Journal: Journal of Fungi

Article Title: The Molecular Basis of the Intrinsic and Acquired Resistance to Azole Antifungals in Aspergillus fumigatus

doi: 10.3390/jof10120820

Figure Lengend Snippet: Substrate binding parameters for AfCYP51B-6×His type I binding of eburicol and lanosterol.

Article Snippet: Codon-optimised A. fumigatus Cyp51A-6×His , AfCyp51B-6×His , AfCprA1-6×His , AfCprA2-6×His, and AfErg6-FLAG gene sequences were purchased from ATUM (Newark, CA, USA) in plasmids.

Techniques: Binding 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