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Commit 5d56d2f0 authored by mk11g11's avatar mk11g11
Browse files

corrections

parent e74fe8cf
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......@@ -16,7 +16,7 @@ The shape and amplitude of a single EPG varies between worms even of the same ge
knitr::include_graphics("fig/results3/basa-epg-individual.png")
```
## Chapter aims
### Chapter aims
In this chapter, the effects of 5-HT and the cholinergic compounds on EPG are investigated. These results are compared to the effects elicited by nicotine and neonicotinoids to further inform on what is their mode of action.
## Results
......@@ -68,9 +68,8 @@ To assess the effects of exogenous drug application on the pharynx an EPG - an e
<!-- ggsave("fig/results3/figure-test.pdf", width = 9, height = 10, units = "cm") -->
<!-- ``` -->
### Effects of 5-HT
### Effects of 5-HT on electropharyngeogram
Application of 5-HT concentrations ranging from 1 nM to 10 $\mu$M led to dose dependent stimulation of the pharyngeal pumping, sustained throughout the 5-minute perfusion (Figure \@ref(fig:epg-5ht-label)). The maximum response achieved was 4.4 Hz by 10 $\mu$M. The EC~50~ was 255 nM. Washing the pharynx for 5 minutes was sufficient to observe recovery from the 5-HT induced stimulation of pumping.
To determine whether 5-HT had an effect on the shape of an EPG, individual EPG waveforms were examined closely (Figure \@ref(fig:5ht-epg-ind-label)a). A visible decrease in duration of the pump in response to 5-HT was noted. To quantify this, the pump duration of EPGs during the basal and treatment recording as well as after 5 minute wash were measured (Figure \@ref(fig:5ht-epg-ind-label)). The direct measurements were made by quantifying the time taken from E to R peak. 1 $\mu$M 5-HT reduced the latency by 27 %. This effect was reversible and returned to basal duration after 5 minutes of washing.
......@@ -100,7 +99,7 @@ knitr::include_graphics("fig/results3/5HT_EPG-all.png")
knitr::include_graphics("fig/results3/5HT_ind_EPG.png")
```
### Effects of Acetylcholine
### Effects of acetylcholine on electropharyngeogram
Acetylcholine was applied at concentrations ranging from 1 to 100 $\mu$M. This led to concentration and time dependent effects on pumping (Figure \@ref(fig:ach-epg-traces-label)). 10 $\mu$M acetylcholine stimulated pumping. This stimulation was sustained for 5 minute perfusion and reversed to basal activity upon washing (Figure \@ref(fig:ach-epg-traces-label) a). Exposure of the pharynx to 15, 25, 50 or 100 $\mu$M acetylcholine led to potent stimulation of the pharyngeal activity before blocking its activity completely. Following, two types of activities were recorded: the pharynxes remained blocked even after 5 minutes of washing (Figure \@ref(fig:ach-epg-traces-label) b), or began pumping again whilst being perfused with acetylcholine (Figure \@ref(fig:ach-epg-traces-label) c). The ratio of pharynxes exhibiting the first or the second type of response to acetylcholine concentrations $\ge$ 15 $\mu$M was 1 : 1 and this was not concentration dependent (data not shown).
......@@ -146,7 +145,7 @@ knitr::include_graphics("fig/results3/ach_train_properties.png")
knitr::include_graphics("fig/results3/epg_ach_graph.png")
```
### Effects of nicotine
### Effects of nicotine on electropharyngeogram
Similarly to ACh, nicotine elicited concentration and time dependent changes to the EPG. At 1 $\mu$M it led to moderate but sustained stimulation of the pharyngeal activity. At higher doses (i.e. 5, 10, 25 and 50 $\mu$M) it caused a potent but transient elevation of pumping frequency followed by an inhibition of the pharyngeal activity (Figure \@ref(fig:epg-nicotine-2-label)). The stimulation by nicotine concentrations ranging from 5 to 50 $\mu$M consisted of a train of EPG spikes which decreased in amplitude with time (Figure \@ref(fig:epg-nicotine-2-label)).
