]

Abstract: Metabolic labeling of rodent proteins with 15N, a heavy stable isotope of nitrogen, provides an efficient way for relative quantitation of differentially expressed proteins. Here we describe a protocol for metabolic labeling of rats with an 15N-enriched spirulina diet. As a case study, we also demonstrate the application of 15N-enriched tissue as a common internal standard in quantitative analysis of differentially expressed proteins in neurodevelopment in rats at two different time points, postnatal day 1 and 45. We briefly discuss the bioinformatics tools, ProLucid and Census, which can easily be used in a sequential manner to identify and quantitate relative protein levels on a proteomic scale. [Copyright &y& Elsevier]

Summary: The primary cilium is a microtubule-based organelle that senses extracellular signals as a cellular antenna []. Primary cilia are found on many types of cells in our body and play important roles in development and physiology. Defects of primary cilia cause a broad class of human genetic diseases called ciliopathies. To gain new insights into ciliary functions and better understand the molecular mechanisms underlying ciliopathies, it is of high importance to generate a catalog of primary cilia proteins. In this study, we isolated primary cilia from mouse kidney cells by using a calcium-shock method and identified 195 candidate primary cilia proteins by MudPIT (multidimensional protein identification technology), protein correlation profiling, and subtractive proteomic analysis. Based on comparisons with other proteomic studies of cilia, around 75% of our candidate primary cilia proteins are shared components with motile or specialized sensory cilia. The remaining 25% of the candidate proteins are possible primary cilia-specific proteins. These possible primary cilia-specific proteins include EVC2, INPP5E, and inversin, several of which have been linked to known ciliopathies. We have performed the first reported proteomic analysis of primary cilia from mammalian cells. These results provide new insights into primary cilia structure and function. [ABSTRACT FROM AUTHOR]

Abstract: Mitotic kinases orchestrate cell cycle processes by phosphorylation of cell cycle regulators. DDA3, a spindle-associated phosphor-protein, is a substrate of mitotic kinases that control chromosome movement and spindle microtubule (MT) dynamics. Through a mass spectrometry analysis, we identified phosphorylation sites on the endogenous mitotic DDA3, which include Ser22, Ser65, Ser70, and Ser223. Phosphorylation of these residues converts interphase form of DDA3 to mitotic form by changing its biochemical activity, as unphosphorylated DDA3 processed both the MT polymerizing and bundling activities, whereas phosphor-mimic mutants lost both activities, only retaining the MT-binding activity. We found that mitotic kinases, such as Cdk1, Aurora A, and Plk1, phosphorylate DDA3 in vitro. Whereas Cdk1 and Aurora A negatively regulate MT-polymerizing and MT-bundling activities, Plk1 does not affect these activities. Interestingly, the phosphorylation of DDA3 by Aurora A and Plk1 inhibits the phosphorylation by other kinases, indicating that sequential phosphorylation is important for the regulation of DDA3 function. We conclude that kinases control the function of DDA3 in the cell cycle by regulating its MT-polymerizing/bundling activities through sequential phosphorylation. [Copyright &y& Elsevier]

Abstract: Ligand engagement by integrins induces receptor clustering and formation of complexes at the integrin cytoplasmic face that controls cell signaling and cytoskeletal dynamics critical for adhesion-dependent processes. This study searches for a subset of integrin effectors that coordinates both tumor cell invasion and resistance to the chemotherapeutic drug cisplatin in oral carcinomas. Candidate integrin effectors were identified in a proteomics screen of proteins recruited to clustered integrin αβ1, αvβ or α6β receptors in oral carcinomas. Proteins with diverse functions including microtubule and actin binding proteins, and factors involved in trafficking, transcription and translation were identified in oral carcinoma integrin complexes. Knockdown of effectors in the oral carcinoma HN12 cells revealed that p130Cas, Dek, Src and talin were required for invasion through Matrigel. Disruption of talin or p130Cas by RNA interference increased resistance to cisplatin, whereas targeting Dek, Src or zyxin reduced HN12 resistance to cisplatin. Analysis of the spreading of HN12 cells on collagen I and laminin I revealed that a decrease in p130Cas or talin expression inhibited spreading on both matrices. Interestingly, a reduction in zyxin expression enhanced spreading on laminin I and inhibited spreading on collagen I. Reduction of Dek, Src, talin or zyxin expression reduced HN12 proliferation by 30%. Proliferation was not affected by a reduction in p130Cas expression. We conclude that p130Cas, Src and talin function in both oral carcinoma invasion and resistance to cisplatin. [Copyright &y& Elsevier]

