Fluorescent DNA Stain Research Articles

Plasmid Stability Analysis with Open-Source Droplet Microfluidics.

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating the construction of biological circuits and pathways within and between cell populations. For many applications, maintaining functional plasmids without antibiotic selection is critical. This study introduces an open-hardware-based microfluidic workflow for analyzing plasmid retention by culturing single cells in gel microdroplets and quantifying microcolonies using fluorescence microscopy. This approach allows for the parallel analysis of numerous droplets and microcolonies, providing greater statistical power compared to traditional plate counting and enabling the integration of the assay into other droplet microfluidic workflows. By using plasmids expressing fluorescent proteins alongside a non-specific fluorescent DNA stain, single colonies can be identified and differentiated based on plasmid loss or fluorescent marker expression. Notably, this advanced workflow, implemented with open-source hardware, offers precise flow control and temperature management of both the sample and the microfluidic chip. These features enhance the workflow's ease of use, robustness, and accessibility. While the study focuses on Escherichia coli as the experimental model, the method's true potential lies in its versatility. It can be adapted for various studies requiring fluorescence signal quantification from plasmids or stains, as well as for other applications. The adoption of open-source hardware broadens the potential for conducting high-throughput bioanalyses using accessible technology in diverse research settings.

Prostaglandin F2A Disturbs Oogenesis by Causing Meiotic Spindle Damage

Abstract According to recent data, prostaglandin F2 alpha can have a negative influence on meiosis during oogenesis. Previously, we have found that this prostaglandin may accelerate in a dosage-dependent way the postovulatory aging in ovulated mature oocytes, compromising the integrity of their meiotic spindles. Aim. The study aimed to investigate the effects of prostaglandin F2α on the course and outcome of oocyte meiosis in a mouse model. Materials and Methods. Mouse oocytes were matured in vitro in the presence of prostaglandin F2α in a concentration of 100 ng/ml. Their meiotic stage, spindle morphology and chromosome arrangement were assessed by immunofluorescent labeling of tubulin and fluorescent staining of DNA. Results. We obtained a higher percentage of immature oocytes in metaphase I after the treatment than in untreated control oocytes. In addition, there were specific morphological changes in the meiotic spindles of oocytes exposed to prostaglandin F2α associated with a reduced number of fibers. Conclusion. It is probable that prostaglandin F2α has an impact on the microtubule dynamics of the meiotic spindle that can prevent the transition of maturing oocytes to the second meiotic division, most likely by triggering the spindle assembly checkpoint.

The Intercalator Ethidium Bromide Generates Covalent Adducts at Apurinic/Apyrimidinic Sites in DNA.

Ethidium bromide was first described as a DNA intercalator 60 years ago and, over the ensuing years, may be the most widely used fluorescent DNA stain in molecular biology, biochemistry, and histology. Noncovalent DNA binding by ethidium has been well characterized, but to date, there have been no reports of covalent DNA adduct formation by ethidium bromide. This report describes the characterization of covalent adducts generated by the reaction of ethidium with apurinic/apyrimidinic (AP) sites in DNA. Adduct formation proceeds via the reaction of the amino group(s) on ethidium with the ring-opened aldehyde residue of the AP site in DNA to yield an imine. Ethidium-AP adducts may form under a variety of circumstances due to the ubiquitous occurrence of AP sites in cellular and synthetic DNA.

The diversity of AMPA receptor inhibition mechanisms among amidine-containing compounds.

Amidine-containing compounds are primarily known as antiprotozoal agents (pentamidine, diminazene, furamidine) or as serine protease inhibitors (nafamostat, sepimostat, camostat, gabexate). DAPI is widely recognized as a fluorescent DNA stain. Recently, it has been shown that these compounds also act as NMDA receptor inhibitors. In this study, we examined the activity of these compounds and analyzed the mechanisms of action in relation to another important class of ionotropic glutamate receptors-calcium-permeable AMPA receptors (CP-AMPARs) and calcium-impermeable AMPA receptors (CI-AMPARs) - using the whole-cell patch-clamp method on isolated male Wistar rat brain neurons. Gabexate and camostat were found to be inactive. Other compounds preferentially inhibited calcium-permeable AMPA receptors with IC50 values of 30-60µM. DAPI and furamidine were also active against CI-AMPARs with IC50s of 50-60μM, while others showed poor activity. All active compounds acted as channel blockers, which are able for permeating into the cytoplasm on both CP- and CI-AMPARs. Specifically, sepimostat showed trapping in the closed CP-AMPAR channel. Furamidine and DAPI demonstrated a voltage-independent action on CI-AMPARs, indicating binding to an additional superficial site. While the majority of compounds inhibited glutamate-activated steady-state currents as well as kainate-activated currents on CI-AMPARs, pentamidine significantly potentiated glutamate-induced steady-state responses. The potentiating effect of pentamidine resembles the action of the positive allosteric modulator cyclothiazide although the exact binding site remains unclear. Thus, this study, together with our previous research on NMDA receptors, provides a comprehensive overview of this novel group of ionotropic glutamate receptors inhibitors with a complex pharmacological profile, remarkable diversity of effects and mechanisms of action.

