Biology subjects

Melcher, K.

Publications and source records attributed to Melcher, K..

4 recordsLinked to original sources

bioRxiv2021

Fast and robust non-negative matrix factorization for single-cell experiments

Non-negative matrix factorization (NMF) is a popular method for analyzing strictly positive data due to its relatively straightforward interpretation. However, NMF has a reputation as a less efficient alternative to the singular value decomposition (SVD), a standard operation that is highly optimized in most linear algebra libraries. Sparse single-cell sequencing assays, now feasible in thousands of subjects and millions of cells, generate data matrices with tens of thousands of strictly non-negative transcript abundance entries. We present an extremely fast NMF implementation made available in the RcppML (Rcpp Machine Learning library) R package that rivals the runtimes of state-of-the-art Singular Value Decomposition (SVD). NMF can now be run quickly on desktop computers to analyze sparse single-cell datasets consisting of hundreds of thousands of cells. Our method improves upon current NMF implementations by introducing a scaling diagonal to increase interpretability, guarantee consistent regularization penalties across different random initializations, and symmetry in symmetric factorizations. We use our method to show how NMF models learned on standard log-normalized count data are interpretable dimensional reductions, describe interpretable patterns of coordinated gene activities, and explain biologically relevant metadata. We believe NMF has the potential to replace PCA in most single-cell analyses, and the presented NMF implementation overcomes previous challenges with long runtime.

bioinformatics↗

bioRxiv2021

Generation of a New Frizzled 2 Flox Mouse Model to Clarify Its Role in Development

It is currently accepted that Wnt receptors, Frizzleds (Fzd), have high functional redundancy, making individual receptors challenging to target therapeutically. Specifically, Fzd2 is believed to be functionally redundant with Fzd1 and Fzd7, findings which were based largely on previously published global knockout mouse studies. Conversely, a Fzd2 global knockout mouse model developed by the International Mouse Phenotype Consortium (IMPC) is early embryonic lethal, suggesting Fzd2 is critical for early embryonic development. If global deletion of Fzd2 leads to early lethality, floxed models are necessary to identify tissue-specific phenotypes. We found that a previously published Fzd2 flox model does not fully delete Fzd2 function. To reconcile the contradictory findings in Fzd2 mouse models and allow for tissue-specific studies of Fzd2, we have generated a new flox model using a modified two-cell homologous recombination CRISPR approach. We demonstrated successful simultaneous insertion of two loxP sites fully surrounding the Fzd2 gene and confirmed cre-mediated recombination deletes the sequence between the loxP sites leading to a Fzd2 null allele. Preliminary studies suggest global knockouts are early embryonic lethal and full characterization of the tissue-specific effects of Fzd2 deletion is currently underway. This work suggests Fzd2 uniquely regulates development and emphasizes the importance of thorough validation of newly generated mouse models.

cell biology↗

bioRxiv2019

A complex structure of arrestin-2 bound to a G protein-coupled receptor

Arrestins comprise a family of signal regulators of G-protein-coupled receptors (GPCRs), which include arrestins 1 to 4. While arrestins 1 and 4 are visual arrestins dedicated to rhodopsin, arrestins 2 and 3 (Arr2 and Arr3) are {beta}-arrestins known to regulate many nonvisual GPCRs. The dynamic and promiscuous coupling of Arr2 to nonvisual GPCRs has posed technical challenges to tackle the basis of arrestin binding to GPCRs. Here we report the structure of Arr2 in complex with neurotensin receptor 1 (NTSR1), which reveals an overall assembly that is strikingly different from the visual arrestin-rhodopsin complex by a 90{degrees} rotation of Arr2 relative to the receptor. In this new configuration, intracellular loop 3 (ICL3) and transmembrane helix 6 (TM6) of the receptor are oriented toward the N-terminal domain of the arrestin, making it possible for GPCRs that lack the C-terminal tail to couple Arr2 through their ICL3. Molecular dynamics simulation and crosslinking data further support the assembly of the Arr2-NTSR1 complex. Sequence analysis and homology modeling suggest that the Arr2-NTSR1 complex structure may provide an alternative template for modeling arrestin-GPCR interactions.

biophysics↗

bioRxiv2019

Molecular mechanisms of fentanyl mediated β-arrestin biased signaling

The development of novel analgesics with improved safety profiles to combat the opioid epidemic represents a central question to G protein coupled receptor structural biology and pharmacology: What chemical features dictate G protein or {beta}-arrestin signaling? Here we use adaptively biased molecular dynamics simulations to determine how fentanyl, a potent {beta}-arrestin biased agonist, activates the -opioid receptor (OR). The resulting fentanyl-bound pose provides rational insight into a wealth of historical structure-activity-relationship on its chemical scaffold. We found that fentanyl and the synthetic opioid peptide DAMGO require M153 to induce {beta}-arrestin coupling, while M153 was dispensable for G protein coupling. We propose and validate a mechanism where the n-aniline ring of fentanyl mediates OR {beta}-arrestin through a novel M153 \"microswitch\" by synthesizing fentanyl-based derivatives that exhibit complete, clinically desirable, G protein biased coupling. Together, these results provide molecular insight into fentanyl mediated {beta}-arrestin biased signaling and a rational framework for further optimization of fentanyl-based analgesics with improved safety profiles.\n\nAuthor SummaryThe global opioid crisis has drawn significant attention to the risks associated with over-use of synthetic opioids. Despite the public attention, and perhaps in-line with the profit-based incentives of the pharmaceutical industry, there is no public structure of mu-opioid receptor bound to fentanyl or fentayl derivatives. A publicly available structure of the complex would allow open-source development of safer painkillers and synthetic antagonists. Current overdose antidotes, antagonists, require natural products in their synthesis which persists a sizable barrier to market and develop better antidotes. In this work we use advance molecular dynamics techniques to obtain the bound geometry of mu-opioid receptor with fentanyl (and derivatives). Based on our in-silico structure, we synthesized and tested novel compounds to validate our predicted structure. Herein we report the bound state of several dangerous fentanyl derivatives and introduce new derivatives with signaling profiles that may lead to lower risk of respiratory depression.

biophysics↗