Purification of Potassium Ion Channels Using Styrene-Maleic Acid Copolymers.

Structural studies require the production of target proteins in large quantities and with a high degree of purity. For membrane proteins, the bottleneck in determining their structure is the extraction of the target protein from the cell membranes. A detergent that improperly mimics the hydrophobic environment of the protein of interest can also significantly alter its structure. Recently, using lipodiscs with styrene-maleic acid (SMA), copolymers became a promising strategy for the purification of membrane proteins. Here, we describe in detail the one-step affinity purification of potassium ion channels solubilized in SMA and sample preparation for future structural studies.

Construction and application of H1R ligand screening materials based on SMA stabilization and his-tag covalent immobilization of membrane proteins.

The histamine H1 receptor (H1R) plays a pivotal role in allergy initiation and undergoes the necessity of devising a high-throughput screening approach centered on H1R to screen novel ligands effectively. This study suggests a method employing styrene maleic acid (SMA) extraction and His-tag covalent bonding to immobilize H1R membrane proteins, minimizing the interference of nonspecific proteins interference while preserving native protein structure and maximizing target exposure. This approach was utilized to develop a novel material for high-throughput ligand screening and implemented in cell membrane chromatography (CMC). An H1R-His-SMALPs/CMC model was established and its chromatographic performance (selectivity, specificity and lifespan) validated, demonstrating a significant enhancement in lifespan compared to previous CMC models. Subsequently, this model facilitated high-throughput screening of H1R ligands in the compound library and preliminary activity verification of potential H1R antagonists. Identification of a novel H1R antagonist laid the foundation for further development in this area.

Entomological surveys and insecticide susceptibility profile of Aedes aegypti during the dengue outbreak in Sao Tome and Principe in 2022.

BACKGROUND: The first dengue outbreak in Sao Tome and Principe was reported in 2022. Entomological investigations were undertaken to establish the typology of Aedes larval habitats, the distribution of Ae. aegypti and Ae. albopictus, the related entomological risk and the susceptibility profile of Ae. aegypti to insecticides, to provide evidence to inform the outbreak response. METHODOLOGY/PRINCIPAL FINDINGS: Entomological surveys were performed in all seven health districts of Sao Tome and Principe during the dry and rainy seasons in 2022. WHO tube and synergist assays using piperonyl butoxide (PBO) and diethyl maleate (DEM) were carried out, together with genotyping of F1534C/V1016I/V410L mutations in Ae. aegypti. Aedes aegypti and Ae. albopictus were found in all seven health districts of the country with high abundance of Ae. aegypti in the most urbanised district, Agua Grande. Both Aedes species bred mainly in used tyres, discarded tanks and water storage containers. In both survey periods, the Breteau (BI > 50), house (HI > 35%) and container (CI > 20%) indices were higher than the thresholds established by WHO to indicate high potential risk of dengue transmission. The Ae. aegypti sampled were susceptible to all insecticides tested except dichlorodiphenyltrichloroethane (DDT) (9.2% mortality, resistant), bendiocarb (61.4% mortality, resistant) and alpha-cypermethrin (97% mortality, probable resistant). A full recovery was observed in Ae. aegypti resistant to bendiocarb after pre-exposure to synergist PBO. Only one Ae. aegypti specimen was found carrying F1534C mutation. CONCLUSIONS/SIGNIFICANCE: These findings revealed a high potential risk for dengue transmission throughout the year, with the bulk of larval breeding occurring in used tyres, water storage and discarded containers. Most of the insecticides tested remain effective to control Aedes vectors in Sao Tome, except DDT and bendiocarb. These data underline the importance of raising community awareness and implementing routine dengue vector control strategies to prevent further outbreaks in Sao Tome and Principe, and elsewhere in the subregion.

Robust construction of polyvinyl alcohol intercalated graphene oxide nanofiltration membrane for desalination via pervaporation.

