Phosphate-buffered Saline Buffer Research Articles

Swelling kinetics of mixtures of soybean phosphatidylcholine and glycerol dioleate

Lipid-based drug delivery systems offer the potential to enhance bioavailability, reduce dosing frequency, and improve patient adherence. In aqueous environment, initially dry lipid depots take up water and form liquid crystalline phases. Variation of lipid composition, depot size and hydration-induced phase transitions will plausibly affect the diffusion in and out of the depot. Lipid depots of soybean phosphatidylcholine (SPC) and glycerol dioleate (GDO) mixtures were hydrated for varying time durations in a phosphate-buffered saline (PBS) buffer and then analyzed with Karl Fischer titration, magnetic resonance imaging (MRI) and gravimetrically. Mathematical modeling of the swelling process using diffusion equations, was used to estimate the parameters of diffusion. Both composition of lipid mixture and depot size affect swelling kinetics… The diffusion parameters obtained in Karl Fischer titration and MRI (with temporal and spatial resolution respectively) are in good agreement. Remarkably, the MRI results show a gradient of water content within the depot even after the end of diffusion process. Apparently contradicting the first Fick’s law in its classical form, these results find an explanation using the generalized Fick’s law that considers the gradient of chemical potential rather than concentration as the driving force of diffusion.

Exploring viral particle, soil, and extraction buffer physicochemical characteristics and their impacts on extractable viral communities

Viruses are expected to be pivotal members of soil ecosystems, and recent advances in viral size fraction metagenomic (viromic) approaches have improved our ability to interrogate soil viral ecology. However, viromics relies on extraction buffers to effectively desorb viral particles from the soil matrix for downstream analysis, and viral extraction efficiency could be affected by the interplay between viral particles, soils, and extraction buffer chemistry. Here, we investigated whether extraction buffer chemistry affected extractable viral community composition measured by viromics from different soil types, for both biological (samples collected 1 m apart) and technical (subsamples from the same soil homogenate) replicates. We investigated pH manipulation of protein-supplemented phosphate-buffered saline buffer (PPBS, pHs 4.5, 5.5, 6.5, and 7.5) on forest, grassland and wetland soils that exhibited different soil edaphic properties, and we tested different buffer chemistries (PPBS, Carbonated Buffer, Glycine, and Saline Magnesium) on just the wetland soil. Spatial distance (i.e., biological replicate) was the primary driver of extractable viral community composition across all buffers and soils tested. Differences in viral community composition according to extraction buffer properties were only observed in the grassland technical replicates at PPBS buffer pH 4.5, and in both the wetland technical and biological replicates treated with different buffer chemistries, but the effects of buffer chemistry were secondary to spatial distance in the biological replicates. The lack of buffering capacity in the grassland soil technical replicates likely increased sorption of viral particles at pH 4.5, but neither protein composition nor isoelectric point explained this phenomenon. Given that most soil viral ecological studies to date include sample collection over distances much farther apart than the 1-m distances considered here, results suggest that extraction buffer chemistry is likely of much lower importance than ecological considerations, such as spatial distance, in the design of future soil viral ecological investigations.

A Study of the Drift Phenomena of Gate-Functionalized Biosensors and Dual-Gate-Functionalized Biosensors in Human Serum.

In this paper, we study the drift behavior of organic electrochemical transistor (OECT) biosensors in a phosphate-buffered saline (PBS) buffer solution and human serum. Theoretical and experimental methods are illustrated in this paper to understand the origin of the drift phenomenon and the mechanism of ion diffusion in the sensing layer. The drift phenomenon is explained using a first-order kinetic model of ion adsorption into the gate material and shows very good agreement with experimental data on drift in OECTs. We show that the temporal current drift can be largely mitigated using a dual-gate OECT architecture and that dual-gate-based biosensors can increase the accuracy and sensitivity of immuno-biosensors compared to a standard single-gate design. Specific binding can be detected at a relatively low limit of detection, even in human serum.

