Optogenetic silencing allows time-resolved functional interrogation of defined neuronal populations. However, the limitations of inhibitory optogenetic tools impose stringent constraints on experimental paradigms. The high light power requirement of light-driven ion pumps and their effects on intracellular ion homeostasis pose unique challenges, particularly in experiments that demand inhibition of a widespread neuronal population in vivo. Guillardia theta anion-conducting channelrhodopsins (GtACRs) are promising in this regard, due to their high single-channel conductance and favorable photon-ion stoichiometry. However, GtACRs show poor membrane targeting in mammalian cells, and the activity of such channels can cause transient excitation in the axon due to an excitatory chloride reversal potential in this compartment. Here, we address these problems by enhancing membrane targeting and subcellular compartmentalization of GtACRs. The resulting soma-targeted GtACRs show improved photocurrents, reduced axonal excitation and high light sensitivity, allowing highly efficient inhibition of neuronal activity in the mammalian brain.
BACKGROUND: Methylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared. METHODOLOGY/PRINCIPAL FINDINGS: The 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name "island integration determinant" (iid). CONCLUSION/SIGNIFICANCE: These two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic lifestyles.
Large-insert genome analysis (LIGAN) is a broadly applicable, high-throughput technology designed to characterize genome-scale structural variation. Fosmid paired-end sequences and DNA fingerprints from a query genome are compared to a reference sequence using the Genomic Variation Analysis (GenVal) suite of software tools to pinpoint locations of insertions, deletions, and rearrangements. Fosmids spanning regions that contain new structural variants can then be sequenced. Clonal pairs of Pseudomonas aeruginosa isolates from four cystic fibrosis patients were used to validate the LIGAN technology. Approximately 1.5 Mb of inserted sequences were identified, including 743 kb containing 615 ORFs that are absent from published P. aeruginosa genomes. Six rearrangement breakpoints and 220 kb of deleted sequences were also identified. Our study expands the "genome universe" of P. aeruginosa and validates a technology that complements emerging, short-read sequencing methods that are better suited to characterizing single-nucleotide polymorphisms than structural variation.
Renibacterium salmoninarum is the causative agent of bacterial kidney disease and a significant threat to healthy and sustainable production of salmonid fish worldwide. This pathogen is difficult to culture in vitro, genetic manipulation is challenging, and current therapies and preventative strategies are only marginally effective in preventing disease. The complete genome of R. salmoninarum ATCC 33209 was sequenced and shown to be a 3,155,250-bp circular chromosome that is predicted to contain 3,507 open-reading frames (ORFs). A total of 80 copies of three different insertion sequence elements are interspersed throughout the genome. Approximately 21% of the predicted ORFs have been inactivated via frameshifts, point mutations, insertion sequences, and putative deletions. The R. salmoninarum genome has extended regions of synteny to the Arthrobacter sp. strain FB24 and Arthrobacter aurescens TC1 genomes, but it is approximately 1.9 Mb smaller than both Arthrobacter genomes and has a lower G+C content, suggesting that significant genome reduction has occurred since divergence from the last common ancestor. A limited set of putative virulence factors appear to have been acquired via horizontal transmission after divergence of the species; these factors include capsular polysaccharides, heme sequestration molecules, and the major secreted cell surface antigen p57 (also known as major soluble antigen). Examination of the genome revealed a number of ORFs homologous to antibiotic resistance genes, including genes encoding beta-lactamases, efflux proteins, macrolide glycosyltransferases, and rRNA methyltransferases. The genome sequence provides new insights into R. salmoninarum evolution and may facilitate identification of chemotherapeutic targets and vaccine candidates that can be used for prevention and treatment of infections in cultured salmonids.
BACKGROUND Francisella tularensis subspecies tularensis and holarctica are pathogenic to humans, whereas the two other subspecies, novicida and mediasiatica, rarely cause disease. To uncover the factors that allow subspecies tularensis and holarctica to be pathogenic to humans, we compared their genome sequences with the genome sequence of Francisella tularensis subspecies novicida U112, which is nonpathogenic to humans.