Abstract: Mass spectrometry-based proteomics is rapidly becoming an essential tool for biologists. One of the most common applications is identifying the components of protein complexes isolated by co-immunoprecipitation. In this review, we discuss the co-immunoprecipitation, mass spectrometry and data analysis techniques that have been used successfully to define protein complexes in C. elegans research. In this discussion, two strategies emerged. One approach is to use stringent biochemical purification methods and attempt to identify a small number of complex components with a high degree of certainty based on MS data. A second approach is to use less stringent purification and identification parameters, and ultimately test a longer list of potential binding partners in biological validation assays. This should provide a useful guide for biologists planning proteomic experiments. [ABSTRACT FROM AUTHOR]

Abstract: DDA3 is a microtubule-associated protein that controls chromosome congression and segregation by regulating the mitotic spindle. Depletion of DDA3 alters spindle structure, generates unaligned chromosomes at metaphase, and delays the mitotic progression. Through a mass spectrometry analysis, we found that DDA3 is phosphorylated on Ser225 during mitosis. Phosphorylation of this residue is important for the mitotic function of DDA3, as the phospho-mimicking DDA3-S225D variant, but not the nonphosphorable DDA3-S225A mutant, rescues the DDA3-knockdown phenotype. We conclude that the mitotic function of DDA3 is regulated by phosphorylation on the Ser225 residue. [Copyright &y& Elsevier]

Mass spectrometry-based proteomics is increasingly used to address basic and clinical questions in biomedical research through studies of differential protein expression, protein-protein interactions, and posttranslational modifications. The complex structural and functional organization of the human brain warrants the application of high-throughput, systematic approaches to understand the functional alterations under normal physiological conditions and the perturbations of neurological diseases. This primer focuses on shotgun-proteomics-based tandem mass spectrometry for the identification of proteins in a complex mixture. It describes the basic concepts of protein differential expression analysis and posttranslational modification analysis and discusses several strategies to improve the coverage of the proteome. [Copyright &y& Elsevier]

Mass spectrometry has been widely used to analyze biological samples and has evolved into an indispensable tool for proteomics research. Our desire to understand the proteome has led to new technologies that push the boundary of mass spectrometry capabilities, which in return has allowed mass spectrometry to address an ever-increasing array of biological questions. The recent development of a novel mass spectrometer (Orbitrap) and new dissociation methods such as electron-transfer dissociation has made possible the exciting new areas of proteomic application. Although bottom-up proteomics (analysis of proteolytic peptide mixtures) remains the workhorse for proteomic analysis, middle-down and top-down strategies (analysis of longer peptides and intact proteins, respectively) should allow more complete characterization of protein isoforms and post-translational modifications. Finally, stable isotope labeling strategies have transformed mass spectrometry from merely descriptive to a tool for measuring dynamic changes in protein expression, interaction, and modification. [Copyright &y& Elsevier]

