Differential Staining Research Articles

Carbon dots as a dual sensor for the selective determination of d-penicillamine and biological applications

Dual mode Nanosensors gained tremendous interest in recent years because of their facile synthesis and dual applications. Herein we have synthesized highly fluorescent carbon dots (CDs) from Mahogany fruit shell by chemical oxidation method. The as prepared CDs exhibited selective and sensitive quenching of fluorescence by Fe3+ and also accompanied by a dramatic increase in absorption intensity. Hence these two processes led to fabricate CDs-Fe3+ system as a dual probe. d-Penicillamine (D-PA) has much affinity toward Fe3+ which resulted in recovery of almost 75% fluorescence intensity and decrease in absorption of the CDs-Fe3+ system. Thus, this tendency has been exploited for the selective detection of D-PA by both Spectrofluorimetrically and UV-Visible spectroscopically and showed a wide linear range of detection 0-48 μg mL−1 and 0-40 μg mL−1 respectively. This developed probe offered low cost, high selectivity, repeatability, facile operation and excellent recovery ratio in detection of D-PA in pharmaceutical samples. Moreover, biological applications CDs were investigated using Saccharomyces cerevisiae strain with confocal microscopy. We found that CDs were a biocompatible and ideal candidate for differential staining of yeast cells.

Effective attenuation of glyphosate-induced oxidative stress and granulosa cell apoptosis by vitamins C and E in caprines.

Pesticides are known to cause a wide range of reproductive problems that possess degenerative effects on mammalian fertility. Glyphosate (GLP), a broad-spectrum organophosphate herbicide, is known to be a potent mammalian toxicant. The present study aims at assessing the GLP-induced (0.1, 2.0, and 4.0 mg/ml) granulosa cells toxicity and evaluating the mitigating effects of vitamins C and E (0.5 mM and 1.0 mM) in healthy caprine antral follicles, cultured in vitro in a dose- and time-dependent manner (24, 48, and 72 hr) and subjected to various cytotoxic and geno-toxic analysis, namely, classic histology, EB/AO differential staining, oxidative stress parameters, and antioxidant enzymatic activity. The histomorphological analysis and EB/AO staining elucidated increase in the incidence of apoptotic attributes within granulosa cells with increasing dose and duration of the GLP treatment. The highest apoptotic frequency was observed at 4.0 mg/ml GLP after 72-hr exposure duration in comparison with the control. GLP exposure also led to a significant decline in the antioxidant enzymes' activity, namely, SOD, catalase, and GST along with enhanced lipid peroxidation and reduced FRAP activity in a dose- and time-dependent manner. Vitamins C and E supplementation decreased oxidative stress-mediated granulosa cells apoptosis, suggesting its efficiency to diminish GLP-mediated GCs cytotoxicity and thereby, preventing associated fertility disorders.

DEVELOPMENT OF AZITHROMYCIN LOADED LEMONGRASS OIL BASED MICROEMULSION AND DETERMINATION OF ANTIBACTERIAL POTENTIAL

Objective: Azithromycin (AZM), an azalide drug is used to treat bacterial infections. It is poorly water-soluble, with low human bioavailability due to partial absorption. This can be improved using a microemulsion drug delivery system using essential oil.Methods: Microemulsion system was prepared with AZM solubilized lemongrass oil (Cymbopogon citratus), Tween 20 and water containing 1% (v/v) 10 mmol sodium hydroxide. In vitro drug release was determined using a 14KDa semipermeable dialysis membrane. The kinetics of bacterial killing was done at MIC concentrations, and viable counts were determined hourly for 24 h. Bacterial cell viability was determined by differential staining with acridine orange and ethidium bromide. In vitro toxicity was determined by the MTT assay, while in vivo toxicity was determined in male Wistar rats.Results: The optimized formulation (5:20:75 %) was thermodynamically stable with drug solubility of 366.90 mg/ml and a droplet diameter of 12.4±3.9 nm, which do not show in vivo or in vitro toxicity. In vitro drug release study in simulated body fluids revealed a controlled drug release from microemulsion-based formulation. The MIC was 1μg/ml and 2μg/ml against Staphylococcus aureus and Escherichia coli respectively. In vitro kill kinetics showed>2 log10 killing by 8 h. Bacterial cell viability assay and scanning electron microscopy analysis further confirmed substantial morphological changes due to alteration in the cell membrane.Conclusion: The reduced droplet size and the inherent antibacterial property of lemongrass oil enhanced the efficacy of the AZM loaded ME system in comparison with the bulk drug, against the bacterial pathogens.

