Agarose Beads Research Articles

BmPxt1 mediated immune response by regulating PGE2 in silkworm, Bombyx mori

Prostaglandins (PGs) mediates the immune response of insects to multiple stimuli. Mammalian cyclooxygenase (COXs) is a key enzyme in the synthesis of PGs, and peroxinectin (Pxt) may have similar functions in some sequenced insect genomes. As a representative of Lepidoptera, the silkworm also contains PGs, but its synthetic pathway is not clear. We cloned a full-length cDNA encoding a Pxt, designated as BmPxt1, from silkworm. Sequence alignment analysis showed that the protein encoded by BmPxt1 has a conserved domain similar to Pxts, and its catalytic site is shared with the Pxt of Manduca sexta, which also produces PGs. The expression of BmPxt1 gene was the highest in the hemocytes and was induced by Nuclear Polyhedrosis Virus (NPV) challenge in the detected tissues. Moreover, we found that dsPxt1 treatment deficiency down-regulated BmPxt1 transcript levels and efficiently inhibiting hemocyte-spreading and nodule formation in silkworm. Hemocyte-spreading, nodule formation, phenoloxidase (PO) and AMP genes (attacin, defencin and moricin) were also inhibited by aspirin, a COX inhibitor. Treatment by PGE2 but not arachidonic acid (AA) rescued the immunosuppression; PGs concentrations was also inhibited by aspirin. PGE2, but not AA, treatment rescued the PGs concentrations. The COX inhibitor, aspirin, impaired the innate immune response including nodulation, encapsulation, and melanization in silkworm, while PGE2, but not arachidonic acid (AA), partially reversed these effects of aspirin. Recombinant BmsPxt1 significantly induced PO activation in larvae hemolymph, PGs concentrations and encapsulation of agarose beads. Injection of recombinant BmsPxt1 into larvae resulted in increased transcript levels of AMP genes. Our results confirmed that BmPxt1 was involved in the synthesis of PGs in the innate immune response of silkworm larvae, and provided new information for the role of BmsPxt1 secreted by silkworm in activating PO and antimicrobial peptides.

Mechanisms of Inflammatory Caspase Activation in Sickle Cell Disease

Background: Sickle cell disease (SCD) is an inherited red blood cell disorder that impacts millions of people worldwide. Patients with SCD experience chronic hemolysis and have elevated serum levels of free heme. Free heme activates a variety of inflammatory pathways that contribute to the serious clinical complications of SCD. One of the inflammatory pathways activated by heme is the inflammatory caspase pathway. Our lab previously showed that heme exposure in macrophages leads to the activation of inflammatory caspase proteins caspase-1 and caspase-4. Activation of these proteins by heme leads to IL-1β release and pyroptosis, a form of inflammatory cell death. Patients with SCD have increased circulating IL-1β levels, indicating this pathway is active in patients. In mouse models, inflammatory caspases are required for hemolysis-induced neutrophil recruitment and lethality, indicating these proteins are central to hemolysis-induced inflammation. Despite the key role these proteins play following hemolysis, it remains unknown how heme activates inflammatory caspases. Interestingly, both caspase-1 and caspase-4 are required for heme-induced IL-1β release while caspase-4 alone is required for heme-induced pyroptosis. In fact, pyroptosis likely is required for IL-1β release from the cell. Based on this data, we hypothesized that caspase-4 is the apical step in this pathway and a key druggable target for controlling unchecked inflammation in SCD. Results: We first sought to determine if caspase-4 activation is upstream of caspase-1 activation. To assess this, we used an imaging-based caspase activation reporter assay. Using this system, we found that siRNA knock-down of caspase-4 in primary human macrophages significantly inhibited heme-induced caspase-1 activation (63.8% vs 26.3%, n =4, p = 0.015). This result shows that caspase-4 is upstream of heme-induced caspase-1 activation and confirmed caspase-4 activation is the initial step in this pathway. Next, we sought to determine how caspase-4 activation occurs. Other known activators of caspase-4 induce activation by directly binding to caspase-4 and inducing self-oligomerization. To determine if heme acts in a similar manner, we used a published heme-binding prediction algorithm called HeMoQuest. We found multiple predicted heme-binding sites in caspase-4. The dissociation constants for these binding sites are less than 1 µM, suggesting strong binding affinity. Importantly, there are no predicted heme binding sites in caspase-1, indicating caspase-4 is the only direct heme receptor in this pathway. To validate these binding predictions, we performed molecular docking experiments and confirmed there are multiple heme-binding sites in caspase-4. Specifically, we found two heme-binding regions in the p10 catalytic subunit of caspase-4 comprising 2-4 essential residues. We next confirmed that heme directly interacts with caspase-4 in vitro. Using a pull-down assay with heme conjugated to agarose beads, we observed heme bound to purified caspase-4 and to casapase-4 in macrophage cell-free extracts. Heme did not interact with proteins structurally related to caspase-4 including caspase-1, caspase-7, and caspase-9 and caspase-4 was not pulled down by control agarose. This confirms the specificity of the heme-caspase-4 interaction. To determine if heme binding to caspase-4 results in activation, we performed native PAGE analysis of purified caspase-4 after exposure to heme. Heme induced formation of a caspase-4 oligomer around 200 kDa in size, four-fold larger than the inactive caspase-4 monomer. Other compounds known to induce IL-1β release from cells, like microbial toxin nigericin, did not induce caspase-4 oligomerization, demonstrating the specificity of heme's effect on caspase-4. Conclusions: We have determined that caspase-4 is a direct intracellular heme receptor and that heme exposure leads to caspase-4 activation, which in turn facilitates caspase-1 activation, IL-1β release and cell death. Our future work will elucidate the functional effects of heme binding to caspase-4 in mouse models of sickle cell disease. Caspase-4 is an attractive therapeutic target as it is required for both functional outputs of inflammatory caspase activation, IL-1β release and pyroptosis. Inhibiting heme-caspase-4 interactions would provide a specific way to block the harmful effects of this pathway in patients.

