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Gallium-68-prostate-specific membrane antigen-11 (68Ga-PSMA-11) is a positron emission tomography radiopharmaceutical that labels a Glu-urea-Lys-based ligand with 68Ga, binding specifically to the PSMA. It is used widely for imaging recurrent prostate cancer and metastases. On the other hand, the preparation and quality control testing of 68Ga-PSMA-11 in medical institutions takes over 60 minutes, limiting the daily capacity of 68Ge/68Ga generators. While the generator provides 1,110 MBq (30 mCi) nominally, its activity decreases over time, and the labeling yield declines irregularly. Consequently, additional preparations are needed, increasing radiation exposure for medical technicians, prolonging patient wait times, and necessitating production schedule adjustments. This study aimed to reduce the 68Ga-PSMA-11 preparation time and optimize the automated synthesis system. By shortening the reaction time between 68Ga and the PSMA-11 precursor and adjusting the number of purification steps, a faster and more cost-effective method was tested while maintaining quality. The final synthesis time was reduced from 30 to 20 minutes, meeting the standards for the HEPES content, residual solvent EtOH content, and radiochemical purity. This optimized procedure minimizes radiation exposure for medical technicians, reduces patient wait times, and maintains consistent production schedules, making it suitable for clinical application.

Purpose/study question: Does piercing oocyte membranes during ICSI allow the influx of surrounding zwitterionic buffer into human oocytes and result in altered developmental competence? Methods: 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). Results: 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). Conclusion: 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. [ABSTRACT FROM AUTHOR]

For decades, surgeons have utilized 0.9% normal saline (NS) for joint irrigation to improve visualization during arthroscopic procedures. This continues despite mounting evidence that NS exposure impairs chondrocyte metabolism and compromises articular cartilage function. We hypothesized that chondrocyte oxidative stress induced by low pH is the dominant factor driving NS toxicity, and that buffering NS to increase its pH would mitigate these effects. Effects on chondrocyte viability, reactive oxygen species (ROS) production, and overall metabolic function were assessed. Even brief exposure to NS caused cell death, ROS overproduction, and disruption of glycolysis, pentose phosphate, and tricarboxylic acid (TCA) cycle pathways. NS also stimulated ROS overproduction in synovial cells that could adversely alter the synovial function and subsequently the entire joint health. Buffering NS with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) significantly increased chondrocyte viability, reduced ROS production, and returned metabolite levels to near control levels while also reducing ROS production in synovial cells. These results confirm that chondrocytes and synoviocytes are vulnerable to insult from the acidic pH of NS and demonstrate that adding a buffering agent to NS averts many of its most harmful effects. [ABSTRACT FROM AUTHOR]

The large-scale culture of human induced pluripotent stem cells (hiPSCs) is essential for developing new pharmaceuticals and regenerative therapy methods. While for the development of cultured meat products, mass production of animal myoblasts is necessary. Both hiPSCs and animal myoblasts consume d-glucose as their energy source and produce l-lactate, which accumulates in cell culture media and inhibits cell proliferation. To make large-scale cell culture economically feasible, l-lactate removal and subsequent reuse of media are of high importance. The adsorption technique is attractive for l-lactate removal due to its low cost, ease of operation, and scalability. The current study is dedicated to 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES) intercalated Mg–Al layered double hydroxide (LDH), which acts as a biocompatible anion-exchanger in media. HEPES‧Mg–Al LDH was able to remove l-lactate from hiPS cells and myoblast-relevant media selectively while mostly retaining d-glucose. Adsorbent exhibited dose-dependent cytotoxicity to hiPSCs and C2C12 cells, mainly related to elevated osmolarity, HEPES, and Mg levels and adsorption of media micro components. By employing alternatively prepared sol-gel derived HEPES‧Mg–Al LDH, the required adsorbent dose for efficient l-lactate removal was reduced to a safe level. The current study thoroughly evaluates Mg–Al layered double hydroxides as suitable adsorbents for cell culture media regeneration and discusses the limitations of Mg–Al LDHs in systems relevant to hiPS cells and C2C12 cells. This work promotes the cost-effective large-scale production of cells and gives insight into the limitations of Mg–Al LDHs applied to systems of biological origin.

[Display omitted] • pH change is necessary for Presynaptic Homeostatic Potentiation at the mouse NMJ. • Extracellular acidification is sufficient for quantal content (QC) upregulation. • Pharmacological inhibition of the PMCA increases QC, contingent on functional ASICs. • Inhibition of PMCA precludes Presynaptic Homeostatic Potentiation. At the vertebrate neuromuscular junction (NMJ), presynaptic homeostatic potentiation (PHP) refers to an increase in neurotransmitter release that restores the strength of synaptic transmission following a blockade of nicotinic acetylcholine receptors (nAChRs). Mechanisms informing the presynaptic terminal of the loss of postsynaptic receptivity remain poorly understood. Previous research at the mouse NMJ suggests that extracellular protons may function as a retrograde signal that triggers an upregulation of neurotransmitter output (measured by quantal content, QC) through the activation of acid-sensing ion channels (ASICs). We further investigated the pH-dependency of PHP in an ex-vivo mouse muscle preparation. We observed that increasing the buffering capacity of the perfusion saline with HEPES abolishes PHP and that acidifying the saline from pH 7.4 to pH 7.2–7.1 increases QC, demonstrating the necessity and sufficiency of extracellular acidification for PHP. We then sought to uncover how the blockade of nAChRs leads to the pH decrease. Plasma-membrane calcium ATPase (PMCA), a calcium-proton antiporter, is known to alkalize the synaptic cleft following neurotransmission in a calcium-dependent manner. We hypothesize that since nAChR blockade reduces postsynaptic calcium entry, it also reduces the alkalizing activity of the PMCA, thereby causing acidosis, ASIC activation, and QC upregulation. In line with this hypothesis, we found that pharmacological inhibition of the PMCA with carboxyeosin induces QC upregulation and that this effect requires functional ASICs. We also demonstrated that muscles pre-treated with carboxyeosin fail to generate PHP. These findings suggest that reduced PMCA activity causes presynaptic homeostatic potentiation by activating ASICs at the mouse NMJ. [ABSTRACT FROM AUTHOR]

