MOPS Buffer Research Articles

Micronutrient availability alters Candida albicans growth and farnesol accumulation: implications for studies using RPMI-1640.

The dimorphic fungus Candida albicans is a major opportunistic pathogen of humans. RPMI-1640 is a chemically defined growth medium commonly used with C. albicans. We identified over 32,000 publications with keywords RPMI and C. albicans. Additionally, Antifungal Susceptibility Testing (AFST) protocols in the United States (CLSI) and Europe (EUCAST) utilize RPMI as a base media to assess drug efficacy against clinical fungal isolates. RPMI contains many nutrients but no added trace metals. We found that the growth characteristics with RPMI were dependent on which MOPS buffer was chosen and the contamination of that buffer by trace levels of Mn(II) and Zn(II). Added Mn(II) was most needed for cell growth while added Zn(II) was most needed for secretion of farnesol and other signaling molecules.

Unraveling the impact of pH, sodium concentration, and medium osmolality on Vibrio natriegens in batch processes

BackgroundVibrio natriegens, a halophilic marine γ-proteobacterium, holds immense biotechnological potential due to its remarkably short generation time of under ten minutes. However, the highest growth rates have been primarily observed on complex media, which often suffer from batch-to-batch variability affecting process stability and performance. Consistent bioprocesses necessitate the use of chemically defined media, which are usually optimized for fermenters with pH and dissolved oxygen tension (DOT) regulation, both of which are not applied during early-stage cultivations in shake flasks or microtiter plates. Existing studies on V. natriegens’ growth on mineral media report partially conflicting results, and a comprehensive study examining the combined effects of pH buffering, sodium concentration, and medium osmolality is lacking.ResultsThis study evaluates the influence of sodium concentration, pH buffering, and medium osmolality on the growth of V. natriegens under unregulated small-scale conditions. The maximum growth rate, time of glucose depletion, as well as the onset of stationary phase were observed through online-monitoring the oxygen transfer rate. The results revealed optimal growth conditions at an initial pH of 8.0 with a minimum of 300 mM MOPS buffer for media containing 20 g/L glucose or 180 mM MOPS for media with 10 g/L glucose. Optimal sodium chloride supplementation was found to be between 7.5 and 15 g/L, lower than previously reported ranges. This is advantageous for reducing industrial corrosion issues. Additionally, an osmolality range of 1 to 1.6 Osmol/kg was determined to be optimal for growth. Under these optimized conditions, V. natriegens achieved a growth rate of 1.97 ± 0.13 1/h over a period of 1 h at 37 °C, the highest reported rate for this organism on a mineral medium.ConclusionThis study provides guidelines for cultivating V. natriegens in early-stage laboratory settings without pH and DOT regulation. The findings suggest a lower optimal sodium chloride range than previously reported and establish an osmolality window for optimal growth, thereby advancing the understanding of V. natriegens’ physiology. In addition, this study offers a foundation for future research into the effects of different ions and carbon sources on V. natriegens.

Electrochemical Studies of Gold in Mops Buffer System

Recent work in this laboratory has investigated interactions of L-cysteine with metal ions.1 For this purpose the MOPS [(3-N-morpholino)propanesulfonic acid] aqueous buffer system has been employed in order to minimize interactions between the metal ions and buffer anions.2 Gold electrodes are expected to be useful in the investigation of L-cysteine and related substances,3 so it is useful to ascertain the background behavior of gold in the aqueous MOPS buffer system. It is the purpose of the present work to determine the voltammetric features in this situation for use in amino acid/metal ion studies.As shown in the accompanying figure, an initial positive-going sweep has a very small oxidation process at +0.45 V vs Ag/AgCl, presumably for the initial oxidation of the gold surface in pH 7.4 MOPS buffer. As this sweep continues, a much larger cathodic process corresponding to bulk gold oxidation is observed. On the return negative-going sweep, there is only a reduction process corresponding to the initial surface oxidation process. It is interesting to note that this reduction process is not observed upon reversing the sweep at only +0.50 V. On the second scan, the initial oxidation process has increased, possibly due to surface roughening in the first scan. Another investigation has involved addition of EDTA to the MOPS solution, resulting in an increase of the second (or bulk) gold oxidation process. This increase is apparently due to the ability of the EDTA to complex the gold cations formed in the oxidation process. This overall behavior is somewhat different compared to that found in other buffer systems such as phosphate,3 and contrasts to such behavior are planned for presentation in this talk. T. Cheek and D. Peña, J. Electrochem. Soc., 167, 155522 (2020).M. H. Ferreira, I. S. S. Pinto, E. V. Soares, and H. M. V. M Soares, RSC Advances, 5(39), 30989 (2015). G.T. Cheek and M.A. Worosz, ECS Trans., 72(27), 1 (2016). doi:10.1149/07227.0001ecst Figure 1

Unveiling the influence of high hydrostatic pressure and protein interactions on the color and chemical stability of cyanidin-3-O-glucoside

