Benjamin L. Miller

HMMER web server: 2015 update

The HMMER website, available at http://www.ebi.ac.uk/Tools/hmmer/, provides access to the protein homology search algorithms found in the HMMER software suite. Since the first release of the website in 2011, the search repertoire has been expanded to include the iterative search algorithm, jackhmmer. The continued growth of the target sequence databases means that traditional tabular representations of significant sequence hits can be overwhelming to the user. Consequently, additional ways of presenting homology search results have been developed, allowing them to be summarised according to taxonomic distribution or domain architecture. The taxonomy and domain architecture representations can be used in combination to filter the results according to the needs of a user. Searches can also be restricted prior to submission using a new taxonomic filter, which not only ensures that the results are specific to the requested taxonomic group, but also improves search performance. The repertoire of profile hidden Markov model libraries, which are used for annotation of query sequences with protein families and domains, has been expanded to include the libraries from CATH-Gene3D, PIRSF, Superfamily and TIGRFAMs. Finally, we discuss the relocation of the HMMER webserver to the European Bioinformatics Institute and the potential impact that this will have.

Synthesis at the molecular frontier

Driven by remarkable advances in the understanding of structure and reaction mechanisms, organic synthesis will be increasingly directed to producing bioinspired and newly designed molecules.

A multimodal RAGE-specific inhibitor reduces amyloid β–mediated brain disorder in a mouse model of Alzheimer disease

In Alzheimer disease (AD), amyloid β peptide (Aβ) accumulates in plaques in the brain. Receptor for advanced glycation end products (RAGE) mediates Aβ-induced perturbations in cerebral vessels, neurons, and microglia in AD. Here, we identified a high-affinity RAGE-specific inhibitor (FPS-ZM1) that blocked Aβ binding to the V domain of RAGE and inhibited Aβ40- and Aβ42-induced cellular stress in RAGE-expressing cells in vitro and in the mouse brain in vivo. FPS-ZM1 was nontoxic to mice and readily crossed the blood-brain barrier (BBB). In aged APPsw/0 mice overexpressing human Aβ-precursor protein, a transgenic mouse model of AD with established Aβ pathology, FPS-ZM1 inhibited RAGE-mediated influx of circulating Aβ40 and Aβ42 into the brain. In brain, FPS-ZM1 bound exclusively to RAGE, which inhibited β-secretase activity and Aβ production and suppressed microglia activation and the neuroinflammatory response. Blockade of RAGE actions at the BBB and in the brain reduced Aβ40 and Aβ42 levels in brain markedly and normalized cognitive performance and cerebral blood flow responses in aged APPsw/0 mice. Our data suggest that FPS-ZM1 is a potent multimodal RAGE blocker that effectively controls progression of Aβ-mediated brain disorder and that it may have the potential to be a disease-modifying agent for AD.

Dynamic combinatorial selection of molecules capable of inhibiting the (CUG) repeat RNA-MBNL1 interaction in vitro: discovery of lead compounds targeting myotonic dystrophy (DM1).

Myotonic dystrophy type 1 (DM1), the most common form of muscular dystrophy in adults, is an RNA-mediated disease. Dramatically expanded (CUG) repeats accumulate in nuclei and sequester RNA-binding proteins such as the splicing regulator MBNL1. We have employed resin-bound dynamic combinatorial chemistry (RBDCC) to identify the first examples of compounds able to inhibit MBNL1 binding to (CUG) repeat RNA. Screening an RBDCL with a theoretical diversity of 11 325 members yielded several molecules with significant selectivity for binding to (CUG) repeat RNA over other sequences. These compounds were also able to inhibit the interaction of GGG-(CUG)(109)-GGG RNA with MBNL1 in vitro, with K(i) values in the low micromolar range.

Nanoscale silicon microcavities for biosensing

We report the design and testing of a versatile biosensor exclusively using silicon. The device structure consists of a microcavity resonator made of various porous silicon layers. Porous silicon contains silicon nanocrystals that can luminesce efficiently in the visible, depending on the size and passivation conditions. When a luminescent porous silicon layer is inserted between two Bragg reflectors (also made of porous silicon), the broad luminescence band is altered and multiple and very narrow peaks are detected. The position of these peaks is extremely sensitive to a small change in refractive index, such as that obtained when a biological object is attached to the large internal surface of porous silicon. We have demonstrated a DNA sensor that displays appropriate sensitivity, selectivity and response speed. The device fabrication procedure and the results of extensive testing are presented. An extension of the DNA biosensor has been made to include the detection of viral DNA. This work will lead to the development of an all-silicon sensor array for the detection of biomacromolecules.

Silicon photonic crystal nanocavity-coupled waveguides for error-corrected optical biosensing.