The lipopolysaccharide (LPS)-triggered horseshoe crab coagulation cascade is composed of three protease zymogens, prochelicerase C (proC), prochelicerase B (proB) and the proclotting enzyme (proCE). In this study, we found that Ca 2+ ions increase the production of the clotting enzyme as a result of a cascade reaction reconstituted by recombinant proteins of wild-type (WT) proC, WT proB and WT proCE. We divided the cascade into three stages: autocatalytic activation of WT proC on the surface of LPS into WT α-chelicerase C (Stage 1); activation of WT proB on the surface of LPS into WT chelicerase B by WT α-chelicerase C (Stage 2) and activation of WT pro ce into WT CE by chelicerase B (Stage 3). Ca2+ ions enhanced the proteolytic activation in Stage 2, but not those in Stages 1 and 3. Moreover, we performed isothermal titration calorimetry to clarify the interaction of LPS or the recombinant zymogens with Ca2+ ions. LPS interacted with Ca2+ ions at an association constant of K a = 4.7 × 104 M−1, but not with any of the recombinant zymogens. We concluded that LPS bound with Ca2+ ions facilitates the chain reaction of the cascade as a more efficient scaffold than LPS itself. [ABSTRACT FROM AUTHOR]

The interaction of the β -coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid (N) protein with genomic RNA is initiated by specific RNA regions and subsequently induces the formation of a continuous polymer with characteristic structural units for viral formation. We hypothesized that oligomeric RNAs, whose sequences are absent in the 29.9-kb genome sequence of SARS-CoV-2, might affect RNA–N protein interactions. We identified two such hexameric RNAs, In-1 (CCGGCG) and G6 (GGGGGG), and investigated their effects on the small filamentous/droplet-like structures (< a few μm) of N protein–genomic RNA formed by liquid–liquid phase separation. The small N protein structures were sequence-specifically enhanced by In-1, whereas G6 caused them to coalesce into large droplets. Moreover, we found that a guanosine 12-mer (G12, GGGGGGGGGGGG) expelled preexisting genomic RNA from the small N protein structures. The presence of G12 with the genomic RNA suppressed the formation of the small N protein structures, and alternatively apparently altered phase separation to induce the formation of large droplets with unclear phase boundaries. We showed that the N-terminal RNA-binding domain is required for the stability of the small N protein structures. Our results suggest that G12 may be a strong inhibitor of the RNA–N protein interaction. [ABSTRACT FROM AUTHOR]

It has been acknowledged for years that compounds containing sulfur (S) are an important source of endogenous acid production. In the metabolism, S is oxidized to sulfate, and therefore the mEq sulfate excreted in the urine is counted as acid retained in the body. In this study we show that pH in fluids with constant [Na] and [HEPES] declines as sulfate ions are added, and we show that titratable acidity increases exactly with the equivalents of sulfate. Therefore, sulfate excretion in urine is also acid excretion per se. This is in accordance with the down-regulation of proximal sulfate reabsorption under acidosis and the observation that children with distal renal tubular acidosis may be sulfate depleted. These results are well explained using charge-balance modeling, which is based only on the three fundamental principles of electroneutrality, conservation of mass, and rules of dissociation as devised from physical chemistry. In contrast, the findings are in contrast to expectations from conventional narratives. These are unable to understand the decreasing pH as sulfate is added since no conventional acid is present. The results may undermine the traditional notion of endogenous acid production since in the case of sulfur balance, S oxidation and its excretion as sulfate exactly balance each other. Possible clinical correlates with these findings are discussed. [ABSTRACT FROM AUTHOR]

X‐ray crystallography is the main experimental method behind ligand–macromolecule complexes found in the Protein Data Bank (PDB). Applying bioinformatics methods to such structural data can fuel drug discovery, albeit under the condition that the information is correct. Regrettably, a small number of structures in the PDB are of suboptimal quality due to incorrectly identified and modeled ligands in protein–ligand complexes. In this paper, we combine a theoretical‐graph approach, nuclear density estimates, bioinformatics methods, and prior chemical knowledge to analyze two non‐physiological ligands, HEPES and MES, that are frequent components of crystallization and purifications buffers. Our analysis includes quantum mechanics calculations and Cambridge Structure Database (CSD) queries to define the ideal conformation of these ligands, geometry analysis of PDB deposits regarding several quality factors, and a search for homologous structures to identify other small molecules that could bind in place of the parasitic ligand. Our results highlight the need for careful refinement of macromolecule–ligand complexes and better validation tools that integrate results from all relevant resources. PDB Code(s): 3K4L, 3PYI, 5T6L, 6BB0, 1PJX, 3O4P, 6WCF, 3DKE, 3E10, 6G38, 4E8R, 4Z91, 3E9F, 1MOS, 1MOQ, 2ESB, 1VHR, 4P66 and 6NNI; [ABSTRACT FROM AUTHOR]

