Browsing by Author "Gryczynski, Zygmunt"
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Item A comparison of photophysical characteristics of rHDL encapsulated anti-cancer drug valrubicin and free valrubicin.(2015-03) Shah, Sunil Ajit; Chib, Rahul; Raut, Sangram; Bermudez, Jaclyn Y.; Sabnis, Nirupama; Duggal, Divya; Kimball, Joseph; Lacko, Andras G.; Borejdo, Julian; Gryczynski, Zygmunt; Gryczynski, IgnacyNanotechnology as a channel for drug delivery is one of the rapidly developing fields in cancer therapeutics. Targeted drug delivery has the advantage of having minimal interaction with healthy tissue, thereby reducing the toxicity of the drug to the rest of the body. rHDL nanoparticles have been found to be an efficient delivery system for highly lipophilic anti-cancer drugs. This is achieved through the interaction of scavenger receptors class B type I (SR-BI), which are highly expressed on cancer cells interact with rHDL nanoparticles for effective drug delivery to the cancer cell and tumor. The drug under investigation is Valrubicin, which, apart from being an effective anti-cancer drug, also has intrinsic fluorescence. This allowed for the comparison of photophysical properties of free Valrubicin and rHDL Valrubicin via steady state and time resolved fluorescence measurements. The steady-state anisotropy of rHDL Valrubicin is higher as compared to free Valrubicin, suggesting its encapsulation in rHDL nanoparticles. A longer rotational correlation time was observed for rHDL Valrubicin in time resolved anisotropy measurements compared to free Valrubicin, further supporting steady state anisotropy data.. We also studied the cellular internalization of free Valrubicin and rHDL Valrubicin using confocal microscopy. This could help track the movement of rHDL nanoparticles within the cancer cells.Item A fluorescent biosensor for the detection and imaging of a cancer biomarker(2016-03-23) Mummert, Mark; Bora, Ilkay; Shah, Sunil; Gryczynski, Ignacy; Borejdo, Julian; Gryczynski, Zygmunt; Fudala, Rafal; Chib, RahulPurpose: Cancer is among the leading cause of death worldwide with approximately 8.2 million deaths. Cancer mortality can be reduced if it can be detected and treated at early stages. Certain biomarkers like hyaluronidase are reported in the literature for the screening and detection of cancer. It is an endoglycosidase that degrades hyaluronic acid (HA) and this enzyme is overexpressed in various cancers. Therefore, it is of great interest to develop a simple, sensitive and fast technique with which one can estimate the activity/level of hyaluronidase. We have developed a fluorescent biosensor for the detection of hyaluronidase activity/level. This probe was developed by labeling hyaluronic acid with a long lifetime fluorophore. Monitoring the cleavage of hyaluronic acid by measuring the changes in the fluorescent properties of the biosensor will be a simple and precise tool reflecting hyaluronidase activity and can be used for the detection of cancer. Methods: This fluorescent biosensor was developed by heavy labeling of hyaluronic acid with an orange/red emitting azadioxatriangulenium (ADOTA) fluorophore. ADOTA in water emits at 560 nm with a long fluorescence lifetime of ~ 20 ns. The hyaluronidase activity was measured as a function of the change in the steady-state fluorescence intensity and fluorescence lifetime of the biosensor. Fluorescence lifetime imaging microscopy (FLIM) was also used to image hyaluronidase activity in cancer cells. Results: The heavily ADOTA labeled hyaluronic acid (HA-ADOTA) has a red shift in the peak emission wavelength (605 nm), a weak fluorescence signal and a short fluorescence lifetime due to efficient self-quenching. In the presence of enzyme hyaluronidase, the brightness and fluorescence lifetime of the sample increases with a shift in the peak emission to its original wavelength at 560 nm. The ratio of the fluorescence intensity of the HA - ADOTA probe at 560 nm and 605 nm can be used as the sensing signal for detecting hyaluronidase. Recovery in the fluorescence lifetime was used for fluorescence lifetime-based imaging of cancer. Conclusions: Our results show the ability of this the HA-ADOTA probe to detect activity/level of hyaluronidase in biological samples. Due to long fluorescence lifetime of the dye, it can be also be used to remove the background in cellular images. In future, this novel technology can be used to design a small device which can be used in primary care settings for the screening of cancer.Item Assessment of WIN 55,212-2 Loaded Reconstituted High-Density Lipoprotein Nanoparticles for Ocular Delivery(2024-03-21) Petty, R. Max; Ranjan, Rajiv; Sabnis, Nirupama; Fudala, Rafal; Lacko, Andras; Gryczynski, Zygmunt; Krishnamoorthy, Raghu; Stankowska, DorotaPurpose: Overcoming challenges in glaucoma therapy, such as biological barriers and retina delivery, led us to develop innovative reconstituted high-density lipoprotein nanoparticles (rHDL NPs) for effective drug delivery. Optic nerve head astrocytes (ONHAs) are vital in maintaining retinal ganglion cell (RGC) axon integrity. This study describes the encapsulation of WIN 55,212-2 (WIN) in rHDL NPs and investigates the delivery mechanism of these nanoparticles in ONHAs. Methods: Using a novel preparation method, a stable rHDL-payload complex was created by combining lipophilic fluorescent dye IR780 or therapeutic agent WIN with phosphatidylcholine and apolipoprotein A-I (Apo A-I). Fluorescent rHDL (rHDL-IR780) was used to assess cellular uptake in human primary