Dye Staining Method Research Articles

A one-way ticket: Wheat roots do not functionally refill xylem emboli following rehydration

Abstract Understanding xylem embolism spread in roots is essential for predicting the loss of function across root systems during drought. However, the lasting relevance of root embolism to plant recovery depends on whether roots can refill xylem emboli and resume function after rehydration. Using MicroCT and optical and dye staining methods, we investigated embolism repair in rehydrated intact roots of wheat (Triticum aestivum L. ‘Krichauff’) exposed to a severe water deficit of −3.5 MPa, known to cause approximately 30% total root network embolism in this species. Air emboli in the xylem vessels of intact roots remained clearly observable using MicroCT after overnight rehydration. This result was verified by xylem staining of the root system and optical quantification of emboli, both of which indicated a lack of functional root xylem recovery 60 h following soil re-saturation. The absence of root xylem refilling in wheat has substantial implications for how we understand plant recovery after drought. Our findings suggest that xylem embolism causes irreversible damage to the soil–root hydraulic connection in affected parts of the root network.

Formation of Ethanolamine-Mediated Surfactant-Free Microemulsions Using Hydrophobic Deep Eutectic Solvents.

Hydrophobic deep eutectic solvents (HDESs) are emerging as versatile, relatively benign, and inexpensive alternatives to conventional organic solvents in a diverse set of applications. In this context, the formation of microemulsions with HDES replacing the oil phase has become an area of active exploration. Because of recent reports on the undesirable toxicity of many common surfactants, efforts are under way to investigate the formation of surfactant-free microemulsions (SFMEs) using HDES as an oil phase. We present SFME formation using HDESs constituted of n-decanoic acid and five (5) structurally different terpenoids [thymol, l(-)-menthol, linalool, β-citronellol, and geraniol] at a 1:1 molar ratio as the oil phase and water as the hydrophilic phase. Ethanolamine (ETA) exhibited the best potential as a hydrotrope among several other similar small molecules. Results showed a drastic increase in water solubility within the HDESs in the presence of ETA. ETA exerted its hydrotropic action at different extent for each DES system via chemical interaction with the H-bond donor (HBD) constituent of the HDES. The optimum hydrotropic concentration (minimum hydrotrope and maximum water retention, XETAOPT) assigned for each DES/ETA/water system and water loading are reported, and the trends are discussed in detail. Ternary phase diagrams are constructed using visual observation and the dye staining method. The area under the single- and multiple-phase regions (assigned in ternary phase diagrams) was estimated. "Pre-Ouzo" enforced by ETA was investigated using dynamic light scattering (DLS) of the DES/ETA/water systems at XETAOPT. A systematic growth in nanoaggregates was observed with the subsequent addition of water in DES/ETA systems while continuously changing the existing microstructure. The presence of a core (oil)-shell (water)-like structure as indicated by the fluorescence response of Nile red in the "pre-Ouzo" region is speculated. We were able to prepare a homogeneous solution of [K3Fe(CN)6] salt in "pre-Ouzo" mixtures with no apparent deviation in the Beer-Lambert law.

New Heterocyclic Organo-Chalcogenide Compounds: Synthesis, Physicochemical Characterization, and Evaluation of Anticancer Activity against Breast Cancer Cells

This work aimed to synthesize, characterize and evaluate the thermal stability of new sulfur and selenium organochalcogenide derivatives and to test the cytotoxic activity against breast adenocarcinoma cell line (MCF-7) through conducting MTT assay and AO/EB dual staining-technique. Two series of ten organo-chalcogen compounds: 4-(substituted)phenylthiomorpholine-3,5-dione and 4-(substituted)phenylselenomorpholine-3,5-dione were prepared by the reaction of Na2S·3H2O and NaHSe with N-(substituted)phenyl-2-chloro-N-(2-chloroacetyl)acetamide, respectively, under nitrogen atmosphere to give the corresponding cyclic chalcogenide ligands. All new compounds were characterized by melting point, FTIR, elemental analysis, UV-Visible, 1H-NMR and 13C-NMR. Meanwhile, TG/DTA analysis of some of these ligands was conducted to evaluate the thermal stability, kinetic, and characteristic thermodynamic parameters. Absorption spectroscopy was used to investigate these compounds with human DNA. The experimental results investigated a hypochromic effect via intercalation binding mode. The role of the prepared ligands in breast cell lines has been investigated by conducting MTT assay via spectroscopic techniques on HBL100 and MCF-7, normal and cancer breast cell lines, respectively. Cell death was seen after AO/EB dye staining method employing the fluorescence microscopy technique. The results revealed that these compounds possess cytotoxic activity on the MCF-7 and HBL-100 cell lines at a fixed concentration.