Background: Nowadays, in Nuclear Medicine, clinically applied radiopharmaceuticals must meet quality release criteria such as high radiochemical purity and radiochemical yield. Many radiopharmaceuticals do not have marketing authorization and have no dedicated monograph within European Pharmacopeia (Ph. Eur.); therefore, general monographs on quality controls (QCs) have to be applied for clinical application. These criteria require standardization and validation in labeling and preparation, including quality controls measurements, according to well defined standard operation procedures. However, QC measurements are often based on detection techniques that are specific to a certain chromatographic system. Several radiosyntheses of [68Ga]Ga-radiopharmaceuticals are more efficient and robust when they are performed with 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES) buffer, which is considered as an impurity to be assessed in the QC procedure, prior to clinical use. Thus, Ph. Eur. has introduced a thin-layer chromatography (TLC) method to quantify the HEPES amount that is present in [68Ga]Ga-radiopharmaceuticals. However, this is only qualitative and has proven to be unreliable. Here we develop and validate a new high-performance liquid chromatography (UV-Radio-HPLC) method to quantify the residual amount of HEPES in 68Ga-based radiopharmaceuticals. Method: To validate the proposed UV-Radio-HPLC method, a stepwise approach was used, as defined in the guidance document that was adopted by the European Medicines Agency (CMP/ICH/381/95 2014). The assessed parameters are specificity, linearity, precision (repeatability), accuracy, and limit of quantification. A range of concentrations of HEPES (100, 80, 60, 40, 20, 10, 5, 3 μg/mL) were analyzed. Moreover, to test the validity and pertinence of our new HPLC method, we analyzed samples of [68Ga]Ga-DOTATOC; [68Ga]Ga-PSMA; [68Ga]Ga-DOTATATE; [68Ga]Ga-Pentixafor; and [68Ga]Ga-NODAGA-Exendin-4 from different batches that were prepared for clinical use. Results: In the assessed samples, HEPES could not be detected by the TLC method that was described in Ph. Eur. within 4 min incubation in an iodine-saturated chamber. Our developed HPLC method showed excellent linearity between 3 and 100 μg/mL for HEPES, with a correlation coefficient (R2) for calibration curves that was equal to 0.999, coefficients of variation (CV%) < 2%, and percent deviation value of bias from 100% to 5%, in accordance with acceptance criteria. The intra-day and inter-day precision of our method was statistically confirmed and the limit-of-quantification (LOQ) was 3 μg/mL, confirming the high sensitivity of the method. The amount of HEPES that was detected with our developed HPLC method in the tested [68Ga]Ga-radiopharmaceuticals resulted well below the Ph. Eur. limit, especially for [68Ga]Ga-NODAGA-Exendin-4. Conclusions: The TLC method that is described in Ph. Eur. to assess residual HEPES in [68Ga]-based radiopharmaceuticals may not be sufficiently sensitive and thus unsuitable for QC release. Our new HPLC method was sensitive, quantitative, reproducible, and rapid for QCs, allowing us to exactly determine the residual HEPES amount in [68Ga]Ga-radiopharmaceuticals for safe patient administration. [ABSTRACT FROM AUTHOR]

Background: The common methods to preserve cell for protein analyses are in cold condition or treated with freeze solution and packaging in dry ice for shipping. Solution which can preserve cervical cells at room temperature is preferable and cost consuming for laboratory testing. Aims and Objective: Research and optimized the storage and transport solution for cervical sample which can preserve cells at room temperature for laboratory testing. Materials and Methods: In this study, cervical specimens were collected in 3 different preservation solutions. Storage and transport of samples was at ambient or refrigerated temperature. The effect of preservation solution and temperature was check by cell visualization under microscope and protein measurement. Results: Presence of cells were detected in all three solutions. Among those, HEPES solution can preserve the highest number of cells and at room temperature. Conclusion: HEPES solution appeared suitable to preserve cervical cytology specimens at ambient temperature for further laboratory testing at protein and DNA level.

Abstract HEPES (4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid) is a buffer that is used in the radiolabelling of gallium-68 compounds. The beneficial effects of HEPES on molar activity in bioconjugates have been well described. Current strict regulations on the HEPES content in radiopharmaceuticals limit its use when intended for parenteral administration. This short communication summarizes data from the literature on the toxicity of HEPES in dogs after intravenous infusion and the subsequent use in humans. We also highlight the use of HEPES in an FDA labelled intravenous drug formulation. Regulatory institutions may consider this data to review current strict limits.

