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.     

A new load cell for measuring normal and shear stresses

A special stress measuring device for determining the normal and shear stresses acting on silo walls was developed and tested. The load cell can be used for measuring simultaneously the normal stress as well as the intensity and direction of the shear stress.     

Microcellular sheet extrusion system process design models for shaping and cell growth control

The feasibility of shaping a nucleated polymer/gas solution represents a significant advancement for microcellular plastics process technology. Through proper design of the foaming die, nucleated solution flows can be shaped to arbitary dimensions while maintaining the functional independence of cell nucleation, cell growth and shaping. To maintain funcational independence, stringent pressure and temperature design specifications, which supersede those of conventional foam processing, must be met by the foaming die design. As a means of aiding the design process, a model is developed for predicting pressure losses and flow rates of nucleated polymer/gas solutions. A comparison of the model predictions and the actual foaming die design performance shows good agreement for limited data. These relatively simple models capture the major physics of the complicated two-phase flow field and provide a sound base from which scale-up of the foaming die concept can be achieved. The nucleated polymer/gas solution flow models predict highly nonlinear volumetric flow rates contrasting constant flow rates predicted for the neat polymer flow. In addition, a convenient method for classifying nucleated polymer/gas solution flow is presented based on a dimensionless ratio of the characteristic flow rate to the characteristic gas diffusion rate.     

PVC汽车脚垫专用料的研究与开发

在研究各种助剂对试样性能影响的基础上,开发出了高弹性、高流动性的汽车脚垫专用料,其基本配方为:PVC S-2500树脂100份,复合铅盐稳定剂6份,增塑剂120份,重质CaCO3份,弹性体10份,流动改性剂10份,其他助剂适量.加工试验表明,PVC汽车脚垫专用料的加工性能优良.     

A fully automatic system for acid-base coulometric titrations

An automatic system for acid-base titrations by electrogeneration of H⁺ and OH^- ions, with potentiometric end-point detection, was developed. The system includes a PC-compatible computer for instrumental control, data acquisition and processing, which allows up to 13 samples to be analysed sequentially with no human intervention.The system performance was tested on the titration of standard solutions, which it carried out with low errors and RSD. It was subsequently applied to the analysis of various samples of environmental and nutritional interest, specifically waters, soft drinks and wines.     

Simultaneous automatic measurement of salt and water fluxes through a charged membrane

A feedback controlled system is proposed which maintains constant concentrations of solutions on both sides of a membrane. The constancy is obtained by feeding each half cell with amounts of suitable adjusting solutions. Both salt flux, J_s and water flux, J_w are deduced from addition rates. It is of special interest that the system allows simultaneous measurement of both fluxes, in absolutely controlled steady state condtions.The system is analyzed on the basis of the methods of automatics. The reliability of the controlling device has been tested by comparing the value indicated for known fluxes imposed through capillaries simulating a membrane.Date are given for a sulfonated polyethylene cation exchange membrane.     

In Situ Rheo-NMR Measurements of Solid Fat Content

The properties of crystallized fats depend on their solid fat content (SFC) and their fractal structures. The SFC and the structures are dramatically affected during crystallization under shear flow. A mini-Couette cell was developed to crystallize fat samples under shear. The cell was tested with blends of canola stearin (CS) in canola oil (CO) in a 20-MHz NMR spectrometer. The blends were placed in the cell, melted at 80 °C, and then crystallized under different shear rates (58–460 s⁻¹) at 40 °C inside the spectrometer for 4 h. Time averaged NMR free induction decay (FID) curves were captured at 20 s intervals. SFC values were calculated using parameters determined by a calibration procedure. The SFC values determined by the direct method with and without the shaft of the Couette device were reasonably close. Similar results were observed with and without shear in the Couette device. The FID curves did not show a significant difference either. Therefore this system is accurate for in-situ time-resolved determination of SFC under shear flow. Furthermore, a combination of the direct and the indirect methods was successfully used to estimate the temperature increase due to viscous heating. The system developed will help in understanding the effects of shear flow on SFC of nanostructured lipid multicomponent systems. This will permit the optimization of the manufacturing processes.     

