Commit 9e297c11 authored by mk11g11's avatar mk11g11
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\phantomsection
\pdfbookmark[0]{Title Page}{title}
<!-- Title, name and date -->
\title{\LARGE {\bf Investigation of the selective toxicity of neonicotinoids using the nematode worm Caenorhabditis elegans}\\
\title{\LARGE \textbf {Investigation of the selective toxicity of neonicotinoids using the nematode worm \textit {\textbf {Caenorhabditis elegans}}}\\
\vspace*{6mm}}
\author{Monika Kudelska}
\maketitle
......
......@@ -41,7 +41,7 @@ library(kableExtra)
In 1970s, the scientists of Shell Development Company Biological Research Centre in California identified alpha- DBPN (2-(dibromonitromethyl)-3-(methylpyridine)), first synthesised by Prof. Henry Feuer [@feuer1986]. This lead compound showed low insecticidal activity against aphid and house fly [@tomizawa2003; @tomizawa2005]. Structural alterations of DBPN resulted in production of nithiazine (Figure \@ref(fig:neonics-structure-label)). Nithiazine showed improved insecticidal activity and was particularly effective as a new housefly repellent [@kollmeyer1999]. Further replacement of the thiazine ring by chloropyridinylmethyl (CPM) group, addition of the imidazolidine or its acyclic counterpart, and retention of the nitromethylene group resulted in generation of more potent compounds, one of which, nitenpyram, exhibited particularly high efficacy. Regrettably, both nithiazine and nitenpyram are not useful in fields, as they are unstable in light. The latter however is successfully used in veterinary medicine as an external parasite treatment for cats and dogs.
To solve the issue of photo-instability, nitromethylene group (CCHNO2) was replaced by nitroguanidine (CNNO2) and cyanoamidine (CNCN) (Figure \@ref(neonics-structure-label) and @kagabu1995). These chemical moieties have absorbance spectra at much shorter wavelengths hence do not degrade upon exposure to sunlight. Further alterations, such as replacement of imidazolidine by thiazolidine or oxadiazinane, and/or chloropyridinylmethyl by chlorothiazole or tetrahydrofuran (THF) did not hinder insecticidal activity [@yamamoto1999]. As a result of these modifications, all 6 currently used neonicotinoids were synthesised. They are grouped according to their pharmacophore into N-nitroguanidines, nitromethylenes and N-cyanoamidines (Figure \@ref(fig:neonics-structure-label)). Generally compounds with acyclic- guanidine or amidine and with nitromethylene are more efficacious against moth- and butterfly- pests than those with cyclic counterparts or nitroimine respectively [@ihara2006], nevertheless all are commonly used in agriculture. Imidacloprid, currently the most widely used neonicotinoid, was synthesised in 1970 in Bayer Agrochemical Japan and introduced to the EU market in 1991. Its trade names include Confidor, Admire and Advantage. Together with thiacloprid (Calypso), imidacloprid is marketed by Bayer CropScience. Thiamethoxam (Actara) is produced by Syngenta, Clothianidin (Poncho, Dantosu, Dantop) and Nitenpyram (Capstar) by Sumitomo Chemical, acetamiprid (Mospilan) by Certis, whereas dinotefuran (Starkle) by Mitsui Chemicals company. Last neonicotinoid (dinotefuran) was launched in the EU in 2008.
