Many theoretical advances have been made in applying probabilistic inference methods to improve the power of sequence homology searches, yet the BLAST suite of programs is still the workhorse for most of the field. The main reason for this is practical: BLAST’s programs are about 100-fold faster than the fastest competing implementations of probabilistic inference methods. I describe recent work on the HMMER software suite for protein sequence analysis, which implements probabilistic inference using profile hidden Markov models. Our aim in HMMER3 is to achieve BLAST’s speed while further improving the power of probabilistic inference based methods. HMMER3 implements a new probabilistic model of local sequence alignment and a new heuristic acceleration algorithm. Combined with efficient vector-parallel implementations on modern processors, these improvements synergize. HMMER3 uses more powerful log-odds likelihood scores (scores summed over alignment uncertainty, rather than scoring a single optimal alignment); it calculates accurate expectation values (E-values) for those scores without simulation using a generalization of Karlin/Altschul theory; it computes posterior distributions over the ensemble of possible alignments and returns posterior probabilities (confidences) in each aligned residue; and it does all this at an overall speed comparable to BLAST. The HMMER project aims to usher in a new generation of more powerful homology search tools based on probabilistic inference methods.

Nitric oxide (NO) is a mediator of immunity to malaria, and genetic polymorphisms in the promoter of the inducible NO synthase gene (NOS2) could modulate production of NO. We postulated that NOS2 promoter polymorphisms would affect resistance to severe malaria.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate the expression of target transcript mRNAs. Many miRNAs have been defined, however their roles and the processes influenced by miRNA pathways are still being elucidated. A role for miRNAs in development and cancer has been described. We recently isolated the miRNA bantam (ban) in a genetic screen for modulators of pathogenicity of a human neurodegenerative disease model in Drosophila. These studies showed that upregulation of ban mitigates degeneration induced by the pathogenic polyglutamine (polyQ) protein Ataxin-3, which is mutated in the human polyglutamine disease spinocerebellar ataxia type 3 (SCA3). To address the broader role for miRNAs in neuroprotection, we also showed that loss of all miRNAs, by dicer mutation, dramatically enhances pathogenic polyQ protein toxicity in flies and in human HeLa cells. These studies suggest that miRNAs may be important for neuronal survival in the context of human neurodegenerative disease. These studies provide the foundation to define the miRNAs involved in neurodegenerative disease, and the biological pathways affected.

An efficient two-step strategy has been developed to access diversely functionalized benzylic sulfonamides. Execution of this strategy required the development of two reaction methods: the palladium-catalyzed cross-coupling of aryl halides with CH-acidic methanesulfonamides and a metathesis reaction between the resulting alpha-arylated sulfonamides and diverse amines. The broad scope of the cross-coupling process combined with a versatile sulfonamide metathesis constitutes an efficient strategy for the synthesis of various benzylic sulfonamides.

Dopamine has been implicated in mediating contextual modulation of motor behaviors and learning in many species. In songbirds, dopamine may act on the basal ganglia nucleus Area X to influence the neural activity that contributes to vocal learning and contextual changes in song variability. Neurons in midbrain dopamine centers, the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA), densely innervate Area X and show singing-related changes in firing rate. In addition, dopamine levels in Area X change during singing. It is unknown, however, how song-related information could reach dopaminergic neurons. Here we report an anatomical pathway that could provide song-related information to the SNc and VTA. By using injections of bidirectionally transported fluorescent tracers in adult male zebra finches, we show that Area X and other song control nuclei do not project directly to the SNc or VTA. Instead, we describe an indirect pathway from Area X to midbrain dopaminergic neurons via a connection in the ventral pallidum (VP). Specifically, Area X projects to the VP via axon collaterals of Area X output neurons that also project to the thalamus. Dual injections revealed that the area of VP receiving input from Area X projects to the SNc and VTA. Furthermore, VP terminals in the SNc and VTA overlap with cells that project back to Area X. A portion of the arcopallium also projects to the SNc and VTA and could carry auditory information. These data demonstrate an anatomical loop through which Area X activity could influence its dopaminergic input.

In mammals, taste perception is a major mode of sensory input. We have identified a novel family of 40-80 human and rodent G protein-coupled receptors expressed in subsets of taste receptor cells of the tongue and palate epithelia. These candidate taste receptors (T2Rs) are organized in the genome in clusters and are genetically linked to loci that influence bitter perception in mice and humans. Notably, a single taste receptor cell expresses a large repertoire of T2Rs, suggesting that each cell may be capable of recognizing multiple tastants. T2Rs are exclusively expressed in taste receptor cells that contain the G protein alpha subunit gustducin, implying that they function as gustducin-linked receptors. In the accompanying paper, we demonstrate that T2Rs couple to gustducin in vitro, and respond to bitter tastants in a functional expression assay.

