Longer Incubation Times Research Articles

Transient cell stiffening triggered by magnetic nanoparticle exposure

BackgroundThe interactions between nanoparticles and the biological environment have long been studied, with toxicological assays being the most common experimental route. In parallel, recent growing evidence has brought into light the important role that cell mechanics play in numerous cell biological processes. However, despite the prevalence of nanotechnology applications in biology, and in particular the increased use of magnetic nanoparticles for cell therapy and imaging, the impact of nanoparticles on the cells’ mechanical properties remains poorly understood.ResultsHere, we used a parallel plate rheometer to measure the impact of magnetic nanoparticles on the viscoelastic modulus G*(f) of individual cells. We show how the active uptake of nanoparticles translates into cell stiffening in a short time scale (< 30 min), at the single cell level. The cell stiffening effect is however less marked at the cell population level, when the cells are pre-labeled under a longer incubation time (2 h) with nanoparticles. 24 h later, the stiffening effect is no more present. Imaging of the nanoparticle uptake reveals almost immediate (within minutes) nanoparticle aggregation at the cell membrane, triggering early endocytosis, whereas nanoparticles are almost all confined in late or lysosomal endosomes after 2 h of uptake. Remarkably, this correlates well with the imaging of the actin cytoskeleton, with actin bundling being highly prevalent at early time points into the exposure to the nanoparticles, an effect that renormalizes after longer periods.ConclusionsOverall, this work evidences that magnetic nanoparticle internalization, coupled to cytoskeleton remodeling, contributes to a change in the cell mechanical properties within minutes of their initial contact, leading to an increase in cell rigidity. This effect appears to be transient, reduced after hours and disappearing 24 h after the internalization has taken place.

Cavitation Erosion Behaviour of Martensitic Stainless Steels SUS 420J2 and SUS440C by High Pressure Liquid Jet

The high-pressure cavitation erosion (CE) behavior of SUS420J2 and SUS440C were studied by a high pressure cavitating liquid jet experiment apparatus, according to ASTM Standard G134-95. The microstructures were investigated by X-ray diffraction and scanning electron microscopy to determine the phases and precipitates. The mass loss vs. exposure time relations indicated that SUS440C had longer incubation time and a decreasing erosion rate. The eroded topographical evolution was investigated, indicating that the initiation of cavitation erosion was accumulated from grain boundaries for SUS420J2, and from M3C precipitates or ferric oxide inclusions for SUS440C. The dominant factors that influenced CE behaviors changed in different cavitation stages. Micro-pitting and initial damage induced by grain boundaries or precipitates were the dominant erosion forms during the incubation stage; however, the matrix hardness started to prevail as the dominant factor after the precipitates had been torn off, or grain boundaries damage disappeared.

The photosensitiser azure A disrupts mitochondrial bioenergetics through intrinsic and photodynamic effects