ONHAs in vitro. Scavenger receptor class B1 (SR-B1) expression was confirmed in retinal cell lysates by SDS-PAGE followed by western blot analysis. Receptor-mediated payload release through SR-B1 was confirmed by receptor blocking using BLT-1 as a specific SR-B1 receptor-blocking agent. Results: Fluorescent rHDL NPs exhibited an IR780 encapsulation efficiency of 68.7% (103 M), a polydispersity index (PDI) of 0.287 ± 0.013, a size of 14.01 ± 4.36 nm, and a zeta potential of -7.44 ± 0.90 mV. Additionally, drug-loaded rHDL-WIN NPs displayed a WIN encapsulation efficiency of 44.6% (341.4 M), a PDI of 0.280 ± 0.011, a size of 62.04 ± 25.06 nm, and a zeta potential of -20.13 ± 0.86 mV. Western blot analysis on human retinal lysates, ONHA lysates, and RGC lysates indicated the expression of SR-B1 (57/82 kDa (unmodified/glycosylated)). Cellular uptake studies confirmed the ability of rHDL to deliver payloads to ONHAs and RGCs. Receptor blocking with 10 nM BLT-1 highlighted the role of SR-B1 in specific cellular uptake from rHDL to ONHAs (p < 0.01). Conclusions: Our study highlights the role of SR-B1 in facilitating the delivery of rHDL payloads to ONHAs, offering the potential for targeted drug delivery in glaucoma. We anticipate that the cellular uptake by RGCs will follow the same SR-B1-mediated pathway. Successful WIN encapsulation in rHDL NPs suggests a potential avenue for targeting therapies to treat and prevent glaucomatous damage. Further studies are needed to determine the neuroprotective effects of rHDL-WIN and develop the potential of rHDL NPs to be used as an agent to target therapies in glaucoma.Item ASSOCIATED ANISOTROPY DECAY OF ETHIDIUM BROMIDE INTERACTING WITH DNA(2014-03) Chib, Rahul; Raut, Sangram; Sabnis, Sarika; Singhal, Preeti; Gryczynski, Zygmunt; Gryczynski, IgnacyEthidium Bromide (EB) is a commonly used dye in a deoxyribonucleic acid (DNA) study. Upon an intercalation, this dye significantly increases its brightness and lifetime. In this report we studied time-resolved fluorescence properties of EB fluorophore existing simultaneously in free and bound forms in the solution. Fluorescence intensity decays were fitted globally to a double exponential model with lifetimes corresponding to free (1.6ns) and bound (22ns) forms, and molar fractions were determined for all used solutions. Anisotropy decays displayed characteristic time dependence with an initial rapid decline followed by recovery and slow decay. This is because of the two existing fractions contributing to a total anisotropy change in time. The short-lived fraction associated with the free EB molecules decreases faster the than the long-lived fraction associated with EB bound to DNA. Consequently, contribution from fast rotation leads to initial rapid decay in anisotropy. On the other hand bound fraction, due to slow rotation helps recover anisotropy in time. This effect of associated anisotropy decays in systems such as EB free/EB-DNA is clearly visible in a wide range of concentrations, and should be taken into account in polarization assays and biomolecular dynamics studies. Purpose (a): To study the interaction of DNA with ethidium bromide. Methods (b): 1) Steady state fluorescence intensity measurement. 2) Time resolved measurements. 3) Steady state anisotropy. Results (c): Increase in fluorescence lifetime and fluorescence anisotropy when ethidium bromide interact with DNA. Conclusions (d): We believe that assays involving EB and DNA should be analyzed with the associated decay model. Neglecting this type of decay pattern can lead to false interpretation of results.Item Differences in Actomyosin Function in the Left and Right Ventricles of Human Hearts(2017-03-14) Nagwekar, Janhavi; Patel, Vipulkumar; Fudala, Rafal; Gryczynski, Zygmunt; Gryczynski, Ignacy; Borejdo, Julian; Requena, SebastianPurpose: In both ventricles of the heart, actin is expressed from the same genes. There are no differences in twitch duration, work performance, and power among the right (RV) and left (LV) ventricles in animals. So there is no expectation that the properties of actin or myosin isolated from either ventricle would be different. Nevertheless, the situation is more complex in human hearts. The LV must pump more powerfully because it has to overcome a larger resistance presented by the systemic system than the RV, which has to overcome a lower resistance offered by the pulmonary system. The question arises whether stronger pumping action of the LV is partially caused by the LV actomyosin developing more force than the RV actomyosin. The goal of this work is too identify if there are any differences in the kinetics rates of the actomyosin mechanochemical cycle in the LV versus the RV. Methods: Such a question is impossible to answer by making macroscopic measurements such as tension or ATPase activity, because the number of molecules involved in these processes is too large, (of the order of 1011) Measurements must be taken from a few molecules. We measured variations in the polarization of fluorescence of a few actomyosin molecules during the contraction cycle using time-resolved single molecule fluorescent microscopy. We obtained molecular kinetic information by calculating its autocorrelation function using R (version 3.3.1). The autocorrelation curve was fitted with a bi-exponential decay model to extract the rate constants using XPFIT (version 1.2.1) The goodness of fit was assessed by chi-squared. Results: The results suggest that actomyosin function is identical in both ventricles. There are no statistically significant differences in the kinetic rates that we obtained. Additionally, the spatial distribution of actomyosin is also the same. Conclusions: Our results suggest that the differences in the LV and RV may not be due to differences at the molecular level between actomyosin from the LV or RV in human hearts. However, our study only involved the use of failing human hearts with a wide variety of clinical parameters. These differences in the type of heart failures and patients may mean that we will not be able to extract statistically different results between kinetic rates. We have begun working on non-failing human hearts and will see if differences are present in that case.Item Evaluating reconstituted high density lipoprotein nanoparticles as target specific Doxorubicin carriers using fluorescence spectroscopy.