Reaction of KHP with excess NaOH or TRIS as standard reactions for calibration of titration calorimeters from 0 to 60 °C.

Calibration of titration calorimeters is an ongoing problem, particularly with calorimeters with reaction vessel volumes < 10 mL in which an electrical calibration heater is positioned outside the calorimetric vessel. Consequently, a chemical reaction with a known enthalpy change must be used to accurately calibrate these calorimeters. This work proposes the use of standard solutions of potassium acid phthalate (KHP) titrated into solutions of excess sodium hydroxide (NaOH) or excess tris(hydroxymethyl)aminomethane (TRIS) as standard reactions to determine the collective accuracy of the relevant variables in a determination of the molar enthalpy change for a reaction. KHP is readily available in high purity, weighable for easy preparation of solutions with accurately known concentrations, stable in solution, not compromised by side reactions with common contaminants such as atmospheric CO2, and non-corrosive to materials used in calorimeter construction. Molar enthalpy changes for these reactions were calculated from 0 to 60 °C from reliable literature data for the pKa of KHP, the molar enthalpy change for protonation of TRIS, and the molar enthalpy change for ionization of water. The feasibility of using these reactions as enthalpic standards was tested in several calorimeters; a 50 mL CSC 4300, a 185 µL NanoITC, a 1.4 mL VP-ITC, and a TAM III with 1 mL reaction vessels. The results from the 50 mL CSC 4300, which was accurately calibrated with an electric heater, verified the accuracy of the calculated standard values for the molar enthalpy changes of the proposed reactions.

Thermal characterization and separation of whey proteins by differential scanning calorimetry.

Most commercially available whey products contain a mixture of 6-7 whey proteins; however, there is an increased focus on using the individual whey proteins for their unique biological activities. Before extracting individual whey proteins for use, it is important to quantify how much of a particular protein is present in whey mixtures as well as if the protein is still structurally folded. We first characterized the denaturation temperature and enthalpy values for the six purified whey proteins at six pHs (3-9) and under ion chelation using a nano-differential scanning calorimeter (DSC). From the individual protein scans, we determined the optimal condition for detecting all 6 proteins on a single DSC scan was whey in an EDTA MOPs pH 6.7 buffer.

Utilizing mixture design response surface methodology to determine effective combinations of plant derived compounds as prostate cancer treatments.

BACKGROUND: Prostate cancer (PC) is estimated to cause 13.1% of all new cancer cases in the United States in 2021. Natural bioactive compounds have drawn the interest of researchers worldwide in their efforts to find novel treatments for PC. Many of these bioactive compounds have been identified from traditional Chinese medicine (TCM) remedies often containing multiple bioactive compounds. However, in vitro studies frequently focus on the compounds in isolation. AIM: We used mixture design response surface methodology (MDRSM) to assess changes in PC cell viability after 48 h of treatment to identify the optimal mixture of all 35 three-compound combinations of seven bioactive compounds from TCM. METHODS AND RESULTS: We used berberine, wogonin, shikonin, curcumin, triptolide, emodin, and silybin to treat PC3 and LNCaP human PC cells at their IC50 concentrations that we calculated. These compounds modulate many chemotherapeutic pathways including intrinsic and extrinsic apoptosis, increasing reactive oxygen species, decreasing metastatic pathways, inhibiting cell cycle progression. We hypothesize that because these compounds bind to unique molecular targets to activate different chemotherapeutic pathways, they will act synergistically to decrease tumor cell viability. Results from MDRSM showed that two-way combinations were more effective than three-way or single compounds. Most notably wogonin, silybin, emodin and berberine responded well in two-compound combinations with each other in PC3 and LNCaP cells. We then conducted cell viability tests combining two bioactive compound ratios with docetaxel (Doc) and found significant results within the LNCaP cell line. In particular, mixtures of berberine and wogonin, berberine and silybin, emodin and berberine, and emodin and silybin reduced LNCaP cell viability up to an average of 90.02%. The two-compound combinations were significantly better than docetaxel treatment of LNCaP cells. CONCLUSION: Within the PC3 cells, we show that a combination of berberine, wogonin and docetaxel is just as effective as docetaxel alone. Thus, we provide new combination treatments that are highly effective in vitro for treating androgen-dependent and androgen-independent PC.