Azure A (AA) is a cationic molecule of the class of phenothiazines that has been applied in vitro as a photosensitising agent in photodynamic antimicrobial chemotherapy. It is a di-demethylated analogue of methylene blue (MB), which has been demonstrated to be intrinsically and photodynamically highly active on mitochondrial bioenergetics. However, as far as we know, there are no studies about the photodynamic effects of AA on mammalian mitochondria. Therefore, this investigation aimed to characterise the intrinsic and photodynamic acute effects of AA (0.540 μM) on isolated rat liver mitochondria, isolated hepatocytes, and isolated perfused rat liver. The effects of AA were assessed by evaluating several parameters of mitochondrial bioenergetics, oxidative stress, cell viability, and hepatic energy metabolism. The photodynamic effects of AA were assessed under simulated hypoxic conditions, a suitable way for mimicking the microenvironment of hypoxic solid tumour cells. AA interacted with the mitochondria and, upon photostimulation (10 min of light exposure), produced toxic amounts of reactive oxygen species (ROS), which damaged the organelle, as demonstrated by the high levels of lipid peroxidation and protein carbonylation. The photostimulated AA also depleted the GSH pool, which could compromise the mitochondrial antioxidant defence. Bioenergetically, AA photoinactivated the complexes I, II, and IV of the mitochondrial respiratory chain and the F1FO–ATP synthase complex, sharply inhibiting the oxidative phosphorylation. Upon photostimulation (10 min of light exposure), AA reduced the efficiency of mitochondrial energy transduction and oxidatively damaged lipids in isolated hepatocytes but did not decrease the viability of cells. Despite the useful photobiological properties, AA presented noticeable dark toxicity on mitochondrial bioenergetics, functioning predominantly as an uncoupler of oxidative phosphorylation. This harmful effect of AA was evidenced in isolated hepatocytes, in which AA diminished the cellular ATP content. In this case, the cells exhibited signs of cell viability reduction in the presence of high AA concentrations, but only after a long time of incubation (at least 90 min). The impairments on mitochondrial bioenergetics were also clearly manifested in intact perfused rat liver, in which AA diminished the cellular ATP content and stimulated the oxygen uptake. Consequently, gluconeogenesis and ureogenesis were strongly inhibited, whereas glycogenolysis and glycolysis were stimulated. AA also promoted the release of cytosolic and mitochondrial enzymes into the perfusate concomitantly with inhibition of oxygen consumption. In general, the intrinsic and photodynamic effects of AA were similar to those of MB, but AA caused some distinct effects such as the photoinactivation of the complex IV of the mitochondrial respiratory chain and a diminution of the ATP levels in the liver. It is evident that AA has the potential to be used in mitochondria-targeted photodynamic therapy, even under low oxygen concentrations. However, the fact that AA directly disrupts mitochondrial bioenergetics and affects several hepatic pathways that are linked to ATP metabolism, along with its ability to perturb cellular membranes and its little potential to reduce cell viability, could result in significant adverse effects especially in long-term treatments.

Streptococcus agalactiae amylomaltase offers insight into the transglycosylation mechanism and the molecular basis of thermostability among amylomaltases

Amylomaltase (AM) catalyzes transglycosylation of starch to form linear or cyclic oligosaccharides with potential applications in biotechnology and industry. In the present work, a novel AM from the mesophilic bacterium Streptococcus agalactiae (SaAM), with 18–49% sequence identity to previously reported AMs, was characterized. Cyclization and disproportionation activities were observed with the optimum temperature of 30 °C and 40 °C, respectively. Structural determination of SaAM, the first crystal structure of small AMs from the mesophiles, revealed a glycosyl-enzyme intermediate derived from acarbose and a second acarbose molecule attacking the intermediate. This pre-transglycosylation conformation has never been before observed in AMs. Structural analysis suggests that thermostability in AMs might be mainly caused by an increase in salt bridges since SaAM has a lower number of salt bridges compared with AMs from the thermophiles. Increase in thermostability by mutation was performed. C446 was substituted with A/S/P. C446A showed higher activities and higher kcat/Km values for starch in comparison to the WT enzyme. C446S exhibited a 5 °C increase in optimum temperature and the threefold increase in half-life time at 45 °C, most likely resulting from H-bonding interactions. For all enzymes, the main large-ring cyclodextrin (LR-CD) products were CD24-CD26 with CD22 as the smallest. C446S produced more CD35-CD42, especially at a longer incubation time.

Effective role of minor silicon addition on crystallization kinetics of Cu50Zr43Al7 bulk metallic glass

The Si addition has a great impact on the glass forming ability of Cu50Zr43Al7 bulk metallic glass (BMG). To investigate the effective role of Si doping on the crystallization behavior of the BMGs, crystallization transformation kinetics of Cu50Zr43Al7 and (Cu50Zr43Al7)99Si1 BMGs were explored at non-isothermal and isothermal conditions by differential scanning calorimetry (DSC) experiments. The Si doping enhances the activation energy of crystallization transformation (resistance against crystallization) under both thermal circumstances. Avrami exponents were obtained by employing the Johnson–Mehl–Avrami–Kolmogorov (JMAK) model. In the isothermal regime, Avrami exponents in both BMGs were 2.2, which reveals that the dominant mechanism of crystallization is the three-dimensional growth with a decreasing nucleation rate of crystals. The time–temperature–transformation or TTT diagram displayed that minor alloying of Si displaced the crystallization transformation to elevated temperatures and longer incubation times.

