This document describes a new method for measuring bufodienolides, which are powerful inhibitors of Na/K ATPase, in plasma using high performance liquid chromatography (HPLC). Bufodienolides were first extracted from pig plasma using methylene chloride and a solid phase extraction column. They were then conjugated to a fluorescent probe, N-methylisatoic anhydride (NMIA), which allowed for detection on HPLC. Standard curves were generated for three bufodienolides (bufalin, cinobufagin, resibufogenin) with high linearity (R2=0.99). This new method allows for accurate measurement of bufodienolides in mammalian plasma and could
It is my journal club presentation on Synthesis, Docking Studies and Anticancer Activity of New Substituted Pyrimidine and Triazolopyrimidine Glycosides.
I sincerely thank the authors Wael A. El-Sayed, Ashraf M. Mohamed , Hemat S. Khalaf, Dina S. EL-Kady, May Al-Manawaty
It is my journal club presentation on Synthesis, Docking Studies and Anticancer Activity of New Substituted Pyrimidine and Triazolopyrimidine Glycosides.
I sincerely thank the authors Wael A. El-Sayed, Ashraf M. Mohamed , Hemat S. Khalaf, Dina S. EL-Kady, May Al-Manawaty
Development and Validation of a Two-Site Immunoradiometric assay for Glypican...Premier Publishers
This work aimed to set-up and evaluate a lab-made immunoradiometric assay for the detection of plasma glypican-3 (GPC3) in comparison with a commercial ELISA kit and to use them to evaluate the diagnostic potential of GPC3 in HCC patients. Anti-GPC3 monoclonal antibodies were radio-iodinated and used with a second antibody in the IRMA assay. The study included 450 subjects in 3 groups, 150 HCC patients, 150 hepatitis-C-virus (HCV) patients and 150 normal healthy subjects. Plasma GPC3 was assayed by our lab-made IRMA and by a commercial ELISA kit, along with alpha-fetoprotein (AFP). We were able to set-up an IRMA assay to measure plasma GPC3 and applied it to diagnose HCC patients. When compared to a ELISA kit, our IRMA assay showed much better performance characteristics on ROC curves with much higher area under the curves, sensitivities and specificities when diagnosing HCC patients from normal controls or HCV patients. Using our IRMA assay, showed that GPC3 is a good diagnostic indicator for HCC with 1.4 ng/ml cutoff for controls and at a cutoff value of 1.55 ng/ml it was able to discriminate HCC and HCV patients. GPC3 measurement was much better than AFP for the diagnosis of HCC.
Massa Is Kassa 2 Gea Boekuitgave Low ResAndré Knol
‘Massa is kassa 2’ is de vernieuwde en uitgebreide editie van een in boekvorm uitgebracht onderzoek in opdracht van het Stimuleringsfonds voor de Pers.
‘Massa is kassa’ vertelt na een grondige analyse van het huidige distributie en losse verkoopmodel van tijdschriften hoe door inzet van nieuwe kanalen, een ketengerichte wijze van organisatie en inzet van technologie, oude wetmatigheden vervangen kunnen worden door nieuwe. De oude ‘Massa is kassa’ van het tijdschriftenschap krijgt er een nieuwe ‘Massa is kassa’ bij!
Het boek ‘Massa is kassa’ gaat over bestaande en nieuwe vormen van distributie en losse verkoop van fysieke tijdschriften en de factoren die bepalen welk kanaal het meest geschikt is om een tijdschrift te vermarkten.
Regular program of the Ministry of Federal Affairs & Local Development (MoFALD) & Local Governance and Community Development (LGCDP), Government of Nepal. This annual Western regional review and progress report meeting was held in Pokhara August 31 - Sept 1, 2015 at UDTC, Pokhara.
This presentation is the summary of the same, and progress report from LGCDP Pokhara office. ICT for Development and e-Governance in Nepal has been depicted in the work.
Clinical diagnosis of chronic myeloid leukemia by real time polymerase chain ...Teboho Mooko
Oncology study i did in my third year (2014). the study was basically about monitoring Chronic Myeloid leukemia (CML) using Real-Time PCR techniques to check how patients from Universitas Hospital responded to the treatment of Gleevec drug.
ST8 micellar/niosomal vesicular nanoformulation for delivery of naproxen in c...Vahid Erfani-Moghadam
Naproxen (NPX) is a non-steroidal anti-inflammatory drug (NSAID) used against a variety of diseases, including autoimmune disorders and chronic inflammations. However, low water solubility limits its therapeutic efficacy and novel nanoformulations are required to bypass its poor bioavailability to reach its therapeutic effect. The purpose of the study was to investigate the role of the nanoformulation of biocompatible molecules; Squalene (S) and Tween 80 (T8) Micellar/Niosomal Vesicles (ST8MNV) prepared, by thin-film hydration method and their potential as a drug delivery system for NPX. The percentage of encapsulation efficiency was calculated to be 99.5 ± 0.2% for 5% of NPX weight in total ingredients of micellar/niosomal vesicles (w/w). The ST8MNV nanoformulation exhibited a slower rate of NPX release from the drug encapsulated over seven days, suggesting a stable complex of NPX. Finally, cell toxicity assay demonstrated that the half-maximal inhibitory concentrations (IC50) of NPX were drastically reduced by ST8MNV nanoformulation in MCF-7, A549, HeLa, and MDA-MB-231 cancer cell lines. Our data show this micellar/niosomal naproxen nanoformulation is a great candidate for the future in vitro and in vivo studies for potential clinical anti-inflammatory and anticancer applications.