To better understand the nicotine-induced pharyngeal events, a video recording and photos of pharynxes perfused with nicotine at 10 $\mu$M was taken (Supplementary video 3, Figure \@ref(fig:nicotine-photo-label)). In agreement with EPG data, nicotine induced a short-lived train of pharyngeal contractions. Cycles of simultaneous contraction-relaxation of the corpus and the grinder in the terminal bulb can be seen. This suddenly ceases. Following, asynchronous contraction of the isthmus and the grinder began which led to contraction and hence opening of the grinder and isthmus. In addition, twitching of the terminal bulb muscle could be observed throughout.
......@@ -155,9 +154,9 @@ To score for the stimulatory effects of nicotine of pumping, the peak pumping ra
Investigations into the recovery from nicotine-induced pharynx stimulation were made. Following stimulation by 1 $\mu$M nicotine, pharynxes returned to the basal pumping rate within 5 minute wash, those exposed to higher nicotine concentrations remained inhibited (Figure \@ref(fig:nic-epg-traces-label)). To see if pharynxes begin to pump after longer wash, experiment were repeated. Cut heads were exposed to nicotine for 5 minutes, but after this time the washing period was extended until the pharyngeal activity was restored. To score for recovery, the time taken from the beginning of wash to the first EPG spike was taken (Figure \@ref(fig:epg-nicotine-3-label)). It takes 8 minutes or longer to recover from the effects induced by 5 and 10 $\mu$M nicotine. The time needed to recover from 50 $\mu$M perfusion was 18 minutes, suggesting longer washing is required to remove residual nicotine after exposure to higher drug concentrations.
(ref:epg-nic-traces) **Concentration dependent effects of nicotine on EPG frequency.** Cut heads were perfused for 5 minutes in each Dent's saline (basal), nicotine and again in Dent's saline for recovery. Example EPG traces from pharynxes exposed to 1 $\mu$M (a) and 25 $\mu$M nicotine (b). Response to 25 $\pm$M represent responses to nicotine concentrations ranging from 5 to 50 $mu$M.
(ref:epg-nic-traces) **The concentration dependent effects of nicotine on EPG frequency.** Cut heads were perfused for 5 minutes in each Dent's saline (basal), nicotine and again in Dent's saline for recovery. Example EPG traces from pharynxes exposed to 1 $\mu$M (a) and 25 $\mu$M nicotine (b). Response to 25 $\pm$M represent responses to nicotine concentrations ranging from 5 to 50 $mu$M.
```{r nic-epg-traces-label, fig.cap="(ref:epg-nic-traces)", fig.scap = "Concentration dependent effects of nicotine on EPG frequency.", fig.align= 'center', echo=FALSE}
```{r nic-epg-traces-label, fig.cap="(ref:epg-nic-traces)", fig.scap = "The concentration dependent effects of nicotine on EPG frequency.", fig.align= 'center', echo=FALSE}
knitr::include_graphics("fig/results3/epg-nicotine-traces.png")
```
......@@ -234,7 +233,7 @@ knitr::include_graphics("fig/results3/nic-epg-trace-long-exposure-comb.png")
\newpage
### Effects of cytisine
### Effects of cytisine on electropharyngeogram
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)).
......@@ -272,7 +271,7 @@ knitr::include_graphics("fig/results3/epg-cyt.png")
\newpage
### Effects of neonicotinoids
### Effects of neonicotinoids on electropharyngeogram
The effects of neonicotinoids on EPG were also examined. Pharynxes 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 a 5 minute wash (Figure \@ref(fig:clo-epg-label)). EPGs from clothianidin-perfused pharynxes 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 pharynxes exposed to clothianidin (Figure \@ref(fig:clo-er-ratio-label) a and c). This effect was not reversed upon 5 minute wash.
