Fast Protein Liquid Chromatography Research Articles

Characterization of a novel model for atherosclerosis imaging: the apolipoprotein E-deficient rat

BackgroundThe apolipoprotein E-deficient (apoE−/−) mouse is a well-established model for studying atherosclerosis. However, its small size limits its use in longitudinal positron emission tomography (PET) imaging studies. Recently, the apoE−/− rat has emerged as an alternative. With this study, we investigate the feasibility of using apoE−/− rats as an in vivo model for longitudinal atherosclerotic PET/CT imaging.ResultsApoE−/− rats showed significantly higher [18F]FDG uptake than controls in the aortic arch (+ 18.5%, p < 0.001) and abdominal aorta (+ 31.0%, p < 0.001) at weeks 12, 26, and 51. ApoE−/− rats exhibited hypercholesterolemia, as evidenced by plasma cholesterol levels that were up to tenfold higher, and total hepatic cholesterol levels that were up to threefold higher than the control rats at the end of the study. Fast protein liquid chromatography cholesterol profiling indicated very high levels of pro-atherogenic apoB-containing very low-density lipoprotein and low-density lipoprotein fractions in the apoE−/− rats. Atherosclerotic lesions cover 19.9% of the surface of the aortic arch (p = 0.0013), and there was a significantly higher subendothelial accumulation of ED1-positive macrophages in the abdominal aorta of the apoE−/− rats compared to control rats (Ctrl) (p = 0.01). No differences in neutral sterols were observed but higher levels of bile acids were found in the apoE−/− rats.ConclusionThese data demonstrate early signs of hypercholesterolemia, high levels of bile acids, the development of atherosclerotic lesions, and macrophage accumulation in apoE−/− rats. Therefore, this model shows promise for atherosclerosis imaging studies.

Expression, purification, and characterization of self-assembly virus-like particles of capsid protein L1 HPV 52 in Pichia pastoris GS115

BackgroundCervical cancer caused by the human papillomavirus (HPV) is one of the most frequent malignances globally. HPV 52 is a high-risk cancer-causing genotype that has been identified as the most prevalent type in Indonesia. Virus-like particles (VLP)-based vaccinations against HPV infection could benefit from self-assembled VLP of L1 capsid protein. ResultThe recombinant HPV 52 L1 was expressed in Pichia pastoris on a shake-flask scale with 0.5% methanol induction in this study. The copy number was used to compare the expression level and stability. The colony that survived on a solid medium containing 2000 μg/ml of Zeocin was selected and cultured to express HPV 52 L1. DNA was extracted from the chosen colony, and the copy was determined using qPCR. HPV 52 L1 protein was then purified through fast performance liquid chromatography. Transmission electron microscopy (TEM) evaluation confirmed the VLP self-assembly. The genomic DNA remained intact after 100 generations of serial cultivation under no selective pressure medium conditions, and the protein produced was relatively stable. However, the band intensity was slightly lower than in the parental colony. In terms of copy number, a low copy transformant resulted in low expression but produced a highly stable recombinant clone. Eventually, the L1 protein expressed in Pichia pastoris can self-assemble into VLP. Therefore, recombinant HPV possesses a stable clone and the ability to self-assemble into VLP. ConclusionThe recombinant L1 HPV 52 protein is successfully expressed in P. pastoris within a size range of approximately 55 kDa and demonstrated favorable stability. The L1 protein expressed in Pichia pastoris successful self-assembled of HPV VLPs, thereby establishing their potential efficacy as a prophylactic vaccine.

A Recombinant Alginate Lyase Algt1 with Potential in Preparing Alginate Oligosaccharides at High-Concentration Substrate.

Alginate lyase has been demonstrated as an efficient tool in the preparation of functional oligosaccharides (AOS) from alginate. The high viscosity resulting from the high concentration of alginate poses a limiting factor affecting enzymatic hydrolysis, particularly in the preparation of the fragments with low degrees of polymerization (DP). Herein, a PL7 family alginate lyase Algt from Microbulbifer thermotolerans DSM 19189 was developed and expressed in Pichia pastoris. The recombinant alginate lyase Algt1 was constructed by adopting the structural domain truncation strategy, and the enzymatic activity towards the alginate was improved from 53.9 U/mg to 212.86 U/mg compared to Algt. Algt1 was stable when incubated at 40 °C for 90 min, remaining with approximately 80.9% of initial activity. The analyses of thin-layer chromatography (TLC), fast protein liquid chromatography (FPLC), and electrospray ionization mass spectrometry (ESI-MS) demonstrated that the DP of the minimum identifiable substrate of Algt1 was five, and the main hydrolysis products were AOS with DP 1-4. Additionally, 1-L the enzymatic hydrolysis system demonstrated that Algt1 exhibited an effective degradation at alginate concentrations of up to 20%, with the resulting products of monosaccharides (14.02%), disaccharides (21.10%), trisaccharides (37.08%), and tetrasaccharides (27.80%). These superior properties of Algt1 make it possible to efficiently generate functional AOS with low DP in industrial processing.