Inhibiting transforming growth factor-β signaling regulates in vitro maintenance and differentiation of bovine bone marrow mesenchymal stem cells.

Bovine bone marrow mesenchymal stem cells (bBMSC) are potential stem cell source which can be used for multipurpose. However, their application is limited because the in vitro maintenance of these cells is usually accompanied by aging and multipotency losing. Considering transforming growth factor-β (TGF-β) pathway inhibitor Repsox is beneficial for cell reprogramming, here we investigated its impacts on the maintenance and differentiation of bBMSC. The bBMSC were enriched and characterized by morphology, immunofluorescent staining, flow cytometry, and multilineage differentiation. The impacts of Repsox on their proliferation, apoptosis, cell cycle, multipotency, and differentiation were examined by Cell Counting Kit-8 (CCK-8), real-time polymerase chain reaction, induced differentiation and specific staining. The results showed that highly purified cluster of diffrentiation 73+ (CD73 + )/CD90 + /CD105 + /CD34 - /CD45 - bBMSC with adipogenic, osteogenic, and chondrogenic differentiation capacities were enriched. Repsox treatments (5 μM, 48 hr) enhanced the messenger RNA mRNA levels of the proliferation gene (telomerase reverse transcriptase [ TERT]; basic fibroblast growth factor [ bFGF]), apoptosis-related gene ( bax and Bcl2), antiapoptosis ratio ( Bcl2/bax), and pluripotency marker gene ( Oct4, Sox2, and Nanog), instead of changing the cell cycle, in bBMSC. Repsox treatments also enhanced the osteogenic differentiation but attenuated the chondrogenic differentiation of bBMSC, concomitant with decreased Smad2 and increased Smad3/4 expressions in TGF-β pathway. Collectively, inhibiting TGF-β/Smad signaling by Repsox regulates the in vitro maintenance and differentiation of bBMSC.

A combined banding method that allows the reliable identification of chromosomes as well as differentiation of AT- and GC-rich heterochromatin.

Сonstitutive heterochromatin areas are revealed by differential staining as C-positive chromosomal regions. These C-positive bands may greatly vary by location, size, and nucleotide composition. CBG-banding is the most commonly used method to detect structural heterochromatin in animals. The difficulty in identification of individual chromosomes represents an unresolved problem of this method as the body of the chromosome is stained uniformly and does not have banding pattern beyond C-bands. Here, we present the method that we called CDAG for sequential heterochromatin staining after differential GTG-banding. The method uses G-banding followed by heat denaturation in the presence of formamide with consecutive fluorochrome staining. The new technique is valid for the concurrent revealing of heterochromatin position due to differential banding of chromosomes and heterochromatin composition (AT-/GC-rich) in animal karyotyping.

A tridecaptin-based fluorescent probe for differential staining of Gram-negative bacteria.