This study explored how high hydrostatic pressure (HHP) and proteins (i.e., BSA and HSA) influence the color and chemical stability of cyanidin-3-O-glucoside (C3G) at neutral pH. HHP treatments (100–500 MPa, 0–20 min, 25 °C) did not affect C3G content in phosphate buffer (PB) and MOPS buffer. However, significant color loss of C3G occurred in PB due to pressure-induced pH reduction (e.g., from 7 to 4.8 at 500 MPa), which accelerated the hydration of C3G, converting it from colored to colorless species. Consequently, MOPS buffer was employed for subsequent stability experiments to assess the impact of protein and HHP on the thermal, storage, and UV light stability of C3G. Initially, rapid color loss occurred during heating and storage, primarily due to the reversible hydration of C3G until equilibrium with colorless species was reached, followed by slower parallel degradation. HSA increased the fraction of colored species at equilibrium but accelerated thermal degradation, while BSA had minimal effects. UV light irradiation accelerated the degradation of C3G colored species, causing direct degradation without conversion to colorless species, a process further intensified by the presence of proteins. HHP exhibited a negligible effect on C3G stability regardless of protein addition. These findings provide insights into anthocyanin stability under HHP and protein interactions, contributing to the development of future formulation and processing strategies for improved stability and broader applications.

Utilization of orange peel waste for sustainable amino acid production by Corynebacterium glutamicum.

Oranges are the most processed fruit in the world-it is therefore apparent that the industrial production of orange juice generates large quantities of orange peel as a by-product. Unfortunately, the management of the orange peel waste leads to economic and environmental problems. Meanwhile, the use of sustainable raw materials for the production of bulk chemicals, such as amino acids, is becoming increasingly attractive. To address both issues, this study focused on the use of orange peel waste as a raw material for media preparation for the production of amino acids by engineered Corynebacterium glutamicum. C. glutamicum grew on pure orange peel hydrolysate (OPH) and growth was enhanced by the addition of a nitrogen source and a pH buffer. Inhibitory effects by the combination of high concentrations of OPH, (NH4)2SO4, and MOPS buffer in the wild-type strain (WT), were overcome in the tyrosine-producing engineered C. glutamicum strain AROM3. Genetic modifications that we identified to allow for improved growth rates under these conditions included the deletions of the vanillin dehydrogenase gene vdh, the ʟ-lactate dehydrogenase gene ldhA and the 19 genes comprising cluster cg2663-cg2686. A growth inhibiting compound present in high concentrations in the OPH is 5-(hydroxymethyl)furfural (HMF). We identified vdh as being primarily responsible for the oxidation of HMF to its acid 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), as the formation of HMFCA was reduced by 97% upon deletion of vdh in C. glutamicum WT. In addition, we showed that growth limitations could be overcome by adjusting the media preparation, using a combination of cheap ammonia water and KOH for pH neutralization after acidic hydrolysis. Overall, we developed a sustainable medium based on orange peel waste for the cultivation of C. glutamicum and demonstrated the successful production of the exemplary amino acids ʟ-arginine, ʟ-lysine, ʟ-serine, ʟ-valine and ʟ-tyrosine.

Nanoscale engineering of gold nanostars for enhanced photoacoustic imaging.

Photoacoustic (PA) imaging is a diagnostic modality that combines the high contrast resolution of optical imaging with the high tissue penetration of ultrasound. While certain endogenous chromophores can be visualized via PA imaging, many diagnostic assessments require the administration of external probes. Anisotropic gold nanoparticles are particularly valued as contrast agents, since they produce strong PA signals and do not photobleach. However, the synthesis of anisotropic nanoparticles typically requires cytotoxic reagents, which can hinder their biological application. In this work, we developed new PA probes based on nanostar cores and polymeric shells. These AuNS were obtained through one-pot synthesis with biocompatible Good's buffers, and were subsequently functionalized with polyethylene glycol, chitosan or melanin, three coatings widely used in (pre)clinical research. Notably, the structural features of the nanostar cores strongly affected the PA signal. For instance, despite displaying similar sizes (i.e. 45nm), AuNS obtained with MOPS buffer generated between 2 and 3-fold greater signal intensities in the region between 700 and 800nm than nanostars obtained with HEPES and EPPS buffers, and up to 25-fold stronger signals than spherical gold nanoparticles. A point source analytical model demonstrated that AuNS synthesized with MOPS displayed greater absorption coefficients than the other particles, corroborating the stronger PA responses. Furthermore, the AuNS shell not only improved the biocompatibility of the nanoconstructs but also affected their performance, with melanin coating enhancing the signal more than 4-fold, due to its own PA capacity, as demonstrated by both in vitro and ex vivo imaging. Taken together, these results highlight the strengths of gold nanoconstructs as PA probes and offer insights into the design rules for the nanoengineering of new nanodiagnostic agents.

