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Platypus Paper, Rewritten

This is a completely rewritten version of a biology paper titled, “A Model for the Evolution of the Mammalian T-cell Receptor α/δ and μ Loci Based on Evidence from the Duckbill Platypus.” It is meant to be a demonstration and a proof of concept for JAWWS, my idea of a science journal that focusses on readability.

You can read the original version on the Molecular Biology and Evolution journal’s website, or here on this blog, with annotations by me.

Why did I choose to rewrite this paper? I wish I had a more principled answer, but the truth is that I simply went to ResearchHub, a website where scientists share papers between themselves and upvote the most interesting one, went to the Evolutionary Biology section (because that used to be my field), and picked the first paper that was open-access and seemed fit for my purposes. In other words, a paper that seemed like it could be improved a lot because it seemed more difficult to understand than was warranted.

Only later did I realize it was a paper from 2012, so not that recent. I don’t think that matters too much for now since it’s just a proof of concept. Nor does the topic, i.e. molecular evolution in the vertebrate immune system. The actual journal will need to pick papers in a more principled way, of course.

What did my rewrite entail? The best way to know is to read (not necessarily closely) the original and rewritten versions. But here’s a sample of my “interventions”:

  • I put almost all citations in collapsible footnotes.
  • I cut up most long paragraphs, including the abstract.
  • I added many context sentences, including at the beginning of sections, to give a better sense of why we’re reading this. One example is the first sentence of the introduction: “How did the immune system of jawed vertebrates evolve?”
  • I reworked some of the paper’s structure. One major change: I put the major contribution of the study, that is, the new evolutionary model, in its own section after the introduction. This way, it is not buried deep in the discussion; readers can start with it and dig into the rest only if they want more details. I also reordered the methods so that they would match the ordering in the Results.
  • I added several subheadings to the sections that didn’t already have subsections (Introduction, and Results and Discussion)
  • I tried my best to avoid abbreviations. One difficulty is that some of them are probably very recognizable by people who  know immunology, and not by me. So I left some in, while trying to make sure they don’t hamper readability. The major example is “TCR”, which means T-cell receptor and was used a lot. It’s still used in my version, but far less often.
  • I removed some jargon words. E.g. “proximal” became “closest”.
  • I formatted some information in point form, such as the T-cell lineages or the protocols in the Methods.
  • I added some text formatting to guide the reader. For instance, bold font for groups of animals in the introduction, and color in the text to match colored elements in figures.
  • I changed one figure by reorganizing its parts to make it clearer (fig. 2, which used to be fig. 5). A lot of additional clarity could potentially be gained from editing the figures, but that’s a lot of work, so I didn’t press it further.
  • I fixed a number of typos and grammatical errors. Mistakes like this are not a big problem, but there were enough that I assume little editing work was done on this paper.

On footnotes: I’m using two different kinds of collapsible footnotes. Those in the usual style of the blog, like this one,1Here you would usually read a citation in short form such as “Rast et al. 1997.” Head to the original paper webpage to see the reference list in full. contain the citations included in the original paper.  Footnotes with brackets like this[1]This is an example comment. are for comments on the rewriting process and are also shown at the bottom of the paper. I suggest you don’t click on the former, unless you want to see a reference, and hover onto the latter to read my comments.

Overall, my rewrite increased the length of the abstract from 267 to 286 words, and of the rest of the paper from about 6000 to about 6400 words. I consider this acceptable.

I will publish some more thoughts on the rewriting process later.

Abstract

Goal: This study presents a new model for the evolution of part of the vertebrate immune system: the genes encoding the T-cell receptor (TCR) δ chains.

Background: T lymphocytes have to recognize specific antigen for the adaptive immune response to work in vertebrates. They perform this using a somatically diversified T-cell receptor. All jawed vertebrates use four T-cell receptor chains called α, β, γ, and δ, but some lineages have nonconventional receptor chains: monotremes and marsupials encode a fifth one, called TCRµ. Its function is unknown, but it is somatically diversified like the conventional chains. Its origins are also unclear. It appears to be distantly related to the TCRδ chain, for which recent evidence from birds and frogs has provided new information that was not available from humans or other placental (eutherian) mammals.

Experiment: We analyzed the genes encoding the δ chains in the platypus. This revealed the presence of a highly divergent variable (V) gene, indistinguishable from immunoglobulin heavy chain V genes (VH), and related to V genes used in the µ chain. This gene is expressed as part of TCRδ repertoire, so it is designated VHδ.

Conclusions: The VHδ gene is similar to what has been found in frogs and birds, but it is the first time such a gene has been found in a mammal. This provides a critical link in reconstructing the evolutionary history of TCRµ. The current structure of the δ and µ genes in tetrapods suggests ancient and possibly recurring translocations of gene segments between the δ and immunoglobulin heavy genes, as well as translocations of δ genes out of the TCRα/δ locus early in mammals, creating the TCRµ locus. We present a detailed model of this evolutionary history.[2]Major changes to the abstract: I split it in four paragraphs with section titles (this is common in some journals; it should be common in most journals). I also added a section at the beginning to … Continue reading

Introduction

How did the immune system of jawed vertebrates evolve? In this study, we use genomic evidence from the platypus to propose a model for the evolution of a specific component of the vertebrate immune system: the receptors on the surface of T lymphocytes.

As a reminder, T lymphocytes (or T cells) are white blood cells that play a critical role in the adaptive immune system. They can be classified into two main lineages based on the receptor they use:2Rast et al. 1997; reviewed in Davis and Chein 2008

  1. αβT cell lineage: The receptor is composed of a heterodimer of α and β chains. Most circulating human T cells are αβT cells, including familiar subsets such as CD4+ helper T cells and regulatory T cells, CD8+ cytotoxic T cells, and natural killer T (NKT) cells.
  2. γδT cell lineage: The receptor is composed of γ and δ chains. The function of these cells is less well defined. They have been associated with a broad range of immune responses including tumor surveillance, innate responses to pathogens and stress, and wound healing.3Hayday 2009 γδT cells are found primarily in epithelial tissues and form a lower percentage of circulating lymphocytes in some species.

αβ and γδ T cells also differ in the way they interact with antigen. The receptors of αβT cells are “restricted” relative to the major histocompatibility complex (MHC), meaning that that they bind antigenic epitopes, such as peptide fragments, bound to, or “presented” by, molecules encoded in the MHC. In contrast, γδT receptors have been found to bind antigens directly in the absence of MHC, as well as self-ligands that are often MHC-related molecules.4Sciammas et al. 1994; Hayday 2009

All gnathostomes (jawed vertebrates) have αβ and γδ T cells. As we will see below, marsupial and monotreme mammals have an additional type of T-cell receptor, denoted with the letter µ. The platypus, a monotreme, further has a non-conventional receptor with δ chains, which is also present in birds and amphibians.

Before presenting our evolutionary model, let’s review these types of T-cell receptors and their structure.

Structure and Genes of Conventional T-Cell Receptors

The chains of conventional T-cell receptors are composed of two extracellular domains, both members of the immunoglobulin domain superfamily of cell surface proteins (fig. 1):5reviewed in Davis and Chein 2008

  • The closest domain to the cellular membrane is called C for constant.[3]Since this abbreviation comes up a lot, I put it first, with its meaning in parentheses. The C domain is largely invariant among T-cell clones expressing the same class of the receptor chain.
  • The domain farthest from the cellular membrane is called V for variable. It is the region that contacts antigen and MHC. Similar to antibodies, the individual clonal diversity in the V domain is generated by somatic DNA recombination.6Tonegawa 1983 [4]I didn’t change much in the figure’s caption, but it seemed pretty trivial to add color to the text to facilitate looking up what the colors mean.
Fig. 1. Cartoon diagram of the T-cell receptor (TCR) forms found in different species. Oblong circles indicate immunoglobulin superfamily domains and are color coded as C domains (blue), conventional V domains (red), and VHδ or Vµ (yellow). The gray shaded chains represent the hypothetical partner chain for µ and δ using VHδ.

While C domains are usually encoded by a single, intact exon, V domains are assembled somatically from germ-line segments in developing T cells. These segments are genes called V (again for variable), D (for diversity), and J (for joining). The assembly process depends on the enzymes encoded by two genes, the recombination activating genes (RAG)-1 and RAG-2.7Yancopoulos et al. 1986; Schatz et al. 1989

The various T-cell receptor chains differ in how their V domains are assembled. β and δ chains are assembled from all three types of gene segments, whereas α and γ chains use only V and J. The different combinations of two or three segments, selected from a large repertoire of germ-line gene segments, along with variation at the junctions due to the addition and deletion of nucleotides during recombination, contribute to a vast diversity of T-cell receptors. It is this diversity that creates the individual antigen specificity of T-cell clones.[5]This is an example of two sentences taken verbatim from the original paper. Not all of it was poorly written!

These genes are highly conserved among species in both their genomic sequence and their organization.8Rast et al. 1997; Parra et al. 2008, 2012; Chen et al. 2009 In all tetrapods examined, the β and γ chains are each encoded at multiple separate loci, whereas the genes encoding the α and δ chains are nested at a single locus, called the TCRα/δ locus.9Chien et al. 1987; Satyanarayana et al. 1988; reviewed in Davis and Chein 2008 The V domains of α and δ chains can use a common pool of V gene segments, but distinct D, J, and C genes.

The recombination of V, J and optionally D genes, referred to as V(D)J recombination, and mediated by RAG, is also known to generate the diversity of antibodies produced by another type of lymphocyte, the B cells.[6]It took me forever to rewrite this part. The original sentence was, “Diversity in antibodies produced by B cells is also generated by RAG-mediated V(D)J recombination and the TCR and Ig genes … Continue reading10Flajnik and Kasahara 2010; Litman et al. 2010

Non-Conventional Receptors Across Vertebrates

T-cell receptor and immunoglobulin genes clearly share a common origin in the jawed vertebrates.11Flajnik and Kasahara 2010; Litman et al. 2010 Usually, the V, D, J, and C coding regions are readily distinguishable from immunoglobulin, at least for conventional T-cell receptors, owing to divergence over the past 400 million years.

Recently, however, the discovery of non-conventional isoforms of the δ chain has blurred the boundary between them. These non-conventional forms use V genes that appear indistinguishable from the immunoglobulin heavy chain V.12Parra et al. 2010, 2012 Such V genes have been named VHδ.[7]The original combined this sentence and the next, even though they’re about quite distinct ideas: the name of the genes, and the species where they’re found.

VHδ genes have been found in both amphibians and birds (see the rightmost part of fig. 1).[8]Why not indicate the part of the figure that is relevant? Whenever you can, provide reader guidance! In the frog Xenopus tropicalis, as well as in a passerine bird, the zebra finch Taeniopygia guttata, the VHδ genes coexist with the conventional Vα and Vδ genes at the TCRα/δ locus.13Parra et al. 2010, 2012

In galliform birds, such as the chicken Gallus gallus, they are instead located at a second TCRδ locus that is unlinked to the conventional TCRα/δ.14Parra et al. 2012 VHδ are the only type of V gene segment present at the second locus and, although closely related to antibody VH genes, the VHδ appear to be used exclusively in δ chains. This is true as well for frogs where the TCRα/δ and IgH (immunoglobulin heavy chain) loci are tightly linked.15Parra et al. 2010

In mammals, a TCRα/δ locus has been characterized in several eutherian species and at least one marsupial, the opossum Monodelphis domestica. VHδ genes have not been found in mammals to date.16Satyanarayana et al.1988; Wang et al. 1994; Parra et al. 2008 However, marsupials do have an additional locus, unlinked to TCRα/δ, that uses antibody-related V genes. This fifth chain is called µ, and the receptor that uses it is referred to as TCRµ. The µ chain is related to the δ chain, but it diverges from it in both sequence and structure.17Parra et al. 2007, 2008 It has also been found in a monotreme, the platypus.[9]The authors like to use “duckbill platypus,” but there’s only one species of platypus, so I took that word out. The platypus and marsupial TCRµ genes are clearly orthologous, which is consistent with the idea that the µ chain is ancient in mammals, but has been lost in the eutherians.18Parra et al. 2008; Wang et al. 2011

TCRµ chains use their own unique set of V genes, called Vµ.19Parra et al. 2007; Wang et al. 2011 So far, no evidence has been found of V(D)J recombination between Vµ genes and genes from other immunoglobulin or T-cell receptor loci.[10]Another horrible sentence from the original, recorded for posterity: “Trans-locus V(D)J recombination of V genes from other Ig and TCR loci with TCRµ genes has not been found.” That … Continue reading  Neither have TCRµ homologues been found in non-mammals.20Parra et al. 2008

The structure of TCRµ chains is atypical. They contain three, rather than two, extra-cellular domains from the immunoglobulin superfamily;[11]The abbreviation IgSF was used in the paper, with no explanation. I assume the people who would read this paper tend to know what that means, but still. this is due to an extra N-terminal V domain (see fig. 1).21Parra et al. 2007; Wang et al. 2011 Both V domains are encoded by a unique set of Vµ genes and are more related to immunoglobulin heavy chain V than to conventional T-cell receptor V domains. The N-terminal one is diverse and encoded by genes that undergo somatic V(D)J recombination, while the second V domain (referred to as “supporting”) has little or no diversity.

The supporting V domain differs between marsupials and monotremes. In marsupials, it is encoded by a germ-line joined, or pre-assembled, V exon that is invariant.22Parra et al. 2007 In the platypus, it is encoded by gene segments requiring somatic DNA recombination, but with limited diversity due in part to the lack of D segments.23Wang et al. 2011

Sharks and other cartilaginous fish also have a T-cell receptor chain that is structurally similar to TCRµ (see middle part of fig. 1).24Criscitiello et al. 2006; Flajnik et al. 2011 The resulting receptor is called NAR-TCR. Like the receptor of marsupials and monotremes, it contains three extracellular domains, but its N-terminal V domain is related to chains used by IgNAR (immunoglobulin new antigen receptor) antibodies, a type of antibody found only in sharks.25Greenberg et al. 1995 In both the TCRµ of marsupials and monotremes and the NAR-TCR of cartilaginous fishes, the current working model is that the N-terminal V domain is unpaired and acts as a single, antigen binding domain. This would be analogous to the V domains of light-chainless antibodies found in sharks and camelids.26Flajnik et al. 2011; Wang et al. 2011

How did the µ chain arise? Phylogenetic analyses support an origin after the avian–mammalian split.27Parra et al. 2007; Wang et al. 2011 Previously, we hypothesized that it originated as a recombination between ancestral immunoglobulin heavy and TCRδ-like loci,28Parra et al. 2008 but this hypothesis is problematic for several reasons. One challenge is the apparent genomic stability and ancient conserved synteny (order of genes on the chromosome) in the region surrounding the TCRα/δ locus; this region has appeared to remain stable over at least the past 350 million years of tetrapod evolution.29Parra et al. 2008, 2010

As a result, we need a new model for the evolution of TCRµ and the TCRα/δ locus. Here we present the best current model, supported by an analysis of the platypus genome—the first to examine a monotreme TCRα/δ locus in detail—as described in the methods and results sections below.

The Model

Our model can be summarized in six stages (fig. 2).[12]Major change from me here. This section was moved here from the discussion, because it is the core and most interesting part of the paper. It is now its own first-level section alongside … Continue reading

Fig. 2. A model of the stages of evolution of the TCRα/δ loci in tetrapods and the origins of TCRµ in mammals. Refer to the text for detailed explanation of stages A-F.
  1. Duplication of the cluster. This occurred early in the evolution of tetrapods, or earlier. The duplication resulted in two copies of the C gene of the δ chain, each with its own set of D and J segments.

