Isotonic Buffer Research Articles

Three-dimensional isotropic imaging of live suspension cells enabled by droplet microvortices

Fast, nondestructive three-dimensional (3D) imaging of live suspension cells remains challenging without substrate treatment or fixation, precluding scalable single-cell morphometry with minimal alterations. While optical sectioning techniques achieve 3D live cell imaging, lateral versus depth resolution differences further complicate analysis. We present a scalable microfluidic method capable of 3D fluorescent isotropic imaging of live, nonadherent cells suspended inside picoliter droplets with high-speed single-cell volumetric readout (800 to 1,200 slices in 5 to 8 s) and near-diffraction limit resolution (~216 nm). The platform features a droplet trap array that leverages flow-induced droplet interfacial shear to generate intradroplet microvortices, which rotate single cells on their axis to enable optical projection tomography (OPT)-based imaging. This allows gentle (~1 mPa shear stress) observation of cells encapsulated inside nontoxic isotonic buffer droplets, facilitating scalable OPT acquisition by simultaneous spinning of hundreds of cells. We demonstrate 3D imaging of live myeloid and lymphoid cells in suspension, including K562 cells, as well as naive and activated T cells-small cells prone to movement in their suspended phenotype. Our fully suspended, orientation-independent cell morphometry, driven by isotropic imaging and spherical harmonic analysis, enabled the study of primary T cells across various immunological activation states. This approach unveiled six distinct nuclear content distributions, contrasting with conventional 2D images that typically portray spheroid and bean-like nuclear shapes associated with lymphocytes. Our arrayed-droplet OPT technology is capable of isotropic, single live-cell 3D imaging, with the potential to perform large-scale morphometry of immune cell effector function states while providing compatibility with microfluidic droplet operations.

Optimization of Cell Membrane Purification for the Preparation and Characterization of Cell Membrane Liposomes.

Cell membrane nanoparticles have attracted increasing interest in nanomedicine because they allow to exploit the complexity of cell membrane interactions for drug delivery. Several methods are used to obtain plasma membrane to generate cell membrane nanoparticles. Here, an optimized method combining nitrogen cavitation in isotonic buffer and sucrose gradient fractionation is presented. The method allows to obtain cell membrane fractions of high purity from both suspension and adherent cells. Comparison with other common methods for membrane extraction, where mechanical lysis using cell homogenizers is performed in isotonic or hypotonic buffers, shows that the optimized procedure yields high purity membrane in a robust and reproducible way. Procedures to mix the purified membrane with synthetic lipids to obtain cell membrane liposomes (CMLs) are presented and indications on how to optimize these steps are provided. CMLs made using crude membrane isolates or the purified membrane fractions show different uptake by cells. The CMLs made with the optimized procedure and liposomes of the same composition but without cell membrane components are thoroughly characterized and compared for their size, zeta potential, bilayer and mechanical properties to confirm membrane protein inclusion in the CMLs. Cell uptake studies confirm that the inclusion of membrane components modifies liposome interactions with cells.

An osmolarity dependent mechanism partially ameliorates retinal cysts and rescues cone function in a mouse model of X-linked retinoschisis.

X-linked retinoschisis (XLRS) is a vitreoretinal dystrophy caused by RS1 gene mutations which disrupt retinoschisin-1 (RS1) function. Vital for retinal architecture, the absence of functional RS1 leads to the development of intraretinal cysts. Intravitreal injection of a gene therapy for treating XLRS caused ocular inflammation in high dose groups in a phase I/II clinical trial. This study investigates a low dose subretinal gene therapy in Rs1 knockout (Rs1-KO) mice compared to injection of buffer alone. Observation of an unexpected therapeutic effect following the subretinal injection of the hypertonic buffer led to novel findings in XLRS. Rs1-KO mice were subretinally injected with an AAV2/4 vector (n = 10) containing the RS1 gene driven by an Ef1α promoter, a hypertonic buffer (n = 15) (180 mM NaCl 0.001% F68/PBS (pH 7.4)), or isotonic buffer (n = 7) (155.2 mM NaCl 0.001% F68/PBS, pH 7.0). A sham puncture group was also included (n = 6). Endpoints included electroretinogram (ERG), optical coherence tomography (OCT), a visually guided swim assay (VGSA), and immunohistochemistry. Unexpectedly, hypertonic buffer-injected eyes had reduced cyst severity at 1-month post-injection (MPI) (p < 0.0001), higher amplitudes in cone-dominant ERGs persisting to 5 MPI (5 Hz flicker; p < 0.0001; 3.0 flash; p = 0.0033) and a trend for improved navigational vision in the light compared to untreated Rs1-KO eyes. To investigate the role of tonicity on this effect, an isotonic buffer-injected cohort was created (155.2 mM NaCl 0.001% F68/PBS, pH 7.0) (n = 7). Surprisingly, hypertonic buffer-injected eyes exhibited a greater reduction in cyst severity and demonstrated improved cone-dominant ERG metrics over isotonic buffer-injected and sham puncture eyes. An immunohistochemistry assay demonstrated greater cone density in hypertonic buffer-injected eyes than untreated Rs1-KO eyes at 5-6 MPI (p = 0.0198), suggesting a possible cone preservation mechanism. Moreover, our findings reveal a negative correlation between the peak severity of cysts and long-term ERG amplitudes in cone-dominant pathways, implying that effectively managing cysts could yield enduring benefits for cone function. This study presents evidence that cyst resolution can be triggered through an osmolarity-dependent pathway, and early cyst resolution has long-term effects on cone signaling and survival, offering potential insights for the development of novel treatments for XLRS patients.