......@@ -384,7 +383,8 @@ knitr::include_graphics("fig/results3/Clo-epg-comb.png")
knitr::include_graphics("fig/results3/clo-ind-epg.png")
```
### Effects of acetylcholine in the presence of nitenpyram and thiacloprid
### Effects of acetylcholine in the presence of nitenpyram and thiacloprid on electropharyngeogram
Neither nitenpyram at 100 $\mu$M nor thiacloprid at 50 $\mu$M impaired on EPG frequency. To determine whether they inhibit the stimulatory effect of acetylcholine on EPG frequency, pharynxes were pre-incubated with nitenpyram or thiacloprid and then exposed to both thiacloprid or nitenpyram and acetylcholine (Figure \@ref(fig:ach-nit-label) and Figure \@ref(fig:ach-thia-label)). Acetylcholine at 10 $\mu$M was tested, because this dose is close to the EC~50~ on EPG frequency. The response of the pharynx to acetylcholine in the presence or absence of neonicotinoids was compared. Pre-exposure of the pharynx to either neonicotinoid did not influence the EPG spike frequency elicited by acetylcholine. In both cases, upon application of acetylcholine, the pumping frequency increased from 0.2 to ~ 1 Hz.
(ref:ach-nit) **Effects of acetylcholine on the EPG frequency in the presence and absence of nitenpyram.** Pharynxes were pre- exposed to 100 $\mu$M nitenpyram. 5 minutes later, 100 $\mu$M nitenpyram and 10$\mu$M acetylcholine were applied. Responses to acetylcholine in the presence of nitenpyram were compared to responses elicited by acetylcholine. Pharyngeal pumping rates were derived by extracting peak response in a 10 second window. Data are mean $\pm$ SEM from 3-8 individual worms collected from paired experiments done on $\ge$ 2 days. Example EPG traces of the pharyngeal responses in the presence or absence of nitenpyram (b and c respectively).
......@@ -438,7 +438,7 @@ knitr::include_graphics("fig/results3/nit+ACH+combined.png")
knitr::include_graphics("fig/results3/thia+ACH+combined.png")
```
### Effects of nicotine on worms deficient in nAChR
### Effects of nicotine on on electropharyngeogram of worms deficient in nAChR
EPG analysis of the effects of nicotine on the pharynx revealed a nicotinic effect not seen in the visual observation experiments. To determine whether the effects of nicotine on EPG on nAChR *eat-2* mutant differs from wild-type, EPG recordings from both strains were obtained. The stimulatory effect of nicotine concentrations ranging from 100 nM to 50 $\mu$M was scored (Figure \@ref(fig:nicotine-epg-eat2-label)). No marked differences in nicotine-sensitivity of *eat-2* mutant vs wild-type worms were noted. The EC~50~ on wild-type was 3 $\mu$M, in comparison to 5 $\mu$M in *eat-2* mutant.
......
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......@@ -6,9 +6,9 @@
knitr::include_graphics("fig/appendix/seq_align_1a.png")
```
(ref:pharacophore-seq) Sequence alignment of the binding pocket of the ligand binding protein and nicotinic acetylcholine receptors. Amino acid sequences from the principal (a) and complementary (b) binding site loops, which form the ligand binding pocket. Residues important for agonist binding are highlighted and color coded as in Figure \@ref(fig:binding-pocket-label). Numbering corresponds to the sequence of the great pond snail acetylcholine binding protein (AChBP). Alignment was generated with MUSCLE [@edgar2004]. Abbreviations used: Ls - *Lymnaela stagnalis* (great pond snail), Am- *Apis mellifera* (honeybee), Mz- *Myzus persicae* (peach aphid), Hs- *Homo sapiens* (human), Gg- *Gallus gallus* (chicken), Ce - *C. elegans*.
(ref:pharacophore-seq) **Sequence alignment of the binding pocket of the ligand binding protein and nicotinic acetylcholine receptors.** Amino acid sequences from the principal (a) and complementary (b) binding site loops, which form the ligand binding pocket. Residues important for agonist binding are highlighted and color coded as in Figure \@ref(fig:binding-pocket-label). Numbering corresponds to the sequence of the great pond snail acetylcholine binding protein (AChBP). Alignment was generated with MUSCLE [@edgar2004]. Abbreviations used: Ls - *Lymnaela stagnalis* (great pond snail), Am- *Apis mellifera* (honeybee), Mz- *Myzus persicae* (peach aphid), Hs- *Homo sapiens* (human), Gg- *Gallus gallus* (chicken), Ce - *C. elegans*.
