RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

Abstract Background The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old) FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR) for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD), testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp. Results Biologically useful RNA (minimum RNA integrity number, RIN, 1.4) was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old") samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp. Conclusion All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of extracted RNA, although it produced similar quality RNA to other protocols. If a chosen protocol fails to extract biologically useful RNA from a given sample in a first attempt, another attempt and then another protocol should be tried before excluding the case from molecular analysis.

Steel fiber reinforced concrete pipes: part 1: technological analysis of the mechanical behavior

This paper is the first part of an extensive work focusing the technological development of steel fiber reinforced concrete pipes (FRCP). Here is presented and discussed the experimental campaign focusing the test procedure and the mechanical behavior obtained for each of the dosages of fiber used. In the second part ("Steel fiber reinforced concrete pipes. Part 2: Numerical model to simulate the crushing test"), the aspects of FRCP numerical modeling are presented and analyzed using the same experimental results in order to be validated. This study was carried out trying to reduce some uncertainties related to FRCP performance and provide a better condition to the use of these components. In this respect, an experimental study was carried out using sewage concrete pipes in full scale as specimens. The diameter of the specimens was 600 mm, and they had a length of 2500 mm. The pipes were reinforced with traditional bars and different contents of steel fibers in order to compare their performance through the crushing test. Two test procedures were used in that sense. In the 1st Series, the diameter displacement was monitored by the use of two LVDTs positioned at both extremities of the pipes. In the 2nd Series, just one LVDT is positioned at the spigot. The results shown a more rigidity response of the pipe during tests when the displacements were measured at the enlarged section of the socket. The fiber reinforcement was very effective, especially when low level of displacement was imposed to the FRCP. At this condition, the steel fibers showed an equivalent performance to superior class pipes made with traditional reinforced. The fiber content of 40 kg/m3 provided a hardening behavior for the FRCP, and could be considered as equivalent to the critical volume in this condition.

Steel fibre reinforced concrete pipes. Part 2: numerical model to simulate the crushing test

This paper is part of an extensive work about the technological development, experimental analysis and numerical modeling of steel fibre reinforced concrete pipes. The first part ("Steel fibre reinforced concrete pipes. Part 1: technological analysis of the mechanical behavior") dealt with the technological development of the experimental campaign, the test procedure and the discussion of the structural behavior obtained for each of the dosages of fibre used. This second part deals with the aspects of numerical modeling. In this respect, a numerical model called MAP, which simulates the behavior of fibre reinforced concrete pipes with medium-low range diameters, is introduced. The bases of the numerical model are also mentioned. Subsequently, the experimental results are contrasted with those produced by the numerical model, obtaining excellent correlations. It was possible to conclude that the numerical model is a useful tool for the design of this type of pipes, which represents an important step forward to establish the structural fibres as reinforcement for concrete pipes. Finally, the design for the optimal amount of fibres for a pipe with a diameter of 400 mm is presented as an illustrating example with strategic interest.

Short steel and concrete columns under high temperatures

The growing demand for knowledge about the effect of high temperatures on structures has stimulated increasing research worldwide. This article presents experimental results for short composite steel and concrete columns subjected to high temperatures in ovens with or without an axial compression load, numerically analyzes the temperature distribution in these columns after 30 and 60 minutes and compares them with experimental results. The models consist of concrete-filled tubes of three different thicknesses and two different diameters, and the concrete fill has conventional properties that remained constant for all of the models. The stress-strain behavior of the composite columns was altered after exposure to high temperatures relative to the same columns at room temperature, which was most evident in the 60-minute tests due to the higher temperatures reached. The computational analysis adopted temperature rise curves that were obtained experimentally.

Effects of cryogenic and stress relief treatments on temper carbide precipitation in AISI D2 tool steel

The effects of cryogenic and stress relief treatments on temper carbide precipitation in the cold work tool steel AISI D2 were studied. For the cryogenic treatment the temperature was −196°C and the holding time was 2, 24 or 30 h. The stress relief heat treatment was carried at 130°C/90 min, when applied. All specimens were compared to a standard thermal cycle. Specimens were studied using metallographic characterisation, X-ray diffraction and thermoelectric power measurements. The metallographic characterisation used SEM (scanning electron microscopy) and SEM-FEG (SEM with field emission gun), besides OM (optical microscopy). No variation in the secondary carbides (micrometre sized) precipitation was found. The temper secondary carbides (nanosized) were found to be more finely dispersed in the matrix of the specimens with cryogenic treatment and without stress relief. The refinement of the temper secondary carbides was attributed to a possible in situ carbide precipitation during tempering.

