The FSHD2 Gene SMCHD1 Is a Modifier of Disease Severity in Families Affected by FSHD1

Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is caused by contraction of the D4Z4 repeat array on chromosome 4 to a size of 1–10 units. The residual number of D4Z4 units inversely correlates with clinical severity, but significant clinical variability exists. Each unit contains a copy of the DUX4 retrogene. Repeat contractions are associated with changes in D4Z4 chromatin structure that increase the likelihood of DUX4 expression in skeletal muscle, but only when the repeat resides in a genetic background that contains a DUX4 polyadenylation signal. Mutations in the structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) gene, encoding a chromatin modifier of D4Z4, also result in the increased likelihood of DUX4 expression in individuals with a rare form of FSHD (FSHD2). Because SMCHD1 directly binds to D4Z4 and suppresses somatic expression of DUX4, we hypothesized that SMCHD1 may act as a genetic modifier in FSHD1. We describe three unrelated individuals with FSHD1 presenting an unusual high clinical severity based on their upper-sized FSHD1 repeat array of nine units. Each of these individuals also carries a mutation in the SMCHD1 gene. Familial carriers of the FSHD1 allele without the SMCHD1 mutation were only mildly affected, suggesting a modifier effect of the SMCHD1 mutation. Knocking down SMCHD1 in FSHD1 myotubes increased DUX4 expression, lending molecular support to a modifier role for SMCHD1 in FSHD1. We conclude that FSHD1 and FSHD2 share a common pathophysiological pathway in which the FSHD2 gene can act as modifier for disease severity in families affected by FSHD1.     

T‐type calcium channel blockers inhibit autophagy and promote apoptosis of malignant melanoma cells

We have recently reported that human melanoma cells express a variety of voltage‐gated calcium (Ca²⁺) channel types, including low‐voltage‐activated T‐type channels that play a significant role in melanoma cell cycle progression. Here, we challenged melanoma metastatic cells with T‐type channel blockers of clinical use and found a dual effect on cell viability: (i) a reduction in the proliferation rate, through a halt in the progression to the G₁‐S phase; and (ii) a promotion of cell death that was partially dependent on the activation of caspases. An in‐depth analysis of the death process showed that the apoptotic pathway is preceded by endoplasmic reticulum stress and the subsequent inhibition of the basal macroautophagy which is active in these cells. The effects of pharmacological blockers on Ca²⁺ homeostasis, autophagy, and cell death were mimicked by T‐type channel gene silencing. These results provide the basis for a new pharmacological and/or gene silencing approach toward tackling melanoma metastasis.     

Four Common Simplifications of Multi-Criteria Decision Analysis do not hold for River Rehabilitation

River rehabilitation aims at alleviating negative effects of human impacts such as loss of biodiversity and reduction of ecosystem services. Such interventions entail difficult trade-offs between different ecological and often socio-economic objectives. Multi-Criteria Decision Analysis (MCDA) is a very suitable approach that helps assessing the current ecological state and prioritizing river rehabilitation measures in a standardized way, based on stakeholder or expert preferences. Applications of MCDA in river rehabilitation projects are often simplified, i.e. using a limited number of objectives and indicators, assuming linear value functions, aggregating individual indicator assessments additively, and/or assuming risk neutrality of experts. Here, we demonstrate an implementation of MCDA expert preference assessments to river rehabilitation and provide ample material for other applications. To test whether the above simplifications reflect common expert opinion, we carried out very detailed interviews with five river ecologists and a hydraulic engineer. We defined essential objectives and measurable quality indicators (attributes), elicited the experts´ preferences for objectives on a standardized scale (value functions) and their risk attitude, and identified suitable aggregation methods. The experts recommended an extensive objectives hierarchy including between 54 and 93 essential objectives and between 37 to 61 essential attributes. For 81% of these, they defined non-linear value functions and in 76% recommended multiplicative aggregation. The experts were risk averse or risk prone (but never risk neutral), depending on the current ecological state of the river, and the experts´ personal importance of objectives. We conclude that the four commonly applied simplifications clearly do not reflect the opinion of river rehabilitation experts. The optimal level of model complexity, however, remains highly case-study specific depending on data and resource availability, the context, and the complexity of the decision problem.     

Transmigration characteristics of breast cancer and melanoma cells through the brain endothelium: Role of Rac and PI3K

Brain metastases are common and devastating complications of both breast cancer and melanoma. Although mammary carcinoma brain metastases are more frequent than those originating from melanoma, this latter has the highest tropism to the brain. Using static and dynamic in vitro approaches, here we show that melanoma cells have increased adhesion to the brain endothelium in comparison to breast cancer cells. Moreover, melanoma cells can transmigrate more rapidly and in a higher number through brain endothelial monolayers than breast cancer cells. In addition, melanoma cells have increased ability to impair tight junctions of cerebral endothelial cells. We also show that inhibition of Rac or PI3K impedes adhesion of breast cancer cells and melanoma cells to the brain endothelium. In addition, inhibition of Rac or PI3K inhibits the late phase of transmigration of breast cancer cells and the early phase of transmigration of melanoma cells. On the other hand, the Rac inhibitor EHT1864 impairs the junctional integrity of the brain endothelium, while the PI3K inhibitor LY294002 has no damaging effect on interendothelial junctions. We suggest that targeting the PI3K/Akt pathway may represent a novel opportunity in preventing the formation of brain metastases of melanoma and breast cancer.     

