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Objective: We conducted the present study to investigate the anticancer effects of curcumin and chrysin loaded in the alginate-chitosan hydrogel on breast cancer (T47D) and lung cancer (A549). Background: Cancer, which is defined as abnormal cell growth, is one of the biggest public health problems in the world. Natural compounds, such as polyphenols, are used as chemo-preventive and chemotherapeutic agents in different types of cancer owing to their antioxidant, antineoplastic, and cytotoxic properties. To improve their bioavailability and releasing behavior, hydrogel systems with high drug loadingg, stability and hydrophilic nature have been designed. Methods: The curcumin-chrysin-loaded alginate-chitosan hydrogels were prepared through the ionic gelation mechanism utilizing CaCl2. The prepared hydrogels were studied by using the Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). The MTT and DAPI staining assays were employed for cytotoxicity and apoptosis studies of curcumin-chrysin- loaded alginate-chitosan hydrogels. The effects of the curcumin-chrysin-loaded alginate-chitosan hydrogels on the cell cycle of cell lines T47D and A549 were also evaluated using the propidium iodide staining. Results: The curcumin-chrysin-loaded alginate-chitosan hydrogels could significantly (p Conclusion: The alginate-chitosan hydrogels could work best as an enhanced anticancer drug delivery system.

Background: Chronic inflammation plays a crucial role in the initiation, promotion, and invasion of tumors, and thus the antiproliferative effects of numerous anti-inflammatory drugs have been frequently reported in the literature. Upregulation of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) has been linked to various human cancers, including breast cancer. Objective: This research aims to investigate the antiproliferative activity of different Non-steroidal anti-inflammatory drugs (NSAIDs), including COX-2 selective and non-selective agents, against various breast cancer cell lines and to elucidate possible molecular pathways involved in their activity. Methods: The antiproliferative and combined effects of NSAIDs with raloxifene were evaluated by MTT assay. Cell migration was assessed using a wound-healing assay. The mechanism of cell death was determined using the Annexin V-FITC/ propidium iodide staining flow cytometry method. A mass spectrometry-based targeted metabolomics approach was used to profile the metabolomic changes induced in the T47d cells upon drug treatment. Results: Our results have demonstrated that celecoxib, a potent and selective COX-2 inhibitor, resulted in significant antiproliferative activity against all examined breast cancer cell lines with IC50 values of 95.44, 49.50. and 97.70 μM against MDA-MB-231, T47d, and MCF-7, respectively. Additionally, celecoxib exhibited a synergistic effect against T47d cells combined with raloxifene, a selective estrogen receptor modulator. Interestingly, celecoxib treatment increased cell apoptosis and resulted in substantial inhibition of cancer cell migration. In addition, the metabolomic analysis suggests that celecoxib may have affected metabolites (n = 43) that are involved in several pathways, including the tricarboxylic acid cycle, amino acids metabolism pathways, and energy production pathways in cancer cells. Conclusion: Celecoxib may possess potential therapeutic utility for breast cancer treatment as monotherapy or in combination therapy. The reported metabolic changes taking place upon celecoxib treatment may shed light on possible molecular targets mediating the antiproliferative activity of celecoxib in an independent manner of its COX-2 inhibition.

Background: In recent biomedical research, the area of cancer and infectious diseases occupies a leading position in the utilization of medicinal plants as a source of drug discovery. Malaysia has a diversity and a large number of underutilized fruits that are rich in phenolic compounds. Artoarpus altilis is considered an underutilized fruit that is rich in phenolic compounds. Methanol extracts of A. altilis have been previously found to contain a high content of antioxidant phytochemicals. Objective: The purpose of the study was to evaluate the cytotoxicity and toxicological effect of methanol fruit extracts against MCF-7 cells. To determine the least concentration that might kill or suppress the growth of the cancer cells was in a concentration-dependent manner. Methods: The variation in the cytotoxic activity among the extracts was indicated by determining the IC50 of each extract against cells at 72 h. The IC50 of the samples was measured using a trypan blue exclusion assay. The methanol extract of the pulp part showed the least inhibition concentration of 15.40±0.91 μg/mL on MCF-7 cells. In the study, the molecular mechanism of methanol extracts-induced apoptosis and cell cycle arrested in human cancer cells were investigated in a time-dependent-manner by using flow cytometry. The treated cells were stained with nexin to detect early and late apoptosis and with propidium iodide (PI) for cell cycle arreste associated with the DNA fragmentation; various cell arrests occurred at G1/S, S, and G2/M phases. Lastly, the gene expression analysis by RT-qPCR method was carried out by analyzing the expression of the gene of interest for the quantification of mRNA levels. Results: Results after cells were treated with IC50 were revealed by upregulating anti-apoptotic genes/downregulated of pro-apoptotic BCL-2 gene expressions triggered the treated cells into CASPASE-3, intrinsic and extrinsic pathways. Conclusion: These findings suggest that the methanol extracts of three parts of A. altilis fruit have potential anticancer activity against MCF-7 cells mainly the pulp part of the fruit.

Background: Fomitopsis officinalis (Vill. ex Fr. Bond. et Sing) is a medicinal mushroom, commonly called ‘Agarikon’; it has traditionally been used to treat cough and asthma in the Mongolian population. Objective: The objective of the study was to examine the significance of biological activity of F. officinalis and evaluation of the antioxidant activity and anticancer activity of six fractions of F. officinalis residues (Fo1-powder form dissolved in ethanol, Fo2-petroleum ether residue, Fo3-chloroformic, Fo4-ethylacetate, Fo5-buthanolic, and Fo6-waterethanolic) against hepatocellular carcinoma cells. Methods: We performed in vitro studies of cell proliferation and viability assay, annexin V-FITC/Propidium Iodide assay, and NF-kB signaling pathway by immunoblot analysis. Results: Our findings revealed that all six fractions/extracts have antioxidant activity, and somehow, they exert anticancerous effects against cancer cells. In cancerous cell lines (HepG2 and LO2), Fo3 chloroformic extract promoted the cancer cell apoptosis and cell viability, activated G2/M-phase cell cycle, and selectively induced NF-kB proteins, revealing as a novel antitumor extract. Conclusion: This study reports that Fo3-chloroformic extract is rich in antitumor activity, which was previously not investigated in cancer. To develop the impact of F. officinalis among natural products to treat/prevent oxidative stress disorders or cancers, further examinations of F. officinalis are needed to develop new natural drugs to treat cancer. However, this study assessed only one extract, Fo3-chloroformic, which has a significant impact against cancer cell lines.

Silicosis is a leading cause of occupational disease-related morbidity and mortality worldwide, but the molecular basis underlying its development remains unclear. An accumulating body of evidence supports gasdermin D (GSDMD)-mediated pyroptosis as a key component in the development of various pulmonary diseases. However, there is little experimental evidence connecting silicosis and GSDMD-driven pyroptosis. In this work, we investigated the role of GSDMD-mediated pyroptosis in silicosis. Single-cell RNA sequencing of healthy and silicosis human and murine lung tissues indicated that GSDMD-induced pyroptosis in macrophages was relevant to silicosis progression. Through microscopy we then observed morphological alterations of pyroptosis in macrophages treated with silica. Measurement of interleukin-1β release, lactic dehydrogenase activity, and real-time propidium iodide staining further revealed that silica induced pyroptosis of macrophages. Additionally, we verified that both canonical (caspase-1-mediated) and non-canonical (caspase-4/5/11-mediated) signaling pathways mediated silica-induced pyroptosis activation, in vivo and in vitro. Notably, Gsdmd knockout mice exhibited dramatically alleviated silicosis phenotypes, which highlighted the pivotal role of pyroptosis in this disease. Taken together, our results demonstrated that macrophages underwent GSDMD-dependent pyroptosis in silicosis and inhibition of this process could serve as a viable clinical strategy for mitigating silicosis.

Background: Low-molecular citrus pectin (LCP) is a pectin polysaccharide with low molec-ular weight, low degree of crux, and no branching. It is obtained by degrading natural citrus pectin (CP) through physical, chemical and enzymatic methods. LCP has received considerable attention in recent years due to its potential applications in the medical and biological fields. Methods: In our previous study, LCP was prepared from CP by using recombinant Bacillus subtilis pectate lyase B. Monosaccharide comparative analysis revealed that the galacturonic acid content of LCP was higher than that of CP. The cell viability effect of LCP was elucidated by using HepG2 cells and the Cell Counting Kit-8 (CCK-8) assay. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, Annexin V-FITC/PI staining, and flow cytometer propidium iodide stain-ing were performed to detect the effects of LCP on apoptosis and cell cycle arrest in HepG2 cells. Mi-tochondrial membrane potential (MMP) was observed through 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine assay. Results & discussion: The Mw of the prepared LCP was 7.6 kDa, which was significantly lower than that of CP (140 kDa). Cell viability decreased with the increase in the concentration of LCP. The half-inhibitory concentration of 1.46 ± 0.02 mg/mL was determined. Treatment with 1.6 mg/mL LCP in-duced the apoptosis of HepG2 cells with the inhibition rate of 83.10% ± 4.72%, and the cell cycle was arrested in the S phase. Furthermore, the MMP of HepG2 cells decreased with the increase in LCP concentration. Conclusion: The enzymatically prepared LCP could inhibit the proliferation of HepG2 cells. This study provided a partial experimental basis and reference for LCP to become a potential functional food for anti-liver cancer.

