Protein Loading Research Articles

In vitro study of the bactericidal activity for new derivatives of quaternary ammonium salts based on pyridoxine against gram-positive/gram-negative bacteria

Background: preventive care and successful treatment of infectious diseases (including hospital-acquired infections) are among the most important tasks of modern healthcare. There are not many effective antiseptics and disinfectants to solve this problem. Quaternary ammonium salts are currently one of the promising classes of disinfectants but their use is limited by the ability of microorganisms to develop resistance to these compounds. Modification of QAS with substituents of various natures is a possible way to solve this problem.
 Aims: in this work a bactericidal activity comparative analysis for two original quaternary ammonium compounds based on pyridoxine was carried out in comparison with benzalkonium chloride.
 Materials and methods: the bactericidal activity of 0.1% and 0.2% aqueous solutions of original QACs based on pyridoxine were studied against two widespread pathogens: S. aureus and E. coli. Antibacterial activity data were obtained using a standard in vitro test (bactericidal activity test on a metal surface, exposure time 1, 5 and 15 minutes). In addition to standard conditions, antibacterial efficacy was also studied under protein loading conditions (adding BSA solution to the surface).
 Results: for the studied compounds the RF (reduction factor - reduction lgCFU of microorganisms) was on average ≥ 3. Complicating the model conditions in the form of protein load led to a decrease in efficiency by 1-3 orders. We have also observed an interesting effect of exposure time-dependent specificity reversal to the S. aureus and E. coli.
 Conclusions: The obtained results showed high activity of the studied compounds (corresponding to the effectiveness criterion RF≥3 for commercial compounds used in this field).

Assessment of subclinical renal dysfunction in post COVID-19 patients

ABSTRACT Introduction Kidney damage in COVID-19 is common and may lead to CKD. Early recognition of progression is mandatory, so there is a need for sensitive markers of kidney damage. Renal functional reserve (RFR) is described as the ability of the kidneys to augment their function in special situations. Aim of Work Was to assess subclinical renal dysfunction post-COVID-19. Methods This cross-sectional study investigates 31 patients previously infected with COVID-19 within 2–3 months and 31 healthy subjects with matched age and sex. Assessment of RFR was done by calculating the estimated GFR using the CKD-EPI equation before and 2 hours after 80 g protein load by intravenous infusion. Results RFR was significantly lower in post-COVID-19 patients (mean of 11.19 ± 4.76) than the control (mean of 25.81 ± 4.42, p < 0.001). Conclusion Despite preserved eGFR, there is a reduction in RFR, indicating progressive loss of nephron mass post-COVID.

Widespread nuclear lamina injuries defeat proteostatic purposes of α-synuclein amyloid inclusions.

Biogenesis of inclusion bodies (IBs) facilitates protein quality control (PQC). Canonical aggresomes execute degradation of misfolded proteins while non-degradable amyloids sequester into insoluble protein deposits. Lewy bodies (LBs) are filamentous amyloid inclusions of α-synuclein, but PQC benefits and drawbacks associated with LB-like IBs remain underexplored. Here, we report that crosstalk between filamentous LB-like IBs and aggresome-like IBs of α-synuclein (Syn-aggresomes) buffer the load, aggregation state, and turnover of the amyloidogenic protein in mouse primary neurons and HEK293T cells. Filamentous LB-like IBs possess unorthodox PQC capacities of self-quarantining α-synuclein amyloids and being degradable upon receding fresh amyloidogenesis. Syn-aggresomes equilibrate biogenesis of filamentous LB-like IBs by facilitating spontaneous degradation of α-synuclein and conditional turnover of disintegrated α-synuclein amyloids. Thus, both types of IB primarily contribute to PQC. Incidentally, the overgrown perinuclear LB-like IBs become degenerative once these are misidentified by BICD2, a cargo-adapter for the cytosolic motor-protein dynein. Microscopy indicates that microtubules surrounding the perinuclear filamentous inclusions are also distorted, misbalancing the cytoskeleton-nucleoskeleton tension leading to widespread lamina injuries. Together, nucleocytoplasmic mixing, DNA damage, and deregulated transcription of stress chaperones defeat the proteostatic purposes of the filamentous amyloids of α-synuclein.

