Biology
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Item Impaired Focal Adhesion Kinase-Grb2 Interaction during Elevated Activity in Hippocampal Neurons(MDPI, 2015-07-10) Murase, SachikoExcitatory/inhibitory imbalances are implicated in many neurological disorders. Previously, we showed that chronically elevated network activity induces vulnerability in neurons due to loss of signal transducer and activator of transcription 3 (STAT3) signaling in response to the impairment of the serine/threonine kinase, extracellular-signal-regulated kinases 1/2 (Erk1/2) activation. However, how phosphorylation of Erk1/2 decreases during elevated neuronal activity was unknown. Here I show the pErk1/2 decrease induced by 4-aminopyridine (4-AP), an A-type potassium channel inhibitor can be blocked by a broad-spectrum matrix-metalloproteinase (MMP) inhibitor, FN-439. Surface expression levels of integrin β1 dramatically decrease when neurons are challenged by chronically elevated activity, which is reversed by FN-439. Treatment with 4-AP induces degradation of focal adhesion kinase (FAK), the mediator of integrin signaling. As a result, interactions between FAK and growth factor receptor-bound protein 2 (Grb2), the adaptor protein that mediates Erk1/2 activation by integrin, are severely impaired. Together, these data suggest the loss of integrin signaling during elevated activity causes vulnerability in neurons.Item Membrane Affinity of Platensimycin and Its Dialkylamine Analogs(MDPI, 2015-08-04) Rowe, Ian; Guo, Min; Yasmann, Anthony; Cember, Abigail; Sintim, Herman O.; Sukharev, SergeiMembrane permeability is a desired property in drug design, but there have been difficulties in quantifying the direct drug partitioning into native membranes. Platensimycin (PL) is a new promising antibiotic whose biosynthetic production is costly. Six dialkylamine analogs of PL were synthesized with identical pharmacophores but different side chains; five of them were found inactive. To address the possibility that their activity is limited by the permeation step, we calculated polarity, measured surface activity and the ability to insert into the phospholipid monolayers. The partitioning of PL and the analogs into the cytoplasmic membrane of E. coli was assessed by activation curve shifts of a re-engineered mechanosensitive channel, MscS, in patch-clamp experiments. Despite predicted differences in polarity, the affinities to lipid monolayers and native membranes were comparable for most of the analogs. For PL and the di-myrtenyl analog QD-11, both carrying bulky sidechains, the affinity for the native membrane was lower than for monolayers (half-membranes), signifying that intercalation must overcome the lateral pressure of the bilayer. We conclude that the biological activity among the studied PL analogs is unlikely to be limited by their membrane permeability. We also discuss the capacity of endogenous tension-activated channels to detect asymmetric partitioning of exogenous substances into the native bacterial membrane and the different contributions to the thermodynamic force which drives permeation.Item Genomic Characterization of a B Chromosome in Lake Malawi Cichlid Fishes(MDPI, 2018-12-05) Clark, Frances E.; Conte, Matthew A.; Kocher, Thomas D.B chromosomes (Bs) were discovered a century ago, and since then, most studies have focused on describing their distribution and abundance using traditional cytogenetics. Only recently have attempts been made to understand their structure and evolution at the level of DNA sequence. Many questions regarding the origin, structure, function, and evolution of B chromosomes remain unanswered. Here, we identify B chromosome sequences from several species of cichlid fish from Lake Malawi by examining the ratios of DNA sequence coverage in individuals with or without B chromosomes. We examined the efficiency of this method, and compared results using both Illumina and PacBio sequence data. The B chromosome sequences detected in 13 individuals from 7 species were compared to assess the rates of sequence replacement. B-specific sequence common to at least 12 of the 13 datasets were identified as the “Core” B chromosome. The location of B sequence homologs throughout the genome provides further support for theories of B chromosome evolution. Finally, we identified genes and gene fragments located on the B chromosome, some of which may regulate the segregation and maintenance of the B chromosome.Item Distribution and Community Assembly of Trees Along an Andean Elevational Gradient(MDPI, 