To solve the issue of photo-instability, nitromethylene group (CCHNO2) was replaced by nitroguanidine (CNNO2) and cyanoamidine (CNCN) (Figure \@ref(fig:neonics-structure-label) and @kagabu1995). These chemical moieties have absorbance spectra at much shorter wavelengths hence do not degrade upon exposure to sunlight. Further alterations, such as replacement of imidazolidine by thiazolidine or oxadiazinane, and/or chloropyridinylmethyl by chlorothiazole or tetrahydrofuran (THF) did not hinder insecticidal activity [@yamamoto1999]. As a result of these modifications, all 6 currently used neonicotinoids were synthesised. They are grouped according to their pharmacophore into N-nitroguanidines, nitromethylenes and N-cyanoamidines (Figure \@ref(fig:neonics-structure-label)). Generally compounds with acyclic- guanidine or amidine and with nitromethylene are more efficacious against moth- and butterfly- pests than those with cyclic counterparts or nitroimine respectively [@ihara2006], nevertheless all are commonly used in agriculture. Imidacloprid, currently the most widely used neonicotinoid, was synthesised in 1970 in Bayer Agrochemical Japan and introduced to the EU market in 1991. Its trade names include Confidor, Admire and Advantage. Together with thiacloprid (Calypso), imidacloprid is marketed by Bayer CropScience. Thiamethoxam (Actara) is produced by Syngenta, Clothianidin (Poncho, Dantosu, Dantop) and Nitenpyram (Capstar) by Sumitomo Chemical, acetamiprid (Mospilan) by Certis, whereas dinotefuran (Starkle) by Mitsui Chemicals company. Last neonicotinoid (dinotefuran) was launched in the EU in 2008.
Research into novel neonicotinoids continues [@shao2013]. In the last decade, several novel insecticides have been characterised and approved for use in the EU. Sulfoxafrol [@zhu2011; @eu2019a] and flupyradifurone [@nauen2015; @eu2019b] have been classified as representatives of new chemical classes, namely sulfoximines and butenolides. However, due to their mode of action and similar biochemical properties, some argue that they are in fact neonicotinoids, whereas their mis-classification has been deliberate to avoid association with neonicotinoids [@pan2019].
......@@ -348,7 +348,7 @@ The effects of neonicotinoids on the neuronal transmission was investigated on i
@sone1994 investigated the effects of imidacloprid on the neuronal activity at the thoracic ganglia of male adult American cockroaches, *Periplaneta americana* using extracurricular recordings. This method allows for a record of changes in spontaneous neuronal activity in response to mechanical or pharmacological interventions. At a very low concentration of 1 nM, imidacloprid induced a sustained for over 2 minutes increase in the rate of neuronal firing. At concentrations ranging from 10 nM to 100 $\mu$M, the following sequence of events was noted: an increase of the rate of spontaneous action potentials of neurons followed by a gradual decline, leading to a complete block of neuronal activity [@sone1994]. Imidacloprid had the same effect on various insect preparations including thoracic ganglion of the Leptinotarsa decemlineata [@tan2008] and on the abdominal ganglion of *Periplaneta americana* [@buckingham1997]. The same observations were made for other neonicotinoids [@thany2009; @schroeder1984]. This provided evidence that neonicotinoids stimulate the nervous system of insects. This conclusion was supported by the behavioural observation, whereby the neonicotinoid intoxication mirrors intoxication seen with cholinergic agents (Section . In response to imidacloprid, insects become hyper excited as evident by excessive pacing. They then collapse and exhibit diminishing uncoordinated leg and abdomen movement until eventual death [@sone1994; @elbart1997; @suchail2001]. Sub-lethal doses (i.e. < 4 nM) have distinct effect, such as an inhibition of feeding leading to starvation [@nauen1995; @elbart1997].
@sattelle1989 used isolated cocroach neuronal preparation to record post-synaptic intracellular currents in response to neonicotinoid prototype 2(nitromethylene) tetrahydro-1, 3-thiazine (NMTHT). NMTHT depolarised the post-synaptic unpaired median neurons and the cell body of motor neurons of the abdominal ganglion. Agriculturally relevant neonicotinoids had the same effect on the post-synaptic membrane of the isolated cocroach thoracic ganglia [@tan2007; @thany2009] potato beetle isolated neurons [@tan2008], and on cultured cocroach [@ihara2006], honeybee [@palmer2013] and fruit fly [@brown2006] neurons. These data provide evidence that neonicotinoids act directly on the post-synaptic neuron in both target and non-target insects.
