RNA sequencing and lipidomics uncovers novel pathomechanisms in recessive X-linked ichthyosis.

Recessive X-linked ichthyosis (RXLI), a genetic disorder caused by deletion or point mutations of the steroid sulfatase (STS) gene, is the second most common form of ichthyosis. It is a disorder of keratinocyte cholesterol sulfate retention and the mechanism of extracutaneous phenotypes such as corneal opacities and attention deficit hyperactivity disorder are poorly understood. To understand the pathomechanisms of RXLI, the transcriptome of differentiated primary keratinocytes with STS knockdown was sequenced. The results were validated in a stable knockdown model of STS, to confirm STS specificity, and in RXLI skin. The results show that there was significantly reduced expression of genes related to epidermal differentiation and lipid metabolism, including ceramide and sphingolipid synthesis. In addition, there was significant downregulation of aldehyde dehydrogenase family members and the oxytocin receptor which have been linked to corneal transparency and behavioural disorders respectively, both of which are extracutaneous phenotypes of RXLI. These data provide a greater understanding of the causative mechanisms of RXLI's cutaneous phenotype, and show that the keratinocyte transcriptome and lipidomics can give novel insights into the phenotype of patients with RXLI.

Proteomics and histological assessment of an organotypic model of human skin following exposure to Naja nigricollis venom.

Snakebite envenoming was reintroduced as a Category A Neglected Tropical Disease by the World Health Organization in 2017. Since then, increased attention has been directed towards this affliction and towards the development of a deeper understanding of how snake venoms exert their toxic effects and how antivenoms can counter them. However, most of our in vivo generated knowledge stems from the use of animal models which do not always accurately reflect how the pathogenic effects of snake venoms manifest in humans. Moreover, animal experiments are associated with pain, distress, and eventually animal sacrifice due to the toxic nature of snake venoms. Related to this, the implementation of the 3Rs principle (Replacement, Reduction, and Refinement) in the use of experimental animals in snakebite envenoming research is recommended by the World Health Organization. Therefore, more humane experimental designs and new in vitro/ex vivo alternatives for experimental animals are sought after. Here, we report the use of an organotypic model of human skin to further elucidate the pathophysiology of the dermonecrotic effects caused by the venom of the black-necked spitting cobra, Naja nigricollis, in humans. The goal of this study is to expand the repertoire of available models that can be used to study the local tissue damages induced by cytotoxic venoms.

Unity Makes Strength: Exploring Intraspecies and Interspecies Toxin Synergism between Phospholipases A2 and Cytotoxins.

Toxin synergism is a complex biochemical phenomenon, where different animal venom proteins interact either directly or indirectly to potentiate toxicity to a level that is above the sum of the toxicities of the individual toxins. This provides the animals possessing venoms with synergistically enhanced toxicity with a metabolic advantage, since less venom is needed to inflict potent toxic effects in prey and predators. Among the toxins that are known for interacting synergistically are cytotoxins from snake venoms, phospholipases A2 from snake and bee venoms, and melittin from bee venom. These toxins may derive a synergistically enhanced toxicity via formation of toxin complexes by hetero-oligomerization. Using a human keratinocyte assay mimicking human epidermis in vitro, we demonstrate and quantify the level of synergistically enhanced toxicity for 12 cytotoxin/melittin-PLA2 combinations using toxins from elapids, vipers, and bees. Moreover, by utilizing an interaction-based assay and by including a wealth of information obtained via a thorough literature review, we speculate and propose a mechanistic model for how toxin synergism in relation to cytotoxicity may be mediated by cytotoxin/melittin and PLA2 complex formation.

ABCB4 missense mutations D243A, K435T, G535D, I490T, R545C, and S978P significantly impair the lipid floppase and likely predispose to secondary pathologies in the human population.

Bile salts are natural detergents required to solubilise dietary fat and lipid soluble vitamins. They are synthesised in hepatocytes and secreted into the luminal space of the biliary tree by the bile salt export pump (BSEP), an ATP-binding cassette (ABC) transporter in the canalicular membrane. BSEP deficiency causes cytotoxic accumulation of bile salts in the hepatocyte that results in mild-to-severe forms of cholestasis. The resulting inflammation can also progress to hepatocellular cancer via a novel mechanism involving upregulation of proliferative signalling pathways. A second ABC transporter of the canalicular membrane is also critical for bile formation. ABCB4 flops phosphatidylcholine into the outer leaflet of the membrane to be extracted by bile salts in the canalicular space. These mixed micelles reduce the detergent action of the bile salts and protect the biliary tree from their cytotoxic activity. ABCB4 deficiency also causes cholestasis, and might be expected to cause cholangitis and predispose to liver cancer. Non-synonymous SNPs in ABCB4 have now been described in patients with liver cancer or with inflammatory liver diseases that are known to predispose to cancer, but data showing that the SNPs are sufficiently deleterious to be an etiological factor are lacking. Here, we report the first characterisation at the protein level of six ABCB4 variants (D243A, K435T, G535D, I490T, R545C, and S978P) previously found in patients with inflammatory liver diseases or liver cancer. All significantly impair the transporter with a range of phenotypes exhibited, including low abundance, intracellular retention, and reduced floppase activity, suggesting that ABCB4 deficiency is the root cause of the pathology in these cases.