Lebanon, NH, United States
Lebanon, NH, United States

Time filter

Source Type

Franzen S.,Linköping University | Pihl L.,Linköping University | Khan N.,Center for Viable Systems | Palm F.,Linköping University | And 2 more authors.
Advances in Experimental Medicine and Biology | Year: 2014

Intrarenal oxygenation is heterogeneous with oxygen levels normally being highest in the superficial cortex and lowest in the inner medulla. Reduced intrarenal oxygenation has been implied in the pathology of several kidney diseases. However, there is currently no method available to repetitively monitor regional renal oxygenation using minimally invasive procedures. We therefore evaluated implantable lithium phthalocyanine (LiPc) probes, which display a close correlation between EPR line width and oxygen availability. LiPc probes were implanted in the kidney cortex and medulla in the same mouse and EPR spectra were acquired using a L band scanner during inhalation of air (21% oxygen) or a mixture of air and nitrogen (10% oxygen). In order to separate the signals from the two probes, a 1 G/cm gradient was applied and the signals were derived from 40 consecutive sweeps. Peak-to-peak comparison of the EPR line was used to convert the signal to an approximate oxygen tension in MATLAB. Kidney cortex as well as medullary oxygenation was stable over the 45 day period (cortex 56 ± 7 mmHg and medulla 43 ± 6 mmHg). However, 10% oxygen inhalation significantly reduced oxygenation in both cortex (56 ± 6 to 34 ± 2 mmHg n = 15 p < 0.05) and medulla (42 ± 5 to 29 ± 3 mmHg n = 7 p < 0.05). In conclusion, L band EPR using LiPc probes implanted in discrete intrarenal structures can be used to repetitively monitor regional renal oxygenation. This minimally invasive method is especially well suited for conditions of reduced intrarenal oxygenation since this increases the signal intensity which facilitates the quantification of the EPR signal to absolute oxygenation values. © Springer Science+Business Media, LLC 2014.


Khan N.,Center for Viable Systems | Khan N.,Norris Cotton Cancer Center | Hou H.,Center for Viable Systems | Hou H.,Norris Cotton Cancer Center | And 7 more authors.
Advances in Experimental Medicine and Biology | Year: 2014

A lack of strategy to counteract hypoxia (pO2 < 10–15 mmHg) and technique to repeatedly measure tumor pO2 has restricted therapeutic optimization. We report the results obtained with an innovative anti-angiogenic strategy of recurrent low-dose (metronomic) chemotherapy to modulate hypoxia and growth of the Head and Neck tumor xenografts. The FaDu tumors were established in the fl ank of immune defi cient mice and EPR oximetry with lithium phthalocyanine crystals was used to follow the temporal changes in tumor pO2 on treatment with gemcitabine including controls for three weeks. The FaDu tumors were hypoxic with a baseline (pre-treatment) pO2 of 2–8 mmHg. A transient increase in the tumor pO2 was evident on day 3 on treatment with a conventional schedule of gemcitabine (150 mg/kg, d1, d8, d15). No signifi cant change in the tumor pO2 on treatment with metronomic gemcitabine (25 mg/kg on d1, d3, d5 for 3 weeks) was observed. However, tumor pO2 increased signifi cantly on d15–d18 during treatment with a metronomic schedule of 15 mg/kg gemcitabine (d1, d3, d5 for 3 weeks). A modest decrease in the tumor growth was evident on treatment with conventional gemcitabine. Notably, tumor growth was signifi cantly inhibited by metronomic (25 and 15 mg/kg) gemcitabine treatment. The immunohistochemistry (IHC) analyses of the tumor samples indicate a decrease in HIF-1α and TSP-1 on treatment with metronomic gemcitabine. In conclusion, a signifi cant inhibition of tumor growth on treatment with metronomic gemcitabine was observed; however, the increase in pO2 was dose dependent. EPR oximetry can be used to follow the temporal changes in tumor pO2 to identify a therapeutic window on treatment with metronomic chemotherapy for potential combination with radiotherapy. © Springer Science+Business Media, LLC 2014.


