Virus-Specific Nanobody-Chimeras Degrade the Human Cytomegalovirus US28 Protein in CD34+ Cells.

After primary infection, human cytomegalovirus (HCMV) establishes lifelong persistence, underpinned by latent carriage of the virus with spontaneous reactivation events. In the immune-competent, primary infection or reactivation from latency rarely causes disease. However, HCMV can cause significant disease in immune-compromised individuals such as immune-suppressed transplant patients.

Repurposing an endogenous degradation domain for antibody-mediated disposal of cell-surface proteins.

The exquisite specificity of antibodies can be harnessed to effect targeted degradation of membrane proteins. Here, we demonstrate targeted protein removal utilising a protein degradation domain derived from the endogenous human protein Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9). Recombinant antibodies genetically fused to this domain drive the degradation of membrane proteins that undergo constitutive internalisation and recycling, including the transferrin receptor and the human cytomegalovirus latency-associated protein US28.

High-level compliance to opt-out HIV testing in the emergency department (ED) of a large teaching hospital using the biochemistry sample as the sample type for HIV screening.

Introduction: HIV remains a key public health issue. National Institute for Health and Care Excellence and British HIV Association guidance recommends that patients should be offered HIV testing when admitted to hospital or attending emergency departments (EDs) in areas with a prevalence ≥ 2 per 1000. We report a novel method of testing and the first 3-year results from our HIV ED testing programme utilizing biochemistry samples for HIV testing, with the aim of improving uptake while ensuring no changes to clinical practice in EDs.

FCHO controls AP2's initiating role in endocytosis through a PtdIns(4,5)P-dependent switch.

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane-localized Fer/Cip4 homology domain-only proteins (FCHO). Here, live-cell enhanced total internal reflection fluorescence-structured illumination microscopy shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform-sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Epidermal growth factor potential receptor substrate number 15 (Eps15) ring.

Architecture of the AP2/clathrin coat on the membranes of clathrin-coated vesicles.

Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell's plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. Adaptor protein complex (AP2) is the key adaptor in CME.

Temporal Ordering in Endocytic Clathrin-Coated Vesicle Formation via AP2 Phosphorylation.

Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime.

Electromagnetic Chirps from Neutron Star-Black Hole Mergers.

We calculate the electromagnetic signal of a gamma-ray flare coming from the surface of a neutron star shortly before merger with a black hole companion. Using a new version of the Monte Carlo radiation transport code Pandurata that incorporates dynamic spacetimes, we integrate photon geodesics from the neutron star surface until they reach a distant observer or are captured by the black hole. The gamma-ray light curve is modulated by a number of relativistic effects, including Doppler beaming and gravitational lensing.

Prompt Electromagnetic Transients from Binary Black Hole Mergers.

Binary black hole (BBH) mergers provide a prime source for current and future interferometric GW observatories. Massive BBH mergers may often take place in plasma-rich environments, leading to the exciting possibility of a concurrent electromagnetic (EM) signal observable by traditional astronomical facilities. However, many critical questions about the generation of such counterparts remain unanswered.

Cellular and viral peptides bind multiple sites on the N-terminal domain of clathrin.

Short peptide motifs in unstructured regions of clathrin-adaptor proteins recruit clathrin to membranes to facilitate post-Golgi membrane transport. Three consensus clathrin-binding peptide sequences have been identified and structural studies show that each binds distinct sites on the clathrin heavy chain N-terminal domain (NTD). A fourth binding site for adaptors on NTD has been functionally identified but not structurally characterised.

Using selenomethionyl derivatives to assign sequence in low-resolution structures of the AP2 clathrin adaptor.

Selenomethionine incorporation is a powerful technique for assigning sequence to regions of electron density at low resolution. Genetic introduction of methionine point mutations and the subsequent preparation and crystallization of selenomethionyl derivatives permits unambiguous sequence assignment by enabling the placement of the anomalous scatterers (Se atoms) thus introduced. Here, the use of this approach in the assignment of sequence in a part of the AP2 clathrin adaptor complex that is responsible for clathrin binding is described.

CALM regulates clathrin-coated vesicle size and maturation by directly sensing and driving membrane curvature.

The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of "open" clathrin-coated pits (CCPs) to "necked"/"closed" CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects.

Clathrin adaptors. AP2 controls clathrin polymerization with a membrane-activated switch.

Clathrin-mediated endocytosis (CME) is vital for the internalization of most cell-surface proteins. In CME, plasma membrane-binding clathrin adaptors recruit and polymerize clathrin to form clathrin-coated pits into which cargo is sorted. Assembly polypeptide 2 (AP2) is the most abundant adaptor and is pivotal to CME.

Endocytic sorting of transmembrane protein cargo.