Fully integrated rapid microfluidic device translated from conventional 96-well ELISA kit

In this work, a fully integrated active microfluidic device transforming a conventional 96-well kit into point-of-care testing (POCT) device was implemented to improve the performance of traditional enzyme-linked immunosorbent assay (ELISA). ELISA test by the conventional method often requires the collection of 96 samples for its operation as well as longer incubation time from hours to overnight, whereas our proposed device conducts ELISA immediately individualizing a 96-well for individual patients. To do that, a programmable and disposable on-chip pump and valve were integrated on the device for precise control and actuation of microfluidic reagents, which regulated a reaction time and reagent volume to support the optimized protocols of ELISA. Due to the on-chip pump and valve, ELISA could be executed with reduced consumption of reagents and shortening the assay time, which are crucial for conventional ELISA using 96-well microplate. To demonstrate highly sensitive detection and easy-to-use operation, this unconventional device was successfully applied for the quantification of cardiac troponin I (cTnI) of 4.88 pg/mL using a minimum sample volume of 30 µL with a shorter assay time of 15 min for each ELISA step. The limit of detection (LOD) thus obtained was significantly improved than the conventional 96-well platform.

Changes on structural characteristics of cellulose pulp fiber incubated for different times in anaerobic digestate

ABSTRACT Background: The objective of the present work was to investigate the influence of a pre-treatment of microbial-rich digestate (liquid mesophilic anaerobic digestate - AD-supernatant) on the morphology, crystallinity, and thermal stability of cellulose pulp fiber. Results: The six most abundant bacteria in the AD-supernatant were determined by 16S analysis. The bacteria population was comprised mostly of Bacteroides graminisolvens (66%) and Parabacteroides chartae (28%). Enzymatic activity from the bacteria partially removed the amorphous components and increased the crystallinity and crystallite size of the cellulose substrate. The fiber pulp was incubated in AD-supernatant for 5, 10, and 20 days. The X-ray diffraction data provided evidence that the amorphous portion of the cellulose was more readily and quickly hydrolyzed than the crystalline portion. The longest incubation times (20 days) resulted in substantial deconstruction of the cellulose fiber structure and decreased the thermal degradation temperature. Conclusion: The anaerobic digestate is inexpensive and could be used to effectively aid in the pre-treatment of cellulose on large scale transformation processes, e.g. for making biofuels, cellulose micro/nanofiber production or engineered fiber-based materials.

The effects of Benjakul extract and its isolated compounds on cell cycle arrest and apoptosis in human non-small cell lung cancer cell line NCI-H226.

Background and purpose:Benjakul, a traditional Thai formulation for cancer treatment, is composed of five plants. This study aimed to assess the cytotoxicity of Benjakul, its five plants, and its isolated compounds against non-small cell lung cancer (NSCLC) by the sulforhodamine B (SRB) assay.Experimental approach:Analyses of cell cycle and membrane asymmetry changes were performed with different fluorescent dyes and analyzed by flow cytometry in NCI-H226 cells. Activation of caspase-3 was measured using a caspase-3 colorimetric assay kit. The pan-caspase inhibitor Z-VAD-FMK was used in analyses of cell cycle and caspase-3 activity.Findings/Results:Benjakul exhibited cytotoxicity against NSCLC with IC50 between 5.56-5.64 μg/mL. Among its five ingredients, Benjakul displayed the highest selectivity with selectivity index values ranging from 2.93 to 6.88, with the exception of Plumbago indica, indicating its protective effects. Plumbagin and 6- shogaol displayed the highest cytotoxicity and underwent molecular studies in NCI-H226 cells. Flow cytometry analysis revealed that Benjakul and 6-shogaol dose-dependently induced G2/M phase arrest, and plumbagin dose-dependently induced S-G2/M phase arrest with the highest percentage in early incubation time (12-24 h). At the highest doses, Benjakul extract, 6-shogaol, and plumbagin time-dependently increased the population of sub-G1 apoptotic cells with the highest percentage in longer incubation time (60-72 h). Similarly, membrane asymmetry changes showed time-dependent increases in the percentage of early and late apoptotic cells. Moreover, the apoptosis-inducing effect of Benjakul, 6-shogaol, and plumbagin at the highest dose, via the caspase cascade was confirmed by time-dependent induction of caspase-3 activity, followed by its complete reduction and abolished sub-G1 peaks upon addition of Z-VAD-FMK.Conclusion and implication:Our findings demonstrated for the first time the effects of Benjakul and its compounds on S-G2/M or G2/M phase arrest and caspase-dependent apoptosis in lung cancer cells.