In an elaborate form of inter-species exploitation, many insects hijack plant development to induce novel plant organs called galls that provide the insect with a source of nutrition and a temporary home. Galls result from dramatic reprogramming of plant cell biology driven by insect molecules, but the roles of specific insect molecules in gall development have not yet been determined. Here, we study the aphid Hormaphis cornu, which makes distinctive "cone" galls on leaves of witch hazel Hamamelis virginiana. We found that derived genetic variants in the aphid gene determinant of gall color (dgc) are associated with strong downregulation of dgc transcription in aphid salivary glands, upregulation in galls of seven genes involved in anthocyanin synthesis, and deposition of two red anthocyanins in galls. We hypothesize that aphids inject DGC protein into galls and that this results in differential expression of a small number of plant genes. dgc is a member of a large, diverse family of novel predicted secreted proteins characterized by a pair of widely spaced cysteine-tyrosine-cysteine (CYC) residues, which we named BICYCLE proteins. bicycle genes are most strongly expressed in the salivary glands specifically of galling aphid generations, suggesting that they may regulate many aspects of gall development. bicycle genes have experienced unusually frequent diversifying selection, consistent with their potential role controlling gall development in a molecular arms race between aphids and their host plants.

Trophic factors are a heterogeneous group of molecules that promote cell growth and survival. In freshwater planarians, the small secreted protein TCEN49 is linked to the regional maintenance of the planarian central body region. To investigate its function in vivo, we performed loss-of-function and gain-of-function experiments by RNA interference and by the implantation of microbeads soaked in TCEN49, respectively. We show that TCEN49 behaves as a trophic factor involved in central body region neuron survival. In planarian tail regenerates, tcen49 expression inhibition by double-stranded RNA interference causes extensive apoptosis in various cell types, including nerve cells. This phenotype is rescued by the implantation of microbeads soaked in TCEN49 after RNA interference. On the other hand, in organisms committed to asexual reproduction, both tcen49 mRNA and its protein are detected not only in the central body region but also in the posterior region, expanding from cells close to the ventral nerve chords. In some cases, the implantation of microbeads soaked in TCEN49 in the posterior body region drives organisms to reproduce asexually, and the inhibition of tcen49 expression obstructs this process, suggesting a link between the central nervous system, TCEN49, regional induction, and asexual reproduction. Finally, the distribution of TCEN49 cysteine and tyrosine residues also points to a common evolutionary origin for TCEN49 and molluscan neurotrophins.

Gene expression depends upon the antagonistic actions of chromatin remodeling complexes. While this has been studied extensively for the enzymes that covalently modify the tails of histones, the mechanism of how ATP-dependent remodeling complexes antagonize each other to maintain the proper level of gene activity is not known. The gene encoding a large subunit of ribonucleotide reductase, RNR3, is regulated by ISW2 and SWI/SNF, complexes that repress and activate transcription, respectively. Here, we studied the functional interactions of these two complexes at RNR3. Deletion of ISW2 causes constitutive recruitment of SWI/SNF, and conditional reexpression of ISW2 causes the repositioning of nucleosomes and reduced SWI/SNF occupancy at RNR3. Thus, ISW2 is required for restriction of access of SWI/SNF to the RNR3 promoter under the uninduced condition. Interestingly, the binding of sequence-specific DNA binding factors and the general transcription machinery are unaffected by the status of ISW2, suggesting that disruption of nucleosome positioning does not cause a nonspecific increase in cross-linking of all factors to RNR3. We provide evidence that ISW2 does not act on SWI/SNF directly but excludes its occupancy by positioning nucleosomes over the promoter. Genetic disruption of nucleosome positioning by other means led to a similar phenotype, linking repressed chromatin structure to SWI/SNF exclusion. Thus, incorporation of promoters into a repressive chromatin structure is essential for prevention of the opportunistic actions of nucleosome-disrupting activities in vivo, providing a novel mechanism for maintaining tight control of gene expression.

We present a novel framework for automatically constraining parameters of compartmental models of neurons, given a large set of experimentally measured responses of these neurons. In experiments, intrinsic noise gives rise to a large variability (e.g., in firing pattern) in the voltage responses to repetitions of the exact same input. Thus, the common approach of fitting models by attempting to perfectly replicate, point by point, a single chosen trace out of the spectrum of variable responses does not seem to do justice to the data. In addition, finding a single error function that faithfully characterizes the distance between two spiking traces is not a trivial pursuit. To address these issues, one can adopt a multiple objective optimization approach that allows the use of several error functions jointly. When more than one error function is available, the comparison between experimental voltage traces and model response can be performed on the basis of individual features of interest (e.g., spike rate, spike width). Each feature can be compared between model and experimental mean, in units of its experimental variability, thereby incorporating into the fitting this variability. We demonstrate the success of this approach, when used in conjunction with genetic algorithm optimization, in generating an excellent fit between model behavior and the firing pattern of two distinct electrical classes of cortical interneurons, accommodating and fast-spiking. We argue that the multiple, diverse models generated by this method could serve as the building blocks for the realistic simulation of large neuronal networks.