Publications by authors named "Tanmay Kulkarni"

14 Publications

  • Page 1 of 1

Nanomechanical Insight of Pancreatic Cancer Cell Membrane during Receptor Mediated Endocytosis of Targeted Gold Nanoparticles.

ACS Appl Bio Mater 2021 Jan 30;4(1):984-994. Epub 2020 Dec 30.

Department of Biochemistry and Molecular Biology and Department of Physiology and Biomedical Engineering, Mayo College of Medicine and Science, Jacksonville, Florida 32224, United States.

Nanoscale alterations in the cellular membrane transpire during cellular interactions with the extracellular environment through the endocytosis processes. Although the biological innuendos as well as alterations in cellular morphology during endocytosis are well-known, nanomechanical amendments in the cellular membrane are poorly understood. In this manuscript, atomic force microscope is employed to demonstrate the nanomechanical alterations in membrane dynamics during receptor mediated endocytosis of gold nanoparticles conjugated with either plectin-1 targeted peptide (PTP-GNP) or scrambled peptide (sPEP-GNP). Plectin-1 is aberrantly overexpressed at cell membrane of pancreatic cancer cells and is known to provide and maintain cellular mechanical integrity. During receptor mediated endocytosis of nanoparticles, we demonstrate temporal nanomechanical changes of cell membrane in both immortal pancreatic cancer Panc1 cells and patient derived primary pancreatic cancer cell, 4911. We further confirm the alterations of plectin-1 expression in Panc1 cell membrane during the receptor mediated endocytosis using classical streptavidin-biotin reaction and establish its association with nanomechanical alteration in membrane dynamics. Withdrawal of PTP-GNPs from the cell culture restores the plectin-1 expression at the membrane and reverses the mechanical properties of Panc1. We also show a distinctly opposite trend in nanomechanical behavior in cancer and endothelial cells when treated with sPEP-GNP and PTP-GNP, respectively, signifying receptor independent endocytosis process. This study illustrates the nanomechanical perspective of cell membrane in receptor mediated endocytosis of nanoparticles designed for organ specific drug delivery.
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http://dx.doi.org/10.1021/acsabm.0c01443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8670601PMC
January 2021

Dissecting VEGF-induced acute versus chronic vascular hyperpermeability: Essential roles of dimethylarginine dimethylaminohydrolase-1.

iScience 2021 Oct 30;24(10):103189. Epub 2021 Sep 30.

Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, 4500 San Pablo Road South, Jacksonville, FL 32224, USA.

Vascular endothelial cell growth factor (VEGF) is a key regulator of vascular permeability. Herein we aim to understand how acute and chronic exposures of VEGF induce different levels of vascular permeability. We demonstrate that chronic VEGF exposure leads to decreased phosphorylation of VEGFR2 and c-Src as well as steady increases of nitric oxide (NO) as compared to that of acute exposure. Utilizing heat-inducible VEGF transgenic zebrafish () and establishing an algorithm incorporating segmentation techniques for quantification, we monitored acute and chronic VEGF-induced vascular hyperpermeability in real time. Importantly, dimethylarginine dimethylaminohydrolase-1 (DDAH1), an enzyme essential for NO generation, was shown to play essential roles in both acute and chronic vascular permeability in cultured human cells, zebrafish model, and Miles assay. Taken together, our data reveal acute and chronic VEGF exposures induce divergent signaling pathways and identify DDAH1 as a critical player and potentially a therapeutic target of vascular hyperpermeability-mediated pathogenesis.
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http://dx.doi.org/10.1016/j.isci.2021.103189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8521174PMC
October 2021

A Fully Integrated Online Platform For Real Time Monitoring Of Multiple Product Quality Attributes In Biopharmaceutical Processes For Monoclonal Antibody Therapeutics.

J Pharm Sci 2022 Feb 14;111(2):358-367. Epub 2021 Sep 14.

CMC Analytical, Product Development & Supply, GlaxoSmithKline, PA 19406, United States.