Purpose Cell cycle checkpoints and DNA repair are important for cell survival after exogenous DNA damage. Both rapid blockage of G2 to M phase transition in the cell cycle and the maintenance of relatively slow G2 arrest are critical to protect cells from lethal ionizing radiation (IR). Checkpoint kinase 1 is pivotal in blocking the transition from G2 to M phases in response to IR. The 14-3-3σ protein is important for IR-induced G2 arrest maintenance in which p53-dependent 14-3-3σ transcription is involved. It has been demonstrated that Ring finger protein 126 (RNF126), an E3 ligase, is required to upregulate checkpoint kinase 1 expression. Thus, our goal was to study the role of RNF126 in the G2/M phase checkpoint. Methods and Materials The transition from G2 to M phases and G2 accumulation in response to IR were determined by flow cytometry through staining with phospho-histone H3 (pS10) antibody and propidium iodide, respectively. The interaction of RNF126 and 14-3-3σ was determined by GST-pulldown and coimmunoprecipitation assays. The stability of RNF126 and 14-3-3σ was determined by cycloheximide-based stability assay and ubiquitination detection by coimmunoprecipitation. The sequestering of cyclin-dependent kinase 1 and cyclin B1 from the nucleus was determined by immunofluorescence staining. Results RNF126 knockdown had no impact on the IR-induced transient blockage of G2 to M but impaired IR-induced G2 arrest maintenance in cells with or without wild-type p53. Mechanistically, RNF126 binds 14-3-3σ and prevents both proteins from ubiquitination-mediated degradation. Last, RNF126 is required for enforcing the cytoplasmic sequestration of cyclin B1 and cyclin-dependent kinase 1 proteins in response to IR. Conclusions RNF126 promotes G2 arrest via interaction with 14-3-3σ in response to IR. Our study revealed a novel role for RNF126 in promoting G2 arrest, providing a new target for cancer treatment.

Background: In the last decades, metformin (Met), an herbal anti-diabetic medicine, has been proposed as an anti-cancer agent. Objective: Thyroid cancers are the most common malignancy of the endocrine system. Therefore, the current study was performed to assess the effects of Met on cell proliferation and activation of the Phosphoinositide 3-Kinase (PI3K)/Protein kinase B (AKT)/Forkhead Box O1 (FOXO1) signaling pathway in the Medullary Thyroid Carcinoma (MTC) cells. The effects of Met on the expression of REarranged during Transfection (RET) proto-oncogene were also investigated. Methods: MTC cell line (TT) was treated with 0, 2.5, 5, 10, 20, 30, 40, 50, and 60 mM concentrations of Met for 24, 48, and 72h. The viability and apoptosis of the treated cells were measured by the 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) and Annexin V- Propidium Iodide (PI) assays. The expression level of PI3K, AKT, FOXO1, and RET genes was investigated by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), and phosphorylation of their proteins was determined by the Enzyme-Linked Immunosorbent Assay (ELISA). Results: Results showed that Met significantly decreased the viability of the MTC cells. Met also reduced the expression level of PI3K, AKT, and FOXO1 genes (P Conclusion : It seems that the Met has cytostatic effect on the TT cells. Our results showed that anti-tumoral effects of Met may be cell type-specific, and according to the induction of RET (as a proto-oncogene) and inhibition of FOXO1 (as a tumor suppressor gene), Met could not be an appropriate agent in treatment of MTC. The antineoplastic activity of Met has been confirmed against several malignancies in "in vitro" and "in vivo" studies. However, its molecular mechanisms in the treatment of different carcinomas particularly in thyroid cancers are not clearly understood and more studies are required to confirm its exact effect on the MTC.

Ghrelin is a neuropeptide that has various physiological functions and has been demonstrated to be neuroprotective in a number of neurological disease models. However, the underlying mechanisms of ghrelin in Parkinson's disease remain largely unexplored. The current study aimed to study the effects of ghrelin in a 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease model and evaluate the potential underlying mechanisms. In the present study, we treated an SH-SY5Y cell model with 6-OHDA, and observed that pretreatment with different concentrations of ghrelin (1, 10, and 100 nM) for 30 minutes relieved the neurotoxic effects of 6-OHDA, as revealed by Cell Counting Kit-8 and Annexin V/propidium iodide (PI) apoptosis assays. Reverse transcription quantitative polymerase chain reaction and western blot assay results demonstrated that 6-OHDA treatment upregulated α-synuclein and lincRNA-p21 and downregulated TG-interacting factor 1 (TGIF1), which was predicted as a potential transcription regulator of the gene encoding α-synuclein (SNCA). Ghrelin pretreatment was able to reverse the trends caused by 6-OHDA. The Annexin V/PI apoptosis assay results revealed that inhibiting either α-synuclein or lincRNA-p21 expression with small interfering RNA (siRNA) relieved 6-OHDA-induced cell apoptosis. Furthermore, inhibiting lincRNA-p21 also partially upregulated TGIF1. By retrieving information from a bioinformatics database and performing both double luciferase and RNA immunoprecipitation assays, we found that lincRNA-p21 and TGIF1 were able to form a double-stranded RNA-binding protein Staufen homolog 1 (STAU1) binding site and further activate the STAU1-mediated mRNA decay pathway. In addition, TGIF1 was able to transcriptionally regulate α-synuclein expression by binding to the promoter of SNCA. The Annexin V/PI apoptosis assay results showed that either knockdown of TGIF1 or overexpression of lincRNA-p21 notably abolished the neuroprotective effects of ghrelin against 6-OHDA-induced neurotoxicity. Collectively, these findings suggest that ghrelin exerts neuroprotective effects against 6-OHDA-induced neurotoxicity via the lincRNA-p21/TGIF1/α-synuclein pathway.

Monitoring bacteria is essential for ensuring microbial safety of water sources, which include river water and treated wastewater. The plate count method is common for monitoring bacterial abundance, although it cannot detect all live bacteria such as viable but non-culturable bacteria, causing underestimation of microbial risks. Live/Dead BacLight kit, involving fluorochromes SYTO 9 and propidium iodide (PI), provides an alternative to assess bacterial viability using flow cytometry or microscopy. However, its application is limited due to the high cost of flow cytometry and the inapplicability of microscopy to most environmental waters. Thus, this study introduces the combination of BacLight Kit and fluorescence spectroscopy for quantifying live bacteria in river water and treated wastewater. Mixtures of live and dead Escherichia coli (E. coli) with various ratios and total cell concentrations were stained with SYTO 9 and PI and measured by fluorescence spectroscopy. The fluorescence emission peak area of SYTO 9 in the range of 500–510 nm at the excitation wavelength of 470 nm correlates linearly with the viable cell counts (R2 > 0.99, p < 0.0001) with only slight variations in the complex water matrix. The tested method can quantify the concentration of live E. coli from 3.67 × 104 to 2.70 × 107 cells/mL. This method is simple, sensitive and reliable for quantifying live bacteria in environmental water, which can be later integrated into real-time monitoring systems

Background: Podophyllotoxin is a natural lignan which possesses anticancer and antiviral activities. Etoposide and teniposide are semisynthetic glycoside derivatives of podophyllotoxin and are increasingly used in cancer medicine. Objective: The present work was aimed to design and synthesize a series of 2, 4, 5-trideoxyhexopyranosides derivatives of 4’-demethylepipodophyllotoxin as novel anticancer agents. Methods: A divergent de novo synthesis of 2, 4, 5-trideoxyhexopyranosides derivatives of 4’-demethylepipodophyllotoxin has been established via palladium-catalyzed glycosylation. The abilities of synthesized glycosides to inhibit the growth of A549, HepG2, SH-SY5Y, KB/VCR and HeLa cancer cells were investigated by MTT assay. Flow cytometric analysis of cell cycle with propidium iodide DNA staining was employed to observe the effect of compound 5b on cancer cell cycle. Results: Twelve D and L monosaccharides derivatives 5a-5l have been efficiently synthesized in three steps from various pyranone building blocks employing de novo glycosylation strategy. D-monosaccharide 5b showed highest cytotoxicity on five cancer cell lines with the IC50 values from 0.9 to 6.7 mM. It caused HepG2 cycle arrest at G2/M phase in a concentration-dependent manner. Conclusion: The present work leads to the development of novel 2, 4, 5-trideoxyhexopyranosides derivatives of 4’- demethylepipodophyllotoxin. The biological results suggested that the replacement of the glucosyl moiety of etoposide with 2, 4, 5-trideoxyhexopyranosyl is favorable to their cytotoxicity. D-monosaccharide 5b caused HepG2 cycle arrest at G2/M phase in a concentration-dependent manner.

Aims Previous work showed that Bacillus subtilis dormant spore killing and germination by dodecylamine take place by different mechanisms. This new work aimed to optimize killing of B. subtilis and other Firmicutes spores and to determine the mechanism of the killing. Methods and Results Spores of seven Firmicute species were killed rapidly by dodecylamine under optimal conditions and more slowly by decylamine or tetradecylamine. The killed spores were not recovered by additions to recovery media, and some of the killed spores subsequently germinated, all indicating that dodecylamine-killed spores truly are dead. Spores of two species treated with dodecylamine were more sensitive to killing by a subsequent heat treatment, and spore killing of at least one species was faster with chemically decoated spores. The cores of dodecylamine-killed spores were stained by the nucleic acid stain propidium iodide, and dodecylamine-killed wild-type and germination-deficient spores released their stores of phosphate-containing small molecules. Conclusions This work indicates that dodecylamine is likely a universal sporicide for Firmicute species, and it kills spores by damaging their inner membrane, with attendant loss of this membrane as a permeability barrier. Significance and Impact of the Study There is a significant need for agents that can effectively kill spores of a number of Firmicute species, especially in wide area decontamination. Dodecylamine appears to be a universal sporicide with a novel mechanism of action, and this or some comparable molecule could be useful in wide area spore decontamination.

In this unprecedented study, the anticancer properties of gold nanoparticles synthesized using Achillea biebersteinii flower extractwere investigated. Nanoparticle synthesis using herbal extracts is low-cost and can, therefore, serve as an economically viable alternative for large‐scale nanoparticle production. The smallest nanoparticles were spherical within a range of 2–30 nm (with an average of 8 nm). The gold nanoparticles were characterized using UV-visible spectrophotometry, x-ray diffraction, Fourier-transform infrared spectroscopy, dynamic light scattering, and transmission electron microscopy. The gold nanoparticles displayed great antioxidant potential similar to the positive control. Their cytotoxic effects on human testicular embryonic carcinoma stem cells were assessed using the MTT assay and acridine orange/propidium iodide. The nanoparticles demonstrated dose‐dependent cell viability against cancer cells, and the half inhibitory concentration (IC50) values were 10 μg/mL. The expression of Bax, Bcl2, caspase-3, caspase-9, p53, and HSPA2 genes was evaluated using reverse transcription-polymerase chain reaction (RT-PCR) analysis to further confirm that gold nanoparticles induced apoptosis. The cytotoxicity analysis results indicated that the gold nanoparticles were toxic for NTERA-2 cells. These positive outcomes can contribute to the rational design of novel anticancer agents.