Here, we report the results of experiments regarding the chemical repositioning of 4‐(2‐hydroxyethyl)‐1‐piperazine ethane sulfonic acid (HEPES), a representative buffering agent. In this work, we confirmed that HEPES could disaggregate amyloid plaques, a hallmark of Alzheimer's disease, and we verified its sustained‐release system (release for up to 2 weeks) via hybridization with a biocompatible polymer (Eudragit S100). [ABSTRACT FROM AUTHOR]

Abstract Background The introduction of a GMP-certified 68Ga-generator spurred the application of 68Ga-radiopharmaceuticals. Several radiosynthesis of 68Ga-radiopharmaceuticals are more efficient and robust when performed with 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES) buffer, which is considered as an impurity in the quality control (QC) procedure. Thus, prior to clinical use, QC must be conducted to ensure that HEPES does not exceed the maximum dose of 200 μg/V Injected as described in European Pharmacopoeia (Ph Eur) for edotreotide. However, when applying the thin-layer chromatography (TLC) method described in the Ph Eur to quantify the HEPES amount present in the 68Ga-octreotide or in the remaining 68Ga-radiopharmaceuticals that were tested, no amount was detectable after 4 min of iodine incubation. Here we tested our modified TLC method and validate a new high-performance liquid chromatography (HPLC) method to quantify HEPES in 68Ga-radiopharmaceuticals and compare it to the TLC-method described in Ph Eur. In addition, samples collected from various institutes were tested to evaluate whether the synthesis of different 68Ga-radiopharmaceuticals or the use of different synthesis methods could affect the amounts of HEPES. Results HEPES could not be detected by the TLC method described in the Ph Eur within 4 min incubation in an iodine-saturated chamber. As for our modified TLC method, only after 2 h, spots were only visible > 1 mg/mL. The HPLC method had a limit-of-quantification (LOQ) of 3 μg/mL and a limit-of-detection (LOD) of 1 μg/mL. From the three 68Ga-radiopharmaceuticals tested, only in the [68Ga]Ga-NODAGA-Exendin samples exceeding amounts of HEPES were found and its concentration in the [68Ga]Ga-NODAGA-Exendin was significantly higher, when compared to [68Ga]Ga-DOTATOC and [68Ga]Ga-PSMA-11. Conclusion The TLC method described in Ph Eur and our modified TLC method may not be sufficiently sensitive and thus unsuitable to use for QC release. The new HPLC method was sensitive, quantitative, reproducible and suitable for QC release. With this method, we were able to determine that some 68Ga-radiopharmaceuticals may exceed the HEPES limit of 200 μg/ V Injected. This new analytical system would allow correcting for the maximum injected dose in order not to exceed this amount.

Metal-organic frameworks (MOFs), a diverse and rapidly expanding class of crystalline materials, present many opportunities for various applications. Within this class, the amino-functionalized Zr-MOF, namely, UiO-66-NH 2 , stands out due to its distinctive chemical and physical properties. In this study, we report on the new unique property where UiO-66-NH 2 nanocrystals exhibited enhanced fluorescence upon heating, which was persistently maintained postcooling. To unravel the mechanism, the changes in the fluorescence signal were monitored by steady-state fluorescence spectroscopy, lifetime measurements, and a fluorescence microscope, which revealed that upon heating, multiple mechanisms could be contributing to the observed enhancement; the MOFs can undergo disaggregation, resulting in a fluorescent enhancement of the colloidally stable MOF nanocrystals and/or surface-induced phenomena that result in further fluorescence enhancement. This observed temperature-dependent photophysical behavior has substantial applications. It not only provides pathways for innovations in thermally modulated photonic applications but also underscores the need for a better understanding of the interactions between MOF crystals and their environments.

Metastable protein-rich microdroplets are produced from liquid-liquid phase separation (LLPS) of protein aqueous solutions. These globules can be intermediates for the formation of other protein-rich phases. Lysozyme aqueous solutions undergo LLPS around 0 °C in the presence of NaCl near physiological conditions. Here, it is shown that insertion of small amounts of 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES, 0.1 M) as a second additive to lysozyme-NaCl-water solutions near physiological ionic strength (0.2 M) is an essential step for triggering conversion of protein-rich droplets into another phase. Specifically, LLPS induced by cooling reproducibly leads to a rapid and high-yield formation of compact tetragonal crystalline microparticles only in the presence of HEPES. These microcrystals exhibit small size (1-3 μm), narrow size distribution and guest-binding properties. The temperature-concentration phase diagram shows a characteristic topology with LLPS boundary metastable with respect to tetragonal microcrystals, which in turn become less stable than rod-shaped orthorhombic crystals above 40 °C. Interestingly, dynamic light scattering, hydrogen-ion titrations and isothermal titration calorimetry reveal that lysozyme-HEPES interactions were found to be weakly attractive and exothermic. Our findings indicate that additives of salting-in type can represent an important factor controlling the fate of metastable protein-rich microdroplets relevant to drug formulations, femtosecond crystallography, and potential implications in protein-driven cytoplasmic compartmentalization. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: The common methods to preserve cell for protein analyses are in cold condition or treated with freeze solution and packaging in dry ice for shipping. Solution which can preserve cervical cells at room temperature is preferable and cost consuming for laboratory testing. Aims and Objective: Research and optimized the storage and transport solution for cervical sample which can preserve cells at room temperature for laboratory testing. Materials and Methods: In this study, cervical specimens were collected in 3 different preservation solutions. Storage and transport of samples was at ambient or refrigerated temperature. The effect of preservation solution and temperature was check by cell visualization under microscope and protein measurement. Results: Presence of cells were detected in all three solutions. Among those, HEPES solution can preserve the highest number of cells and at room temperature.Conclusion: HEPES solution appeared suitable to preserve cervical cytology specimens at ambient temperature for further laboratory testing at protein and DNA level. [ABSTRACT FROM AUTHOR]