Influx of zwitterionic buffer after intracytoplasmic sperm injection (ICSI) membrane piercing alters the transcriptome of human oocytes.

Does piercing oocyte membranes during ICSI allow the influx of surrounding zwitterionic buffer into human oocytes and result in altered developmental competence? Human oocytes directed to IRB-approved research were used to determine the unrestricted influx of surrounding buffer into the oocyte after piercing of membranes via confocal fluorescence microscopy (n = 80 human MII oocytes) and the influence of the select buffer influx of HEPES, MOPS, and bicarbonate buffer on the oocyte transcriptome using ultra-low input RNA sequencing (n = 40 human MII oocytes). Piercing membranes of human MII oocytes during sham-ICSI resulted in the unrestricted influx of surrounding culture buffer into the oocyte that was beyond technician control. Transcriptome analysis revealed statistically significant decreased cytoskeletal transcripts in the pierced buffer cohorts, higher levels of embryo competency transcripts (IGF2 and G6PD) in the bicarbonate buffer cohort, higher levels of stress-induced transcriptional repressor transcripts (MAF1) in the HEPES and MOPS cohorts, and decreased levels of numerous chromosomal maintenance transcripts (SMC3) in the HEPES buffer cohort. The HEPES buffer cohort also revealed higher levels of transcripts suggesting increased oxidative (GPX1) and lysosomal stress (LAMP1). The influence of zwitterionic buffer on intrinsic cellular mechanisms provides numerous concerns for their use in IVF clinical applications. The primary concern is the ICSI procedure, in which the surrounding buffer is allowed influx into the oocytes after membrane piercing.Selecting a physiological bicarbonate buffer may reduce imposed stress on oocytes, resulting in improved embryo development and clinical results because intracellular MOPS, and especially HEPES, may negatively impact intrinsic biological mechanisms, as revealed by transcriptome changes. These findings further support the utilization of bicarbonate buffer as the oocyte-holding medium during ICSI.

Zinc complexes of chloroquine and hydroxychloroquine versus the mixtures of their components: Structures, solution equilibria/speciation and cellular zinc uptake

The zinc complexes of chloroquine (CQ; [Zn(CQH+)Cl3]) and hydroxychloroquine (HO-CQ; [Zn(HO-CQH+)Cl3]) were synthesized and characterized by X-Ray structure analysis, FT-IR, NMR, UV–Vis spectroscopy, and cryo-spray mass spectrometry in solid state as well as in aqueous and organic solvent solutions, respectively. In acetonitrile, up to two Zn2+ ions bind to CQ and HO-CQ through the tertiary amine and aromatic nitrogen atoms (KN-aminCQ = (3.8 ± 0.5) x 104 M−1 and KN-aromCQ = (9.0 ± 0.7) x 103 M−1 for CQ, and KN-aminHO-CQ = (3.3 ± 0.4) x 104 M−1 and KN-aromHO-CQ = (1.6 ± 0.2) x 103 M−1 for HO-CQ). In MOPS buffer (pH 7.4) the coordination proceeds through the partially deprotonated aromatic nitrogen, with the corresponding equilibrium constants of KN-arom(aq)CQ = (3.9 ± 1.9) x 103 M−1and KN-arom(aq)HO-CQ = (0.7 + 0.4) x 103 M−1 for CQ and HO-CQ, respectively. An apparent partition coefficient of 0.22 was found for [Zn(CQH+)Cl3]. Mouse embryonic fibroblast (MEF) cells were treated with pre-synthesized [Zn((HO-)CQH+)Cl3] complexes and corresponding ZnCl2/(HO-)CQ mixtures and zinc uptake was determined by application of the fluorescence probe and ICP-OES measurements. Administration of pre-synthesized complexes led to higher total zinc levels than those obtained upon administration of the related zinc/(hydroxy)chloroquine mixtures. The differences in the zinc uptake between these two types of formulations were discussed in terms of different speciation and character of the complexes. The obtained results suggest that intact zinc complexes may exhibit biological effects distinct from that of the related zinc/ligand mixtures.

Electrochemical and Spectroscopic Investigations of L-Glutathione with Bismuth Pharmaceuticals