Comparison of Reasoner’s 2A Agar and Muller Hinton Agar Media for Microbiological Monitoring of Dialysis Water

Background and Aim: Microbiological culture of dialysis water is a routine safety measure. In, Khorramabad laboratories perform these cultures on Muller Hinton Agar (MHA) at 35–378C for 48 h, not on the Reasoner’s 2A agar (R2A agar) at 17–238oC for 7 days recommended by international standards, the objective of the present study was the comparison of the efficiency of R2A and MHA media in the counting of heterotrophic bacteria in the samples of water collected in dialysis centers from 2 hospitals in Khorramabad, from September to November 2019. Methods: A total of 165 samples of treated water in dialysis centers were collected aseptically and then transported in ice‑packs to the Department of Medical Microbiology of the Lorestan University of Medical Sciences and the pour plate technique was carried out for the enumerating of heterotrophic bacteria. Finally, bacterial colonies were counted after incubation at 34±2oC for 48 hours on MHA and 25oC for 1 week on R2A. Results: Results showed heterotrophic bacterial counts in R2A were greater than those in MHA in 89% of the samples, so enumeration of heterotrophic bacteria should be carried out in R2A agar associated with longer incubation times, because of the greater sensitivity. The proportion of water samples yielding colony counts ≥200 CFU/mL by R2A -7d was significantly different from the proportion by MHA-48h (p<0.001). Conclusion: The results proposed using R2A agar combined with relative low culture temperature (20-25°C), and an extended incubation time (7-10 days) is more efficient. However, as the spectrum of bacterial contamination is not similar for dialysis centers and countries, many studies using different media and culture parameters are required to confirm this. *Corresponding Author: Faranak Rezaei; Email: Rezaei.f@lums.ac.ir Please cite this article as: Pouladi I, Delfani S, Hadian B, Soroush S, Anbari K, Rezaei F. Comparison of Reasoner’s 2A Agar and Muller Hinton Agar Media for Microbiological Monitoring of Dialysis Water. Arch Med Lab Sci. 2020;6:1-5 (e10). https://doi.org/10.22037/amls.v6.32905

Proof of Concept of Culturomics Use of Time of Care.

Culturomics, a high throughput culture method with rapid identification of the colonies by Matrix Assisted Laser Desorption Ionization/Time Of Flight Mass Spectrometry (MALDI-TOF MS), has demonstrated its contribution to the exploration of the gut microbiota over the past 10 years. However, the cost, work time and workload, considerably limit its use on a large scale or emergency context. Here, by testing two different stool samples, including a stool sample from a patient requiring rapid immunotherapy treatment, we tested a new fast culturomic protocol using two pre-incubation media, blood culture bottle and YCFA modified medium. Both media were supplemented with 2 ml of rumen fluid filtered at 0.2 μm and 2 ml of defibrinated and sterile sheep blood. Unlike the standard culturomics, subculturing of blood culture bottle were performed at reduced incubation time (3 h, 6 h, 9 h, 24 h) and at a longer incubation time (3 days, 7 days, and 10 days) at 37°C. By testing 5,200 colonies per MALDI-TOF MS and obtaining a comparable number of cultured bacterial species (131 to 143) in a stool sample, this new protocol reduced the number of colonies tested by 57%, working time by 78.6% and cost by 72.2%. In addition, we highlighted that the proportion of strict anaerobic species has increased by 24%, known to be the preferential targets for biotherapy, including Faecalibacterium prausnitzii, Akkermansia muciniphila, Christensenella minuta, and Phascolarctobacterium faecium. Finally, this work showed that some bacterial species grew earlier but disappeared with prolonged incubation times.