Immobilization of Lipase on Agarose Beads for Enzymatic Hydrolysis/Transesterification of Castor Oil

Lipase was immobilized on agarose beads and used for enzyme-catalyzed hydrolysis/transesterification of castor oil at room temperature without surfactants and salts. The immobilization yield was evaluated by reacting p-nitrophenyl butyrate and immobilized lipase. The reaction was performed in a 25 mL phosphate buffer medium (pH 7.0) and incubated for 2 hours. The absorbance of the formed p-nitrophenol was measured at 346 nm. The highest immobilization rate was obtained using 5.0 mg lipase with 2.5 mL glyoxal agarose beads. The reaction was performed in 25.0 mL sodium bicarbonate buffer pH 10 for 12 h. The hydrolysis/transesterification of castor oil was performed in phosphate buffer (pH 7) – ethanol medium. The main products were ricinoleic acid and ethyl ricinoleate. The highest hydrolysis/transesterification yield was 87% using 1.0 g castor oil and 2.5 mL of lipase immobilized agarose beads in the phosphate buffer (pH 7): ethanol (1: 9, V: V) at 72 hours. The reaction products were analyzed using FTIR and RP-HPLC.

Development of UV-Vis Imaging Compatible Chromatographic Matrix with Application for Injectable Formulation Characterization.

Transport within human tissue matrices, e.g., the subcutaneous tissue, exhibits some resemblance to chromatographic processes. Here, a porous matrix comprising agarose beads compatible with UV-vis imaging was developed for a parallel piped rectangular flow cell (4 mm light path). Introduction of high-molecular weight dextrans (Mr ∼ 200000 and ∼500000) at 10% (w/v) rendered imaging possible by providing optical clearing of the turbid porous matrix, resulting in improved transmittance as well as resolution (from 400 to 180 μm) at 280 nm, as well as 520 nm. The interplay between diffusive and convective transport at 0 < Pe ≤ 28 was visualized at 280 nm upon injection of dexamethasone suspensions. Real-time UV-vis imaging showed in-flow cell the effect of incorporating ion-exchange resins on the retention of infliximab, lysozyme, and α-lactalbumin. The ion-exchange matrix may serve as a surrogate for polyelectrolytes in the subcutaneous tissue, assessing the potential role of electrostatic interactions of biotherapeutics upon injection. UV-vis imaging of size-exclusion chromatographic matrixes may be of interest in its own right and potentially develop into a characterization tool for injectables.

