Evaluating and optimizing Acid-pH and Direct Lysis RNA extraction for SARS-CoV-2 RNA detection in whole saliva.

COVID-19 has been a global public health and economic challenge. Screening for the SARS-CoV-2 virus has been a key part of disease mitigation while the world continues to move forward, and lessons learned will benefit disease detection beyond COVID-19. Saliva specimen collection offers a less invasive, time- and cost-effective alternative to standard nasopharyngeal swabs. We optimized two different methods of saliva sample processing for RT-qPCR testing. Two methods were optimized to provide two cost-efficient ways to do testing for a minimum of four samples by pooling in a 2.0 mL tube and decrease the need for more highly trained personnel. Acid-pH-based RNA extraction method can be done without the need for expensive kits. Direct Lysis is a quick one-step reaction that can be applied quickly. Our optimized Acid-pH and Direct Lysis protocols are reliable and reproducible, detecting the beta-2 microglobulin (B2M) mRNA in saliva as an internal control from 97 to 96.7% of samples, respectively. The cycle threshold (Ct) values for B2M were significantly higher in the Direct Lysis protocol than in the Acid-pH protocol. The limit of detection for N1 gene was higher in Direct Lysis at ≤ 5 copies/µL than Acid-pH. Saliva samples collected over the course of several days from two COVID-positive individuals demonstrated Ct values for N1 that were consistently higher from Direct Lysis compared to Acid-pH. Collectively, this work supports that each of these techniques can be used to screen for SARS-CoV-2 in saliva for a cost-effective screening platform.

Age-related defects in autophagy alter the secretion of paracrine factors from bone marrow mononuclear cells.

Bone marrow mononuclear cell therapy improves cardiac repair after myocardial infarction (MI), in-part through signaling to resident cardiac cells, such as fibroblasts, which regulate scar formation. The efficacy of cell therapy declines with age, as aging of both donor and recipient cells decreases repair responses. Autophagy regulates the microenvironment by both extracellular vesicle (EV)-dependent and independent secretion pathways. We hypothesized that age-related autophagy changes in bone marrow cells (BMCs) alter paracrine signaling, contributing to lower cell therapy efficacy. Here, we demonstrate that young Sca-1+ BMCs exhibited a higher LC3II/LC3I ratio compared to old Sca-1+ BMCs, which was accentuated when BMCs were cultured under hypoxia. To examine the effect on paracrine signaling, old cardiac fibroblasts were cultured with conditioned medium (CM) from young and old Sca-1+ BMCs. Young, but not old CM, enhanced fibroblast proliferation, migration, and differentiation, plus reduced senescence. These beneficial effects were lost when autophagy or EV secretion in BMCs was blocked pharmacologically, or by siRNA knockdown of Atg7. Therefore, both EV-dependent and -independent paracrine signaling from young BMCs is responsible for paracrine stimulation of old cardiac fibroblasts. In vivo, bone marrow chimerism of old mice with young BMCs increased the number of LC3b+ cells in the heart compared to old mice reconstituted with old BMCs. These data suggest that the deterioration of autophagy with aging negatively impacts the paracrine effects of BMCs, and provide mechanistic insight into the age-related decline in cell therapy efficacy that could be targeted to improve the function of old donor cells.

Cellular senescence contributes to age-dependent changes in circulating extracellular vesicle cargo and function.

Extracellular vesicles (EVs) have emerged as important regulators of inter-cellular and inter-organ communication, in part via the transfer of their cargo to recipient cells. Although circulating EVs have been previously studied as biomarkers of aging, how circulating EVs change with age and the underlying mechanisms that contribute to these changes are poorly understood. Here, we demonstrate that aging has a profound effect on the circulating EV pool, as evidenced by changes in concentration, size, and cargo. Aging also alters particle function; treatment of cells with EV fractions isolated from old plasma reduces macrophage responses to lipopolysaccharide, increases phagocytosis, and reduces endothelial cell responses to vascular endothelial growth factor compared to cells treated with young EV fractions. Depletion studies indicate that CD63+ particles mediate these effects. Treatment of macrophages with EV-like particles revealed that old particles increased the expression of EV miRNAs in recipient cells. Transfection of cells with microRNA mimics recapitulated some of the effects seen with old EV-like particles. Investigation into the underlying mechanisms using bone marrow transplant studies revealed circulating cell age does not substantially affect the expression of aging-associated circulating EV miRNAs in old mice. Instead, we show that cellular senescence contributes to changes in particle cargo and function. Notably, senolytic treatment of old mice shifted plasma particle cargo and function toward that of a younger phenotype. Collectively, these results demonstrate that senescent cells contribute to changes in plasma EVs with age and suggest a new mechanism by which senescent cells can affect cellular functions throughout the body.