Snider G.W.,University of Vermont | Ruggles E.,University of Vermont | Khan N.,Center for Viable Systems | Hondal R.J.,University of Vermont
Biochemistry | Year: 2013

Mammalian thioredoxin reductase (TR) is a selenocysteine (Sec)-containing homodimeric pyridine nucleotide oxidoreductase which catalyzes the reduction of oxidized thioredoxin. We have previously demonstrated the full-length mitochondrial mammalian TR (mTR3) enzyme to be resistant to inactivation from exposure to 50 mM H2O2. Because a Sec residue oxidizes more rapidly than a cysteine (Cys) residue, it has been previously thought that Sec-containing enzymes are "sensitive to oxidation" compared to Cys-orthologues. Here we show for the first time a direct comparison of the abilities of Sec-containing mTR3 and the Cys-orthologue from D. melanogaster (DmTR) to resist inactivation by oxidation from a variety of oxidants including H2O2, hydroxyl radical, peroxynitrite, hypochlorous acid, hypobromous acid, and hypothiocyanous acid. The results show that the Sec-containing TR is far superior to the Cys-orthologue TR in resisting inactivation by oxidation. To further test our hypothesis that the use of Sec confers strong resistance to inactivation by oxidation, we constructed a chimeric enzyme in which we replaced the active site Cys nucleophile of DmTR with a Sec residue using semisynthesis. The chimeric Sec-containing enzyme has similar ability to resist inactivation by oxidation as the wild type Sec-containing TR from mouse mitochondria. The use of Sec in the chimeric enzyme "rescued" the enzyme from oxidant-induced inactivation for all of the oxidants tested in this study, in direct contrast to previous understanding. We discuss two possibilities for this rescue effect from inactivation under identical conditions of oxidative stress: (i) Sec resists overoxidation and inactivation, whereas a Cys residue can be permanently overoxidized to the sulfinic acid form, and (ii) Sec protects the body of the enzyme from harmful oxidation by allowing the enzyme to metabolize (turnover) various oxidants much better than a Cys-containing TR. © 2013 American Chemical Society.


Hou H.,Dartmouth Hitchcock Medical Center | Hou H.,Center for Viable Systems | Khan N.,Dartmouth Hitchcock Medical Center | Chen E.Y.,Dartmouth Hitchcock Medical Center | And 5 more authors.
Advances in Experimental Medicine and Biology | Year: 2014

The feasibility of EPR oximetry using a single-probe implantable oxygen sensor (ImOS) was tested for repeated measurement of pO2 in skeletal muscle and ectopic 9L tumors in rats. The ImOS (50 mm length) were constructed using nickel– chromium alloy wires, with lithium phthalocyanine (LiPc, oximetry probe) crystals loaded in the sensor loop and coated with AF 2400 ® Tefl on. These ImOS were implanted into the skeletal muscle in the thigh and subcutaneous 9L tumors. Dynamic changes in tissue pO2 were assessed by EPR oximetry at baseline, during tumor growth, and repeated hyperoxygenation with carbogen breathing. The mean skeletal muscle pO2 of normal rats was stable and signifi cantly increased during carbogen inhalation in experiments repeated for 12 weeks. The 9L tumors were hypoxic with a tissue pO2 of 12.8 ± 6.4 mmHg on day 1; however, the response to carbogen inhalation varied among the animals. A signifi cant increase in the glioma pO2 was observed during carbogen inhalation on day 9 and day 14 only. In summary, EPR oximetry with ImOS allowed direct and longitudinal oxygen measurements in deep muscle tissue and tumors. The heterogeneity of 9L tumors in response to carbogen highlights the need to repeatedly monitor pO2 to confi rm tumor oxygenation so that such changes can be taken into account in planning therapies and interpreting results. © Springer Science+Business Media, LLC 2014.


Constantinou C.,Harvard University | Constantinou C.,University of Patras | Apidianakis Y.,Harvard University | Psychogios N.,Harvard University | And 8 more authors.
International Journal of Molecular Medicine | Year: 2016

