Survival Journals Download Easy
Abra Pabst
Survival analysis, also called time-to-event analysis, is a common approach to handling event data in cardiovascular nursing and health-related research. Survival analysis is used to describe, explain, and/or predict the occurrence and timing of events. There is a specific language used and methods designed to handle the unique nature of event data. In this methods paper, we provide an 'easy start guide' to using survival analysis by (i) providing a step-by-step guide and (ii) applying the steps with example data. Specifically, we analyse cardiovascular event data over 6 months in a sample of patients with heart failure.
Recent studies have identified a feedback loop whereby MiT/TFE family members transcriptionally up-regulate mTOR complex components during the starvation phase to permit mTOR reactivation upon refeeding [14] to channel the resumption of nutrient supply towards growth. Interestingly, prior studies in worms indicate that RNA polymerase accumulates at the promoters of growth genes in preparation for prompt activation of transcription upon resumption of nutrient supply [15]. Whether the MiT/TFE family members are critical for organismal survival with refeeding following starvation is not known.
To understand the role of hlh-30 in sustaining survival during starvation and preparing for refeeding, we modeled starvation-induced arrest of L1 stage worms deficient in hlh-30 using the strong loss-of-function tm1978 mutation (a deletion allele lacking two exons, heretofore termed as hlh-30(loss-of-function) or hlh-30(lf); S1 Table) [18]. Upon hatching in a nutrient-free salt solution, L1 worms were starved for variable times, and aliquots of starved worms were refed on nematode growth medium (NGM) dishes containing Escherichia coli OP50. Survival was scored immediately by visual observation for spontaneous movement, defined as alive after starvation (Fig 1A). Fig 1B shows that periods of starvation of 48 hours or more significantly increased lethality in hlh-30(lf) worms. By contrast, wild-type worms were able to withstand nutrient deficient conditions for much longer (Fig 1B, S1A Fig).
This metabolomics analysis showed that linoleic acid moieties are enriched among lipids (7/24) in the subset of metabolites with the highest importance to group separation in the random forest analysis (S8B Fig; see S4 Table for the list of metabolites evaluated). Also, the abundance of linoleoyl glycerolphosphocholine (linoleoyl-GPC), the metabolite with the highest importance to group separation (S8B Fig), was significantly increased in refed hlh-30(lf) worms compared to their starved counterparts (Fig 3B). While linoleate levels were not different (S10 Fig), the abundance of α- and γ-linolenate (α- and γ-FFA; downstream metabolites of linoleic acid) was significantly increased in refed hlh-30(lf) worms compared to starved hlh-30(lf) worms (Fig 3C), indicating that administered linoleic acid was being metabolized. Taken together, these findings indicate that ω-6 fatty acids are required (along with glucose) to confer survival in the setting of hlh-30 deficiency under starvation conditions.
To investigate the functional importance of vha genes, we used the specific V-ATPase inhibitors concanamycin A or bafilomycin A1 (Baf-A1) [39, 40]. Both drugs reduced survival of starved hlh-30(lf) worms refed with CeMM (Fig 6D). In addition, the method of RNAi was used to reduce the activity of vha-12, a proton pump gene that was restored to wild-type levels by CeMM, but not E. coli OP50, refeeding in starved hlh-30(lf) worms (Fig 5H). Reducing vha-12 activity also attenuated the CeMM-mediated rescue of starved hlh-30(lf) worms refed with CeMM (Fig 6E), indicating the proton pump encoded by vha-12 is necessary for this rescue. Interestingly, nuclear hormone receptor 31 (nhr-31), which encodes a C. elegans ortholog of mammalian hepatocyte nuclear factor 4α (HNF-4α) (with linoleic acid as an endogenous ligand [41]), has been identified as a specific transcriptional activator of vha gene transcription in worms [42]. RNAi targeting of nhr-31 transcripts reduced CeMM-induced rescue of starved hlh-30(lf) mutants (Fig 6E), indicating the nuclear receptor encoded by nhr-31 is necessary for this rescue. To further evaluate nhr-31, we determined whether overexpression of nhr-31 was sufficient to rescue hlh-30(lf) mutants supplemented with either glucose or linoleic acid. A significant increase in survival was not observed, pointing to a critical role for both the nutrients (glucose and linoleic acid) in entraining nhr-31 signaling to confer rescue (S21 Fig).
In mammals, activation of TOR signaling on lysosomes in response to refeeding is mediated via sensing amino acids and/or cholesterol and is essential for survival and resumption of growth [7, 30, 43]. Recent studies have ascribed a critical role for TFEB in transcriptionally up-regulating RagD during starvation to permit mTOR activation with refeeding [14]. Our data demonstrate that while amino acids and cholesterol are not essential for conferring rescue in hlh-30(lf) mutants (Fig 2C, S9B Fig), two different classes of nutrients, glucose and linoleic acid, drive increased transcript levels of V-ATPase genes, which may facilitate LYNUS to TOR activation.