Renal vascular control during normothermia and passive heat stress in healthy younger men and women.

We tested the hypothesis that the renal vasoconstrictor and vasodilator responses will be greater in younger women compared to men during passive heat stress. Twenty-five healthy adults [12 women (early follicular phase), 13 men] completed two experimental visits, heat stress or normothermic time-control, assigned in a block-randomized crossover design. During heat stress, core temperature was increased by ~0.8°C in the first hour before commencing a 2-min cold pressor test (CPT). Core temperature remained clamped and at one-hour post-CPT, subjects ingested a whey protein shake (1.2 g of protein/kg body weight), and measurements were taken pre-, 75-, and 150-min post-protein. Segmental artery vascular resistance (VR, Doppler ultrasound) was calculated as segmental artery blood velocity ÷ mean arterial pressure. CPT-induced increases in segmental artery VR did not differ between trials (trial effect: p=0.142) nor between men (heat stress: 1.5 ± 1.0 mmHg/cm/s, normothermia: 1.4 ± 1.0 mmHg/cm/s) and women (heat stress: 1.4 ± 1.2 mmHg/cm/s, normothermia: 2.1 ± 1.1 mmHg/cm/s) (group effect: p=0.429). Reductions in segmental artery VR following oral protein loading did not differ between trials (trial effect: p=0.080) nor between men (heat stress: -0.6 ± 0.8 mmHg/cm/s, normothermia: -0.6 ± 0.6 mmHg/cm/s) and women (heat stress: -0.5 ± 0.5 mmHg/cm/s, normothermia: -1.1 ± 0.6 mmHg/cm/s) (group effect: p=0.204). Renal vasoconstrictor responses to the cold pressor test and vasodilator responses following an oral protein load during heat stress or normothermia do not differ between younger men and younger women in the early follicular phase of the menstrual cycle.

Inter-coat protein loading of active ingredients into Tobacco mild green mosaic virus through partial dissociation and reassembly of the virion

Chemical pesticide delivery is a fundamental aspect of agriculture. However, the extensive use of pesticides severely endangers the ecosystem because they accumulate on crops, in soil, as well as in drinking and groundwater. New frontiers in nano-engineering have opened the door for precision agriculture. We introduced Tobacco mild green mosaic virus (TMGMV) as a viable delivery platform with a high aspect ratio and favorable soil mobility. In this work, we assess the use of TMGMV as a chemical nanocarrier for agriculturally relevant cargo. While plant viruses are usually portrayed as rigid/solid structures, these are “dynamic materials,” and they “breathe” in solution in response to careful adjustment of pH or bathing media [e.g., addition of solvent such as dimethyl sulfoxide (DMSO)]. Through this process, coat proteins (CPs) partially dissociate leading to swelling of the nucleoprotein complexes—allowing for the infusion of active ingredients (AI), such as pesticides [e.g., fluopyram (FLP), clothianidin (CTD), rifampicin (RIF), and ivermectin (IVM)] into the macromolecular structure. We developed a “breathing” method that facilitates inter-coat protein cargo loading, resulting in up to  ~ 1000 AIs per virion. This is of significance since in the agricultural setting, there is a need to develop nanoparticle delivery strategies where the AI is not chemically altered, consequently avoiding the need for regulatory and registration processes of new compounds. This work highlights the potential of TMGMV as a pesticide nanocarrier in precision farming applications; the developed methods likely would be applicable to other protein-based nanoparticle systems.

Abstract 1588: Identification of new signaling pathways between fibroblasts &amp; tumor cells in pancreatic cancer