@sattelle1989 used isolated cocroach neuronal preparation to record post-synaptic intracellular currents in response to neonicotinoid prototype 2(nitromethylene) tetrahydro-1, 3-thiazine (NMTHT). NMTHT depolarised the post-synaptic unpaired median neurons and the cell body of motor neurons of the abdominal ganglion. Agriculturally relevant neonicotinoids had the same effect on the post-synaptic membranes in the isolated cocroach thoracic ganglia [@tan2007; @thany2009] potato beetle isolated thoracic ganglion [@tan2008], terminal abdominal ganglion of the americal cocroach [@ihara2006] and in the honeybee [@palmer2013] and fruit fly [@brown2006] neurons.
Pharmacological characterisation of neonicotinoids-induced currents provided further evidence for their mode of action. The inward current elicited by neonicotinoids were dose-dependent, whereby the higher the concentration, the grater the depolarisation. EC50 values (concentrations at which the half of the maximum current was observed) are in the region of 1 - 5 $\mu$M [@thany2009; @tan2007]. Such low values indicate highly potent action of neonicotinoids on insects, in agreement with toxicological data (Section \@ref(potentpests)). Neonicotinoid-induced currents were reminiscent of those induced by acetylcholine and nicotine, and were prevented by the application of nAChRs antagonists ($\alpha$-bungarotoxin, methyllycaconitine, mecamylamine or d-tubocurarine) not by muscarinic receptor antagonists (atropine, pirenzepine), suggesting neonicotinoid-induced currents are due to the activation of nicotinic receptors.
......@@ -586,7 +586,7 @@ Recombinant insect nAChR are notoriously difficult to express. Several intervent
## General biology ##{#genbiology}
*C. elegans* exists as a male and hermaphrodite, with the latter sex being the more prevalent one. In the lab, 99.9 % of worms are hermaphrodites, which self-fertilize their eggs. *C. elegans* has a fast life-cycle (www.wormbook.org), which is temperature-dependent. At 15^o^C, it takes 5.5 days from egg-fertilization to the development of a worm into an adult. This process is shortened to 3.5 and 2.5 days at 20 and 25 deg;C, respectively (Figure \@ref(fig:life-cycle-label)). At 20 degrees, hermaphrodite lay eggs 2.5 hours after the fertilisation. 8 hours later the embryo hatches as a larvae in the first stage of its development (L1). In the presence of food, larvae develops into an adult through three further developmental stages, namely L2, L3 and L4. The transition between each larval stage is marked by a process of maulting, during which the old cuticle is shed and replaced by a new one. In the absence of food, developing L2 and L3 worms enter the dauer stage. The worms can remain arrested at this low metabolic activity state for up to several weeks, and will develop into adults, should the food re-appear. Hermaphrodites remain fertile for the first three days of their adulthood. Their eggs can be fertilised internally with the sperm produced by the hermaphrodite, or, if there are males available, by mating. Unmated worm can lay up to 350 eggs, whereas mated over a 1000 eggs. Figure \@ref(fig:life-cycle-label) illustrated the full *C. elegans* life cycle.
*C. elegans* exists as a male and hermaphrodite, with the latter sex being the more prevalent one. In the lab, 99.9 % of worms are hermaphrodites, which self-fertilize their eggs. *C. elegans* has a fast life-cycle (www.wormbook.org), which is temperature-dependent. At 15^o^C, it takes 5.5 days from egg-fertilization to the development of a worm into an adult. This process is shortened to 3.5 and 2.5 days at 20 and 25 $^\circ$C, respectively (Figure \@ref(fig:life-cycle-label)). At 20 degrees, hermaphrodite lay eggs 2.5 hours after the fertilisation. 8 hours later the embryo hatches as a larvae in the first stage of its development (L1). In the presence of food, larvae develops into an adult through three further developmental stages, namely L2, L3 and L4. The transition between each larval stage is marked by a process of maulting, during which the old cuticle is shed and replaced by a new one. In the absence of food, developing L2 and L3 worms enter the dauer stage. The worms can remain arrested at this low metabolic activity state for up to several weeks, and will develop into adults, should the food re-appear. Hermaphrodites remain fertile for the first three days of their adulthood. Their eggs can be fertilised internally with the sperm produced by the hermaphrodite, or, if there are males available, by mating. Unmated worm can lay up to 350 eggs, whereas mated over a 1000 eggs. Figure \@ref(fig:life-cycle-label) illustrated the full *C. elegans* life cycle.
