We demonstrate the feasibility of a nanopore based single-molecule DNA sequencing method, which employs multi-color readout. Target DNA is converted according to a binary code, which is recognized by molecular beacons with two types of fluorophores. Solid-state nanopores are then used to sequentially strip off the beacons, leading to a series of detectable photon bursts, at high speed. We show that signals from multiple nanopores can be detected simultaneously, allowing straightforward parallelization to large nanopore arrays.Nanopore based DNA sequencing is widely considered to be a promising next generation sequencing platform [1,2]. Two main features of the nanopore method make it exceptionally useful for single molecule-based genome analyses: First, the method's ability to electrophoretically focus and thread extremely long DNA molecules from the bulk into the pore, making it possible to analyze minute DNA samples [3]. Second, sub-5 nm pores are now routinely used to linearize long DNA coils, thus in principle, nanopores can be used to effectively 'scan' information along a long genome. These features, as well as the fact that solid-state nanopores can be fabricated in a highly dense array [4,5], allow the development of massively parallel detection, and are crucial for the realization of an amplification-free, lowcost and high-throughput sequencing [2,[6][7][8][9].A nanopore is a nanometer-sized pore in an ultra-thin membrane that separates two chambers containing an ionic solution. An external electrical field applied across the membrane creates an ionic current and a local electrical potential gradient near the pore, which draws in and threads biopolymers through the pore in a single file manner [3,10]. As a biopolymer enters the pore, it displaces a fraction of the electrolytes, giving rise to a change in the pore conductivity, which can be measured directly using an electrometer. A number of nanopore based DNA sequencing methods have recently been proposed and highlight two major challenges [1,11]: 1) The ability to discriminate among individual nucleotides (nt). The system must be capable of differentiating among the four bases on a single-molecule level.2) The method must enable parallel readout. As a single nanopore can probe only a single molecule at a time, a strategy for manufacturing an array of nanopores and simultaneously monitoring them is needed. To date, parallel readout through any nanopore-based method has not yet been demonstrated. A large number of current, and future, generation sequencing methodologies rely on the use of an enzyme (polymerase, exonuclease, etc) in the readout process. The kinetics of enzymatic activity, however, is a major bottleneck for increasing readout speed, and these + Corresponding author. Email: ameller@bu.edu. * These authors contributed equally to this work. NIH Public AccessAuthor Manuscript Nano Lett. Author manuscript; available in PMC 2011 June 9. Published in final edited form as:Nano Lett. 2010 June 9; 10(6): 2237-2244. doi:10.1021/nl1012147. NI...

Multidrug resistance in pathogens is an increasingly significant threat for human health. Indeed, some strains are resistant to almost all currently available antibiotics, leaving very limited choices for antimicrobial clinical therapy. In many such cases, polymyxins are the last option available, although their use increases the risk of developing resistant strains. This review mainly aims to discuss advances in unraveling the mechanisms of antibacterial activity of polymyxins and bacterial tolerance together with the description of polymyxin structure, synthesis, and structural modification. These are expected to help researchers not only develop a series of new polymyxin derivatives necessary for future medical care, but also optimize the clinical use of polymyxins with minimal resistance development.

We present a novel method for integrating two single-molecule measurement modalities, namely, total internal reflection microscopy and electrical detection of biomolecules using nanopores. Demonstrated here is the electrical measurement of nanopore based biosensing performed simultaneously and in-sync with optical detection of analytes. This method makes it possible, for the first time, to visualize DNA and DNA-protein complexes translocating through a nanopore with high temporal resolution ͑1000 frames/s͒ and good signal to background. This paper describes a detailed experimental design of custom optics and data acquisition hardware to achieve simultaneous high resolution electrical and optical measurements on labeled biomolecules as they traverse through a ϳ4 nm synthetic pore. In conclusion, we discuss new directions and measurements, which this technique opens up.

Single nucleotide polymorphisms in type A gamma-aminobutyric acid (GABA(A)) receptor beta2 subunit gene (GABRB2) were found to be associated with schizophrenia in Chinese, German, Japanese and Portuguese. To explore potential functional consequences of these DNA sequence polymorphisms, this study examined the expression and electrophysiological properties of two alternatively spliced products of GABRB2 along with genotypical disease association analysis. Real-time quantitative polymerase chain reaction, performed with a cohort of 31 schizophrenics and 31 controls of US population, showed 21.7% reduction in the expression of the long isoform beta(2L), 13.4% in the short isoform beta(2S) and 15.8% in the sum of the two isoforms beta(2T) in postmortem schizophrenic brain. Furthermore, two independent mRNA quantitation methods showed that the relative expression of the long over the short isoforms was significantly decreased, suggesting the occurrence of altered splicing, in schizophrenia. In male schizophrenics, the heterozygous genotypes of rs1876071 (T/C) and rs1876072 (A/G) were correlated with reduced expression of beta(2L), beta(2S) and beta(2T), and the heterozygous of rs2546620 (A/G) and homozygous-minor of rs1876071 (C/C) and rs1876072 (G/G) were correlated with reduced expression of beta(2T). Significant correlations of expression levels with different alleles and haplotypes were also indicated by quantitative trait analysis. Recombinant GABA(A) receptors expressed in HEK293 human cells containing beta(2L) underwent a steeper current rundown upon repetitive GABA activation than receptors containing beta(2S). The results thus revealed genotype-dependent expression of the alternatively spliced isoforms of GABA(A) receptor beta2 subunit, giving rise to electrophysiological consequences that could play an important role in the pathogenesis mechanism of schizophrenia.

The gamma-aminobutyric acid type-A (GABAA) receptor plays a major role in inhibitory neurotransmissions. Intronic SNPs and haplotypes in GABRB2, the gene for GABAA receptor β2 subunit, are associated with schizophrenia and correlated with the expression of two alternatively spliced β2 isoforms. In the present study, using chimpanzee as an ancestral reference, high frequencies were observed for the derived (D) alleles of the four SNPs rs6556547, rs187269, rs1816071 and rs1816072 in GABRB2, suggesting the occurrence of positive selection for these derived alleles. Coalescence-based simulation showed that the population frequency spectra and the frequencies of H56, the haplotype having all four D alleles, significantly deviated from neutral-evolution expectation in various demographic models. Haplotypes containing the derived allele of rs1816072 displayed significantly less diversity compared to haplotypes containing its ancestral allele, further supporting positive selection. The variations in DD-genotype frequencies in five human populations provided a snapshot of the evolutionary history, which suggested that the positive selections of the D alleles are recent and likely ongoing. The divergence between the DD-genotype profiles of schizophrenic and control samples pointed to the schizophrenia-relevance of positive selections, with the schizophrenic samples showing weakened selections compared to the controls. These DD-genotypes were previously found to increase the expression of β2, especially its long isoform. Electrophysiological analysis showed that this long β2 isoform favored by the positive selections is more sensitive than the short isoform to the inhibition of GABAA receptor function by energy depletion. These findings represent the first demonstration of positive selection in a schizophrenia-associated gene.

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L-amino acid oxidase (LAAO) is attracting increasing attention due to its important functions. Diverse detection methods with their own properties have been developed for characterization of LAAO. In the present study, a simple, rapid, sensitive, cost-effective and reproducible method for quantitative in-gel determination of LAAO activity based on the visualization of Prussian blue-forming reaction is described. Coupled with SDS-PAGE, this Prussian blue agar assay can be directly used to determine the numbers and approximate molecular weights of LAAO in one step, allowing straightforward application for purification and sequence identification of LAAO from diverse samples.