Trauma is the most common cause of mortality among individuals aged between 1 and 44 years and the third leading cause of mortality overall in the US. In this study, we examined the effects of trauma on the expression of genes in Drosophila melanogaster, a useful model for investigating genetics and physiology. After trauma was induced by a non-lethal needle puncture of the thorax, we observed the differential expression of genes encoding for mitochondrial uncoupling proteins, as well as those encoding for apoptosis-related and insulin signaling-related proteins, thus indicating muscle functional dysregulation. These results prompted us to examine the link between insulin signaling and mitochondrial dysfunction using in vivo nuclear magnetic resonance (NMR) with complementary electron paramagnetic resonance (EPR) spectroscopy. Trauma significantly increased insulin resistance biomarkers, and the NMR spectral profile of the aged flies with trauma-induced thoracic injury resembled that of insulin-resistant chico mutant flies. In addition, the mitochondrial redox status, as measured by EPR, was significantly altered following trauma, indicating mitochondrial uncoupling. A mitochondria-targeted compound, Szeto-Schiller (SS)-31 that promotes adenosine triphosphate (ATP) synthesis normalized the NMR spectral profile, as well as the mitochondrial redox status of the flies with trauma-induced thoracic injury, as assessed by EPR. Based on these findings, we propose a molecular mechanism responsible for trauma-related mortality and also propose that trauma sequelae in aging are linked to insulin signaling and mitochondrial dysfunction. Our findings further suggest that SS-31 attenuates trauma-associated pathological changes.


Hashem M.,Technion - Israel Institute of Technology | Weiler-Sagie M.,Weizmann Institute of Science | Kuppusamy P.,Center for Viable Systems | Neufeld G.,Technion - Israel Institute of Technology | And 2 more authors.
Journal of Magnetic Resonance | Year: 2015

Oxygen (O2) plays a central role in most living organisms. The concentration of O2 is important in physiology and pathology. Despite the importance of accurate knowledge of the O2 levels, there is very limited capability to measure with high spatial resolution its distribution in millimeter-scale live biological samples. Many of the current oximetric methods, such as oxygen microelectrodes and fluorescence lifetime imaging, are compromised by O2 consumption, sample destruction, invasiveness, and difficulty to calibrate. Here, we present a new method, based on the use of the pulsed electron spin resonance (ESR) microimaging technique to obtain a 3D mapping of oxygen concentration in millimeter-scale biological samples. ESR imaging requires the incorporation of a suitable stable and inert paramagnetic spin probe into the desirable object. In this work, we use microcrystals of a paramagnetic spin probe in a new crystallographic packing form (denoted tg-LiNc-BuO). These paramagnetic species interact with paramagnetic oxygen molecules, causing a spectral line broadening that is linearly proportional to the oxygen concentration. Typical ESR results include 4D spatial-spectral images that give an indication about the oxygen concentration in different regions of the sample. This new oximetry microimaging method addresses all the problems mentioned above. It is noninvasive, sensitive to physiological oxygen levels, and easy to calibrate. Furthermore, in principle, it can be used for repetitive measurements without causing cell damage. The tissue model used in this research is spheroids of Human Colorectal carcinoma cell line (HCT-116) with a typical diameter of ∼600 μm. Most studies of the microenvironmental O2 conditions inside such viable spheroids carried out in the past used microelectrodes, which require an invasive puncturing of the spheroid and are also not applicable to 3D O2 imaging. High resolution 3D oxygen maps could make it possible to evaluate the relationship between morphological and physiological alterations in the spheroids, which would help understand the oxygen metabolism in solid tumors and its correlation with the susceptibility of tumors to various oncologic treatments. © 2015 Elsevier Inc. All rights reserved.


PubMed | Stanford University, Cornell University, Center for Viable Systems and Harvard University
Type: Journal Article | Journal: International journal of molecular medicine | Year: 2016

Trauma is the most common cause of mortality among individuals aged between 1 and 44 years and the third leading cause of mortality overall in the US. In this study, we examined the effects of trauma on the expression of genes in Drosophila melanogaster, a useful model for investigating genetics and physiology. After trauma was induced by a non-lethal needle puncture of the thorax, we observed the differential expression of genes encoding for mitochondrial uncoupling proteins, as well as those encoding for apoptosis-related and insulin signaling-related proteins, thus indicating muscle functional dysregulation. These results prompted us to examine the link between insulin signaling and mitochondrial dysfunction using in vivo nuclear magnetic resonance (NMR) with complementary electron paramagnetic resonance (EPR) spectroscopy. Trauma significantly increased insulin resistance biomarkers, and the NMR spectral profile of the aged flies with trauma-induced thoracic injury resembled that of insulin-resistant chico mutant flies. In addition, the mitochondrial redox status, as measured by EPR, was significantly altered following trauma, indicating mitochondrial uncoupling. A mitochondria-targeted compound, Szeto-Schiller (SS)-31 that promotes adenosine triphosphate (ATP) synthesis normalized the NMR spectral profile, as well as the mitochondrial redox status of the flies with trauma-induced thoracic injury, as assessed by EPR. Based on these findings, we propose a molecular mechanism responsible for trauma-related mortality and also propose that trauma sequelae in aging are linked to insulin signaling and mitochondrial dysfunction. Our findings further suggest that SS-31 attenuates trauma-associated pathological changes.