Synthesis of micron-sized magnetic agarose beads chelated with nickel ions towards the affinity-based separation of histidine-tagged/rich proteins

Developing high-performance magnetic particles for the effective separation and purification of target proteins has become an important topic in the area of biomedical research. In this work, a simple and novel strategy was proposed for fabricating magnetic Fe3O4@agarose-iminodiacetic acid-Ni microspheres (MAIN), which can efficiently and selectively isolate histidine-tagged/rich proteins (His-proteins). Based on the thermoreversible sol-gel transition of agarose, basic magnetic agarose microspheres were prepared through the inverse emulsion method, in which the emulsion contained agarose and amine-modified Fe3O4 nanoparticles. The size of the emulsion was controlled by the emulsification of a high-speed shear machine, which improved the specific surface area of MAIN. Subsequently, the amine-modified Fe3O4 nanoparticles were covalently crosslinked with agarose through epichlorohydrin, which could avoid leakage of the magnetic source during use and increase the stability of MAIN. The microsized MAIN exhibited a clearly visible spherical core-shell structure with a diameter range from 3.4 μm to 9.8 μm, and excellent suspension ability in aqueous solution. The maximum adsorption capacity of MAIN for histidine-rich bovine hemoglobin was 1069.2 mg g−1 at 35 °C, which was higher than those of commercialized and most reported magnetic agarose microspheres/nanoparticles. The MAIN showed excellent adsorption ability and selectivity toward His-proteins in a mixture of histidine-rich bovine serum albumin (BSA) and histidine-poor lysozyme (LYZ). When the amount of LYZ was 5-fold higher than that of BSA, the recovery of BSA reached 75.0%. To prove its practicability, MAIN was successfully employed for the enrichment of histidine-tagged RSV-F0 from the cell culture medium supernatant. According to the optimized conditions, MAIN could enrich approximately 0.1 mg of RSV-F0 from 1 mL of complex biological sample. Therefore, we believe that the novel MAIN could be applicable for efficient separation and purification of His-proteins from complex biological systems.

Proof of concept of using a membrane-sensing peptide for sEVs affinity-based isolation.

Introduction: One main limitation in biomarker studies using EVs is the lack of a suitable isolation method rendering high yield and purity samples in a quick and easily standardized procedure. Here we report an affinity isolation method with a membrane-sensing peptide (MSP) derived from bradykinin. Methods: We designed a protocol based on agarose beads carrying cation chelates to specifically bind to the 6His-tagged membrane-sensing peptide. This approach presents several advantages: 1) cation-carrying agaroses are widely used and standardized for His-tagged protein isolation, 2) the affinity protocol can be performed in small volumes, feasible and manageable for clinical routine and 3) elution with imidazole or EDTA allows a gentle and easy recovery without EV damage, facilitating subsequent characterization and functional analyses. Results: The optimized final procedure incubates 0.5mg of peptide for 10min with 10µL of Long-arm Cobalt agarose before an overnight incubation with concentrated cell conditioned medium. EV downstream analyses can be directly performed on the agarose beads adding lysis or nucleic-acid extraction buffers, or gently eluted with imidazole or EDTA, rendering a fully competent EV preparation. Discussion: This new isolation methodology is based on the recognition of general membrane characteristics independent of surface markers. It is thus unbiased and can be used in any species EV sample, even in samples from animal or plant species against which no suitable antibodies exist. Being an affinity method, the sample handling protocol is very simple, less time-consuming, does not require specialized equipment and can be easily introduced in a clinical automated routine. We demonstrated the high purity and yield of the method in comparison with other commercially available kits. This method can also be scale up or down, with the possibility of analyzing very low amounts of sample, and it is compatible with any downstream analyses thanks to the gentle elution procedure.

