Cell Protein Extracts Research Articles

A Streamlined Sample Preparation Method for Mass Spectrometric Analysis.

Mass spectrometry-based proteomic technology experienced remarkable advancement in the past decades. However, their application was still hampered by the complexity of sample preparation. Conventional strategies for sample preparation incorporate multiple time-consuming steps, including cell lysis, protein extraction, protease cleavage, and desalting. Thus, we explored a simplified method (the cell-absorb method) during which living cells were absorbed into vacuum-dried polyacrylamide gel and directly digested in gel into peptides for subsequent LC-MS/MS analysis. As a consequence, both of the steps for cell lysis and protein extraction involved in traditional protocol were skipped. In addition to the decrease in time, more proteins were identified. Indeed, 3022 proteins were identified by the cell-absorb method. Meanwhile, only 2642 and 2420 proteins were identified by the classical SDS-PAGE based method and the reported gel absorption-based method, respectively. The cell-absorb method exhibited apparent advantage in terms of the depth of proteome coverage. Furthermore, the number of proteins identified show excellent reproducibility with a CV (coefficient of variation) of 0.03 among three replicates using the cell-absorb method. These advantages suggest that cell-absorb method is a promising choice for mapping the whole proteome of cells. © 2018 by John Wiley & Sons, Inc.

Synthesis of novel (−)-epicatechin derivatives as potential endothelial GPER agonists: Evaluation of biological effects

To potentially identify proteins that interact (i.e. bind) and may contribute to mediate (−)-epicatechin (Epi) responses in endothelial cells we implemented the following strategy: 1) synthesis of novel Epi derivatives amenable to affinity column use, 2) in silico molecular docking studies of the novel derivatives on G protein-coupled estrogen receptor (GPER), 3) biological assessment of the derivatives on NO production, 4) implementation of an immobilized Epi derivative affinity column and, 5) affinity column based isolation of Epi interacting proteins from endothelial cell protein extracts. For these purposes, the Epi phenol and C3 hydroxyl groups were chemically modified with propargyl or mesyl groups. Docking studies of the novel Epi derivatives on GPER conformers at 14 ns and 70 ns demostrated favorable thermodynamic interactions reaching the binding site. Cultures of bovine coronary artery endothelial cells (BCAEC) treated with Epi derivatives stimulated NO production via Ser1179 phosphorylation of eNOS, effects that were attenuated by the use of the GPER blocker, G15. Epi derivative affinity columns yielded multiple proteins from BCAEC. Proteins were electrophoretically separated and inmmunoblotting analysis revealed GPER as an Epi derivative binding protein. Altogether, these results validate the proposed strategy to potentially isolate and identify novel Epi receptors that may account for its biological activity.

<i>N</i>-Nitrosation of Dimethylamine by <i>Bacillus cereus</i> Isolated from Fermented Palm Sap (<i>Elaeis guineensis</i>)

This study investigated the nitrosating activities of intact cells and cell fractions of Bacillus cereus isolated from fermented palm wine ( Elaeis guineensis ) during incubation with dimethylamine and nitrite or nitrate. The pH values of the incubation mixtures were 7.2 ± 0.07 and 7.1 ± 0.07 in the presence of nitrite and nitrate, respectively. The N -Nitrosodimethylamine (NDMA) level in cell debris was significantly higher compared with cell extract, cell suspension and sterile controls. The time-course of NDMA formation in cell debris showed a significant (p m ) value was 37.5% lower, while initial velocity (V max ) was 20.0% higher in cell debris relative to cell extract, when a fixed DMA concentration was used against varying nitrite concentrations. However, at a fixed nitrite concentration against varying DMA concentration, K m value was 44.4% lower, while V max was 166.7% higher in cell debris compared with cell extract. Investigation of nitrosation mechanism showed that V max values in the cell debris were 41.7, 50.0 and 55.6 μmol NDMA/mg protein, while the values were 34.5, 37.0 and 43.5 μmol NDMA/mg protein in cell extract for the respective three nitrite concentrations. This study has shown that intact Bacillus cereus cells catalyzed nitrosation of dimethylamine at near neutral pH, and the nitrosation, which was higher in cell debris than cell extract of the bacterium, followed a sequential mechanism of enzyme catalysis.

