Implanted gradient index lenses have extended the reach of standard multiphoton microscopy from t... more Implanted gradient index lenses have extended the reach of standard multiphoton microscopy from the upper layers of the mouse cortex to the lower cortical layers and even subcortical regions. These lenses have the clarity to visualize dynamic activities, such as calcium transients, with subcellular and millisecond resolution and the stability to facilitate repeated imaging over weeks and months. In addition, behavioral tests can be used to correlate performance with observed changes in network function and structure that occur over time. Yet, this raises the questions, does an implanted microlens have an effect on behavioral tests, and if so, what is the extent of the effect? To answer these questions, we compared the performance of three groups of mice in three common behavioral tests. A gradient index lens was implanted in the prefrontal cortex of experimental mice. We compared their performance with mice that had either a cranial window or a sham surgery. Three presurgical and five postsurgical sets of behavioral tests were performed over seven weeks. Behavioral tests included rotarod, foot fault, and Morris water maze. No significant differences were found between the three groups, suggesting that microlens implantation did not affect performance. The results for the current study clear the way for combining behavioral studies with gradient index lens imaging in the prefrontal cortex, and potentially other regions of the mouse brain, to study structural, functional, and behavioral relationships in the brain.
We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of ␣... more We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of ␣7 and 2 subunits. We measured optical and electrophysiological properties of wildtype and mutant subunits expressed in cell lines and Xenopus laevis oocytes. Laser scanning confocal microscopy indicated that fluorescently tagged ␣7 and 2 subunits colocalize. Fö rster resonance energy transfer between fluorescently tagged subunits strongly suggested that ␣7 and 2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function ␣7 and 2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, ␣72 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of ␣7 nAChRs, although amplitudes of ␣72 nAChRmediated, agonist-evoked currents were generally ϳ2-fold lower than those for ␣7 nAChRs. It is noteworthy that ␣72 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro--erythroidine that was not observed for ␣7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the ␣7-2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower ␣72 nAChR current amplitudes and challenges in identifying the function of native ␣72 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of 2 subunits within the ␣72 nAChRs.
... the author. TAM Author is a PhD candidate in Bioengineering at Arizona State University (ASU)... more ... the author. TAM Author is a PhD candidate in Bioengineering at Arizona State University (ASU) and affiliated with the Biodesign Instituteat ASU, Tempe, AZ 85287-5201 USA (e-mail: teresa.murray@ asu.edu.). hippocampus ...
The influence of the protein environment on the primary electron donor, P, a bacteriochlorophyll ... more The influence of the protein environment on the primary electron donor, P, a bacteriochlorophyll a dimer, of reaction centers from Rhodobacter sphaeroides, has been investigated using electron paramagnetic resonance and electron nuclear double resonance spectroscopy. These techniques were used to probe the effects on P that are due to alteration of three amino acid residues, His L168, Asn L170, and Asn M199. The introduction of Glu at L168, Asp at L170, or Asp at M199 changes the oxidation/ reduction midpoint potential of P in a pH-dependent manner (Williams et al. (2001) Biochemistry 40, 15403-15407). For the double mutant His L168 to Glu and Asn at L170 to Asp, excitation results in electron transfer along the Aside branch of cofactors at pH 7.2, but at pH 9.5, a long-lived state involving B-side cofactors is produced (Haffa et al. (2004) J Phys Chem B 108, 4-7). Using electron paramagnetic resonance spectroscopy, the mutants with alterations of each of the three individual residues and a double mutant, with changes at L168 and L170, were found to have increased linewidths of 10.1-11.0 G compared to the linewidth of 9.6 G for wild type. The Special TRIPLE spectra were pH dependent, and at pH 8, the introduction of aspartate at L170 increased the spin density ratio, q L /q M , to 6.1 while an aspartate at the symmetry related position, M199, decreased the ratio to 0.7 compared to the value of 2.1 for wild type. These results indicate that the energy of the two halves of P changes by about 100 meV due to the mutations and are consistent with the interpretation that electrostatic interactions involving these amino acid residues contribute to the switch in pathway of electron transfer. Keywords Reaction centers Á Purple bacteria Á Magnetic resonance Á Bacteriochlorophyll Á Oxidized bacteriochlorophyll dimer Á Electron paramagnetic resonance Abbreviations EPR Electron paramagnetic resonance ENDOR Electron nuclear double resonance TRIPLE Electron-nuclear-nuclear triple resonance Rb.
Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent pro... more Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR α7 subunit without compromising formation of functional receptors. Methods: A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR α7 subunit (α7Y). SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits (α7) or with α7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125 I-labeled α-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy. Results: Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC 50 values for the α7 nAChR-selective agonist, choline, and IC 50 values for the α7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs. Conclusion: nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs. α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.
Hippocampal gamma oscillations, as a form of neuronal network synchronization, are speculated to ... more Hippocampal gamma oscillations, as a form of neuronal network synchronization, are speculated to be associated with learning, memory and attention. Nicotinic acetylcholine receptor alpha7 subtypes (alpha7-nAChRs) are highly expressed in hippocampal neurons and play important roles in modulating neuronal function, synaptic plasticity, learning and memory. However, little is known about the role of alpha7-nAChRs in hippocampal gamma oscillations. Here, we examined the effects of selective alpha7- and non-alpha7-nAChR antagonists on tetanic gamma oscillations in rat hippocampal slices. We found that brief tetanic stimulation-induced gamma oscillations (30-80 Hz) and pharmacological blockade of alpha7-nAChRs using the relatively selective alpha7-nAChR antagonists, methyllycaconitine (10 or 100 nM) or alpha-bungarotoxin (10 nM), significantly reduced the frequency spectrum power, the number of spikes, and burst duration of evoked gamma oscillations. Neither mecamylamine nor dihydro-beta-erythroidine, which are selective antagonists of non-alpha7-nAChRs, demonstrated significant effects on tetanic gamma oscillations. Nicotine exposure promotes hippocampal gamma oscillations in a methyllycaconitine-sensitive manner. It is concluded that alpha7-nAChRs in hippocampal slices play important roles in regulation of gamma oscillations, thus potentially helping to explain roles of nAChRs in cognitive functions such as learning, memory and attention.
The main purpose of this paper is to share the Mentoring for INnovative Design Solutions (MINDS) ... more The main purpose of this paper is to share the Mentoring for INnovative Design Solutions (MINDS) Scholars Program developed by Alpha Eta Mu Beta, the International Biomedical Engineering Honor Society. The program’s goals are to (1) introduce biomedical engineering students to an open-ended design experience as part of interuniversity teams with industry and faculty mentors, and (2) develop the ability to create designs considering clinical translatability on teams with different backgrounds and areas of expertise. MINDS uses an experiential learning approach to (1) enrich student curricular experiences through inter-institutional collaboration, (2) build engineering design skills, including three key design considerations for clinical/commercial success: intellectual property protection, regulatory strategy, and market identification; and (3) emphasize the importance of end-user considerations. From 2015 to 2022, MINDS has involved 131 students from 50 universities and 22 faculty a...
Imbalances in levels of glutamate (GLU) and gamma-aminobutyric acid (GABA) and their sub-second s... more Imbalances in levels of glutamate (GLU) and gamma-aminobutyric acid (GABA) and their sub-second signaling dynamics occur in several brain disorders including traumatic brain injury, epilepsy, and Alzheimer’s disease. The present work reports on the optimization and in vivo testing of a silicon (Si) multifunctional biosensor probe for sub-second simultaneous real-time detection of GLU and GABA. The Si probe features four surface-functionalized platinum ultramicroelectrodes (UMEs) for detection of GLU and GABA, a sentinel site, and integrated microfluidics for in-situ calibration. Optimal enzyme concentrations, size-exclusion phenylenediamine layer and micro spotting conditions were systematically investigated. The measured GLU sensitivity for the GLU and GABA sites were as high as 219 ± 8 nA μM−1 cm−2 (n = 3). The measured GABA sensitivity was as high as 10 ± 1 nA μM−1 cm−2 (n = 3). Baseline recordings (n = 18) in live rats demonstrated a useful probe life of at least 11 days with GL...