In response to FDA's call for Quality by Design (QbD) in biopharmaceutical product development, the biopharmaceutical industry has been developing highly sensitive and specific technologies in the monitoring and controlling of product quality attributes for bioprocesses. We previously published the successful application of an off-line multi-attribute method (MAM) to monitor more than 20 critical quality attributes (CQA) with superior sensitivity for the upstream process. To further remove the hurdles of laborious process sampling and sample preparation associated with the offline method, we present here a fully integrated MAM based online platform for automated real time online process monitoring. This integrated system includes Modular Automated Sampling Technology (MAST) based aseptic sampling, multi-function Sequential Injection Analysis (SIA) sample preparation, UHPLC separation and high-resolution mass spectrometry (HRMS) analysis. Continuous automated daily monitoring of a 17-day cell culture process was successfully demonstrated for a model monoclonal antibody (mAb) molecule with similar specificity and sensitivity as we reported earlier. To the best of our knowledge, this is the first report of an end-to-end automated online MAM system, which would allow the MAM to be applied to routine bioprocess monitoring, potentially replacing multiple conventional low resolution and low sensitivity off-line methods. The online HPLC or HPLC/MS platform could be easily adapted to support other processing steps such as downstream purification with minimal software re-configuration.
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http://dx.doi.org/10.1016/j.xphs.2021.09.011DOI Listing
February 2022

The Immunopathobiology of SARS-CoV-2 Infection.

FEMS Microbiol Rev 2021 11;45(6)

Department of Pharmacology and Experimental Neuroscience, College of Medicine, University of Nebraska Medical Center, NE 68198, USA.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can lead to coronavirus disease 2019 (COVID-19). Virus-specific immunity controls infection, transmission and disease severity. With respect to disease severity, a spectrum of clinical outcomes occur associated with age, genetics, comorbidities and immune responses in an infected person. Dysfunctions in innate and adaptive immunity commonly follow viral infection. These are heralded by altered innate mononuclear phagocyte differentiation, activation, intracellular killing and adaptive memory, effector, and regulatory T cell responses. All of such affect viral clearance and the progression of end-organ disease. Failures to produce effective controlled antiviral immunity leads to life-threatening end-organ disease that is typified by the acute respiratory distress syndrome. The most effective means to contain SARS-CoV-2 infection is by vaccination. While an arsenal of immunomodulators were developed for control of viral infection and subsequent COVID-19 disease, further research is required to enable therapeutic implementation.
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http://dx.doi.org/10.1093/femsre/fuab035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8632753PMC
November 2021

Lipophilic nanocrystal prodrug-release defines the extended pharmacokinetic profiles of a year-long cabotegravir.

Nat Commun 2021 06 8;12(1):3453. Epub 2021 Jun 8.

Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA.

A once every eight-week cabotegravir (CAB) long-acting parenteral is more effective than daily oral emtricitabine and tenofovir disoproxil fumarate in preventing human immunodeficiency virus type one (HIV-1) transmission. Extending CAB dosing to a yearly injectable advances efforts for the elimination of viral transmission. Here we report rigor, reproducibility and mechanistic insights for a year-long CAB injectable. Pharmacokinetic (PK) profiles of this nanoformulated CAB prodrug (NM2CAB) are affirmed at three independent research laboratories. PK profiles in mice and rats show plasma CAB levels at or above the protein-adjusted 90% inhibitory concentration for a year after a single dose. Sustained native and prodrug concentrations are at the muscle injection site and in lymphoid tissues. The results parallel NM2CAB uptake and retention in human macrophages. NM2CAB nanocrystals are stable in blood and tissue homogenates. The long apparent drug half-life follows pH-dependent prodrug hydrolysis upon slow prodrug nanocrystal dissolution and absorption. In contrast, solubilized prodrug is hydrolyzed in hours in plasma and tissues from multiple mammalian species. No toxicities are observed in animals. These results affirm the pharmacological properties and extended apparent half-life for a nanoformulated CAB prodrug. The report serves to support the mechanistic design for drug formulation safety, rigor and reproducibility.
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http://dx.doi.org/10.1038/s41467-021-23668-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8187380PMC
June 2021

Myosin 10 Regulates Invasion, Mitosis, and Metabolic Signaling in Glioblastoma.

iScience 2020 Dec 13;23(12):101802. Epub 2020 Nov 13.

Department of Cancer Biology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224, USA.

