Rumbaugh, K. P. | Search bioRxiv

bioRxiv2019

NemaLife: A structured microfluidic culture device optimized for aging studies in crawling C. elegans

Caenorhabditis elegans is a powerful animal model in aging research. Standard longevity assays on agar plates involve the tedious task of picking and transferring animals to prevent younger progeny from contaminating age-synchronized adult populations. Large-scale studies employ progeny-blocking drugs or sterile mutants to avoid progeny contamination, but such manipulations change adult physiology and alter the influence of reproduction on normal aging. Moreover, for some agar growth-based technology platforms, such as automated lifespan machines, reagents such as food or drugs cannot be readily added/removed after initiation of the study. Current microfluidic approaches are well-suited to address these limitations, but in their liquid-based environments animals swim rather than crawl, introducing swim-induced stress in the lifespan analysis. Here we report a simple microfluidic device that we call NemaLife that features: 1) an optimized micropillar arena in which animals can crawl, 2) sieve channels that separate progeny and prevent the loss of adults from the arena during culture maintenance, and 3) ports which allow rapid accessibility to feed the adult-only population and introduce reagents as needed. Culture maintenance and liquid manipulation are performed with simple hand-held syringes to facilitate integration of our technology into general laboratory protocols. Additionally, device geometry and feeding protocols were designed to emulate the body gait, locomotion, and lifespan of animals reared on agar. We validated our approach with longevity analyses of classical aging mutants (daf-2, age-1, eat-2, and daf-16) and animals subjected to RNAi knockdown of age-related genes (age-1 and daf-16). We also showed that healthspan measures such as pharyngeal pumping and tap-induced stimulated reversals can be scored across the lifespan. Overall, the capacity to generate reliable lifespan and physiological data from the NemaLife chip underscores the potential of this device to accelerate healthspan and lifespan investigations in C. elegans.

bioengineering↗

bioRxiv2019

Specific Disruption of Established P. aeruginosa Biofilms Using Polymer-Attacking Enzymes

Biofilms are communities of bacteria embedded in an extracellular matrix of self-produced polymeric substances. This polymer matrix lends the bacteria protection against a wide array of chemical and mechanical stresses that they may experience in their environment, which might be a location in the human body in the case of a biofilm infection, or a surface immersed in fluid in an industrial setting. Breaking down the matrix network renders biofilms more susceptible to physical disruption and to treatments. Different species of bacteria, and different strains within the same species, produce different types of matrix polymers - this suggests that targeting specific polymers for disruption may be more effective than non-specific approaches to disrupting biofilm matrices. In this study, we treated Pseudomonas aeruginosa biofilms with enzymes that are specific to different matrix polymers. We used bulk rheology to measure the resulting alteration in biofilm mechanics, and scanning electron microscopy to visualize the alteration in the matrix network upon treatment. Different lab strains of P. aeruginosa form biofilms that can be dominated by one of three main extracellular polysaccharides: Psl, alginate, and Pel, which binds electrostatically to extracellular DNA in the matrix. We applied enzymes to biofilms dominated by different extracellular polysaccharides and found that, for biofilms grown in vitro, the effect of enzymatic treatment is maximized when the enzyme is specific to a dominant matrix polymer - for such a case, specifically-matched enzymatic treatment tends to: reduce yield strain and yield stress; reduce or eliminate long-range structure and shorten or eliminate connecting network fibers in the biofilm as seen under scanning electron microscopy; and increase the rate of biofilm drying, most likely due to increased diffusivity as a result of network compromise. However, for ex vivo biofilms grown in murine wounds, we find that generic glycoside hydrolases have more profound disruptive effects than specifically-matched enzymes, even though they had no measurable effect for biofilms grown in vitro. This highlights the importance of the environment in which the biofilms are grown, the need to take this into account when developing treatments for biofilms, and the possibility that effective approaches to eradicating biofilms in environmental or industrial settings may need to be very different from effective treatments of infection.

microbiology↗