Novel Anthra[1,2-c][1,2,5]Thiadiazole-6,11-Diones as Promising Anticancer Lead Compounds: Biological Evaluation,Characterization & Molecular Targets Determination

The novel compounds NSC745885 and NSC757963 developed at our laboratory were tested against a panel of 60 cancer cell lines at the National Cancer Institute, USA, and a panel of 39 cancer cell lines at the Japanese Foundation of Cancer Research. Both compounds demonstrated selective unique multi-log differential patterns of activity, with GI₅₀ values in the sub-micro molar range against cancer cells rather than normal cardiac cells. NSC757963 showed high selectivity towards the leukemia subpanel. Activities of both compounds strongly correlated to expression of NFKB1 and CSNK2B genes, implying that they may inhibit the NF-κB pathway. Immunocytochemical microscopy of OVCAR-3 cells showed clear cytosolic accumulation of the NF-κB p65 subunit following treatment. Western blotting showed dose dependent inhibition of the nuclear expression of the NF-κB p65 subunit with subsequent accumulation in the cytosol following treatment. Docking experiments showed binding of both compounds to the NF-κB activator IKKβ subunit preventing its translocation to the nucleus. Collectively, these results confirm the ability of our compounds to inhibit the constitutively active NF-κB pathway of OVCAR-3 cells. Furthermore, COMPARE analysis indicated that the activity of NSC757963 is similar to the antituberculosis agent rifamycin SV, this was confirmed by testing the antimycobacterial activity of NSC757963 against Mycobacterium tuberculosis, results revealed potent activity suitable for use in clinical practice. Molecular properties and Lipinski’s parameters predicted acceptable bioavailability properties with no indication of mutagenicity, tumorigenicity, irritability and reproductive effects. Oral absorption experiments using the human Caco-2 model showed high intestinal absorption of NSC745885 by passive transport mechanism with no intestinal efflux or active transport mechanisms. The unique molecular characterization as well as the illustrated anticancer spectra of activity and bioavailability properties warrant further development of our compounds and present a foundation brick in the pre-clinical investigations to implement such compounds in clinical practice.     

Characterization of Genomic Inheritance of Intergeneric Hybrids between Ascocenda and Phalaenopsis Cultivars by GISH,PCR-RFLP and RFLP

BackgroundThe intergeneric hybrids between Ascocenda John De Biase ‘Blue’ and Phalaenopsis Chih Shang''s Stripes have been generated to introduce the blue color into the Phalaenopsis germplasm in prior study. In order to confirm the inheritance in hybrid progenies, genomic in situ hybridization (GISH) and restriction fragment length polymorphism (RFLP) analysis were conducted to confirm the intergeneric hybridization status.Methods/ResultsGISH analysis showed the presence of both maternal and paternal chromosomes in the cells of the putative hybrids indicating that the putative hybrid seedlings were intergeneric hybrids of the two parents. Furthermore, twenty-seven putative hybrids were randomly selected for DNA analysis, and the external transcribed spacer (ETS) regions of nrDNA were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and RFLP analyses to identify the putative hybrids. RFLP analysis showed that the examined seedlings were intergeneric hybrids of the two parents. However, PCR-RFLP analysis showed bias to maternal genotype.ConclusionsBoth GISH and RFLP analyses are effective detection technology to identify the intergeneric hybridization status of putative hybrids. Furthermore, the use of PCR-RFLP analysis to identify the inheritance of putative hybrids should be carefully evaluated.     

Atrial Fibrillation Management Strategies in Routine Clinical Practice: Insights from the International RealiseAF Survey

Background

Atrial fibrillation (AF) can be managed with rhythm- or rate-control strategies. There are few data from routine clinical practice on the frequency with which each strategy is used and their correlates in terms of patients’ clinical characteristics, AF control, and symptom burden.