Investigating the influence of solvent type and pH on protein adsorption onto silica surface by evanescent-wave cavity ring-down spectroscopy.

Several studies have explored the adsorption of various proteins onto solid-liquid interfaces, revealing the crucial role of buffer solutions in biological processes. However, a comprehensive evaluation of the buffer's influence on protein absorption onto fused silica is still lacking. This study employs evanescent-wave cavity ring-down spectroscopy (EW-CRDS) to assess the influence of buffer solutions and pH on the adsorption kinetics of three globular proteins: hemoglobin (Hb), myoglobin (Mb), and cytochrome c (Cyt-C) onto fused silica. The EW-CRDS tool, with a ring-down time of and a minimum detectable absorbance of , enabled precise optical measurements at solid-liquid interfaces. The three heme proteins' adsorption behavior was investigated at pH 7 in three different solvents: deionized (DI) water, tris(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl), and phosphate buffered saline (PBS). For each protein, the surface coverage, the adsorption and desorption constants, and the surface equilibrium constant were optically measured by our EW-CRDS tool. Depending on the nature of each solvent, the proteins showed a completely different adsorption trend on the silica surface. The adsorption of Mb on the silica surface was depressed in the presence of both Tris-HCl and PBS buffers compared with unbuffered (DI water) solutions. In contrast, Cyt-C adsorption appears to be relatively unaffected by the choice of buffer, as it involves strong electrostatic interactions with the surface. Notably, Hb exhibits an opposite trend, with enhanced protein adsorption in the presence of Tris-HCl and PBS buffer. The pH investigations demonstrated that the electrostatic interactions between the proteins and the surface had a major influence on protein adsorption on the silica surface, with adsorption being greatest when the pH values were around the protein's isoelectric point. This study demonstrated the ability of the highly sensitive EW-CRDS tool to study the adsorption events of the evanescent-field-confined protein species in real-time at low surface coverages with fast resolution, making it a valuable tool for studying biomolecule kinetics at solid-liquid interfaces.

Measuring Bacterial Colonization on Arabidopsis thaliana Roots in Hydroponic Condition.

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized method for measuring bacterial colonization in the rhizosphere is necessary to improve reproducibility. We first cultured sterile A.thaliana in hydroponic conditions and then inoculated the bacterial cells in the rhizosphere at a final concentration of OD600 of 0.01. At 2 days post-inoculation, the root tissue was harvested and washed three times in sterile water to remove the uncolonized bacterial cells. The roots were then weighed, and the bacterial cells colonized on the root were collected by vortex. The cell suspension was diluted in a gradient with a phosphate-buffered saline (PBS) buffer, followed by plating onto a Luria-Bertani (LB) agar medium. The plates were incubated at 37 °C for 10 h, and then, the single colonies on LB plates were counted and normalized to indicate the bacterial cells colonized on roots. This method is used to detect bacterial colonization in the rhizosphere in mono-interaction conditions, with good reproducibility.

Medicinal potential of antimicrobial peptides from two plants against <i>Bacillus cereus</i> and <i>Staphylococcus aureus</i>

Bacillus cereus and Staphylococcus aureus are the most important bacteria that cause nosocomial infection and are resistant to antibiotics. Crude proteins from Cassia fistula and Ricinus communis were isolated to study their medicinal potential against Bacillus cereus, and Staphylococcus aureus. Extraction of the crude proteins from plants was done by phosphate buffer saline (PBS) and Tris NaCl buffer by using the roots and seeds of both plants. Antimicrobial activity was checked against bacterial strains by using agar disc diffusion and agar well diffusion methods. Zones of inhibitions were measured. On well diffusion method, PBS buffer protein extract of C. fistula roots showed a maximum zone of inhibition of 25 mm against B. cereus. Tris NaCl buffer extracts of C. fistula roots and seeds showed zones of inhibition of 12mm and 5mm respectively against S. aureus while Ricinus communis roots showed a zone of 12mm against B. cereus. Because the protein of the plants showed good antimicrobial activity, we can use these plants against various diseases caused by Bacillus cereus and Staphylococcus aureus.