A Simple Protocol for Sample Preparation for Scanning Electron Microscopic Imaging Allows Quick Screening of Nanomaterials Adhering to Cell Surface

Preparing biological specimens for scanning electron microscopy (SEM) can be difficult to implement, as it requires specialized equipment and materials as well as the training of dedicated personnel. Moreover, the procedure often results in damage to the samples to be analyzed. This work presents a protocol for the preparation of biological samples to evaluate the adherence of nanomaterials on the cell surface using SEM. To this end, we used silicon wafers as a substrate to grow cells and replaced difficult steps such as the critical point drying of the samples in order to make the method quicker and easier to perform. The new protocol was tested using two different types of cells, i.e., human osteosarcoma cells and adipose-derived mesenchymal stem cells, and it proved that it can grossly preserve cell integrity in order to be used to estimate nanomaterials’ interaction with cell surfaces.     

疫苗诱导的特异性细胞杀伤活性检测方法的建立

目的建立一种基于流式细胞术的评价疫苗诱导的特异性细胞杀伤活性的方法,以完善疫苗及基因治疗药物的评价方法。方法用羧基荧光素二乙酸盐琥珀酰亚胺酯(Carboxyfluorescein diacetate,succinimidyl ester,CFSE)标记淋巴细胞,利用肿瘤坏死因子(Tumor necrosis factor,TNFα)模拟杀伤淋巴细胞,用碘化丙啶(Propidium iodide,PI)染色,流式细胞仪检测,确定CFSE和PI的浓度及作用时间。利用已确知有较强细胞免疫作用的治疗性乙型肝炎疫苗免疫小鼠,分离特异性淋巴细胞并用特异性肽刺激,分离未免疫小鼠的淋巴细胞作为靶细胞,并用特异性抗原肽致敏,并从靶细胞的标记、效应细胞培养时间,效靶作用时间、效靶比几方面进行优化,确定试验方法的操作流程。结果采用CFSE/PI双标记能有效分离实验所需各组群,细胞分为CFSE+PI-、CFSE+PI+、CFSE-PI+、CFSE-PI-4个组群,可区分活细胞和凋亡细胞。CFSE标记靶细胞的时间为6 h;效应细胞培养时间为72 h;效靶作用时间为6 h;效靶比可使用100∶1和50∶1。结论建立了基于流式细胞术的评价疫苗诱导的特异性细胞杀伤活性的方法,该方法可有效和精确地评价CTL杀伤效应,完善了疫苗及基因治疗药物的评价方法。     

Automatic system for the determination of boron in ceramic frits

An automatic system for the potentiometric determination of boron in ceramic frits was developed. The system includes a personal computer for instrumental control, data acquisition and processing, which allows up to 13 samples to be analysed sequentially with no human intervention.The system performance was tested on the titration of standard solutions, which it carried out with low errors and RSD. It was subsequently applied to the determination of the B₂0₃ content in various types of ceramic frits with good results.     

First Steps Towards Fuel Cells Testing Harmonisation: Procedures and Parameters for Single Cell Performance Evaluation

The great interest in Fuel Cell Systems, combined with the innovation of the device itself, has led to a huge developmental effort to make the steps necessary for future FC plant commissioning. The clearest and most effective way to evaluate the performance of a fuel cell is to measure it directly and, since few fuel cell test rigs are available at the moment, standard experimental procedures have not been realized so far. Our research group is currently performing single cell testing at the University of Perugia fuel cell laboratory and particular emphasis has been put on the definition of procedures and the testing of parameterisation. The work team strongly believes that this is the key to effective system testing and reliable performance evaluation. In this work, the test parameterisation developed by the team, and the resulting advanced control procedure used for a single MCFC experimental characterization are presented. Efforts have been dedicated to obtain some relevant non‐dimensional parameters to allow an easy understanding of the results and quick comparisons with other tests under different operating conditions, or with results obtained on different cells eventually tested in different laboratories. The authors strongly emphasise this topic to avoid the data that developers and research institutions collect being of no practical use due to a lack of shared rules.     

In situ X-ray analysis under controlled potential conditions: An innovative setup and its application to the investigation of ultrathin films electrodeposited on Ag(1 1 1)

An innovative setup to combine electrochemical and in situ surface X-ray diffraction (SXRD) measurements is described. This electrochemical cell has a different design from the other ones commonly used for X-ray diffraction studies. It allows the sample surface to stay always completely immersed into the solution under controlled potential conditions even during the SXRD measurements. The X-ray beam crosses the liquid (about 1 cm) and the cell walls. Because of the high X-ray energy, the beam attenuation is negligible and by an appropriate positioning of the detector arm slits it is possible to minimize the diffuse scattering induced by the liquid and cell walls in order to still detect the minima of the crystal truncation rods (CTRs). The liquid solution in the cell is managed by a special device, which allows the controlled exchange of the electrolyte solutions necessary in the electrochemical atomic layer epitaxy (ECALE) growth. The whole setup can be remotely controlled from outside the experimental hutch by a dedicated computer. As an example we report measurements on S layers deposited at underpotential on the Ag(1 1 1) surface, and on CdS films of increasing thickness.     

