Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput. Video LinkThe video component of this article can be found at

The greatest diversity of influenza A virus (IAV) is found in wild aquatic birds of the orders Anseriformes and Charadriiformes. In these birds, IAV replication occurs mostly in the intestinal tract. Fecal, cloacal, and/or tracheal swabs are typically collected and tested by real‐time RT‐PCR (rRT‐PCR) and/or by virus isolation in embryonated chicken eggs in order to determine the presence of IAV. Virus isolation may impose bottlenecks that select variant populations that are different from those circulating in nature, and such bottlenecks may result in artifactual representation of subtype diversity and/or underrepresented mixed infections. The advent of next‐generation sequencing (NGS) technologies provides an opportunity to explore to what extent IAV subtype diversity is affected by virus isolation in eggs. In the present work, we evaluated the advantage of sequencing by NGS directly from swab material of IAV rRT‐PCR‐positive swabs collected during the 2013–14 surveillance season in Guatemala and compared to results from NGS after virus isolation. The results highlight the benefit of sequencing IAV genomes directly from swabs to better understand subtype diversity and detection of alternative amino acid motifs that could otherwise escape detection using traditional methods of virus isolation. In addition, NGS sequencing data from swabs revealed reduced presence of defective interfering particles compared to virus isolates. We propose an alternative workflow in which original swab samples positive for IAV by rRT‐PCR are first subjected to NGS before attempting viral isolation. This approach should speed the processing of samples and better capture natural IAV diversity. OPEN RESEARCH BADGES This article has earned an Open Data Badge for making publicly available the digitally‐shareable data necessary to reproduce the reported results. The data is available at https://doi.org/10.5061/dryad.3h2n106.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

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Bcl‐2 family proteins regulate mitochondrial apoptosis and have been shown to be involved in a variety of diseases. In sepsis, systemic inflammatory response to pathogens, apoptosis is triggered in immune cells as a result of the cytokine storm induced by sepsis. Immune cell death is not the only damage caused by sepsis and the mechanism of cellular injury on end organs is unknown despite advances in modern medicine. Much of the mortality associated with sepsis is a result of end organ failure, particularly the kidneys. Research has shown that a small amount of apoptotic cell death is characteristic in acute renal injury associated with sepsis, thus Bcl‐2 family proteins should be involved in renal death as a result of septic shock. Use of a two‐step cell culture model stresses immune cells to stimulate cytokine release comparable to the cytokine storm found in sepsis, which can then be harvested and used to treat cultured kidney cells to induce death. Examination of Bcl‐2 proteins after treatment with stimulated cytokines showed a decrease in cell viability; an increase of BH3 only, pro‐apoptotic proteins; and a decrease in anti‐apoptotic Bcl‐2 confirming their hypothesized involvement. Results provide hope for elucidation of the mechanism of sepsis from which more efficient treatments can be developed. Research funded by Elon University URP, Department of Chemistry, Elon College Fellows, and the Provost Award to VDGM.