Mechanism study on the influences of buffer osmotic pressure on microfluidic chip-based cell electrofusion.

Cell electrofusion is a key process in many research fields, such as genetics, immunology, and cross-breeding. The electrofusion efficiency is highly dependent on the buffer osmotic pressure properties. However, the mechanism by which the buffer osmotic pressure affects cell electrofusion has not been theoretically or numerically understood. In order to explore the mechanism, the microfluidic structure with paired arc micro-cavities was first evaluated based on the numerical analysis of the transmembrane potential and the electroporation induced on biological cells when the electrofusion was performed on this structure. Then, the numerical model was used to analyze the effect of three buffer osmotic pressures on the on-chip electrofusion in terms of membrane tension and cell size. Compared to hypertonic and isotonic buffers, hypotonic buffer not only increased the reversible electroporation area in the cell-cell contact zone by 1.7 times by inducing a higher membrane tension, but also significantly reduced the applied voltage required for cell electroporation by increasing the cell size. Finally, the microfluidic chip with arc micro-cavitieswas fabricated and tested for electrofusion of SP2/0 cells. The results showed that no cell fusion occurred in the hypertonic buffer. The fusion efficiency in the isotonic buffer was about 7%. In the hypotonic buffer, the fusion efficiency was about 60%, which was significantly higher compared to hypertonic and isotonic buffers. The experimental results were in good agreement with the numerical analysis results.

Disrupting Na+ ion homeostasis and Na+/K+ ATPase activity in breast cancer cells directly modulates glycolysis in vitro and in vivo

BackgroundGlycolytic flux is regulated by the energy demands of the cell. Upregulated glycolysis in cancer cells may therefore result from increased demand for adenosine triphosphate (ATP), however it is unknown what this extra ATP turnover is used for. We hypothesise that an important contribution to the increased glycolytic flux in cancer cells results from the ATP demand of Na+/K+-ATPase (NKA) due to altered sodium ion homeostasis in cancer cells.MethodsLive whole-cell measurements of intracellular sodium [Na+]i were performed in three human breast cancer cells (MDA-MB-231, HCC1954, MCF-7), in murine breast cancer cells (4T1), and control human epithelial cells MCF-10A using triple quantum filtered 23Na nuclear magnetic resonance (NMR) spectroscopy. Glycolytic flux was measured by 2H NMR to monitor conversion of [6,6-2H2]d-glucose to [2H]-labelled l-lactate at baseline and in response to NKA inhibition with ouabain. Intracellular [Na+]i was titrated using isotonic buffers with varying [Na+] and [K+] and introducing an artificial Na+ plasma membrane leak using the ionophore gramicidin-A. Experiments were carried out in parallel with cell viability assays, 1H NMR metabolomics of intracellular and extracellular metabolites, extracellular flux analyses and in vivo measurements in a MDA-MB-231 human-xenograft mouse model using 2-deoxy-2-[18F]fluoroglucose (18F-FDG) positron emission tomography (PET).ResultsIntracellular [Na+]i was elevated in human and murine breast cancer cells compared to control MCF-10A cells. Acute inhibition of NKA by ouabain resulted in elevated [Na+]i and inhibition of glycolytic flux in all three human cancer cells which are ouabain sensitive, but not in the murine cells which are ouabain resistant. Permeabilization of cell membranes with gramicidin-A led to a titratable increase of [Na+]i in MDA-MB-231 and 4T1 cells and a Na+-dependent increase in glycolytic flux. This was attenuated with ouabain in the human cells but not in the murine cells. 18FDG PET imaging in an MDA-MB-231 human-xenograft mouse model recorded lower 18FDG tumour uptake when treated with ouabain while murine tissue uptake was unaffected.ConclusionsGlycolytic flux correlates with Na+-driven NKA activity in breast cancer cells, providing evidence for the ‘centrality of the [Na+]i-NKA nexus’ in the mechanistic basis of the Warburg effect.