High resolution, in vivo optical imaging of the mouse brain over time often requires anesthesia, ... more High resolution, in vivo optical imaging of the mouse brain over time often requires anesthesia, which necessitates maintaining the animal's body temperature and level of anesthesia, as well as securing the head in an optimal, stable position. Controlling each parameter usually requires using multiple systems. Assembling multiple components into the small space on a standard microscope stage can be difficult and some commercially available parts simply do not fit. Furthermore, it is time-consuming to position an animal in the identical position over multiple imaging sessions for longitudinal studies. This is especially true when using an implanted gradient index (GRIN) lens for deep brain imaging. The multiphoton laser beam must be parallel with the shaft of the lens because even a slight tilt of the lens can degrade image quality. In response to these challenges, we have designed a compact, integrated in vivo imaging support system to overcome the problems created by using sepa...
The quarterly Women in Engineering and Science Luncheons provide focused professional development... more The quarterly Women in Engineering and Science Luncheons provide focused professional development for women faculty and graduate students, while also providing a platform for peer-to-peer and student-to-faculty networking. This session will discuss the structure of the luncheons, the professional development training, the interactive activities, networking, and present assessment findings.
Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter that is essential for norma... more Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter that is essential for normal brain function. It is involved in multiple neuronal activities, including plasticity, information processing, and network synchronization. Abnormal GABA levels result in severe brain disorders and therefore GABA has been the target of a wide range of drug therapeutics. GABA being non-electroactive is challenging to detect in real-time. To date, GABA is detected mainly via microdialysis with a high-performance liquid chromatography (HPLC) system that employs electrochemical (EC) and spectroscopic methodology. However, these systems are bulky and unsuitable for real-time continuous monitoring. As opposed to microdialysis, biosensors are easy to miniaturize and are highly suitable for studies; they selectively oxidize GABA into a secondary electroactive product (usually hydrogen peroxide, HO) in the presence of enzymes, which is then detected by amperometry. Unfortunately, this method req...
Nanomedicine : nanotechnology, biology, and medicine, Apr 26, 2016
A solvent-free microsphere sintering technique was developed to fabricate scaffolds with pore siz... more A solvent-free microsphere sintering technique was developed to fabricate scaffolds with pore size gradient for tissue engineering applications. Poly(D,L-Lactide) microspheres were fabricated through an emulsification method where TiO2 nanoparticles were employed both as particulate emulsifier in the preparation procedure and as surface modification agent to improve bioactivity of the scaffolds. A fine-tunable pore size gradient was achieved with a pore volume of 30±2.6%. SEM, EDX, XRD and FTIR analyses all confirmed the formation of bone-like apatite at the 14th day of immersion in Simulated Body Fluid (SBF) implying the ability of our scaffolds to bond to living bone tissue. In vitro examination of the scaffolds showed progressive activity of the osteoblasts on the scaffold with evidence of increase in its mineral content. The bioactive scaffold developed in this study has the potential to be used as a suitable biomaterial for bone tissue engineering and hard tissue regeneration.
Implanted gradient index lenses have extended the reach of standard multiphoton microscopy from t... more Implanted gradient index lenses have extended the reach of standard multiphoton microscopy from the upper layers of the mouse cortex to the lower cortical layers and even subcortical regions. These lenses have the clarity to visualize dynamic activities, such as calcium transients, with subcellular and millisecond resolution and the stability to facilitate repeated imaging over weeks and months. In addition, behavioral tests can be used to correlate performance with observed changes in network function and structure that occur over time. Yet, this raises the questions, does an implanted microlens have an effect on behavioral tests, and if so, what is the extent of the effect? To answer these questions, we compared the performance of three groups of mice in three common behavioral tests. A gradient index lens was implanted in the prefrontal cortex of experimental mice. We compared their performance with mice that had either a cranial window or a sham surgery. Three presurgical and fi...
Nicotine via nicotinic acetylcholine receptors (nAChRs) stimulates non-small cell lung cancer (NS... more Nicotine via nicotinic acetylcholine receptors (nAChRs) stimulates non-small cell lung cancer (NSCLC) cell invasion and epithelial to mesenchymal transition (EMT) which underpin the cancer metastasis. However, the receptor subtypedependent effects of nAChRs on NSCLC cell invasion and EMT, and the signaling pathway underlying the effects remain not fully defined. We identified that nicotine induced NSCLC cell invasion, migration, and EMT; the effects were suppressed by pharmacological intervention using a7-nAChR selective antagonists or by genetic intervention using a7-nAChR knockdown via RNA inference. Meanwhile, nicotine induced activation of MEK/ERK signaling in NSCLC cells; a7-nAChR antagonism or MEK/ERK signaling pathway inhibition suppressed NSCLC cell invasion and EMT marker expression. These results indicate that nicotine induces NSCLC cell invasion, migration, and EMT; the effects are mediated by a7-nAChRs and involve MEK/ERK signaling pathway. Delineating the effect of nicotine on the NSCLC cell invasion and EMT at receptor subtype level would improve the understanding of cancer biology and offer potentials for the exploitation of selective ligands for the control of the cancer metastasis.