The accurate distribution and recycling of transmembrane proteins amongst the membrane-bound organelles of the cell is vital to ensure its correct functioning. Transmembrane protein cargo destined for clathrin-mediated endocytosis and transport along the endocytic pathway is sorted into transport vesicles by interactions with adaptors, which simultaneously link clathrin to the membrane. Clathrin adaptors recognize a variety of signals present in the cytoplasmic portions of cargo proteins; recent structural, biophysical and cell biological studies have elucidated new types of cargo-adaptor interactions and probed the molecular mechanisms regulating cargo selection and vesicle maturation.

A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex.

The AP2 adaptor complex (alpha, beta2, sigma2, and mu2 subunits) crosslinks the endocytic clathrin scaffold to PtdIns4,5P(2)-containing membranes and transmembrane protein cargo. In the "locked" cytosolic form, AP2's binding sites for the two endocytic motifs, YxxPhi on the C-terminal domain of mu2 (C-mu2) and [ED]xxxL[LI] on sigma2, are blocked by parts of beta2. Using protein crystallography, we show that AP2 undergoes a large conformational change in which C-mu2 relocates to an orthogonal face of the complex, simultaneously unblocking both cargo-binding sites; the previously unstructured mu2 linker becomes helical and binds back onto the complex.

Genome remodelling in a basal-like breast cancer metastasis and xenograft.

Massively parallel DNA sequencing technologies provide an unprecedented ability to screen entire genomes for genetic changes associated with tumour progression. Here we describe the genomic analyses of four DNA samples from an African-American patient with basal-like breast cancer: peripheral blood, the primary tumour, a brain metastasis and a xenograft derived from the primary tumour. The metastasis contained two de novo mutations and a large deletion not present in the primary tumour, and was significantly enriched for 20 shared mutations.

A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex.

Most transmembrane proteins are selected as transport vesicle cargo through the recognition of short, linear amino acid motifs in their cytoplasmic portions by vesicle coat proteins. In the case of clathrin-coated vesicles (CCVs) the motifs are recognised by clathrin adaptors. The AP2 adaptor complex (subunits α,β2,μ2,σ2) recognises both major endocytic motifs: YxxΦ motifs and [DE]xxxL[LI] acidic dileucine motifs.

Selective gene amplification.

We describe a system for directed evolution based on in vitro compartmentalisation in which amplification of a gene is coupled to the formation of product by the enzyme it encodes. This approach mimics the process of natural selection; 'fitter' genes--encoding more efficient enzymes--have more 'offspring'. It allows selection for any activity so long as a product-specific ligand (e.

Consistency of post-Newtonian waveforms with numerical relativity.

General relativity predicts the gravitational wave signatures of coalescing binary black holes. Explicit waveform predictions for such systems, required for optimal analysis of observational data, have so far been achieved primarily using the post-Newtonian (PN) approximation. The quality of this treatment is unclear, however, for the important late-inspiral portion.

Miniaturizing chemistry and biology in microdroplets.

By compartmentalizing reactions in aqueous microdroplets of water-in-oil emulsions, reaction volumes can be reduced by factors of up to 10(9) compared to conventional microtitre-plate based systems. This allows massively parallel processing of as many as 10(10) reactions in a total volume of only 1 ml of emulsion. This review describes the use of emulsions for directed evolution of proteins and RNAs, and for performing polymerase chain reactions (PCRs).

Evolutionary and biomedical insights from the rhesus macaque genome.

The rhesus macaque (Macaca mulatta) is an abundant primate species that diverged from the ancestors of Homo sapiens about 25 million years ago. Because they are genetically and physiologically similar to humans, rhesus monkeys are the most widely used nonhuman primate in basic and applied biomedical research. We determined the genome sequence of an Indian-origin Macaca mulatta female and compared the data with chimpanzees and humans to reveal the structure of ancestral primate genomes and to identify evidence for positive selection and lineage-specific expansions and contractions of gene families.

Droplets as microreactors for high-throughput biology.

Inspired by the principles of biological evolution, biologists--and others--have in recent decades harnessed the power of "natural" selection to sift through huge libraries of genes and find those with desirable properties. At the same time, the demand for high-throughput biochemical and genetic assays and screens has driven the development of increasingly miniaturised assay systems. An exciting synergy is now emerging between these two fields, whereby the tools of ultrahigh-throughput screening promise to open up new directions in molecular engineering.

High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions.

We describe a completely in vitro high-throughput screening system for directed evolution of enzymes based on in vitro compartmentalization (IVC). Single genes are transcribed and translated inside the aqueous droplets of a water-in-oil emulsion. Enzyme activity generates a fluorescent product and, after conversion into a water-in-oil-in-water double emulsion, fluorescent droplets are sorted using a fluorescence-activated cell sorter (FACS).

Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization.

In vitro compartmentalization (IVC) has previously been used to evolve protein enzymes. Here, we demonstrate how IVC can be applied to select RNA enzymes (ribozymes) for a property that has previously been unselectable: true intermolecular catalysis. Libraries containing 10(11) ribozyme genes are compartmentalized in the aqueous droplets of a water-in-oil emulsion, such that most droplets contain no more than one gene, and transcribed in situ.