Evaluating Bacterial Viability in Faecal Microbiota Transplantation: A Comparative Analysis of InVitro Cultivation and Membrane Integrity Methods.

Faecal microbiota transplantation (FMT) is a developing therapy for disorders related to gut dysbiosis. Despite its growing application, standardised protocols for FMT filtrate preparation and quality assessment remain undeveloped. The viability of bacteria in the filtrate is crucial for FMT's efficacy and for validating protocol execution. We compared two methods-in vitro cultivation and membrane integrity assessment-for their accuracy, reproducibility and clinical applicability in measuring bacterial viability in frozen FMT stool filtrate. Bacterial viability in stool filtrate was evaluated using (i) membrane integrity through fluorescent DNA staining with SYTO9 and propidium iodide, followed by flow cytometry and (ii) culturable bacteria counts (colony-forming units, CFU) under aerobic or anaerobic conditions. Using different types of samples (pure bacterial culture, stool of germ-free and conventionally bred mice, native and heat-treated human stool), we refined the bacterial DNA staining protocol integrated with flow cytometry for assessment of bacterial viability in frozen human stool samples. Both the membrane integrity-based and cultivation-based methods exhibited significant variability in bacterial viability across different FMT filtrates, without correlation. The cultivation-based method showed a mean coefficient of variance of 30.3%, ranging from 7.4% to 60.1%. Conversely, the membrane integrity approach yielded more reproducible results, with a mean coefficient of variance for viable cells of 6.4% ranging from 0.2% to 18.2%. Bacterial viability assessment in stool filtrate using the membrane integrity method offers robust and precise data, making it a suitable option for faecal material evaluation in FMT. In contrast, the cultivation-dependent methods produce inconsistent outcomes.

Using computer vision libraries to streamline nuclei quantification: A validation study

Valid analyses of cell function-based experiments depend on accuracy and reproducibility of the experimental protocol, often requiring normalization to cell count or other measures. The process of counting cells manually is laborious and susceptible to human error. Although cell counting programs exist, they are not preferred by all users, may be cost-prohibitive, and validation is minimal. Therefore, we aimed to validate an object visualization effciency tool that we developed to provide users with an alternative means of automated cell counting for the very specific, necessary, and widely performed task: quantifying nuclei to assess cell count in images. We hypothesized strong agreement between manual and automated counts. To ensure validation across cell densities, C2C12 myoblasts were seeded at 7.3x104 cells per cm2, proliferated for various timeframes (24, 48, and 72 hours; 6 wells across 2 plates per time point), and fixed with ice-cold methanol for 10 minutes. The nuclei were then stained using the fluorescent DNA stain, 4’,6-diamidino-2-phenylindole (DAPI), and 5 images per well were captured. The number of nuclei in each image were manually quantified by 2 separate raters (MC1 and MC2) using ImageJ, and with our automated program developed using computer vision libraries in Python (AI). To minimize risk for observer bias, AI counts and counts completed by each rater were kept separately until all 3 counts for each image were complete. To validate the program, we assessed interrater reliability between 1) MC1 and MC2 to ensure manual count agreement and 2) the average of MC1 and MC2 (MCavg) with AI to assess agreement between manual and AI counts. Spearman’s Rho (rs) was utilized to assess the strength of the association between counts across all images and separately for each quartile (Q) where Q1 had the fewest and Q4 had the most nuclei by manual counts. Nuclei per image ranged from 44 to 1166. Interrater reliability was nearly perfect between MC1 and MC2 (ICC=0.999) and between MCavg and AI (ICC=0.991). A significant, strong correlation was observed between AI and MCavg (rs=0.998, p<0.001). This strong correlation between persisted regardless of cell density (MCavg vs AI: Q1: rs=.998, p<0.001; Q2: rs=0.999, p<0.001; Q3: rs=1.000, p<0.001; Q4: rs=1.000, p<0.001). The results indicate strong agreement between nuclei counts obtained manually and using our automated object recognition program, which remained consistent regardless of cell quantity, demonstrating the validity of this program. Therefore, the automated object recognition program analyzed in the present study can be considered an accurate, rapid, user-friendly, and inexpensive resource for quantifying nuclei to assess cell count across a range of cell densities. Use of this software will save the user valuable time during the critically important but often time-consuming task of quantifying nuclei. Danielle Levitt's start-up funding through Texas Tech University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

