---
title: "3D Cell Culture"
output:
  html_vignette:
    mathjax: null
vignette: >
  %\VignetteIndexEntry{3D Cell Culture}
  %\VignetteEngine{knitr::rmarkdown}
  %\VignetteEncoding{UTF-8}
bibliography: cpdat.bib
csl: peerj.csl
---

```{r setup, include=FALSE}
library(canprot)
library(knitr)
## use pngquant to reduce size of PNG images
knit_hooks$set(pngquant = hook_pngquant)
pngquant <- "--speed=1 --quality=0-25"
# in case pngquant isn't available (R-Forge?)
if (!nzchar(Sys.which("pngquant"))) pngquant <- NULL 
# read 'basis' option
basis <- getOption("basis")
```

This vignette from the R package [canprot](https://github.com/jedick/canprot) version `r packageDescription("canprot")$Version` shows compositional metrics and phylostrata for proteins that are differentially expressed in 3D compared to 2D culture conditions.
The analysis is described in more detail in a manuscript ([<span title="Water as a reactant in the differential expression of proteins in cancer">Dick, 2020</a>](https://doi.org/10.1101/2020.04.09.035022)).
Abbreviations:

  * <i>Z</i><sub>C</sub> &ndash; average oxidation state of carbon.
  * <i>n</i><sub>H<sub>2</sub>O</sub> &ndash; stoichiometric hydration state per residue ([<span title="Uncovering chemical signatures of salinity gradients through compositional analysis of protein sequences">Dick et al., 2020</span>](https://doi.org/10.5194/bg-2020-146)) computed using the **`r basis`** basis species (`r basis.text(basis)`, H~2~O, O~2~).
  * PS &ndash; phylostratum; <i>n</i><sub>AA</sub> &ndash; protein length; MW &ndash; molecular weight per residue.

Gene ages, expressed as phylostratum numbers, are taken from [<span title="Altered interactions between unicellular and multicellular genes drive hallmarks of transformation in a diverse range of solid tumors">Trigos et al., 2017</span>](https://doi.org/10.1073/pnas.1617743114) (TPPG17) or [<span title="Towards consensus gene ages ">Liebeskind et al, 2016</span>](https://doi.org/10.1093/gbe/evw113) (LMM16).

<style type="text/css">
body {
  max-width: 800px;
  margin-left: auto;
  margin-right: auto;
}
</style>

```{r datasets}
datasets <- pdat_3D(2020)
```

```{r comptab, results="hide", message=FALSE, echo = FALSE}
pdat1 <- lapply(datasets, pdat_3D)
comptab1 <- lapply(pdat1, get_comptab)
comptab2 <- lapply(pdat1, get_comptab, "PS_TPPG17", "PS_LMM16")
comptab3 <- lapply(pdat1, get_comptab, "nAA", "MW")
```

Differences are calculated as (median value for up-regulated proteins) - (median value for down-regulated proteins), except for phylostrata, where mean values are used.
Dashed lines enclose the 50% confidence region for highest probability density.

```{r diffplot, fig.width=9, fig.height=8, fig.align = "center", echo = FALSE, pngquant = pngquant}
layout(matrix(c(0, 2, 1, 2, 1, 3, 0, 3), nrow = 2))
par(cex = 1, mar = c(4, 4, 1, 1), mgp = c(2.5, 1, 0))
diffplot(comptab1, labtext = NA)
diffplot(comptab2, c("PS_TPPG17", "PS_LMM16"))
diffplot(comptab3, c("nAA", "MW"), labtext = NA)
```

In the table, values of &Delta;<i>Z</i><sub>C</sub> and &Delta;<i>n</i><sub>H<sub>2</sub>O</sub> are multiplied by 1000, values of &Delta;PS and &Delta;MW are multiplied by 100, and negative values are shown in bold.
Abbreviations:

  * _n_~down~ &ndash; number of down-regulated proteins; _n_~up~ &ndash; number of up-regulated proteins; P2 &ndash; passage 2; P5 &ndash; passage 5; MSC &ndash; mesenchymal stromal cells.

```{r xsummary, results="asis", echo = FALSE}
library(xtable)
out <- xsummary2(comptab1, comptab2, comptab3)
# round values and include dataset tags
tags <- sapply(sapply(strsplit(datasets, "="), "[", -1), paste, collapse = ";")
out <- cbind(out[, 1:2], tags = tags, out[, 3:19])
out[, 6:20] <- round(out[, 6:20], 4)
file <- paste0("3D_", basis, ".csv")
write.csv(out, file, row.names = FALSE, quote = 2)
```

## Data Sources
Gene names or other identifiers were converted to UniProt accession numbers using the <a href="https://www.uniprot.org/mapping/">UniProt mapping tool</a>.

__a__. Gene names from Supporting Information Table 1S of @PLC+10.
__b__. __c__. Sheets 2 and 3 in Table S1 of @MHG+12.
__d__. __e__. Supplemental Table 1C of @MVC+12, filtered to include proteins with expression ratios < 0.77 or > 1.3.
__f__. Supplemental Table S5 of @YYW+13.
__g__. __h__. Supplemental Table S4 of @ZMH+13.
__i__. Table S1 of @HKX+14.
__j__. __k__. __l__. Supplemental Table S1 of @KDS+14 (hESC: human embryonic stem cells; hiPSC: human induced pluripotent stem cells; hPSC: human pluripotent stem cells).
__m__. Table S2 of @RKP+14, filtered to include proteins that have differences in spectral counts recorded in at least two of three experiments, absolute overall fold change is ≥ 1.5 or ≤ 2/3, and _p_-value is < 0.05.
__n__. Supplementary Figure 2 of @SAS+14.
__o__. P1&lowbar;Data sheet in the Supporting Information file of @WRK+14.
__p__. Supplemental Table S1 of @MTK+15, filtered to exclude marked contaminants and reverse sequences and to include proteins with "Ratio H/L normalized" > 1.5 or < 2/3.
__q__. Tables S1a and S1b of @YLW+16.
__r__. Tables S2 and S3 of @KJK+18.
__s__. __t__. Supplementary Table 3 of @TGD18. 
__u__. Extracted from proteinGroups.txt in ProteomeXchange Dataset [PXD008244](ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/04/PXD008244)/txt.zip [@EWK+19], including proteins quantified in at least two replicates in each condition (adherent and spheroid) and with median fold change > 2 or < 0.5.
__v__. Extracted from Supplementary Table 1 of @GADS19. Values were quantile normalized, then ratios were calculated between 3D and 2D cultures for each treatment (control, CBD, UVA, UVA+CBD, UVB, UVB+CBD). Ratios > 1.2 or < 1/1.2 in at least 4 treatments were used to identify differentially expressed proteins.
__w__. Supplementary Data I, sheet "Volcano plot 2Dv3D" of @HLC19.
__x__. __y__. __z__. Supporting Table 1 of @LPK+19, sheets "3P2P" (mono-cultured preadipocytes), "3A2A" (mono-cultured adipocytes), "3C2C" (co-cultured adipocytes with macrophages), filtered to include proteins marked as "T-test Significant" and with absolute value of "N: T-test Difference" > log~2~(0.5).
__A__. Supplementary Table S2 of @DKM+20, filtered to include proteins with fold change > 2 or < 0.5 for all donors.

## References