The traditional Gram-staining method, which was invented more than a century ago for differentiating bacteria as Gram positive or Gram negative, is still widely practiced in microbiology. However, Gram staining suffers from several problems which can affect the accuracy of the diagnosis. Here, we report a new Gram-negative-specific fluorescent probe, which is based on a narrow-spectrum antibiotic, tridecaptin A1, and allows selective staining of Gram-negative bacteria in different fixed bacterial samples. Solid-phase peptide synthesis was used to prepare the tridecaptin A1-fluorophore conjugate with a single structure. Labeling selectivity of the probe toward Gram-negative bacteria was confirmed by testing against a panel of bacterial species. By combining the use of a previously reported Gram-positive-specific fluorescent probe, we then further showed the capability of the new probe in differential labeling of a number of complex bacterial samples, which included a mouse gut microbiota cultured in vitro, as well as microbiotas collected from the human oral cavity, soil, and crude oil. High labeling selectivity and coverage were observed in most samples. This method offers a new Gram-negative-specific probe with a defined structure, which allows facile fluorescence-based differentiation of Gram-positive and Gram-negative bacteria for further microbial studies.

Isolation and characterization of stem cells from differentially degenerated human lumbar zygapophyseal articular cartilage

The present study aimed to verify the presence of stem cells with multilineage differentiation potential in human lumbar zygapophyseal articular cartilage (LZAC) and to compare the chondrogenic potential of cells obtained from differentially degenerated articular cartilage samples. Surgically obtained human lumbar zygapophyseal joint tissues were classified into the normal, mildly degenerated and severely degenerated groups, according to their pathological characteristics. Primary chondrocytes from these groups were cultured, and stem cells were selected using a monoclonal cell culture method. Differences in stem cell morphology between the three groups were observed using inverted microscopy and phalloidin staining. In addition, stem cell chondrogenic potential was determined through induced differentiation and cellular staining. Gene and protein expression levels of the chondrogenic‑specific markers aggrecan, collagen type‑II and SRY‑related high‑mobility‑group box9 were determined using reverse transcription‑quantitative polymerase chain reaction and western blotting. The clonogenic ability of stem cells in the three groups was determined using a clonogenic assay. It was revealed that stem cells with multilineage differentiation potential were isolated from all three cartilage groups; however, the cells obtained from severely degenerated articular cartilage resulted in severe fibrosis, whilst those obtained from mildly degenerated articular cartilage possessed stronger chondrogenic and clonogenic abilities. Taken together, stem cells with multilineage differentiation potential and clonal properties were identified in human LZAC, and these characteristics were more prominent in mildly degenerated as compared with severely degenerated articular cartilage.

MSC-derived exosomes ameliorate erectile dysfunction by alleviation of corpus cavernosum smooth muscle apoptosis in a rat model of cavernous nerve injury

BackgroundThis study investigated the therapeutic effects of MSC-derived exosomes (MSC-Exos) on erectile function in a rat model of cavernous nerve injury (CNI).MethodsMSCs were isolated from rat bone marrow and exosomes were isolated from the supernatants by ultracentrifugation. The tissue explant adherent method was used to isolate and culture corpus cavernosum smooth muscle cells (CCSMCs). MSCs and CCSMCs were identified by flow cytometry, in vitro differentiation or immunofluorescence staining. Thirty-two 10-week-old male Sprague Dawley (SD) rats were divided into four groups: a sham operation group and bilateral CNI groups that received intracavernosal (IC) injection of either PBS, MSCs or MSC-Exos. Four weeks after CNI and treatment, the erectile function of the rats was measured by electrically stimulating the cavernous nerve. The penile tissues were harvested for blinded histologic analysis and western blotting. H2O2 was used to induce apoptosis in the CCSMCs, and a flow cytometer was used to measure the cell viability of the CCSMCs treated with or without exosomes in vitro.ResultsRecovery of erectile function was observed in the MSC-Exos group. The MSC-Exos treatment significantly enhanced smooth muscle content and neuronal nitric oxide synthase in the corpus cavernosum. The ratio of smooth muscle to collagen in the corpus cavernosum was significantly improved in the MSC-Exos treatment group compared to the PBS vehicle group. WB confirmed these biological changes. Cell viability of the CCSMCs was increased in the MSC-Exos-treated groups, and caspase-3 expression was decreased after the MSC-Exos treatment in vivo and in vitro.ConclusionsExosomes isolated from MSCs culture supernatants by ultracentrifugation could ameliorate CNI-induced ED in rats by inhibiting apoptosis in CCSMCs, with similar potency to that observed in the MSCs-treated group. Therefore, this cell-free therapy has great potential for application in the treatment of CNI-induced ED for replacing cell therapy.Graphical abstractMSC-derived exosomes ameliorate erectile dysfunction in a rat model of cavernous nerve injury

