RSC Advances

Biological sample clearing techniques are a potent tool for three-dimensional biological imaging, among which delipidation is an essential step in achieving high-quality biological sample transparency. Detergents and organic solvents can both be used for lipids removal. The former has been extensively investigated in biological sample clearing, while the delipidation process based on organic solvents remains to be further elucidated. Recently, organic solvents also served as a delipidation reagent in aqueous-based clearing methods and exhibited very fast clearing speed. To explain the high efficiency of organic solvents, we described the delipidation process of both detergents and organic solvents with a simple diffusion model, we proposed a possible mechanism of the delipidation process of water-miscible polar organic solvents based on the clearing results of brain samples. Both our results and model revealed that polar or non-polar organic solvents with a certain molecular structure could achieve a much faster clearing speed than detergents which could be a guide for establishing a rapid clearing protocol for biological samples with large volumes.

Microbial-induced calcium carbonate precipitation (MICP) is a biological process inducing biomineralization of CaCO3. This can be used to form a solid, concrete-like material. To be able to use MICP successfully for producing solid materials, it is important to understand the formation process of the material in detail. It is well known, that crystallization surfaces can influence the precipitation process. Therefore, we present in this contribution a systematic study investigating the influence of calcite seeds on the MICP processes. We focus on the pH changes during the crystallization process measured with absorption spectroscopy and on the optical density (OD) signal to analyze the precipitation process. Furthermore, optical microscopy was used to visualize the precipitation processes in the sample and connect them to changes in pH and OD. We show that there is a significant difference in the pH evolution between samples with and without calcite seeds present and that the shape of the pH evolution and the changes in OD can give detailed information about the mineral precipitation and transformations. In the presented experiments we show that amorphous calcium carbonate (ACC) can also precipitate in the presence of initial calcite seeds, which can have consequences for consolidated MICP materials.

Plant diseases pose threat to global food security. The excessive use of synthetic agro-chemical engender pesticide resistance. The exploration of alternative sustainable diseases management practices are crucial to overcome the devastative plant diseases. In this study, a facile innocuous approach was adopted for biogenic synthesis of Fe2O3-CsNPs via Crocus sativus corm aqueous extract and was evaluated for their antifungal efficacy against the Verticillium wilt pathogen Verticillium dahliae. The physico-chemical characterization of biosynthesized nanoparticles were performed through UV-visible Spectroscopy, Fourier Transform Infrared Spectroscopy, Energy Dispersive X-ray Spectroscopy, X-Ray Diffraction, and Scanning Electron Microscopy. The fungus mycelium growth was significantly inhibited in the media containing 3mg/mL Fe2O3-CsNPs. Degenerated, concentrated and shriveled hyphae were revealed in Scanning Electron Microscopy. The overall results demonstrated that the biogenic Fe2O3-CsNPs have the efficacy to control devastative phytopathogens.

Studying the interactions between microorganisms and plant roots is crucial for understanding a variety of phenomena concerning crop yield and health. The role of root surface properties in these interactions, is rarely addressed. To this end, we previously built a synthetic system, from the inert polymer polydimethyl siloxane (PDMS), mimicking the root surface microstructure, using a replication technique. This replica enables the study of isolated effects of surface structure on microorganism-plant interactions. Since the root surface is composed mostly of cellulose, using cellulose-like materials as our replica, instead of PDMS, is the next logical step. This will enable following the hydrolysis of such surfaces as a result of microorganisms secreting Plant Cell Wall Degrading Enzymes (PCWDE), and in particular, cellulase. Visualization of such hydrolysis in a synthetic system can assist in studying the localization and activity of microorganisms and how they correlate with surface microtopography, separately from chemical plant signals. In this work, we modified the known carboxymethyl cellulase (CMC) hydrolysis visualization method to enable real-time tracking of cellulase activity of microorganisms on the surface. Surface was formed from pure CMC, rather than CMC incorporated in agar as is often done, and by that, eliminating diffusion issues. Acridine orange dye, which is compatible, at low concentrations, with microorganisms, as opposed to other routinely used dyes, was incorporated into the film. The dye disassociated from the film when hydrolysis occurred, forming a halo surrounding the point of hydrolysis. This enabled real-time visualization since the common need for post hydrolysis dyeing was negated. Using Root Knot Nematode (RKN) as a model organism that penetrates the plant root, we showed it was possible to follow microorganism cellulase secretion on the surface in the form of CMC film hydrolysis. Furthermore, the addition of natural additives, in the form of root extract was also shown to be an option and resulted in an increased RKN response. We tested our newly developed method by changing temperature and pH conditions and by characterization of the hydrolyzed surface using both Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). This method will be implemented in the future on a root surface microstructure replica. We believe the combination of this new method with our previously developed root surface microstructure replication technique can open a new avenue of research in the field of plant root-microorganism interactions.

