Chronic exposure to odors at naturally occurring concentrations triggers limited plasticity in early stages of Drosophila olfactory processing.

In insects and mammals, olfactory experience in early life alters olfactory behavior and function in later life. In the vinegar fly Drosophila, flies chronically exposed to a high concentration of a monomolecular odor exhibit reduced behavioral aversion to the familiar odor when it is reencountered. This change in olfactory behavior has been attributed to selective decreases in the sensitivity of second-order olfactory projection neurons (PNs) in the antennal lobe that respond to the overrepresented odor. However, since odorant compounds do not occur at similarly high concentrations in natural sources, the role of odor experience-dependent plasticity in natural environments is unclear. Here, we investigated olfactory plasticity in the antennal lobe of flies chronically exposed to odors at concentrations that are typically encountered in natural odor sources. These stimuli were chosen to each strongly and selectively excite a single class of primary olfactory receptor neuron (ORN), thus facilitating a rigorous assessment of the selectivity of olfactory plasticity for PNs directly excited by overrepresented stimuli. Unexpectedly, we found that chronic exposure to three such odors did not result in decreased PN sensitivity but rather mildly increased responses to weak stimuli in most PN types. Odor-evoked PN activity in response to stronger stimuli was mostly unaffected by odor experience. When present, plasticity was observed broadly in multiple PN types and thus was not selective for PNs receiving direct input from the chronically active ORNs. We further investigated the DL5 olfactory coding channel and found that chronic odor-mediated excitation of its input ORNs did not affect PN intrinsic properties, local inhibitory innervation, ORN responses or ORN-PN synaptic strength; however, broad-acting lateral excitation evoked by some odors was increased. These results show that PN odor coding is only mildly affected by strong persistent activation of a single olfactory input, highlighting the stability of early stages of insect olfactory processing to significant perturbations in the sensory environment.

Lineages to circuits: the developmental and evolutionary architecture of information channels into the central complex.

The representation and integration of internal and external cues is crucial for any organism to execute appropriate behaviors. In insects, a highly conserved region of the brain, the central complex (CX), functions in the representation of spatial information and behavioral states, as well as the transformation of this information into desired navigational commands. How does this relatively invariant structure enable the incorporation of information from the diversity of anatomical, behavioral, and ecological niches occupied by insects? Here, we examine the input channels to the CX in the context of their development and evolution. Insect brains develop from ~ 100 neuroblasts per hemisphere that divide systematically to form "lineages" of sister neurons, that project to their target neuropils along anatomically characteristic tracts. Overlaying this developmental tract information onto the recently generated Drosophila "hemibrain" connectome and integrating this information with the anatomical and physiological recording of neurons in other species, we observe neuropil and lineage-specific innervation, connectivity, and activity profiles in CX input channels. We posit that the proliferative potential of neuroblasts and the lineage-based architecture of information channels enable the modification of neural networks across existing, novel, and deprecated modalities in a species-specific manner, thus forming the substrate for the evolution and diversification of insect navigational circuits.

Restructuring of olfactory representations in the fly brain around odor relationships in natural sources.

A core challenge of olfactory neuroscience is to understand how neural representations of odor are generated and progressively transformed across different layers of the olfactory circuit into formats that support perception and behavior. The encoding of odor by odorant receptors in the input layer of the olfactory system reflects, at least in part, the chemical relationships between odor compounds. Neural representations of odor in higher order associative olfactory areas, generated by random feedforward networks, are expected to largely preserve these input odor relationships1-3. We evaluated these ideas by examining how odors are represented at different stages of processing in the olfactory circuit of the vinegar fly D. melanogaster. We found that representations of odor in the mushroom body (MB), a third-order associative olfactory area in the fly brain, are indeed structured and invariant across flies. However, the structure of MB representational space diverged significantly from what is expected in a randomly connected network. In addition, odor relationships encoded in the MB were better correlated with a metric of the similarity of their distribution across natural sources compared to their similarity with respect to chemical features, and the converse was true for odor relationships encoded in primary olfactory receptor neurons (ORNs). Comparison of odor coding at primary, secondary, and tertiary layers of the circuit revealed that odors were significantly regrouped with respect to their representational similarity across successive stages of olfactory processing, with the largest changes occurring in the MB. The non-linear reorganization of odor relationships in the MB indicates that unappreciated structure exists in the fly olfactory circuit, and this structure may facilitate the generalization of odors with respect to their co-occurence in natural sources.