  2. Insertion of VH. Recall that VH refers to the variable chain of immunoglobulin heavy (IgH). One or more genes were translocated from the IgH locus and inserted into the TCRα/δ locus, most likely to a location between the existing Vα/δ genes and the 5′-proximal cluster. This is the configuration found today in the zebra finch genome.30Parra et al. 2012

  3. Inversion of the VHδcluster in amphibians. This cluster of genes was translocated and inverted, and the number of VHδ genes increased. The frog X. tropicalis currently has the greatest number of VHδ genes, where they make up the majority of V genes available in the germ-line for T-cell receptor δ chains.31Parra et al. 2010

  4. Translocation of the VHδcluster to another site in galliforms. In chickens and turkeys, the same cluster that was inverted in amphibians instead moved out of the TCRα/δ locus and is now found on another chromosome. There are no or genes at this second TCRδ locus in chickens, and only a single gene remains at the conventional TCRα/δ locus.32Parra et al. 2012

  5. Translocation of the VHδcluster to another site (TCRµ) in mammals. A similar process to step D in galliforms happened in a common ancestor of mammals, giving rise to TCRµ. Internal duplications of the VH, D, and J genes gave rise to the current [(VDJ) − (VDJ) − C] organization that can encode chains with double V domains.33Parra et al. 2007, Wang et al. 2011

  6. Further changes in the three mammalian lineages. 

    • In the platypus, the second VDJ cluster, which encodes the supporting (non-terminal) V chain, lost its D segments and generates V domains with short complementarity-determining region-3 (CDR3) encoded by direct V to J recombination.34Wang et al. 2011

    • Meanwhile, in therians (marsupials and placentals), the VHδ gene disappeared from the TCRα/δ locus (not shown in fig. 2).35Parra et al. 2008

    • Then, in placentals, the TCRµ locus was also lost.36Parra et al. 2008

    • The marsupials kept TCRµ, but the second set of V and J segments (which encode the supporting V domain) was replaced with a germ-line joined V gene (fused yellowgreen segment in fig. 2), probably due to germ-line V(D)J recombination and retro-transposition.37Parra et al. 2007, 2008

    • In both monotremes and marsupials, the whole cluster from VH to C appears to have undergone additional tandem duplication as it exists in multiple copies in the opossum and probably in the platypus.38Parra et al. 2007, 2008; Wang et al. 2011

The rest of the paper explains the analyses that gave us with the evidence to build this model. Additional discussion of the model is provided in the last section.

Materials and Methods

There are three parts to the analyses and experiments that allowed us to gather evidence and build our evolutionary model. First, find the TCRα/δ locus in platypus genome data. Second, perform phylogenetic analyses with the relevant genes. Third, confirm from a live specimen that the platypus expresses VHδ.[13]This new paragraph is important! It gives context to the experiments below and it guides the reader for the entire section. Also notice this is a case of an enumeration without point form. I like … Continue reading

1) Identification and Annotation of the Platypus TCRα/δ Locus

We analyzed the genome of the platypus, Ornithorhynchus anatinus, using the assembly version 5.0.1 (http://www.ncbi.nlm.nih.gov/genome/guide/platypus/). We used two genome alignment tools: whole-genome BLAST from NCBI (www.ncbi.nlm.nih.gov/) and BLAST/BLAT from Ensembl (www.ensembl.org).

We located the V and J gene segments by looking for similarity with the corresponding segments of other species, and by identifying flanking conserved recombination signal sequences. (RSS). We annotated V segments in the 5′ to 3′ direction as either Vα or Vδ, followed by the family number and the gene segment number if there were more than one in the family. For example, Vα15.7 is the seventh Vα gene in family 15.

As for the D segments, we identified them from cDNA clones using VHδ, using complementarity-determining region-3 (CDR3) sequences that represent the V-D-J junctions.

We labeled the platypus T-cell receptor gene segments according to the IMGT nomenclature (http://www.imgt.org/). We provide the location for the TCRα/δ genes of the platypus genome version 5.0.1 in supplementary table S1, available online.

2) Phylogenetic Analyses

We used BioEdit39Hall 1999 as well as the accessory application ClustalX40Thompson et al. 1997 to align the nucleotide sequences of the V genes regions, from the framework region FR1 to FR3, including the complementarity-determining regions CDR1 and CDR2. We established the codon position of the alignments using amino acid sequences.41Hall 1999 When necessary, we corrected the alignments through visual inspection. We then analyzed them with MEGA Software.42Kumar et al. 2004

We generated phylogenetic trees using two methods: Neighbor Joining (NJ) with uncorrected nucleotide differences (p-distance), and Minimum Evolution distances.

We evaluated support for the generated trees using bootstrap values from 1000 replicates. Supplementary table S2 contains the GenBank accession numbers for the sequences used in tree construction.[14]In the original paper, this section comes after the Confirmation of Expression section below, but in the results section, the phylogenetic results are discussed first. I don’t know if there was … Continue reading

3) Confirmation of Expression of Platypus VHδ

As described with more detail in the Results and Discussion section below, the annotation step allowed us to find an atypical VHδ gene in the platypus genome. To confirm that it was not an artifact of the genome assembly process, we looked at the expression of this gene in a live specimen, a male platypus from the Upper Barnard River in New South Wales, Australia. The platypus was collected under the same permits as in Warren et al. 2008.

We performed reverse transcription PCR (RT-PCR) on the RNA from the spleen of this New South Wales specimen. As a second point of comparison, we also used a previously described platypus spleen cDNA library that was constructed from RNA extracted from a Tasmanian animal.43Vernersson et al. 2002 The protocols and products used at every step are as follows:

  • cDNA synthesis: Invitrogen Superscript III-first strand synthesis kit, using the manufacturer’s recommended protocol44Invitrogen, Carlsbad, CA, USA
  • PCR amplification: we used the QIAGEN HotStar HiFidelity Polymerase Kit45BD Biosciences, CLONTECH Laboratories, Palo Alto, CA, USA in total volume of 20 µl containing:
    • 1× Hotstar Hifi PCR Buffer (containing 0.3 mM dNTPs)
    • 1µM of primers: we identified these from the platypus genome assembly step.46Warren et al. 2008 We targeted T-cell receptor δ transcripts with two primers, one for VHδ and one for Cδ:
        • 5′-GTACCGCCAACCACCAGGGAAAG-3′ for VHδ
        • 5′-CAGTTCACTGCTCCATCGCTTTCA-3′ for Cδ
    • 1.25U Hotstar Hifidelity DNA polymerase
  • PCR product cloning: TopoTA cloning® kit 47Invitrogen
  • Sequencing: BigDye terminator cycle sequencing kit version 348Applied Biosystems, Foster City, CA, USA according to the manufacturer recommendations.
  • Analysis of sequencing reactions: ABI Prism 3100 DNA automated sequences.49PerkinElmer Life and Analytical Sciences, Wellesley, MA, USA
  • Chromatogram analysis: Sequencher 4.9 software50Gene Codes Corporation, Ann Arbor, MI, USA

We archived the sequence on GenBank under the accession numbers JQ664690–JQ664710.

Results and Discussion

Results of the TCRα/δ Locus Identification in the Platypus

Here are the results of our analysis of the platypus genome from part 1 of the Materials and Methods section, which allowed us to identify the TCRα/δ locus and annotate its V, D, J and C gene segments, as well as the exons. Refer to fig. 3 below for the annotation map.

Fig. 3. Annotated map of the platypus TCRα/δ locus, showing the locations of the Vα and Vδ (red), VHδ (yellow), Dδ (orange), Jα and Jδ (green), Cδ (dark blue), and Cα (light blue). Conserved syntenic genes are in gray. The scaffold and contig numbers are indicated.

Most of the locus is present on a single scaffold. The remainder is on a shorter contig. On either sides of the locus, we find the genes SALL2, DAD1, and several olfactory receptor genes (OR). All of these genes share conserved synteny with the TCRα/δ locus in amphibians, birds, and mammals.51Parra et al. 2008, 2010, 2012

The platypus locus has many typical features common to TCRα/δ loci in other tetrapods.52Satyanarayana et al. 1988; Wang et al. 1994; Parra et al. 2008, 2010, 2012 Two C region genes are present: a Cα (light blue in fig. 3) at the 3′ end of the locus, and a Cδ (dark blue) oriented 5′ of the Jα genes. These Jα genes occur in a large number (32) of fragments (in green) located between Cδ and Cα. A large array of Jα genes like this is believed to facilitate secondary Vα to Jα rearrangements in developing αβT cells if the primary rearrangements are nonproductive or need replacement.53Hawwari and Krangel 2007 Primary TCRα V–J rearrangements generally use Jα segments towards the 5′-end of the array and can progressively use downstream Jα in subsequent rearrangements. There is also a single Vδ gene (the last red segment in fig. 3) in reverse transcriptional orientation between the platypus Cδ gene and the Jα array that is conserved in mammalian TCRα/δ both in location and orientation.54Parra et al. 2008

There are 99 conventional T-cell receptor V gene segments in the platypus TCRα/δ locus (red in fig. 3). The vast majority, 89, share nucleotide identity with Vα in other species; the other 10 share identity with Vδ genes. The Vδ genes are clustered towards the 3′-end of the locus. Based on nucleotide identity shared among the platypus V genes, they can be classified into 17 different Vα families and two different Vδ families, based on the criteria of a V family sharing >80% nucleotide identity (the family and segment numbers are annotated in fig. 3). This is a typical level of complexity for mammalian Vα and Vδ genes.55Giudicelli et al. 2005; Parra et al. 2008

Also present were two Dδ (orange) and seven Jδ (green) gene segments oriented upstream of the Cδ. All gene segments were flanked by canonical recombination signal sequences (RSS), which are the recognition substrate of the RAG recombinase. The D segments were asymmetrically flanked by an RSS containing at 12 bp spacer on the 5′-side and 23 bp spacer on the 3′-side, as has been shown previously for T-cell receptor D gene segments in other species.56Carroll et al. 1993; Parra et al. 2007, 2010 In summary, the overall content and organization of the platypus TCRα/δ locus appeared fairly generic, with one exception.

This atypical feature of the platypus locus is an additional V gene that shares greater identity to antibody VH genes than to T-cell receptor V genes. Among V genes, this segment is the closest to the D and J genes (see the yellow segment in fig. 3). We tentatively designated it as VHδ. 

VHδ Phylogenetics

VHδ genes are, by definition, V genes that are indistinguishable from immunoglobulin heavy V (Ig VH) genes, but used in encoding T-cell receptor δ chains. Recall from the introduction[15]Yes, you are allowed to make links between the sections of your paper like this! that they have previously been found only in the genomes of birds and frogs.57Parra et al. 2008, 2010, 2012

To put the platypus VHδ gene in context, let us examine the phylogeny of VH genes. In mammals and other tetrapods, VH genes have been shown to cluster into three ancient clans (shown in fig. 4). Individual species differ in the presence of one or more of these clans in their germ-line immunoglobulin heavy locus.58Tutter and Riblet 1989; Ota and Nei 1994 For example, humans, mice, echidnas, and frogs have VH genes from all three clans,59Schwager et al. 1989; Ota and Nei 1994; Belov and Hellman 2003 whereas rabbits, opossums, and chickens have only a single clan.60McCormack et al. 1991; Butler 1997; Johansson et al. 2002; Baker et al. 2005

Fig. 4. Phylogenetic tree of mammalian VH genes, including the platypus VHδ and monotreme Vµ. The three major VH clans are bracketed. A box indicates the platypus VHδ, and bolding indicates the clade containing platypus VHδ along with platypus and echidna Vµ within clan III. The three-digit numbers following the VH gene labels are the last three digits of the GenBank accession number referenced in supplementary table S2. The numbers following the platypus and echidna Vµ labels are clone numbers. The tree shown here was generated using the Minimum Evolution method; the Neighbor Joining method yielded a similar topology.

Our phylogenetic analyses showed that the platypus VHδ was most related to the platypus Vµ genes found at the TCRµ locus (see the boxed and bolded parts of fig. 4). Platypus VHδ, however, shares only 51–61% nucleotide identity (average 56.6%) with the platypus Vµ genes. Both the platypus Vµ and VHδ clustered within clan III.61Wang et al. 2011 This is noteworthy since VH genes in the platypus IgH locus are also from clan III and, in general, clan III is the most ubiquitous and conserved lineage of VH.62Johansson et al. 2002; Tutter and Riblet 1989 Although clearly related to platypus VH, the VHδ gene shares only 34–65% nucleotide identity (average 56.9%) with the “authentic”[16]I’m not sure about this but the original phrase was bona fide, which I had to look up. Maybe “authentic” between quotes isn’t the best translation, but a translation is better … Continue reading VH used in antibody heavy chains in this species.

Results of the Confirmation of VHδ Expression

It was necessary to rule out that the VHδ gene present in the platypus TCRα/δ locus was not an artifact of the genome assembly process. This is why we performed a “wet lab” verification step on cDNA synthesized from the splenic RNA of two platypuses, one from New South Wales and one from Tasmania (see Materials and Methods). We performed RT-PCR with primers that were specific for VHδ and Cδ. We were successful in amplifying the PCR products of the NSW specimen, but not for the Tasmanian one.

One piece of supporting evidence for the expression of VHδ would be the demonstration that it is recombined to downstream Dδ and Jδ segments, and expressed with Cδ in complete T-cell receptor δ transcripts. This is what we found from the twenty sequenced clones we obtained from PCR in the New South Wales platypus. Each clone contained a unique nucleotide sequence that comprised the VHδ gene recombined to the Dδ and Jδ gene segments (see fig. 4A). Of these 20, 11 had unique V, D, and J combinations that would therefore encode 11 different complementarity-determining regions-3 (CDR3; see fig. 4B). More then half of these (8 out of 11) contained evidence of using both D genes, giving a VDDJ pattern. This is a common feature of δ V domains where multiple D genes can be incorporated into the recombination due to the presence of asymmetrical RSS.63Carroll et al. 1993

Fig. 4. (A) Alignment of predicted protein sequence of transcripts containing a recombined VHδ gene isolated from platypus spleen RNA. The individual clones are identified by the last three digits of their GenBank accession numbers (JQ664690–JQ664710). Shown is the region from FR3 of the VHδ through the beginning of the Cδ domain. The sequence in bold at the top of the alignment is the germ-line VHδ and Cδ gene sequence. The double cysteines at the end of FR3 and unpaired cysteines in CDR3 are shaded, as is the canonical FGXG in FR4. (B) Nucleotide sequence of the CDR3 region of the eleven unique V(D)J recombinants using VHδ described in the text. The germ-line sequence of the 3′-end of VHδ, the two Dδ, are shown at the top. The germ-line Jδ sequences are shown on the right-hand side of the alignment interspersed amongst the cDNA sequences using each. Nucleotides in the junctions between the V, D, and J segments, shown italicized, are most likely N-nucleotides added by TdT.

The region corresponding to the junctions between the V, D, and J segments contained an additional sequence that could not be accounted for by the germ-line gene segments (fig. 4B). There are two possible sources of such a sequence. One is palindromic nucleotides that are created during V(D)J recombination when the RAG generates hairpin structures that are resolved asymmetrically during the re-ligation process.64Lewis 1994 The second is non-templated nucleotides that can be added by the enzyme terminal deoxynucleotidyl transferase (TdT) during the V(D)J recombination process.

An unusual feature of the platypus VHδ is the presence of a second cysteine encoded near the 3′-end of the gene, directly next to the cysteine predicted to form the intra-domain disulfide bond in immunoglobulin domains (fig. 4A). Additional cysteines in the complementarity-determining region 3 of VH domains have been thought to provide stability to unusually long CDR3 loops, as has been described for cattle and the platypus previously.65Johansson et al. 2002 The CDR3 of T-cell receptor δ using VHδ are only slightly longer than conventional δ chains (ranging 10–20 residues).66Rock et al. 1994; Wang et al. 2011 Furthermore, the stabilization of CDR3 generally involves multiple pairs of cysteines, which were not present in the platypus VHδ clones (fig. 4A). 

The Tasmanian specimen

The above concerns the animal collected from New South Wales. With the Tasmanian specimen, we were unable to amplify T-cell receptor δ transcripts containing VHδ from its splenic cDNA. We did, however, successfully isolate transcripts containing conventional Vα/δ segments, which provides a positive control.

