The Future of Mixing Research

This paper is the result of a plenary discussion session held at the 11^th European Conference on Mixing. Three perspectives on mixing research are explored: that of the industrialist, the equipment manufacturer, and the academic researcher. There was strong agreement that, while the one dimensional problems are reasonably well understood and many engineers thus perceive that mixing is simple, current practice actually requires us to address complex, multi‐dimensional problems with interactions between mixing, reaction, multi‐phase physics, surface phenomena, and transport phenomena. Understanding these multi‐scale, multi‐mechanism problems requires models which include interactions between the phenomena, and allow the effects of these interactions to emerge. Developing these models will require us to shift our perspective on mixing from one of equipment design to one of the mixing field as a fundamental physical mechanism.     

Coating evaluation and validation of accelerated test conditions using an in-situ corrosion sensor

An in-situ corrosion sensor was used to obtain electrochemical impedance spectroscopy (EIS) measurements on coated panels under a variety of accelerated laboratory test conditions as well as ambient exposure at a Florida beach. Three studies are reported. The first compared the sensor (EIS) measurements taken in a salt fog chamber to those obtained using a clamp-on cell and the conventional remote electrode/immersion approach. For coatings with minimal edge defects, the two methods gave equivalent results. For coatings with edge defects, the sensor was able to detect the defects provided the surface was wet, as in the salt fog chamber. In contrast, the conventional approach was unable to detect defects unless they were within the confines of the clamp-on cell. In the second case, sensor measurements were used to compare coating degradation during salt fog, a cyclic corrosion test, humidity, and immersion to that occurring at a Florida beach. The cyclic corrosion test showed an excellent correlation with beach exposure while the salt fog and other test showed very little correlation, suggesting that the cyclic test is more valid for discriminating coating performance for seacoast exposure. The sensor also indicated that the test could be short-ened by up to 40% without significantly reducing the validity of the test. In the final example, a series of primers and appliqués were evaluated using the cyclic corrosion test. The sensor EIS results allowed a discrimination between the materials sets even though there was little or no visual difference between the specimens. 10260 Old Columbia Rd., Columbia, MD 21046.     

The complexity of intersecting finite automata having few final states

The problem of determining whether several finite automata accept a word in common is closely related to the well-studied membership problem in transformation monoids. We raise the issue of limiting the number of final states in the automata intersection problem. For automata with two final states, we show the problem to be \({\oplus}\)L-complete or NP-complete according to whether a nontrivial monoid other than a direct product of cyclic groups of order 2 is allowed in the automata. We further consider idempotent commutative automata and (Abelian, mainly) group automata with one, two, or three final states over a singleton or larger alphabet, elucidating (under the usual hypotheses on complexity classes) the complexity of the intersection nonemptiness and related problems in each case.     

Evaluating meeting support tools

Many attempts are underway for developing meeting support tools, but less attention is paid to the evaluation of meetingware. This article describes the development and testing of an instrument for evaluating meeting tools. First, we specified the object of evaluation—meetings—by means of a set of input, process, and outcome factors. Then, we designed the process of evaluation, consisting of, first, the generation of meeting behavior in the form of a controlled series of meetings within the context of a project and, second, the measurement of the identified meeting factors. To measure these factors, a rating scale, questionnaires, and information flow analysis were used. Next, the instrument was tested, and the factors for successful meetings were determined in 13 projects in which four participants had to meet four times. The evaluation instrument proved to be a reliable and useful aid for the development and improvement of meeting tools.     

Counting Paths in VPA Is Complete for #NC 1

We give a #NC ¹ upper bound for the problem of counting accepting paths in any fixed visibly pushdown automaton. Our algorithm involves a non-trivial adaptation of the arithmetic formula evaluation algorithm of Buss, Cook, Gupta and Ramachandran (SIAM J. Comput. 21:755?C780, 1992). We also show that the problem is #NC ¹ hard. Our results show that the difference between #BWBP and #NC ¹ is captured exactly by the addition of a visible stack to a nondeterministic finite-state automaton.     

Dye linked conjugated homopolymers: using conjugated polymer electroluminescence to optically pump porphyrin-dye emission

Zinc-porphyrin dye molecules were incorporated into the backbone of a conjugated polymer material by a method, which allowed for the incorporation of only one zinc-porphyrin dye molecule into the backbone of each conjugated polymer molecule. The electronic properties of the homopolymer were established using ultraviolet photoelectron spectroscopy (UPS) for the determination of the electronic energy levels and the injection barrier for holes into the valence band. Pulse radiolysis time resolved microwave conductivity (PR-TRMC) was used to determine the sum of charge carrier mobilities. Electroluminescent devices of the homopolymer itself and of the zinc-porphyrin containing polymer were prepared and the nature of the electroluminescence was characterized. The homopolymer segments were found to optically pump the emission of the zinc-porphyrin dye moities. The homopolymer exhibits blue-green emission and the zinc-porphyrin linked homopolymers emit near-infrared/infrared light. This was demonstrated to be due to electroluminescence pumping of the zinc-porphyrin moieties that were covalently linked to homopolymer material. When only one zinc-porphyrin dye was incorporated into the backbone of each homopolymer molecule complete suppression of homopolymer electroluminescence was observed with exclusive near-infrared emission.     

