Extraction of redox extracellular vesicles using exclusion-based sample preparation.

Studying specific subpopulations of cancer-derived extracellular vesicles (EVs) could help reveal their role in cancer progression. In cancer, an increase in reactive oxygen species (ROS) happens which results in lipid peroxidation with a major product of 4-hydroxynonenal (HNE). Adduction by HNE causes alteration to the structure of proteins, leading to loss of function. Blebbing of EVs carrying these HNE-adducted proteins as a cargo or carrying HNE-adducted on EV membrane are methods for clearing these molecules by the cells. We have referred to these EVs as Redox EVs. Here, we utilize a surface tension-mediated extraction process, termed exclusion-based sample preparation (ESP), for the rapid and efficient isolation of intact Redox EVs, from a mixed population of EVs derived from human glioblastoma cell line LN18. After optimizing different parameters, two populations of EVs were analyzed, those isolated from the sample (Redox EVs) and those remaining in the original sample (Remaining EVs). Electron microscopic imaging was used to confirm the presence of HNE adducts on the outer leaflet of Redox EVs. Moreover, the population of HNE-adducted Redox EVs shows significantly different characteristics to those of Remaining EVs including smaller size EVs and a more negative zeta potential EVs. We further treated glioblastoma cells (LN18), radiation-resistant glioblastoma cells (RR-LN18), and normal human astrocytes (NHA) with both Remaining and Redox EV populations. Our results indicate that Redox EVs promote the growth of glioblastoma cells, likely through the production of H2O2, and cause injury to normal astrocytes. In contrast, Remaining EVs have minimal impact on the viability of both glioblastoma cells and NHA cells. Thus, isolating a subpopulation of EVs employing ESP-based immunoaffinity could pave the way for a deeper mechanistic understanding of how subtypes of EVs, such as those containing HNE-adducted proteins, induce biological changes in the cells that take up these EVs.

Development and Validation of a Simplified Method for Analysis of SARS-CoV-2 RNA in University Dormitories.

Over the course of the COVID-19 pandemic, wastewater surveillance has become a useful tool for describing SARS-CoV-2 prevalence in populations of varying size, from individual facilities (e.g., university residence halls, nursing homes, prisons) to entire municipalities. Wastewater analysis for SARS-CoV-2 RNA requires specialized equipment, expensive consumables, and expert staff, limiting its feasibility and scalability. Further, the extremely labile nature of viral RNA complicates sample transportation, especially in regions with limited access to reliable cold chains. Here, we present a new method for wastewater analysis, termed exclusion-based sample preparation (ESP), that substantially simplifies workflow (at least 70% decrease in time; 40% decrease in consumable usage compared with traditional techniques) by targeting the labor-intensive processing steps of RNA purification and concentration. To optimize and validate this method, we analyzed wastewater samples from residence halls at the University of Kentucky, of which 34% (44/129) contained detectible SARS-CoV-2 RNA. Although concurrent clinical testing was not comprehensive, student infections were identified in the 7 days following a positive wastewater detection in 68% of samples. This pilot study among university residence halls validated the performance and utility of the ESP method, laying the foundation for future studies in regions of the world where wastewater testing is not currently feasible.

Mechanical analysis of avian feet: multiarticular muscles in grasping and perching.

The grasping capability of birds' feet is a hallmark of their evolution, but the mechanics of avian foot function are not well understood. Two evolutionary trends that contribute to the mechanical complexity of the avian foot are the variation in the relative lengths of the phalanges and the subdivision and variation of the digital flexor musculature observed among taxa. We modelled the grasping behaviour of a simplified bird foot in response to the downward and upward forces imparted by carrying and perching tasks, respectively. Specifically, we compared the performance of various foot geometries performing these tasks when actuated by distally inserted flexors only, versus by both distally inserted and proximally inserted flexors. Our analysis demonstrates that most species possess relative phalanx lengths that are conducive to grasps actuated only by a single distally inserted tendon per digit. Furthermore, proximally inserted flexors are often required during perching, but the distally inserted flexors are sufficient when grasping and carrying objects. These results are reflected in differences in the relative development of proximally and distally inserted digital flexor musculature among 'perching' and 'grasping' taxa. Thus, our results shed light on the relative roles of variation in phalanx length and digit flexor muscle distribution in an integrative, mechanical context.

A novel device to preserve intestinal tissue ex-vivo by cold peristaltic perfusion.

In the past two decades, much advancement has been made in the area of organ procurement and preservation for the transplant of kidneys, livers, and lungs. However, small intestine preservation remains unchanged. We propose a new preservation system for intestinal grafts that has the potential to increase the viability of the organ during transport. When experimented with porcine intestine, our device resulted in superior tissue quality than tissue in standard of care.