Chih-kuan Tung

Acceleration of Emergence of Bacterial Antibiotic Resistance in Connected Microenvironments

Gradients of antibiotics generated in a microfluidic device provoke selection of ciprofloxacin resistance in Escherichia coli. 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.

Upconverting nanophosphors for bioimaging.

Upconverting nanoparticles (UCNPs) when excited in the near-infrared (NIR) region display anti-Stokes emission whereby the emitted photon is higher in energy than the excitation energy. The material system achieves that by converting two or more infrared photons into visible photons. The use of the infrared confers benefits to bioimaging because of its deeper penetrating power in biological tissues and the lack of autofluorescence. We demonstrate here sub-10 nm, upconverting rare earth oxide UCNPs synthesized by a combustion method that can be stably suspended in water when amine modified. The amine modified UCNPs show specific surface immobilization onto patterned gold surfaces. Finally, the low toxicity of the UCNPs is verified by testing on the multi-cellular C. elegans nematode.

Microgrooves and fluid flows provide preferential passageways for sperm over pathogen Tritrichomonas foetus

Significance Mammalian females must selectively allow sperm with normal morphology and motility to ascend the reproductive tract while rejecting invasion of pathogens. By using microfluidic modeling, we revealed that microgrooves and gentle fluid flows, two major biophysical characteristics of the female tract, synergistically provide preferential pathways for sperm. In contrast, the sexually transmitted pathogen, Tritrichomonas foetus, is swept away from these pathways. These findings are not only valuable to dairy and beef industries for maximizing fertility and suppressing disease, but also to human medicine, because the morphology and movement of bull sperm closely resemble those of human sperm, and T. foetus closely resembles Trichomonas vaginalis, which infects 170 million people annually worldwide. Successful mammalian reproduction requires that sperm migrate through a long and convoluted female reproductive tract before reaching oocytes. For many years, fertility studies have focused on biochemical and physiological requirements of sperm. Here we show that the biophysical environment of the female reproductive tract critically guides sperm migration, while at the same time preventing the invasion of sexually transmitted pathogens. Using a microfluidic model, we demonstrate that a gentle fluid flow and microgrooves, typically found in the female reproductive tract, synergistically facilitate bull sperm migration toward the site of fertilization. In contrast, a flagellated sexually transmitted bovine pathogen, Tritrichomonas foetus, is swept downstream under the same conditions. We attribute the differential ability of sperm and T. foetus to swim against flow to the distinct motility types of sperm and T. foetus; specifically, sperm swim using a posterior flagellum and are near-surface swimmers, whereas T. foetus swims primarily via three anterior flagella and demonstrates much lower attraction to surfaces. This work highlights the importance of biophysical cues within the female reproductive tract in the reproductive process and provides insight into coevolution of males and females to promote fertilization while suppressing infection. Furthermore, the results provide previously unidentified directions for the development of in vitro fertilization devices and contraceptives.

Fluid viscoelasticity promotes collective swimming of sperm

From flocking birds to swarming insects, interactions of organisms large and small lead to the emergence of collective dynamics. Here, we report striking collective swimming of bovine sperm in dynamic clusters, enabled by the viscoelasticity of the fluid. Sperm oriented in the same direction within each cluster, and cluster size and cell-cell alignment strength increased with viscoelasticity of the fluid. In contrast, sperm swam randomly and individually in Newtonian (nonelastic) fluids of low and high viscosity. Analysis of the fluid motion surrounding individual swimming sperm indicated that sperm-fluid interaction was facilitated by the elastic component of the fluid. In humans, as well as cattle, sperm are naturally deposited at the entrance to the cervix and must swim through viscoelastic cervical mucus and other mucoid secretions to reach the site of fertilization. Collective swimming induced by elasticity may thus facilitate sperm migration and contribute to successful fertilization. We note that almost all biological fluids (e.g. mucus and blood) are viscoelastic in nature, and this finding highlights the importance of fluid elasticity in biological function.

