Biology subjects

Kling, S.

Publications and source records attributed to Kling, S..

5 recordsLinked to original sources

bioRxiv2025

Integration of mechanical testing, in vivo optical coherence elastography and personalized finite element modeling to predict geometrical outcomes of corneal cross-linking

PurposeCorneal cross-linking (CXL) induces both mechanical and geometrical changes in the cornea, which are typically overlooked in pre-operative planning. We propose a patient-specific finite element model (FEM) to predict the topographic alterations resulting from CXL. MethodsTo calibrate the model, we performed nanoindentation and ex vivo OCE inflation tests before and after CXL on five human donor corneas. Nanoindentation results tuned the visco-hyperelastic parameters, while ex vivo OCE axial strains were used for validation. Personalized corneal models were generated from the topographies of three keratoconus patients, with regional stiffness in the affected areas adjusted based on axial strain measured by in vivo pressure-modulated OCE. Simulated CXL outcomes were then compared to 6-month clinical results. ResultsCXL induces a 16-fold increase in the fiber-related mechanical parameters and reduces the viscoelasticity time constant by three. In vivo OCE measurements showed an average mechanical weakening of 57% in the KC regions. When compared to the clinical topography at the 6-month follow-up, the CXL-induced curvature changes predicted by the model were -1.5 D vs. -1.76 D, -1.65 D vs. - 1.91 D, and -1.76 D vs. -1.57 D, for the three patients, respectively. ConclusionBy combining FEM with in vivo corneal mechanical characterization, patient-specific topographic changes can be predicted, which can be used to improve the planning of CXL treatments.

bioengineering↗

bioRxiv2025

Automated, high-throughput in-situ hybridization of Lytechinus pictus embryos

Despite the reach of in situ hybridization (ISH) in developmental biology, it has rarely been used at scale. The major limitation has been the throughput of the assay, which typically relies upon labor intensive manual steps. The goal of this study was to develop a fully automated hybridization chain reaction (HCR) pipeline capable of large-scale gene expression pattern profiling, with dramatically reduced cost and effort, in the sea urchin Lytechinus pictus. Our resulting pipeline, which we term high throughput (HT)-HCR, can process 192 gene probe sets on whole-mount embryos within 32 hours. The unique qualities of the sea urchin embryo enabled us to automate the entire HCR assay in a 96-well plate format, and utilize highly miniaturized reaction volumes, a general purpose robotic liquid handler, and automated confocal microscopy. From this approach we produced high quality localization data for 101 target genes across three developmental stages of L. pictus. The results reveal the localization of previously undescribed physiological genes, as well as canonical developmental transcription factors. HT-HCR represents a log order increase in the rate at which spatial transcriptomic data can be resolved in the sea urchin. This study paves the way for localization of understudied genes and for sophisticated perturbation analysis. Summary StatementWe developed an automated high-throughput HCR pipeline to rapidly map expression of 101 genes in sea urchin embryos, enabling large-scale discovery of novel developmental gene expression patterns.

developmental biology↗

bioRxiv2024

Stable germline transgenesis using the Minos Tc1/mariner element in the sea urchin, Lytechinus pictus.

Stable transgenesis is a transformative tool in model organism biology. While the sea urchin is one of the oldest animal models in cell and developmental biology, it has relied on transient manipulations of wild animals, and has lacked a strategy for stable transgenesis. Here we build on recent progress to develop a more genetically tractable sea urchin species, Lytechinus pictus, to establish a robust transgene integration method. Three commonly used transposons (Minos, Tol2, piggyBac) were tested for non-autonomous transposition, using plasmids containing a polyubiquitin promoter upstream of a H2B-mCerulean nuclear marker. Minos was the only transposable element that resulted in significant expression past metamorphosis. F0 animals were raised to sexual maturity and spawned to determine germline integration, transgene inheritance frequency, and to characterize expression patterns of the transgene in F1 progeny. The results demonstrated transgene transmission through the germline, the first example of a germline transgenic sea urchin, and indeed of any echinoderm. This milestone paves the way for the generation of diverse transgenic resources that will dramatically enhance the utility, reproducibility, and efficiency of sea urchin research. Significance StatementTransgenic tools are essential for effective utilization of animal models. Despite being an established model for cell and developmental biology, the sea urchin has not previously benefited from transgenic technology. This study reports the generation of the first germline transgenic sea urchin and opens new avenues for this organism.

developmental biology↗

bioRxiv2020

A Wnt-specific astacin proteinase controls head formation in Hydra

The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report the identification of an astacin family proteinase as a Wnt processing factor. Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt levels as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by co-injection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.

developmental biology↗

bioRxiv2019

Profiling cellular diversity in sponges informs animal cell type and nervous system evolution

The evolutionary origin of metazoan cell types such as neurons, muscles, digestive, and immune cells, remains unsolved. Using whole-body single-cell RNA sequencing in a sponge, an animal without nervous system and musculature, we identify 18 distinct cell types comprising four major families. This includes nitric-oxide sensitive contractile cells, digestive cells active in macropinocytosis, and a family of amoeboid-neuroid cells involved in innate immunity. We uncover presynaptic genes in an amoeboid-neuroid cell type, and postsynaptic genes in digestive choanocytes, suggesting asymmetric and targeted communication. Corroborating this, long neurite-like extensions from neuroid cells directly contact and enwrap choanocyte microvillar collars. Our data indicate a link between neuroid and immune functions in sponges, and suggest that a primordial neuro-immune system cleared intruders and controlled ciliary beating for feeding.

developmental biology↗