Invasion and proliferation are defining phenotypes of cancer, and in glioblastoma blocking one stimulates the other, implying that effective therapy must inhibit both, ideally through a single target that is also dispensable for normal tissue function. The molecular motor myosin 10 meets these criteria. Myosin 10 knockout mice can survive to adulthood, implying that normal cells can compensate for its loss; its deletion impairs invasion, slows proliferation, and prolongs survival in murine models of glioblastoma. Myosin 10 deletion also enhances tumor dependency on the DNA damage and the metabolic stress responses and induces synthetic lethality when combined with inhibitors of these processes. Our results thus demonstrate that targeting myosin 10 is active against glioblastoma by itself, synergizes with other clinically available therapeutics, may have acceptable side effects in normal tissues, and has potential as a heretofore unexplored therapeutic approach for this disease.
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http://dx.doi.org/10.1016/j.isci.2020.101802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702012PMC
December 2020

A year-long extended release nanoformulated cabotegravir prodrug.

Nat Mater 2020 08 27;19(8):910-920. Epub 2020 Apr 27.

Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA.

Long-acting cabotegravir (CAB) extends antiretroviral drug administration from daily to monthly. However, dosing volumes, injection site reactions and health-care oversight are obstacles towards a broad usage. The creation of poloxamer-coated hydrophobic and lipophilic CAB prodrugs with controlled hydrolysis and tissue penetrance can overcome these obstacles. To such ends, fatty acid ester CAB nanocrystal prodrugs with 14, 18 and 22 added carbon chains were encased in biocompatible surfactants named NMCAB, NM2CAB and NM3CAB and tested for drug release, activation, cytotoxicity, antiretroviral activities, pharmacokinetics and biodistribution. Pharmacokinetics studies, performed in mice and rhesus macaques, with the lead 18-carbon ester chain NM2CAB, showed plasma CAB levels above the protein-adjusted 90% inhibitory concentration for up to a year. NM2CAB, compared with NMCAB and NM3CAB, demonstrated a prolonged drug release, plasma circulation time and tissue drug concentrations after a single 45 mg per kg body weight intramuscular injection. These prodrug modifications could substantially improve CAB's effectiveness.
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http://dx.doi.org/10.1038/s41563-020-0674-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384935PMC
August 2020

Nanomechanical insights: Amyloid beta oligomer-induced senescent brain endothelial cells.

Biochim Biophys Acta Biomembr 2019 12 9;1861(12):183061. Epub 2019 Sep 9.

Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, Jacksonville, FL, USA; Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine and Science, Jacksonville, FL, USA. Electronic address:

Senescent cells accumulate in various peripheral tissues during aging and have been shown to exacerbate age-related inflammatory responses. We recently showed that exposure to neurotoxic amyloid β (Aβ1-42) oligomers can readily induce a senescence phenotype in human brain microvascular endothelial cells (HBMECs). In the present work, we used atomic force microscopy (AFM) to further characterize the morphological properties such as cell membrane roughness and cell height and nanomechanical properties such as Young's modulus of the membrane (membrane stiffness) and adhesion resulting from the interaction between AFM tip and cell membrane in Aβ1-42 oligomer-induced senescent human brain microvascular endothelial cells. Morphological imaging studies showed a flatter and spread-out nucleus in the senescent HBMECs, both characteristic features of a senescent phenotype. Furthermore, the mean cell body roughness and mean cell height were lower in senescent HBMECs compared to untreated normal HBMECs. We also observed increased stiffness and alterations in the adhesion properties in Aβ1-42 oligomer-induced senescent endothelial cells compared to the untreated normal HBMECs suggesting dynamic reorganization of cell membrane. We then show that vascular endothelial growth factor receptor 1 (VEGFR-1) knockdown or overexpression of Rho GTPase Rac 1 in the endothelial cells inhibited senescence and reversed these nanomechanical alterations, confirming a direct role of these pathways in the senescent brain endothelial cells. These results illustrate that nanoindentation and topographic analysis of live senescent brain endothelial cells can provide insights into cerebrovascular dysfunction in neurodegenerative diseases such as Alzheimer's disease.
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http://dx.doi.org/10.1016/j.bbamem.2019.183061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791778PMC
December 2019

AFM study: Cell cycle and probe geometry influences nanomechanical characterization of Panc1 cells.

Biochim Biophys Acta Gen Subj 2019 05 11;1863(5):802-812. Epub 2019 Feb 11.

