Fluorescent Staining Technique Research Articles

The Anti-oxidative Effects of Encapsulated Cysteamine During Mice In Vitro Matured Oocyte/Morula-Compact Stage Embryo Culture Model: a Comparison of High-Efficiency Nanocarriers for Hydrophilic Drug Delivery-a Pilot Study.

Although it is well-recognized that antioxidant nano-encapsulation has many benefits such as minimizing side effects (e.g., high-dose toxicity), the most attention was paid to the hydrophobic antioxidant not hydrophilic. In this regard, we sought to compare two hydrophilic model nanocarriers to deliver the optimal dose of cystamine (Cys) into the in vitro matured oocyte and the first cleavage stages until morula-compact stage embryonic cells. The formation of Cys-loaded solid self-emulsifying lipid (Cys + SLN) and Cys-loaded chitosan shell (Cys-CS-NC) were confirmed by FT-IR and UV-Vis spectrophotometry, dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) technologies. In two experiments, the oocytes/presumptive zygotes were cultured under various concentrations of Cys-SLN and Cys-CS-NC. The results of nuclear staining (aceto-orcein and Hoechst 33342), H2DCFDA fluorescent staining, chemiluminescence test, and quantitative reverse transcription-PCR (qRT-PCR) technique as in vitro toxicity studies demonstrated that adding the lowest dose of Cys-encapsulated in both nanocarriers [Cys-SLN (5μM) and Cys-CS-NC (10μM)] to maturation or culture medium could accumulate a strong anti-oxidative effect in oocyte/embryo by controlled release and enhanced intracellular penetration of Cys. In comparison, Cys-SLN (5μM) is more effective than Cys-CS-NC (10μM) groups to improve the expression of antioxidant genes (SOD, CAT, GPx) or anti-apoptotic (BCL-2) gene and decreased apoptosis (BAX and caspase-3) or intra-/extracellular ROS levels. In a nutshell, both nanocarriers (CS-NC or SLN) can deliver the lowest dose of Cys into the oocyte/embryo, thus encouraging a better expansion of antioxidant genes and enhancing the development of in vitro oocyte/embryo.

Zingerone suppresses cell proliferation via inducing cellular apoptosis and inhibition of the PI3K/AKT/mTOR signaling pathway inhuman prostate cancer PC-3 cells.

Prostate cancer (PCa) is both the foremost and second cause of cancer death in the male population. Patients with hormone-dependent PCa are initially sensitive to androgen-deprivation therapy, later the cancer progress to a hormone-independent state and fails to respond and progress to the metastatic stage, where the cells gain the ability to escape cell death and develop resistance to current therapies, thereby leadingto migration, invasion, and metastasis of cancer. Many clinical trialsusing nutraceuticals on cancer using human subjects have also been extensively studied, these studies confirm the efficacy of drugs tested in in vitro and in vivo preclinical models. Among various dietary phytochemicals, ginger is commonly used in the diet and possesses manyactive principles that act against cancer. Among various active principles, zingerone isa key active phenolic compound present in Zingiber officinale (Ginger), it has potent antioxidant property and it acts against carcinogens. The present study evaluated the efficacy of zingerone at different doses on the PCa cell line regarding apoptosis, upstream signing molecules such as Akt/mTOR, and migration metastasis. A cell viability assay using MTT wasperformed to estimate the percentage of viability of zingerone-treated PC-3 cells. The mitochondrial membrane potential, intracellular reactive oxygen species, and apoptosis induction in the zingerone-treated PC-3 cells were studied by using different fluorescencestaining techniques. The expression patterns of PI3K, AKT, p-AKT, mTOR, and p-mTOR were investigated through the Western blot analysis assay. Zingerone induces apoptosis and alters Akt/mTOR molecules; it also inhibits cell adhesion andmigration of PCa cells. From the present study, it is concluded thatzingerone effectively induces apoptosis and inhibits cancer signaling, thereby acting as a potent drug against PCa.

