Journal: Scientific Reports

Article Title: Quantitative analysis of intrinsic and extrinsic factors in the aggregation mechanism of Alzheimer-associated Aβ-peptide

doi: 10.1038/srep18728

Figure Lengend Snippet: ( A , B ) Normalized time courses (ThT fluorescence data points) for aggregation reactions starting from 0.8 (grey) to 10 μM (red) Aβ42-A2V monomer in 20 mM NaP, 0.2 mM EDTA, 0.02% NaN 3 , pH 8.0. Each colour shows four technical replicates at each concentration. The solid lines represents the best fits of model 3a (A) and model 4a (B) and plots for all models are shown in . ( A ) The best fit with a model consistent with the Aβ42 wt data: primary nucleation, secondary nucleation and elongation with fixed reaction orders n c = n 2 = 2 (as for wt) (model 3a) and ( B ) the best fit with a model including primary nucleation and multi-step secondary nucleation (model 4a). ( C ) The half time as a function of peptide concentration from the best fit of each model is compared to the experimental data. ( D ) Error square sum, a measure of the goodness of the fit, of each model relative to model 4a. The models, with the number of fitting parameters given in brackets, are: Model1b Primary nucleation and elongation (2). Model2b Primary nucleation, fragmentation and elongation (3). Model3a Primary nucleation, secondary nucleation and elongation (2). Model3b Primary nucleation, secondary nucleation and elongation (4). Model4a Primary nucleation, multi-step secondary nucleation and elongation (3). Although involving one less free parameter than model 3b, model 4a yields a lower error. See for the processes and parameters for each model. ( E ) Normalized aggregation kinetics data for samples that initially contain 2.3 μM monomer supplemented with 0.03, 0.1, 0.3, 1, 3, 10 or 30% seeds (in monomer equivalents) confirm the strong role of surface-catalyzed secondary nucleation. The solid lines are fits of model 4a, using the parameter values found above and one free parameter, the elongation rate constant k + . ( F ) Comparison of k 2 k + and k 2 /kn for Aβ42-A2V relative to Aβ42-wt. In particular note the large increase in the relative importance of secondary over primary nucleation.

Article Snippet: A synthetic gene with E. coli optimised codons for Aβ(M1-42) with the A2V mutation cloned into the Pet3a vector was purchased from Genscript (Piscataway; New Jersey, USA).

Techniques: Fluorescence, Concentration Assay, Comparison

Journal: bioRxiv

Article Title: The specificity of ParR binding determines the compatibility of conjugative plasmids in Clostridium perfringens

doi: 10.1101/462192

Figure Lengend Snippet: Replication and parMRC locus of pCW3: parM C is shown in yellow, parR C is shown in blue, the parC C site (four direct repeats shown in green) is upstream of parM C , the five inverted repeats (IR) of oriV (IR1 in orange, IR2 in pink, IR3 in lavender, IR4 in blue, IR5 in purple) are shown upstream of rep , which is indicated by the red arrow.

Article Snippet: The parR C gene from pCW3 was codon optimised for expression in E. coli , synthesised by GenScript and cloned into the EcoRV site of pUC57-Kan. Codon optimised parR C then was subcloned into the NdeI/XhoI sites of pET22b(+). parR D (pJIR3118) was PCR amplified from CN1020 gDNA isolated as before (O’Connor et al ., 2006) and cloned into the NdeI/Xhol site of pET22b (+) for expression. parR B (pJIR4165), parR B (pJGS1987B) parR C (pJGS1987C) and parR D (pJGS1987D) were codon optimised and synthesised before being cloned into pET22b(+) NdeI/XhoI sites by GenScript.

Techniques:

Journal: bioRxiv

Article Title: The specificity of ParR binding determines the compatibility of conjugative plasmids in Clostridium perfringens

