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    Structured Review

    ATCC c tetani strains
    Petabase-scale screen of the NCBI sequence read archive reveals <t>C.</t> <t>tetani</t> related genomes in ancient human archeological samples. ( A ) Analysis of 43,620 samples from the NCBI sequence read archive. Each sample is depicted according to its C. tetani k -mer abundance (y-axis) versus the overall dataset size (x-axis). A threshold was used to distinguish samples with high detected C. tetani DNA content, and these data points are colored by sample origin: modern C. tetani genomes (red), non-human (light blue), modern human (blue), ancient human (black). The pie chart displays a breakdown of identified SRA samples with a high abundance of C. tetani DNA signatures. ( B ) Geographical locations and timeline of ancient DNA samples. The 76 ancient DNA datasets are associated with 38 distinct samples (BioSample IDs), which are represented as individual data points. Four samples lack date information and are absent from (B). ( C ) C. tetani chromosomal coverage and; ( D ) plasmid coverage detected for 38 a ncient DNA associated c lostridial m etagenome- a ssembled g enomes (acMAGs) using the C. tetani E88 genome as a reference. Samples are numbered in order of their average percent sequence identity to the reference (E88) <t>strain,</t> from ‘1’ (closest to reference) to ‘38’ (most dissimilar from reference). See table S2 for information on associated BioSample IDs and sample names. The tent and colT genes are indicated on the plasmid in red and blue, respectively. ( E ) Average persample coverage of C. tetani chromosome, plasmid, and key virulence genes, tent and colT . Also shown is the estimated completeness and contamination of C. tetani related MAGs assembled from aDNA samples. MAG quality estimates were performed using CheckM.
    C Tetani Strains, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Ancient Clostridium DNA and variants of tetanus neurotoxins associated with human archaeological remains"

    Article Title: Ancient Clostridium DNA and variants of tetanus neurotoxins associated with human archaeological remains

    Journal: bioRxiv

    doi: 10.1101/2022.06.30.498301

    Petabase-scale screen of the NCBI sequence read archive reveals C. tetani related genomes in ancient human archeological samples. ( A ) Analysis of 43,620 samples from the NCBI sequence read archive. Each sample is depicted according to its C. tetani k -mer abundance (y-axis) versus the overall dataset size (x-axis). A threshold was used to distinguish samples with high detected C. tetani DNA content, and these data points are colored by sample origin: modern C. tetani genomes (red), non-human (light blue), modern human (blue), ancient human (black). The pie chart displays a breakdown of identified SRA samples with a high abundance of C. tetani DNA signatures. ( B ) Geographical locations and timeline of ancient DNA samples. The 76 ancient DNA datasets are associated with 38 distinct samples (BioSample IDs), which are represented as individual data points. Four samples lack date information and are absent from (B). ( C ) C. tetani chromosomal coverage and; ( D ) plasmid coverage detected for 38 a ncient DNA associated c lostridial m etagenome- a ssembled g enomes (acMAGs) using the C. tetani E88 genome as a reference. Samples are numbered in order of their average percent sequence identity to the reference (E88) strain, from ‘1’ (closest to reference) to ‘38’ (most dissimilar from reference). See table S2 for information on associated BioSample IDs and sample names. The tent and colT genes are indicated on the plasmid in red and blue, respectively. ( E ) Average persample coverage of C. tetani chromosome, plasmid, and key virulence genes, tent and colT . Also shown is the estimated completeness and contamination of C. tetani related MAGs assembled from aDNA samples. MAG quality estimates were performed using CheckM.
    Figure Legend Snippet: Petabase-scale screen of the NCBI sequence read archive reveals C. tetani related genomes in ancient human archeological samples. ( A ) Analysis of 43,620 samples from the NCBI sequence read archive. Each sample is depicted according to its C. tetani k -mer abundance (y-axis) versus the overall dataset size (x-axis). A threshold was used to distinguish samples with high detected C. tetani DNA content, and these data points are colored by sample origin: modern C. tetani genomes (red), non-human (light blue), modern human (blue), ancient human (black). The pie chart displays a breakdown of identified SRA samples with a high abundance of C. tetani DNA signatures. ( B ) Geographical locations and timeline of ancient DNA samples. The 76 ancient DNA datasets are associated with 38 distinct samples (BioSample IDs), which are represented as individual data points. Four samples lack date information and are absent from (B). ( C ) C. tetani chromosomal coverage and; ( D ) plasmid coverage detected for 38 a ncient DNA associated c lostridial m etagenome- a ssembled g enomes (acMAGs) using the C. tetani E88 genome as a reference. Samples are numbered in order of their average percent sequence identity to the reference (E88) strain, from ‘1’ (closest to reference) to ‘38’ (most dissimilar from reference). See table S2 for information on associated BioSample IDs and sample names. The tent and colT genes are indicated on the plasmid in red and blue, respectively. ( E ) Average persample coverage of C. tetani chromosome, plasmid, and key virulence genes, tent and colT . Also shown is the estimated completeness and contamination of C. tetani related MAGs assembled from aDNA samples. MAG quality estimates were performed using CheckM.