RESULTS:
Comparison of the genomes of human pathogenic Francisella strains with the genome of U112 identifies genes specific to the human pathogenic strains and reveals pseudogenes that previously were unidentified. In addition, this analysis provides a coarse chronology of the evolutionary events that took place during the emergence of the human pathogenic strains. Genomic rearrangements at the level of insertion sequences (IS elements), point mutations, and small indels took place in the human pathogenic strains during and after differentiation from the nonpathogenic strain, resulting in gene inactivation.
CONCLUSION:
The chronology of events suggests a substantial role for genetic drift in the formation of pseudogenes in Francisella genomes. Mutations that occurred early in the evolution, however, might have been fixed in the population either because of evolutionary bottlenecks or because they were pathoadaptive (beneficial in the context of infection). Because the structure of Francisella genomes is similar to that of the genomes of other emerging or highly pathogenic bacteria, this evolutionary scenario may be shared by pathogens from other species.
After the completion of a draft human genome sequence, the International Human Genome Sequencing Consortium has proceeded to finish and annotate each of the 24 chromosomes comprising the human genome. Here we describe the sequencing and analysis of human chromosome 3, one of the largest human chromosomes. Chromosome 3 comprises just four contigs, one of which currently represents the longest unbroken stretch of finished DNA sequence known so far. The chromosome is remarkable in having the lowest rate of segmental duplication in the genome. It also includes a chemokine receptor gene cluster as well as numerous loci involved in multiple human cancers such as the gene encoding FHIT, which contains the most common constitutive fragile site in the genome, FRA3B. Using genomic sequence from chimpanzee and rhesus macaque, we were able to characterize the breakpoints defining a large pericentric inversion that occurred some time after the split of Homininae from Ponginae, and propose an evolutionary history of the inversion.
Human chromosome 7 has historically received prominent attention in the human genetics community, primarily related to the search for the cystic fibrosis gene and the frequent cytogenetic changes associated with various forms of cancer. Here we present more than 153 million base pairs representing 99.4% of the euchromatic sequence of chromosome 7, the first metacentric chromosome completed so far. The sequence has excellent concordance with previously established physical and genetic maps, and it exhibits an unusual amount of segmentally duplicated sequence (8.2%), with marked differences between the two arms. Our initial analyses have identified 1,150 protein-coding genes, 605 of which have been confirmed by complementary DNA sequences, and an additional 941 pseudogenes. Of genes confirmed by transcript sequences, some are polymorphic for mutations that disrupt the reading frame.
AIM: To examine the effect of overexpression of human intracellular copper-zinc superoxide dismutase (CuZnSOD1) gene on fetal mice brain exposed to in-utero ischemic reperfusion injury. DESIGN: Transient in-utero ischemia (7 min) was induced in pregnant transgenic mice overexpressing human CuZnSOD1 and wild-type mice by occluding the blood supply to the uterine artery on day 17 of pregnancy, followed by 24 hours of reperfusion. The level of lipid peroxidation in fetal mice brains was compared between the transgenic and non-transgenic (control) fetal mice. Motor and coordination skills of transgenic and control adult mice (six to eight months old) which were exposed to ischemic reperfusion injury in-utero were compared by the rope grip test and visible platform task. RESULTS: We first measured CuZnSOD1 activity in the brains of the transgenic fetal mice and confirmed that the enzyme activity is 4.2-fold higher than control. We also established that ischemia reperfusion on day 17 of pregnancy led to increased level of TBARS (Thiobarbituric acid reactive substance) in brains of wild-type fetal mice when compared to sham operated mice (72.5 +/- 3.4 vs. 49.4 +/- 1.5 nmol/mg. p < 0.001). The increase was markedly accentuated in the CuZnSOD1 transgenic mice, and significantly higher compared to control mice exposed to ischemia-reperfusion (85.6 +/- 4.0 vs. 69.5 +/- 2.3 nmol/mg, p < 0.001). Moreover, we found that the transgenic mice that were subjected to in-utero ischemia reperfusion exhibited a significantly higher rate of failures in the rope grip test and poorer performance in the visible platform task, when compared to non-transgenic mice exposed to identical insult. CONCLUSIONS: Oxygen free radicals play an important role in the pathogenesis of perinatal hypoxia. Overexpression of the enzyme CuZnSOD1 in transgenic mice exposed their brains to increased damage during ischemic-reperfusion insult.