Laparoscopic Portal Territory Hepatectomy (Extended Segment 5) by an Indocyanine Green Fluorescent Dual Staining Technique (Video)

BackgroundAnatomical liver resections (ALR), which both remove the tumor and the corresponding segments, meet more with the surgical oncology’s radical nature as reported by Makuuchi.1 Nevertheless, laparoscopic ALR remains a highly specialized procedure due to technical difficulty in the selection of the transection plane. This video aimed to present a novel laparoscopic ALR strategy of tumor-bearing portal territory hepatectomy with an indocyanine green (ICG) fluorescent dual staining technique. Video: A 42-year-old man admitted to our center for a single hepatic mass at the end of segment 5. To test liver reserve function and locate the tumor, we intravenously administrated ICG (0.5 mg/kg) 5 days before the operation. The ICG-R15 of the patient was 4.1%. The Glissonian pedicles of target portal territory were approached and temporally clamped with Takasaki’s Glissonian method as discussed by Takasaki.2 Then, we intravenously administered ICG (1 mL, 5 mg/L) to negative stain the portal territory of segment 5 and expose biliary leak from the cut surface. Fusion ICG imaging of the tumor and positive fluorescent region were obtained using the PINPOINT image system (Stryker, Kalamazoo, MI). Liver parenchyma transection was performed alongside the boundary of negative fluorescence region using an ultrasonic scalpel and L-PMOD as reported by Cai et al..3 Since the tumor located at the junction of segments 5 and 8, we did some extended resection. ResultsOperative time was 195 min, and the estimated intraoperative blood loss was 100 mL. The patient was discharged on the seventh day, without any complications. HCC was confirmed in histopathology with a free margin (over 1.3 cm). No recurrence was noted in the follow-up period for 6 months after the operation. ConclusionLaparoscopic anatomical S5 segmentectomy using portal territory hepatectomy strategy in a dual staining method was technically feasible and safe for patients with HCC located in segment 5.

Cucurbita ficifolia Fruit Extract Induces Tp53/Caspase-Mediated Apoptosis in MCF-7 Breast Cancer Cells.

The second most biggest cancer worldwide is breast cancer. There is an increasing need for safer, effective, and affordable drug candidates from natural sources to treat breast cancer. In the present investigation, the anticancer effect of Cucurbita ficifolia Bouché (C. ficifolia) fruit extract was tested on the human breast cancer cells such as MCF-7. The cells were exposed with different doses of C. ficifolia, for the assessment of IC50 concentrations on the MCF-7 cell lines for 24 hs. The effect of C. ficifolia fruit extract on morphological and apoptotic changes were evaluated by specific fluorescence staining techniques and real-time PCR in a time-dependent manner for 24 hs and 48 hs. The IC50 value for C. ficifolia fruit extract was found to be 90 μg/mL. Morphological alteration and apoptotic distinctiveness aspect like chromatin condensation and nuclear fragmentation were noticed in C. ficifolia extract exposed breast cancer cells. Further, we observed that C. ficifolia extract-induced programmed cell death in the MCF-7 cells were mediated with the elevated expression of the tumor suppressor gene such as p53 and apoptotic markers such as caspase-8, caspase-9, caspase-3, fatty acid synthase (FAS), Fas-associated protein with death domain (FADD), Bcl-2 homologous antagonist/killer (BAK), and Bcl-2-associated X protein (BAX). These observations established that C. ficifolia significantly concealed the cell division and provoked p53/caspase-mediated programmed cell death. Further, we noticed that this cell death in MCF-7 cells is concentration and time dependent. As evaluated through the comet assay, C. ficifolia induced DNA damage; further upon increasing the duration of the treatment, the DNA damage was higher than before. Thus, our study concludes that C. ficifolia could serve as an effective anticancer agent through vital gene modulation.