Sertoli Cell Preparation for Co-immunoprecipitation.

Most techniques to study protein-protein interactions, gene expression or signal transduction, among others, in Sertoli cells, depend on obtaining a protein extract of such cells. This is accomplished by lysing the Sertoli cells and solubilizing the intracellular proteins. Depending on the purpose of your study, the technique used to lyse and consequently obtain protein extracts from Sertoli cells must be considered. In this chapter, we will focus on how to obtain a protein Sertoli cell extract suitable for co-immunoprecipitation technique.

Novel fluorochromes label tonoplast in living plant cells and reveal changes in vacuolar organization after treatment with protein phosphatase inhibitors.

The recently synthesized isocyanonaphtalene derivatives ACAIN and CACAIN are fluorochromes excitable at wavelengths of around 366nm and bind cysteine-rich proteins with hydrophobic motifs. We show that these compounds preferentially label tonoplasts in living Arabidopsis and tobacco (Nicotiana tabacum SR1) cells. ACAIN-labeled membranes co-localized with the GFP signal in plants expressing GFP-δ-TIP (TIP2;1) (a tonoplast aquaporin) fusion protein. ACAIN preserved the dynamics of vacuolar structures. tip2;1 and triple tip1;1-tip1;2-tip2;1 knockout mutants showed weaker ACAIN signal in tonoplasts. The fluorochrome is also suitable for the labeling and detection of specific (cysteine-rich, hydrophobic) proteins from crude cell protein extracts following SDS-PAGE and TIP mutants show altered labeling patterns; however, it appears that ACAIN labels a large variety of tonoplast proteins. ACAIN/CACAIN could be used for the detection of altered vacuolar organization induced by the heptapeptide natural toxin microcystin-LR (MCY-LR), a potent inhibitor of both type 1 and 2A protein phosphatases and a ROS inducer. As revealed both in plants with GFP-TIP2;1 fusions and in wild-type (Columbia) plants labeled with ACAIN/CACAIN, MCY-LR induces the formation of small vesicles, concomitantly with the absence of the large vegetative vacuoles characteristic for differentiated cells. TEM studies of MCY-LR-treated Arabidopsis cells proved the presence of multimembrane vesicles, with characteristics of lytic vacuoles or autophagosomes. Moreover, MCY-LR is a stronger inducer of small vesicle formation than okadaic acid (which inhibits preferentially PP2A) and tautomycin (which inhibits preferentially PP1). ACAIN and CACAIN emerge as useful novel tools to study plant vacuole biogenesis and programmed cell death.

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method.

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and α-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an α-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and α-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an α-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

Screening of Neem extracts for microbial anti-chaperone activity by employing in vitro enzyme refolding assay.

Microbial heat shock proteins(Hsps) play an important role in pathogenesis and development of resistance to existing drugs. New compounds that target microbial molecular chaperones have the potential of combating the challenge of anti-microbial resistance. The present study was aimed at assessing the employment of in vitro enzyme refolding assay to detect anti-chaperone activity of Neem (Azadirachta indica) extracts. Protein extracts of thermotolerant Escherichia coli cells were used as a source of Hsps or chaperones. Thermotolerance was found to be induced by pre-treating E. coli cells at 47°C before subjecting them to a lethal temperature of 55°C. This thermotolerance correlated with over-expression of specific proteins and reduced aggregation as evident from the SDS-PAGE profiles. Refolding assays of denatured enzymes exhibited 45% activity regain in presence of cell protein extracts containing chaperones compared to less than 5% regain in BSA negative controls. The chaperone activity was found to be ATP dependent. Addition of Neem extracts to refolding reaction mixtures distinctly reduced the activity regain (20%) in a dose dependent manner (500 and 1000ppm). The negative influence of plant extract on refolding of the enzyme in the presence of chaperones gives evidence to its anti-chaperone activity. We propose that the employment of in vitro enzyme refolding assays will help not only to analyze the activity of known and putative chaperones but also to screen natural compounds for anti-microbial-Hsp activity.