The nature of the buffer alters the effects of the chemical modification on the stability of immobilized lipases

The objective of this paper was to analyze whether an interaction between the effects of the buffer nature and chemical modification on enzyme stability exists. For this, the lipase B from Candida antarctica (CALB) and the lipase from Thermomyces lanuginosus (TLL) were immobilized on octyl agarose beads and modified with picryl sulfonic acid (TNBS) and ethylenediamine via the carbodiimide route. The obtained biocatalysts were them inactivated in different buffers (2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris)-HCl, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) or phosphate). A significant interaction was found between the chemical modification of the enzyme surfaces and their stabilities in these different buffers. While phosphate was the buffer where the lowest enzyme stabilities were found for both unmodified immobilized enzymes, the differences on enzyme stabilities become much smaller after some chemical modifications. In many instances, chemical modification improves enzyme stability when using a buffer and was negative when using the other one (e.g., TNBS modification of TLL was positive using Tris-HCl or negative using the other buffers, amination of CALB decreased its stability when inactivated in Tris-HCl or HEPES while it almost had no effect in phosphate). Thus, clear co-interactions of the effects on enzyme stability between chemical modification and buffer nature were established.

Glyoxyl-ficin: An example where a more intense multipoint covalent attachment may decrease enzyme stability.

The immobilization of ficin extract (sulfidryl proteases) on glyoxyl agarose beads stabilized it, but there is a clear stability maximum after 3 h of immobilization, being the 24 h biocatalyst less stable when thermally inactivated in presence of air. The incubation of the thermally inactivated biocatalysts in the presence of dithiothreitol permitted to recover almost total activity of the biocatalysts prepared for 24 h of immobilization, while the enzyme immobilized for 3 h recovered less activity. This suggested that the main cause of the faster enzyme inactivation of the 24 h biocatalyst was the Cys oxidation. This was confirmed by inactivating the Cys blocked biocatalysts by using 2,2′-dipyridyldisulfide. Again, a higher percentage of the initial activity (when unblocked by incubation in mercaptoethanol the thermally inactivated biocatalysts) was observed for the 24 h biocatalyst. The use of helium atmosphere enabled to greatly improve the enzyme stability of all immobilized biocatalysts, confirming the highest stability of the biocatalyst immobilized for 24 h. Results suggested that this biocatalyst was in fact the most rigid one, but that the distortion induced by the immobilization produced a higher exposition of the catalytic Cys, and this way, its oxidation becomes faster in the presence of oxygen.

In vivo impact of tubulin polymerization promoting protein (Tppp) knockout to the airway inflammatory response

Microtubule dysfunction has been implicated as a mediator of inflammation in multiple diseases such as disorders of the cardiovascular and neurologic systems. Tubulin polymerization promoting protein (Tppp) facilitates microtubule elongation and regulates tubulin acetylation through inhibition of cytosolic deacetylase enzymes. Pathologic alterations in microtubule structure and dynamics have been described in cystic fibrosis (CF) and associated with inflammation, however the causality and mechanism remain unclear. Likewise, Tppp has been identified as a potential modifier of CF airway disease severity. Here we directly assess the impact of microtubule dysfunction on infection and inflammation by interrogating wild type and a Tppp knockout mouse model (Tppp − / −). Mice are challenged with a clinical isolate of Pseudomonas aeruginosa-laden agarose beads and assessed for bacterial clearance and inflammatory markers. Tppp − / − mouse model demonstrate impaired bacterial clearance and an elevated inflammatory response compared to control mice. These data are consistent with the hypothesis microtubule dysregulation is sufficient to lead to CF-like airway responses in mice.

Glutaraldehyde modification of lipases immobilized on octyl agarose beads: Roles of the support enzyme loading and chemical amination of the enzyme on the final enzyme features

Lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) have been immobilized on octyl agarose at low loading and at a loading exceeding the maximum support capacity. Then, the enzymes have been treated with glutaraldehyde and inactivated at pH 7.0 in Tris-HCl, sodium phosphate and HEPES, giving different stabilities. Stabilization (depending on the buffer) of the highly loaded biocatalysts was found, very likely as a consequence of the detected intermolecular crosslinkings. This did not occur for the lowly loaded biocatalysts. Next, the enzymes were chemically aminated and then treated with glutaraldehyde. In the case of TLL, the intramolecular crosslinkings (visible by the apparent reduction of the protein size) increased enzyme stability of the lowly loaded biocatalysts, an effect that was further increased for the highly loaded biocatalysts due to intermolecular crosslinkings. Using CALB, the intramolecular crosslinkings were less intense, and the stabilization was lower, even though the intermolecular crosslinkings were quite intense for the highly loaded biocatalyst. The stabilization detected depended on the inactivation buffer. The interactions between enzyme loading and inactivating buffer on the effects of the chemical modifications suggest that the modification and inactivation studies must be performed under the target biocatalysts and conditions.

