Biology subjects

Kempf, H.

Publications and source records attributed to Kempf, H..

3 recordsLinked to original sources

bioRxiv2026

Hydration and H/D exchange-dependent infrared signatures of the GCN4 leucine zipper

Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy of proteins in aqueous solution is often limited by water absorption and other optical artifacts. To overcome these limitations, we evaluated the structural features and hydrogen-deuterium exchange (HDX) kinetics of the -helical protein GCN4 in both hydrated (wet) and vacuum-dried (dry) states. While solvent heavily mask the second-derivative spectra of wet samples, vacuum drying yielded a thin, protein-rich film on the ATR crystal, significantly enhancing the signal-to-noise ratio and resolving the protein features without altering the native structure. Dry-state analysis clearly resolved the Amide I, Amide II, and deuterium-shifted Amide II (1450 cm-1) bands. Notably, second-derivative analysis of the dry spectra of the HDX samples revealed a bimodal Amide I distribution consisting of a stationary band at 1653 cm-1 from the solvent-inaccessible regions and an isotopically sensitive band shifting from 1648 cm-1 to 1644 cm-1 from solvent-accessible regions. These results demonstrate that vacuum-dried ATR-FTIR spectroscopy effectively eliminates solvent masking, providing the spectral clarity required to resolve discrete -helical sub-populations after deuteration.

biochemistry↗

bioRxiv2020

Scalable production of tissue-like vascularised liver organoids from human PSCs.

A lack of physiological parity between 2D cell culture and in vivo, has paved the way towards more organotypic models. Organoids exist for a number of tissues, including the liver. However, current approaches to generate hepatic organoids suffer drawbacks, including a reliance on extracellular matrices (ECM), the requirement to pattern in 2D culture, costly growth factors and a lack of cellular diversity, structure and organisation. Current hepatic organoid models are generally simplistic, composed of hepatocytes or cholangiocytes, which renders them less physiologically relevant when compared to native tissue. Here we aim to address these drawbacks. To address this, we have developed an approach that does not require 2D patterning, is ECM independent combined with small molecules to mimic embryonic liver development that produces massive quantities of liver like organoids. Using single-cell RNA sequencing and immunofluorescence we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, innervation and a population of resident macrophage - the Kupffer cells. The organoids exhibit key liver functions including drug metabolism, serum protein production, coagulation factor production, bilirubin uptake and urea synthesis. The organoids can be transplanted and maintained in mice producing human albumin long term. The organoids exhibit a complex cellular repertoire reflective of the organ, have de novo vascularization and innervation, enhanced function and maturity. This is a pre-requisite for a myriad of applications from cellular therapy, tissue engineering, drug toxicity assessment, disease modeling, to basic developmental biology.

cell biology↗

bioRxiv2020

Airway Basal Cells show a dedifferentiated KRT17high Phenotype and promote Fibrosis in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with limited treatment options. In this study we focus on the profibrotic properties of airway basal cells (ABC) obtained from patients with IPF (IPF-ABC). Single cell RNA sequencing of bronchial brushes revealed extensive reprogramming of IPF-ABC towards a KRT17high PTENlow dedifferentiated cell type. In the 3D organoid model, compared to ABC obtained from healthy volunteers, IPF-ABC give rise to more bronchospheres, de novo bronchial structures resembling lung developmental processes, induce fibroblast proliferation and extracellular matrix deposition in co-culture. Intratracheal application of IPF-ABC into minimally injured lungs of Rag2-/- or NRG mice causes severe fibrosis, remodeling of the alveolar compartment, and formation of honeycomb cyst-like structures. Connectivity MAP analysis of scRNA seq of bronchial brushings suggested that gene expression changes in IPF-ABC can be reversed by SRC inhibition. After demonstrating enhanced SRC expression and activity in these cells, and in IPF lungs, we tested the effects of saracatinib, a potent SRC inhibitor previously studied in humans. We demonstrated that saracatinib modified in-vitro and in-vivo the profibrotic changes observed in our 3D culture system and novel mouse xenograft model.

cell biology↗