Quantifying the Integration of Quorum-Sensing Signals with Single-Cell Resolution

Cell-to-cell communication in bacteria is a process known as quorum sensing that relies on the production, detection, and response to the extracellular accumulation of signaling molecules called autoinducers. Often, bacteria use multiple autoinducers to obtain information about the vicinal cell density. However, how cells integrate and interpret the information contained within multiple autoinducers remains a mystery. Using single-cell fluorescence microscopy, we quantified the signaling responses to and analyzed the integration of multiple autoinducers by the model quorum-sensing bacterium Vibrio harveyi. Our results revealed that signals from two distinct autoinducers, AI-1 and AI-2, are combined strictly additively in a shared phosphorelay pathway, with each autoinducer contributing nearly equally to the total response. We found a coherent response across the population with little cell-to-cell variation, indicating that the entire population of cells can reliably distinguish several distinct conditions of external autoinducer concentration. We speculate that the use of multiple autoinducers allows a growing population of cells to synchronize gene expression during a series of distinct developmental stages.     

Characterization of the MUC1-C Cytoplasmic Domain as a Cancer Target

Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly expressed in diverse human carcinomas and certain hematologic malignancies. The oncogenic MUC1 transmembrane C-terminal subunit (MUC1-C) functions in part by transducing growth and survival signals from cell surface receptors. However, little is known about the structure of the MUC1-C cytoplasmic domain as a potential drug target. Using methods for structural predictions, our results indicate that a highly conserved CQCRRK sequence, which is adjacent to the cell membrane, forms a small pocket that exposes the two cysteine residues for forming disulfide bonds. By contrast, the remainder of the MUC1-C cytoplasmic domain has no apparent structure, consistent with an intrinsically disordered protein. Our studies thus focused on targeting the MUC1 CQCRRK region. The results show that L- and D-amino acid CQCRRK-containing peptides bind directly to the CQC motif. We further show that the D-amino acid peptide, designated GO-203, blocks homodimerization of the MUC1-C cytoplasmic domain in vitro and in transfected cells. Moreover, GO-203 binds directly to endogenous MUC1-C in breast and lung cancer cells. Colocalization studies further demonstrate that GO-203 predominantly binds to MUC1-C at the cell membrane. These findings support the further development of agents that target the MUC1-C cytoplasmic domain CQC motif and thereby MUC1-C function in cancer cells.     

Lesions of the Basal Forebrain Cholinergic System in Mice Disrupt Idiothetic Navigation

Loss of integrity of the basal forebrain cholinergic neurons is a consistent feature of Alzheimer’s disease, and measurement of basal forebrain degeneration by magnetic resonance imaging is emerging as a sensitive diagnostic marker for prodromal disease. It is also known that Alzheimer’s disease patients perform poorly on both real space and computerized cued (allothetic) or uncued (idiothetic) recall navigation tasks. Although the hippocampus is required for allothetic navigation, lesions of this region only mildly affect idiothetic navigation. Here we tested the hypothesis that the cholinergic medial septo-hippocampal circuit is important for idiothetic navigation. Basal forebrain cholinergic neurons were selectively lesioned in mice using the toxin saporin conjugated to a basal forebrain cholinergic neuronal marker, the p75 neurotrophin receptor. Control animals were able to learn and remember spatial information when tested on a modified version of the passive place avoidance test where all extramaze cues were removed, and animals had to rely on idiothetic signals. However, the exploratory behaviour of mice with cholinergic basal forebrain lesions was highly disorganized during this test. By contrast, the lesioned animals performed no differently from controls in tasks involving contextual fear conditioning and spatial working memory (Y maze), and displayed no deficits in potentially confounding behaviours such as motor performance, anxiety, or disturbed sleep/wake cycles. These data suggest that the basal forebrain cholinergic system plays a specific role in idiothetic navigation, a modality that is impaired early in Alzheimer’s disease.     