Inactivation of Listeria monocytogenes on paperboard, a food packaging material, using 410 nm light emitting diodes

Light emitting diodes of wavelength 410 nm were used to inactivate Listeria monocytogenes stains on paperboard, an increasingly popular food packaging material. The integrity of the cell membranes was examined using differential fluorescent staining. Scanning electron microscopy (SEM) was used to obtain a deeper understanding of L. monocytogenes stain formation on paperboard and the damage caused to the cells by the LEDs. While the planktonic L. monocytogenes population could be completely inactivated following a brief lag phase that lasted about 20 min, the illumination of the sessile population left some persisters despite immediate commencement of the inactivation. Planktonic populations of inocula sized 3, 5 and 7 log CFU/mL were reduced below the detection limit in 54, 80 and 84 min respectively, whereas it took 120 and 390 min to reach constancy in the sessile populations of inocula sized 5 and 7 log CFU/cm2. The number of membrane-damaged cells was seen to increase with the illumination time. SEM images provided evidence of the protection conferred by the stain on the underlying cells. This study demonstrates that blue LEDs have the potential to reduce the risk of L. monocytogenes contamination from paperboard cartons under refrigeration.

Cytogenetic Trends in Two Families of the Neotropical Catfishes: Heptapteridae and Pseudopimelodidae (Siluriformes).

Several neotropical Siluriformes groups suffered important taxonomic revisions based on the evaluation of morphological and molecular characteristics that allow the construction of new phylogenetic hypothesis. In the present study were cytogenetically analyzed six species belonging to Heptapteridae (Cetopsorhamdia iheringi, Phenacorhamdia tenebrosa, Rhamdella eriarcha, Pimelodella meeki, Pimelodella australis, Heptapterus mustelinus) and two to Pseudopimelodidae families (Microglanis cottoides and Microglanis cibelae) by means of differential staining techniques to describe more precisely cytogenetic similarities and differences. The diploid number of R. eriarcha with 2n = 58 and M. cibelae with 2n = 56 were reported for the first time. Also, the lowest chromosome number (2n = 48) for P. tenebrosa was described. The chromosome-banding techniques for to put in evidence nucleolar organizers impregnated by silver nitrate ([AgNORs], chromomycin A3 [CMA3], and rDNA 18S) showed for all studied species conserved patterns, characteristic for each family. The rDNA 5S showed high variability among species or populations of both families, these regions could be simple or multiple, syntenic, or not with rDNA18S. The chromosome markers showed that both families are related not only from a morphologic point of view but also by their karyotypic characteristics, however, some of the present cytogenetic results evidence the importance of new morphologic, molecular, and phylogenetic studies to improve the knowledge of these fish groups.

Interleukin 17 (IL-17)-Induced Mesenchymal Stem Cells Prolong the Survival of Allogeneic Skin Grafts.

BackgroundMesenchymal stem cells (MSCs) have the potential of self-renewal and multi-differentiation and have a wide application prospect in organ transplantation for the effect of inducing immune tolerance. It has found that interleukin 17 (IL-17) could enhance the inhibition effect of MSCs on T cell proliferation and increase the immunosuppressive effect of MSCs. In this study, we aimed to investigate the effect of IL-17-induced MSCs on allograft survival time after transplantation.Material/MethodsBMSCs were characterized by differential staining. The allogenic skin transplantations were performed and the BMSCs pre-treated by IL-17 were injected. To assess the immunosuppressive function of IL-17-induced BMSCs, the morphology of the grafts, the homing ability of the BMSCs, and the survival time of the grafts were analyzed.ResultsBMSCs from BALB/c have multidirectional differentiation potential to differentiate into osteogenic, chondrogenic, and adipogenic lineage cells. IL-17-induced BMSCs prolonged the survival time of allogeneic skin grafts dramatically. We found that there were more labeled MSCs in the skin grafts, and the Treg subpopulations percentage, IL-10, and TGF-β were significantly increased, while the IFN-γ level was decreased compared to the control group and MSCs group. In conclusion, IL-17 can enhance the homing ability of MSCs and regulate the immunosuppressive function of MSC.ConclusionsOur data demonstrate that IL-17 plays the crucial role in MSC homing behaviors and promotes immunosuppression of MSCs during transplantation procedures, suggesting that IL-17-pre-treated MSCs have potential to prolong graft survival and reduce transplant rejection.