doi: 10.1101/462192

Figure Lengend Snippet: ParR C (pCW3) binds to a cognate parC C (pCW3) sequence. (A) Schematic of the parC C (pCW3) fragment array that consists of 30 bp fragments that overlap by 20 bp, direct repeats are indicated above the fragment array in red. (B) Representative ParR C (pCW3) binding to the parC C (pCW3) fragment array as determined by SPR. (C) Representative SPR binding curves for ParR C (pCW3) and parC C (pCW3) fragments, ParR C (pCW3) + C3 binding curve is shown in blue, and ParR C (pCW3) + C12 binding curve is shown in red. AUC sedimentation velocity experiments were also conducted on ParR C (pCW3), parC C (pCW3) fragment C5 and ParR C (pCW3) and parC C (pCW3) fragment C5 in combination. (D) The continuous sedimentation coefficient distribution [ c ( s )] as a function of normalised sedimentation coefficient ( s 20,W ) for ParR C (pCW3). (E) The continuous mass distribution c ( M ) distribution as a function of molecular mass (Da) for ParR C (pCW3). (F) The continuous sedimentation coefficient distribution [ c ( s )] as a function of s 20,W for parC C (pCW3) C5 (red), ParR C (pCW3) (blue) and, ParR C (pCW3) and parC C (pCW3) C5 in combination (green). Residuals for each fit are shown as insets, confirming the validity of the fit of the data.

Article Snippet: The parR C gene from pCW3 was codon optimised for expression in E. coli , synthesised by GenScript and cloned into the EcoRV site of pUC57-Kan. Codon optimised parR C then was subcloned into the NdeI/XhoI sites of pET22b(+). parR D (pJIR3118) was PCR amplified from CN1020 gDNA isolated as before (O’Connor et al ., 2006) and cloned into the NdeI/Xhol site of pET22b (+) for expression. parR B (pJIR4165), parR B (pJGS1987B) parR C (pJGS1987C) and parR D (pJGS1987D) were codon optimised and synthesised before being cloned into pET22b(+) NdeI/XhoI sites by GenScript.

Techniques: Sequencing, Binding Assay, Sedimentation

Journal: bioRxiv

Article Title: The specificity of ParR binding determines the compatibility of conjugative plasmids in Clostridium perfringens

doi: 10.1101/462192

Figure Lengend Snippet: Surface plasmon resonance analysis demonstrated that ParR homologues bind to their cognate parC sites. A) Schematic of parC overlapping fragments. parC B (pJIR4165), parC C (pCW3) and parC D (pJIR3118) fragment arrays were constructed to test binding of ParR homologues to each parC region. All fragment arrays consisted of 30 bp oligonucleotides with 20 bp of overlapping sequence and were designed using POOP. Antisense oligonucleotides were constructed with the ReDCaT linker sequence present at the 3’ end of each fragment in the diagram above. Oligonucleotides were annealed before being captured onto the ReDCaT primed Streptavidin (SA) chip via the complementary base pairing between the ReDCaT linker and the complementary ReDCaT sequence on the Biacore T200 chip. B) SPR profiles obtained when ParR B (pJIR4165) was tested against parC B (pJIR4165) (blue), parC C (pCW3) (Green) and parC D (pJIR3118) (Orange) C) Shows ParR C (pCW3) binding profiles, D) Shows ParR D (pJIR3118) binding profiles. The first lane in every binding graph shows a no protein control with the fragments C1, B1 and D1.

Article Snippet: The parR C gene from pCW3 was codon optimised for expression in E. coli , synthesised by GenScript and cloned into the EcoRV site of pUC57-Kan. Codon optimised parR C then was subcloned into the NdeI/XhoI sites of pET22b(+). parR D (pJIR3118) was PCR amplified from CN1020 gDNA isolated as before (O’Connor et al ., 2006) and cloned into the NdeI/Xhol site of pET22b (+) for expression. parR B (pJIR4165), parR B (pJGS1987B) parR C (pJGS1987C) and parR D (pJGS1987D) were codon optimised and synthesised before being cloned into pET22b(+) NdeI/XhoI sites by GenScript.

Techniques: SPR Assay, Construct, Binding Assay, Sequencing

Journal: bioRxiv

Article Title: The specificity of ParR binding determines the compatibility of conjugative plasmids in Clostridium perfringens

doi: 10.1101/462192

Figure Lengend Snippet: JGS1987 ParR homologues bind to non-cognate parC from the same family. ParR B , ParR C and ParR D homologues from the C. perfringens isolate, JGS1987, were tested against parC B (pJIR4165), parC C (pCW3) and parC D (pJIR3118) fragment arrays, and binding stability was measured using surface plasmon resonance. A) Shows ParR B (pJGS1987B) binding profiles when used to challenge parC B (pJIR4165) (blue), parC C (pCW3) and parC D (pJIR3118). B) Shows ParR C (pJGS1987C) binding profiles (blue) C) Shows ParR D (pJGS1987D) binding profiles (orange). The first fragment in every graph shows a no protein control

Article Snippet: The parR C gene from pCW3 was codon optimised for expression in E. coli , synthesised by GenScript and cloned into the EcoRV site of pUC57-Kan. Codon optimised parR C then was subcloned into the NdeI/XhoI sites of pET22b(+). parR D (pJIR3118) was PCR amplified from CN1020 gDNA isolated as before (O’Connor et al ., 2006) and cloned into the NdeI/Xhol site of pET22b (+) for expression. parR B (pJIR4165), parR B (pJGS1987B) parR C (pJGS1987C) and parR D (pJGS1987D) were codon optimised and synthesised before being cloned into pET22b(+) NdeI/XhoI sites by GenScript.