The construction and modification of a Graphene Oxide (GO) membrane, incorporating polyvinyl alcohol (PVA) cross-linked with maleic acid (MA) and supported by a nylon membrane, have been successfully completed. Systematic variations in PVA and MA concentrations were conducted to achieve membranes with favorable characteristics, stability, and excellent desalination performance. Optimization studies utilizing the Central Composite Design (CCD) revealed that the most optimal desalination results were obtained with 10 mL of PVA (0.1 mg mL-1) and 0.9 M of MA (GO-MA0.9-PVA10/Nylon membrane). Experimental findings demonstrated that the inclusion of PVA and MA resulted in an increased interlayer distance of GO and enhanced membrane stability. The addition of PVA increases GO membrane hydrophilicity, while the addition of MA reduces membrane hydrophilicity. The GO-MA0.9-PVA10/Nylon membrane exhibited the highest desalination performance, boasting a rejection value exceeding >99.9% and a permeance of 18.76 kg m-2.h-1 under 1% NaCl feed at a temperature of 50 °C. This membrane demonstrated consistent desalination performance stability over an extended period of up to 70 h. Moreover, it exhibited durability through 8 cycles of 24-h usage with washing treatment. In conclusion, the GO-MA0.9-PVA10/Nylon membrane is strongly recommended for practical applications, outperforming other membrane options based on the comprehensive evaluation of its stability and desalination efficiency.

Maleic acid crosslinked starch/polyvinyl alcohol blend films with improved barrier properties for packaging applications.

Incorporating starch, which is a potential biodegradable substitute for petroleum-based polymers, into conventional polymers is challenging owing to limitations in processability and weak-performing resulting materials. Herein, corn starch/polyvinyl alcohol (PVA) blend films (starch: PVA ratio of 50:50) were prepared via the solvent casting method using glycerol as a plasticizer and with varying concentrations of maleic acid as the crosslinking agent. Fourier transform infrared spectroscopy revealed the molecular interactions of the maleic acid crosslinker with the polymeric network of starch and PVA through an ester linkage. The properties of the films were strongly dependent on the maleic acid concentration. An increasing maleic acid concentration imparted hydrophobicity to the film; therefore, water swelling was significantly reduced, and water resistance was enhanced. The film containing 20 wt% maleic acid exhibited excellent barrier properties, with the lowest oxygen and water vapor transmission rates of 0.5 ± 0.2 cc/m2⋅day and 232.3 ± 5.4 g/m2⋅day, respectively. Moreover, the mechanical properties of the film improved with increasing crosslinking. This study demonstrates that the addition of maleic acid leads to an improvement in the overall performance of starch/PVA blend films. Therefore, maleic acid-crosslinked films can be used as barrier materials in food packaging applications.

Synthesis of guar gum maleate under dry conditions: Reaction kinetics and characterization.

In the present study, a novel environment friendly dry method for preparation of guar gum maleate (GGM) with varying degrees of substitution (DS; 0.02-1.04) was optimized. GGM with a maximum DS of 1.04 was successfully synthesized using guar gum (GG) and maleic anhydride (MA) in proportion of 1: 1 at 80 °C with 4 h of reaction time. The activation energy for the reaction was determined to be 36.91 ± 3.61 kJ mol-1 with pre-exponential factor of 1392 min-1. Esterification of GG was confirmed by FT-IR and 13C NMR. Analysis using size exclusion chromatography (SEC) indicated a decrease in weight average molecular weight (Mw) of the polymer with an increase in polydispersity index (PDI) due to esterification. In comparison with GG, GGM displayed increased hydrophobicity and reduced thermal stability, as analysed by differential scanning calorimetry (DSC). Rheological studies of GGM revealed that initial apparent viscosity decreased with increasing DS. For the first time, the study offered valuable insights on GGM synthesis under dry solvent-less reaction conditions enabling simpler and scalable synthesis process.

Integrative Salt Selection and Formulation Optimization: Perspectives of Disproportionation and Microenvironmental pH Modulation.

We report a novel utilization of a pH modifier as a disproportionation retardant in a tablet formulation. The drug molecule of interest has significant bioavailability challenges that require solubility enhancement. In addition to limited salt/cocrystal options, disproportionation of the potential salt(s) was identified as a substantial risk. Using a combination of Raman spectroscopy with chemometrics and quantitative X-ray diffraction in specially designed stress testing, we investigated the disproportionation phenomena. The learnings and insight drawn from crystallography drove the selection of the maleate form as the target API. Inspired by the fumarate form's unique stability and solubility characteristics, we used fumaric acid as the microenvironmental pH modulator. Proof-of-concept experiments with high-risk (HCl) and moderate-risk (maleate) scenarios confirmed the synergistic advantage of fumaric acid, which interacts with the freebase released by disproportionation to form a more soluble species. The resultant hemifumarate helps maintain the solubility at an elevated level. This work demonstrates an innovative technique to mediate the solubility drop during the "parachute" phase of drug absorption using compendial excipients, and this approach can potentially serve as an effective risk-mitigating strategy for salt disproportionation.