Formulation and Evaluation of Betulin Loaded Transdermal Patches

Objectives: To develop and evaluate Transdermal patches of Betulin along with various polymers for controlled release action. Method: Suitable method such as Solvent Casting Technique of Film Casting Technique are used for preparation of Transdermal patch. Result: The prepared Transdermal patches were transparent, smooth, uniform and flexible. The method adopted for preparation of system was found satisfactory. Conclusion: Various formulations were developed by using hydrophilic and hydrophobic polymers like HPMC E5 and EC respectively in single and combinations by solvent evaporation technique with incorporation of penetration enhancer such as dimethylsulfoxide and dibutyl phthalate as plasticizer. Formulation F7 containing equal ratio of HPMC E5: EC (5:5) showed maximum percentage drug content 95.44%. A suitable UV Spectroscopy method for the analysis of Betulin was developed. Betulin showed maximum absorption at wave length 215 nm in isotonic phosphate buffer (pH 7.4) solutions. The pre-formulation studies involving description, solubility, melting point, partition coefficient of the drug were found to be comparable with the standard. The Transdermal patch of Betulin was prepared successfully by solvent evaporation method. Transdermal patch of Betulin for Transdermal drug delivery was evaluated. Calibration curve was obtained; Transdermal film was prepared, overcome limitations regarding bioavailability of drug was done. Keywords: Controlled DDS, Transdermal DDS, Betulin, Transdermal Patch, solvent evaporation method.

Hypertonicity and/or Acidosis Induce Marked Rheological Changes Under Hypoxic Conditions in Sickle Trait Red Blood Cells

Hemoglobin S (HbS) is an abnormal hemoglobin resulting from a single nucleotide substitution in the beta-globin gene. Individuals who are heterozygous for the HbS genotype are regarded as carriers of sickle cell anemia (SCA; HbSS) and are said to have sickle cell trait (SCT; HbAS). Recent data have established SCT as a risk factor for venous thromboembolism (VTE), chronic kidney disease (CKD), and end-stage renal disease (ESRD) (Naik RP, 2018). These complications have a relative risk of approximately 1.5 - 2.0 compared to race- and age-matched controls, but the mechanisms that account for these complications are not well understood. To study potential mechanisms for renal disease, we evaluated red blood cell (RBC) deformability from SCT donors under conditions designed to mimic the hypertonic, acidemic, and hypoxemic environment of the inner renal medulla. RBC deformability was measured using the Laser Optical Rotational Red Cell Analyzer (LORRCA; RR Mechatronics, The Netherlands). In SCA, LORRCA has been used to monitor RBC rigidity at baseline and under progressive hypoxia by calculating the Elongation Index (EI; the longitudinal dimension of the RBC along the axis of applied shear stress divided by transverse dimension of the cell along the same axis). The OxygenScan function measures EI during a cycle of continuously decreasing pO2 from about 150 mmHg (ambient air) to near 20 mm Hg, before reversing in a re-oxygenation phase. In SS RBCs, EI decreases from a stable baseline as RBCs start to sickle; this ‘point of sickling (PoS)‘ is defined as the pO2 at which there is a &amp;gt;5% decrease in EI. Similarly, the Osmoscan function can record the RBC EI at ambient oxygenation while osmolality of the blood progressively increases from approximately 100 to 600 mOsm. First, OxygenScan profiles were performed utilizing modified LORRCA buffers to evaluate four separate conditions: isotonic + neutral pH, isotonic + pH 6.9, 460 mOsm + neutral pH, and 460 mOsm + pH 6.9. Buffers were modified by the addition of sodium chloride, hydrochloric acid, or both. HbS percentage (HbS%) and RBC indices were recorded for each subject. HbS% and RBC indices were compared to PoS for all samples and conditions. EI from AS and AA samples were compared at physiologic serum osmolality (290 mOsm) and at 460 mOsm. AA samples demonstrated no measurable PoS under any condition. Some (11/21) AS samples demonstrated sickling under hypoxia alone, with mean PoS of 32.26 mmHg. Mean PoS for AS samples in isotonic buffer + pH 6.9 was 40.73 mmHg, but with sickling detected in all samples. Mean PoS in hypertonic buffer (460 mOsm) significantly increased to 60.68 mmHg, with notable inter-individual heterogeneity (Fig. 1). Mean PoS in hypertonic buffer + pH 6.9 was 79.04 mmHg, with less inter-sample heterogeneity. In exploratory experiments, we have observed similar changes in the Townes AS murine model of SCT (not shown). PoS was compared to HbS% in the human samples under all conditions. A positive correlation was observed between HbS% and PoS in isotonic buffer at pH 6.9 (r=0.566, p=0.012). PoS was also compared to RBC indices, with a trend towards a positive correlation with mean corpuscular hemoglobin concentration (MCHC) under hypertonic + neutral pH conditions ( p=0.11). Next, utilizing the Osmoscan function, no difference in EI at physiologic osmolality (290 mOsm) was observed between AA (n= 7) vs. AS (n= 8). However, at 460 mOsm, EI trended lower in AS RBCs ( p=0.12) (Fig. 2). Given the presence of severe tissue hypoxia (as low as 10 mmHg), hypertonicity (as high as 1,200 mOsm), and acidemia (as low as pH 5.5) in the inner renal medulla, these data provide an explanation for RBC-mediated occlusion of the vasa recta that is known to occur in the kidneys of subjects with SCT. We demonstrate that extracellular hypertonicity contributes significantly to SCT RBC sickling in the presence of hypoxemia, and that there is significant inter-individual susceptibility to sickling under these harsh biophysical conditions that is only partially explained by HbS%.