Detection of Biofilm Formation on Material Surfaces by Ag+ Coating

The evaluation of biofilm formation is important, given the ubiquity and problematic nature of biofilms in industrial and medical settings, as well as in everyday life. Basically, biofilms are formed on substrates. Therefore, it is essential to consider the properties of the substrates during biofilm evaluation. The common dye staining method to evaluate biofilm formation requires a short evaluation time and enables the evaluation of a large area of the sample. Furthermore, it can be easily determined visually, and quantitative evaluation is possible by quantifying color adsorption. Meanwhile, the dye staining method has the problem of adsorption even on substrate surfaces where no biofilm has formed. Therefore, in this study, we focused on Ag+ reduction reaction to devise a novel biofilm evaluation method. Ag+ is highly reductive and selectively reacts with organic substances, such as saccharides, aldehydes, and proteins contained in biofilms, depositing as metallic Ag. First, to simply evaluate biofilm formation, we used a glass substrate as a smooth, transparent, and versatile oxide material. We observed that the amount of Ag deposited on the substrate was increased proportionally to the amount of biofilm formed under light irradiation. Upon comparing the Ag deposition behavior and adsorption behavior of crystal violet, we discovered that for short immersion times in AgNO3 solution, Ag deposition was insufficient to evaluate the amount of biofilm formation. This result suggests that the Ag reduction reaction is more insensitive than the crystal violet adsorption behavior. The results of the Ag deposition reaction for 24 h showed a similar trend to the crystal violet dye adsorption behavior. However, quantitative biofilm evaluation using the proposed method was difficult because of the Ag+ exchange with the alkali metal ions contained in the glass substrate. We addressed this issue by using the basic solution obtained by adding an ammonia solution to aqueous AgNO3. This can cause Ag+ to selectively react with the biofilm, thus enabling a more accurate quantitative evaluation. The optimum was determined at a ratio of distilled water to aqueous ammonia solution of 97:3 by weight. This biofilm was also evaluated for materials other than ceramics (glass substrate): organic material (polyethylene) and metal material (pure iron). In the case of polyethylene, a suitable response and evaluation of biofilm formation was successfully achieved using this method. Meanwhile, in the case of pure iron, a significantly large lumpy deposit of Ag was observed. The likely reason is that Ag precipitation occurred along with the elution of iron ions because of the difference in ionization tendency. It could be concluded that the detection of biofilm formation using this method was effective to evaluate biofilm formation on materials, in which the reduction reaction of [Ag(NH3)2]+ does not occur. Thus, a simple and relatively quantitative evaluation of biofilms formed on substrates is possible using this method.

Microplastics generation and concentration during mechanical-biological treatment of mixed municipal solid waste

Mechanical-biological treatment (MBT) is a popular solution for the processing of mixed municipal solid waste (MSW). However, it is assumed that the treatment processes can lead to the generation of microplastics in large quantities and their concentration in the organic output. Organic outputs from MBT as a source of microplastics are still poorly understood. The current article aims to fill this gap and investigate microplastics formation during MBT and their abundance in ready stabilized organic output. Seasonal samples were taken from the four stages of the possible microplastics pathway in MBT to study changes in microplastics numerical and mass concentration, shape and size. Large microplastics were identified by Fourier transform infrared spectroscopy, and small microplastics by Nile Red dye staining method. The results showed that both mechanical pre-treatment and aerobic treatment had a significant impact on microplastics formation, while mechanical post-treatment only resulted in the enrichment of the output with microplastics. Moreover, microplastics became finer during treatment. Microplastics abundance in ready organic output ranged from 8925 ± 1344 particles/kg in winter 2021 to 17407 ± 4319 particles/kg in summer 2020, and up to 160.5 t of microplastics were emitted from the Kaunas MBT treatment facility during the study year. In addition, a relationship between the microplastics abundance and plastic content of the incoming waste was found by a regression analysis. Therefore, to reduce the formation and emission of microplastics by MBT, the organic fraction of MSW should be collected and treated separately.