Comparison of different DNA preservation solutions for oral cytological samples

ObjectiveThe objective of this study was to compare the DNA preservation capacity of buccal mucosa exfoliated cells when stored in different solutions under varying time and temperature conditions. DesignDNA preservation solutions, including Dimethyl sulphoxide disodium-EDTA-saturated NaCl (DESS), Tris-EDTA-NaCl-Tween20 buffer (TENT), Nucleic Acid Preservation Buffer (NAP), and phosphate-buffered saline (PBS), were prepared. Buccal mucosa cells from a single patient were collected, dispensed into these solutions, and stored at room temperature (RT) and 4 °C for 24 h, 72 h, 30 days, 90 days, and 180 days. DNA was extracted using the salting-out method and the QIAamp DNA Mini Kit. DNA concentration and purity were determined using the QuBit device and NanoDrop, while DNA integrity was assessed using the Agilent 4200 TapeStation system. The ability to amplify the IFNA primer was also evaluated by PCR. ResultsThe salting-out method yielded better concentration and purity results, with PBS, TENT, and DESS buffers demonstrating superior concentration values when stored at 4 °C, resulting in mean values exceeding 10 ng/μL for up to 30 days. DESS consistently exhibited the best integrity values over time for both temperature conditions. Amplification capacity was enhanced when samples were stored at 4 °C. When stored at RT, PBS achieved 100% amplification within 24 h. NAP yielded the poorest results. ConclusionIn the context of long-term preservation, the DESS buffer emerges as the most effective solution, maintaining requisite DNA quality and quantity standards for up to 30 days at RT and up to 3 months at 4 °C.

Cyclic and differential pulse voltammetric measurements on fibrils formation of alpha synuclein in Parkinson’s disease by a gold interdigitated tetraelectrodes

Parkinson's disease is a neurodegenerative disorder characterized by the aggregation and deposition of alpha-synuclein protein, which are pathological hallmarks. To understand the fibril formation of alpha-synuclein in Parkinson's disease, this study uses cyclic and differential pulse voltammetric measurements. These measurements analyze the electrochemical properties and behavior of alpha-synuclein during its fibril formation process. By applying a potential sweep or pulse to the alpha-synuclein sample, it is possible to gain insights into its redox activity and structural changes during fibril formation. This could lead to the development of therapeutic strategies to prevent or disrupt this pathological event in Parkinson's disease. To detect Parkinson's disease, a 15 nm sized gold conjugated antibody was used as the probe and seeded on gold interdigitated tetraelectrodes (AuIDTE). Alpha synuclein variations (fibriled and non-fibriled) were detected using phosphate-buffer saline and glycine buffer based on cyclic voltammetry and differential pulse voltammetry techniques. Discriminated by Tau protein measurement that was employed as a control. The linear regression for detecting alpha synuclein aggregation using differential pulse voltammetry was R2 = 0.9987 [y = 9E-06x - 4E-07], with a limit of detection of 10 aM, on a linear range of 1 aM-1 pM. Cyclic voltammetry determined the limit of detection of aggregated alpha synuclein to be 100 aM, with a linear relationship of R2 = 0.9939 [y = 7E-06x - 2E-06]. The sensor has excellent analytical performance in terms of detection limit, sensitivity, selectivity, repeatability, and stability.

Effect of vascular endothelial growth factor 165 on dopamine level in the retinas of guinea pigs with form-deprivation myopia.