Prediction of PP2A Holoenzymes Guided By the Structural Characterization of Methylation Independent PP2A Assembly

Introduction: The dysregulation of phosphorylation-dependent signaling is a hallmark of leukemogenesis. Kinase inhibitors have revolutionized the outcomes of patients with chronic myeloid leukemia. An orthogonal, but less explored way to target phosphorylation pathways is to activate phosphatases, such as the ubiquitously expressed protein- phosphatase 2A (PP2A). PP2A is an essential serine/threonine phosphatase that regulates various cellular processes, including cell growth, division, and differentiation. Dysregulation of PP2A has been linked to leukemia. PP2A is a modular multi-subunit enzyme: One scaffold subunit (A) binds to a catalytic subunit (C) to form a core AC heterodimer, which together with one of many regulatory (B) subunits forms the active enzyme. The regulatory (B) subunits are classified into 4 distinct subfamilies without significant homology. The combinatorial assembly of these subunits generates a wide range of PP2A complexes, resulting in diverse substrate specificity and subcellular localization. The detailed mechanism of PP2A regulation remains elusive and reports about a governing role of methylation of the carboxy-terminal Leucin of PP2A C are conflicting. Understanding the underlying molecular regulation of PP2A activity is of significant interest for future therapeutic development for acute myeloid leukemia. Methods: Recombinant PP2A was expressed in insect cells, and purified by affinity purification, anion exchange chromatography and size exclusion chromatography (SEC). Extensive quality control, including liquid chromatography-mass spectrometry, SDS-PAGE gel and fast protein liquid chromatography was performed. The degree of methylation of PP2A C Leucine 309 was confirmed by mass spectrometry. We validated protein stability with differential scanning fluorimetry. Pull-down assays and size exclusion chromatography characterized complex formation. A calorimetric assay using p-nitrophenyl phosphate as a substrate functionally assessed PP2A in vitro. Structural information was obtained by X-ray crystallography and AlphaFold Multimer. Results: Using in vitro biochemistry, including pull down and size exclusion chromatography, we demonstrate that methylation of the carboxy terminus of the PP2A C subunit is dispensable for PP2A assembly in vitro. Absence of methylation of Leucine 309 was confirmed by mass-spectrometry. We show by SEC that instead PP2A complex formation is dependent on B subunit concentration. Carboxy-terminal methylation of the PP2A C subunit was also not required for functional activity in a calorimetric assay using p-nitrophenyl phosphate as a substrate. PP2A formed spontaneously in vitro and was enzymatically active independent of methylation of Leucine 309. To corroborate these findings, we determined the X-ray crystal structure of the PP2A:PPP2B56E:PPP2C complex to 2.7 Å resolution with the unmethylated Leucine 309. Interestingly our experimental structure superimposed with an AlphaFold Multimer model of the PP2A trimer. Building on this finding, we predicted the models of all 64 possible PP2A complexes providing a framework for further study and structural analysis of PP2A complexes and their activation. These studies provide key information for the structural design and development of novel drugs for hematologic malignancies by targeting of selected PP2A complexes and clarified the role of Leucine 309 methylation in PP2A activation. Conclusion: Methylation of the carboxy-terminal Leucine 309 of the PP2A C subunitis dispensable for PP2A activity in vitro. Integrative structural biology studies, combining biochemistry, X-ray crystallography and AlphaFold Multimer provide a comprehensive map of PP2A complexes. These studies have the potential to accelerate the drug discovery process and unlock PP2A activation as a new therapeutic modality for leukemia.

Industrialization possibilities of purified pig sperm hyaluronidase.