From 1967 through 1973, 80 consecutive patients un- derwent simultaneous aortic valve replacement... more From 1967 through 1973, 80 consecutive patients un- derwent simultaneous aortic valve replacement (AVR) and coronary bypass grafting. Fourteen (18%) experienced no angina pectoris and had no history or electrocardiographic evidence of coronary athero- sclerosis. Seven of these 14 had severe multiple vessel disease. All operations were performed under normothermic conditions without coronary perfusion. Seven patients (9%) died during operation. Intra- operative myocardial infarction was documented in eight (10%). THE FREQUENT FINDING of coronary atherosclerosis in adults with aortic valve disease has influenced the practice of coronary arteriography in the preoperative examination. Patients with aortic stenosis (AS) who experienced angina pectoris have a prevalence of 58% severe coronary obstruc- tion(s) reported by Lewis and Creus' and 64% reported by Hancock2 in series documented by arteriography. Those without angina and/or patients with aortic regurgitation (AR) have a lower prevalence of associated coronary artery stenosis. Atherosclerotic heart disease may severely jeopardize the prognosis after valve replacement. To offset this risk, with proper angiographic indications, bypass grafts have been combined with all forms of valve repair or replacement. Aortic valve replacement performed simultaneously with direct myocardial revascularization is the subject of this report. In reviewing the Cleveland Clinic experience, we From the Department of Thoracic and Cardiovascular Surgery, The Cleveland Clinic Foundation and the Cleveland Clinic Educational Founda- tion, Cleveland, Ohio.
Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent pro... more Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR α7 subunit without compromising formation of functional receptors. Methods: A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR α7 subunit (α7Y). SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits (α7) or with α7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125 I-labeled α-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy. Results: Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC 50 values for the α7 nAChR-selective agonist, choline, and IC 50 values for the α7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs. Conclusion: nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs. α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.
The surface of halloysite nanotubes (HNTs) was bifunctionalized with two ligands—folic acid and a... more The surface of halloysite nanotubes (HNTs) was bifunctionalized with two ligands—folic acid and a fluorochrome. In tandem, this combination should selectively target cancer cells and provide a means for imaging the nanoparticle. Modified bi-functionalized HNTs (bi-HNTs) were then doped with the anti-cancer drug methotrexate. bi-HNTs were characterized and subjected to in vitro tests to assess cellular growth and changes in cellular behavior in three cell lines—colon cancer, osteosarcoma, and a pre-osteoblast cell line (MC3T3-E1). Cell viability, proliferation, and cell uptake efficiency were assessed. The bi-HNTs showed cytocompatibility at a wide range of concentrations. Compared with regular-sized HNTs, reduced HNTs (~6 microns) were taken up by cells in more significant amounts, but increased cytotoxicity lead to apoptosis. Multi-photon images confirmed the intracellular location of bi-HNTs, and the method of cell entry was mainly through caveolae-mediated endocytosis. The bi-HNT...
A high-resolution, three-dimensional, optical imaging technique for the murine brain was develope... more A high-resolution, three-dimensional, optical imaging technique for the murine brain was developed to identify the effects of different therapeutic windows for preclinical brain research. This technique tracks the same cells over several weeks. We conducted a pilot study of a promising drug to treat diffuse axonal injury (DAI) caused by traumatic brain injury, using two different therapeutic windows, as a means to demonstrate the utility of this novel longitudinal imaging technique. DAI causes immediate, sporadic axon damage followed by progressive secondary axon damage. We administered minocycline for three days commencing one hour after injury in one treatment group and beginning 72 hours after injury in another group to demonstrate the method’s ability to show how and when the therapeutic drug exerts protective and/or healing effects. Fewer varicosities developed in acutely treated mice while more varicosities resolved in mice with delayed treatment. For both treatments, the drug...
