Critical Steps in Data Management During a Crisis.

Data management is essential in a flow cytometry (FCM) shared resource laboratory (SRL) for the integrity of collected data and its long-term preservation, as described in the Cytometry publication from 2016, ISAC Flow Cytometry Shared Resource Laboratory (SRL) Best Practices (Barsky et al.: Cytometry Part A 89A(2016): 1017-1030). The SARS-CoV-2 pandemic introduced an array of challenges in the operation of SRLs. The subsequent laboratory shutdowns and access restrictions brought to the forefront well-established practices that withstood the impact of a sudden change in operations and illuminated areas that need improvement. The most significant challenges from a data management perspective were data access for remote analysis and workstation management. Notably, lessons learned from this challenge emphasize the importance of safeguarding collected data from loss in various emergencies such as fire or natural disasters where the physical hardware storing data could be directly affected. Here, we describe two data management systems that have been successful during the current emergency created by the pandemic, specifically remote access and automated data transfer. We will discuss other situations that could arise and lead to data loss or challenges in interpreting data. © 2020 International Society for Advancement of Cytometry.

High-Resolution Single-Cell Sequencing of Malaria Parasites.

Single-cell genomics is a powerful tool for determining the genetic architecture of complex communities of unicellular organisms. In areas of high transmission, malaria patients are often challenged by the activities of multiple Plasmodium falciparum lineages, which can potentiate pathology, spread drug resistance loci, and also complicate most genetic analysis. Single-cell sequencing of P. falciparum would be key to understanding infection complexity, though efforts are hampered by the extreme nucleotide composition of its genome (∼80% AT-rich). To counter the low coverage achieved in previous studies, we targeted DNA-rich late-stage parasites by Fluorescence-Activated Cell Sorting and whole genome sequencing. Our method routinely generates accurate, near-complete capture of the 23 Mb P. falciparum genome (mean breadth of coverage 90.7%) at high efficiency. Data from 48 single-cell genomes derived from a polyclonal infection sampled in Chikhwawa, Malawi allowed for unambiguous determination of haplotype diversity and recent meiotic events, information that will aid public health efforts.

Targeted therapy of human osteosarcoma with 17AAG or rapamycin: characterization of induced apoptosis and inhibition of mTOR and Akt/MAPK/Wnt pathways.

Osteosarcoma is highly resistant to current chemotherapy regimens. Novel therapeutic approaches, potentially involving targeting of specific survival pathways, are needed. We used 17-AAG to inhibit Hsp90 and rapamycin to inhibit mTOR, in the osteosarcoma cell lines, HOS and KHOS/NP. HOS and KHOS cells were treated for 24 and 48 h with 17-AAG or rapamycin and studied drug-induced apoptosis, cell cycle, mitochondrial membrane potential and levels of reduced glutathione (GSH), dephosphorylation of signal transduction proteins in the Akt/MAP kinase pathway and mTOR signaling. 17-AAG was a potent inducer of apoptosis, involving effective depletion of GSH and mitochondrial membrane (MM) depolarization, strong activation of caspase-8 and -9 and release of AIF from mitochondria to the cytosol. Furthermore, 17-AAG down-regulated pAkt, p44Erk, p-mTOR, p70S6, TSC1/2 and pGSK-3beta. Treatment with 17-AAG also caused down-regulation of cyclin D1, GADD45a, GADD34 and pCdc2 and upregulation of cyclin B1 and mitotic block. A decrease in Hsp90 and increase in Hsp70 and Hsp70 C-terminal fragments were also observed. Rapamycin was a less potent inducer of apoptosis, involving a small decrease in GSH and MM potential with no activation of caspases or release of AIF. Rapamycin strongly inhibited cell growth with an increase in G1 and a decrease in S-phase of the cell cycle concomitant with down-regulation of cyclin D1. Rapamycin also down-regulated the activity of p70S6, pAkt and p-mTOR, but had no effect on pGSK-3beta, p44Erk, pCdc2, TSC1/2 or Hsp70 or Hsp90. We conclude that Hsp90 inhibition merits further study in the therapy of osteosarcoma.