updates

00-preface.Rmd

 \phantomsection
 \pdfbookmark[0]{Title Page}{title}
  <!-- Title, name and date -->
- \title{\LARGE {\bf My awesome thesis dissertaion title}\\
+ \title{\LARGE {\bf Investigation of the selective toxicity of neonicotinoids using the nematode worm Caenorhabditis elegans}\\
   \vspace*{6mm}}
  \author{Monika Kudelska}
 \maketitle

02-methods.Rmd

@@ -433,7 +433,7 @@ Protein samples were subject to denaturing SDS-PAGE and stained with the Commass
 
 BioRad electrophoresis apparatus was used. Electrophoresis chamber was filled with the running buffer (25mM Tris, 192mM glycine, 0.1 % SDS) and the electrophoresis proceeded at 80 V for 1 hour and then 120V for 2 hours. The gel was either stained to visualise all proteins present in samples, or used in Western Blot to detect the presence of a specific protein.
 
-#### Coomassie staining and imaging #### {#coomassiestaining}
+#### Coomassie staining and imaging ####{#coomassiestaining}
 Following SDS-PAGE electrophoresis, the stack and resolving gel were incubated in fixing buffer (10% acetic acid, 40% ethanol, 50 % dH2O) placed on an oscillating platform for at least 1 hour to remove gel buffer components. The fixed gel was then washed with dH2O and incubated with Coomassie stain (14 mg of Coomassie Blue R-250 (ThermoScientific) /L of dH~2~O) overnight. The gel was de-stained by incubation with dH~2~O for at least 8 hours and imaged with Gel Doc^TM^ XR+ (Bio-Rad). 
 
 ### Analysis of protein sizes using one-dimensional non-denaturing polyacrylamide gel electrophoresis 
@@ -445,7 +445,7 @@ Two protein eluate samples were collected from the Immobilized metal affinity ch
 Nondenaturing gel (12 % Acrylamide/bisacrylamide, 1 M Tris pH = 8.4, 0.06 % ammonium persulfate,  0.13 % TEMED) was used to resolve the size of proteins. 
 
 ##### Gel electrophoresis
-BioRad electrophoresis apparatus was used. Electrophoresis chamber was filled with the running buffer (25mM Tris, 192mM glycine) and the electrophoresis proceeded at 80 V for 1 hour and then 120V for 2 hours. The gel was stained and imaged as described in Section @\ref(coomassiestaining) 
+BioRad electrophoresis apparatus was used. Electrophoresis chamber was filled with the running buffer (25mM Tris, 192mM glycine) and the electrophoresis proceeded at 80 V for 1 hour and then 120V for 2 hours. The gel was stained and imaged as described in Section \@ref(coomassiestaining) 
 
 ### Western Blots ###{#western}
 
@@ -505,7 +505,7 @@ kable("latex", booktabs = T, escape = F,
 
 (ref:protein-standard) **Gel filtration of protein markers. Data derived from the spectra and normalised to the dextran void.**
 