Olfactory coding.

Modern humans live in real and digital environments dominated by sight and sound, but the vast majority of organisms on the planet rely on information received through air- or water-borne molecules to find food, avoid danger, and reproduce. Olfaction is at once both the primitive sensory modality and one of the hardest to understand, in large part due to the complexity of olfactory stimulus space. Whereas light and sound are easily ordered along natural physical axes that are reflected in their respective sensory codes, the organizational axes of odor space are not obvious. The search for systematic relationships between physicochemical characteristics of monomolecular odorants (carbon chain length, bond numbers, functional groups, etc.) and human perception of odorants suggests that olfactory perceptual space is a relatively low-dimensional structure. Odor descriptors provided by human observers are often significantly correlated. For instance, odors perceived as 'woody' are also likely to be described as 'warm', and many studies converge on hedonic valence or 'pleasantness' as being one of the most important dimensions of how people perceive odors. The identification of additional perceptual 'primaries' around which olfaction is organized is an active area of investigation, and a useful account of olfactory coding must explain this transformation of odor stimuli from the high dimensional chemical space to a lower dimensional perceptual space.

Parallel encoding of CO2 in attractive and aversive glomeruli by selective lateral signaling between olfactory afferents.

We describe a novel form of selective crosstalk between specific classes of primary olfactory receptor neurons (ORNs) in the Drosophila antennal lobe. Neurotransmitter release from ORNs is driven by two distinct sources of excitation: direct activity derived from the odorant receptor and stimulus-selective lateral signals originating from stereotypic subsets of other ORNs. Consequently, the level of presynaptic neurotransmitter release from an ORN can be significantly dissociated from its firing rate. Stimulus-selective lateral signaling results in the distributed representation of CO2-a behaviorally important environmental cue that directly excites a single ORN class-in multiple olfactory glomeruli, each with distinct response dynamics. CO2-sensitive glomeruli coupled to behavioral attraction respond preferentially to fast changes in CO2 concentration, whereas those coupled to behavioral aversion more closely follow absolute levels of CO2. Behavioral responses to CO2 also depend on the temporal structure of the stimulus: flies walk upwind to fluctuating, but not sustained, pulses of CO2. Stimulus-selective lateral signaling generalizes to additional odors and glomeruli, revealing a subnetwork of lateral interactions between ORNs that reshapes the spatial and temporal structure of odor representations in a stimulus-specific manner.

Therapeutic applications of trans-splicing.

BACKGROUND: RNA trans-splicing joins exons from different pre-mRNA transcripts to generate a chimeric product. Trans-splicing can also occur at the protein level, with split inteins mediating the ligation of separate gene products to generate a mature protein. SOURCES OF DATA: Comprehensive literature search of published research papers and reviews using Pubmed. AREAS OF AGREEMENT: Trans-splicing techniques have been used to target a wide range of diseases in both in vitro and in vivo models, resulting in RNA, protein and functional correction. AREAS OF CONTROVERSY: Off-target effects can lead to therapeutically undesirable consequences. In vivo efficacy is typically low, and delivery issues remain a challenge. GROWING POINTS: Trans-splicing provides a promising avenue for developing novel therapeutic approaches. However, much more research needs to be done before developing towards preclinical studies. AREAS TIMELY FOR DEVELOPING RESEARCH: Increasing trans-splicing efficacy and specificity by rational design, screening and competitive inhibition of endogenous cis-splicing.

Mapping Odor to Action: (Dopaminergic) Timing Is Everything.

Animal brains use the relative timing between sensory cues and behaviorally salient events to form predictive associations about their environment. Handler and colleagues provide new mechanistic insights into how differential signaling downstream of dopamine receptors couples this timing to the dynamic reweighting of synapses that link sensation to action.

Tracing neuronal circuits in transgenic animals by transneuronal control of transcription (TRACT).

Understanding the computations that take place in brain circuits requires identifying how neurons in those circuits are connected to one another. We describe a technique called TRACT (TRAnsneuronal Control of Transcription) based on ligand-induced intramembrane proteolysis to reveal monosynaptic connections arising from genetically labeled neurons of interest. In this strategy, neurons expressing an artificial ligand ('donor' neurons) bind to and activate a genetically-engineered artificial receptor on their synaptic partners ('receiver' neurons). Upon ligand-receptor binding at synapses the receptor is cleaved in its transmembrane domain and releases a protein fragment that activates transcription in the synaptic partners. Using TRACT in Drosophila we have confirmed the connectivity between olfactory receptor neurons and their postsynaptic targets, and have discovered potential new connections between neurons in the circadian circuit. Our results demonstrate that the TRACT method can be used to investigate the connectivity of neuronal circuits in the brain.

