Purification and Characterization of Nickel Uptake Regulator (NUR) and Single NUR Mutants from \u3cem\u3eStreptomyces coelicolor\u3c/em\u3e

The Nickel Uptake Regulator (NUR) is a metalloregulatory protein found in the microorganism Streptomyces coelicolor. S.coelicolor is a gram-positive bacterium that plays an important role in antibiotic production. NUR is responsible for the uptake of nickel and the regulation of the enzyme Superoxide Dismutase (SOD) within S.coelicolor. NUR regulates Fe-and Ni-SODs through direct and indirect transcription processes, respectively. The goal of this research is to purify and characterize the metal and DNA binding affinities in Wild Type (WT) NUR. There are two metal binding sites within NUR that is believed to contribute to the function of this protein. These sites are the M-site and the Ni-site; both sites are highlighted and enlarged in Figure 1. Our focus in characterizing this protein is to understand the distinct roles each specific metal binding site plays in NUR’s functionality. Using optimized purification strategies, we are able to successfully purify protein that is characterized by quantification of original metal content bound to NUR, metal binding affinity, and DNA binding affinity. One experiment, represented in Figure 2 below, shows a titration of NUR and DNA into a solution of a known metal chelator, Fura-2. This experiment presents evidence of our hypothesis that one M-site has a lesser affinity than the other for metal, and in order to remove the other metal, DNA must be bound to NUR. Other experiments will be performed to reproduce this data and give more information to help support this hypothesis

Cloning and characterization of nickel uptake regulator NUR mutants from \u3cem\u3eStreptomyces coelicolor\u3c/em\u3e

Sufficient concentrations of metal within a cell are required for proper cellular function; however, metals become toxic at very high concentrations. Therefore, it is important for an organism to have a mechanism for maintaining metal homeostasis within its cells. Streptomyces coelicolor, a soil-dwelling bacterium important in the production of antibiotics, utilizes the nickel uptake regulator (NUR) to maintain nickel homeostasis and oxidative response. NUR functions as a transcriptional repressor that responds to changing cytosolic concentrations of Ni2+. Previous research describes two key metal-binding sites per NUR monomer. Our research seeks to analyze how changes to these binding sites affect the ability of NUR to coordinate metals and bind to DNA. NUR mutants containing single or multiple amino acid substitutions at each binding site were cloned. Biophysical characterization of these mutants and the wild-type protein will aid in understanding the role each metal site plays in the function of NUR

Controlling Oct4 Expression Levels Using Invitrogen’s GeneSwitch™ System

Oct4 is a protein that is involved in the retention of pluripotency in adipose derived stem cells (ADSCs). Despite this knowledge, Oct4’s exact role in the complex system used in maintaining pluripotency is not known. One approach to explore Oct4’s role would be through the use of cellular assays to control the expression of Oct4. This can possibly be accomplished by introducing a biological switch and the gene of interest into ADSCs. In this project, the GeneSwitch™ System is used to ultimately induce Oct4 expression. Before the GeneSwitch™ System can be used, the Oct4 gene is extracted from murine embryonic stem cell (ES) RNA. This ES RNA is then used as a template to create complimentary DNA (cDNA) that can then be used to create an insert with the Oct4 gene. In addition to the cDNA, recognition sites for endonucleases must be added on to fully create the Oct4 insert. This insert could then be placed into one of the GeneSwitch™ System plasmids that have the same recognition sites and placed into ADSCs along with the plasmid that will act as a biological switch. With this system put into ADSCs, it is expected that Oct4 levels will be successfully controlled. Once controlled, Oct4 expression can be tested and investigations can be completed to determine how Oct4 expression levels influence pluripotency of ADSCs. This may have significant impact on the creation of regenerative medicine