Department of Biochemistry and Molecular Biology, Mayo Clinic, Jacksonville, FL, USA; Department of Pathology and Biomedical Engineering, Mayo Clinic, Jacksonville, FL, USA. Electronic address:

Atomic force microscope (AFM) is emerging as an immensely promising tool to study the cellular morphology with a nanometer scale resolution and to analyze nanomechanical properties (NPs) at various physiological conditions. Advancement of AFM technology enables studying living cells and differentiating cancer cell from normal cells based on topography and NPs. Though the trend overlaps from different literature; numerical values of nanomechanical readouts depict variations over a wide range. These anomalies are associated with the experimental setup under study. In this manuscript, we have identified heterogeneity in cell culture system in addition to the selection of AFM probe with specific tip geometry as the major contributors to the above mentioned anomalies. To test our hypothesis, we have used Panc1 cells, which is a pancreatic ductal adenocarcinoma cell type. Our results suggest that the cellular morphology, membrane roughness and NPs calculated from AFM study are distinctly influenced by cell cycle. Furthermore, we found that the NPs readout is also significantly associated with AFM tip geometries. The cells were found to be softer in their early resting phase when indented with pyramidal probe and became increasingly stiffer as they progressed through the cell cycles. On the contrary, when indented with the spherical probe, cells in G0/G1 phase were observed to be the stiffest. Such an exhaustive study of the role of cell cycle in influencing the NPs in Panc1 cell line along with the impact of tip geometry on NPs is the first of its kind, to the best of our knowledge.
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http://dx.doi.org/10.1016/j.bbagen.2019.02.006DOI Listing
May 2019

A self-powered glucose biosensor based on pyrolloquinoline quinone glucose dehydrogenase and bilirubin oxidase operating under physiological conditions.

Annu Int Conf IEEE Eng Med Biol Soc 2017 Jul;2017:5-8

A novel biosensing system capable of simultaneously sensing glucose and powering portable electronic devices such as a digital glucometer is described. The biosensing system consists of enzymatic glucose biofuel cell bioelectrodes functionalized with pyrolloquinoline quinone glucose dehydrogenase (PQQ-GDH) and bilirubin oxidase (BOD) at the bioanode and biocathode, respectively. A dual-stage power amplification circuit is integrated with the single biofuel cell to amplify the electrical power generated. In addition, a capacitor circuit was incorporated to serve as the transducer for sensing glucose. The open circuit voltage of the optimized biofuel cell reached 0.55 V, and the maximum power density achieved was 0.23 mW/ cm at 0.29 V. The biofuel cell exhibited a sensitivity of 0.312 mW/mM.cm with a linear dynamic range of 3 mM - 20 mM glucose. The overall self-powered glucose biosensor is capable of selectively screening against common interfering species, such as ascorbate and urate and exhibited an operational stability of over 53 days, while maintaining 90 % of its activity. These results demonstrate the system's potential to replace the current glucose monitoring devices that rely on external power supply, such as a battery.
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http://dx.doi.org/10.1109/EMBC.2017.8036749DOI Listing
July 2017

Highly Selective and Sensitive Self-Powered Glucose Sensor Based on Capacitor Circuit.

Sci Rep 2017 05 3;7(1):1471. Epub 2017 May 3.

University of Maryland Baltimore County and Bioelectronics Laboratory, Department of Computer Science and Electrical Engineering, Maryland, USA.

Enzymatic glucose biosensors are being developed to incorporate nanoscale materials with the biological recognition elements to assist in the rapid and sensitive detection of glucose. Here we present a highly sensitive and selective glucose sensor based on capacitor circuit that is capable of selectively sensing glucose while simultaneously powering a small microelectronic device. Multi-walled carbon nanotubes (MWCNTs) is chemically modified with pyrroloquinoline quinone glucose dehydrogenase (PQQ-GDH) and bilirubin oxidase (BOD) at anode and cathode, respectively, in the biofuel cell arrangement. The input voltage (as low as 0.25 V) from the biofuel cell is converted to a stepped-up power and charged to the capacitor to the voltage of 1.8 V. The frequency of the charge/discharge cycle of the capacitor corresponded to the oxidation of glucose. The biofuel cell structure-based glucose sensor synergizes the advantages of both the glucose biosensor and biofuel cell. In addition, this glucose sensor favored a very high selectivity towards glucose in the presence of competing and non-competing analytes. It exhibited unprecedented sensitivity of 37.66 Hz/mM.cm and a linear range of 1 to 20 mM. This innovative self-powered glucose sensor opens new doors for implementation of biofuel cells and capacitor circuits for medical diagnosis and powering therapeutic devices.
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http://dx.doi.org/10.1038/s41598-017-01665-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5431189PMC
May 2017

Application of Semipermeable Membranes in Glucose Biosensing.