Optimization of extracellular vesicle isolation and their separation from lipoproteins by size exclusion chromatography

AbstractInterest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. However, one main unsolved issue in the EV field is finding a technique able to eliminate non‐EV contaminants present in biofluid samples in a one‐step isolation protocol. Due to the expansion and value of size exclusion chromatography (SEC) as one of the best EV isolation methods, we have tested several agarose resins with different agarose percentages, bead sizes and crosslinking features to optimize EV isolation. For this optimization of SEC, we first employed conditioned media from a melanoma cell culture, a simpler sample in comparison to biological fluids, but which also contains abundant contaminants such as soluble protein and lipoproteins (LPPs). The distinct agaroses and the combinations of resins with different agarose percentages in the same column were tested. Soluble protein, EVs and LPPs levels from the different eluted fractions were quantitated by immunodetection or absorbance measurements. Samples were also analysed by NTA and TEM to verify the yield and the LPP contamination. Different percentages of agarose resins (2%, 4% and 6%) yielded samples with increasing LPP contamination respectively, which was not improved in the columns that combined them. Crosslinking of the agarose did not affect EV isolation yield nor the LPP contamination. In contrast, reducing the bead size greatly improved EV purity. We thus selected 4% Rapid Run Fine agarose beads as the resin that more efficiently isolated EVs with almost no contamination of other particles. Using blood plasma samples, this resin also demonstrated an improved capacity in the isolation of EVs from LPPs in comparison to the agaroses most commonly used in the field and differential ultracentrifugation.

A minimally invasive bronchoscopic approach for direct delivery to murine airways and application to models of pulmonary infection.

The laboratory mouse is used extensively for human disease modeling and preclinical therapeutic testing for efficacy, biodistribution, and toxicity. The variety of murine models available, and the ability to create new ones, eclipses all other species, but the size of mice and their organs create challenges for many in vivo studies. For pulmonary research, improved methods to access murine airways and lungs, and track substances administered to them, would be desirable. A nonsurgical endoscopic system with a camera, effectively a bronchoscope, coupled with a cryoimaging fluorescence microscopy technique to view the lungs in 3D, is described here that allows visualization of the procedure, including the anatomical location at which substances are instilled and fluorescence detection of those substances. We have applied it to bacterial infection studies to characterize better and optimize a chronic lung infection murine model in which we instill bacteria-laden agarose beads into the airways and lungs to extend the duration of the infection and inflammation. The use of the endoscope as guidance for placing a catheter into the airways is simple and quick, requiring only momentary sedation, and reduces post-procedural mortality compared with our previous instillation method that includes a trans-tracheal surgery. The endoscopic method improves speed and precision of delivery while reducing the stress on animals and the number of animals generated and used for experiments.

Highly stable integration of graphene Hall sensors on a microfluidic platform for magnetic sensing in whole blood

The detection and analysis of rare cells in complex media such as blood is increasingly important in biomedical research and clinical diagnostics. Micro-Hall detectors (μHD) for magnetic detection in blood have previously demonstrated ultrahigh sensitivity to rare cells. This sensitivity originates from the minimal magnetic background in blood, obviating cumbersome and detrimental sample preparation. However, the translation of this technology to clinical applications has been limited by inherently low throughput (<1 mL/h), susceptibility to clogging, and incompatibility with commercial CMOS foundry processing. To help overcome these challenges, we have developed CMOS-compatible graphene Hall sensors for integration with PDMS microfluidics for magnetic sensing in blood. We demonstrate that these graphene μHDs can match the performance of the best published μHDs, can be passivated for robust use with whole blood, and can be integrated with microfluidics and sensing electronics for in-flow detection of magnetic beads. We show a proof-of-concept validation of our system on a silicon substrate and detect magnetic agarose beads, as a model for cells, demonstrating promise for future integration in clinical applications with a custom CMOS chip.

