Sex-specific microglia state in the Neuroligin-4 knock-out mouse model of autism spectrum disorder.

Neuroligin-4 (NLGN4) loss-of-function mutations are associated with monogenic heritable autism spectrum disorder (ASD) and cause alterations in both synaptic and behavioral phenotypes. Microglia, the resident CNS macrophages, are implicated in ASD development and progression. Here we studied the impact of NLGN4 loss in a mouse model, focusing on microglia phenotype and function in both male and female mice. NLGN4 depletion caused lower microglia density, less ramified morphology, reduced response to injury and purinergic signaling specifically in the hippocampal CA3 region predominantly in male mice. Proteomic analysis revealed disrupted energy metabolism in male microglia and provided further evidence for sexual dimorphism in the ASD associated microglial phenotype. In addition, we observed impaired gamma oscillations in a sex-dependent manner. Lastly, estradiol application in male NLGN4-/- mice restored the altered microglial phenotype and function. Together, these results indicate that loss of NLGN4 affects not only neuronal network activity, but also changes the microglia state in a sex-dependent manner that could be targeted by estradiol treatment.

SubCellBarCode: integrated workflow for robust spatial proteomics by mass spectrometry.

The molecular functions of a protein are defined by its inherent properties in relation to its environment and interaction network. Within a cell, this environment and network are defined by the subcellular location of the protein. Consequently, it is crucial to know the localization of a protein to fully understand its functions. Recently, we have developed a mass spectrometry- (MS) and bioinformatics-based pipeline to generate a proteome-wide resource for protein subcellular localization across multiple human cancer cell lines ( www.subcellbarcode.org ). Here, we present a detailed wet-lab protocol spanning from subcellular fractionation to MS-sample preparation and analysis. A key feature of this protocol is that it includes all generated cell fractions without discarding any material during the fractionation process. We also describe the subsequent quantitative MS-data analysis, machine learning-based classification, differential localization analysis and visualization of the output. For broad applicability, we evaluated the pipeline by using MS data generated by two different peptide pre-fractionation approaches, namely high-resolution isoelectric focusing and high-pH reverse-phase fractionation, as well as direct analysis without pre-fractionation by using long-gradient liquid chromatography-MS. Moreover, an R package covering the dry-lab part of the method was developed and made available through Bioconductor. The method is straightforward and robust, and the entire protocol, from cell harvest to classification output, can be performed within 1-2 weeks. The protocol enables accurate classification of proteins to 15 compartments and 4 neighborhoods, visualization of the output data and differential localization analysis including treatment-induced protein relocalization, condition-dependent localization or cell type-specific localization. The SubCellBarCode package is freely available at https://bioconductor.org/packages/devel/bioc/html/SubCellBarCode.html .

Proteogenomics of non-small cell lung cancer reveals molecular subtypes associated with specific therapeutic targets and immune evasion mechanisms.

Despite major advancements in lung cancer treatment, long-term survival is still rare, and a deeper understanding of molecular phenotypes would allow the identification of specific cancer dependencies and immune evasion mechanisms. Here we performed in-depth mass spectrometry (MS)-based proteogenomic analysis of 141 tumors representing all major histologies of non-small cell lung cancer (NSCLC). We identified six distinct proteome subtypes with striking differences in immune cell composition and subtype-specific expression of immune checkpoints. Unexpectedly, high neoantigen burden was linked to global hypomethylation and complex neoantigens mapped to genomic regions, such as endogenous retroviral elements and introns, in immune-cold subtypes. Further, we linked immune evasion with LAG3 via STK11 mutation-dependent HNF1A activation and FGL1 expression. Finally, we develop a data-independent acquisition MS-based NSCLC subtype classification method, validate it in an independent cohort of 208 NSCLC cases and demonstrate its clinical utility by analyzing an additional cohort of 84 late-stage NSCLC biopsy samples.

Elucidation of Selectivity for Silver Ion Based on Metal-Induced H-Type Aggregation of Fluorescent Receptor.