    Techniques Used: Sequencing, Ancient DNA Assay, Plasmid Preparation

    Phylogenetic analysis reveals known and novel lineages of C. tetani in ancient DNA samples, as well as a previously unidentified Clostridium species (“X”). ( A ) Whole genome phylogenetic tree of acMAGs from ancient samples and modern C. tetani genomes along with previously labeled phylogenetic lineages. Novel lineages are labeled “X” and “Y”, which are phylogenetically distinct from existing C. tetani genomes. ( B) Geographic clustering of newly identified lineage 1H acMAGs in ancient samples from the Americas, and ( C ) of newly identified clade X species in ancient samples from Europe. ( D ) Average nucleotide identity (ANI) between clade X MAGs recovered from ancient DNA and genomes of modern Clostridium species. Clade X MAGs show the highest ANI to C. tetani and C. cochlearium at a level that is sufficient to classify them as a novel Clostridium species. Note that one sample (Vac-Mummy-Tissue) was removed due to insufficient data required for fastANI. See table S10 for ANI values and genome IDs. ( E ) Distributions of damage levels for acMAGs from each phylogenetic group.
    Figure Legend Snippet: Phylogenetic analysis reveals known and novel lineages of C. tetani in ancient DNA samples, as well as a previously unidentified Clostridium species (“X”). ( A ) Whole genome phylogenetic tree of acMAGs from ancient samples and modern C. tetani genomes along with previously labeled phylogenetic lineages. Novel lineages are labeled “X” and “Y”, which are phylogenetically distinct from existing C. tetani genomes. ( B) Geographic clustering of newly identified lineage 1H acMAGs in ancient samples from the Americas, and ( C ) of newly identified clade X species in ancient samples from Europe. ( D ) Average nucleotide identity (ANI) between clade X MAGs recovered from ancient DNA and genomes of modern Clostridium species. Clade X MAGs show the highest ANI to C. tetani and C. cochlearium at a level that is sufficient to classify them as a novel Clostridium species. Note that one sample (Vac-Mummy-Tissue) was removed due to insufficient data required for fastANI. See table S10 for ANI values and genome IDs. ( E ) Distributions of damage levels for acMAGs from each phylogenetic group.