-```{r protein-standard-label, fig.cap = "(ref:protein-standard)", fifg.scap = "Gel filtration of protein markers.", fig.align = 'center',  echo=FALSE, fig.pos = 'H'}
+```{r protein-standard-label, fig.cap = "(ref:protein-standard)", fig.scap = "Gel filtration of protein markers.", fig.align = 'center',  echo=FALSE, fig.pos = 'H'}
 knitr::include_graphics("fig/methods/gel-filtration-spectra.png")
 ```
 
@@ -556,7 +556,7 @@ All experiments, with the exception of the development assay, were performed on
 All assays were performed in M9 medium. M9 buffer composition is (g/litre): 6g Na~2~HPO~4~, 3g KH~2~PO~4~, 5g NaCl, 0.25g MgSO~4~.H~2~O. Worms were exposed to varying indicated concentrations of nicotine or neonicotinoids for a maximum period of 2 hours. The effects of these compounds on locomotion and feeding was scored. 
 10 x stock concentrations nicotine, nitenpyram and 5-HT were added to the assay to give the indicated final concentration. To keep the concentration of DMSO below the concentration that have known effects the stocks of thiacloprid and clothianidin in 100 % DMSO were used in 1 in 200 (0.5%), dilution and mixed vigorously with buffer.
 
-#### Effects of drugs on of intact *C. elegans* in liquid #### {#thrashing}
+#### Effects of drugs on of intact *C. elegans* in liquid ####{#thrashing}
 Whilst in liquid, worms exhibit rhythmical swimming-like behaviour known as thrashing. A single thrash was defined as a complete bend in the mid-point of the body. Experiments were performed in a 24-well plate filled with 450 or 497.$\mu$L buffer. 50 $\mu$L nicotine/ nitenpyram/vehicle or 2.5 $\mu$L thiacloprid/clothianidin was added to the final volume of 500 $\mu$L to achieve final desired concentration.  
  
 Worms were picked off the food and transferred to the experimental arena. After 5 minutes of acclimatization to allow recovery from mechanical transfer, the first thrashing count was performed (time 0). This provided baseline thrashing for each worm. Only thrashing worms were included in the analysis. After estimation control thrashing wells were supplemented with drug / vehicle. For experiments with 1.5 mM thiacloprid, worms were transferred in a small volume of liquid (~2 $\mu$L) from the control to the experimental well by pipetting. This method was adopted due to drug’s limited solubility. It must be noted that after a period of about 40 minutes, 1.5 mM thiacloprid began to visibly precipitate. Measurements were taken for 30 seconds typically at time points: 10, 30, 40, 60 and 120 post-addition of the drug/drug vehicle. At least three independent repeats for each condition were carried out. The number of worms in each experiment varied from 2-6.
@@ -659,11 +659,11 @@ knitr::include_graphics("fig/results3/liquid_basal_modified.png")
 
 ### Stimulatory effects of drugs on pharyngeal pumping of dissected *C. elegans*
 
-#### Effects of 5-HT #### {#cuthead}
+#### Effects of 5-HT ####{#cuthead}
 Following dissection, the heads were placed in Dent's solution. After 5 minutes the initial count of pharyngeal pumping was made and heads were transferred to a drug containing well. Pharyngeal pumping was estimated at 10, 20, 30, 60 minutes after being placed in the drug. Heads were transferred to Dent’s solution for recovery and pumping measured 30 mins later (90 minutes after starting the measurements). As a negative control, worms were incubated in buffer throughout the duration of the experiment. 
 
 #### Effects of neonicotinoids and nicotine 
-Experiments were set up as described above (\@ref(cuthead-5ht)) but the time points were: 0 (Dent's), 2, 5, 10, 15, 20, 30, 45, 60 65, 75, 90 and 120 (treatment) and 130 (recovery). As a control, cut heads were incubated with a 5-HT concentration eliciting maximal response. That is 1 $\mu$ for wild-type N2 worms and 50 $\mu$M for *eat-2* mutant worms. 
+Experiments were set up as described above (Section \@ref(cuthead-5ht)) but the time points were: 0 (Dent's), 2, 5, 10, 15, 20, 30, 45, 60 65, 75, 90 and 120 (treatment) and 130 (recovery). As a control, cut heads were incubated with a 5-HT concentration eliciting maximal response. That is 1 $\mu$ for wild-type N2 worms and 50 $\mu$M for *eat-2* mutant worms. 
 
 ### Inhibitory effects of drugs on pharmacologically induced pharyngeal pumping of dissected *C. elegans*
 The effects of compounds on 5-HT stimulated pharyngeal pumping was tested.

03-results-01.Rmd

@@ -15,11 +15,10 @@ library(knitr)
 ## Introduction
 Neonicotinoids are the most commonly used insecticides worldwide due to their high efficacy against pest insects (Section \@ref(potentpests)), selective toxicity to insect pests over mammals (Section \@ref(seltox)) and advantageous physicochemical attributes (Section \@ref(physchem)). The main disadvantage of these compounds is that they can be toxic to non-target species, including bees (Section \@ref(sublethal)). This undesired ecotoxicological effect spurred a debate over their environmental impact and revealed a necessity to further investigate their effects on other ecologically important organisms  such as worms. 
 
-## Soil worms and nematodes
-Soil worms includes segmented worms representing Phylum Annelida and round worms from Nematoda phylum. The most common representatives of this phylum are the redworm *Eisenia fetida* and the common earthworm *Lumbricus terrestris*. All annelids are free-living, whereas Nematodes are classified as parasitic and non-parasitic. Parasitic worms living in the soil can be divided into those that infect plans or animals. These nematodes can cause large health, life-stock and agricultural loses. For example, *Melyidogyne* species including *Meloidogyne incognita*, account for 95 % of all plant infestation species and cause a loss of 5 % of global crops [@taylor1978] including loss tomatoes, cowpea and blackpepper. 
 
-### Ecological role of non-parasitic worms
-Non parasitic earth worms and nematodes, play an important biological role. They are the most abundant multicellular organisms on earth and are significant biomass contributors. In addition, they cycle nutrients contributing as much as 1/5 of all bioavailable nitrogen in soil [@neher2001], promoting plant growth [@ingham1985] and soil fertility. They are also valuable bioindicators and have been used in assessment of contaminated soil [@lecomte-pradines2014]. 
+## Ecological role of non-parasitic worms 
+
+non parasitic earth worms and nematodes, play an important biological role. They are the most abundant multicellular organisms on earth and are significant biomass contributors. In addition, they cycle nutrients contributing as much as 1/5 of all bioavailable nitrogen in soil [@neher2001], promoting plant growth [@ingham1985] and soil fertility. They are also valuable bioindicators and have been used in assessment of contaminated soil [@lecomte-pradines2014]. 
 
 ### Residues of neonicotinoids in soil 
 Neonicotinoids are commonly applied as a seed dressing [@jeschke2011; @alford2017], due to a benefit of extended crops protection resulting in a reduction of the insecticide application frequency. However, on average, only 5 % of the active ingredient is taken up by and distributed throughout the developing plant [@sur2003]. The remainder enters the wider environment, including soils, where they can have a negative effect on inhabiting worm species. 
@@ -62,7 +61,7 @@ Egg-laying is controlled by the contraction of vulvar muscles under the influenc
 knitr::include_graphics("fig/intro_2/vulva.jpg")
 ```
 
