Minimal-Invasive 3D Laser Printing of Microimplants in Organismo.

Multi-photon 3D laser printing has gathered much attention in recent years as a means of manufacturing biocompatible scaffolds that can modify and guide cellular behavior in vitro. However, in vivo tissue engineering efforts have been limited so far to the implantation of beforehand 3D printed biocompatible scaffolds and in vivo bioprinting of tissue constructs from bioinks containing cells, biomolecules, and printable hydrogel formulations. Thus, a comprehensive 3D laser printing platform for in vivo and in situ manufacturing of microimplants raised from synthetic polymer-based inks is currently missing. Here, a platform for minimal-invasive manufacturing of microimplants directly in the organism is presented by one-photon photopolymerization and multi-photon 3D laser printing. Employing a commercially available elastomeric ink giving rise to biocompatible synthetic polymer-based microimplants, first applicational examples of biological responses to in situ printed microimplants are demonstrated in the teleost fish Oryzias latipes and in embryos of the fruit fly Drosophila melanogaster. This provides a framework for future studies addressing the suitability of inks for in vivo 3D manufacturing. The platform bears great potential for the direct engineering of the intricate microarchitectures in a variety of tissues in model organisms and beyond.

Deficiency of acyl-CoA synthetase 5 is associated with a severe and treatable failure to thrive of neonatal onset.

Failure to thrive (FTT) causes significant morbidity, often without clear etiologies. Six individuals of a large consanguineous family presented in the neonatal period with recurrent vomiting and diarrhea, leading to severe FTT. Standard diagnostic work up did not ascertain an etiology. Autozygosity mapping and whole exome sequencing identified homozygosity for a novel genetic variant of the long chain fatty acyl-CoA synthetase 5 (ACSL5) shared among the affected individuals (NM_203379.1:c.1358C>A:p.(Thr453Lys)). Autosomal recessive genotype-phenotype segregation was confirmed by Sanger sequencing. Functional in vitro analysis of the ACSL5 variant by immunofluorescence, western blotting and enzyme assay suggested that Thr453Lys is a loss-of-function mutation without any remaining activity. ACSL5 belongs to an essential enzyme family required for lipid metabolism and is known to contribute the major activity in the mouse intestine. Based on the function of ACSL5 in intestinal long chain fatty acid metabolism and the gastroenterological symptoms, affected individuals were treated with total parenteral nutrition or medium-chain triglyceride-based formula restricted in long-chain triglycerides. The patients responded well and follow up suggests that treatment is only required during early life.

Lipid droplet quantification based on iterative image processing.

Lipid droplets (LDs) are ubiquitous and highly dynamic subcellular organelles required for the storage of neutral lipids. LD number and size distribution are key parameters affected not only by nutrient supply but also by lipotoxic stress and metabolic regulation. Current methods for LD quantification lack general applicability and are either based on time consuming manual evaluation or show limitations if LDs are high in numbers or closely clustered. Here, we present an ImageJ-based approach for the detection and quantification of LDs stained by neutral lipid dyes in images acquired by conventional wide-field fluorescence microscopy. The method features an adjustable preprocessing procedure that resolves LD clusters. LD identification is based on their circular edges and central fluorescence intensity maxima. Adaptation to different cell types is mediated by a set of interactive parameters. Validation was done for three different cell lines using manual evaluation of LD numbers and volume measurement by 3D rendering of confocal datasets. In an application example, we show that overexpression of the acyl-CoA synthetase, FATP4/ACSVL5, in oleate-treated COS7 cells increased the size of LDs but not their number.