Recombinase-driver rat lines: tools, techniques, and optogenetic application to dopamine-mediated reinforcement

Currently there is no general approach for achieving specific optogenetic control of genetically defined cell types in rats, which provide a powerful experimental system for numerous established neurophysiological and behavioral paradigms. To overcome this challenge we have generated genetically restricted recombinase-driver rat lines suitable for driving gene expression in specific cell types, expressing Cre recombinase under the control of large genomic regulatory regions (200-300 kb). Multiple tyrosine hydroxylase (Th)::Cre and choline acetyltransferase (Chat)::Cre lines were produced that exhibited specific opsin expression in targeted cell types. We additionally developed methods for utilizing optogenetic tools in freely moving rats and leveraged these technologies to clarify the causal relationship between dopamine (DA) neuron firing and positive reinforcement, observing that optical stimulation of DA neurons in the ventral tegmental area (VTA) of Th::Cre rats is sufficient to support vigorous intracranial self-stimulation (ICSS). These studies complement existing targeting approaches by extending the generalizability of optogenetics to traditionally non-genetically-tractable but vital animal models.     

Normalization, testing, and false discovery rate estimation for RNA-sequencing data

We discuss the identification of genes that are associated with an outcome in RNA sequencing and other sequence-based comparative genomic experiments. RNA-sequencing data take the form of counts, so models based on the Gaussian distribution are unsuitable. Moreover, normalization is challenging because different sequencing experiments may generate quite different total numbers of reads. To overcome these difficulties, we use a log-linear model with a new approach to normalization. We derive a novel procedure to estimate the false discovery rate (FDR). Our method can be applied to data with quantitative, two-class, or multiple-class outcomes, and the computation is fast even for large data sets. We study the accuracy of our approaches for significance calculation and FDR estimation, and we demonstrate that our method has potential advantages over existing methods that are based on a Poisson or negative binomial model. In summary, this work provides a pipeline for the significance analysis of sequencing data.     

Strategies for evaluating the environment-public health interaction of long-term latency disease: the quandary of the inconclusive case-control study

Environmental links to disease are difficult to uncover because environmental exposures are variable in time and space, contaminants occur in complex mixtures, and many diseases have a long time delay between exposure and onset. Furthermore, individuals in a population have different activity patterns (e.g., hobbies, jobs, and interests), and different genetic susceptibilities to disease. As such, there are many potential confounding factors to obscure the reasons that one individual gets sick and another remains healthy. An important method for deducing environmental associations with disease outbreak is the retrospective case-control study wherein the affected and control subject cohorts are studied to see what is different about their previous exposure history. Despite success with infectious diseases (e.g., food poisoning, and flu), case-control studies of cancer clusters rarely have an unambiguous outcome. This is attributed to the complexity of disease progression and the long-term latency between exposure and disease onset. In this article, we consider strategies for investigating cancer clusters and make some observations for improving statistical power through broader non-parametric approaches wherein sub-populations (i.e., whole towns), rather than individuals, are treated as the cases and controls, and the associated cancer rates are treated as the dependent variable. We subsequently present some ecological data for tungsten and cobalt from studies by University of Arizona researchers who document elevated levels of tungsten and cobalt in Fallon, NV. These results serve as candidates for future hybrid ecologic case-control investigations of childhood leukemia clusters.     

Exposure to sodium tungstate and Respiratory Syncytial Virus results in hematological/immunological disease in C57BL/6J mice

The etiology of childhood leukemia is not known. Strong evidence indicates that precursor B-cell Acute Lymphoblastic Leukemia (Pre-B ALL) is a genetic disease originating in utero. Environmental exposures in two concurrent, childhood leukemia clusters have been profiled and compared with geographically similar control communities. The unique exposures, shared in common by the leukemia clusters, have been modeled in C57BL/6 mice utilizing prenatal exposures. This previous investigation has suggested in utero exposure to sodium tungstate (Na2WO4) may result in hematological/immunological disease through genes associated with viral defense. The working hypothesis is (1) in addition to spontaneously and/or chemically generated genetic lesions forming pre-leukemic clones, in utero exposure to Na2WO4 increases genetic susceptibility to viral influence(s); (2) postnatal exposure to a virus possessing the 1FXXKXFXXA/V9 peptide motif will cause an unnatural immune response encouraging proliferation in the B-cell precursor compartment. This study reports the results of exposing C57BL/6J mice to Na2WO4 in utero via water (15 ppm, ad libetum) and inhalation (mean concentration PM5 3.33 mg/m3) and to Respiratory Syncytial Virus (RSV) within 2 weeks of weaning. Inoculation of C57BL/6J mice with RSV was associated with a neutrophil shift in 56% of 5-month old mice. When the RSV inoculation was combined with Na2WO4-exposure, significant splenomegaly resulted (p=0.0406, 0.0184, 0.0108 for control, Na2WO4-only and RSV-only, respectively) in addition to other hematological pathologies which were not significant. Exposure to Na2WO4 and RSV resulted in hematological/immunological disease, the nature of which is currently inconclusive. Further research is needed to characterize this potential leukemia mouse model.     