(2016-03-23) Requena, Sebastian; Sabnis, Nirupama; Chib, Rahul; Lacko, Andras G.; Borejdo, Julian; Gryczynski, Zygmunt; Gryczynski, Ignacy; Shah, Sunil AjitDoxorubicin, also known as Adriamycin, is an anthracycline antibiotic which first gained clinical prominence in the early 1970’s as an effective antitumor agent. It is still used today to treat a spectrum of cancers like lymphoma, bladder, stomach, lung, breast, ovarian, and several others. Due to its production of free radicals to attack tumor cells, Doxorubicin interferes with mitochondrial phosphorylation and also induces cardiotoxicity. Thus, efficient and biocompatible delivery methods are needed for targeted drug delivery to overcome systemic toxicity. To maintain a high level of growth, tumor cells overexpress Scavenger receptors type B-1 (SR-B1). This cellular characteristic can be used to target and selectively deliver doxorubicin to tumor cells by packing it in reconstituted high density lipoprotein (rHDL) nanoparticles, which bind selectively to SR-B1 receptors. Nanoparticles as target-specific drug delivery agents are increasingly used in cancer therapy to enhance bioavailability and to reduce off target toxicity of anti-cancer agents. Several different formulations of rHDL nanoparticles to incorporate doxorubicin were synthesized. Doxorubicin’s intrinsic fluorescence was used to photophysically characterize the properties of loaded rHDL nanoparticles including absorption, emission, excitation, steady-state and time resolved anisotropy measurements, and quenching to study drug shielding by nanoparticles. Overall the fluorescence properties of the rHDL: doxorubicin complex may reveal valuable novel characteristics of this drug delivery vehicle that may be particularly applicable when used in systemic (intravenous) therapy.Item Fluorescence polarization based detection of hyaluronidase activity as a biomarker for skin cancer.(2015-03) Chib, Rahul; Shah, Sunil; Mummert, Mark; Raut, Sangram; Bora, Ilkay; Pedro, Bobby; Gryczynski, Ignacy; Gryczynski, Zygmunt; Fudala, RafalPurpose: Malignant melanoma (MM) is a type of skin cancer that has a high potential to metastasize to distant organs and cause death. MM is the third most common skin cancer in the United States and has an incidence of 18 new cases per 100,000. It is more lethal compared to other type of skin cancers due to its higher rate of metastasis and has a 5 years survival rate of 7-18%. In MM, the levels of hyaluronidase are overexpressed. Hyaluronidase is an endoglycosidase that degrades glycosaminoglycan and the hyaluronic acid (HA). Therefore, monitoring the hyaluronidase activity can be used as a contrasting mechanism for its detection. A novel fluorescence polarization based detection of enzyme activity using a fluorophore with a long fluorescence lifetime can enable a simple wide field molecular analysis of the cancer activities at the cellular and tissue level. Hyaluronic acid is a large biopolymer (over 1MDa) that is cleaved by the hyaluronidase enzyme to smaller elements. Monitoring kinetics of HA degradation by fluorescence polarization will be a simple and precise tool reflecting hyaluronidase activity and can be used for the detection, diagnosis and monitoring of the malignant melanoma. Methods: A long lifetime fluorophore (ADOTA) with a fluorescence lifetime of ̴ 20 ns was used to label hyaluronic acid. The hyaluronidase activity was measured as a function of change in the steady state fluorescence intensity and fluorescence polarization. Results: An intact HA will rotate slowly and the fluorescence polarization will be high, on the other hand, the cleaved HA ( ̴300KDa) will depolarize within the fluorescence lifetime of ADOTA yielding low polarization. The change in the polarization directly reflects hyaluronidase activity. We found an increase in the fluorescence intensity with increasing time. This is due to release of HOMO-FRET. We also observed a decrease in the fluorescence correlation time as cleaved hyaluronic acid fragments needs smaller time to rotate. Conclusions: Thus, preliminary results show the ability of HA-ADOTA probe to efficiently detect hyaluronidase activity. This study will develop a new technology for the non-invasive detection of the molecular activity of tumor in situ. In future, this technology can be used to construct a devise which can be used in the primary care setting for the detection of melanoma and monitoring the therapy progress.Item Forensics, Touch DNA, Genetic Profiling, Fluorescence, DNA detection(2023-05) Duque, Tiffany M.; Coble, Michael D.; Gryczynski, Zygmunt; Cihlar, Jennifer C.Current methods for forensic DNA typing focus on the identification and processing of bodily fluids and