Development of a pressure–volume–temperature measurement method for thermoplastic materials based on compression injection molding

The pressure–volume–temperature (pvT) relationship of polymers is vitally important information in designing and manufacturing polymers. Because of the special behavior of polymers, however, it is extremely difficult to accurately measure the data in a way that matches the thermal conditions of injection molding, which is one of the most widely used processing methods. As neither widely used, commercially available measuring devices, nor special equipment mentioned in the literature can fully satisfy this need, it was decided to build a new device able to determine the pvT relationship during injection molding of the polymer. The new device consists of a special mold that can be used on an injection molding machine and a data collection system connected to it. With the help of this device, pvT data can be measured during processing according to the thermal conditions of injection molding, and also considerably faster, and even in an industrial setting. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 41140.     

Preparation of lot samples of nut meats for mycotoxin assay

A procedure has been devised for preparing lot samples of mycotoxin-contaminated nut meats so that a representative analytical sample may be removed. The sample is rapidly reduced to coarse size. A relatively large portion (about 1/10 of total sample) of subsample is then split out and further comminuted to a fine particle size with the aid of a fat solvent (meat-solvent, w/v, 3:2). The analytical sample is removed from this mixture. The procedure was tested with shelled almonds and shelled walnuts using radioactive nuts to simulate the mycotoxin contamination and provide a simple, precise measure of the contaminated nut meat distribution. The pooled coefficient of variation was 18% for the subsamples and 4.4% for the analytical samples. Considering the dilution factors used (1.50 and 2.14 contaminated nuts/10⁴ nuts) and the low degree of reliability of the lot sample, the sample preparation methods tested appear to be practical and reliable.     

Human iPSC-Derived 2D and 3D Platforms for Rapidly Assessing Developmental,Functional, and Terminal Toxicities in Neural Cells

With increasing global health threats has come an urgent need to rapidly develop and deploy safe and effective therapies. A common practice to fast track clinical adoption of compounds for new indications is to repurpose already approved therapeutics; however, many compounds considered safe to a specific application or population may elicit undesirable side effects when the dosage, usage directives, and/or clinical context are changed. For example, progenitor and developing cells may have different susceptibilities than mature dormant cells, which may yet be different than mature active cells. Thus, in vitro test systems should reflect the cellular context of the native cell: developing, nascent, or functionally active. To that end, we have developed high-throughput, two- and three-dimensional human induced pluripotent stem cell (hiPSC)-derived neural screening platforms that reflect different neurodevelopmental stages. As a proof of concept, we implemented this in vitro human system to swiftly identify the potential neurotoxicity profiles of 29 therapeutic compounds that could be repurposed as anti-virals. Interestingly, many compounds displayed high toxicity on early-stage neural tissues but not on later stages. Compounds with the safest overall viability profiles were further evaluated for functional assessment in a high-throughput calcium flux assay. Of the 29 drugs tested, only four did not modulate or have other potentially toxic effects on the developing or mature neurospheroids across all the tested dosages. These results highlight the importance of employing human neural cultures at different stages of development to fully understand the neurotoxicity profile of potential therapeutics across normal ontogeny.     

Effect of temperatures on the mechanical and morphological properties of polyphenylene sulfide composites

Summary Poly(styrene-b-paramethylstyrene) block copolymer samples have been prepared in a mechanical press, cooled under pressure below the glass transition temperature and investigated by small angle neutron scattering. We observe a microphase separation transition from an orderd to a disorderd state with ascending pressure. The observed effect is not expected solely due to pressure, but the situation in the press is more complicated: (i) Ascending pressure leads to an increase of the glass transition temperature which slows down relaxations and hinders the evolution of thermodynamic equilibrium, (ii) the uniaxial movement of the piston leads to an uniaxial shear field and samples get oriented. An adiabatic heating during the pressing procedure has been found to be of minor influence. The situation is discussed with respect to common sample preparation conditions and processing.     