Porcine RACK1 negatively regulates the infection of classical swine fever virus and the NF-κB activation in PK-15 cells

Classical swine fever (CSF) is one of the main viral diseases of swine worldwide. The causative pathogen is CSF virus (CSFV), a small enveloped RNA virus of the genus Pestivirus. Activation of NF-κB is a hallmark of most viral infections and the viral pathogens frequently kidnap NF-κB pathway for their own advantages, however, it is unclear or even controversial about whether CSFV infection can activate NF-κB signal pathway. RACK1 was shown as an interacting host protein with CSFV NS5A protein, but no studies so far have clearly defined the role of RACK1 during CSFV infection and NF-κB activation. In this study, to properly address these open questions, using RT-qPCR, western blot, indirect fluorescence staining, siRNA knockdown and protein overexpression techniques, we demonstrated that CSFV infection reduced the RACK1 expression at both mRNA and protein levels in PK-15 cells. Downregulation of cellular RACK1 enhanced CSFV infection and subsequent NF-κB activation, while RACK1 overexpression inhibited CSFV infection and the NF-κB activation. In conclusion, RACK1 is a negative cellular regulator for CSFV infection and NF-κB activation in PK-15 cells. Our work addressed a novel aspect concerning the regulation of innate antiviral immune response during CSFV infection. This study may provide some insights into the molecular mechanisms of CSFV infection in swine. However, the elaborate mechanism by which CSFV regulates NF-κB activation and how RACK1 plays its roles in CSFV infection and NF-κB induction require further in-depth studies.

Piperine loaded zinc oxide nanocomposite inhibits the PI3K/AKT/mTOR signaling pathway via attenuating the development of gastric carcinoma: In vitro and in vivo studies

Gastric cancer (GC) is the fifth most cancer type and the third most cause of cancer-associated deaths worldwide along with the 5-year survival rate is less the 30%. This investigation was aimed to synthesis the piperine-loaded zinc oxide nanocomposite (ZnO-Pip-NC) and investigating its anticancer activity against the GC by in vitro and in vivo models by the inhibiting the apoptotic and PI3K/Akt/mTOR signaling pathways. The synthesized ZnO-Pip-NC was characterized by different techniques. The cytotoxicity of zinc oxide, piperine and the formulated ZnO-Pip-NC was tested against the AGS cells by MTT assay. The intracellular ROS level, mitochondrial membrane potential, and apoptotic cell necrosis in the AGS cells was examined by fluorescent staining techniques. The expression of apoptotic and PI3K/Akt/mTOR signaling markers were inspected by western blotting and the expression of pro0inflammatory markers analyzed by RT-PCR technique. The antioxidant levels were examined by standard methods and histopathology of gastric mucosa was analyzed. The ZnO-Pip-NC treatment appreciably inhibited the AGS cell viability. ZnO-Pip-NC treated cells also exhibited excessive intracellular ROS, diminished MMP, nuclear damages, and apoptosis induction in AGS cells. The enhanced expression of pro-apoptotic proteins and inhibition of PI3K/Akt/mTOR signaling pathway was noted in ZnO-Pip-NC treated cells. In vivo studies proved that the ZnO-Pip-NC noticeably restored the antioxidants in the GC animals and also prevented the gastric mucosa and inhibited the GC tumor formation. In conclusion, the findings of this investigation confirmed the anticancer potential of ZnO-Pip-NC against the GC via inhibiting the PI3K/Akt/mTOR signaling pathway.

Betulinic Acid-Nitrogen Heterocyclic Derivatives: Design, Synthesis, and Antitumor Evaluation in Vitro.

Betulinic acid (BA) is a star member of the pentacyclic triterpenoid family, which exhibits great prospects for antitumor drug development. In an attempt to develop novel antitumor candidates, 21 BA-nitrogen heterocyclic derivatives were synthetized, in addition to four intermediates, 23 of which were first reported. Moreover, they were screened for in-vitro cytotoxicity against four tumor cell lines (Hela, HepG-2, BGC-823 and SK-SY5Y) by a standard methylthiazol tetrazolium (MTT) assay. The majority of these derivatives showed much stronger cytotoxic activity than BA. Remarkably, the most potent compound 7e (the half maximal inhibitory concentration (IC50) of which was 2.05 ± 0.66 μM) was 12-fold more toxic in vitro than BA-treated Hela. Furthermore, multiple fluorescent staining techniques and flow cytometry collectively revealed that compound 7e could induce the early apoptosis of Hela cells. Structure–activity relationships were also briefly discussed. The present study highlighted the importance of introducing nitrogen heterocyclic rings into betulinic acid in the discovery and development of novel antitumor agents.

