Axonally transported proteins associated with axon growth in rabbit central and peripheral nervous systems

In an effort to determine whether the “growth state” and the “mature state” of a neuron are differentiated by different programs of gene expression, we have compared the rapidly transported (group I) proteins in growing and nongrowing axons in rabbits. We observed two polypeptides (GAP-23 and GAP-43) which were of particular interest because of their apparent association with axon growth. GAP-43 was rapidly transported in the central nervous system (CNS) (retinal ganglion cell) axons of neonatal animals, but its relative amount declined precipitously with subsequent development. It could not be reinduced by axotomy of the adult optic nerves, which do not regenerate; however, it was induced after axotomy of an adult peripheral nervous system nerve (the hypoglossal nerve, which does regenerate) which transported only very low levels of GAP-43 before axotomy. The second polypeptide, GAP-23 followed the same pattern of growth-associated transport, except that it was transported at significant levels in uninjured adult hypoglossal nerves and not further induced by axotomy. These observations are consistent with the “GAP hypothesis” that the neuronal growth state can be defined as an altered program of gene expression exemplified in part by the expression of GAP genes whose products are involved in critical growth-specific functions. When interpreted in terms of GAP hypothesis, they lead to the following conclusions: (a) the growth state can be subdivided into a “synaptogenic state” characterized by the transport of GAP-23 but not GAP-43, and an “axon elongation state” requiring both GAPs; (b) with respect to the expression of GAP genes, regeneration involves a recapitulation of a neonatal state of the neuron; and (c) the failure of mammalian CNS neurons to express the GAP genes may underly the failure of CNS axons to regenerate after axon injury.     

Use of Mutated Self-Cleaving 2A Peptides as a Molecular Rheostat to Direct Simultaneous Formation of Membrane and Secreted Anti-HIV Immunoglobulins

In nature, B cells produce surface immunoglobulin and secreted antibody from the same immunoglobulin gene via alternative splicing of the pre-messenger RNA. Here we present a novel system for genetically programming B cells to direct the simultaneous formation of membrane-bound and secreted immunoglobulins that we term a “Molecular Rheostat”, based on the use of mutated “self-cleaving” 2A peptides. The Molecular Rheostat is designed so that the ratio of secreted to membrane-bound immunoglobulins can be controlled by selecting appropriate mutations in the 2A peptide. Lentiviral transgenesis of Molecular Rheostat constructs into B cell lines enables the simultaneous expression of functional b12-based IgM-like BCRs that signal to the cells and mediate the secretion of b12 IgG broadly neutralizing antibodies that can bind and neutralize HIV-1 pseudovirus. We show that these b12-based Molecular Rheostat constructs promote the maturation of EU12 B cells in an in vitro model of B lymphopoiesis. The Molecular Rheostat offers a novel tool for genetically manipulating B cell specificity for B-cell based gene therapy.     

The “next-generation” knowledge of papillary thyroid carcinoma

The application of Next-Generation Sequencing for studying the genetics of papillary thyroid carcinomas (PTC) has recently revealed new somatic mutations and gene fusions as potential new tumor-initiating events in patients without any known driver lesion. Gene and miRNA expression analyses defined clinically relevant subclasses correlated to tumor progression. In addition, it has been shown that tumor driver mutations in BRAF, and RET rearrangements - altogether termed “BRAF-like” carcinomas - have a very similar expression pattern and constitute a distinct category. Conversely, “RAS-like” carcinomas have a different genomic, epigenomic, and proteomic profile. These findings justify the need to reconsider PTC classification schemes.     