Techniques: Binding Assay, SPR Assay

Journal: bioRxiv

Article Title: Genomic, Functional and Structural Analyses Reveal Mechanisms of Evolutionary Innovation within the Sea Anemone 8 Toxin Family

doi: 10.1101/2022.12.08.518931

Figure Lengend Snippet: Schematic of pET32a expression vector used for expression of SA8 in the periplasm of E. coli . Abbreviations: RBS= ribosome binding site; MalESS= MalE signal sequence; His 6 = poly-histidine affinity tag; TEV= TEV protease recognition site; SA8= sea anemone 8

Article Snippet: A nucleotide sequence encoding the mature peptide sequence of SA8, containing codons optimised for expression in Escherichia coli , was synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Expressing, Plasmid Preparation, Binding Assay, Sequencing

Journal: bioRxiv

Article Title: Genomic, Functional and Structural Analyses Reveal Mechanisms of Evolutionary Innovation within the Sea Anemone 8 Toxin Family

doi: 10.1101/2022.12.08.518931

Figure Lengend Snippet: (A) Genomic arrangement and orientation of SA8 genes in T. stephensoni and A. tenebrosa . The inverted SA8 gene identified in the genome of T. stephensoni is depicted in blue. Numbers in square brackets indicate the scaffold number for A. tenebrosa SA8 genes. (B) Sequence space of the SA8 family. Sequence space of mature SA8 peptides reveals that they fall into four clusters, with the inverted SA8 T. stephensoni peptide found in the largest cluster (black)

Article Snippet: A nucleotide sequence encoding the mature peptide sequence of SA8, containing codons optimised for expression in Escherichia coli , was synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Genomic, Functional and Structural Analyses Reveal Mechanisms of Evolutionary Innovation within the Sea Anemone 8 Toxin Family

doi: 10.1101/2022.12.08.518931

Figure Lengend Snippet: Expression of SA8 genes across functional regions in A. tenebrosa (A) and T. stephensoni (B). Each graph shows the tissue-specific expression pattern of a transcript that corresponds to a SA8 gene. One transcript corresponds to two SA8 genes (SA8_clustered_2&7) in T. stephensoni . Bars represent the mean TMM-normalised expression value of the SA8 transcript in each anatomical structure, with error bars representing the standard deviation. Abbreviations: A= acrorhagi; C= club-tips; T= tentacle; P=actinopharynx; M= mesenterial filaments; B= body column; PD= pedal disc

Article Snippet: A nucleotide sequence encoding the mature peptide sequence of SA8, containing codons optimised for expression in Escherichia coli , was synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Expressing, Functional Assay, Standard Deviation

Journal: bioRxiv

Article Title: Genomic, Functional and Structural Analyses Reveal Mechanisms of Evolutionary Innovation within the Sea Anemone 8 Toxin Family

doi: 10.1101/2022.12.08.518931

Figure Lengend Snippet: Cluster dendrogram of putative toxin transcripts from T. stephensoni (A) and A. tenebrosa (B) constructed using WGCNA. Each color represents one module. (A) Most T. stephensoni SA8 sequences were assigned to the turquoise and brown modules: turquoise = higher expression in the tentacles; brown = higher expression in the epidermis. The inverted T. stephensoni SA8 sequence was assigned to the black module, which was not associated with a specific expression pattern. Other modules: blue = higher expression in the mesenterial filaments; green= higher expression in the epidermis and tentacles; red= higher expression in the actinopharynx; yellow= higher expression in the tentacles and mesenterial filaments. (B) A. tenebrosa SA8 sequences were assigned to all four modules: blue = higher expression in the acrorhagi; brown= higher expression in the acrorhagi and tentacles; yellow= higher expression in the tentacles; turquoise = no distinct expression pattern. Putative toxin transcripts not assigned to a module are shown in grey

Article Snippet: A nucleotide sequence encoding the mature peptide sequence of SA8, containing codons optimised for expression in Escherichia coli , was synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Construct, Expressing, Sequencing