Abstract We explore the unstudied role of nanoparticles (NPs), recently discovered secreted non-vesicular nanoparticles, in signaling between pre-cancer associated fibroblasts (preCAF), CAFs, and tumor cells. We generated two novel primary lines preCAF PFUM1 from a patient with high-grade intraductal papillary mucinous neoplasms and CAF FUM2 from a patient with pancreatic ductal adenocarcinoma, all tests performed between passages 2 and 3, fibroblast purity validated by RT-PCR and ACTA2 immunofluorescence. Small extracellular vesicles (sEVs), exomeres, and supermeres (the two main forms of NPs) were isolated by the ultracentrifugation method from PF-UM1 and F-UM2 with n=2-3 experiments. To validate the isolation, we used nanoparticle tracking, electron, and fluid-phase atomic force microscopy. Proteins were quantified by BCA assay. Liquid chromatography-mass spectrometry characterized the cargo from each fraction. Human cancer lines MIAPACA2 and SU8686 were treated with 10 μg of each fraction and cell proliferation was tested by phase-contrast images (Cytation 5), analyzed by Ilastik-based 1.4.0 machine-learning. We performed fluorescent Ki-67 and vimentin staining and quantified in QuPath 0.4.3. Non-parametric statistics were performed in Prism GraphPad. NPs constitute the major fraction of small particles released by fibroblasts, containing 80.5% (PFUM1) and 77.5% (FUM2) of the protein load by quantification. Mass spectrometry reveals enrichment of growth factors, epithelial-to-mesenchymal (EMT) transition, and metabolism-associated proteins in the NPs compared to sEVs. At 72 hours, cell proliferation for Su8686 reveals a proliferative effect for all fractions from both PFUM1 and FUM2 (19-63% increase compared to controls, p=0.049 to &amp;lt;0.0001). For MIAPACA2, only supermeres from PFUM1 and FUM2 consistently increased proliferation (9.5-35% increase, p=0.0051 to &amp;lt;0.0001). Ki-67 expression in MIAPACA2, increased by 9.6-23.0% following treatment by NPs (all p&amp;lt;0.0001) and by 2.9-9.2% with sEVs (p=0.011 to &amp;lt;0.0001). EMT was estimated by vimentin in MIAPACA2, as all preCAF fractions from PFUM1 reduced vimentin expression (p&amp;lt;0.001), while CAF FUM2 NPs increased vimentin expression (p&amp;lt;0.0001) while sEVs had no effect (p=0.075). In conclusion, the majority of pancreatic tumor-associated fibroblast small secretome is composed of NPs, exhibiting significant functional pro-tumoral effects. Citation Format: Ayman Al Shoukari, Maelle Batardiere, Nooshin Movahed, Camille beaussier, Jumanah Baig, Melissa gonzalez, Patricia Moraille, Eric Bonneil, Louise Rousseau, Simon turcotte, Kathleen Delgiorno, Marcus Tan, Anna Means, Elham Dianati Ajibisheh, Quoc-huy Trinh. Identification of new signaling pathways between fibroblasts &amp; tumor cells in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1588.

Engineered Metal‐Loaded Biohybrids to Promote the Attachment and Electron‐Shuttling between Enzymes and Carbon Electrodes

AbstractThe inorganic content and the catalytic performance pose metal‐loaded enzyme nanoflowers as promising candidates for developing bioelectrodes capable of functioning without the external addition of a redox mediator. However, these protein‐inorganic hybrids have yet to be successfully applied in combination with electrode materials. Herein, the synthesis procedure of these bionanomaterials is reproposed to precisely control the morphology, composition, and performance of this particular protein‐mineral hybrid, formed by glucose oxidase and cobalt phosphate. This approach aims to enhance the adherence and electron mobility between the enzyme and a carbon electrode. The strategy relies on dressing the protein in a tailored thin nanogel with multivalent chemical motifs. The functional groups of the polymer facilitate the fast protein sequence‐independent biomineralization. Furthermore, the engineered enzymes enable the fabrication of robust cobalt‐loaded enzyme inorganic hybrids with exceptional protein loads, exceeding 90% immobilization yields. Notably, these engineered biohybrids can be readily deposited onto flat electrode surfaces without requiring chemical pre‐treatment. The resulting bioelectrodes are robust and exhibit electrochemical responses even without the addition of a redox mediator, suggesting that cobalt complexes promote electron wiring between the active site of the enzyme and the electrode.

Antibody desolvation with sodium chloride and acetonitrile generates bioactive protein nanoparticles.