The goals of the present study were to develop a simple method for obtain highly purified pig sperm hyaluronidase (pHyase) and to assess its activity, function, and safety. In mammals, sperm-specific glycophosphatidylinositol (GPI)-anchored Hyase assists sperm penetration through the cumulus mass surrounding the egg and aids in the dispersal of the cumulus-oocyte complex. Recently, Purified bovine sperm hyaluronidase (bHyase) has been shown to enhance therapeutic drug transport by breaking down the hyaluronan barrier to the lymphatic and capillary vessels, thereby facilitating tissue absorption. Commercially available Hyase is typically isolated from bovine or ovine; which have several disadvantages, including the risk of bovine spongiform encephalopathy, low homology with human Hyase, and the requirement for relatively complex isolation procedures. This study successfully isolated highly purified pHyase in only two steps, using ammonium sulfate precipitation and fast protein liquid chromatography. The isolated Hyase had activity equal to that of commercial bHyase, facilitated in vitro fertilization, and effectively dissolved high molecule hyaluronic acid. This simple, effective isolation method could improve the availability of pHyase for research and clinical applications.

ANGPTL3 (Angiopoietin-Like 3) Preferentially Resides on High-Density Lipoprotein in the Human Circulation, Affecting Its Activity.

Background ANGPTL3 (angiopoietin-like protein 3) is an acknowledged crucial regulator of lipid metabolism by virtue of its inhibitory effect on lipoprotein lipase and endothelial lipase. It is currently unknown whether and to which lipoproteins ANGPTL3 is bound and whether the ability of ANGPTL3 to inhibit lipase activity is affected by binding to lipoproteins. Methods and Results Incubation of ultracentrifugation-isolated low-density lipoprotein (LDL) and high-density lipoprotein (HDL) fractions from healthy volunteers with recombinant ANGPTL3 revealed that ANGPTL3 associates with both HDL and LDL particles ex vivo. Plasma from healthy volunteers and a patient deficient in HDL was fractionated by fast protein liquid chromatography, and ANGPTL3 distribution among lipoprotein fractions was measured. In healthy volunteers, ≈75% of lipoprotein-associated ANGPTL3 resides in HDL fractions, whereas ANGPTL3 was largely bound to LDL in the patient deficient in HDL. ANGPTL3 activity was studied by measuring lipolysis and uptake of 3H-trioleate by brown adipocyte T37i cells. Unbound ANGPTL3 did not suppress lipase activity, but when given with HDL or LDL, ANGPTL3 suppressed lipase activity by 21.4±16.4% (P=0.03) and 25.4±8.2% (P=0.006), respectively. Finally, in a subset of the EPIC (European Prospective Investigation into Cancer) Norfolk study, plasma HDL cholesterol and amount of large HDL particles were both positively associated with plasma ANGPTL3 concentrations. Moreover, plasma ANGPTL3 concentrations showed a positive association with incident coronary artery disease (odds ratio, 1.25 [95% CI, 1.01-1.55], P=0.04). Conclusions Although ANGPTL3 preferentially resides on HDL, its activity was highest once bound to LDL particles.

Natural Wood-Derived Macroporous Cellulose for Highly Efficient and Ultrafast Elimination of Double-Stranded RNA from In Vitro-Transcribed mRNA.

Double-stranded RNA (dsRNA) is a major impurity that can induce innate immune responses and cause adverse drug reactions. Removing dsRNA is an essential and non-trivial process in manufacturing mRNA. Current methods for dsRNA elimination use either high-performance liquid chromatography or microcrystalline cellulose, rendering the process complex, expensive, toxic, and/or time-consuming. This study introduces a highly efficient and ultrafast method for dsRNA elimination using natural wood-derived macroporous cellulose (WMC). With a naturally formed large total pore area and low tortuosity, WMC removes up to 98% dsRNA within 5min. This significantly shortens the time for mRNA purification and improves purification efficiency. WMC can also be filled into chromatographic columns of different sizes and integrates with fast-protein liquid chromatography for large-scale mRNA purification to meet the requirements of mRNA manufacture. This study further shows that WMC purification improves the enhanced green fluorescent protein mRNA expression efficiency by over 28% and significantly reduces cytokine secretion and innate immune responses in the cells. Successfully applying WMC provides an ultrafast and efficient platform for mRNA purification, enabling large-scale production with significant cost reduction.