Detecting Legionella pneumophila in Cooling Tower Water Samples with a DNAzyme/Bead‐Based Fluorescence Assay

AbstractLegionella pneumophila is the causative agent behind the deadly waterborne disease Legionnaires’, which is commonly transmitted by the spread of contaminated droplets from cooling tower water samples. The lack of effective detection methods presents a challenge for L. pneumophila outbreak control. Previously, an RNA‐cleaving DNAzyme called LP1 was reported to specifically target L. pneumophila. In this study, LP1 was immobilized onto agarose beads via streptavidin‐biotin interaction to develop a bead‐based fluorescence assay for L. pneumophila detection. This bead‐based assay demonstrated excellent stability and functionality in various cooling tower water samples. To improve L. pneumophila monitoring in real‐world samples, a lysozyme treatment was used to enhance L. pneumophila recognition. The limit of detection of this DNAzyme‐based bead assay can reach 103 CFUs in cell‐spiked cooling tower water samples without cell culturing or signal amplification steps.

Magnetic DNA random access memory with nanopore readouts and exponentially-scaled combinatorial addressing

The storage of data in DNA typically involves encoding and synthesizing data into short oligonucleotides, followed by reading with a sequencing instrument. Major challenges include the molecular consumption of synthesized DNA, basecalling errors, and limitations with scaling up read operations for individual data elements. Addressing these challenges, we describe a DNA storage system called MDRAM (Magnetic DNA-based Random Access Memory) that enables repetitive and efficient readouts of targeted files with nanopore-based sequencing. By conjugating synthesized DNA to magnetic agarose beads, we enabled repeated data readouts while preserving the original DNA analyte and maintaining data readout quality. MDRAM utilizes an efficient convolutional coding scheme that leverages soft information in raw nanopore sequencing signals to achieve information reading costs comparable to Illumina sequencing despite higher error rates. Finally, we demonstrate a proof-of-concept DNA-based proto-filesystem that enables an exponentially-scalable data address space using only small numbers of targeting primers for assembly and readout.

Efficient Purification of Polyhistidine-Tagged Recombinant Proteins Using Functionalized Corundum Particles.

Immobilized metal affinity chromatography (IMAC) is a popular and valuable method for the affinity purification of polyhistidine-tagged recombinant proteins. However, it often shows practical limitations, which might require cumbersome optimizations, additional polishing, and enrichment steps. Here, we present functionalized corundum particles for the efficient, economical, and fast purification of recombinant proteins in a column-free format. The corundum surface is first derivatized with the amino silane APTES, then EDTA dianhydride, and subsequently loaded with nickel ions. The Kaiser test, well known in solid-phase peptide synthesis, was used to monitor amino silanization and the reaction with EDTA dianhydride. In addition, ICP-MS was performed to quantify the metal-binding capacity. His-tagged protein A/G (PAG), mixed with bovine serum albumin (BSA), was used as a test system. The PAG binding capacity was around 3 mg protein per gram of corundum or 2.4 mg per 1 mL of corundum suspension. Cytoplasm obtained from different E. coli strains was examined as examples of a complex matrix. The imidazole concentration was varied in the loading and washing buffers. As expected, higher imidazole concentrations during loading are usually beneficial when higher purities are desired. Even when higher sample volumes, such as one liter, were used, recombinant protein down to a concentration of 1 µg/mL could be isolated selectively. Comparing the corundum material with standard Ni-NTA agarose beads indicated higher purities of proteins isolated using corundum. His6-MBP-mSA2, a fusion protein consisting of monomeric streptavidin and maltose-binding protein in the cytoplasm of E. coli, was purified successfully. To show that this method is also suitable for mammalian cell culture supernatants, purification of the SARS-CoV-2-S-RBD-His8 expressed in human Expi293F cells was performed. The material cost of the nickel-loaded corundum material (without regeneration) is estimated to be less than 30 cents for 1 g of functionalized support or 10 cents per milligram of isolated protein. Another advantage of the novel system is the corundum particles' extremely high physical and chemical stability. The new material should be applicable in small laboratories and large-scale industrial applications. In summary, we could show that this new material is an efficient, robust, and cost-effective purification platform for the purification of His-tagged proteins, even in challenging, complex matrices and large sample volumes of low product concentration.