Retinitis pigmentosa (RP) is a major cause of blindness that is difficult to diagnose and treat. PKM2, a subtype of pyruvate kinase, is strongly associated with oxidative stress and is expressed in photoreceptors. We investigated whether PKM2 reduces photoreceptor cell apoptosis and evaluated possible antiapoptotic mechanisms in RP. We established RP models by exposing 661W cells to blue light and modulated PKM2 activity using a PKM2 inhibitor. We measured the apoptosis rates using calcein-acetoxymethyl ester/propidium iodide double staining and Cell Counting Kit-8, the oxidative stress levels using a reactive oxygen species assay, and the changes in protein expression by western blotting. Photodamage increased PKM2 expression, cellular oxidative stress, and apoptosis of 661W cells. PKM2 inhibition significantly reduced the levels of apoptosis and oxidative stress induced by photodamage. Our data suggest that PKM2 is a potential disease marker and therapeutic target for RP.

Background: Ligustrazine and chalcones have been reported previously for various biological activities including anticancer effects. Objectives: Based on the multitargeted biological activities approach of ligustrazine-based chalcones, in the current study 18 synthetic ligustrazine-containing α, β-unsaturated carbonyl-based 1, 3- Diphenyl-2-propen-1-one derivatives were evaluated for their inhibitory effects on the growth of five different types of cancer cells. Methods: All the compounds were evaluated for anticancer effects on various cancer cell lines by propidium iodide fluorescence assay and various other assays were performed for mechanistic studies. Results: A majority of compounds exhibited strong inhibition of cancer cells, especially synthetic compounds 4a and 4b, bearing 1-Pyridin-3-yl-ethanone as a ketone moiety in the main structural backbone were found to be most powerful inhibitors of cancer cell growth. Nine most active compounds among the whole series were selected for further studies related to different cancer targets, including EGFR TK kinases, tubulin polymerization, KAF and BRAFV600E. Conclusion: Synthetic derivatives, including 4a-b and 5a-b showed a multitarget approach and strong inhibitory effects on EGFR, FAK and BRAF while three compounds, including 3e bearing methoxy substitution, 4a and 4b with 1-pyridin-3-yl-ethanone moiety showed the inhibition of tubulin polymerization.

Research question Are there any differences in viability and ultrastructure amongst embryos biopsied on Day 5 versus Day 3 following vitrification in open and closed systems and compared to fresh embryos? Design One hundred human embryos (40 blastocysts biopsied on Day 5 and subsequently vitrified in open or closed systems and 60 Day 3 biopsied embryos that developed to blastocysts but were rejected for transfer following preimplantation genetic testing for monogenic/single gene defects and for aneuploidies were either treated fresh [n = 20] or vitrified [n = 40] in open or closed systems) and following warming and culture for 4 h were subjected to viability staining with carboxyfluorescein-diacetate succinimidylester/propidium iodide or processed for transmission electron microscopy. Results No statistically significant differences were observed in the viability of human biopsied embryos following vitrification in open and closed systems. Compared to fresh embryos, vitrified ones had a higher incidence of damage (propidium iodide-stained cells) irrespective of the vitrification method (P = 0.005). These damaged cells were more prominent in Day 5 biopsied blastocysts and mainly located at the position of cutting. Characteristic lipofuscin droplets (representative of apoptosis) and a higher number of vacuoles and distension of mitochondria were also more evident in vitrified embryos, although this was not statistically assessed. Conclusions Vitrification in open and closed systems does not adversely affect the viability and ultrastructure of Day 5 and Day 3 biopsied embryos as revealed by the minimal yet statistically significant cell damage observed. This damage may be compensated by the embryos, which in their attempt to fully recover following vitrification, potentially enable ‘rescue’ processes to eliminate it.

Background and Objectives: Cancer is one of the leading causes of death in the world affecting millions of people. The commercially available anticancer drugs lack the selectivity and show several undue side effects during the biologically targeted therapy, thus calling for the exploration of wider chemical space to furnish new structural leads with promising anticancer potential. In this endeavor, we synthesized a series of coumarinyl thiazolotriazoles with diverse functional group tolerance and will be tested for their anticancer properties against cancer cell lines (HeLa and MCF-7) and a normal cell line (BHK-21). Materials and Methods: To overcome such complications, in the current study, we evaluated the cytotoxic effects of coumarinyl thiazolotriazole hybrids on human breast adenocarcinoma (MCF-7), cervical adenocarcinoma (HeLa) cells and normal cells i.e., Baby Hamster Kidney cells (BHK-21) using MTT (dimethyl-2-thiazolyl- 2,5-diphenyl-2H-tetrazolium bromide) assay. DNA binding studies of compound 6c was performed on Herring- Sperm DNA (HS-DNA) and docking studies were also carried out. The mechanistic studies were performed on potent compounds by fluorescent microscopic studies, release of Lactate Dehydrogenase (LDH) and mitochondrial membrane potential, activation of caspase-9 and -3 and flow cytometric analysis. Results: As revealed by MTT assay, compounds 6m and 6c were identified as the most potent derivatives among the tested series with IC50 values of 5.64 and 29.1 μM against HeLa and MCF cells, respectively as compared to cisplatin which gave IC50 values of 11.3 and 6.20 μM, respectively. DNA binding studies of compound 6c showed the binding of compound in DNA with Gibbs free energy of ‒17 KJ/mol and docking studies validated the DNA binding studies. Fluorescent microscopic studies using 4′,6-diamidino-2-phenylindole (DAPI) and Propidium Iodide (PI) staining confirmed the occurrence of apoptosis in HeLa cells treated with the most active compound 6m. Moreover, compounds 6m and 6c also triggered the release of Lactate Dehydrogenase (LDH) in treated HeLa and MCF-7 cells while a luminescence assay displayed a remarkable increase in the activity of caspase-9 and -3. Moreover, flow cytometric results revealed that compound 6m caused G0 /G1 arrest in the treated HeLa cells. Conclusion: Our results suggested that the compound possesses chemotherapeutic properties against breast cancer and cervical adenocarcinoma cells, thus warranting further research to test the anticancer efficacy of this compound at clinical level.

Background We discovered that pure positive electrostatic charges (PECs) have an intrinsic suppressive effect on the proliferation and metabolism of invasive cancer cells (cell lines and animal models) without affecting normal tissues. Methods We interacted normal and cancer cell lines and animal tumors with PECs by connecting a charged patch to cancer cells and animal tumors. many biochemical, molecular and radiological assays were carried out on PEC treated and control samples. Results Correlative interactions between electrostatic charges and cancer cells contain critical unknown factors that influence cancer diagnosis and treatment. Different types of cell analyses prove PEC‐based apoptosis induction in malignant cell lines. Flowcytometry and viability assay depict selective destructive effects of PEC on malignant breast cancer cells. Additionally, strong patterns of pyknotic apoptosis, as well as downregulation of proliferative‐associated proteins (Ki67, CD31, and HIF‐1α), were observed in histopathological and immunohistochemical patterns of treated mouse malignant tumors, respectively. Quantitative real‐time polymerase chain reaction results demonstrate up/down‐regulated apoptotic/proliferative transcriptomes (P21, P27, P53/CD34, integrin α5, vascular endothelial growth factor, and vascular endothelial growth factor receptor) in treated animal tumors. Expression of propidium iodide in confocal microscopy images of treated malignant tissues was another indication of the destructive effects of PECs on such cells. Significant tumor size reduction and prognosis improvement were seen in over 95% of treated mouse models with no adverse effects on normal tissues. Conclusion We discovered that pure positive electrostatic charges (PECs) have an intrinsic suppressive effect on the proliferation and metabolism of invasive cancer cells (cell lines and animal models) without affecting normal tissues. The findings were statistically and observationally significant when compared to radio/chemotherapy‐treated mouse models. As a result, this nonionizing radiation may be used as a practical complementary approach with no discernible side effects after passing future human model studies., The effect of pure electrostatic charges has been investigated on cultured normal and cancerous breast cell lines for the first time. The selective effect of positive electrostatic charges (PECs) on metastatic cell lines and animal tumors has been reported with no effect on normal tissues. PECs induce apoptosis in metastatic breast cancer cell lines and tumors by suppressing proifration pathways. More than 400 mouse models have been tested to investigate the effects of PECs on cancerous tumors and animal organs by different analyses. Survival analyses depict a high survival rate of treated mice with positive electrostatic charges compared to other conventional cancer treatments such as chemotherapy and radiotherapy.

Background: Pseudo-ginsenoside-Rh2 (pseudo-G-Rh2), a novel derivative of ginsenoside Rh2, is reported to exert a pro-apoptotic effect on various malignancies. However, whether this anti-cancer action of pseudo-G-Rh2 involves autophagy remains to be determined and explored. Objectives: The objective of this study was to investigate the pseudo-G-Rh2-induced apoptosis and autophagy and the underlying mechanism. Methods: In the present study, the MTT assay was used for evaluating cell viability, and the lactate dehydrogenase (LDH) assay was performed to assess cell toxicity. Autophagy evaluation was performed using monodansylcadaverine (MDC) staining and transmission electron microscopy (TEM). The levels of autophagy-associated and apoptosis-associated proteins were determined using Western blotting. The Annexin V-FITC/propidium iodide (PI) assay was used to assess apoptosis. Results: The Annexin V-FITC/PI assay revealed that the percentage of apoptotic cells in HepG2 cells at concentrations 0, 20, 40, and 60 μM was 3.75%±1.37%, 5.70%±1.04%, 12.30%±2.10%, and 34.26%±4.73%, respectively. Pseudo-G-Rh2 was observed to significantly increase the expressions of BAX, cleaved-caspase-3, and cleaved-caspase-9, while it decreased the Bcl-2 expression. MDC and TEM analysis revealed that pseudo-G-Rh2 at concentrations 20, 40, and 60 μM significantly facilitated the accumulation of autophagosomes and autolysosomes within the HepG2 cells. Moreover, pseudo-G-Rh2 significantly increased the expressions of LC3 II/LC3 I and Beclin-1 and decreased the expression of p62. The Annexin V-FITC/PI assay also revealed that in comparison to the pseudo-G-Rh2 group, the concurrent treatment with pseudo-G-Rh2 and an autophagy inhibitor (CQ or 3-MA) significantly induced distinct apoptosis. In addition, pseudo-G-Rh2 activated AMPK and inhibited the PI3K/Akt/mTOR pathway in a concentration-dependent manner. Pseudo- G-Rh2 is similar to the current patents, which enhanced its anti-cancer activity by combining with autophagy inhibitors. Conclusion: Pseudo-G-Rh2 could induce protective autophagy in HepG2 cells, at least in part, via AMPK and the PI3K/Akt/mTOR pathway.