Column generation for a multitrip vehicle routing problem with time windows, driver work hours, and heterogeneous fleet

This study addresses a vehicle routing problem with time windows, accessibility restrictions on customers, and a fleet that is heterogeneous with regard to capacity and average speed. A vehicle can performmultiple routes per day, all starting and ending at a single depot, and it is assigned to a single driverwhose totalwork hours are limited.Acolumn generation algorithmis proposed.The column generation pricing subproblem requires a specific elementary shortest path problem with resource constraints algorithm to address the possibility for each vehicle performingmultiple routes per day and to address the need to set the workday’s start time within the planning horizon. A constructive heuristic and a metaheuristic based on tabu search are also developed to find good solutions.

Thermal analysis of the chip formation in austenitic stainless steel

The most important property of austenitic stainless steels is corrosion resistance. In these steels, the transition between paramagnetic and ferromagnetic conditions occurs at low temperatures. Therefore, the use of austenitic stainless steels in conditions in which ferromagnetism absence is important can be considered. On the other hand, the formation of strain-induced martensite is detected when austenitic stainless steels are deformed as well as machined. The strain-induced martensite formed especially in the machining process is not uniform through the chip and its formation can also be related to the Md temperature. Therefore, both the temperature distribution and the gradient during the cutting and chip formation are important to identify regions in which martensite formation is propitiated. The main objective here is evaluate the strain-induced martensite formation throughout machining by observing microstructural features and comparing these to thermal results obtained through finite element method analysis. Results show that thermal analysis can give support to the martensite identified in the microstructural analysis.

Influence of HSM cutting parameters on the surface integrity characteristics of hardened AISI H13 steel

The purpose of this study is to evaluate the influence of the cutting parameters of high-speed machining milling on the characteristics of the surface integrity of hardened AISI H13 steel. High-speed machining has been used intensively in the mold and dies industry. The cutting parameters used as input variables were cutting speed (v c), depth of cut (a p), working engagement (a e) and feed per tooth (f z ), while the output variables were three-dimensional (3D) workpiece roughness parameters, surface and cross section microhardness, residual stress and white layer thickness. The subsurface layers were examined by scanning electron and optical microscopy. Cross section hardness was measured with an instrumented microhardness tester. Residual stress was measured by the X-ray diffraction method. From a statistical standpoint (the main effects of the input parameters were evaluated by analysis of variance), working engagement (a e) was the cutting parameter that exerted the strongest effect on most of the 3D roughness parameters. Feed per tooth (f z ) was the most important cutting parameter in cavity formation. Cutting speed (v c) and depth of cut (a p) did not significantly affect the 3D roughness parameters. Cutting speed showed the strongest influence on residual stress, while depth of cut exerted the strongest effect on the formation of white layer and on the increase in surface hardness.

Impact of thermal bridging on the performance of buildings using Light Steel Framing in Brazil

The Light Steel Framing building technology was introduced in Brazil in the late 1990s for the construction of residential houses. Because the design system was imported from the United States and is optimised to work well in that temperate climate, some modi fi cations must be made to adapt it for the Brazilian climate. The objective of this paper was to assess the impact of thermal bridging across enclosure elements on the thermal performance of buildings designed with Light Steel Framing in Brazil. The numerical simulation program EnergyPlus and a speci fi c method that considered the effects of metallic structures in the hourly simulations were used for the analysis. Two air-conditioned commercial buildings were used as case studies. The peak thermal load increased approximately 10% when an interior metal frame was included in the numerical simulations compared to non-metallic structures. Even when a metal frame panel was used only for vertical elements in the facade of a building with a conventional concrete structure, the simulations showed a 5% increase in annual energy use.

Friction and wear of performance of MoDTC and ester-containing lubrificants over steel surfaces under reciprocating conditions

Since the earliest developments of human history, friction has been a major issue. From the invention of the wheel and the use of the first lubricants to the studiesof coated and microtexturized surfaces, significant effort has been put on improvements that couldovercome the resistance to motion. Areview by Holmberg, Andersson and Erdemir[1] shows that, in an average passenger car, about one third of the total energy consumptionis due to friction losses. Of these, another one third is consumed in the engine system. The optimization of the lubricating oil formulation used ininternal combustion enginesis an important way to reduce friction, therefore improving energeticefficiencyand controllingemissions.Lubrication is also a way to assure the required protection to the system by maintaining wear rates in an adequate level, which helps to minimize maintenance costs.