Metabolomics of acid whey derived from Greek yogurt.

Acid whey, a byproduct of Greek yogurt production, has little commercial value due to its low protein content and is also environmentally harmful when disposed of as waste. However, as a product of microbial fermentation, acid whey could be a rich source of beneficial metabolites associated with fermented foods. This study increases understanding of acid whey composition by providing a complete metabolomic profile of acid whey. Commercial and laboratory-made Greek yogurts, prepared with 3 different bacterial culture combinations, were evaluated. Samples of uncultured milk and cultured whey from each batch were analyzed. Ultra-high-performance liquid chromatography-tandem mass spectrometry metabolomics were used to separate and identify 477 metabolites. Compared with uncultured controls, acid whey from fermented yogurt showed decreases in some metabolites and increases in others, presumably due to the effects of microbial metabolism. Additional metabolites appeared in yogurt whey but not in the uncultured control. Therefore, the effect of microbial fermentation is complex, leading to increases or decreases in potentially bioactive bovine metabolites while generating new microbial compounds that may be beneficial. Metabolite production was significantly affected by combinations of culturing organisms and production location. Differences between laboratory-made and commercial samples could be caused by different starting ingredients, environmental factors, or both.

Gluten cross-contact from common food practices and preparations.

Patients with celiac disease continue to be exposed to gluten despite efforts to maintain a gluten-free diet (GFD). Gluten exposure in those with celiac disease leads to pathological changes in the small intestine that may or may not be associated with gastrointestinal distress. While several studies have investigated a GFD, little is known about sources of gluten contamination that prevent proper maintenance of such a diet by celiac patients. In this study, we investigate common food practices that could lead to gluten cross-contact. Three different practices were examined for gluten cross-contact: gluten-free foods fried in a fryer also used for gluten containing foods, gluten-free bread toasted in a toaster also used for gluten-containing bread, and popular sandwich spreads applied with a knife used on gluten-containing bread (mayonnaise, jam, and peanut butter). We used the ALLER-TEK™ Gluten ELISA test kit and the sandwich ELISA RIDASCREEN Gliadin test kit, which is endorsed for determination of gluten content and used for the evaluation of food cross-contact. Using both kits gave the advantage of using the 401.2 antibody as well as the better established R5 antibody, providing increased confidence in our results. We found these practices resulted in small amounts of gluten cross-contact, although the majority of the results (93.6%) showed no significant cross-contact. Mayonnaise and peanut butter samples were contaminated with gluten above the limit designated by the FDA as gluten-free <20 kg/mg (ppm).

γ-Tocotrienol and α-Tocopherol Ether Acetate Enhance Docetaxel Activity in Drug-Resistant Prostate Cancer Cells.

Prostate cancer is the second most commonly diagnosed cancer in men, and metastatic prostate cancer is currently incurable. Prostate cancer frequently becomes resistant to standard of care treatments, and the administration of chemotherapeutic drugs is often accompanied by toxic side effects. Combination therapy is one tool that can be used to combat therapeutic resistance and drug toxicity. Vitamin E (VE) compounds and analogs have been proposed as potential non-toxic chemotherapeutics. Here we modeled combination therapy using mixture design response surface methodology (MDRSM), a statistical technique designed to optimize mixture compositions, to determine whether combinations of three chemotherapeutic agents: γ-tocotrienol (γ-T3), α-tocopherol ether acetate (α-TEA), and docetaxel (DOC), would prove more effective than docetaxel alone in the treatment of human prostate cancer cells. Response surfaces were generated for cell viability, and the optimal treatment combination for reducing cell viability was calculated. We found that a combination of 20 µM γ-T3, 30 µM α-TEA, and 25 nm DOC was most effective in the treatment of PC-3 cells. We also found that the combination of γ-T3 and α-TEA with DOC decreased the amount of DOC required to reduce cell viability in PC-3 cells and ameliorated therapeutic resistance in DOC-resistant PC-3 cells.

