Teaching an old dog new tricks: The case of Fenbendazole.

The objective of this study is the assessment of the cytotoxic effect of fenbendazole and its commercially available formulation, which is used for its antihelmintic properties. The formulation was tested for its efficacy as well as the determination of the ingredients with proliferation assays and analytical techniques. HPLC, LC-MS and NMR confirmed the stated amount of active ingredient on the label. Dissolution studies were performed to simulate the ability of fenbendazole to dissolve adequately in the fluids of the Gastrointestinal tract, be absorbed in the circulation and reach certain areas of the human body. However, dissolution studies showed that both brands possess issues in their distribution. The in vitro drug screening exhibited potential cytotoxic effect in different types of human cancer cell lines and MDA-MB-231 human breast adenocarcinoma cells appeared to be the most sensitive with IC50 value lower than 10 µM.

Targeting SARS-CoV-2 Polymerase with New Nucleoside Analogues.

Despite the fact that COVID-19 vaccines are already available on the market, there have not been any effective FDA-approved drugs to treat this disease. There are several already known drugs that through drug repositioning have shown an inhibitory activity against SARS-CoV-2 RNA-dependent RNA polymerase. These drugs are included in the family of nucleoside analogues. In our efforts, we synthesized a group of new nucleoside analogues, which are modified at the sugar moiety that is replaced by a quinazoline entity. Different nucleobase derivatives are used in order to increase the inhibition. Five new nucleoside analogues were evaluated with in vitro assays for targeting polymerase of SARS-CoV-2.

Genistein as a dietary supplement; formulation, analysis and pharmacokinetics study.

The objective of this study is to improve and optimize the formulation of Genistein in capsules in order to result in a better pharmacokinetic profile comparing to existing commercial products. In order to do this, five different formulations of Genistein capsules were developed and examined by reviewing their disintegration and dissolution properties. Furthermore, flowability of the powder along with potent incompatibilities between Genistein and its excipients were monitored through their thermal properties. The final formulation of Genistein was quantified using HPLC analysis and then its stability was evaluated thoroughly in real time and accelerated conditions. Finally, with the target to have a product with actual results, in vitro and in vivo studies were conducted. The final product proved to have better results in disintegration and dissolution. Moreover, R.G.C.C.'s capsules exhibited enhanced action in human cell lines as well as impressive pharmacokinetic results in animal models. The in vitro results showed an advantage of the R.G.C.C. product compared to the commercial one, whereas its maximum concertation in vivo was determined 34% higher than the commercial one.

Evaluation of Tegaran Formula ZhenHua cytotoxicity against human cancer cell lines.

The aim of this study is to evaluate the potential health effects of Tegaran Formula ZhenHua, a nutritional supplement used mainly by cancer patients. Its active ingredients and cytotoxicity was assessed with analytical methods and viability assays, respectively. The analytical methods consisted of dissolution, disintegration, HPLC, LC/MS, GC/MS and NMR. Cytotoxicity was assessed by MTT, SRB, CVE colorimetric viability assays in 0, 24, 48 and 72h time points. The results indicate that Tegaran Formula ZhenHua supplement did not present any cytotoxic effects due to issues related to the capsules' solubility, distribution and identification of the active ingredient.

Design, Synthesis, and In Vitro Activity of Pyrazine Compounds.

Despite the fact that there are several anticancer drugs available, cancer has evolved using different pathways inside the cell. The protein tyrosine phosphatases pathway is responsible for monitoring cell proliferation, diversity, migration, and metabolism. More specifically, the SHP2 protein, which is a member of the PTPs family, is closely related to cancer. In our efforts, with the aid of a structure-based drug design, we optimized the known inhibitor SHP099 by introducing 1-(methylsulfonyl)-4-prolylpiperazine as a linker. We designed and synthesized three pyrazine-based small molecules. We started with prolines as cyclic amines, confirming that our structures had the same interactions with those already existing in the literature, and, here, we report one new hydrogen bond. These studies concluded in the discovery of methyl (6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)prolylprolinate hydrochloride as one of the final compounds which is an active and acceptable cytotoxic agent.