Isolation, Screening and Identification of Cellulolytic Streptomyces corchorusii (Mn244066) From Soil Sample of Visakhapatnam

Fifty one isolates of cellulolytic bacteria were isolated from cow dung and two soil samples were obtained from Kambalakonda Wildlife Sanctuary and RCD Biodiversity Park in Visakhapatnam, Andhra Pradesh by Enrichment method in basal salt medium with cellulose as substrate for degradation. The cellulolytic activity of the isolated bacteria was determined by the diameter of the zone of hydrolysis by Gram’s iodine dye staining method. After primary screening, a total of fifty one isolates showed cellulolytic activity. Out of fifty one strains of cellulolytic bacteria, twenty three isolates from Kambalakonda Wildlife Sanctuary, seventeen isolates from RCD Biodiversity Park and Eleven isolates from cow dung sample obtained from cattle ranch, Visakhapatnam showed cellulase activity. Seven strains showed maximum hydrolytic value greater than 4.0 cm, nineteen strains showed average hydrolytic value between 3.0 and 3.9 cm and twenty one strains showed minimum hydrolytic value between 1.5 and2.9 cm. The potential isolates were obtained from Kambalakonda Wildlife Sanctuary and RCD Biodiversity Park than cow dung sample. The 13 C strain exhibited maximum hydrolytic value of 5.6 cm which was designated as KKV1. The strain KKVI was identified as Streptomyces corchorusii (MN244066) by morphological, cultural, biochemical and 16S rRNA sequence. The CMCase and FPase activity of the crude sample were examined by DNS method and found to be 0.21 U/ml and 0.041 U/ml respectively and the specific activity was 4.38 U/mg proteins and 0.86 U/mg proteins respectively. The present study emphasizes that the Streptomyces corchorusii have a higher cellulase activity and the soils of bio reserves have a lot of scope for isolating high cellulolytic bacteria which can be exploited for different industrial purposes.

Isolation and characterization of Rhodococcus qingshengii, a cellulolytic bacteria from Cnaphalocrocis medinalis (Lepidoptera: Crambidae) gut

Herbivorous Lepidopteran insects depend on microbial cellulases for cellulose digestion. The present study aimed to investigate the herbivorous Lepidoptera Cnaphalocrocis medinalis gut for isolation of cellulose degrading bacteria. A Gram positive rod, non-motile, mesophilic, neutrophile bacterial strain TCI11 was isolated from C. medinalis (Lepidoptera: Crambidae) 5th instar larvae gut. The isolate was found to be a potent cellulose degrader, which was isolated on carboxymethylcellulose agar medium using carboxymethylcellulose as a sole carbon source and screened by Congo red and Gram’s iodine dye staining method. The cellulose degrading strain was characterized on the basis of Gram staining, morphological and biochemical properties. Based on 16S rRNA gene analysis, the strain was found to be most closely related to Rhodococcus baikonurensis, Rhodococcus erythropolis, Rhodococcus globerulus (99.77, 99.35 and 99.06% similarity, respectively) and was identified as Rhodococcus qingshengii. R. qingshengii is being reported for the first time as a potent cellulose degrader. The source of isolation of this particular bacterial strain is also novel.

Computed tomography-guided marking using a dye-staining method for preoperative localization of tiny pulmonary lesions in children.

It is important to confirm the existence of pulmonary metastases in pediatric patients with malignancies. Therefore, we aimed to investigate if computed tomography CT-guided marking is a feasible and safe method for the identification and resection of tiny pulmonary lesions in pediatric cancer patients. We retrospectively reviewed the medical records of pediatric cancer patients who underwent CT-guided marking procedures in our institutions between Jan 2011 and Apr 2020. After 2015, these procedures were combined with an indocyanine green (ICG) navigation-guided surgery for hepatoblastoma cases. We targeted a total of 22 nodules in 12 patients. Of these, marking was successful in 18 (81.8%) nodules, 10 of which contained viable malignant cells. Complications caused by the marking procedures included mild pneumothorax and mild atelectasis in two patients, respectively. Of the eight resected nodules in patients with hepatoblastoma, four were ICG-positive and contained viable malignant cells. Two additional ICG-positive nodules, which were unidentified before surgery, were observed intraoperatively. CT-guided marking is a feasible and safe method that can be used to identify and resect tiny pulmonary lesions in pediatric cancer patients. An ICG navigation-guided surgery is useful when combined with CT-guided marking, particularly in hepatoblastoma cases.