Production and characterization of a highly stable laccase from extreme thermophile Geobacillus stearothermophilus MB600 isolated from hot spring of Gilgit Balitistan (Pakistan)

Extracellular thermophilic multicopper oxidase i.e. laccase was purified, identified and characterized from Geobacillus stearothermophilus MB600. Fast protein liquid chromatography (FPLC) of lacasse showed a prominent peak at 610 nm. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for laccase presented a molecular weight of 59.13 kDa. Analysis via Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) confirmed the presence of laccase similar to in Geobacillus thermoleovorans B23 and Geobacillus sp. LEMMY01. Laccase showed Km 0.532 mM for guaiacol and 0.307 mM for ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)). Its optimum activity was witnessed at 90°C, at pH 5.0. Laccase catalytic activity was completely inhibited by sodium dodecyl-sulfate (SDS), sodium azide (NaN3), and ethylenediaminetetraacetic acid (EDTA). Among the metal salts against Cu, Ca, Cd, and Li enhanced while Mg inhibited the enzyme activity. It was stable against Cl, I and solvents (Ethanol > Methanol > Dimethyl sulfoxide > Acetonitrile). Thermophilic laccase effectively degraded 93% of Remazol brilliant blue R, 92% Bisphenol A diglycidyle ether without mediator.

Chromatographic purification of small extracellular vesicles using an affinity column for phospholipid membranes

ObjectivesThis study aimed to investigate whether chromatography using an ExoPUA column, an affinity column for phospholipid membranes, could potentially serve as an efficient, rapid, scalable, and reproducible method for purifying small extracellular vesicles (sEVs).ResultsWe used the ExoPUA column connected to a fast-performance liquid chromatography system. One-step chromatographic purification of sEVs from culture supernatant using the ExoPUA protocol resulted in an 82 ± 16-fold increase in purity with a yield of 38 ± 5% of sEVs. The purified sEVs contained CD9, CD63, TSG101, and miRNA (miR-21), but not the endoplasmic reticulum protein Calnexin. Transmission electron microscopy indicated that the purified sEVs were intact. The purification performance of the ExoPUA protocol showed superior results in terms of yield compared to that of the differential ultracentrifugation method, the most commonly used method for purifying sEVs in laboratories, and purity compared to that of the DEAE chromatography protocol.ConclusionThe sEVs were effectively purified in the bind-elute mode and the ExoPUA column can be refreshed and sterilized with sodium hydroxide (NaOH), having high potential for multiple sEV purification in a scalable and industrial manner.

Protein Hydrolysate from Underutilized Legumes: Unleashing the Potential for Future Functional Foods.

Proteins play a vital role in human development, growth, and overall health. Traditionally, animal-derived proteins were considered the primary source of dietary protein. However, in recent years, there has been a remarkable shift in dietary consumption patterns, with a growing preference for plant-based protein sources. This shift has resulted in a significant increase in the production of plant proteins in the food sector. Consequently, there has been a surge in research exploring various plant sources, particularly wild, and underutilized legumes such as Canavalia, Psophocarpus, Cajanus, Lablab, Phaseolus, and Vigna, due to their exceptional nutraceutical value. This review presents the latest insights into innovative approaches used to extract proteins from underutilized legumes. Furthermore, it highlights the purification of protein hydrolysate using Fast Protein Liquid Chromatography. This review also covers the characterization of purified peptides, including their molecular weight, amino acid composition, and the creation of three-dimensional models based on amino acid sequences. The potential of underutilized legume protein hydrolysates as functional ingredients in the food industry is a key focus of this review. By incorporating these protein sources into food production, we can foster sustainable and healthy practices while minimizing environmental impact. The investigation of underutilized legumes offers exciting possibilities for future research and development in this area, further enhancing the utilization of plant-based protein sources.

Production of Recombinant Zika Virus Envelope Protein by Airlift Bioreactor as a New Subunit Vaccine Platform.

The Zika Virus (ZIKV) is an emerging arbovirus of great public health concern, particularly in the Americas after its last outbreak in 2015. There are still major challenges regarding disease control, and there is no ZIKV vaccine currently approved for human use. Among many different vaccine platforms currently under study, the recombinant envelope protein from Zika Virus (rEZIKV) constitutes an alternative option for vaccine development and has great potential for monitoring ZIKV infection and antibody response. This study describes a method to obtain a bioactive and functional rEZIKV using an E. coli expression system, with the aid of a 5-L airlift bioreactor and following an automated fast protein liquid chromatography (FPLC) protocol, capable of obtaining high yields of approximately 20 mg of recombinant protein per liter of bacterium cultures. The purified rEZIKV presented preserved antigenicity and immunogenicity. Our results show that the use of an airlift bioreactor for the production of rEZIKV is ideal for establishing protocols and further research on ZIKV vaccines bioprocess, representing a promising system for the production of a ZIKV envelope recombinant protein-based vaccine candidate.

