Detection of Cancer Cell Death Mediated by a Synthetic Granzyme B-like Peptide Fluorescent Conjugate and the same Peptide Binding in Bacteria

Granzyme-mediated apoptosis, supported by pore-forming perforin, plays an important role in CD8+ T lymphocytes (CTL)-dependent cellular immunity protection against both cancer and viral infection. Quantitative and qualitative problems with CTL are potential contributing factors to disease progression. The feasibility of developing CTL-independent cellular immunity is desired but must first overcome the barrier of CTL-independent target cell recognition. Granzyme B with its strong pro-apoptotic activity in many different target cells is investigated for use in the CTL-independent cellular immunity approach, and granzyme B or its bioactive peptides without the enzymatic activity are more desirable for use. Native granzyme B with enzymatic activity is usually investigated in cancer cells for its mediation of apoptosis by detection of DNA fragmentation. Detection of cell death mediated by such peptides in cancer cells is needed to demonstrate the potential therapeutic purposes. We show with never-before-seen microscopic images using fluorescence microscopy that a synthetic granzyme B-like peptide fluorescent conjugate (GP1R) can: 1) mediate cell death of different cancer cells via membrane extrusion, 2) bind to constitutively expressed binding targets in different cancer cells and bacteria, and 3) promote bacterial phagocytosis. The putative binding targets may serve as a universal pathologic biomarker detectable by GP1R. Our data taken together demonstrate the potential applications of GP1R for use in CTL-independent target cell recognition and target cell death induction. It may lead to development of rapid targeted detection and new treatment of cancer, viral and bacterial infections. The new treatment may show mutual benefits for two or more diseases.     

Use of thermal imaging to characterize laser light reflection from thermoplastics as a function of thickness,laser incidence angle and surface roughness

Laser light reflection during the laser transmission welding (LTW) of thermoplastics has the potential to overheat and/or cause unintentional welding of adjacent features of the part being welded. For this reason, and in order to assess how much light is being absorbed by the transparent part (after measurement of the light transmitted through the transparent part), it is important to be able to quantify the magnitude and distribution of reflected light. The magnitude and distribution of the reflected light depends on the total laser input power as well as its distribution, the laser incidence angle (angle between the normal to the transparent part surface and the laser beam), the laser light polarization as well as the surface and optical properties of the transparent part. A novel technique based on thermal imaging of the reflected light was previously developed by the authors. It is used in this study to characterize the magnitude and distribution of reflected light from thermoplastics as a function of thickness (1–3.1 mm), laser incidence angle (20–40°) and surface roughness (0.04–1.04 μm). Results from reflection tests on nearly polished nylon 6 (surface roughness between 0.04 and 0.05 μm) have shown that, for the various thicknesses tested (1–3.1 mm), the total reflection was larger than the specular top surface reflection predicted via the Fresnel relation. From these observations, it is conjectured that, in addition to top surface reflection, the bulk and/or bottom surface also contribute to the total reflection. The results also showed that reflection decreased slightly with increasing thickness. As expected, for the p-polarized light used in this study, the reflection decreased with increasing angle of incidence for the range of angles studied. It was also found that when the surface roughness was close to zero and when it was close to the wavelength of the input laser beam (i.e. 940 nm), the reflectance values were close and reached a minimum between these two roughness values.     

Enhanced Methylarginine Characterization by Post-Translational Modification-Specific Targeted Data Acquisition and Electron-Transfer Dissociation Mass Spectrometry