Starch-capped sulphur nanoparticles synthesised from bulk powder sulphur and their anti-phytopathogenic activity against Clavibacter sepedonicus.

Water‐soluble, stable nanoparticles of elemental sulphur with a size of 9‐52 nm have been synthesised using the stabilising potential of starch. Sulphide anions were used as sulphur precursors that were generated earlier from the bulk powder sulphur in the base‐reduction system NaOH‐N2H4·H2O followed by their oxidation with molecular oxygen to element sulphur atoms. Using a set of modern spectral and microscopic methods (XRD, optical spectroscopy, DLS, TEM), the phase state, elemental composition of the nanocomposites and their nanomorphological characteristics have been investigated. It was found that nanocomposites are formed as sulphur particles with the shape which is nearly spherical dispersed in the polysaccharide starch matrix with a pronounced tendency to cluster into ring formations. Water solubility and stability of the obtained nanoparticles is ensured by sorption of starch macromolecules on the surface of sulphur nanoparticles, with the thickness of the stabilising shell in a range of 10‐171 nm. In vitro experiments were carried out to study the anti‐microbial activity of the obtained sulphur nanocomposite (1.6% S) using the propidium iodide fluorescent dye staining method and the diffusion method. It showed that the water solution of the starch‐capped sulphur nanoparticles at the concentration of 6.25 µg/ml had a pronounced anti‐phytopathogenic activity against the potato ring rot pathogen Clavibacter michiganensis subsp. sepedonicus.

Characterizing Preferential Flow Paths in Texturally Similar Soils under Different Land Uses by Combining Drainage and Dye-Staining Methods

Preferential flow paths have been widely characterized by many visualization methods. However, the differences in preferential flow paths under various land uses and their relationships to hydraulic properties remain uncertain. The objectives of this study are to (1) characterize preferential flow paths under various land uses (forest and orchard) by combining drainage and dye-staining methods and to (2) build a connection between preferential flow paths and hydraulic-related parameters and extract the proportion of preferential flow paths from the compounding effects of matrix flow and preferential flow. The dye-staining experiments were conducted in five sandy soils and one sandy clay loam in situ, including four soils from forest and two soils from orchards. A total of 47 soil cores, 4 cm in height and 9 cm in diameter, were collected in each layer of the dye-stained soils for drainage experiments in the laboratory. Dye coverage and hydraulically equivalent macropore parameters (macroporosity, pore size distribution, and number of macropores) and their relationships were analyzed. The results show that the volume of preferential flow is partly affected by the total macropore volume. The effect of macropores on preferential flow varies by macropore size distribution. Dye coverage exhibited a significant (P < 0.01) correlation with macroporosity (correlation coefficient 0.83). Based on the value of macroporosity or steady effluent rates, the part of the dye coverage that was due to preferential flow on the surface dye-stained soil (resulting from both matrix and preferential flow) could be identified in this study. Compared with orchards, forestland has more preferential flow paths in both surface soil and subsoil. Further studies are needed to quantify the 3-D preferential flow paths and build a connection between preferential flow paths and hydraulic properties.

Tumor cell network integration in glioma represents a stemness feature.

Malignant gliomas including glioblastomas are characterized by a striking cellular heterogeneity, which includes a subpopulation of glioma cells that becomes highly resistant by integration into tumor microtube (TM)-connected multicellular networks. A novel functional approach to detect, isolate, and characterize glioma cell subpopulations with respect to in vivo network integration is established, combining a dye staining method with intravital two-photon microscopy, Fluorescence-Activated Cell Sorting (FACS), molecular profiling, and gene reporter studies. Glioblastoma cells that are part of the TM-connected tumor network show activated neurodevelopmental and glioma progression gene expression pathways. Importantly, many of them revealed profiles indicative of increased cellular stemness, including high expression of nestin. TM-connected glioblastoma cells also had a higher potential for reinitiation of brain tumor growth. Long-term tracking of tumor cell nestin expression in vivo revealed a stronger TM network integration and higher radioresistance of the nestin-high subpopulation. Glioblastoma cells that were both nestin-high and network-integrated were particularly able to adapt to radiotherapy with increased TM formation. Multiple stem-like features are strongly enriched in a fraction of network-integrated glioma cells, explaining their particular resilience.