Effects of Early Life Oral Arsenic Exposure on Intestinal Tract Development and Lipid Homeostasis in Neonatal Mice: Implications for NAFLD Development.

Newborns can be exposed to inorganic arsenic (iAs) through contaminated drinking water, formula, and other infant foods. Epidemiological studies have demonstrated a positive association between urinary iAs levels and the risk of developing nonalcoholic fatty liver disease (NAFLD) among U.S. adolescents and adults. The present study examined how oral iAs administration to neonatal mice impacts the intestinal tract, which acts as an early mediator for NAFLD. Neonatal mice were treated with a single dose of iAs via oral gavage. Effects on the small intestine were determined by histological examination, RNA sequencing, and biochemical analysis. Serum lipid profiling was analyzed by fast protein liquid chromatography (FPLC), and hepatosteatosis was characterized histologically and biochemically. Liver X receptor-alpha () knockout () mice and liver-specific activating transcription factor 4 (ATF4)-deficient () mice were used to define their roles in iAs-induced effects during the neonatal stage. Neonatal mice exposed to iAs via oral gavage exhibited accumulation of dietary fat in enterocytes, with higher levels of enterocyte triglycerides and free fatty acids. These mice also showed accelerated enterocyte maturation and a longer small intestine. This was accompanied by higher levels of liver-derived very low-density lipoprotein and low-density lipoprotein triglycerides, and a lower level of high-density lipoprotein cholesterol in the serum. Mice exposed during the neonatal period to oral iAs also developed hepatosteatosis. Compared with the control group, iAs-induced fat accumulation in enterocytes became more significant in neonatal mice, accompanied by accelerated intestinal growth, hypertriglyceridemia, and hepatosteatosis. In contrast, regardless of enterocyte fat accumulation, hepatosteatosis was largely reduced in iAs-treated neonatal mice. Exposure to iAs in neonatal mice resulted in excessive accumulation of fat in enterocytes, disrupting lipid homeostasis in the serum and liver, revealing the importance of the gut-liver axis and endoplasmic reticulum stress in mediating iAs-induced NAFLD at an early age. https://doi.org/10.1289/EHP12381.

Production and Purification of Cysteine-Rich Leptospiral Virulence-Modifying Proteins with or Without mCherry Fusion.

Recombinant fluorescent fusion proteins are fundamental to advancing many aspects of protein science. Such proteins are typically used to enable the visualization of functional proteins in experimental systems, particularly cell biology. An important problem in biotechnology is the production of functional, soluble proteins. Here we report the use of mCherry-fusions of soluble, cysteine-rich, Leptospira-secreted exotoxins in the PF07598 gene family, the so-called virulence modifying (VM) proteins. The mCherry fusion proteins facilitated the visual detection of pink colonies of the VM proteins (LA3490 and LA1402) and following them through lysis and sequential chromatography steps. CD-spectroscopy analysis confirmed the stability and robustness of the mCherry-fusion protein, with a structure comparable to AlphaFold structural predictions. LA0591, a unique member of the PF07598 gene family that lacks N-terminal ricin B-like domains, was produced without mCherry tag that strengthens the recombinant protein production protocol without fusion protein as well. The current study provides the approaches for the synthesis of 50-125kDa soluble, cysteine-rich, high-quality fast protein liquid chromatography (FPLC)-purified protein, with and without a mCherry tag. The use of mCherry-fusion proteins enables a streamlined, efficient process of protein production and qualitative and quantitative downstream analytical and functional studies. Approaches for troubleshooting and optimization were evaluated to overcome difficulties in recombinant protein expression and purification, demonstrating biotechnology utility in accelerating recombinant protein production.