Biochemical, microbiological, and sensory properties of probiotic yogurt made from Iranian native strains compared to commercial strains

This study was performed to evaluate the effects of using native and commercial probiotic strains on biochemical, microbiological, and sensory properties of yogurt. The viability of probiotic bacteria in all samples exceeded 6 log CFU/ml required to provide health benefits. The native Iranian Lactobacillus casei showed the highest viability throughout storage (8.5 log CFU/ml at the end of storage). Three distinct phases were observed in pH, acidity, and redox potential during the fermentation of all samples. Commercial L. casei strain had the lowest mean acidity increase rates and the longest incubation times. The growth of Lactobacillus delbrueckii ssp. Bulgaricus and Streptococcus thermophilus in probiotic yogurt was improved compared to the control yogurt. The control sample had the lowest score in all sensory parameters evaluated. Overall, yogurt containing native Iranian L. casei strain is recommended because of its good sensory and microbial properties. Practical applications Among different functional food products, probiotic dairy products are of great interest and yogurt is the most popular probiotic carrier. In most cases, commercial strains of Lactobacillus and bifidobacteria are incorporated into yogurt and native strains have not been studied widely. In this regard, isolation, characterization, and investigation of the effect of their application in dairy products can be promising in the development of functional dairy products with acceptable qualitative characteristics.

Effect of Interaction between 17β-Estradiol, 2-Methoxyestradiol and 16α-Hydroxyestrone with Chromium (VI) on Ovary Cancer Line SKOV-3: Preliminary Study.

Ovarian cancer is the leading cause of death from gynecologic malignancies. Some estrogens, as well as xenoestrogens, such as chromium (VI) (Cr(VI)), are indicated as important pathogenic agents. The objective of this study was to evaluate the role of estradiol and some its metabolites upon exposure to the metalloestrogen Cr(VI) in an in vitro model. The changes in cell viability of malignant ovarian cancer cells (SKOV-3 resistant to cisplatin) exposed to 17β-estradiol (E2) and its two metabolites, 2-methoxyestradiol (2-MeOE2) and 16α-hydroxyestrone (16α-OHE1), upon exposure to potassium chromate (VI) and its interactions were examined. The single and mixed models of action, during short and long times of incubation with estrogens, were applied. The different effects (synergism and antagonism) of estrogens on cell viability in the presence of Cr(VI) was observed. E2 and 16α-OHE1 caused a synergistic effect after exposure to Cr(VI). 2-MeOE2 showed an antagonistic effect on Cr(VI). The examined estrogens could be ranked according to the most protective effect or least toxicity in the order: 2-MeOE2 > E2 > 16α-OHE1. Early pre-incubation (24 h or 7 days) of cells with estrogens caused mostly an antagonistic effect—protective against the toxic action of Cr(VI). The beneficial action of estrogens on the toxic effect of Cr(VI), in the context of the risk of ovarian cancer, seems to be important and further studies are needed.

Biological Resistance of Elm (Ulmus carpinifolia var. Umbelifera) Trees against Fungal Endophytes and White Rot Decay Fungi

Urban trees are one of the valuable storage in metropolitan areas. Nowadays, a particular attention is paid to the trees and spends million dollars per year to their maintenance. Trees are often subjected to abiotic factors, such as fungi, bacteria, and insects, which lead to decline mechanical strength and wood properties. The objective of this study was to determine the potential degradation of Elm tree wood by Phellinus pomaceus fungi, and Biscogniauxia mediteranae endophyte. Biological decay tests were done according to EN 113 standard and impact bending test in accordance with ASTM-D256-04 standard. The results indicated that with longer incubation time, weight loss increased for both sapwood and heartwood. Fungal deterioration leads to changes in the impact bending. In order to manage street trees, knowing tree characteristics is very important and should be regularly monitored and evaluated in order to identify defects in the trees.

