Role of the gradient slope during the product internal recycling for the multicolumn countercurrent solvent gradient purification of PEGylated proteins.

Protein PEGylation, i.e. functionalization with poly(ethylene glycol) chains, has been demonstrated an efficient way to improve the therapeutic index of these biopharmaceuticals. We demonstrated that Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) is an efficient process for the separation of PEGylated proteins (Kim et al., Ind. and Eng. Chem. Res. 2021, 60, 29, 10764-10776), thanks to the internal recycling of product-containing side fractions. This recycling phase plays a critical role in the economy of MCSGP as it avoids wasting valuable product, but at the same time impacts its productivity extending the overall process duration. In this study, our aim is to elucidate the role of the gradient slope within this recycling stage on the yield and productivity of MCSGP for two case-studies: PEGylated lysozyme and an industrially relevant PEGylated protein. While all the examples of MCSGP in the literature refer to a single gradient slope in the elution phase, for the first time we systematically investigate three different gradient configurations: i) a single gradient slope throughout the entire elution, ii) recycling with an increased gradient slope, to shed light on the competition between volume of the recycled fraction and required inline dilution and iii) an isocratic elution during the recycling phase. The dual gradient elution proved to be a valuable solution for boosting the recovery of high-value products, with the potential for alleviating the pressure on the upstream processing.

Urinary Exosomal Cystatin C and Lipopolysaccharide Binding Protein as Biomarkers for Antibody-Mediated Rejection after Kidney Transplantation.

We aimed to discover and validate urinary exosomal proteins as biomarkers for antibody-mediated rejection (ABMR) after kidney transplantation. Urine and for-cause biopsy samples from kidney transplant recipients were collected and categorized into the discovery cohort (n = 36) and a validation cohort (n = 65). Exosomes were isolated by stepwise ultra-centrifugation for proteomic analysis to discover biomarker candidates for ABMR (n = 12). Of 1820 exosomal proteins in the discovery cohort, four proteins were specifically associated with ABMR: cystatin C (CST3), serum paraoxonase/arylesterase 1, retinol-binding protein 4, and lipopolysaccharide-binding protein (LBP). In the validation cohort, the level of urinary exosomal LBP was significantly higher in the ABMR group (n = 25) compared with the T-cell-mediated rejection (TCMR) group and the no major abnormality (NOMOA) group. Urinary exosomal CST3 level was significantly higher in the ABMR group compared with the control and NOMOA groups. Immunohistochemical staining showed that LBP and CST3 in the glomerulus were more abundant in the ABMR group compared with other groups. The combined prediction probability of urinary exosomal LBP and CST3 was significantly correlated with summed LBP and CST3 intensity scores in the glomerulus and peritubular capillary as well as Banff g + ptc scores. Urinary exosomal CST3 and LBP could be potent biomarkers for ABMR after kidney transplantation.

Design and economic investigation of a Multicolumn Countercurrent Solvent Gradient Purification unit for the separation of an industrially relevant PEGylated protein.

Conjugation of biopharmaceuticals to polyethylene glycol chains, known as PEGylation, is nowadays an efficient and widely exploited strategy to improve critical properties of the active molecule, including stability, biodistribution profile, and reduced clearance. A crucial step in the manufacturing of PEGylated drugs is the purification. The reference process in industrial settings is single-column chromatography, which can meet the stringent purity requisites only at the expenses of poor product recoveries. A valuable solution to this trade-off is the Multicolumn Countercurrent Solvent Gradient Purification (MCSGP), which allows the internal and automated recycling of product-containing side fractions that are typically discarded in the batch processes. In this study, an ad hoc design procedure was applied to the single-column batch purification of an industrially relevant PEGylated protein, with the aim of defining optimal collection window, elution duration and elution buffer ionic strength to be then transferred to the MCSGP. This significantly alleviates the design of the continuous operation, subjected to manifold process parameters. The MCSGP designed by directly transferring the optimal parameters allowed to improve the yield and productivity by 8.2% and 17.8%, respectively, when compared to the corresponding optimized batch process, ensuring a purity specification of 98.0%. Once the efficacy of MCSGP was demonstrated, a detailed analysis of its cost of goods was performed and compared to the case of single-column purification. To the best of our knowledge, this is the first example of a detailed economic investigation of the MCSGP across different manufacturing scenarios and process cadences of industrial relevance, which demonstrated not only the viability of this continuous technology but also its flexibility.