    Techniques Used: Ancient DNA Assay, Labeling

    C. tetani DNA from a subset of ancient samples show hallmarks of ancient DNA. ( A ) MapDamage misincorporation plots for five acMAGs displaying the highest damage levels. The plot shows the frequency of C→T (red) and G→A (blue) misincorporations at the first and last 25 bases of sequence fragments. Increased misincorporation frequency at the edges of reads is characteristic of ancient DNA, and this pattern is not observed in a representative modern C. tetani genomic dataset ( B ). ( C ) Correlation between damage levels of acMAGs and corresponding human mtDNA from the same sample.
    Figure Legend Snippet: C. tetani DNA from a subset of ancient samples show hallmarks of ancient DNA. ( A ) MapDamage misincorporation plots for five acMAGs displaying the highest damage levels. The plot shows the frequency of C→T (red) and G→A (blue) misincorporations at the first and last 25 bases of sequence fragments. Increased misincorporation frequency at the edges of reads is characteristic of ancient DNA, and this pattern is not observed in a representative modern C. tetani genomic dataset ( B ). ( C ) Correlation between damage levels of acMAGs and corresponding human mtDNA from the same sample.

    Techniques Used: Ancient DNA Assay, Sequencing

    Analysis and experimental testing of a novel TeNT lineage identified from ancient DNA. ( A ) Maximum-likelihood phylogenetic tree of tent genes including novel tent sequences assembled from ancient DNA samples and a non-redundant set of tent sequences from existing strains in which duplicates have been removed (see Methods for details). The phylogeny has been subdivided into four subgroups. Sequences are labeled according to sample followed by their associated clade in the genome-based tree ( Fig. 3A ), except for the Barcelona-3031-Tooth sequence (*) as it fell below the coverage threshold. ( B ) Visualization of tent sequence variation, with vertical bars representing nucleotide substitutions found uniquely in tent sequences from ancient DNA samples. On the right, a barplot is shown that indicates the number of unique substitutions found in each sequence, highlighting the uniqueness of subgroup 2. ( C ) Structural model of TeNT/Chinchorro indicating all of its unique amino acid substitutions, which are not observed in modern TeNT sequences. Also shown is a segment of the translated alignment for a specific N-terminal region of the TeNT protein (residues 141-149, uniprot ID P04958). This sub-alignment illustrates a segment containing a high density of unique amino acid substitutions, four of which are shared in TeNT/El-Yaral and TeNT/Chinchorro. ( D ) MapDamage analysis of the tent /Chinchorro gene, and associated C. tetani contigs and mtDNA from the Chinchorro-Mummy-Bone sample. ( E ) Cultured rat cortical neurons were exposed to full-length toxins in culture medium at indicated concentration for 12 hrs. Cell lysates were analyzed by immunoblot. WT TeNT and TeNT/Chinchorro (“ch”) showed similar levels of activity in cleaving VAMP2 in neurons. ( F - G ), Full-length toxins ligated by sortase reaction were injected into the gastrocnemius muscles of the right hind limb of mice. Extent of muscle rigidity was monitored and scored for 4 days (means ± se; n=3). TeNT/Chinchorro (“ch”) induced typical spastic paralysis and showed a potency similar to WT TeNT.
    Figure Legend Snippet: Analysis and experimental testing of a novel TeNT lineage identified from ancient DNA. ( A ) Maximum-likelihood phylogenetic tree of tent genes including novel tent sequences assembled from ancient DNA samples and a non-redundant set of tent sequences from existing strains in which duplicates have been removed (see Methods for details). The phylogeny has been subdivided into four subgroups. Sequences are labeled according to sample followed by their associated clade in the genome-based tree ( Fig. 3A ), except for the Barcelona-3031-Tooth sequence (*) as it fell below the coverage threshold. ( B ) Visualization of tent sequence variation, with vertical bars representing nucleotide substitutions found uniquely in tent sequences from ancient DNA samples. On the right, a barplot is shown that indicates the number of unique substitutions found in each sequence, highlighting the uniqueness of subgroup 2. ( C ) Structural model of TeNT/Chinchorro indicating all of its unique amino acid substitutions, which are not observed in modern TeNT sequences. Also shown is a segment of the translated alignment for a specific N-terminal region of the TeNT protein (residues 141-149, uniprot ID P04958). This sub-alignment illustrates a segment containing a high density of unique amino acid substitutions, four of which are shared in TeNT/El-Yaral and TeNT/Chinchorro. ( D ) MapDamage analysis of the tent /Chinchorro gene, and associated C. tetani contigs and mtDNA from the Chinchorro-Mummy-Bone sample. ( E ) Cultured rat cortical neurons were exposed to full-length toxins in culture medium at indicated concentration for 12 hrs. Cell lysates were analyzed by immunoblot. WT TeNT and TeNT/Chinchorro (“ch”) showed similar levels of activity in cleaving VAMP2 in neurons. ( F - G ), Full-length toxins ligated by sortase reaction were injected into the gastrocnemius muscles of the right hind limb of mice. Extent of muscle rigidity was monitored and scored for 4 days (means ± se; n=3). TeNT/Chinchorro (“ch”) induced typical spastic paralysis and showed a potency similar to WT TeNT.