Importance of phosphoinositide-dependent signaling pathways in the control of gene expression in resting cells and in response to phytohormones

“Phosphoinositide” refers to phosphorylated forms of phosphatidylinositol, including phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. Both of these molecules could be in vivo substrates of plant phospholipase C. These phosphoinositides can also be biologically active “per se,” by directly binding to proteins and thus altering their location and/or activity. The use of pharmacological agents in Arabidopsis suspension cells allowed us to identify genes whose expression was positively or negatively controlled, in the basal state, by products of phosphoinositide-dependent phospholipase C. In this basal state, it seems that no genes exhibit a phosphoinositide-dependent expression “per se.” However, many genes whose expression is altered in the presence of phospholipase C inhibitors appeared to be responsive to salicylic acid. This allowed us to show that salicylic acid acts both by increasing the phosphoinositide pool and by inhibiting the phospholipase C. In response to salicylic acid it is possible to identify genes whose expression is controlled by products of PI-PLC, but also genes whose expression is controlled by phosphoinositides “per se.” Our data highlight the importance of phosphoinositide-dependent pathways in gene expression in resting cells and in response to phytohormones.     

Interleukin-8 Responses of Multi-Layer Gingival Epithelia to Subgingival Biofilms: Role of the “Red Complex” Species

Periodontitis is an infectious inflammatory disease that results in the destruction of the tooth-supporting (periodontal) tissues. The Gram-negative anaerobic species Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, (also known as the “red complex” species) are highly associated with subgingival biofilms at periodontitis-affected sites. A major chemokine produced by the gingival epithelium in response to biofilm challenge, is interleukin (IL)-8. The aim of this in vitro study was to investigate the relative effect of the “red complex” species as constituents of subgingival biofilms, on the regulation of IL-8 by gingival epithelia. Multi-layered organotypic human gingival epithelial cultures were challenged with a 10-species in vitro subgingival biofilm model, or its 7-species variant, excluding the “red complex”. IL-8 gene expression and secretion analyses were performed by qPCR and ELISA, respectively. After 3 h, both biofilms up-regulated IL-8 gene expression, but the presence of the “red complex” resulted in 3-fold greater response. IL-8 secretion was also up-regulated by both biofilms, with no differences between them. After 24 h, the 10-species biofilm reduced IL-8 secretion to 50% of the control, but this was not affected when the “red complex” was absent. In conclusion, as part of biofilms, “red complex” species differentially regulate IL-8 in gingival epithelia, potentially affecting the chemotactic responses of the tissue.     

Evolution and Functional Characterisation of Melanopsins in a Deep-Sea Chimaera (Elephant Shark,Callorhinchus milii)

Non-visual photoreception in mammals is primarily mediated by two splice variants that derive from a single melanopsin (OPN4M) gene, whose expression is restricted to a subset of retinal ganglion cells. Physiologically, this sensory system regulates the photoentrainment of many biological rhythms, such as sleep via the melatonin endocrine system and pupil constriction. By contrast, melanopsin exists as two distinct lineages in non-mammals, opn4m and opn4x, and is broadly expressed in a wide range of tissue types, including the eye, brain, pineal gland and skin. Despite these findings, the evolution and function of melanopsin in early vertebrates are largely unknown. We, therefore, investigated the complement of opn4 classes present in the genome of a model deep-sea cartilaginous species, the elephant shark (Callorhinchus milii), as a representative vertebrate that resides at the base of the gnathostome (jawed vertebrate) lineage. We reveal that three melanopsin genes, opn4m1, opn4m2 and opn4x, are expressed in multiple tissues of the elephant shark. The two opn4m genes are likely to have arisen as a result of a lineage-specific duplication, whereas “long” and “short” splice variants are generated from a single opn4x gene. By using a heterologous expression system, we suggest that these genes encode functional photopigments that exhibit both “invertebrate-like” bistable and classical “vertebrate-like” monostable biochemical characteristics. We discuss the evolution and function of these melanopsin pigments within the context of the diverse photic and ecological environments inhabited by this chimaerid holocephalan, as well as the origin of non-visual sensory systems in early vertebrates.     