```{r pharacophore-seq-label, fig.cap="(ref:pharacophore-seq)", fig.scap="Sequence alignment of the ligand binding pocket of the AchBPs and nAChRs.", fig.align='center', echo=FALSE, fig.pos='H'}
```{r pharacophore-seq-label, fig.cap="(ref:pharacophore-seq)", fig.scap="Sequence alignment of the ligand binding pocket of the AChBPs and nAChRs.", fig.align='center', echo=FALSE, fig.pos='H'}
knitr::include_graphics("fig/appendix/seq_align_1b.png")
```
......@@ -2,7 +2,7 @@
\newpage
(ref:app2) Sequencing of *myo-2-eat-2* from the *pDEST* vector. Myo-2::eat-2 nucleotide fragment from the expression vector used to generate *C. elegans* transgenic strains was sequenced following cloning. 3 forward (Fw) and a reverse (Rev) primer were used to generate overlaping sequencing fragments spaning the entire sequence of interets (a). Sequencing results authenticated the identity of the construct (b) and confirmed the amino acid sequence of the *eat-2* gene.
(ref:app2) **Sequencing of *myo-2-eat-2* from the *pDEST* vector.** Myo-2::eat-2 nucleotide fragment from the expression vector used to generate *C. elegans* transgenic strains was sequenced following cloning. 3 forward (Fw) and a reverse (Rev) primer were used to generate overlaping sequencing fragments spaning the entire sequence of interets (a). Sequencing results authenticated the identity of the construct (b) and confirmed the amino acid sequence of the *eat-2* gene.
```{r echo=FALSE, out.height = '80%'}
knitr::include_graphics("fig/results4/PNG/1-myo2-eat-2.png")
......@@ -21,7 +21,7 @@ knitr::include_graphics("fig/results4/PNG/4-myo2-eat2.png")
```
\newpage
```{r app2-label, fig.cap="(ref:app2)", fig.scap= "Sequencing of \\textit{myo-2-eat-2} from the \\textit{pDEST} vector", include="TRUE", results="show", echo=FALSE}
```{r app2-label, fig.cap="(ref:app2)", fig.scap= "Sequencing of \\textit{myo-2-eat-2} from the \\textit{pDEST} vector", fig.align='center', include="TRUE", results="show", echo=FALSE}
knitr::include_graphics("fig/results4/PNG/5-myo2-eat2.png")
```
......@@ -2,7 +2,7 @@
\newpage
(ref:app1) Sequencing of *pmyo2-CHRNA7* from the *pDEST* expression vector.