Implanted gradient index lenses have extended the reach of standard multiphoton microscopy from t... more Implanted gradient index lenses have extended the reach of standard multiphoton microscopy from the upper layers of the mouse cortex to the lower cortical layers and even subcortical regions. These lenses have the clarity to visualize dynamic activities, such as calcium transients, with subcellular and millisecond resolution and the stability to facilitate repeated imaging over weeks and months. In addition, behavioral tests can be used to correlate performance with observed changes in network function and structure that occur over time. Yet, this raises the questions, does an implanted microlens have an effect on behavioral tests, and if so, what is the extent of the effect? To answer these questions, we compared the performance of three groups of mice in three common behavioral tests. A gradient index lens was implanted in the prefrontal cortex of experimental mice. We compared their performance with mice that had either a cranial window or a sham surgery. Three presurgical and five postsurgical sets of behavioral tests were performed over seven weeks. Behavioral tests included rotarod, foot fault, and Morris water maze. No significant differences were found between the three groups, suggesting that microlens implantation did not affect performance. The results for the current study clear the way for combining behavioral studies with gradient index lens imaging in the prefrontal cortex, and potentially other regions of the mouse brain, to study structural, functional, and behavioral relationships in the brain.
We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of ␣... more We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of ␣7 and 2 subunits. We measured optical and electrophysiological properties of wildtype and mutant subunits expressed in cell lines and Xenopus laevis oocytes. Laser scanning confocal microscopy indicated that fluorescently tagged ␣7 and 2 subunits colocalize. Fö rster resonance energy transfer between fluorescently tagged subunits strongly suggested that ␣7 and 2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function ␣7 and 2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, ␣72 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of ␣7 nAChRs, although amplitudes of ␣72 nAChRmediated, agonist-evoked currents were generally ϳ2-fold lower than those for ␣7 nAChRs. It is noteworthy that ␣72 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro--erythroidine that was not observed for ␣7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the ␣7-2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower ␣72 nAChR current amplitudes and challenges in identifying the function of native ␣72 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of 2 subunits within the ␣72 nAChRs.
... the author. TAM Author is a PhD candidate in Bioengineering at Arizona State University (ASU)... more ... the author. TAM Author is a PhD candidate in Bioengineering at Arizona State University (ASU) and affiliated with the Biodesign Instituteat ASU, Tempe, AZ 85287-5201 USA (e-mail: teresa.murray@ asu.edu.). hippocampus ...
The influence of the protein environment on the primary electron donor, P, a bacteriochlorophyll ... more The influence of the protein environment on the primary electron donor, P, a bacteriochlorophyll a dimer, of reaction centers from Rhodobacter sphaeroides, has been investigated using electron paramagnetic resonance and electron nuclear double resonance spectroscopy. These techniques were used to probe the effects on P that are due to alteration of three amino acid residues, His L168, Asn L170, and Asn M199. The introduction of Glu at L168, Asp at L170, or Asp at M199 changes the oxidation/ reduction midpoint potential of P in a pH-dependent manner (Williams et al. (2001) Biochemistry 40, 15403-15407). For the double mutant His L168 to Glu and Asn at L170 to Asp, excitation results in electron transfer along the Aside branch of cofactors at pH 7.2, but at pH 9.5, a long-lived state involving B-side cofactors is produced (Haffa et al. (2004) J Phys Chem B 108, 4-7). Using electron paramagnetic resonance spectroscopy, the mutants with alterations of each of the three individual residues and a double mutant, with changes at L168 and L170, were found to have increased linewidths of 10.1-11.0 G compared to the linewidth of 9.6 G for wild type. The Special TRIPLE spectra were pH dependent, and at pH 8, the introduction of aspartate at L170 increased the spin density ratio, q L /q M , to 6.1 while an aspartate at the symmetry related position, M199, decreased the ratio to 0.7 compared to the value of 2.1 for wild type. These results indicate that the energy of the two halves of P changes by about 100 meV due to the mutations and are consistent with the interpretation that electrostatic interactions involving these amino acid residues contribute to the switch in pathway of electron transfer. Keywords Reaction centers Á Purple bacteria Á Magnetic resonance Á Bacteriochlorophyll Á Oxidized bacteriochlorophyll dimer Á Electron paramagnetic resonance Abbreviations EPR Electron paramagnetic resonance ENDOR Electron nuclear double resonance TRIPLE Electron-nuclear-nuclear triple resonance Rb.
Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent pro... more Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR α7 subunit without compromising formation of functional receptors. Methods: A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR α7 subunit (α7Y). SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits (α7) or with α7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125 I-labeled α-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy. Results: Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC 50 values for the α7 nAChR-selective agonist, choline, and IC 50 values for the α7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs. Conclusion: nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs. α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.
Hippocampal gamma oscillations, as a form of neuronal network synchronization, are speculated to ... more Hippocampal gamma oscillations, as a form of neuronal network synchronization, are speculated to be associated with learning, memory and attention. Nicotinic acetylcholine receptor alpha7 subtypes (alpha7-nAChRs) are highly expressed in hippocampal neurons and play important roles in modulating neuronal function, synaptic plasticity, learning and memory. However, little is known about the role of alpha7-nAChRs in hippocampal gamma oscillations. Here, we examined the effects of selective alpha7- and non-alpha7-nAChR antagonists on tetanic gamma oscillations in rat hippocampal slices. We found that brief tetanic stimulation-induced gamma oscillations (30-80 Hz) and pharmacological blockade of alpha7-nAChRs using the relatively selective alpha7-nAChR antagonists, methyllycaconitine (10 or 100 nM) or alpha-bungarotoxin (10 nM), significantly reduced the frequency spectrum power, the number of spikes, and burst duration of evoked gamma oscillations. Neither mecamylamine nor dihydro-beta-erythroidine, which are selective antagonists of non-alpha7-nAChRs, demonstrated significant effects on tetanic gamma oscillations. Nicotine exposure promotes hippocampal gamma oscillations in a methyllycaconitine-sensitive manner. It is concluded that alpha7-nAChRs in hippocampal slices play important roles in regulation of gamma oscillations, thus potentially helping to explain roles of nAChRs in cognitive functions such as learning, memory and attention.
The main purpose of this paper is to share the Mentoring for INnovative Design Solutions (MINDS) ... more The main purpose of this paper is to share the Mentoring for INnovative Design Solutions (MINDS) Scholars Program developed by Alpha Eta Mu Beta, the International Biomedical Engineering Honor Society. The program’s goals are to (1) introduce biomedical engineering students to an open-ended design experience as part of interuniversity teams with industry and faculty mentors, and (2) develop the ability to create designs considering clinical translatability on teams with different backgrounds and areas of expertise. MINDS uses an experiential learning approach to (1) enrich student curricular experiences through inter-institutional collaboration, (2) build engineering design skills, including three key design considerations for clinical/commercial success: intellectual property protection, regulatory strategy, and market identification; and (3) emphasize the importance of end-user considerations. From 2015 to 2022, MINDS has involved 131 students from 50 universities and 22 faculty a...
Imbalances in levels of glutamate (GLU) and gamma-aminobutyric acid (GABA) and their sub-second s... more Imbalances in levels of glutamate (GLU) and gamma-aminobutyric acid (GABA) and their sub-second signaling dynamics occur in several brain disorders including traumatic brain injury, epilepsy, and Alzheimer’s disease. The present work reports on the optimization and in vivo testing of a silicon (Si) multifunctional biosensor probe for sub-second simultaneous real-time detection of GLU and GABA. The Si probe features four surface-functionalized platinum ultramicroelectrodes (UMEs) for detection of GLU and GABA, a sentinel site, and integrated microfluidics for in-situ calibration. Optimal enzyme concentrations, size-exclusion phenylenediamine layer and micro spotting conditions were systematically investigated. The measured GLU sensitivity for the GLU and GABA sites were as high as 219 ± 8 nA μM−1 cm−2 (n = 3). The measured GABA sensitivity was as high as 10 ± 1 nA μM−1 cm−2 (n = 3). Baseline recordings (n = 18) in live rats demonstrated a useful probe life of at least 11 days with GL...