Good's buffer or HEPES has advantages over other buffers commonly used in radiopharmaceutical preparation as it exhibits significantly lower complexation tendency with metal ions. However, use of HEPES buffer for radiolabeling reactions, meant for clinical applications, has been underrated due to the non-availability of sufficient toxicity data. The objective of the present study is to find the evidences towards safety of intravenous administration of HEPES through systemic toxicological studies in small animal model to support its safe application for clinical exploitation. A pilot study was performed to investigate the lethal dose of HEPES in female Sprague Dawley rats by administering seven different doses of HEPES solution (150 to 2000 mg/kg), through intravenous pathway. Similarly, for determining maximum tolerated dose (MTD), gradually increasing doses of HEPES (50 to 950 mg/kg) were administered in the same species via similar pathway. Various hematological and clinical pathological investigations were carried out in order to find out the safe administration dose of HEPES in rats. No mortality was observed up to 2000 mg/kg doses of HEPES. The doses beyond 300 mg/kg resulted few temporary adverse effects, though these were found to disappear within 4–5 days of dosing. The amount of HEPES to be administered during clinical intervention is usually much lower (typically 1–2.5 mg per kg of body weight of healthy adult) than the MTD determined in rat model during present report. Hence, the utilization of this buffer for preparation of radiolabeled drugs for human investigation may be safe. However, further detailed investigations may be warranted for supporting the candidature of Good's buffer for regular clinical exploitation. [Display omitted] [ABSTRACT FROM AUTHOR]

• HEPES acts as a salting-out agent for lysozyme crystallization and a salting-in agent for liquid–liquid phase separation (LLPS) of lysozyme-NaCl aqueous solutions. • HEPES increases metastability gap between crystal solubility and LLPS. This explains observed enhancement of LLPS-induced protein crystallization. • The opposite effects of HEPES on LLPS and protein crystallization are consistent with ability of HEPES to stabilize lysozyme crystal lattice. • Changing HEPES protonation state has no qualitative effect on observed LLPS-induced protein crystallization. • Changing HEPES with other similar organic molecules significantly reduce LLPS-induced protein crystallization. • Changing NaCl with phosphate buffer as LLPS inducer significantly reduce LLPS-induced protein crystallization. Aqueous solutions of lysozyme undergo LLPS by cooling in the presence of NaCl (0.1–0.2 M) around neutral pH. This phase transition is metastable with respect to protein crystallization. Addition of 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES, 0.1 M) to these solutions triggers a rapid, reproducible and high-yield formation of tetragonal lysozyme microcrystals through LLPS even if NaCl concentration is quite low. In this work, we demonstrate that HEPES causes opposite thermodynamic effects on solubility and LLPS boundaries in the protein phase diagram, leading to broadening of metastability gap. This increases thermodynamic driving force for crystal nucleation inside protein-rich liquid phase. The observed enhancement of LLPS-based crystallization is independent of HEPES protonation state. However, crystallization output is dramatically reduced when HEPES is replaced by other organic molecules sharing same functional groups. We attribute HEPES effect on crystallization to its ability to bind lysozyme inside crystal. Replacing NaCl with phosphate buffer as LLPS inducer radically reduces crystallization, consistent with NaCl being a more effective agent for lysozyme crystallization. This work shows that protein crystallization from metastable LLPS can be successfully enhanced by combining an additive that induces LLPS with another additive that increases metastability gap. [ABSTRACT FROM AUTHOR]

The blood-brain barrier (BBB) is the main entry route for chemicals into the mammalian central nervous system (CNS). Two transmembrane transporters of the ATP-binding cassette (ABC) family - breast cancer resistance protein (ABCG2 in humans, Abcg2 in rodents) and P-glycoprotein (ABCB1 in humans, Abcb1 in rodents) - play a key role in mediating this process. Pharmacological and genetic evidence suggests that Abcg2 prevents CNS access to a group of highly potent and selective O-arylcarbamate fatty-acid amidohydrolase (FAAH) inhibitors, which include the compound URB937 (cyclohexylcarbamic acid 3'-carbamoyl-6-hydroxybiphenyl-3-yl ester). To define structure-activity relationships of the interaction of these molecules with Abcg2, in the present study we tested various peripherally restricted and non-restricted O-arylcarbamate FAAH inhibitors for their ability to serve as transport substrates in monolayer cultures of Madin-Darby Canine Kidney-II (MDCKII) cells over-expressing Abcg2. Surprisingly, we found that the majority of compounds tested - even those able to enter the CNS in vivo - were substrates for Abcg2 in vitro. Additional experiments in MDCKII cells overexpressing ABCB1 revealed that only those compounds that were dual substrates for ABCB1 and Abcg2 in vitro were also peripherally restricted in vivo. The extent of such restriction seems to depend upon other physicochemical features of the compounds, in particular the polar surface area. Consistent with these in vitro results, we found that URB937 readily enters the brain in dual knockout mice lacking both Abcg2 and Abcb1, whereas it is either partially or completely excluded from the brain of mice lacking either transporter alone. The results suggest that Abcg2 and Abcb1 act together to restrict the access of URB937 to the CNS.