Myopia is the most common refractive error because excessive increase in the axial length of a myopic eye leads to the thinning of the posterior scleral pole and can cause serious complications resulting in blindness. Thus, myopia has become a great concern worldwide. Dopamine (DA) plays a role in the development of myopia. Moreover, in Parkinson's disease, it has been proved that vascular endothelial growth factor 165 (VEGF165) can promote the survival and recovery of DA neurons, resulting in increased DA secretion in the striatum, thereby treating neuropathy. Therefore, we speculate that VEGF165 can also promote the release of DA in the retina to inhibit the occurrence and development of myopia. We aimed to investigate the effect of VEGF165 on DA levels in the retinas of guinea pigs with form-deprivation myopia (FDM) and the effects of DA on myopia prevention and control. Healthy 3-week-old pigmented guinea pigs were randomly divided into blank, FDM, phosphate buffer saline (PBS), 1, 5, and 10 ng groups. The FDM model was established by covering the right eye continuously with a translucent latex balloon pullover for 14 days. The pigs in the PBS, 1, 5, and 10 ng groups were injected with PBS buffer and 1, 5, and 10 ng of VEGF165 recombinant human protein, respectively, in the vitreous of the right eye before masking. The refractive error and axial length were measured before and after modeling. All retinas were used for biomolecular analyses after 14 days. We found that the intravitreal injection of VEGF165 elevated DA levels in the retina and was effective in slowing the progression of myopia, and 1 ng of VEGF165 was the most effective. Moreover, the number of vascular endothelial cell nuclei in the 1 ng group was lower than that in the other VEGF165 groups. Our data suggest that VEGF165 has a promoting effect on DA in the retinas of guinea pigs with FDM, potentially controlling the development of myopia.

Optimizing Exosome Preparation Based on Size and Morphology: Insights From Electron Microscopy.

Extracellular vesicles (EVs), including exosomes, are crucial in intercellular communication, but differentiating between exosomes and microvesicles is challenging due to their similar morphology and size. This study focuses on multivesicular bodies (MVBs), where exosomes mature, and optimizes exosome isolation using transmission electron microscopy (TEM) for size information. Considering that EVs are nanocolloidal particles, a salt-free Bis-Tris buffer is found to maintain EV integrity better than phosphate-buffered saline (PBS). Dynamic light scattering (DLS) and TEM analysis confirm that intact exosome fractions under the salt-free Bis-Tris buffer condition exhibit polydispersity, including a unique population of <50 nm vesicles resembling intraluminal membrane vesicles (ILVs) in MVBs, alongside larger populations. This <50 nm population disappears in PBS or Bis-Tris buffer with 140 mM NaCl, transforming into a monodisperse population >100 nm. Immunoelectron microscopy also validates the presence of CD63, an exosome biomarker, on approximately 50 nm EVs. These findings provide valuable insights into exosome characterization and isolation, essential for future biomedical applications in diagnostics and drug delivery.

Bacterial Leaf Spot of Plum Caused by Sphingomonas spermidinifaciens in Guangxi, China.