Transient Receptor Potential Channel A1 (TRPA1) Regulates Sulfur Mustard-Induced Expression of Heat Shock 70 kDa Protein 6 (HSPA6) In Vitro.

The chemosensory transient receptor potential ankyrin 1 (TRPA1) ion channel perceives different sensory stimuli. It also interacts with reactive exogenous compounds including the chemical warfare agent sulfur mustard (SM). Activation of TRPA1 by SM results in elevation of intracellular calcium levels but the cellular consequences are not understood so far. In the present study we analyzed SM-induced and TRPA1-mediated effects in human TRPA1-overexpressing HEK cells (HEKA1) and human lung epithelial cells (A549) that endogenously exhibit TRPA1. The specific TRPA1 inhibitor AP18 was used to distinguish between SM-induced and TRPA1-mediated or TRPA1-independent effects. Cells were exposed to 600 µM SM and proteome changes were investigated 24 h afterwards by 2D gel electrophoresis. Protein spots with differential staining levels were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nano liquid chromatography electrospray ionization tandem mass spectrometry. Results were verified by RT-qPCR experiments in both HEKA1 or A549 cells. Heat shock 70 kDa protein 6 (HSPA6) was identified as an SM-induced and TRPA1-mediated protein. AP18 pre-treatment diminished the up-regulation. RT-qPCR measurements verified these results and further revealed a time-dependent regulation. Our results demonstrate that SM-mediated activation of TRPA1 influences the protein expression and confirm the important role of TRPA1 ion channels in the molecular toxicology of SM.

CellDetect® – A Urine-based Differential Staining Assay for Non-invasive Detection of Prostate Cancer

CellDetect® – A Urine-based Differential Staining Assay for Non-invasive Detection of Prostate Cancer

New Properties of a Bioinspired Pyridine Benzimidazole Compound as a Novel Differential Staining Agent for Endoplasmic Reticulum and Golgi Apparatus in Fluorescence Live Cell Imaging.

In this study, we explored new properties of the bioinspired pyridine benzimidazole compound B2 (2,4-di-tert-butyl-6-(3H-imidazo[4,5-c]pyridine-2-yl)phenol) regarding its potential use as a differential biomarker. For that, we performed 1D 1HNMR (TOCSY), UV-Vis absorption spectra in different organic solvents, voltammetry profile (including a scan-rate study), and TD-DFT calculations that including NBO analyses, to provide valuable information about B2 structure and luminescence. In our study, we found that the B2 structure is highly stable, where the presence of an intramolecular hydrogen bond (IHB) seems to have a crucial role in the stability of luminescence, and its emission can be assigned as fluorescence. In fact, we found that the relatively large Stokes Shift observed for B2 (around 175 nm) may be attributed to the stability of the B2 geometry and the strength of its IHB. On the other hand, we determined that B2 is biocompatible by cytotoxicity experiments in HeLa cells, an epithelial cell line. Furthermore, in cellular assays we found that B2 could be internalized by passive diffusion in absence of artificial permeabilization at short incubation times (15 min to 30 min). Fluorescence microscopy studies confirmed that B2 accumulates in the endoplasmic reticulum (ER) and Golgi apparatus, two organelles involved in the secretory pathway. Finally, we determined that B2 exhibited no noticeable blinking or bleaching after 1 h of continuous exposure. Thus, B2 provides a biocompatible, rapid, simple, and efficient way to fluorescently label particular organelles, producing similar results to that obtained with other well-established but more complex methods.