tissues, while touch DNA evidence has remained less used due to the inability to detect where this type of DNA is deposited. This study utilizes four DNA-binding fluorescent dyes (Diamond™ Nucleic Acid Dye, SYBR™ Green I Nucleic Acid Stain, Ethidium Bromide, and YOYO™) to detect DNA at three concentrations (100, 500, and 1000 pg/μL) and explores the interactions and effects the dye may have on subsequent genetic profiling. Genetic profiling was conducted at three time points (0, 1, and 4 weeks,) via STR amplification and capillary electrophoresis (CE). DNA was detected using all dyes and increases in fluorescence were observed in response to increases in DNA concentration or dye volume added. Most samples produced full genetic profiles, all of which were stable over the 4-week time period.Item Imaging viscosity of intragranular mucin matrix in cystic fibrosis cells(2019-03-05) Ponomarchuk, Olga; Castillo, Marlius; Rebik, Jonathan; Brochiero, Emmanuelle; Borejdo, Julian; Gryczynski, Ignacy; Dzyuba, Sergei; Gryczynski, Zygmunt; Grygorczyk, Ryszard; Fudala, Rafal; Requena, SebastianPurpose: Abnormalities of mucus viscosity play a critical role in the athogenesis of several respiratory diseases, including cystic fibrosis (CF). Currently, there are no approaches to assess the rheological properties of mucin granule matrices in live cells. This is the first example of the use of a molecular rotor, a BODIPY dye, to quantitatively visualize the viscosity of intragranular mucin matrices in a large population of individual granules in differentiated primary bronchial epithelial cells using fluorescence lifetime imaging microscopy. Methods: We use a simple fluorescent phenyl-BODIPY rotor molecule which is readily uptaken into mucin granules and exhibits dramatic changes in its fluorescent lifetime as a function of its environments viscosity. To measure the distribution of viscosities in intracellular mucin, we use fluorescence lifetime microscopy (FLIM) to image the non-CF and CF. We employ a machine learning algorithm to analyze the pictures and use a combination of Python and ImageJ to compute the size and viscosity distribution of intracellular mucin granules. Results: In this work, we demonstrate the use of a simple BODIPY rotor to measure the apparent viscosity of intracellular mucin granules in human bronchial epithelial cells with and without CF. The molecular rotor is readily taken up into mucin granules and can be used to quantify the intracellular viscosity of mucin granules. Additionally, as a control, we use a non-rotor analog of the phenyl-BODIPY probe which is demonstrates little or no change in its fluorescent lifetime. Our results indicate the molecular rotor can be a valuable tool to study and quantify mucus pathology in diseased cells. Conclusion: We demonstrated that BODIPY-rotor could probe intragranular viscosities of CF and non-CF cells. Importantly, two different populations of viscosities were identified in the CF granules as opposed to a single population of viscosities in non-CF granules. This indicates a heterogeneous nature of the CF granules, which might be related to the pathology. Overall, our results suggest that BODIPY viscometers could be viable tools for assessing the viscoelastic properties of mucin matrix within intact granules in live cells. Combining FLIM studies with such molecular viscometers should provide valuable insight into various stages of CF mucus pathogenesis, and potentially could aid in the development of efficient therapeutic approaches to combat the disease.Item Involvement of Caspase-2 in Cisplatin-Induced Cell Death in 2008 Ovarian Cancer Cells(2008-04-01) Adkins, Brett T.; Basu, Alakananda; Berg, Rance E.; Gryczynski, ZygmuntAdkins, B., Involvement of caspase-2 in cisplatin-induced cell death in 2008 ovarian cancer cells. Master of Science (Molecular Biology and Immunology) April, 2008, 59 pp., 12 illustrations, bibliography, 73 titles. Cisplatin, one of the most effective anticancer drugs in the treatment of ovarian cancer, causes DNA damage and leads to apoptosis. Caspases, a family of cysteine proteases, are essential for the induction of apoptosis. Initiator caspases activate effector caspases to trigger apoptosis. Caspase-2 can function as both an initiator and effector caspase although there are controversies regarding its role in DNA damage-induced apoptosis. Caspase-2 is the only caspase constitutively located in the nucleus, although its function there is unknown. In the present study we have investigated if caspase-2 is important during cisplatin-induced apoptosis and whether cisplatin treatment affects the localization of caspase-2. Caspase-2 depletion suggested that caspase-2 acts upstream of caspase-2 acts upstream of caspase-9 in cisplatin-induced apoptosis. We also made a novel observation that rottlerin, an inhibitor of DNA damage-induced apoptosis, specifically downregulates caspase-2 via the ubiquitin proteamose-mediated pathway. We further show that cisplatin induces caspase-2 translocation out of the nucleus. Moreover, translocation of caspase-2 is more important for cisplatin-induced cell death.Item Long Range Forster Resonance Energy Transfer for rHDL Nanoparticles Drug Payload Estimation(2020) Raut, Sangram; Shah, Sunil; Sabnis, Nirupama; Gryczynski, Zygmunt; Borejdo, Julian; Lacko, Andras G.; Gryczynski, Ignacy; Fudala, RafalPurpose: Over the last 30 years, there has been a dramatic increase in the application of fluorescence technology in the biomedical sciences. These advances include the application of