Clinical studies have shown that agonist-antagonist opioid analgesics that produce their analgesic effect via action on the kappa-opioid receptor, produce a delayed-onset anti-analgesia in men but not women, an effect blocked by co-administration of a low dose of naloxone. We now report the same time-dependent anti-analgesia and its underlying mechanism in an animal model. Using the Randall-Selitto paw-withdrawal assay in male rats, we found that nalbuphine, pentazocine, and butorphanol each produced analgesia during the first hour followed by anti-analgesia starting at ∼90min after administration in males but not females, closely mimicking its clinical effects. As observed in humans, co-administration of nalbuphine with naloxone in a dose ratio of 12.5:1 blocked anti-analgesia but not analgesia. Administration of the highly selective kappa-opioid receptor agonist U69593 produced analgesia without subsequent anti-analgesia, and confirmed by the failure of the selective kappa antagonist nor-binaltorphimine to block nalbuphine-induced anti-analgesia, indicating that anti-analgesia is not mediated by kappa-opioid receptors. We therefore tested the role of other receptors in nalbuphine anti-analgesia. Nociceptin/orphanin FQ (NOP) and sigma-1 and sigma-2 receptors were chosen on the basis of their known anti-analgesic effects and receptor binding studies. The selective NOP receptor antagonists, JTC801, and J-113397, but not the sigma receptor antagonist, BD 1047, antagonized nalbuphine anti-analgesia. Furthermore, the NOP receptor agonist NNC 63-0532 produced anti-analgesia with the same delay in onset observed with the three agonist-antagonists, but without producing preceding analgesia and this anti-analgesia was also blocked by naloxone. These results strongly support the suggestion that clinically used agonist-antagonists act at the NOP receptor to produce anti-analgesia.

Background: Nowadays, in Nuclear Medicine, clinically applied radiopharmaceuticals must meet quality release criteria such as high radiochemical purity and radiochemical yield. Many radiopharmaceuticals do not have marketing authorization and have no dedicated monograph within European Pharmacopeia (Ph. Eur.); therefore, general monographs on quality controls (QCs) have to be applied for clinical application. These criteria require standardization and validation in labeling and preparation, including quality controls measurements, according to well defined standard operation procedures. However, QC measurements are often based on detection techniques that are specific to a certain chromatographic system. Several radiosyntheses of [68Ga]Ga-radiopharmaceuticals are more efficient and robust when they are performed with 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES) buffer, which is considered as an impurity to be assessed in the QC procedure, prior to clinical use. Thus, Ph. Eur. has introduced a thin-layer chromatography (TLC) method to quantify the HEPES amount that is present in [68Ga]Ga-radiopharmaceuticals. However, this is only qualitative and has proven to be unreliable. Here we develop and validate a new high-performance liquid chromatography (UV-Radio-HPLC) method to quantify the residual amount of HEPES in 68Ga-based radiopharmaceuticals. Method: To validate the proposed UV-Radio-HPLC method, a stepwise approach was used, as defined in the guidance document that was adopted by the European Medicines Agency (CMP/ICH/381/95 2014). The assessed parameters are specificity, linearity, precision (repeatability), accuracy, and limit of quantification. A range of concentrations of HEPES (100, 80, 60, 40, 20, 10, 5, 3 μg/mL) were analyzed. Moreover, to test the validity and pertinence of our new HPLC method, we analyzed samples of [68Ga]Ga-DOTATOC; [68Ga]Ga-PSMA; [68Ga]Ga-DOTATATE; [68Ga]Ga-Pentixafor; and [68Ga]Ga-NODAGA-Exendin-4 from different batches that were prepared for clinical use. Results: In the assessed samples, HEPES could not be detected by the TLC method that was described in Ph. Eur. within 4 min incubation in an iodine-saturated chamber. Our developed HPLC method showed excellent linearity between 3 and 100 μg/mL for HEPES, with a correlation coefficient (R2) for calibration curves that was equal to 0.999, coefficients of variation (CV%) < 2%, and percent deviation value of bias from 100% to 5%, in accordance with acceptance criteria. The intra-day and inter-day precision of our method was statistically confirmed and the limit-of-quantification (LOQ) was 3 μg/mL, confirming the high sensitivity of the method. The amount of HEPES that was detected with our developed HPLC method in the tested [68Ga]Ga-radiopharmaceuticals resulted well below the Ph. Eur. limit, especially for [68Ga]Ga-NODAGA-Exendin-4. Conclusions: The TLC method that is described in Ph. Eur. to assess residual HEPES in [68Ga]-based radiopharmaceuticals may not be sufficiently sensitive and thus unsuitable for QC release. Our new HPLC method was sensitive, quantitative, reproducible, and rapid for QCs, allowing us to exactly determine the residual HEPES amount in [68Ga]Ga-radiopharmaceuticals for safe patient administration.

HEPES (4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid) is a buffer that is used in the radiolabelling of gallium-68 compounds. The beneficial effects of HEPES on molar activity in bioconjugates have been well described. Current strict regulations on the HEPES content in radiopharmaceuticals limit its use when intended for parenteral administration. This short communication summarizes data from the literature on the toxicity of HEPES in dogs after intravenous infusion and the subsequent use in humans. We also highlight the use of HEPES in an FDA labelled intravenous drug formulation. Regulatory institutions may consider this data to review current strict limits. [ABSTRACT FROM AUTHOR]