Multivalent dipeptidyl peptidase IV fragment-nanogold complex inhibits cancer metastasis by blocking pericellular fibronectin

Inhibition of cancer metastasis is a fundamental challenge in cancer treatment. We have previously shown that metastasis of cancer cells in the lung is critically promoted by the interaction between the superficial dipeptidyl peptidase IV (DPP IV) expressed on lung endothelial cells and the pericellular polymeric fibronectin (polyFN) of circulating cancer cells. In the present study, we aimed to search for DPP IV fragments with high avidity to polyFN and develop FN-targeted gold nanoparticles (AuNPs) conjugated with DPP IV fragments for treating cancer metastasis. We first identified a DPP IV fragment encompassing amino acids 29-130 of DPP IV, designated DP4A, which contained FN-binding sites and could specifically bind to FN immobilized on gelatin agarose beads. Furthermore, we conjugated maltose binding protein (MBP)-fused DP4A proteins to AuNPs for fabricating a DP4A-AuNP complex and evaluated its FN-targeted activity in vitro and anti-metastatic efficacy in vivo. Our results show that DP4A-AuNP exhibited higher binding avidity to polyFN than DP4A by 9 folds. Furthermore, DP4A-AuNP was more potent than DP4A in inhibiting DPP IV binding to polyFN. In terms of polyFN-targeted effect, DP4A-AuNP interacted with FN-overexpressing cancer cells and was endocytosed into cells 10 to 100 times more efficiently than untargeted MBP-AuNP or PEG-AuNP with no noticeable cytotoxicity. Furthermore, DP4A-AuNP was superior to DP4A in competitive inhibition of cancer cell adhesion to DPP IV. Confocal microscopy analysis revealed that binding of DP4A-AuNP to pericellular FN induced FN clustering without altering its surface expression on cancer cells. Notably, intravenous treatment with DP4A-AuNP significantly reduced metastatic lung tumor nodules and prolonged the survival in the experimental metastatic 4T1 tumor model. Collectively, our findings suggest that the DP4A-AuNP complex with potent FN-targeted effects may have therapeutic potential for prevention and treatment of tumor metastasis to the lung.

Prolonged intermittent hypoxia causes mitochondrial dysfunction due to O-GlcNacylation of DRP1 in models of COPD (chronic obstructive pulmonary disease)

Background: Sarcopenia, or skeletal muscle loss, is common complication in COPD and contributes to adverse clinical outcomes including mortality. As the disease progresses, a significant proportion of COPD patients develop nocturnal hypoxemia which causes tissue-level hypoxia in the skeletal muscle which we have termed prolonged intermittent hypoxia (PIH). We have demonstrated sarcopenia, mitochondrial dysfunction, and dysregulated hypoxia-inducible factor 1-α (HIF1-α) responses both in vitro and in vivo in response to PIH. We also noted multiple transcriptional targets of HIF1-α were upregulated in the glycolytic pathway. We hypothesized that PIH upregulates the Hexosamine Biosynthetic (HB) pathway, a branch of glycolysis responsible for post-translational glycosylation of proteins, to cause O-GlcNAcylation of DRP1 leading to increased mitochondrial fission, fragmented mitochondrial networks, mitochondrial dysfunction, and sarcopenia. Methods: Our in vitro model of PIH exposed differentiated, murine C2C12 myotubes to 16h normoxia/8h hypoxia [1% oxygen] for 3 days, while our in vivo model exposed C57BL/6 mice to 12h normoxia/12h hypoxia [10% oxygen] for 21 days. Protein extraction and immunoblots were performed to probe for enzymes in the HB, mTORC1 pathways, O-GlcNAcylation, and puromycin incorporation. Imaging of mitochondrial structure was determined by staining with MitoTracker orange and electron microscopy. Immunoprecipitation and detection of Drp1 O-GlcNAcylation was performed by incubating protein lysates with Wheat Germ Agglutinin coated Agarose beads. To determine responses to inhibition of O-GlcNAcylation, we performed CRISPR-Cas9 RNP deletion of GFAT, the first enzyme in the HB pathway. Results: We found significant increase in O-GlcNAcylated proteins in our PIH models . This was accompanied by increased expression of HIF1-α, mitochondrial dysfunction, and upregulated enzymes in the HB pathway. O-GlcNAcylated modification of DRP1 was significantly upregulated due to PIH in vitro. GFAT KO myotubes demonstrated significant reductions in global O-GlcNAcylation of proteins and O-GlcNAcylated Drp1 with PIH. Interestingly, protein synthesis was rescued in GFAT KO myotubes as determined by puromycin incorporation, restoration of mTORC1 signaling, and reversal of a sarcopenic phenotype. Conclusion: Our findings demonstrate a potential mechanism for mitochondrial dysfunction and sarcopenia due to PIH which may be translationally relevant for patients with COPD. Future studies focused on O-GlcNAcylated modification of DRP1 and the functional consequences are needed to identify novel therapeutic targets to reverse sarcopenia in COPD. Supported in part by: NIH RO1 GM119174; RO1 DK113196; P50 AA024333; RO1 AA021890; 3U01AA026976 - 03S1; UO1 AA 026976; R56HL141744;UO1 DK061732; 5U01DK062470-17S2; R21 AR 071046; Howard and Helen Trevey Endowment; (SD); K12 HL141952 (AA) and the American College of Gastroenterology Clinical Research Award and NIH KO8 AAAA028794 (NW). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Heterologous Expression, Purification and Structural Characterization of Ribonuclease E from Mycobacterium smegmatis

