A recommended and verified procedure for in situ tryptic digestion of formalin‐fixed paraffin‐embedded tissues for analysis by matrix‐assisted laser desorption/ionization imaging mass spectrometry

Matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a molecular imaging technology uniquely capable of untargeted measurement of proteins, lipids, and metabolites while retaining spatial information about their location in situ. This powerful combination of capabilities has the potential to bring a wealth of knowledge to the field of molecular histology. Translation of this innovative research tool into clinical laboratories requires the development of reliable sample preparation protocols for the analysis of proteins from formalin‐fixed paraffin‐embedded (FFPE) tissues, the standard preservation process in clinical pathology. Although ideal for stained tissue analysis by microscopy, the FFPE process cross‐links, disrupts, or can remove proteins from the tissue, making analysis of the protein content challenging. To date, reported approaches differ widely in process and efficacy. This tutorial presents a strategy derived from systematic testing and optimization of key parameters, for reproducible in situ tryptic digestion of proteins in FFPE tissue and subsequent MALDI IMS analysis. The approach describes a generalized method for FFPE tissues originating from virtually any source.     

Back to basics: label-free technologies for small molecule screening

Small molecule high-throughput screening in drug discovery today is dominated by techniques which are dependent upon artificial labels or reporter systems. While effective, these approaches can be affected by certain experimental limitations, such as conformational restrictions imposed by the selected label or compound fluorescence/quenching. Label-free approaches potentially address many of these issues by allowing researchers to investigate more native systems without fluorescence- or luminescence-based readouts. However, due to throughput and expense constraints, label-free methods have been largely relegated to a supporting role as the basis of secondary assays. In this review, we describe recent improvements in impedance-based, optical biosensor-based, automated patch clamp and mass spectrometry technologies that have enhanced their ease of use and throughput and, hence, their utility for primary screening of small- to medium-sized compound libraries. The ultimate maturation of these techniques will enable drug discovery researchers to screen large chemical libraries against minimally manipulated biological systems.     

Tool replacement policy for one-sided processes with low fraction defective

In most manufacturing industries, tool replacement policy is essential for minimizing the fraction defective and the manufacturing cost. Tool wear is caused by the action of sliding chips in the shear zone, and the friction generated between the tool flank and workpiece. This wear, apparently, is a dominant and irremovable component of variability in many machining processes, which is a systematic assignable cause. As the tool wear occurs in the machining processes, the fraction of defectives would gradually become significant. When the fraction defective reaches a certain level, the tool must be replaced. Therefore, detecting suitable time for tool replacement operation becomes essential. In this paper, we present an analytical approach for unilateral processes based on the one-sided process capability index C _PU (or C _PL ) to find the appropriate time for tool replacement. Accurate process capability must be calculated, particularly, when the data contains assignable cause variation. By calculating the index C _PU (or C _PL ) in a dynamical environment, we propose estimators of C _PU (or C _PL ) and obtain exact form of the sampling distribution in the presence of systematic assignable cause. The proposed procedure is then applied to a real manufacturing process involving tool wear problem, to demonstrate the applicability of the proposed approach.     

A Probability Model of Star‐Branched Polymers Formed by Connecting Polydispersed Primary Chains Onto a Multifunctional Coupling Agent

Summary: A probability model, based on the “in‐out” recursive analysis, is developed for obtaining the average molecular weights of star polymers formed by connecting polydispersed primary chains onto a multifunctional coupling agent. The average properties and the polydispersity index of the formed star polymers can be described as a function of the reaction conversion and the average properties of the polydispersed primary chains without the knowledge of the whole distribution. The results indicate that, although PI of the resulting star polymers might increase at the intermediate conversion for the higher functionalities of the core molecules, the resulting star polymers generally have narrower molecular weight distributions at the complete conversion compared to the initial polydispersed polymer chains.

A schematic illustration of the star polymer formation.     

Synthesis and structural studies of <Emphasis Type="Italic">tris</Emphasis>-2-chlorobenzylamine and <Emphasis Type="Italic">tris</Emphasis>-2-bromobenzylamine

The tris-2-chloro and 2-bromotribenzylamines are prepared from aqueous ammonia and 2-chlorobenzyl chloride and 2-bromobenzyl bromide, respectively, in ethanol. Recrystallization yielded colorless cubes of each product. The crystal structures are each solved in space group P , and are isostructural. The tris-2-chloro compound, 1, has a = 7.4226(5) Å, b = 9.0825(7) Å, c = 14.529(1) Å, = 78.279(1), = 82.389(1), = 84.661(1), and V = 948.41(12) ų with Z = 2, and d_calc = 1.368 Mg/m³. The tris-2-bromo analog, 2, has a = 7.6569(11) Å, b = 9.0922(13) Å, c = 14.614(2) Å, = 79.286(2), = 81.777(2), = 85.401(2), and V = 987.9(2) ų with Z = 2, and d_calc = 1.762 Mg/m³. Lithium–halogen exchange experiments conducted in tetrahydrofuran at –78C using n-butyl lithium revealed that no exchange occurred for the tris-2-chloro compound, but did occur for the tris-2-bromo analog to yield tribenzylamine upon quench and work-up.     