Introduction The electrochemical and spectroscopic investigation of bismuth(III) compounds with L-cysteine has been carried out in this laboratory since our initial publication.1 Cyclic voltammetry has shown that the interaction of L-cysteine with bismuth nitrate, bismuth salicylate, and bismuth citrate increases the solubility of these compounds by complexation with L-cysteine, thereby increasing the reduction current for the bismuth complexes formed. The appearance of a UV-VIS spectroscopic band at 340 nm indicates the formation of a Bi-S bond, the sulfur being supplied by the L-cysteine. These investigations have been conducted at pH 1.00 and 3.00 to simulate conditions in the human stomach and at pH 7.40 MOPS [(3-N-morpholino)propanesulfonic acid] to simulate general physiological conditions, in particular the intestinal tract. In this presentation, continuing studies of L-glutathione, a tripeptide incorporating L-cysteine, under these conditions shall be discussed. This work is applicable to the important field of bismuth pharmaceuticals as a means of understanding the interaction of these compounds under various conditions in the human digestion system.2 Results and Discussion Initial studies of L-glutathione with bismuth nitrate, bismuth salicylate, and bismuth citrate have been carried out under conditions similar to those of our initial work.1 It has been found that the general characteristics of the cyclic voltammograms for L-glutathione (GSH) are similar to those for L-cysteine; however, the slower rate of complexation of GSH with these bismuth compounds is evident in the changes in voltammetric features in successive sweeps. In the case of GSH addition to bismuth citrate in pH 7.4 MOPS buffer, for instance, the first sweep shows reduction of the bismuth:GSH complex at -0.71 V vs Ag/AgCl at a gold electrode, whereas a reduction process at -0.60 V is observed on the second sweep. Similar results are obtained at glassy carbon. The bismuth:GHS complex is destroyed during the reduction process as a bismuth film forms on the electrode surface. The following sweep, then, is indicative of the complexation environment in solution at that point. The bismuth ion, restored in the stripping step, apparently does not have time in the few seconds after the stripping process to fully form a complex with GSH. The observed reduction potential around -0.60 V is due to the reduction of another bismuth complex, perhaps with citrate. Further investigations are in progress to more fully describe this situation.References G. T. Cheek and D. Peña, J. Electrochem. Soc., 167, 155522 (2020)H. Li, R. Wang, and H. Sun, Acc. Chem. Res., 52, 216 (2019).

A simple method for oriented immobilization of HydSL hydrogenase of Thiocapsa bogorovii on carbon electrodes

HydSL hydrogenase from Thiocapsa bogorovii BBS (formerly Thiocapsa roseopersicina BBS) is NiFe hydrogenase belonging to 1e subgroup. Recently it was shown that C-terminal domain of HydS subunit is important for hydrogenase docking to transmembrane protein Isp1 for electron transfer (https://doi.org/10.1016/j.bbabio.2021.148492.) It is suggested that this C-domain is a hydrophobic transmembrane alpha helix located near distal FeS claster. Using this peculiarity, a simple method for oriented HydSL hydrogenase immobilization on a hydrophobic carbon electrode is developed. The method is based on the incubation of the hydrogenase with carbon paper Freudenberg H24C3 or Vulcan nanoparticles in the solution of MOPS buffer and isopropanol alcohol with ratio 1/2 (v/v) with slow (15 h) evaporation of the solution in humidified atmosphere. The developed electrodes were applied to the fuel cell with Pt catalyst as the oxygen electrode and produced 2 mW cm−2 power with current at this power more than 6 mA cm−2. Furthermore, the fuel cell was stable after 49 days of incubation under the air atmosphere with periodic measurements with some power and current increase.

P-157 Improved embryo development and clinical outcome using bicarbonate buffer as the oocyte holding medium during Intracytoplasmic sperm injection (ICSI) compared to MOPS buffer