Plum (Prunus salicina L.) is a traditional fruit in Southern China and is ubiquitous throughout the world. In August 2021, leaves of plum trees showed water-soaking spots and light yellow-green halos with incidence exceeding 50% in Babu district in Hezhou, Guangxi (N23°49'-24°48', E111°12'-112°03'). To isolate the causal agent, three diseased leaves collected from three different trees growing in different orchards were cut into 5 mm × 5 mm pieces, disinfected with 75% ethanol for 10 sec, 2% sodium hypochlorite for 1 min and rinsed three times in sterile water. The diseased pieces were ground in sterile water and then kept static for about 10 min. Ten-fold serial dilutions in water were prepared and 100 µL of each dilution from 10-1 to 10-6 were plated on Luria-Bertani (LB) Agar. After incubation at 28℃ for 48 h, the proportion of isolates with similar morphology was 73%. Three representative isolates (GY11-1, GY12-1 and GY15-1) were selected for further study. The colonies were non-spore-forming, yellow, round, opaque, rod shaped, convex with smooth and bright neat edges. Biochemical test results showed that the colonies were strictly aerobic and gram-negative. The isolates were able to grow on LB agar containing 0-2% (w/v) NaCl and could utilize glucose, lactose, galactose, mannose, sucrose, maltose and rhamnose as a carbon source. They displayed a positive reaction for H2S production, oxidase, catalase and gelatin, but negative for starch. Genomic DNA of the three isolates was extracted for amplification of the 16S rDNA with primers 27F and 1492R. The resulting amplicons were sequenced. Additionally, five housekeeping genes atpD, dnaK, gap, recA, and rpoB of the three isolates were amplified using the corresponding primer pairs and sequenced. The sequences were deposited in GenBank (16S rDNA, OP861004-OP861006; atpD, OQ703328-OQ703330; dnaK, OQ703331-OQ703333; gap, OQ703334-OQ703336; recA, OQ703337-OQ703339; and rpoB, OQ703340-OQ703342). The isolates were identified as Sphingomonas spermidinifaciens based on the phylogenetic tree inferred by maximum-likelihood using MegaX 7.0 of the concatenated six sequences (multilocus sequence analysis, MLSA) compared with sequences from different Sphingomonas type strains . Pathogenicity of the isolates was tested on healthy leaves of the two-year-old plum plants in a greenhouse. The leaves were wounded by a sterilized needle and sprayed with bacterial suspensions prepared in PBS (Phosphate buffer saline) at OD600=0.5. PBS buffer solution was used as negative control. Each isolate was used to inoculate on 20 leaves per plum tree. The plants were covered with plastic bags to maintain high humidity. Dark brown-to-black lesions were observed on leaves 3 days post incubation at 28℃ with constant light. The average diameter of lesions was 1 cm after seven days, but the negative controls were symptomless. Bacteria reisolated from the diseased leaves were the same as the ones used for inoculation on the basis of morphological and molecular identification, fulfilling Koch's postulates. Plant disease caused by a Sphingomonas species has been reported on mango, pomeand Spanish melon. However, this is the first report of S. spermidinifaciens causing leaf spot disease of plum in China. This report will help to develop effective disease control strategies in the future.

Accurate method for value assignment of carcinoembryonic antigen reference materials.

In this study, we explored the commutability of reference materials (RMs) for carcinoembryonic antigen (CEA), selected the appropriate diluent matrix of the first International Reference Preparation (IRP) 73/601 of the World Health Organization (WHO 73/601) for CEA, and improved the comparability of CEA measurement results among different assay systems. Forty serum samples were divided into five aliquots. WHO 73/601 was diluted into nine concentrations using five diluents with different components, and the candidate RMs for CEA at five concentrations (C1-C5) were prepared by the Beijing Clinical Laboratory Center (BCCL). The samples were analyzed via five automated CEA immunoassays. Carcinoembryonic antigen candidate RMs were commutable among all immunoassays based on the CLSI approach and among 7 of 10 assay combinations based on the IFCC approach. WHO 73/601 diluted in phosphate-buffered saline (PBS) was commutable among all assays based on the CLSI approach and among 5 of 10 pairwise comparisons based on the IFCC approach with correction of bias at diluted concentrations, except for the lowest concentration, which had the smallest variation among systems. The median percentage biases among assays were decreased after calibration. The BCCL candidate RMs (C2-C5) for CEA were commutable among all immunoassays. WHO 73/601 RMs diluted in a PBS buffer matrix were selected as common calibrators for five immunoassays, which reduced bias, thereby effectively improving the harmonization of CEA detection; therefore, they could be used to assign values to CEA candidate RMs developed by BCCL. Our findings promote the harmonization of CEA detection in immunoassays.

Conformational Parameters and Hydrodynamic Behavior of Poly(2-Methyl-2-Oxazoline) in a Broad Molar Mass Range.