Methods

RealiseAF was an international, cross-sectional, observational survey of 11,198 patients with AF. The aim of this analysis was to describe patient profiles and symptoms according to the AF management strategy used. A multivariate logistic regression identified factors associated with AF management strategy at the end of the visit.

Results

Among 10,497 eligible patients, 53.7% used a rate-control strategy, compared with 34.5% who used a rhythm-control strategy. In 11.8% of patients, no clear strategy was stated. The proportion of patients with AF-related symptoms (EHRA Class > = II) was 78.1% (n = 4396/5630) for those using a rate-control strategy vs. 67.8% for those using a rhythm-control strategy (p<0.001). Multivariate logistic regression analysis revealed that age <75 years or the paroxysmal or persistent form of AF favored the choice of a rhythm-control strategy. A change in strategy was infrequent, even in patients with European Heart Rhythm Association (EHRA) Class > = II.

Conclusions

In the RealiseAF routine clinical practice survey, rate control was more commonly used than rhythm control, and a change in strategy was uncommon, even in symptomatic patients. In almost 12% of patients, no clear strategy was stated. Physician awareness regarding optimal management strategies for AF may be improved.     

The Prevalence and Incidence of Atrial Fibrillation in Patients with Acute Pulmonary Embolism

Background

Symptomatic pulmonary embolism (PE) is a major cause of cardiovascular death and morbidity. Estimated prevalence and incidence of atrial fibrillation (AF) in developed countries are between 388–661 per 100,000, and 90–123 per 100,000 person-years respectively. However, the prevalence and incidence of AF in patients presenting with an acute PE and its predictors are not clear.

Methods

Individual patient clinical details were retrieved from a database containing all confirmed acute PE presentations to a tertiary institution from 2001–2012. Prevalence and incidence of AF was tracked from a population registry by systematically searching for AF during any hospital admission (2000–2013) based on International Classification of Disease (ICD-10) code.

Results

Of the 1,142 patients included in this study, 935 (81.9%) had no AF during index PE admission whilst 207 patients had documented baseline AF (prevalence rate 18,126 per 100,000; age-adjusted 4,672 per 100,000). Of the 935 patients without AF, 126 developed AF post-PE (incidence rate 2,778 per 100,000 person-years; age-adjusted 984 per 100,000 person-years). Mean time from PE to subsequent AF was 3.4 ± 2.9 years. Total mortality (mean follow-up 5.0 ± 3.7 years) was 42% (n = 478): 35% (n = 283), 59% (n = 119) and 60% (n = 76) in the no AF, baseline AF and subsequent AF cohorts respectively. Independent predictors for subsequent AF after acute PE include age (hazard ratio [HR] 1.06, 95% confidence interval [CI] 1.04–1.08, p<0.001), history of congestive cardiac failure (HR 1.88, 95% CI 1.12–3.16, p = 0.02), diabetes (HR 1.72, 95% CI 1.07–2.77, p = 0.02), obstructive sleep apnea (HR 4.83, 1.48–15.8, p = 0.009) and day-1 serum sodium level during index PE admission (HR 0.94, 95% CI 0.90–0.98, p = 0.002).

Conclusions

Patients presenting with acute PE have a markedly increased age-adjusted prevalence and subsequent incidence of AF. Screening for AF may be of importance post-PE.     

Measuring triamcinolone acetonide in aqueous humor by gas chromatography-electron-capture negative-ion mass spectrometry

