Haploinsufficiency of KDM6A is associated with severe psychomotor retardation, global growth restriction, seizures and cleft palate

We describe a female subject (DGAP100) with a 46,X,t(X;5)(p11.3;q35.3)inv(5)(q35.3q35.1)dn, severe psychomotor retardation with hypotonia, global postnatal growth restriction, microcephaly, globally reduced cerebral volume, seizures, facial dysmorphia and cleft palate. Fluorescence in situ hybridization and whole-genome sequencing demonstrated that the X chromosome breakpoint disrupts KDM6A in the second intron. No genes were directly disrupted on chromosome 5. KDM6A is a histone 3 lysine 27 demethylase and a histone 3 lysine 4 methyltransferase. Expression of KDM6A is significantly reduced in DGAP100 lymphoblastoid cells compared to control samples. We identified nine additional cases with neurodevelopmental delay and various other features consistent with the DGAP100 phenotype with copy number variation encompassing KDM6A from microarray databases. We evaluated haploinsufficiency of kdm6a in a zebrafish model. kdm6a is expressed in the pharyngeal arches and ethmoid plate of the developing zebrafish, while a kdm6a morpholino knockdown exhibited craniofacial defects. We conclude KDM6A dosage regulation is associated with severe and diverse structural defects and developmental abnormalities.     

Molecular and structural insight into the role of key residues of thrombospondin-1 and calreticulin in thrombospondin-1-calreticulin binding

Thrombospondin-1 (TSP1) binding to calreticulin (CRT) on the cell surface signals focal adhesion disassembly, leading to the intermediate adhesive phenotype, cell migration, anoikis resistance, and collagen stimulation. Residues Lys 24 and 32 in TSP1 and amino acids 24-26 and 32-34 in CRT have been shown through biochemical and cell-based approaches to be critical for TSP1-CRT binding and signaling. This study investigated the molecular and structural basis for these key TSP1 and CRT residues in TSP1-CRT binding. On the basis of a validated TSP1-CRT complex structure, we adopted steered molecular dynamics simulations to determine the effect of mutation of these key residues on TSP1-CRT binding and validated the simulation results with experimental observations. We further performed 30 ns molecular dynamics simulations for wild-type TSP1, CRT, K24A/K32A mutant TSP1, and mutant CRT (residues 24-26 and 32-34 mutated to Ala) and studied the conformational and structural changes in TSP1 and CRT as the result of mutation of these critical residues. Results showed that mutation of residues 24 and 32 to Ala in TSP1 and of amino acids 24-26 and 32-34 to Ala in CRT results in a shortened β-strand in the binding site, decreased hydrogen bond occupancy for β-strand pairs that are located within or near the binding site, increased conformational flexibility of the binding site, a changed degree of dynamically correlated motion between the residues in the binding site and the other residues in protein, and a changed degree of overall correlated motion between the residues in the protein. These changes could directly contribute to the loss or weakened binding between TSP1 and CRT and the resultant effects on TSP1-CRT binding-induced cellular activities. Results from this study provide a molecular and structural insight into the role of these critical residues of TSP1 and CRT in TSP1-CRT binding.     

Bacillus subtilis class Ib ribonucleotide reductase is a dimanganese(III)-tyrosyl radical enzyme

Bacillus subtilis class Ib ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, providing the building blocks for DNA replication and repair. It is composed of two proteins: α (NrdE) and β (NrdF). β contains the metallo-cofactor, essential for the initiation of the reduction process. The RNR genes are organized within the nrdI-nrdE-nrdF-ymaB operon. Each protein has been cloned, expressed, and purified from Escherichia coli. As isolated, recombinant NrdF (rNrdF) contained a diferric-tyrosyl radical [Fe(III)(2)-Y(?)] cofactor. Alternatively, this cluster could be self-assembled from apo-rNrdF, Fe(II), and O(2). Apo-rNrdF loaded using 4 Mn(II)/β(2), O(2), and reduced NrdI (a flavodoxin) can form a dimanganese(III)-Y(?) [Mn(III)(2)-Y(?)] cofactor. In the presence of rNrdE, ATP, and CDP, Mn(III)(2)-Y(?) and Fe(III)(2)-Y(?) rNrdF generate dCDP at rates of 132 and 10 nmol min(-1) mg(-1), respectively (both normalized for 1 Y(?)/β(2)). To determine the endogenous cofactor of NrdF in B. subtilis, the entire operon was placed behind a Pspank(hy) promoter and integrated into the B. subtilis genome at the amyE site. All four genes were induced in cells grown in Luria-Bertani medium, with levels of NrdE and NrdF elevated 35-fold relative to that of the wild-type strain. NrdE and NrdF were copurified in a 1:1 ratio from this engineered B. subtilis. The visible, EPR, and atomic absorption spectra of the purified NrdENrdF complex (eNrdF) exhibited characteristics of a Mn(III)(2)-Y(?) center with 2 Mn/β(2) and 0.5 Y(?)/β(2) and an activity of 318-363 nmol min(-1) mg(-1) (normalized for 1 Y(?)/β(2)). These data strongly suggest that the B. subtilis class Ib RNR is a Mn(III)(2)-Y(?) enzyme.     