Two Subgroups within the GH43_36 α-l-Arabinofuranosidase Subfamily Hydrolyze Arabinosyl from Either Mono-or Disubstituted Xylosyl Units in Wheat Arabinoxylan

Fungal arabinofuranosidases (ABFs) catalyze the hydrolysis of arabinosyl substituents (Ara) and are key in the interplay with other glycosyl hydrolases to saccharify arabinoxylans (AXs). Most characterized ABFs belong to GH51 and GH62 and are known to hydrolyze the linkage of α-(1→2)-Ara and α-(1→3)-Ara in monosubstituted xylosyl residues (Xyl) (ABF-m2,3). Nevertheless, in AX a substantial number of Xyls have two Aras (i.e., disubstituted), which are unaffected by ABFs from GH51 and GH62. To date, only two fungal enzymes have been identified (in GH43_36) that specifically release the α-(1→3)-Ara from disubstituted Xyls (ABF-d3). In our research, phylogenetic analysis of available GH43_36 sequences revealed two major clades (GH43_36a and GH43_36b) with an expected substrate specificity difference. The characterized fungal ABF-d3 enzymes aligned with GH43_36a, including the GH43_36 from Humicola insolens (HiABF43_36a). Hereto, the first fungal GH43_36b (from Talaromyces pinophilus) was cloned, purified, and characterized (TpABF43_36b). Surprisingly, TpABF43_36b was found to be active as ABF-m2,3, albeit with a relatively low rate compared to other ABFs tested, and showed minor xylanase activity. Novel specificities were also discovered for the HiABF43_36a, as it also released α-(1→2)-Ara from a disubstitution on the non-reducing end of an arabinoxylooligosaccharide (AXOS), and it was active to a lesser extent as an ABF-m2,3 towards AXOS when the Ara was on the second xylosyl from the non-reducing end. In essence, this work adds new insights into the biorefinery of agricultural residues.     

Synthesis of an Anti-CD7 Recombinant Immunotoxin Based on PE24 in CHO and E. coli Cell-Free Systems

Recombinant immunotoxins (RITs) are an effective class of agents for targeted therapy in cancer treatment. In this article, we demonstrate the straight-forward production and testing of an anti-CD7 RIT based on PE24 in a prokaryotic and a eukaryotic cell-free system. The prokaryotic cell-free system was derived from Escherichia coli BL21 Star^TM (DE3) cells transformed with a plasmid encoding the chaperones groEL/groES. The eukaryotic cell-free system was prepared from Chinese hamster ovary (CHO) cells that leave intact endoplasmic reticulum-derived microsomes in the cell-free reaction mix from which the RIT was extracted. The investigated RIT was built by fusing an anti-CD7 single-chain variable fragment (scFv) with the toxin domain PE24, a shortened variant of Pseudomonas Exotoxin A. The RIT was produced in both cell-free systems and tested for antigen binding against CD7 and cell killing on CD7-positive Jurkat, HSB-2, and ALL-SIL cells. CD7-positive cells were effectively killed by the anti-CD7 scFv-PE24 RIT with an IC₅₀ value of 15 pM to 40 pM for CHO and 42 pM to 156 pM for E. coli cell-free-produced RIT. CD7-negative Raji cells were unaffected by the RIT. Toxin and antibody domain alone did not show cytotoxic effects on either CD7-positive or CD7-negative cells. To our knowledge, this report describes the production of an active RIT in E. coli and CHO cell-free systems for the first time. We provide the proof-of-concept that cell-free protein synthesis allows for on-demand testing of antibody–toxin conjugate activity in a time-efficient workflow without cell lysis or purification required.     

An Innovative Approach to Tissue Processing and Cell Sorting of Fixed Cells for Subsequent Single-Cell RNA Sequencing

Although single-cell RNA sequencing (scRNA-seq) is currently the gold standard for the analysis of cell-specific expression profiles, the options for processing, staining, and preserving fresh cells remain very limited. Immediate and correct tissue processing is a critical determinant of scRNA-seq success. One major limitation is the restricted compatibility of fixation approaches, which must not destabilize or alter antibody labeling or RNA content or interfere with cell integrity. An additional limitation is the availability of expensive, high-demand cell-sorting equipment to exclude debris and dead or unwanted cells before proceeding with sample sequencing. The goal of this study was to develop a method that allows cells to be fixed and stored prior to FACS sorting for scRNA-seq without compromising the quality of the results. Finally, the challenge of preserving as many living cells as possible during tissue processing is another crucial issue addressed in this study. Our study focused on pancreatic ductal adenocarcinoma samples, where the number of live cells is rather limited, as in many other tumor tissues. Harsh tissue dissociation methods and sample preparation for analysis can negatively affect cell viability. Using the murine pancreatic cancer model Pan02, we evaluated the semi-automated mechanical/enzymatic digestion of solid tumors by gentleMACS Dissociator and compared it with mechanical dissociation of the same tissue. Moreover, we investigated a type of cell fixation that is successful in preserving cell RNA integrity yet compatible with FACS and subsequent scRNA-sequencing. Our protocol allows tissue to be dissociated and stained in one day and proceeds to cell sorting and scRNA-seq later, which is a great advantage for processing clinical patient material.