KIAA1462, A Coronary Artery Disease Associated Gene,Is a Candidate Gene for Late Onset Alzheimer Disease in APOE Carriers

Alzheimer disease (AD) is a devastating neurodegenerative disease affecting more than five million Americans. In this study, we have used updated genetic linkage data from chromosome 10 in combination with expression data from serial analysis of gene expression to choose a new set of thirteen candidate genes for genetic analysis in late onset Alzheimer disease (LOAD). Results in this study identify the KIAA1462 locus as a candidate locus for LOAD in APOE4 carriers. Two genes exist at this locus, KIAA1462, a gene associated with coronary artery disease, and “rokimi”, encoding an untranslated spliced RNA The genetic architecture at this locus suggests that the gene product important in this association is either “rokimi”, or a different isoform of KIAA1462 than the isoform that is important in cardiovascular disease. Expression data suggests that isoform f of KIAA1462 is a more attractive candidate for association with LOAD in APOE4 carriers than “rokimi” which had no detectable expression in brain.     

Primary Events, Energy Transfer, and Reactions in Photosynthetic Units: A Connected Model of the Photosynthetic Unit

The concept of photosynthetic unit (PSU) is reviewed in the light of the authors' results in the fields of fluorescence and luminescence (delayed light). Models of PSU are mainly distinguished by the amount of exciton exchange which is allowed between units. The “separate” model, with its “first-order” character, is not consistent with fluorescence kinetic data. The sigmoidal rise of fluorescence under actinic light is best explained by “nonseparate” models; however, most of these models assume a delocalization of excitons or centers. The “connected” model introduced here is not subject to this criticism. It discloses a new effect (the “îlot” effect): a nonrandom grouping of fluorescent units the consequences of which are discussed. It is noted that a “two-quantum” model for the photochemical reaction gives results very similar to those of the connected model. A relation between luminescence intensity and fluorescence yield is seen as a necessary consequence of the PSU concept. Its meaning is different in separate and nonseparate models. This relation is discussed in connection with the true system II fluorescence emission.     

Conformational states of syntaxin-1 govern the necessity of N-peptide binding in exocytosis of PC12 cells and Caenorhabditis elegans

Syntaxin-1 is the central SNARE protein for neuronal exocytosis. It interacts with Munc18-1 through its cytoplasmic domains, including the N-terminal peptide (N-peptide). Here we examine the role of the N-peptide binding in two conformational states (“closed” vs. “open”) of syntaxin-1 using PC12 cells and Caenorhabditis elegans. We show that expression of “closed” syntaxin-1A carrying N-terminal single point mutations (D3R, L8A) that perturb interaction with the hydrophobic pocket of Munc18-1 rescues impaired secretion in syntaxin-1–depleted PC12 cells and the lethality and lethargy of unc-64 (C. elegans orthologue of syntaxin-1)-null mutants. Conversely, expression of the “open” syntaxin-1A harboring the same mutations fails to rescue the impairments. Biochemically, the L8A mutation alone slightly weakens the binding between “closed” syntaxin-1A and Munc18-1, whereas the same mutation in the “open” syntaxin-1A disrupts it. Our results reveal a striking interplay between the syntaxin-1 N-peptide and the conformational state of the protein. We propose that the N-peptide plays a critical role in intracellular trafficking of syntaxin-1, which is dependent on the conformational state of this protein. Surprisingly, however, the N-peptide binding mode seems dispensable for SNARE-mediated exocytosis per se, as long as the protein is trafficked to the plasma membrane.     

Molecular Evidence for the Broad Distribution of Anaerobic Ammonium-Oxidizing Bacteria in Freshwater and Marine Sediments

Previously available primer sets for detecting anaerobic ammonium-oxidizing (anammox) bacteria are inefficient, resulting in a very limited database of such sequences, which limits knowledge of their ecology. To overcome this limitation, we designed a new primer set that was 100% specific in the recovery of ~700-bp 16S rRNA gene sequences with >96% homology to the “Candidatus Scalindua” group of anammox bacteria, and we detected this group at all sites studied, including a variety of freshwater and marine sediments and permafrost soil. A second primer set was designed that exhibited greater efficiency than previous primers in recovering full-length (1,380-bp) sequences related to “Ca. Scalindua,” “Candidatus Brocadia,” and “Candidatus Kuenenia.” This study provides evidence for the widespread distribution of anammox bacteria in that it detected closely related anammox 16S rRNA gene sequences in 11 geographically and biogeochemically diverse freshwater and marine sediments.     