Rhodamine B dye staining for visualizing microplastics in laboratory-based studies.

In the recent years, microplastics have attracted much attention as new emerging environmental pollutants. Previously, several studies were performed to understand the source and fate of microplastics in the environment, organisms, and food webs. To track microplastics and improve their legibility, labeling them is a very effective method during laboratory experiments. This study presents an effective Rhodamine B dye (RhB) staining method for microplastics. The method is crucial for the visual observation of white or transparent plastics by dyeing them in purple or pink, as well as makes the microplastics to fluoresce under common microscope fluorescence filter ranges. Five types of microplastic polymers, namely polyethylene, polypropylene, polystyrene, polyvinyl chloride, and polyurethane were used as the test materials. The efficiencies of ethanol, acetone, and distilled water as possible solvents for dissolving RhB were investigated. Next, the fluorescence stability in various conditions was assessed. The results indicated that ethanol was the most appropriate solvent in dissolving RhB used in staining the microplastics. RhB was fluorescently stable under varying conditions (light and gut fluid) or different solutions (KOH, nitric acid, and saturated NaCl). Additionally, RhB staining exhibited an insignificant effect on the Raman spectra of the microplastics. Our proposed method is simple and robust and helps to visualize the different types of microplastic polymers tested in laboratory experiments, particularly the transparent, white, and small size microplastics.

Excited-State Properties of Metal-Free ((Z)-2-Cyano-3-(4-((E)-2-(6-(4-methoxyphenyl)-9-octyl-9H-carbazol-3-yl)vinyl)phenyl)acrylic Acid and (E)-2-Cyano-3-(4-((E)-4-(diphenylamino)styryl)phenyl)acrylic Acid) and Ru-Based (N719 and Z907) Dyes and Photoinduced Charge Transfer Processes in FTO/TiCl4/TiO2/Dye Photoanodes Fabricated by Conventional Staining and Potential-Assisted Adsorption.

Excited-state properties of two novel metal-free custom-made dyes D2d [(Z)-2-cyano-3-(4-((E)-2-(6-(4-methoxyphenyl)-9-octyl-9H-carbazol-3-yl)vinyl)phenyl)acrylic acid] and T-SB-C [(E)-2-cyano-3-(4-((E)-4-(diphenylamino)styryl)phenyl)acrylic acid] and two commercially available Ruthenium-based N719 and Z907 dyes were investigated with application of time-resolved absorption and emission. Singlet excited state lifetimes of D2d and T-SB-C were determined in acetonitrile and are 1.4 and 2.45 ns, respectively. The 3MLCT state lifetimes of N719 and Z907 dyes determined in methanol are 9.25 and 8.85 ns, respectively. Subsequently, photoexcited processes like electron injection and charge recombination were studied for those dyes adsorbed on the FTO/TiCl4/TiO2 photoanodes and fabricated via a conventional staining technique and innovative potential-assisted fast dye staining method. The dynamics of the spectro-temporal data was determined with application of single-wavelength and global fitting. All dye-TiO2 systems showed fast picosecond injection of excited electrons to the conduction band of the TiO2 layer and in complex multiphasic charge recombination processes. The dynamics of those processes is not altered by the dye adsorption method.

The effect of composite interface morphology on wetting states for nanocomposite superhydrophobic coating

This paper focuses on the effect of composite interface morphology on the wetting behavior of a nanocomposite coating based on an organosilane binder. Experimental results supported by modeling demonstrated that a sharp change in contact angle hysteresis occurred at air fractions in the range of 40–60%, as controlled by particle loading. The air-liquid interface evolution with particle loading was visualized using the fluorescent dye staining method. A correlation between topography and this air-liquid-solid interface was done by overlapping the optical images and fluorescence dye stained images of the stained superhydrophobic surface, which allowed the visualization of microscale features. The results showed the structures supporting solid-liquid contacts were 20 to 40 μm in size and had an edge-to-edge spacing that decreased with increasing particle loading. In this system a critical spacing to transition from the Wenzel to the Cassie-Baxter state was found to be approximately five times the average width of these microscale structures. Identification of critical surface topography can aid in the development of coatings that provide anti-corrosive and/or anti-icing features for marine vessels, bridges, and buildings.