Rapid adaptations to various environmental changes are key to the survival of bacterial cells, which is mediated at the level of transcription initiation and RNA degradation/processing. Ribonuclease E (RNase E), an essential endoribonuclease gene in Mycobacterium species, is a key mediator in most reactions of RNA metabolism such as messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA) processing and their degradation qualifying it as an important drug target. Here, we first report the cloning of full-length Mycobacterium smegmatis mc2155 (M. smegmatis) RNase E [MSMEG_4626] and its expression with isopropyl β-D-1-thiogalactopyranoside (IPTG) in Escherichia coli (E. coli) as a glutathione S-transferase (GST) fusion protein. The best expression of the recombinant protein was optimized with respect to different parameters, such as E. coli strains, temperature, IPTG concentrations and time points of induction. Despite the use of protease inhibitors, we observed low-molecular-weight degradation products along with full-length fusion proteins. Maximum expression of soluble and least degraded full-length RNase E was obtained at 32 °C/0.1-mM IPTG, further purified by affinity chromatography using glutathione agarose beads and confirmed by immunoblotting technique using anti-GST antibody. In silico characterization using multiple sequence alignment, homology modeling and docking studies revealed four amino acid residues, Asp613, Phe657, Ile662 and Glu688, in the catalytic domain of RNase E that are completely conserved in both E. coli and Mycobacterium species, also involved in RNA binding and might act as candidate drug targets to combat antibiotic-resistant mycobacterial diseases. Additionally, recombinant GST-RNase E protein can unravel the mechanism of RNA degradation/processing through protein–protein interaction studies.

Cloning, expression, purification, and immunoblotting analysis of recombinant type III fibronectin domains of human oncostatin M receptor.

The human oncostatin M receptor subunit , commonly known as the oncostatin M receptor (OSMR), is a cell surface protein and belongs to the family of type I cytokine receptors. It is highly expressed in several cancers and is a potential therapeutic target. Structurally, OSMR consists of three major domains: the extracellular, transmembrane, and cytoplasmic domains. The extracellular domain further comprises four Type III fibronectin subdomains. The functional relevance of these type III fibronectin domains is not known yet, and it is of great interest to us to understand their role in OSMR-mediated interactions with other oncogenic proteins. The four type III fibronectin domains of hOSMR were amplified by PCR using the pUNO1-hOSMR construct as a template. The molecular size of the amplified products was confirmed by agarose gel electrophoresis. The amplicons were then cloned into a pGEX4T3 vector containing GST as an N-terminal tag. Positive clones with domain inserts were identified by restriction digestion and overexpressed in E. coli Rosetta (DE3) cells. The optimum conditions for overexpression were found to be 1 mM IPTG and an incubation temperature of 37°C. The overexpression of the fibronectin domains was confirmed by SDS-PAGE, and they are affinity purified by using glutathione agarose beads in three repetitive steps. The purity of the isolated domains analyzed by SDS-PAGE and western blotting showed that they were exactly at their corresponding molecular weights as a single distinct band. In this study, we have successfully cloned, expressed, and purified four Type III fibronectin subdomains of hOSMR.