A photonic crystal (PhC) waveguide based optical biosensor capable of label-free and error-corrected sensing was investigated in this study. The detection principle of the biosensor involved shifts in the resonant mode wavelength of nanocavities coupled to the silicon PhC waveguide due to changes in ambient refractive index. The optical characteristics of the nanocavity structure were predicted by FDTD theoretical methods. The device was fabricated using standard nanolithography and reactive-ion-etching techniques. Experimental results showed that the structure had a refractive index sensitivity of 10(-2) RIU. The biosensing capability of the nanocavity sensor was tested by detecting human IgG molecules. The device sensitivity was found to be 2.3±0.24×10(5) nm/M with an achievable lowest detection limit of 1.5 fg for human IgG molecules. Additionally, experimental results demonstrated that the PhC devices were specific in IgG detection and provided concentration-dependent responses consistent with Langmuir behavior. The PhC devices manifest outstanding potential as microscale label-free error-correcting sensors, and may have future utility as ultrasensitive multiplex devices.

Haptoglobin Genotype Is a Regulator of Reverse Cholesterol Transport in Diabetes In Vitro and In Vivo

Two common alleles exist at the haptoglobin (Hp) locus, and the Hp2 allele is associated with an increased incidence of cardiovascular disease, specifically in diabetes mellitus (DM). Oxidative stress is increased in Hp2 mice and humans with DM. Oxidative modification of the apolipoprotein A-I inhibits reverse cholesterol transport. We sought to test the hypothesis that reverse cholesterol transport is impaired in Hp2 DM mice and humans. In vitro, using serum from non-DM and DM individuals, we measured cholesterol efflux from 3H-cholesterol–labeled macrophages. In vivo, we injected 3H-cholesterol–loaded macrophages intraperitoneally into non-DM and DM mice with the Hp1-1 or Hp2-2 genotype and monitored 3H-tracer levels in plasma, liver, and feces. In vitro, in DM individuals only, we observed significantly decreased cholesterol efflux from macrophages incubated with serum from Hp2-1 or Hp2-2 as compared with Hp1-1 individuals (P<0.01). The interaction between Hp type and DM was recapitulated using purified Hp and glycated Hb. In vivo, DM mice loaded with 3H-cholesterol–labeled macrophages had a 40% reduction in 3H-cholesterol in plasma, liver, and feces as compared with non-DM mice (P<0.01). The reduction in reverse cholesterol transport associated with DM was significantly greater in Hp2-2 mice as compared with Hp1-1 mice (54% versus 25% in plasma; 52% versus 27% in liver; 57% versus 32% in feces; P<0.03). reverse cholesterol transport is decreased in Hp2-2 DM. This may explain in part the increased atherosclerotic burden found in Hp2-2 DM individuals.

Generation of novel DNA-binding compounds by selection and amplification from self-assembled combinatorial libraries

Abstract We describe a general method for the selection of compounds from self-assembled libraries which employs an immobilized receptor (i.e., and affinity reagent) to effect the selection. Using commercially available oligo d(A·T) DNA-cellulose resin, a set of three setereoisomeric coordination complexes are identified as DNA binding compounds from an equilibrating, self-assembled library of 36 bis(salicyladiminato)-zinc coordination complexes.

From dynamic combinatorial ‘hit’ to lead: in vitro and in vivo activity of compounds targeting the pathogenic RNAs that cause myotonic dystrophy

The myotonic dystrophies (DM) are human diseases in which the accumulation of toxic RNA (CUG or CCUG) repeats in the cell causes sequestration of splicing factors, including MBNL1, leading to clinical symptoms such as muscle wasting and myotonia. We previously used Dynamic Combinatorial Chemistry to identify the first compounds known to inhibit (CUG)-MBNL1 binding in vitro. We now report transformation of those compounds into structures with activity in vivo. Introduction of a benzo[g]quinoline substructure previously unknown in the context of RNA recognition, as well as other modifications, provided several molecules with enhanced binding properties, including compounds with strong selectivity for CUG repeats over CAG repeats or CAG–CUG duplex RNA. Compounds readily penetrate cells, and improve luciferase activity in a mouse myoblast assay in which enzyme function is coupled to a release of nuclear CUG–RNA retention. Most importantly, two compounds are able to partially restore splicing in a mouse model of DM1.

Selection of DNA-binding compounds via multistage molecular evolution

Abstract Combinatorial libraries incorporating multiple equilibria offer opportunities to study molecular evolution, and are a novel method of identifying ligands for biological receptors. We describe the construction and evaluation of a multi-equilibrium combinatorial library, in which structural diversity and structural mutation are accomplished via reversible imine formation and transition-metal complexation. We demonstrate that oligo d(A·T)-cellulose resin can select subsets of this libray, in accord with measured solution-phase affinities.