Anticarcinogenic effect of zinc oxide nanoparticles synthesized from Rhizoma paridis saponins on Molt-4 leukemia cells

Leukemia is considered as a multistep progression by progressively genetic modifications that make the conversion of normal human hematopoietic progenitor/stem cells into leukemic cells. In this current investigation, we optimized the reaction of parameters to control the nanoparticle size that was examined via SEM. A various characterization techniques, for example UV–visible spectroscopy, FTIR and EDX were carried out for the synthesized ZnONPs from Rhizoma paridis saponins plant extract. The RPS-ZnONPs were evaluated to inspect their cytotoxic activity against Molt-4 cells via MTT test. Apoptosis was determined through the ROS, MMP and AO/EtBr fluorescent staining techniques. Moreover, pro and anti-apoptotic signaling protein expression was inspected through RT-PCR method. We observed the fabricated RPS-ZnONPs have a crystalline nature, spherical formand various biomolecules were exist. As well, we detected the cytotoxicity effect of synthesized RPS-ZnONPs at a range of 23.5 μg followed to 24hrs treatment. Apoptosis was triggered via the RPS-ZnONPs along with augmented ROS, decreased MMP and altered dual staining. Moreover, the apoptosis were identified through significant up-regulation of Bax, caspase-9 and caspase-3, but the anti-apoptotic protein expression i.e., Bcl-2 was down regulated. Though, this study, we investigated the synthesis, characterization and anticancer activity of ZnONPs of Rhizoma paridis saponins plant extract (RPS-ZnONPs) were useful for treatments of leukemia.

Rapid On-Site Monitoring of Bacteria in Freshwater Environments Using a Portable Microfluidic Counting System.

Freshwater environments and natural water parks are important as recreation areas; however, people enjoying recreation at the river- or lake-side are sometimes infected with pathogenic microbes. Microbiological monitoring is fundamental for the routine evaluation of water quality. Fluorescent staining techniques are regarded as among the most useful rapid microbiological methods; however, preparation of samples for fluorescence microscopy is often labor-intensive, and one usually has to take the samples to a laboratory for measurement, which often alters the culturability of bacteria in the samples. These factors have created demand for a rapid and simple method of bacterial quantification in freshwater that can be performed on-site. In this study, we applied our microfluidic device, which was originally designed for on-chip fluorescent staining and semi-automated counting of target microbial cells with fluorescent antibody-staining, to enumerate bacterial cells in freshwater. This was combined with a self-made portable system for rapid on-site monitoring of the bacterial cells. Numbers of both esterase-active bacteria and total bacteria in pond water samples could be successfully determined by on-chip staining with 6-carboxyfluorescein diacetate and SYBR Green II, respectively, using the portable microfluidic counting system. The counting was completed within 1 h (30 min for pre-filtration of freshwater and 30 min for on-chip staining and counting). These results indicate that rapid and accurate counting of bacterial cells in freshwater can be performed and this technique could be applied for "on-site first screening" purposes in microbial quality control of freshwater.

In-vitro Capacitation of spermatozoa as assessed by chlortetracycline staining in camels (Camelus dromedarius)