Summary: In eukaryotes, exit from mitosis occurs through the inactivation of the Cdk1-cyclin B kinase complex and the reversal of its phosphorylation events. These late mitotic events are tightly regulated to occur only after the onset of anaphase and prior to cytokinesis. Central to this regulation is the conserved Cdc14 family of protein phosphatases, whose activity reverses Cdk-dependent phosphorylation events. S. cerevisiae Cdc14 activity is restrained from dephosphorylating Cdk substrates and inactivating Cdk1 through its nucleolar sequestration prior to anaphase. Here, we describe a unique mode of Cdc14 regulation that operates prior to anaphase in fission yeast. Cdk1 phosphorylates and inhibits the catalytic activity of the Cdc14 family member, Clp1/Flp1. As Cdk1 activity declines during anaphase progression, Clp1/Flp1 autocatalytically reverses these phosphorylation events to stimulate its own activity. These findings point to a simple regulatory circuit that couples Cdk1 activation with its inactivation mediated through phosphorylation-dependent regulation of Clp1/Flp1 phosphatase activity. [Copyright &y& Elsevier]

Summary: The G protein-coupled receptor Gpr1 and associated Gα subunit Gpa2 govern dimorphic transitions in response to extracellular nutrients by signaling coordinately with Ras to activate adenylyl cyclase in the yeast Saccharomyces cerevisiae. Gpa2 forms a protein complex with the kelch Gβ mimic subunits Gpb1/2, and previous studies demonstrate that Gpb1/2 negatively control cAMP-PKA signaling via Gpa2 and an unknown second target. Here, we define these targets of Gpb1/2 as the yeast neurofibromin homologs Ira1 and Ira2, which function as GTPase activating proteins of Ras. Gpb1/2 bind to a conserved C-terminal domain of Ira1/2, and loss of Gpb1/2 results in a destabilization of Ira1 and Ira2, leading to elevated levels of Ras2-GTP and unbridled cAMP-PKA signaling. Because the Gpb1/2 binding domain on Ira1/2 is conserved in the human neurofibromin protein, an analogous signaling network may contribute to the neoplastic development of neurofibromatosis type 1. [Copyright &y& Elsevier]

Vertebrates produce multiple chondroitin sulfate proteoglycans that play important roles in development and tissue mechanics. In the nematode Caenorhabditis elegans, the chondroitin chains lack sulfate but nevertheless play essential roles in embryonic development and vulval morphogenesis. However, assignment of these functions to specific proteoglycans has been limited by the lack of identified core proteins. We used a combination of biochemical purification, Western blotting, and mass spectrometry to identify nine C. elegans chondroitin proteoglycan core proteins, none of which have homologues in vertebrates or other invertebrates such as Drosophila melanogaster or Hydra vulgaris. CPG-1/CEJ-1 and CPG-2 are expressed during embryonic development and bind chitin, suggesting a structural role in the egg. RNA interference (RNAi) depletion of individual CPGs had no effect on embryonic viability, but simultaneous depletion of CPG-1/CEJ-1 and CPG-2 resulted in multinucleated single-cell embryos. This embryonic lethality phenocopies RNAi depletion of the SQV-5 chondroitin synthase, suggesting that chondroitin chains on these two proteoglycans are required for cytokinesis. [ABSTRACT FROM AUTHOR]

Summary: During chromosome segregation, kinetochores form dynamic connections with spindle microtubules. In vertebrates, these attachments require the activities of a number of outer kinetochore proteins, including CENP-F [] and the widely conserved microtubule-associated protein CLASP []. Here, we investigate the functional relationship between HCP-1/2, two redundant CENP-F-like proteins, and CLASPCLS-2 in Caenorhabditis elegans. HCP-1/2 and CLASPCLS-2 localize transiently to mitotic C. elegans kinetochores with nearly identical kinetic profiles, and biochemical purifications demonstrate that they also associate physically. In embryos depleted of HCP-1/2, CLASPCLS-2 no longer localizes to chromosomes, whereas CLASPCLS-2 depletion does not prevent HCP-1/2 targeting. Consistent with the localization dependency and biochemical association, depletion of HCP-1/2 or CLASPCLS-2 resulted in virtually identical defects in mitotic chromosome segregation characterized by a failure of sister-chromatid biorientation. This phenotype could be partially suppressed by disrupting the astral forces that pull spindle poles apart in the 1 cell embryo, indicating that CLASPCLS-2 is required for biorientation when chromosome-spindle attachments are subjected to poleward force. Our results establish that the key role of HCP-1/2 is to target CLASPCLS-2 to kinetochores, and they support the recently proposed model that CLASP functions to promote the polymerization of kinetochore bound microtubules [4]. [Copyright &y& Elsevier]