Novel class of peptides disintegrating biological membranes to aid in the characterization of membrane proteins.

Styrene-maleic acid (SMA) and similar amphiphilic copolymers are known to cut biological membranes into lipid nanoparticles/nanodiscs containing membrane proteins apparently in their relatively native membrane lipid environment. Our previous work demonstrated that membrane raft microdomains resist such disintegration by SMA. The use of SMA in studying membrane proteins is limited by its heterogeneity and the inability to prepare defined derivatives. In the present paper, we demonstrate that some amphiphilic peptides structurally mimicking SMA also similarly disintegrate cell membranes. In contrast to the previously used copolymers, the simple peptides are structurally homogeneous. We found that their membrane-disintegrating activity increases with their length (reaching optimum at 24 amino acids) and requires a basic primary structure, that is, (XXD)n, where X represents a hydrophobic amino acid (optimally phenylalanine), D aspartic acid, and n is the number of repeats of these triplets. These peptides may provide opportunities for various well-defined potentially useful modifications in the study of membrane protein biochemistry. Our present results confirm a specific character of membrane raft microdomains.

Characterization of two novel hydrolases from Sphingopyxis sp. DBS4 for enantioselective degradation of chiral herbicide diclofop-methyl.

Diclofop-methyl, an aryloxyphenoxypropionate (AOPP) herbicide, is a chiral compound with two enantiomers. Microbial detoxification and degradation of various enantiomers is garnering immense research attention. However, enantioselective catabolism of diclofop-methyl has been rarely explored, especially at the molecular level. This study cloned two novel hydrolase genes (dcmA and dcmH) in Sphingopyxis sp. DBS4, and characterized them for diclofop-methyl degradation. DcmA, a member of the amidase superfamily, exhibits 26.1-45.9% identity with functional amidases. Conversely, DcmH corresponded to the DUF3089 domain-containing protein family (a family with unknown function), sharing no significant similarity with other biochemically characterized proteins. DcmA exhibited a broad spectrum of substrates, with preferential hydrolyzation of (R)-(+)-diclofop-methyl, (R)-(+)-quizalofop-ethyl, and (R)-(+)-haloxyfop-methyl. DcmH also preferred (R)-(+)-quizalofop-ethyl and (R)-(+)-haloxyfop-methyl degradation while displaying no apparent enantioselective activity towards diclofop-methyl. Using site-directed mutagenesis and molecular docking, it was determined that Ser175 was the fundamental residue influencing DcmA's activity against the two enantiomers of diclofop-methyl. For the degradation of AOPP herbicides, DcmA is an enantioselective amidase that has never been reported in research. This study provided novel hydrolyzing enzyme resources for the remediation of diclofop-methyl in the environment and deepened the understanding of enantioselective degradation of chiral AOPP herbicides mediated by microbes.

High-throughput BCRP inhibitors screening system based on styrene maleic acid polymer membrane protein stabilization strategy and surface plasmon resonance biosensor.

Multidrug resistance (MDR) is a dominant challenge in cancer chemotherapy failure. The over-expression of breast cancer resistance protein (BCRP) in tumorous cells, along with its extensive substrate profile, is a leading cause of tumor MDR. Herein, on the basis of styrene maleic acid (SMA) polymer membrane protein stabilization strategy and surface plasmon resonance (SPR) biosensor, a novel high-throughput screening (HTS) system for BCRP inhibitors has been established. Firstly, LLC-PK1 and LLC-PK1/BCRP cell membranes were co-incubated with SMA polymers to construct SMA lipid particles (SMALPs). PK1-SMALPs were thus immobilized in channel 1 of the L1 chip as the reference channel, and BCRP-SMALPs were immobilized in channel 2 as the detection channel to establish the BCRP-SMALPs-SPR screening system. The methodological investigation demonstrated that the screening system was highly specific and stable. Three active compounds were screened out from 26 natural products and their affinity constants with BCRP were determined. The KD of xanthotoxin, bergapten, and naringenin were 5.14 µM, 4.57 µM, and 3.72 µM, respectively. The in vitro cell verification experiments demonstrated that xanthotoxin, bergapten, and naringenin all significantly increased the sensitivity of LLC-PK1/BCRP cells to mitoxantrone with possessing reversal BCRP-mediated MDR activity. Collectively, the developed BCRP-SMALPs-SPR screening system in this study has the advantages of rapidity, efficiency, and specificity, providing a novel strategy for the in-depth screening of BCRP inhibitors with less side effects and higher efficacy.