Twelve ejaculates were collected from 6 adult healthy dromedary camels during the rutting season to study the effect of heparin, caffeine and calcium-ionophore on the induction of capacitation in dromedary spermatozoa. Each semen sample was evaluated (sperm progressive motility % and sperm concentration × 106/mL). Nine ejaculates out of twelve were diluted with Shotor buffer to obtain 15 aliquots of 5-10 × 106 motile spermatozoa/ 990μl. Five aliquots were mixed with 10 pl of heparin in concentrations of 0 control, 10 IU (2.5 μl/mL), 25 IU (5 μl/ mL), 50 IU (10 μl/mL) and 100 IU (20 μl/mL). Caffeine (10 μl) was added to another 5 aliquots in concentrations of 0 control, 2.5 mM (0.00485g/mL), 5 mM (0.0097 g/mL), 10 mM (0.0194g/mL) and 20 mM (0.0388g/mL). The last 5 aliquots were mixed with 10 pl of calcium-ionophore A23187 in concentrations of 0 control, 0.05 mM (3.75 μl/mL), 0.1 mM (7.53 μl/mL), 0.2 mM (14.95 μl/mL) and 0.3 mM (20 μl/mL). All aliquots were incubated at 38°C in a 5% CO2 atmosphere and 90% relative humidity for 60 min. Aliquots from replications were taken at 0, 5, 15, 30 and 60 min and evaluated for percentages of sperm motility, live sperm and spermatozoa with reacted acrosomes using eosin nigrosin and Chlortetracycline staining. Results revealed differences in viability indices (VI) between camel semen incubated with calcium-ionophore and both semen incubated with heparin and caffeine. Heparin 100 IU, caffeine 5 mM and calcium-ionophore 0.05 mM were the best capacitating factors. A marked increase existed in B (capacitated and acrosome intact) and AR (capacitated and acrosome reacted) patterns cells accompanied with a large decrease in F pattern (uncapacitated and acrosome intact) cells in aliquots with the capacitating factors than in control. In conclusion, heparin (100 IU), caffeine (5 mM) and calcium-ionophore A23187 (0.05 mM) are convenient capacitating factors for dromedary camels' semen. CTC fluorescent staining technique can be used for assessing capacitation status and acrosome reaction in dromedary camels.

Применение методов флюоресцентного окрашивания для определения микроорганизмов в фармацевтическом анализе

Применение методов флюоресцентного окрашивания для определения микроорганизмов в фармацевтическом анализе

Adsorption interaction between cement hydrates minerals with fluid loss additive investigated by fluorescence technique

The mechanism of synthetic polymer fluid loss additive (FLA) was based on adsorption of FLA on the surface of hydrating cement particles and the formation of a complex network structure, resulting in decline of cement slurry filtrate. However, due to the complexity of early hydration environment of cement slurry, there was lack of effective and intuitive way to characterize the adsorption interaction between FLA and cement particles. In this study we described our quest in furthering understanding of the adsorption action of fluid loss additive on the cement minerals. By fluorescence staining technique (combined polymer additives with organic fluorescent dyes), we observed that the skin of the hydrated particles was coated with dyed polymer, which led to a successful fluid loss additive. More importantly, the analysis of the fluorescence intensity of luminous polymer in cement pore solution distinguished the respective adsorption capacity of various admixtures (dyed and undyed) on the surface of the cement particles, so as to solve the problem of the traditional method in characterizing the competitive adsorption between polymers, thus gave a convenient way to analyze the interaction mechanism between polymer admixture and cement particles from the theory and technology.

Biological Impacts of Metal(II) Complex-Based DNA Probes Derived from Bidentate N,O Donor Schiff Base Ligand.

In this article, we have reported the preparation and structural characterization of a new Schiff base ligand (E)-2-(((2,6-difluorophenyl)imino)methyl)phenol (HSBL) and its derived metal(II) complexes [Cu(SBL)2] (1), [Ni(SBL)2] (2) and [Pd(SBL)2] (3). Using various analytical and spectroscopic techniques, their structural properties have been appraised. The proposed chemical structure of HSBL has been confirmed by Single crystal XRD studies. Bidentate characteristic of HSBL and its coordination with metal(II) ions through the oxygen atom of the phenolic group and nitrogen atom of the azomethine group have been evaluated from the FT-IR spectral analysis. Pd(II) complex of HSBL (complex 3) has found to be efficient in bringing about the interaction with DNA as well as BSA molecules. The in vitro antimicrobial studies have been demonstrated that complex 3 has a superior antimicrobial activity than HSBL, complexes 1 and 2. According to the values of zone of inhibition, the antimicrobial ability has been increased in the order of 3 > 1 > 2 > HSBL. A significant decrease in percent cell viability has been suggested that complex 3 has remarkable cytotoxicity (IC50 = 15.7 ± 0.6μg/mL) on human breast cancer (MCF-7) cells. Besides, their induced apoptosis pathway of cytotoxicity has been demonstrated by fluorescence staining techniques using AO/EB staining method. We hope this article will be very helpful for future research on the development of new anticancer agents.