-### Pharmacology 
+### Pharmacology ###{#pharmacelegans}
 Pharmacological experiments in which nAChR agonists were tested against *C. elegans* behaviours provide evidence for the important role of these receptors in the regulation of locomotion and egg laying. 
 
 #### Levamisole
@@ -75,11 +74,13 @@ Nicotine is an exhogenous agonist, naturally occurring in Tobacco plant. Nicotin
 
 <!-- Most ecotoxicological studies focused on the effects of neonicotinoids on behaviours governed by the cholinergic neurotransmission and worms mortility. These were conducted on earthworms, Lumbricus terrestris and Eisenia fetida, which reflects the pivotoal ecological role of these “undrgrdound dwellers”. @basley2017 showed that field realistic concentrations of clothianidin have no effects on Lumbricus terrestris mortality, feeding and worm population [@basley2017], but it is toxic to Eisenia fetida with LC50 value of ~1 mM [@wang2012]. -->
 
-#### Neonicotinoids 
+#### Neonicotinoids ####{#chapter3effectsofneonics}
 <!-- There is a limited literature regarding the effects of neonicotinoids on nematodes. Studies by [@dong2014; @dong2017] revealed antiparasytic potential of neonicotinoids. Thiacloprid kills plant parasite *Meloidogyne incognita* with the LC50 of 24 $\mu$M [@dong2014]  -->
 