Among urological tumors, renal cell carcinoma (RCC) is the third-highest mortality rate tumor, and 20%–30% of RCC patients present with metastases at the time of diagnosis. While the treatment of RCC has been improved over the last few years, its mortality stays high. Y-box binding protein 1 (YBX1) is a well-known oncoprotein that has tumor-promoting functions. YBX1 is widely considered to be an attractive therapeutic target in cancer. To develop novel therapeutics to target YBX1, it is of great importance to understand how YBX1 is finely regulated in cancer. Our previous studies showed that YBX1 in RCC cells significantly promoted cell adhesion, migration, and invasion. However, the role of YBX1 in RCC cells apoptosis has not been reported. In this study, we investigated the effect of YBX1 on cell apoptosis and elucidated the mechanisms involved. Results showed that YBX1 regulated RCC cells apoptosis and reactive oxygen species (ROS) generation via Kindlin-2. These findings indicated that YBX1 inhibited RCC cells apoptosis and may serve as a candidate RCC prognostic marker and a potential therapeutic target. Abbreviations: RCC: Renal cell carcinoma; YBX1: Y-box binding protein 1; ROS: Reactive oxygen species; ccRCC: Clear cell renal cell carcinoma; mccRCC: Metastatic clear cell renal cell carcinoma; G3BP1: Ras-GTPase activating protein SH3 domain-binding proteins 1; SPP1: Secreted phosphoprotein 1; NF-κB: Nuclear factor kappa beta; ECM: Extracellular matrix; EMT: Epithelial-mesenchymal transition; PYCR1: Pyrroline-5-carboxylate reductase 1; MEM: Eagle’s Minimum Essential Medium; DMEM: Dulbecco’s modified Eagle medium; FBS: Fetal bovine serum; PCR: Polymerase chain reaction; shRNA: Short hairpin RNA; siRNA: Small interfering RNA; BSA: Bovine serum albumin; DCFH-DA: 2,7-Dichlorodihydrofluorescein diacetate; FITC: Fluorescein isothiocyanate; PI: Propidium iodide

Made available in DSpace on 2022-05-01T10:18:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Polymicrobial biofilms comprising Candida albicans and Staphylococcus aureus can increase the frequency and severity of oral diseases. This study assessed oral keratinocyte cell death, apoptosis and/or necrosis, promoted by soluble factors from single and dual biofilms of S. aureus and C. albicans. The soluble factors were obtained from the 16-h biofilm growth media. Cell viability was assessed by MTT and cell membrane damage by LDH. SEM was used for morphology changes. Assessment of apoptosis and necrosis was performed using annexin V and propidium iodide and caspases -2, -3, -6, -8 and -9. Statistical analysis was conducted with ANOVA and Tukey tests (α = 5%). Dual biofilms promoted the greatest harmful effect on oral cells, with a viability rate of 31.76%, damage to cell membranes and LDH released. Dual biofilms also induced higher percentages of necrotic cells (24.95%). Apoptosis was associated with caspases -2, -3, -6 and -8 activation. Department of Dental Materials and Prosthodontics School of Dentistry São Paulo State University (UNESP) Department of Dental Materials and Prosthodontics School of Dentistry São Paulo State University (UNESP)

Discovery of novel anticancer agents is of crucial importance to expand the therapeutic options for cancer patients. In this study, a series of 49 5-oxo-hexahydroquinoline and 5-oxo-tetrahydrocyclopentapyridine analogs, containing different pyridine alkyl carboxylates at C3 and various aliphatic, aromatic, and heteroaromatic substitutions at the C4 position of the central core, were synthesized. The target compounds were tested for antiproliferative effect against three human cancer cell lines including MOLT-4 (acute lymphoblastic leukemia), K562 (chronic myelogenous leukemia), and MCF-7 (breast adenocarcinoma) by MTT assay, and the effect of the most potent derivatives on cell cycle was evaluated by RNase/propidium iodide (PI) flow cytometric assay. Generally, 5-oxo-hexahydroquinoline derivatives (E series) possessed superior antiproliferative activities compared to their 5-oxo-tetrahydrocyclopentapyridine counterparts (F series). 5-Oxo-hexahydroquinoline compounds bearing 2-pyridyl propyl carboxylate (group D) and 3-pyridyl propyl carboxylate (group E) were better antiproliferative agents than those bearing other pyridyl alkyl carboxylates. Five best compounds with IC50 values in the range of 9.5-22.9 µM against MOLT-4 cells were selected for cell-cycle analysis, which revealed that derivatives D5, E3, and E5 with 2,3-dichlorophenyl, 3-nitrophenyl, and 2-nitrophenyl substitutions at C4 position, respectively, may induce apoptosis in MOLT-4 cells. Molecular docking analysis, which was employed to make some predictions on the interaction of the most active derivatives with the binding site of Bcl-2 and Bcl-xL proteins, suggested that the compounds may be well accommodated within the binding sites of these anti-apoptotic proteins via hydrogen-bonding and hydrophobic interactions. The findings of this study present 5-oxo-hexahydroquinoline derivatives as antiproliferative agents with potential apoptosis-inducing ability in cancer cells.

International audience; Transgenic Nicotiana tabacum cells constitutively expressing the C-terminal domain (hub domain) of clathrin heavy chain, as a dominant negative mutation for the onset of endocytosis, were examined. We first analyzed the morphometry parameters (length, width, surface area) of cells from Xanthi and BY-2 callus and suspensions and we calculated their shape coefficient. Cells expressing the hub domain tended to be more elongated than the wild-type cells. Then, we performed flow cytometry characterization after staining with the A–T specific dye 4,6-diamidino-2-phenylindole dihydrochloride to assess their relative DNA content, with propidium iodide to determine their genome size, and with chromomycin A3 to assess their G–C content. The A–T level was reduced in callus, but variable in cell suspensions. On the other hand, genome size was variable for callus and cell suspensions of both Xanthi and BY-2 cultivars. Xanthi cells being more affected than BY-2 cells in general, this prompted a detailed study with cell suspensions of Xanthi lines which showed that DNA content, genome size, nucleus and cell morphometry are correlated. These results suggest that disturbing clathrin process(es) impacts flow cytometry and morphometry traits of cells in culture.

Propionibacterium acnes plays a major role in acne vulgaris. In the pre-experiment, the growth of P. acnes was inhibited effectively using surfactin; however, the antibacterial mechanism has not been described. Therefore, the aim of this study was to evaluate antibacterial activity and analyse the mechanism of surfactin against P. acnes. Minimum inhibitory concentration, time-killing kinetics and scanning electron microscopy were used to evaluate the activity of surfactin against P. acnes, which showed that 128 μg ml-1 effectively inhibited growth. Cell wall permeability was evaluated by detecting the extracellular alkaline phosphatase activity, which increased to 1·83- and 2·32-fold after incubating with 128 and 256 μg ml-1 of surfactin for 10 h, respectively. Propidium iodide fluorescence, leakage of nucleic acid, protein, K+ , and Ca2+ , membrane potential and the leakage of calcein from small unilamellar vesicles all increased after incubation with surfactin, indicating that its strong biological activities act mainly by altering membrane integrity. In a mouse model of acne, surfactin significantly reduced P. acnes-induced epidermal swelling and erythema. These results indicate that surfactin effectively inhibited the growth of P. acnes by destroying the cell wall and membrane, and is a potential candidate for acne treatment.

This study presents a straightforward and reliable method for determining the viability of Acanthamoeba cysts. A standard method for determining Acanthamoeba cyst viability in an in vitro cytotoxicity analysis is required to ensure that the double-walled and sturdy cysts are affected by the substance tested. In this study, a new approach was used to determine the cysticidal potential of redox Cleland’s reagent, dithiothreitol (DTT), against Acanthamoeba cysts. This approach constitutes a significant breakthrough, as the cyst form of Acanthamoeba is known for its high resistance to various chemicals and drugs used to treat infections of the central nervous system and eyes caused by Acanthamoeba. Cyst viability was evaluated based on the intensity of the cyst population under fluorescence produced by propidium iodide (PI) dye and measured using an enzyme-linked immunosorbent assay (ELISA) reader at an absorbance of 636 nm. The results were validated using high-content screening (HCS). For analysis, an individual cell was imaged and examined for phenotypic changes in the Acanthamoeba cyst at the cyst population level. Fluorescence intensity of the cysts in each well in a 96-well plate was measured using Image J software. HCS is an automated technique that uses fluorescence microscopy to produce quantitative data.

Some clinical studies reported that glucose variability increased the risk of developing diabetes-related late complications more than constant hyperglycemia, while others claimed that the evidence was not strong enough to support such a conclusion. A few in vitro studies investigated the effect of constantly high or variable glucose levels (VGLs) on endothelial cells (EC). The first aim of this work was to review these studies and demonstrate that most of them support the notion that viability and other metabolic parameters of EC deteriorate faster in cell cultures with VGLs than in cultures with stable normal or high glucose concentration. The second aim was to verify whether the effect of glucose concentration is the same regardless of other culture conditions such as the substrate on which the cells are grown. We cultured Human Umbilical Vein Endothelial Cells (HUVECs) for 7 or 14 days in constant (5 mM or 20 mM) or variable (switching between 5 mM and 20 mM once a day) glucose concentration in culture plates, which were either not-covered with any additional substrate or were covered with fibronectin or gelatin. We assessed the cell viability using a propidium iodide test. The ANOVA revealed that HUVECs viability was affected not only by glucose concentration and duration of the cell culturing but also by the type of substrate and interactions of these three factors. In conclusion, the effect of glucose level on EC viability should not be analyzed in isolation from other culture conditions that may amplify or attenuate this effect.