    Techniques Used: Ancient DNA Assay, Labeling, Sequencing, Cell Culture, Concentration Assay, Activity Assay, Injection, Mouse Assay

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    ATCC c tetani strains
    Petabase-scale screen of the NCBI sequence read archive reveals <t>C.</t> <t>tetani</t> related genomes in ancient human archeological samples. ( A ) Analysis of 43,620 samples from the NCBI sequence read archive. Each sample is depicted according to its C. tetani k -mer abundance (y-axis) versus the overall dataset size (x-axis). A threshold was used to distinguish samples with high detected C. tetani DNA content, and these data points are colored by sample origin: modern C. tetani genomes (red), non-human (light blue), modern human (blue), ancient human (black). The pie chart displays a breakdown of identified SRA samples with a high abundance of C. tetani DNA signatures. ( B ) Geographical locations and timeline of ancient DNA samples. The 76 ancient DNA datasets are associated with 38 distinct samples (BioSample IDs), which are represented as individual data points. Four samples lack date information and are absent from (B). ( C ) C. tetani chromosomal coverage and; ( D ) plasmid coverage detected for 38 a ncient DNA associated c lostridial m etagenome- a ssembled g enomes (acMAGs) using the C. tetani E88 genome as a reference. Samples are numbered in order of their average percent sequence identity to the reference (E88) <t>strain,</t> from ‘1’ (closest to reference) to ‘38’ (most dissimilar from reference). See table S2 for information on associated BioSample IDs and sample names. The tent and colT genes are indicated on the plasmid in red and blue, respectively. ( E ) Average persample coverage of C. tetani chromosome, plasmid, and key virulence genes, tent and colT . Also shown is the estimated completeness and contamination of C. tetani related MAGs assembled from aDNA samples. MAG quality estimates were performed using CheckM.
    C Tetani Strains, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/c tetani strains/product/ATCC
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    ATCC c tetani reference strain
    Macroscopic aspect of <t>Clostridium</t> <t>tetani</t> strain TV1277. ( a ) Lipase-positive (white arrows) and lipase-negative (black arrows) colonies in a 48 h-old pure culture (dimorphism). ( b ) After one week of incubation all colonies appear lipase-positive.
    C Tetani Reference Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Petabase-scale screen of the NCBI sequence read archive reveals C. tetani related genomes in ancient human archeological samples. ( A ) Analysis of 43,620 samples from the NCBI sequence read archive. Each sample is depicted according to its C. tetani k -mer abundance (y-axis) versus the overall dataset size (x-axis). A threshold was used to distinguish samples with high detected C. tetani DNA content, and these data points are colored by sample origin: modern C. tetani genomes (red), non-human (light blue), modern human (blue), ancient human (black). The pie chart displays a breakdown of identified SRA samples with a high abundance of C. tetani DNA signatures. ( B ) Geographical locations and timeline of ancient DNA samples. The 76 ancient DNA datasets are associated with 38 distinct samples (BioSample IDs), which are represented as individual data points. Four samples lack date information and are absent from (B). ( C ) C. tetani chromosomal coverage and; ( D ) plasmid coverage detected for 38 a ncient DNA associated c lostridial m etagenome- a ssembled g enomes (acMAGs) using the C. tetani E88 genome as a reference. Samples are numbered in order of their average percent sequence identity to the reference (E88) strain, from ‘1’ (closest to reference) to ‘38’ (most dissimilar from reference). See table S2 for information on associated BioSample IDs and sample names. The tent and colT genes are indicated on the plasmid in red and blue, respectively. ( E ) Average persample coverage of C. tetani chromosome, plasmid, and key virulence genes, tent and colT . Also shown is the estimated completeness and contamination of C. tetani related MAGs assembled from aDNA samples. MAG quality estimates were performed using CheckM.