Resveratrol derivatives increase cytosolic calcium by inhibiting plasma membrane ATPase and inducing calcium release from the endoplasmic reticulum in prostate cancer cells.

Resveratrol (RES) is a putative chemotherapeutic naturally found in grapes, peanuts, and Japanese knotweed. Previous studies demonstrate that RES modulates calcium signaling as part of its chemotherapeutic activity. In this study, we determined the chemotherapeutic activity of three RES esters that have been modified at the 4' hydroxyl by the addition of pivalate, butyrate, and isobutyrate. All of the RES derivatives disrupted the calcium signaling in prostate cancer cells more than the parent compound, RES. Further, we demonstrate that the RES derivatives may disrupt the calcium homeostasis by activating calcium release from the endoplasmic reticulum and inhibiting plasma membrane Ca2+-ATPase. The pivalated and butyrated RES derivatives decreased cell viability significantly more than RES. Because pivalated and butyrated RES are more effective than RES at targeting calcium signaling pathways, pivalated and butyrated RES may serve as more effective chemotherapeutics.

Measuring Enzymatic Stability by Isothermal Titration Calorimetry.

This work demonstrates a new method for measuring the stability of enzyme activity by isothermal titration calorimetry (ITC). The peak heat rate observed after a single injection of the substrate solution into an enzyme solution is correlated with enzyme activity. Multiple injections of the substrate into the same enzyme solution over time show the loss of enzyme activity. The assay is autonomous, requiring very little personnel time, and is applicable to most media and enzymes.

Application of Mixture Design Response Surface Methodology for Combination Chemotherapy in PC-3 Human Prostate Cancer Cells.

Combining chemotherapeutics to treat malignant tumors has been shown to be effective in preventing drug resistance, tumor recurrence, and reducing tumor size. We modeled combination drug therapy in PC-3 human prostate cancer cells using mixture design response surface methodology (MDRSM), a statistical technique designed to optimize compositions that we applied in a novel manner to design combinations of chemotherapeutics. Conventional chemotherapeutics (mitoxantrone, cabazitaxel, and docetaxel) and natural bioactive compounds (resveratrol, piperlongumine, and flavopiridol) were used in 12 different combinations containing three drugs at varying concentrations. Cell viability and cell cycle data were collected and used to plot response surfaces in MDRSM that identified the most effective concentrations of each drug in combination. MDRSM allows for extrapolation of data from three or more compounds in variable ratio combinations, unlike the Chou-Talalay method. MDRSM combinations were compared with combination index data from the Chou-Talalay method and were found to coincide. We propose MDRSM as an effective tool in devising combination treatments that can improve treatment effectiveness and increase treatment personalization, because MDRSM measures effectiveness rather than synergism, potentiation, or antagonism.

Calorimetric Methods for Measuring Stability and Reusability of Membrane Immobilized Enzymes.

The aim of this work is to develop calorimetric methods for characterizing the activity and stability of membrane immobilized enzymes. Invertase immobilized on a nylon-6 nanofiber membrane is used as a test case. The stability of both immobilized and free invertase activity was measured by spectrophotometry and isothermal titration calorimetry (ITC). Differential scanning calorimetry was used to measure the thermal stability of the structure and areal concentration of invertase on the membrane. This is the 1st demonstration that ITC can be used to determine activity and stability of an enzyme immobilized on a membrane. ITC and spectrophotometry show maximum activity of free and immobilized invertase at pH 4.5 and 45 to 55 °C. ITC determination of the activity as a function of temperature over an 8-h period shows a similar decline of activity of both free and immobilized invertase at 55 °C. PRACTICAL APPLICATION: Enzyme-catalyzed reactions occur in mild and environmentally friendly conditions, but are usually too costly to use in food manufacturing. When free enzymes are used, they are used once and replaced for each reaction, but enzymes immobilized on a solid support can be reused and have the additional advantage of being removed from the product. In this study, new calorimetric methods that are universally applicable to characterizing immobilized enzymes are used to determine the activity, stability, and reusability of invertase immobilized on a nanofiber support.