The use of serum extracellular vesicles for novel small molecule inhibitor cell delivery.

In Research Genetic Cancer Center (RGCC), we are in the process of synthesizing a novel ERK inhibitor. We have currently synthesized an intermediate molecule, RGCC169, that needed to be tested to confirm we are using the appropriate synthetic pathways. Because of the limited solubility the compound exhibits, a strategy had to be devised for the free entrance of the molecule into the cell. Extracellular vesicles (EVs) were isolated by polyethylene glycol precipitation and identified by western blot and scanning electron microscopy. Loading was determined by high-performance liquid chromatography, differential scanning calorimetry, and scanning electron microscopy. EV uptake was determined by fluorescent microscopy. The effect of EV-encapsulated RGCC169 was determined by MTT viability assay on MCF7 cells. RGCC169 was incorporated into EVs as shown by high-performance liquid chromatography (26.6%) and scanning electron microscopy. Differential scanning calorimetry peaks shifted from 100.84 to 108.79°C upon encapsulation. EVs were taken up by cells as evident from CD63 fluorescent signal inside the cell's cytoplasm. RGCC169 decreased MCF proliferation (93.5±2.2, P=0.02). EV-encapsulated RGCC169 decreased cell proliferation even further (93.5±2.2 vs. 81.6±2.8, P=0.0002). RGCC169 was successfully loaded into serum EVs possibly by incorporation into the lipid membrane. EVs were taken up by MCF7 cells possibly by endocytic pathways. Although RGCC169 significantly reduced MCF7 viability at 3 µmol/l, the same concentration of RGCC169 encapsulated into EVs decreased cells viability even further. Our findings validate the correctness of our methods and are very promising for the achievement of our final goal, that is, the synthesis of a novel cytotoxic agent.

Involvement of retrotransposon L1 in stemness and cellular plasticity.

Epithelial-to-mesenchymal transition (EMT) as well as the reverse process, mesenchymal-to-epithelial transition (MET) is important during embryogenesis. EMT is also involved in cancer invasion and metastasis, and can generate cells with properties similar to those of stem cells. Retrotransposons can rearrange the genome by inserting DNA in new loci, thus inducing mutations. This study examines the gene expression of transcription factors involved in EMT and MET. In the second experimental panel, the gene expression of L1 retrotransposon was studied. L1-open reading frame (ORF) 2 mRNA was found to be expressed both in cancer and cancer stem cells, while L1-ORF1 mRNA was expressed only in cancer cells. The suppression of L1-ORF2 gene expression demonstrated that this retrotransposon might affect EMT in colon cancer stem cells. This study highlights that the EMT process seems to differ between cancer cells and cancer stem cells, and that transposable elements seem to be involved in the process, influencing cellular plasticity.

Potential role for the Metnase transposase fusion gene in colon cancer through the regulation of key genes.

The Metnase fusion gene consists of a SET histone methyltransferase domain and a transposase domain from Mariner transposase. This transposable element is involved in chromosome decatenation, enhances DNA repair, promotes foreign DNA integration, and assists topoisomerase II function. This study investigates the role of Metnase in colon cancer homeostasis and maintenance of the stemness phenotype in colon cancer stem cells (CSCs). Silencing of Metnase was performed in human cancer cell lines before and after treatment with cisplatin, and in colon CSCs. Subsequent changes in the expression of genes involved in repair mechanisms, DNA synthesis, topoisomerase II function, and metastasis as well stemness transcription factors were studied with RT-qPCR experiments. Cellular viability and apoptosis were evaluated by flow cytometry. The results suggest that Metnase influences the expression of many genes involved in the above processes. Furthermore, Metnase levels appear to impact upon expression of NANOG, OCT3/4, and SOX2. Suppression of Metnase also led to an increase in apoptosis. Therefore, Metnase may possess an important role in DNA repair, topoisomerase II function, and the maintenance of stemness during colon cancer development.

Use of the comet assay technique for quick and reliable prediction of in vitro response to chemotherapeutics in breast and colon cancer.