Abstract 1938: Exportin-1 (XPO1) is a novel therapeutic biomarker for patients with neuroblastoma

Abstract Background: Half of patients with high-risk neuroblastoma succumb to disease, yet these patients with inferior outcome cannot be identified at diagnosis despite contemporary risk stratification that integrates MYCN copy number status, tumor histology, and patient age. We have shown that overexpression of Exportin-1 (XPO1) is associated with poor survival in neuroblastoma, affirming what has been identified across a range of malignancies. Selinexor (KPT-330, Karyopharm Therapeutics), an XPO1 inhibitor in early phase clinical trials, inhibits the nuclear to cytoplasmic translocation of tumor suppressor and growth regulatory proteins implicated in oncogenesis. We hypothesized that protein expression level of XPO1 in neuroblastoma cell lines predict the effective response of treatment with selinexor. Methods: We used a panel of early passage patient derived neuroblastoma cell lines including IMR5, NLF, KELLY, NB-EBC1, and SKNSH that are representative of the spectrum and genetic diversity of human disease. Cell lines were treated with varying concentrations of selinexor. RNA and whole cell protein extract as well as nuclear and cytoplasmic fractionated protein was obtained from untreated and treated conditions. Treated and untreated cells were also pelleted, and, along with patient tumor samples, formalin-fixed and paraffin-embedded for microscopic and immunohistochemical (IHC) evaluation. Results: Compared to noncancerous cell lines, neuroblastoma cell lines have high protein and mRNA expression of XPO1; higher expression correlates with greater sensitivity to selinexor. Selinexor treatment reduced XPO1 protein and increased XPO1 mRNA expression, and it lead to nuclear retention of XPO1 cargo such as p53 and survivin. Comparison of microscopic features and protein staining intensity of patient samples with known XPO1 expression is ongoing. Conclusions: This study provides rationale for XPO1 protein abundance as a potential biomarker of therapeutic response to the drug selinexor. By using patient derived neuroblastoma cell lines, we are able to evaluate the response of selinexor and XPO1 expression in the context of this enigmatic disease. These data suggest that initial drug response to selinexor can be predicted early in the course of disease and more investigation of the high expression of XPO1 seen in patients with poor outcome will provide strategies for combinatorial treatment approaches. Citation Format: Basia Galinski, Marcus Luxemburg, Michelle Ewart, Yosef Landesman, Daniel Weiser. Exportin-1 (XPO1) is a novel therapeutic biomarker for patients with neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1938. doi:10.1158/1538-7445.AM2017-1938

Abstract 1798: Mortalin precursor as potential marker for chemoprevention with SHetA2