It is known that the circadian rhythm of melatonin production depends on the intensity of illumination. Violation of the light regime leads to suppression of melatonin synthesis and the development of desynchronosis, which increases the risk of developing a number of pathologies. In this regard, it is relevant to search for opportunities to restore disturbed circadian rhythms and, especially, to correct immune dysfunctions that occur in these situations.The aim of this study was to examine the effect of a complex of melatonin, aluminum oxide and polymethylsiloxane on the lymphocytes of the spleen of mice kept under round-the-clock lighting.Materials and methods. Mice of the C57Bl/6J line were kept under round-the-clock lighting for 14 days, against which they were intragastrically injected with distilled water, aluminum oxide with polydimethylsiloxane, melatonin and a complex of melatonin, aluminum oxide and polymethylsiloxane (a new drug developed by the Research Institute of Clinical and Experimental Lymphology – Branch of the Federal Research Center Institute of Cytology and Genetics SB RAS; Patent of Russian Federation No. 2577580, 2016), represented by a complex of porous material (aluminum oxide with polydimethylsiloxane) and melatonin, immobilized in the pores, from which it is gradually released in a liquid medium. Intact animals kept under the light regime of ST 12/12 and under round-the-clock lighting served as a control. Immunophenotyping of spleen B- and T-lymphocytes was performed on a flow cytofluorimeter with monoclonal antibodies APC CD3 and FITC CD19. For studying the distribution of cells by stages of the cell cycle in splenocytes, the amount of intracellular DNA was measured by the level of inclusion of propidium iodide.Results. Flow cytometry of the distribution of B- and T-lymphocytes of the spleen in male mice of the C57Bl/6J line kept under round-the-clock lighting conditions (KO 24/0 h) revealed a decrease in the percentage of B-lymphocytes and an increase in the number of T-lymphocytes, compared with animals kept under standard lighting conditions (the light/dark photoperiod – 14/10 hours). The ratio of CD19+/CD3+ lymphocytes of the spleen in mice under the conditions of KO significantly decreases (1.5 times) compared to intact animals (p ≤ 0.001). The administration of pure and modified melatonin (Complex M) to animals kept under round-the-clock lighting conditions has an equally pronounced normalizing effect on the cellular composition of B- (CD19) and T- (CD3) lymphocytes of the spleen, bringing the values of the studied parameters to the control values of the intact animals (p ≤ 0.001) Round-the-clock lighting affects the proliferative potential of splenocytes, reducing the number of cells in the G2/M phase, compared with animals treated with melatonin (p ≤ 0.050). The introduction of melatonin leads to an increase in the percentage of cells in the G2/M phase relative to the placebo group (p ≤ 0.050). In the group of mice treated with Complex M, the greatest increase in cells at the S + G2/M phases and the highest percentage of cells at the G2/M phase were revealed compared to the placebo control group (p ≤ 0.050).Conclusion. The complex of melatonin, aluminum oxide and polymethylsiloxane has additional immunotropic properties in relation to the modifier molecule, which, apparently, are due to the joint immunostimulating effect of melatonin and the lymphostimulating effect of the sorbent. Melatonin in the composition of the complex shows its properties more stably.

Bone metastasis is a significant cause of morbidity and mortality in patients with prostate cancer (PCa). This study is aimed at illustrating the mechanism of sweroside-mediated regulation in bone metastasis in PCa cells. Owing to the limitations of antitumor drugs in terms of their physical and chemical properties, making them into nanomaterials can effectively improve drug stability and bioavailability. Apoptosis was assessed with flow cytometry using the annexin V/propidium iodide binding assay; proteins, including p53, P21, Bcl-2, and Bax; and induction of intracellular reactive oxygen species (ROS). Using colony formation assay, sphere formation assay, and the expression changes in CD133 and CD44, stem cell characteristics were assessed. Epithelial–mesenchymal transition (EMT) activity was accessed by levels of the expression changes of EMT-related markers, vimentin and E-cadherin. Wnt/β-catenin signaling pathway was examined to detect the levels of the expression changes of snail and β-catenin. PC-3 cells were treated with lithium chloride (LiCl), which is an agonist of Wnt/β-catenin signaling, and the levels of CD133, CD44, vimentin, E-cadherin, snail, and β-catenin were detected. T-cell factor/lymphocyte enhancer factor (TCF/LEF) activity in cells overexpressing β-catenin was used to detect the effects on β-catenin transcription, and the expression of c-myc, Cyclin D1, Survivin, and MMP-7 were used to detect Wnt downstream target genes. Our results suggest that sweroside induces apoptosis and intracellular ROS; upregulates apoptotic proteins; and suppresses proliferation, invasion, and migration, preventing stem cell characteristics, including sphere formation, colony formation, and CD133 and CD44 expressions. Furthermore, sweroside nanoparticles exerts inhibitory effects on β-catenin transcription by suppressing TTCF/LEF activity in cells overexpressing β-catenin and downregulation of the expression of Wnt downstream target genes, including c-myc, Cyclin D1, Survivin, and MMP-7. The potential therapeutic effect of sweroside nanoparticles on bone metastatis of PCa was suggested, by these findings.

Background/aim We investigated the cytotoxic effects of plumbagin on metastatic retinoblastoma, using the highly metastatic cell line Y79. Materials and methods Effect of plumbagin on cell growth was assessed with water-soluble tetrazolium 1 (WST-1) cell proliferation assay and automated hemocytometry with trypan blue-exclusion assay. Cell death was studied with acridine orange/ethidium bromide live-dead assay and annexin-V-fluorescein isothiocyanate/propidium iodide microscopy. Loss of mitochondrial membrane potential was studied with JC-10 dye and caspase activation was investigated using CellEvent Caspase-3/7 Green detection reagent. Results Plumbagin highly significantly reduced the growth of Y79 cells treated for 24 h with 2.5 μM or more. Plumbagin also induced significantly high levels of cell death which was associated with loss of mitochondrial membrane potential and caspase activation. Conclusion At very low concentration (2.5 μM), plumbagin potently induced cytotoxicity in metastatic retinoblastoma cells via loss of mitochondrial membrane potential and caspase activation.

Circular RNAs (circRNAs) titrate the function of microRNAs (miRNAs), regulate transcription, and interfere with splicing. This study attempted to confirm the role of a novel circRNA circ_0128846 during osteoarthritis (OA) progression. Tissues and chondrocytes were isolated from OA patients. Overexpression and knockdown of target genes were generated using cell transfection and siRNA interference. Expression levels of genes were measured by qRT-PCR, Western blot, and immunohistochemistry, respectively. The interactions among circ_0128846, miR-140-3p, and JAK2 were verified by bioinformatics prediction, a dual-luciferase reporter assay, and RNA immunoprecipitation assay. The role of circ_0128846 in vivo was confirmed by the construction of experimental OA rats. Pathological changes were evaluated by hematoxylin and eosin and Safranin O staining. In OA patients, the level of circ_0128846 and JAK2 were up-regulated with down-regulated level of miR-140-3p. Circ_0128846 was principally located in the cytoplasm. Circ_0128846 silence enhanced cells viability, but reduced apoptosis rate and inflammatory response, which was obviously reversed by miR-140-3p knockdown. The overexpression of JAK2 reversed the effects of miR-140-3p on cell phenotypes. Circ_0128846 silence suppressed the level of MMP-13 and promoted the expression of collagen II by up-regulating miR-140-3p and down-regulating JAK2 in OA cells. Results of animal experiments demonstrated that circ_0128846 silence promoted collagen II expression and attenuated the OA progression by regulating the miR-140-3p/JAK2 axis. Circ_0128846 contributes to OA development through acting as a sponge RNA for miR-140-3p and thereby increasing JAK2 expression. Results indicated that targeting circ_0128846 may have the potential to alleviate OA progression.Abbreviations:circRNAs: Circular RNAs; miRNAs: microRNAs; OA: osteoarthritis; RIP: RNA immunoprecipitation; HE ncRNAs: noncoding RNAs; ceRNA: competitive endogenous RNA; DMEM: Dulbecco's modified Eagle's medium; PBS: phosphate buffered saline; OE-circ_0128846: overexpression vector for circ_0128846; pcDNA3.1-JAK2: pcDNA3.1 overexpression vector for Janus kinase 2; NC: negative control; CCK-8: Cell Counting Kit-8; PI: propidium iodide; WT: Wild-type; mutants (MUT); SD rats: Sprague Dawley rats; DMM: destabilization of medial meniscus; IHC: immunohistochemistry; DAB: diaminobenzene; pre-Mrna: precursor mRNA.

Introduction: Cytotoxic T-lymphocytes play an important role in a specific immune response via a lytic effect in relation to abnormal cells. The number of these cells greatly increases sin pathological states. In addition, an increased cytotoxic activity is a characteristic of the immune response of people living in the Far NorthTherefore, it is important to understand what function cytotoxic T-lymphocytes predominantly perform in healthy people in the North - cytolytic or suppressor? Aim: To assess associations between CD8+ concentration and immune response and apoptotic deaths of the lymphocytes in healthy individuals. Methods: Ninety-three healthy adult residents of the Russian North comprised the sample. Apoptotic lymphocyte death was studied by flow cytometry. FITC-annexine-V and propidium iodide labelled cells were detected. Concentrations of cytokines and apoptosis mediators were assessed by a solid-phase enzyme immunoassay. Neutrophiles, monocytes and phagocytic activity of neutrophils were studied in blood smears stained by Romanowsky's - Giemsa. The level of phenotypic activity of lymphocytes was assessed by double peroxidase labeling using monoclonal antibodies. The data were presented using means, standard deviations, medians, the 1st and the 3rd quartiles. All study participants were divided into two groups: with normal- (0.2-0,4 х109 kl/l) and elevated (more 0,6 х109 kl/l) blood cytotoxic lymphocyte levels. The groups were similar in terms of by age- and gender distribution. Continuous variables were analyzed using Mann-Whitney tests. Results: In individuals with an increased level of cytotoxic CD8+ lymphocytes in peripheral venous blood had greater concentrations of leukocytes (7.4 ± 0.49 х109 cells/l vs. 5.5 ± 0.23 х109, p = 0.003), lymphocytes (2.8 ± 0.17 х109 cells/l vs.1.8 ± 0.07 х109 cells/t p = 0.005), and mature neutrophils (4.1 ± 0.19 х109 cells/l vs. 3.4 ± 0.49 х109 cells/l, p = 0.013). No associations between the level of apoptosis of lymphocytes (AnV+/PI-) and concentrations of sFasL, TRAIL, TNFa, and cytochrome C were observed. Conclusions: Lymphocytes CD8+ in healthy residents of the Russian North perform mainly cytotoxic function, which is not related to apoptotic cellular death.