The current study was carried out to compare the antileishmanial potential of the characterized chemically and plant mediated (Trigonella foenum-graecum) FeO-NPs (FeO-NPs) against L.tropica KWH23. The promastigotes and amastigotes mortality, ROS generation, and biocompatibility assessment were estimated in time-dose dependent manner by PDT. LED exposed bio-inspired FeO-NPs after 72 hours, express significant suppressive effects by exhibiting 50% inhibitory concentration (IC50) 0.001572{+/-}0.02g/ml and 0.011408{+/-}0.02g/ml for promastigotes and amastigotes, respectively. Leishmanial DNA damage and membrane integrity caused by FeO-NPs were owe to high production of reactive oxygen species (ROS). By applying different ROS scavengers (mannitol and sodium azide) hydroxyl radical and singlet oxygen were found as main moieties for cell death by giving quantum yield 0.15 and 0.28 through chemically and green synthesized FeO-NPs, respectively. The light exposed green synthesized nanoparticles were found biocompatible on human red blood cells (RBCs), by exhibiting LD50 value 2659g/mL. From these findings, it can be concluded that plant mediated FeO-NPs can be used as promising antiprotozoal agent.

The bottom-up constructed artificial cells help to understand the cell working mechanism and provide the evolution clues for organisms. Cyanobacteria are believed to be the ancestors of chloroplasts according to endosymbiosis theory. Herein we demonstrate an artificial cell containing cyanobacteria to mimic endosymbiosis phenomenon. The cyanobacteria sustainably produce glucose molecules by converting light energy into chemical energy. Two downstream "metabolic" pathways starting from glucose molecules are investigated. One involves enzyme cascade reaction to produce H2O2 (assisted by glucose oxidase) first, followed by converting Amplex red to resorufin (assisted by horseradish peroxidase). The more biological one involves nicotinamide adenine dinucleotide (NADH) production in the presence of NAD+ and glucose dehydrogenase. Further, NADH molecules are oxidized into NAD+ by pyruvate catalyzed by lactate dehydrogenase, meanwhile, lactate is obtained. Therefore, the sustainable cascade cycling of NADH/NAD+ is built. The artificial cells built here simulate the endosymbiosis phenomenon, meanwhile pave the way for investigating more complicated sustainable energy supplied metabolism inside artificial cells.

Guanine based particle is one of very efficient material to control light especially near the surface of animal body. Past studies reported that platelets of anhydrous guanin crystal are utilized by living creatures, and they can change their colour and light reflection intensity when the arrangement of platelets. Guanine has relatively high reflective index and its particle can exhibit a unique optical property. Under higher pH which can be provided by a high concentration of NaOH aqueous solution, guanine molecules formed a sodium salt particle which contain guanine. Here this work presents that particles containing guanine in basic aqueous solution with NaOH exhibited very strong shining and light reflection switching by magnetic fields. In addition, adding an extracted solution of a fish iridophore enhanced the formation of intensively light reflecting guanine particles floating in a strong basic solution.