21 Fluorescent protein-based DNA staining dyes

21 Fluorescent protein-based DNA staining dyes

Boron Clusters Alter the Membrane Permeability of Dicationic Fluorescent DNA-Staining Dyes.

Membrane-permeable fluorescent dyes that stain DNA are useful reagents for microscopic imaging, as they can be introduced into living cells to label DNA. However, the number of these dyes, such as Hoechst 33342, is limited. Here, we show that the icosahedral dodecaborate B12Br122-, a superchaotropic carrier that delivers different types of molecules into cells, functions as an excellent carrier for membrane-impermeable fluorescent dyes. Propidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI), dicationic membrane-impermeable fluorescent dyes that stain DNA, can permeate cell membranes in the presence of boron clusters. Methyl green (MG), a dicationic dye used in the histological and fluorescent staining of DNA, permeated cell membranes in the presence of boron clusters. In contrast, monocationic membrane-permeable fluorescent dyes, such as acridine orange and pyronin Y, exhibited reduced fluorescence in cells in the presence of boron clusters. Boron clusters do not quench dicationic fluorescent dyes in water in vitro but have quenching effects on monocationic fluorescent dyes. We have demonstrated that the addition of B12Br122- to impermeable dicationic fluorescent DNA-staining dyes, such as DAPI, PI, and MG, which have been widely used for numerous years, imparts membrane permeability to introduce these dyes into living cells.

Investigation of In-vitro Antiproliferative activity of Ampelocissus latifolia root extract on MCF-7 breast cancer cells Ampelocissus latifolia root extract against MCF-7 breast cancer cells.

Background: Breast cancer is one of the leading causes of cancer related death in women worldwide. Treatment of cancer has been plagued with toxic side effects of anticancer drugs. The need of the hour is the development of novel compounds with maximum cytotoxic effect on cancer cells with minimal toxicity to normal cells. The current direction of researchers worldwide is to identify anticancer compounds from natural sources. In India, traditional medicine has employed use of herbaceous climbers of grape family for treatment of ailments ranging from snakebites to diabetes. In the current study, an attempt has been made to explore the in vivo antiproliferative property of ethanolic extract of root of Ampelocissus latifolia against MCF-7 breast cancer cell line. Materials and Methods: The antiproliferative effect of Ethanolic extract of root of Ampelocissus latifolia (ERAL) was estimated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl--tetrazolium bromide (MTT) assay. The MCF -7 cell viability in various concentrations of ERAL including 7.8mcg/ml, 15.6mcg/ml, 31.2mcg/ml, 62.5mcg/ml, 125mcg/ml, 250mcg/ml, 500mcg/ml and 1000mcg/ml was tested. The IC50 value was calculated. All the experiments were done in triplicates. This was followed up with DNA fragmentation assay and fluorescent staining and microscopy. Results: The MCF -7 cell viability in various concentration of ERAL including 7.8mcg/ml, 15.6mcg/ml, 31.2mcg/ml ,62.5mcg/ml, 125mcg/ml, 250mcg/ml, 500mcg/ml and 1000mcg/ml was found to be 69.01%, 62.39%, 55.04%, 48.00%, 41.17%, 33.82%, 26.78% and 20.06% respectively .The IC50 concentration was found to be in the range of 62.5mcg/ml.The cell viability was found to be dose dependant.DNA fragmentation assay and DAPI and PI staining of cells treated with IC50 concentration of ERAL were indicative of significant cell death. Conclusion: The concentration dependent inhibition of MCF-7 cells supported by DNA fragmentation and fluorescent staining indicate that Ampelocissus latifolia can be a source of novel anticancer molecules.