In this work, we report our results on the hydrodynamic behavior of poly(2-methyl-2-oxazoline) (PMeOx). PMeOx is gaining significant attention for use as hydrophilic polymer in pharmaceutical carriers as an alternative for the commonly used poly(ethylene glycol) (PEG), for which antibodies are found in a significant fraction of the human population. The main focus of the current study is to determine the hydrodynamic characteristics of PMeOx under physiological conditions, which serves as basis for better understanding of the use of PMeOx in pharmaceutical applications. This goal was achieved by studying PMeOx solutions in phosphate-buffered saline (PBS) as a solvent at 37 °C. This study was performed based on two series of PMeOx samples; one series is synthesized by conventional living cationic ring-opening polymerization, which is limited by the maximum chain length that can be achieved, and a second series is obtained by an alternative synthesis strategy based on acetylation of well-defined linear poly(ethylene imine) (PEI) prepared by controlled side-chain hydrolysis of a defined high molar mass of poly(2-ethyl-2-oxazoline). The combination of these two series of PMeOx allowed the determination of the Kuhn-Mark-Houwink-Sakurada equations in a broad molar mass range. For intrinsic viscosity, sedimentation and diffusion coefficients, the following expressions were obtained: η=0.015M0.77, s0=0.019M0.42 and D0=2600M-0.58, respectively. As a result, it can be concluded that the phosphate-buffered saline buffer at 37 °C represents a thermodynamically good solvent for PMeOx, based on the scaling indices of the equations. The conformational parameters for PMeOx chains were also determined, revealing an equilibrium rigidity or Kuhn segment length, (A) of 1.7 nm and a polymer chain diameter (d) of 0.4 nm. The obtained value for the equilibrium rigidity is very similar to the reported values for other hydrophilic polymers, such as PEG, poly(vinylpyrrolidone) and poly(2-ethyl-2-oxazoline), making PMeOx a relevant alternative to PEG.

Optimized Ebselen-Based Inhibitors of Bacterial Ureases with Nontypical Mode of Action.

Screening of 25 analogs of Ebselen, diversified at the N-aromatic residue, led to the identification of the most potent inhibitors of Sporosarcina pasteurii urease reported to date. The presence of a dihalogenated phenyl ring caused exceptional activity of these 1,2-benzisoselenazol-3(2H)-ones, with Ki value in a low picomolar range (<20 pM). The affinity was attributed to the increased π-π and π-cation interactions of the dihalogenated phenyl ring with αHis323 and αArg339 during the initial step of binding. Complementary biological studies with selected compounds on the inhibition of ureolysis in whole Proteus mirabilis cells showed a very good potency (IC50 < 25 nM in phosphate-buffered saline (PBS) buffer and IC90 < 50 nM in a urine model) for monosubstituted N-phenyl derivatives. The crystal structure of S. pasteurii urease inhibited by one of the most active analogs revealed the recurrent selenation of the Cys322 thiolate, yielding an unprecedented Cys322-S-Se-Se chemical moiety.

Ice Nucleation Promotion Impact on the Ice Recrystallization Inhibition Activity of Polyols.

Heterogeneous ice nucleation occurs vis-à-vis nucleating agents already present in solution yet can occur within a rather broad range of temperatures (0 to ca. -38 °C). Controlling this temperature and the subsequent growth of resulting ice crystals is crucial for the survival of biological organisms (certain insects, fish, and plants that endure subzero temperatures), as well as in the context of medical cryopreservation and food science. In these environments, uncontrolled crystal shape and size can rupture the cell membrane causing irreversible and catastrophic damage. Antifreeze (AF) proteins and synthetic AF analogs address this issue to restrict crystal growth and to shape ice crystals. Yet, if the nucleation temperature is not controlled and occurs in a lower temperature range, nascent ice crystals will have grown to a significantly larger size before the AF agents can be active on their surface to halt or slow the Ostwald ripening process during recrystallization. At a higher nucleation temperature, diffusion of AF macromolecules is enhanced, and dynamic crystal shaping can start earlier, producing smaller crystals overall. While antifreeze proteins, the inspiration for these synthetic analogs, are always applied in a salt buffer aqueous environment (most typically phosphate-buffered saline (PBS) buffer), the heterogeneous nucleation events are stochastic and occur within a wide temperature range. Silver iodide (AgI), however, is a highly effective ice nucleation promoter as its crystal lattice structure is a 98% lattice match to the basal plane of hexagonal ice (Ih) crystals acting as a template for water molecule orientation and decreasing the interfacial free energy. Here, we expose the advantage of purposely seeding such nascent ice crystals with AgI at a defined and higher temperature (-7 °C) in ultrapure water (UPW) such that nucleation can only come from AgI (and also in AgI/PBS), resulting in the most potent synthetic IRI observed to date (at concentrations as low as 0.001 mg·mL-1).

