Biology subjects

Gunnels, T.

Publications and source records attributed to Gunnels, T..

4 recordsLinked to original sources

bioRxiv2026

A massively parallel synthetic gene atlas for learning compact cis-regulatory grammar across cellular contexts

Virtual-cell models increasingly learn from large perturbation atlases, but their view of cis regulation remains limited to endogenous genes embedded in broad native regulatory contexts. Here we introduce Therna Biosciences' Chronos platform and its first public dataset release, comprising two complementary modules of a massively parallel synthetic gene atlas: Penta-47x27K for 5' UTRs/internal-promoter elements and Tria-47x28K for 3' UTR stability elements. Together, these datasets measure ~60,000 compact cis-regulatory elements across approximately 50 cell lines in one pooled experiment. The choice of cell lines here, provided to us courtesy of Tahoe Tx, was rooted in our aim to make this dataset maximally useful for the virtual-cell modeling community. At Therna, we routinely apply Chronos -- part of our RNA-Logix(TM) platform -- beyond cancer cell-line pools, to systems such as primary cells and organoids, via LNP-formulated mRNA delivery. Chronos captures both transcriptional and post-transcriptional gene expression control. Episomal DNA delivery measures DNA-normalized mRNA output, while direct RNA delivery with longitudinal sampling reliably quantifies RNA decay. The RNA-delivery arm uses chemically modified synthetic mRNA incorporating N1-methylpseudouridine, a modification widely used in mRNA therapeutics. To resolve high-complexity libraries of up to 30,000 elements, Chronos pushes the sensitivity limits of single-cell RNA sequencing to quantify individual RNA molecules within single cells. By massively expanding gene regulatory networks with synthetic genes whose variable regulatory code is short and defined, Chronos provides an auxiliary cis-regulatory lens for virtual-cell modeling and enables context-specific cis-trans regulatory interactions to be learned directly.

systems biology↗

bioRxiv2022

Binding of ATP to GroEL: a Case for Variable-Temperature Native Mass Spectrometry Thermodynamic Studies

Understanding how large, oligomeric protein complexes respond to the binding of small ligands and other proteins is essential for describing the molecular basis of life. This in turn requires a complete characterization of the binding energetics and correlation of thermodynamic data with interacting structures, including effects of small molecules and solvent. However, the size of many protein oligomers, the myriad intermediate ligation states they can populate, and their often complex allosteric regulation typically restrict analysis by traditional methods to low resolution, ensemble averages. Here, we employ variabletemperature electrospray ionization native mass spectrometry to determine the thermodynamics for stepwise binding of up to ATP molecules to the 801 kDa GroEL complex, a tetradecamer chaperonin complex. Binding thermodynamics reveal strong enthalpy-entropy compensation (EEC) and high degrees of cooperativity are observed for formation of GroEL-ATP7 and GroEL-ATP14. These are evidenced by entropically favored ATP binding to the cis ring (formation of GroEL-ATP1-7), with variations in EEC for subsequent binding of ATP to the trans ring (GroEL-ATP8-14), as expected for negative inter-ring cooperativity. Entropy driven ATP binding to the GroEL tetradecamer is consistent with ligand induced conformational changes of the GroEL tetradecamer, though the magnitude of the entropy change suggests that reorganization of GroEL-hydrating water molecules and/or expulsion of water from the GroEL cavity may also play a key role. By determining the thermodynamic signatures for individual ligand binding reactions to the large, nearly MDa GroEL complex, we expand our fundamental view of chaperonin functional chemistry. Moreover, this work and related studies of protein-ligand interactions illustrate unparalleled capabilities of vT-ESI-nMS for thermodynamics studies of protein interactions with ligands, and other molecules, such as proteins and drugs.

biophysics↗

bioRxiv2020

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs

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.

molecular biology↗

bioRxiv2020

The ITS region provides a reliable DNA barcode for identifying reishi/lingzhi (Ganoderma) from herbal supplements

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.

genetics↗