Human Macrophage- and Osteoclast-Based Constructs Do Not Induce Ectopic Bone Formation

PurposeAn increasing body of evidence suggests that bone resorbing osteoclasts are important—but as yet underrated—cellular initiators of bone formation. Furthermore, macrophages also have shown stimulatory effects on the osteogenic differentiation of mesenchymal stromal cells (MSCs). Consequently, we here investigated whether human macrophage- and osteoclast-laden carrier materials can induce ectopic bone formation upon subcutaneous implantation in nude mice.MethodsHuman osteoclast precursors were isolated and differentiated toward macrophages. Subsequently, these macrophages were seeded onto two types of cell carrier materials (i.e., electrospun polymeric scaffolds and devitalized bovine bone granules) and differentiated for 14 days toward osteoclasts. DNA assay and fluorescent nuclei staining were performed. Osteoclast differentiation was assessed by a tartrate-resistant acid phosphatase (TRAP)-activity assay, TRAP, and immunocytochemical staining for β3 integrin. After 60 days of implantation into nude mice, specimens were retrieved, histologically processed, and stained with hematoxylin and eosin (HE) as well as for TRAP to study ectopic bone formation and osteoclast activity, respectively.ResultsOsteoclast precursors limitedly adhered to both material types. Osteoclast-laden samples showed increased intracellular gross TRAP-activity on both cell carrier types, TRAP staining on polymeric electrospun scaffolds, and positive β3 integrin staining on decellularized bovine bone granules compared to the macrophage-laden materials. We observed that only the positive control samples loaded with bone morphogenetic protein-2 (BMP-2) induced ectopic bone formation and TRAP signal.ConclusionWe conclude that neither human macrophage- nor osteoclast-laden constructs are capable to induce ectopic bone formation under the current experimental set-up.Lay summaryInterestingly, increasing amounts of evidence suggest that osteoclasts—the cells responsible for breaking down bone tissue—can trigger bone formation. Therefore, we here aimed to study whether blood-derived macrophages and osteoclasts can induce bone formation in vivo. Consequently, we generated human macrophage- and osteoclast-laden constructs using two types of scaffold materials and implanted them underneath the skin of nude mice. Although we confirmed the presence of macrophages and osteoclasts on the materials, we found no signs of bone formation.

21 Fluorescent Protein-Based DNA Staining Dyes.

Fluorescent protein–DNA-binding peptides or proteins (FP-DBP) are a powerful means to stain and visualize large DNA molecules on a fluorescence microscope. Here, we constructed 21 kinds of FP-DBPs using various colors of fluorescent proteins and two DNA-binding motifs. From the database of fluorescent proteins (FPbase.org), we chose bright FPs, such as RRvT, tdTomato, mNeonGreen, mClover3, YPet, and mScarlet, which are four to eight times brighter than original wild-type GFP. Additionally, we chose other FPs, such as mOrange2, Emerald, mTurquoise2, mStrawberry, and mCherry, for variations in emitting wavelengths. For DNA-binding motifs, we used HMG (high mobility group) as an 11-mer peptide or a 36 kDa tTALE (truncated transcription activator-like effector). Using 21 FP-DBPs, we attempted to stain DNA molecules and then analyzed fluorescence intensities. Most FP-DBPs successfully visualized DNA molecules. Even with the same DNA-binding motif, the order of FP and DBP affected DNA staining in terms of brightness and DNA stretching. The DNA staining pattern by FP-DBPs was also affected by the FP types. The data from 21 FP-DBPs provided a guideline to develop novel DNA-binding fluorescent proteins.