(ref:app1) **Sequencing of *pmyo2-CHRNA7* from the *pDEST* expression vector.**
```{r out.height = '80%', echo=FALSE}
knitr::include_graphics("fig/results4/PNG/1-myo2-chrna7.png")
......@@ -20,6 +20,6 @@ knitr::include_graphics("fig/results4/PNG/3-myo2-chrna7.png")
knitr::include_graphics("fig/results4/PNG/4-myo2-chrna7.png")
```
```{r app1-lbl, fig.cap="(ref:app1)",, fig.scap = "Sequencing of \\textit{myo-2-$\\alpha$-7} from the pDEST vector", fig.align='center', out.height = '80%', echo=FALSE}
```{r app1-lbl, fig.cap="(ref:app1)", fig.scap = "Sequencing of \\textit{pmyo2-CHRNA7} from the \\textit{pDEST} vector", fig.align='center', out.height = '80%', echo=FALSE}
knitr::include_graphics("fig/results4/PNG/5-myo2-chrna7.png")
```
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# DNA sequence used for the expression of human $\alpha7$ extracellular domain in *E. coli*
@nauen1996
@neher1995
@nguyen1995
@niacaris2003
@noda1982
@noda1983
@okkema1993
@orr1990
@partridge2008
@pereira2015
@perry2008
@petzold2011
@planson2003
@putrenko2005
@raizen1994
@raizen1995
@rand1984
@rand1985
@rand1989
@reynolds1978
@richmond1999
@rogers2006
@rosano2014
@ruan2009
@ruaud2006
@salom2012
@sattelle1981
@sattelle1983
@sattelle2005
<!-- ```{r} -->
<!-- library(kableExtra) -->
<!-- library(dplyr) -->
<!-- footnotew <- ("Receptors were expressed in Xenopus oocytes") -->
<!-- footnotex <- ("Rn = \\textit{Rattus norvegicus} (rat), Gg = \\textit{Gallus gallus} (chicken), Dm = \\textit{Drosophila melanogaster} (fruit fly), Nl = \\textit{Nilaparvata lugens} (planthopper), Cf = \\textit{Ctenocephalides felis} (cat flea)), Lc = \\textit{Lucilia cuprina} (sheep blowfly)") -->
<!-- potencyrecombinant <- data.frame( -->
<!-- Receptor = c("Nl$\\alpha1$/Rn$\\beta2$", "Nl$\\alpha2$/Rn$\\beta2$", "Nl$\\alpha3$/Rn$\\beta2$", "Nl$\\alpha3\\alpha8$/Rn$\\beta2$", "", "", "", "Dm$\\alpha1$/Gg$\\beta2$", "", "", "", "Dm$\\alpha2$/Gg$\\beta2$", "", "", "", "Cf$\\alpha1$/Gg$\\beta2$", "", "", "", "Cf$\\alpha2$/Gg$\\beta2$", "", "", "", "Cf$\\alpha4$/Gg$\\beta2$", "", "", ""), -->
<!-- Compound = c("Imidacloprid", "Imidacloprid", "Imidaclorprid", "Imidacloprid", "Clothianidin", "Thiacloprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram", "Imidacloprid", "Clothianidin", "Acetamiprid", "Nitenpyram"), -->
<!-- EC50 = c("61", "870", "350", "3.2", "5.1", "2.8", "5.6", "0.04", "0.34", "0.23", "0.4", "0.84", "5.4", "2", "35.4", "0.02", "0.15", "0.11", "0.63", "1.31", "1.65", "2.63", "24.4", "13.8", "21.3", "9.4", "45.8"), -->
<!-- References = c("Liu et al. 2009", "", "", "Yixi et al. 2009", "", "", "", "Dederer et al. 2011", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "", "")) -->
<!-- potencyrecombinant %>% -->
<!-- mutate_all(linebreak) %>% -->
<!-- kable("latex", align = "c", booktabs = TRUE, escape = F, -->
<!-- col.names = linebreak(c("Receptor", "Compound", "EC50\n($\\mu$M)", "Rerefence")), -->
<!-- caption = 'The potency of neonicotinoids on recombinantly expressed insect hybrid nAChRs.') %>% -->
<!-- kable_styling(position = "center", full_width = FALSE, latex_options = "hold_position") %>% -->
<!-- add_footnote(notation = "none", c(footnotew, footnotex), -->
<!-- threeparttable = T) -->
<!-- ``` -->
<!-- (ref:appe) **Sequencing of *pelB-3C* cloned into *pET27* expression vector.** Inserted into *pET27 pelB-3C* sequence was sequenced using universal T7 forward and T7 terminator primers (a). The cloned sequence (Query) was compared to the expected sequence (Subject) (b). Single nucleotide mutation from A to C occured, highlighted in red, changing the codon from GCC to GCA, both of which encode for alanine. The cloned nucleotide sequence was translated (c) and major functional domains, as well highlighted. -->
(ref:appe) **Sequencing of *pelB-3C* cloned into *pET27* expression vector.** Inserted into *pET27 pelB-3C* sequence was sequenced using universal T7 forward and T7 terminator primers (a). The cloned sequence (Query) was compared to the expected sequence (Subject) (b). Single nucleotide mutation from A to C occured, highlighted in red, changing the codon from GCC to GCA, both of which encode for alanine. The cloned nucleotide sequence was translated (c) and major functional domains, as well highlighted.