fluorescence anisotropy for the assessment of the location and rigidity of the drug payload within the lipoprotein nanocomplexes. Forster Resonance Energy Transfer (FRET) is the long range through space interaction between the two chromophores (one energy donor and one energy acceptor) that very strongly depends on the chromophores separation. In the studies presented here, we selected the optimal combination and synthesized a FRET pair using doxorubicin within the rHDL nanoparticles as acceptor, and carboxyfluorescein linked to the rHDL via an 18-carbon lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) [18:1 PE-CF]) as the donor. The emission spectra of donor (18:1 PE-CF) has a good overlap with the absorption spectra of an acceptor (doxorubicin), making this combination suitable for FRET studies. In this communication, we used our previous findings and FRET methodology for the assessment of the localization of the payload (doxorubicin) within the respective nanoparticles and their corresponding encapsulation/entrapment efficiencies. These findings validate the diameter and the three-dimensional array of the drug transporting (rHDL) nanoparticles, while additional measurements also allowed the estimation of the number of drug molecules carried by the individual nano-assemblies using non-destructive sampling procedure. The approach described here could likely to be extended to the investigation of the loading capacity of a variety of drug carrying nanostructures, including micellar nanoparticles and exosomes.Item Measuring Intracellular Mucin Viscosity in Human Bronchial epithelial cells with Cystic Fibrosis(2017-03-14) Ponomarchuk, Olga; Castillo, Marlius; Rebik, Jonathan; Brochiero, Emmanuelle; GrygorczYk, Ryszard; Dzyuba, Sergei; Gryczynski, Ignacy; Gryczynski, Zygmunt; Borejdo, Julian; Fudala, Rafal; Requena, SebastianBackground: Cystic fibrosis (CF) is a genetic disease which causes mucus to be abnormally thick and viscous. The thick mucus harbors bacteria and particulates and is unable to be cleared by the mucociliary system resulting in respiratory disease. Understanding mucus pathology is critical to understanding and treating diseases like CF. While many factors that influence CF mucus to be unusually thick are known, the question remains if there are differences in the viscosity of the mucus before secretion. Before being secreted, mucus exists as granules in the cell known as mucin. In this work, we examine the viscosity of intracellular mucin of human bronchial epithelial cells with and without cystic fibrosis. Methods: We use a simple fluorescent phenyl-BODIPY rotor molecule which is readily uptaken into mucin granules and exhibits dramatic changes in its fluorescent lifetime as a function of its environments viscosity. To measure the distribution of viscosities in intracellular mucin, we use time-resolved fluorescent microscopy to image the non-CF and CF cells and measure the fluorescent lifetime of the probe in intracellular mucin. We employ a machine learning algorithm to analyze the pictures and use a combination of Python and ImageJ to compute the size and viscosity distribution of intracellular mucin granules. Results: Our results show that our molecular rotor is readily uptaken into mucin granules of human epithelial cells. The changes in fluorescent lifetime are substantial enough to determine the apparent viscosity distribution of intracellular mucin granules. The non-CF cells have a single normally distributed peak in the viscosity distribution centered at 560 cP. The CF cells have a bimodal distribution with a peak at 560 cP and an additional peak at 210 cP. The origin and implications this second low viscosity group of mucin granules in is unclear but may provide biophysical insight into CF mucus pathology. Conclusions: Our phenyl-BODIPY molecular rotor in combination with fluorescent lifetime imaging microscopy is a promising method to study the intracellular viscosity distribution of cells. Our results suggest that there is a distinct difference in the viscosity of mucin granules in non-CF cells versus CF cells. We believe our work will provide a new tool for investigators to study intracellular mucin and examine a variety of mucus related diseases.Item METHYLENE BLUE PROTECTS ASTROCYTES FROM HYPOXIA-REOXYGENATION INJURY BY IMPROVING CELLULAR BIOENERGETICS(2014-03) Roy Choudhury, Gourav; Winters, Ali; Rich, Ryan; Ryou, Myoung-Gwi; Gryczynski, Zygmunt; Yang, ShaohuaIn ischemic stroke, which is caused by a clot that blocks a brain blood vessel, a portion of the brain is deprived of oxygen and nutrients that are carried in the blood. When this clot is removed either by time or drugs, the affected region is flooded with an overabundance of nutrients and oxygen to a degree that is larger than the cells in the region can process and this unbalances the system and causes further damage. In our current study, we are testing a chemical compound called Methylene Blue to see if it can improve the cells’ ability to handle this excess of nutrients and oxygen available to them at the time of clot dissolution and thereby improve their survival after ischemic stroke. Purpose (a): Ischemic Stroke inflicts a double blow to the affected brain region by characteristically presenting a period of acute ischemia during which the cells are completely deprived of valuable nutrients (Glucose & oxygen) resulting in cell death, however as the blood flow is restored (Spontaneously/surgically) the surviving cells are exposed to an overwhelming levels of glucose and oxygen