PubMed | Dartmouth Hitchcock Medical Center and Center for Viable Systems
Type: Journal Article | Journal: Medical gas research | Year: 2016

The effect of hyperoxygenation with carbogen (95% O


PubMed | Center for Viable Systems and Pharmacology & Toxicology
Type: | Journal: Clinical cancer research : an official journal of the American Association for Cancer Research | Year: 2016

Determine the roles of the phosphatidylinositol 3-kinase (PI3K) isoforms p110 and p110 in PTEN-deficient, estrogen receptor (ER)-positive breast cancer, and the therapeutic potential of isoform-selective inhibitors.Anti-estrogen-sensitive and -resistant PTEN-deficient, ER+ human breast cancer cell lines, and mice bearing anti-estrogen-resistant xenografts were treated with the anti-estrogen fulvestrant, the p110 inhibitor BYL719, the p110 inhibitor GSK2636771, or combinations. Temporal response to growth factor receptor-initiated signaling, growth, apoptosis, predictive biomarkers, and tumor volumes were measured.p110 primed cells for response to growth factor stimulation. While p110 inhibition suppressed cell and tumor growth, dual targeting of p110/ enhanced apoptosis and provided sustained tumor response. The growth of anti-estrogen-sensitive cells was inhibited by fulvestrant, but fulvestrant inconsistently provided additional therapeutic effects beyond PI3K inhibition alone. Treatment-induced decreases in phosphorylation of AKT and Rb were predictive of therapeutic response. Short-term drug treatment induced tumor cell apoptosis and proliferative arrest to induce tumor regression, while long-term treatment only suppressed proliferation to provide durable regression.p110 is the dominant PI3K isoform in PTEN-deficient, ER+ breast cancer cells. Upon p110 inhibition, p110 did not induce significant reactivation of AKT, but combined targeting of p110/ most effectively induced apoptosis in vitro and in vivo and provided durable tumor regression. Since apoptosis and tumor regression occurred early but not late in the treatment course, and proliferative arrest was maintained throughout treatment, p110/ inhibitors may be considered short-term cytotoxic agents and long-term cytostatic agents.


PubMed | Center for Viable Systems
Type: Journal Article | Journal: Molecular medicine reports | Year: 2011

Using a mouse model, we tested the hypotheses that severe burn trauma causes metabolic disturbances in skeletal muscle, and that these can be measured and repeatedly followed by in vivo electron paramagnetic resonance (EPR). We used a 1.2-GHz (L-band) EPR spectrometer to measure partial pressure of oxygen (pO(2)) levels, redox status and oxidative stress following a non-lethal burn trauma model to the left hind limbs of mice. Results obtained in the burned mouse gastrocnemius muscle indicated a significant decrease in tissue pO(2) immediately (P=0.032) and at 6 h post burn (P=0.004), compared to the gastrocnemius of the unburned hind limb. The redox status of the skeletal muscle also peaked at 6 h post burn (P=0.027) in burned mice. In addition, there was an increase in the EPR signal of the nitroxide produced by oxidation of the hydroxylamine (CP-H) probe at 12 h post burn injury, indicating a burn-induced increase in mitochondrial reactive oxygen species (ROS). The nitroxide signal continued to increase between 12 and 24 h, suggesting a further increase in ROS generation post burn. These results confirm genomic results, which indicate a downregulation of antioxidant genes and therefore strongly suggest the dysfunction of the mitochondrial oxidative system. We believe that the direct measurement of tissue parameters such as pO(2), redox and ROS by EPR may be used to complement measurements by nuclear magnetic resonance (NMR) in order to assess tissue damage and the therapeutic effectiveness of antioxidant agents in severe burn trauma.

Loading Center for Viable Systems collaborators
Loading Center for Viable Systems collaborators