Abstract Study question Does bicarbonate buffer used as the oocyte holding medium during ICSI result in improved embryo development and clinical outcome compared to the zwitterionic buffer MOPS? Summary answer Pre-equilibrated bicarbonate buffer is acceptable for ICSI use in atmospheric conditions, and results in superior sibling-oocyte cohort study outcomes compared to ICSI with MOPS buffer. What is known already Maintaining proper pH is of the utmost importance during IVF micromanipulation procedures. However, currently used zwitterionic buffers, HEPES and MOPS, may significantly influence intrinsic biological mechanisms outside pH maintenance, impacting oocyte competence, embryo development, and clinical outcome. Zwitterionic buffer influence is of concern during ICSI because piercing the oolemma allows the influx of the surrounding buffer into the oocyte (Mendola unpublished data, 2022). Bicarbonate is the physiological buffer for pH maintenance in all mammals providing numerous benefits for cellular development. Study design, size, duration An iSTAT handheld blood analyzer was used to analyze the pH shift of pre-equilibrated bicarbonate buffer dishes to determine the acceptablity for use during ICSI in atmospheric conditions. A clinical sibling-oocyte cohort study consisting of 115 patients and 1,997 oocytes (1,016 bicarbonate/981 MOPS) was analyzed to compare the embryo development and clinical outcomes of bicarbonate buffer vs. MOPS as the oocyte-holding medium during ICSI. Morphokinetic time points for all PGT-A tested embryos were also compared. Participants/materials, setting, methods We analyzed the pH shift of pre-equilibrated bicarbonate buffer in atmospheric conditions using 50x9mm culture dishes (Falcon, cat#351006) comparing light and high viscosity paraffin oil overlay, OvOil and Heavy OvOil (Vitrolife), after 5-minute, 10-minute, and 15-minute intervals. We analyzed all data points in triplicate. We analyzed the sibling-oocyte cohort study comparing fertilization rate, abnormal fertilization rate, useable blastocyst rate, mosaicism rate, euploidy/aneuploidy rate, morphokinetic time points, pregnancy rate, and ongoing pregnancy rate. Main results and the role of chance Pre-equilibrated (6.5%CO2/5.0%O2/7.301pH) bicarbonate buffer in 50 × 9mm ICSI culture dishes, with 5.0mL of OvOil overlay, maintained proper pH (7.301/7.328/7.354pH) for the time required to complete the ICSI procedure in atmospheric conditions (0min/5min/10min). The use of the high viscosity paraffin oil overlay, Heavy OvOil, reduced pH drift of the bicarbonate buffer ICSI dishes (7.301/7.323/7.342pH). In this clinical sibling-oocyte cohort study, ICSI was performed in pre-equilibrated bicarbonate buffer culture drops (6µL) in the 50x9mm dishes with 5.0mL Heavy OvOil overlay. Utilization of bicarbonate buffer as the oocyte-holding medium during ICSI is superior to MOPS buffer. Bicarbonate buffer used with ICSI resulted in higher fertilization rates (85.4% vs. 78.8%, P<0.0001), higher useable blastocyst rate per ICSI oocyte (50.6% vs. 46.2%, P<0.05), lower abnormal fertilization rate (4.5% vs. 6.7%, P<0.05), and a lower whole chromosome mosaicism rate (4.2% vs. 8.2%, P<0.03) compared to the MOPS buffer. Pregnancy rates were higher in the bicarbonate group compared to MOPS but not statistically significant. Morphokinetic analysis revealed a slight delay in all MOPS developmental time points compared to bicarbonate, but only statistically significant in the time to compaction (P<0.03). Limitations, reasons for caution During this study, different genetic testing companies, with differing mosaicism thresholds and decisions on included ploidy results, were used for PGT-A analysis of patient samples. Wider implications of the findings We determined that pre-equilibrated bicarbonate buffer can be used for ICSI in atmospheric conditions, therefore avoiding the potential detrimental effect of zwitterionic buffer after influx into the oocyte from the ICSI procedure. Bicarbonate buffer used during ICSI was determined to be superior to MOPS buffer. Trial registration number Not applicable

P-061 In situ microfluidics of fluidic walls: a novel deviceless and cost-effective approach for sperm selection in the same ICSI-dish

Abstract Study question Is our novel deviceless method based on in-situ microfluidics a valuable strategy to select suitable sperm for ICSI? Summary answer The novel protocol allows selecting sperm for ICSI within 15 minutes, in the same ICSI dish and disregarding fungible supplies, centrifugation and washing steps. What is known already Microfluidics is an innovative operation in ART which integrates sperm guidance biomimicry during the in vitro sperm selection process. In contrast to other sperm preparation methods such as Wash-Swim-Up and Density- Gradients-Centrifugation, microfluidics disregards washing and centrifugation steps along the procedure. In addition, microfluidics are time-efficient methods which reduce risks associated with handling, gamete mix-up and ROS production. However, microfluidics devices are costly and likely to fail to select motile sperm in dispermic samples. With the aim of outlining the application of microfluidics in ART, we conceived a lab-on-a-chip approach for sperm selection by in-situ handmade microfluidics of fluidic walls. Study design, size, duration The microfluidics circuit conforms to Laplace and hydrostatic pressure laws and integrates the sperm guidance mechanisms of rheotaxis and boundary-following behavior. System’s efficacy is based on the volume differences, the distances between microdoplets, and their arrangement. It divides into Section 1 (dispersion and seminal plasma removal) and Section 2 (sperm sorting in response to positive rheotaxis). The system was adjusted to recover at least 20 selected progressive sperm for ICSI in less than 15 minutes. Participants/materials, setting, methods We prepared the system on a conventional IVF polystyrene round dish (15 mm diameter) using MOPS buffer, PVP and mineral oil only. We verified the flow-driven dynamics by registering the horizontal displacement (direction) and flow velocity (µm/s) using eosin-Y and methylene blue dyes. We confirmed the removal of the seminal plasma in a PSA assay. The proof-of-concept analysis was performed using fresh semen samples from 150 patients (Caucasian; mean age: 33,53±8,19). Main results and the role of chance Our design includes three main points to allow sperm separation: 1 (initial), 2 (rebalancing), 3 (final) with a total distance of 1,5 cm between 1 and 3. Our system assures a continuous microfluidic flow, with two opposed directions (from point 1 to point 2 and from point 3 to point 2). PSA assay showed a significant decrease in PSA levels >200000 ng/ml in the fresh sample to undeterminable levels (<0,0091 ng/ml) at point 3. To assess the utility to select a suitable number of sperm for ICSI we used sperm samples with the following characteristics: volume 3,6 (1,0-7,6) ml, concentration 68 (0,29-150) ×106/ml, progressive motility 24 (2-41) % and morphology 6 (1-15) % normal. The minimum and maximum times for sperm recovery at point 3 were recorded at 5 and 35 minutes, respectively. Normozoospermic samples required lower recovery times compared to dispermic (p < 0,001), although according to the median, samples with at least 1 × 106/ml of motile sperm need less than ten minutes recovery time. Sperm parameters: concentration, motility and morphology were inversely correlated with the recovery time (p < 0,0001), being morphology the parameter with the lesser influence on the outcome. Limitations, reasons for caution The present protocol depends on a free-hand preparation. Although the use of templates and the training reduces inter and intra-operator variability, a ready-to-use surface would benefit the operability. Non-inferiority studies are required to compare our method with the currently used ones (Wash-Swim-Up or Density-Gradients-Centrifugation) before clinical implementation. Wider implications of the findings Our deviceless methodology streamlines sperm selection for ICSI and reduces risks along the procedure. This novel approach represents a paradigm shift in sperm preparation, since it dispenses with centrifugation, washing and plasticware. The present protocol can be implemented to the clinic in the near-term, particularly due to its simplicity. Trial registration number na