Structures of two cockroach neuropeptides assigned by fast atom bombardment mass spectrometry

Amino acid sequences have been assigned to two cockroach neuropeptides (Glu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH₂, M I, and Glu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH₂, M II) by application of fast atom bombardment mass spectrometry, including high resolution and linked scan (metastable) studies. The peptides show considerable homology with two other invertebrate neuropeptides, adipokinetic hormone (AKH, from a locust) and red pigment concentrating hormone (RPCH, from a prawn), whose fast atom bombardment spectra were also studied. M I and M II are thus members of a family of structurally-related invertebrate neuropeptides.     

Mycobacterial lipoarabinomannan: an extraordinary lipoheteroglycan with profound physiological effects

Detailed structural and functional studies over the last decade have led to current recognition of the mycobacterial lipoarabinomannan (LAM) as a phosphatidylinositol anchored lipoglycan with diverse biological activities. Fatty acylation has been demonstrated to be essential for LAM to maintain its functional integrity although the focus has largely been on the arabinan motifs and the terminal capping function. It has recently been shown that the mannose caps may be involved not only in attenuating host immune response, but also in mediating the binding of mycobacteria to and subsequent entry into macrophages. This may further be linked to an intracellular trafficking pathway through which LAM is thought to be presented by CD1 to subsets of T-cells. The implication of LAM as major histocompatibility complex (MHC)-independent T-cell epitope and the ensuing immune response is an area of intensive studies. Another recent focus of research is the biosynthesis of arabinan which has been shown to be inhibitable by the anti- tuberculosis drug, ethambutol. The phenomenon of truncated LAM as synthesized by ethambutol resistant strains provides an invaluable handle for dissecting the array of arabinosyltransferases involved, as well as generating much needed structural variants for further structural and functional studies. It is hoped that with more systematic investigations based on clinical isolates and human cell lines, the true significance of LAM in the immunopathogenesis of tuberculosis and leprosy can eventually be explained.      

Some generalized conjugacy theorems and the concepts of fitness and survival in logistic growth models

Current research into the dynamics of iterative ecological and biological models has lead to a number of theorems concerning the existence of various types of iterative dynamical behavior. In particular, much study has been done on the dynamical behavior of the “simplest dynamical system”f _b(x)=bx(1−x), which is just the canonical discrete form of logistic growth equations found in ecology, sociobiology, and population biology. In this paper, we make use of some of the techniques and concepts of topological dynamics to construct a number of generalized conjugacy theorems. These theorems are then used to demonstrate that the mappingf _b has a number of conjugacy classes in which the dynamics of the iterates is equivalent to within a change of variables. The concepts of fitness and survival in logistic equations are then shown to be independent, if we follow certain intuitive definitions for these concepts. This conclusion follows from a comparison of the conjugacy classes of the functionf _b and the extinction sets off _b.     

D-Trp(34)] neuropeptide Y is a potent and selective neuropeptide Y Y(5) receptor agonist with dramatic effects on food intake

The neuropeptide Y (NPY) Y(5) receptor has been proposed to mediate several physiological effects of NPY, including the potent orexigenic activity of the peptide. However, the lack of selective NPY Y(5) receptor ligands limits the characterization of the physiological roles of this receptor. Screening of several analogs of NPY revealed that [D-Trp(34)]NPY is a potent and selective NPY Y(5) receptor agonist. Unlike the prototype selective NPY Y(5) receptor agonist [D-Trp(32)]NPY, [D-Trp(34)]NPY markedly increases food intake in rats, an effect that is blocked by the selective NPY Y(5) receptor antagonist CGP 71683A. These data demonstrate that [D-Trp(34)]NPY is a useful tool for studies aimed at determining the physiological roles of the NPY Y(5) receptor.