Abstract Introduction: The flexible heteroarotinoid (Flex-Het), SHetA2, is a novel anticancer drug that induces both intrinsic and extrinsic apoptosis and autophagy in cancer cells, but not in normal cells. Protein isolation / mass spectrometry analysis using SHetA2-coupled polystyrene magnetic beads yielded three SHetA2-binding proteins all belonging to HSPA family namely, HSPA9/mortalin, HSPA8/Hsc70 and HSPA5/BiP. Mortalin, in addition to its vital chaperoning roles in other organelles of the cell, is essential for import and folding of nuclear-encoded mitochondrial proteins. The precursor form of mortalin has a 46-amino acid N-terminal region that functions as a mitochondrial localization sequence (MLS) and is cleaved by proteases after import into the mitochondrial matrix. Hypothesis: SHetA2 binding to mortalin causes alterations that can be measured to study mechanism and monitor drug effects in animal models and clinical trials. Methods: Western blots of whole cell or subcellular protein extracts made from cultures of normal human fallopian tube secretory or mammary epithelial cells, rat mammary tumors or human ovarian cancer cell lines treated with SHetA2 or solvent were probed with an antibody that recognizes total mortalin, a custom-made rabbit polyclonal antibody specific for the mortalin MLS (PA8238) or antibodies to loading control proteins. Immunocytochemistry using these antibodies was performed with an automated (Leica Bond III) IHC work station on cells treated with SHetA2 or solvent. Results: Using a commercial antibody against total mortalin, we observed a lower-mobility band closely moving above the specific (mature) mortalin band in Western blots. Our mortalin MLS-specific antibody recognized only the lower-mobility band confirming it as the precursor form of mortalin in the SHetA2-treated cell extracts. Subcellular fractionation of the drug-treated cells revealed that the precursor protein accumulated only in the cytoplasm and not in the mitochondria. Staining of whole cells with an antibody to total mortalin showed no effect of SHetA2 treatment on the punctate pattern of expression consistent with mitochondrial localization. In contrast, staining with the mortalin MLS-specific antibody demonstrated that SHetA2 increased the intensity of the diffuse cytoplasmic staining consistent with localization of mortalin throughout the cytoplasm. These results were consistently observed among the various cell types. Conclusions: SHetA2 is the only known drug that blocks the translocation of the precursor mortalin to mitochondria and its processing to mature protein. Such cytoplasmic accumulation of the precursor form of mortalin can potentially serve as pharmacodynamic endpoint to study SHetA2’s effect in laboratory experimental tumor models as well as in human clinical trials. Funding: NCI PREVENT Task Order HHSN26100002 Citation Format: Elangovan Thavathiru, Vishal Chandra, Rajani Rai, Doris Benbrook. Mortalin precursor as potential marker for chemoprevention with SHetA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1798. doi:10.1158/1538-7445.AM2017-1798

Proteomic analysis of organic sulfur compound utilisation in Advenella mimigardefordensis strain DPN7T.

2-Mercaptosuccinate (MS) and 3,3´-ditiodipropionate (DTDP) were discussed as precursor substance for production of polythioesters (PTE). Therefore, degradation of MS and DTDP was investigated in Advenella mimigardefordensis strain DPN7T, applying differential proteomic analysis, gene deletion and enzyme assays. Protein extracts of cells cultivated with MS, DTDP or 3-sulfinopropionic acid (SP) were compared with those cultivated with propionate (P) and/or succinate (S). The chaperone DnaK (ratio DTDP/P 9.2, 3SP/P 4.0, MS/S 6.1, DTDP/S 6.2) and a Do-like serine protease (DegP) were increased during utilization of all organic sulfur compounds. Furthermore, a putative bacterioferritin (locus tag MIM_c12960) showed high abundance (ratio DTDP/P 5.3, 3SP/P 3.2, MS/S 4.8, DTDP/S 3.9) and is probably involved in a thiol-specific stress response. The deletion of two genes encoding transcriptional regulators (LysR (MIM_c31370) and Xre (MIM_c31360)) in the close proximity of the relevant genes of DTDP catabolism (acdA, mdo and the genes encoding the enzymes of the methylcitric acid cycle; prpC,acnD, prpF and prpB) showed that these two regulators are essential for growth of A. mimigardefordensis strain DPN7T with DTDP and that they most probably regulate transcription of genes mandatory for this catabolic pathway. Furthermore, proteome analysis revealed a high abundance (ratio MS/S 10.9) of a hypothetical cupin-2-domain containing protein (MIM_c37420). This protein shows an amino acid sequence similarity of 60% to a newly identified MS dioxygenase from Variovorax paradoxus strain B4. Deletion of the gene and the adjacently located transcriptional regulator LysR, as well as heterologous expression of MIM_c37420, the putative mercaptosuccinate dioxygenase (Msdo) from A. mimigardefordensis, showed that this protein is the key enzyme of MS degradation in A. mimigardefordensis strain DPN7T (KM 0.2 mM, specific activity 17.1 μmol mg-1 min-1) and is controlled by LysR (MIM_c37410).