Differentiation of HSA and BSA and Instantaneous Detection of HSO3 - Using Confined Space of Serum Albumins and Live Cell Imaging of Exogenous/Endogenous HSO3.

The limitations of prevailing probes for the detection of human serum albumin (HSA) and HSO3 - make it challenging to apprehend the cooperative effect of both HSA and HSO3 - in biological systems. Herein, we present a multi-responsive fluorescent probe MGTP, which distinguishes HSA from bovine serum albumin (BSA) through an ∼104-fold fluorescence enhancement at an emission maximum of 595 nm with HSA and only an ∼10-fold increase at an emission maximum of 615 nm with a shoulder at 680 nm with BSA. The absorbance spectrum of MGTP also discriminates HSA and BSA with the respective absorption maxima at 543 nm and at 580 nm. MGTP in the confined space of HSA or BSA undergoes instantaneous conjugate addition of HSO3 - and results in a ratiometric change in fluorescence intensity with diminishing of red fluorescence (600 nm) and emergence of green fluorescence (515 nm). MGTP in the absence of SAs does not react with HSO3 - in phosphate-buffered saline buffer and reacts sluggishly in the dimethyl sulfoxide-water 1:1 mixture. The limit of detection values for the detection of HSA and HSO3 - are 4 and 6.88 nM, respectively. The drug binding studies reveal that MGTP preferably confines itself at the bilirubin site of HSA. In MCF-7 cancer cells, MGTP is localized into mitochondria and reveals both exogenous and endogenous visualization of HSO3 - through a change in fluorescence from the red to green channel.

Exosomes derived from lung cancer cells: Isolation, characterization, and stability studies

Recent advances in nanomedicine have paved the way for developing targeted drug delivery systems. Nanoscale exosomes are present in almost every body fluid and represent a novel mechanism of intercellular communication. Because of their membrane origin, they easily fuse with cells, acting as a natural delivery system and maintaining the bioactivity and immunotolerance of cells. To develop a reconstitutable exosome-based drug candidate for clinical applications, quality assurance by preserving its physical and biological properties during storage is necessary. Therefore, this study aimed to determine the best storage conditions for exosomes derived from lung cancer cells (A549). This study established that the phosphate-buffered saline buffer enriched with 25 mM trehalose is an optimal cryoprotectant for A549-derived exosomes stored at −80°C. Under these conditions, the concentration, size distribution, zeta potential, and total cargo protein levels of the preserved exosomes remained constant.

Cross-Linked Gamma Polyglutamic Acid/Human Hair Keratin Electrospun Nanofibrous Scaffolds with Excellent Biocompatibility and Biodegradability.