Low-Noise Fluorescent Infrared Detection of Single-Cell Cardiac Action Potentials

Optical recordings with voltage-sensitive fluorescent dyes enable non-invasive action potential (AP) measurements, which are useful for high-throughput drug screening. However, optical measurements are hampered by dye rundown, photobleaching, and phototoxicity. Long-lasting infrared dyes overcome these limitations, but optical recordings from single-cells are difficult due to low signal-to-noise ratios (SNR). Using the voltage-sensitive infrared dye, Di-4-ANBDQBS (Cytovolt1), we developed an optical system consisting of matched hardware, biological, and dye components to produce a low-noise fluorescent infrared detection system for single-cell AP recording. We combined a high-sensitivity photodiode-based detector, a high-stability LED excitation source, and an optimized optical filter set for enhanced single-cell optical AP recordings. We validated our system by correlating membrane voltage to fluorescence under single-cell patch clamp, and showed the fluorescence signal is highly-linear with membrane voltage. We also optimized dye concentration and staining method along with emission/excitation wavelengths to yield maximum ΔF/F and thus maximum SNR. To correlate membrane voltages to optical voltage signals, we performed simultaneous patch clamp and optical recordings on HEK293 cells and human induced pluripotent stem cell derived cardiomyocytes (hIPSC-CMs). Low-noise recordings, with minimal dye rundown, were obtained from single-cells. Optical signals were recorded in response to a voltage ramp, and averaged R2 values from linear fits of fluorescence versus voltage were 0.88+0.04, 0.97+0.007, 0.97+0.004 and 0.98+0.004 for 50, 75, 100 and 150 μM of Di‑4‑ANBDQBS, respectively, in HEK293 cells. ΔF/F measurements were 10.5+0.3%, 14.9+1.8%, 13.8+2.5% and 13.6+3.5% for 50, 75, 100 and 150 μM of dye, respectively. For hiPSC-CMs, ΔF/F measurements from simultaneous voltage and fluorescence of APs were up to 23.1% per 100 mV. Taken together, these data demonstrate the feasibility of high fidelity single cell voltage measurements in a simple system not requiring photomultiplier tubes or laser excitation.

In vitro anti‐proliferative and in silico docking studies of heteroleptic copper(II) complexes of pyridazine‐based ligands and ciprofloxacin

Three heteroleptic copper(II) complexes of the type [Cu(L1–3)(cf)(ClO4)] (1–3), where cf = ciprofloxacin, have been synthesized using pyridazine‐based ligands 3‐chloro‐6‐(salicylidenehydrazinyl)pyridazine (HL1), 3‐chloro‐6‐(4‐diethylaminosalicylidenehydrazinyl)pyridazine (HL2) and 3‐chloro‐6‐(5‐bromosalicylidenehydrazinyl)pyridazine (HL3). Electronic spectral data and magnetic moment values suggest octahedral geometry for the synthesized copper(II) complexes. Electrochemical data of the copper(II) complexes present an irreversible one‐electron reduction wave in the cathodic potential region (Epc) between −0.631 and −0.670 V. Frontier molecular orbital calculations were carried out, and the obtained low‐energy gap supports the bio‐efficacy of the complexes. All the complexes were screened for their in vitro cytotoxicity activity against three human cancerous (breast adenocarcinoma (MCF‐7), hepatoma (HepG‐2) and cervical (HeLa)) and one non‐cancerous (non‐tumorigenic human dermal fibroblast (NHDF)) cell lines using MTT assay, in which complex 2 exhibited higher activity. The apoptosis induction by the complexes was analysed using the Hoechst dye staining method with MCF‐7 cell line, which indicates higher apoptotic activity of complex 2. A molecular docking study was carried out to ascertain the binding affinity of the synthesized heteroleptic copper(II) complexes with phosphoinositide 3‐kinase gamma (PI3Kγ) receptor.

Detection of incidence of Babesia spp. in sheep and goats by parasitological diagnostic techniques.