(ref:life-cycle) **The life cycle of *C. elegans*.** *C. elegans* develops into an adult through 4 larval stages L1- L4. These stages are separated by molts associated with shedding of an old and exposure of a new cuticle. Adults emerge can lay over a 1000 eggs a day which hatch within several hours. Dauer stage is a metabolic compromised worm stage entered in the absence of food. Upon re-appearance of food, worms develop into L4 and adults normally. Figure taken from www.wormatlas.org.
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## Introduction
Previous chapter describes the effects of nicotine, 5-HT and neonicotinoids on pharyngeal pumping of the cut-head worm, scored by visual observation. An alternative pharyngeal assay is an extracellular recording of an electrical function of the pharynx, or electropharyngeogram (EPG). Unlike scoring pharyngeal pumping by visual observation, EPG recordings allow for resolution of early time points. Additionally, investigations into drug-induced changes of the EPG waveform may inform on the potential mode of action of compounds.
Previous chapter describes the effects of nicotine, 5-HT and neonicotinoids on pharyngeal pumping of the cut-head worm, scored by visual observation (Chapter 4). An alternative pharyngeal assay is an extracellular recording of an electrical function of the pharynx, or electropharyngeogram (EPG). Unlike scoring pharyngeal pumping by visual observation, EPG recordings allow for resolution of early time points. Additionally, investigations into drug-induced changes of the EPG waveform may inform on the potential mode of action of compounds.
In an EPG assay, a worm's head is placed in a recording chamber. The chamber can be perfused, exposing the pharynx to different drug solutions. A tight seal between the electrode and the tip of the *C. elegans* nose is made. Contracting pharyngeal muscle produces currents which flows out of the worm's mouth; this is detected by the electrode. Each cycle of contraction and relaxation gives rise to a single waveform on the trace, known as an EPG. There are several phases constituting an individual EPG (Figure \@ref(fig:example-epg-label)). The beginning of the EPG signal marks excitatory phase. This phase mirrors depolarisation and contraction of the corpus and the terminal bulb and constitutes from 2 spikes. e spike arises due to the release of ACh from MC neurons and an activation of of nAChRs [@raizen1995]. E peak is due to a subsequent calcium channels activation [@lee1997; @shtonda2005]. e is often unseen on the EPG trace due to merging with the larger E. I or inhibitory spikes are diverse in number and amplitude and arise as a result of the inhibitory currents. These currents are produced by ligand-gated chloride channels in response to glutamate release from M3 neurons [@dent1997; @li1997]. Lastly, R and r reflect relaxation of the corpus and the terminal bulb, due to a repolarisation of the terminal bulb muscle cells caused by the flow of potassium through the potassium ion channel [@shtonda2005]. r spikes are frequently merged with larger in amplitude R.
......@@ -233,6 +232,8 @@ Comparing the effects of nicotine on the pharynx as revealed by EPG and visual s
knitr::include_graphics("fig/results3/nic-epg-trace-long-exposure-comb.png")
```
\newpage
### Effects of cytisine
The effects of cytisine, an agonist of nAChR was tested. Cytisine was applied at concentrations ranging from 1 to 100 $\mu$M. As in case of acetylcholine and nicotine, two types of responses were observed. Moderate but sustained stimulation of the pharyngeal activity was elicited by 5 $\mu$M, whereas at concentrations $\ge$ 10 $\mu$M, the pharynx was stimulated and subsequently inhibited (Figure \@ref(fig:cyt-epg-label)). The EC~50~ of cytisine on EPG was 3 $\mu$M (Figure \@ref(fig:cyt-epg-graph-label)).