Label-free imaging and evaluation of characteristic properties of asthma-derived eosinophils using optical diffraction tomography.

Optical diffraction tomography (ODT), an emerging imaging technique that does not require fluorescent staining, can measure the three-dimensional distribution of the refractive index (RI) of organelles. In this study, we used ODT to characterize the pathological characteristics of human eosinophils derived from asthma patients presenting with eosinophilia. In addition to morphological information about organelles appearing in eosinophils, including the cytoplasm, nucleus, and vacuole, we succeeded in imaging specific granules and quantifying the RI values of the granules. Interestingly, ODT analysis showed that the RI (i.e., molecular density) of granules was significantly different between eosinophils from asthma patients and healthy individuals without eosinophilia, and that vacuoles were frequently found in the cells of asthma patients. Our results suggest that the physicochemical properties of eosinophils derived from patients with asthma can be quantitatively distinguished from those of healthy individuals. The method will provide insight into efficient evaluation of the characteristics of eosinophils at the organelle level for various diseases with eosinophilia.

Characterizing Organelles in Live Stem Cells Using Label-Free Optical Diffraction Tomography.

Label-free optical diffraction tomography (ODT), an imaging technology that does not require fluorescent labeling or other pre-processing, can overcome the limitations of conventional cell imaging technologies, such as fluorescence and electron microscopy. In this study, we used ODT to characterize the cellular organelles of three different stem cells-namely, human liver derived stem cell, human umbilical cord matrix derived mesenchymal stem cell, and human induced pluripotent stem cell-based on their refractive index and volume of organelles. The physical property of each stem cell was compared with that of fibroblast. Based on our findings, the characteristic physical properties of specific stem cells can be quantitatively distinguished based on their refractive index and volume of cellular organelles. Altogether, the method employed herein could aid in the distinction of living stem cells from normal cells without the use of fluorescence or specific biomarkers.

Exosomal miR-1260b derived from non-small cell lung cancer promotes tumor metastasis through the inhibition of HIPK2.

Tumor-derived exosomes (TEXs) contain enriched miRNAs, and exosomal miRNAs can affect tumor growth, including cell proliferation, metastasis, and drug resistance through cell-to-cell communication. We investigated the role of exosomal miR-1260b derived from non-small cell lung cancer (NSCLC) in tumor progression. Exosomal miR-1260b induced angiogenesis by targeting homeodomain-interacting protein kinase-2 (HIPK2) in human umbilical vein endothelial cells (HUVECs). Furthermore, exosomal miR-1260b or suppression of HIPK2 led to enhanced cellular mobility and cisplatin resistance in NSCLC cells. In patients with NSCLC, the level of HIPK2 was significantly lower in tumor tissues than in normal lung tissues, while that of miR-1260b was higher in tumor tissues. HIPK2 and miR-1260b expression showed an inverse correlation, and this correlation was strong in distant metastasis. Finally, the expression level of exosomal miR-1260b in plasma was higher in patients with NSCLC than in healthy individuals, and higher levels of exosomal miR-1260b were associated with high-grade disease, metastasis, and poor survival. In conclusion, exosomal miR-1260b can promote angiogenesis in HUVECs and metastasis of NSCLC by regulating HIPK2 and may serve as a prognostic marker for lung cancers.

Correlative Light and Electron Microscopy for Nanoparticle-Cell Interaction and Protein Localization.