Dynamics of Bovine Sperm Interaction with Epithelium Differ Between Oviductal Isthmus and Ampulla

ABSTRACT In mammals, many sperm that reach the oviduct are held in a reservoir by binding to epithelium. To leave the reservoir, sperm detach from the epithelium; however, they may bind and detach again as they ascend into the ampulla toward oocytes. In order to elucidate the nature of binding interactions along the oviduct, we compared the effects of bursts of strong fluid flow (as would be caused by oviductal contractions), heparin, and hyperactivation on detachment of bovine sperm bound in vitro to epithelium on intact folds of isthmic and ampullar mucosa. Intact folds of oviductal mucosa were used to represent the strong attachments of epithelial cells to each other and to underlying connective tissue that exist in vivo. Effects of heparin on binding were tested because heparin binds to the Binder of SPerm (BSP) proteins that attach sperm to oviductal epithelium. Sperm bound by their heads to beating cilia on both isthmic and ampullar epithelia and could not be detached by strong bursts of fluid flow. Addition of heparin immediately detached sperm from isthmic epithelium but not ampullar epithelium. Addition of 4-aminopyridine immediately stimulated hyperactivation of sperm but did not detach them from isthmic or ampullar epithelium unless added with heparin. These observations indicate that the nature of binding of sperm to ampullar epithelium differs from that of binding to isthmic epithelium; specifically, sperm bound to isthmic epithelium can be detached by heparin alone, while sperm bound to ampullar epithelium requires both heparin and hyperactivation to detach from the epithelium.

Nanochannels for Genomic DNA Analysis: The Long and the Short of It

This review will discuss the theory of confined polymers in nanochannels and present our experiments, which test the theory and explore use of nanochannels for genomic analysis. Genomic length DNA molecules contained in nanochannels, which are less than one persistence length in diameter, are highly elongated. Thus, nanochannels can be used to analyze genomic length DNA molecules with very high linear spatial resolution. Also, nanochannels can be used to study the position and dynamics of proteins such as transcription factors that bind to DNA with high specificity. In order to realize these goals not only must nanochannels be constructed whose radius is less than the persistence length of DNA, but it is also necessary to understand the dynamics of polymers within nanochannels and develop experimental tools to study the dynamics of polymers in such confined volumes, tools which we review here.

SENSING DNA WITH ALTERNATING CURRENTS USING A NANOGAP SENSOR EMBEDDED IN A NANOCHANNEL DEVICE

We report an integrated nanochannel/nanoelectrode sensor for the detection of DNA using alternating currents. We find that DNA can be detected using platinum as the metal for the detecting electrodes, with a signal to noise ratio exceeding 10. We argue that the signal is at least in part electrochemical in nature, thus holds the promise to yield a sequence-dependent signal. However, we also find that for large voltages, DNA attaches irreversibly to the driving electrodes.

Effects of index-mismatch-induced spherical aberration on two-photon imaging in skin and tissue-like constructs

Index mismatch induced spherical aberration is studied by comparing imaging ability of different immersion objectives in both uniformly fluorescent solutions and fluorescently labeled skin samples using scanning two-photon fluorescence microscopy. We investigated the performances of the objectives (air, water, glycerin, and oil immersion) by measuring the fluorescence profiles at different depths. In homogeneous fluorescent samples, we found that immersion medium with compatible refractive index as the samples yields better results, and small differences in refractive indices did not cause noticeable effects. Similar results were found in skin samples. Except for the air objective, we found that the choice of immersion medium did not have significant effects for in-depth imaging in the fluorescent solutions or skin samples.

Use of sub-10 nm Diameter Upconversion Nanophosphors as Bio-labels

We have synthesized rare-earth doped sub-10 nm diameter upconverting yttrium oxide based nanophosphors by flame spray pyrolysis. We have investigated the emitted visible fluorescence of the sub-10nm nanophosphors under both infrared excitation and electron excitation, and observed comparable narrow band emission spectra. The viability of the nanoparticles for biological imaging was confirmed by imaging the digestive system of the nematode worm C. elegans in the upconversion mode. We have surface functionalized the nanophosphors making them suitable for bio labeling.