The Mechanisms of SAA/TLR4 Inducing Angiogenesis in Rheumatoid Arthritis through NETs Formation

Objective: Rheumatoid arthritis (RA) is an autoimmune disease in which angiogenesis represents a critical early event of synovial inflammation. The present study aimed to reveal the potential molecular mechanisms of SAA/TLR4 induction of angiogenesis through NETs in RA. Materials and methods: Firstly, immunohistochemistry and immunofluorescence were used to determinate TLR4 and NETs expression in synovial tissue, respectively. ELISA was used to detect the content of SAA, MPO and NE in serum and synovial fluid of patients. DNA quantification was done by fluorescence. DNA fluorescence staining was used to compare NETs formation in RA and HC sera, and to investigate the mechanism of NETs formation induced by SAA stimulation. PicoGreen DNA testing was used to characterize the DNA in the supernatants. Also, DNA fluorescence staining to explore whether NETs formation induced by SAA was dependent or independent on NADPH oxidase pathway. MTT assay, Wound healing assay, Tube formation assay were performed to analyze human veins umbilical cells (HUVECs) proliferation, migration, and tube vessels formation, respectively under NETs or NETs + DNase stimulants. Results: Firstly, we demonstrated that TLR4 was predominantly and widely expressed in synovial tissues with elevated serum levels of SAA, compared to osteoarthritis (OA) patients, and the similar results were observed for NETs formation. Afterwards, in a series of in vitro experiments, we reported an increased MPO and NE levels, and a relatively decreased DNA level in the sera of RA patients. Set apart, the levels of MPO and NE in RA were correlated to the disease activity. Moreover, an increased spontaneous NETs formation was observed in RA patients, enhanced under SAA stimulation and regulated by TLR4 activation. And the total DNA expressed in RA patients was partly composed of NET-DNA. Also, SAA induced NETs formation dependent on NADPH pathway. Finally, our results indicated that extracted SAA-induced NETs promoted endothelial cells (ECs) migration, proliferation, and vascular tube formation. Conclusion: Our current study highlighted the role of SAA/TLR4 interaction in the induction of angiogenesis through formed NETs. Therefore, this study offers new perspectives in the understanding of RA pathogenicity and its management.

Synthesis and Spectral Characterisation of (E)-3-(3-(4 (Dimethylamino)Phenyl)Acrylo-yl)-4-Hydroxy-2H-Chromen-2-One and Their Antibacterial Activity and Acetylcholinesterase Inhibition

A new coumarin derivative, (E)-3-(3-(4-(dimethylamino) phenyl) acrylo-yl)-4-hydroxy-2H-chromen-2-one (3), was synthesized by the condensation of 3-acetyl-4-hydroxycoumarin (1) with 4-N,N-dimethylaminobenzaldehyde (2) in the presence of piperidine in ethanol. The structure of the synthesized compound was characterized using spectroscopic data (IR and 1H NMR) and elemental analysis. The antimicrobial properties and acetylcholinesterase inhibition activity (AChEI) of coumarin 3 were investigated, with the highest observed AChEI activity providing 48.25% inhibition. The electronic absorption and emission spectra revealed that 3 exists as two, main keto-enol tautomers. The ratios of these tautomers in both protic and aprotic solvents with different polarities and dielectric constants were calculated. The fluorescence of coumarin 3 was enhanced upon increasing the medium viscosity, which was due to the resultant molecular rigidity. This criterion was further investigated using DNA, whereby 3 showed enhanced fluorescence upon its uptake in DNA grooves and was therefore tested as a novel DNA fluorescent stain.

Self-assembled chitosan derived microparticles inhibit tumor angiogenesis and induce apoptosis in Ehrlich-ascites-tumor bearing mice

Self-assembled microparticles from chitosan (SAMC) was prepared by depolymerization induced by potassium persulfate. Particle size distribution data showed averaged around 5μm size and SEM indicated the sequential formation of "RBC" shaped particles. Soluble SAMC consists of 'deacetylated' residues as revealed by 13C NMR. SAMC showed antitumor efficacy in human breast cancer cell lines through mitigation in cell proliferation, colony formation and cell migration. Anti-tumor and anti-angiogenic properties of SAMC was found in vivo Ehrlich ascites tumor (EAT) bearing mice model resulting in tumor growth inhibition (EAT control, 17.4ml; SAMC treated, 6.8ml) and improved survival potency (15days). Moreover, the decrease in ascites VEGF secretion (EAT control, 1354ng; SAMC treated, 351ng) accompanied with reduction in neovessel formation. Apoptosis induction by SAMC was confirmed by DNA fragmentation, caspase activities and fluorescence staining methods respectively. SAMC may be a safe candidate for anti-tumor dietary supplement production in food industry.