In Situ Monitoring of Competitive Coformer Exchange Reaction by 1H MAS Solid-State NMR.

In a competitive coformer exchange reaction, a recent topic of interest in pharmaceutical research, the coformer in a pharmaceutical cocrystal is exchanged with another coformer that is expected to form a cocrystal that is more stable. There will be a competition between coformers to form the most stable product through the formation of hydrogen bonds. This will cause destabilization of the pharmaceutical products during processing or storage. Therefore, it is important to develop a mechanistic understanding of this transformation by monitoring each and every step of the reaction, employing a technique such as 1H nuclear magnetic resonance (NMR). In this study, an in situ monitoring of a coformer exchange reaction is carried out by 1H magic angle spinning (MAS) solid-state NMR (SSNMR) at a spinning frequency of 60 kHz. The changes in caffeine maleic acid cocrystals on addition of glutaric acid and caffeine glutaric cocrystals on addition of maleic acid were monitored. In all of the reactions, it has been observed that caffeine glutaric acid Form I is formed. When glutaric acid was added to 2:1 caffeine maleic acid, the formation of metastable 1:1 caffeine glutaric acid Form I was observed at the start of the experiment, indicating that the centrifugal pressure is enough for the formation. The difference in the end product of the reactions with a similar reaction pathway of 1:1 and 2:1 reactant stoichiometry indicates that a complete replacement of maleic acid has occurred only in the 1:1 stoichiometry of the reactants. The polymorphic transition of caffeine glutaric acid Form II to Form I at higher temperatures was a crucial reason that triggered the exchange of glutaric acid with maleic acid in the reaction of caffeine glutaric acid and maleic acid. Our results are novel since the new reaction pathways in competitive coformer exchange reactions enabled understanding the remarkable role of stoichiometry, polymorphism, temperature, and centrifugal pressure.

Solubilization of Oligomeric Cell-Free Synthesized Proteins Using SMA Copolymers.

Although membrane proteins are abundant in nature, their investigation is limited due to bottlenecks in heterologous overexpression and consequently restricted accessibility for downstream applications. In this chapter, we address these challenges by presenting a fast and straightforward synthesis platform based on eukaryotic cell-free protein synthesis (CFPS) and an efficient solubilization strategy using styrene-maleic acid (SMA) copolymers. We demonstrate CFPS of TWIK-1, a dimeric ion channel, based on Sf21 (Spodoptera frugiperda) insect lysate showing homooligomerization and N-glycosylation enabled by endoplasmic reticulum-derived microsomes. Furthermore, we employ SMA copolymers for protein solubilization, which preserves the native-like microsomal environment. This approach not only retains the solubilized protein's suitability for downstream applications but also maintains the oligomerization and glycosylation of TWIK-1 post-solubilization. We validate the solubilization procedure using autoradiography, particle size analysis, and biomolecular fluorescence assay and confirm the very efficient, structurally intact solubilization of cell-free synthesized TWIK-1.

Study on tissue distribution, metabolite profiling, and excretion of [14C]-labeled flonoltinib maleate in rats.

Flonoltinib Maleate (FM) is a dual-target inhibitor that selectively suppresses Janus kinase 2/FMS-like tyrosine kinase 3 (JAK2/FLT3), which is currently in phase I/IIa clinical trial in China for the treatment of myeloproliferative neoplasms (MPNs). In this research, we used [14C]-labeled FM (14C-FM) to investigate the distribution, metabolism, and excretion of FM in rats using High-Performance Liquid Chromatography coupled with High-Resolution Mass Spectrometry/Radioactivity Monitoring (HPLC-HRMS/RAM) and liquid scintillation counter. The results revealed that FM displayed widespread distribution in rats. Furthermore, FM demonstrated rapid clearance without any observed risk of organ toxicity attributed to accumulation. Profiling of FM metabolites in rat plasma, feces, urine, and bile identified a total of 17 distinct metabolites, comprising 7 phase I metabolites and 10 phase II metabolites. The major metabolic reactions involved oxygenation, dealkylation, methylation, sulfation, glucuronidation and glutathione conjugation. Based on these findings, a putative metabolic pathway of FM in rats was proposed. The overall recovery rate in the excretion experiment ranged from 93.04 % to 94.74 %. The results indicated that FM undergoes extensive hepatic metabolism in SD rats, with the majority being excreted through bile as metabolites and ultimately eliminated via feces. A minor fraction of FM (<10 %) was excreted through renal excretion in the form of urine. Integration of the current results with previous pharmacokinetic investigations of FM in rats and dogs enables a comprehensive elucidation of the in vivo ADME processes and characteristics of FM, thereby establishing a solid foundation for subsequent clinical investigations of FM.