Feasibility of Extracorporeal Delivery of Fusosomes to Generate CAR T Cells In Vivo

Fusosomes are viral vectors pseudotyped with modified paramyxovirus envelopes targeting specific cell types. A CD8-targeted fusosome delivering a CD19CAR transgene has the potential to provide an off-the-shelf therapeutic approach to generate in vivo CD19-directed chimeric antigen receptor (CAR) T cells in patients. For an in vivo gene delivery platform there are several approaches for administration that could be considered including direct intravenous (IV) administration or extracorporeal delivery (ECD). With ECD, patients would first undergo apheresis, then fusosomes would be retained with patient peripheral blood mononuclear cells (PBMCs) in the apheresis bag for a brief period of time before the entire contents of the bag is IV delivered into the same patient. Here, we demonstrate the feasibility of this approach to generate in vivo CAR T cells in a clinical setting with a mock infusion and discuss the translation of these findings to direct IV administration. A healthy donor was apheresed to collect PBMCs using standard apheresis equipment. No further cell enrichment, separation, or washing was performed. CD8-targeted fusosome formulated in an isotonic cryoprotective buffer was administered into the apheresis bag by gravity via an infusion set at a dose of ~0.5 IU/cell, gently mixed and retained for 30 min. The contents of the bag were mock infused into sample collection bags at a controlled flow rate using an infusion pump. Samples were collected before and after fusosome addition, after 30 min retention, and every 30 min during the mock infusion. Cell counts and viability were measured by complete blood count (CBC) and flow cytometry analyses. Fusosome binding to CD8 + T cells was measured by flow cytometry. Cytokines and complement components in supernatants were measured by ELISA or MSD. Residual fusosome in the supernatant was measured by ddPCR. Transduction of CD8 + T cells was measured by flow cytometry and vector copy number (VCN) analysis after PBMCs were washed, activated with CD3/CD28 beads, and cultured for 8 days. Early transcription events were measured by ddPCR using specific amplicons targeting different DNA species generated during reverse transcription. The CD8-targeted fusosome was successfully deliveredusing aseptic techniques to a freshly collected apheresis bag.PBMC viability stayed high (&amp;gt;%95) throughout the process. There were no major changes in white blood cell, platelet, or red blood cell levels as measured by CBC analysis. There were no changes in CD8 + T cell phenotype throughout the time course. Fusosome binding to CD8 + T cells was detected as early as fusosome addition to the apheresis bag (~5 min) and increased over the course of the process. Analysis of vector genomes in the supernatants indicated presence of unbound residual fusosome. Exposure of PBMC to fusosome did not induce cytokine secretion or complement activation throughout the process. Early events of transduction of cells could be detected by analysis of reverse transcription intermediates. Analysis of cultured cells via both flow cytometry and VCN analysis indicated generation of CD8 + CAR T cells upon short-term exposure to fusosome. In vivo delivery ofCD8-targeted fusosome encoding a CD19CAR transgene enables a novel CAR T cell approach. This study demonstrated that administration of fusosomes via ECD is operationally feasible in a clinical setting and scale as a novel method of administration to generate CD8 + CD19CAR T cells in vivo. ECD setup also served as an in vitro pharmacokinetic model for direct IV infusion. In scenarios where in vivo exposure is limited due to very rapid clearance (e.g. few minutes), ECD has the benefit of reducing the volume of distribution and bringing T cells and fusosomes in close proximity for efficient gene delivery. The findings of study suggest that with rapid binding of fusosomes to CD8 + T cell leading to early transcription events, and an in vivo half-life of more than 30 min, fusosome may generate CAR T cells successfully via direct IV administration.

SLC22A11 Inserts the Uremic Toxins Indoxyl Sulfate and P-Cresol Sulfate into the Plasma Membrane.

Chronic kidney disease (CKD) is a global health concern affecting millions worldwide. One of the critical challenges in CKD is the accumulation of uremic toxins such as p-cresol sulfate (pCS) and indoxyl sulfate (IS), which contribute to systemic damage and CKD progression. Understanding the transport mechanisms of these prominent toxins is essential for developing effective treatments. Here, we investigated whether pCS and IS are routed to the plasma membrane or to the cytosol by two key transporters, SLC22A11 and OAT1. To distinguish between cytosolic transport and plasma membrane insertion, we used a hyperosmolarity assay in which the accumulation of substrates into HEK-293 cells in isotonic and hypertonic buffers was measured in parallel using LC-MS/MS. Judging from the efficiency of transport (TE), pCS is a relevant substrate of SLC22A11 at 7.8 ± 1.4 µL min-1 mg protein-1 but not as good as estrone-3-sulfate; OAT1 translocates pCS less efficiently. The TE of SLC22A11 for IS was similar to pCS. For OAT1, however, IS is an excellent substrate. With OAT1 and p-aminohippuric acid, our study revealed an influence of transporter abundance on the outcomes of the hyperosmolarity assay; very high transport activity confounded results. SLC22A11 was found to insert both pCS and IS into the plasma membrane, whereas OAT1 conveys these toxins to the cytosol. These disparate transport mechanisms bear profound ramifications for toxicity. Membrane insertion might promote membrane damage and microvesicle release. Our results underscore the imperative for detailed structural inquiries into the translocation of small molecules.