Various silica-based fluorescent nanoparticles ((Si-FNP)) with magnetic or metal cores represent a standard class of nanoparticles offering new opportunities for high-resolution cellular imaging and biomedicine applications, such as drug delivery. Their high solubility, homogeneity, biocompatibility, and chemical inertness Si-FNPs make them attractive probes for correlative light and electron microscopy (CLEM) studies, offering novel insights into nanoparticle-cell interactions in detail. In the present chapter, we present a procedure for imaging silica-based fluorescent magnetic core-shell nanoparticles (Si-FMNP) at the single-particle scale in cells. Our method facilitates the acquisition of information on the extracellular and intercellular distribution of nanoparticles and their various interactions with various cellular organelles when cells are cultured and electroporated by NPs. In addition, such information could facilitate the evaluation of the efficacy of nanocarriers designed for drug delivery.

Immuno-gold Techniques in Biomedical Sciences.

Since their development in the 1960s, immuno-gold techniques have been steadily used in biomedical science, because these techniques are applicable to all kinds of antigens, from viruses to animal tissues. Immuno-gold staining exploits antigen-antibody reactions and is used to investigate locations and interactions of components in the ultrastructure of tissues, cells, and particles. These methods are increasingly used with advanced technologies, such as correlative light and electron microscopy and cryo-techniques. In this protocol, we introduce the principles and technical details of recent advances in this area and discuss their advantages and limitations.

Enhancement of Wound Healing Efficacy by Increasing the Stability and Skin-Penetrating Property of bFGF Using 30Kc19α-Based Fusion Protein.

The instability of recombinant basic fibroblast growth factor (bFGF) is a major disadvantage for its therapeutic use and means frequent applications to cells or tissues are required for sustained effects. Originating from silkworm hemolymph, 30Kc19α is a cell-penetrating protein that also has protein stabilization properties. Herein, it is investigated whether fusing 30Kc19α to bFGF can enhance the stability and skin penetration properties of bFGF, which may consequently increase its therapeutic efficacy. The fusion of 30Kc19α to bFGF protein increases protein stability, as confirmed by ELISA. 30Kc19α-bFGF also retains the biological activity of bFGF as it facilitates the migration and proliferation of fibroblasts and angiogenesis of endothelial cells. It is discovered that 30Kc19α can improve the transdermal delivery of a small molecular fluorophore through the skin of hairless mice. Importantly, it increases the accumulation of bFGF and further facilitates its translocation into the skin through follicular routes. Finally, when applied to a skin wound model in vivo, 30Kc19α-bFGF penetrates the dermis layer effectively, which promotes cell proliferation, tissue granulation, angiogenesis, and tissue remodeling. Consequently, the findings suggest that 30Kc19α improves the therapeutic functionalities of bFGF, and would be useful as a protein stabilizer and/or a delivery vehicle in therapeutic applications.

Mitotic Chromosomes in Live Cells Characterized Using High-Speed and Label-Free Optical Diffraction Tomography.

The cell nucleus is a three-dimensional, dynamic organelle organized into subnuclear compartments such as chromatin and nucleoli. The structure and function of these compartments are maintained by diffusion and interactions between related factors as well as by dynamic and structural changes. Recent studies using fluorescent microscopic techniques suggest that protein factors can access and are freely mobile in heterochromatin and in mitotic chromosomes, despite their densely packed structure. However, the physicochemical properties of the chromosome during cell division are not fully understood. In the present study, characteristic properties such as the refractive index (RI), volume of the mitotic chromosomes, and diffusion coefficient (D) of fluorescent probes inside the chromosome were quantified using an approach combining label-free optical diffraction tomography with complementary confocal laser-scanning microscopy and fluorescence correlation spectroscopy. Variations in these parameters correlated with osmotic conditions, suggesting that changes in RI are consistent with those of the diffusion coefficient for mitotic chromosomes and cytosol. Serial RI tomography images of chromosomes in live cells during mitosis were compared with three-dimensional confocal micrographs to demonstrate that compaction and decompaction of chromosomes induced by osmotic change were characterized by linked changes in chromosome RI, volume, and the mobilities of fluorescent proteins.