Solution- and solid-state NMR studies of GPCRs and their ligands

G protein-coupled receptors (GPCRs) represent one of the major targets of new drugs on the market given their roles as key membrane receptors in many cellular signalling pathways. Structure-based drug design has potential to be the most reliable method for novel drug discovery. Unfortunately, GPCR-ligand crystallisation for X-ray diffraction studies is very difficult to achieve. However, solution- and solid-state NMR approaches have been developed and have provided new insights, particularly focussing on the study of protein-ligand interactions which are vital for drug discovery. This review provides an introduction for new investigators of GPCRs/ligand interactions using NMR spectroscopy. The guidelines for choosing a system for efficient isotope labelling of GPCRs and their ligands for NMR studies will be presented, along with an overview of the different sample environments suitable for generation of high resolution structural information from NMR spectra.     

Differential thermal tolerance between algae and corals may trigger the proliferation of algae in coral reefs

Marine heatwaves can lead to rapid changes in entire communities, including in the case of shallow coral reefs the potential overgrowth of algae. Here we tested experimentally the differential thermal tolerance between algae and coral species from the Red Sea through the measurement of thermal performance curves and the assessment of thermal limits. Differences across functional groups (algae vs. corals) were apparent for two key thermal performance metrics. First, two reef‐associated algae species (Halimeda tuna and Turbinaria ornata) had higher lethal thermal limits than two coral species (Pocillopora verrucosa and Stylophora pistillata) conferring those species of algae with a clear advantage during heatwaves by surpassing the thermal threshold of coral survival. Second, the coral species had generally greater deactivation energies for net and gross primary production rates compared to the algae species, indicating greater thermal sensitivity in corals once the optimum temperature is exceeded. Our field surveys in the Red Sea reefs before and after the marine heatwave of 2015 show a change in benthic cover mainly in the southern reefs, where there was a decrease in coral cover and a concomitant increase in algae abundance, mainly turf algae. Our laboratory and field observations indicate that a proliferation of algae might be expected on Red Sea coral reefs with future ocean warming.     

Mating patterns influence vulnerability to the extinction vortex

Earth's biodiversity is undergoing mass extinction due to anthropogenic compounding of environmental, demographic and genetic stresses. These different stresses can trap populations within a reinforcing feedback loop known as the extinction vortex, in which synergistic pressures build upon one another through time, driving down population viability. Sexual selection, the widespread evolutionary force arising from competition, choice and reproductive variance within animal mating patterns could have vital consequences for population viability and the extinction vortex: (a) if sexual selection reinforces natural selection to fix ‘good genes’ and purge ‘bad genes’, then mating patterns encouraging competition and choice may help protect populations from extinction; (b) by contrast, if mating patterns create load through evolutionary or ecological conflict, then population viability could be further reduced by sexual selection. We test between these opposing theories using replicate populations of the model insect Tribolium castaneum exposed to over 10 years of experimental evolution under monogamous versus polyandrous mating patterns. After a 95‐generation history of divergence in sexual selection, we compared fitness and extinction of monogamous versus polyandrous populations through an experimental extinction vortex comprising 15 generations of cycling environmental and genetic stresses. Results showed that lineages from monogamous evolutionary backgrounds, with limited opportunities for sexual selection, showed rapid declines in fitness and complete extinction through the vortex. By contrast, fitness of populations from the history of polyandry, with stronger opportunities for sexual selection, declined slowly, with 60% of populations surviving by the study end. The three vortex stresses of (a) nutritional deprivation, (b) thermal stress and (c) genetic bottlenecking had similar impacts on fitness declines and extinction risk, with an overall sigmoid decline in survival through time. We therefore reveal sexual selection as an important force behind lineages facing extinction threats, identifying the relevance of natural mating patterns for conservation management.     

Phylogenetic and Molecular Epidemiological Studies Reveal Evidence of Multiple Past Recombination Events between Infectious Laryngotracheitis Viruses

In contrast to the RNA viruses, the genome of large DNA viruses such as herpesviruses have been considered to be relatively stable. Intra-specific recombination has been proposed as an important, but underestimated, driving force in herpesvirus evolution. Recently, two distinct field strains of infectious laryngotracheitis virus (ILTV) have been shown to have arisen from independent recombination events between different commercial ILTV vaccines. In this study we sequenced the genomes of additional ILTV strains and also utilized other recently updated complete genome sequences of ILTV to confirm the existence of a number of ILTV recombinants in nature. Multiple recombination events were detected in the unique long and repeat regions of the genome, but not in the unique short region. Most recombinants contained a pair of crossover points between two distinct lineages of ILTV, corresponding to the European origin and the Australian origin vaccine strains of ILTV. These results suggest that there are two distinct genotypic lineages of ILTV and that these commonly recombine in the field.