Impact of Cuff Over Inflation on Blood Pressure Readings in Adults

Purpose Physical therapists regularly use blood pressure (BP) readings when making clinical decisions. Inaccuracies in BP readings may occur when not following standard procedure. False or missed diagnoses of hypertension (HTN) may lead to improper medical management. Objective To determine the effects of cuff over inflation on BP readings compared to the standardized 20 mmHg above the loss of Korotkoff sounds when taking manual BP in adults. Participants 109 adult participants recruited at the MN State Fair on August 28, 2019. Methods Data Collection Utilized American Heart Association (AHA) standardized positioning and procedures Baseline BP measurement taken using standard cuff inflation of 20 mmHg above loss of systolic Korotkoff sounds BP measured three subsequent times inflating the cuff to 40, 60, 80 mmHg above the lost of Korotkoff sounds in randomized order Data Analysis Friedman\u27s ANOVA performed to analyze differences in BP measurements Point biserial correlation performed to determine effects of demographics on change in BP measurements Results Significant difference found in systolic readings between standardized measurement and cuff inflation level of greater than or equal to 60 mmHg above loss of Korotkoff sounds Significant correlation found between body mass index (BMI) and BP change Conclusion Over inflating the blood pressure cuff created statistically significant differences in BP readings as compared to standard procedures Higher cuff inflation levels impact accuracy of clinical decisions Participants with higher BMI have a greater incidence of inaccurate readings with increasing cuff inflation level Clinical Relevance Over inflating the blood pressure cuff when manually taking an adult\u27s blood pressure may lead to significantly elevated systolic readings. Cuff inflation level for manual blood pressure assessment needs to be standardized. The determination of care being delivered can be significantly affected when standard procedures are not followed

A minimalist chemical model of matrix metalloproteinases- Can small peptides mimic the more rigid metal binding sites of proteins?

In order to develop a minimalist chemical model of matrix metalloproteinases (MMPs), we synthesized a pentadecapeptide (Ac-KAHEFGHSLGLDHSK-NH2) corresponding to the catalytic zinc(II) binding site of human MMP-13. The multi-domain structural organization of MMPs fundamentally determines their metal binding affinity, catalytic activity and selectivity. Our potentiometric, UV-VIS, CD, EPR, NMR, ESI-MS and kinetic study are aimed to explore the usefulness of flexible peptides to mimic the more rigid metal binding sites of proteins, to examine the intrinsic metal binding properties of this naked sequence, as well as to contribute the development of a minimalist, peptide-based chemical model of MMPs, including the catalytic properties. Since multiimidazole environment is also characteristic for copper(II), and recently copper(II) containing variants of MMPs have been identified, we also studied the copper(II) complexes of the above peptide. Around pH 6-7 the peptide, similarly to MMPs, offers {3Nim} coordinated binding site for both zinc(II) and copper(II). In the case of copper(II), the formation of amide coordinated species at higher pH ceased the analogy with the copper(II) containing MMP variant. On the other hand, the zinc(II)-peptide system mimics some basic features of the MMP active sites: the main species around pH 7 (ZnH2L) possesses {3Nim,H2O} coordination environment, the deprotonation of the zinc-bound water takes place near to the physiological pH, it forms relatively stable ternary complexes with hydroxamic acids, and the species ZnH2L(OH) and ZnH2L(OH)2 have notable hydrolytic activity between pH 7-9

Dyadic adjustment and spiritual activities in parents of children with cystic fibrosis

Children's diseases can negatively impact marital adjustment and contribute to poorer child health outcomes. To cope with increased marital stress and childhood diseases severity, many people turn to spirituality. While most studies show a positive relationship between spirituality and marital adjustment, spirituality has typically been measured only in terms of individual behaviors. Using the Dyadic Adjustment Scale (DAS) and Daily Phone Diary data from a sample of 126 parents of children with cystic fibrosis as a context for increased marital stress, spiritual behavior of mother-father dyads and of whole families were used as predictors of marital adjustment. Frequency and duration of individual, dyadic and familial spiritual activities correlated positively with dyadic adjustment. Significant differences in spiritual activities existed between couples with marital adjustment scores above and below the cutoff for distress. The only significant factors in regressions of spiritual activities on marital adjustment scores were number of pulmonary exacerbations and parent age. Higher odds of maintaining a marital adjustment score greater than 100 were significantly associated with spending approximately twelve minutes per day in individual, but not conjugal or familial, spiritual activities. The Daily Phone Diary is a feasible tool to study conjugal and familial activities and their relationships with beliefs and attitudes, including spirituality