resulting in reperfusion injury which further aggravates the cellular injury inflicted by ischemia. Methylene Blue (MB) is a heterocyclic aromatic compound shown to function as an alternative electron carrier and improve glucose uptake, cerebral blood flow (CBF), and cerebral metabolic rate of oxygen in the brain. In our current study we aim to delineate if MB is protective in astrocyte against hypoxia-reoxygenation injury and determine its underlying mechanism. Methods (b): Primary astrocytes cultures isolated from day old C57BL6 were used in the current study. Protective role of Methylene blue (MB) in primary astrocyte cultures was evaluated in an in vitro model of cellular hypoxia (0.1% O2, 6h) and re-oxygenation (24h). The effect of MB on glucose uptake was determined by using the 2-NBDG assay. Oxygen sensitive dye Tris (2,2′-bipyridyl) dichloro Ruthenium(II) hexahydrate and Fluorescence Life Time Imaging (FLTI) was used to determine the effect of MB on intracellular oxygen concentration. ATP assay was used to determine the effect of MB on cellular energy status. Results (c): Results from cell viability assay showed that MB treatment significantly protected astrocytes from hypoxia-reoxygenation induced cell death. MB treatment significantly increased cellular glucose uptake in primary astrocyte cultures. FLTI showed that MB significantly increased intracellular oxygen concentration in primary astrocytes. Astrocytes treated with MB also had significantly higher ATP concentration compared to non-treated cells. Conclusions (d): Methylene Blue protects astrocytes against hypoxia-reoxygenation injury by improving astrocyte bioenergetics.Item MYOSIN REGULATORY LIGHT CHAIN A13T MUTATION ASSOCIATED WITH CARDIAC HYPERTROPHY IMPOSES DIFFERENCES ON KINETICS AND SPATIAL DISTRIBUTIONS OF CROSS-BRIDGES IN HEALTHY AND DISEASED VENTRICLES(2014-03) Nagwekar, Janhavi; Duggal, Divya; Midde, Krishna; Kazmierczak, Katarzyna; Huang, Weiwen; Fudala, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Szczesna-Cordary, Danuta; Borejdo, JulianThe study is performed at protein and myofibrilar level to identify key sub-steps of ATP induced XB cycle deregulation in FHC. Humans being heterozygous (50%) for FHC, our experiments with 10% penetrance in the mice will analyze subtle changes in the XB mechanisms to which humans have 40% more chances to develop those defects. This project may also provide insights into other RLC mutations (e.g. R58Q, E22K, D166V, P95A) that may involve the same alterations. The project aims to identify drugs to alter specific rate constants, affect ordering of XBs or reverse A13T effect and thus treating patients with personalized therapy. Purpose (a): Muscle is organized into regular periodic thick myosin and thin actin filaments. Myosin tails interact with each other to form a tight coiled coil rod, and the heads protrude out to interact with actin. Myosin head referred to as cross-bridge has ATPase activity and actin binding domain. The tail has a site (Regulatory Light Chain (RLC) domain) which when mutated at A13T site cause myosin heads to bind slowly to the actin molecules affecting the overall ATPase cycle and power strokes necessary for a muscle to contract in the process. Methods (b): Rabbit ventricle muscle is the source of sample for experiments in this project. Glycerinated muscle bundles were homogenized and myofibrils were extracted. Myofibrils were labeled with 0.1 nM rhodamine-phalloidin (RP) + 10 μM unlabeled-phalloidin (UP) in Ca2+-rigor solution in the the ratio of 1:100,000 fluorescent to non-fluorescent phalloidin to ensure 1 in ~105 actin monomers carry a fluorophore. Labeled myofibrils were analyzed for error of the mean of polarized fluorescence to determine kinetic rate constants in the ATPase cycle and distribution of orientations emanating from myosin cross-bridges. Results (c): Histograms were plotted from the polarized fluorescence data and the Full Width at Half Maximum (FWHM) of the mean was calculated. The mean polarization of a contracting WT myofibril was -0.176±0.018 and that of contracting A13T Mutated myofibril was -0.247±0.017. Significant differences in rate constants k1, k2 and k3 of the ATPase cycle were observed with WT values being 325±34, 0.16±0.03, 0.32±0.08 and A13T mutated values being 54±80*, 0.25±0.04, 0.57±0.12 respectively. On comparing the peaks of the fit of the data, peak 1 assumed to be the pre power stroke was lost in the A13T mutated myofibrils while peak 2 (post power stroke) almost remained constant in both muscle types. Conclusions (d): The study suggest that the functional differences between ventricles containing WT myosin and myosin in which the RLC contains the A13T mutation are caused by a change in the rate of binding of myosin cross-bridges to the thin filaments. Differences in the polarization, FWHM and peaks indicate that pre-power strokes are necessary for myosin cross-bridges and that any alterations in its functions may lead to cardiomyopathy.Item Novel Role of Bone-Targeted Cabazitaxel Nanoparticles: Beyond the Classical Taxane Function in Metastatic Prostate Cancer Cells(2023-05) Lampe, Jana B.; Vishwanatha, Jamboor K.; Hsieh, Jer-Tsong; Gryczynski, ZygmuntBone-metastatic prostate cancer symbolizes the beginning of the end-stage disease. Bone is the primary metastatic site for prostate cancer, a condition correlated to low five-year overall survival rates, extreme pain, and poor quality of life. We designed a cabazitaxel-loaded, poly (lactic-co-glycolic acid) (PLGA) nanoparticle using an