 <!-- Thiaclopand inhibits its egg hatching with the EC~50~ of 300 $\mu$M [@dong2014; @dong2017]. -->
-Insecticides neonicotinoids have variable effects on *C. elegans*. mM concentrations of thiacloprid and clothianidin have a negative effect on reproduction [@gomez-eyles2009]. Imidacloprid at 20 nM increases the rate of egg-laying [@ruan2009], whereas at 120 $\mu$M to 2 mM it inhibits motility [@mugova2018]. In contrast, 2 to 40 $\mu$M thiacloprid elevates motility [@hopewell2017]. 
+Neonicotinoids have variable effects on *C. elegans*. @mugova2018 reports an inhibitory effect on motility of imidacloprid at concentration ranging from 120 $\mu$M to 2 mM. Thiacloprid seems to have an opposite effect. At concentrations ranging from 2 to 40 $\mu$M it elevates locomotion in liquid of mixed developmental stage population of *C. elegans* [@hopewell2017]. 
+
+Variable effects of neonicotinoids on egg-laying are also reported. Low mM concentrations of clothianidin and thiacloprid inhibit egg-laying [@gomez-amaro2015]. In contrast, imidacloprid at a single concentration of 20 nM, elevates the number of egg-laid, but has no effect at 120 $\mu$M - 2 mM, suggesting this effect is not dose-dependent [@ruan2009]. 
 
 #### *C. elegans* nAChRs ####{#muscletypenachr} 
 The identity of two muscle-type nAChRs was depicted in a combination of behavioural, pharmacological and electrophysiological approaches. 
@@ -140,10 +141,9 @@ knitr::include_graphics("fig/results2/final/pngpdf/fig2.png")
 
 \newpage
 
-### Effects of neonicotinoids on thrashing
+### Effects of neonicotinoids on thrashing ###{#effectsofneonicsonthrashing}
 To assess the effects of neonicotinoids on motility of worms in liquid, the thrashing experiment was repeated with nitenpyram, thiacloprid and clothianidin. Out of the three compounds tested, only nitenpyram at concentrations ranging from 1 to 100 mM induced concentration-dependent paralysis of N2 wild-type worms. Low water solubility of thiacloprid and clothianidin limited the maximum testable doses to 1.5 and 2.5 mM, respectively. Results in (Figure \@ref(fig:thrashing-tc-comp-label), left panel), show that at these doses neither of the two have an effect on thrashing of wild-type worm.
-To determine whether a cuticle also limits the bioavailability of neonicotinoids, experiments  were repeated on *bus-17* mutant. Shift in potency of all compounds was noted (Figure \@ref(fig:thrashing-tc-comp-label) and \@ref(fig:DR-neonics-label)). The EC~50~ of nitenpyram on wild-type increased by almost 12-fold on mutant worm (195.8 (95% CI= 133.9 to 313.9) and 16.6 (95% CI= 12.0 to 22.6) mM). Thiacloprid and clothianidin were with no effects on wild-type worms, but induced paralysis of the *bus-17* mutant with the EC~50~ of 377.6 $\mu$M (95% CI= 311.8 to 454.0 μM) and 3.5 mM (95% CI= 24.1 to 53.5mM), respectively. The time course for both clothianidin and thiacloprid have similar features: gradual increase in inhibition of thrashing with the maximal effect achieved after 1 hour followed by a slow retrieval. The gradual recovery might represent adaptation of the neuronal circuit for locomotion, desensitization of receptors mediating the response or precipitation of a drug (although no visual sigh of this were observed with exception of 1.5 mM thiacloprid after 45 minutes). The  breakdown in liquid is unlikely, as both compounds have long half-live in water (ref to table in the general introduction) [@gilbert2010].
-SIMILARITIES IN THE INHIBITION WAS ALSO NOTED - UNCOORDINATED MOVEMENT WITH TWITCHING MORIONS SEEN ON THE VIDEO 
+To determine whether a cuticle also limits the bioavailability of neonicotinoids, experiments  were repeated on *bus-17* mutant. Shift in potency of all compounds was noted (Figure \@ref(fig:thrashing-tc-comp-label) and \@ref(fig:DR-neonics-label)). The EC~50~ of nitenpyram on wild-type increased by almost 12-fold on mutant worm (195.8 (95% CI= 133.9 to 313.9) and 16.6 (95% CI= 12.0 to 22.6) mM). Thiacloprid and clothianidin were with no effects on wild-type worms, but induced paralysis of the *bus-17* mutant with the EC~50~ of 377.6 $\mu$M (95% CI= 311.8 to 454.0 $\mu$M) and 3.5 mM (95% CI= 24.1 to 53.5mM), respectively. The time course for both clothianidin and thiacloprid have similar features: gradual increase in inhibition of thrashing with the maximal effect achieved after 1 hour followed by a slow retrieval. The gradual recovery might represent adaptation of the neuronal circuit for locomotion, desensitization of receptors mediating the response or precipitation of a drug (although no visual sigh of this were observed with exception of 1.5 mM thiacloprid after 45 minutes). The breakdown in liquid is unlikely, as both compounds have long half-live in water (Table \@ref(tab:properties)) [@gilbert2010].
 
