Monitoring potentially pathogenic protists in sewage sludge using Metataxonomics.

Intestinal parasites continue to pose a significant threat to human health worldwide, particularly among children. Contaminated water and soil serve as major transmission vehicles for these parasites and intestinal protists are among the most prevalent parasites in both developed and developing nations. Traditionally, parasites have been studied using human or animal fecal samples, while studying them in environmental samples has been challenging due to technical limitations. However, advancements in Next-Generation Sequencing (NGS) and bioinformatic approaches now enable the detection of parasite DNA in environmental samples. In this study, we applied a metataxonomic and phylogenetic strategy to detect and classify DNA of protists present in sewage sludge from two major cities in Colombia: Medellin and Cali. We successfully detected several human pathogenic parasites including Giardia intestinalis, Entamoeba histolytica, and Blastocystis sp., among other protists, in all sludge samples examined. We also investigated the entry and exit of parasite DNA from the San Fernando wastewater treatment plant (WWTP). We observed a higher number of parasite DNA sequences in the plant's influent wastewater, but we also detected the discharge of DNA from pathogenic parasites in both effluent waters and biosolids.

MechanoBioTester: A Decoupled Multistimulus Cell Culture Device for Studying Complex Microenvironments In Vitro.

Increasingly being recognized is the role of the complex microenvironment to regulate cell phenotype; however, the cell culture systems used to study these effects in vitro are lagging. The complex microenvironment is host to a combination of biological interactions, chemical factors, and mechanical stimuli. Many devices have been designed to probe the effects of one mechanical stimulus, but few are capable of systematically interrogating all combinations of mechanical stimuli with independent control. To address this gap, we have developed the MechanoBioTester platform, a decoupled, multi-stimulus cell culture model for studying the cellular response to complex microenvironments in vitro. The system uses an engineered elastomeric chamber with a specially defined region for incorporating different target materials to act as the cell culture substrate. We have tested the system with several target materials including: polydimethylsiloxane elastomer, polyacrylamide gel, poly(1,8-octanediol citrate) elastomer, and type I collagen gel for both 2D and 3D co-culture. Additionally, when the chamber is connected to a flow circuit and our stretching device, stimuli in the form of fluid flow, cyclic stretch, and hydrostatic pressure are able to be imparted with independent control. We validated the device using experimental and computational methods to define a range of capabilities relevant to physiological microenvironments. The MechanoBioTester platform promises to function as a model system for mechanobiology, biomaterial design, and drug discovery applications that focus on probing the impact of a complex microenvironment in an in vitro setting. The protocol described within provides the details characterizing the MechanoBioTester system, the steps for fabricating the MechanoBioTester chamber, and the procedure for operating the MechanoBioTester system to stimulate cells.