emulsion-diffusion-evaporation technique. Bis (sulfosuccinimidyl) suberate (BS3) was non-covalently inserted into the nanoparticle as a linker for the conjugation of a bone-targeting moiety to the outside of the nanoparticle. I hypothesized that the nanoparticles would have the ability to inhibit the epithelial-to-mesenchymal transition (EMT), invasion, and migration in prostate cancer cells. Targeted, cabazitaxel-loaded nanoparticles attenuate the EMT marker, Vimentin, and lead to an increase in E-cadherin expression, which imparts epithelial characteristics to cells and inhibits cancer progression of advanced prostate cancer to distant sites. I observed the reduction of phosphorylated Src at tyrosine 416 along with increased expression of phosphorylated cofilin, cofilin at serine 3, which could affect migration and invasion pathways in cancer cells. Both increased expression of p-120 catenin and inhibition in IL-8 expression were seen in targeted, cabazitaxel-loaded nanoparticles. Overall, our data show that the targeted, cabazitaxel-loaded nanoparticles can act as a promising treatment for prostate cancer by inhibiting invasion, migration, and EMT in prostate cancer cells. Finally, I discuss an ongoing project that also targets bone metastatic PCa with a different strategy, a liposomal drug delivery system.Item Photophysical characterization of oligopeptide linked FRET system in PVA matrix and buffer to detect levels of matrix metalloprotease-9.(2017-03-14) Mandecki, Wlodeck; Li, Ji; Gryczynski, Zygmunt; Borejdo, Julian; Gryczynski, Ignacy; Fudala, Rafal; Shah, Sunil AjitPurpose: Matrix metalloproteinases (MMP’s) are a group of zinc dependent peptidases which can be classified based on their structural differences. So far, over 26 MMP’s have been identified. Out of these, MMP-9 is of particular interest in many biomedical applications. MMP-9, also known as gelatinase B, plays an important role in degrading the basement membrane of the extracellular matrix (ECM). Levels of MMP-9 have been found to be upregulated in several types of cancer, including breast, bladder, colon, ovarian etc. and are generally associated with poor prognosis. The basement degradation activity of MMP-9 allows for tumor growth. Thus, the overall goal of this project is to develop applications for detecting MMP-9 enzyme levels. This would result in rapid, non-invasive detection, and possibly early treatment for several cancers. Approach: We can use Forster resonance energy transfer (FRET) to come up with a custom peptide that is cleaved by MMP-9 enzyme, leading to easy detection and diagnosis. FRET is a well-known phenomenon being used today in studying molecular interactions. Briefly; FRET is the energy transfer between two fluorophores when they are within 1-10nms of each other. The fluorophore with emission at the shorter wavelength acts as the donor, and instead of emitting florescence, transfers its energy to an acceptor molecule, whose emission is generally at a longer wavelength. It is a very sensitive technique which can be used as a precise measurement and detection tool. Materials and Methods: We were able to successfully demonstrate FRET with a custom peptide whose partial sequence was recognized and selectively cleaved by MMP-9 enzyme. The probe uses 5,6 TAMRA and HiLyte 647 as a donor and acceptor respectively. The target peptide sequence is Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys-NH2, and was optimized by Kridel et al. The fluorophores were attached to the peptide at the Ser-Leu bond, labeled on the ε-NH2 groups of lysine with donor (5, 6 TAMRA) and acceptor (HiLyte647) dye. Peptide labelled with 5,6-TAMRA only was used as the donor control, and free HiLyte 647 was used as the control for acceptor. The probe and donor control were dissolved in 10% (w/w) poly-vinyl alcohol, and dried on glass slides. This produced films 200 microns in thickness. Furthermore, the probe was dissolved in buffer and upon addition of MMP-9 enzyme, showed a gradual decrease in energy transfer over time. These measurements were done by using a 1cmx1cm quartz cuvette and a square geometry set-up with 470nm as the excitation wavelength. Results: Absorption spectra and other steady state measurements indicate successful energy transfer between donor and acceptor fluorophores which gradually reduces over time as it gets cleaved by MMP-9. This showed that the peptide is functional, and also being recognized and cleaved by MMP-9 enzyme. Conclusions: It is possible to synthesize a functional FRET probe that is selectively cleaved by the enzyme MMP-9, which shows elevated levels in several cancers due to its role in basement degradation. We successfully demonstrated using FRET as a precise technique to detect and measure MMP-9 enzyme activity.Item R21C Mutation in Cardiac Troponin I Imposes Differences in the Degree of Order and Kinetics of Myosin Cross-bridges of Left and Right Ventricles(2015-03) Duggal, Divya; Nagwekar, Janhavi; Rich, Ryan; Raut, Sangram; Fudala, Rafal; Das, Hriday; Gryczynski, Zygmunt; Gryczynski, Ignacy; Szczesna-Cordary, Danuta; Borejdo, JulianThe effect of the TnI R21C mutation in the human cardiac troponin I, the mutation that is linked to hypertrophic cardiomyopathy, on muscles of the left (LV) and right (RV) ventricles was examined in knock-in mice. Experiments probed 3-4 actin molecules in ex-vivo myofibrils prepared from LV and RV muscles. Control anisotropy experiments revealed that the orientation of actin reflected orientation of cross-bridges (XBs). It was found that the mutation