Addition of formaldehyde releaser imidazolidinyl urea and MOPS buffer to urine samples enables delayed processing for flow cytometric analysis of urinary cells: A simple, two step conservation method of urinary cells for flow cytometry.

Kidney diseases are a major health concern worldwide. Currently there is a large unmet need for novel biomarkers to non-invasively diagnose and monitor kidney diseases. Urinary cells are promising biomarkers and their analysis by flow cytometry has demonstrated its utility in diverse clinical settings. However, up to date this methodology depends on fresh samples, as cellular event counts and the signal-to-noise-ratio deter over time. Here we developed an easy-to-use two-step preservation method for conservation of urine samples for subsequent flow cytometry. The protocol utilizes a combination of the formaldehyde releasing agent imidazolidinyl urea (IU) and MOPS buffer, leading to gentle fixation of urinary cells. The preservation method increases acceptable storing time of urine samples from several hours to up to 6 days. Cellular event counts and staining properties of cells remain comparable to fresh untreated samples. The hereby presented preservation method facilitates future investigations on flow cytometry of urinary cells as potential biomarkers and may enable broad implementation in clinical practice.

Radiolabeling and in vivo evaluation of lanmodulin with biomedically relevant lanthanide isotopes.

Short-lived, radioactive lanthanides comprise an emerging class of radioisotopes attractive for biomedical imaging and therapy applications. To deliver such isotopes to target tissues, they must be appended to entities that target antigens overexpressed on the target cell's surface. However, the thermally sensitive nature of biomolecule-derived targeting vectors requires the incorporation of these isotopes without the use of denaturing temperatures or extreme pH conditions; chelating systems that can capture large radioisotopes under mild conditions are therefore highly desirable. Herein, we demonstrate the successful radiolabeling of the lanthanide-binding protein, lanmodulin (LanM), with medicinally relevant radioisotopes: 177Lu, 132/135La and 89Zr. Radiolabeling of the endogenous metal-binding sites of LanM, as well exogenous labeling of a protein-appended chelator, was successfully conducted at 25 °C and pH 7 with radiochemical yields ranging from 20-82%. The corresponding radiolabeled constructs possess good formulation stability in pH 7 MOPS buffer over 24 hours (>98%) in the presence of 2 equivalents of natLa carrier. In vivo experiments with [177Lu]-LanM, [132/135La]-LanM, and a prostate cancer targeting-vector linked conjugate, [132/135La]-LanM-PSMA, reveal that endogenously labeled constructs produce bone uptake in vivo. Exogenous, chelator-tag mediated radiolabeling to produce [89Zr]-DFO-LanM enables further study of the protein's in vivo behavior, demonstrating low bone and liver uptake, and renal clearance of the protein itself. While these results indicate that additional stabilization of LanM is required, this study establishes precedence for the radiochemical labeling of LanM with medically relevant lanthanide radioisotopes.

Molecular interaction of benzo-a-pyrene inhibits the catalytic activity of catalase: Insights from biophysical and computational studies

Benzo-a-pyrene (BaP) is a polycyclic aromatic hydrocarbon, which alters the redox balance and results into oxidative stress. Among antioxidant enzymes, catalase plays a vital role in neutralizing the harmful effects of pollutant-induced oxidative stress. However, the molecular mechanism underlying the interaction of catalase with BaP is poorly understood. In this communication, structural and functional alterations of bovine liver catalase (BLC) in presence of BaP have been investigated at molecular and cellular levels. Reduced catalase activity is evident after BaP treatment in mouse embryonic fibroblast cell lines. Nevertheless, the enzyme activity of BLC in response to BaP was gradually decreased in Phosphate, HEPES, MOPS, and Tris buffers. The secondary conformation of BLC has altered considerably (decrease in α-helix and increase in other secondary structures) due to BaP exposure. In addition, molecular interaction study shows BaP binds near proximity to the active site of BLC with binding affinity of -11.2 kcal/mol. Molecular simulation determined the structural modifications in the protein, which indicate maximum fluctuations of BLC occured between 20-60, 160-180 and 380-440 amino acid residues. Amino acids like Leu175, Ala249, Ala250 and Ala253 are observed to interact with BaP over 100 ns trajectory through hydrophobic interaction. In summary, this integrated study comprising experimental, in vitro and in silico approaches presents a mechanistic insights towards BaP driven compromised conformation and catalytic activity of catalase.