BACKGROUND: Determination of response to chemotherapy is a major requirement of personalized medicine. Resistance, whether developed or native, critically affects a treatment's success. Single Cell Gel lectrophoresis - also known as a comet assay - is used to detect DNA damage at the level of individual eukaryotic cells. We assessed the use of comet assays in determining response to chemotherapeutic drugs that are widely used in breast and colon cancer. RESULTS: We treated human breast and colon cancer cell lines with melphalan, cisplatin, mechlorethamine or doxorubicin, as monotherapies. Drug activities varied even in the same cancer types, further demonstrating the heterogeneity of different cancer types. CONCLUSION: The comet assay technique can provide reliable and quick results with minimum requirements and is applicable to a wide variety of drugs.

Development of a generic assay for the determination of total trihydroxybenzoate derivatives based on gold-luminol chemiluminescence.

A selective assay for the determination of one of the most important class of phenolic compounds, namely trihydroxybenzoates (monomeric and polymeric compounds having at least one gallate moiety) based on their enhancing effect on the chemiluminogenic reaction between gold ions and luminol is described for the first time. In the presence of trihydroxybenzoate derivatives, the light emission generated when alkaline luminol is oxidized by gold ions is amplified several orders of magnitude compared to other common phenolic compounds which exhibit minor reactivity or no reactivity at all (e.g. hydroxycinnamates, flavonols, benzenediols). Based on this property, the experimental conditions were optimized in order to enable the determination of total trihydroxybenzoates in complex mixtures without resorting to separation techniques. The method was applied to samples of different composition (teas, herbal infusions and wines) with satisfactory analytical features yielding detection limits at the 10(-7) mol L(-1) level, intra-day precision of 3.1%, inter-day precision less than 10% and recoveries between 88.7 and 97.6%. The strengths and weaknesses of the method were identified and discussed in relation to its application in real samples.

AP-1 Gene Expression Levels May Be Correlated with Changes in Gene Expression of Some Stemness Factors in Colon Carcinomas.

The AP-1 transcription factor is a heterodimer protein that regulates gene expression in response to a variety of extrinsic stimuli through signal transduction. It is involved in processes including differentiation, proliferation, and apoptosis. Among the genes it regulates are transcription factors that contribute to the stemness phenotype. Cancer stem cells have the ability to self-renew and initiate differentiation into heterogenic cancer cells, which may cause metastasis and relapses. In the present study, we evaluated the effect of AP-1 complexes, as well as the C-FOS and C-JUN genes, in relation to NANOG, OCT3/4, and SOX2 transcription factors. All assays were undertaken with colon cancer stem cells. Knockdown experiments with siRNA were performed for each individual gene as well as their combination. Changes in gene expression were calculated with quantitative polymerase chain reaction experiments, while the effect on cell cycle distribution and apoptosis was studied by flow cytometry. The results differed depending on the percentage of repression, as well as the gene that was suppressed. In all cases, the number of apoptotic cells was increased. These findings indicate that AP-1 may have a crucial role in the maintenance of cancer stem cells.

Is routine ultrasound examination of the gallbladder justified in critical care patients?

Objective. We evaluated whether routine ultrasound examination may illustrate gallbladder abnormalities, including acute acalculous cholecystitis (AAC) in the intensive care unit (ICU). Patients and Methods. Ultrasound monitoring of the GB was performed by two blinded radiologists in mechanically ventilated patients irrespective of clinical and laboratory findings. We evaluated major (gallbladder wall thickening and edema, sonographic Murphy's sign, pericholecystic fluid) and minor (gallbladder distention and sludge) ultrasound criteria. Measurements and Results. We included 53 patients (42 males; mean age 57.6 ± 2.8 years; APACHE II score 21.3 ± 0.9; mean ICU stay 35.9 ± 4.8 days). Twenty-five patients (47.2%) exhibited at least one abnormal imaging finding, while only six out of them had hepatic dysfunction. No correlation existed between liver biochemistry and ultrasound results in the total population. Three male patients (5.7%), on the grounds of unexplained sepsis, were diagnosed with AAC as incited by ultrasound, and surgical intervention was lifesaving. Patients who exhibited ≥2 ultrasound findings (30.2%) were managed successfully under the guidance of evolving ultrasound, clinical, and laboratory findings. Conclusions. Ultrasound gallbladder monitoring guided lifesaving surgical treatment in 3 cases of AAC; however, its routine application is questionable and still entails high levels of clinical suspicion.

