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Correction to: Genetics in Medicine 22:2020 https://doi.org/10.1038/s41436-019-0743-3 published online 17 January 2020. An incorrect reference was cited in the fourth sentence of the Results section. The correct citation should have been Al-Gazali LI, Hamid Z, Hertecant J et al. An autosomal recessive syndrome of choanal atresia, hypothelia/athelia and thyroid gland anomalies overlapping Bamforth syndrome, ANOTHER syndrome and methimazole embryopathy. Clin Dysmorphol 2002;2:79–85. This has now been corrected in both the PDF and HTML versions of the Article.

The endocytosis of ligand-bound receptors and their eventual recycling to the plasma membrane (PM) are processes that have an influence on signalling activity and therefore on many cell functions, including migration and proliferation. Like other tyrosine kinase receptors (TKR), the insulin receptor (INSR) has been shown to be endocytosed by clathrin-dependent and -independent mechanisms. Once at the early endosome (EE), the sorting of the receptor, either to the late endosome (LE) for degradation or back to the PM through slow or fast recycling pathways, will determine the intensity and duration of insulin effects. Both the endocytic and the endosomic pathways are regulated by many proteins, the Arf and Rab families of small GTPases being some of the most relevant. Here, we argue for a specific role for the slow recycling route, whilst we review the main molecular mechanisms involved in INSR endocytosis, sorting and recycling, as well as their possible role in cell functions.

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into sterile culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to interested researchers worldwide as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been widely applied across at least 5 continents, 23 countries and in 62 amphibian species, and have been successfully used to isolate chytrids in remote field locations. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this important group of emerging fungal pathogens.

Host-associated microbes are vital for combatting infections and maintaining health. In amphibians, certain skin-associated bacteria inhibit the fungal pathogen Batrachochytrium dendrobatidis (Bd), yet our understanding of host microbial ecology and its role in disease outbreaks is limited. We sampled skin-associated bacteria and Bd from Pyrenean midwife toad populations exhibiting enzootic or epizootic disease dynamics. We demonstrate that bacterial communities differ between life stages with few shared taxa, indicative of restructuring at metamorphosis. We detected a significant effect of infection history on metamorph skin microbiota, with reduced bacterial diversity in epizootic populations and differences in community structure and predicted function. Genome sequencing of Bd isolates supports a single introduction to the Pyrenees and reveals no association between pathogen genetics and epidemiological trends. Our findings provide an ecologically relevant insight into the microbial ecology of amphibian skin and highlight the relative importance of host microbiota and pathogen genetics in predicting disease outcome.

Elevated blood levels of endogenous digitalis-like factors (EDLF) may decrease erythrocyte sodium pump activity in preeclampsia. As the highest EDLF levels might be expected in severe preeclampsia, we investigated sodium pump activity in that group of patients.Erythrocyte sodium pump activity was determined by (86)Rubidium uptake (in nM per hour per 10(6) cells) in women with severe preeclampsia and those with normal pregnancies, matched for gestational age, and in healthy nonpregnant women (n=12 in each group). Differences between groups were analyzed by a two-sided Student t-test.Sodium pump activity was significantly increased in normotensive pregnancies as compared with normotensive non-pregnant women (81.4+/-8.4 vs 61.1+/-7.4, mean+/-s.d., p0.05), and was decreased 43% in severe preeclamptic pregnancies as compared with normotensive pregnancies (46.4+/-14.1 vs 81.4+/-8.4, p0.05).Severe preeclampsia is associated with significantly lower erythrocyte sodium pump activity than normotensive pregnancy. These data suggest that plasma levels of a biologically active EDLF are elevated in patients with severe preeclampsia.

