Daniel R. Kennedy

Single chemical modifications of the C-1027 enediyne core, a radiomimetic antitumor drug, affect both drug potency and the role of ataxia-telangiectasia mutated in cellular responses to DNA double-strand breaks

The radiomimetic enediyne C-1027 induces almost exclusively DNA double-strand breaks (DSB) and is extremely cytotoxic. Unique among radiomimetics, ataxia-telangiectasia mutated (ATM) is dispensable for cellular responses to C-1027-induced DNA damage. This study explores the biological activity of three recently bioengineered C-1027 analogues: 7-desmethyl-C-1027 (desmethyl), 20-deschloro-C-1027 (deschloro), and 22-deshydroxy-C-1027 (deshydroxy). Each compound maintains the characteristic ability of radiomimetics to cleave DNA in cell-free systems, varying in activity from 2-fold (deschloro) to 55-fold (desmethyl) less than C-1027. The induction of cellular DNA breaks based on pulsed field gel electrophoresis, comet analysis, and gammaH2AX activation was in the same rank order as cell-free DNA break induction, although the amount of breaks induced by desmethyl is greatly reduced compared with the other analogues. Despite the disparity in inducing DNA DSBs, all of the analogues produced G2-M cell cycle arrest and activated DNA DSB damage response proteins, such as p53-Ser15 and Chk2-Thr68, at concentrations in concordance with their ability to inhibit cell growth. Interestingly, of the three analogues, only the desmethyl-induced DNA damage response was similar to C-1027, as it did not cause hypersensitive cell growth inhibition in the absence of ATM nor require the kinase to phosphorylate p53 or Chk2. These findings show that simple modifications of the chromophore of C-1027 can result in varied induction of, and cellular response to, DNA DSBs.

Designer enediynes generate DNA breaks, interstrand cross-links, or both, with concomitant changes in the regulation of DNA damage responses

The ability of the radiomimetic anticancer enediyne C-1027 to induce ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR)-independent damage responses was discovered to reside in its unique ability to concurrently generate robust amounts of double-strand breaks (DSBs) and interstrand cross-links (ICLs) in cellular DNA. Furthermore, a single substitution to the chromophores benzoxazolinate moiety shifted DNA damage to primarily ICLs and an ATR- but not ATM-dependent damage response. In contrast, single substitutions of the chromophores β-amino acid component shifted DNA damage to primarily DSBs, consistent with its induction of conventional ATM-dependent damage responses of the type generated by ionizing radiation and other radiomimetics. Thus, phosphatidylinositol 3-kinase-like protein kinase regulation of DNA damage responses is dictated by the relative proportions of DSBs and ICLs.

Macro-scale acoustophoretic separation of lipid particles from red blood cells.

Autologous blood salvage is frequently used in cardiac surgery. However, shed mediastinal blood contains lipid particles ranging in size from 10 to 60 μm. Lipid emboli flow and subsequently lodge in the brain capillaries resulting in strokes, leading to neurocognitive dysfunction and death. A novel acoustophoretic filtration system has been developed to separate the lipids from the red blood cells (RBCs). The system works at the macro-scale, supporting flow rates in excess of 2 L/hr. The system is designed such that the acoustic radiation force is able to overcome the combined effects of fluid drag and buoyancy forces. Both RBCs and lipid particles are therefore trapped in the ultrasonic standing wave. Due to the opposite contrast factors of lipids and RBCs, the two components separate at opposite nodes within the standing wave, with lipids concentrating at pressure anti-nodes and RBCs at pressure nodes. Subsequent gravitational separation is used to separate the lipids and RBCs. Preliminary results were ...

Revisiting the mechanistic basis of the French Paradox: Red wine inhibits the activity of protein disulfide isomerase in vitro.

INTRODUCTIONnAlthough epidemiologic evidence points to cardioprotective activity of red wine, the mechanistic basis for antithrombotic activity has not been established. Quercetin and related flavonoids are present in high concentrations in red but not white wine. Quercetin-glycosides were recently shown to prevent thrombosis in animal models through the inhibition of extracellular protein disulfide isomerase (PDI). We evaluated whether red or white wine inhibited PDI activity in vitro.nnnMETHODSnQuercetin levels in red and white wines were measured by HPLC analysis. Inhibition of PDI activity by red and white wines was assessed by an insulin reduction turbidity assay at various concentrations of wine. PDI inhibition was confirmed using a reduced peptide that contained a disulfide containing peptide as a substrate. The inhibition of PDI related thiol isomerases ERp5 and ERp57 was also assessed.nnnRESULTSnWe observed a dose-dependent decrease of PDI activity for a variety of red but not white wines. Red wine diluted to 3% final concentration resulted in over 80% inhibition of PDI activity by insulin reductase assay for all varieties tested. This inhibition was also observed in the peptide based assay. Red grape juice yielded similar results but ethanol alone did not affect PDI activity. Interestingly, red wine also inhibited the PDI related thiol isomerases ERp5 and ERp57, albeit to a lesser degree than PDI.nnnCONCLUSIONSnPDI activity is inhibited by red wine and grape juice, identifying a potentially novel mechanism underlying the cardiovascular benefits attributed to wine consumption.

