The core structure of motile cilia and flagella, the axoneme, is built from a stable population of doublet microtubules. This unique stability is brought about, at least in part, by a network of Microtubule Inner Proteins (MIPs) that are bound to the luminal side of the microtubule walls. Rib72A and Rib72B were identified as MIPs in the motile cilia of the protist Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of additional MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout Tetrahymena axonemes. We identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

The dietary supplement industry is rapidly growing yet, a recent study revealed that up to 60% of supplements may have substituted ingredients, some of which can be harmful contaminants or additives. When ingredients cannot be verified morphologically or biochemically, DNA barcoding complemented with a molecular phylogenetic analysis can be a powerful method for species authentication. We employed a molecular phylogenetic analysis for species authentication of the commonly used fungal supplement, reishi (Ganoderma lingzhi), by amplifying and sequencing the nuclear ribosomal internal transcribed spacer regions (ITS) with genus-specific primers. PCR of six powdered samples and one dried sample all sold as G. lucidum representing independent suppliers produced single, strong amplification products in the expected size-range for Ganoderma. Both best-hit BLAST and molecular phylogenetic analyses clearly identified the presence of G. lingzhi DNA in all seven herbal supplements. We detected variation in the ITS sequences among our samples, but all herbal supplement samples fall within a large clade of G. lingzhi ITS sequences. ITS-based phylogenetic analysis is a successful and cost-effective method for DNA-based species authentication that could be used in the herbal supplement industry for this and other fungal and plant species that are otherwise difficult to identify.

Motile cilia and flagella are built from stable populations of doublet microtubules that comprise their axonemes. Their unique stability is brought about, at least in part, by a network of Microtubule Inner Proteins (MIPs) found in the lumen of their doublet microtubules. Rib72A and Rib72B were identified as microtubule inner proteins (MIPs) in the motile cilia of Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of multiple MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout (KO) Tetrahymena cells. From this analysis we identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

The dietary supplement industry is a growing enterprise, valued at over $100 billion by 2025 yet, a recent study revealed that up to 60% of herbal supplements may have substituted ingredients not listed on their labels, some with harmful contaminants. Substituted ingredients make rigorous quality control testing a necessary aspect in the production of supplements. Traditionally, species have been verified morphologically or biochemically, but this is not possible for all species if the identifying characteristics are lost in the processing of the material. One approach to validating plant and fungal ingredients in herbal supplements is through DNA barcoding complemented with a molecular phylogenetic analysis. This method provides an efficient, objective, rigorous and repeatable method for species identification. We employed a molecular phylogenetic analysis for species authentication of the commonly used fungal supplement, reishi (Ganoderma lingzhi), by amplifying and sequencing the nuclear ribosomal internal transcribed spacer regions (ITS) with genus-specific primers. PCR of six powdered samples and one dried sample sold as G. lucidum representing independent suppliers produced single, strong amplification products in the expected size-range for Ganoderma. Both best-hit BLAST and molecular phylogenetic analyses using a reference panel assembled from Genbank clearly identified the predominant fungal DNA was G. lingzhi in all seven herbal supplements. We detected variation in ITS among our samples, but all samples still fall within a large clade of G. lingzhi. ITS is a successful and cost-effective method for DNA-based species authentication that could be used in the herbal supplement industry for this and other fungal and plant species that are otherwise difficult to identify.