Detailed Analysis of Gene Polymorphisms Associated with Ischemic Stroke in South Asians

The burden of stroke is disproportionately high in the South Asian subcontinent with South Asian ethnicity conferring a greater risk of ischemic stroke than European ancestry regardless of country inhabited. While genes associated with stroke in European populations have been investigated, they remain largely unknown in South Asians. We conducted a comprehensive meta-analysis of known genetic polymorphisms associated with South Asian ischemic stroke, and compared effect size of the MTHFR C677T-stroke association with effect sizes predicted from homocysteine-stroke association. Electronic databases were searched up to August 2012 for published case control studies investigating genetic polymorphisms associated with ischemic stroke in South Asians. Pooled odds ratios (OR) for each gene-disease association were calculated using a random-effects model. We identified 26 studies (approximately 2529 stroke cases and 2881 controls) interrogating 33 independent genetic polymorphisms in 22 genes. Ten studies described MTHFR C677T (108 with TT genotype and 2018 with CC genotype) -homocysteine relationship and six studies (735 stroke cases and 713 controls) described homocysteine-ischemic stroke relationship. Risk association ORs were calculated for ACE I/D (OR 5.00; 95% CI, 1.17–21.37; p = 0.03), PDE4D SNP 83 (OR 2.20; 95% CI 1.21–3.99; p = 0.01), PDE4D SNP 32 (OR 1.57; 95% CI 1.01–2.45, p = 0.045) and IL10 G1082A (OR 1.44; 95% CI, 1.09–1.91, p = 0.01). Significant association was observed between elevated plasma homocysteine levels and MTHFR/677 TT genotypes in healthy South Asians (Mean difference (ΔX) 5.18 µmol/L; 95% CI 2.03–8.34: p = 0.001). Our results demonstrate that the genetic etiology of ischemic stroke in South Asians is broadly similar to the risk conferred in Europeans, although the dataset is considerably smaller and warrants the same clinical considerations for risk profiling.     

Modulation of PPAR subtype selectivity. Part 2: Transforming PPARα/γ dual agonist into α selective PPAR agonist through bioisosteric modification

A novel series of oxime containing benzyl-1,3-dioxane-r-2-carboxylic acid derivatives (6a-k) were designed as selective PPARα agonists, through bioisosteric modification in the lipophilic tail region of PPARα/γ dual agonist. Some of the test compounds (6a, 6b, 6c and 6f) showed high selectivity towards PPARα over PPARγ in vitro. Further, highly potent and selective PPARα agonist 6c exhibited significant antihyperglycemic and antihyperlipidemic activity in vivo, along with its improved pharmacokinetic profile. Favorable in-silico interaction of 6c with PPARα binding pocket correlate its in vitro selectivity profile toward PPARα over PPARγ. Together, these results confirm discovery of novel series of oxime based selective PPARα agonists for the safe and effective treatment of various metabolic disorders.     

ABL/BCR gene variant with two-step mechanism: Unusual localization and rare/novel chromosomal rearrangements in CML patients

Aims: Variant translocations involving 9q, 22q and at least one additional genomic locus occur in 5-10% of the patients with chronic myeloid leukemia (CML). The mechanisms for the formation of these variant translocations are not fully characterized. Here we report CML cases presenting a variant translocation indicating two-step mechanism with rare/novel chromosomal rearrangement. Methods: Karyotype analysis was performed on metaphases obtained through short-term cultures of bone marrow and blood. Detection of BCR-ABL fusion gene was performed using dual-color dual-fusion (D-FISH) and extra signal (ES) translocation probes. BAC-FISH was also carried out. Results: In Patient 1, the third partner chromosome was der(11)(p15) with a 2F2G1R signal pattern, which is an unusual signal pattern with the two-step mechanism. Patients 2 and 3 showed typical positive (2F1G1R) signal pattern. In Patient 2, both the chromosome 22s were involved in variant formation. The second fusion was observed below the BCR gene of the second homologue. In Patient 3 the third chromosome was der(13)(q14). The fourth patient showed a variant pattern with BCR/ABL-ES probe involving der(X)(q13) region. Conclusion: The presence of different rearrangements of both 9q34 and 22q11 regions highlights the genetic heterogeneity of this subgroup of CML. In each case with variants, further studies with FISH, BAC-FISH or more advanced technique such as microarray should be performed. Future studies should be performed to confirm the presence of true breakpoint hot spots and assess their implications in CML with variant Ph.     