The radiomimetic enediyne, 20'-deschloro-C-1027 induces inter-strand DNA crosslinks in hypoxic cells and overcomes cytotoxic radioresistance.

The ability of the radiomimetic anti-tumor enediyne C-1027 to induce DNA inter-strand crosslinks (ICLs), in addition to the expected DNA strand breaks, is unique among traditional DNA targeted cancer therapies. Importantly, radiation therapy and most radiomimetic drugs have diminished effect in hypoxic environments due to decreased induction of DNA strand breaks, which is an oxygen requiring process. However, C-1027s induction of ICLs is enhanced under hypoxia and it is actually more potent against hypoxic cells, overcoming this common tumor resistance mechanism. In this study, an analog of C-1027, 20-deschloro-C-1027 was examined for its ability to induce DNA ICLs under hypoxic conditions. Deschloro-induced ICLs were detected under hypoxic cell-free conditions, with a concomitant reduction in the induction of DNA strand breaks. In cells deschloro behaved similarly, inducing cellular ICLs under hypoxic conditions with a reduction in DNA breaks. The cytotoxicity of deschloro treatment was similar in normoxic and hypoxic cells, suggesting that the ICL induction allows deschloro to retain its cytotoxic activity under hypoxia. It appears that rational engineering of the C-1027 family of radiomimetics holds promise toward overcoming the radioresistance associated with the hypoxic environment associated with solid tumors.

A Novel Macro-scale Acoustic Device for Blood Filtration

Retransfusion of a patients own shed blood during cardiac surgery is attractive since it reduces the need for allogeneic transfusion, minimizes cost, and decreases transfusion related morbidity. Evidence suggests that lipid micro-emboli associated with the retransfusion of the shed blood are the predominant causes of the neurocognitive disorders. We have developed a novel acoustophoretic filtration system that can remove lipids from blood at clinically relevant flow rates. Unlike other acoustophoretic separation systems, this ultrasound technology works at the macroscale, and is therefore able to process larger flow rates than typical micro-electromechanical system (MEMS) scale acoustophoretic separation devices. In this work, we have first demonstrated the systematic design of the acoustic device and its optimization, followed by examining the feasibility of the device to filter lipids from the system. Then, we demonstrate the effects of the acoustic waves on the shed blood; examining hemolysis using both haptoglobin formation and lactate dehydrogenase release, as well as the potential of platelet aggregation or inflammatory cascade activation. Finally, in a porcine surgical model, we determined the potential viability of acoustic trapping as a blood filtration technology, as the animal responded to redelivered blood by increasing both systemic and mean arterial blood pressure.

A novel application of radiomimetic compounds as antibiotic drugs

This study aims to examine the potential of radiomimetic compounds as antimicrobial therapeutics, as the recent advances in radiomimetic targeting as well as rapid increase of multidrug resistant bacteria make these compounds attractive for future development.

P & T and Me

Very few occasions bring more stress to a faculty member than the promotion and/or tenure (P&T) process. In this commentary, two recent chairs of P&T committees give their advice to future petitioners. Subtopics of the editorial discuss knowing the expectations, finding mentors to guide you, understanding the role of peer reviewers and preparing your dossier.

Integration of a temperature sensor for in situ monitoring of an acoustophoretic separation system

A novel device that uses acoustics to separate contaminants from blood is currently being developed. One potential application of the technology is to remove lipids that are inadvertently introduced into circulation during retransfusion of shed blood in cardiopulmonary bypass surgery, thus helping to avoid post-operative impairments such as stroke. However, the acoustic energy that is supplied to the system to achieve the separation may be heating the blood above the physiologically safe temperature limit. Therefore, a temperature sensor is being developed for in situ monitoring of the acoustophoretic separation system to determine if the filtered blood is viable for retransfusion back into the patient.

Concentration of blood components by acoustic radiation force

We have recently reported on macro-scale separation of lipids from red blood cells (J. Acoust. Soc. Am. 133(5, Pt. 2), 3279). In this system, the acoustic radiation force is used to trap red blood cells and lipid particles in a standing wave. Subsequent clumping of red blood cells results in gravitational settling of the red blood cells. Similarly, coalescing of lipids results in rising of the lipids out of solution. We now report on more detailed measurements of the concentration and separation of various blood components, such as red blood cells, white blood cells, and platelets. Porcine blood was used in the experiments and diluted by a factor of ten in phosphate buffered saline. A VetScan HM5 hematology analyzer was used to do the blood count. Inlet flow rate through the device was 16 ml/min, the concentrate flow rate of the blood components was typically about 1 ml/min. The transducer was a 2 MHz PZT-8 operating at 10 W. No lysing was observed in any of the experiments. Results indicate successful ca...