The Effects of 4'-Esterified Resveratrol Derivatives on Calcium Dynamics in Breast Cancer Cells.

Triple-negative breast cancer is a highly aggressive subtype of breast cancer. Frequently, breast cancer cells modulate their calcium signaling pathways to optimize growth. Unique calcium pathways in breast cancer cells could serve as a way to target tumorigenic cells without affecting normal tissue. Resveratrol has previously been shown to activate calcium signaling pathways. We use cell viability, single-cell calcium microscopy, and RT-PCR assays to determine the activity and mechanism of three different 4'-esterified resveratrol derivatives. We demonstrate that two of the derivatives reduce cell viability more effectively than resveratrol in MDA-MB-231 human breast cancer cells. The derivatives also activate similar pro-apoptotic calcium signaling pathways. In particular, the pivalated and butyrated resveratrol derivatives are intriguing putative chemotherapeutics because they are more effective at decreasing cell viability in vitro and inhibiting the plasma membrane Ca2+-ATPase, a protein that is often modulated in breast cancer.

Avenanthramide-C reduces the viability of MDA-MB-231 breast cancer cells through an apoptotic mechanism.

BACKGROUND: Avenanthramides (AVN) are a relatively unstudied family of phytochemicals that could be novel chemotherapeutics. These compounds, found in oats, are non-toxic to healthy cells and have been shown to reduce viability of human colon and liver cancers in vitro. However, these studies do not elucidate a molecular mechanism for individual AVN. In this study we aim to see the effects of AVN on MDA-MB-231 breast cancer cells. METHODS: An MTT assay was used to determine cell viability. Staining and analysis with a flow cytometer was used to identify cell cycle progression and apoptosis. FloJo software was used to analyze the cytometric data. In all experiments, statistical significance was determined by a two-tailed t test. RESULTS: This study demonstrates that AVN-A, B, and C individually reduce viability in the MDA-MB-231 breast cancer cell line. AVN-C has the most potent decrease in tumor cell viability, decreasing viable cells to below 25% at 400 µM when compared to control after 96 h. We demonstrate that treatment with AVN-C causes DNA fragmentation and accumulation of over 90% of cells into a sub G1 cell cycle population. Further, we conclude that AVN-C treated cells activate apoptosis because 97% of treated cells stain positive for annexin V while 91% have caspase-3/7 activity, a late marker of apoptosis. CONCLUSIONS: Breast cancer cells treated with AVN-C have a decrease in cell viability, an increase in the sub G1 population, and stain positive for both annexin V and caspase activity, indicating that AVN-C induces apoptosis in breast cancer cells. These compounds may be able to act as chemotherapeutics as demonstrated through future in vivo studies.

Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium.

Plasma membrane Ca2+-ATPase (PMCA) plays a vital role in maintaining cytosolic calcium concentration ([Ca2+] i ). Given that many diseases have modified PMCA expression and activity, PMCA is an important potential target for therapeutic treatment. This study demonstrates that the non-toxic, naturally-occurring polyphenol resveratrol (RES) induces increases in [Ca2+] i via PMCA inhibition in primary dermal fibroblasts and MDA-MB-231 breast cancer cells. Our results also illustrate that RES and the fluorescent intracellular calcium indicator Fura-2, are compatible for simultaneous use, in contrast to previous studies, which indicated that RES modulates the Fura-2 fluorescence independent of calcium concentration. Because RES has been identified as a PMCA inhibitor, further studies may be conducted to develop more specific PMCA inhibitors from RES derivatives for potential therapeutic use.

Natural products induce a G protein-mediated calcium pathway activating p53 in cancer cells.

Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cells to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death.

Small Retinoprotective Peptides Reveal a Receptor-binding Region on Pigment Epithelium-derived Factor.