......@@ -269,6 +270,8 @@ knitr::include_graphics("fig/results3/cyt-traces.png")
knitr::include_graphics("fig/results3/epg-cyt.png")
```
\newpage
### Effects of neonicotinoids
The effects of neonicotinoids on EPG were also examined. Pharynges were exposed to 100 $\mu$M nitenpyram, 50 $\mu$M thiacloprid and 75 $\mu$M clothianidin. Neither nitenpyram (Figure \@ref(fig:Nit-EPG-label)), nor thiacloprid had an effect on the frequency of pharyngeal activity (Figure \@ref(fig:epg-thia-label)). In contract, clothianidin stimulated pharyngeal activity. The frequency increased from 0.6 to 1.1 Hz and returned to basal following 5 minute wash (Figure \@ref(fig:clo-epg-label)). EPGs from clothianidin-perfused pharynges were examined and a reduction of R peak in relation to E peak was noted. Clothianidin significantly increased the E/R ratio from 1.3 to 1.8 (Figure \@ref(fig:clo-er-ratio-label) a and b). 5-minute wash did not reverse this effect. A change in duration of pumping activity was also observed. The latency of EPG decreased from 130 ms to 110 ms when pharynges exposed to clothianidin (Figure \@ref(fig:clo-er-ratio-label) a and c). This effect was not reversed upon 5 minute wash.
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......@@ -107,24 +107,26 @@ It needs to be noticed that although this chapter describes the expression of hu
Two further ECDs, namely honey bee $\alpha5$ and *C. elegans* ACR-21 subunit were cloned. Their expression was driven from plasmids containing 2GSC, as well as two other proteins: 1VR4 and 2GUV. 1VR4 and 2GUV are bacterial proteins of unknown function, shown to form pentamers in *E. coli*. Out of 9 constructs tested, the results from the $\alpha7$ were the most promising.
```{r tesing-constructs, echo=FALSE}
testconstructs <- data.frame(
Construct = c("$\\alpha7$-1VR4", "$\\alpha7$2GUV", "ACR21-2GSC", "ACR21-1VR4", "ACR21-2GUV", "$\\alpha5$-2GSC", "$\\alpha5$-1VR4", "$\\alpha5$-2GUV"),
Expression = c("", "~10 % purified. Remainer lost after 1st spin", "", "", "", "", "", ""),
Purification = c("", "", "", "", "", "", "", ""),
Gel = c("ND", "ND", "ND", "ND", "Aggregates", "ND", "ND", "ND")
)
testconstructs %>%
mutate_all(linebreak) %>%
kable("latex", align = "l", booktabs = TRUE, escape = F,
col.names = linebreak(c("Construct", "Expression", "Purification", "Gel\npurification")),
caption = 'Expression of human, honeybee and C. elegans nAChR extracellular domains in E. coli',
) %>%
kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position")
<!-- ```{r tesing-constructs, echo=FALSE} -->
<!-- library(kableExtra) -->
<!-- library(dplyr) -->
```
<!-- testconstructs <- data.frame( -->
<!-- Construct = c("$\\alpha7$-1VR4", "$\\alpha7$2GUV", "ACR21-2GSC", "ACR21-1VR4", "ACR21-2GUV", "$\\alpha5$-2GSC", "$\\alpha5$-1VR4", "$\\alpha5$-2GUV"), -->
<!-- Expression = c("", "~10 % purified. Remainer lost after 1st spin", "", "", "", "", "", ""), -->
<!-- Purification = c("", "", "", "", "", "", "", ""), -->
<!-- Gel = c("ND", "ND", "ND", "ND", "Aggregates", "ND", "ND", "ND") -->
<!-- ) -->
<!-- testconstructs %>% -->
<!-- mutate_all(linebreak) %>% -->
<!-- kable("latex", align = "l", booktabs = TRUE, escape = F, -->
<!-- col.names = linebreak(c("Construct", "Expression", "Purification", "Gel\npurification")), -->
<!-- caption = 'Expression of human, honeybee and C. elegans nAChR extracellular domains in E. coli', -->
<!-- ) %>% -->
<!-- kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") -->
<!-- ``` -->
\newpage
......@@ -170,7 +172,7 @@ Western Blot was examined closely to establish which condition resulted in the p
knitr::include_graphics("fig/results5/png/induction-of-hua7-2gsc-WB-and-SDS.png")
