Development of SimCells as a novel chassis for functional biosensors

This work serves as a proof-of-concept for bacterially derived SimCells (Simple Cells), which contain the cell machinery from bacteria and designed DNA (or potentially a simplified genome) to instruct the cell to carry out novel, specific tasks. SimCells represent a reprogrammable chassis without a native chromosome, which can host designed DNA to perform defined functions. In this paper, the use of Escherichia coli MC1000 ∆minD minicells as a non-reproducing chassis for SimCells was explored, as demonstrated by their ability to act as sensitive biosensors for small molecules. Highly purified minicells derived from E. coli strains containing gene circuits for biosensing were able to transduce the input signals from several small molecules (glucarate, acrylate and arabinose) into the production of green fluorescent protein (GFP). A mathematical model was developed to fit the experimental data for induction of gene expression in SimCells. The intracellular ATP level was shown to be important for SimCell function. A purification and storage protocol was developed to prepare SimCells which could retain their functions for an extended period of time. This study demonstrates that SimCells are able to perform as 'smart bioparticles' controlled by designed gene circuits

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X's gene content, gene expression, and evolution

Functional Fcgamma Receptor Polymorphisms Are Associated with Human Allergy

<div><p>Objective</p><p>IgG Fc receptors (FcγRs) play important roles in immune responses. It is not clear whether FcγR receptors play a role in human asthma and allergy. The aim of current study was to investigate whether functional single nucleotide polymorphisms (SNPs) of FcγR genes (<i>FCGR</i>) are associated with human asthma and allergy.</p><p>Methods</p><p>Functional SNPs of <i>FCGR2A</i> (FcγRIIA-131His>Arg, rs1801274), <i>FCGR2B</i> (FcγRIIB-187Ile>Thr, rs1050501), <i>FCGR2C (</i>FcγRIIC-13Gln>Stop, rs10917661), <i>FCGR3A</i> (FcγRIIIA-158Val>Phe, rs396991), and <i>FCGR3B</i> variants (FcγRIIIB NA1 and NA2) were genotyped in an asthma family cohort including 370 atopy positive, 239 atopy negative, and 169 asthma positive subjects. The genotype and phenotype data (asthma, bronchial hyper-responsiveness, and atopy) of subjects were analyzed using family-based association tests (FBAT) and logistic regression adjusted for age and sex.</p><p>Result</p><p>The FcγRIIA-131His>Arg SNP is significantly associated with atopy in a family-based association test (<i>P</i> = 0.00287) and in a logistic regression analysis (<i>P</i> = 0.0269, OR 0.732, 95% CI: 0.555–0.965). The FcγRIIA-131His (or rs1801274-A) allele capable of binding human IgG2 has a protective role against atopy. In addition, the rare FcγRIIB-187Thr (or rs1050501-C) allele defective for the receptor-mediated inhibitory signals is a risk factor for atopy (<i>P</i> = 0.0031, OR 1.758, 95% CI: 1.209–2.556) and IgE production (<i>P</i><0.001). However, variants of activating FcγRIIIA (rs396991), and FcγRIIIB (NA1 and NA2), and FcγRIIC (rs10917661) are not associated with asthma, BHR, and atopy (<i>P</i>>0.05).</p><p>Conclusions</p><p>FcγRIIA and FcγRIIB functional polymorphisms may have a role in the pathogenesis of allergy.</p></div

Primers and probes of TaqMan <i>FCGR</i> gene SNP assays.

<p>Italic and underlined nucleotides are SNP sites in respective <i>FCGR</i> genes.</p

Functional SNPs of <i>FCGR3A, FCGR3B,</i> and <i>FCGR2C</i> are not associated with asthma, BHR, and atopy.

<p>SNPs of <i>FCGR3A</i> SNP (FcγRIIIA-158Val>Phe, rs396991), <i>FCGR3B</i> allele (FcγRIIIB-NA1/NA2), and <i>FCGR2C</i> SNP (FcγRIIC-13Gln>Stop, rs10917661) are not associated with asthma, BHR, and atopy in family-based association test (FBAT) analyses (<i>P</i>>0.05) and logistic regression analyses adjusted for age and sex (<i>P</i>>0.05, data not listed).</p><p><b>MAF</b>: minor allele frequency.</p

Association of SNP <i>FCGR2B</i>-187Ile>Thr with IgE levels.

<p><b>A.</b> The genotypes of <i>FCGR2B</i>-187Ile>Thr were significantly associated with IgE levels in ANOVA (<i>P</i><0.0001). The rare <i>FCGR2B</i>-187Thr carriers (187Ile/Thr and 187Thr/Thr genotypes) produced significantly more IgE than the homozygous subjects for common allele (187Ile) in Mann-Whitney tests. <b>B.</b> The genotypes of <i>FCGR2B</i>-131His>Arg were not associated with IgE levels in ANOVA (<i>P</i> = 0.817). No significant differences were found between <i>FCGR2A</i>-131Arg carriers (131his/Arg and 131Arg/Arg genotypes) and the 131His homozygous subjects in Mann-Whitney tests (<i>P</i>>0.05).</p

5-HT and DOI induce rolling of AML14.3D10 human Eos-like cells.

<p>(<b>A</b>) Expression of 5-HT2A by AML14.3D10 cells by RT-PCR with β-actin as the internal control. (<b>B</b>) Rolling of AML14.3D10 cells on rh VCAM-1-coated cover-slips under conditions of flow <i>in vitro</i> after treatment with 5-HT, DOI or DMSO (vehicle) for 5 min prior to infusion into flow chamber. (<b>C</b>) Confocal microscopy of FITC-phalloidin stained AML14.3D10 cells adhered to rh VCAM-1 on cover-slips in the presence of vehicle (DMSO), 5-HT or DOI as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054840#pone-0054840-g001" target="_blank">Figure 1</a>. Magnification ×600. Combined data (Mean ± SEM) of 3 (for 5-HT)-5 (for DOI) independent experiments in duplicate is shown for B. *p<0.03 in B for comparison of 5-HT- or DOI-treated versus vehicle-treated cells. Data shown in C is representative of three independent experiments.</p