About 30% of the FDA approved drugs in 2021 were protein-based therapeutics. However, therapeutic proteins can be unstable and rapidly eliminated from the blood, compared to conventional drugs. Furthermore, on-target but off-tumor protein binding can lead to off-tumor toxicity, lowering the maximum tolerated dose. Thus, for effective treatment therapeutic proteins often require continuous or frequent administration. To improve protein stability, delivery and release, proteins can be encapsulated inside drug delivery systems. These drug delivery systems protect the protein from degradation during (targeted) transport, prevent premature release and allow for long-term, sustained release. However, thus far achieving high protein loading in drug delivery systems remains challenging. Here, the use of protein desolvation with acetonitrile as an intermediate step to concentrate monoclonal antibodies for use in drug delivery systems is reported. Specifically, trastuzumab, daratumumab and atezolizumab were desolvated with high yield (∼90%) into protein nanoparticles below 100 nm with a low polydispersity index (<0.2). Their size could be controlled by the addition of low concentrations of sodium chloride between 0.5 and 2 mM. Protein particles could be redissolved in aqueous solutions and redissolved antibodies retained their binding activity as evaluated in cell binding assays and exemplified for trastuzumab in an ELISA.

Polydopamine-Coated Polymer Nanofibers for In Situ Protein Loading and Controlled Release.

Nanofibrous polymeric materials, combined with protein therapeutics, play a significant role in biomedical and pharmaceutical applications. However, the upload of proteins into nanofibers with a high yield and controlled release has been a challenging issue. Here, we report the in situ loading of a model protein (bovine serum albumin) into hydrophilic poly(vinyl alcohol) nanofibers via ice-templating, with a 100% protein drug loading efficiency. These protein-loaded nanofibers were further coated by polydopamine in order to improve the nanofiber stability and achieve a controlled protein release. The mass ratio between poly(vinyl alcohol) and bovine serum albumin influenced the percentage of proteins in composite nanofibers and fiber morphology. More particles and less nanofibers were formed with an increasing percentage of bovine serum albumin. By varying the coating conditions, it was possible to produce a uniform polydopamine coating with tunable thickness, which acted as an additional barrier to reduce burst release and achieve a more sustained release profile.

Enhancing biocatalyst performance through immobilization of lipase (Eversa® Transform 2.0) on hybrid amine-epoxy core-shell magnetic nanoparticles

Magnetic nanoparticles were functionalized with polyethylenimine (PEI) and activated with epoxy. This support was used to immobilize Lipase (Eversa® Transform 2.0) (EVS), optimization using the Taguchi method. XRF, SEM, TEM, XRD, FTIR, TGA, and VSM performed the characterizations. The optimal conditions were immobilization yield (I.Y.) of 95.04 ± 0.79 %, time of 15 h, ionic load of 95 mM, protein load of 5 mg/g, and temperature of 25 °C. The maximum loading capacity was 25 mg/g, and its stability in 60 days of storage showed a negligible loss of only 9.53 % of its activity. The biocatalyst demonstrated better stability at varying temperatures than free EVS, maintaining 28 % of its activity at 70 °C. It was feasible to esterify free fatty acids (FFA) from babassu oil with the best reaction of 97.91 % and ten cycles having an efficiency above 50 %. The esterification of produced biolubricant was confirmed by NMR, and it displayed kinematic viscosity and density of 6.052 mm2/s and 0.832 g/cm3, respectively, at 40 °C. The in-silico study showed a binding affinity of −5.8 kcal/mol between EVS and oleic acid, suggesting a stable substrate-lipase combination suitable for esterification.

Tailoring the pore accessibility using proteins in laser induced porous graphitic structures: Correlating the physicochemical & electrochemical characteristics

While the popular means of enhancing the sensor's electroanalytical performance involves novel nanomaterial modifications and functionalization, these are multistep procedures which require several material/electrode characterization studies and are resource intensive. Alternatively, the sensor's performance can also be greatly enhanced by appropriately choosing the electroanalytical technique and by tuning its associated parameters. While such optimization has been popularly addressed via one-parameter-at-a-time strategies, this method demands greater number of experiments and fails to factor in the interaction between variables. Such issues can be addressed via the DoE multivariate statistical technique, which, however has not been widely employed in the field of electroanalysis. Moreover, there is a significant dearth in DoE based works for optimizing the LIG electrode's electroanalytical performance. Hence, in this work we have performed the DoE, FFD-based SWV technique optimization in bare LIG electrodes and have demonstrated a label-free affinity sensing application. While the optimized parameters significantly improved the output signals, we observed counterintuitive traits of increasing current values with protein loading. Hence, to explain these contrary trends we evaluate the electrochemical and physicochemical traits of model protein, BSA modified LIG electrodes, wherein we report the eccentric porous electrode behavior ensuant upon protein modification, associated with pore accessibility in these electrodes. Subsequently, we attribute the pore accessibility traits to hydrophilicity & steric hindrance aspects, and correlate it to the CPE effects, relaxation times and diffusion resistance. While few LIG-based works have reported such contrary trends in label-free affinity sensing, they have not provided the plausible explanation, and this is the first report to comprehensively elucidate the reasons.