Dislodgement resistance and structural changes of tricalcium silicate-based cements after exposure to different chelating agents.

This study aimed to evaluate the dislodgement resistance and structural changes of different mineral trioxide aggregate cements (MTA) like Pro-Root MTA, Ortho MTA, and Retro MTA after exposure to sodium hypochlorite (NaOCl), NaOCl-Ethylenediaminetetraacetic acid (EDTA), 1-hydroxyethylidene-1, 1-bisphosphonate (Dual Rinse HEDP), and NaOCl-Maleic acid (MA). The root canal spaces of 150 dentine slices were obturated using tricalcium silicate cements and divided into 3 groups (n = 50): Group1: ProRoot MTA, Group2: Retro MTA, and Group3: Ortho MTA. The samples in each group were further subdivided into four experimental (n = 10) and one control groups (n = 10): 2.5% NaOCl-17% EDTA, Dual Rinse HEDP, 2.5% NaOCl-7% Maleic acid, 2.5% NaOCl, distilled water (control). The dislodgement resistance and structural changes of cements were measured. Use of DR HEDP resulted in higher dislodgement resistance compared to17% EDTA and 7% MA in the samples obturated with Ortho MTA and Pro-Root MTA (p<0.001). In Retro MTA group, samples treated with DR HEDP and 17% EDTA had higher dislodgment resistance compared to 7% MA (p<0.001). On microstructural and elemental analysis of all the three MTA cements, samples treated with 17% EDTA and 7% MA were more amorphous and granular when compared to DR HEDP, which was pettle shaped. Calcium level was decreased more in samples treated with 17% EDTA and 7% MA when compared to DR HEDP.

Critical roles of tubular mitochondrial ATP synthase dysfunction in maleic acid-induced acute kidney injury.

Maleic acid (MA) induces renal tubular cell dysfunction directed to acute kidney injury (AKI). AKI is an increasing global health burden due to its association with mortality and morbidity. However, targeted therapy for AKI is lacking. Previously, we determined mitochondrial-associated proteins are MA-induced AKI affinity proteins. We hypothesized that mitochondrial dysfunction in tubular epithelial cells plays a critical role in AKI. In vivo and in vitro systems have been used to test this hypothesis. For the in vivo model, C57BL/6 mice were intraperitoneally injected with 400 mg/kg body weight MA. For the in vitro model, HK-2 human proximal tubular epithelial cells were treated with 2 mM or 5 mM MA for 24 h. AKI can be induced by administration of MA. In the mice injected with MA, the levels of blood urea nitrogen (BUN) and creatinine in the sera were significantly increased (p < 0.005). From the pathological analysis, MA-induced AKI aggravated renal tubular injuries, increased kidney injury molecule-1 (KIM-1) expression and caused renal tubular cell apoptosis. At the cellular level, mitochondrial dysfunction was found with increasing mitochondrial reactive oxygen species (ROS) (p < 0.001), uncoupled mitochondrial respiration with decreasing electron transfer system activity (p < 0.001), and decreasing ATP production (p < 0.05). Under transmission electron microscope (TEM) examination, the cristae formation of mitochondria was defective in MA-induced AKI. To unveil the potential target in mitochondria, gene expression analysis revealed a significantly lower level of ATPase6 (p < 0.001). Renal mitochondrial protein levels of ATP subunits 5A1 and 5C1 (p < 0.05) were significantly decreased, as confirmed by protein analysis. Our study demonstrated that dysfunction of mitochondria resulting from altered expression of ATP synthase in renal tubular cells is associated with MA-induced AKI. This finding provides a potential novel target to develop new strategies for better prevention and treatment of MA-induced AKI.