2019-09-05) Worthy, Samantha J.; Jiménez Paz, Rosa A.; Pérez, Álvaro J.; Reynolds, Alex; Cruse-Sanders, Jennifer; Valencia, Renato; Barone, John A.; Burgess, Kevin S.Highlighting patterns of distribution and assembly of plants involves the use of community phylogenetic analyses and complementary traditional taxonomic metrics. However, these patterns are often unknown or in dispute, particularly along elevational gradients, with studies finding different patterns based on elevation. We investigated how patterns of tree diversity and structure change along an elevation gradient using taxonomic and phylogenetic diversity metrics. We sampled 595 individuals (36 families; 53 genera; 88 species) across 15 plots along an elevational gradient (2440–3330 m) in Ecuador. Seventy species were sequenced for the rbcL and matK gene regions to generate a phylogeny. Species richness, Shannon–Weaver diversity, Simpson’s Dominance, Simpson’s Evenness, phylogenetic diversity (PD), mean pairwise distance (MPD), and mean nearest taxon distance (MNTD) were evaluated for each plot. Values were correlated with elevation and standardized effect sizes (SES) of MPD and MNTD were generated, including and excluding tree fern species, for comparisons across elevation. Taxonomic and phylogenetic metrics found that species diversity decreases with elevation. We also found that overall the community has a non-random phylogenetic structure, dependent on the presence of tree ferns, with stronger phylogenetic clustering at high elevations. Combined, this evidence supports the ideas that tree ferns have converged with angiosperms to occupy the same habitat and that an increased filtering of clades has led to more closely related angiosperm species at higher elevations.Item Cooperativity and Steep Voltage Dependence in a Bacterial Channel(MDPI, 2019-09-11) Lin, Shang H.; Chang, Kai-Ti; Cherian, Nuval; Wu, Benjamin; Phee, Hyo; Cho, Christy; Colombini, MarcoThis paper reports on the discovery of a novel three-membrane channel unit exhibiting very steep voltage dependence and strong cooperative behavior. It was reconstituted into planar phospholipid membranes formed by the monolayer method and studied under voltage-clamp conditions. The behavior of the novel channel-former, isolated from Escherichia coli, is consistent with a linearly organized three-channel unit displaying steep voltage-gating (a minimum of 14 charges in the voltage sensor) that rivals that of channels in mammalian excitable membranes. The channels also display strong cooperativity in that closure of the first channel permits the second to close and closure of the second channel permits closure of the third. All three have virtually the same conductance and selectivity, and yet the first and third close at positive potentials whereas the second closes at negative potentials. Thus, is it likely that the second channel-former is oriented in the membrane in a direction opposite to that of the other two. This novel structure is named “triplin.” The extraordinary behavior of triplin indicates that it must have important and as yet undefined physiological roles.Item Genomic characterization of the Yersinia genus(Springer Nature, 2010-01-04) Chen, Peter E; Cook, Christopher; Stewart, Andrew C; Nagarajan, Niranjan; Sommer, Dan D; Pop, Mihai; Thomason, Brendan; Thomason, Maureen P Kiley; Lentz, Shannon; Nolan, Nichole; Sozhamannan, Shanmuga; Sulakvelidze, Alexander; Mateczun, Alfred; Du, Lei; Zwick, Michael E; Read, Timothy DNew DNA sequencing technologies have enabled detailed comparative genomic analyses of entire genera of bacterial pathogens. Prior to this study, three species of the enterobacterial genus Yersinia that cause invasive human diseases (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) had been sequenced. However, there were no genomic data on the Yersinia species with more limited virulence potential, frequently found in soil and water environments. We used high-throughput sequencing-by-synthesis instruments to obtain 25- to 42-fold average redundancy, whole-genome shotgun data from the type strains of eight species: Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. kristensenii, Y. intermedia, Y. mollaretii, Y. rohdei, and Y. ruckeri. The deepest branching species in the genus, Y. ruckeri, causative agent of red mouth disease in fish, has the smallest genome (3.7 Mb), although it shares the same core set of approximately 2,500 genes as the other members of the species, whose genomes range in size from 4.3 to 4.8 Mb. Yersinia genomes had a similar global partition