Detection of Methicillin-Resistant Staphylococcus Aureus Bacteria Using Liquid Crystals

Methicillin-resistant Staphylococcus aureus (MRSA) is an essential pathogen for public health and this bacteria commonly cause serious infectious in humans. In recent years, MRSA bacteria are detected by the bacterial culture and nucleic acid-based methods which are time-consuming and labor-intensive. In this study, a novel liquid crystal (LC)-based biosensing system was developed to overcome these limitations. The objective of this study was to detect the presence of MRSA bacteria which prepared within the isotonic water and phosphate buffer saline (PBS). In this system, the binding of MRSA bacteria to the dimethyloctadecyl [3-(trimethoxysilyl) propyl] ammonium chloride (DMOAP)-coated surface disrupted the orientation of LCs, triggering a transition from a homeotropic orientation to a random one. This transition in the orientation of the LCs was observed as a change from a dark optical LC image to a bright one under a polarized light microscope (POM) and the reflection values of LC molecules were determined by using a spectrometer. Through this sensing mechanism, MRSA bacteria prepared within the isotonic water was detected ranging from the 9.2x103 CFU/mL to 9.2x107 CFU/mL concentration. Furthermore, MRSA bacteria prepared in PBS was detected in the concentration range of 7.1x104 CFU/mL to 7.1x108 CFU/mL by using this system.

Preparation and Characterization of Prednisolone Acetate Microemulsion for Ophthalmic Use

Background: Prednisolone acetate is an ester form of prednisolone. It is used topically as an ophthalmic suspension to treat many inflammatory ocular conditions, where its absorption from suspension is highly variable and has poor dose accuracy. Objectives: The main objective of this research is to formulate and evaluate prednisolone acetate microemulsion for ophthalmic use to increase solubility, residence time, and corneal permeability of the drug to enhance patient compliance and treatment efficacy. Methods: Twenty-four prednisolone acetate-loaded microemulsion (0.5%w/w) formulas were prepared using oleic acid, isopropyl myristate as (oil phase) (1:1), tween 80, labrasol, and cremophor EL as (surfactant), ethanol, polyethylene glycol 400, propylene glycol, transcutol P as co-surfactant and Sörensen isotonic phosphate buffer saline pH 7.4 as the aqueous phase at different Smix ratios (1:1), (1:2) and (2:1) by aqueous titration method to construct pseudoternary phase diagram to determine the existence of microemulsion region. All the prepared formulas were subjected to different evaluation tests to determine the optimum formula. Results: observations of the microemulsion showed that it had a clear and transparent yellowish color, formulation F9 composed of oleic acid and isopropyl myristate in a ratio (1:1) as oil, twee80 as a surfactant, and propylene glycol: ethanol (1:1) in a ratio (2:1) as cosurfactant gave the best particle size (10.18nm), polydispersity index (0.2216), zeta potential (-25,91), % of transmittance (99.382%±0. 09), and drug content (100±0.16). Microemulsion formulation provided considerably higher permeability than the marketed eye drop suspension (Optipred®) and improved bioavailability. Conclusions: The microemulsion-containing prednisolone acetate is a promising ocular carrier for the controlled release of prednisolone acetate in treating anterior segment inflammation. Received: Dec. 2022 Accepted March 2023 Published: Oct.2023

Enhancement of the viability of T cells electroporated with DNA via osmotic dampening of the DNA-sensing cGAS-STING pathway.

Viral delivery of DNA for the targeted reprogramming of human T cells can lead to random genomic integration, and electroporation is inefficient and can be toxic. Here we show that electroporation-induced toxicity in primary human T cells is mediated by the cytosolic pathway cGAS-STING (cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase-stimulator of interferon genes). We also show that an isotonic buffer, identified by screening electroporation conditions, that reduces cGAS-STING surveillance allowed for the production of chimaeric antigen receptor (CAR) T cells with up to 20-fold higher CAR T cell numbers than standard electroporation and with higher antitumour activity in vivo than lentivirally generated CAR T cells. The osmotic pressure of the electroporation buffer dampened cGAS-DNA interactions, affecting the production of the STING activator 2'3'-cGAMP. The buffer also led to superior efficiencies in the transfection of therapeutically relevant primary T cells and human haematopoietic stem cells. Our findings may facilitate the optimization of electroporation-mediated DNA delivery for the production of genome-engineered T cells.

Hypoosmosis alters hepatocyte mitochondrial morphology and induces selective release of carbamoyl phosphate synthetase 1.

