Exploring Intra- and Intermolecular Interactions in Selected N-Oxides-The Role of Hydrogen Bonds.

Intra- and intermolecular interactions have been explored in selected N-oxide derivatives: 2-(N,N-dimethylamino-N-oxymethyl)-4,6-dimethylphenyl (1) and 5,5'-dibromo-3-diethylaminomethyl-2,2'-biphenol N-oxide (2). Both compounds possess intramolecular hydrogen bonding, which is classified as moderate in 1 and strong in 2, and resonance-assisted in both cases. Density Functional Theory (DFT) in its classical formulation as well as Time-Dependent extension (TD-DFT) were employed to study proton transfer phenomena. The simulations were performed in the gas phase and with implicit and explicit solvation models. The obtained structures of the studied N-oxides were compared with experimental data available. The proton reaction path was investigated using scan with an optimization method, and water molecule reorientation in the monohydrate of 1 was found upon the proton scan progress. It was found that spontaneous proton transfer phenomenon cannot occur in the electronic ground state of the compound 1. An opposite situation was noticed for the compound 2. The changes of nucleophilicity and electrophilicity upon the bridged proton migration were analyzed on the basis of Fukui functions in the case of 1. The interaction energy decomposition of dimers and microsolvation models was investigated using Symmetry-Adapted Perturbation Theory (SAPT). The simulations were performed in both phases to introduce polar environment influence on the interaction energies. The SAPT study showed rather minor role of induction in the formation of homodimers. However, it is worth noticing that the same induction term is responsible for the preference of water molecules' interaction with N-oxide hydrogen bond acceptor atoms in the microsolvation study. The Natural Bond Orbital (NBO) analysis was performed for the complexes with water to investigate the charge flow upon the polar environment introduction. Finally, the TD-DFT was applied for isolated molecules as well as for microsolvation models showing that the presence of solvent affects excited states, especially when the N-oxide acceptor atom is microsolvated.

IMPERFECTIVE EXINE FORMATION (IEF) is required for exine formation and male fertility in Arabidopsis.

KEY MESSAGE: IEF, a novel plasma plasma membrane protein, is important for exine formation in Arabidopsis. Exine, an important part of pollen wall, is crucial for male fertility. The major component of exine is sporopollenin which are synthesized and secreted by tapetum. Although sporopollenin synthesis has been well studied, the transportation of it remains elusive. To understand it, we analyzed the gene expression pattern in tapetal microdissection data, and investigated the potential transporter genes that are putatively regulated by ABORTED MICROSPORES (AMS). Among these genes, we identified IMPERFECTIVE EXINE FORMATION (IEF) that is important for exine formation. Compared to the wild type, ief mutants exhibit severe male sterility and pollen abortion, suggesting IEF is crucial for pollen development and male fertility. Using both scanning and transmission electron microscopes, we showed that exine structure was not well defined in ief mutant. The transient expression of IEF-GFP driven by the 35S promoter indicated that IEF-GFP was localized in plasma membrane. Furthermore, AMS can specifically activate the expression of promoterIEF:LUC in vitro, which suggesting AMS regulates IEF for exine formation. The expression of ATP-BINDING CASSETTE TRANSPORTER G26 (AGCB26) was not affected in ief mutants. In addition, SEM and TEM data showed that the sporopollenin deposition is more defective in abcg26/ief-2 than that of in abcg26, which suggesting that IEF is involved in an independent sporopollenin transportation pathway. This work reveal a novel gene, IEF regulated by AMS that is essential for exine formation.

Participation in civil society organizations and ethnic minorities' interethnic friendships in Britain.

In Britain, civil society organizations (CSOs) have garnered much praise for promoting interethnic friendships (IEF) and strengthening community cohesion. Yet, there is very little empirical evidence to suggest that participation in CSOs promotes ethnic minorities' IEF. Using nationally representative longitudinal (2011-2019) and cross-sectional (2010) data, this article explores the association between participation in CSOs and IEF formation among five British ethnic minority groups and analyses how this relationship is affected by the ethnic composition of CSOs. Overall, fixed effects models show that participation in CSOs only significantly promotes IEF for Indians. For other minority groups it has either no effect or, in the case of Pakistanis, significantly decreases IEF. Further analyses show that compared with ethnic minorities that do not participate in any CSOs, those who participate in mostly interethnic CSOs tend to have significantly more IEF, whereas those who participate in mostly co-ethnic CSOs tend to have significantly less IEF. Taken together, these findings suggest that the association between civic participation and ethnic minorities' IEF is much more nuanced than previously thought and policy interventions seeking to improve ethnic integration should, therefore, take the ethnic background of participants and the ethnic composition of CSOs into account.