It is possible that Tasmanian platypuses, which have been separated from the mainland population at least 14,000 years ago, either have a divergent VHδ or have deleted this single V gene altogether.67Lambeck and Chappell 2001

Sequence variation in VHδ

Although there is only a single VHδ in the current platypus genome assembly, there was sequence variation in the region corresponding to FR1 through FR3 of the V domains (see fig. 4A; the sequence data are not shown here, but are available in GenBank). We have three potential explanations for this variation:

  1. Two alleles of a single VHδ gene
  2. Somatic mutation of expressed VHδ genes
  3. Allelic variation in gene copy number

The two-allele explanation makes sense given that the RNA used in this experiment is from a wild-caught individual from the same population that was used to generate the whole-genome sequence, which was found to contain substantial heterozygosity.68Warren et al. 2008 However, the variation was too large to be fully explained by this. 

The second possibility, somatic mutation (i.e. mutation not occurring in germ cells), is considered controversial for T-cell receptor chains. Nonetheless, it has been invoked in sharks and postulated in salmonids to explain the variation that exceeds the apparent gene copy number in these vertebrates.69Yazawa et al. 2008; Chen et al. 2009 Therefore, it seems possible[17]I kind of like the original phrasing “it does not seem to be out of the realm of possibility” but that could be easily simplified, so I did. that somatic mutation is occurring in platypus VHδ. One piece of evidence in favor of this is that the mutations appear to be localized to the V region with no variation in the C region (fig. 4A). This may be due to the relatedness between VHδ and immunoglobulin VH genes where somatic hyper-mutation is well documented. Somatic mutation in immunoglobulin VH contributes to overall affinity maturation in secondary antibody responses.70Wysocki et al. 1986 However, this means that the evidence is mixed: the pattern of mutation seen in the platypus is found in the complementarity-determining region 3, which would be indicative of selection for affinity maturation, but was also found in the framework regions, which does not indicate this. As further evidence against the somatic mutation explanation, there is no evidence of somatic mutation in the V regions of birds, which also have only a single VHδ.71Parra et al. 2012 The contribution of mutation to the platypus TCRδ repertoire, if it is occurring, remains to be determined.

Alternatively, the sequence polymorphism may be due to VHδ gene copy number variation between individual TCRα/δ alleles.

Irrespective of the number of VHδ genes in the platypus TCRα/δ locus, the results clearly support T-cell receptor δ transcripts containing VHδ recombined to Dδ and Jδ gene segments in the TCRα/δ locus (fig. 4). A VHδ gene or genes in the platypus TCRα/δ locus in the genome assembly, therefore, does not appear to be an assembly artifact. Rather, it is present and functional, and contributes to the expressed T-cell receptor δ chain repertoire. The possibility that some platypus TCRα/δ loci contain more than a single VHδ does not alter the principal conclusions of this study.

Discussion of our Model of the Evolution of TCRα/δ and TCRµ

The results above make up the evidence that allowed us to construct the model shown after the introduction section (see fig. 2). Here we discuss various considerations about the model.

Previous hypothesis of the origin of TCRµ in mammals

Our previous hypothesis72Parra et al. 2008 about the origin of T-cell receptor µ (TCRµ) in mammals involved the recombination between an ancestral TCRα/δ locus and an immunoglobulin heavy (IgH) locus. The IgH locus would have contributed the V gene segments at the 5′-end, while the T-cell receptor δ would have contributed the D, J, and C genes at the 3′-end of the locus.

The difficulty with this hypothesis was the clear stability of the genome region surrounding the TCRα/δ locus. In other words, the chromosomal region containing the TCRα/δ locus appears to have remained relatively undisrupted for at least the past 360 million years.73Parra et al. 2008, 2010, 2012

VHδ in different vertebrate lineages

An alternative model for the origins of TCRµ emerged from the discovery, in amphibians and birds, of VHδ genes inserted into the TCRα/δ locus. This model involves the insertion of VH (fig. 2B) followed by the duplication and translocation of T-cell receptor genes (fig. 2C-E).

The insertion in the TCRα/δ locus seems to occur without disrupting the local syntenic region, as we know from zebra finches and frogs. In frogs, the IgH and TCRα/δ loci are tightly linked, which may have facilitated the translocation of VH genes into the TCRα/δ locus.74Parra et al. 2010

But close linkage is not a requirement. The genomes of birds and platypuses do not show such linkage, and the translocation of VH genes to the TCRα/δ locus appears to have occurred independently from frogs in these two lineages. We know this from the lack of similarity and relatedness between the VHδ genes of frogs, birds, and monotremes.75Parra et al. 2012 As can be seen in the phylogenetic tree of fig. 4, they appear derived each from different, ancient VH clans:

  • Clan I for birds
  • Clan II for frogs
  • Clan III for platypuses

Therefore, we suggest that the transfer of VHδ occurred independently in the different lineages. Another possibility is that transfers of VHδ may have occurred frequently and repeatedly in the past. Gene replacement may be the best explanation for the current content of these genes in the different tetrapod lineages.

The new evidence of platypus VHδ from this study allows us to update the model.

Updating the model for mammalian TCRµ

Let us contrast the evidence from marsupials with the evidence we have gathered from the platypus. In marsupials, there is no VHδ; the Vµ genes are highly divergent; and at least in the opossum, there is no conserved synteny with genes linked to TCRµ. These facts provide little insight into the origins of T-cell receptor µ and its relationship to other T-cell receptor chains like δ or the conventional ones.76Parra et al. 2008

In the platypus genome, however, we notice a striking similarity between VH, VHδ, and Vµ. These genes are all in clan III. In particular, the close relationship between the platypus VHδ and Vµ genes lends greater support for the model presented in fig. 2E, with TCRµ having been derived from TCRδ genes.

The similarity that we found here between the platypus VHδ and V genes in the TCRµ locus is, so far, the clearest evolutionary association between the µ and δ loci in one species.

Evolution of chains with three extracellular domains

TCRµ is an example of a T-cell receptor form with three extracellular domains (refer back to fig. 1). These forms have evolved at least twice in vertebrates. The first was in the ancestors of the cartilaginous fish in the form of NAR-TCR.77Criscitiello et al. 2006 The second was in the mammals as TCRµ.78Parra et al. 2007

As we discussed in the introduction, NAR-TCR uses an N-terminal V domain that is related to the V domains found in IgNAR antibodies, which are unique to cartilaginous fish,79Greenberg et al. 1995; Criscitiello et al. 2006 and not closely related to antibody VH domains. Therefore, it appears that NAR-TCR and TCRµ are more likely the result of convergent evolution rather than being related by direct descent.80Parra et al. 2007; Wang et al. 2011

Evolution of chains with antibody-like V domains

T-cell receptor chains that use antibody-like V domains, such as TCRδ using VHδ, NAR-TCR, or TCRµ (i.e. the receptors with yellow ovals in fig. 1) are widely distributed in vertebrates. Only the bony fish and placental mammals lack them.

In addition to NAR-TCR, some shark species appear to generate T-cell receptor chains using antibody V genes. This occurs via trans-locus V(D)J recombination between immunoglobulin IgM and IgW heavy chain V genes and TCRδ and TCRα D and J genes.81Criscitiello et al. 2010 This may be possible, in part, due to the multiple clusters of immunoglobulin genes found in the cartilaginous fish. It also illustrates that there have been independent solutions to generating T-cell receptor chains with antibody V domains in different vertebrate lineages.

In the tetrapods, the VH genes were trans-located into the T-cell receptor loci where they became part of the germ-line repertoire. By comparison, in cartilaginous fish, something equivalent may occur somatically during V(D)J recombination in developing T cells. Either mechanism suggests there has been selection for having T-cell receptors using antibody V genes over much of vertebrate evolutionary history.

What function do the antibody V chains serve? The current working hypothesis is that they are able to bind native antigen directly. This is consistent with a selective pressure for T-cell receptor chains that may bind or recognize antigen in ways similar to antibodies in many different lineages of vertebrates.

In the case of NAR-TCR and TCRµ, the N-terminal V domain (the “third” one) is likely to be unpaired and bind antigen as a single domain (see fig. 1), as has been described for IgNAR and some IgG antibodies in camels (recently reviewed in Flajnik et al. 2011). This model of antigen binding is consistent with the evidence that the N-terminal V domains in TCRµ are somatically diverse, while the second, supporting V domains have limited diversity and presumably perform a structural role rather than one of antigen recognition.82Parra et al. 2007; Wang et al. 2011

There is no evidence of double V domains in TCRδ chains using VHδ in frogs, birds, or platypus (rightmost part of fig. 1).83Parra et al. 2010, 2012 Rather, the complex containing VHδ would likely be structured similar to a conventional γδ receptors with a single V domain on each chain. It is possible that such receptors also bind antigen directly, but this remains to be determined.

A compelling model for the evolution of the immunoglobulin and T-cell receptor loci has been one of internal duplication, divergence and deletion. This is the so-called birth-and-death model of evolution of immune genes and was promoted by Nei and colleagues.84Ota and Nei 1994; Nei et al. 1997 Our results do not contradict that the birth-and-death mode of gene evolution has played a significant role in shaping these complex loci. However, our results do support the role of horizontal transfer of gene segments between the loci that had not been previously appreciated. With this mechanism, T cells may have been able to acquire the ability to recognize native, rather than processed antigen, much like B cells.

Notes

Notes
1 This is an example comment.
2 Major changes to the abstract: I split it in four paragraphs with section titles (this is common in some journals; it should be common in most journals). I also added a section at the beginning to state the main point of the paper. Scientists have the bad habit of starting with background information before we even know why we’re supposed to care. This fixes that.

The abstract is longer now, but not terribly so (286 vs. 267 words), so I think it’s fine; it could be shortened some more, but that would be a question of picking what to remove, which I’m less confident to do as I’m not the author.

Note that I rewrote the abstract after rewriting the rest.

3 Since this abbreviation comes up a lot, I put it first, with its meaning in parentheses.
4 I didn’t change much in the figure’s caption, but it seemed pretty trivial to add color to the text to facilitate looking up what the colors mean.
5 This is an example of two sentences taken verbatim from the original paper. Not all of it was poorly written!
6 It took me forever to rewrite this part. The original sentence was, “Diversity in antibodies produced by B cells is also generated by RAG-mediated V(D)J recombination and the TCR and Ig genes clearly share a common origin in the jawed-vertebrates.” Soooo many things wrong here.

First, the weirdly formatted term “V(D)J” was not defined anywhere. I assume it means “V, J, and optionally D,” but it’s not as obvious as the authors seem to think.

Second, why are we talking about B cells? They don’t come up anywhere else except in the very last sentence of the paper. We’ve been talking about T cells; if you’re going to switch to a different but similarly named type of cell, then you should tell the reader explicitly.

Third, this is two different ideas linked together with an “and”. I have no clue why it was written as a single sentence, except maybe for the bad reason of having the citations refer to both ideas. They’re so different that it made sense to split them into not only distinct sentences or paragraphs, but actual sections!

7 The original combined this sentence and the next, even though they’re about quite distinct ideas: the name of the genes, and the species where they’re found.
8 Why not indicate the part of the figure that is relevant? Whenever you can, provide reader guidance!
9 The authors like to use “duckbill platypus,” but there’s only one species of platypus, so I took that word out.
10 Another horrible sentence from the original, recorded for posterity: “Trans-locus V(D)J recombination of V genes from other Ig and TCR loci with TCRµ genes has not been found.” That distance between the subject (recombination) and the verb (has). Ugh.
11 The abbreviation IgSF was used in the paper, with no explanation. I assume the people who would read this paper tend to know what that means, but still.
12 Major change from me here. This section was moved here from the discussion, because it is the core and most interesting part of the paper. It is now its own first-level section alongside Introduction, Materials and Methods, etc.

I also simplified the contents. The six stages used to be identified with letters A-F in the figure, and 1-6 in the text. I changed that to use letters everywhere. I removed most of the figure’s caption since it repeats the text.

There was an inconsistency in calling the same thing the Dδ–Jδ–Cδ cluster in the figure and D–J–Cδ cluster in the text. I fixed that. I also color-coded the elements in the text according to the figure.

One thing I didn’t like about the original figure is that the six stages aren’t sequential. The figure presented steps A to F as if they followed one another, but steps C, D and E-F describe the evolution in different animal lineages. So I reorganized the contents and added some arrows for clarification. It also seems that the steps 5-6 in the text and E-F in the figure didn’t quite match, with some parts illustrated in step F being explained in step 5; I edited the text so that they do match. I think the figure could be improved much more, notably by splitting the complex F stage in multiple steps, but I don’t want to change it too much.

13 This new paragraph is important! It gives context to the experiments below and it guides the reader for the entire section. Also notice this is a case of an enumeration without point form. I like point form, but it must not be overused.
14 In the original paper, this section comes after the Confirmation of Expression section below, but in the results section, the phylogenetic results are discussed first. I don’t know if there was a reason for this (maybe they performed the phylogenetic analysis later) but it seems better to keep the same order in both sections, which is why I’m placing this part here.
15 Yes, you are allowed to make links between the sections of your paper like this!
16 I’m not sure about this but the original phrase was bona fide, which I had to look up. Maybe “authentic” between quotes isn’t the best translation, but a translation is better than a Latin phrase that many people will not get.
17 I kind of like the original phrasing “it does not seem to be out of the realm of possibility” but that could be easily simplified, so I did.
Categories
guidelines

Science Style Guide: Links

This post is part of my ongoing scientific style guideline series.

Go to Wikipedia and start reading an article on some topic you don’t know much about. For example, the umami taste.

Chances are that by the end of the first few paragraph, you will have clicked on several links, either because they referred to terms you didn’t know (glutamateinosine monophosphate), or because you were curious (what does Wikipedia have to say about the five basic tastes?). Now these links might be open in new tabs for you to check later. Or maybe you’ve already given up reading the original umami article, and are now exploring some new rabbit hole (e.g. the Scoville scale of spiciness).

Wikipedia is a great resource for many reasons, but one of them is this constant hyperlinking to other relevant Wikipedia articles. This has a major advantage: it allows the reader to create their own reading experience. Advanced readers on some topic can keep reading without having to go through the basics they already know. Beginner readers can look up technical terms easily. Wikipedia is a choose-your-own-adventure book, where you can wander according to your own character level.

Links are even an answer to some degree to the four-way tradeoff I wrote about here. It’s difficult to write something that is clear, brief, complex, and information-rich. But Wikipedia articles come closer to the golden middle, and that’s thanks to links. With no need to explain every difficult term directly in the text, articles can be more brief without sacrificing clarity. By packaging complex information in other articles, and showing only the link, Wikipedia articles can contain more complexity and richness of information.

(The reason it doesn’t falsify my four-way tradeoff theory is that Wikipedia as a whole cannot be called succinct. To understand a topic well, you still have to read a lot of articles. But Wikipedia packages this information in relatively brief articles. In other words, information architecture is a pretty good solution to the tradeoff.)

Links make matters easier to readers, but they also help writers. You don’t have to do as much guessing about what your audience knows; your audience will decide for themselves. And you can just reuse existing information written by others.

This suggests that our two principles are satisfied:

  • Minimum reading friction: links give agency to readers. They make it easier to look up complex terms (which readers will tend to google anyway).
  • Low-hanging fruit: adding links to existing public resources like Wikipedia, other encyclopedias, or open-access papers, is an easy thing to do for a writer.

Links in scientific papers

Given all of the above, we’d expect scientific papers — which are almost always at the frontier of the tradeoff, trying to cram a lot of complex information within a word limit without being too difficult to read — to use hyperlinks heavily. Right?

Nope. They rarely do. At most they include citations that link to the reference section, which may include a link to the original paper, which may or may not be openly accessible to you, and which may or may not be a 10-page difficult read in which the explanation you seek is buried in page three of the discussion with no hint to tell you where to look. And they definitely never include links to Wikipedia or anything like it.

Why is that? I’m guessing part of the explanation is the high importance of those citations. It is considered vital to put your work in relation to existing literature, so scientists have an incentive to reference as many relevant papers as they can, and no incentive to link to anything else. The respectability of the sources comes into play; Wikipedia isn’t a reputable source (it can be edited by anyone! It’s not peer-reviewed!!), nor are a lot of the other websites you could link to. So they tend to be avoided.

Then there’s the requirements of proper information management. References must be written in standard format. So if for some reason you do need to link to a website, then you’ll have to use a format like:

“Questions and Answers on Monosodium glutamate (MSG)”. Silver Spring, Maryland: United States Food and Drug Administration. 19 November 2012. Retrieved 19 February 2017.