Effects of radish powder concentration and incubation time on the physicochemical characteristics of alternatively cured pork products.

Previous research has indicated that radish powder could be a suitable replacement for chemical nitrite sources in alternatively cured meat products. However, the effects of radish powder level on the physicochemical properties of cured meat have not been systematically studied. In this study, we aimed to investigate the effects of varying concentrations of radish powder and incubation time on the physicochemical properties and cured meat pigments of alternatively cured meat products. We divided our experimental setup into seven groups with different radish powder concentrations and incubation times: control (0.01% sodium nitrite), treatment 1 (0.15% radish powder and 2 h incubation), treatment 2 (0.15% radish powder and 4 h incubation), treatment 3 (0.30% radish powder and 2 h incubation), treatment 4 (0.30% radish powder and 4 h incubation), treatment 5 (0.30% celery powder and 2 h incubation), and treatment 6 (0.30% celery powder and 4 h incubation). The cooking yield, CIE a* values (redness), and total pigment levels were not significantly different (p > 0.05) between any of the alternatively cured treatments and the control. However, when 0.30% radish powder or celery powder was added to the products, the CIE b* values increased significantly (p < 0.05) with incubation time. At the same vegetable concentration, the nitrite content, nitrosyl hemochrome, and curing efficiency also increased significantly (p < 0.05) as the incubation time increased from 2 to 4 h, regardless of the types of vegetable powder. Among the meat products cured with radish powder, treatment 4 showed the highest increase in residual nitrite content, nitrosyl hemochrome content, and curing efficiency, but showed decreased lipid oxidation. Our results suggest that increased concentrations of radish powder and longer incubation times would be more suitable for producing alternatively cured meat products comparable to traditionally cured products treated with synthetic nitrite.

Dry heat treatment of skim milk powder greatly improves the heat stability of recombined evaporated milk emulsions

In this study, skim milk powder (SMP) was subjected to a controlled dry heat treatment at 60 °C and relative humidity of 74% to improve the heat stability of recombined evaporated milk (REM) emulsions. Free amino group determination and SDS-PAGE analysis confirmed that the glycation of both whey proteins (WP) and caseins with lactose mainly occurred in the early period (<8 h). An increase in surface hydrophobicity and changes in the structure of the SMP proteins by circular dichroism (CD) and FT-IR were found after a long time of incubation (>16 h). The non-sedimentable protein analysis indicated that WP-casein interaction and casein micelle dissociation happened during (prolonged) dry heating. The heat stability of REM emulsions stabilized by dry-heated SMP was greatly improved: dry-heated SMP-stabilized REM emulsions could withstand at least 30 min of heating at 120 °C without significant increase in particle size and viscosity (P > 0.05). However, excessive dry heating (>20 h) adversely affected the reconstitution ability of SMP as well as the heat stability of REM emulsions.

Influence of the forming method on flash sintering of ZnO ceramics

This work aimed to investigate different forming techniques used in the ceramic industry when applied to flash sintering. The way the density gradient interferes with the final densification and microstructural heterogeneity development of ZnO specimens was studied. For this purpose, ZnO cylindrical specimens were formed by uniaxial pressing, isostatic pressing, and slip casting. All experiments were conducted isothermally at 800 °C in an adapted tube furnace, with an applied electric field of 60 V/cm and maximum current density of 200 mA/mm2. Samples formed by isostatic pressing showed better control in the development of microstructural homogeneity. The ones formed by slip casting had lower apparent densities and longer incubation times among all the forming methods. Also, slip casting samples showed the largest detachment of average grain size in the central region compared to the other regions. One outstanding result is related to differences between the regions close to the upper and lower electrodes in the samples formed by uniaxial pressing, even when using alternating current mode. These results showed that the density distribution along the green specimen should be considered a decisive factor when evaluating flash sintered samples. The chosen forming technique causes significant differences in incubation time, densification, and the development of microstructural heterogeneity.