Carbamoyl phosphate synthetase 1 (CPS1) is the most abundant hepatocyte mitochondrial matrix protein. Hypoosmotic stress increases CPS1 release in isolated mouse hepatocytes without cell death. We hypothesized that increased CPS1 release during hypoosmosis is selective and associates with altered mitochondrial morphology. Both ex vivo and in vivo models were assessed. Mouse hepatocytes and livers were challenged with isotonic or hypoosmotic (35 mosM) buffer. Mice were injected intraperitoneally with water (10% body weight) with or without an antidiuretic. Mitochondrial and cytosolic fractions were isolated using differential centrifugation, then analyzed by immunoblotting to assess subcellular redistribution of four mitochondrial proteins: CPS1, ornithine transcarbamylase (OTC), pyrroline-5-carboxylate reductase 1 (PYCR1), and cytochrome c. Mitochondrial morphology alterations were examined using electron microscopy. Hypoosmotic treatment of whole livers or hepatocytes led to preferential or increased mitochondrial release, respectively, of CPS1 as compared with two mitochondrial matrix proteins (OTC/PYCR1) and with the intermembrane space protein, cytochrome c. Mitochondrial apoptosis-induced channel opening using staurosporine in hepatocytes led to preferential CPS1 and cytochrome c release. The CPS1-selective changes were accompanied by dramatic alterations in ultrastructural mitochondrial morphology. In mice, hypoosmosis/hyponatremia led to increased liver vascular congestion and increased CPS1 in bile but not blood, coupled with mitochondrial structural alterations. In contrast, isotonic increase of intravascular volume led to a decrease in mitochondrial size with limited change in bile CPS1 compared with hypoosmotic conditions and absence of the hypoosmosis-associated histological alterations. Taken together, hepatocyte CPS1 is selectively released in response to hypoosmosis/hyponatremia and provides a unique biomarker of mitochondrial injury.NEW & NOTEWORTHY Exposure of isolated mouse livers, primary cultured hepatocytes, or mice to hypoosmosis/hyponatremia conditions induces significant mitochondrial shape alterations accompanied by preferential release of the mitochondrial matrix protein CPS1, a urea cycle enzyme. In contrast, the intermembrane space protein, cytochrome c, and two other matrix proteins, including the urea cycle enzyme ornithine transcarbamylase, remain preferentially retained in mitochondria. Therefore, hepatocyte CPS1 manifests unique mitochondrial stress response compartmentalization and is a sensitive sensor of mitochondrial hypoosmotic/hyponatremic injury.

Syringic acid Attenuates Oxidative Stress in Plasma and Peripheral Blood Mononuclear Cells of Patients with Acute Myeloid Leukemia

Syringic acid (SA) is a natural phenolic acid that possesses antioxidant properties. The current study aimed to assess the possible ameliorative effects of SA on oxidative stress in patients with acute myeloid leukemia (AML). Twenty-two healthy donors as well as 22 sex- and age-matched AML patients participated in the study. AML patients were at the time of diagnosis and before remission. The peripheral blood mononuclear cells (PBMCs) and plasma samples were obtained and divided into four groups. The groups include: 1) buffer (B), containing isotonic phosphate buffer saline (100 mM, pH 7.4, 1 hr); 2) OX, containing solution subjected to iron-mediated oxidation (2.7 µM, 1 hr); 3) SA, containing SA solution (10 µM, 1 h) as ROS quencher and 4) SA + OX in which samples were pretreated with 10 µM of SA for 1 h, and then exposed to OX solution (2.7 µM) for 1 h. The results indicated that SA caused a significant increase in the activity of glutathione peroxidase (GPX) in PBMCs. Of note, the treatment of PBMCs and plasma samples of AML patients with SA was able to normalize the altered levels of GPX, superoxide dismutase (SOD), and catalase (CAT). The antioxidant effect of SA was further confirmed by analyzing the total oxidant status, lipid peroxidation, and protein carbonylation in both plasma samples and PBMCs of AML patients. According to the results, it seems that SA has strong protective effects on oxidative stress by elevating the total antioxidant status (TAS) of PBMCs and plasma specimens from AML patients.