Summary: Microtubules regulate actin-based processes such as cell migration and cytokinesis, but molecular mechanisms are not understood. In the fission yeast Schizosaccharomyces pombe, microtubule plus ends regulate cell polarity in part by transporting the kelch repeat protein tea1p to cell ends. Here, we identify tea4p, a SH3 domain protein that binds directly to tea1p. Like tea1p, tea4p localizes to growing microtubule plus ends and to cortical sites at cell ends, and it is necessary for the establishment of bipolar growth. Tea4p binds directly to and recruits the formin for3p, which nucleates actin cable assembly. During “new end take off” (NETO), formation of a protein complex that includes tea1p, tea4p, and for3p is necessary and sufficient for the establishment of cell polarity and localized actin assembly at new cell ends. Our results suggest a molecular mechanism for how microtubule plus ends regulate the spatial distribution of actin assembly. [Copyright &y& Elsevier]

The dynamic responses of the standard Charpy impact machine were studied experimentally using strain gauges and accelerometer attached to the striker and the rotary position sensor fixed at the rotating axis and numerically with the finite element analysis. The fracture propagation was simulated with the cellular automata finite element approach developed earlier. A series of low velocity as well as full capacity Charpy tests were analysed. It was found that the strain gauge signal recorded close to the tup edge and the acceleration recorded at the back of the striker do not match. The energy calculated with the strain gauge data agrees well with the dial reading, while the energy calculated with the accelerometer signal is never near it. Frequencies close to the first natural \hbox{frequency} of the Charpy sample have high modal magnitudes in the acceleration signal but are effectively damped in the strain gauge response. Vibrations of the striker arm have highest modal magnitudes in the rotary position sensor. A low-pass filter is used to obtain the striker movements. The finite element analysis partly supports the experimental observations but also suggests that acceleration at the tup edge suffers higher oscillations than strain. [ABSTRACT FROM AUTHOR]

The purpose of this work is to develop and verify statistical models for protein identification using peptide identifications derived from the results of tandem mass spectral database searches. Recently we have presented a probabilistic model for peptide identification that uses hyper-geometric distribution to approximate fragment ion matches of database peptide sequences to experimental tandem mass spectra. Here we apply statistical models to the database search results to Validate protein identifications. For this we formulate the protein identification problem in terms of two independent models, two- hypothesis binomial and multinomial models, which use the hypergeometric probabilities and cross-correlation scores, respectively. Each database search result is assumed to be a probabilistic event The Bernoulli event has two outcomes: a protein is either identified or not. The probability of identifying a protein at each Bernoulli event is determined from relative length of the protein in the database (the null hypothesis) or the hypergeometric probability scores of the protein's peptides (the alternative hypothesis). We then calculate the binomial probability that the protein will be observed a certain number of times (number of database matches to its peptides) given the size of the data set (number of spectra) and the probability of protein identification at each Bernoulli event The ratio of the probabilities from these two hypotheses (maximum likelihood ratio) is used as a test statistic to discriminate between true and false identifications. The significance and confidence levels of protein identifications are calculated from the model distributions. The multinomial model combines the database search results and generates an observed frequency distribution of cross-correlation scores (grouped into bins) between experimental spectra and identified amino acid sequences. [ABSTRACT FROM AUTHOR]

We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins. [ABSTRACT FROM AUTHOR]