A new stability-indicating HPLC-UV method for determination of amlodipine besylate and its impurities in drug substance

A new fast stability-indication high performance liquid chromatography method was developed and validated for the determination of amlodipine besylate and its organic impurities in drug substance. The separation of amlodipine and its seven impurities was achieved on a core shell C18 column, 100 mm × 4.6 mm; 2.6 μm, within 15 min. The mobile phase comprised of 0.4% ammonium hydroxide in water and methanol delivered in a gradient mode; the method detection wavelength is 237 nm. The selected column is stable at high pH and provided a good peak shape for basic compounds. Amlodipine besylate was subject to acid, base, oxidative, thermal, and photolytic stress conditions. The degradation products were well resolved from the amlodipine peak and its impurities. Major degradants were analyzed by liquid chromatography coupled with single-quadrupole mass detector. Amlodipine peak was shown to be free of co-elution by mass spectral analysis in all stress conditions. The method was validated in terms of specificity, linearity, accuracy, precision, and robustness. The developed method could be applied for routine quality control analysis of amlodipine besylate drug substance.

A fast protein liquid chromatography method for purification of myoglobin from different species

The aim of this study is to describe a fast method for the purification of high-purity myoglobin for Mass Spectroscopy analyses and to use it as standard-grade material. A three-step Fast Protein Liquid Chromatography (FPLC) method was used to produce high-purity myoglobin. SEC 650 gel filtration followed by an Enrich Q anion exchange chromatography was used to produce myoglobin in acceptable purity for most research methods. A second filtration step was carried out by narrow field SEC 70 gel to prepare high-purity myoglobin at standard grade purity and capable of Mass Spectroscopy analyses. At least 90% pure myoglobin was obtained by applying two chromatography steps in the samples of three species, and over 99% pure myoglobin was obtained in standard material quality and suitable for mass spectroscopy when the additional narrow field SEC 70 chromatography step was applied. The proposed method provides higher purity compared to other methods and can be applied in a shorter time. FPLC columns significantly reduce the duration of the chromatography steps. At the same time, the use of solid extraction columns instead of dialysis reduced the long overnight dialysis process to a few minutes.

Establishment of a sensitive UPLC-MS/MS method to quantify safinamide in rat plasma.

A fast, simple, and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established for the quantification of safinamide in rat plasma. Plasma samples were treated with acetonitrile for protein precipitation, and diazepam was used as an internal standard (IS). The analytes were separated on an Acquity UPLC C18 (2.1mm × 50mm, 1.7μm) chromatographic column with gradient elution using a mobile phase (0.1% formic acid-acetonitrile). Then, the eluates were detected by electrospray ionization (ESI) in positive ion mode. The analytes were quantified by multiple reaction monitoring (MRM) using the transition m/z 303.3→215.0 of safinamide and m/z 285.0→154.0 of IS. Safinamide had good linearity in the concentration range of 1.0-2000ng/mL, and the lower limit of quantitation (LLOQ) was 1.0ng/mL. The intra- and inter-day precision and accuracy of safinamide were less than 7.63%, while the average recovery rate was 92.98%-100.29%. The method was validated to be stable and had low noise, short chromatographic run time, wide linear range, small sample volumes, low sample injection volumes, and high sensitivity. Therefore, it can be used in pharmacokinetics and preclinical and clinical studies.

SPECT/CT imaging for tracking subendothelial retention of electronegative low-density lipoprotein in vivo

The fifth subfraction of low-density lipoprotein (L5 LDL) can be separated from human LDL using fast-protein liquid chromatography with an anion exchange column. L5 LDL induces vascular endothelial injury both in vitro and in vivo through the lectin-like oxidized LDL receptor-1 (LOX-1). However, no in vivo evidence shows the tendency of L5 LDL deposition on vascular endothelium and links to dysfunction. This study aimed to investigate L5 LDL retention in vivo using SPECT/CT imaging, with Iodine-131 (131I)-labeled and injected into six-month-old apolipoprotein E knockout (apoE−/−) mice through tail veins. Besides, we examined the biodistribution of L5 LDL in tissues and analyzed the intracellular trafficking in human aortic endothelial cells (HAoECs) by confocal microscopy. The impacts of L5 LDL on HAoECs were analyzed using electron microscopy for mitochondrial morphology and western blotting for signaling. Results showed 131I-labeled-L5 was preferentially deposited in the heart and vessels compared to L1 LDL. Furthermore, L5 LDL was co-localized with the mitochondria and associated with mitofusin (MFN1/2) and optic atrophy protein 1 (OPA1) downregulation, leading to mitochondrial fission. In summary, L5 LDL exhibits a propensity for subendothelial retention, thereby promoting endothelial dysfunction and the formation of atherosclerotic lesions.