Stable structures or PABP1 loading protects cellular and viral RNAs against ISG20-mediated decay.

ISG20 is an IFN-induced 3'-5' RNA exonuclease that acts as a broad antiviral factor. At present, the features that expose RNA to ISG20 remain unclear, although recent studies have pointed to the modulatory role of epitranscriptomic modifications in the susceptibility of target RNAs to ISG20. These findings raise the question as to how cellular RNAs, on which these modifications are abundant, cope with ISG20. To obtain an unbiased perspective on this topic, we used RNA-seq and biochemical assays to identify elements that regulate the behavior of RNAs against ISG20. RNA-seq analyses not only indicate a general preservation of the cell transcriptome, but they also highlight a small, but detectable, decrease in the levels of histone mRNAs. Contrarily to all other cellular ones, histone mRNAs are non-polyadenylated and possess a short stem-loop at their 3' end, prompting us to examine the relationship between these features and ISG20 degradation. The results we have obtained indicate that poly(A)-binding protein loading on the RNA 3' tail provides a primal protection against ISG20, easily explaining the overall protection of cellular mRNAs observed by RNA-seq. Terminal stem-loop RNA structures have been associated with ISG20 protection before. Here, we re-examined this question and found that the balance between resistance and susceptibility to ISG20 depends on their thermodynamic stability. These results shed new light on the complex interplay that regulates the susceptibility of different classes of viruses against ISG20.

Chestnut-Inspired Hollow Hydroxyapatite 3D Printing Scaffolds Accelerate Bone Regeneration by Recruiting Calcium Ions and Regulating Inflammation.

This study aims to overcome the drawbacks associated with hydroxyapatite (HAP) dense structures after sintering, which often result in undesirable features such as large grain size, reduced porosity, high crystallinity, and low specific surface area. These characteristics hinder osseointegration and limit the clinical applicability of the material. To address these issues, a new method involving the preparation of hollow hydroxyapatite (hHAP) microspheres has been proposed. These microspheres exhibit distinctive traits including weak crystallization, high specific surface area, and increased porosity. The weak crystallization aligns more closely with early mineralization products found in the human body and animals. Moreover, the microspheres' high specific surface area and porosity offer advantages for protein loading and facilitating osteoblast attachment. This innovative approach not only mitigates the limitations of conventional HAP structures but also holds the potential for improving the effectiveness of hydroxyapatite in biomedical applications, particularly in enhancing osseointegration. Three-dimensional printed hHAP/chitosan (CS) scaffolds with different hHAP concentration gradients were manufactured, and the physical and biological properties of each group were systematically evaluated. In vitro and in vivo experiments show that the hHAP/CS scaffold has excellent performance in bone remodeling. Furthermore, in-scaffold components, hHAP and CS were cocultured with bone marrow mesenchymal stem cells to explore the regulatory role of hHAP and CS in the process of bone healing and to reveal the cell-level specific regulatory network activated by hHAP. Enrichment analysis showed that hHAP can promote bone regeneration and reconstruction by recruiting calcium ions and regulating inflammatory reactions.

"The Influence of Protein Loading in Emptied Yeast on its Bactericidal and Anticancer Effectiveness"

"The Influence of Protein Loading in Emptied Yeast on its Bactericidal and Anticancer Effectiveness"

Protein Glycosylation Patterns Shaped By the IRE1‐XBP1s Arm of the Unfolded Protein Response

