Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability.

Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Despite their importance as epigenetic markers and as central players in cellular processes, it is not well understood how these modifications influence physical properties of DNA and chromatin. Here we report a comprehensive survey of the effect of cytosine modifications on DNA flexibility. We find that even a single copy of 5-fC increases DNA flexibility markedly. 5-mC reduces and 5-hmC enhances flexibility, and 5-caC does not have a measurable effect. Molecular dynamics simulations show that these modifications promote or dampen structural fluctuations, likely through competing effects of base polarity and steric hindrance, without changing the average structure. The increase in DNA flexibility increases the mechanical stability of the nucleosome and vice versa, suggesting a gene regulation mechanism where cytosine modifications change the accessibility of nucleosomal DNA through their effects on DNA flexibility.

Ancient hot and cold genes and chemotherapy resistance emergence.

We use a microfabricated ecology with a doxorubicin gradient and population fragmentation to produce a strong Darwinian selective pressure that drives forward the rapid emergence of doxorubicin resistance in multiple myeloma (MM) cancer cells. RNA sequencing of the resistant cells was used to examine (i) emergence of genes with high de novo substitution densities (i.e., hot genes) and (ii) genes never substituted (i.e., cold genes). The set of cold genes, which were 21% of the genes sequenced, were further winnowed down by examining excess expression levels. Both the most highly substituted genes and the most highly expressed never-substituted genes were biased in age toward the most ancient of genes. This would support the model that cancer represents a revision back to ancient forms of life adapted to high fitness under extreme stress, and suggests that these ancient genes may be targets for cancer therapy.

Asymmetric unwrapping of nucleosomes under tension directed by DNA local flexibility.

Dynamics of the nucleosome and exposure of nucleosomal DNA play key roles in many nuclear processes, but local dynamics of the nucleosome and its modulation by DNA sequence are poorly understood. Using single-molecule assays, we observed that the nucleosome can unwrap asymmetrically and directionally under force. The relative DNA flexibility of the inner quarters of nucleosomal DNA controls the unwrapping direction such that the nucleosome unwraps from the stiffer side. If the DNA flexibility is similar on two sides, it stochastically unwraps from either side. The two ends of the nucleosome are orchestrated such that the opening of one end helps to stabilize the other end, providing a mechanism to amplify even small differences in flexibility to a large asymmetry in nucleosome stability. Our discovery of DNA flexibility as a critical factor for nucleosome dynamics and mechanical stability suggests a novel mechanism of gene regulation by DNA sequence and modifications.

RNA biochemistry. Determination of in vivo target search kinetics of regulatory noncoding RNA.

Base-pairing interactions between nucleic acids mediate target recognition in many biological processes. We developed a super-resolution imaging and modeling platform that enabled the in vivo determination of base pairing-mediated target recognition kinetics. We examined a stress-induced bacterial small RNA, SgrS, which induces the degradation of target messenger RNAs (mRNAs). SgrS binds to a primary target mRNA in a reversible and dynamic fashion, and formation of SgrS-mRNA complexes is rate-limiting, dictating the overall regulation efficiency in vivo. Examination of a secondary target indicated that differences in the target search kinetics contribute to setting the regulation priority among different target mRNAs. This super-resolution imaging and analysis approach provides a conceptual framework that can be generalized to other small RNA systems and other target search processes.

Emergence of antibiotic resistance from multinucleated bacterial filaments.

Bacteria can rapidly evolve resistance to antibiotics via the SOS response, a state of high-activity DNA repair and mutagenesis. We explore here the first steps of this evolution in the bacterium Escherichia coli. Induction of the SOS response by the genotoxic antibiotic ciprofloxacin changes the E. coli rod shape into multichromosome-containing filaments. We show that at subminimal inhibitory concentrations of ciprofloxacin the bacterial filament divides asymmetrically repeatedly at the tip. Chromosome-containing buds are made that, if resistant, propagate nonfilamenting progeny with enhanced resistance to ciprofloxacin as the parent filament dies. We propose that the multinucleated filament creates an environmental niche where evolution can proceed via generation of improved mutant chromosomes due to the mutagenic SOS response and possible recombination of the new alleles between chromosomes. Our data provide a better understanding of the processes underlying the origin of resistance at the single-cell level and suggest an analogous role to the eukaryotic aneuploidy condition in cancer.

You cannot tell a book by looking at the cover: Cryptic complexity in bacterial evolution.

Do genetically closely related organisms under identical, but strong selection pressure converge to a common resistant genotype or will they diverge to different genomic solutions? This question gets at the heart of how rough is the fitness landscape in the local vicinity of two closely related strains under stress. We chose a Growth Advantage in Stationary Phase (GASP) E scherichia coli strain to address this question because the GASP strain has very similar fitness to the wild-type (WT) strain in the absence of metabolic stress but in the presence of metabolic stress continues to divide and does not enter into stationary phase. We find that under strong antibiotic selection pressure by the fluoroquinolone antibiotic ciprofloxacin in a complex ecology that the GASP strain rapidly evolves in under 20 h missense mutation in gyrA only 2 amino acids removed from the WT strain indicating a convergent solution, yet does not evolve the other 3 mutations of the WT strain. Further the GASP strain evolves a prophage e14 excision which completely inhibits biofilm formation in the mutant strain, revealing the hidden complexity of E. coli evolution to antibiotics as a function of selection pressure. We conclude that there is a cryptic roughness to fitness landscapes in the absence of stress.