There are good reasons to this formalism. But it also means that adding any link to a paper requires some work. As a result, I suspect it leads to less hyperlinking in science papers than would be useful to readers.

If you’ve ever read a scientific paper, it’s likely that you have googled complicated terms and looked up Wikipedia articles to help you. Scientists shouldn’t pretend that this isn’t happening. They should not hesitate to add link to resources like Wikipedia, blogs, Twitter threads, and other papers, in order to guide readers and reduce friction.

Drawbacks

There are a few drawbacks to hyperlinked text. None of them invalidate the idea that links should be used more, but we should keep them in mind.

One drawback is that links can be distracting. A barrage of links in a paragraph might be somewhat annoying to read. (Although links also have the benefit of providing novelty to text — even a simple thing like the color of a hyperlink can be useful to make a piece of writing less boring.) And having to open links may drive readers away from the original paper, and require some more effort on their part as opposed to a piece written in a way that beginners don’t need to look up extra information.

Another major drawback is link rot. A webpage may stop existing at any moment, and then your link becomes useless. Also, Wikipedia articles can change and stop fulfilling their original purpose. (Although in practice Wikipedia contributors are mindful of that and use redirections a lot.) One way to circumvent this is to link to archived pages, such as the Wayback Machine.

And of course, links don’t work offline. But my contention is that fewer and fewer people are reading papers in print or without internet access. We shouldn’t make it impossible for these modes of reading to happen, but it’s time we make full use of the web’s possibilities to improve science publishing.

Recommendations

  • Do not hesitate to add links to various resources, including encyclopedias, your own content (whether formally published or not), etc.
  • Try to find the correct balance between too few and too many links.
    • Your paper should be readable without clicking any links, so do explain the crucial parts directly in the text.
    • Too many links can be distracting, so choose carefully when to add one.
  • Link to archived webpages when possible.
  • Links shouldn’t replace formal citations, but it’s good practice to pair citations with direct links to make it easier to look up the reference.
Categories
guidelines

Science Style Guide: Bullet Points

This post is part of my ongoing scientific style guideline series.

Writing with bullet points (or bullet numbers, letters etc.) has several advantages:

  • It provides clear guidance to readers.
  • It forces the writer to think about the structure of what they’re trying to say.
  • It comes with built-in line breaks, which tends to create shorter, more readable paragraphs.
  • It breaks the flow of normal prose, which makes reading less monotonous.
  • It is another channel to communicate emphasis (in addition to italics, bold, caps, subheadings, etc.).

Not everything in a piece of writing should follow point form format. Regular prose, organized in paragraphs, is better for most things. But when you are trying to express something that’s highly structured, like a list of steps (in a recipe, or in an experimental protocol), then not using bulleted lists can work against you.

Science papers being a weirdly conservative genre, bulleted lists are somewhat uncommon in them. Papers will quite often use quasi-point form formats, like (1) having numbers or letters in the middle of a paragraph, like this; (2) using “first,” “second,” “third”; or (3) separating ideas with quasi-titles written in italics or bold, but without a line break.

You see this a lot in figures. Many scientific figures are complex and contain multiple parts. Each part is identified with a letter, as in the following example from the platypus paper:

And the caption will have long explanations separated by very easy to miss letters, like this. (A) The first part of the figure, including some colors and shapes. (B) The second part. Note that this part comes after the first part, and before part C. (C) The third part. I’m running out pointless things to write, but I want this to be a wall of text, so I’m gonna keep writing. (D) Have you lost attention yet? (E) That’s a lot of parts, isn’t it? Funny thing, there’s a limitation of how I do image captions that wouldn’t even let me use bullet points even if I wanted to. (F) At last, the final part. So much information needed to make sense of this picture, right? It’s good to be exhaustive, but there’s no point in making it difficult for readers.

A lot of these habits come, I assume, from the fact that journals used to be available only in print. Space was very limited, and there’s often a lot of scientific information to display, so you’re not going to waste any with bullet points.

Today we don’t have these limitations. Using bullet points where appropriate is nice to your readers, so use them. They’re an easy way to reduce reading friction.

They’re also clearly a Low-Hanging Fruit. Similar to breaking paragraphs, it takes very little work to turn a piece of text into a list, if it’s already presenting the information in something close to a list. (If it isn’t, then bullet points probably won’t work well anyway.) It’s also one of those interventions that can be done almost mechanistically.

Recommendations

  • Use bullet points liberally when it is appropriate, e.g. for:
    • Steps in a process, experiment, protocol, etc.
    • Lists of materials used, substances, etc.
    • Enumerations (e.g. “the five characteristics of X are : …”)
  • Nested bullet points (just like the above) can be useful, but don’t overuse them.
    • At more than two levels, the information structure is probably too complex for the bullet points to improve readability.
  • Pick bullet points instead of numbers/letters when the order does not matter. Pick numbers (for simplicity, prefer Arabic numerals, but Roman numerals can work) or letters when the order does matter (e.g. for steps in a protocol).
  • Bullet points are useful to break the monotony of reading paragraphs, but when there’s too much point form, the reverse becomes true. Use bulleted lists less than normal prose.
Categories
guidelines

Science Style Guide: Giving Examples

This post is part of my ongoing scientific style guideline series.

Imagine you’re writing a science paper. The journal you’re going to submit it to specifies a word limit: 5,000 max. You open the stats in your finished draft — 5,523 words. You’re going to have to cut.

Problem is: everything you wrote is important! You can’t take out anything from the Methods or Results sections: that would make the study weaker and less likely to be accepted for publication. You can’t take out any of the background information in the introduction: you already included the bare minimum for readers to understand the rest.

Although, on closer inspection, perhaps not quite the bare minimum. You reread this sentence:

Most models of trait evolution are based on Brownian motion, which assumes that a trait (say, beak size in some group of bird species) changes randomly, with some species evolving a larger beak, some a smaller one, etc.

What if you removed the parts that talk about beak size? That’s not strictly necessary.

Most models of trait evolution are based on Brownian motion, which assumes that a trait changes randomly.

There we go. More concise, more to the point, and most importantly, you shaved off 23 words from that word count. Of course, the sentence is less illustrative, but whatever: your readers are smart, they’ll be able to figure out an example on their own. Right?

Wrong.

Well, okay, not quite wrong, your readers probably are smart. But this goes against the Minimum Reading Friction principle. The point of most writing, including science papers, is to do the work so that readers don’t need to. If readers need to think of an illustrative example themselves to fully understand your abstract idea, then you’re asking a big effort of them.

Picking good, concrete, relevant examples is a lot of work, whether as a reader or writer.1Here’s an aside that’s not directly related to science but, instead, to computer programming.

When coding, you’ll usually refer a lot to developer documentation about whatever preexisting code you’re using. E.g. you want to convert a date to a different format, so you look up the docs for the function
convertFormat(someDate) -> convertedDate. The docs will describe how the function works, what its input (someDate) and outputs (convertedDate) exactly are, and so on — but very often they will not include an example of using convertFormat() in code. If there is an example, it’s often trivial and not very helpful. When I worked as a programmer, I was commonly frustrated by the lack of examples, both because I wanted to figure out quickly how to use a complicated function, and because I wanted to know about any usage conventions.

I suspect that writing documentation would be a lot more work if it included clear and relevant examples everywhere, which is probably why it’s rarely done.
I realize this constantly when I write. It’s very tempting to just state an abstract idea and not bother finding a good example to illustrate it. After all, the abstract idea is more general and therefore more valuable — provided that your readers understand it.

I struggled with example-finding in this very essay. It took me a while to think of the opening about cutting examples to respect a word count limit. And I’m not even that happy with this example. For one thing, it’s not very concrete. For another, it’s not even the most common reason for lack of examples: usually, we don’t cut them out, we simply fail to come up with them in the first place.

And so, unfortunately, this piece of guidance is less of a Low-Hanging Fruit than others: adding good examples is a skill that takes some practice. At the very least, it’s not difficult from the point of view of structure, since it doesn’t require you to rethink your argument — you usually just need to add a sentence or two.

Here are a few other minor points:

Where should examples be placed relative to the main idea?

It’s most intuitive to place an example right after the idea it supports, and that’s probably fine most of the time. But there are benefits to placing an example first.

Consider:

Left-handedness seems to be somewhat correlated with extraordinary success, including political success. For example, despite a base rate of about 10% left-handedness in the general population, four of the seven last United States presidents — Barack Obama, Bill Clinton, George H.W. Bush, and Ronald Reagan — were left-handed.

vs.

What do US presidents Barack Obama, Bill Clinton, George H.W. Bush, and Ronald Reagan have in common? They were all left-handed. In other words, four of the last seven presidents were left-handed, compared to a base rate in the general population of about 10%. This suggests that left-handedness is correlated with extraordinary success, at least in politics.

I find the second version more engaging. You see an interesting fact, you’re drawn in, and then the writer tells you the more general point when you’re most receptive.

Journalists do this a lot. They opens with a story, and then proceed to make their point.

What types of scientific writing does this apply to?

Anything that deals mostly with abstract ideas. Highly concrete writing, such as the sections describing the methods or results of a study, aren’t concerned. Thus, in a typical experiment paper, this advice is mostly relevant to the discussion section and some of the introductory background.

Authors of literature reviews may need to be more careful. These papers integrate a lot of ideas from reviewed studies; it can be tempting to skip examples in order to include more content in less space. The paragraph I worked on here was from a literature review.

What about word limits, though?

Sometimes you really are constrained by externally imposed word limits, and sometimes the examples really are the the least problematic thing to take out. In those cases, well, do what you have to do.

In JAWWS, I don’t want to be strict about word limits. They often force writers to sacrifice clarity to satisfy other components of the four-way tradeoff. They’re also not as relevant in an age where papers are rarely printed on, well, paper. On the other hand, I imagine that many other publications first think that and then have to implement limits to avoid very long submissions. I wonder if the solution could be to make concrete examples not count, provided it’s not too difficult to identify them.

Recommendations

  • Support each abstract idea with at least one example
    • Complicated abstract idea may benefit from multiple examples
  • Choose concrete, specific examples that can be grasped immediately
  • When possible, put the example before stating the underlying idea

See also

Categories
guidelines

Science Style Guide: Paragraph Length

This post is part of my ongoing scientific style guideline series.

There are famous words from Gary Provost that go like this. Pay attention to the rhythm:

This sentence has five words. Here are five more words. Five-word sentences are fine. But several together become monotonous. Listen to what is happening. The writing is getting boring. The sound of it drones. It’s like a stuck record. The ear demands some variety.

Now listen. I vary the sentence length, and I create music. Music. The writing sings. It has a pleasant rhythm, a lilt, a harmony. I use short sentences. And I use sentences of medium length. And sometimes when I am certain the reader is rested, I will engage him with a sentence of considerable length, a sentence that burns with energy and builds with all the impetus of a crescendo, the roll of the drums, the crash of the cymbals—sounds that say listen to this, it is important.

So write with a combination of short, medium, and long sentences. Create a sound that pleases the reader’s ear. Don’t just write words. Write music.

This is legendary advice for writing sentences. It is delightfully illustrative; we grasp it immediately. And it is correct: diversity in sentence length is a necessity of good writing, just like it is for musical notes.

I claim that the same is true of paragraph length.

Science papers usually feature many long paragraphs. Often, all or almost all paragraphs in a paper are long.

Put negatively, we might call them Walls of Text. This is a good metaphor because Walls of Text, just like regular walls, serve as obstacles. They make information less accessible. How often have you looked at a Wall of Text and simply decided it wasn’t worth the effort?

Walls of Text are bad because:

  1. They make it more difficult for readers to take breaks.
  2. They provide no hints about the structure of the underlying ideas.

We’ll examine both in more detail below. But first I want to tie my ideas about paragraphs with my two major writing style principles.

Minimum Reading Friction: The point of having paragraphs at all, as opposed to perfectly continuous text with no line breaks, is to provide some help for readers. If you don’t do that, you’re essentially telling your readers that they’re on their own. This is the opposite of what we want — the effort should be made once, by the writer, so that the many readers don’t have to.

Low-Hanging Fruit: Cutting up paragraphs is a relatively easy task. If the sentences are structured well already, it’s just a matter of finding the “joints” in the written text where it makes sense to add a line break. If the ideas are structured in a confusing manner, then it’s more work, but there’s also greater room for improvement.

In the interest of not making this post too long, I won’t include a full-fledged example, but this past post in which I rewrote a paragraph (into several ones) is a good illustration.

1. Rewarding the reader with breaks

Humans aren’t computers. We can’t work continuously without resting. Reading science papers text is work, so we’re always on the lookout for opportunities to take breaks — sometimes microbreaks on the order of a few seconds, sometimes longer breaks like a full day.

Paragraphs, like chapters, sections, and sentences, serve the purpose of telling readers, “hey, good job, you read a thing, now you can take a break if you want.” It’s rewarding. It indicates that it’s safe(r) to take a break after a paragraph because it’ll be less work to find a reentry point later, and because you expect the next paragraph to be about a different idea.

I don’t know if it’s a coincidence that the word break is used for both concepts, but if so, it’s a fortuitous one.

Walls of Text are often bad because when they loom ahead, you brace yourself. You wonder if you’ll have the energy and time to read it all. If not, maybe you quit reading (and it’s anybody’s guess whether you’ll come back to it later). If yes, then you come out at the other end with less energy and time, and good luck if the next paragraph is also a Wall of Text. And that’s assuming you do reach the end. It’s quite likely that you quit halfway — because you had to stop to think about something you read, or you needed to look up a word, or you clicked on a link, or some random distraction outside the text grabbed your attention.

At the most extreme, you could imagine an entire book that consists of a single paragraph, with no chapters or line breaks at all.1In fact very old books, from centuries or millennia ago, are often like that, probably because back then paper or parchment were expensive. You wouldn’t want to waste precious space with line breaks. This is really lazy on the part of the writer — the reader has to do all the work!

Now, that’s not to say long paragraphs are always wrong. Sometimes it really does make sense to package a lot of ideas together in a single Wall of Text. Also, long paragraphs can be easy to read if the sentences are good and logically connected. But this also means that if you do choose to write a Wall of Text, then you should be extra careful with how you structure the writing inside it.

2. Providing structure

Speaking of structure: line breaks are one of the most useful tools to communicate structure to readers.

We expect paragraphs to contain a single idea. You may have learned in school that a paragraph should have a “topic sentence” with additional sentences to provide “supporting detail.” This is somewhat too rigid, but the principle is sound.

The worst kinds of Walls of Texts are those that have multiple competing ideas inside them. Find where the boundaries are, and cut them up! The ideas don’t even have to be very different. Suppose you have a transition word like “Similarly” or “Alternatively” in the middle of a paragraph. The next sentence if probably closely related to the previous one, but the transition word does indicate a shift, so it’s a nice spot for adding a line break.

Of course, sometimes you really have a single idea with lots of supporting detail that it makes to sense to break up. This is why Walls of Text are sometimes useful.

In fact, as the Gary Provost quote at the top illustrates for sentences, diversity in paragraph length is a good thing.

Having only very short paragraphs is bad.

Think of low-quality newspaper pieces where there’s a line break after each sentence.

It’s jarring.

This is almost as bad as Walls of Text, from the point of view of structure.

Okay, that was annoying, right? The reason is that sentences already provide structure. So using only single-sentence paragraphs amounts to not using line breaks as an extra channel for reader guidance.

Strive to have a mix of short, medium, and long paragraphs. Heterogeneity is good. It carries more information.

Recommendations

  • If you’re ever debating whether or not to end the paragraph and add a line break, err on the side of “yes”.
    • Verbatim from Slate Star Codex’s Nonfiction Writing Advice, an excellent essay whose section 1 heavily inspired this post.
  • Balance your piece between short, medium, and long paragraphs.
  • Cut up existing Walls of Text by finding the boundaries between different ideas.
  • This advice generalizes to section breaks:
    • Err on the side of more shorter sections rather than few long ones.
    • Split sections that are long and contain many distinct ideas.