Predator–prey interactions often lead to the coevolution of adaptations associated with avoiding predation and, for predators, overcoming those defences. Antagonistic coevolutionary relationships are often not simple interactions between a single predator and prey but rather a complex web of interactions between multiple coexisting species. Coevolution between venomous rattlesnakes and small mammals has led to physiological venom resistance in several mammalian taxa. In general, viperid venoms contain large quantities of snake venom metalloproteinase toxins (SVMPs), which are inactivated by SVMP inhibitors expressed in resistant mammals. We explored variation in venom chemistry, SVMP expression, and SVMP resistance across four co‐distributed species (California Ground Squirrels, Bryant's Woodrats, Southern Pacific Rattlesnakes, and Red Diamond Rattlesnakes) collected from four different populations in Southern California. Our aim was to understand phenotypic and functional variation in venom and venom resistance in order to compare coevolutionary dynamics of a system involving two sympatric predator–prey pairs to past studies that have focused on single pairs. Proteomic analysis of venoms indicated that these rattlesnakes express different phenotypes when in sympatry, with Red Diamonds expressing more typical viperid venom (with a diversity of SVMPs) and Southern Pacifics expressing a more atypical venom with a broader range of non‐enzymatic toxins. We also found that although blood sera from both mammals were generally able to inhibit SVMPs from both rattlesnake species, inhibition depended strongly on the snake population, with snakes from one geographic site expressing SVMPs to which few mammals were resistant. Additionally, we found that Red Diamond venom, rather than woodrat resistance, was locally adapted. Our findings highlight the complexity of coevolutionary relationships between multiple predators and prey that exhibit similar offensive and defensive strategies in sympatry. [ABSTRACT FROM AUTHOR]

Purpose The purpose of this descriptive report is to share experiences in crisis response planning and risk mitigation at a university health system department of pharmacy with an integrated clinical practice model in the early months of the coronarvirus disease 2019 (COVID-19) pandemic. Summary The department of pharmacy's COVID-19 pandemic response included successful planning and implementation of measures to maintain pharmacy operations and minimize COVID-19 exposure of patients and staff. These measures included ensuring adequate personnel staffing using flexible staffing solutions, ongoing assessment of supply chain integrity, and continuation of integrated clinical pharmacy services 24/7 throughout the initial phase of the COVID-19 pandemic. Information technology (IT) and educational program modifications are also discussed. Conclusion This report describes successful crisis planning and risk mitigation in the setting of COVID-19, which was facilitated by the department of pharmacy's integrated clinical practice model. This model enabled uninterrupted personnel scheduling, supply chain integrity, continued provision of 24/7 integrated clinical services, adaptive use of IT tools, and continuation of educational programs. The experiences described may be instructive to other pharmacy departments in evaluating their response to the COVID-19 pandemic and in planning for similar pandemic or other emergency scenarios. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are cell‐derived membrane vesicles that are released into the extracellular space. EVs encapsulate key proteins and mediate intercellular signalling pathways. Recently, primary cilia have been shown to release EVs under fluid‐shear flow, but many proteins encapsulated in these vesicles have never been identified. Primary cilia are ubiquitous mechanosensory organelles that protrude from the apical surface of almost all human cells. Primary cilia also serve as compartments for signalling pathways, and their defects have been associated with a wide range of human genetic diseases called ciliopathies. To better understand the mechanism of ciliopathies, it is imperative to know the distinctive protein profiles of the differently sourced EVs (cilia vs cytosol). Here, we isolated EVs from ciliated wild‐type (WT) and non‐ciliated IFT88 knockout (KO) mouse endothelial cells using fluid‐shear flow followed by a conventional method of EV isolation. EVs isolated from WT and KO exhibited distinctive sizes. Differences in EV protein contents were studied using liquid chromatography with tandem mass spectrometry (LC‐MS‐MS) and proteomic comparative analysis, which allowed us to classify proteins between ciliary EVs and cytosolic EVs derived from WT and KO, respectively. A total of 79 proteins were exclusively expressed in WT EVs, 145 solely in KO EVs, and 524 in both EVs. Our bioinformatics analyses revealed 29% distinct protein classes and 75% distinct signalling pathways between WT and KO EVs. Based on our statistical analyses and in vitro studies, we identified NADPH‐cytochrome P450 reductase (POR), and CD166 antigen (CD166) as potential biomarkers for ciliary and cytosolic EVs, respectively. Our protein‐protein interaction network analysis revealed that POR, but not CD166, interacted with either established or strong ciliopathy gene candidates. This report shows the unique differences between EVs secreted from cilia and the cytosol. These results will be important in advancing our understanding of human genetic diseases. [ABSTRACT FROM AUTHOR]