Inorganic phosphorus homeostasis during the first hour of dialysis.

BACKGROUND/AIM: Hyperphosphatemia is a well-recognized complication of chronic kidney disease, and phosphorus kinetics during hemodialysis (HD) remains a vague area of investigation. We studied the inorganic phosphorus homeostasis during the first hour of an HD session. MATERIALS/METHODS: Twelve patients were studied twice, in two consecutive HD sessions. Total (TPR), extracellular (EPR), and intracellular (IPR) phosphorus mass removal was determined using the direct dialysate quantification (DDQ) method. Alterations of serum inorganic phosphorus (sP), erythrocyte intracellular phosphorus (P(ERY)), and 2,3-diphosphoglycerate (2,3-DPG) concentrations were measured before HD initiation and at 1, 2, 3, 4, 5, 10, 30, and 60 min. RESULTS: The contribution of IPR to TPR was negative in the first 10 min of both HD sessions (-27.2 ± 6.5 and -26.4 ± 58 mmol, respectively, p = ns) while the contribution of the IPR to TPR increased as the time elapsed. Intracellular phosphorus and 2,3-DPG remained almost unchanged during the 60 min of HD session. CONCLUSIONS: Unchanged P(ERY) concentration during the first hour of an HD session does not reject the hypothesis of a simultaneous efflux and influx of phosphorus from/to intracellular compartment.

Circulating matrix metalloproteinase 3 due to myocardial ischemia.

BACKGROUND: Experimental data suggest that matrix metalloproteinases (MMPs) such as MMP-3 have a central role in the remodeling period after a myocardial infarction (MI). The aim of this study was to use an experimental small-animal model to investigate the fluctuation in MMP-3 levels occurring in vivo after an acute MI. METHODS: We studied 13 New Zealand white rabbits weighing between 3 and 4 kg. After anesthetizing the animals, we performed a tracheotomy and induced an acute MI in 10 of the animals by occluding the left anterior descending coronary artery for 45 minutes. The remaining 3 rabbits constituted the control group. Three hours after reperfusion, blood samples were taken for biomedical analyses. RESULTS: Three hours after the artificially induced acute MI, serum MMP-3 levels were decreased by almost 50%. Cardiac troponin I (cTnI) concentrations were increased greatly (90-fold) after MI, further validating the efficiency of our experimental in vivo model of acute MI. CONCLUSION: Combining the data, we demonstrated that acute MI caused an early reduction in MMP-3 levels. The range of MMP-3 reduction is limited compared with other factors predicting MI, such as cTnI, which increases its usefulness. We demonstrated, however, that plasma fluctuation in MMP-3 levels could be used as a supplementary independent predictor of cardiovascular events in patients with stable coronary artery disease. This acute MI model used in our controlled setting proved to be a reliable and safe method for conducting in vivo studies.

Effects of black and green tea consumption on blood glucose levels in non-obese elderly men and women from Mediterranean Islands (MEDIS epidemiological study).