DIFFERENTIAL STABILIZING EFFECTS OF BUFFERS ON STRUCTURAL STABILITY OF BOVINE SERUM ALBUMIN AGAINST UREA DENATURATION

The conformational stability of bovine serum albumin (BSA) against urea denaturation was investigated in aqueous solutions both in the absence and presence of buffers. Various buffers differing in polar and nonpolar characters such as sodium phosphate, Tris-HCl, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HEPES and [3-(N-morpholino)propanesulfonic acid] MOPS buffers were used in this study. Urea-induced structural changes were analyzed using different probes, i.e., intrinsic fluorescence, ANS fluorescence and UV-difference spectral signal. Presence of different buffers in the incubation medium offered different degrees of resistance to the protein against urea-induced structural changes compared to those obtained in water (in the absence of buffers). A similar trend of buffer-induced structural resistance was noticed with three different probes. The stabilizing effect of these buffers followed the order: MOPS > HEPES > sodium phosphate > Tris-HCl > water. As found in MOPS and HEPES buffers, the highest stability of BSA can be attributed to the presence of morpholine and piperazine rings, respectively, in their structures. These groups might have produced a hydrophobic environment around the protein surface, thus stabilizing protein conformation against urea denaturation.

Modifications of antifungal sensibility testing as suggested by CLSI document M27-A4: proposal for using different culture medium and buffer

A common strategy in antifungal susceptibility testing is the utilization of the standardized protocol based on the microbroth dilution assay approach as described by the Clinical Laboratory Standards Institute (CLSI) (M27-A4). One major problem for laboratories in resource-limited countries with this protocol arises from the use of expensive culture media like RPMI-1640 and 3-N-morpholinopropanesulfonic acid (MOPS) buffer. One approach of circumventing this problem in cases of economic need is the evaluation of alternative culture media and buffers. The overall goal of this work was to investigate the influence of modifications in the protocol M27-A4 on diagnostic reliability. We performed univariate analyses evaluating (1) 2 different culture media (YNB and modified SAB); (2) three different buffers (sodium bicarbonate, Tris-HCL, and phosphate), as well as the influence of inoculum concentration (102, 103, 104, 105 cells/mL), the influence of incubation time, and the influence of the assessment mode (visual, biological dye, and spectrophotometer). Our results suggested that (1) RPMI-1640 may be substituted by modified SAB and (2) MOPS buffer may be substituted by Tris-HCl buffer for defined analyses. By comparing the CLSI protocol and the alternative protocol proposed in the present study (modified SAB and Tris-HCl buffer) for the assessment of fluconazole susceptibility of eighteen yeasts (clinical isolates), similar results with both methodologies were recorded. We feel that this study should stimulate a discussion on the feasibility and evolution of the M27-A4 protocol in order to include pragmatic alternatives for resource-limited settings.

Development and validation of a liquid chromatography high-resolution mass spectrometry orbitrap method for the sensitive quantification of amoxicillin, piperacillin, tazobactam and meropenem in human faeces

Beta-lactam antibiotics are of vital importance for the treatment of infections in a broad range of patients. Although most systemically administered antibiotics will be excreted renally, a fraction will reach the gastro-intestinal tract, affecting the intestinal microbiome by eradicating a wide range of bacterial species while facilitating the growth of antimicrobial-resistant species. A better understanding of the kinetics of beta-lactam antibiotics in the gastro-intestinal tract is essential to study their role in the development of antibiotic resistance in bacteria and to help develop future therapies to prevent damage to, or restore, the intestinal microbiome.Analysis of beta-lactam antibiotics in faeces is particularly challenging due to the heterogeneous nature of the matrix, rapid degradation of some beta-lactam antibiotics in faeces and very strong ion suppression when using mass spectrometry. Sample preparation was optimized using a sequential strategy of experimental designs. It resulted in lyophilization, a MOPS buffer system and the addition of the beta-lactamase inhibitor avibactam to minimize degradation of antibiotics allowing sensitive quantification. The developed liquid chromatography method with high-resolution mass spectrometric detection was successfully validated according to bioanalytical EMA guidelines and had a linear range of 1–200 μg g−1 lyophilized faeces for amoxicillin, piperacillin and meropenem; and 0.5–100 μg g−1 lyophilized faeces for tazobactam. Despite the highly complex and heterogeneous composition of faeces, the accuracy (0.1–15%) and precision (1.7–12.1%) were in line with those obtained for quantification methods of beta-lactam antibiotics in plasma, the golden standard matrix for therapeutic drug monitoring. The applicability of the method was illustrated by successful quantification of piperacillin and tazobactam in faeces from an intensive care unit patient receiving piperacillin/tazobactam in a continuous intravenous infusion. Both piperacillin and tazobactam were still present six days after discontinuation of the therapy.