Journal: bioRxiv

Article Title: Genomic, Functional and Structural Analyses Reveal Mechanisms of Evolutionary Innovation within the Sea Anemone 8 Toxin Family

doi: 10.1101/2022.12.08.518931

Figure Lengend Snippet: Peptide corresponding to calculated mass of the T. stephensoni SA8 venom peptide is abundant in the gastrodermis and epidermis (A) H&E stain of T. stephensoni section. (B) putative peptide observed at 5409 m/z with greatest abundance in the mesenterial filaments (m) and pedal disc (pd)

Article Snippet: A nucleotide sequence encoding the mature peptide sequence of SA8, containing codons optimised for expression in Escherichia coli , was synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Staining

Journal: bioRxiv

Article Title: Genomic, Functional and Structural Analyses Reveal Mechanisms of Evolutionary Innovation within the Sea Anemone 8 Toxin Family

doi: 10.1101/2022.12.08.518931

Figure Lengend Snippet: Phylogenetic relationships among 71 sea anemone sequences from the SA8 gene family were inferred under maximum likelihood in IQ-TREE. The inverted SA8 gene from T. stephensoni is found in a well-supported clade on a branch sister to a Metridioidea-specific clade

Article Snippet: A nucleotide sequence encoding the mature peptide sequence of SA8, containing codons optimised for expression in Escherichia coli , was synthesised by GenScript (Piscataway, NJ, USA).

Techniques:

Journal: bioRxiv

Article Title: Genomic, Functional and Structural Analyses Reveal Mechanisms of Evolutionary Innovation within the Sea Anemone 8 Toxin Family

doi: 10.1101/2022.12.08.518931

Figure Lengend Snippet: Sequence space of the SA8 family (A) and disulfide connectivities of venom SA8 (B) and ShK (C). (A) The SA8 family (yellow) forms a distinct cluster from the ShKT domains of ShK-like (red) and CRISP (black) peptides . (B) The C1-C5, C2-C6, C3-C4 disulfide connectivity of the mature venom SA8 peptide from T. stephensoni . (C) The C1-C6, C2-C4, C3-C5 disulfide connectivity of ShK toxin from S. helianthus

Article Snippet: A nucleotide sequence encoding the mature peptide sequence of SA8, containing codons optimised for expression in Escherichia coli , was synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Genomic, Functional and Structural Analyses Reveal Mechanisms of Evolutionary Innovation within the Sea Anemone 8 Toxin Family

doi: 10.1101/2022.12.08.518931

Figure Lengend Snippet: SA8-induced inhibition of hK V 1.2 and selectivity profile of SA8. (A) Representative whole-cell current traces were recorded for hK V 1.2 using the voltage protocol shown above the raw current traces every 15 s in the absence (black, control) and presence of 100 nM SA8 (red) and 14 nM charybdotoxin (ChTx, green) as positive control. (B) Low affinity, concentration-dependent block of hK V 1.2 channels by SA8 was determined fitting a straight line to the reciprocal of the remaining current fraction (1/RCF) plotted as a function of SA8 concentration. Remaining current fraction (RCF) was calculated as I/I 0 , where I 0 is the peak current in the absence and I is the peak current at equilibrium block in the presence of SA8 at concentrations of 0.01, 0.1, 1, and 10 µM (filled circles). Points on the linear concentration-response curve represent the mean of four independent measurements where the error bars represent SEM. The line was drawn using linear least squares fit and the reciprocal of the slope of the best fit yielded an IC 50 of 40.8 ± 4.9 µM. (C) The effect of SA8 (100nM, except for K V 10.1 which was tested at 1µM) on the peak currents was reported as the remaining current fraction (RCF). Bars represent the mean of 4–6 independent measurements; error bars indicate the SEM. Data are shown for the following channels: Hk V 1.1, hK V 1.2, hK V 1.3, hK V 10.1, hK V 11.1, mK Ca 1.1, hKCa3.1, hTRPA1, hTRPV1, and hNa V 1.7 (for details of the expression systems, solutions, and voltage protocols, see Materials and Methods, and for raw current traces see Supplementary Figure 9). SA8 did not inhibit either of the investigated channels at the applied concentrations

Article Snippet: A nucleotide sequence encoding the mature peptide sequence of SA8, containing codons optimised for expression in Escherichia coli , was synthesised by GenScript (Piscataway, NJ, USA).

Techniques: Inhibition, Control, Positive Control, Concentration Assay, Blocking Assay, Expressing