AbstractThe unfolded protein response (UPR) is a sensing and signaling pathway that surveys the endoplasmic reticulum (ER) for protein folding challenges and responds whenever issues are detected. UPR activation leads to upregulation of secretory pathway chaperones and quality control factors, as well as reduces the nascent protein load on the ER, thereby restoring and maintaining proteostasis. This paradigm‐defining view of the role of the UPR is accurate, but it elides additional key functions of the UPR in cell biology. In particular, recent work has revealed that the UPR can shape the structure and function of N‐ and O‐glycans installed on ER client proteins. This crosstalk between the UPR's reaction to protein misfolding and the regulation of glycosylation remains insufficiently understood. Still, emerging evidence makes it clear that the UPR, and particularly the IRE1‐XBP1s arm of the UPR, may be a central regulator of protein glycosylation, with important biological consequences. In this review, we discuss the crosstalk between proteostasis, the UPR, and glycosylation, present progress towards understanding biological functions of this crosstalk, and examine potential roles in diseases such as cancer.

Carrier-based immobilization of Aerococcus viridansl-lactate oxidase

l-Lactate oxidase has important applications in biosensing and finds increased use in biocatalysis. The enzyme has been characterized well, yet its immobilization has not been explored in depth. Here, we studied immobilization of Aerococcus viridansl-lactate oxidase on porous carriers of variable matrix material (polymethacrylate, polyurethane, agarose) and surface functional group (amine, Ni2+-loaded nitrilotriacetic acid (NiNTA), epoxide). Carrier activity (Ac) and immobilized enzyme effectiveness (ɳ) were evaluated in dependence of protein loading. Results show that efficient immobilization (Ac: up to 1450 U/g carrier; ɳ: up to 65%) requires a hydrophilic carrier (agarose) equipped with amine groups. The value of ɳ declines sharply as Ac increases, probably due to transition into diffusional regime. Untagged l-lactate oxidase binds to NiNTA carrier similarly as N-terminally His-tagged enzyme. Lixiviation studies reveal quasi-irreversible enzyme adsorption on NiNTA carrier while partial release of activity (≤ 25%) is shown from amine carrier. The desorbed enzyme exhibits the same specific activity as the original l-lactate oxidase. Collectively, our study identifies basic requirements of l-lactate oxidase immobilization on solid carrier and highlights the role of ionic interactions in enzyme-surface adsorption.

Development of a New Affinity Gold Polymer Membrane with Immobilized Protein A.

New and highly selective stationary phases for affinity membrane chromatography have the potential to significantly enhance the efficiency and specificity of therapeutic protein purification by reduced mass transfer limitations. This work developed and compared different immobilization strategies for recombinant Protein A ligands to a gold-sputtered polymer membrane for antibody separation in terms of functionalization and immobilization success, protein load, and stability. Successful, functionalization was validated via X-ray photoelectron spectroscopy (XPS). Here, a recombinant Protein A ligand was coupled by N-hydroxysuccinimide (NHS)/N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) chemistry to carboxy-functionalized, gold-sputtered membranes. We achieved a binding capacity of up to 104 ± 17 mg of the protein ligand per gram of the gold-sputtered membrane. The developed membranes were able to successfully capture and release the monoclonal antibody (mAb) Trastuzumab, as well as antibodies from fresh frozen human blood plasma in both static and dynamic setups. Therefore, they demonstrated successful functionalization and immobilization strategies. The antibody load was tested using bicinchoninic acid (BCA), ultraviolet-visible spectroscopy (UV-vis) measurements, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The outcome is a fully functional affinity membrane that can be implemented in a variety of different antibody purification processes, eliminating the need for creating individualized strategies for modifying the surface to suit different substrates or conditions.

A comparison of dual-enzyme immobilization by magnetic nanoparticles and magnetic enzyme aggregates for cascade enzyme reactions

Immobilized enzymes have been widely used for their efficiency, economy, and environment friendly. In this case, a simpler and effective immobilization method has been developed. In this study, the enzyme immobilized by magnetic beads with genipin (MGE) was prepared by mixing genipin-modified nano-Fe3O4 with (R)− 1-phenylethanol dehydrogenase ((R)-PEDH) and recombinant glucose dehydrogenase (rGDH), and the magnetic cross-linked enzyme aggregates (MCLEAs) was prepared by stirring a mixture of the sulfur precipitate of the (R)-PEDH and rGDH, poly-L-lysine, and cross-linkers. When o-phthalaldehyde (o-PA) was used as a cross-linking agent for the MCLEAs preparation, a protein loading of 99.9% and 62.93% recovery activity of (R)-PEDH and 70.12% of rGDH were obtained. Compared to the MGE, the MCLEAs possessed better enzyme activity recovery. Furthermore, this study tried the synthesis of (R)− 1-(furan-2-yl) ethanol by the immobilized enzymes and obtained a concentration of 1.10 mM for the MCLEAs and 0.24 mM for the MGE at 48 h. This study explored a facile method for co-immobilization of (R)-PEDH and rGDH to synthesize (R)− 1-(furan-2-yl) ethanol.