of protein functions, as measured by the distribution of Cluster of Orthologous Groups families. Genome to genome variation in islands with genes encoding functions such as ureases, hydrogeneases and B-12 cofactor metabolite reactions may reflect adaptations to colonizing specific host habitats. Rapid high-quality draft sequencing was used successfully to compare pathogenic and non-pathogenic members of the Yersinia genus. This work underscores the importance of the acquisition of horizontally transferred genes in the evolution of Y. pestis and points to virulence determinants that have been gained and lost on multiple occasions in the history of the genus.Item Acoel and platyhelminth models for stem-cell research(Springer Nature, 2010-02-16) Bely, Alexandra E; Sikes, James MAcoel and platyhelminth worms are particularly attractive invertebrate models for stem-cell research because their bodies are continually renewed from large pools of somatic stem cells. Several recent studies, including one in BMC Developmental Biology, are beginning to reveal the cellular dynamics and molecular basis of stem-cell function in these animals. See research article http://www.biomedcentral.com/1471-213X/9/69 .Item A toolkit for rapid gene mapping in the nematode Caenorhabditis briggsae(Springer Nature, 2010-04-13) Koboldt, Daniel C; Staisch, Julia; Thillainathan, Bavithra; Haines, Karen; Baird, Scott E; Chamberlin, Helen M; Haag, Eric S; Miller, Raymond D; Gupta, Bhagwati PThe nematode C. briggsae serves as a useful model organism for comparative analysis of developmental and behavioral processes. The amenability of C. briggsae to genetic manipulations and the availability of its genome sequence have prompted researchers to study evolutionary changes in gene function and signaling pathways. These studies rely on the availability of forward genetic tools such as mutants and mapping markers. We have computationally identified more than 30,000 polymorphisms (SNPs and indels) in C. briggsae strains AF16 and HK104. These include 1,363 SNPs that change restriction enzyme recognition sites (snip-SNPs) and 638 indels that range between 7 bp and 2 kb. We established bulk segregant and single animal-based PCR assay conditions and used these to test 107 polymorphisms. A total of 75 polymorphisms, consisting of 14 snip-SNPs and 61 indels, were experimentally confirmed with an overall success rate of 83%. The utility of polymorphisms in genetic studies was demonstrated by successful mapping of 12 mutations, including 5 that were localized to sub-chromosomal regions. Our mapping experiments have also revealed one case of a misassembled contig on chromosome 3. We report a comprehensive set of polymorphisms in C. briggsae wild-type strains and demonstrate their use in mapping mutations. We also show that molecular markers can be useful tools to improve the C. briggsae genome sequence assembly. Our polymorphism resource promises to accelerate genetic and functional studies of C. briggsae genes.Item An EST resource for tilapia based on 17 normalized libraries and assembly of 116,899 sequence tags(Springer Nature, 2010-04-30) Lee, Bo-Young; Howe, Aimee E; Conte, Matthew A; D'Cotta, Helena; Pepey, Elodie; Baroiller, Jean-Francois; di Palma, Federica; Carleton, Karen L; Kocher, Thomas DLarge collections of expressed sequence tags (ESTs) are a fundamental resource for analysis of gene expression and annotation of genome sequences. We generated 116,899 ESTs from 17 normalized and two non-normalized cDNA libraries representing 16 tissues from tilapia, a cichlid fish widely used in aquaculture and biological research. The ESTs were assembled into 20,190 contigs and 36,028 singletons for a total of 56,218 unique sequences and a total assembled length of 35,168,415 bp. Over the whole project, a unique sequence was discovered for every 2.079 sequence reads. 17,722 (31.5%) of these unique sequences had significant BLAST hits (e-value < 10-10) to the UniProt database. Normalization of the cDNA pools with double-stranded nuclease allowed us to efficiently sequence a large collection of ESTs. These sequences are an important resource for studies of gene expression, comparative mapping and annotation of the forthcoming tilapia genome sequence.Item Between a chicken and a grape: estimating the number of human genes(Springer Nature, 2010-05-05) Pertea, Mihaela; Salzberg, Steven LMany people expected the question 'How many genes in the human genome?' to be resolved with the publication of the genome sequence in 2001, but estimates continue to fluctuate.