The cytoprotective effects of pigment epithelium-derived factor (PEDF) require interactions between an as of a yet undefined region with a distinct ectodomain on the PEDF receptor (PEDF-R). Here we characterized the area in PEDF that interacts with PEDF-R to promote photoreceptor survival. Molecular docking studies suggested that the ligand binding site of PEDF-R interacts with the neurotrophic region of PEDF (44-mer, positions 78-121). Binding assays demonstrated that PEDF-R bound the 44-mer peptide. Moreover, peptide P1 from the PEDF-R ectodomain had affinity for the 44-mer and a shorter fragment within it, 17-mer (positions 98-114). Single residue substitutions to alanine along the 17-mer sequence were designed and tested for binding and biological activity. Altered 17-mer[R99A] did not bind to the P1 peptide, whereas 17-mer[H105A] had higher affinity than the unmodified 17-mer. Peptides 17-mer, 17-mer[H105A], and 44-mer exhibited cytoprotective effects in cultured retina R28 cells. Intravitreal injections of these peptides and PEDF in the rd1 mouse model of retinal degeneration decreased the numbers of dying photoreceptors, 17-mer[H105A] being most effective. The blocking peptide P1 hindered their protective effects both in retina cells and in vivo. Thus, in addition to demonstrating that the region composed of positions 98-114 of PEDF contains critical residues for PEDF-R interaction that mediates survival effects, the findings reveal distinct small PEDF fragments with neurotrophic effects on photoreceptors.

The sustained delivery of resveratrol or a defined grape powder inhibits new blood vessel formation in a mouse model of choroidal neovascularization.

The objective of this study was to determine whether resveratrol or a defined, reconstituted grape powder can attenuate the formation of new blood vessels in a mouse model of choroidal neovascularization (CNV). To accomplish this objective, C57BL/6J mice were randomized into control or treatment groups which received either resveratrol or grape powder by daily oral gavage, resveratrol or grape powder delivered ad libitum through the drinking water, or resveratrol by slow release via implanted osmotic pumps. A laser was used to rupture Bruch's membrane to induce CNV which was then detected in sclerochoroidal eyecups stained with antibodies against intercellular adhesion molecule-2. CNV area was measured using fluorescence microscopy and Image J software. Ad libitum delivery of both resveratrol and grape powder was shown to significantly reduce the extent of CNV by 68% and 57%, respectively. Parallel experiments conducted in vitro demonstrated that resveratrol activates p53 and inactivates Akt/protein kinase B in choroidal endothelial cells, contributing to its anti-proliferative and anti-migratory properties. In addition resveratrol was shown to inhibit the formation of endothelial cell networks, augmenting its overall anti-angiogenic effects. The non-toxic nature of resveratrol makes it an especially attractive candidate for the prevention and/or treatment of CNV.

Pigment epithelium-derived factor (PEDF) prevents retinal cell death via PEDF Receptor (PEDF-R): identification of a functional ligand binding site.

The extracellular pigment epithelium-derived factor (PEDF) displays retina survival activity by interacting with receptor proteins on cell surfaces. We have previously reported that PEDF binds and stimulates PEDF receptor (PEDF-R), a transmembrane phospholipase. However, the PEDF binding site of PEDF-R and its involvement in survival activity have not been identified. The purpose of this work is to identify a biologically relevant ligand-binding site on PEDF-R. PEDF bound the PEDF-R ectodomain L4 (Leu(159)-Met(325)) with affinity similar to the full-length PEDF-R (Met(1)-Leu(504)). Binding assays using synthetic peptides spanning L4 showed that PEDF selectively bound E5b (Ile(193)-Leu(232)) and P1 (Thr(210)-Leu(249)) peptides. Recombinant C-terminal truncated PEDF-R4 (Met(1)-Leu(232)) and internally truncated PEDF-R and PEDF-R4 (ΔHis(203)-Leu(232)) retained phospholipase activity of the full-length PEDF-R. However, PEDF-R polypeptides without the His(203)-Leu(232) region lost the PEDF affinity that stimulated their enzymatic activity. Cell surface labeling showed that PEDF-R is present in the plasma membranes of retina cells. Using siRNA to selectively knock down PEDF-R in retina cells, we demonstrated that PEDF-R is essential for PEDF-mediated cell survival and antiapoptotic activities. Furthermore, preincubation of PEDF with P1 and E5b peptides blocked the PEDF·PEDF-R-mediated retina cell survival activity, implying that peptide binding to PEDF excluded ligand-receptor interactions on the cell surface. Our findings establish that PEDF-R is required for the survival and antiapoptotic effects of PEDF on retina cells and has determinants for PEDF binding within its L4 ectodomain that are critical for enzymatic stimulation.