Establishment of Polydopamine-Modified HK-2 Cell Membrane Chromatography and Screening of Active Components from Plantago asiatica L.

Cell membrane chromatography (CMC) has been widely recognized as a highly efficient technique for in vitro screening of active compounds. Nevertheless, conventional CMC approaches suffer from a restricted repertoire of cell membrane proteins, making them susceptible to oversaturation. Moreover, the binding mechanism between silica gel and proteins primarily relies on intermolecular hydrogen bonding, which is inherently unstable and somewhat hampers the advancement of CMC. Consequently, this investigation aimed to establish a novel CMC column that could augment protein loading, enhance detection throughput, and bolster binding affinity through the introduction of covalent bonding with proteins. This study utilizes polydopamine (PDA)-coated silica gel, which is formed through the self-polymerization of dopamine (DA), as the carrier for the CMC column filler. The objective is to construct the HK-2/SiO2-PDA/CMC model to screen potential therapeutic drugs for gout. To compare the quantity and characteristics of Human Kidney-2 (HK-2) cell membrane proteins immobilized on SiO2-PDA and silica gel, the proteins were immobilized on both surfaces. The results indicate that SiO2-PDA has a notably greater affinity for membrane proteins compared to silica gel, resulting in a significant improvement in detection efficiency. Furthermore, a screening method utilizing HK-2/SiO2-PDA/CMC was utilized to identify seven potential anti-gout compounds derived from Plantago asiatica L. (PAL). The effectiveness of these compounds was further validated using an in vitro cell model of uric acid (UA) reabsorption. In conclusion, this study successfully developed and implemented a novel CMC filler, which has practical implications in the field.

Quantification of AMPA receptor subunits and RNA editing-related proteins in the J20 mouse model of Alzheimer's disease by capillary western blotting.

Accurate modelling of molecular changes in Alzheimer's disease (AD) dementia is crucial for understanding the mechanisms driving neuronal pathology and for developing treatments. Synaptic dysfunction has long been implicated as a mechanism underpinning memory dysfunction in AD and may result in part from changes in adenosine deaminase acting on RNA (ADAR) mediated RNA editing of the GluA2 subunit of AMPA receptors and changes in AMPA receptor function at the post synaptic cleft. However, few studies have investigated changes in proteins which influence RNA editing and notably, AD studies that focus on studying changes in protein expression, rather than changes in mRNA, often use traditional western blotting. Here, we demonstrate the value of automated capillary western blotting to investigate the protein expression of AMPA receptor subunits (GluA1-4), the ADAR RNA editing proteins (ADAR1-3), and proteins known to regulate RNA editing (PIN1, WWP2, FXR1P, and CREB1), in the J20 AD mouse model. We describe extensive optimisation and validation of the automated capillary western blotting method, demonstrating the use of total protein to normalise protein load, in addition to characterising the optimal protein/antibody concentrations to ensure accurate protein quantification. Following this, we assessed changes in proteins of interest in the hippocampus of 44-week-old J20 AD mice. We observed an increase in the expression of ADAR1 p110 and GluA3 and a decrease in ADAR2 in the hippocampus of 44-week-old J20 mice. These changes signify a shift in the balance of proteins that play a critical role at the synapse. Regression analysis revealed unique J20-specific correlations between changes in AMPA receptor subunits, ADAR enzymes, and proteins that regulate ADAR stability in J20 mice, highlighting potential mechanisms mediating RNA-editing changes found in AD. Our findings in J20 mice generally reflect changes seen in the human AD brain. This study underlines the importance of novel techniques, like automated capillary western blotting, to assess protein expression in AD. It also provides further evidence to support the hypothesis that a dysregulation in RNA editing-related proteins may play a role in the initiation and/or progression of AD.