Direct digestion of living cells via a gel-based strategy for mass spectrometric analysis.

A novel sample preparation method was established for proteomic analysis, during which living cells were absorbed into vacuum-dried polyacrylamide gel and directly digested into peptides for subsequent LC-MS/MS assays. As a consequence, both of the steps for cell lysis and protein extraction involved in a conventional digestion method were skipped.

Intravenous Administration of Adipose-Derived Stem Cell Protein Extracts Improves Neurological Deficits in a Rat Model of Stroke.

Treatment of adipose-derived stem cell (ADSC) substantially improves the neurological deficits during stroke by reducing neuronal injury, limiting proinflammatory immune responses, and promoting neuronal repair, which makes ADSC-based therapy an attractive approach for treating stroke. However, the potential risk of tumorigenicity and low survival rate of the implanted cells limit the clinical use of ADSC. Cell-free extracts from ADSC (ADSC-E) may be a feasible approach that could overcome these limitations. Here, we aim to explore the potential usage of ADSC-E in treating rat transient middle cerebral artery occlusion (tMCAO). We demonstrated that intravenous (IV) injection of ADSC-E remarkably reduces the ischemic lesion and number of apoptotic neurons as compared to other control groups. Although ADSC and ADSC-E treatment results in a similar degree of a long-term clinical beneficial outcome, the dynamics between two ADSC-based therapies are different. While the injection of ADSC leads to a relatively mild but prolonged therapeutic effect, the administration of ADSC-E results in a fast and pronounced clinical improvement which was associated with a unique change in the molecular signature suggesting that potential mechanisms underlying different therapeutic approach may be different. Together these data provide translational evidence for using protein extracts from ADSC for treating stroke.

Beyond bread and beer: whole cell protein extracts from baker's yeast as a bulk source for 3D cell culture matrices.

Here, we present a novel approach to form hydrogels from yeast whole cell protein. Countless hydrogels are available for sophisticated research, but their fabrication is often difficult to reproduce, with the gels being complicated to handle or simply too expensive. The yeast hydrogels presented here are polymerized using a four-armed, amine reactive crosslinker and show a high chemical and thermal resistance. The free water content was determined by measuring swelling ratios for different protein concentrations, and in a freeze-drying approach, pore sizes of up to 100μm in the gel could be created without destabilizing the 3D network. Elasticity was proofed to be adjustable with the help of atomic force microscopy by merely changing the amount of used protein. Furthermore, the material was tested for possible cell culture applications; diffusion rates in the network are high enough for sufficient supply of human breast cancer cells and adenocarcinomic human alveolar basal epithelial cells with nutrition, and cells showed high viabilities when tested for compatibility with the material. Furthermore, hydrogels could be functionalized with RGD peptide and the optimal concentration for sufficient cell adhesion was determined to be 150μM. Given that yeast protein is one of the cheapest and easiest available protein sources and that hydrogels are extremely easy to handle, the developed material has highly promising potential for both sophisticated cell culture techniques as well as for larger scale industrial applications.

TET2 binds the androgen receptor and loss is associated with prostate cancer.