Nanocomposite multifunctional polyelectrolyte thin films with copper nanoparticles as the antimicrobial coatings

The work presents the formation and physicochemical characterization of polyelectrolyte-copper nanocomposite coatings using: poly(diallyldimethylammonium chloride) (PDADMAC) as a polycation, poly(sodium 4-styrenesulfonate)(PSS) as a polyanion and negatively charged copper nanoparticles (CuNPs) to obtain biocompatible surfaces with an antibacterial functionality. The mass and thickness of composite films were investigated by the quartz crystal microbalance with dissipation monitoring (QCM-D) and the ellipsometry whereas, the structure and morphology of coatings were examined using scanning electron microscopy (SEM). The increase of the UV–Vis absorption confirmed the formation of the consecutive layers of the film. Antibacterial activity of the coatings was tested on a representative Gram-positive bacteria strain, Staphylococcus aureus. The microbiological tests were performed and bacteria visualized using fluorescent staining and microscopic technique. It was demonstrated that nanostructured films had antibacterial properties, which makes polyelectrolyte multilayer films containing copper an interesting material in biomedical applications area, e.g., for the prevention of microbial deposition on surfaces.

Diagnostic Yield of Fluorescence and Ziehl-Neelsen Staining Techniques in the Diagnosis of Pulmonary Tuberculosis: A Comparative Study in a District Health Facility

Background Despite the recent advancement in diagnostic methods, the smear microscopy remains the gold standard for the diagnosis of pulmonary tuberculosis in high burden countries like Ghana. Notwithstanding, fluorescence staining technique provides a more efficient option for the detection of Mycobacterium tuberculosis positive smears. This study therefore aimed at assessing the diagnostic performance of fluorescence microscopy (FM) and Ziehl-Neelsen (ZN) staining techniques in the diagnosis of pulmonary tuberculosis. Methods A comparative study was carried out on 100 patients who reported at the Out Patients Department (OPD) or the Directly Observed Therapy (DOT) center of the Kade Government Hospital and were suspected of having pulmonary tuberculosis. Two (2) sputum samples each were collected. This included one spot and one morning sample. The smears were prepared and stained with FM and ZN staining techniques. Xpert MTB/RIF assay was also performed. Results Of the 200 samples analyzed, 71 (35.5%), 46 (23.0%), and 84 (42.0%) were positive for pulmonary tuberculosis when FM, ZN, and XPERT MTB/RIF assays were used, respectively. The mean reading time of FM was three times faster than the ZN technique with very good acceptance (1.5min: 4.6min). The sensitivity and specificity of fluorescent staining to that of XPERT MTB/RIF assay were 84.5% and 100%, respectively, while those of ZN staining were 54.8% and 100%, respectively. Conclusion For a routine laboratory test in a resource-limited setting, our study has demonstrated that fluorescence staining technique is a more sensitive test for the diagnosis of pulmonary tuberculosis as compared to the conventional ZN technique.