```
Following overnight protein expression at 18 &deg;C, induced by 0.2 mM IPTG, and 6-hour expression at 37 &deg;C induced with 0.5 mM IPTG, the protein was purified. (Section \@ref:(purification-general-methods)). At each stage of purification the samples were collected to run them on the SDS-PAGE gel. Briefly, purification was done is a three-step process. The cells were precipitated and broken down by sonication to release their content. Homogenised cells were then spun down (low speed spin) to remove the unbroken cells, nucleic acids, organelles and large insoluble cellular particles, such as inclusion bodies (precipant was colected as a Whole Cell sample). The supernatant from this low speed spin was then spun at 100 000g to precipitate cellular organelles. $\alpha7$ ECD in the 100 000g soluble fraction was subsequently resolved using solid phase Ni^2+^-NTA IMAC purification (Section \@ref(his)). Briefly, the soluble fraction was incubated with Ni2+-NTA resin for 2 hours at 4 &dec;C to allow binding of the expressed HIS-tagged $\alpha7$ ECD chimera to beads. The mixture was decanted into the chromatography column. The unbound proteins were collected in the flow-through, before the the beads were washed three times. The wash fractions were pooled and run as “Wash” on the gel. The resin-bound protein was then eluted by washing the column with 5mL of 0.2 mM imidazole, to displace the HIS-tagged protein from the imobilized Nickel by competion. The eluted proteins were collected in two eluate fractions (Eluate 1 and 2).
Following overnight protein expression at 18 &deg;C, induced by 0.2 mM IPTG, and 6-hour expression at 37 &deg;C induced with 0.5 mM IPTG, the protein was purified. (Section \@ref:(purification-general-methods)). At each stage of purification the samples were collected to run them on the SDS-PAGE gel. Briefly, purification was done is a three-step process. The cells were precipitated and broken down by sonication to release their content. Homogenised cells were then spun down (low speed spin) to remove the unbroken cells, nucleic acids, organelles and large insoluble cellular particles, such as inclusion bodies (precipant was colected as a Whole Cell sample). The supernatant from this low speed spin was then spun at 100 000g to precipitate cellular organelles. $\alpha7$ ECD in the 100 000g soluble fraction was subsequently resolved using solid phase Ni^2+^-NTA IMAC purification (Section \@ref(his)). Briefly, the soluble fraction was incubated with Ni2+-NTA resin for 2 hours at 4 $^\circ$C to allow binding of the expressed HIS-tagged $\alpha7$ ECD chimera to beads. The mixture was decanted into the chromatography column. The unbound proteins were collected in the flow-through, before the the beads were washed three times. The wash fractions were pooled and run as “Wash” on the gel. The resin-bound protein was then eluted by washing the column with 5mL of 0.2 mM imidazole, to displace the HIS-tagged protein from the imobilized Nickel by competion. The eluted proteins were collected in two eluate fractions (Eluate 1 and 2).
Representative samples from each of the fractions indicated above were resolved on the SDS-PAGE gel (Figure \@ref(fig:expression-conditions-test-label)) and the presence of recombinant protein was detected by Western Blot with anti-HIS-tag antibodies (Figure \@ref(fig:expression-conditions-test-label) b). Since most of the purification features are common following expression at 18 and 37 &deg;C, some general comments are be made first. Then the comparison between the total purified protein will be made between the two.