Arrayed isoelectric focusing using photopatterned multi-domain hydrogels.

Isoelectric focusing (IEF) is a powerful separation method, useful for resolving subtle changes in the isoelectric point of unlabeled proteins. While microfluidic IEF has reduced the separation times from hours in traditional benchtop IEF to minutes, the enclosed devices hinder post-separation access to the sample for downstream analysis. The two-layer open IEF device presented here comprises a photopatterned hydrogel lid layer containing the chemistries required for IEF and a thin polyacrylamide bottom layer in which the analytes are separated. The open IEF device produces comparable minimum resolvable difference in isoelectric point and gradient stability to enclosed microfluidic devices while providing post-separation sample access by simple removal of the lid layer. Further, using simulations, we determine that the material properties and the length of the separation lanes are the primary factors that affect the electric field magnitude in the separation region. Finally, we demonstrate self-indexed photomasks for alignment-free fabrication of multi-domain hydrogels. We leverage this approach to generate arrayed pH gradients with a total of 80 concurrent separation lanes, which to our knowledge is the first demonstration of multiple IEF separations in series addressed by a single pair of electrodes.

Simultaneous pre-concentration and separation on simple paper-based analytical device for protein analysis.

In this work, fast isoelectric focusing (IEF) was successfully implemented on an open paper fluidic channel for simultaneous concentration and separation of proteins from complex matrix. With this simple device, IEF can be finished in 10 min with a resolution of 0.03 pH units and concentration factor of 10, as estimated by color model proteins by smartphone-based colorimetric detection. Fast detection of albumin from human serum and glycated hemoglobin (HBA1c) from blood cell was demonstrated. In addition, off-line identification of the model proteins from the IEF fractions with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was also shown. This PAD IEF is potentially useful either for point of care test (POCT) or biomarker analysis as a cost-effective sample pretreatment method.

Latent viruses can cause disease by disrupting the competition for the limiting factor p300/CBP.

CBP and p300 are histone acetyltransferase coactivators that control the transcription of numerous genes in humans, viruses, and other organisms. Although two separate genes encode CBP and p300, they share a 61% sequence identity, and they are often mentioned together as p300/CBP. Zhou et al. showed that under hypoxic conditions, HIF1α and the tumor suppressor p53 compete for binding to the limiting p300/CBP coactivator. Jethanandani & Kramer showed that δEF1 and MYOD genes compete for the limited amount of p300/CBP in the cell. Bhattacharyya et al. showed that the limiting availability of p300/CBP in the cell serves as a checkpoint for HIF1α activity. Here, we use the microcompetition model to explain how latent viruses with a specific viral cis-regulatory element in their promoter/enhancer can disrupt this competition, causing diseases such as cancer, diabetes, atherosclerosis, and obesity.

The class III peroxidase PRX17 is a direct target of the MADS-box transcription factor AGAMOUS-LIKE15 (AGL15) and participates in lignified tissue formation.

Several physiological functions have been attributed to class III peroxidases (PRXs) in plants, but the in planta role of most members of this family still remains undetermined. Here, we report the first functional characterization of PRX17 (At2g22420), one of the 73 members of this family in Arabidopsis thaliana. Localization of PRX17 was examined by transient expression in Nicotiana benthamiana. Loss- and gain-of-function mutants in A. thaliana were studied. Regulation at the gene and protein levels was analyzed using ß-glucuronidase (GUS) activity, quantitative reverse transcriptase (qRT)-PCR, zymography, and chromatin immunoprecipitation. Phenotypes were characterized including lignin and xylan contents. PRX17 was expressed in various tissues, including vascular tissues, and PRX17 was localized to the cell wall. In prx17, the lignin content was reduced in the stem and siliques and bolting was delayed, while the opposite phenotype was observed in 35S:PRX17 plants, together with a significant increase of lignin and xylan immunofluorescence signal. Finally, we demonstrated that the transcription factor AGAMOUS-LIKE15 (AGL15) binds to the PRX17 promoter and regulates PRX17 expression level. This converging set of structural, transcriptomic and physiological data suggests that PRX17, under the control of AGL15, contributes to developmental programs by playing an essential role in regulating age-dependent lignified tissue formation, including changes in cell wall properties.

Steady-state protein focusing in carrier ampholyte based isoelectric focusing: Part I-Analytical solution.