BACKGROUND: Obesity and diabetes are metabolic disorders that affect a large amount of the elderly population and are related to increased cardiovascular risk. Tea intake has been associated with lower risk of mortality and morbidity in some, but not all studies. We evaluated the association between tea intake, blood glucose levels, in a sample of elderly adults. METHODS: During 2005-2006, 300 men and women from Cyprus, 142 from Mitilini and 100 from Samothraki islands (aged 65-100 years) were enrolled. Dietary habits (including tea consumption) were assessed through a food frequency questionnaire. Among various factors, fasting blood glucose and body mass index (BMI) were measured. RESULTS: Fifty-four percent of the participants reported that they consume tea at least once a week (mean intake 1.6 +/- 1.1 cup/day). A significant interaction was observed between tea intake, obesity status on glucose levels (P < 0.001). After adjusting for various confounders, tea intake was associated with lower blood glucose levels in non-obese (P for trend <0.001), but not in obese people (P = 0.24). Multiple logistic regression analysis revealed that moderate tea consumption (1-2 cups/day) was associated with 88% (95% CI 76-98%) lower odds of having diabetes among non-obese participants, irrespective of age, sex, smoking, physical activity status, dietary habits and other clinical characteristics. CONCLUSION: Tea consumption is associated with reduced levels of fasting blood glucose only among non-obese elderly people.

Food pattern analysis and prevalence of cardiovascular disease risk factors among elderly people from Mediterranean islands.

The effect of various foods on the development of cardiovascular disease (CVD) has already been investigated. We performed a food pattern analysis and evaluated the association between the consumption of various patterns and the prevalence of CVD risk factors among elderly people from Mediterranean islands (the MEDIS study). During 2005-2006, 300 men and women from Cyprus, 142 from Mitilini, 100 from Samothraki, and 104 from Kefalonia islands (65-100 years old) were enrolled. CVD risk factors (i.e., hypertension, diabetes, hypercholesterolemia, and obesity) were assessed through standard procedures. All participants were asked about their usual frequency of consumption of various foods through a semiquantitative food frequency questionnaire, and food pattern analysis using the principal components analysis (PCA) method was then performed. PCA extracted five components that explained the 56.53% of the total variation in intake: i.e., a food pattern (component 1) that was loaded mainly on low-fat products, a high glycemic index and high-fat pattern (component 2), a pattern that included consumption of cereals and sweets (component 3), a pattern that was characterized by the intake of dairy products and fruits (component 4), and a pattern that was characterized by the consumption of alcoholic beverages (component 5). Ordinal logistic regression analysis revealed that component 1, component 3, and component 5 were associated with lower likelihood of having increased burden of CVD (P < .01), irrespective of various potential confounders. Food pattern analysis revealed the current nutritional status of our elderly participants, and provided a pathway for reducing the burden of CVD risk factors among these people.

Long-term, moderate coffee consumption is associated with lower prevalence of diabetes mellitus among elderly non-tea drinkers from the Mediterranean Islands (MEDIS Study).

BACKGROUND: We evaluated the association between coffee drinking and the prevalence of type 2 diabetes mellitus in elderly people from the Mediterranean islands. METHODS: During 2005-2007, 500 men and 437 women (aged 65 to 100 years) from the islands of Cyprus (n = 300), Mitilini (n = 142), Samothraki (n = 100), Cephalonia (n = 104), Corfu (n = 160) and Crete (n = 131) participated in the survey. Cardiovascular disease (CVD) risk factors (i.e. hypertension, diabetes, hypercholesterolemia and obesity), as well as behavioral, lifestyle and dietary characteristics were assessed using face-to-face interviews and standard procedures. Among various factors, fasting blood glucose was measured and prevalence of type 2 diabetes mellitus was estimated, according to the established American Diabetes Association (ADA) criteria, while all participants were asked about the frequency of any type of coffee consumption over the last year. RESULTS: Coffee drinking was reported by 84% of the participants, the majority of whom drank boiled coffee. The participants reported that they had consumed coffee for at least 30 years of their life. Data analysis adjusted for various potential confounders, revealed that, compared to non-consumption, the multi-adjusted odds ratio for having diabetes was 0.47 (95%, CI 0.32 to 0.69) for 1-2 cups/day, while it was 1.05 (95%, CI 0.70 to 1.55) for >3 cups/day, after adjusting for various potential confounders. The association of coffee drinking with diabetes was significant only among non-tea drinkers. Increased coffee intake was not associated with diabetes prevalence. CONCLUSION: The data presented suggest that moderate coffee drinking is associated with a lower likelihood of having diabetes, after adjusting for various potential confounders.