Interaction of lentil lectin with human platelets. Evidence against glycoprotein II as aggregation mediator

Human platelets bind on an average of 5 × 10⁵ molecules of lentil lectin/cell with an apparent dissociation constant of 3 × 10^?7 M. The lectin binds mainly to surface glycoprotein II with an apparent molecular weight of 125,000. Lentil lectin neither caused aggregation nor did it inhibit platelet aggregation by other agents. It had no influence on the binding of thrombin to platelets or on thrombin-induced clot retraction. The hypothesis that glycoprotein II mediates platelet aggregation needs reevaluation.     

Saccharomyces cerevisiae Hop1 protein zinc finger motif binds to the Holliday junction and distorts the DNA structure: implications for holliday junction migration

Saccharomyces cerevisiae HOP1, which encodes a component of synaptonemal complex, plays an important role in crossing over between homologues. Hop1p contains a zinc finger motif, and substitution of a conserved Cys371 by Ser rendered the hop1 mutant allele defective in sporulation and meiosis. However, the molecular mechanism underlying the function of Hop1 zinc finger motif (ZnF) remains obscure. Here we show that wild-type Hop1 ZnF binds significantly better to the Holliday junction compared with other recombination intermediates. Consequently, the salt titration midpoint for dissociation of the Holliday junction-ZnF complex was higher than the complexes containing flush-ended linear or tailed duplex DNA. Although DNase I footprinting showed that Hop1 ZnF binds to each of the four arms of the junction, KMnO4 probing and 2-aminopurine fluorescence emission data disclosed that it distorts the DNA structure along a pair of symmetrical arms. Molecular modeling studies show that Hop1 ZnF forms a unique zinc-binding fold, reminiscent of the basic helix-loop-helix motif. In the presence of Zn2+, docking studies show that alpha helix 1, which is replete with basic amino acid residues, makes stabilizing contacts with the sugar-phosphate backbone. Structural comparison revealed a striking similarity between RecG wedge domain and Hop1 ZnF motif. We propose that Hop1 ZnF motif plays a key role in the physical monitoring of recombination intermediates and branch migration of the Holliday junction.     

Spliceosomal intron insertions in genome compacted ray-finned fishes as evident from phylogeny of MC receptors, also supported by a few other GPCRs

Background

Insertions of spliceosomal introns are very rare events during evolution of vertebrates and the mechanisms governing creation of novel intron(s) remain obscure. Largely, gene structures of melanocortin (MC) receptors are characterized by intron-less architecture. However, recently a few exceptions have been reported in some fishes. This warrants a systematic survey of MC receptors for understanding intron insertion events during vertebrate evolution.

Methodology/Principal Findings

We have compiled an extended list of MC receptors from different vertebrate genomes with variations in fishes. Notably, the closely linked MC2Rs and MC5Rs from a group of ray-finned fishes have three and one intron insertion(s), respectively, with conserved positions and intron phase. In both genes, one novel insertion was in the highly conserved DRY motif at the end of helix TM3. Further, the proto-splice site MAG↑R is maintained at intron insertion sites in these two genes. However, the orthologs of these receptors from zebrafish and tetrapods are intron-less, suggesting these introns are simultaneously created in selected fishes. Surprisingly, these novel introns are traceable only in four fish genomes. We found that these fish genomes are severely compacted after the separation from zebrafish. Furthermore, we also report novel intron insertions in P2Y receptors and in CHRM3. Finally, we report ultrasmall introns in MC2R genes from selected fishes.

Conclusions/Significance

The current repository of MC receptors illustrates that fishes have no MC3R ortholog. MC2R, MC5R, P2Y receptors and CHRM3 have novel intron insertions only in ray-finned fishes that underwent genome compaction. These receptors share one intron at an identical position suggestive of being inserted contemporaneously. In addition to repetitive elements, genome compaction is now believed to be a new hallmark that promotes intron insertions, as it requires rapid DNA breakage and subsequent repair processes to gain back normal functionality.