<!-- \newpage -->
\newpage
<!-- ```{r out.height = '90%', fig.align='center', echo=FALSE} -->
<!-- knitr::include_graphics("fig/results5/png/pelb-3c_seq_1.png") -->
<!-- ``` -->
```{r out.height = '90%', fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results5/png/pelb-3c_seq_1.png")
```
<!-- ```{r fig.align='center', echo=FALSE} -->
<!-- knitr::include_graphics("fig/results5/png/pelb-3c_seq_2.png") -->
<!-- ``` -->
```{r fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results5/png/pelb-3c_seq_2.png")
```
<!-- ```{r pelb-3c-lbl, fig.cap = "(ref:appe)", fig.scap= "Sequencing of \\textit{pelB-3C} cloned into \\textit{pET27} expression vector", fig.align='center', echo=FALSE} -->
<!-- knitr::include_graphics("fig/results5/png/pelb-3c_seq_3.png") -->
<!-- ``` -->
\ No newline at end of file
```{r pelb-3c-lbl, fig.cap = "(ref:appe)", fig.scap= "Sequencing of \\textit{pelB-3C} cloned into \\textit{pET27} expression vector", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results5/png/pelb-3c_seq_3.png")
```
\ No newline at end of file
......@@ -3,11 +3,11 @@ output:
pdf_document: default
html_document: default
---
# Sequencing of the DNA sequence used for the expression of of the $\alpha7$ ECD-2GSC
# Sequencing of the DNA sequence used for the expression of of the $\alpha7$ chimera protein
\newpage
(ref:appf) **Sequencing of $\alpha7$ ECD-2GSC cloned into pET27-pelB-3C expression vector.** Inserted into pET27-pelB-3C vector $\alpha7$ ECD-2GSC was sequenced using universal T7 Terminator reverse primer and primer within the 3' region of MBP (a). The cloned sequence (Query) was compared to the expected sequence (Subject) (b). The sequence was translated and merged with the pel-3C to depict the entire protein expressed (c). The major domains are highlighted, as well as the mutated Cys-loop (in red).
(ref:appf) **Sequencing of *$\alpha7$ ECD-2GSC* cloned into *pET27-pelB-3C* expression vector.** Inserted into *pET27-pelB-3C* vector *$\alpha7$ ECD-2GSC* was sequenced using universal T7 Terminator reverse primer and primer within the 3' region of MBP (a). The cloned sequence (Query) was compared to the expected sequence (Subject) (b). The sequence was translated and merged with the pel-3C to depict the entire protein expressed (c). The major domains are highlighted, as well as the mutated Cys-loop (in red).
```{r echo=FALSE}
knitr::include_graphics("fig/results5/png/alpha7-2gsc-seq1.png")
......@@ -17,6 +17,6 @@ knitr::include_graphics("fig/results5/png/alpha7-2gsc-seq1.png")
knitr::include_graphics("fig/results5/png/alpha7-2gsc-seq2.png")
```
```{r appe-label, fig.cap = "(ref:appf)", fig.scap = "Sequence of the $\\alpha7$ ECD-2GSC", echo=FALSE}
```{r appe-label, fig.cap = "(ref:appf)", fig.scap = "Sequence of the $\\alpha7$ \\textit{ECD-2GSC} cloned into \\textit{pET27-pelB-3C} expression vector.", fig.align='center', echo=FALSE}
knitr::include_graphics("fig/results5/png/alpha7-2gsc-seq3.png")
```
book_filename: "thesis-dissertaion" # Change this to the actual title
delete_merged_file: true
rmd_files: ["index.Rmd", "00-preface.Rmd", "26-appendix-f.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_2.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", "27-appendix-g.Rmd" "99-references.Rmd"]
language:
ui:
chapter_name: "Chapter "
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