The determination of an analytical solution to find the steady-state protein concentration distribution in IEF is very challenging due to the nonlinear coupling between mass and charge conservation equations. In this study, approximate analytical solutions are obtained for steady-state protein distribution in carrier ampholyte based IEF. Similar to the work of Svensson, the final concentration profile for proteins is assumed to be Gaussian, but appropriate expressions are presented in order to obtain the effective electric field and pH gradient in the focused protein band region. Analytical results are found from iterative solutions of a system of coupled algebraic equations using only several iterations for IEF separation of three plasma proteins: albumin, cardiac troponin I, and hemoglobin. The analytical results are compared with numerically predicted results for IEF, showing excellent agreement. Analytically obtained electric field and ionic conductivity distributions show significant deviation from their nominal values, which is essential in finding the protein focusing behavior at isoelectric points. These analytical solutions can be used to determine steady-state protein concentration distribution for experiment design of IEF considering any number of proteins and ampholytes. Moreover, the model presented herein can be used to find the conductivity, electric field, and pH field.

Quantitation of CRM197 using imaged capillary isoelectric focusing with fluorescence detection and capillary Western.

Maurice is a new instrument that can perform imaged capillary isoelectric focusing (icIEF). The standard detection for icIEF is UV absorbance at 280 nm, which limits its application to high protein concentration samples and non-complex samples. Here we describe an icIEF instrument with fluorescence detection. We demonstrate the advantage of using either icIEF with fluorescence detection or quantitative Western Blot to measure diphtheria toxin mutant CRM197 protein titer in crude cell lysates and purified samples. These two techniques have great potentials to become standard methods to analyze protein titers in crude cell lysate or other complex samples types.

Combining high-throughput MALDI-TOF mass spectrometry and isoelectric focusing gel electrophoresis for virtual 2D gel-based proteomics.

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.

Fractionation-dependent improvements in proteome resolution in the mouse hippocampus by IEF LC-MS/MS.

An assessment of fractionated mouse hippocampal peptides was conducted. Protein extract from a single mouse hippocampus was enzymatically digested and fractionated by IEF. Aliquots of fractions were pooled into fewer, more complex samples. The unfractionated lysate, fractions, and pooled fractions were subjected to LC-MS/MS analysis. Samples consisting of many individual fractions had more protein identifications, greater protein sequence coverage, and quantified proteins with more spectral counts than protein extract that was unfractionated or pooled into fewer LC-MS/MS samples. Additionally, prefractionation reduced the median CV for spectral counts as much as 33%. However, the relative gain in proteome resolution was found to saturate with increasing fractionation extent. This study demonstrates how prefractionation by offline IEF can improve the resolution of proteomic investigations of the mouse hippocampus, and that a data-driven pooling methodology can reduce excessive and cost-ineffective fractionation.

Normal human mitral valve proteome: A preliminary investigation by gel-based and gel-free proteomic approaches.

The mitral valve is a highly complex structure which regulates blood flow from the left atrium to the left ventricle (LV) avoiding a significant forward gradient during diastole or regurgitation during systole. The integrity of the mitral valve is also essential for the maintenance of normal LV size, geometry, and function. Significant advances in the comprehension of the biological, functional, and mechanical behavior of the mitral valve have recently been made. However, current knowledge of protein components in the normal human mitral valve is still limited and complicated by the low cellularity of this tissue and the presence of high abundant proteins from the extracellular matrix. We employed here an integrated proteomic approach to analyse the protein composition of the normal human mitral valve and reported confident identification of 422 proteins, some of which have not been previously described in this tissue. In particular, we described the ability of pre-MS separation technique based on liquid-phase IEF and SDS-PAGE to identify the largest number of proteins. We also demonstrated that some of these proteins, e.g. αB-Crystallin, septin-11, four-and-a-half LIM domains protein 1, and dermatopontin, are synthesised by interstitial cells isolated from human mitral valves. These initial results provide a valuable basis for future studies aimed at analysing in depth the mitral valve protein composition and at investigating potential pathogenetic molecular mechanisms. Data are available via ProteomeXchange with identifier PXD004397.

Purification of plant complex protein extracts in non-denaturing conditions by in-solution isoelectric focusing.

An alternative approach for plant complex protein extracts pre-purification by in-solution isoelectric focusing in non-denaturing conditions is presented. The separation of biologically active proteins, in narrow ranges of isoelectric point (pI) was obtained by a modified OFFGEL electrophoresis. Two different water-soluble protein extracts from Phragmites leaves were fractionated into 24 fractions within a 3-10 pI range at 10 °C in the absence of denaturing/reducing agents. One-dimensional electrophoretic analysis revealed different protein distribution patterns and the effective fractionation of both protein extracts. Peroxidase activity of each fraction confirmed that proteins remained active and pre-purification occurred. Biological triplicates assured the needed reproducibility.