Genetic alterations associated with prostate cancer (PCa) may be identified by sequencing metastatic tumor genomes to identify molecular markers at this lethal stage of disease. Previously, we characterized somatic alterations in metastatic tumors in the methylcytosine dioxygenase ten-eleven translocation 2 (TET2), which is altered in 5–15% of myeloid, kidney, colon and prostate cancers. Genome-wide association studies previously identified non-coding risk variants associated with PCa and melanoma. We performed fine-mapping of PCa risk across TET2 using genotypes from the PEGASUS case-control cohort and identified six new risk variants in introns 1 and 2. Oligonucleotides containing two risk variants were bound by the transcription factor octamer-binding protein 1 (Oct1/POU2F1) and TET2 and Oct1 expression were positively correlated in prostate tumors. TET2 is expressed in normal prostate tissue and reduced in a subset of tumors from the Cancer Genome Atlas (TCGA). Small interfering RNA (siRNA)-mediated TET2 knockdown (KD) increases LNCaP cell proliferation, migration, and wound healing, verifying loss drives a cancer phenotype. Endogenous TET2 bound the androgen receptor (AR) and AR-coactivator proteins in LNCaP cell extracts, and TET2 KD increases prostate-specific antigen (KLK3/PSA) expression. Published data reveal TET2 binding sites and hydroxymethylcytosine (hmC) proximal to KLK3. A gene co-expression network identified using TCGA prostate tumor RNA-sequencing identifies co-regulated cancer genes associated with 2-oxoglutarate (2-OG) and succinate metabolism, including TET2, lysine demethylase (KDM) KDM6A, BRCA1-associated BAP1, and citric acid cycle enzymes IDH1/2, SDHA/B, and FH. The co-expression signature is conserved across 31 TCGA cancers suggesting a putative role for TET2 as an energy sensor (of 2-OG) that modifies aspects of androgen-AR signaling. Decreased TET2 mRNA expression in TCGA PCa tumors is strongly associated with reduced patient survival indicating reduced expression in tumors maybe an informative biomarker of disease progression and perhaps metastatic disease.

Chaperonin-containing T-complex Protein 1 Subunit ζ Serves as an Autoantigen Recognized by Human Vδ2 γδ T Cells in Autoimmune Diseases

Human γδ T cells recognize conserved endogenous and stress-induced antigens typically associated with autoimmune diseases. However, the role of γδ T cells in autoimmune diseases is not clear. Few autoimmune disease-related antigens recognized by T cell receptor (TCR) γδ have been defined. In this study, we compared Vδ2 TCR complementarity-determining region 3 (CDR3) between systemic lupus erythematosus (SLE) patients and healthy donors. Results show that CDR3 length distribution differed significantly and displayed oligoclonal characteristics in SLE patients when compared with healthy donors. We found no difference in the frequency of Jδ gene fragment usage between these two groups. According to the dominant CDR3δ sequences in SLE patients, synthesized SL2 peptides specifically bound to human renal proximal tubular epithelial cell line HK-2; SL2-Vm, a mutant V sequence of SL2, did not bind. We identified the putative protein ligand chaperonin-containing T-complex protein 1 subunit ζ (CCT6A) using SL2 as a probe in HK-2 cell protein extracts by affinity chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry analysis. We found CCT6A expression on the surface of HK-2 cells. Cytotoxicity of only Vδ2 γδ T cells to HK-2 cells was blocked by anti-CCT6A antibody. Finally, we note that CCT6A concentration was significantly increased in plasma of SLE and rheumatoid arthritis patients. These data suggest that CCT6A is a novel autoantigen recognized by Vδ2 γδ T cells, which deepens our understanding of mechanisms in autoimmune diseases.

Selectivity Determination of a Small Molecule Chemical Probe Using Protein Microarray and Affinity Capture Techniques.

Small molecule selectivity is an essential component of candidate drug selection and target validation. New technologies are required to better understand off-target effects, with particular emphasis needed on broad protein profiling. Here, we describe the use of a tritiated chemical probe and a 9000 human protein microarray to discern the binding selectivity of an inhibitor of the mRNA decapping scavenger enzyme DcpS. An immobilized m7GTP resin was also used to assess the selectivity of a DcpS inhibitor against mRNA cap-associated proteins in whole cell extracts. These studies confirm the exquisite selectivity of diaminoquinazoline DcpS inhibitors, and highlight the utility of relatively simple protein microarray and affinity enrichment technologies in drug discovery and chemical biology.

Protein-Induced Pluripotent Stem Cells Ameliorate Cognitive Dysfunction and Reduce Aβ Deposition in a Mouse Model of Alzheimer's Disease.