Thermal stress induces pyknosis in pigeon erythrocytes: digital image analysis

Pyknosis or hypercondensation of chromatin is informative in the understanding of nucleosomal packing in translationally inactive chromatin and in the compression of cell death. However, mechanisms that result in the formation of avian erythrocytes with variant nuclear morphology are poorly understood. Purpose: In this work, we evaluated pyknosis in pigeon erythrocytes treated with thermal stress using Digital Image Analysis (DIA). Materials and methods: Pigeon erythrocytes were treated at thermal stress (33 °C, 43 °C, and 53 °C), and nuclear modifications were analyzed by DIA. Results: Our results showed that thermal stress induced DNA condensation. Based on DNA fluorescent staining and compaction, four subclasses with progressively more pyknotic nuclei each could be distinguished. Alkaline comet assay showed that the presence of pyknotic nuclei was associated with the DNA fragmentation typical of apoptosis. DIA analysis showed a decrease of nuclear area and a significant increase of fluorescence intensity with respect to non-pyknotic nucleus. Additionally we observed nuclear dissolution events associated with swell and loose membrane integrity. Conclusion: These findings can contribute to the evaluation of health and metabolic status in diagnostic cytology, especially in neoplastic conditions and infection by microorganisms.

Increased levels of NETosis in myeloproliferative neoplasms are not linked to thrombotic events

Morbidity and mortality of Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are mainly determined by thromboembolic complications. Thrombus formation is facilitated by a neutrophil-specific form of cell death linked to neutrophil extracellular trap (NET) formation (NETosis). Preclinical and clinical data suggested a potential link between NETosis and thrombosis in MPNs. In this study, we aimed to define the impact of NETosis on clinical end points in a large MPN cohort. NETosis was induced in vitro by ionomycin and quantified by enzyme-linked immunosorbent assay-based nucleosome release assays as well as fluorescent staining of free DNA in samples from 103 MPN patients and 28 healthy donors. NETosis rate was correlated with a broad set of clinical data, such as MPN subtype, mutational status, laboratory variables, history of thrombotic events, and treatment types. Triggered NETosis levels were clearly higher in MPN patients than in healthy donors. Positivity for JAK2 V617F or exon 12 as well as CALR mutations correlate with increased NET formation. However, neither JAK2 allelic burden nor history of thromboembolic complication nor the presence of other risk factors for thrombosis (eg, leukocytosis) were associated with the rate of NETosis. In addition, none of the analyzed laboratory parameters nor the type of treatment significantly impacted the rate of NETosis formation. The biology of MPNs has an impact on NET formation because genetic driver mutations favor induction of NETosis, but this does not seems to translate into important clinical end points such as thromboembolic complications. Therefore, NETosis may play a role in facilitating thrombosis, but it is not a sole causative determinant in MPN-associated thrombophilia.

Inhibitory effect of lignans from Ailanthus altissima on proliferation of human lung cancer cells and its mechanism.

e15059 Background: Plants are thought to contain compounds that inhibit the proliferation of cancer cells in vitro, and many attempts have been made to isolate anti-cancer drugs from plants. Ailanthus altissima is a family of Simaroubaceae, the bark is gray to grayish-black, because of the smell of the gland patches at the base of the leaves. 3,5,3-trimethoxy-8,1-neoligna-4,2,7,9,9-pentol (TNP) isolated from Ailanthus altissima. Methods: (1) Apoptosis was detected by flow cytometry: the cells were incubated with 10 mmol/L TNP for 24 h, and the cells were collected. The cell concentration was adjusted to 5×105 ̃ 5×106/ L. While propionate iodide was added for DNA fluorescence staining (Propidium Iodide, PI: 50 mg/L，triton-x100 1.0%). (2) The changes of p53, Bax and caspase-3 were detected by Western blot. The cells were cultured in 10 μmol/L cisplatin and TNP medium for 24 hours. Image J image analysis software is used for semi quantitative analysis. The data were expressed by mean standard deviation, and the mean values of each group were compared by analysis of variance and the least significant difference. There was a significant difference in P< 0.05. (3) A549 cells were treated with 10 μmol/L cisplatin and TNP. A549 cells were incubated with cisplatin and TNP, and then treated with solvent control (final concentration 0.1% dimethyl sulfoxide) with or without caspase inhibitor for 48 hours The OD value was determined by MTT method. Cell survival rate (%) = (OD value of control group /OD value of control group) 100%. Results: (1) The reverse effect of caspase inhibitor on the proliferation of A549 cells: the cell survival rates of A549 cells treated with 0.1, 1 and 10 μmol/L cisplatin and TNP were 96.11%, 72.36%, 27.57% and 71.42%, 28.97% and 4.34% respectively. The survival rate of A549 cells incubated with cisplatin and TNP 20 μmol/L increased to 100.00%, 81.92%, 57.54% and 85.69%, 48.57% and 16.95% respectively. Caspase inhibitor reversed the inhibitory effect of TNP on A549 cells. (2) The effect of TNP on apoptosis of A549 cells: after treatment with 10 μmol/L TNP for 24 h, the apoptosis rate of A549 cells was (24.01)%, which was significantly higher than that of the blank control group (6.79)% ( P< 0.05). (3) The results showed that the up regulation of p53, Bax and caspase-3 protein by TNP was 10 μmol/L. Conclusions: TNP can induce apoptosis of human lung cancer cell line A549, and it can up regulate the expression of p53, Bax and Caspase-3, which may be one of the mechanisms of TNP inducing apoptosis of A549 cells. The experiment also proves that caspase inhibitor can reverse the effect of TNP. TNP inhibits the proliferation of A549 cells.