Preparation of cryoelectron microscopy (cryo-EM) grids for imaging of amyloid fibrils is notoriously challenging. The human islet amyloid polypeptide (hIAPP) serves as a notable example, as the majority of reported structures have relied on the use of nonphysiological pH buffers, N-terminal tags, and seeding. This highlights the need for more efficient, reproducible methodologies that can elucidate amyloid fibril structures formed under diverse conditions. In this work, we demonstrate that the distribution of fibrils on cryo-EM grids is predominantly determined by the solution composition, which is critical for the stability of thin vitreous ice films. We discover that, among physiological pH buffers, HEPES uniquely enhances the distribution of fibrils on cryo-EM grids and improves the stability of ice layers. This improvement is attributed to direct interactions between HEPES molecules and hIAPP, effectively minimizing the tendency of hIAPP to form dense clusters in solutions and preventing ice nucleation. Furthermore, we provide additional support for the idea that denatured protein monolayers forming at the interface are also capable of eliciting a surfactant-like effect, leading to improved particle coverage. This phenomenon is illustrated by the addition of nonamyloidogenic rat IAPP (rIAPP) to a solution of preaggregated hIAPP just before the freezing process. The resultant grids, supplemented with this "spectator protein", exhibit notably enhanced coverage and improved ice quality. Unlike conventional surfactants, rIAPP is additionally capable of disentangling the dense clusters formed by hIAPP. By applying the proposed strategies, we have resolved the structure of the dominant hIAPP polymorph, formed in vitro at pH 7.4, to a final resolution of 4 Å. The advances in grid preparation presented in this work hold significant promise for enabling structural determination of amyloid proteins which are particularly resistant to conventional grid preparation techniques., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

A highly sensitive detection platform for heparin was constructed via the utilization of a commercially available cationic fluorescent dye (cresyl violet acetate, CV) as a fluorescence probe. The electrostatic binding between CV and heparin quenched the fluorescence in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic (HEPES) buffer solution (10 mM, pH 7.1). CV was highly selective towards heparin over other potential inferring substances. The detection limit of heparin detection was 5.19 ng/mL, and the linear working range was 0 ∼ 1 μg/mL in HEPES solution. In 1 % serum, the detection platform based on the fluorescence "turn-off" behavior of CV was also successfully constructed with a detection limit of 5.86 ng/mL in the linear range of 0 ∼ 0.8 μg/mL. Moreover, the CV-heparin complex was considered a potential sensor platform for the detection of protamine because of its stronger affinity for heparin and protamine., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Good’s buffers are commonly used for cell culture and, although developed to have minimal to no biological impact, they cause alterations in cellular processes such as autophagy and lysosomal enzyme activity. Using Chinese hamster ovary cells and induced pluripotent stem cell-derived neurons, this study explores the effect of zwitterionic buffers, specifically HEPES, on lysosomal volume and Ca2+ levels. Certain zwitterionic buffers lead to lysosomal expansion and reduced lysosomal Ca2+. Care should be taken when selecting buffers for growth media to avoid detrimental impacts on lysosomal function.

Abstract Background HEPES is a favorable buffer for 68Ga-complexations in radiochemical laboratories. The drawback of this buffer is its prescribed limit of 200 μg per recommended application volume in the final formulation. Currently, a TLC test according to the European Pharmacopoeia (Ph. Eur.) has to be performed for quantification, but this analysis suffers from low reliability and reproducibility and is based on a subjective, semi-quantitative visual evaluation. In this study, the TLC method according to the Ph. Eur. and two literature-known HPLC assays for HEPES quantification were evaluated. Additionally, the development of an improved TLC method was performed. Results The assay according to Antunes et al. provided a reasonable quantification of HEPES using HPLC. Additionally, a reliable and conclusive TLC method was developed, which facilitates quantitative analysis by means of a pixel-based evaluation. A comparison of those two methods with the Ph. Eur. TLC assay pinpoints the superiority of the HPLC as well as the new TLC assay. Furthermore, evaluation of HEPES contents using both TLC assays by 28 subjects supported the conclusion that the newly developed TLC method is clearly favorable. Conclusion The TLC method according to the Ph. Eur. provides unsatisfactory results in terms of conclusiveness and reproducibility. In contrast, a reported HPLC assay showed valid results, with the drawback of high technical effort. An optimized alternative is provided by the improved TLC method described in this work that results in reliable outcomes and additionally offers quantitative analysis.

Background: The introduction of a GMP-certified 68Ga-generator spurred the application of 68Ga-radiopharmaceuticals. Several radiosynthesis of 68Ga-radiopharmaceuticals are more efficient and robust when performed with 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES) buffer, which is considered as an impurity in the quality control (QC) procedure. Thus, prior to clinical use, QC must be conducted to ensure that HEPES does not exceed the maximum dose of 200 μg/V Injected as described in European Pharmacopoeia (Ph Eur) for edotreotide. However, when applying the thin-layer chromatography (TLC) method described in the Ph Eur to quantify the HEPES amount present in the 68Ga-octreotide or in the remaining 68Ga-radiopharmaceuticals that were tested, no amount was detectable after 4 min of iodine incubation. Here we tested our modified TLC method and validate a new high-performance liquid chromatography (HPLC) method to quantify HEPES in 68Ga-radiopharmaceuticals and compare it to the TLC-method described in Ph Eur. In addition, samples collected from various institutes were tested to evaluate whether the synthesis of different 68Ga-radiopharmaceuticals or the use of different synthesis methods could affect the amounts of HEPES. Results: HEPES could not be detected by the TLC method described in the Ph Eur within 4 min incubation in an iodine-saturated chamber. As for our modified TLC method, only after 2 h, spots were only visible > 1 mg/mL. The HPLC method had a limit-of-quantification (LOQ) of 3 μg/mL and a limit-of-detection (LOD) of 1 μg/mL. From the three 68Ga-radiopharmaceuticals tested, only in the [68Ga]Ga-NODAGA-Exendin samples exceeding amounts of HEPES were found and its concentration in the [68Ga]Ga-NODAGA-Exendin was significantly higher, when compared to [68Ga]Ga-DOTATOC and [68Ga]Ga-PSMA-11. Conclusion: The TLC method described in Ph Eur and our modified TLC method may not be sufficiently sensitive and thus unsuitable to use for QC release. The new HPLC method was sensitive, quantitative, reproducible and suitable for QC release. With this method, we were able to determine that some 68Ga-radiopharmaceuticals may exceed the HEPES limit of 200 μg/ V Injected. This new analytical system would allow correcting for the maximum injected dose in order not to exceed this amount. [ABSTRACT FROM AUTHOR]