......@@ -290,7 +292,7 @@ In adition, to the band representing $\alpha7$ LBD - chimera there was also an i
Western Blot with anti-HIS antibodies detected the presence of HIS-tagged proteins with immunoreactivity consistent with the expressed size of $\alpha7$ ECD - chimera. However, purification results showed that the proportion of induced protein availble for binding was disappointing. The expressed protein was lost during the purifcation procedure, some was precipitated following centrifugation of sonicated cells (Figure \@ref(fig:expression-conditions-test-label) Whole Cell sample), suggesting the formation of inclusion bodies. In addition, some expressed protein failed to bind to the nickel resin, potentially due to misfolding or aggregation. The expression of remaining contstruct was even more challenging, with a smaller proportion of the ECD - chimera purified.
### Analysis of canbdiate expressed protein quaternary structure.
### Analysis of the quaternary structure.
To determine whether the protein was purified as a pentamer, Native-PAGE gel was run, which enables separation of folded and assembled proteins on the gel. Two samples were prepared: one containing denatured by boiling proteins and the other containing non-denatured, un-boiled proteins. A clear staining of high molecular weight proteins was observed in the un-boiled sample, but not in the boiled sample, suggesting purification of multimeric proteins. The presence of high molecular weight protein was not confirmed by the gel filtration. Therefore further experiments are needed to investigate whether expressed $\alpha7$ ECD chimera forms pentameric structures. This could include binding of radio labelled ligands, such as $\alpha-bgtx$ [@barnard1971; @carbonetto1979; @clarke1985].
......
......@@ -2,6 +2,6 @@
(ref:appd) **Structures of soluble homopentameric soluble domains**. Single subunits are color coded and the termini of a single subunits (in purple) are shown. 2GUV and 1VR4 subunts were used as C-terminal tags of nAChR expressed in *E. coli* to prompte pentamerisation. Images generated in USCF Chimera (PDB codes: 2GUV and 1VR4).
```{r eppd-label, fig.cap="(ref:appd)", fig.scap="Structures of soluble homopentameric soluble domains", fig.align='center', echo=FALSE}
```{r appd-label, fig.cap="(ref:appd)", fig.scap="Structures of soluble homopentameric soluble domains", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results5/png/HPSD.png")
```
\ No newline at end of file
book_filename: "thesis-dissertaion" # Change this to the actual title
delete_merged_file: true
rmd_files: ["index.Rmd","00-preface.Rmd", "01-intro.Rmd","02-methods.Rmd", "03-results-01.Rmd", "04-results-02.Rmd", "05-results-03.Rmd", "06-results-04.Rmd", "19-discussion.Rmd", "20-appendix.Rmd","21-appendix-a.Rmd","22-appendix-b.Rmd", "23-appendix-c.Rmd", "24-appendix-d.Rmd", "25-appendix-e.Rmd", "26-appendix-f.Rmd", "99-references.Rmd"]
rmd_files: ["index.Rmd","00-preface.Rmd", "01-intro_2.Rmd","02-methods.Rmd", "03-results-01.Rmd", "04-results-02.Rmd", "05-results-03.Rmd", "06-results-04.Rmd", "19-discussion.Rmd", "20-appendix.Rmd", "21-appendix-a.Rmd", "22-appendix-b.Rmd", "23-appendix-c.Rmd", "24-appendix-d.Rmd", "25-appendix-e.Rmd", "26-appendix-f.Rmd", "99-references.Rmd"]
#rmd_files: ["index.Rmd","00-preface.Rmd", "01-intro.Rmd","02-methods.Rmd", "03-results-01.Rmd", "04-results-02.Rmd", "05-results-03.rmd", "06-results-04.Rmd", "19-discussion.Rmd", "20-appendix.Rmd","21-appendix-a.Rmd","22-appendix-b.Rmd", "23-appendix-c.Rmd", "24-appendix-d.Rmd", "25-appendix-e.Rmd", "26-appendix-f.Rmd", "99-references.Rmd"]
language:
ui:
......
......@@ -17,3 +17,5 @@ curl
usethis
devtools
knitr
extrafont
magick
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