Intravitreal triamcinolone acetonide (IVTA) injection has been used in the treatment of various posterior segment diseases. One of the side effects of IVTA is raised intraocular pressure, which may be secondary to triamcinolone acetonide (TAA)'s effects on the trabecular meshwork that affects aqueous outflow. In order to study the biological effects of TAA on the trabecular meshwork, we firstly need to reliably and accurately detect the concentration of TAA in tissue cells or fluids. In this study we have described a technique of using gas chromatography-electron-capture-negative-ion mass spectrometry (GC-NCI-MS) to develop a simple, sensitive, selective and validated method to detect TAA in aqueous humor (AH) of rabbits following IVTA and subconjunctival TAA injections. We derivatized TAA from extracted aqueous sample by acetic anhydride and BSTFA, respectively, and analyzed by GC-NCI-MS. The detection limit was 0.3ng/ml, linearity over 0.995 from 0 to 300ng/ml. The reproducibility ranged from 10.4 to 3.9 for concentrations from 3 to 300ng/ml, and recovery was over 95% for the concentrations 10, 60, and 200ng/ml. No interference was found from 159 aqueous samples. There was no TAA residue carried to the next injection from previously high concentration injection, 10,000ng/ml. We have provided an alternative, rapid, and robust method other than LC-MS-MS for TAA detection in AH.     

Medium optimization for the production of recombinant nattokinase by Bacillus subtilis using response surface methodology

Nattokinase is a potent fibrinolytic enzyme with the potential for fighting cardiovascular diseases. Most recently, a new Bacillus subtilis/Escherichia coli (B. subtilis/E. coli) shuttle vector has been developed to achieve stable production of recombinant nattokinase in B. subtilis (Chen; et al. 2007, 23, 808-813). With this developed B. subtilis strain, the design of an optimum but cost-effective medium for high-level production of recombinant nattokinase was attempted by using response surface methodology. On the basis of the Plackett-Burman design, three critical medium components were selected. Subsequently, the optimum combination of selected factors was investigated by the Box-Behnken design. As a result, it gave the predicted maximum production of recombinant nattokinase with 71 500 CU/mL for shake-flask cultures when the concentrations of soybean hydrolysate, potassium phosphate, and calcium chloride in medium were at 6.100, 0.415, and 0.015%, respectively. This was further verified by a duplicated experiment. Moreover, the production scheme based on the optimum medium was scaled up in a fermenter. The batch fermentation of 3 L was carried out by controlling the condition at 37 degrees C and dissolved oxygen reaching 20% of air saturation level while the fermentation pH was initially set at 8.5. Without the need for controlling the broth pH, recombinant nattokinase production with a yield of 77 400 CU/mL (corresponding to 560 mg/L) could be obtained in the culture broth within 24 h. In particular, the recombinant B. subtilis strain was found fully stable at the end of fermentation when grown on the optimum medium. Overall, it indicates the success of this experimental design approach in formulating a simple and cost-effective medium, which provides the developed strain with sufficient nutrient supplements for stable and high-level production of recombinant nattokinase in a fermenter.     

Stable isotope analysis reveals ontogenetic feeding shifts in Pacific blue marlin (Makaira nigricans) off eastern Taiwan

To gain a better understanding of the trophic ecology of Pacific blue marlin Makaira nigricans off eastern Taiwan, nitrogen and carbon stable isotopes (δ¹⁵N and δ¹³C) and Bayesian mixing models were used to explore trophic dynamics and potential ontogenetic feeding shifts across M. nigricans of different size classes. Makaira nigricans samples from east of Taiwan (n = 213) and Palau (n = 37), as well as their prey (n = 70), were collected during 2012 and 2013. Results indicated increases in δ¹⁵N with size, with values of larger size classes (> 200 cm eye-to-fork length; L_EF) significantly higher than those < 200 cm L_EF. Values of δ¹³C were negatively correlated with size. Makaira nigricans > 200 cm L_EF had the highest estimated trophic position (4.44) and also exhibited ontogenetic changes in trophic position. Large M. nigricans fed more on dolphinfish Coryphaena hippurus and hairtail Trichiurus lepturus, while smaller M. nigricans consumed smaller forage fish (e.g., moonfish Mene maculata) and cephalopods. These changes may relate to greater swimming speeds and vertical habitat use in larger M. nigricans, allowing capture and consumption of larger prey items at higher trophic positions. The high trophic level of M. nigricans east of Taiwan confirms its important role as an apex predator in marine food webs and how ecological role changes with size.