    Journal: bioRxiv

    Article Title: Ancient Clostridium DNA and variants of tetanus neurotoxins associated with human archaeological remains

    doi: 10.1101/2022.06.30.498301

    Figure Lengend Snippet: Petabase-scale screen of the NCBI sequence read archive reveals C. tetani related genomes in ancient human archeological samples. ( A ) Analysis of 43,620 samples from the NCBI sequence read archive. Each sample is depicted according to its C. tetani k -mer abundance (y-axis) versus the overall dataset size (x-axis). A threshold was used to distinguish samples with high detected C. tetani DNA content, and these data points are colored by sample origin: modern C. tetani genomes (red), non-human (light blue), modern human (blue), ancient human (black). The pie chart displays a breakdown of identified SRA samples with a high abundance of C. tetani DNA signatures. ( B ) Geographical locations and timeline of ancient DNA samples. The 76 ancient DNA datasets are associated with 38 distinct samples (BioSample IDs), which are represented as individual data points. Four samples lack date information and are absent from (B). ( C ) C. tetani chromosomal coverage and; ( D ) plasmid coverage detected for 38 a ncient DNA associated c lostridial m etagenome- a ssembled g enomes (acMAGs) using the C. tetani E88 genome as a reference. Samples are numbered in order of their average percent sequence identity to the reference (E88) strain, from ‘1’ (closest to reference) to ‘38’ (most dissimilar from reference). See table S2 for information on associated BioSample IDs and sample names. The tent and colT genes are indicated on the plasmid in red and blue, respectively. ( E ) Average persample coverage of C. tetani chromosome, plasmid, and key virulence genes, tent and colT . Also shown is the estimated completeness and contamination of C. tetani related MAGs assembled from aDNA samples. MAG quality estimates were performed using CheckM.

    Article Snippet: We then aligned the 20 ancient tent gene sequences with 30 tent sequences from modern C. tetani strains, which reduced to 12 representative modern tent sequences after duplicates were removed using Jalview v2.9.0b2. tent/E88 was identical with tent from 11 strains (1586-U1, CN655, 641.84, C2, Strain_3, 75.97, 89.12, 46.1.08, A, 4784A, Harvard), tent /132CV with 1 other (Mfbjulcb2), tent /63.05 with 2 others (3483, 184.08), tent /1337 with 2 others (B4, 1240), tent /ATCC_453 with 1 other (3582), and tent /202.15 with 1 other (358.99).

    Techniques: Sequencing, Ancient DNA Assay, Plasmid Preparation

    Phylogenetic analysis reveals known and novel lineages of C. tetani in ancient DNA samples, as well as a previously unidentified Clostridium species (“X”). ( A ) Whole genome phylogenetic tree of acMAGs from ancient samples and modern C. tetani genomes along with previously labeled phylogenetic lineages. Novel lineages are labeled “X” and “Y”, which are phylogenetically distinct from existing C. tetani genomes. ( B) Geographic clustering of newly identified lineage 1H acMAGs in ancient samples from the Americas, and ( C ) of newly identified clade X species in ancient samples from Europe. ( D ) Average nucleotide identity (ANI) between clade X MAGs recovered from ancient DNA and genomes of modern Clostridium species. Clade X MAGs show the highest ANI to C. tetani and C. cochlearium at a level that is sufficient to classify them as a novel Clostridium species. Note that one sample (Vac-Mummy-Tissue) was removed due to insufficient data required for fastANI. See table S10 for ANI values and genome IDs. ( E ) Distributions of damage levels for acMAGs from each phylogenetic group.