The Ehlers-Danlos syndrome (EDS) is a heterogeneo US group of inherited connective tissue disorders in which cutaneous fragility and ligamentous laxity often combine with vascular, gastrointestinal, and skeletal deformities. There is considerable phenotypic overlap between the more common fonas of EDS (types I and II), in which specific molecular defects have not yet been identified. Recently, genetic linkage has been demonstrated between the COL5A1 gene, which encodes the α1 chain of type V collagen, and EDS type II in a large British kindred. Using a polymorphic intragenic simple sequence repeat at the COL5A1 locus, we now demonstrate tight linkage to EDS type I/II in a three-generation family, giving a LOD score (log10 of the odds for linkage) of 4.07 at zero recombination. The variation in expression in this family suggests that EDS types I and II are allelic, and the linkage data support the hypothesis that mutation in COL5A1 can cause both phenotypes. [ABSTRACT FROM AUTHOR]

CD22, a member of Siglec family of sialic acid binding proteins, has restricted expression on B cells. Antibody‐based agents targeting CD22 or CD20 on B lymphoma and leukemia cells exhibit clinical efficacy for treating these malignancies, but also attack normal B cells leading to immune deficiency. Here, we report a chemoenzymatic glycocalyx editing strategy to introduce high‐affinity and specific CD22 ligands onto NK‐92MI and cytokine‐induced natural killer cells to achieve tumor‐specific CD22 targeting. These CD22‐ligand modified cells exhibited significantly enhanced tumor cell binding and killing in vitro without harming healthy B cells. For effective lymphoma cell killing in vivo, we further functionalized CD22 ligand‐modified NK‐92MI cells with the E‐selectin ligand sialyl Lewis X to promote trafficking to bone marrow. The dual‐functionalized cells resulted in the efficient suppression of B lymphoma in a xenograft model. Our results suggest that natural killer cells modified with glycan ligands to CD22 and selectins promote both targeted killing of B lymphoma cells and improved trafficking to sites where the cancer cells reside, respectively. [ABSTRACT FROM AUTHOR]

CD22, a member of Siglec family of sialic acid binding proteins, has restricted expression on B cells. Antibody‐based agents targeting CD22 or CD20 on B lymphoma and leukemia cells exhibit clinical efficacy for treating these malignancies, but also attack normal B cells leading to immune deficiency. Here, we report a chemoenzymatic glycocalyx editing strategy to introduce high‐affinity and specific CD22 ligands onto NK‐92MI and cytokine‐induced natural killer cells to achieve tumor‐specific CD22 targeting. These CD22‐ligand modified cells exhibited significantly enhanced tumor cell binding and killing in vitro without harming healthy B cells. For effective lymphoma cell killing in vivo, we further functionalized CD22 ligand‐modified NK‐92MI cells with the E‐selectin ligand sialyl Lewis X to promote trafficking to bone marrow. The dual‐functionalized cells resulted in the efficient suppression of B lymphoma in a xenograft model. Our results suggest that natural killer cells modified with glycan ligands to CD22 and selectins promote both targeted killing of B lymphoma cells and improved trafficking to sites where the cancer cells reside, respectively. [ABSTRACT FROM AUTHOR]