Physicochemical Properties of Nucleoli in Live Cells Analyzed by Label-Free Optical Diffraction Tomography.

The cell nucleus is three-dimensionally and dynamically organized by nuclear components with high molecular density, such as chromatin and nuclear bodies. The structure and functions of these components are represented by the diffusion and interaction of related factors. Recent studies suggest that the nucleolus can be assessed using various protein probes, as the probes are highly mobile in this organelle, although it is known that they have a densely packed structure. However, physicochemical properties of the nucleolus itself, such as molecular density and volume when cellular conditions are changed, are not yet fully understood. In this study, physical parameters such as the refractive index (RI) and volume of the nucleoli in addition to the diffusion coefficient (D) of fluorescent probe protein inside the nucleolus are quantified and compared by combining label-free optical diffraction tomography (ODT) with confocal laser scanning microscopy (CLSM)-based fluorescence correlation spectroscopy (FCS). 3D evaluation of RI values and corresponding RI images of nucleoli in live HeLa cells successfully demonstrated varying various physiological conditions. Our complimentary method suggests that physical property of the nucleolus in live cell is sensitive to ATP depletion and transcriptional inhibition, while it is insensitive to hyper osmotic pressure when compared with the cytoplasm and nucleoplasm. The result demonstrates that the nucleolus has unique physicochemical properties when compared with other cellular components.

Safety and efficacy of a metal stent covered with a silicone membrane containing integrated silver particles in preventing biofilm and sludge formation in endoscopic drainage of malignant biliary obstruction: a phase 2 pilot study.

BACKGROUND AND AIMS: Membrane-covered self-expandable metal stents (SEMSs) have been developed to prolong the patency of stents by reducing tissue hyperplasia or tumor ingrowth. However, their effectiveness is attenuated by stent clogging as a result of biofilm formation on the inner surface of the membrane. The aim of this pilot study was to evaluate the efficacy and safety of SEMSs covered with a silicone membrane containing integrated silver particles (Ag-P) in malignant distal biliary obstruction. METHODS: Twenty-four patients who underwent SEMS placement because of malignant distal biliary obstruction were enrolled in this single-center pilot study. The main outcomes were technical success, clinical success, adverse events, stent patency, and survival. RESULTS: The technical and clinical success rates were 100% and 91.7% (22 of 24), respectively. The rates of early and late adverse events were 22.7% and 36.4%, respectively. The primary reintervention rate was 27.3% (6 of 22). Only 1 case involving stent malfunction was associated with sludge impaction. Median stent patency was 179 days. During follow-up, there were no serious adverse events or mortality related to the stents or Ag-P. Serum and urine silver concentrations before and after stent placement and at 32 weeks after placement did not differ. All serum and urine silver concentrations were <3 µg/L (3 ppb) and 5 µg/L (5 ppb), respectively. CONCLUSIONS: SEMSs covered with a silicone membrane containing integrated Ag-Ps may be effective and safe in malignant distal biliary obstruction. Stent dysfunction related to sludge impaction may be less frequent using this new stent. (Clinical Research Information Service identifier: KCT 0002310.).

Large-scale synthesis of uniform hexagonal boron nitride films by plasma-enhanced atomic layer deposition.

Hexagonal boron nitride (h-BN) has been previously manufactured using mechanical exfoliation and chemical vapor deposition methods, which make the large-scale synthesis of uniform h-BN very challenging. In this study, we produced highly uniform and scalable h-BN films by plasma-enhanced atomic layer deposition, which were characterized by various techniques including atomic force microscopy, transmission electron microscopy, Raman spectroscopy, and X-ray diffraction. The film composition studied by X-ray photoelectron spectroscopy and Auger electron spectroscopy corresponded to a B:N stoichiometric ratio close to 1:1, and the band-gap value (5.65 eV) obtained by electron energy loss spectroscopy was consistent with the dielectric properties. The h-BN-containing capacitors were characterized by highly uniform properties, a reasonable dielectric constant (3), and low leakage current density, while graphene on h-BN substrates exhibited enhanced electrical performance such as the high carrier mobility and neutral Dirac voltage, which resulted from the low density of charged impurities on the h-BN surface.