    Journal: bioRxiv

    Article Title: Ancient Clostridium DNA and variants of tetanus neurotoxins associated with human archaeological remains

    doi: 10.1101/2022.06.30.498301

    Figure Lengend Snippet: Phylogenetic analysis reveals known and novel lineages of C. tetani in ancient DNA samples, as well as a previously unidentified Clostridium species (“X”). ( A ) Whole genome phylogenetic tree of acMAGs from ancient samples and modern C. tetani genomes along with previously labeled phylogenetic lineages. Novel lineages are labeled “X” and “Y”, which are phylogenetically distinct from existing C. tetani genomes. ( B) Geographic clustering of newly identified lineage 1H acMAGs in ancient samples from the Americas, and ( C ) of newly identified clade X species in ancient samples from Europe. ( D ) Average nucleotide identity (ANI) between clade X MAGs recovered from ancient DNA and genomes of modern Clostridium species. Clade X MAGs show the highest ANI to C. tetani and C. cochlearium at a level that is sufficient to classify them as a novel Clostridium species. Note that one sample (Vac-Mummy-Tissue) was removed due to insufficient data required for fastANI. See table S10 for ANI values and genome IDs. ( E ) Distributions of damage levels for acMAGs from each phylogenetic group.

    Article Snippet: We then aligned the 20 ancient tent gene sequences with 30 tent sequences from modern C. tetani strains, which reduced to 12 representative modern tent sequences after duplicates were removed using Jalview v2.9.0b2. tent/E88 was identical with tent from 11 strains (1586-U1, CN655, 641.84, C2, Strain_3, 75.97, 89.12, 46.1.08, A, 4784A, Harvard), tent /132CV with 1 other (Mfbjulcb2), tent /63.05 with 2 others (3483, 184.08), tent /1337 with 2 others (B4, 1240), tent /ATCC_453 with 1 other (3582), and tent /202.15 with 1 other (358.99).

    Techniques: Ancient DNA Assay, Labeling

    C. tetani DNA from a subset of ancient samples show hallmarks of ancient DNA. ( A ) MapDamage misincorporation plots for five acMAGs displaying the highest damage levels. The plot shows the frequency of C→T (red) and G→A (blue) misincorporations at the first and last 25 bases of sequence fragments. Increased misincorporation frequency at the edges of reads is characteristic of ancient DNA, and this pattern is not observed in a representative modern C. tetani genomic dataset ( B ). ( C ) Correlation between damage levels of acMAGs and corresponding human mtDNA from the same sample.

    Journal: bioRxiv

    Article Title: Ancient Clostridium DNA and variants of tetanus neurotoxins associated with human archaeological remains

    doi: 10.1101/2022.06.30.498301

    Figure Lengend Snippet: C. tetani DNA from a subset of ancient samples show hallmarks of ancient DNA. ( A ) MapDamage misincorporation plots for five acMAGs displaying the highest damage levels. The plot shows the frequency of C→T (red) and G→A (blue) misincorporations at the first and last 25 bases of sequence fragments. Increased misincorporation frequency at the edges of reads is characteristic of ancient DNA, and this pattern is not observed in a representative modern C. tetani genomic dataset ( B ). ( C ) Correlation between damage levels of acMAGs and corresponding human mtDNA from the same sample.