 

Categories
original research

An Annotated Reading of a Paper about Platypuses

Here I present a paper I chose to rewrite as a demonstration for the JAWWS project. The original text and figures are reproduced below,1the paper has a Creative Commons non-commercial license interspersed with my comments in the following format:

hello I am a blue comment in a quote-block

Feel free to just read the comments. Annotating the paper was a first step in the process. Next I will focus on the rewriting per se. Should be fun!

I didn’t have a particularly strict selection procedure — I went on ResearchHub, in the evolutionary biology section (since that used to be my field), and picked one that seemed appropriate. A cursory skimming showed it had plenty of abbreviations and long paragraphs, which suggested there was a lot of room for improvement.

Also, it’s about platypuses. Or platypi. Platypodes. Whatever.

Here are the metadata:

  • Title: “A Model for the Evolution of the Mammalian T-cell Receptor α/δ and μ Loci Based on Evidence from the Duckbill Platypus”
  • Authors: Zuly E. Parra, Mette Lillie, Robert D. Miller
  • Journal: Molecular Biology and Evolution
  • Link to original version
  • Word count: 5,800 words.

A disclaimer: some of the comments below will be harsh. Again, I don’t mean to attack the authors, who did their job as well as they could, and in fact succeeded at it — after all, they managed to publish their work!

With that, let’s pretend we’re semi-aquatic platypuses and dive in.

A Model for the Evolution of the Mammalian T-cell Receptor α/δ and μ Loci Based on Evidence from the Duckbill Platypus

Comments: Okay, this paper is going to be about T cells (I vaguely remember this being about immunity?), platypuses, and evolution. Sounds good.

Abstract

The specific recognition of antigen by T cells is critical to the generation of adaptive immune responses in vertebrates. T cells recognize antigen using a somatically diversified T-cell receptor (TCR). All jawed vertebrates use four TCR chains called α, β, γ, and δ, which are expressed as either a αβ or γδ heterodimer. Nonplacental mammals (monotremes and marsupials) are unusual in that their genomes encode a fifth TCR chain, called TCRµ, whose function is not known but is also somatically diversified like the conventional chains. The origins of TCRµ are also unclear, although it appears distantly related to TCRδ. Recent analysis of avian and amphibian genomes has provided insight into a model for understanding the evolution of the TCRδ genes in tetrapods that was not evident from humans, mice, or other commonly studied placental (eutherian) mammals. An analysis of the genes encoding the TCRδ chains in the duckbill platypus revealed the presence of a highly divergent variable (V) gene, indistinguishable from immunoglobulin heavy (IgH) chain V genes (VH) and related to V genes used in TCRµ. They are expressed as part of TCRδ repertoire (VHδ) and similar to what has been found in frogs and birds. This, however, is the first time a VHδ has been found in a mammal and provides a critical link in reconstructing the evolutionary history of TCRµ. The current structure of TCRδ and TCRµ genes in tetrapods suggests ancient and possibly recurring translocations of gene segments between the IgH and TCRδ genes, as well as translocations of TCRδ genes out of the TCRα/δ locus early in mammals, creating the TCRµ locus.

Comments: That’s a pretty dense abstract. There’s a lot of acronyms in there, which I find distracting. Also, it’s not immediately obvious why we should be interested in this paper. It seems to be this: studying platypuses uncovered new information about how T cells evolved. But that info is buried in the fourth sentence and beyond.

Introduction

T lymphocytes are critical to the adaptive immune system of all jawed vertebrates and can be classified into two main lineages based on the T-cell receptor (TCR) they use (Rast et al. 1997; reviewed in Davis and Chein 2008). The majority of circulating human T cells are the αβT cell lineage which use a TCR composed of a heterodimer of α and β TCR chains. αβT cells include the familiar T cell subsets such as CD4+ helper T cells and regulatory T cells, CD8+ cytotoxic T cells, and natural killer T (NKT) cells. T cells that are found primarily in epithelial tissues and a lower percentage of circulating lymphocytes in some species express a TCR composed of γ and δ TCR chains. The function of these γδ T cells is less well defined and they have been associated with a broad range of immune responses including tumor surveillance, innate responses to pathogens and stress, and wound healing (Hayday 2009). αβ and γδ T cells also differ in the way they interact with antigen. αβTCR are major histocompatibility complex (MHC) “restricted” in that they bind antigenic epitopes, such as peptide fragments, bound to, or “presented” by, molecules encoded in the MHC. In contrast, γδTCR have been found to bind antigens directly in the absence of MHC, as well as self-ligands that are often MHC-related molecules (Sciammas et al. 1994Hayday 2009).

I can hardly think of a less exciting introduction. I’m expecting talk of platypuses, of puzzling questions about evolution or the immune system — and all I get is a boring lecture on T cells. Make no mistake: all of this information is important. We need to know a T cell is, what’s a T-cell receptor, and that there exist at least two kinds (αβ and γδ).

But this information shouldn’t be put first. And it could definitely be split up into more paragraphs.

The conventional TCR chains are composed of two extracellular domains that are both members of the immunoglobulin (Ig) domain super-family (reviewed in Davis and Chein 2008) (fig. 1). The membrane proximal domain is the constant (C) domain, which is largely invariant amongst T-cell clones expressing the same class of TCR chain, and is usually encoded by a single, intact exon. The membrane distal domain is called the variable (V) domain and is the region of the TCR that contacts antigen and MHC. Similar to antibodies, the individual clonal diversity in the TCR V domains is generated by somatic DNA recombination (Tonegawa 1983). The exons encoding TCR V domains are assembled somatically from germ-line gene segments, called the V, diversity (D), and joining (J) genes, in developing T cells, a process dependent upon the enzymes encoded by the recombination activating genes (RAG)-1 and RAG-2 (Yancopoulos et al. 1986Schatz et al. 1989). The exons encoding the V domains of TCR β and δ chains are assembled from all three types of gene segments, whereas the α and γ chains use only V and J. The different combinations of V, D, and J or V and J, selected from a large repertoire of germ-line gene segments, along with variation at the junctions due to addition and deletion of nucleotides during recombination, contribute to a vast TCR diversity. It is this diversity that creates the individual antigen specificity of T-cell clones.

Fig. 1.
Cartoon diagram of the TCR forms found in different species. Oblong circles indicate Ig super-family domains and are color coded as C domains (blue), conventional TCR V domains (red), and VHδ or Vµ (yellow). The gray shaded chains represent the hypothetical partner chain for TCRµ and TCRδ using VHδ.
The figure helps, but again, why are we reading this? This paper seems to follow the common pattern in which the introduction gradually “zooms into” the main point. This is not a good pattern, because it doesn’t tell us the reason for this information. Sure, we suspect it’s relevant to understand what comes next, but without any mystery to anchor this to, it’s hard to be really engaged.

The TCR genes are highly conserved among species in both genomic sequence and organization (Rast et al. 1997Parra et al. 20082012Chen et al. 2009). In all tetrapods examined, the TCRβ and γ chains are each encoded at separate loci, whereas the genes encoding the α and δ chains are nested at a single locus (TCRα/δ) (Chien et al. 1987Satyanarayana et al. 1988; reviewed in Davis and Chein 2008). The V domains of TCRα and TCRδ chains can use a common pool of V gene segments, but distinct D, J, and C genes.

Diversity in antibodies produced by B cells is also generated by RAG-mediated V(D)J recombination and the TCR and Ig genes clearly share a common origin in the jawed-vertebrates (Flajnik and Kasahara 2010Litman et al. 2010). However, the V, D, J, and C coding regions in TCR have diverged sufficiently over the past >400 million years (MY) from Ig genes that they are readily distinguishable, at least for the conventional TCR. Recently, the boundary between TCR and Ig genes has been blurred with the discovery of non-conventional TCRδ isoforms that have been found that use V genes that appear indistinguishable from Ig heavy chain V (VH) (Parra et al. 20102012). Such V genes have been designated as VHδ and have been found in both amphibians and birds (fig. 1). In the frog Xenopus tropicalis, and a passerine bird, the zebra finch Taeniopygia guttata the VHδ are located within the TCRα/δ loci where they co-exist with conventional Vα and Vδ genes (Parra et al. 20102012). In galliform birds, such as the chicken Gallus gallus, VHδ are present but located at a second TCRδ locus that is unlinked to the conventional TCRα/δ (Parra et al. 2012). VHδ are the only type of V gene segment present at the second locus and, although closely related to antibody VH genes, the VHδ appear to be used exclusively in TCRδ chains. This is true as well for frogs where the TCRα/δ and IgH loci are tightly linked (Parra et al. 2010).

Okay… different species have slightly different genes… Cool.

Also, “MY” for million years, really? Do we really need that, especially when there are already about five abbreviations per sentence?

The TCRα/δ loci have been characterized in several eutherian mammal species and at least one marsupial, the opossum Monodelphis domestica, and VHδ genes have not been found to date (Satyanarayana et al.1988Wang et al. 1994Parra et al. 2008). However, marsupials do have an additional TCR locus, unlinked to TCRα/δ, that uses antibody-related V genes. This fifth TCR chain is called TCRµ and is related to TCRδ, although it is highly divergent in sequence and structure (Parra et al. 20072008). A TCRµ has also been found in the duckbill platypus and is clearly orthologous to the marsupial genes, consistent with this TCR chain being ancient in mammals, although it has been lost in the eutherians (Parra et al. 2008Wang et al. 2011). TCRµ chains use their own unique set of V genes (Vµ) (Parra et al. 2007Wang et al. 2011). Trans-locus V(D)J recombination of V genes from other Ig and TCR loci with TCRµ genes has not been found. So far, TCRµ homologues have not been found in non-mammals (Parra et al. 2008).

After an overview of non-mammal tetrapods (frogs, birds), we’re now talking about mammals: platypuses, marsupials, eutherians. It seems like the zooming in is coming to an end…

TCRµ chains are atypical in that they contain three extra-cellular IgSF domains rather than the conventional two, due to an extra N-terminal V domain (fig. 1) (Parra et al. 2007Wang et al. 2011). Both V domains are encoded by a unique set of Vµ genes and are more related to Ig VH than to conventional TCR V domains. The N-terminal V domain is diverse and encoded by genes that undergo somatic V(D)J recombination. The second or supporting V domain has little or no diversity. In marsupials this V domain is encoded by a germ-line joined, or pre-assembled, V exon that is invariant (Parra et al. 2007). The second V domain in platypus is encoded by gene segments requiring somatic DNA recombination; however, only limited diversity is generated partly due to the lack of D segments (Wang et al. 2011). A TCR chain structurally similar to TCRµ has also been described in sharks and other cartilaginous fish (fig. 1) (Criscitiello et al. 2006Flajnik et al. 2011). This TCR, called NAR-TCR, also contains three extracellular domains, with the N-terminal V domain being related to those used by IgNAR antibodies, a type of antibody found only in sharks (Greenberg et al. 1995). The current working model for both TCRµ and NAR-TCR is that the N-terminal V domain is unpaired and acts as a single, antigen binding domain, analogous to the V domains of light-chainless antibodies found in sharks and camelids (Flajnik et al. 2011Wang et al. 2011).

I’ve tried reading this paragraph like five times and I’m still not sure what it’s trying to say. It feels like it’s mostly disjointed sentences that had to be included so the authors can assume you know this, but since we still don’t have a vision of the larger picture, it’s really hard to pay attention.

Phylogenetic analyses support the origins of TCRµ occurring after the avian–mammalian split (Parra et al. 2007Wang et al. 2011). Previously, we hypothesized the origin of TCRµ being the result of a recombination between ancestral IgH and TCRδ-like loci (Parra et al. 2008). This hypothesis, however, is problematic for a number of reasons. One challenge is the apparent genomic stability and ancient conserved synteny in the region surrounding the TCRα/δ locus; this region has appeared to remain stable over at least the past 350 MY of tetrapod evolution (Parra et al. 20082010). The discovery of VHδ genes inserted into the TCRα/δ locus of amphibians and birds has provided an alternative model for the origins of TCRµ; this model involves both the insertion of VH followed by the duplication and translocation of TCR genes. Here we present the model along with supporting evidence drawn from the structure of the platypus TCRα/δ locus, which is also the first analysis of this complex locus in a monotreme.

The last sentence is the first interesting one of the entire paper. It could have come earlier. Technically we should know this from the abstract, but the abstract was pretty difficult to read too.

Also, this is definitely at least two paragraphs merged into one: the first about the previous hypothesis, and the second about the alternative model that is going to be presented.

Materials and Methods

The intro was painful, and usually materials and methods are even worse. We’ll see! 🙂

Identification and Annotation of the Platypus TCRα/δ Locus

The analyses were performed using the platypus (Ornithorhynchus anatinus) genome assembly version 5.0.1 (http://www.ncbi.nlm.nih.gov/genome/guide/platypus/). The platypus genome was analyzed using the whole-genome BLAST available at NCBI (www.ncbi.nlm.nih.gov/) and the BLAST/BLAT tool from Ensembl (www.ensembl.org). The V and J segments were located by similarity to corresponding segments from other species and by identifying the flanking conserved recombination signal sequences (RSS). V gene segments were annotated 5′ to 3′ as Vα or Vδ followed by the family number and the gene segment number if there were greater than one in the family. For example, Vα15.7 is the seventh Vα gene in family 15. The D segments were identified using complementarity-determining region-3 (CDR3) sequences that represent the V–D–J junctions, from cDNA clones using VHδ. Platypus TCR gene segments were labeled according to the IMGT nomenclature (http://www.imgt.org/). The location for the TCRα/δ genes in the platypus genome version 5.0.1 is provided in supplementary table S1Supplementary Material online.

Actually, this isn’t that bad: it’s easier to follow than the introduction because it tells us sequential actions. They make sense together.

But there are a few things wrong here. First, the use of the dreaded passive voice. “The analyses were performed …” No! Tell us who performed it! Second, it’s a pretty dense paragraph and the only one in its section (Identification and Annotation …), which means there’s no benefit to bundling all these sentences together: the title already serves this purpose. Third, it lacks some sentence to tell us what the goal is. The intro was not clear enough to assume readers know what the end point of these analyses is.

Confirmation of Expression of Platypus VHδ

Reverse transcription PCR (RT–PCR) was performed on total splenic RNA extracted from a male platypus from the Upper Barnard River, New South Wales, Australia. This platypus was collected under the same permits as in Warren et al. (2008). The cDNA synthesis step was carried out using the Invitrogen Superscript III-first strand synthesis kit according to the manufacturer’s recommended protocol (Invitrogen, Carlsbad, CA, USA). TCRδ transcripts containing VHδ were targeted using primers specific for the Cδ and VHδ genes identified in the platypus genome assembly (Warren et al. 2008). PCR amplification was performed using the QIAGEN HotStar HiFidelity Polymerase Kit (BD Biosciences, CLONTECH Laboratories, Palo Alto, CA, USA) in total volume of 20 µl containing 1× Hotstar Hifi PCR Buffer (containing 0.3 mM dNTPs), 1µM of primers, and 1.25U Hotstar Hifidelity DNA polymerase. The PCR primers used were 5′-GTACCGCCAACCACCAGGGAAAG-3′ and 5′-CAGTTCACTGCTCCATCGCTTTCA-3′ for the VHδ and Cδ, respectively. A previously described platypus spleen cDNA library constructed from RNA extracted from tissue from a Tasmanian animal was also used (Vernersson et al. 2002).

PCR products were cloned using TopoTA cloning® kit (Invitrogen). Sequencing was performed using the BigDye terminator cycle sequencing kit version 3 (Applied Biosystems, Foster City, CA, USA) and according to the manufacturer recommendations. Sequencing reactions were analyzed using the ABI Prism 3100 DNA automated sequences (PerkinElmer Life and Analytical Sciences, Wellesley, MA, USA). Chromatograms were analyzed using the Sequencher 4.9 software (Gene Codes Corporation, Ann Arbor, MI, USA). Sequences have been archived on GenBank under accession numbers JQ664690–JQ664710.

This seems to be mostly a list of the machines, substances, protocols etc. that were used. Accordingly, it should be formatted as a list. It doesn’t read well as a paragraph (nor should it be expected to).