To investigate the role of TOR activation, we attempted CeMM rescue in the presence of rapamycin, a known inhibitor of TOR activity [44]. Treatment with rapamycin significantly attenuated CeMM-mediated rescue of the starved hlh-30(lf) mutants (Fig 6F), pointing to a critical role for TOR reactivation in survival of starved hlh-30(lf) mutants.
To investigate regulation of TOR pathway transcripts, we employed quantitative PCR. Transcripts for let-363 (the C. elegans ortholog of mammalian mTOR, S22A Fig), daf-15 (C. elegans ortholog of mammalian raptor, S22B Fig), rsks-1 (the C. elegans ortholog of mammalian S6 kinase, a TOR target; S22F Fig), and ragc-1 (the C. elegans ortholog of RAG-C/D required for TOR activation [14], Fig 7A) displayed significantly lower levels in starved hlh-30(lf) worms compared to the starved wild type. These findings suggest hlh-30 is necessary to promote transcription of these genes during starvation. Importantly, refeeding with CeMM but not E. coli OP50 restored the expression of let-363 and ragc-1 to wild-type levels in hlh-30(lf) mutants (S22A Fig and Fig 7A). We focused on the function of ragc-1 because its regulatory control was strongly correlated with survival: E. coli OP50 refeeding significantly increased ragc-1 transcript levels in starved wild-type but not hlh-30(lf) worms, whereas CeMM exposure increased ragc-1 transcript levels in hlh-30(lf) worms. We analyzed a strong loss-of-function ragc-1(tm1974) mutation caused by a deletion spanning multiple exons (S3 Table), termed ragc-1(lf). ragc-1(lf) animals displayed striking sensitivity to starvation-induced lethality (Fig 7B). Refeeding either with E. coli OP50 or CeMM did not restore viability (Fig 7C and 7D). Overexpression of ragc-1 in hlh-30(lf) mutants was sufficient to confer partial rescue on starved animals fed E. coli OP50 (Fig 7E, S23 Fig). Taken together, these data indicate that TOR activation was downstream of the observed CeMM-mediated rescue.
To deliver dsRNA to worms by feeding, gravid wild-type and hlh-30(lf) adults were bleached, and eggs were placed on NGM dishes containing 5 mM IPTG and 25 μg/ml carbenicillin. An HT-115(DE3) bacterial colony containing L4440 or bec-1 plasmid [70] was inoculated in LB broth containing 25 μg/mL carbenicillin, and grown for eight hours in a 37 C shaker. The bacteria were plated on the NGM dish containing IPTG and carbenicillin an hour prior to addition of the worms. The bacterial solution was permitted to dry on the surface of the dish. Worms were added to NGM dishes containing IPTG, carbenicillin, and freshly plated bacteria. To starve worms, we washed L4 worms off dishes with M9, washed the worms seven times, then resuspended them at a density of one worm per μL in a 15 mL tube containing M9. The tubes were laid flat on a horizontal shaker at 20 C for 36 hours, then the worms were incubated in CeMM for 15 hours and placed on NGM dishes seeded with bacteria containing L4440 or bec-1 plasmid. Worms were scored alive after refeeding 48 hours later. For RNAi to nhr-31, we employed dsRNA soaking. An nhr-31 RNA construct was generated through PCR amplification of 1,569 bp from GE Dharmacon C. elegans clone C26B2.3 ORF. To check for potential cross interference, defined by greater than 80% identity over 200 bp, a nucleotide Blast search was performed using WormBase. There were no matches greater than 200 bp except for exon sequences found in nhr-31. The amplimer was subcloned into the multiple cloning site of L4440 at the HindIII and NotI restriction site, between the T7 RNA polymerase binding sites, which was verified with sequencing. The clone for expressing RNAi targeting vha-12 was obtained from the Ahringer RNAi library. To generate dsRNA from these clones, a PCR produced a linear template of dsDNA that included the subcloned nhr-31 fragment or the vha-12 fragment and the flanking T7 RNA polymerase binding sites. The product of the DNA polymerase reaction was purified using the Monarch PCR and DNA Cleanup Kit (New England BioLabs [NEB], T10305; Ipswich, MA, USA). Subsequently, in vitro transcription was performed with 500 ng of purified dsDNA using the HiScribe T7 RNA Polymerase Kit (NEB, E2040S). The product of the RNA polymerase reaction was purified using the RNeasy MinElute Kit (Qiagen, 74204; Hilden, Germany). The starvation and refeeding assay for L1 stage larvae was performed as described above in the presence of 1 μg of L4440, nhr-31, or vha-12 dsRNA.
In many medical studies, time to death is the event of interest. However, in cancer, another important measure is the time between response to treatment and recurrence or relapse-free survival time (also called disease-free survival time). It is important to state what the event is and when the period of observation starts and finishes. For example, we may be interested in relapse in the time period between a confirmed response and the first relapse of cancer.
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