Analysis of Contact Allergens in Polyvinyl Chloride Examination Gloves in the United States.

Background: Polyvinyl chloride (PVC) gloves are recommended as a safe alternative for patients with rubber accelerator allergy. However, allergic contact dermatitis to other chemicals in PVC gloves has been reported. Objective: To analyze single-use PVC medical examination gloves in the United States for the presence of potential contact allergens. Methods: Using liquid chromatography-mass spectrometry, 20 unique PVC gloves were analyzed in triplicate for 6 chemicals: benzisothiazolinone, bisphenol A, mono(2-ethylhexyl) maleate, tricresyl phosphate, triphenyl phosphate, and triphenyl phosphite. Results: All 20 PVC gloves contained detectable quantities of benzisothiazolinone (range, 0.001-1.48 parts per million [ppm]), bisphenol A (0.01-0.11 ppm), triphenyl phosphate (0.01-2.11 ppm), and triphenyl phosphite (0.001-0.22 ppm). Eighteen (90%) gloves contained mono(2-ethylhexyl) maleate (0.001-0.14 ppm) and 3 (15%) contained tricresyl phosphate (0.001-0.002 ppm). Conclusions: Known allergens were present in all 20 PVC gloves. However, the detected levels were mostly low and their relationship with sensitization and elicitation thresholds requires further study.

A randomized controlled trial assessing denture adhesive efficacy on denture retention across 13 hours.

PURPOSE: To compare the effects of two denture adhesive formulations on the bite force required to dislodge a maxillary denture in adult participants during a 13-h test period. MATERIALS AND METHODS: Twenty-two participants with a fair-to-poor fitting maxillary denture opposed by natural dentition or a stable mandibular denture were enrolled in this single-center, randomized, double-blind, two-treatment, 4-period crossover study. Participants were randomly assigned a product usage sequence so that each participant used each product twice during the 4-day test period. The test product was a denture cream adhesive formulated with an optimized calcium/zinc partial salt of polyvinyl methyl ether/maleic acid (Fixodent Ultra technology); the control product was a cream adhesive formulated with a calcium/zinc partial salt of polyvinyl methyl ether/maleic acid (Fixodent Original technology). On each study day, bite force at dislodgement was measured with a gnathodynamometer at baseline, representing the "no adhesive" score. Then, after standardized product application to the participant's existing maxillary denture by site staff, bite force measurements were retaken at 1, 3, 5, 7, 9, 11, and 13 h. The change from baseline and the 13-h area under the bite-force-change-from-baseline curve were analyzed via an analysis of variance. RESULTS: Twenty-one participants completed all test periods; one additional participant completed three test periods so 22 participants were included in the analysis. There were 15 females and 7 males with a mean age of 70 years. The mean 13-h area under the bite-force-change-from-baseline curve was 8% greater (p = 0.010) for the test adhesive (114.3 lb) than for the control adhesive (105.9 lb). Both adhesives showed a statistically significant increase in bite force (p < 0.001) at each time point compared to no adhesive. CONCLUSIONS: The optimized calcium/zinc partial salt of polyvinyl methyl ether/maleic acid test adhesive provided superior maxillary denture retention relative to that of the control adhesive across 13 h. Both adhesives increased bite force at dislodgement compared to no adhesive.

Uptake Pathway of Styrene Maleic Acid Copolymer-Coated Lipid Emulsions Under Acidic Tumor Microenvironment.

The purpose of this study was to elucidate and compare styrene maleic acid copolymer (SMA)-coated lipid emulsions (SMA emulsions) uptake pathway in vascular endothelial cells and surrounding cancer cells under not only neutral but also acidic pH, which is often observed in tumor microenvironment. DiI-labeled SMA emulsions were prepared using 1-palmitoyl-2-oleoyl-sn­glycero-3-phosphocholine and triolein. In murine melanoma B16-BL6 (B16) cells and human umbilical vein endothelial cells (HUVEC), DiI-labeled SMA emulsions uptake under near-neutral (pH 7.4) and acidic (pH 6.0) conditions was determined by fluorescent analysis. SMA emulsions were taken up more efficiently into HUVEC than B16 cells under acidic condition in a temperature-dependent manner. Uptake study using endocytosis inhibitors showed that SMA emulsions were taken up by macropinocytosis and clathrin-mediated endocytosis in B16 cells. In HUVEC, however, they were taken up by clathrin- and caveolae-independent, but dynamin-dependent pathway. SMA emulsions would be internalized efficiently into vascular endothelial cells as well as cancer cells under acidic microenvironment via different endocytosis pathways. SMA emulsions could be a promising drug delivery carrier for anti-angiogenic drugs.

