Significance: Although sequencing of the human genome was completed years ago, we still do not know about the physiological significance of thousands of predicted proteins, particularly of predicted membrane proteins. On the other hand, for approximately 100 human enzymes, no coding gene is known even though their enzymatic reaction has been well characterized. In this work, we assign one of those predicted membrane proteins (transmembrane protein 189; TMEM189) to one of the enzymatic reactions with an uncharacterized gene (plasmanylethanolamine desaturase). This enzyme catalyzes the final step in the biosynthesis of plasmalogens, an abundant class of glycerophospholipids that is depleted in such diseases as Alzheimer’s. Our findings enable interpretation of the previously characterized impaired growth phenotype of Tmem189 -deficient mice.

The molecular assembly of cells depends not only on the balance between anabolism and catabolism but to a large degree on the building blocks available in the environment. For cultured mammalian cells, this is largely determined by the composition of the applied growth medium. Here, we study the impact of lipids in the medium on mitochondrial membrane architecture and function by combining LC-MS/MS lipidomics and functional tests with lipid supplementation experiments in an otherwise serum-free and lipid-free cell culture model. We demonstrate that the composition of mitochondrial cardiolipins strongly depends on the lipid environment in cultured cells and favors the incorporation of essential linoleic acid over other fatty acids. Simultaneously, the mitochondrial respiratory complex I activity was altered, whereas the matrix-localized enzyme citrate synthase was unaffected. This raises the question on a link between membrane composition and respiratory control. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium. This underlines the importance of considering these factors when using and establishing cell culture models in biomedical research. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium.

Background: Genome editing in mice using either classical approaches like homologous recombination or CRISPR/Cas9 has been reported to harbor off target effects (insertion/deletion, frame shifts or gene segment duplications) that lead to mutations not only in close proximity to the target site but also outside. Only the genomes of few engineered mouse strains have been sequenced. Since the role of the ether-lipid cleaving enzyme alkylglycerol monooxygenase (AGMO) in physiology and pathophysiology remains enigmatic, we created a knockout mouse model for AGMO using EUCOMM stem cells but unforeseen genotyping issues that did not agree with Mendelian distribution and enzyme activity data prompted an in-depth genomic validation of the mouse model. Results: We report a gene segment tandem duplication event that occurred during the generation of an Agmo knockout-first allele by homologous recombination. Only low homology was seen between the breakpoints. While a single copy of the recombinant 18 kb cassette was integrated correctly around exon 2 of the Agmo gene, whole genome nanopore sequencing revealed a 94 kb duplication in the Agmo locus that contains Agmo wild-type exons 1–3. The duplication fooled genotyping by routine PCR, but could be resolved using qPCR-based genotyping, targeted locus amplification sequencing and nanopore sequencing. Despite this event, this Agmo knockout mouse model lacks AGMO enzyme activity and can therefore be used to study its physiological role. Conclusions: A duplication event occurred at the exact locus of the homologous recombination and was not detected by conventional quality control filters such as FISH or long-range PCR over the recombination sites. Nanopore sequencing provides a cost convenient method to detect such underrated off-target effects, suggesting its use for additional quality assessment of gene editing in mice and also other model organisms.

Deficiency of the transacylase tafazzin due to loss of function variants in the X‐chromosomal TAFAZZIN gene causes Barth syndrome (BTHS) with severe neonatal or infantile cardiomyopathy, neutropenia, myopathy, and short stature. The condition is characterized by drastic changes in the composition of cardiolipins, a mitochondria‐specific class of phospholipids. Studies examining the impact of tafazzin deficiency on the metabolism of other phospholipids have so far generated inhomogeneous and partly conflicting results. Recent studies showed that the cardiolipin composition in cells and different murine tissues is highly dependent on the surrounding lipid environment. In order to study the relevance of different lipid states and tafazzin function for cardiolipin and phospholipid homeostasis we conducted systematic modulation experiments in a CRISPR/Cas9 knock‐out model for BTHS. We found that—irrespective of tafazzin function—the composition of cardiolipins strongly depends on the nutritionally available lipid pool. Tafazzin deficiency causes a consistent shift towards cardiolipin species with more saturated and shorter acyl chains. Interestingly, the typical biochemical BTHS phenotype in phospholipid profiles of HEK 293T TAZ knock‐out cells strongly depends on the cellular lipid context. In response to altered nutritional lipid compositions, we measured more pronounced changes on phospholipids that were largely masked under standard cell culturing conditions, therewith giving a possible explanation for the conflicting results reported so far on BTHS lipid phenotypes.

Long-term results following solid organ transplantation do not mirror the excellent short-term results achieved in recent decades. It is therefore clear that current immunosuppressive maintenance protocols primarily addressing the adaptive immune system no longer meet the required clinical need. Identification of novel targets addressing this shortcoming is urgently needed. There is a growing interest in better understanding the role of the innate immune system in this context. In this review, we focus on macrophages, which are known to prominently infiltrate allografts and, during allograft rejection, to be involved in the surge of the adaptive immune response by expression of pro-inflammatory cytokines and direct cytotoxicity. However, this active participation is janus-faced and unspecific targeting of macrophages may not consider the different subtypes involved. Under this premise, we give an overview on macrophages, including their origins, plasticity, and important markers. We then briefly describe their role in acute allograft rejection, which ranges from sustaining injury to promoting tolerance, as well as the impact of maintenance immunosuppressants on macrophages. Finally, we discuss the observed immunosuppressive role of the vitamin-like compound tetrahydrobiopterin and the recent findings that suggest the innate immune system, particularly macrophages, as its target.

Alkylglycerol monooxygenase (AGMO) and plasmanylethanolamine desaturase (PEDS1) are enzymes involved in ether lipid metabolism. While AGMO degrades plasmanyl lipids by oxidative cleavage of the ether bond, PEDS1 exclusively synthesizes a specific subclass of ether lipids, the plasmalogens, by introducing a vinyl ether double bond into plasmanylethanolamine phospholipids. Ether lipids are characterized by an ether linkage at the sn-1 position of the glycerol backbone and they are found in membranes of different cell types. Decreased plasmalogen levels have been associated with neurological diseases like Alzheimer's disease. Agmo-deficient mice do not present an obvious phenotype under unchallenged conditions. In contrast, Peds1 knockout mice display a growth phenotype. To investigate the molecular consequences of Agmo and Peds1 deficiency on the mouse lipidome, five tissues from each mouse model were isolated and subjected to high resolution mass spectrometry allowing the characterization of up to 2013 lipid species from 42 lipid subclasses. Agmo knockout mice moderately accumulated plasmanyl and plasmenyl lipid species. Peds1-deficient mice manifested striking changes characterized by a strong reduction of plasmenyl lipids and a concomitant massive accumulation of plasmanyl lipids resulting in increased total ether lipid levels in the analyzed tissues except for the class of phosphatidylethanolamines where total levels remained remarkably constant also in Peds1 knockout mice. The rate-limiting enzyme in ether lipid metabolism, FAR1, was not upregulated in Peds1-deficient mice, indicating that the selective loss of plasmalogens is not sufficient to activate the feedback mechanism observed in total ether lipid deficiency.