Biological synthesis reflects as an eco-friendly, nontoxic and easy method of nanoparticle preparation. Present investigation deals with biosynthesis of silver nanoparticles using Drimia indica leaf extract. Initially the synthesized silver nanoparticles were characterized and confirmed by UV-Vis spectroscopy, X-ray diffraction (XRD) and SEM. The synthesized nanoparticles when used for determination of antibacterial activity, by Microtitre Broth Dilution method exhibited remarkable activity against Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli.

In this study, we describe the cytotoxic and antibacterial capabilities of gold nanoparticles (GNPs) synthesized from Dillenia indica leaves. Here, we used an environmentally friendly method to synthesize GNPs and then characterized them using techniques such as transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-visible spectroscopy, XRD, and Fourier transform infrared spectroscopy. The Surface Plasmon Resonance of GNPs was identified by an absorption peak at 533 nm in the UV-visible spectroscopic studies. The biosynthesized GNPs had an average size of 83 nm and were spherical in shape, according to TEM examination. Its interesting to note that the biosynthesized GNPs exhibited antibacterial action against a variety of bacterial isolates, both Gram-positive and negative. By using an MTT assay, growth inhibition and the cytotoxic effect of the synthesized GNPs against Daltons lymphoma cell lines were also evaluated.

To study the effect of electrospinning nanofiber mats synthesized using hydroxypropyl chitosan in conjugation with poly (vinyl alcohol) on the osteogenesis in MC3T3 cell, we fabricated different ratios of hydroxypropyl chitosan (HPCH) and poly (vinyl alcohol) to electrospin the nanofiber mats, the mechanical and physical properties of nanofiber mats were tested by using SEM, FT-IR and conventional methods. Then the effect of the electrospinning mats on osteogenesis in MC3T3 cell were evaluated by Alizarin Red stain, ALP stain and expression of relevant genes. We found that the nanofiber mats fabricated by hydroxypropyl chitosan (50%) and poly (vinyl alcohol)(50%) can induce osteoblast differentiation apparently in MC3T3 cell.

In this paper, we report for the first time, the synthesis of a semiconducting biofilm. Photosynthetic bacterial biofilm has been used to weave together MoS2 nanosheets into an adherent film grown on interdigitated electrodes. Liquid-phase exfoliation of bulk MoS2 powder was used to obtain MoS2 nanosheets. A synchronous-fluorescence scan revealed the presence of two emission maxima at 682nm and 715nm for the MoS2 suspension. Such maxima with bandgap energy 1.82 and 1.73 eV corresponded to the single and double layer of MoS2. The presence of such single and multi-layered structures was confirmed by Raman spectroscopy, FTIR studies, and electron microscopy. The current-voltage (I-V) studies of such a bio-nano hybrid revealed the emergence of the gated nature of the current flow. This Schottky diode like behavior, reported earlier for Graphene-biofilm junctions, is also observed in this case. Gating voltage depended on the composition of the biofilm. The semiconductor biofilms, when studied using electrochemical impedance spectroscopy, revealed characteristic Nyquist and Bode plots, suggesting special circuit-equivalence for each film. While Mos2 was marked with stability with respect to variations in RMS voltage and bias voltage, the graphene biofilm was unique by the absence of any Warburg element.

Our work presented here showed that MelB can be crystallized in the conditions as similar as that of other membrane transporter protein of known structure. To identify a rigid protein by modifying the protein structure is the critical factor for facilitating MelB crystallization. It is necessary to perform extensive crystallization screens to obtain crystals. MelB-MelB interaction in the DDM containing solution will be affect by protein preparation, which may lead to reduce in reproducibility of crystallization experiment. Using a detergent mixture is essential for improve protein contact in the crystals, then improve crystallizability. R149C MelB crystal can be obtained in DDM, but these crystals were only diffracted to about 8Å resolution limit. MelB wide type crystal also can be obtained from the condition as that of R149C mutant, but the resolution is weaker than that of mutant. Although MelB and other transporters of known structure share common feature of the crystallization, the emphasis was as much on the protein itself, as it was on detergent type or efficient screening and refinement of the crystallization conditions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Hundreds of hydrophobic substances: alkanes, alcohols, benzodiacepines, barbiturates, ethers and even gases, can induce General Anaesthesia (GA) in mammal animals. Moreover, it has been suggested that the primary site of action of such agents is on the spinal cord. Here, we investigate a scenario that is in double measure important to understand the mechanisms behind GA: its induction under water in invertebrate animals. We evaluate the capacity of xenon, nitrous oxide and krypton to suppress harmful sensations, provoked by intense light, in the crustacean D. magna. Due to the hydrophobic characteristics of those gases, we designed a special chamber to force them to dissolve in water at pressures up to to 50 atmospheres, whereas at the same time measure in real time the motility of the animals. Surprisingly, the aquatic animals are immobilized with xenon and nitrous oxide. Under this condition, they dont respond to a noxious stimulus. Our results are crucial to understand the action of inert gases in GA and the role of the spinal cord.