Paradoxical induction of growth arrest and apoptosis by EGF via the up-regulation of PTEN by activating Redox factor-1/Egr-1 in human lung cancer cells.

Epidermal growth factor (EGF) signaling promotes cell proliferation and survival in several types of cancer. Here, however, we showed that EGF inhibits proliferation and promotes programmed cell death in non-small cell lung cancer (NSCLC) cells. In A549 cells, EGF increased redox factor-1 (Ref-1) expression and the association of Ref-1 with zinc finger-containing transcriptional regulator (EGR1) via activation of p22phox, RAC1, and an NADPH oxidase subunit. EGF increased p22phox and RAC1 expression through activation of purinergic receptors (P2Y). Elevated Ref-1/EGR1 levels increased phosphatase and tensin homolog (PTEN) levels, leading to inhibition of the Akt pathway. EGF-induced PTEN upregulation increased apoptosis and autophagy-induced damage in A549 cells, whereas Ref-1 knockdown blocked EGF-induced PTEN upregulation in an NADPH oxidase p22phox subunit-independent manner. In addition, p22phox knockdown restored EGF-induced effects, implying that changes in P2Y activity caused by EGF, which activates NADPH oxidase via RAC1, influenced Ref-1-mediated redox regulation. Finally, EGF similarly attenuated cell proliferation and promoted autophagy and apoptosis in vivo in a xenograft model using A549 cells. These findings reveal that EGF-induced redox signaling is linked to Ref-1-induced death in NSCLC cells.

Comparison of foot pressure in stretching exercises according to the type of ankle ramp.

[Purpose] This study compared and analyzed use of an existing ankle ramp and a newly developed ankle ramp for stretching exercises. [Subjects] Fourteen subjects were included; they were stroke patients more than 6 months after onset, with no orthopedic or biological problems in the legs, so independent gait was possible. [Methods] The subjects performed stretching exercises for 5 min with an existing ankle ramp and a newly developed ankle ramp; foot pressure was then measured. [Results] The averaged percentage and kilopascal data for weight bearing and foot pressure on the affected side with the newly developed ankle ramp for stretching exercises were significantly higher than those with the existing ankle ramp. [Conclusion] Our results suggest that stretching exercises using the newly developed ankle ramp more effectively increase foot pressure than the existing ankle ramp.

Sub-100-as timing jitter optical pulse trains from mode-locked Er-fiber lasers.

We demonstrate sub-100-as timing jitter optical pulse trains generated from free-running, 77.6 MHz repetition-rate, mode-locked Er-fiber lasers. At -0.002(±0.001) ps2 net cavity dispersion, the rms timing jitter is 70 as (224 as) integrated from 10 kHz (1 kHz) to 38.8 MHz offset frequency, when measured by a 24 as resolution balanced optical cross correlator. To our knowledge, this result corresponds to the lowest rms timing jitter measured from any mode-locked fiber lasers so far. The measured result also agrees fairly well with the Namiki-Haus analytic model of quantum-limited timing jitter in stretched-pulse fiber lasers.

Measurement of the polarization of high-order harmonics from aligned N(2) molecules by spatial interferometry.

The polarization of high-harmonics from aligned N(2) molecules was measured by observing the visibility of spatial interference between two high-harmonics generated separately. The minimum visibility was observed at an angle of 60 degrees between the polarization of the harmonic generation laser field and the molecular orientation. In this case, the angular shift of harmonic polarization is 15 degrees from the molecular orientation. Our measurement of the visibility variation matches the theoretical prediction based on the harmonic field calculation for aligned N(2) molecules.