The present study was undertaken on epidemiology and diagnosis of babesiosis in sheep and goats in Bengaluru Urban and Rural districts of Karnataka state from November 2017 to May 2018. Out of 343 (225 sheep and 118 goats) blood smears examined by Giemsa and acridine orange (AO) fluorescent dye staining methods, 3.55 and 4.0 per cent of sheep and 0.84 and 1.69 per cent of goat samples were found positive for Babesia organisms, respectively. The sensitivity and specificity was found to be higher in AO fluorescent dye staining method. In agewise susceptibility, the percent positivity was found to be higher in animals > 6months old. In genderwise susceptibility, the percent positivity was found to be higher in females than males. Hence, AO fluorescent dye staining method is found to be very rapid and cost effective diagnostic method for treatment and control of babesiosis.

Assessment of antibacterial efficacy of a biocompatible nanoparticle PC@AgNPs against Staphylococcus aureus

A plant, Priva cordifolia mediated silver nanoparticle was prepared and characterized by UV-Vis spectroscopy, fourier-transform infrared spectroscopy (FT-IR), energy dispersive X-ray (EDX), scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS) and x-ray diffraction (XRD) analysis. The minimum inhibitory concentration of the synthesized nanoparticle against Staphylococcus aureus was found to be 100 ± 0.80 μg/mL with 9.38 ± 0.04 mm zone of inhibition. The bactericidal activity was shown primarily due to membrane damage evident from SEM, atomic force microscopy (AFM), potassium efflux, cellular material leakage, and bio-electrochemical changes in electron transport chain data. It was also of interest to find PC@AgNPs interfering with biofilm formation by S.aureus, assessed qualitatively by SEM, confocal laser scanning microscopy (CLSM) and quantitatively by dye staining method. The bio-compatibility of PC@AgNPs was established by anti-coagulant, thrombolytic, partial thromboplastin time, thrombin-like activity and fibrinolytic activity that suggested its good maintenance of hemostatic conditions. PC@AgNPs also prevented the coagulation of rabbit plasma which as per the standard drug Dabigatran reaction was indicative of the down-regulation of virulence Coa gene expression.

Synergistic anticancer and antibacterial activities of cordycepin and selected natural bioactive compounds

Purpose: To investigate if cordycepin (Cor) exerts synergistic anticancer and antibacterial activities with curcumin (Cur), resveratrol (Res), genistein (Gen), and anthocyanidin (Ant) in order to improve its therapeutic effect. Methods: Antibacterial activity was assayed against Escherichia coli BL21 (DE3) cultured in the Luria-Bertani (LB) medium, while anticancer activity against Hela cells was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Immunofluorescence assay was used to measure the expressions of p53 and caspase-3, while apoptosis was assayed in Hela cells using Wright-Giemsa dye staining method. Results: The combination of Cor and Gen produced the most significant synergistic antibacterial activity, while combination of Cor, Cur and Gen exerted significant and synergistic anticancer activity against Hela cells. Among all three-compound combinations, Cor + Res + Gen had the highest synergistic anticancer activity. Immunofluorescence data indicate that Cor functioned by up-regulating p53-dependent apoptosis, while Res exerted its effect by p53- and caspase-3-dependent apoptosis, and Gen functioned by inhibiting adenosine deaminase and Cor degradation. Conclusion: The three compounds function via different mechanisms, and synergistically induce Hela cell apoptosis. This work provides a strong basis for the development of highly active and multicomponent anticancer drugs from natural sources. Keywords: Cordycepin, Natural phenolic compounds, Caspase, Anticancer, Synergy

Counterion Dye Staining of Proteins in One- and Two-Dimensional Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Tryptic Gel Digestion of Stained Protein for Mass Spectrometry.

A fast and matrix-assisted laser desorption/ionization-mass spectrometry compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, zincon and ethyl violet to form an ion-pair complex. The protocol including fixing, staining, and quick washing steps can be completed in 1-1.5h depending upon gel thickness. It has the sensitivity comparable to the colloidal Coomassie Brilliant Blue G stain using phosphoric acid as a component of staining solution (4-8ng). The counterion dye stain does not induce protein modifications that complicate interpretation of peptide mapping data from mass spectrometry. Considering the speed, sensitivity, and compatibility with mass spectrometry, the counterion dye stain may be more practical than any other dye-based protein stains for routine proteomic researches.