Phylogenetic Analyses

Nucleotide sequences from FR1 to FR3 of the V genes regions, including CDR1 and CDR2, were aligned using BioEdit (Hall 1999) and the accessory application ClustalX (Thompson et al. 1997). Nucleotide alignments analyzed were based on amino acid sequence to establish codon position (Hall 1999). Alignments were corrected by visual inspection when necessary and were then analyzed using the MEGA Software (Kumar et al. 2004). Neighbor joining (NJ) with uncorrected nucleotide differences (p-distance) and minimum evolution distances methods were used. Support for the generated trees was evaluated based on bootstrap values generated by 1000 replicates. GenBank accession numbers for sequences used in the tree construction are in supplementary table S2Supplementary Material online.

I have a graduate degree in evolutionary biology, I’ve done plenty of phylogenetic analyses (building trees of life), and somehow I hadn’t understood yet that this is what this paper was about. Maybe that’s really obvious to practicing evolutionary biologists, but it seems to me that the kind of analysis could have been made more obvious earlier.

Results and Discussion

Not a bad idea to merge results and discussion together IMO, as long as it doesn’t hinder comprehension.

The TCRα/δ locus was identified in the current platypus genome assembly and the V, D, J, and C gene segments and exons were annotated and characterized (fig. 2). The majority of the locus was present on a single scaffold, with the remainder on a shorter contig (fig. 2). Flanking the locus were SALL2DAD1 and several olfactory receptor (OR) genes, all of which share conserved synteny with the TCRα/δ locus in amphibians, birds, and mammals (Parra et al. 200820102012). The platypus locus has many typical features common to TCRα/δ loci in other tetrapods (Satyanarayana et al. 1988Wang et al. 1994Parra et al. 200820102012). Two C region genes were present: a Cα that is the most 3′ coding segment in the locus, and a Cδ oriented 5′ of the Jα genes. There is a large number of Jα gene segments (n = 32) located between the Cδ and Cα genes. Such a large array of Jα genes are believed to facilitate secondary Vα to Jα rearrangements in developing αβT cells if the primary rearrangements are nonproductive or need replacement (Hawwari and Krangel 2007). Primary TCRα V–J rearrangments generally use Jα segments towards the 5′-end of the array and can progressively use downstream Jα in subsequent rearrangements. There is also a single Vδ gene in reverse transcriptional orientation between the platypus Cδ gene and the Jα array that is conserved in mammalian TCRα/δ both in location and orientation (Parra et al. 2008).

Fig. 2.
Annotated map of the platypus TCRα/δ locus showing the locations of the Vα and Vδ (red), VHδ (yellow), Dδ (orange), Jα and Jδ (green), Cδ (dark blue), and Cα (light blue). Conserved syntenic genes are in gray. The scaffold and contig numbers are indicated.
Oof. I had to actually add line breaks to this paragraph to parse it. It mostly says the same things as the figure, which isn’t too bad. Repeating important info in multiple formats is a good idea. The figure itself could have been clearer, though — it took me a few minutes to understand that the multiple lines in it represent contiguous segments of the chromosome (at least that’s what I think it means). I also had to look up what “synteny” means: it’s having the same order for genetic elements across species. 
There are 99 conventional TCR V gene segments in the platypus TCRα/δ locus, 89 of which share nucleotide identity with Vα in other species and 10 that share identity with Vδ genes. The Vδ genes are clustered towards the 3′-end of the locus. Based on nucleotide identity shared among the platypus V genes they can be classified into 17 different Vα families and two different Vδ families, based on the criteria of a V family sharing >80% nucleotide identity (not shown, but annotated in fig. 2). This is also a typical level of complexity for mammalian Vα and Vδ genes (Giudicelli et al. 2005Parra et al. 2008). Also present were two Dδ and seven Jδ gene segments oriented upstream of the Cδ. All gene segments were flanked by canonical RSS, which are the recognition substrate of the RAG recombinase. The D segments were asymmetrically flanked by an RSS containing at 12 bp spacer on the 5′-side and 23 bp spacer on the 3′-side, as has been shown previously for TCR D gene segments in other species (Carroll et al. 1993Parra et al. 20072010). In summary, the overall content and organization of the platypus TCRα/δ locus appeared fairly generic.

The last sentence seems to be the main takeaway. I would have put it first.

What is atypical in the platypus TCRα/δ locus was the presence of an additional V gene that shared greater identity to antibody VH genes than to TCR V genes (figs. 2 and 3). This V gene segment was the most proximal of the V genes to the D and J genes and was tentatively designated as VHδ. VHδ are, by definition, V genes indistinguishable from Ig VH genes but used in encoding TCRδ chains and have previously been found only in the genomes of birds and frogs (Parra et al. 200820102012).

Shortish paragraph, intriguing first sentence — good job!

Fig. 3.
Phylogenetic tree of mammalian VH genes including the platypus VHδ and monotreme Vµ. The three major VH clans are bracketed. The platypus VHδ is boxed and the clade containing platypus VHδ along with platypus and echidna Vµ is in bold and indicated by a smaller bracket in VH clan III. The three-digit numbers following the VH gene labels are the last three digits of the GenBank accession number referenced in supplementary table S2, Supplementary Material online. The numbers following the platypus and echidna Vµ labels are clone numbers. The tree presented was generated using the Minimum Evolution method. Similar topology was generation using the Neighbor Joining method.

Maybe that’s the ex-biologist speaking, but I personally really like phylogenetic trees. I find them quite illustrative. On the other hand, I, uh, didn’t remember at all what a VH gene is, so I had to go back to the introduction. There should have been a way to make it clearer, since VH genes play a big role in the results.

Also, not important, but there’s a big typo in the last sentence (generation should have been generated).

VH genes from mammals and other tetrapods have been shown to cluster into three ancient clans and individual species differ in the presence of one or more of these clans in their germ-line IgH locus (Tutter and Riblet 1989Ota and Nei 1994). For example, humans, mice, echidnas, and frogs have VH genes from all three clans (Schwager et al. 1989Ota and Nei 1994Belov and Hellman 2003), whereas rabbits, opossums, and chickens have only a single clan (McCormack et al. 1991Butler 1997Johansson et al. 2002Baker et al. 2005). In phylogenetic analyses, the platypus VHδ was most related to the platypus Vµ genes found in the TCRµ locus in this species (fig. 3). Platypus VHδ, however, share only 51–61% nucleotide identity (average 56.6%) with the platypus Vµ genes. Both the platypus Vµ and VHδ clustered within clan III (fig. 3) (Wang et al. 2011). This is noteworthy given that VH genes in the platypus IgH locus are also clan III and, in general, clan III VH are the most ubiquitous and conserved lineage of VH (Johansson et al. 2002Tutter and Riblet 1989). Although clearly related to platypus VH, the VHδ gene share only 34–65% nucleotide identity (average 56.9%) with the bona fide VH used in antibody heavy chains in this species.

Okay, this explains the three VH parts in the tree. It’s pretty clear.

It was necessary to rule out that the VHδ gene present in the platypus TCRα/δ locus was not an artifact of the genome assembly process. One piece of supporting evidence would be the demonstration that the VHδ is recombined to downstream Dδ and Jδ segments and expressed with Cδ in complete TCRδ transcripts. PCR using primers specific for VHδ and Cδ was performed on cDNA synthesized from splenic RNA from two different platypuses, one from New South Wales and the other from Tasmania. PCR products were successfully amplified from the NSW animal and these were cloned and sequenced. Twenty clones, each containing unique nucleotide sequence, were characterized and found to contain the VHδ recombined to the Dδ and Jδ gene segments (fig. 4A). Of these 20, 11 had unique V, D, and J combinations that would encode 11 different complementarity-determining regions-3 (CDR3) (fig. 4B). More than half of the CDR3 (8 out of 11) contained evidence of using both D genes (VDDJ) (fig. 4B). This is a common feature of TCRδ V domains where multiple D genes can be incorporated into the recombination due to the presence of asymmetrical RSS (Carroll et al. 1993). The region corresponding to the junctions between the V, D, and J segments, contained additional sequence that could not be accounted for by the germ-line gene segments (fig. 4B). There are two possible sources of such sequence. One are palindromic (P) nucleotides that are created during V(D)J recombination when the RAG generates hairpin structures that are resolved asymmetrically during the re-ligation process (Lewis 1994). The second are non-templated (N) nucleotides that can be added by the enzyme terminal deoxynucleotidyl transferase (TdT) during the V(D)J recombination process. An unusual feature of the platypus VHδ is the presence of a second cysteine encoded near the 3′-end of the gene, directly next to the cysteine predicted to form the intra-domain disulfide bond in Ig domains (fig. 4A). Additional cysteines in the CDR3 region of VH domains have been thought to provide stability to unusually long CDR3 loops, as has been described for cattle and the platypus previously (Johansson et al. 2002). The CDR3 of TCRδ using VHδ are only slightly longer than conventional TCRδ chains (ranging 10–20 residues) (Rock et al. 1994Wang et al. 2011). Furthermore, the stabilization of CDR3 generally involves multiple pairs of cysteines, which were not present in the platypus VHδ clones (fig. 4A). Attempts to amplify TCRδ transcripts containing VHδ from splenic RNA obtained from the Tasmanian animal were unsuccessful. As a positive control, TCRδ transcripts containing conventional Vα/δ were successfully isolated, however. It is possible that Tasmanian platypuses, which have been separated from the mainland population at least 14,000 years either have a divergent VHδ or have deleted this single V gene altogether (Lambeck and Chappell 2001).

I like the thought process: “hey, our results may have been an artifact, here’s what we did to prove it wasn’t.” But why is this paragraph so long? Seems like it could have been multiple smaller ones, perhaps with a section subheading.

Fig. 4.
(A) Alignment of predicted protein sequence of transcripts containing a recombined VHδ gene isolated from platypus spleen RNA. The individual clones are identified by the last three digits of their GenBank accession numbers (JQ664690–JQ664710). Shown is the region from FR3 of the VHδ through the beginning of the Cδ domain. The sequence in bold at the top of the alignment is the germ-line VHδ and Cδ gene sequence. The double cysteines at the end of FR3 and unpaired cysteines in CDR3 are shaded, as is the canonical FGXG in FR4. (B) Nucleotide sequence of the CDR3 region of the eleven unique V(D)J recombinants using VHδ described in the text. The germ-line sequence of the 3′-end of VHδ, the two Dδ, are shown at the top. The germ-line Jδ sequences are shown on the right-hand side of the alignment interspersed amongst the cDNA sequences using each. Nucleotides in the junctions between the V, D, and J segments, shown italicized, are most likely N-nucleotides added by TdT.

This figure is probably good to visualize what their results actually looked like, but it also seems like a way to cram as much information in a visual and its caption as humanly possible… I’ll let it pass. It’s fine that some parts of the paper go more in depth, if they can be easily ignored, as I think is the case here.

Small nitpick: This is two figures, and I would preferred that this fact would have been clearer. A small “(A)” and “(B)” in the paragraph doesn’t really help the reader.

Although there is only a single VHδ in the current platypus genome assembly, there was sequence variation in the region corresponding to FR1 through FR3 of the V domains (fig. 4A and sequence data not shown but available in GenBank). Some of this variation could represent two alleles of a single VHδ gene. Indeed, the RNA used in this experiment is from a wild-caught individual from the same population that was used to generate the whole-genome sequence and was found to contain substantial heterozygosity (Warren et al. 2008). There was greater variation in the transcribed sequences, however, than could be explained simply by two alleles of a single gene (fig. 4A). Two alternative explanations are the occurrence of somatic mutation of expressed VHδ genes or allelic variation in gene copy number. Somatic mutation in TCR chains is controversial. Nonetheless, it has been invoked to explain the variation in expressed TCR chains that exceeds the apparent gene copy number in sharks, and has also been postulated to occur in salmonids (Yazawa et al. 2008Chen et al. 2009). Therefore, it does not seem to be out of the realm of possibility that somatic mutation is occurring in platypus VHδ. Indeed, the mutations appear to be localized to the V region with no variation in the C region (fig. 4A). This may be due to its relatedness of VHδ to Ig VH genes where somatic hyper-mutation is well documented. Such somatic mutation contributes to overall affinity maturation in secondary antibody responses (Wysocki et al. 1986). The pattern of mutation seen in platypus VHδ however, is not localized to the CDR3, which would be indicative of selection for affinity maturation, but was also found in the framework regions. Furthermore, in the avian genomes where there is also only a single VHδ, there was no evidence of somatic mutation in the V regions (Parra et al. 2012). The contribution of mutation to the platypus TCRδ repertoire, if it is occurring, remains to be determined. Alternatively, the sequence polymorphism may be due to VHδ gene copy number variation between individual TCRα/δ alleles.

Not the worst paragraph, but again, doesn’t need to be a Wall of Text.

Irrespective of the number of VHδ genes in the platypus TCRα/δ locus, the results clearly support TCRδ transcripts containing VHδ recombined to Dδ and Jδ gene segments in the TCRα/δ locus (fig. 4). A VHδ gene or genes in the platypus TCRα/δ locus in the genome assembly, therefore, does not appear to be an assembly artifact. Rather it is present, functional and contributes to the expressed TCRδ chain repertoire. The possibility that some platypus TCRα/δ loci contain more than a single VHδ does not alter the principal conclusions of this study.

Previously, we hypothesized the origin of TCRµ in mammals involving the recombination between and ancestral TCRα/δ locus and an IgH locus (Parra et al. 2008). The IgH locus would have contributed the V gene segments at the 5′-end of the locus, with the TCRδ contributing the D, J, and C genes at the 3′-end of the locus. The difficulty with this hypothesis was the clear stability of the genome region surrounding the TCRα/δ locus. In other words, the chromosomal region containing the TCRα/δ locus appears to have remained relatively undisrupted for at least the past 360 million years (Parra et al. 200820102012). The discovery of VHδ genes within the TCRα/δ loci of frog and zebra finch is consistent with insertions occurring without apparently disrupting the local syntenic region. In frogs, the IgH and TCRα/δ loci are tightly linked, which may have facilitated the translocation of VH genes into the TCRα/δ locus (Parra et al. 2010). However, close linkage is not a requirement since the translocation of VH genes appears to have occurred independently in birds and monotremes, due to the lack of similarity between the VHδ in frogs, birds, and monotremes (Parra et al. 2012). Indeed, it would appear is if the acquisition of VH genes into the TCRα/δ locus occurred independently in each lineage.

The similarity between the platypus VHδ and V genes in the TCRµ locus is, so far, the clearest evolutionary association between the TCRµ and TCRδ loci in one species. From the comparison of the TCRα/δ loci in frogs, birds, and monotremes, a model for the evolution of TCRµ and other TCRδ forms emerges (fig. 5), which can be summarized as follows:

Oooh, exciting! The title promised a model, and at last we get it. Also it seems that below we get point-form stuff! I like point-form stuff. It’s often really helpful to guide the reader.

  1. Early in the evolution of tetrapods, or earlier, a duplication of the D–J–Cδ cluster occurred resulting in the presence of two Cδ each with its own set of Dδ and Jδ segments (fig. 5A).

  2. Subsequently, a VH gene or genes was translocated from the IgH locus and inserted into the TCRα/δ locus, most likely to a location between the existing Vα/Vδ genes and the 5′-proximal D–J–Cδ cluster (fig. 5B). This resulted in the configuration like that which currently exists in the zebra finch genome (Parra et al. 2012).

  3. In the amphibian lineage there was an inversion of the region containing VHδ–Dδ–Jδ–Cδ cluster and an expansion in the number of VHδ genes (fig. 5C). Currently, X. tropicalis has the greatest number of VHδ genes, where they make up the majority of V genes available in the germ-line for use in TCRδ chains (Parra et al. 2010).

  4. In the galliform lineage (chicken and turkey), the VHδ–Dδ–Jδ–Cδ cluster was trans-located out of the TCRα/δ locus where it currently resides on another chromosome (fig. 5D). There are no Vα or Vδ genes at the site of the second chicken TCRδ locus and only a single Cδ gene remains in the conventional TCRα/δ locus (Parra et al. 2012).