Membranes (Basel) 2016 Dec 14;6(4). Epub 2016 Dec 14.

Bioelectronics Laboratory, Department of Computer Science and Electrical Engineering, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA.

Glucose biosensors have received significant attention in recent years due to the escalating mortality rate of diabetes mellitus. Although there is currently no cure for diabetes mellitus, individuals living with diabetes can lead a normal life by maintaining tight control of their blood glucose levels using glucose biosensors (e.g., glucometers). Current research in the field is focused on the optimization and improvement in the performance of glucose biosensors by employing a variety of glucose selective enzymes, mediators and semipermeable membranes to improve the electron transfer between the active center of the enzyme and the electrode substrate. Herein, we summarize the different semipermeable membranes used in the fabrication of the glucose biosensor, that result in improved biosensor sensitivity, selectivity, dynamic range, response time and stability.
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http://dx.doi.org/10.3390/membranes6040055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192411PMC
December 2016

A self-powered glucose biosensing system.

Biosens Bioelectron 2016 Apr 11;78:45-50. Epub 2015 Nov 11.

Bioelectronics Laboratory, Department of Computer Science and Electrical Engineering, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA.

A self-powered glucose biosensor (SPGS) system is fabricated and in vitro characterization of the power generation and charging frequency characteristics in glucose analyte are described. The bioelectrodes consist of compressed network of three-dimensional multi-walled carbon nanotubes with redox enzymes, pyroquinoline quinone glucose dehydrogenase (PQQ-GDH) and laccase functioning as the anodic and cathodic catalyst, respectively. When operated in 45 mM glucose, the biofuel cell exhibited an open circuit voltage and power density of 681.8 mV and 67.86 µW/cm(2) at 335 mV, respectively, with a current density of 202.2 µA/cm(2). Moreover, at physiological glucose concentration (5mM), the biofuel cell exhibits open circuit voltage and power density of 302.1 mV and 15.98 µW/cm(2) at 166.3 mV, respectively, with a current density of 100 µA/cm(2). The biofuel cell assembly produced a linear dynamic range of 0.5-45 mM glucose. These findings show that glucose biofuel cells can be further investigated in the development of a self-powered glucose biosensor by using a capacitor as the transducer element. By monitoring the capacitor charging frequencies, which are influenced by the concentration of the glucose analyte, a linear dynamic range of 0.5-35 mM glucose is observed. The operational stability of SPGS is monitored over a period of 63 days and is found to be stable with 15.38% and 11.76% drop in power density under continuous discharge in 10mM and 20mM glucose, respectively. These results demonstrate that SPGSs can simultaneously generate bioelectricity to power ultra-low powered devices and sense glucose.
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http://dx.doi.org/10.1016/j.bios.2015.11.022DOI Listing
April 2016

Hyperglycemic Challenge and Distribution of Adipose Tissue in Obese Baboons.

Int J Diabetol Vasc Dis Res 2014 Feb;2(1)

Texas Biomedical Research Institute, San Antonio, TX ; Department of Nutrition and Nutrition Research Institute, University of North Carolina at Chapel Hill, Kannapolis, NC.

Background: Blood glucose levels regulate the rate of insulin secretion, which is the body's mechanism for preventing excessive elevation in blood glucose. Impaired glucose metabolism and insulin resistance have been linked to excess body fat composition. Here, we quantify abdominal muscle and abdominal adipose tissue compartments in a large nonhuman primate, the baboon, and investigate their relationship with serum glucose response to a hyperglycemic challenge.

Methods: Five female baboons were fasted for 16 hours prior to 90 minute body imaging experiment that consisted of a 20-min baseline, followed by a bolus infusion of glucose (500mg/kg). The blood glucose was sampled at regular intervals. The total volumes of the muscle, visceral and subcutaneous adipose tissue were measured.

Results And Discussion: We found that adipose tissue composition predicted fluctuations in glucose responses to a hyperglycemic challenge of a non-human primate. Animals with higher visceral adiposity showed significantly reduced glucose elimination. The glucose responses were positively correlated with body weight, visceral and muscle fat (p < 0.005). Polynomial regression analysis showed that body weight, visceral and muscle were significant.

Conclusions: These results reveal the similarity between humans and baboons with respect to glucose metabolism and strengthen the utility of baboon for biomedical research.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4241571PMC
February 2014
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