It is well known that some hydrophobic atomic and molecular gases provoke anaesthetic effects in mammal animals. Depending on the gas, there is a Minimum Alveolar Concentration (MAC) to produce anaesthesia. The gas enters in the lungs, dissolve in the blood and reaches the brain. Where are the targets and which are the action mechanisms are subjects not fully understood yet. Very recently, we reported the effects of local anaesthetics on the swimming behaviour of the water flea Daphnia magna (STOTEN 691, 278-283, 2019). Our aim now is to report new studies on the behaviour of this aquatic invertebrate in the presence of three hydrophobic gases: xenon, nitrous oxide and krypton. However, if local anaesthetics easily dissolve in water, these gases do not. Therefore, we designed a chamber to dissolve the gases using pressures up to 50 atmospheres. Simultaneously, we were able to measure in real time the response of the animals through transparent windows able to support such high pressures. Xenon and nitrous oxide effectively induce lack of movement in the daphnids. The effective pressures EP50 for xenon and nitrous oxide were and 5.2 atmospheres, respectively. Krypton does not present clear effects on the motile suppression, even after the exposure to 44 atmospheres. Our findings provide insight on the physiological effects important gases used in human medicine produce in aquatic invertebrate animals considered as potential models to study anesthesia.

Green synthesis of nanoparticles (NPs) involves the use of diverse extracts of biological origin as substrates to synthesize nanoparticles and can overcome the hazards associated with chemical methods. Coconut inflorescence sap, which is unfermented phloem sap obtained by tapping of coconut inflorescence, is a rich source of sugars and secondary metabolites. In this study, coconut inflorescence sap was used to synthesize silver nanoparticles (AgNPs). We have initially undertaken metabolomic profiling of coconut inflorescence sap from West Coast Tall cultivar to delineate its individual components. Secondary metabolites constituted the major portion of the inflorescence sap along with sugars, lipids and, peptides. The concentration of silver nitrate, inflorescence sap and incubation temperature for synthesis of AgNPs were optimized. Incubating the reaction mixture at 40{degrees}C was found to enhance AgNP synthesis. The AgNPs synthesized were characterized using UV-Visible spectrophotometry, X-Ray Diffraction (XRD), Fourier Transform Infrared spectroscopy (FTIR), Field Emission Scanning Electron Microscopy (FESEM) and Transmission Electron Microscopy (TEM). Antimicrobial property of AgNP was tested in tissue culture of arecanut (Areca catechu L.) where bacterial contamination (Bacillus pumilus) was a frequent occurrence. Significant reduction in the contamination was observed when plantlets were treated with aqueous solutions of 0.01, 0.02 and 0.03% of AgNPs for one hour. Notably, treatment with AgNPs did not affect growth and development of the arecanut plantlets. Cytotoxicity of AgNPs was quantified in HeLa cells. Viability (%) of HeLa cells declined significantly at 10 ppm concentration of AgNP and complete mortality was observed at 60 ppm. Antimicrobial properties of AgNPs synthesized from inflorescence sap were also evaluated and confirmed in human pathogenic bacteria viz., Salmonella sp., Vibrio parahaemolyticus, and Escherichia coli. The study concludes that unfermented inflorescence sap, with above neutral pH, serves as an excellent reducing agent to synthesize AgNPs from Ag+.\n\nO_FIG O_LINKSMALLFIG WIDTH=177 HEIGHT=200 SRC=\"FIGDIR/small/775940v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (64K):\norg.highwire.dtl.DTLVardef@3c3f1forg.highwire.dtl.DTLVardef@1540f6borg.highwire.dtl.DTLVardef@1c069aborg.highwire.dtl.DTLVardef@159772c_HPS_FORMAT_FIGEXP M_FIG Graphical abstract\n\nC_FIG