Development and Validation of a Two-Site Immunoradiometric assay for Glypican...Premier Publishers
This work aimed to set-up and evaluate a lab-made immunoradiometric assay for the detection of plasma glypican-3 (GPC3) in comparison with a commercial ELISA kit and to use them to evaluate the diagnostic potential of GPC3 in HCC patients. Anti-GPC3 monoclonal antibodies were radio-iodinated and used with a second antibody in the IRMA assay. The study included 450 subjects in 3 groups, 150 HCC patients, 150 hepatitis-C-virus (HCV) patients and 150 normal healthy subjects. Plasma GPC3 was assayed by our lab-made IRMA and by a commercial ELISA kit, along with alpha-fetoprotein (AFP). We were able to set-up an IRMA assay to measure plasma GPC3 and applied it to diagnose HCC patients. When compared to a ELISA kit, our IRMA assay showed much better performance characteristics on ROC curves with much higher area under the curves, sensitivities and specificities when diagnosing HCC patients from normal controls or HCV patients. Using our IRMA assay, showed that GPC3 is a good diagnostic indicator for HCC with 1.4 ng/ml cutoff for controls and at a cutoff value of 1.55 ng/ml it was able to discriminate HCC and HCV patients. GPC3 measurement was much better than AFP for the diagnosis of HCC.
Massa Is Kassa 2 Gea Boekuitgave Low ResAndré Knol
‘Massa is kassa 2’ is de vernieuwde en uitgebreide editie van een in boekvorm uitgebracht onderzoek in opdracht van het Stimuleringsfonds voor de Pers.
‘Massa is kassa’ vertelt na een grondige analyse van het huidige distributie en losse verkoopmodel van tijdschriften hoe door inzet van nieuwe kanalen, een ketengerichte wijze van organisatie en inzet van technologie, oude wetmatigheden vervangen kunnen worden door nieuwe. De oude ‘Massa is kassa’ van het tijdschriftenschap krijgt er een nieuwe ‘Massa is kassa’ bij!
Het boek ‘Massa is kassa’ gaat over bestaande en nieuwe vormen van distributie en losse verkoop van fysieke tijdschriften en de factoren die bepalen welk kanaal het meest geschikt is om een tijdschrift te vermarkten.
Regular program of the Ministry of Federal Affairs & Local Development (MoFALD) & Local Governance and Community Development (LGCDP), Government of Nepal. This annual Western regional review and progress report meeting was held in Pokhara August 31 - Sept 1, 2015 at UDTC, Pokhara.
This presentation is the summary of the same, and progress report from LGCDP Pokhara office. ICT for Development and e-Governance in Nepal has been depicted in the work.
Clinical diagnosis of chronic myeloid leukemia by real time polymerase chain ...Teboho Mooko
Oncology study i did in my third year (2014). the study was basically about monitoring Chronic Myeloid leukemia (CML) using Real-Time PCR techniques to check how patients from Universitas Hospital responded to the treatment of Gleevec drug.
ST8 micellar/niosomal vesicular nanoformulation for delivery of naproxen in c...Vahid Erfani-Moghadam
Naproxen (NPX) is a non-steroidal anti-inflammatory drug (NSAID) used against a variety of diseases, including autoimmune disorders and chronic inflammations. However, low water solubility limits its therapeutic efficacy and novel nanoformulations are required to bypass its poor bioavailability to reach its therapeutic effect. The purpose of the study was to investigate the role of the nanoformulation of biocompatible molecules; Squalene (S) and Tween 80 (T8) Micellar/Niosomal Vesicles (ST8MNV) prepared, by thin-film hydration method and their potential as a drug delivery system for NPX. The percentage of encapsulation efficiency was calculated to be 99.5 ± 0.2% for 5% of NPX weight in total ingredients of micellar/niosomal vesicles (w/w). The ST8MNV nanoformulation exhibited a slower rate of NPX release from the drug encapsulated over seven days, suggesting a stable complex of NPX. Finally, cell toxicity assay demonstrated that the half-maximal inhibitory concentrations (IC50) of NPX were drastically reduced by ST8MNV nanoformulation in MCF-7, A549, HeLa, and MDA-MB-231 cancer cell lines. Our data show this micellar/niosomal naproxen nanoformulation is a great candidate for the future in vitro and in vivo studies for potential clinical anti-inflammatory and anticancer applications.
BIOLOGICAL ACTIVITY OF THE OXIDIZED POLYSACCHARIDES
Bio-reactive polysaccharides have been most commonly used as drugs or drug delivery systems. The present paper describes the biological activity of some artificial and natural polyanionic polysaccharides.