In vitro microbial culture models and their application in drug development.

Drug development faces its nemesis in the form of drug resistance. The rate of bacterial resistance to antibiotics, or tumor resistance to chemotherapy decisively depends on the surrounding heterogeneous tissue. However, in vitro drug testing is almost exclusively done in well stirred, homogeneous environments. Recent advancements in microfluidics and microfabrication introduce opportunities to develop in vitro culture models that mimic the complex in vivo tissue environment. In this review, we will first discuss the design principles underlying such models. Then we will demonstrate two types of microfluidic devices that combine stressor gradients, cell motility, large population of competing/cooperative cells and time varying dosage of drugs. By incorporating ideas from how natural selection and evolution move drug resistance forward, we show that drug resistance can occur at much greater rates than in well-stirred environments. Finally, we will discuss the future direction of in vitro microbial culture models and how to extend the lessons learned from microbial systems to eukaryotic cells.

Superconductivity in bundles of double-wall carbon nanotubes.

We present electrical and thermal specific heat measurements that show superconductivity in double-wall carbon nanotube (DWCNT) bundles. Clear evidence, comprising a resistance drop as a function of temperature, magnetoresistance and differential resistance signature of the supercurrent, suggest an intrinsic superconducting transition below 6.8 K for one particular sample. Additional electrical data not only confirm the existence of superconductivity, but also indicate the T(c) distribution that can arise from the diversity in the diameter and chirality of the DWCNTs. A broad superconducting anomaly is observed in the specific heat of a bulk DWCNT sample, which yields a T(c) distribution that correlates well with the range of the distribution obtained from the electrical data. As quasi one dimensionality of the DWCNTs dictates the existence of electronic density of state peaks, confirmation of superconductivity in this material system opens the exciting possibility of tuning the T(c) through the application of a gate voltage.

Applications of Microfluidics in Stem Cell Biology.

Stem cell research can significantly benefit from recent advances of microfluidics technology. In a rationally designed microfluidics device, analyses of stem cells can be done in a much deeper and wider way than in a conventional tissue culture dish. Miniaturization makes analyses operated in a high-throughput fashion, while controls of fluids help to reconstruct the physiological environments. Through integration with present characterization tools like fluorescent microscope, microfluidics offers a systematic way to study the decision-making process of stem cells, which has attractive medical applications. In this paper, recent progress of microfluidics devices on stem cell research are discussed. The purpose of this review is to highlight some key features of microfluidics for stem cell biologists, as well as provide physicists/engineers an overview of how microfluidics has been and could be used for stem cell research.

The Goldilocks principle and antibiotic resistance in bacteria.

We have designed and fabricated a microecology to mimic a naturally occurring bacterial culture, which includes the stress gradient, metapopulation, and cellular motility. In this microecology, we show that it is possible to fix the resistance to the mutagenic antibiotic Ciprofloxacin in wild-type Escherichia coli within 10 h. We found the evolution of resistance is further accelerated in microecology if bacteria have already acquired the phenotype of growth advantage at the stationary phase (GASP).

Acceleration of emergence of bacterial antibiotic resistance in connected microenvironments.

The emergence of bacterial antibiotic resistance is a growing problem, yet the variables that influence the rate of emergence of resistance are not well understood. In a microfluidic device designed to mimic naturally occurring bacterial niches, resistance of Escherichia coli to the antibiotic ciprofloxacin developed within 10 hours. Resistance emerged with as few as 100 bacteria in the initial inoculation. Whole-genome sequencing of the resistant organisms revealed that four functional single-nucleotide polymorphisms attained fixation. Knowledge about the rapid emergence of antibiotic resistance in the heterogeneous conditions within the mammalian body may be helpful in understanding the emergence of drug resistance during cancer chemotherapy.

Superconducting characteristics of 4-A carbon nanotube-zeolite composite.

We have fabricated nanocomposites consisting of 4-A carbon nanotubes embedded in the 0.7-nm pores of aluminophosphate-five (AFI) zeolite that display a superconducting specific heat transition at 15 K. MicroRaman spectra of the samples show strong and spatially uniform radial breathing mode (RBM) signals at 510 cm(-1) and 550 cm(-1), characteristic of the (4, 2) and (5, 0) nanotubes, respectively. The specific heat transition is suppressed at >2 T, with a temperature dependence characteristic of finite-size effects. Comparison with theory shows the behavior to be consistent with that of a type II BCS superconductor, characterized by a coherence length of 14 +/- 2 nm and a magnetic penetration length of 1.5 +/- 0.7 mum. Four probe and differential resistance measurements have also indicated a superconducting transition initiating at 15 K, but the magnetoresistance data indicate the superconducting network to be inhomogeneous, with a component being susceptible to magnetic fields below 3 T and other parts capable of withstanding a magnetic field of 5 T or beyond.