20 Jahre Entwicklung einer bipolaren Membranzelle für die Alkalichlorid-Elektrolyse vom Labor bis zur weltweiten Anwendung

First samples of perfluorinated cation exchange membranes were obtained in 1971. They were tested for use in the diaphragm process as a replacement for asbestos diaphragms. However, the special features of these membranes led to the decision to develop special new membrane electrolysis cells. A patent was sought for the first new cell design in 1975. After extensive material testing and many improvements, the HOECHST bipolar membrane cell was produced, consisting of an electrolyzer made up of individual elements, i.e. individual sealed electrolysis cells. Starting in 1982, this cell type was developed further in cooperation with UHDE GmbH. In 1983 it was installed for the first time in an industrial electrolysis plant. Despite strong competition, the bipolar HOECHST -UHDE membrane cell has now gained an important market share. In 1998 it will be installed worldwide in 43 electrolysis plants, with a total NaOH capacity then amounting to 2.5 million mtpa.     

Prediction of mechanical properties of polyethylene mouldings based on laminate theory and thermomechanical indices

Abstract

The properties of moulded plastic products are dependent on the processing technology used in their manufacture and in particular on the structural morphology resulting from the thermomechanical environment imposed on the melt. This paper presents a unified approach to describe the behaviour of the products based on knowledge of the thermomechanical conditions imposed during processing. A linear medium density polyethylene has been processed using rotational moulding, compression moulding, and injection moulding in order to achieve different thermomechanical conditions (i.e. shear rates and cooling rates). The processing conditions used were typical of those in common use in the respective industries. The moulding parts were mechanically tested to determine the tensile, flexural, and impact properties. These measurements were performed both on samples corresponding to the entire thickness of the moulding and on slices taken from across the section of the mouldings. On the basis of these measurements, two models were developed. One is based on laminate theory, in which, from a knowledge of the mechanical properties of the individual layers through the wall thickness, it is possible to predict the tensile and flexural properties of the full thickness moulding. The other is an empirical model that predicts the tensile modulus of a plastic part as a function of two thermomechanical indices. It is shown that the type of dependence of the mechanical performance on the thermomechanical conditions imposed during processing is similar for the three moulding techniques used. A good agreement is achieved between the experimental data and those predicted by the thermomechanical model. It is also shown that via the combined use of the thermomechanical indices concept and the laminate analysis, good predictions of the mechanical behaviour of plastic mouldings with complex microstructures can be achieved. It is proposed that this approach could provide a very valuable addition to existing melt flow simulation packages. This would enable not only processing conditions to be optimised but the properties of the end product could be predicted.     

Quality control of gasoline by H NMR: Aromatics, olefinics, paraffinics, and oxygenated and benzene contents

A simple and fast ¹H NMR method, without any pretreatment, was developed for quality control of gasoline. It is based on the average group molecular weight approach and relative-content concept involving aromatics, olefinics and paraffinics, including also ethanol and benzene contents. The ethanol content was evaluated for Brazilian samples, but the method can be easily adapted to any oxygenated compound (ex. MTBE), and to gasoline from other countries. Twenty two laboratory prepared gasoline samples (gasoline from Brazilian refineries plus hydrocarbon solvents) and thirty four real (i.e., Brazilian gas stations) gasoline samples were tested. The routine quality control carried out through the usual physicochemical analyses reached a level of confidence of 75% and 73% in detecting non conformity in laboratory and real gasoline samples, respectively. The NMR method was very superior reaching 100% and 97% of confidence, respectively. It was better suited for laboratories with high sample throughput since measurement time is short and only one NMR experiment is needed per sample.     

Flow Cytometry Analysis of Circulating Extracellular Vesicle Subtypes from Fresh Peripheral Blood Samples

Extracellular vesicles (EVs) are released by shedding during different physiological processes and are increasingly thought to be new potential biomarkers. However, the impact of pre-analytical processing phases on the final measurement is not predictable and for this reason, the translation of basic research into clinical practice has been precluded. Here we have optimized a simple procedure in combination with polychromatic flow cytometry (PFC), to identify, classify, enumerate, and separate circulating EVs from different cell origins. This protocol takes advantage of a lipophilic cationic dye (LCD) able to probe EVs. Moreover, the application of the newly optimized PFC protocol here described allowed the obtainment of repeatable EVs counts. The translation of this PFC protocol to fluorescence-activated cell sorting allowed us to separate EVs from fresh peripheral blood samples. Sorted EVs preparations resulted particularly suitable for proteomic analyses, which we applied to study their protein cargo. Here we show that LCD staining allowed PFC detection and sorting of EVs from fresh body fluids, avoiding pre-analytical steps of enrichment that could impact final results. Therefore, LCD staining is an essential step towards the assessment of EVs clinical significance.