Green synthesis of silver nanoparticles has gained much interest over few decades in the field of biomedical research due to their ease of synthesis, cost effectiveness, enhanced bioactivity and biocompatibility compared to the chemical synthesis. Recent studies on silver nanoparticles have shown their potential in various fields like antimicrobial, anticancer, larvicidal, catalytic, and wound healing properties. In the present study, entomopathogenic fungi Metarhizium anisopliae was used to synthesize silver nanoparticles. These silver nanoparticles were synthesized and characterized using UV-Vis spectroscopy, FESEM, FETEM and FTIR. Compared to the chemically synthesized silver nanoparticles, the mycosynthesized silver nanoparticles (MaAgNP) showed high yield. The size of mycosynthesized silver nanoparticles was found to be 5-20 nm and was spherical in shape. FTIR results confirmed the possible functional groups that are responsible for the reduction of silver ions. The mycosynthesized silver nanoparticles showed cytotoxicity on human breast cancer cell line (MCF-7) and the calculated IC50 value for MaAgNP was found to be 16.50 g ml-1 whereas the chemically synthesized silver nanoparticles showed cytotoxicity but with increasing concentration, no further significant reduction in cell viability was observed. The possible reason behind improved cytotoxicity of MaAgNP can be the presence of extracellular secondary metabolites present in the fungal filtrate used to synthesize the nanoparticles. The MaAgNP was also observed to induce cell death through reactive oxygen species (ROS) generation.

Wedelia chinensis is a medicinal herb of Asteraceae family. Preliminary phytochemical screening of internodal extract was done by using its aqueous, methanolic and ethanolic extracts. Preliminary phytochemical screening showed the presence of several pharmaceutically important compounds such as alkaloids, tannins, anthraquinons, saponins, flavonoids, terpenoids, phenols, etc. Aqueous internodal extract was used for the biosynthesis of gold nanoparticles. The characterization of gold nanoparticles were done by using a range of analytical techniques, such as energy-dispersive X-ray spectroscopy (EDS, EDX), X-ray diffraction (XRD), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transformed infrared spectroscopy (FTIR), scanning electron microscopy (SEM). The stability and other characteristics were ascertained using a UV-Vis spectrophotometer. The plasmon resonance occurred at the wavelength of 530 nm, thus showing the formation of gold nanoparticles. X-ray diffraction demonstrated the existence of Au-rich (fcc) phases in gold nanoparticles. FTIR analysis clearly indicated the participation of active constituents of internodal extract in the conversion of gold ions into AuNPs. DLS showed the size distribution (99.71 nm) of the suspended particles. There was variations in the sizes and shapes of the nanoparticles. The zeta potential of synthesized AuNPs was observed as -5.12 mV, thus showing the stability of the synthesized nanoparticles. In TEM study, uniform distribution of nanoparticles was seen with some agglomerates and average size of nanoparticles was observed in between 30-40 nm. The antioxidant potential of internodal extract was slightly higher than the synthesized gold nanoparticles. The green-synthesized AuNPs along with antibiotics demonstrated strong synergistic effect against several bacterial strains such as Escherichia coli and Bacillus subtilis. Gold nanoparticles showed good hemolytic activity against rat erythrocytes (90%). Gold nanoparticles also showed catalytic activity that reduces methylene blue at low concentration (1 mg). Highlights[tpltrtarr] Internodal extracts showed the presence of several active constituents in preliminary phytochemical screening. [tpltrtarr]Internodal extract was used for the synthesis of gold nanoparticles in one step process. [tpltrtarr]Synergistic action of AuNPs and antibiotics were reported against many pathogenic bacterial strains. [tpltrtarr]AuNPs showed its potential as antioxidant. [tpltrtarr]Hemolytic effect of nanoparticles was reported on rat blood sample. [tpltrtarr]Nanoparticles showed dye reducing activity thus enhancing its application in the purification of water bodies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/660090v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@12a27e3org.highwire.dtl.DTLVardef@a0d1b6org.highwire.dtl.DTLVardef@28178aorg.highwire.dtl.DTLVardef@216935_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig.C_FLOATNO Graphical abstract of AuNPs C_FIG