Meiotic Cas9 expression mediates gene conversion in the male and female mouse germline

Highly efficient gene conversion systems have the potential to facilitate the study of complex genetic traits using laboratory mice and, if implemented as a “gene drive,” to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of gene conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double-strand DNA break (DSB) is feasible in the female mouse germline. In the male germline, however, all DSBs were instead repaired by end joining (EJ) mechanisms to form an “insertion/deletion” (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology-directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate gene conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient DSB formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of gene conversion and further increase the rate of “super-mendelian” inheritance from both male and female mice.

This study shows that while Cas9 expression during meiosis I promotes genotype conversion - the mechanism underlying CRISPR ’gene drive’ - in both male and female mice, timing and high levels of Cas9 protein are critical to achieve robust efficiency.     

M tuberculosis in the Adjuvant Modulates Time of Appearance of CNS-Specific Effector T Cells in the Spleen through a Polymorphic Site of TLR2

DC deliver information regulating trafficking of effector T cells along T-cell priming. However, the role of pathogen-derived motives in the regulation of movement of T cells has not been studied. We hereinafter report that amount of M tuberculosis in the adjuvant modulates relocation of PLP139-151 specific T cells. In the presence of a low dose of M tuberculosis in the adjuvant, T cells (detected by CDR3 BV-BJ spectratyping, the so-called “immunoscope”) mostly reach the spleen by day 28 after immunization (“late relocation”) in the SJL strain, whereas T cells reach the spleen by d 14 with a high dose of M tuberculosis (“early relocation”). The C57Bl/6 background confers a dominant “early relocation” phenotype to F1 (SJL×C57Bl/6) mice, allowing early relocation of T cells in the presence of low dose M tuberculosis. A single non-synonymous polymorphism of TLR2 is responsible for “early/late” relocation phenotype. Egress of T lymphocytes is regulated by TLR2 expressed on T cells. Thus, pathogens engaging TLR2 on T cells regulate directly T-cell trafficking, and polymorphisms of TLR2 condition T-cell trafficking upon a limiting concentration of ligand.     

“Candidatus Accumulibacter” Population Structure in Enhanced Biological Phosphorus Removal Sludges as Revealed by Polyphosphate Kinase Genes

We investigated the fine-scale population structure of the “Candidatus Accumulibacter” lineage in enhanced biological phosphorus removal (EBPR) systems using the polyphosphate kinase 1 gene (ppk1) as a genetic marker. We retrieved fragments of “Candidatus Accumulibacter” 16S rRNA and ppk1 genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and ppk1 were largely congruent, with ppk1 granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the “Candidatus Accumulibacter” lineage. Sequences from at least five clades of “Candidatus Accumulibacter” were recovered by ppk1-targeted PCR, and subsequently, specific primer sets were designed to target the ppk1 gene for each clade. Quantitative real-time PCR (qPCR) assays using “Candidatus Accumulibacter”-specific 16S rRNA and “Candidatus Accumulibacter” clade-specific ppk1 primers were developed and conducted on three laboratory-scale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total “Candidatus Accumulibacter” lineage and the relative distributions and abundances of the five “Candidatus Accumulibacter” clades. The qPCR-based estimation of the total “Candidatus Accumulibacter” fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using ppk1, demonstrating the power of ppk1 as a genetic marker for detection of all currently defined “Candidatus Accumulibacter” clades. The relative distributions of “Candidatus Accumulibacter” clades varied among different EBPR systems and also temporally within a system. Our results suggest that the “Candidatus Accumulibacter” lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.