High resolution, in vivo optical imaging of the mouse brain over time often requires anesthesia, ... more High resolution, in vivo optical imaging of the mouse brain over time often requires anesthesia, which necessitates maintaining the animal's body temperature and level of anesthesia, as well as securing the head in an optimal, stable position. Controlling each parameter usually requires using multiple systems. Assembling multiple components into the small space on a standard microscope stage can be difficult and some commercially available parts simply do not fit. Furthermore, it is time-consuming to position an animal in the identical position over multiple imaging sessions for longitudinal studies. This is especially true when using an implanted gradient index (GRIN) lens for deep brain imaging. The multiphoton laser beam must be parallel with the shaft of the lens because even a slight tilt of the lens can degrade image quality. In response to these challenges, we have designed a compact, integrated in vivo imaging support system to overcome the problems created by using sepa...
The quarterly Women in Engineering and Science Luncheons provide focused professional development... more The quarterly Women in Engineering and Science Luncheons provide focused professional development for women faculty and graduate students, while also providing a platform for peer-to-peer and student-to-faculty networking. This session will discuss the structure of the luncheons, the professional development training, the interactive activities, networking, and present assessment findings.
Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter that is essential for norma... more Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter that is essential for normal brain function. It is involved in multiple neuronal activities, including plasticity, information processing, and network synchronization. Abnormal GABA levels result in severe brain disorders and therefore GABA has been the target of a wide range of drug therapeutics. GABA being non-electroactive is challenging to detect in real-time. To date, GABA is detected mainly via microdialysis with a high-performance liquid chromatography (HPLC) system that employs electrochemical (EC) and spectroscopic methodology. However, these systems are bulky and unsuitable for real-time continuous monitoring. As opposed to microdialysis, biosensors are easy to miniaturize and are highly suitable for studies; they selectively oxidize GABA into a secondary electroactive product (usually hydrogen peroxide, HO) in the presence of enzymes, which is then detected by amperometry. Unfortunately, this method req...
Nanomedicine : nanotechnology, biology, and medicine, Apr 26, 2016
A solvent-free microsphere sintering technique was developed to fabricate scaffolds with pore siz... more A solvent-free microsphere sintering technique was developed to fabricate scaffolds with pore size gradient for tissue engineering applications. Poly(D,L-Lactide) microspheres were fabricated through an emulsification method where TiO2 nanoparticles were employed both as particulate emulsifier in the preparation procedure and as surface modification agent to improve bioactivity of the scaffolds. A fine-tunable pore size gradient was achieved with a pore volume of 30±2.6%. SEM, EDX, XRD and FTIR analyses all confirmed the formation of bone-like apatite at the 14th day of immersion in Simulated Body Fluid (SBF) implying the ability of our scaffolds to bond to living bone tissue. In vitro examination of the scaffolds showed progressive activity of the osteoblasts on the scaffold with evidence of increase in its mineral content. The bioactive scaffold developed in this study has the potential to be used as a suitable biomaterial for bone tissue engineering and hard tissue regeneration.
Implanted gradient index lenses have extended the reach of standard multiphoton microscopy from t... more Implanted gradient index lenses have extended the reach of standard multiphoton microscopy from the upper layers of the mouse cortex to the lower cortical layers and even subcortical regions. These lenses have the clarity to visualize dynamic activities, such as calcium transients, with subcellular and millisecond resolution and the stability to facilitate repeated imaging over weeks and months. In addition, behavioral tests can be used to correlate performance with observed changes in network function and structure that occur over time. Yet, this raises the questions, does an implanted microlens have an effect on behavioral tests, and if so, what is the extent of the effect? To answer these questions, we compared the performance of three groups of mice in three common behavioral tests. A gradient index lens was implanted in the prefrontal cortex of experimental mice. We compared their performance with mice that had either a cranial window or a sham surgery. Three presurgical and fi...
Nicotine via nicotinic acetylcholine receptors (nAChRs) stimulates non-small cell lung cancer (NS... more Nicotine via nicotinic acetylcholine receptors (nAChRs) stimulates non-small cell lung cancer (NSCLC) cell invasion and epithelial to mesenchymal transition (EMT) which underpin the cancer metastasis. However, the receptor subtypedependent effects of nAChRs on NSCLC cell invasion and EMT, and the signaling pathway underlying the effects remain not fully defined. We identified that nicotine induced NSCLC cell invasion, migration, and EMT; the effects were suppressed by pharmacological intervention using a7-nAChR selective antagonists or by genetic intervention using a7-nAChR knockdown via RNA inference. Meanwhile, nicotine induced activation of MEK/ERK signaling in NSCLC cells; a7-nAChR antagonism or MEK/ERK signaling pathway inhibition suppressed NSCLC cell invasion and EMT marker expression. These results indicate that nicotine induces NSCLC cell invasion, migration, and EMT; the effects are mediated by a7-nAChRs and involve MEK/ERK signaling pathway. Delineating the effect of nicotine on the NSCLC cell invasion and EMT at receptor subtype level would improve the understanding of cancer biology and offer potentials for the exploitation of selective ligands for the control of the cancer metastasis.