Determination of the True Inclination Angle of the Main Bronchi Relative to the Median Sagittal Plane for Placement of a Left-Sided Double-Lumen Tube.

OBJECTIVES: This study aimed to determine the true inclination angle of the main bronchi relative to the median sagittal plane, using CT imaging to help increase accuracy of double-lumen tube (DLT) placement. DESIGN: In this retrospective study, 2 investigators independently measured normal chest CT scans from 50 male and 50 female patients. To determine the true AP axis, a mid-sagittal plane reference line (MSPRL) was drawn, intersecting the midsternum and the vertebral spinous process at the level of mid-carina. Lines were drawn through the center of each main bronchus to determine the inclination angle with regard to the MSPRL. SETTING: Research was conducted at a single institution, the Los Angeles County and University of Southern California Medical Center. PARTICIPANTS: Normal chest CT images from 50 women and 50 men. MAIN RESULTS: The mean true inclination angle between the main bronchi and trachea in the mid-sagittal plane was 108.4° on the left compared with 96.2° on the right (p<0.0001). INTERVENTIONS: No specific interventions were done because this was a retrospective study and CT scan analysis. CONCLUSION: The data suggested that the trachea does not merely branch in the horizontal plane but branches posteriorly as well, with a true mean anatomic angle between the left main bronchus and trachea of 108.4°. This finding concurred with the authors' suggestion that the DLT be rotated to 110° counterclockwise instead of the routine practice of 90°. The authors suggest clinicians rotate the DLT an additional 20° counterclockwise and direct the top of the DLT to the 11 o'clock position.

Intravenously Administered Recombinant Human Type VII Collagen Derived from Chinese Hamster Ovary Cells Reverses the Disease Phenotype in Recessive Dystrophic Epidermolysis Bullosa Mice.

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited disorder characterized by skin fragility, blistering, and multiple skin wounds with no currently approved or consistently effective treatment. It is due to mutations in the gene encoding type VII collagen (C7). Using recombinant human C7 (rhC7) purified from human dermal fibroblasts (FB-rhC7), we showed previously that intravenously injected rhC7 distributed to engrafted RDEB skin, incorporated into its dermal-epidermal junction (DEJ), and reversed the RDEB disease phenotype. Human dermal fibroblasts, however, are not used for commercial production of therapeutic proteins. Therefore, we generated rhC7 from Chinese hamster ovary (CHO) cells. The CHO-derived recombinant type VII collagen (CHO-rhC7), similar to FB-rhC7, was secreted as a correctly folded, disulfide-bonded, helical trimer resistant to protease degradation. CHO-rhC7 bound to fibronectin and promoted human keratinocyte migration in vitro. A single dose of CHO-rhC7, administered intravenously into new-born C7-null RDEB mice, incorporated into the DEJ of multiple skin sites, tongue and esophagus, restored anchoring fibrils, improved dermal-epidermal adherence, and increased the animals' life span. Furthermore, no circulating or tissue-bound anti-C7 antibodies were observed in the mice. These data demonstrate the efficacy of CHO-rhC7 in a preclinical murine model of RDEB.

Simultaneous encoding of odors by channels with diverse sensitivity to inhibition.

Odorant receptors in the periphery map precisely onto olfactory glomeruli ("coding channels") in the brain. However, the odor tuning of a glomerulus is not strongly correlated with its spatial position. This raises the question of whether lateral inhibition between glomeruli is specific or nonspecific. Here we show that, in the Drosophila brain, focal activation of even a single glomerulus recruits GABAergic interneurons in all glomeruli. Moreover, the relative level of interneuron activity in different glomeruli is largely odor invariant. Although interneurons are recruited nonspecifically, glomeruli differ dramatically in their sensitivity to interneuron activity, and this is explained by their varying sensitivity to GABA. Interestingly, a stimulus is typically encoded in parallel by channels having high and low sensitivity to inhibition. Because lateral inhibition confers both costs and benefits, the brain might rely preferentially on "high" and "low" channels in different behavioral contexts.

Synaptic and circuit mechanisms promoting broadband transmission of olfactory stimulus dynamics.

