Journal: BMC Microbiology

Article Title: First human cell-based cultivation system for the syphilis spirochete Treponema pallidum

doi: 10.1186/s12866-026-04856-5

Figure Lengend Snippet: Human foreskin fibroblast cell lines support the growth of T. pallidum in vitro . A Human foreskin fibroblast cell lines (MoNa, HFF1, and HFFC) support the growth of T. pallidum in long-term, continuous, in vitro cultivation. While cultivation of strain SS14 was discontinued after 10 weeks, in vitro cultures of the DAL-1 strain have been ongoing for over one year. During this period, scheme of subcultures was optimized (up to 1:20). A booster passage (*), extending the standard 7-day subculture to a 14-day period with a cultivation medium exchange on day 7, was employed to enrich the treponemal culture during critical declines. In vitro cultivation was performed in triplicate (i.e., in three cultivation wells) and the mean values for each subculture are presented in graph. Source data from individual wells are shown in Supplementary Table . B Parallel cultivation for seven days ( n = 15) with defined DAL-1 inoculum (10 6 treponemes) validated the differences in growth support of individual foreskin cell lines. Treponemal growth on human foreskin fibroblast cells was slower compared to growth on rabbit epithelial Sf1Ep cells. Red bar, mean. C Human foreskin-based cultivation system supports the growth of various T. pallidum strains ( n = 8), from the Nichols-like as well as the SS14-like cluster. Note that human foreskin fibroblast cells were prepared as an equal mixture of three tested cell lines. Each T. pallidum strain was cultivated in a single in vitro well, representing a sole experimental replicate used for data acquisition

Article Snippet: Human foreskin fibroblasts HFF1 (SCRC-1041; ATCC) and HFFC (300715; Cytion) were purchased, while the third cell line (MoNa) was kindly provided by Dr. Vladimir Rotrekl (Masaryk University), and was originally obtained from the National Tissue Centre (Czech Republic).

Techniques: In Vitro

Journal: Nucleic Acids Research

Article Title: RNA duplexes with abasic substitutions are potent and allele-selective inhibitors of huntingtin and ataxin-3 expression

doi: 10.1093/nar/gkt594

Figure Lengend Snippet: Effects of chemically modified duplex RNAs with mismatched bases on HTT expression. Duplex RNAs were transfected into HD patient-derived cells (GM04281, CAG 69/17) ( A ) Representative western blot images of HTT expression after transfection of duplex RNAs at 25 nM. ( B ) Western blot images and dose curves of duplex RNAs CMOD1, CMOD12 and CMOD13. Dose curves are averaged data from three independent experiments. ( C ) Effects of chemically modified RNA CMOD13 and analogous unmodified RNA duplex PM4 on HTT expression. Duplex RNAs were added at 25 nM and harvested at indicated days after transfection. The bar graph shows quantification of mutant HTT protein levels relative to treatment with a control duplex MM4 that contained mismatches within the seed sequence.

Article Snippet: Patient-derived fibroblast cell lines GM04281 and GM06151 were obtained from the Coriell Institute.

Techniques: Modification, Expressing, Transfection, Derivative Assay, Western Blot, Mutagenesis, Control, Sequencing

Journal: Nucleic Acids Research

Article Title: RNA duplexes with abasic substitutions are potent and allele-selective inhibitors of huntingtin and ataxin-3 expression

doi: 10.1093/nar/gkt594

Figure Lengend Snippet: Effects of duplex RNAs with abasic modifications on expression of HTT. Duplexes were transfected into HD patient-derived fibroblast cells (GM04281, CAG 69/17). ( A ) Representative western blot images and ( B ) averaged protein levels of HTT expression after transfection of duplex RNAs at 25 nM. Western blot images and dose curves of ( C ) duplex RNA AB5 and ( D ) duplex RNA AB6. Dose curves are averaged data from three independent experiments. MM:noncomplementary duplex RNA.

Article Snippet: Patient-derived fibroblast cell lines GM04281 and GM06151 were obtained from the Coriell Institute.

Techniques: Expressing, Transfection, Derivative Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: RNA duplexes with abasic substitutions are potent and allele-selective inhibitors of huntingtin and ataxin-3 expression

doi: 10.1093/nar/gkt594

Figure Lengend Snippet: Effects of siRNAs with multiple abasic modifications on expression of HTT. Duplex RNAs were tested in HD patient-derived fibroblast cells (GM04281, CAG 69/17). ( A ) Representative western blot images of HTT expression after treating with 25 nM of siRNAs. Averaged dose curves showing HTT expression after treating with increased concentrations of duplex RNAs ( B ) AB9 and ( C ) AB12.

Article Snippet: Patient-derived fibroblast cell lines GM04281 and GM06151 were obtained from the Coriell Institute.

Techniques: Expressing, Derivative Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: RNA duplexes with abasic substitutions are potent and allele-selective inhibitors of huntingtin and ataxin-3 expression

doi: 10.1093/nar/gkt594

Figure Lengend Snippet: Effects of longer abasic duplex RNAs (22 mers) on expression of HTT. Duplex RNAs were tested in HD patient-derived fibroblast cells (GM04281, CAG 69/17). Averaged dose–response curves of RNA duplexes ( A ) AB18(Y33), ( B ) AB19(Y33), ( C ) AB20(Y16) and ( D ) AB21(Y16) containing three abasic substitutions.

Article Snippet: Patient-derived fibroblast cell lines GM04281 and GM06151 were obtained from the Coriell Institute.

Techniques: Expressing, Derivative Assay

Journal: Nucleic Acids Research

Article Title: RNA duplexes with abasic substitutions are potent and allele-selective inhibitors of huntingtin and ataxin-3 expression

doi: 10.1093/nar/gkt594

Figure Lengend Snippet: Mechanistic studies of abasic siRNAs. ( A ) HTT mRNA levels after treating with 25 nM of abasic duplex RNAs in HD patient fibroblasts (GM04281, CAG 69/17). siHdh1 is a positive control siRNA targeting HTT mRNA at a sequence outside the trinucleotide repeat region. ( B ) In vitro cleavage assay using RNA antisense strands and recombinant human AGO2 protein. Ladder: radiolabeled 10-nt DNA markers; NT: no treatment; REP: fully complementary anti-CAG siRNA; H153: fully complementary siRNA targeting HTT upstream region of CAG repeat; AB3 and AB6 are abasic-substituted siRNAs. ( C ) RNA immunoprecipitation (RIP) using an anti-AGO2 antibody reveals association of AGO2/siRNA complexes with HTT mRNA (GM04281). ( D ) Abasic siRNA AB8 selectively inhibits mutant HTT or ataxin-3 expression in a cooperative manner.

Article Snippet: Patient-derived fibroblast cell lines GM04281 and GM06151 were obtained from the Coriell Institute.

Techniques: Positive Control, Sequencing, In Vitro, Cleavage Assay, Recombinant, RNA Immunoprecipitation, Mutagenesis, Expressing