Molecular imprinted based microcryogels for thrombin purification

In addition to understanding and explaining the functions of proteins, the need for low-cost, easy and efficient purification methods has been increasing in the field of protein purification, which is also important for enzyme production. In this context, an alternative approach has been developed for the purification of thrombin, which has a crucial role in the hemostatic process, via thrombin imprinted microcryogels that allow reuse and have high selectivity. The characterization studies of the microcryogels were accomplished with micro-computed tomography (µCT), scanning electron microscopy (SEM), optical microscope, surface area measurements (BET analyses) and swelling test measurements. By scanning various parameters affecting thrombin adsorption, the maximum thrombin adsorption capacity (Qmax) was found to be 55.86 mg/g. Also, the selectivity of microcryogels was investigated with the competitive agents and reusability studies were performed. The purity of thrombin was evaluated by Fast Performance Liquid Chromatography (FPLC) method. Experimental results indicated that adsorption of thrombin by the developed microcryogels fit the Langmuir isotherm model (Qmax: 55.86 mg/g, R2: 0.9505) and pseudo-second order for three different thrombin concentrations (R2: 0.9978, R2: 0.9998, R2: 0.9999).

Proteomic and functional analysis of HDL subclasses in humans and rats: a proof-of-concept study

BackgroundThe previous study investigated whether the functions of small, medium, and large high density lipoprotein (S/M/L-HDL) are correlated with protein changes in mice. Herein, the proteomic and functional analyses of high density lipoprotein (HDL) subclasses were performed in humans and rats.MethodsAfter purifying S/M/L-HDL subclasses from healthy humans (n = 6) and rats (n = 3) using fast protein liquid chromatography (FPLC) with calcium silica hydrate (CSH) resin, the proteomic analysis by mass spectrometry was conducted, as well as the capacities of cholesterol efflux and antioxidation was measured.ResultsOf the 120 and 106 HDL proteins identified, 85 and 68 proteins were significantly changed in concentration among the S/M/L-HDL subclasses in humans and rats, respectively. Interestingly, it was found that the relatively abundant proteins in the small HDL (S-HDL) and large HDL (L-HDL) subclasses did not overlap, both in humans and in rats. Next, by searching for the biological functions of the relatively abundant proteins in the HDL subclasses via Gene Ontology, it was displayed that the relatively abundant proteins involved in lipid metabolism and antioxidation were enriched more in the medium HDL (M-HDL) subclass than in the S/L-HDL subclasses in humans, whereas in rats, the relatively abundant proteins associated with lipid metabolism and anti-oxidation were enriched in M/L-HDL and S/M-HDL, respectively. Finally, it was confirmed that M-HDL and L-HDL had the highest cholesterol efflux capacity among the three HDL subclasses in humans and rats, respectively; moreover, M-HDL exhibited higher antioxidative capacity than S-HDL in both humans and rats.ConclusionsThe S-HDL and L-HDL subclasses are likely to have different proteomic components during HDL maturation, and results from the proteomics-based comparison of the HDL subclasses may explain the associated differences in function.

Solubilization and Hydrolysis of Porcine Coagulated Blood Protein Using Sub-Critical Solvent Extraction

Pork represents a major fraction of the meat consumed worldwide but only 30% of the blood generated in slaughterhouses is re-used as raw material for food and feed. Innovative technologies and efficient processing strategies capable of generating added-value products from it are now attracting attention. In this study, the hydrolysis of porcine coagulated blood using sub-critical solvent extraction was investigated. Biomass was hydrolyzed using different temperatures (120–210 °C), applying only water (sub-critical water; SCW) or water with a low concentration of alkali (0.1 mol L−1 NaOH) and different reaction times (30–90 min). Resultant hydrolysates were analyzed for crude and soluble protein, peptide profile, and bioactivity by combining protein quantification, antioxidant activity, and fast protein liquid chromatography measurements. Results showed that increasing temperature increases the degree of hydrolysis and that the addition of NaOH enhances the solubilization of peptides with high molecular weights. Also, hydrolysates showed interesting antioxidant activity, being 60 min the time of reaction with best antioxidant activity. Nevertheless, using only water (SCW) as solvent, without chemical additives, allows the delivering of interesting protein-based bioactive fractions. Sub-critical solvent treatment of porcine blood resulted in added-value fractions with potential bioactivities through a simple and environmentally friendly process.