  5. Similar to galliform birds, the VHδ–Dδ–Jδ–Cδ cluster was trans-located out of the TCRα/δ locus in presumably the last common ancestor of mammals, giving rise to TCRµ (fig. 5E). Internal duplications of the VHδ–Dδ–Jδ genes gave rise to the current [(V–D–J) − (V–D–J) − C] organization necessary to encode TCR chains with double V domains (Parra et al. 2007Wang et al. 2011). In the platypus, the second V–D–J cluster, encoding the supporting V, has lost its D segments and generates V domains with short CDR3 encoded by direct V to J recombination (Wang et al. 2011). The whole cluster appears to have undergone additional tandem duplication as it exists in multiple tandem copies in the opossum and also likely in the platypus (Parra et al. 20072008Wang et al. 2011).

  6. In the therian lineage (marsupials and placentals), the VHδ was lost from the TCRα/δ locus (Parra et al. 2008). In placental mammals, the TCRµ locus was also lost (Parra et al. 2008). The marsupials retained TCRµ, however the second set of V and J segments, encoding the supporting V domain in the protein chain, were replaced with a germ-line joined V gene, in a process most likely involving germ-line V(D)J recombination and retro-transposition (fig. 5F) (Parra et al. 20072008).

Yeah, this was good. These point-form paragraphs, combined with Fig. 5 (below) did more to help me understand the paper than anything else so far. I kind of wish the paper had just opened with this, and then proceeded to explain the reasoning behind.

TCR forms such as TCRµ, which contain three extracellular domains, have evolved at least twice in vertebrates. The first was in the ancestors of the cartilaginous fish in the form of NAR-TCR (Criscitiello et al. 2006) and the second in the mammals as TCRµ (Parra et al. 2007). NAR-TCR uses an N-terminal V domain related to the V domains found in IgNAR antibodies, which are unique to cartilaginous fish (Greenberg et al. 1995Criscitiello et al. 2006), and not closely related to antibody VH domains. Therefore, it appears that NAR-TCR and TCRµ are more likely the result of convergent evolution rather than being related by direct descent (Parra et al. 2007Wang et al. 2011). Similarly, the model proposed in fig. 5 posits the direct transfer of VH genes from an IgH locus to the TCRα/δ locus. But it should be pointed out the VHδ found in frogs, birds, and monotremes are not closely related (fig. 3); indeed, they appear derived each from different, ancient VH clans (birds, VH clan I; frogs VH clan II; platypus VH clan III). This observation would suggest that the transfer of VHδ into the TCRα/δ loci occurred independently in the different lineages. Alternatively, the transfer of VH genes into the TCRα/δ locus may have occurred frequently and repeatedly in the past and gene replacement is the best explanation for the current content of these genes in the different tetrapod lineages. The absence of VHδ in marsupials, the highly divergent nature of Vµ genes in this lineage, and the absence of conserved synteny with genes linked to TCRµ in the opossum, provide little insight into the origins of TCRµ and its relationship to TCRδ or the other conventional TCR (Parra et al. 2008). The similarity between VH, VHδ, and Vµ genes in the platypus genome, which are all clan III, however is striking. In particular, the close relationship between the platypus VHδ and Vµ genes lends greater support for the model presented in fig. 5E, with TCRµ having been derived from TCRδ genes.

My comments are getting repetitive. This could have been multiple paragraphs etc. etc. It’s easy enough to find the joints where it should be carved, by the way: right before the sentences that start with “Similarly” and “Alternatively” would be a good start, since these words indicate that we’re switching to a new idea.

Fig. 5.
A model of the stages of evolution of the TCRα/δ loci in tetrapods and the origins of TCRµ in mammals. A color key of the gene segments is presented at the bottom. (A) Depiction of the Dδ-Jδ-Cδ duplication in an ancestral TCRα/δ locus that provides a second Cδ gene found in frogs and zebra finch. (B) Depiction of the insertion of a VH gene into the TCRα/δ locus producing a current organization as it is found in zebra finch. (C) Depiction of the inversion/translocation and VHδ gene duplication that yielded the current organization found in frogs. (D) Depiction of the translocation of a VHδ–Dδ–Jδ–Cδ cluster to a location outside the TCRα/δ locus generating a second TCRδ locus as it is currently found in chicken and turkey. (E) Depiction the translocation that took place in mammals giving rise to the TCRµ locus. (F) Loss of TCRµ in placental mammals, loss of D gene segments in cluster encoding the support V domain, retro-transpostion to form a germ-line joined V in marsupials, and duplication of TCRµ clusters in both monotremes and marsupials.

Super helpful figure. Although I’m generally in favor of repeating important info, I do feel that the caption could have simply referred to the 6-point model in the text. The caption as it stands doesn’t add much and looks like a Wall of Text. But that’s not a big deal.

The presence of TCR chains that use antibody like V domains, such as TCRδ using VHδ, NAR-TCR or TCRµ are widely distributed in vertebrates with only the bony fish and placental mammals missing. In addition to NAR-TCR, some shark species also appear to generate TCR chains using antibody V genes. This occurs via trans-locus V(D)J recombination between IgM and IgW heavy chain V genes and TCRδ and TCRα D and J genes (Criscitiello et al. 2010). This may be possible, in part, due to the multiple clusters of Ig genes found in the cartilaginous fish. It also illustrates that there has been independent solutions to generating TCR chains with antibody V domains in different vertebrate lineages. In the tetrapods, the VH genes were trans-located into the TCR loci where they became part of the germ-line repertoire. Whereas in cartilaginous fish something equivalent may occur somatically during V(D)J recombination in developing T cells. Either mechanism suggests there has been selection for having TCR using antibody V genes over much of vertebrate evolutionary history.

The current working hypothesis for such chains is that they are able to bind native antigen directly. This is consistent with a selective pressure for TCR chains that may bind or recognize antigen in ways similar to antibodies in many different lineages of vertebrates. In the case of NAR-TCR and TCRµ, the N-terminal V domain is likely to be unpaired and bind antigen as a single domain (fig. 1), as has been described for IgNAR and some IgG antibodies in camels (recently reviewed in Flajnik et al. 2011). This model of antigen binding is consistent with the evidence that the N-terminal V domains in TCRµ are somatically diverse, while the second, supporting V domains have limited diversity with the latter presumably performing a structural role rather than one of antigen recognition (Parra et al. 2007Wang et al. 2011). There is no evidence of double V domains in TCRδ chains using VHδ in frogs, birds, or platypus (fig. 1) (Parra et al. 20102012). Rather, the TCR complex containing VHδ would likely be structured similar to a conventional γδTCR with a single V domain on each chain. It is possible that such receptors also bind antigen directly, however this remains to be determined.

Not much to add except that I just had a thought that subheadings would have greatly eased this section (like they did the Methods section).

A compelling model for the evolution of the Ig and TCR loci has been one of internal duplication, divergence and deletion; the so-called birth-and-death model of evolution of immune genes promoted by Nei and colleagues (Ota and Nei 1994Nei et al. 1997). Our results in no way contradict that the birth-and-death mode of gene evolution has played a significant role in shaping these complex loci. However, our results do support the role of horizontal transfer of gene segments between the loci that has not been previously appreciated. With this mechanism T cells may have been able to acquire the ability to recognize native, rather than processed antigen, much like B cells.

Pretty good conclusion, opening on new ideas and showing the significance of this work in the field.


Phew. I’m done.

Reading this paper took me several days, although I could have been more focussed in general. But this shows how much work is required to read papers! I had to push myself to read. Many times I caught myself skimming paragraphs without understanding anything, and I had to read again. Right now I think I would benefit from reading it all a second time, but I resist the thought, because it’s work.

But I think it’s a good candidate for my rewriting project. It should be relatively easy to cut down the number of abbreviations, split long paragraphs, and add subheadings. More thorough rewriting will probably involve clarifying the main points and claims right at the start. At the most extreme (I’m not sure I’ll go there), it could be beneficial to change the entire structure: give the detailed model first, and only then explain the background and methods.

Stay tuned!

Categories
guidelines

Science Style Guide: Abbreviations

This post is part of my ongoing scientific style guideline series.

Textual compression techniques (TCDs) are used more and more in science writing. TCDs come in various forms, including truncation (e.g. mi for mile), acronyms (lol for laughing out loud), syllabic acronyms (covid for coronavirus disease), contraction (int’l for international), and others. The primary benefit of using TCDs in writing is to reduce text length. This is especially useful in contexts where space is limited, such as tables and charts, as well as when a long word or phrase is repeated multiple times. Another reason to use a TCD is to create a new semantic unit that is more practical to use than the long version. For instance, the TCD laser is both more convenient and more recognizable than the original light amplification by the stimulated emission of radiation.

Okay. Look at that paragraph without reading it. Does anything stand out?

I made up the phrase textual compression device and its acronym TCD. They simply mean “abbreviation,” which is what I would have used if I weren’t trying to illustrate the following points:

  • Abbreviations can be distracting. Readers expect words, and things that look less like words — capitalized acronyms, random apostrophes in the middle of a word — will stand out, as does TCD above. Used sparingly, that can be good, to draw attention to something. But in large quantities, it’s jarring.
    • It’s even worse when multiple different abbreviations are in close proximity, or when similar abbreviations are used (e.g. TCRµ and TCRδ, which come up all the time in a paper I’m reading).
  • More importantly, abbreviations demand cognitive effort. If the reader doesn’t already know an abbreviation (for instance because you made it up), they have to spend some energy learning it. You’d probably prefer them to expend that energy understanding your paper instead.
    • Worse, they might have to interrupt their reading to go back to where you defined the abbreviation, or to look it up online. (A nice opportunity to quit reading your paper altogether.)

Humans don’t read like computers. You can’t just “declare” an abbreviation as you would a variable in code, and assume that from now on your reader knows what it stands for. It’s quite likely that readers will skim your piece, or jump directly to a specific section (e.g. results), in which case they can miss the definition. Even if they do read it, they might forget — in a typical paper, there’s a lot of information to remember.

Of course, abbreviations can be useful, as the TCD paragraph laboriously explains. But the benefits are rather minor. On computer screens, which is where your scientific writing will almost always be read, space is virtually unlimited. (Figures and tables remain a good use case, as long as the abbreviations are easily readable in the caption.) Creating a new, more practical way to call a thing (e.g. laser) can be quite useful, but again, only if used sparingly, for important concepts.

Overall, the benefits of abbreviations are much greater for the writer than for the reader — which is exactly the opposite of what we want as per the Minimum Reading Friction principle.

The other principle, Low-Hanging Fruit, says that the best improvements are those that require little writing skill to implement. Abbreviation minimization fits the bill. In most cases, you can improve the text just by replacing the abbreviation with:

  • The spelled-out version (textual compression device instead of TCD)
  • A synonym (abbreviation instead of TCD / textual compression device)
  • The core noun of the abbreviated phrase (e.g. device; not the best example but you get the idea. It will usually be clear in context what you refer to, unless you’re talking about many different types of devices).

Sometimes you’ll need to perform a bit more rephrasing, but rarely will you have to perform major rephrasing due to abbreviations. If you do, that’s probably a sign that the original text was awfully painful to read.

Recommendations

  • Coin new abbreviations as rarely as possible.
    • If you must coin new abbreviations, make sure they’re short, pronounceable, and memorable. Don’t hesitate to repeat their meaning multiple times — you’re teaching your readers a new word.
  • Generally prefer the use of spelled-out versions, core nouns, or synonyms.
  • Avoid using multiple different abbreviations in close proximity.
  • Abbreviations that are generally well-known, such as DNA, can be used as much as you want. A good way to tell is if they’re included in dictionaries.
  • If you can’t avoid using several uncommon or new abbreviations, it can be helpful to draw attention to them, so that readers are warned that they will have a better time if they make sure they learn the new terms.
    • This could take the form of a short glossary at the beginning, making it easy to look up definitions during reading.
Categories
guidelines

Proposal for a New Scientific Writing Guide

Scientific writing is in bad shape. Realizing that, and wanting to do something about it, was the starting point for my essay on the creation of a new journal, one that would rewrite some science papers in a better style and kickstart a movement to ultimately change the writing norms.

Since I published the essay last July, the Journal of Actually Well-Written Science (yeah, it needs a better name) has gone from “cool idea” to “project I’m actually trying to bring to life.” Many questions remain unanswered as to how best to proceed. But one important thing I must figure out is: What should the writing norms be changed to?

Today I’m committing to publish several short posts over the course of the next month to answer exactly that.

Below is some brief discussion of the two principles that will guide my thinking. They both center on the idea of minimizing effort, for the reader as well as for the writer. Writers should make some effort to ensure readers don’t have to (that’s the basic job of a writer), but I’ll focus on improvements that don’t require a lot of time and effort from writers, since those tend to be busy scientists.

I’ll also include a table of contents to easily access the posts as they are published.

Two effort-minimization principles

1) Minimum Reading Friction: Demand less cognitive resources from the reader

Science papers are usually technical. They deal with complex questions. They assume specialized background knowledge. They may involve math.

It is expected that papers be difficult to read. But we can at least make sure the writing doesn’t get in the way.

The first principle of this style guide says that you should do everything feasible to reduce the amount of effort readers will need to make when reading your paper. In other words, your job is to make their job easier.

If something — e.g. finding a good example to illustrate a point1like I just did! — asks some effort from you but reduces the effort readers will need to make when reading, then do it. Conversely, don’t make your own life easier if it’s going to make the reader’s life harder. An example would be using an abbreviation to spend less time typing at the cost of increasing the cognitive demands on the reader.

The larger your readership, the more important this principle is. If you write for one person (e.g. an email), then it doesn’t matter that much if it takes some work to read (although it might hurt your chances of getting a reply). But if you expect to be read by 1,000 people, then every abbreviation that saved you some inconvenience is now multiplied into an inconvenience for 1,000 people.

2) Low-Hanging Fruit: Focus on improvements that are easy to apply

“Writing well” is a complicated art. Developing it can be the project of a lifetime. Scientists are typically too busy for that.

Fortunately (for me), science writing is so bad that there’s a lot of low-hanging fruit to pick. Many improvements need little effort. For example, using fewer abbreviations results in less demanding reading without requiring advanced writing skills — you can often just replace the abbreviations with the unabridged terms. Splitting long paragraphs into smaller chunks is often as easy as adding a line break when you notice a shift to a different idea.

Such improvements can also be applied almost mechanistically, which is ideal for someone who rewrites a paper without being as intimate with the topic as the author is.

The second principle therefore says to focus primarily (but not exclusively) on the elements of style that require the least effort and skill relative to how much they improve the writing.

Other things to keep in mind

  • Keep the good current norms. The goal of this project is not to burn scientific writing down and rebuild it from scratch. For example, it is good that scientists, by default, try to avoid ornamented writing. This helps with precision and objectivity.
  • Formatting is an area that can be improved, but it’s a less tractable problem because it differs a lot between publications. For instance, citation style (e.g. footnotes vs. inline) can help or hinder reading. Still, I’ll eventually need to develop guidelines for formatting in JAWWS, so I will probably discuss it a few times.
  • Focus on the classic paper format. There are a lot of new, exotic ways that science could be communicated, but at first we’ll assume that papers — usually with traditional structures like intro-methods-results-discussion — will remain the main format in the foreseeable future.
  • Personal preferences can be hard to distinguish from objective quality measures. Of course, everything I propose will reflect what I personally look for in science writing. I think and hope most guidelines will be broadly popular, but I’m always open for feedback and I’ll tweak them if others make convincing arguments.

Table of contents

I will update this list as I publish the posts.

In the meantime, here’s a very informal list of topics I might cover:

  • Abbreviations
  • Paragraph length
  • Giving examples
  • Bullet points
  • Links
  • Citations and references
  • Point of view (1st vs 3rd person) and voice (active vs passive)
  • Humor
  • Flourishes, ornamentation
  • Paper structure
  • Length vs. clarity vs. density tradeoff
  • Figures
  • Reading guidance (e.g. “read the methods section to understand what we did, but feel free to skip the more technical section 2.3”)
  • Jargon, vocabulary, word choice
  • Writing in narrative form (a difficult skill!)