International Study of Chaplains’ Attitudes About Research

An online survey was conducted by twelve professional chaplain organizations to assess chaplains’ attitudes about and involvement in research. A total of 2,092 chaplains from 23 countries responded to the survey. Over 80% thought research was definitely important and nearly 70% thought chaplains should definitely be research literate. Just over 40% said they regularly read research articles and almost 60% said they occasionally did. The respondents rated their own research literacy as 6.5 on a 0–10 scale. Significant positive inter-correlations were found among all four measures: importance of (a) research and (b) research literacy; (c) frequency of reading articles; and (d) research literacy rating. Approximately 35% were never involved, 37% had been involved, 17% were currently involved, and 11% expected to be involved in research. The last three groups were significantly more likely to think research and research literacy were important and to read research articles than chaplains who were never involved in research. Given chaplains’ interest in research, actions should be undertaken to facilitate further research engagement

Is This Relevant? Physician Perceptions, Clinical Relevance, and Religious Content in Clinical Interactions

Despite wide support among physicians for practicing patient-centered care, clinical interactions are primarily driven by physicians’ perception of relevance. While some will perceive a connection between religion and patient health, this relevance will be less apparent for others. I argue that physician responses when religious/spiritual topics come up during clinical interactions will depend on their own religious/spiritual background. The more central religion is for the physician, the greater his or her perception of religion\u27s impact on health outcomes and his or her inclusion of religion/spirituality within clinical interactions. Using a nationally representative sample of physicians in the United States and mediated path models, I estimate models for five different physician actions to evaluate these relationships. I find that a physician\u27s religious background is strongly associated with whether or not he or she thinks religion impacts health outcomes, which is strongly predictive of inclusion. I also find that not all of the association between inclusion and physicians’ religious background is mediated by thinking religion impacts health outcomes. Issues of religion\u27s relevance for medicine are important to the degree that religious beliefs are an important dimension of patients’ lives

Calorimetric Investigation of Copper Binding in the N-Terminal Region of the Prion Protein at Low Copper Loading: Evidence for an Entropically Favorable First Binding Event

Although the Cu²⁺-binding sites of the prion protein have been well studied when the protein is fully saturated by Cu²⁺, the Cu²⁺-loading mechanism is just beginning to come into view. Because the Cu²⁺-binding modes at low and intermediate Cu²⁺ occupancy necessarily represent the highest-affinity binding modes, these are very likely populated under physiological conditions, and it is thus essential to characterize them in order to understand better the biological function of copper–prion interactions. Besides binding-affinity data, almost no other thermodynamic parameters (e.g., Δ<i>H</i> and Δ<i>S</i>) have been measured, thus leaving undetermined the enthalpic and entropic factors that govern the free energy of Cu²⁺ binding to the prion protein. In this study, isothermal titration calorimetry (ITC) was used to quantify the thermodynamic parameters (<i>K</i>, Δ<i>G</i>, Δ<i>H</i>, and <i>T</i>Δ<i>S</i>) of Cu²⁺ binding to a peptide, PrP­(23–28, 57–98), that encompasses the majority of the residues implicated in Cu²⁺ binding by full-length PrP. Use of the buffer <i>N</i>-(2-acetomido)-aminoethanesulfonic acid (ACES), which is also a well-characterized Cu²⁺ chelator, allowed for the isolation of the two highest affinity binding events. Circular dichroism spectroscopy was used to characterize the different binding modes as a function of added Cu²⁺. The <i>K</i>_d values determined by ITC, 7 and 380 nM, are well in line with those reported by others. The first binding event benefits significantly from a positive entropy, whereas the second binding event is enthalpically driven. The thermodynamic values associated with Cu²⁺ binding by the Aβ peptide, which is implicated in Alzheimer’s disease, bear striking parallels to those found here for the prion protein