Fluorescence titration using magnetic nanoparticles (FTMN) was performed as a rapid, inexpensive, and simple method for quantifying the amount of fluorophore-intercalated plasmid DNA on these DNA attractive nanoparticles. Binding of the propidium iodide (PI)-intercalated DNA (PI/DNA) to polyethylenimine (PEI)-coated monodisperse iron oxide magnetic nanoparticles (PEI-MNs) was confirmed with transmission electron microscopy after the two species were mixed in water for less than a minute. The amount of DNA on PEI-MNs in aqueous solution, however, could not be easily determined using direct fluorescence measurements due to strong scattering by aggregated MNs, especially at high nanoparticle concentrations. Instead, fluorescence measurements were taken immediately after the solution of PI/DNA and PEI-MN mixtures was treated with a magnet to pull the PEI-MNs out of the solution. The detected fluorescence signal of the remaining free PI/DNA in the solution decreased as the concentration of PEI-MNs in the pre-treated solutions increased, resulting in a titration curve, which was used to determine the amount of DNA on MNs, the dissociation constant, and binding energy after the concentration of PEI-MNs was calibrated with microwave-plasma atomic emission spectroscopy. Quantitative polymerase chain reaction was used to understand the binding of DNA to MNs and to measure the amount of free PI/DNA in solution, and the results were similar to those obtained with the FTMN method.

Litsea elliptica is traditionally believed to prevent and treat stomach ulcers, cancer, fever and headaches. This study investigates the phytochemical composition, antioxidant and cytotoxic effects of L. elliptica leaf extracts. The phytochemical content was determined via GCMS analysis and total phenolic content (TPC) and total flavonoid content (TFC) were analysed using the Folin-Ciocalteu and aluminium-chloride assays. Antioxidant activities were determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging and ferric-ion reducing antioxidant power (FRAP) assays, whereas cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and calcein/ethidium viability assays. The mechanism of cytotoxicity was investigated using Annexin V/propidium iodide. Modifications in the mitochondria were investigated using MitoTracker Red CMXRos. Ten and twenty-six compounds were characterized in the young-leaf and mixed-leaves extracts, respectively. The young-leaf methanolic extract demonstrated the highest antioxidant capacity of at least four-folds greater than the mixed-leaves and ethanolic extracts. The methanolic extract also had higher TPC and TFC values compared to the ethanolic extract. Although the mixed L. elliptica leaves had lower antioxidant capacities compared to the young leaves, the mixed leaves extract has demonstrated greater cytotoxicity against the A549 cancer cell line. Further investigation revealed that the L. elliptica leaves-induced cytotoxicity on A549 cells was possibly via the non-inflammatory mitochondria-mediated apoptotic pathway. Overall, our results showed the potential of the L. elliptica leaves possessing cytotoxic activities against carcinoma cells where the compounds present can be further investigated for its therapeutic application.

T-lymphoblast-like human leukemia cells of the Jurkat line (Jurkat T cells) form polyploid forms with a high DNA content in suspension culture. Due to pronounced genetic instability, this contributes to further transformation and development of clonal diversity (polyclonal) of the cell line. Little information is available on the adherent subpopulation of Jurkat T cells. In this work, we analyzed the content of nucleic acids in suspension (DNA) and adhesive (DNA, RNA) subpopulations of Jurkat T cells using flow cytometry and propidium iodide dye, as well as confocal laser microscopy and acridine orange dye. The morphology and mobility of large (with a diameter of more than 15 μm) Jurkat T cells adhering to plastic were studied using Cell-IQ phase-contrast microscopy in real-time. According to the intensity of fluorescence in the conditionally green wavelength range (300–530 nm: from UV to green) and conditionally red (565–800 nm: from red to far-red), three subpopulations of adherent Jurkat T cells were identified: with high, medium, and low nucleic acid content. Thus, Jurkat-T cells adhering to the plastic surface of the plates retain a pronounced heterogeneity in the DNA content characteristic of the suspension fraction, which suggests a difference in the morphofunctional properties (polyclonicity) of this subpopulation of cell culture. With a sharp increase in the total cell mass, the proportion of large (giant, 15–50 μm or more) cells attached to the plastic remained constant for 21 days of cultivation and amounted to 1% of the adhesive fraction. It was found that large Jurkat T cells (with a median diameter of 31 µm) moved along the plastic at a linear (median) speed of 38 µm/h. The polynuclear Jurkat T cells on plastic are morphologically identified, with a linear increase being revealed in the mobility of adherent cells with an increase in their diameter (regression coefficient r = 0.33, p < 0.02, n = 52). Possible cellular and molecular mechanisms of an increased number of DNA copies in some adhering Jurkat T cells are discussed. It is assumed that the discovered new property (locomotor activity) can provide polyploid (multinucleated) adhering Jurkat T cells with a significant advantage—directed migration (chemotaxis) in a growing cell population under conditions of nutrient deficiency due to a change in the nutrient medium after 3–4 days of cultivation.

Tendinopathy is a common disease in elite and recreational athletes, and Eriocitrin is a flavonoid compound, which has antioxidant properties. The present study was undertaken to investigate the effect of Eriocitrin on tendon stem cells in vitro. Different concentrations of Eriocitrin (0, 25, 50, 75 µM) were used to treat tendon stem cells, cell proliferation was determined by CCK8 assay, apoptosis was detected by propidium iodide (PI) staining assays and caspase-3 activity assessment, wound-healing assaywas selected to detect the migration, and RT-PCR was chosen to detect the expression levels of scar formation-related gene markers (COMP, Fibronectin, and Biglycan). Eriocitrin treatment promoted cell proliferation of tendon stem cells in dose-dependent manner, and it reduced the apoptotic activities and improved the migration of tendon stem cells. It inhibited COMP, Biglycan, and Fibronectin expression. In summary, eriocitrin promoted cell proliferation of tendon stem cells, improved the migration of tendon stem cells, and inhibited the expression levels of some scar formation-related gene markers.

Introduction: Somatostatin and dopamine (DA) receptors have a pivotal role in controlling hormone secretion and cell proliferation in different neuroendocrine neoplasms, including medullary thyroid cancer (MTC). In the present preclinical study, we evaluated the anti-tumor activity of TBR-065 (formerly BIM-23B065), a second-generation somatostatin-DA chimera, in 2 human MTC cell lines. Methods: The effects of lanreotide (LAN) and TBR-065 on cell growth and proliferation, calcitonin (CT) secretion, cell cycle, apoptosis, cell migration, and tumor-induced angiogenesis have been evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, DNA flow cytometry with propidium iodide (PI), Annexin V-FITC/PI staining, electrochemiluminescence immuno assay, wound-healing assay, and zebrafish platform, respectively. Results: TBR-065 exerted a more prominent anti-tumor activity than LAN in both MTC cell lines, as shown by inhibition of cell proliferation (maximal inhibition in TT: −50.3 and −37.6%, respectively; in MZ-CRC-1: −58.8 and −27%, respectively) and migration (in TT: −42.7 and −22.9%, respectively; in MZ-CRC-1: −75.5 and −58.2%, respectively). Only the new chimera decreased significantly the fraction of cells in S phase (TT: −33.8%; MZ-CRC-1: −18.8%) and increased cells in G2/M phase (TT: +13%; MZ-CRC-1: +30.5%). In addition, TBR-065 exerted a more prominent pro-apoptotic effect than LAN in TT cells. A concomitant decrease in CT secretion was observed after 2 days of incubation with both drugs, with a more relevant effect of TBR-065. However, neither LAN nor TBR-065 showed any effect on tumor-induced angiogenesis, as evaluated using a zebrafish/tumor xenograft model. Discussion/Conclusion: In MTC cell lines, a second-generation somatostatin-DA analog, TBR-065, exerts a more relevant anti-tumor activity than LAN, through modulation of cell cycle, induction of apoptosis, and reduction in migration. Further studies are required to establish whether TBR-065 has comparable potent inhibitory effects on tumor growth in vivo.

Erythropoietin (EPO) is widely used to treat anemia in patients undergoing chemotherapy for cancers. The main objective of this study was to investigate the effect of rHuEPO on the response of spheroid breast cancer, MCF-7, cells to tamoxifen treatment. The MCF-7 spheroids were treated with 10 mg/mL tamoxifen in combination with either 0, 10, 100 or 200 IU/mL rHuEPO for 24, 48 or 72 h. The viability of the MCF-7 cells was determined using the annexin-V, cell cycle, caspases activation and acridine orange/propidium iodide staining. rHuEPO-tamoxifen combination significantly (p greater than 0.05) increased the number of spheroid MCF-7 cells entering early apoptotic phase after 12 h and late apoptotic phase after 24 h of treatment; primarily the result of the antiproliferative effect tamoxifen. Tamoxifen alone significantly (p

Background and Aims: Nickel oxide nanoparticles (NiO-NPs) are one of the most used nanoparticles, especially as photosensitizers. Although some studies evaluate their toxicity in the liver, the information about their toxicity at the cellular and molecular levels is still controversial. In the present study, it was aimed to investigate the in vitro toxic potentials of NiO-NPs (average size 15.0 nm) in the liver (HepG2) cell Line. Methods: NiO-NPs were characterized by Transmission Electron Microscopy (TEM), the cellular uptake of NPs and the morphologic changes were evaluated by TEM and Inductively Coupled Plasma-Mass Spectrometry (ICP-MS), the cytotoxicity was evaluated by MTT and neutral red uptake (NRU) tests, comet assay was used for genotoxicity, Annexin V-FITC/propidium iodide (PI) apoptosis detection kit was used for apoptosis/ necrosis evaluation and Enzyme-Linked Immune Sorbent Assays (ELISA) kits were used for the potential of oxidative damage. Results: Our results showed that cellular uptake of NiO-NPs led to morphological changes in the cells, and caused cell death (1050 was 146.7 mu g/mL by MTT) mainly by apoptosis. Genotoxicity and oxidative damage were observed to be in a dosedependent manner. Conclusion: Results confirm previous data and draw attention to the toxic effects of NiO-NPs; further in vivo and in vitro studies need to be done to clarify the safety or toxicity of NiO-NPs. Research Fund of Istanbul UniversityIstanbul University [37785] This work was supported by the Research Fund of Istanbul University (Project No: 37785) .