 (ref:thrashing-tc-comp-capt) **The concentration and time dependence of neonicotinoids inhibition of *C. elegans* thrashing.** Wild type (left panel) and *bus-17* (right panel) worms were acutely exposed to varying concentrations of nitenpyram, thiacloprid, clothianidin or drug vehicle (0, Ctr). The number of thrashed over 30 seconds at indicated time points was scored. Data are mean  $\pm$ SEM of $\ge$ 6 individual worms collected from paired experiments done on $\ge$ 2 days.
 
@@ -284,7 +284,7 @@ avoid <-  on_plate_dat_trans_1 %>%
 
 \newpage
 
-#### Effects on body bends 
+#### Effects on body bends ####{#bodybendsneonics}
 Whilst on solid medium, *C. elegans* exhibits sinusoidal movement (ref to intro). This can be quantified by counting a number of forward body bends per unit of time and is a measure of the motor function. The presence of food modifies this behavior (ref to intro), therefore the measurements were made on treatment- soaked solid medium containing no OP50 food patch (reference to method).
 
 Untreated wild-type worms move at a rate of 39 body bends per minute (Figure \@ref(fig:BB-plot-label), left panel). This is reduced to 33 bends per minute in *bus-17* mutant (Figure \@ref(fig:BB-plot-label) right panel), due to a reduced traction of the body on agar medium [@yook2007]. The  body bends of wild-type *C. elegans* was altered by nicotine with with the EC~50~ of 3.6 mM (95 % CI= 2.6 to 4.4 mM), whereas nitenpyram, thiacloprid and clothianidin had no effect (Figure \@ref(fig:BB-plot-label) and \@ref(fig:DR-body-bends-label)). In contrast, the body bends rate of *bus-17* mutant was reduced by all compounds, except for up to the 1 mM nitenpyram. The EC~50~ for the effects of nicotine and clothianidin was 1.6 (CI= ) and 3.3 (CI = ) mM, respectively. Thiacloprid was the most potent with the EC~50~ of 721.2 $\mu$M (95 % CL= 502.4 μM to 1.0 mM) (Figure \@ref(fig:DR-body-bends-label)).
@@ -840,4 +840,3 @@ Egg-hatching      Nicotine             N2        > 1mM
 ------------------------------------------------------
 
 Table: (\#tab:discuss1-summary-table) Summary table of the effects of nicotine and neonicotinoids on *C. elegans*. 
-First results chapter.