The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.
Title: Alzheimer's disease and Lewy body dementia Levy R Ref: British Journal of Psychiatry, 164:268, 1994 : PubMed
Activity of the enzyme which synthesizes acetylcholine, choline acetyltransferase, was estimated in the neocortex of three series of control and demented cases. Clinically demented cases were divided into those with the classical neuropathological features of Alzheimer's disease (numerous neocortical plaques and tangles) and those with Lewy bodies in the brain stem and cortex (together with plaques and variable neurofibrillary pathology). In the Lewy body cases neocortical choline acetyltransferase was consistently lower than in the classical Alzheimer-type cases. Two of the Lewy body cases with extremely low cholinergic activity were responders in therapeutic trials of the cholinesterase inhibitor, tacrine, and the combined data suggest that cholinergic therapy may be particularly relevant to patients with Lewy body type dementia.
Title: Tacrine in Alzheimer's disease Levy R Ref: N Engl J Med, 328:808; author reply 809, 1993 : PubMed
Results of a placebo controlled cross-over trial (N = 89) of the anticholinesterase drug THA as a treatment for dementia of the Alzheimer's type (DAT) are reported, with reference to previous trials of the drug and the cholinergic hypothesis of aging and dementia. Using computerised tests sensitive to specific aspects of memory and attention, evidence is found for improvements in attentional function rather than memory, in patients with mild to moderate DAT. Although these improvements were significant, they were small and restricted to certain tests of attentional function. Nevertheless, they add to the growing body of evidence that the cholinergic system is involved in the control of attentional processes. The results will be relevant to future investigations into the therapeutic effects of enhancement of the cholinergic system in DAT sufferers.
Title: Tacrine in Alzheimer's disease. Time course of changes in cognitive function and practice effects Eagger S, Morant N, Levy R, Sahakian B Ref: British Journal of Psychiatry, 160:36, 1992 : PubMed
This paper concerns certain questions which arose during the analysis of a trial showing positive effects of tacrine in Alzheimer's disease. Cognitive improvement occurred during the first two weeks, reached a maximum at one month and was maintained during the rest of the three-month treatment period. Rebound effects were not detected in any of the key outcome variables, but were suggested by one of the supporting cognitive tests and other measures. Practice effects occurred on tests which were repeated at short intervals or too frequently. The paper discusses the significance of these findings for the interpretation of other trials and for practical management.
Title: Tacrine in Alzheimer's disease Eagger SA, Levy R, Sahakian BJ Ref: Acta Neurologica Scandinavica Supplementum, 139:75, 1992 : PubMed
Title: Effects of tetrahydroaminoacridine on liver function in patients with Alzheimer's disease O'Brien JT, Eagger S, Levy R Ref: Age Ageing, 20:129, 1991 : PubMed
Detailed liver function test analysis is reported for 30 patients with Alzheimer's disease who were treated with tetrahydroaminoacridine. Results show that a benign elevation of aspartate transaminase occurs in up to 50% cases, that the reaction can be a symptomatic one and that clinical hepatitis can occasionally result. Liver function test changes appear dose-dependent and normalize within 2-4 weeks of stopping the drug or of reducing the dose. Women appear more likely to develop hepatotoxicity than men. Rechallenge with THA in patients previously showing abnormalities in liver function shows that some patients are able to tolerate the drug a second time.
Title: Are drugs targeted at Alzheimer's disease useful? 1. Useful for what? Levy R Ref: BMJ, 300:1131, 1990 : PubMed