 

Last updated: November 12, 2021

Categories
essay

Leveling Up the Skill of Friendship: Deepening Friendship

A little less than a year ago, in the midst of the very depressing covid lockdown fall, I decided I would write about friendship.

It was a good topic. I had been lamenting about the state of my social life for a while, and writing on the topic seemed like it would be a good idea to improve that part of my life. You could see friendship not just as something that happens, but as a skill that can be practiced and improved. And so I wrote an essay arguing for the importance of developing the friendship skill.

The people who read it liked my essay, but they wanted to know how you could develop that skill. Well — why not write about that too? My single essay grew into a series of three or five essays, depending on how you count. It was going to be the Comprehensive Guide About How To Become Better at Friendship. It was going to cover everything: making friends, keeping them, getting close friends, and dealing with friendship problems.

Here’s some advice: Don’t do this.

Specifically, don’t commit on writing a Comprehensive Guide of any sort, unless you really know what you’re doing.

Writing the first part of my guide took two whole months. I published it on 30 December 2020 because I really wanted it to be done before the end of the year. Then it took me a while to write the second part, which I published in April 2021. In fact it was only half of the second part, because by then I realized it was too long and I had to split it. (Actually, the entire project was originally supposed to be contained in one post. Ah, the naïveté of thinking I could cover everything in a single blog post! I’m pretty sure that one post would have had the length of a book.)

Part 3 never happened until now. There was a draft, lingering from before I split Part 2. There’s some valuable stuff in there, so I’m publishing it today, although, to be honest, this introduction is probably the more interesting section of this post.

What happened? Mostly, I lost interest. Friendship did not turn out to be a topic I wanted to spend months thinking about. Also, I took too long to write it. The excitement waned. There were better topics to focus on.

Here’s some more advice: Write on what you’re interested right now, and don’t count on being still interested weeks, months or years from now.

We do have long (sometimes lifelong) obsessions. You probably already know what most of yours are. Write about those all you want, now, later, whenever. But don’t commit to writing multiple blog posts about a topic that you got into only a few days ago.

With this, here are the contents of Leveling Up the Skill of Friendship: Part 3. It is not comprehensive. Read at your own risk.

Part 4, which was going to be about the problems and the end of friendship, will not be written.

Deepening a friendship

The mark of perfect Friendship is not that help will be given when the pinch comes (of course it will) but that, having been given, it makes no difference at all.

C.S. Lewis

All right, you’ve got a bunch of friends. People you like and who like you. People who are kind and interesting, who are fun to hang out with. People you can rely on for help… to an extent.

But as we saw earlier, there’s a higher level after strangers, acquaintances, and friends. The elite caste: close friends.

Close friends are friends you can rely on for help to a (sometimes surprisingly) large extent. They’re the one you can tell your secrets to. They’re this nourishing presence that will make you feel lucky to be alive. They’re the friends of virtue Aristotle was talking about. The friends for whom the C.S. Lewis quote above is true.

Close friends are much rarer than casual friends, and require much more effort. But they’re incredibly important. And yet not everyone has them; some have only a few. Let’s figure out how to remedy this.

Is there an optimal number of close friends?

Is there an equivalent of Dunbar’s number for close friendships? Yes. In fact, Dunbar’s number should be seen as the size of only one of several concentric circles of relationships. 150 is the value for casual friends. According to Dunbar’s research, the other values are:

  • 50 for close friends that you would invite to a group dinner, for instance
  • 15 for friends you can turn to for help
  • 5 for your core support group (best friends, romantic partners, and family members)

We can immediately see that there are competing definitions of “close friend” here. So the precise number will depend on what exactly you mean.

For instance, the number could be 1. The French philosopher and essayist Michel de Montaigne, describing his relationship with his BFF Étienne de la Boétie, wrote in 1580 that

the perfect friendship I speak of is indivisible; each one gives himself so entirely to his friend, that he has nothing left to distribute to others.

I think we can agree that Montaigne describes a very special kind of friendship, one that approaches the strength and significance of a (fulfilling) romantic relationship. Perhaps indeed you can’t expect to have more than one such friendship in your life.

Most people define close friends differently, though. For instance, in a 2004 poll, Americans said they had, on average, 8 to 9 close friends. 2% had none; 27% had more than 10.

The takeaway from this section is that (again) the numerical value doesn’t matter much. What matter is that you’re satisfied with what you have. If you have fewer close friends that you think is ideal, go ahead and make some.

No threshold, no ceiling

Regardless of how exactly you define a “close friend,” there is no clear-cut point at which a casual friend reaches that level. Friendship exists on a spectrum. This is why I titled the section “Deepening a friendship” rather than “Making close friends.”

There also isn’t a maximum. You can always get to know a friend better and get closer to them. In practice, to get really close to someone, you’ll have to share a significant part of your life with them. That happens mostly in romantic relationships, as well as in familial ones, for instance when growing up with siblings. Or in rare intense bromance situations like with Montaigne and de la Boétie.

So there’s no threshold or ceiling, but is there an optimal level of friendship for any given pair of people? I think so. It seems plausible that two people may do great as casual friends but not make excellent close friends. Or that a pair will be only lukewarm acquaintances until some event brings them close together, at which point they realize how important they are for each other.

I’m not sure, however, that there’s any way to know this optimal level until you reach it — and try to go beyond.

How to create close friendship

To make a close friend, you’ll need time, proximity, repeated interactions, and so on. They’re the basic ingredients — but you’ll need a lot of them. Since the list includes time, you’ll most likely require a lot of patience. The formation of a close friendship generally can’t be rushed.

My friend Daniel Golliher writes that this is accompanied by a feeling of frustration. When you meet someone who’s a lot like you, you wish you could speed through the phase of getting to know each other. It’s like watching a friend discover a book or TV series you love: you can’t wait until they’re done with it so you can discuss it. Daniel continues:

This might otherwise be called the frustration with acquiring old friends. You can’t just make an old friend who’s known you for a while, and consequently understands you more than others might (and vice versa). If you want more friends like this, you have to start today and let them mature over the course of years.

I think this is generally true, but there are some steps we can take to at least make sure we don’t stall the process.

First, as a prerequisite for the rest, let’s mention spending some 1-on-1 time with your friend. That may sound obvious, but you’ll never get very close to anyone if you only ever see them in company of other people.

Be kind, and be kind at a higher level than before. This means providing help at critical moments, especially if their other friends aren’t doing that. Offer your help, and be there when the need arises. Be trustworthy. Don’t be judgmental, except when you need to.

Be interesting. At this level, be interesting in a way that you aren’t with others. Share your weirdness. Share your dreams and ambitions. Become intimate with your friend; show your vulnerability. Trust them, and let strong bonds forge themselves out of your shared secrets.

Create memories between the two of you. Having a history of common experiences allows you to build a solid foundation on which to add the new. It also gives you more conversation options! Sometimes, you might even have not much in common with an old friend, because your interests diverged over time — but the common foundation is sufficient to keep the friendship going and fulfilling.

Catalyzing the process

Is it truly impossible to speed up the deepening of a friendship?

Well, not quite. There are some ways; they boil down to compressing the required time into a shorter period.

The best example might be traveling. Spend a week or two planning day trips, sitting next to each other in a train, maybe even sharing a bedroom with a friend, and you’ll get close, very fast. In such a setting, you have no choice but to become intimate. Plus you create tons of shared memories.

Co-living is another way to set up a life of spending time together. Yet another is to start a collective project — a music band, a startup, whatever.

You may have noticed something in common with all these examples. In each of them, there’s a risk that it will make your relationship worse. Maybe you’ll find that your friend is not reliable when you ask them to plan your travel itinerary. Maybe you’ll find them unbelievably annoying to spend pre-coffee mornings with. Maybe your startup or band will fail and you’ll be certain it’s their fault, and fall out, and that’ll be the end of it.

I think that the idea of risk is inherent to a close friendship. You build trust by showing vulnerability — and that can backfire!

Just like it’s possible to lose a friend by attempting to become a lover, it’s possible to lose a friend by trying to become too close. When that happens, it’s probably best to think that the intimate friendship was never meant to be anyway.

But they1no idea who, this was in my draft with no source lol say that if you’re friends with someone for 7 years, you will probably remain lifelong friends. So if you can’t catalyze the process, don’t worry. Things will get clearer with time.

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essay

Feelings of Infinity

I grew up in a small suburb of a mid-size city. The suburb was my world — regular, recognizable, bounded. The city was a vastly richer world. Going downtown meant leaving what I knew to experience something infinitely bigger.

It is a great feeling, the feeling of infinity. It is the feeling that anything is possible. That you can lose yourself in something greater than you are. It is, I think, a precious feeling.

Eventually I went to school in a more urban part of Quebec City. I gained more autonomy, and explored more of the city, and met more of its people. The feeling of infinity subsided. Now it was just a place, and one that I knew fairly well.

I moved to Montreal. Montreal is the core of Quebec’s culture, its nucleus, the city where all the important and interesting people live, the place that you see on TV. It is three to five times more populous than Quebec City, depending on how you count. When I began university, Montreal felt big. It was bustling, overwhelming, brimming with people (and more diverse people, linguistically and ethnically and politically and economically and so on). It was incredibly alive. It was the Place Where Things Happen.

Montreal was the place you saw on TV — but TV gives you just a glimpse of anything, and lets your imagination fill the rest. And yet imagination is limited, so when you move there, it feels gigantic, truly infinite.

I’ve lived in Montreal for about ten years. Today it doesn’t feel infinite at all. It doesn’t quite feel small, and it objectively isn’t. It just feels finite.

Quebec City feels even more finite. Quebec as a whole feels finite now — because Montreal is by far its biggest place, and so there’s nothing else to “conquer,” so to speak. To get the feeling again, I would need to move someplace else. I could move to Toronto, the biggest place in Canada. I could move to the US — a big country, with big cities, surely able to sustain the feeling for a while. Perhaps I should try living in the giant metropolises of Asia.

Maybe focussing on cities isn’t the answer. I love cities, because they offer a far richer experience than other places, but there is plenty of nature to lose oneself into, too. Yet that doesn’t ring very true to me. In part because I’m not a very outdoorsy person, but mostly, I think, because the closest we can get to infinity is through interaction with other humans. After a while, the natural world gets a little boring, a little predictable, in a way that grand landscapes can’t compensate for. I suppose that’s why I eventually quit biology.

When I traveled to a sparsely populated region of Quebec, earlier this month, I was struck by the finiteness of it all. The villages there often have about 200 inhabitants. With such small populations, everyone is the cousin or the sibling of everyone else — there is no illusion of an infinite number of people. The territory was big, and felt more infinite, but not very much more. After all, we have good maps of everything, so there is very little room for the unknown. And the landscapes, while grand and beautiful, are rarely surprising.

In any case, both living and traveling somewhere “consume” your feelings of infinity. After you have known a place, it’s impossible for that place to generate the feeling again. No longer does Europe, where I lived for a year and a half, feel overwhelming. Gorgeous, interesting, yes, but not infinitely so.

And outer space won’t save us. The universe certainly is infinite in space and time, at least relative to the scales of a human life, but it is also mostly empty. What isn’t empty is mostly homogeneous: star plasma and smaller rock. After reading about astronomy for a bit, there isn’t that much new stuff to see. The vastness of the universe can grant you feelings of infinity, but only for a time, like everything else.

In a finite world, the supply of infinity is necessarily finite.


Geography is the easiest way to illustrate what I’m talking about, but it generalizes far beyond that.

There is a meta layer to every human experience. Traveling to Paris and traveling to Tokyo may feel like two very different activities, but they’re both traveling. Recall the first time you traveled somewhere far and different (of, if you never did that, imagine the first time that you will). The excitement of being in a plane, right before takeoff, for your first flight; the exoticness of seeing everything written in a language you don’t understand. Those things can only be lived once. So even though I have traveled only in a limited part of the world,1a quadrilateral whose extremes are Vancouver in the west, northern Sweden in the north, Israel in the east and Nicaragua in the south I don’t get a strong feeling of infinity anymore when I travel (I still love travelling, however).

And so it goes with every human activity — watching a movie, going to a party, having sex with someone, making a new friend, learning a language, practicing a sport, writing a blog post. You can do these things infinitely many times, and there will always be variations, but they will not provide you with an overwhelmingly new experience each time. For every new type of activity you experience, your world gets a little more finite.

The question becomes: are there enough sufficiently distinct types of activities to sustain a person’s feelings of infinity for their entire life?

It’s possible that there is. Unless something major happens, I expect to be alive for 50 to 60 more years. There are still plenty of experiences I’ve never tried, like raising a child, going to space, trying almost any drug, or leading a company, and it’s conceivable that these things have the potential to fill the rest of my life.

But sometimes, especially when I’m feeling down, I worry that they won’t. That eventually, perhaps soon, I’ll have exhausted my supply, and will almost never feel infinity anymore. It scares me.


I don’t want this post to be depressing, though. Let’s explore some possible ways out of this.

One first path is to manage the resource. If a person’s lifetime feelings of infinity come in a limited supply, it implies that we can decide when and how to consume it, like we can manage a limited supply of money.

This suggests that we should be smart about seeking new experiences. We shouldn’t try to sample all the foods and visit all the countries too fast — lest we become jaded and feel the world shrink. We should carefully manage how much we are exposed to the vastness of the world, so that it remains vast for a long time.

This sounds… fine, I guess. Smart management is good, but it’s also kind of lame. Carefully managing a small amount of money is better than spending it all and cornering yourself into a bad situation — but if you can, it’s far better to make more money.

But perhaps the analogy with money isn’t good, because while money is necessary for good living, feelings of infinity are only a luxury. You can simply accept that at some point you will run out. So another path to dealing with the finiteness of the world is to embrace it. Find beauty and meaning in smallness. Enjoy the minute variations between different instances of an experience — movies, travels, sex, whatever you want — instead of seeking completely novel types of experiences.

I think in general it’s smart to be able to find happiness from a variety of sources, including the mundane. But feelings of awe, of infinity, can be transformative experiences; it would be sad if we got less and less of them as we grow; it would be a great loss.

Maybe there is a way to engineer infinity, to add it to our lives when we lack it. Psychedelic drugs, which I’ve never tried, sound like they may be able to do that. Meditation, too. In fact that may be the main purpose of religion: to construct stories about something so much greater than we are — the divine — in which we can get lost.

Another possible way to engineer infinity is through art. Each new piece of literature, of film, of painting, can be a little fragment of infinity that we get to experience. And there is more art being created than we can consume; it is like the expanding universe, growing faster than we could ever travel even if we reached the speed of light.

Art forms are finite and can feel so after a time. But one great thing about art is that it attempts, by its very nature, to surprise and expand our experiences. The most successful artists make us see in ways we couldn’t before. New forms of art get invented, providing the new types of experiences I was talking about. So art is in some sense infinite — at least as long as artists make sufficient amounts of it.

And this brings us to the only true way to avoid shortages of infinity: expanding human civilization. Make more art. Invent more things. Make more humans, too.

Children seem like a good way to vicariously get feelings of infinity (among other things). Everything is big and impressive for a child; watching them experience the world can give us a glimpse of what we once felt. But the impact of having children is far greater than this.

Having children means continuing and augmenting human civilization. It means more people who will make art and create new ways to experience life. It means more people who will create new subcultures and colonize planets and make more children in their turn to keep the cycle going, until one day we have reached all the stars that we can.

I didn’t expect this essay to become a defence of pronatalism, but here we are. More people is a good thing for many reasons; creating infinity may not be the main argument, but it’s a poetic one, which is maybe more convincing than any of the others ones.


It is night, at the end of summer.

I am at my computer finishing an essay, or perhaps, rather, a meditation, about infinity.

I sit in a fairly small room, with only one window. It doesn’t get enough sunlight, but that doesn’t matter right now.

Outside is a city. Several million people, living complex and beautiful lives. I will never meet all of them.

The city is on a big rock. That rock is the Earth. It is finite, but it is also vast. I will never see all of it.

The Earth is set in a mind-bogglingly large universe. I will never even know what happens in most of it.

Inside my computer is a portal to a vast hypertext library of human knowledge and art. I will never read or see all of it.

I am feeling infinity again. It is a good feeling; I hope I can sustain it for a while.