Replacement of Oxygen by Sulfur in Small Organic Molecules. 3. Theoretical Studies on the Tautomeric Equilibria of the 2OH and 4OH-Substituted Oxazole and Thiazole and the 3OH and 4OH-Substituted Isoxazole and Isothiazole in the Isolated State and in Solution.

This follow-up paper completes the author's investigations to explore the in-solution structural preferences and relative free energies of all OH-substituted oxazole, thiazole, isoxazole, and isothiazole systems. The polarizable continuum dielectric solvent method calculations in the integral-equation formalism (IEF-PCM) were performed at the DFT/B97D/aug-cc-pv(q+(d))z level for the stable neutral tautomers with geometries optimized in dichloromethane and aqueous solution. With the exception of the predictions for the predominant tautomers of the 3OH isoxazole and isothiazole, the results of the IEF-PCM calculations for identifying the most stable tautomer of the given species in the two selected solvents agreed with those from experimental investigations. The calculations predict that the hydroxy proton, with the exception for the 4OH isoxazole and 4OH isothiazole, moves preferentially to the ring nitrogen or to a ring carbon atom in parallel with the development of a C=O group. The remaining, low-fraction OH tautomers will not be observable in the equilibrium compositions. Relative solvation free energies obtained by the free energy perturbation method implemented in Monte Carlo simulations are in moderate accord with the IEF-PCM results, but consideration of the ΔGsolv/MC values in calculating ΔG(s)tot maintains the tautomeric preferences. It was revealed from the Monte Carlo solution structure analyses that the S atom is not a hydrogen-bond acceptor in any OH-substituted thiazole or isothiazole, and the OH-substituted isoxazole and oxazole ring oxygens may act as a weak hydrogen-bond acceptor at most. The molecules form 1.0-3.4 solute-water hydrogen bonds in generally unexplored numbers at some specific solute sites. Nonetheless, hydrogen-bond formation is favorable with the NH, C=O and OH groups.

Epithelial splicing regulatory proteins 1 (ESRP1) and 2 (ESRP2) suppress cancer cell motility via different mechanisms.

ESRP1 (epithelial splicing regulatory protein 1) and ESRP2 regulate alternative splicing events associated with epithelial phenotypes of cells, and both are down-regulated during the epithelial-mesenchymal transition. However, little is known about their expression and functions during carcinogenesis. In this study, we found that expression of both ESRP1 and ESRP2 is plastic: during oral squamous cell carcinogenesis, these proteins are up-regulated relative to their levels in normal epithelium but down-regulated in invasive fronts. Importantly, ESRP1 and ESRP2 are re-expressed in the lymph nodes, where carcinoma cells metastasize and colonize. In head and neck carcinoma cell lines, ESRP1 and ESRP2 suppress cancer cell motility through distinct mechanisms: knockdown of ESRP1 affects the dynamics of the actin cytoskeleton through induction of Rac1b, whereas knockdown of ESRP2 attenuates cell-cell adhesion through increased expression of epithelial-mesenchymal transition-associated transcription factors. Down-regulation of ESRP1 and ESRP2 is thus closely associated with a motile phenotype of cancer cells.

Introduction to opportunities and pitfalls in functional mass spectrometry based proteomics.

With the advent of mass spectrometry based proteomics, the identification of thousands of proteins has become commonplace in biology nowadays. Increasingly, efforts have also been invested toward the detection and localization of posttranslational modifications. It is furthermore common practice to quantify the identified entities, a task supported by a panel of different methods. Finally, the results can also be enriched with functional knowledge gained on the proteins, detecting for instance differentially expressed gene ontology terms or biological pathways. In this study, we review the resources, methods and tools available for the researcher to achieve such a quantitative functional analysis. These include statistics for the post-processing of identification and quantification results, online resources and public repositories. With a focus on free but user-friendly software, preferably also open-source, we provide a list of tools designed to help the researcher manage the vast amount of data generated. We also indicate where such applications currently remain lacking. Moreover, we stress the eventual pitfalls of every step of such studies. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.

Utilization of light by fucoxanthin-chlorophyll-binding protein in a marine centric diatom, Chaetoceros gracilis.