Emulsion Polymerization of Vinyl Acetate at Low Temperatures

Polymerization of vinyl acetate (VAc) was carried out with potassium persulfate (KS) and ammonium sulfite as redox initiator in the presence of acetonitrile for the purpose of preparation of polyvinyl alcohol (PVA) of desired degree of polymerization (DP). The PVA obtained by this method at 0°C showed higher DP and lower solubility in water. It was observed that acetonitrile was a useful chain transfer agent in this polymerization system. It was also observed that ZnCl₂ accelerated the polymerization of VAc and the over-all activation energy of polymerization was 9.73 Kcal/mol. However, the stereoregularity of PVA obtained by the saponification of PVAc in this system was not improved by the presence of ZnCl₂.     

Effects of packing structure on the optoelectronic and charge transport properties in poly(9,9-di-n-octylfluorene-alt-benzothiadiazole)

Spin-coated poly(9,9-di-n-octylfluorene-alt-benzothiadiazole) (F8BT) films of different molecular weights (Mn= 9-255 kg/mol), both in the pristine and annealed state, were studied in an effort to elucidate changes in the polymer packing structure and the effects this structure has on the optoelectronic and charge transport properties of these films. A model based on quantum chemical calculations, wide-angle X-ray scattering, atomic force microscopy, Raman spectroscopy, photoluminescence, and electron mobility measurements was developed to describe the restructuring of the polymer film as a function of polymer chain length and annealing. In pristine high molecular weight films, the polymer chains exhibit a significant torsion angle between the F8 and BT units, and the BT units in neighboring chains are close to one another. Annealing films to sufficiently high transition temperatures allows the polymers to adopt a lower energy configuration in which the BT units in one polymer chain are adjacent to F8 units in a neighboring chain ("alternating structure"), and the torsion angle between F8 and BT units is reduced. This restructuring, dictated by the strong dipole on the BT unit, subsequently affects the efficiencies of interchain electron transfer and exciton migration. Films exhibiting the alternating structure show significantly lower electron mobilities than those of the pristine high molecular weight films, due to a decrease in the efficiency of interchain electron transport in this structure. In addition, interchain exciton migration to low energy weakly emissive states is also reduced for these alternating structure films, as observed in their photoluminescence spectra and efficiencies.     

Reactions of 2-aminothiobenzamide with isocyanates: A new synthesis of 2, 3-dihydroimidazo[1, 2-c]quinazolin-5(6H)-one and 3, 4-Dihydro-2H-pyrimido[1, 2-c]quinazolin-6(7H)-one

2-(2-Chloroethylureido)- and 2-(3-chloropropylureido)thiobenzamides 5a, b were prepared in good yields by treating 2-aminothiobenzamide with 2-chloroethyl and 3-chloropropyl isocyanates respectively. Subsequent treatment of compound 5a and 5b either with alkali or mineral acid led to the formation of 2, 3-dihydro-imidazo[1, 2-c]quinazolin-5(6H)-one 7a and 3, 4-Dihydro-2H-pyrimido[1, 2-c]quinazolin-6(7H)-one 7b .     

Studies on Polymerization of Vinyl Compounds Initiated by Metal Chelate II Kinetics of Polymerization of Methyl Methacrylate Initiated by Nickel Ethyl Acetoacetate in the Presence and the Absence of Aromatic Amines

The kintetic studies of polymerization of methyl methacrylate initiated with nickel ethyl acetoacetate gave the following equations. R_p=K₁[Monomer]^1.4[Chelate]^0.5, in the absence ox aniline R_p=K₂[Monomer]^1.2[Chelate]^0.5 [Aniline]^0,5, in the presence of aniline. Some aromatic amines such as aniline markedly accelerated the polymerization, while pyridine had no such effect. The activation energy of initiation was 24.8 kcal/mol in the absence of aniline, and 8.8 kcal/mol in the presence of aniline. Both the kinetic data and the infrared spectra of the polymer indicated that the polymerization was radical in nature. The reaction order against monomer varied, as the temperature differed. The spectrophotometric investigation indicated formation of a complex between the chelate and the monomer, or amines. It also showed that the formation of a complex was not a factor which controlled the rate of initiation. A scheme of initiation mechanism was presented on the basis of the above experimental evidence.