Same-day sputum smear microscopy for the diagnosis of pulmonary tuberculosis – Light-emitting diode fluorescent staining with direct sputum versus Ziehl–Neelsen staining with concentration sputum

OBJECTIVE: The objective of the study was to evaluate the utility of fluorescent staining (FS) technique with direct sputum for the diagnosis of pulmonary tuberculosis (TB). MATERIALS AND METHODS: Three sputum specimens (spot, second spot, and morning) were collected from the study participants. One smear was prepared from the direct sample and one using the N-acetyl-L-cysteine-sodium hydroxide concentration method. The direct smears were stained using FS, and Ziehl–Neelsen (ZN) staining was used to stain smears prepared after concentration method. Smear results were pooled and compared in two categories: standard spot-morning and same-day smears. The Chi-square test was used to evaluate the statistical difference in smear-positive cases. RESULTS: Among 2922 participants included in the study, smear positivity was, respectively, 10.5% and 10.2% for FS and ZN smears using spot-morning approach. Using the same-day approach, the smear positivity was, respectively, 10.4% and 9.9% for FS and ZN smears; statistically, the difference was not statistically significant (P > 0.05) between the staining techniques in both the approaches. CONCLUSION: More smear positivity is the advantage of using FS technique. Given this advantage, National TB Control Programs of high-TB-burden countries should consider installing light-emitting diode FS technique in peripheral settings also.

Cytotoxic and apoptotic potential of Phyllodium elegans extracts on human cancer cell lines

ABSTRACT The extract of Phyllodium (P.) elegans was investigated for its anti-cancer properties on brain astroglioma cells (U251-MG), colorectal carcinoma cells (HCT116), and malignant melanoma cells (A375). P. elegans methanolic extract (PeME) showed cytotoxicity on all three cancer cell lines tested. The cell viability assay revealed that PeME significantly reduced the viability of these cells. Clear apoptotic features such as cellular morphology, cell shrinkage, and augmentation of dead cells were observed. Flow cytometry and fluorescence staining techniques confirmed the apoptotic property of PeME. In vitro scratch invasion assay showed that cell migration rate was significantly reduced. Fluorescence microscopic studies using 4′,6-diamidino-2-phenylindole staining showed early and late signs of apoptosis after PeME treatment. Upon PeME stimulation, activation of caspase-3/-9 and Mu-2-related death-inducing gene (MUDENG, MuD) was observed by western blot analysis. JC-1 staining analysis by flow cytometry showed that PeME depolarized the mitochondria membrane potential (MMP). Collectively, these findings, for the first time, point to the fact that PeME has anti-cancer properties against brain, colon, and skin cancer cell lines by depolarizing the MMP and activating apoptotic signaling through the activation of caspase-3/-9 as well as MuD. This is the first report reporting the anticancer activity of this specific plant extract.

Anticancer mechanism of troxerutin via targeting Nrf2 and NF-κB signalling pathways in hepatocarcinoma cell line

Troxerutin (TX), a bioflavonoid widely present in various fruits and vegetables, has shown to exhibit numerous pharmacological properties including anti-neoplastic and anti-cancer activities. Nrf2 and NF-κB are the key transcription factors that regulate oxidative stress and inflammation, therefore we assessed whether TX modulate these pathways and its downstream proteins in HuH-7 hepatocarcinoma cells. TX induced apoptotic cellular and nuclear changes were examined by fluorescence staining techniques, agarose gel electrophoresis and flow cytometry. Oxidative stress was determined through biochemical analysis of antioxidant enzymes and lipid peroxidation profile. The protein expressions of NF-κB and Nrf2 pathway regulators, cell proliferation markers and apoptotic pathway mediators were evaluated by performing immunoblotting, immunocytochemistry and molecular docking. Our results revealed that TX inhibits the growth of HuH-7 cells in a concentration and time-dependent manner. TX treated HuH-7 cells exhibited increased heme oxygenase (HO)-1 protein expression, augmented nuclear translocation of Nrf2, and reduced oxidative stress. Furthermore, TX suppressed the expression of IKKβ which subsequently inhibited the nuclear translocation of NF-κB (p65 subunit), and thus downregulated NF-κB mediated inflammatory responses, proliferation and cell survival. Collectively, our results indicate that TX exerts anti-cancer effect in HuH-7 hepatocarcinoma cells possibly through simultaneous regulation of the molecular signalling pathways, Nrf2 and NF-κB.