Chemically-Induced Lipoprotein Breakdown for Improved Extracellular Vesicle Purification.

Extracellular vesicles (EVs) are nanosized biomolecular packages involved in intercellular communication. EVs are released by all cells, making them broadly applicable as therapeutic, diagnostic, and mechanistic components in (patho)physiology. Sample purity is critical for correctly attributing observed effects to EVs and for maximizing therapeutic and diagnostic performance. Lipoprotein contaminants represent a major challenge for sample purity. Lipoproteins are approximately six orders of magnitude more abundant in the blood circulation and overlap in size, shape, and density with EVs. This study represents the first example of an EV purification method based on the chemically-induced breakdown of lipoproteins. Specifically, a styrene-maleic acid (SMA) copolymer is used to selectively breakdown lipoproteins, enabling subsequent size-based separation of the breakdown products from plasma EVs. The use of the polymer followed by tangential flow filtration or size-exclusion chromatography results in improved EV yield, preservation of EV morphology, increased EV markers, and reduced contaminant markers. SMA-based EV purification enables improved fluorescent labeling, reduces interactions with macrophages, and enhances accuracy, sensitivity, and specificity to detect EV biomarkers, indicating benefits for various downstream applications. In conclusion, SMA is a simple and effective method to improve the purity and yield of plasma-derived EVs, which favorably impacts downstream applications.

Mutations in DNAJC19 cause altered mitochondrial structure and increased mitochondrial respiration in human iPSC-derived cardiomyocytes.

BACKGROUND: Dilated cardiomyopathy with ataxia (DCMA) is an autosomal recessive disorder arising from truncating mutations in DNAJC19, which encodes an inner mitochondrial membrane protein. Clinical features include an early onset, often life-threatening, cardiomyopathy associated with other metabolic features. Here, we aim to understand the metabolic and pathophysiological mechanisms of mutant DNAJC19 for the development of cardiomyopathy. METHODS: We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) of two affected siblings with DCMA and a gene-edited truncation variant (tv) of DNAJC19 which all lack the conserved DnaJ interaction domain. The mutant iPSC-CMs and their respective control cells were subjected to various analyses, including assessments of morphology, metabolic function, and physiological consequences such as Ca2+ kinetics, contractility, and arrhythmic potential. Validation of respiration analysis was done in a gene-edited HeLa cell line (DNAJC19tvHeLa). RESULTS: Structural analyses revealed mitochondrial fragmentation and abnormal cristae formation associated with an overall reduced mitochondrial protein expression in mutant iPSC-CMs. Morphological alterations were associated with higher oxygen consumption rates (OCRs) in all three mutant iPSC-CMs, indicating higher electron transport chain activity to meet cellular ATP demands. Additionally, increased extracellular acidification rates suggested an increase in overall metabolic flux, while radioactive tracer uptake studies revealed decreased fatty acid uptake and utilization of glucose. Mutant iPSC-CMs also showed increased reactive oxygen species (ROS) and an elevated mitochondrial membrane potential. Increased mitochondrial respiration with pyruvate and malate as substrates was observed in mutant DNAJC19tv HeLa cells in addition to an upregulation of respiratory chain complexes, while cellular ATP-levels remain the same. Moreover, mitochondrial alterations were associated with increased beating frequencies, elevated diastolic Ca2+ concentrations, reduced sarcomere shortening and an increased beat-to-beat rate variability in mutant cell lines in response to ß-adrenergic stimulation. CONCLUSIONS: Loss of the DnaJ domain disturbs cardiac mitochondrial structure with abnormal cristae formation and leads to mitochondrial dysfunction, suggesting that DNAJC19 plays an essential role in mitochondrial morphogenesis and biogenesis. Moreover, increased mitochondrial respiration, altered substrate utilization, increased ROS production and abnormal Ca2+ kinetics provide insights into the pathogenesis of DCMA-related cardiomyopathy.