Cellular metal ion sensing using DNAzymes

Metal ions are important elements in biology and are involved in numerous reactions essential for maintaining life on earth. Most commonly, metal ions confer their role as structural elements or catalytic cofactors within metalloproteins. Metalloproteins comprise more than half of all known proteins and are at the heart of several important biological processes including photosynthesis, respiration, and nitrogen fixation. Another equally important role of metal ions is as signaling molecules. It has been shown that changes in concentrations of calcium, zinc, or copper can trigger downstream signaling effect in neurons. Despite these important functions, high levels of many metal ions, especially transition metal ions, are known to be toxic to cells. Thus, cells have adapted strategies to finely regulate the uptake, storage, and distribution of metal ions within different compartments. The importance of these mechanisms for maintaining metal ion homeostasis within cells and the key role of metal ions in life have been of major interest for years. However, the understanding of these mechanisms and how metal ions play their role is largely unknown in many cases. An important step towards advancing our understanding of metal ions is developing the ability to measure their concentrations in different cellular compartments with accuracy and sensitivity. To achieve this goal, several techniques are available that have greatly advanced our understanding of metal ions. However, current methods suffer from a number of limitations that have slowed progress. Analytical tools such as ICP-MS and AAS, while effective at measuring the cocnentrations of metal ions very sensitively, usually work in bulk and thus are not easily amenable to single cell studies let alone the distribution in different cellular compartments. Moreover, these techniques are often unable to distinguish between different oxidation states of a metal ion or between bound and mobile forms. Techniques based on X-ray absorption, such as X-ray fluorescence microscopy (XFM) can simultaneously detect multiple metal ions and can distinguish between different oxidation states. However, these techniques require the use of highly focused X-ray beams limiting more widespread availability of such techniques. Furthermore, biological samples cannot be examined in a real time manner, and the obtained distribution of metal ions represents only total metal content, without regard to whether the metals are easily exchangeable or are tightly bound, a major factor determining biological activity. To overcome these issues several metal ion sensors have been designed based on either small molecules or proteins with great sensitivities of detection. The use of such sensors has provided great insight into the functions of metal ions. However, as these sensors are usually rationally designed through a trial and error process, it is challenging to generalize the successful designs to sense other metal ions or to meet new desired criteria. DNAzymes, DNA sequences with catalytic activity, have recently emerged as an alternative class of sensors for metal ions. As selection of DNAzymes for different metal ions is carried out through a combinatorial process, new sequences with desired activity and selectivity can be easily obtained by changing the selection conditions. Many DNAzymes have been selected with high sensitivity for different metal ions including zinc, copper, lead, and uranyl. Surprisingly, despite the promise of using DNAzymes as sensors for cellular metal ions, almost all applications of DNAzyme-based sensors until now have been in environmental metal ion detection and DNAzyme-based sensors for cellular metal detection have only very recently been explored as an option. The goal of my PhD research was to establish novel strategies to make DNAzymes viable sensors for cellular metal ion detection. Chapter 1 briefly describes the background and overall aims of my research. In chapter 2, I describe my efforts in designing "caged" DNAzyme-based sensors that can be activitated by light when they reach the desired cellular compartments. This method reduces the off-target signals and as demonstrated in this chapter is highly generalizable to other DNAzyme-based sensors. In chapter 3, I explain my efforts in designing a ratiometric DNAzyme-based sensor to enable quantitative measurement within the cells. Finally, chapter 4 summarizes my efforts in enhancing the caging strategy to enable better stability, faster decaging, or the use of long-wavelength light for decaging by using lanthanide-doped metal nanoparticles

Comparing clinician knowledge and online information regarding Alli (Orlistat)

BACKGROUND: Many consumers join online communities focused on health. Online forums are a popular medium for the exchange of health information between consumers, so it is important to determine the accuracy and completeness of information posted to online forums. OBJECTIVE: We compared the accuracy and completeness of information regarding the FDA-approved over-the counter weight-loss drug Alli (Orlistat) from forums and from clinicians. METHODS: We identified Alli-related questions posted on online forums and then posed the questions to 11 primary care providers. We then compared the clinicians\u27 answers to the answers given on the forums. A panel of blinded experts evaluated the accuracy and completeness of the answers on a scale of 0-4. Another panel of blinded experts categorized questions as being best answered based on clinical experience versus review of the literature. RESULTS: The accuracy and completeness of clinician responses was slightly better than forum responses, but there was no significant difference (2.3 vs. 2.1, p=0.5). Only one forum answer contained information that could potentially cause harm if the advice was followed. CONCLUSIONS: Forum answers were comparable to clinicians\u27 answers with respect to accuracy and completeness, but answers from both sources were unsatisfactory

Rebuilding CHO again and again: Development of a species agnostic modular cell line development platform for cultivated meat

Please click Additional Files below to see the full abstract

Adsorbate structures and catalytic reactions studied in the torrpressure range by scanning tunneling microscopy

High-pressure, high-temperature scanning tunneling microscopy (HPHTSTM) was used to study adsorbate structures and reactions on single crystal model catalytic systems. Studies of the automobile catalytic converter reaction [CO + NO {yields} 1/2 N{sub 2} + CO{sub 2}] on Rh(111) and ethylene hydrogenation [C{sub 2}H{sub 4} + H{sub 2} {yields} C{sub 2}H{sub 6}] on Rh(111) and Pt(111) elucidated information on adsorbate structures in equilibrium with high-pressure gas and the relationship of atomic and molecular mobility to chemistry. STM studies of NO on Rh(111) showed that adsorbed NO forms two high-pressure structures, with the phase transformation from the (2 x 2) structure to the (3 x 3) structure occurring at 0.03 Torr. The (3 x 3) structure only exists when the surface is in equilibrium with the gas phase. The heat of adsorption of this new structure was determined by measuring the pressures and temperatures at which both (2 x 2) and (3 x 3) structures coexisted. The energy barrier between the two structures was calculated by observing the time necessary for the phase transformation to take place. High-pressure STM studies of the coadsorption of CO and NO on Rh(111) showed that CO and NO form a mixed (2 x 2) structure at low NO partial pressures. By comparing surface and gas compositions, the adsorption energy difference between topsite CO and NO was calculated. Occasionally there is exchange between top-site CO and NO, for which we have described a mechanism for. At high NO partial pressures, NO segregates into islands, where the phase transformation to the (3 x 3) structure occurs. The reaction of CO and NO on Rh(111) was monitored by mass spectrometry (MS) and HPHTSTM. From MS studies the apparent activation energy of the catalytic converter reaction was calculated and compared to theory. STM showed that under high-temperature reaction conditions, surface metal atoms become mobile. Ethylene hydrogenation and its poisoning by CO was also studied by STM on Rh(111) and Pt(111). Poisoning was found to coincide with decreased adsorbate mobility. Under ethylene hydrogenation conditions, no order is detected by STM at 300 K, as hydrogen and ethylidyne, the surface species formed by gas-phase ethylene, are too mobile. When CO is introduced, the reaction stops, and ordered structures appear on the surface. For Rh(111), the structure is predominantly a mixed c(4 x 2), though there are some areas of (2 x 2). For Pt(111), the structure is hexagonal and resembles the Moire pattern seen when Pt(111) is exposed to pure CO. From these studies it is concluded that CO poisons by stopping adsorbate mobility. This lack of adsorbate mobility prevents the adsorption of ethylene from the gas phase by hindering the creation of adsorption sites