Olfactory dysfunction, one of the earliest non-motor symptoms of Parkinson disease (PD), is accompanied by abnormal deposition of SNCA/α-synuclein in the olfactory bulb (OB). The macroautophagy/autophagy-lysosome pathway (ALP) plays an important role in degrading pathological SNCA and modulating this pathway may be a promising treatment strategy. P2RX4 (purinergic receptor P2X, ligand-gated ion channel 4), a member of the purinergic receptor X family, is a key molecule regulating ALP. Piperine (PIP) is a Chinese medicine with anti-inflammatory and anti-oxidant effects. The present study investigated the neuroprotective effects of PIP on SNCA overexpression-induced PD cell and mouse models. We found that PIP oral administration (25, 50 and 100 mg/kg) for 6 weeks attenuated olfactory deficits and delayed motor deficits in Thy 1-SNCA transgenic mice overexpressing human SNCA. This was accompanied by a degradation of pathological SNCA in OB. In addition, PIP improved cell viability and promoted degradation of human SNCA in SK-N-SH cells. These protective effects were exerted via autophagy flux promotion by enhancing autophagosome-lysosome membrane fusion. Furthermore, tandem mass tag proteomics analyses showed that P2RX4 plays an important role in PIP treatment-induced activation of autophagy flux. These findings demonstrate that PIP exerts neuroprotective effects in PD models via promotion of autophagy flux and may be an effective agent for PD treatment.Abbreviations: 6-OHDA, 6-hydroxydopamine; ALP, autophagy-lysosome pathway; BafA1, bafilomycin A1; CoQ10, coenzyme Q10; DMSO: dimethyl sulfoxide; HPLC, high-performance liquid chromatography; IVE, ivermectin; LDH, lactate dehydrogenase; MAP1LC3/LC3-II, lipid-conjugated microtubule-associated protein 1 light chain 3; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; mRFP-GFP, tandem monomeric red fluorescent protein-green fluorescent protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OB, olfactory bulb; P2RX4, purinergic receptor P2X, ligand-gated ion channel 4; PD, Parkinson disease; PBS: phosphate-buffered saline; PI: propidium iodide; PIP, piperine; PLG, piperlongumine; p-SNCA, SNCA phosphorylated at Ser129; Rap, rapamycin; RT-PCR: quantitative real-time PCR; SNARE, soluble N-ethylmaleimide-sensitive factor-attachment protein receptor; SNCA/α-synuclein, synuclein, alpha; STX17, syntaxin17; TG, transgenic; TH, tyrosine hydroxylase; UPS, ubiquitin-proteasome system; WT, wild-type.

Landfill soil leachates, containing myriad of xenobiotics, increase genotoxic and cytotoxic stress-induced cell death. However, the underlying mechanism involved in the elimination of the damaged cells is yet to be fully elucidated. This study investigated the apoptotic processes induced in lymphoma (Jurkat) cells by landfill soil leachates from Olusosun (OSL, Nigeria) and Nagpur (NPL, India). Jurkat was incubated with sub-lethal concentrations of OSL and NPL for 24 h and analyzed for DNA fragmentation and apoptosis using agarose gel electrophoresis and Hoechst 33258-PI staining, respectively. Complementary DNA expression profiling of some pro-apoptotic and anti-apoptotic genes regulating apoptosis was also analyzed using real-time PCR (RT-PCR) method. Agarose gel electrophoresis revealed DNA fragmentations in OSL and NPL-treated cells. Hoecsht-33258 - Propidium Iodide (PI) based apoptotic analysis confirmed apoptotic cell death in exposed Jurkat. RT-PCR analysis revealed different fold changes in the pro- and anti-apoptotic genes in OSL and NPL-treated Jurkat. There was significant increase in fold change of the up-regulated genes; apoptosis inducing factor mitochondrion-associated 2 (AIFM2), Fas-associated death domain (FADD), Caspase-2, Caspase-6, BH3 interacting domain death agonist (BID), tumor suppressor (p53), and BCL2 associated agonist of cell death (BAD) and down-regulation of apoptosis inhibitor 5 (API5). Results suggest that OSL and NPL elicited genotoxic stress-related apoptosis in Jurkat. The dysregulation in the expression of genes involved in apoptotic processes in wildlife and human exposed to landfill emissions may increase aetiology of various pathological diseases including cancer.

Breast cancer, the most common malignancy among women, is closely associated with mutations in the tumor suppressor gene BRCA. DSS1, a component of the TRanscription-EXport-2 (TREX-2) complex involved in transcription and mRNA nuclear export, stabilizes BRCA2 expression. DSS1 is also related to poor prognosis in patients with breast cancer owing to the induction of chemoresistance. Recently, BRCA2 was shown to be associated with the TREX-2 component PCID2, which prevents DNA:RNA hybrid R-loop formation and transcription-coupled DNA damage. This study aimed to elucidate the involvement of these TREX-2 components and BRCA2 in the chemosensitivity of breast carcinomas. Our results showed that compared with that in normal breast tissues, DSS1 expression was upregulated in human breast carcinoma, whereas PCID2 expression was comparable between normal and malignant tissues. We then compared patient survival time among groups divided by high or low expressions of DSS1, BRCA2, and PCID2. Increased DSS1 expression was significantly correlated with poor prognosis in recurrence-free survival time, whereas no differences were detected in the high and low BRCA2 and PCID2 expression groups. We performed in vitro analyses, including propidium iodide nuclear staining, single-cell gel electrophoresis, and clonogenic survival assays, using breast carcinoma cell lines. The results confirmed that DSS1 depletion significantly increased chemosensitivity, whereas overexpression conferred chemoresistance to breast cancer cell lines; however, BRCA2 expression did not affect chemosensitivity. Similar to DSS1, PCID2 expression was also inversely correlated with chemosensitivity. These results strongly suggest that DSS1 and PCID2 depletion is closely associated with increased chemosensitivity via BRCA2-independent DNA damage. Together with the finding that DSS1 is not highly expressed in normal breast tissues, these results demonstrate that DSS1 depletion confers a druggable trait and may contribute to the development of novel chemotherapeutic strategies to treat DSS1-depleted breast carcinomas independent of BRCA2 mutations.

Summary LncRNA PVT1 has been demonstrated to be upregulated in acute myeloid leukaemia (AML) patients and indicates a poor prognosis. Nevertheless, its role in AML remains obscure. This study investigated the regulatory role and potential mechanisms of PVT1 in the progression of AML. Expression of PVT1, miR-29 family and WAVE1 was detected by quantitative real-time polymerase chain reaction. CCK8 and EdU assays were performed to assess the proliferation of AML cells. Cell cycle and apoptosis were determined by propidium iodide (PI) staining and Annexin V/PI staining on a flow cytometer. Transwell assay was carried out to evaluate the migration and invasion abilities. The interaction between miR-29 family and PVT1/WAVE1 was confirmed by dual luciferase reporter assay and RNA immunoprecipitation assay. The protein levels of WAVE1, Bcl-2, Bax, cleaved Caspase 3, cyclin D1, and p21 were detected by western blotting. Xenograft transplantation was performed to determine the tumourigenicity of AML cell in vivo. PVT1 expression was significantly increased in AML patient samples and cells, which positively correlated with WAVE1 expression. Silencing of PVT1 restrained growth, migration and invasion, while inducing apoptosis of AML cells. Moreover, PVT1 acted as a sponge for miR-29 family to increase WAVE1 expression in AML cells. Overexpression of WAVE1 partly counteracted PVT1 knockdown-induced anti-tumour effects on AML cells in vitro and xenograft tumour in vivo. PVT1 facilitated the progression of AML via regulating miR-29 family/WAVE1 axis, which supported the conclusion that PVT1 may be a promising therapeutic target for AML.

Objective We previously identified propionic acid as a microbially-produced volatile organic compound with fungicidal activity against several pathogenic fungi. The purpose of this work is to better understand how propionic acid affects fungi by examining some of the effects of this compound on the yeast cell. Results We show that propionic acid causes a dramatic increase in the uptake of lucifer yellow in yeast cells, which is consistent with enhanced endocytosis. Additionally, using a propidium iodide assay, we show that propionic acid treatment causes a significant increase in the proportion of yeast cells in G1 and a significant decrease in the proportion of cells in G2, suggesting that propionic acid causes a cell cycle arrest in yeast. Finally, we show that the reduction of MTT is attenuated in yeast cells treated with propionic acid, indicating that propionic acid disrupts cellular respiration. Understanding the effects of propionic acid on the yeast cell may aid in assessing the broader utility of this compound.

In this study, it was aimed to determine the antioxidant and anticancer activities of Sideritis perfoliata methanolic extract (SPE) on cervical cancer cells (HeLa). Different doses (25, 50, 100 and 200 µg/mL) of SPE were used to determine proliferation of HeLa cells by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) staining method. Induction of apoptosis was determined by Annexine-V and propidium iodide staining method. Interleukin (IL) 6-8 levels were measured by ELISA method. Antioxidant activities of SPE were determined by DPPH, DNA (plasmid pBR322) protecting and cellular antioxidant activity tests. Some phytochemicals of SPE were also screened by LC-MS-MS. It was determined that SPE reduced the proliferation of HeLa cells and also induced apoptosis. IL6-8 levels importantly decreased at 200 µg/mL. SPE exhibited moderately antioxidant activities in tests used. Among the phenolics identified, vanillic acid had the highest amount. As a result, it was determined to have the anticancer activity of SPE by decreasing cell proliferation, inducing apoptosis and decreasing IL6-8 in HeLa cells.