Molecular structure, DNA binding mode, photophysical properties and recommendations for use of SYBR Gold.

SYBR Gold is a commonly used and particularly bright fluorescent DNA stain, however, its chemical structure is unknown and its binding mode to DNA remains controversial. Here, we solve the structure of SYBR Gold by NMR and mass spectrometry to be [2-(4-{[diethyl(methyl)ammonio]methyl}phenyl)-6-methoxy-1-methyl-4-{[(2Z)-3-methyl-1,3-benzoxazol-2-ylidene]methyl}quinolin-1-ium] and determine its extinction coefficient. We quantitate SYBR Gold binding to DNA using two complementary approaches. First, we use single-molecule magnetic tweezers (MT) to determine the effects of SYBR Gold binding on DNA length and twist. The MT assay reveals systematic lengthening and unwinding of DNA by 19.1° ± 0.7° per molecule upon binding, consistent with intercalation, similar to the related dye SYBR Green I. We complement the MT data with spectroscopic characterization of SYBR Gold. The data are well described by a global binding model for dye concentrations ≤2.5 μM, with parameters that quantitatively agree with the MT results. The fluorescence increases linearly with the number of intercalated SYBR Gold molecules up to dye concentrations of ∼2.5 μM, where quenching and inner filter effects become relevant. In summary, we provide a mechanistic understanding of DNA-SYBR Gold interactions and present practical guidelines for optimal DNA detection and quantitative DNA sensing applications using SYBR Gold.

Explorations into the Effect of meso‐Substituents in Tricarbocyanine Dyes: A Path to Diverse Biomolecular Probes and Materials

AbstractPolymethine cyanine dyes have been widely recognized as promising chemical tools for a range of life science and biomedical applications, such as fluorescent staining of DNA and proteins in gel electrophoresis, fluorescence guided surgery, or as ratiometric probes for probing biochemical pathways. The photophysical properties of such dyes can be tuned through the synthetic modification of the conjugated backbone, for example, by altering aromatic cores or by varying the length of the conjugated polymethine chain. Alternative routes to shaping the absorption, emission, and photostability of dyes of this family are centered around the chemical modifications on the polymethine chain. This Minireview aims to discuss strategies for the introduction of substituents in the meso‐position, their effect on the photophysical properties of these dyes and some structure–activity correlations which could help overcome common limitations in the state of the art in the synthesis.

Explorations into the Effect of meso-Substituents in Tricarbocyanine Dyes: A Path to Diverse Biomolecular Probes and Materials.

Polymethine cyanine dyes have been widely recognized as promising chemical tools for a range of life science and biomedical applications, such as fluorescent staining of DNA and proteins in gel electrophoresis, fluorescence guided surgery, or as ratiometric probes for probing biochemical pathways. The photophysical properties of such dyes can be tuned through the synthetic modification of the conjugated backbone, for example, by altering aromatic cores or by varying the length of the conjugated polymethine chain. Alternative routes to shaping the absorption, emission, and photostability of dyes of this family are centered around the chemical modifications on the polymethine chain. This Minireview aims to discuss strategies for the introduction of substituents in the meso‐position, their effect on the photophysical properties of these dyes and some structure–activity correlations which could help overcome common limitations in the state of the art in the synthesis.