In present work, a new substituted phthalonitrile derivative was prepared by the nucleophilic displacement reaction and then highly soluble zinc phthalocyanine (ZnPc) with four peripheral 1-hydroxyhexan-3-ylthio groups was synthesized by cyclotetramerization and characterized by FTIR, 1H and 13C NMRs spectroscopies, fluorescence and UV-vis measurements. The optical property and quantum yield of ZnPc were elucidated in mixed solvent of ethanol/water with varying compositions. The pH-dependent fluorescence and absorbance spectra of ZnPc in the absence and presence of Ag+ ions were obtained to elucidate the optimum pH value that is convenient for stable complex formation in predetermined mixture. A comparative study for recognition of Ag+ ion has been carried out to evaluate the effect of the solution parameters on selective sensing ability of ZnPc as a fluorescent receptor. Interference effect was investigated by spectrofluorometric titration based on fluorescence quenching of ZnPc upon addition of Ag+ ion in the presence of foreign metal ions. Stepwise complexation/decomplexation cycles with Na2S-titration by fluorescence quenching/enhancement were investigated to establish the reversible response rate and reusability of ZnPc toward Ag+ ions. Graphical Abstract Selective determination of silver ion based on H-type aggregation of zinc phthalocyanine.

Ratiometric sensor based on imprinted quantum dots-cationic dye nanohybrids for selective sensing of dsDNA.

A ratiometrically responsive sensor for dsDNA is reported, based on molecularly imprinted polymer coated quantum dots (MIP-QDs). A new platform is described for probing dsDNA by tracing the "turn on" fluorescence signal of malachite green (MG) as a cationic dye and "turn off-on" room temperature phosphorescence (RTP) signal of MIP-QDs/MG nanohybrids. The interaction between MIP-QDs surface and MG discloses an intense quenching in RTP (turning off) by a phosphorescence resonance energy transfer (PRET) process. After the addition of dsDNA, MG molecules escape from the MIP-QDs surface and intercalate into the dsDNA, resulting in the restoration of RTP intensity of MIP-QDs (turning on) and also enhancing in fluorescence of MG. This outcome hereby can be employed for the selective sensing of dsDNA via optical response. The ratio of fluorescence enhancement of MG to RTP intensity of MIP-QDs is proportional to the concentration of dsDNA in the range of 0.089-1.79 µg/mL with a detection limit (3σ/K) of 19.48 ng/mL under the optimized experimental conditions.

SubCellBarCode: Proteome-wide Mapping of Protein Localization and Relocalization.

Subcellular localization is a main determinant of protein function; however, a global view of cellular proteome organization remains relatively unexplored. We have developed a robust mass spectrometry-based analysis pipeline to generate a proteome-wide view of subcellular localization for proteins mapping to 12,418 individual genes across five cell lines. Based on more than 83,000 unique classifications and correlation profiling, we investigate the effect of alternative splicing and protein domains on localization, complex member co-localization, cell-type-specific localization, as well as protein relocalization after growth factor inhibition. Our analysis provides information about the cellular architecture and complexity of the spatial organization of the proteome; we show that the majority of proteins have a single main subcellular location, that alternative splicing rarely affects subcellular location, and that cell types are best distinguished by expression of proteins exposed to the surrounding environment. The resource is freely accessible via www.subcellbarcode.org.

Engineering filamentous bacteriophages for enhanced gold binding and metallization properties.

Filamentous bacteriophages are nanowire-like virion molecules consisting of a single stranded DNA (ssDNA) as the genomic material packed in a protein cage. In this study, Tyr containing 5-mer peptides were displayed on phage filaments for enhanced Au binding and reduction properties. Wild type fd (AEGDD) and engineered YYYYY, AYSSG and AYGDD phages were investigated by Quartz crystal microbalance (QCM), Atomic force microscopy (AFM), Scanning electron microscopy (SEM) and Energy dispersive X-ray spectroscopy (EDX) analyses. Presence of only one Tyr unit on five aa flexible region of p8 coat proteins increased Au binding affinities of engineered phages. YYYYY phages were shown to have the strongest Au surface and AuNP binding affinities. Recombinant phages were shown to be coated with Au clusters after one-step metallization reaction. With further genetic modifications, phages can be programmed to function as site specific self-assembling biotemplates for bottom-up manufacturing in nanoelectronics and biosensor application studies.

