Capturing cancer cells using aptamer-immobilized square capillary channels

We report a simple square capillary-based cell affinity chromatography device that utilizes a coating of aptamers for selective capture of target cancer cells from a flowing suspension. The device consists of a square capillary with an inner diameter of roughly five cell diameters, connected via Teflon tubing to a syringe. Aptamers are immobilized on the inner surface of the capillary through biotin-avidin chemistry, the extent of which can be controlled by adjusting the aptamer concentration. Introduction of different cell types into separate devices, as well as mixtures of target and non-target cells, demonstrated that aptamer-target cells can be captured in significantly higher concentrations compared to non-target cells. Once optimized, 91.1 ± 3.5% capture efficiency of target leukemia cells was reported, as well as 97.2 ± 2.8% and 83.6 ± 5.8% for two different colon cancer cell lines. In addition, cells captured in the device were imaged, and the square capillary exhibited better optical properties than standard cylindrical capillaries, leading to the detection of leukemia cells in blood samples. Compared to current microfluidic cell affinity devices, this capture device requires no complicated design or fabrication steps. By providing a simple means of detecting and imaging cancer cells in the blood, this work has potential to directly assist clinicians in determining disease prognosis and measuring therapeutic response.     

Programming microbial population dynamics by engineered cell-cell communication

A major aim of synthetic biology is to program novel cellular behavior using engineered gene circuits. Early endeavors focused on building simple circuits that fulfill simple functions, such as logic gates, bistable toggle switches, and oscillators. These gene circuits have primarily focused on single-cell behaviors since they operate intracellularly. Thus, they are often susceptible to cell-cell variations due to stochastic gene expression. Cell-cell communication offers an efficient strategy to coordinate cellular behavior at the population level. To this end, we review recent advances in engineering cell-cell communication to achieve reliable population dynamics, spanning from communication within single species to multispecies, from one-way sender-receiver communication to two-way communication in synthetic microbial ecosystems. These engineered systems serve as well-defined model systems to better understand design principles of their naturally occurring counterparts and to facilitate novel biotechnology applications.     

An atypical epigenetic mechanism affects uniparental expression of Pol IV-dependent siRNAs

Background

Small RNAs generated by RNA polymerase IV (Pol IV) are the most abundant class of small RNAs in flowering plants. In Arabidopsis thaliana Pol IV-dependent short interfering (p4-si)RNAs are imprinted and accumulate specifically from maternal chromosomes in the developing seeds. Imprinted expression of protein-coding genes is controlled by differential DNA or histone methylation placed in gametes. To identify epigenetic factors required for maternal-specific expression of p4-siRNAs we analyzed the effect of a series of candidate mutations, including those required for genomic imprinting of protein-coding genes, on uniparental expression of a representative p4-siRNA locus.

Results

Paternal alleles of imprinted genes are marked by DNA or histone methylation placed by DNA METHYLTRANSFERASE 1 or the Polycomb Repressive Complex 2. Here we demonstrate that repression of paternal p4-siRNA expression at locus 08002 is not controlled by either of these mechanisms. Similarly, loss of several chromatin modification enzymes, including a histone acetyltransferase, a histone methyltransferase, and two nucleosome remodeling proteins, does not affect maternal expression of locus 08002. Maternal alleles of imprinted genes are hypomethylated by DEMETER DNA glycosylase, yet expression of p4-siRNAs occurs irrespective of demethylation by DEMETER or related glycosylases.

Conclusions

Differential DNA methylation and other chromatin modifications associated with epigenetic silencing are not required for maternal-specific expression of p4-siRNAs at locus 08002. These data indicate that there is an as yet unknown epigenetic mechanism causing maternal-specific p4-siRNA expression that is distinct from the well-characterized mechanisms associated with DNA methylation or the Polycomb Repressive Complex 2.     

Bivariate genome-wide association analyses of femoral neck bone geometry and appendicular lean mass

Objective

Femoral neck geometric parameters (FNGPs), such as periosteal diameter (W), cross-sectional area (CSA), cortical thickness (CT), buckling ratio (BR), and section modulus (Z), are highly genetically correlated with body lean mass. However, the specific SNPs/genes shared by these phenotypes are largely unknown.

Methods

To identify the specific SNPs/genes shared between FNGPs and appendicular lean mass (ALM), we performed an initial bivariate genome-wide association study (GWAS) by scanning ∼690,000 SNPs in 1,627 unrelated Han Chinese adults (802 males and 825 females) and a follow-up replicate study in 2,286 unrelated US Caucasians.

Results

We identified 13 interesting SNPs that may be important for both FNGPs and ALM. Two SNPs, rs681900 located in the HK2 (hexokinase 2) gene and rs11859916 in the UMOD (uromodulin) gene, were bivariately associated with FNGPs and ALM (p = 7.58×10⁻⁶ for ALM-BR and p = 2.93×10⁻⁶ for ALM-W, respectively). The associations were then replicated in Caucasians, with corresponding p values of 0.024 for rs681900 and 0.047 for rs11859916. Meta-analyses yielded combined p values of 3.05×10⁻⁶ and 2.31×10⁻⁶ for rs681900 and rs11859916, respectively. Our findings are consistent with previous biological studies that implicated HK2 and UMOD in both FNGPs and ALM. Our study also identified a group of 11 contiguous SNPs, which spanned a region of ∼130 kb, were bivariately associated with FNGPs and ALM, with p values ranging from 3.06×10⁻⁷ to 4.60×10⁻⁶ for ALM-BR. The region contained two neighboring miRNA coding genes, MIR873 (MicroRNA873) and MIR876 (MicroRNA876).