Evaluating Different Counter Stains in Fluorescent Staining Technique for Detecting Acid Fast Bacilli: Best Amongst The Better

Tuberculosis is a global emergency needing an early and accurate diagnosis for management and control. Smear microscopy is a rapid diagnostic method for detecting acid fast bacilli (AFB) and improvising this method would contribute in better detection rates. The present study compares the efficacy of various counter stains used in Auramine O staining method in examining sputum smears under fluorescence microscope. This was a prospective study conducted in the Department of Microbiology from January to March, 2017. 102 sputum specimens were collected during this period from patients clinically suspected with pulmonary tuberculosis. Five smears were prepared from each specimen, where each smear was stained with Auramine O staining technique using five different counter stains, 0.1% potassium permanganate, 0.1% methylene blue, 0.1% toluidine blue, 0.1% malachite green and undiluted blue ink. Among the 102 sputum specimens, 50(49.01%), 50(49.01%), 49(48.03%), 44(43.13%) and 48(47.05%) smears showed positive results using potassium permanganate, methylene blue, toluidine blue, malachite green and blue ink respectively. On comparing the degree of fluorescence exhibited by debris in these smears, using potassium permanganate, methylene blue, toluidine blue, malachite green and blue ink, fluorescence was observed in 22(21.6%), 11(10.8%), 46(45.1%), 0(0%) and 31(30.4%) smears respectively. When the counter stains were analyzed, there was a statistically significant difference for debris getting fluoresced (p<0.001).In conclusion, methylene blue as a counter stain has almost equivalent effect compared to routinely used potassium permanganate. It has shown to have the best sensitivity and specificity,reduced debris fluorescence and better contrast in appreciating AFB.

Pterospermum acerifolium (L.) wild bark extract induces anticarcinogenic effect in human cancer cells through mitochondrial-mediated ROS generation.

Plants have many medicinal properties including anticancer activity due to the presence of several secondary metabolites. Current cancer treatment policies are not much effective because of side effects and resistance development. Therefore, the discovery of new phytotherapeutics with no or fewer side effects is highly needed. Pterospermum acerifolium (L.) wild, an angiosperm has a broad application in traditional Indian medicinal system including cancer treatment. Despite, there is no study available on the cytotoxic and apoptotic effect of P. acerifolium in human cancer cells. Exploring the medicinal properties of P. acerifolium plant by its traditional use will be helpful towards developing novel cancer therapeutics. Hence, we decided to demonstrate the anti-carcinogenic property of P. acerifolium ethanolic bark extract against lung (A549) and pancreatic (PANC-1) cancer cells. The cytotoxicity was demonstrated by MTT assay, morphological changes, and scratch invasion assay. Flow cytometry, fluorescence staining techniques, and cell cycle analysis were confirmed the apoptotic property of P. acerifolium plant. The cell viability assay revealed that P. acerifolium ethanolic bark extract significantly reduced the viability of both A549 and PANC-1 cells. Moreover, PANC-1 cells showed more sensitivity towards P. acerifolium ethanolic bark extract than A549 at higher concentrations. Clear visualization of changes such as cytoplasmic condensation, cellular morphology, cell shrinkage, and augmented number of dead cells in both the cancer cells was observed after treatment. Scratch and invasion assay showed that cell migration and invasion rate of both the cancer cells were significantly reduced. Fluorescence microscopic studies using acridine orange/ethidium bromide and DAPI (4', 6-diamidino-2-phenylindole) staining showed early and late apoptotic symptoms after treatment with bark extract. Rhodamine-123 and DCFH-DA staining analysis by fluorescence and flow cytometry showed that bark extract depolarized the mitochondria membrane potential and induced reactive oxygen species (ROS) generation. Cell cycle analysis through flow cytometry using propidium iodide stain showed that P. acerifolium bark extract arrested A549 and PANC-1 cells in sub-G1 phase stated early apoptosis. These findings collectively point to the fact that P. acerifolium bark extract induced cell cytotoxicity in lung and pancreatic cancer cells by modulating mitochondrial-mediated ROS generation, and cell cycle checkpoints.