Polymersomes as Nanoreactors Enabling the Application of Solvent-Sensitive Enzymes in Different Biphasic Reaction Setups.

There is an increasing interest in biocatalysis to perform chemical reactions in biphasic systems, consisting of an aqueous phase and a water-immiscible organic solvent or ionic liquid. In most cases, the hydrophobic phase is used as reservoir for poorly water-soluble substrates or for in situ product removal. However, many enzymes are solvent-sensitive and cannot be used in such systems. In this study, the solvent-sensitive enzyme mandelate racemase is exemplarily protected from the organic phase by its entrapment in (crosslinked) polymersomes. The covalent crosslinking of the individual chains of the block copolymer poly(2-methyloxazoline)15 -poly(dimethylsiloxane)68 -poly(2-methyloxazoline)15 via terminal methacrylates leads to enhanced membrane stability. This effect is especially pronounced for long-time incubation in the presence of organic solvents and ionic liquids. By using a gentle polymerization initiator at its minimal necessary concentration, the prior encapsulated enzymes remain intact during crosslinking. Although the insertion of natural channel proteins into the membrane improves the mass transport into the vesicles, it is non-essential. Mandelate racemase in (crosslinked) polymersomes remains active in different highly dispersed biphasic systems for more than 24h. The free enzyme, on the other hand, gets completely inactivated within 1h, thus illustrating the potential of polymersomes as nanoreactors in biphasic reaction setups.

Fabrication of Biopassive Surfaces Using Poly(2‐alkyl‐2‐oxazoline)s: Recent Progresses and Applications

AbstractPoly(2‐alkyl‐2‐oxazoline)s (PAOXAs) are emerging among the most promising nonionic alternatives to poly(ethylene glycol)s (PEGs), specifically in the modification and functionalization of biomaterials. Due to their chemical tailorability and robustness, coupled to their relatively easy synthesis, PAOXAs are increasingly applied as adsorbates to generate bioinert surfaces that prevent nonspecific contamination by proteins, cells and bacteria. Passivation of medical devices, sensors and cell‐sensitive platforms with PAOXAs enables a nearly quantitative suppression of nonspecific biological contamination, while biopassivity is maintained over longer incubation times than those recorded for more degradable PEG‐based coatings. Thanks to these unique advantages, surface modification strategies involving PAOXAs are expanding to nanomaterials, especially those applied in the fabrication of nanomedicine and bioimaging tools.

Supramolecular clusters clarification in ethanol-water mixture by using fluorescence spectroscopy and 2D correlation analysis

Due to the importance of ethanol-water solutions in many fields the different supramolecular clusters involved in this binary system is studied in detail by using fluorescence spectroscopy, at different concentration and incubation time after mixing. Two-dimensional (2D) correlation analysis is performed on the concentration-dependent mixtures (10–100%) at each incubation time, which find that the correlation intensity of emission peak at 330 nm for (H2O)m(EtOH)n not only related to the concentration but also gradually increase with the incubation time. Such results indicate that the cluster of (H2O)m(EtOH)n is the most stable one because it tends to be formed with longer incubation time. In addition, the correlation results indicate that at low concentration range of 10–45%, the dominated cluster is (H2O)m(EtOH) corresponding to a peak at 373 nm; while at high concentration range of 80–100%, the main cluster is (H2O)(EtOH)n corresponding to an emission peak at 308 nm; however, the most stable cluster of (H2O)m(EtOH)n, corresponding to the peak at 330 nm, not only predominate at the concentration range of 50–75% but also exist in all mixture at longer incubation time. Therefore, the present study provides a new approach to clarify the different supramolecular clusters in ethanol-water mixtures, which would be applied to other binary solution system.

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks.

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16-24 h), and higher incubation temperature (37 °C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10 min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.