Karyomorphology of Justicia adhatoda L. by differential staining

Karyomorphological features of Justicia adhatoda L. were investigated in this study after differential staining with orcein, CMA and DAPI for proper characterization. In this species "Simple Chromocenter Type" of interphase nuclei was observed with many small heterochromatin blocks after orcein staining. Prophase chromosomes showed "Interstitial Type" of staining pattern which indicated the tendency of aggregation of heterochromatin in interstitial regions of chromosome. This species was found to possess 2n = 34 metacentric chromosomes. The total length of 2n chromosome complement was 132.17 μm. Individual chromosome length ranged from 2.57 to 5.43 μm. The relative length of each chromosome ranged from 0.02 to 0.04. A total of seven CMA-positive bands were observed in this species whereas no band was found after DAPIstaining. This result suggested the absence of AT-rich portions and occurrence of GC-rich portions in the respective banded chromosomes. Except 2n chromosome number, the other karyological and fluorescent banding information probably was the first report for this species and it might help to enhance the chromosomal data base with detailed karyomorphological information of this plant species in Bangladesh.
 Dhaka Univ. J. Biol. Sci. 27(2): 175-181, 2018 (July)

Infection process of Gilbertella persicaria in papaya (Carica papaya L.) fruits

Gilbertella persicaria is a pathogenic fungus recently reported as a causative agent of soft rot in papaya fruits. Here the interactions between G. persicaria and papaya fruits was analyzed under laboratory conditions using histological techniques and optical microscopy to elucidate the process of pathogenesis. Healthy and disinfested fruits of papaya cv. Maradol were also inoculated with a suspension of sporangiospores of G. persicaria. Tissue sections were cut, which were subjected to differential staining with safranin-fast green for different times. Sporangiospores presumably adhered to the cuticle of the fruit by 3 h post inoculation (hpi) and germinated by 6 hpi; invasive intracellular hyphae were growing in host cells by 9 hpi. By 15 hpi, fruit epidermis was macerated, presumably by enzymatic activity reported for mucoral fungal species and appeared as a wet-looking lesion on the cuticle. Fruit mesocarp was colonized by 30 hpi, and asexual reproduction structures had formed by 48 hpi. This process of infection and disease development of G. persicaria in papaya fruits corresponds to that used by pathogens with a necrotrophic lifestyle.

Molecular verification of transplacental transmission of Theileria lestoquardi in goat.

Ovine and caprine malignant theileriosis (OCMT), a critical condition in small ruminant production, causes lethal infections. In September 2016, a total number of 400 goats of Marghoz breed (the largest population of goat breed in Iran), in northwest of Iran, were examined for approximately 25 pre-partum abortions and 7 goats' mortality for a period of 3days. A dead goat and her aborted fetus were brought into the Urmia Veterinary Hospital for further diagnostic investigations. The microbiological assessments including direct microscopical examination of the vaginal discharges and placentome with respect to the differential staining (Gram's staining method), conventional pure culturing, fetal abomasal contents, and the liver were negative. Microscopic examinations of blood smears of the goat and the fetus revealed characteristic of Theileria spp. piroplasms and impression smear samples from goat liver and fetal spleen were positive for Theileria Koch blue bodies. DNA analysis was performed using PCR technique and specific primers derived from the nucleotide sequences of 18S rRNA gene of T. lestoquardi; following extraction from blood samples, placentome, goat liver, and spleen of the fetus. The amplified DNA was sequenced afterwards and the corresponding sequence was registered under GenBank accession number MG208059. The sequence alignment showed that the products of PCR had a homology of 99% to known T. lestoquardi sequence registered under accession numbers of KY352037.1, KC778786.1, and JQ917458.1 in the GenBank. To our knowledge, this is a report demonstrating molecular verification of T. lestoquardi transplacental transmission in a neonatal kid of Marghoz breed of goats, its feasible role in induction of perinatal deaths and abortion in goat flocks.

