Protocol Improvement for RNA Extraction From Compromised Frozen Specimens Generated in Austere Conditions: A Path Forward to Transcriptomics-Pathology Systems Integration.

At the heart of the phenome-to-genome approach is high throughput assays, which are liable to produce false results. This risk can be mitigated by minimizing the sample bias, specifically, recycling the same tissue specimen for both phenotypic and genotypic investigations. Therefore, our aim is to suggest a methodology of obtaining robust results from frozen specimens of compromised quality, particularly if the sample is produced in conditions with limited resources. For example, generating samples at the International Space Station (ISS) is challenging because the time and laboratory footprint allotted to a project can get expensive. In an effort to be economical with available resources, snap-frozen euthanized mice are the straightforward solution; however, this method increases the risk of temperature abuse during the thawing process at the beginning of the tissue collection. We found that prolonged immersion of snap frozen mouse carcass in 10% neutral buffered formalin at 4°C yielded minimal microscopic signs of ice crystallization and delivered tissues with histomorphology that is optimal for hematoxylin and eosin (H&E) staining and fixation on glass slides. We further optimized a method to sequester the tissue specimen from the H&E slides using an incubator shaker. Using this method, we were able to recover an optimal amount of RNA that could be used for downstream transcriptomics assays. Overall, we demonstrated a protocol that enables us to maximize scientific values from tissues collected in austere condition. Furthermore, our protocol can suggest an improvement in the spatial resolution of transcriptomic assays.

Gene-Metabolite Network Linked to Inhibited Bioenergetics in Association With Spaceflight-Induced Loss of Male Mouse Quadriceps Muscle.

Prolonged residence of mice in spaceflight is a scientifically robust and ethically ratified model of muscle atrophy caused by continued unloading. Under the Rodent Research Program of the National Aeronautics and Space Administration (NASA), we assayed the large-scale mRNA and metabolomic perturbations in the quadriceps of C57BL/6j male mice that lived in spaceflight (FLT) or on the ground (control or CTR) for approximately 4 weeks. The wet weights of the quadriceps were significantly reduced in FLT mice. Next-generation sequencing and untargeted mass spectroscopic assays interrogated the gene-metabolite landscape of the quadriceps. A majority of top-ranked differentially suppressed genes in FLT encoded proteins from the myosin or troponin families, suggesting sarcomere alterations in space. Significantly enriched gene-metabolite networks were found linked to sarcomeric integrity, immune fitness, and oxidative stress response; all inhibited in space as per in silico prediction. A significant loss of mitochondrial DNA copy numbers in FLT mice underlined the energy deprivation associated with spaceflight-induced stress. This hypothesis was reinforced by the transcriptomic sequencing-metabolomics integrative analysis that showed inhibited networks related to protein, lipid, and carbohydrate metabolism, and adenosine triphosphate (ATP) synthesis and hydrolysis. Finally, we discovered important upstream regulators, which could be targeted for next-generation therapeutic intervention for chronic disuse of the musculoskeletal system. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

The responses of lungs and adjacent lymph nodes in responding to Yersinia pestis infection: A transcriptomic study using a non-human primate model.

Initiation of treatment during the pre-symptomatic phase of Yersinia pestis (Y. pestis) infection is particularly critical. The rapid proliferation of Y. pestis typically couples with the manifestation of common flu-like early symptoms that often misguides the medical intervention. Our study used African green monkeys (AGM) that did not exhibit clear clinical symptoms for nearly two days after intranasal challenge with Y. pestis and succumbed within a day after showing the first signs of clinical symptoms. The lung, and mediastinal and submandibular lymph nodes (LN) accumulated significant Y. pestis colonization immediately after the intranasal challenge. Hence, organ-specific molecular investigations are deemed to be the key to elucidating mechanisms of the initial host response. Our previous study focused on the whole blood of AGM, and we found early perturbations in the ubiquitin-microtubule-mediated host defense. Altered expression of the genes present in ubiquitin and microtubule networks indicated an early suppression of these networks in the submandibular lymph nodes. In concert, the upstream toll-like receptor signaling and downstream NFκB signaling were inhibited at the multi-omics level. The inflammatory response was suppressed in the lungs, submandibular lymph nodes and mediastinal lymph nodes. We posited a causal chain of molecular mechanisms that indicated Y. pestis was probably able to impair host-mediated proteolysis activities and evade autophagosome capture by dysregulating both ubiquitin and microtubule networks in submandibular lymph nodes. Targeting these networks in a submandibular LN-specific and time-resolved fashion could be essential for development of the next generation therapeutics for pneumonic plague.