Resveratrol metabolites do not elicit early pro-apoptotic mechanisms in neuroblastoma cells.

Resveratrol, a nontoxic polyphenol, has been shown to inhibit tumor growth in a xenograft mouse model of neuroblasoma. However, resveratrol is rapidly metabolized, mainly to its glucuronidated and sulfated derivatives. This study demonstrates that resveratrol alone, and not the glucuronidated or sulfated metabolites, is taken up into tumor cells, induces a rise in [Ca(2+)](i), and ultimately leads to a decrease in tumor cell viability. A new water-soluble resveratrol formulation was delivered directly at the site of the tumor in a neuroblastoma mouse model. The amount of unmodified resveratrol associated with the tumor increased more than 1000-fold. The increase of unmodified resveratrol associated with the tumor resulted in tumor regression. The number of residual tumor cells that remained viable also decreased as the ratio of the metabolites relative to unmodified resveratrol declined.

Non-reductive iron release from horse spleen ferritin using desferoxamine chelation.

The rate of Fe(3+) release from horse spleen ferritin (HoSF) was measured using the Fe(3+)-specific chelator desferoxamine (DES). The reaction consists of two kinetic phases. The first is a rapid non-linear reaction followed by a slower linear reaction. The overall two-phase reaction was resolved into three kinetic events: 1) a rapid first-order reaction in HoSF (k(1)); 2) a second slower first-order reaction in HoSF (k(2)); and 3) a zero-order slow reaction in HoSF (k(3)). The zero-order reaction was independent of DES concentration. The two first-order reactions had a near zero-order dependence on DES concentration and were independent of pH from 6.8 to 8.2. The two first-order reactions accounted for 6-9 rapidly reacting Fe(3+) ions. Activation energies of 10.5±0.8, 13.5±2.0 and 62.4±2.1kJ/mol were calculated for the kinetic events associated with k(1), k(2), and k(3), respectively. Iron release occurs by: 1) a slow zero-order rate-limiting reaction governed by k(3) and corresponding to the dissociation of Fe(3+) ions from the FeOOH core that bind to an Fe(3+) binding site designated as site 1 (proposed to be within the 3-fold channel); 2) transfer of Fe(3+) from site 1 to site 2 (a second binding site in the 3-fold channel) (k(2)); and 3) rapid iron loss from site 2 to DES (k(1)).

Resveratrol: challenges in translation to the clinic--a critical discussion.

Low cancer survival rates and the serious side effects often associated with current chemotherapeutics highlight the need for new and effective nontoxic anticancer agents. Since 1997 when Jang and colleagues first described resveratrol's ability to inhibit carcinogenesis, it has consistently proven effective at tumor inhibition in diverse human cancer models. This finding has raised the hope that resveratrol would pioneer a novel class of nontoxic chemotherapeutics. As a consequence of initial basic and preclinical studies, resveratrol is now being extensively promoted in the unregulated nutraceutical sector. However, some fundamental aspects of resveratrol's action need to be understood before it can be developed into a clinically viable anticancer drug. These areas pertain to the key mechanism(s) by which resveratrol potentiates its antitumor effects. Current research suggests that these mechanisms might be through novel pathways, requiring an understanding of cellular uptake, sentinel targets, and in vivo biological networks. The metabolism of resveratrol and its bioavailability also warrant further consideration in light of recent in vitro and in vivo studies. Finally, we need to appreciate the sorts of information about resveratrol that may translate between different disease entities. We present a critical discussion of these issues and suggest important experiments that could pave the way to the successful translation of resveratrol to the clinic.