When localizing protein post-translational modifications (PTMs) using liquid-chromatography (LC)-tandem mass spectrometry (MS/MS), existing implementations are limited by inefficient selection of PTM-carrying peptides for MS/MS, particularly when PTM site occupancy is sub-stoichiometric. The present contribution describes a method by which peptides carrying specific PTMs of interest-in this study, methylarginines-may be selectively targeted for MS/MS: peptide features are extracted from high mass accuracy single-stage MS data, searched against theoretical PTM-carrying peptide masses, and matching features are subjected to targeted data acquisition LC-MS/MS. Using trypsin digested Saccharomyces cerevisiae Npl3, in which evidence is presented for 18 methylarginine sites-17 of which fall within a glycine-arginine-rich (GAR) domain spanning <120 amino acids-it is shown that this approach outperforms conventional data dependent acquisition (DDA): when applied to a complex protein mixture featuring in vivo methylated Npl3, 95% more (P=0.030) methylarginine-carrying peptides are selected for MS/MS than DDA, leading to an 86% increase (P=0.044) in the number of methylated peptides producing Mascot ion scores ≥20 following electron-transfer dissociation (ETD). Notably, significantly more low abundance arginine methylated peptides (maximum ion intensities <6×10(4) cps) are selected for MS/MS using this approach relative to DDA (50% more in a digest of purified in vitro methylated Npl3). It is also demonstrated that relative to collision-induced dissociation (CID), ETD facilitates a 586% increase (P=0.016) in average Mascot ion scores of methylarginine-carrying peptides. The present PTM-specific targeted data acquisition approach, though described using methylarginine, is applicable to any ionizable PTM of known mass.     

Detection and Characterization of Low Abundance Glycopeptides Via Higher-Energy C-Trap Dissociation and Orbitrap Mass Analysis

Broad-scale mass spectrometric analyses of glycopeptides are constrained by the considerable complexity inherent to glycoproteomics, and techniques are still being actively developed to address the associated analytical difficulties. Here we apply Orbitrap mass analysis and higher-energy C-trap dissociation (HCD) to facilitate detailed insights into the compositions and heterogeneity of complex mixtures of low abundance glycopeptides. By generating diagnostic oxonium product ions at mass measurement errors of <5 ppm, highly selective glycopeptide precursor ion detections are made at sub-fmol limits of detection: analyses of proteolytic digests of a hen egg glycoprotein mixture detect 88 previously uncharacterized glycopeptides from 666 precursor ions selected for MS/MS, with only one false positive due to co-fragmentation of a non-glycosylated peptide with a glycopeptide. We also demonstrate that by (1) identifying multiple series of glycoforms using high mass accuracy single stage MS spectra, and (2) performing product ion scans at optimized HCD collision energies, the identification of peptide + N-acetylhexosamine (HexNAc) ions (Y1 ions) can be readily achieved at <5 ppm mass measurement errors. These data allow base peptide sequences and glycan compositional information to be attained with high confidence, even for glycopeptides that produce weak precursor ion signals and/or low quality MS/MS spectra. The glycopeptides characterized from low fmol abundances using these methods allow two previously unreported glycosylation sites on the Gallus gallus protein ovoglycoprotein (amino acids 82 and 90) to be confirmed; considerable glycan heterogeneities at amino acid 90 of ovoglycoprotein, and amino acids 34 and 77 of Gallus gallus ovomucoid are also revealed.     

Chelating and Tellurolate Ligand‐Transfer Studies of the Complex fac‐[Fe(CO)3(TePh)3]−: Crystal Structures of Heterodinuclear (CO)3Mn(μ‐TePh)3Fe(CO)3 and CpNi(TePh)(PPh3)

Complex fac‐[Fe(CO)₃(TePh)₃]^? was employed as a “metallo chelating” ligand to synthesize the neutral (CO)₃Mn(μ‐TePh)₃Fe(CO)₃ obtained in a one‐step synthesis by treating fac‐[Fe(CO)₃(TePh)₃]^? with fac‐[Mn‐(CO)₃(CH₃CN)₃]⁺. It seems reasonable to conclude that the d⁶ Fe(II) [(CO)₃Fe(TePh)₃]^? fragment is isolobal with the d⁶ Mn(I) [(CO)₃Mn(TePh)₃]^2? fragment in complex (CO)₃Mn(μ‐TePh)₃Fe(CO)₃. Addition of fac‐[Fe(CO)₃(TePh)₃]^? to the CpNi(I)(PPh₃) in THF resulted in formation of the neutral CpNi(TePh)(PPh₃) also obtained from reaction of CpNi(I)(PPh₃) and [Na][TePh] in MeOH. This investigation shows that fac‐[Fe(CO)₃(TePh)₃]^? serves as a tridentate metallo ligand and tellurolate ligand‐transfer reagent. The study also indicated that the fac‐[Fe(CO)₃(SePh)₃]^? may serve as a better tridentate metallo ligand and chalcogenolate ligand‐transfer reagent than fac‐[Fe(CO)₃(TePh)₃]^? in the syntheses of heterometallic chalcogenolate complexes.     