Strong phenotypic divergence in spite of low genetic structure in the endemic Mangrove Warbler subspecies (Setophaga petechia xanthotera) of Costa Rica

Despite the enormous advances in genetics, links between phenotypes and genotypes have been made for only a few nonmodel organisms. However, such links can be essential to understand mechanisms of ecological speciation. The Costa Rican endemic Mangrove Warbler subspecies provides an excellent subject to study differentiation with gene flow, as it is distributed along a strong precipitation gradient on the Pacific coast with no strong geographic barriers to isolate populations. Mangrove Warbler populations could be subject to divergent selection driven by precipitation, which influences soil salinity levels, which in turn influences forest structure and food resources. We used single nucleotide polymorphisms (SNPs) and morphological traits to examine the balance between neutral genetic and phenotypic divergence to determine whether selection has acted on traits and genes with functions related to specific environmental variables. We present evidence showing: (a) associations between environmental variables and SNPs, identifying candidate genes related to bill morphology (BMP) and osmoregulation, (b) absence of population genetic structure in neutrally evolving markers, (c) divergence in bill size across the precipitation gradient, and (d) strong phenotypic differentiation (P_ST) which largely exceeds neutral genetic differentiation (F_ST) in bill size. Our results indicate an important role for salinity, forest structure, and resource availability in maintaining phenotypic divergence of Mangrove Warblers through natural selection. Our findings add to the growing body of literature identifying the processes involved in phenotypic differentiation along environmental gradients in the face of gene flow.     

Membrane fusogenic lysine type lipid assemblies possess enhanced NLRP3 inflammasome activation potency

Lysine (K) type cationic lipid with a propyl spacer and ditetradecyl hydrophobic moieties composing liposomes, K3C14, previously studied for gene delivery, were reported to activate the NLRP3 inflammasomes in human macrophages via the conventional phagolysosomal pathway. In this study, K3C16, a propyl spacer bearing lysine type lipids with dihexadecyl moieties (an extension of two hydrocarbon tail length) were compared with K3C14 as liposomes. Such a small change in tail length did not alter the physical properties such as size distribution, zeta potential and polydispersity index (PDI). The NLRP3 activation potency of K3C16 was shown to be 1.5-fold higher. Yet, the toxicity was minimal, whereas K3C14 has shown to cause significant cell death after 24 h incubation. Even in the presence of endocytosis inhibitors, cytochalasin D or dynasore, K3C16 continued to activate the NLRP3 inflammasomes and to induce IL-1β release. To our surprise, K3C16 liposomes were confirmed to fuse with the plasma membrane of human macrophages and CHO-K1 cells. It is demonstrated that the change in hydrophobic tail length by two hydrocarbons drastically changed a cellular entry route and potency in activating the NLRP3 inflammasomes.     

Active site closure facilitates juxtaposition of reactant atoms for initiation of catalysis by human dUTPase

Human dUTPase, essential for DNA integrity, is an important survival factor for cancer cells. We determined the crystal structure of the enzyme:alpha,beta-imino-dUTP:Mg complex and performed equilibrium binding experiments in solution. Ordering of the C-terminus upon the active site induces close juxtaposition of the incoming nucleophile attacker water oxygen and the alpha-phosphorus of the substrate, decreasing their distance below the van der Waals limit. Complex interactions of the C-terminus with both substrate and product were observed via a specifically designed tryptophan sensor, suitable for further detailed kinetic and ligand binding studies. Results explain the key functional role of the C-terminus.     

The human cytochrome c oxidase assembly factors SCO1 and SCO2 have regulatory roles in the maintenance of cellular copper homeostasis

Human SCO1 and SCO2 are metallochaperones that are essential for the assembly of the catalytic core of cytochrome c oxidase (COX). Here we show that they have additional, unexpected roles in cellular copper homeostasis. Mutations in either SCO result in a cellular copper deficiency that is both tissue and allele specific. This phenotype can be dissociated from the defects in COX assembly and is suppressed by overexpression of SCO2, but not SCO1. Overexpression of a SCO1 mutant in control cells in which wild-type SCO1 levels were reduced by shRNA recapitulates the copper-deficiency phenotype in SCO1 patient cells. The copper-deficiency phenotype reflects not a change in high-affinity copper uptake but rather a proportional increase in copper efflux. These results suggest a mitochondrial pathway for the regulation of cellular copper content that involves signaling through SCO1 and SCO2, perhaps by their thiol redox or metal-binding state.