Recently, human hair keratin has been widely studied and applied in clinical fields due to its good histocompatibility, biocompatibility, and biodegradability. However, the regenerated keratin from human hair cannot be electrospun alone because of its low molecular weight. Herein, gamma polyglutamic acid (γ-PGA) was first selected to fabricate smooth and uniform γ-PGA/keratin composite scaffolds with excellent biocompatibility and biodegradability by electrospinning technology and a chemical cross-linking method in this study. The effect of electrospinning parameters on the structure and morphology, the mechanism of chemical cross-linking, biocompatibility in vitro cell culture experiments, and biodegradability in phosphate-buffered saline buffer solution and trypsin solution of the γ-PGA/keratin electrospun nanofibrous scaffolds (ENS) was studied. The results show that the cross-linked γ-PGA/keratin ENSs had excellent water stability and biodegradability. The γ-PGA/keratin ENSs showed better biocompatibility in promoting cell adhesion and cell growth compared with the γ-PGA ENSs. It is expected that γ-PGA/keratin ENSs will be easily and significantly used in tissue engineering to repair or regenerate materials.

An alternating current electrokinetics biosensor for rapid on-site serological screening of Taenia solium cysticercosis infection.

Cysticercosis, caused by Taenia solium infection, is a leading cause of acquired epilepsy in many developing countries. Several types of immunoassays have been developed for the detection of Taenia solium infection in both infected humans and livestock animals. However, these methods require central laboratory facilities and are both time- and labor-consuming with longer than desired turnaround time. In this work, we demonstrated that AC electrokinetics (ACEK) capacitive sensing can be used to realize point-of-care immunosensor in general, with the on-site screening of Taenia solium infection as an example here. The sensor employs interdigitated microelectrodes (IDME) functionalized with a recombinant Taenia solium antigen, rT24H, to detect anti-rT24H antibodies in clinical serum samples. ACEK capacitive sensing method interrogates the IDME sensors with a special AC signal, which serves the dual purposes of enriching target antibodies by ACEK effects and directly measuring the capacitance change induced by specific binding. First, to characterize the ACEK biosensor as an immunosensor in general, IgG in phosphate-buffered saline buffer was tested against IDME sensors functionalized with anti-IgG. The limit of detection of the sensor was 24.1fg/mL, and the linear dynamic range was 0.1-100pg/mL. To test the clinical usage of this sensor, ACEK capacitive sensors with rT24H probe were used to test clinical serum samples from patients with or without Taenia solium infection. The diagnostic sensitivity of the ACEK capacitive sensor for Taenia solium infection was found to be 88.24%. ACEK capacitive immunosensors have shown good potential for point-of-care diagnostics.

Purification of recombinant endoglucanase GH5-CBM72-CBM72 expressed in Escherichia coli

In a previous study, a gene GL0694641 coding for endoglucanase containing 3 domains GH5-CBM72-CBM72 was exploited from metagenomic DNA data of bacteria in Vietnamese goats’ rumen. The gene (eg5) encoding the mature enzyme (without signal peptide coding sequence) was optimized codons, artificially synthesized, and inserted into the pET22b(+) vector at NcoI and XhoI to generate expression vector pET22-eg5 for expression of the gene in Eschrichia coli. In this study, the gene eg5 was well expressed in E. coli BL21 and Rosetta 1 strains to produce recombinant endoglucanase of 77 kDa. The recombinant enzymes were expressed mainly in the soluble fractions of both strains. However, the enzyme expressed in E. coli BL21 was precipitated by imidazole at even a low concentration of 20 mM, whereas endoglucanase produced from E. coli Rosetta 1 strain was well soluble in buffers containing imidazole at concentrations of 20, 50, 200, and 250 mM. To our knowledge, this is the first study showing the negative effect of imidazole on recombinant protein. Endoglucanase expressed from strain E. coli Rosetta 1 was successfully purified by His-tag affinity chromatography using phosphate buffer saline (PBS). Protein contaminations were washed out by PBS buffer containing 20 mM and 75 mM imidazole then the target protein was harvested by the buffer containing 200 mM imidazole. After purification and desalting by the PD10 column, the recombinant endoglucanase had purity up to 97%. The pure enzyme exhibited endoglucanase activity hydrolyzing carboxymethyl cellulose in agar plate and by zymogram. The purified enzyme can be used as a material for its characterization.