From 1967 through 1973, 80 consecutive patients un- derwent simultaneous aortic valve replacement... more From 1967 through 1973, 80 consecutive patients un- derwent simultaneous aortic valve replacement (AVR) and coronary bypass grafting. Fourteen (18%) experienced no angina pectoris and had no history or electrocardiographic evidence of coronary athero- sclerosis. Seven of these 14 had severe multiple vessel disease. All operations were performed under normothermic conditions without coronary perfusion. Seven patients (9%) died during operation. Intra- operative myocardial infarction was documented in eight (10%). THE FREQUENT FINDING of coronary atherosclerosis in adults with aortic valve disease has influenced the practice of coronary arteriography in the preoperative examination. Patients with aortic stenosis (AS) who experienced angina pectoris have a prevalence of 58% severe coronary obstruc- tion(s) reported by Lewis and Creus' and 64% reported by Hancock2 in series documented by arteriography. Those without angina and/or patients with aortic regurgitation (AR) have a lower prevalence of associated coronary artery stenosis. Atherosclerotic heart disease may severely jeopardize the prognosis after valve replacement. To offset this risk, with proper angiographic indications, bypass grafts have been combined with all forms of valve repair or replacement. Aortic valve replacement performed simultaneously with direct myocardial revascularization is the subject of this report. In reviewing the Cleveland Clinic experience, we From the Department of Thoracic and Cardiovascular Surgery, The Cleveland Clinic Foundation and the Cleveland Clinic Educational Founda- tion, Cleveland, Ohio.
Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent pro... more Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR α7 subunit without compromising formation of functional receptors. Methods: A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR α7 subunit (α7Y). SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits (α7) or with α7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125 I-labeled α-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy. Results: Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC 50 values for the α7 nAChR-selective agonist, choline, and IC 50 values for the α7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs. Conclusion: nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs. α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.
The surface of halloysite nanotubes (HNTs) was bifunctionalized with two ligands—folic acid and a... more The surface of halloysite nanotubes (HNTs) was bifunctionalized with two ligands—folic acid and a fluorochrome. In tandem, this combination should selectively target cancer cells and provide a means for imaging the nanoparticle. Modified bi-functionalized HNTs (bi-HNTs) were then doped with the anti-cancer drug methotrexate. bi-HNTs were characterized and subjected to in vitro tests to assess cellular growth and changes in cellular behavior in three cell lines—colon cancer, osteosarcoma, and a pre-osteoblast cell line (MC3T3-E1). Cell viability, proliferation, and cell uptake efficiency were assessed. The bi-HNTs showed cytocompatibility at a wide range of concentrations. Compared with regular-sized HNTs, reduced HNTs (~6 microns) were taken up by cells in more significant amounts, but increased cytotoxicity lead to apoptosis. Multi-photon images confirmed the intracellular location of bi-HNTs, and the method of cell entry was mainly through caveolae-mediated endocytosis. The bi-HNT...
A high-resolution, three-dimensional, optical imaging technique for the murine brain was develope... more A high-resolution, three-dimensional, optical imaging technique for the murine brain was developed to identify the effects of different therapeutic windows for preclinical brain research. This technique tracks the same cells over several weeks. We conducted a pilot study of a promising drug to treat diffuse axonal injury (DAI) caused by traumatic brain injury, using two different therapeutic windows, as a means to demonstrate the utility of this novel longitudinal imaging technique. DAI causes immediate, sporadic axon damage followed by progressive secondary axon damage. We administered minocycline for three days commencing one hour after injury in one treatment group and beginning 72 hours after injury in another group to demonstrate the method’s ability to show how and when the therapeutic drug exerts protective and/or healing effects. Fewer varicosities developed in acutely treated mice while more varicosities resolved in mice with delayed treatment. For both treatments, the drug...
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Papers by Teresa Murray