Brain photobiomodulation (PBM) is an innovative treatment for a variety of neurological conditions, including cerebral ischemia. However, the capability of PBM for ischemic stroke needs to be further explored and its mechanisms of action remain currently unclear. The aim of the present research was to identify a treatment protocol capable of inducing neuroprotection and to investigate the molecular mechanisms activated by a dual-wavelength near infrared (NIR) laser source in an organotypic hippocampal slice model of hypoxia/ischemia. Hippocampal slices were exposed to oxygen and glucose deprivation (OGD) for 30 min followed by NIR laser light (fluence 3.71, 7.42, or 14.84 J/cm2; wavelengths 808 nm and 905 nm) delivered immediately or 30 min or 60 min after OGD, in order to establish a therapeutic window. Neuronal injury was assessed by propidium iodide fluorescence 24 h later. Our results show that NIR laser irradiation attenuates OGD neurotoxicity once applied immediately or 30 min after OGD. Western blot analysis of proteins involved in neuroinflammation (iNOS, COX-2, NFkB subunit p65, and Bcl-2) and in glutamatergic-mediated synaptic activity (vGluT1, EAAT2, GluN1, and PSD95) showed that the protein modifications induced by OGD were reverted by NIR laser application. Moreover, CA1 confocal microscopy revealed that the profound morphological changes induced by OGD were reverted by NIR laser radiation. In conclusion, NIR laser radiation attenuates OGD neurotoxicity in organotypic hippocampal slices through attenuation of inflammatory mechanisms. These findings shed light on molecular definition of NIR neuroprotective mechanisms, thus underlining the potential benefit of this technique for the treatment of cerebral ischemia.

Background. Gastric adenocarcinoma is the fifth most diagnosed malignancy in the world. The immune system consists of a heterogeneous mixture of macrophages that defense the body through phagocytosis and the production of different cytokines and chemokines. Tumors cause macrophages to polarize differently in the manner of their favorite growth and angiogenesis. Umbelliprenin, a natural sesquiterpene coumarin, has been shown to have anticancer properties against some tumors, including gastric adenocarcinoma. The aim of our study was to investigate the effect of umbelliprenin on the polarization of macrophages in addition to the measurement of some of the soluble factors they produce. Method. The values of IC5 and IC50 for umbelliprenin in the AGS and THP-1 cells were estimated using the MTT assay. THP-1 cells were treated with 10 μM umbelliprenin, either alone or cocultured with AGS cells. Flow cytometry analysis of treated THP-1 cells was performed for CD68, CD86, and CD206 markers to evaluate M0, M1, and M2 macrophages polarization, respectively. AGS cells were assessed for apoptosis and necrosis by flow cytometry after labeling with Annexin V-FITC and propidium iodide. Interleukin- (IL-) 10 and IL-12 contents were measured in the supernatant by the ELISA method. Griess Reaction assay technique was used to determine nitric oxide (NO) concentration. Results. The results of the MTT showed lower toxicity of umbelliprenin in THP-1 (IC50 = 75.79) compared to the AGS cell line (IC50 = 48.81). Umbelliprenin significantly increased the M1/M2 ratio. IL-10 content decreased significantly in the supernatant of M1 and M2 cells after umbelliprenin treatment, while IL-12 increased in the supernatant of M1 cells and decreased in the supernatant of the M2 cells. Umbelliprenin caused an increase in the NO in the supernatant of the M1 cells. Conclusion. Umbelliprenin alters the macrophage’s secretions and its phenotypes in favor of tumor suppression.

BACKGROUND Cirrhosis-related intestinal hyperpermeability and endotoxemia are characterized by intestinal epithelial cell apoptosis, impaired restitution (proliferation and migration), decreased tight junction protein levels, and subsequent barrier dysfunction. In addition to endotoxin and tumor necrosis factor-α (TNFα), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) plays crucial roles in the regulation of apoptosis, restitution, tight junction protein-maintained barrier function of intestinal epithelial cells. METHODS This study aims to explore the roles and underlying mechanisms of CEACAM1 in cirrhosis-related intestinal hyperpermeability through in vitro approach. RESULTS In cirrhotic patients, high serum levels of intestinal hyperpermeability (zonulin and endotoxin) markers were accompanied by elevated serum levels of TNFα and soluble CEACAM1. In in vitro experiments, we evaluated the individual and interacted roles of TNFα and human recombinant CEACAM1 (hrCEACAM1) in LC-sera (sera of cirrhotic patients)-induced intestinal hyperpermeability-related pathogenic signals. In the cell Line human from human colon (Caucasian colon adenocarcinoma) (Caco-2) cell culture, LC-sera, TNFα, and hrCEACAM1 increased apoptosis (measured by Terminal deoxynucleotidyl transferase [TdT] dUTP nick end labeling+/annexin-5+propidium iodide+ cells and caspase-3 activity), decreased restitution capacity (proliferation and migration), and disrupted tight junction protein-maintained barrier function in Caco-2 cells. The pathogenic changes mentioned above were accompanied by an increase in intracellular reactive oxygen species (ROS) levels, lactate dehydrogenase release, and endoplasmic reticulum stress-related signals in the LC-sera or TNFα-pretreated Caco-2 cells. Concomitant incubation of Caco-2 cells with anti-CEACAM1 suppressed these LC-sera or TNFα-induced negative effects on restitution, barrier function, and cell viability. CONCLUSION This study demonstrated that sera from cirrhotic patients contain soluble CEACAM1, which is involved in the pathogenesis of intestinal hyperpermeability. Accordingly, it is noteworthy to explore the potential use of anti-CEACAM1 treatment for cirrhosis-related intestinal hyperpermeability and endotoxemia.

This report describes a combined immunofluorescence and fluorescence viability stain applied as one staining solution for rapid detection of live Legionella pneumophila in mixed bacterial populations. Instead of sequential viability staining with the Invitrogen BacLight LIVE/DEAD staining kit followed by antibody-Alexa Fluor (AF) 647 conjugate staining to identify live L. pneumophila, a combined single cocktail solution staining protocol was developed to simplify and accelerate the time to detection of viable L. pneumophila serogroup-1 (SG-1) in mixed species populations on a filter membrane. The stain cocktail will aid in accelerating fluorescence microscopic analysis of cooling tower, air conditioner and water fountain or other liquid samples for the presence of L. pneumophila and its viability status. Visibly red stained cells were identified as dead non-L. pneumophila SG-1 cells, while green fluorescing cells represented viable non-L. pneumophila SG-1 cells. Due to also staining red with antibody-AF 647, L. pneumophila SG-1 cells were pseudocolorized as blue to distinguish them from other dead cells. Fluorescence color emission mixing from the viability dyes (SYTO 9 and propidium iodide) with antibody-AF 647 stained L. pneumophila led to other fluorescent colors. For example, green plus pseudocolorized blue AF 647-antibody- labeled cells were identified as live cyan-colored L. pneumophila SG-1 cells. Magenta-colored cells resulted from dead L. pneumophila cells that combined red propidium iodide with blue pseudocolorized AF 647-antibody emissions. Analysis of measured RGB (red, green, blue) color values in microscopic images of mixed bacterial populations suggests the possibility of facile automated discrimination of subpopulations of live and dead L. pneumophila and non-L. pneumophila species by computers in 3-dimensional RGB color space after staining in the combined cocktail which will save time for more rapid microscopic detection of potential sources of Legionnaire’s disease.

Multidrug resistance (MDR) is a critical reason for cancer chemotherapy failure. Babaodan (BBD) is a famous traditional Chinese patent medicine reported to have antigastric cancer activity. However, the roles and molecular mechanisms of the reversal of MDR of gastric cancer by BBD have not been well described until now. Therefore, the purpose of this study was to elucidate further the role of BBD in reversing the MDR of gastric cancer cells and its specific regulatory mechanism via in vitro experiments. To verify our results, MTT, Doxorubicin (DOX) staining, Rhodamin123 (Rho123) staining, DAPI staining, Annexin V-FITC, propidium iodide (PI), Cyto-ID, and western blot assays were performed. To determine whether BBD triggers apoptosis and autophagy through the PI3K/AKT/mTOR signaling, we also applied 3-methyladenine (3-MA), chloroquine (CQ), and 740Y-P (an activator of PI3K). The results showed that BBD reversed the MDR and induced apoptosis and autophagy of SGC7901/DDP cells. Pathway analyses suggested BBD inhibits PI3K/AKT/mTOR pathway activity and subsequent apoptosis-autophagy induction. Inhibition of autophagy with 3-MA and chloroquine (CQ) was performed to confirm that BBD promoted autophagy. PI3K agonist, 740Y-P, further verified BBD inhibition of PI3K/AKT/mTOR pathway activation. In conclusion, BBD may reverse the MDR of gastric cancer cells, induce apoptosis, and promote autophagy via inactivation of the PI3K/AKT/mTOR signaling pathway.

Ultraviolet (UV)-B is a component of sunlight and shows a significant effect on DNA damage, which can be regulated by proliferating cell nuclear antigen (PCNA). The role of TaPCNA in wheat (Triticum aestivum L.) programmed cell death (PCD) under UV-B has not been investigated previously. Here, we explored the function of TaPCNA in wheat exposed to UV-B utilising Barley Stripe Mosaic Virus–virus-induced gene silencing (VIGS). The results showed that the expression of TaPCNA was downregulated, and curly wheat leaves with several spots were determined by VIGS. The growth rate and mesophyll cell length were significantly inhibited after TaPCNA was silenced. The activity of superoxide dismutase and the contents of soluble sugar and soluble protein decreased, whereas the activities of peroxidase and catalase and malondialdehyde content increased in TaPCNA-silenced and UV-B treatment groups. DNA laddering and propidium iodide staining results showed that DNA fragments and micronucleus accumulated after TaPCNA silencing with or without UV-B. Thus, TaPCNA participates in plant growth and DNA damage and PCD under UV-B. This study suggests an idea for the exploration of the function of certain genes in such complex wheat genomes and offers a theoretical basis to improve wheat agronomic traits.