Sensory stimuli fluctuate on many timescales. However, short-term plasticity causes synapses to act as temporal filters, limiting the range of frequencies that they can transmit. How synapses in vivo might transmit a range of frequencies in spite of short-term plasticity is poorly understood. The first synapse in the Drosophila olfactory system exhibits short-term depression, but can transmit broadband signals. Here we describe two mechanisms that broaden the frequency characteristics of this synapse. First, two distinct excitatory postsynaptic currents transmit signals on different timescales. Second, presynaptic inhibition dynamically updates synaptic properties to promote accurate transmission of signals across a wide range of frequencies. Inhibition is transient, but grows slowly, and simulations reveal that these two features of inhibition promote broadband synaptic transmission. Dynamic inhibition is often thought to restrict the temporal patterns that a neuron responds to, but our results illustrate a different idea: inhibition can expand the bandwidth of neural coding.

Quantitative profiling of peptides from RNAs classified as noncoding.

Only a small fraction of the mammalian genome codes for messenger RNAs destined to be translated into proteins, and it is generally assumed that a large portion of transcribed sequences--including introns and several classes of noncoding RNAs (ncRNAs)--do not give rise to peptide products. A systematic examination of translation and physiological regulation of ncRNAs has not been conducted. Here we use computational methods to identify the products of non-canonical translation in mouse neurons by analysing unannotated transcripts in combination with proteomic data. This study supports the existence of non-canonical translation products from both intragenic and extragenic genomic regions, including peptides derived from antisense transcripts and introns. Moreover, the studied novel translation products exhibit temporal regulation similar to that of proteins known to be involved in neuronal activity processes. These observations highlight a potentially large and complex set of biologically regulated translational events from transcripts formerly thought to lack coding potential.

Olfactory neuroscience: normalization is the norm.

A recent study shows that neural circuits from vertebrates and invertebrates use common strategies to stabilize odor representations across a wide range of concentrations.

A reference methylome database and analysis pipeline to facilitate integrative and comparative epigenomics.

DNA methylation is implicated in a surprising diversity of regulatory, evolutionary processes and diseases in eukaryotes. The introduction of whole-genome bisulfite sequencing has enabled the study of DNA methylation at a single-base resolution, revealing many new aspects of DNA methylation and highlighting the usefulness of methylome data in understanding a variety of genomic phenomena. As the number of publicly available whole-genome bisulfite sequencing studies reaches into the hundreds, reliable and convenient tools for comparing and analyzing methylomes become increasingly important. We present MethPipe, a pipeline for both low and high-level methylome analysis, and MethBase, an accompanying database of annotated methylomes from the public domain. Together these resources enable researchers to extract interesting features from methylomes and compare them with those identified in public methylomes in our database.

Palliative care nursing interventions in Thailand.

PURPOSE: This study aimed to describe the nursing interventions that nurses in Thailand identify as most important in promoting dignified dying. DESIGN: This study used a cross-sectional descriptive design. METHOD: A total of 247 Thai nurses completed a paper-and-pencil survey written in Thai. The survey included both demographic questions and palliative care interventions, listed with summative rating scales, from the International Classification for Nursing Practice (ICNP) catalogue Palliative Care for Dignified Dying. Descriptive statistics were used to analyze the data. FINDINGS: The five most important nursing interventions to promote dignified dying, ranked by average importance rating, were (a) maintain dignity and privacy, (b) establish trust, (c) manage pain, (d) establish rapport, and (e) manage dyspnea. CONCLUSIONS: This research identified the palliative care nursing interventions considered most important by nurses in Thailand to promote dignified dying. IMPLICATIONS: The ICNP catalogue Palliative Care for Dignified Dying can be used for planning and managing palliative nursing care in Thailand.

MLML: consistent simultaneous estimates of DNA methylation and hydroxymethylation.

MOTIVATION: The two major epigenetic modifications of cytosines, 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC), coexist with each other in a range of mammalian cell populations. Increasing evidence points to important roles of 5-hmC in demethylation of 5-mC and epigenomic regulation in development. Recently developed experimental methods allow direct single-base profiling of either 5-hmC or 5-mC. Meaningful analyses seem to require combining these experiments with bisulfite sequencing, but doing so naively produces inconsistent estimates of 5-mC or 5-hmC levels. RESULTS: We present a method to jointly model read counts from bisulfite sequencing, oxidative bisulfite sequencing and Tet-Assisted Bisulfite sequencing, providing simultaneous estimates of 5-hmC and 5-mC levels that are consistent across experiment types. AVAILABILITY: http://smithlab.usc.edu/software/mlml

Regionally specific and genome-wide analyses conclusively demonstrate the absence of CpG methylation in human mitochondrial DNA.