A theoretical study on the molecular structure and vibrational (FT-IR and Raman) spectra of cyano-bridged heteronuclear polymeric complex of triethylenetetramine.

The cyano bridged complex of triethylenetetramine was characterized by FT-IR, Raman spectroscopy and X-ray single crystal diffraction analysis. The molecular geometry and vibrational frequencies of the complex in the ground state have been calculated by using B3LYP density functional method with LANL2DZ basis set. A good correlation was found via comparison of the experimental and theoretical vibrational frequencies of complex. The complex of the type [Zn(teta)Ni(µ-CN)(2)(CN)(2)](n) has been studied in the 4000-250cm(-1) region and assignment of all the observed bands were made. The analysis of the FT-IR and Raman spectra indicates that there are some structure spectra correlations.

Approach to interfacial and intramolecular electron transfer of the diheme protein cytochrome c4 assembled on Au(111) surfaces.

Intramolecular electron transfer (ET) between metal centers is a core feature of large protein complexes in photosynthesis, respiration, and redox enzyme catalysis. The number of microscopic redox potentials and ET rate constants is, however, prohibitive for experimental cooperative ET mapping, but two-center proteins are simple enough to offer complete communication networks. At the same time, multicenter redox proteins operate in membrane environments where conformational dynamics may lead to gated ET features different from conditions in homogeneous solution. The bacterial respiratory diheme protein Pseudomonas stutzeri cytochrome c(4) has been a target for intramolecular, interheme ET. We report here voltammetric and in situ scanning tunneling microscopy (STM) data for P. stutzeri cyt c(4) at single-crystal, atomically planar Au(111)-electrode surfaces modified by variable-length omega-mercapto-alkanoic carboxylic acids. As evidenced by in situ STM, the strongly dipolar protein is immobilized in a close to vertical orientation at this surface with the positively charged high-potential heme domain adjacent to the electrode. This orientation gives asymmetric voltammograms with two one-ET peaks in the cathodic direction and a single two-ET peak in the anodic direction. Intramolecular, interheme ET with high, 8,000-30,000 s(-1), rate constants is notably an essential part of this mechanism. The high rate constants are in striking contrast to ET reactions of P. stutzeri cyt c(4) with small reaction partners in homogeneous solution for which kinetic analysis clearly testifies to electrostatic cooperative effects but no intramolecular, interheme ET higher than 0.1-10 s(-1). This difference suggests a strong gating feature of the process. On the basis of the three-dimensional structure of P. stutzeri cyt c(4), gating is understandable due to the through-space, hydrogen-bonded electronic contact between the heme propionates which is highly sensitive to environmental configurational fluctuations.

Theoretical studies on the molecular structure and vibrational spectra of some dimethyl substituted pyridine derivatives.

The molecular geometries, normal mode frequencies, intensities and corresponding infrared assignments of monomeric and dimeric 2,3-dimethylpyridine, 2,4-dimethylpyridine, 3,4-dimethylpyridine, 3,5-dimethylpyridine and monomeric 2,6-dimethylpyridine in the ground state were investigated at the density functional theory (DFT)-B3LYP level using the 6-311+G(d, p) basis set. The vibrational frequencies and geometric parameters of C-H stretching and bending in the fundamental region were calculated and compared to the Fourier transform infrared (FT-IR) data obtained. In the studied monomeric and dimeric dimethyl substituted pyridine derivatives, the C-H stretching and bending frequency shifts that occur between the dimer and the monomer may be diagnostic of the magnitude of dimerization energy. As supported by data in the literature, the most stable dimeric form was obtained for the 3,4-dimethylpyridine molecule.

A theoretical study of substituent effects on tautomerism of 2-hydroxybenzimidazoles.

The geometries, relative stabilities of some 4(7) and 5(6) substituted 2-hydroxybenzimidazole derivatives were calculated with full geometry optimization using AM1 and PM3 in aqueous phase. With the exception of molecules 4, 6 and 7 for all the 4(7) and 5(6) substituted 2-hydroxybenzimidazole derivatives the 3H and keto forms were found to be favored.