An improved method for vitrification of in vitro matured ovine oocytes; beneficial effects of Ethylene Glycol Tetraacetic acid, an intracellular calcium chelator

Vitrification affects fertilization ability and developmental competence of mammalian oocytes. This effect may be more closely associated with an intracellular calcium rise induced by cryoprotectants. The present study aimed to assess whether addition of Ethylene Glycol Tetraacetic acid (EGTA) to vitrification solution could improve quality and developmental competence of in vitro matured ovine oocytes. Vitrified groups were designed according to the presence or absence of EGTA and/or calcium in base media, including: mPB1+ (modified PBS with Ca2+), mPB1- (modified PBS without Ca2+), mPB1+/EGTA (mPB1+ containing EGTA), mPB1-/EGTA (mPB1- containing EGTA). In vitro development, numerical chromosome abnormalities, hardening of zona pellucida, mitochondrial distribution and function of viable oocytes were evaluated and compared between groups. Quality of blastocysts was assessed by differential and TUNEL staining. Also, mRNA expression levels of six candidate genes (KIF11, KIF2C, CENP-E, KIF20A, KIF4A and KIF2A), were quantitatively evaluated by RT-PCR. Our results showed that calcium-free vitrification and EGTA supplementation can significantly increase the percentage of normal haploid oocytes and maintain normal distribution and function of mitochondria in vitrified ovine oocytes, consequently improving developmental rate after in vitro fertilization. qRT-PCR analysis showed no significant difference in mRNA expression levels of kinesin genes between vitrified and fresh oocytes. Also, the presence of calcium in vitrification solution significantly increased zona hardening. In conclusion, we have shown for the first time that supplementation of vitrification solution with EGTA, as a calcium chelator, improved the ability of vitrified ovine oocytes to preserve mitochondrial distribution and function, as well as normal chromosome segregation.

Abstract 5813: Thymoquinone influences the anticancer properties of paclitaxel and gemcitabine against breast cancer cells

Abstract The use of anti-cancer adjuvant therapy is rationalized by potentiating the efficacy, and/or protecting from major side effects of chemotherapeutics. Thymoquinone is naturally occurring compound with cumulative evidences of anticancer properties. In this study, we assessed the role of thymoquinone as a potential adjuvant therapeutic in human breast cancer cell lines. Sulfa-Rhodamine-B assay was used to evaluate the effect of thymoquinone on the cytotoxic profile of paclitaxel and gemcitabine in breast cancer cell lines (MCF-7 and T47D). After 72 h of exposure, thymoquinone showed cytotoxic effects against MCF-7 and T47D with IC50's of 64.93±14 µM and 165±2 µM respectively. Combining thymoquinone with paclitaxel increased its IC50 from 0.28±0.1 to 0.3±0.004 µM in MCF-7 cells, with combination index indicative of antagonism (CI-value = 1.53). Similarly, the combination of thymoquinone with paclitaxel in T47D resulted in increasing its IC50 by approximately 1.6 folds (0.09±8 to 0.14±0.02 µM); and the CI-value was 1.66 indicating an antagonistic effect. On the other hand, the IC50 of gemcitabine was significantly reduced after the combination with thymoquinone in both cell lines; MCF-7, T47D, by 15 folds (0.9±0.1 to 0.058±12 µM) and 6 folds (14.3±2 to 2.3±0.2 µM), respectively. In this combination the CI- values were indicative of strong synergism in both cell lines; MCF-7, T47D (0.15 and 0.30 respectively). Further investigation showed that thymoquinone did not affect the cell cycle distribution, however, paclitaxel showed a significant accumulation of cells at G2/M phase in both cell lines after 24, 48h of exposure. On the other hand, gemcitabine caused significant anti-proliferative effect reflected by increasing cell population in S-phase with reciprocal decrease in G2/M cells in both cell lines. The combination of thymoquinone with paclitaxel in T47D didn't enhance paclitaxel-induced G2/M arrest, however it caused significant increase in cell death attributed to this arrest from 38.1% to 69.5%; and from 44.5% to 60.4% after 24 and 48 h of exposure, respectively. Moreover, the combination of thymoquinone with gemcitabine increased S-phase arrest induced by gemcitabine from 44.3% to 49.9%. Accordingly, thymoquinone increased total cell death induced by gemcitabine against T47D from 23.1% to 49.3% and from 15.1 to 64.5 after 24 h and 48 h of exposure, respectively. Using annexin-V/FITC differential staining, thymoquinone significantly increased the percent of apoptotic/necrotic cell death after combination with paclitaxel or gemcitabine. In conclusion, thymoquinone possesses potential chemomodulatory effects to paclitaxel and gemcitabine against breast cancer cells. Despite the promising anti-proliferative activity of thymoquinone against breast cancer, better understanding of these effects are needed to provide a novel approach for the treatment of breast cancer as a disease. Citation Format: Hanan A. Bashmil, Aliaa A. Alamoudi, Abdulwahab Noorwali, Gehan A. Hegazy, Ghada Ajabnoor, Ahmed M. Al-Abd. Thymoquinone influences the anticancer properties of paclitaxel and gemcitabine against breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5813.