Transplantation of stem cells into the brain attenuates functional deficits in the central nervous system via cell replacement, the release of specific neurotransmitters, and the production of neurotrophic factors. To identify patient‐specific and safe stem cells for treating Alzheimer's disease (AD), we generated induced pluripotent stem cells (iPSCs) derived from mouse skin fibroblasts by treating protein extracts of embryonic stem cells. These reprogrammed cells were pluripotent but nontumorigenic. Here, we report that protein‐iPSCs differentiated into glial cells and decreased plaque depositions in the 5XFAD transgenic AD mouse model. We also found that transplanted protein‐iPSCs mitigated the cognitive dysfunction observed in these mice. Proteomic analysis revealed that oligodendrocyte‐related genes were upregulated in brains injected with protein‐iPSCs, providing new insights into the potential function of protein‐iPSCs. Taken together, our data indicate that protein‐iPSCs might be a promising therapeutic approach for AD. Stem Cells Translational Medicine 2017;6:293–305

Measurement of O(6)-alkylguanine-DNA alkyltransferase activity in tumour cells using stable isotope dilution HPLC-ESI-MS/MS.

The repair of DNA mediated by O(6)-alkylguanine-DNA alkyltransferase (AGT) provides protection against DNA damage from endogenous or exogenous alkylation of the O(6) position of guanine. However, this repair acts as a double-edged sword in cancer treatment, as it not only protects normal cells from chemotherapy-associated toxicities, but also results in cancer cell resistance to guanine O(6)-alkylating antitumour agents. Thus, AGT plays an important role in predicting the individual susceptibility to guanine O(6)-alkylating carcinogens and chemotherapies. Accordingly, it is necessary to establish a quantitative method for determining AGT activity with high accuracy, sensitivity and practicality. Here, we describe a novel nonradioactive method for measuring AGT activity using stable isotope dilution high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). This method is based on the irreversibility of the removal of the O(6)-alkyl group from guanine by AGT and on the high affinity of O(6)-benzylguanine (O(6)-BG) as an AGT substrate. HPLC-ESI-MS/MS was used to measure the AGT activities in cell protein extracts from eight tumour lines, demonstrating that AGT activity was quite variable among different cell lines, ranging from nondetectable to 1021 fmol/mg protein. The experiments performed in intact tumour cells yielded similar results but exhibited slightly higher activities than those observed in cell protein extracts. The accuracy of this method was confirmed by an examination of AGT expression levels using western blotting analysis. To our knowledge, this method is the first mass spectrometry-based AGT activity assay, and will likely provide assistance in the screening of cancer risk or the application of chemotherapies.

Evaluation of single cell protein from yeast for the development of wood adhesives

The use of synthetic adhesives in the wood-based panels industry has been influenced by environmental and health restrictions. The main adhesives used in the industry have been urea–formaldehyde and phenol–formaldehyde. However, as these raw materials are petroleum products and natural gas, their processes are related to global warming and they may not be available in the future. Even formaldehyde affects human health. These issues have promoted the use of natural raw materials from sustainable origin in the manufacture of adhesives, such as lignin, tannins and proteins. This research proposes the use of single cell protein extract from yeast (SCPey) as a raw material to produce novel wood adhesives. The protein contents, amino acidic composition and the molecular weight distribution of protein extracts from Rhodotorula rubra, Pichia pastoris and Sacharomyces cerevisiae were investigated. Adhesive solutions were characterized and tested through a dynamic thermo mechanical analysis (DMA). Lap joints of wood veneers were assembled using adhesive solutions in order to perform mechanical tests and evaluate the adhesive performance. Protein content of R. rubra, P. pastoris and S. cerevisiae were 72.4, 71.0 and 64.6 %, respectively. The amino acidic composition of SCPey samples promotes the content of reactive functional groups with the ones present in wood polymers. Molecular weight distribution of proteins from protein extracts ranged from 8 to 130 kDa. DMA showed that the adhesive solutions of the SCPey have a proper performance enhancing the wood adhesion. The joint strength under dry conditions with adhesives based on SCPey ranged from 2.40 to 2.99 MPa, which is similar to those obtained with UF and based on a protein isolated soy flour adhesive. Hence, the results showed that SCPey is a promising natural raw material to produce alternative wood adhesives with superior performance.