The major light-harvesting pigment protein complex (fucoxanthin-chlorophyll-binding protein complex; FCP) was purified from a marine centric diatom, Chaetoceros gracilis, by mild solubilization followed by sucrose density gradient centrifugation, and then characterized. The dynamic light scattering measurement showed unimodality, indicating that the complex was highly purified. The amount of chlorophyll a (Chl a) bound to the purified FCP accounted for more than 60 % of total cellular Chl a. The complex was composed of three abundant polypeptides, although there are nearly 30 FCP-related genes. The two major components were identified as Fcp3 (Lhcf3)- and Fcp4 (Lhcf4)-equivalent proteins based on their internal amino acid sequences and a two-dimensional isoelectric focusing electrophoresis analysis developed in this work. Compared with the thylakoids, the FCP complex showed higher contents of fucoxanthin and chlorophyll c but lower contents of the xanthophyll cycle pigments diadinoxanthin and diatoxanthin. Fluorescence excitation spectra analyses indicated that light harvesting, rather than photosystem protection, is the major function of the purified FCP complex, which is associated with more than 60 % of total cellular Chl a. These findings suggest that the huge amount of Chl bound to the FCP complex composed of Lhcf3, Lhcf4, and an unidentified minor protein has a light-harvesting function to allow efficient photosynthesis under the dim-light conditions in the ocean.

Capillary isoelectric focusing immunoassay as a new nanoscale approach for the detection of oligoclonal bands.

The detection of oligoclonal bands (OCBs) in cerebrospinal fluid is an indicator of intrathecal synthesis of immunoglobulins which is a neurochemical sign of chronic inflammatory brain diseases. Intrathecally synthesized IgGs are typically observed in patients with multiple sclerosis. The current standard protocol for the detection of OCBs is IEF on agarose or polyacrylamide gels followed by immunoblotting or silver staining. These methods are time consuming, show substantial interlaboratory variation and cannot be used in a high throughput-approach. We have developed a new nanoscale method for the detection of OCBs based on automated capillary IEF followed by immunological detection. Evidence for intrathecal IgG synthesis was found in all tested patients (n = 27) with multiple sclerosis, even in two subjects who did not have oligoclonal bands according to standard methods. The test specificity was at 97.5% (n = 19). Our findings indicate that the novel OCB-CIEF-immunoassay is suitable for the rapid and highly sensitive detection of OCBs in clinical samples. Furthermore, the method allows for a higher sample throughput than the current standard methods.

Capillary isoelectric focusing-mass spectrometry: Coupling strategies and applications.

IEF on immobilized pH gradient strips is a widespread tool for protein separation, especially as first dimension in commonly utilized 2DE. In the latter arrangement, separations are based on two orthogonal molecular characteristics according to pI in the first and molecular weight in the second dimension. However, the approach is time consuming, quantification is difficult and MS can be applied only offline. Capillary IEF and related IEF techniques in combination with MS provide similar information. The major benefits are high mass resolution and mass accuracy, reproducibility, speed, automation, and quantification by using a high-resolution mass spectrometer. However, online hyphenation of CIEF with MS is interfered by the ampholytes, acids, and bases needed for high-resolution IEF. This review will give an overview about important coupling techniques, like low ampholyte concentration, interim separation by chromatography, or the use of a dialysis interface to separate the analytes from interfering substances. It is focused on strategies which allow sensitive MS detection of CIEF-separated analytes. In addition, proteomic and biopharmaceutical applications of capillary IEF techniques combined with MS are briefly summarized.

Reduced net charge and heterogeneity of pI isoforms in familial amyotrophic lateral sclerosis mutants of copper/zinc superoxide dismutase.

Familial cases of amyotrophic lateral sclerosis (fALS) are related to mutations of copper/zinc superoxide dismutase 1 (SOD1). Aggregation of SOD1 plays a central role in the pathogenesis of fALS and altered metallation of SOD1 mutants could be involved in this process. Using IEF gel electrophoresis under non-denaturating conditions and particle induced X-ray emission (PIXE) analysis, we studied the pI distribution and metallation status of fALS SOD1 mutants (A4V, G93A, D125H) compared to human wild-type (hWT). SOD1 fALS mutants are characterized by a variable number of isoforms and higher pI compared to hWT, reflecting a reduced net charge that might explain their greater propensity to precipitation and aggregation. Cu/Zn ratios were slightly different for the predominant expressed isoforms of A4V, G93A, and D125H mutants compared to hWT. Differences in metallation were observed within each genotype, the more basic isoforms exhibiting lower Cu/Zn ratios. Moreover, we revealed the existence of a pool of fALS mutants SOD1 pI isoforms, slightly expressed (<10%), with a low Cu/Zn ratio and high pI values. Overall, IEF-PIXE results suggest that the toxicity of SOD1 mutants should be studied at the pI isoform level with a particular attention to the species with the lowest charges.