21-appendix-a.Rmd

-# Title of appendix
+# Pharmacophore of the nicotinic acetylcholine receptor 
+
+\newpage
+
+```{r echo=FALSE, fig.pos='H', fig.align='center', out.width="70%"}
+knitr::include_graphics("fig/appendix/seq_align_1a.png")
+```
+
+(ref:pharacophore-seq) Sequence alignment of the ligand binding pocket of the AChBPs and nAChRs. Amino acid sequences from the principal (a) and complementary (b) binding site loops, which form the ligand binding pocket. Residues important for binding are highlighted and color coded as in Figure \@ref(fig:binding-pocket-label). Numering corresponds to the sequence of Ls 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'}
+knitr::include_graphics("fig/appendix/seq_align_1b.png")
+```
 
-First appendix

22-appendix-b.Rmd

-# Title of appendix
+# DNA sequence used for the expression of *eat-2* nAChR subunit in the pharyngeal muscle of *C. elegans*. 
+
+\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.   
+
+```{r echo=FALSE, out.height = '80%'}
+knitr::include_graphics("fig/results4/PNG/1-myo2-eat-2.png")
+```
+
+```{r echo=FALSE}
+knitr::include_graphics("fig/results4/PNG/2-myo2-eat2.png")
+```
+
+```{r echo=FALSE}
+knitr::include_graphics("fig/results4/PNG/3-myo2-eat2.png")
+```
+
+```{r echo=FALSE}
+knitr::include_graphics("fig/results4/PNG/4-myo2-eat2.png")
+```
+
+```{r app2-label, fig.cap="(ref:app2)", fig.scap= "Sequencing of myo-2-eat-2 from the pDEST vector", fig.align='center', include="TRUE", results="show"}
+knitr::include_graphics("fig/results4/PNG/5-myo2-eat2.png")
+```
+
 
-Second appendix 
\ No newline at end of file

23-appendix-c.Rmd

+# DNA sequence used for the expression of human $\alpha$ 7 nAChR subunit in the pharyngeal muscle of *C. elegans*. 
+
+\newpage
+
+(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")
+```
+
+```{r out.height = '80%', echo=FALSE}
+knitr::include_graphics("fig/results4/PNG/2-myo2-chrna7.png")
+```
+
+```{r out.height = '80%', echo=FALSE}
+knitr::include_graphics("fig/results4/PNG/3-myo2-chrna7.png")
+```
+
+```{r out.height = '80%', echo=FALSE}
+knitr::include_graphics("fig/results4/PNG/4-myo2-chrna7.png")
+```
+
+```{r app1-lbl, fig.cap="(ref:app1)",, fig.scap = "Sequencing of myo-2-$\\alpha$-7 from the pDEST vector",  fig.align='center', out.height = '80%', echo=FALSE}
+knitr::include_graphics("fig/results4/PNG/5-myo2-chrna7.png")
+```
\ No newline at end of file

25-appendix-e.Rmd

+# Bacterial homopentameric soluble domains. 
+
+(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}
+knitr::include_graphics("fig/results5/png/HPSD.png")
+```
\ No newline at end of file

26-appendix-f.Rmd

+# Sequence of the DNA sequence used for the expression of human $\alpha$ 7 ECD in E. coli 
+
+(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
+
+```{r out.height = '80%', echo=FALSE}
+knitr::include_graphics("fig/results5/png/pelb-3c_seq_1.png")
+```
+
+```{r 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 pelB-3C cloned into 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

27-appendix-g.Rmd

+# Sequencing of the DNA sequence used for the expression of of the $\alpha7$ ECD-2GSC
+
+\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). 
+
+```{r echo=FALSE}
+knitr::include_graphics("fig/results5/png/alpha7-2gsc-seq1.png")
+```
+
+```{r echo=FALSE}
+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}
+knitr::include_graphics("fig/results5/png/alpha7-2gsc-seq3.png")
+```

_bookdown.yml

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+#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:
     chapter_name: "Chapter "
\ No newline at end of file

_bookdown_files/thesis-dissertaion_cache/docx/__packages

+base
+tidyverse
+ggplot2
+tibble
+tidyr
+readr
+purrr
+dplyr
+stringr
+forcats
+kableExtra