Conclusion

Our study implicated HK2, UMOD, MIR873 and MIR876, as pleiotropic genes underlying variation of both FNGPs and ALM, thus suggesting their important functional roles in co-regulating both FNGPs and ALM.     

Gene expression changes in GABA(A) receptors and cognition following chronic ketamine administration in mice

Ketamine is a well-known anesthetic agent and a drug of abuse. Despite its widespread use and abuse, little is known about its long-term effects on the central nervous system. The present study was designed to evaluate the effect of long-term (1- and 3-month) ketamine administration on learning and memory and associated gene expression levels in the brain. The Morris water maze was used to assess spatial memory and gene expression changes were assayed using Affymetrix Genechips; a focus on the expression of GABA(A) receptors that mediate a tonic inhibition in the brain, was confirmed by quantitative real-time PCR and western blot. Compared with saline controls, there was a decline in learning and memory performance in the ketamine-treated mice. Genechip results showed that 110 genes were up-regulated and 136 genes were down-regulated. An ontology analysis revealed the most significant effects of ketamine were on GABA(A) receptors. In particular, there was a significant up-regulation of both mRNA and protein levels of the alpha 5 subunit (Gabra5) of the GABA(A) receptors in the prefrontal cortex. In conclusion, chronic exposure to ketamine impairs working memory in mice, which may be explained at least partly by up-regulation of Gabra5 subunits in the prefrontal cortex.     

Infrequent detection of KI, WU and MC polyomaviruses in immunosuppressed individuals with or without progressive multifocal leukoencephalopathy

Conflicting prevalence of newly identified KI (KIPyV), WU (WUPyV) and Merkel Cell Carcinoma (MCPyV) polyomaviruses have been reported in progressive multifocal leukoencephalopathy (PML) patient samples, ranging from 0 to 14.3%. We analyzed the prevalence of these polyomaviruses in cerebrospinal fluid (CSF), peripheral blood mononuclear cells (PBMC), and bone marrow samples from PML patients, immunosuppressed individuals with or without HIV, and multiple sclerosis (MS) patients. Distinct PCR tests for KIPyV, WUPyV and MCPyV DNA performed in two independent laboratories detected low levels of MCPyV DNA only in 1/269 samples. The infrequent detections of these viruses in multiple samples from immunosuppressed individuals including those with PML suggest that their reactivation mechanisms may be different from that of JC polyomavirus (JCPyV) and that they do not play a role in the pathogenesis of PML.     

Preparation of cyclodextrin chiral stationary phases by organic soluble catalytic 'click' chemistry

We describe an effective and simple protocol that uses click chemistry to attach native β-cyclodextrin (β-CD) to silica particles, resulting in a chiral stationary phase (CCNCSP) that can be used for the enantioseparation of chiral drugs by high-performance liquid chromatography (HPLC). Starting from β-CD, the CCNCSP is prepared in several steps: (i) reaction of β-CD with 1-(p-toluenesulfonyl)-imidazole to afford mono-6-toluenesulfonyl-β-CD; (ii) azidolysis of mono-6-toluenesulfonyl-β-CD in dimethylformamide to give mono-6-azido-β-CD (N(3)-CD); (iii) reaction of cuprous iodide with triphenylphosphine to form an organic soluble catalyst CuI(PPh(3)); (iv) preparation of alkynyl-modified silica particles; and (v) click chemistry immobilization of N(3)-CD onto alkynyl-modified silica to afford the desired chiral stationary phase. Synthesis of the stationary phase and column packing takes ～1 week.     

Emergence and transmission pathways of rapidly evolving evolutionary branch C4a strains of human enterovirus 71 in the Central Plain of China

Background

Large-scale outbreaks of hand, foot, and mouth disease (HFMD) occurred repeatedly in the Central Plain of China (Shandong, Anhui, and Henan provinces) from 2007 until now. These epidemics have increased in size and severity each year and are a major public health concern in mainland China.

Principal Findings

Phylogenetic analysis was performed and a Bayesian Markov chain Monte Carlo tree was constructed based on the complete VP1 sequences of HEV71 isolates. These analyses showed that the HFMD epidemic in the Central Plain of China was caused by at least 5 chains of HEV71 transmission and that the virus continued to circulate and evolve over the winter seasons between outbreaks. Between 1998 and 2010, there were 2 stages of HEV71 circulation in mainland China, with a shift from evolutionary branch C4b to C4a in 2003–2004. The evolution rate of C4a HEV71 was 4.99×10^-3 substitutions per site per year, faster than the mean of all HEV71 genotypes. The most recent common ancestor estimates for the Chinese clusters dated to October 1994 and November 1993 for the C4a and C4b evolutionary branches, respectively. Compared with all C4a HEV71 strains, a nucleotide substitution in all C4b HEV71 genome (A to C reversion at nt2503 in the VP1 coding region, which caused amino acid substitution of VP1–10: Gln to His) had reverted.

Conclusions

The data suggest that C4a HEV71 strains introduced into the Central Plain of China are responsible for the recent outbreaks. The relationships among HEV71 isolates determined from the combined sequence and epidemiological data reveal the underlying seasonal dynamics of HEV71 circulation. At least 5 HEV71 lineages circulated in the Central Plain of China from 2007 to 2009, and the Shandong and Anhui lineages were found to have passed through a genetic bottleneck during the low-transmission winter season.