Parkinsons disease (PD) is a neurodegenerative condition that degenerates dopaminergic neurons and is characterized by motor disabilities like rigidity, bradykinesia, postural instability, and resting tremors. Although the etiology of PD remains uncertain, familial and sporadic forms of PD are known to be predominately caused by the G2019S mutation present in the kinase domain of LRRK2. Therefore, it might be possible to treat Parkinsons by inhibiting the kinase action of the mutated LRRK2 protein. In order to find possible inhibitors, 3069 phytochemicals were examined for their ability to bind ROCO4 kinase, which has structural and functional similarities to the LRRK2 protein. Open-source bioinformatics tools were used to determine the binding affinities of phytochemicals to the native and mutant variants of the protein. Mongolicain-A exhibited high specificity and selectivity towards the G2019S mutation of the ROCO4 protein with -12.3 Kcal/mol binding affinity, whereas Bacoside-A displayed high affinity (11.4 Kcal/mol) for the target protein, but lacked specificity towards the mutant form of the protein. Based on molecular simulation studies., RMSD, RMSF, SASA, potential energy, and hydrogen bond analysis, it was suggested that Mongolicain-A may be an effective inhibitor of the G2019S mutation and a promising drug molecule to address PD.

In the current work, biosynthesis of silver nanoparticle (Ag NPs) and interaction study between alpha amylase and Ag NPs/nanocluster has been performed via wet-lab as well as in silico approach. We have synthesized Ag NPs using alpha amylase enzyme which reduces the silver nitrate precursor forming the stable Ag NPs. UV-Visible spectroscopy and fluorescence spectroscopies were performed for optical characterization of Ag NPs. UV-Vis spectra showed the wide absorption band centered around 475 nm due to surface plasmon resonance. We have also observed gradual decrease in fluorescence intensity with the increase in incubation time. Also, shift in {lambda}max of the emission spectra was recorded which clearly suggested the formation of nano-bio-conjugate. Circular dichroism spectra show the initial decrease in the ellipticity, when we added the silver nitrate, but after incubating for different time, there are no major changes in secondary structure of protein. In computational study we have modelled ground state configuration of (Ag)24 nanocluster using in silico approach. Further docking of the modelled optimized nanocluster with alpha amylase was performed and found that Ag-nanocluster showing non-covalent interaction with alpha amylase and forming stable docking complex.

The present study clearly explains the dependency of inhibitory activities in SrtA inhibitors is closely related to protein conformational changes of SrtA from Bacillus anthracis B. anthracisSortase A (SrtA) protein anchors proteins by recognizing a cell wall sorting signal containing the amino acid sequence LPXTG In order to analyze conformational changes and the role of SrtA enzyme, especially the loop motions which situated proximal to the active site molecular dynamic simulation was carried out for 100ns. Particular loop is examined for its various conformations from the MD trajectories and the open/close lid conformations are considered for the enzyme activity validations. Experimentally verified SrtA inhibitors activity was analyzed through 3D-QSAR and Molecular docking approaches. Results indicate that, biological activity of SrtA inhibitors is closely related to the closed lid conformation of SrtA from Bacillus anthracis. This work may lead to a better understanding of the mechanism of action and aid to design a novel and more potent SrtA inhibitors.