Background—Mammalian tissues contain a presumed endogenous Na+, K+-ATPase inhibitor that binds reversibly to the Na+pump with high affinity and specificity. The inhibitor has been linked to the pathogenesis of experimental volume-expanded and human essential hypertension. This compound has been isolated from mammalian hypothalamus and appears to be an isomer of the plant-derived cardiac glycoside ouabain, if not ouabain itself. The objective of this study was to test the hypothesis that a biosynthetic pathway exists in mammalian tissues to produce a steroid derivative closely related to plant cardiac glycosides.Methods and Results—Using bioinformatics and genomic techniques, Milan hypertensive rat tissues were studied because this strain has a 10-fold increase in hypothalamic ouabain-like compound that is linked to the pathogenesis of the hypertension. A putative steroid biosynthetic pathway was constructed and candidate genes encoding enzymes in this pathway were identified from sequence databases. Differential expression of selected genes in the pathway was studied by microarray analysis and quantitative polymerase chain reaction, with functional validation by gene silencing using small interfering RNAs. Marked upregulation of genes coding for P450 side chain cleavage and Δ5-3β-hydroxysteroid dehydrogenase/Δ5-Δ4- isomerase enzymes in hypertensive hypothalamus but not adrenal was found, compared with normotensive Milan rats. Knockdown of the latter gene decreased production of ouabain-like factor from neural tissue.Conclusions—Our findings support the possibility that a unique steroid biosynthetic circuit exists in Milan rat brain, functioning independently from adrenal, which could account for the overproduction of the hypothalamic ouabain-like compound in this species.

Conservation of the binding site on mammalian Na+,K+-ATPase for cardiac glycosides and the importance of the Na+pump in mammalian cellular physiology has stimulated the search for a mammalian analog of these plant compounds. One candidate, isolated from brain and blood, appears to be ouabain itself or a closely related isomer, the ouabain-like compound. Little is known about the circulating form. Because human steroid hormones circulate with carrier proteins, we produced a ouabain-specific monoclonal antibody (mAb 1-10) and used it to probe normal human plasma for ouabain-protein carrier complex. Ouabain-like biological activity was isolated in association with protein bands of 80, 50, and 25 kDa. These proteins appear to be human immunoglobulins or immunoglobulin-like because they are recognized by anti-human immunoglobulin antibodies, but not by anti-mouse immunoglobulin antibodies. The protein-containing fractions inhibit the binding of mAb 1-10 to immobilized ouabain, and with further purification on protein A, the immunoglobulin-like protein binds radioactive ouabain with an IC50of 200 to 600 nmol/L, but binds digoxin with 100-fold less affinity, suggesting specificity for ouabain or its isomer. Active protein fractions after purification on C18inhibit Na+pump activity in human erythrocytes (IC50≈4 nmol/L, ouabain equivalents), and this chromatography appears to dissociate the ouabain-like compound from the immunoglobulin protein(s). These immunoglobulin-like molecules may represent a subset of immunoglobulins (≤0.5% of total protein A immunoglobulin) that function as a reservoir and delivery system for ouabain-like compounds in the modulation of human Na+, K+-ATPase in vivo.

The exogenous digitalis glycosides, ouabain and digoxin, have been widely used in humans to treat congestive heart failure and cardiac arrhythmias. Several reports have also pointed to the existence of endogenous ouabain- and digoxin-like compounds, but their precise roles in mammalian physiology and various disorders of the circulation are not clear. In an attempt to produce specific Abs for the purification and identification of endogenous ouabain-like compounds, somatic cell fusion was used to produce mAbs specific for ouabain. Our attempts to produce ouabain-specific mAbs were unsuccessful when ouabain was coupled to exogenous proteins such as bovine γ-globulins, BSA, and human serum albumin. However, when ouabain was coupled to an Ab of A/J mice origin and the same strain of mouse was used for immunization with ouabain-Ab conjugate, three Abs (1-10, 5A12, and 7-1) specific for ouabain were obtained. In assays of fluorescence quenching and saturation equilibrium with tritiated ouabain, Ab 1-10 exhibited 200 nM affinity for ouabain. These three mAbs are distinguished from existing Abs to ouabain and digoxin by their specificity for ouabain and lack of cross-reactivity with digoxin. Specificity studies showed that the loss of cross-reactivity was correlated with the presence of a hydroxyl group at either position 12β (digoxin) or 16β (gitoxin) of the steroid ring. These Abs can be used to develop assays for detection and characterization of ouabain-like molecules in vivo.