Leveraging gene correlations in single cell transcriptomic data.

BACKGROUND: Many approaches have been developed to overcome technical noise in single cell RNA-sequencing (scRNAseq). As researchers dig deeper into data-looking for rare cell types, subtleties of cell states, and details of gene regulatory networks-there is a growing need for algorithms with controllable accuracy and fewer ad hoc parameters and thresholds. Impeding this goal is the fact that an appropriate null distribution for scRNAseq cannot simply be extracted from data when ground truth about biological variation is unknown (i.e., usually). RESULTS: We approach this problem analytically, assuming that scRNAseq data reflect only cell heterogeneity (what we seek to characterize), transcriptional noise (temporal fluctuations randomly distributed across cells), and sampling error (i.e., Poisson noise). We analyze scRNAseq data without normalization-a step that skews distributions, particularly for sparse data-and calculate p-values associated with key statistics. We develop an improved method for selecting features for cell clustering and identifying gene-gene correlations, both positive and negative. Using simulated data, we show that this method, which we call BigSur (Basic Informatics and Gene Statistics from Unnormalized Reads), captures even weak yet significant correlation structures in scRNAseq data. Applying BigSur to data from a clonal human melanoma cell line, we identify thousands of correlations that, when clustered without supervision into gene communities, align with known cellular components and biological processes, and highlight potentially novel cell biological relationships. CONCLUSIONS: New insights into functionally relevant gene regulatory networks can be obtained using a statistically grounded approach to the identification of gene-gene correlations.

Tunneling nanotube formation promotes survival against 5-fluorouracil in MCF-7 breast cancer cells.

Tunneling nanotubes (TNTs) are F-actin-based open-ended tubular extensions that form following stresses, such as nutritional deprivation and oxidative stress. The chemotherapy agent 5-fluorouracil (5-FU) represents a significant stressor to cancer cells and induces thymidine deficiency, a state similar to nutritional deprivation. However, the ability of 5-FU to induce TNT formation in cancer cells and potentially enhance survival has not been explored. In this study, we examined whether 5-FU can induce TNT formation in MCF-7 breast cancer cells. Cytotoxic doses of 5-FU (150-350 µm) were observed to significantly induce TNT formation beginning at 24 h after exposure. TNTs formed following 5-FU treatment probably originated as extensions of gap junctions as MCF-7 cells detach from cell clusters. TNTs act as conduits for exchange of cellular components and we observed mitochondrial exchange through TNTs following 5-FU treatment. 5-FU-induced TNT formation was inhibited by over 80% following treatment with the F-actin-depolymerizing agent, cytochalasin B (cytoB). The inhibition of TNTs by cytoB corresponded with increased 5-FU-induced cytotoxicity by 30-62% starting at 48 h, suggesting TNT formation aides in MCF-7 cell survival against 5-FU. Two other widely used chemotherapy agents, docetaxel and doxorubicin induced TNT formation at much lower levels than 5-FU. Our work suggests that the therapeutic targeting of TNTs may increase 5-FU chemotherapy efficacy and decrease drug resistance in cancer cells, and these findings merits further investigation.

Mitochondrial remodeling in the liver following chronic alcohol feeding to rats.

The feeding of alcohol orally (Lieber-DeCarli diet) to rats has been shown to cause declines in mitochondrial respiration (state III), decreased expression of respiratory complexes, and decreased respiratory control ratios (RCR) in liver mitochondria. These declines and other mitochondrial alterations have led to the hypothesis that alcohol feeding causes "mitochondrial dysfunction" in the liver. If oral alcohol feeding leads to mitochondrial dysfunction, one would predict that increasing alcohol delivery by intragastric (IG) alcohol feeding to rats would cause greater declines in mitochondrial bioenergetics in the liver. In this study, we examined the mitochondrial alterations that occur in rats fed alcohol both orally and intragastrically. Oral alcohol feeding decreased glutamate/malate-, acetaldehyde- and succinate-driven state III respiration, RCR, and expression of respiratory complexes (I, III, IV, V) in liver mitochondria, in agreement with previous results. IG alcohol feeding, on the other hand, caused a slight increase in glutamate/malate-driven respiration, and significantly increased acetaldehyde-driven respiration in liver mitochondria. IG feeding also caused liver mitochondria to experience a decline in succinate-driven respiration, but these decreases were smaller than those observed with oral alcohol feeding. Surprisingly, oral and IG alcohol feeding to rats increased mitochondrial respiration using other substrates, including glycerol-3-phosphate (which delivers electrons from cytoplasmic NADH to mitochondria) and octanoate (a substrate for beta-oxidation). The enhancement of glycerol-3-phosphate- and octanoate-driven respiration suggests that liver mitochondria remodeled in response to alcohol feeding. In support of this notion, we observed that IG alcohol feeding also increased expression of mitochondrial glycerol phosphate dehydrogenase-2 (GPD2), transcription factor A (TFAM), and increased mitochondrial NAD+-NADH and NADP+-NADPH levels in the liver. Our findings suggest that mitochondrial dysfunction represents an incomplete picture of mitochondrial dynamics that occur in the liver following alcohol feeding. While alcohol feeding causes some mitochondrial dysfunction (i.e. succinate-driven respiration), our work suggests that the major consequence of alcohol feeding is mitochondrial remodeling in the liver as an adaptation. This mitochondrial remodeling may play an important role in the enhanced alcohol metabolism and other adaptations in the liver that develop with alcohol intake.