imposed significant difference on the XB kinetics cycle of the LV and RV: XBs from RV displayed a 3-fold decrease in the rate of power stroke and a 2-fold decrease in the rate of dissociation from thin filaments as compared to LV. The mutation also imposed significant differences in the distribution of angles that actin makes with thin filament axis: during contraction, actin angles from LV were more tightly distributed compared to actin angles from RV. We speculate that molecular differences between ventricles are caused by inability of XBs to dissociate promptly from thin filaments. This work reveals phenotypic differences of the R21C mutation in the left versus right mouse ventricles even though both ventricles express the same isoform of the cardiac TnI and highlights the importance of functional differences between the two ventricles of cardiac disease.Item Sensing and imaging of hyaluronidase activity using a long-lived fluorophore(2016-05-01) Chib, Rahul; Gryczynski, Zygmunt; Fudala, Rafal; Borejdo, JulianThis dissertation explores the synthesis, characterization and biomedical applications of a fluorescent probe for sensing and imaging of hyaluronidase activity. The enzyme hyaluronidase is overexpressed in various cancer including bladder cancer, prostate cancer, melanoma, head and neck carcinoma etc. Fluorescence-based sensing and imaging have tremendous applications in biomedical sciences. A fluorescent probe specific to a disease biomarker can help in the diagnosis and treatment of various diseases like cancer. Fluorescence emission in the red region of the electromagnetic spectrum provides the best optical window for sensing and imaging, as the contribution of autofluorescence decreases in this region. To distinguish the signal from the fluorophore and autofluorescence, efforts have been focused on developing red-emitting fluorophores, preferentially with a long fluorescence lifetime (significantly longer than autofluorescence). This improves signal-to-noise ratio and opens the possibility for time-gated detection. However, the commercially available red fluorophores have a very short fluorescence lifetime. The groups of currently developed triangulenium fluorophores like Azadioxatriangulenium (ADOTA), which emits in the orange/red region with a long fluorescence lifetime and high quantum yield presents great opportunities for sensing and imaging applications. The goal of this study was to characterize the photophysical properties of Azadioxatriangulenium (ADOTA) fluorophore and explore its properties for biomedical sensing and imaging. A sensor for the enzyme hyaluronidase was developed by using ADOTA fluorophore. This sensor was developed by heavy labeling of hyaluronic acid with ADOTA fluorophore. Results from these studies show the applications of ADOTA in fluorescence-based sensing, as a contrast agent in fluorescence-lifetime imaging microscopy (FLIM), and its application in time-gated detection for background-free cellular imaging.Item Single Molecule Fluorescence Studies of Ribosome Dynamics: An Application of Metal Enhanced Fluorescence(2010-05-01) Bharill, Shashank; Gryczynski, ZygmuntItem Spatial Distribution of Actin and Mechanical Cycle of Myosin are Different in Right and Left Ventricles of Healthy Mouse Hearts(2015-03) Nagwekar, Janhavi; Duggal, Divya; Rich, Ryan; Raut, Sangram; Fudala, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Borejdo, JulianThe contraction of the right ventricle (RV) expels blood into the pulmonary circulation, and the contraction of the left ventricle (LV) pumps blood into the systemic circulation through the aorta. The respective afterloads imposed on the LV and RV by aortic and pulmonary artery pressures create very different mechanical requirements for the two ventricles. In spite of these functional differences, it is commonly believed that the right and left ventricular muscles are identical because there were no differences in stress development, twitch duration, work performance and power among the RV and LV. This report shows that the two ventricles in rigor differ in the degree of orientational disorder of actin within thin filaments, and during contraction they differ in the kinetics of the cross-bridge cycle. Mouse ventricle muscle is the source of sample for experiments in this project. Glycerinated muscle bundles were homogenized and myofibrils were extracted. Myofibrils were labeled with 1 nM rhodamine-phalloidin (RP) + 10 nM unlabeled-phalloidin (UP) in Ca2+-rigor solution in the ratio of 1:1000 fluorescent to non-fluorescent phalloidin to ensure 1 in ~105actin monomers carry a fluorophore. Labeled myofibrils were analyzed for error of the mean of polarized fluorescence to determine kinetic rate constants in the ATPase cycle and distribution of orientations emanating from myosin cross-bridges. Histograms were plotted from the polarized fluorescence data and the Full Width at Half Maximum (FWHM) of the mean was calculated. The mean polarization of a contracting WT LV myofibril power stroke 0.159±0.086 was higher as compared to 0.085±0.035 for RV. Similarly, dissociation of myosin from actin was significantly faster in LV compared to RV. The FWHM of actins of RVs were significantly narrower (better ordered) than those of LVs which shows that the LV and RV of the heart are different. The study suggest that the differences in the rate constants during contraction and orientation of cross bridges during rigor signify the functional differences between left and right ventricles of the healthy mouse heart.