    Article Snippet: We then aligned the 20 ancient tent gene sequences with 30 tent sequences from modern C. tetani strains, which reduced to 12 representative modern tent sequences after duplicates were removed using Jalview v2.9.0b2. tent/E88 was identical with tent from 11 strains (1586-U1, CN655, 641.84, C2, Strain_3, 75.97, 89.12, 46.1.08, A, 4784A, Harvard), tent /132CV with 1 other (Mfbjulcb2), tent /63.05 with 2 others (3483, 184.08), tent /1337 with 2 others (B4, 1240), tent /ATCC_453 with 1 other (3582), and tent /202.15 with 1 other (358.99).

    Techniques: Ancient DNA Assay, Sequencing

    Analysis and experimental testing of a novel TeNT lineage identified from ancient DNA. ( A ) Maximum-likelihood phylogenetic tree of tent genes including novel tent sequences assembled from ancient DNA samples and a non-redundant set of tent sequences from existing strains in which duplicates have been removed (see Methods for details). The phylogeny has been subdivided into four subgroups. Sequences are labeled according to sample followed by their associated clade in the genome-based tree ( Fig. 3A ), except for the Barcelona-3031-Tooth sequence (*) as it fell below the coverage threshold. ( B ) Visualization of tent sequence variation, with vertical bars representing nucleotide substitutions found uniquely in tent sequences from ancient DNA samples. On the right, a barplot is shown that indicates the number of unique substitutions found in each sequence, highlighting the uniqueness of subgroup 2. ( C ) Structural model of TeNT/Chinchorro indicating all of its unique amino acid substitutions, which are not observed in modern TeNT sequences. Also shown is a segment of the translated alignment for a specific N-terminal region of the TeNT protein (residues 141-149, uniprot ID P04958). This sub-alignment illustrates a segment containing a high density of unique amino acid substitutions, four of which are shared in TeNT/El-Yaral and TeNT/Chinchorro. ( D ) MapDamage analysis of the tent /Chinchorro gene, and associated C. tetani contigs and mtDNA from the Chinchorro-Mummy-Bone sample. ( E ) Cultured rat cortical neurons were exposed to full-length toxins in culture medium at indicated concentration for 12 hrs. Cell lysates were analyzed by immunoblot. WT TeNT and TeNT/Chinchorro (“ch”) showed similar levels of activity in cleaving VAMP2 in neurons. ( F - G ), Full-length toxins ligated by sortase reaction were injected into the gastrocnemius muscles of the right hind limb of mice. Extent of muscle rigidity was monitored and scored for 4 days (means ± se; n=3). TeNT/Chinchorro (“ch”) induced typical spastic paralysis and showed a potency similar to WT TeNT.

    Journal: bioRxiv

    Article Title: Ancient Clostridium DNA and variants of tetanus neurotoxins associated with human archaeological remains

    doi: 10.1101/2022.06.30.498301

    Figure Lengend Snippet: Analysis and experimental testing of a novel TeNT lineage identified from ancient DNA. ( A ) Maximum-likelihood phylogenetic tree of tent genes including novel tent sequences assembled from ancient DNA samples and a non-redundant set of tent sequences from existing strains in which duplicates have been removed (see Methods for details). The phylogeny has been subdivided into four subgroups. Sequences are labeled according to sample followed by their associated clade in the genome-based tree ( Fig. 3A ), except for the Barcelona-3031-Tooth sequence (*) as it fell below the coverage threshold. ( B ) Visualization of tent sequence variation, with vertical bars representing nucleotide substitutions found uniquely in tent sequences from ancient DNA samples. On the right, a barplot is shown that indicates the number of unique substitutions found in each sequence, highlighting the uniqueness of subgroup 2. ( C ) Structural model of TeNT/Chinchorro indicating all of its unique amino acid substitutions, which are not observed in modern TeNT sequences. Also shown is a segment of the translated alignment for a specific N-terminal region of the TeNT protein (residues 141-149, uniprot ID P04958). This sub-alignment illustrates a segment containing a high density of unique amino acid substitutions, four of which are shared in TeNT/El-Yaral and TeNT/Chinchorro. ( D ) MapDamage analysis of the tent /Chinchorro gene, and associated C. tetani contigs and mtDNA from the Chinchorro-Mummy-Bone sample. ( E ) Cultured rat cortical neurons were exposed to full-length toxins in culture medium at indicated concentration for 12 hrs. Cell lysates were analyzed by immunoblot. WT TeNT and TeNT/Chinchorro (“ch”) showed similar levels of activity in cleaving VAMP2 in neurons. ( F - G ), Full-length toxins ligated by sortase reaction were injected into the gastrocnemius muscles of the right hind limb of mice. Extent of muscle rigidity was monitored and scored for 4 days (means ± se; n=3). TeNT/Chinchorro (“ch”) induced typical spastic paralysis and showed a potency similar to WT TeNT.