Although CpG methylation clearly distributes genome-wide in vertebrate nuclear DNA, the state of methylation in the vertebrate mitochondrial genome has been unclear. Several recent reports using immunoprecipitation, mass spectrometry, and enzyme-linked immunosorbent assay methods concluded that human mitochondrial DNA (mtDNA) has much more than the 2 to 5% CpG methylation previously estimated. However, these methods do not provide information as to the sites or frequency of methylation at each CpG site. Here, we have used the more definitive bisulfite genomic sequencing method to examine CpG methylation in HCT116 human cells and primary human cells to independently answer these two questions. We found no evidence of CpG methylation at a biologically significant level in these regions of the human mitochondrial genome. Furthermore, unbiased next-generation sequencing of sodium bisulfite treated total DNA from HCT116 cells and analysis of genome-wide sodium bisulfite sequencing data sets from several other DNA sources confirmed this absence of CpG methylation in mtDNA. Based on our findings using regionally specific and genome-wide approaches with multiple human cell sources, we can definitively conclude that CpG methylation is absent in mtDNA. It is highly unlikely that CpG methylation plays any role in direct control of mitochondrial function.

Asymmetric neurotransmitter release enables rapid odour lateralization in Drosophila.

In Drosophila, most individual olfactory receptor neurons (ORNs) project bilaterally to both sides of the brain. Having bilateral rather than unilateral projections may represent a useful redundancy. However, bilateral ORN projections to the brain should also compromise the ability to lateralize odours. Nevertheless, walking or flying Drosophila reportedly turn towards the antenna that is more strongly stimulated by odour. Here we show that each ORN spike releases approximately 40% more neurotransmitter from the axon branch ipsilateral to the soma than from the contralateral branch. As a result, when an odour activates the antennae asymmetrically, ipsilateral central neurons begin to spike a few milliseconds before contralateral neurons, and at a 30 to 50% higher rate than contralateral neurons. We show that a walking fly can detect a 5% asymmetry in total ORN input to its left and right antennal lobes, and can turn towards the odour in less time than it requires the fly to complete a stride. These results demonstrate that neurotransmitter release properties can be tuned independently at output synapses formed by a single axon onto two target cells with identical functions and morphologies. Our data also show that small differences in spike timing and spike rate can produce reliable differences in olfactory behaviour.

High concordance between HercepTest immunohistochemistry and ERBB2 fluorescence in situ hybridization before and after implementation of American Society of Clinical Oncology/College of American Pathology 2007 guidelines.

Human epidermal growth factor receptor 2 (HER2, ERBB2) is an important critical predictive marker in patients with invasive breast cancer. It is thus imperative to ensure accuracy and precision in HER2 and ERBB2 testing. In 2007, the American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP) proposed new guidelines for immunohistochemistry and fluorescence in-situ hybridization scoring in an effort to improve accuracy and utility of these companion diagnostic tests. The goal of the 2007 guidelines was to improve concordance rates between the diagnostic tests and decrease the number of inconclusive cases. This study examines the impact in concordance rates and number of inconclusive cases based on the recent change in guidelines in a large study cohort. HER2 immunohistochemistry and ERBB2 fluorescence in-situ hybridization were performed on all specimens from our facility from years 2003 through 2010 (n=1437). Cases from 2003-2007 (n=1016) were scored using Food and Drug Administration guidelines, with immunohistochemical 3+ cases staining >10% of tumor cells and fluorescence in-situ hybridization amplification cutoff value of 2.0. The 2007 guidelines were implemented and scored accordingly for cases from 2008-2010 (n=421), with immunohistochemical 3+ cases staining >30% of tumor cells and fluorescence in-situ hybridization amplification cutoff value of 2.2. We compared concordance rates before and after 2007 guidelines. For the 2003-2007 study population, the concordance rate between the assays was 97.6% with a corresponding kappa coefficient (k) of 0.90. For the 2008-2010 study population, concordance rate was 97.6% with a corresponding k of 0.89. There was no significant difference in number of inconclusive rates before and after 2007 guidelines. In our study, implementation of the new ASCO/CAP 2007 HER2 guidelines did not show a significant difference in concordance rates and did not decrease the number of inconclusive cases.