Peptide Brush Polymers for Efficient Delivery of a Gene Editing Protein to Stem Cells

The scarcity of effective means to deliver functional proteins to living cells is a central problem in biotechnology and medicine. Herein, we report the efficient delivery of an active DNA‐modifying enzyme to human stem cells through high‐density cell penetrating peptide brush polymers. Cre recombinase is mixed with a fluorophore‐tagged polymer carrier and then applied directly to induced pluripotent stem cells or HEK293T cells. This results in efficient delivery of Cre protein as measured by activation of a genomically integrated Cre‐mediated recombination reporter. We observed that brush polymer formulations utilizing cell penetrating peptides promoted Cre delivery but oligopeptides alone or oligopeptides displayed on nanoparticles did not. Overall, we report the efficient delivery of a genome‐modifying enzyme to stem cells that may be generalizable to other, difficult‐to‐transduce cell types.     

The existence,uniqueness, and instability of spherically symmetric solutions of a system of reaction—Diffusion equations

The system $\text{?x}\text{?t}\text{= Δx + F(x,y),}\text{?y}\text{?t}\text{= G(x,y)}$ is investigated, where x and y are scalar functions of time (t ? 0), and n space variables $\text{(ξ}{}_1\text{,…, ξ}{}_{\mathrm{n}}\text{), Δx ≡ ∑}{}_{\mathrm{i\; =\; 1}}{}^{\mathrm{n}}\text{?}{}^2\text{x}\text{?ξ}{}_{\mathrm{i}}{}^2$, and F and G are nonlinear functions. Under certain hypotheses on F and G it is proved that there exists a unique spherically symmetric solution $\text{(x(r),y(r)),}\text{where}\text{r = (ξ}{}_1{}^2\text{+ … + ξ}{}_{\mathrm{n}}{}^2\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, which is bounded for r ? 0 and satisfies x(0) >x₀, y(0) > y₀, x′(0) = 0, y′(0) = 0, and x′ < 0, y′ > 0, ?r > 0. Thus, (x(r), y(r)) represents a time independent equilibrium solution of the system. Further, the linearization of the system restricted to spherically symmetric solutions, around (x(r), y(r)), has a unique positive eigenvalue. This is in contrast to the case n = 1 (i.e., one space dimension) in which zero is an eigenvalue. The uniqueness of the positive eigenvalue is used in the proof that the spherically symmetric solution described is unique.     

Convergence properties of preconditioned Hermitian and skew-Hermitian splitting methods for non-Hermitian positive semidefinite matrices

For the non-Hermitian and positive semidefinite systems of linear equations, we derive necessary and sufficient conditions for guaranteeing the unconditional convergence of the preconditioned Hermitian and skew-Hermitian splitting iteration methods. We then apply these results to block tridiagonal linear systems in order to obtain convergence conditions for the corresponding block variants of the preconditioned Hermitian and skew-Hermitian splitting iteration methods.

     

Equations of fluctuating nonlinear hydrodynamics for normal fluids

The full set of fluctuating nonlinear hydrodynamic equations for normal fluids is derived from the conventional Langevin equations extended to include multiplicative noise. The equations describing the set of conserved variables (the mass density, the momentum densityg, the energy density) agree with those found by Morozov for a case of a driving free energy which is a local function of the hydrodynamic variables. We show here that if the standard form of the hydrodynamic equations is to hold in the absence of noise, then the driving free energy must be a local function ofg and, but it may have to be a nonlocal function of the mass density.