Abstract 4624: The sTRA glycan is complementary to CA19-9 as a serologic biomarker of pancreatic cancer

Abstract The CA19-9 biomarker is elevated in a substantial group of patients with pancreatic cancer, but not enough to be reliable for detection, diagnosis, or monitoring of the disease. We hypothesized that a glycan called sTRA complements CA19-9, and that the two biomarkers in combination provide added information over either biomarker individually. As an individual marker, the sTRA assay performed equivalently with CA19-9 for distinguishing cancers from benign pancreatic diseases, but in combination, CA19-9 and sTRA showed exclusive elevations in significantly different groups of patients. Using high-specificity cutoffs giving one false-positive detection in each marker, sTRA was elevated in 34/46 (74%) patients with low CA19-9 in stage III/IV cancers and 28/70 (40%) with low CA19-9 in stage I/II cancers. A three-marker panel consisting of CA19-9 and two versions of the sTRA assay gave 81% sensitivity and 88% specificity (84% accuracy) in a training set of 72 stage I-IV cancers and 75 benign disease controls, compared to 57% sensitivity and 92% specificity (75% accuracy) for CA19-9 alone. The application of the fixed three-marker classifier to a test set of 25 cancers and 25 benign controls gave 80% sensitivity and 84% specificity, compared to 56% sensitivity and 96% specificity for CA19-9 alone. In an analysis of patient-matched tumor tissue and plasma samples, we found that the tumors secreting both sTRA and CA19-9 into the blood tended to be conventional PDAC with well-to-moderate differentiation and heavy marker staining in the ductal lumens, but the tumors secreting only sTRA showed sTRA staining in glands with extremely large and unpolarized nuclei and vacuolated or foamy cytoplasm. Those without plasma elevations in either marker did not have glandular features of typical adenocarcinoma. Surprisingly, about half of the patients without plasma elevations of CA19-9 nevertheless showed strong tumor staining. Such tumors expressed CA19-9 mostly in poorly differentiated patches of cells with compact nuclei and cytoplasm. Thus, sTRA and CA19-9 are complementary serologic biomarkers that together could increase the diagnostic accuracy for PDAC and potentially identify distinct subgroups of tumors. Citation Format: Ying Liu, Daniel Barnett, Ben Staal, Ying Huang, Katie Partyka, Herbert Zeh, Aatur Singhi, Richard R. Drake, Randall E. Brand, Brian B. Haab. The sTRA glycan is complementary to CA19-9 as a serologic biomarker of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4624.