Axial - Inventors #20
Surveying great inventors and businesses
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A set of ideas and observations on inventions and discoveries in life sciences.
The immune system and everything in it.
Triple RNA-Seq Reveals Synergy in a Human Virus-Fungus Co-infection Model - https://www.cell.com/cell-reports/fulltext/S2211-1247(20)31378-4?sf240095472=1#%20 - the Loeffler Lab at the University of Würzburg used RNA-seq on dendritic cells and simultaneously on 2 pathogens to detect to measure new interactions between the 3:
Dual-sequencing had been established already and enabled by mapping sequencing reads to reference genomes of the host and pathogen. The triple RNA-seq method developed in this paper used similar techniques but added a viral genome (CMV), which had minimal genome overlap with the other two cell types.
Performing RNA-seq on several organisms is fraught with issues ranging from poor RNA extraction protocols generate too much noise in the data and cDNA preparation
The paper challenged monocyte-derived dendritic cells (moDC) with Aspergillus fumigatus and human cytomegalovirus (CMV) separately and together. Both of these pathogens are known to cause post-transplant mortality.
Microscopy and flow cytometry was used to measure infection rates and moDC viability.
Dual RNA-seq found that moDCs overexpressed IL1A/B, CCL3, and TLR2 upon A. fumigatus infection and IFNB and CCL2 activation for CMV infection.
For the triple RNA-seq approach, moDCs were infected in two ways: CMV then A. fumigatus 4.5 hours later, A. fumigatus then CMV 2 hours later, or simultaneous infection; all samples were harvested after 9 hours and sequenced.
After confirming the triple-seq data matched reference datasets, the group found that CMV regulates A. fumigatus-mediated activation of host genes through NF-kB and NFAT and the fungus by inhibiting host response to slow down clearance of CMV.
This paper demonstrates a new way to measure the transcriptomes of a 3-organism system with future applications to the effects of pathogens on T-cells and how macrophages interact with various microenvironments
Biochemistry and structural biology
The granddaddy of them all.
A chemical probe for the methyltransferase PRMT5 with a novel binding mode - https://pubs.acs.org/doi/abs/10.1021/acsmedchemlett.0c00355 - a group at Janssen led by Jan Willem Thuring invented a new probe for a methyltransferase that modifies histones among other proteins:
Protein arginine methyltransferase 5 (PRMT5) is well characterized histone modifier
The enzyme relies on S-adenosyl methionine (SAM) to donate a methyl group
With the observation that adenosine inhibits PRMT5 weakly, the group synthesized analogs that bind to the enzyme but have structural dissimilarities from SAM
Using a mass spec assay, the analogs were screened against PRMT5 measuring the decrease of SAM to SAH conversion. A cellular assay to measure enzyme activity in cells was also used to confirm the hits.
The paper discovered two compounds that inhibited PRMT5 that act as probes
The work is pretty straightforward but validated the premise that adenosine analogs are a useful starting point to drug methyltransferases
Roughly 20 years behind but set up to transform the concept of human.
Integrated Morphoelectric and Transcriptomic Classification of Cortical GABAergic Cells - https://www.cell.com/cell/fulltext/S0092-8674(20)31254-X#%20 - out of the Zeng Lab at the the Allen Institute for Brain Science, the group conducted a large-scale transcriptomic study on over 3K mouse visual cortical GABAergic interneurons:
Relying on Patch-seq, which combines whole-cell patch clamp recording with single-cell RNA-seq and immunohistochemistry, the group measured single cells from the primary visual cortex of a mouse (VISp) and mapped their location according to the Allen Mouse Common Coordinate Framework (CCF)
From the data, 3,708 interneurons had both transcriptomic and electrophysiological data measured with 350 of them successfully reconstructed to access their relative locations. The hard part of this analysis was minimizing noise from non-interneurons, especially glia cells.
The work established at least 20 different interneuron subtypes based on a combination of morphology, electrophysiological features, and their transcriptome. The addition of RNA-seq was essential to distinguish known met-types of interneurons (met = morphology + electrophysiology).
Historically neurons have been classified by their structures and how to fire/wire together
The application of single-cell sequencing, as shown in this paper, has the potential to add another layer of resolution to neuroscience
Cell structure and function.
A novel setup for simultaneous two‑photon functional imaging and precise spectral and spatial visual stimulation in Drosophila - https://www.nature.com/articles/s41598-020-72673-5 - the Wardill Lab at the University of Minnesota developed a new way to imaging method, relying on two-photon microscopy, to measure spatial responses in fruit flies as a model:
The paper sought to understand how visual systems process color
The group designed a clever experiment using a monochromator produced light, which enabled narrow spectrums and little interference with calcium signalling, in combination with two-photon imaging to study how the medulla within the Drosophila brain responds to different colors. This allows for both visual stimulation and brain imaging at the same time.
The paper showed that after 50 different wavelengths were given to the fruit fly model, they saw significant activation changes in the medulla versus other regions but still have to identify particular neurons and layers that respond to a particular color
However, this paper established a useful method to generate brain data in models with both high spatial and spectral resolution. The idea is to map out which neural circuits process specific colors.
Genetics, genomics, and developmental biology
Heredity and variation.
MicroRNA-mediated inhibition of transgene expression reduces dorsal root ganglion toxicity by AAV vectors in primates - https://stm.sciencemag.org/content/12/569/eaba9188.full - out of the Wilson Lab at Penn, a pioneer in adeno-associated virus (AAV) therapies, the group designed AAVs that would be inhibited by microRNAs (miRNA) specific to cells that are specific to neurons involved in dorsal root ganglion (DRG) toxicity:
A major limitation for AAV gene therapies for CNS diseases is DRG toxicity
As a result, the group designed an AAV taking advantage of the fact that DRG neurons selectively express microRNA 183 (miR183). Sequence targets for miR183 were put into the AAV at the 3’ UTR of the transgene.
In non-human primate, the miR183-responsive AAV was injected into brain tissue found that it showed lower transgene expression in DRG neurons and lower toxicity versus an equivalent AAV without the miRNA modification
This proof-of-concept study establishes the ability to imbue AAV expression selectivity based on elements within the host genome, not just within the AAV genome
The ability to mitigate AAV-induced toxicity is pretty important, since we’re in a mini-dark age in the field right now. Next steps are to see if this approach works for other AAV vectors and for other cell-specific miRNAs.