Alzheimer brains: increased soluble amyloid peptides, but decreased lipid rafts

AbstractBackgroundPET imaging studies of AD patients show progressive increases of fibrillar Aβ‐ amyloid. Because current PET ligands underestimate non‐fibrillar forms, we assayed soluble Aβ in AD and controls. To identify mechanisms responsible for soluble Aβ in AD brains, we examined lipid rafts (LR), where APP is enzymatically processed. Frontal cortex was compared with cerebellum, which has minimal AD pathology.MethodPostmortem samples (N=48) were age‐and sex matched for Braak scores. Soluble Aβ was extracted from homogenates of full thickness frontal cortex by isotonic buffer and mild detergent (RIPA; 1% NP40) and centrifuged (10,000g/1h). Supernates were assayed by dot blots with antibodies for Aβ40 and Aβ42 (Biolegend, San Diego, CA). Aβ was assayed as relative fluorescent units per gm brain. LR fractions were isolated from these same brains by kit (Invent Biotechnologies; LR‐039), validated by ultracentrifugal fractionation.ResultCompared with cognitively normal controls (Braak 0‐1), elevations of soluble Aβ40 and Aβ42 were similar for intermediate and later stage AD (Braak 2‐3 and 4‐6). Clinical grade AD had greater increase of soluble Aβ40 (+400%) than Aβ42 (+50%) (p&lt;0.05). LR raft yield per gram AD frontal cortex was 20% below controls (p&lt;0.05), while cerebellar LR did not differ by Braak score. The composition of LR had a similar pattern, with 35% AD decreases of total protein and cholesterol (p&lt;0.05); cerebellar LR again did not differ by Braak score. However, LR 4‐HNE, a marker of oxidative damage, was similarly increased in AD cortex and AD cerebellum.ConclusionThe extensive overlap of soluble Aβ levels in controls with AD contrasts with the PET findings on fibrillar Aβ. These findings further support fibrillar Aβ as a biomarker for AD treatments and show the need for more detailed postmortem analysis of diverse soluble and insoluble Aβ aggregates in relation to PET. The decreased LR in AD frontal cortex is consistent with its extensive neuronal loss, in contrast to the cerebellum. Further studies will examine LR enzymes of APP processing as a source of the increased soluble Aβ in AD, and subcellular clearance processes, which may be impaired in association with the oxidative damage indicated by increased 4‐HNE.

Internalization of Angiotensin‐(1‐12) in Adult Retinal Pigment Epithelial‐19 Cells

IntroductionThe characterization, by this laboratory, of angiotensin‐(1‐12) [Ang‐(1‐12)] as a primary angiotensin II (Ang II)‐forming substrate may be an unrecognized source of Ang II‐mediated microvascular complication in hypertension‐mediated retinopathy. We found that the plasma Ang‐(1‐12) level was 12‐66‐fold higher when compared to the angiotensin I in normal human subjects. For the first time, we investigated the endogenous expression and incorporation of Ang‐(1‐12) in cultured adult retinal pigment epithelial‐19 (ARPE‐19) cells. We also investigated the internalization of Ang‐(1‐12) in the presence of a highly specific monoclonal antibody (mAb) that was developed by us against the C‐terminus of the human Ang‐(1‐12) sequence.MethodARPE‐19 cells (passage 28‐30) were grown to post‐confluence in 10% serum supplemented DMEM/F12 medium for 2‐3 days. An immunofluorescence (IF) staining was performed to detect the internalized Ang‐(1‐12) using the human Ang‐(1‐12) mAb (1:5000 dilution) and the Alexa Flour 488 secondary antibody (1:1000 dilution). Internalization was also confirmed in ARPE‐19 using radiolabeled Ang‐(1‐12) [125I‐Ang‐(1‐12), purity &gt;99%] in the presence and absence of the mAb by HPLC. For this, the APRE‐19 cells were incubated (4 h) with Ang‐(1‐12) alone or Ang‐(1‐12) + mAb. Afterward, cells were washed and harvested in isotonic buffer. The lysed cells were centrifuged at 28,000 g and the membrane pellet and supernatants (cytosolic fractions) were counted on a gamma counter. The cytosolic fractions were further analyzed by HPLC using a C18 column to confirm the internalization of intact Ang‐(1‐12) sequence.ResultsOur IF staining shows that Ang‐(1‐12) is endogenously expressed in ARPE‐19 cells. Further, an increased intensity of staining was detected in ARPE‐19 cells exposed to Ang‐(1‐12) (100 nM). High Ang‐(1‐12) radiolabeled counts were detected in the cytosolic fractions (3.1% of the total loaded radiolabeled) and very low counts in the membrane pellet (0.2%). We found that the internalization of 125I‐Ang‐(1‐12) was significantly decreased (~40%, P&lt;0.0001) in the presence of the human Ang‐(1‐12) mAb (Fig. 1). The detection of a large peak (88%) of 125I‐Ang‐(1‐12) in the cytosolic fraction in the HPLC chromatogram further confirms that the intact Ang‐(1‐12) sequence is internalized by the cells (Fig. 2).ConclusionsAng‐(1‐12) is internalized by ARPE‐19 cells from extracellular spaces of the retina and the internalization of Ang‐(1‐12) decreased following exposure to the human Ang‐(1‐12) mAb. Overall our data suggests the human Ang‐(1‐12) mAb may be used as a novel therapeutic agent to prevent circulating Ang‐(1‐12) internalization into the retinal cells and subsequently reduce the intracellular generation of the Ang II from the Ang‐(1‐12) substrate.