Results regarding oxidized cellulose and carboxymethylcellulose
modified with benzocaine or N – hydroxy – 3,4-dihydroxybenzamide
(Didox) complete the picture of antiviral and antitumoral effects of polysaccharides. The biological tests regarding antiviral and antitumor activity showed that the introduction of benzocaine as a spacer unit between the main chain and a CMC carboxylic group enhances the antiviral and antitumor activity of carboxymethylcellulose.
A comparative bioavailability study of aceclofenac products in healthy human ...
David Ocon ISR ABSTRACT -
1. NEW METHOD FOR MEASURING ANTHRANILOYL-BUFODIENOLIDES IN
PLASMA BY HPLC
David A. Ocon*1
, Cassandra Moreno *2
, and Daniel N. Darlington, PhD*2
.
1*
Summer Intern, Pittsburgh Tissue Engineering Initiative.
2*
US Army Institute of Surgical Research, Fort Sam Houston, Texas.
A critical issue for both the military and civilian population is hemorrhagic shock. About 85% of
potentially survivable deaths in the war in Iraq were due to hemorrhage (1). Recent studies show
that normal restoration of plasma volume fails after inhibition of Na/K ATPase and turn a
normally non-lethal hemorrhage into a lethal one. Bufodienolides, a large family of powerful
Na/K ATPase inhibitors, originally isolated from amphibia, have recently been found in human,
mouse, and rat plasma (2-4). The purpose of this study is to develop a method to measure plasma
levels of bufodienolides in hemorrhagic shock. The method will begin by extracting the
bufodienolides out of plasma, conjugating the bufodienolides to a fluorescent probe, and then
measuring the bufodienolide-fluorescent conjugate on High Pressure Liquid Chromatography
(HPLC).
Bufodienolides were extracted by the following process: 1mL of plasma was spiked with 4, 2, 1,
0.5, and 0.2 µg of bufalin, cinobufagin, and resibufogenin. 10mL of Methylene Chloride was
added and centrifuged at 2000 rpm. The solution phase-separated, and the bottom phase
(Methylene Chloride) was removed and air dried. This portion was then re-dissolved in 10%
Acetonitrile (MeCN) and extraction continued using a HLB OASIS Column (Waters Inc.). The
column was washed in 2ml of MeCN and 1ml of H2O. The sample was added to the column and
washed in 1ml 10% MeCN / 2% Formic Acid and 1ml 10% MeCN. The sample was eluted in
3ml of MeCN and dried under air.
Bufodienolides were conjugated to a fluorescent probe: 400ul of N-methylisatoic anhydride
(NMIA) solution was added to the sample and incubated at 90°C for 24 hours. The NMIA
solution contained 0.50 µl of 0.1M NMIA, dimethyl aminopyridine, 4-pyrrolidino pyridine, and
3.85ml MeCN. The NMIA chemically combined with the bufodienolides to create an
anthraniloyl conjugate.
The anthraniloyl conjugates were measured on HPLC. A Beckman Coulter HPLC with
fluorescent and UV-Visible Multi-array detectors was used. The HPLC gradient was set at 85-
100% MeCN over five minutes. The conjugates were separated and peak heights were measured
at λ = 355 nm (UV) and fluorescence (λa = 355 nm, λe = 435 nm). Standard curves were
generated for bufalin, cinobufagin, and resibufogenin that fit a straight line with a R2
= 0.99.
Bufalin, Cinobufagin, and Resibufogenin were successfully extracted from pig plasma. We were
also able to successfully conjugate NMIA to these bufodienolides. The bufodienolide-conjugates
were then separated and measured on HPLC. Varying concentrations of the bufodienolides led to
a dose-dependent rise in the peak height on the HPLC. Standard curves were developed for each
bufodienolide. These standard curves should allow us to accurately measure bufodienolides in
mammalian plasma.
We have now developed a method that can extract and measure bufodienolides in plasma. This
method can be extended to measure bufodienolides in plasma of patients with hemorrhagic or
other forms of shock.
2. REFERENCES:
1. Kelly, J.F., et al. Injury Severity and Causes of Death From Operation Iraqi Freedom and
Operation Enduring Freedom: 2003-2004 Versus 2006. J Trauma. 2008;64:S21-S27.
2. Bagrov, A. Y., R. I. Dmitrieva, et al. "Endogenous marinobufagenin-like immunoreactive
substance. A possible endogenous Na, K-ATPase inhibitor with vasoconstrictor activity."
Am J Hypertens 1996:9(10bPt 1): 982-90.
3. Bagrov, A. Y., O. V. Fedorova, et al. "Endogenous marinobufagenin-like
immunoreactive factor and Na+, K+ ATPase inhibition during voluntary
hypoventilation." Hypertension 1995:26(5): 781-8.
4. Butler, V. P., Jr., J. F. Morris, et al. "Heterogeneity and lability of endogenous digitalis-
like substances in the plasma of the toad, Bufo marinus." Am J Physiol 1996:271(2 Pt 2):
R325-32.