Electrochemical and Spectroscopic Investigations of Bismuth Pharmaceuticals

IntroductionThe use of bismuth compounds as pharmaceutical agents for gastric maladies has a long history.1 Many techniques have been used to characterize the mode of operation of these compounds; however, electrochemical methods have not been fully utilized in this regard. The sulfhydryl side chain of the amino acid L-cysteine and related compounds can form complexes with bismuth(III) ions,1 so that the effect of L-cysteine added to solutions of bismuth(III) compounds is expected to greatly affect the electrochemical behavior of this system. Previous work in this laboratory has involved electrochemical investigation of both L-cysteine in various media2, and the interaction of bismuth(III) compounds with L-cysteine.3 The present work involves the interaction of L-cysteine with bismuth-containing pharmaceuticals.Results and DiscussionBismuth(III) nitrate has been chosen for the initial studies, followed by work with bismuth(III) citrate and bismuth(III) salicylate, both of which are used in commonly available pharmaceuticals. Our studies have been carried out in 0.100 M HCl and 0.100 M HNO3 in order to simulate the pH level of gastric contents. The electrochemical techniques cyclic voltammetry and square wave voltammetry were used in this work, and some comparisons between the results are intended for presentation. Voltammetric scans have been carried out at both glassy carbon and boron-doped diamond electrodes in order to avoid the reduction of protons, which would occur at platinum and gold electrodes. Although the solubility of Bi(NO3)3 in 0.10 M HNO3 is rather low, initial studies revealed that the addition of L-cysteine to the solution greatly increased the solubility of bismuth(III) nitrate, as seen by an increase in the bismuth(III) reduction current as well as the subsequent stripping current of the bismuth deposit.Bismuth(III) salicylate was found to be soluble at the 1 mM level in 0.100 HNO3, and L-cysteine additions produced potential shifts in the voltammetric processes for the bismuth(III) system. These results support the complexation of bismuth(III) salicylate by L-cysteine, even under these very acidic conditions. This work has now been extended to bismuth(III) citrate in 0.10 M HCl, with overall similar results. Studies in pH 7.4 MOPS buffer have also been carried out to simulate pH levels in other parts of the human body. The electrochemical behavior of bismuth(III) nitrate in pH 7.4 MOPS (3-(N-morpholino)propanesulfonic acid)4 buffer was investigated in this regard. Whereas bismuth(III) nitrate (nominal 1.0 mM) was essentially insoluble in this medium, additions of L-cysteine produced voltammetric currents typical of the 1 mM concentration level. As at pH 1.00, initial reduction of bismuth(III) was followed by bismuth stripping peaks at more positive potentials. This behavior supports the complexation of bismuth(III) ion by L-cysteine. Further evidence of this complexation was found by UV-VIS studies of bismuth(III) nitrate in pH 7.4 MOPS buffer with incremental additions of L-cysteine. An absorbance band at 325 nm appeared as L-cysteine was added, the absorbance of which remained constant after the 1:2 Bi3+: L-cysteine point. These results indicate formation of Bi(Cys)2 under these conditions. As a whole, the present studies indicate the ability of bismuth(III) compounds to interact with L-cysteine at pH 1.0 and pH 7.4.References Li, R. Wang, and H. Sun, Acc. Chem. Res., 52, 216 (2019).T. Cheek and M. A. Worosz, ECS Trans., 164(12), D707 (2016).T. Cheek and D. Pena, J. Electrochem. Soc., 167, 155522 (2020).M. H. Ferreira, I. S. S. Pinto, E. V. Soares, and H. M. V. M Soares, RSC Advances, 5(39), 30989 (2015).

A New Coumarin-Acridone Compound as a Fluorescence Probe for Fe3+ and Its Application in Living Cells and Zebrafish.

A new coumarin-acridone fluorescent probe S was designed and synthesized, and the structure was confirmed with 1H/13C NMR spectrometry, single-crystal X-ray diffraction, and high-resolution mass spectrometry. This probe has high sensitivity and selectivity for Fe3+ over other testing metal ions at 420 or 436 nm in acetonitrile–MOPS (3-Morpholinopropanesulfonic Acid) buffer solution (20.0 μM, pH = 6.9, 8:2 (v/v)). Under physiological conditions, the probe displayed satisfying time stability with a detection limit of 1.77 µM. In addition, probe S was successfully used to detect intracellular iron changes through a fluorescence-off mode, and the imaging results of cells and zebrafish confirmed their low cytotoxicity and satisfactory cell membrane permeability, as well as their potential biological applications.