Storage conditions determine the characteristics of red blood cell derived extracellular vesicles

Extracellular vesicles (EVs) are released during the storage of red blood cell (RBC) concentrates and might play adverse or beneficial roles throughout the utilization of blood products (transfusion). Knowledge of EV release associated factors and mechanism amends blood product management. In the present work the impact of storage time and medium (blood preserving additive vs isotonic phosphate buffer) on the composition, size, and concentration of EVs was studied using attenuated total reflection infrared (ATR-IR) spectroscopy, microfluidic resistive pulse sensing (MRPS) and freeze-fraction combined transmission electron micrography (FF-TEM). The spectroscopic protein-to-lipid ratio based on amide and the C–H stretching band intensity ratio indicated the formation of various vesicle subpopulations depending on storage conditions. After short storage, nanoparticles with high relative protein content were detected. Spectral analysis also suggested differences in lipid and protein composition, too. The fingerprint region (from 1300 to 1000 cm−1) of the IR spectra furnishes additional information about the biomolecular composition of RBC-derived EVs (REVs) such as adenosine triphosphate (ATP), lactose, glucose, and oxidized hemoglobin. The difference between the vesicle subpopulations reveals the complexity of the REV formation mechanism. IR spectroscopy, as a quick, cost-effective, and label-free technique provides valuable novel biochemical insight and might be used complementary to traditional omics approaches on EVs.

Anti Ro52 antibodies: One step further, but still not there yet

The term extractable nuclear antigens (ENAs) is a historical denomination, though still in use, derived from the observation that several antigens from the cell nuclei are soluble and can be isolated or “extracted” in isotonic buffers, which facilitated the characterisation of several autoantibodies. Nowadays, the term has broadened and it also includes other antibodies directed against antigens from the cytoplasm and other cell compartments, such as ribonucleoproteins, histones or antigens only expressed when the cell is found in its mitotic state.

Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis.

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing. This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 µm thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling. OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

Chloride gradient is the driving force for ammonia/ammonium influx in human red blood cells

Ammonium/ammonia (AM) Rhesus transporters are expressed in tissues involved in AM generation, secretion and excretion, however, the physiological role of erythroid RhAG glycoprotein is not fully defined yet. AM concentration in red blood cells (RBCs) is approximately three times higher than in plasma and RBCs are the only blood cells that swell and lyse in isotonic ammonium (NH4+) buffer indicating that AM is transported inside the cell. Two main possible mechanisms of AM transport, including diffusion and active transport, are described, however, the real driving force for AM influx in RBCs is still under debate. RhAG and anion exchanger 1 (AE1, band 3, Cl-/HCO3--exchanger) form a structural complex in RBC membrane therefore we hypothesized that AE1 might be involved in AM influx in RBCs via the interplay with RhAG, and suggested that RhAG uses chloride (Cl-) gradient mediated by AE1 to import NH4+ against the gradient. Washed human RBCs were analyzed by flow cytometry, spectrophotometry and laser diffraction methods. For the evaluation of the interplay between RhAG and AE1 transporters HEPES buffer (in mM: NaCl, 140; KCl, 2; HEPES, 10, MgCl2, 2; Glucose, 5) and isotonic NH4+ buffer (in mM: NH4Cl, 140; KCl, 2; HEPES, 10; MgCl2, 2; Glucose, 5) were used. For the analysis of the role of Cl-gradient NaCl and NH4Cl were substituted by sodium glutamate and ammonium glutamate. Calcein-AM was used for cell vitality registration, hemoglobin (Hb) forms were analyzed spectrally, AM import was assessed by enzymatic and colorimetric AM assay kits, AE1 was inhibited by DIDS. In isotonic NH4+ buffer RBCs rapidly swelled and lysed within 3min at 37°C. The addition of AE1 substrate HCO3- (25mM) to isotonic NH4+ buffer increased the cell swelling rate 49±5 fold (n=11, Leven's test p>0.05, Tukey HSD post hoc, p<0.001). NH4+ import in media containing HCO3- increased by 74±11% (n=9, Leven's test p<0.05, Tamhane T2 post hoc, p<0.001). AE1 inhibition completely prevented hemolysis in isotonic NH4+ buffer and decreased NH4+ import in RBCs by 66±9% (n=9, Leven's test p<0.05, Tamhane T2 post hoc, p<0.001). The lack of Cl- gradient in ammonium glutamate and sodium glutamate isotonic buffers had no effects on cell viability (esterase activity) or Hb conformation. In isotonic ammonium glutamate buffer Cl- substitution prevented cell swelling and completely inhibited hemolysis in the same manner as AE1 inhibition by DIDS in NH4+buffer. AM import in conditions lacking Cl- gradient decreased by 62±11% (n=9, Leven's test p<0.05, Tamhane T2 post hoc, p<0.001) which is consistent with AE1 inhibition data. Our data clearly indicated that RhAG and AE1 are functionally connected, and AE1-mediatedCl-gradient drives RhAG-facilitated AM transport in RBCs. Therefore, we suggest that NH4+ is imported in human RBCs against its gradient by a secondary active transport mechanism, rather than via facilitated diffusion. Taken together our data revealed the new mechanism of AM transport against the gradient in RBCs and suggested that RBCs in addition to main functions are involved in the AM concentration regulation in plasma.