In this study, gold nanoparticles were supported at graphene derivatives (AuNPs@GDs) from chloroauric acid and graphene oxide (GO) by using sodium citrate, L-ascorbic acid, and 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES) as green reductants to evaluate the catalytic activity in the 4-nitrophenol (4-NP) reduction system. Among the three reductants, HEPES which derived AuNP@GD (3) exhibited up to 8.44-fold higher catalytic activity compared with sodium citrate and L-ascorbic acid. To evaluate the influence of mass ratio between Au and GO on the catalytic activity, we controlled adding amounts of chloroauric acid while the other conditions using HEPES were fixed. Notably, decreasing the mass ratio of Au/GO from 0.59 to 0.10 increased the rate constant in the range of 7.6 × 10−3 to 16.1 × 10−3/s. However, when the turnover frequency (TOF) was considered, the maximum value was obtained when Au/GO was 0.39. It can be concluded that AuNPs@GD (4) synthesized using HEPES significantly improved the catalytic activity in both rate constant (10.4 × 10−3/s) and TOF (8.08 × 10−5 mol/g·s) in 4-NP reduction. These results suggest that the type of reductants used to synthesize AuNPs@GDs is a significant factor on the catalytic activity and that GO can help increase the catalytic activity, but has an effective ratio with Au. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the remineralizing effects of aqueous phosphate-buffered solutions using various saturations with respect to octacalcium phosphate or brushite (OCP/ DCPD) on bovine enamel subsurface lesions. Method and Materials: Demineralized specimens (n = 18 per group) were exposed to one of six phosphate-buffered solutions with theoretical OCP saturations of S0.83, S1.17, S1.43, S1.64, S1.83, or S1.99 (pH, 6.3; calcium concentration, 0.53 to 3.18 mM). One aqueous solution without calcium was used as a negative control (S0; pH, 6.3); one without calcium and phosphate was a reference (S; pH, 4.3). HEPES-buffered Buskes solution served as a reference (B2.46; pH, 7.0); a pH-adjusted one was a positive control (B1.21; pH, 6.3). Mineral losses (whole lesion and surface area/inner part of the lesion) before and after storage (2 and 5 weeks, 37° C) were evaluated from microradiographs. Results: The pH values of all solutions remained stable. Compared to baseline, S0.83 to S1.99 and B2.46 to B1.21 showed significantly increased mineral gains after both storage periods (P < .05, paired t test). S0 showed neutral effects (P = .190), whereas S demineralized the specimens (P < .001). Storage in S1.64, S1.83, and S1.99 revealed no differences compared to B2.46 (P > .997, ANOVA and Tukey), but a mineral gain of S1.64 to S1.99 was significantly increased compared to B1.21 (P < .012). Similar results could be observed for surface areas and inner lesion parts. Conclusion: The in vitro conditions chosen revealed that the used phosphate buffer system was suitable to maintain stable pH values. The higher saturated (OCP) solutions S1.64, S1.83, and S1.99 revealed mineral gains comparable to B2.46; thus, saturations of 1.64 (OCP) or 1 (DCPD) might be preferable for remineralization studies. (Quintessence Int 2011;42:501-514) [ABSTRACT FROM AUTHOR]

Gold nanostars are being used more regularly in the biosensing field. Despite their useful attributes, there is still a need to optimize aspects of the synthesis and stability. The seedless, synthetic method comprising 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) is a facile, rapid method; however, it produces heteromorphic nanostars. The modification of a HEPES method resulted in a silver-assisted, seedless gold nanostar synthesis method. The nanostars resulting from this method were monodispersed, multi-branched and approximately 37 ± 2 nm in diameter. It proved to be a repeatable method that produced homogeneous and robust nanostars. Once functionalized with polyvinylpyrrolidone 10 000, the new nanostars were observed to be stable in various environments such as salt, ionic strength and cell culture medium. In conclusion, the addition of the silver nitrate improved the morphology of the reported HEPES nanostars for the purpose of nanobiosensor development.

HEPES is commonly used in cell culture media as a buffering substance. Compared to the bicarbonate/CO2 buffer system, it does not require a CO2 atmosphere, thereby ensuring stable pH values during handling of cell culture media outside of an incubator. Due to its intrinsic charge, HEPES is considered not to be taken up by cells, which was a prerequisite during buffer development for cell culture by Good and colleagues. However, during the last years, evidence has emerged that HEPES seems to be taken up into cells and that it has major effects on cellular functions. Investigating three different cell lines (MCF-7, U2OS, HeLa) showed that all of them accommodated HEPES-containing medium, i.e., they survive and proliferate in the presence of HEPES. Determination of intracellular metabolites revealed the presence of HEPES for all cell lines. Further analysis of MCF-7 cells showed that even 48 h after medium exchange from HEPES-containing medium to HEPES-free medium, intracellular HEPES could still be detected. Thus, contrary to the common view, HEPES is taken up by cells which should be taken into consideration for studies of specific cellular functions.ᅟ [ABSTRACT FROM AUTHOR]