Guidelines for the use of cell lines in biomedical research

Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise

A strategy for efficiently collecting aerosol condensate using silica fibers:application to carbonyl emissions from e-cigarettes

Analysing harmful constituents in e-cigarette aerosols typically involves adopting a methodology used for analysing tobacco smoke. Cambridge filter pads (CFP) are the basis of numerous protocols for analysing the various classes of compounds representing 93 harmful and potentially harmful constituents identified in tobacco smoke by the FDA. This paper describes a simplified method for trapping the low volatility components of e-cigarette aerosols using a single trapping procedure followed by physical extraction. The trap is a plug of amorphous silica fibres (0.75 g of 4 µm diameter) within a 10mL syringe inserted between the e-cigarette mouthpiece and the pump of the vaping machine. The method is evaluated for emissions from three generations of e-cigarette device (Kangertech CE4, EVOD and Subox Mini-C). On average the silica wool traps about 94% of the vapourised liquid mass in the three devices and higher levels of condensate is retained before reaching saturation compared with CFP. The condensate is then physically extracted from the silica wool plug using a centrifuge. Condensate is then available for use directly in multiple analytical procedures or toxicological experiments. The method is tested by comparison with published analyses of carbonyls, among the most potent toxicants and carcinogens in e-cigarette emissions. Ranges for HPLC-DAD analyses of carbonyl-DNPH derivatives in a laboratory formulation of e-liquid are formaldehyde (0.182±0.023 to 9.896±0.709 µg puff-1), acetaldehyde (0.059±0.005 to 0.791±0.073 µg puff-1) and propionaldehyde (0.008±0.0001 to 0.033±0.023 µg puff-1); other carbonyls are identified and quantified. Carbonyls concentrations are also consistent with published experiments showing marked increases in with variable power settings (10W - 50W). Compared with CFPs, e-cigarette aerosol collection by silica wool requires only one vaping session for multiple analyte groups, traps more condensate per puff, collects more condensate before saturating

De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo hACE2 decoys to neutralize SARS-CoV-2. The best decoy, CTC-445.2, binds with low nanomolar affinity and high specificity to the RBD of the spike protein. Cryo-EM shows that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, shows ~10-fold improvement in binding. CTC-445.2d potently neutralizes SARS-CoV-2 infection of cells in vitro and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge

Analysis and Derivation of Allocations for Fiber Contaminants in Liquid Bipropellant Systems

An analysis was performed to identify the engineering rationale for the existing particulate limits in MSFC-SPEC-164, Cleanliness of Components for Use in Oxygen, Fuel, and Pneumatic Systems, determine the applicability of this rationale to fibers, identify potential risks that may result from fiber contamination in liquid oxygen/fuel bipropellant systems, and bound each of these risks. The objective of this analysis was to determine whether fiber contamination exceeding the established quantitative limits for particulate can be tolerated in these systems and, if so, to derive and recommend quantitative allocations for fibers beyond the limits established for other particulate. Knowledge gaps were identified that limit a complete understanding of the risk of promoted ignition from an accumulation of fibers in a gaseous oxygen system

Development of Bacteriophage Based Detection Technique for Food Safety and Environment Monitoring

Foodborne or waterborne pathogens which are responsible for numerous worldwide outbreaks of disease have caused serious health problems and enormous economic cost. Development of new bacterial detection technologies with high sensitivity and specificity is of great importance for food safety and public health. This has highlighted the significance of developing rapid and effective techniques for pathogen screening. Thus we aim to explore a bacteriophage mediated detection system for foodborne or waterborne bacteria monitoring. Bacteriophage as a novel recognition probe has been applied in various biosensor by immobilizing phage particles on solid sensor surface. They possess high specificity for targeted microorganism and a rapid infection between phage and its host cell. Their characteristics including low cost, robustness, fast and easy production plus high tolerance under extreme conditions make phages superior for bacteria detection at resource-limited settings. We utilize biotin-streptavidin interaction system for an engineered phage immobilization on biosensor surface. The fusion of biotin acceptor peptide (BAP) gene and biotin ligase (BirA) gene to phage capsid protein gene enabled the display of BAP ligand and the expression of protein BirA during the replication cycle of phage infection. Then produced phage progenies could be biotinylated in vivo and immobilized on the streptavidin coated magnetic beads. Compared with wild type phage, the recombinant phage showed a significantly higher immobilization efficiency of 82.8% on the magnetic bead and the resulting bio-probe showed a capture efficiency of 86.2% of E.coli within 20 min. This phage based biomagnetic separation coupled PCR detection provided a detection limit of 102 CFU/mL bacteria without additional pre-enrichment. For the detection of pathogenic Escherichia coli O157:H7, we compared phage-based biosorbent and antibody-based one and found that the capture efficiency of our phage assay was about 20% higher than that of antibody based magnetic separation assay under extreme conditions. Then we developed a membrane filter based colorimetric detection assay to offer a convenient and rapid way for the identification of general E.coli from field water on-site. This phage mediated detection provided a convenient and quantitative detection of bacteria number as the colored signal present at the site corresponding to the located bacterial colony

Decentralized Digital Fabrication of Flexible Micro/Nano Devices Using a Laserjet Printer

The fabrication of traditional micro/nano devices requires access to cleanrooms, complex and expensive tools, and highly-skilled labor. A facile and digital do-it-yourself (DIY) technique for the fabrication of low-cost devices on flexible substrates (paper, cloth, and plastic films) is presented in this work. A set of office-grade equipment (i.e., laserjet printer, thermal laminator, computer-aided paper cutter), and commercially available supplies (i.e., baking wax paper, furniture restoration metal-leaf) are utilized. The presented technology enables community-embedded production by removing a high technological barrier. The validity of the proposed technology was proved by designing three levels of experiments, i.e., patterns, devices, and systems. The performance was evaluated at each level to cover various application domains in environmental monitoring and biomedical diagnostics utilizing conductometric, colorimetric, biochemical, and chemoresistive detection principles. Devices with features of varying sizes, from nanometers to centimeters, were fabricated and characterized. Expanding the concept further, a copper oxide (CuO) nano-sorbent cloth-based filter was designed, fabricated, and tested to demonstrate the application in the fabrication of a water filtration system. An inexpensive and robust filtration system for real-time arsenic removal from polluted water, which could easily be embedded into the existing water pipes, showed the effective removal rate without requiring any power source for operation. To demonstrate the pervasiveness of the laserjet printing-based fabrication, a novel print-and-release method to produce color-tagged microplastics was presented. A lack of reliable methods to replicate the microplastic samples is one of the main challenges in the design of experiments for systematic studies. The newly developed fabrication techniques in this work provide an alternative route to decentralized production of low-cost flexible sensors and functional devices, with minimal steps, time, cost, and facilities. The operation of such devices is simple and can be further empowered by ubiquitous smartphones for data analysis and transmission