    Article Snippet: We then aligned the 20 ancient tent gene sequences with 30 tent sequences from modern C. tetani strains, which reduced to 12 representative modern tent sequences after duplicates were removed using Jalview v2.9.0b2. tent/E88 was identical with tent from 11 strains (1586-U1, CN655, 641.84, C2, Strain_3, 75.97, 89.12, 46.1.08, A, 4784A, Harvard), tent /132CV with 1 other (Mfbjulcb2), tent /63.05 with 2 others (3483, 184.08), tent /1337 with 2 others (B4, 1240), tent /ATCC_453 with 1 other (3582), and tent /202.15 with 1 other (358.99).

    Techniques: Ancient DNA Assay, Labeling, Sequencing, Cell Culture, Concentration Assay, Activity Assay, Injection, Mouse Assay

    Macroscopic aspect of Clostridium tetani strain TV1277. ( a ) Lipase-positive (white arrows) and lipase-negative (black arrows) colonies in a 48 h-old pure culture (dimorphism). ( b ) After one week of incubation all colonies appear lipase-positive.

    Journal: Toxins

    Article Title: Detection of Clostridium tetani Neurotoxins Inhibited In Vivo by Botulinum Antitoxin B: Potential for Misleading Mouse Test Results in Food Controls

    doi: 10.3390/toxins10060248

    Figure Lengend Snippet: Macroscopic aspect of Clostridium tetani strain TV1277. ( a ) Lipase-positive (white arrows) and lipase-negative (black arrows) colonies in a 48 h-old pure culture (dimorphism). ( b ) After one week of incubation all colonies appear lipase-positive.

    Article Snippet: The ELISA showed that both trivalent (A, B, E) and monovalent (B) antitoxins reacted with TeNTs produced by the C. tetani reference strain (ATCC 10779) ( ).

    Techniques: Incubation

    The peak list of the strain TV1277 spectrum is displayed in the upper half of the graphic. The color of the peaks reflects the degree of matching with the reference MSP (green = full match, yellow = partial match, red = no match). The lower half of the graphic displays the peak list of the reference MSP ( C. tetani DSM 11745) in blue using an inverted intensity scale.

    Journal: Toxins

    Article Title: Detection of Clostridium tetani Neurotoxins Inhibited In Vivo by Botulinum Antitoxin B: Potential for Misleading Mouse Test Results in Food Controls

    doi: 10.3390/toxins10060248

    Figure Lengend Snippet: The peak list of the strain TV1277 spectrum is displayed in the upper half of the graphic. The color of the peaks reflects the degree of matching with the reference MSP (green = full match, yellow = partial match, red = no match). The lower half of the graphic displays the peak list of the reference MSP ( C. tetani DSM 11745) in blue using an inverted intensity scale.

    Article Snippet: The ELISA showed that both trivalent (A, B, E) and monovalent (B) antitoxins reacted with TeNTs produced by the C. tetani reference strain (ATCC 10779) ( ).

    Techniques: