Free Protein Research Articles

Large-scale Purification of Type III Toxin-antitoxin Ribonucleoprotein Complex and its Components from Escherichia coli for Biophysical Studies.

Toxin-antitoxin (TA) systems are widespread bacterial immune systems that confer protection against various environmental stresses. TA systems have been classified into eight types (I-VIII) based on the nature and mechanism of action of the antitoxin. Type III TA systems consist of a noncoding RNA antitoxin and a protein toxin, forming a ribonucleoprotein (RNP) TA complex that plays crucial roles in phage defence in bacteria. Type III TA systems are present in the human gut microbiome and several pathogenic bacteria and, therefore, could be exploited for a novel antibacterial strategy. Due to the inherent toxicity of the toxin for E. coli, it is challenging to overexpress and purify free toxins from E. coli expression systems. Therefore, protein toxin is typically co-expressed and co-purified with antitoxin RNA as an RNP complex from E. coli for structural and biophysical studies. Here, we have optimized the co-expression and purification method for ToxIN type III TA complexes from E. coli that results in the purification of TA RNP complex and, often, free antitoxin RNA and free active toxin in quantities required for the biophysical and structural studies. This protocol can also be adapted to purify isotopically labelled (e.g., uniformly 15N- or 13C-labelled) free toxin proteins, free antitoxin RNAs, and TA RNPs, which can be studied using multidimensional nuclear magnetic resonance (NMR) spectroscopy methods. Key features Detailed protocol for the large-scale purification of ToxIN type III toxin-antitoxin complexes from E. coli. The optimized protocol results in obtaining milligrams of TA RNP complex, free toxin, and free antitoxin RNA. Commercially available plasmid vectors and chemicals are used to complete the protocol in five days after obtaining the required DNA clones. The purified TA complex, toxin protein, and antitoxin RNA are used for biophysical experiments such as NMR, ITC, and X-ray crystallography.

Comparative study of serum free light chain assay and urine protein immunofixation in the diagnosis of patients with monoclonal gammopathy

Serum protein electrophoresis and urine protein immunofixation (IF) are biological techniques for the diagnosis and monitoring of hematological malignancies. The advent of serum free light chain (FLC) assay has provided a new diagnostic element for monoclonal gammopathies. The study conducted in the medical biology laboratory of the Cheikh Zaid University Hospital in Rabat between 2020 and 2022 showed that the determination of serum free light chains (FLCs) provides more critical and accurate information for both the diagnosis and monitoring of hematological malignancies such as light chain myeloma.

Germinated and non-germinated cooked whole millet (Pennisetum glaucum (L.) R. Br.) flours show a promising effect on protein quality, biochemical profile and intestinal health in vivo.

Millet is a promising cereal with high amounts of dietary fibre and protein, and in addition, bioactive compounds with health-promoting functional properties. This study aimed to evaluate the effect of germinated and cooked whole millet flour (Pennisetum glaucum (L.) R. Br.) on protein quality, biochemical profile and intestinal health in vivo. Thirty-two male Wistar rats (21 days old) were separated into four groups, which received a casein control diet (CC; n = 8), a free protein diet (aproteic; n = 8) and two treatment diets: non-germinated millet (NM; n = 8) and germinated millet (GM; n = 8) for 29 days. The whole millet flours presented an adequate essential amino acid profile, except for lysine. The GM group presented a higher protein efficiency ratio and net protein ratio compared to the NM group. Weight gain, Lee index, and food efficiency ratio were lower in the treatment groups, compared to the control group. The GM group had lower plasma glucose, uric acid, cholesterol, and faecal pH compared to the other groups. The treatment groups presented lower triglyceride levels, higher levels of acetic and propionic acids, a larger thickness and depth of the colonic crypts, and a higher expression of PepT1 genes than the CC group. In conclusion, the millet flours demonstrated potential for controlling the lipid profile and biometric measurements. Additionally, the whole germinated millet flour provided better protein quality and improved intestinal morphology and functionality. These results indicate that the consumption of millet could be increased in human food, and considering its potential health benefits, it could be an alternative for dietary diversification, and germination is a good processing option.

The influence of lower-leg injury and knee arthroscopy on natural anticoagulants and fibrinolysis

BackgroundPatients with lower-leg injuries and those undergoing knee arthroscopy are at increased risk of developing venous thromboembolism. The mechanism is unknown, including the influence of lower-leg injury and knee arthroscopy on natural anticoagulant factors and fibrinolysis. ObjectivesTo study the effect of lower-leg injury and knee arthroscopy on plasma levels of anticoagulant and fibrinolytic factors. MethodsWe applied the following 2 designs to investigate this effect: a cross-sectional study for lower-leg trauma and a before-and-after study for knee arthroscopy. Plasma samples of POT-CAST– and POT-KAST–randomized clinical trial participants (collected shortly after lower-leg trauma or before or after arthroscopy) were analyzed for clot lysis time and levels of antithrombin, tissue factor pathway inhibitor, protein C, free protein S, plasminogen, tissue plasminogen activator, plasminogen activator inhibitor 1, antiplasmin, thrombin activatable fibrinolysis inhibitor, plasmin-antiplasmin, and D-dimer. For the effect of lower-leg injury, samples of 289 patients were compared with preoperative samples of 293 arthroscopy patients, acting as controls using linear regression and adjusting for age, sex, body mass index, comorbidities, and diurnal variation. For the effect of knee arthroscopy, mean changes were calculated for 277 patients using linear mixed models adjusted for diurnal variation. Parameters other than CLT and D-dimer were measured in smaller subsets. ResultsIn lower-leg injury patients, most parameters were stable, whereas D-dimer increased. After arthroscopy, most parameters decreased (especially clot lysis time, D-dimer, plasminogen, and anticoagulant factors), whereas tissue plasminogen activator and thrombin activatable fibrinolysis inhibitor slightly increased. ConclusionIn contrast to lower-leg injury, knee arthroscopy was associated with decreased natural anticoagulant factor levels. Neither lower-leg injury nor knee arthroscopy affected in vivo fibrinolysis.

Virus-like Particles for TEM Regulation and Antitumor Therapy.

Tumor development and metastasis are intimately associated with the tumor microenvironment (TME), and it is difficult for vector-restricted drugs to act on the TME for long-term cancer immunotherapy. Virus-like particles (VLPs) are nanocage structures self-assembled from nucleic acid free viral proteins. Most VLPs range from 20-200 nm in diameter and can naturally drain into lymph nodes to induce robust humoral immunity. As natural nucleic acid nanocarriers, their surfaces can also be genetically or chemically modified to achieve functions such as TME targeting. This review focuses on the design ideas of VLP as nanocarriers and the progress of their research in regulating TME.

Small-angle x-ray and neutron scattering of MexR and its complex with DNA supports a conformational selection binding model

In this work, we used small-angle x-ray and neutron scattering to reveal the shape of the protein-DNA complex of the Pseudomonas aeruginosa transcriptional regulator MexR, a member of the multiple antibiotics resistance regulator (MarR) family, when bound to one of its native DNA binding sites. Several MarR-like proteins, including MexR, repress the expression of efflux pump proteins by binding to DNA on regulatory sites overlapping with promoter regions. When expressed, efflux proteins self-assemble to form multiprotein complexes and actively expel highly toxic compounds out of the host organism. The mutational pressure on efflux-regulating MarR family proteins is high since deficient DNA binding leads to constitutive expression of efflux pumps and thereby supports acquired multidrug resistance. Understanding the functional outcome of such mutations and their effects on DNA binding has been hampered by the scarcity of structural and dynamic characterization of both free and DNA-bound MarR proteins. Here, we show how combined neutron and x-ray small-angle scattering of both states in solution support a conformational selection model that enhances MexR asymmetry in binding to one of its promoter-overlapping DNA binding sites.

Characterization of endotoxin free protein production of brain-derived neurotrophic factor (BDNF) for the study of Parkinson model in SH-SY5Y differentiated cells

Human neuronal cells are a more appropriate cell model for neurological disease studies such as Alzheimer and Parkinson's disease. SH-SY5Y neuroblastoma cells have been widely used for differentiation into a mature neuronal cell phenotype. The cellular differentiation process begins with retinoic acid incubation, followed by incubation with brain-derived neurotrophic factor (BDNF), a recombinant protein produced in E. coli cells. Endotoxin or lipopolysaccharide (LPS) is the major component of the outer membrane of bacterial cells that triggers the activation of pro-inflammatory cytokines and ultimately cell death. Consequently, any endotoxin contamination of the recombinant BDNF used for cell culture experiments would impact on data interpretation. Therefore, in this study, we expressed the BDNF recombinant protein in bacterial endotoxin-free cells that were engineered to modify the oligosaccharide chain of LPS rendering the LPS unable to trigger the immune response of human cells. The expression of DCX and MAP-2 in differentiated cells indicate that in-house and commercial BDNF are equally effective in inducing differentiation. This suggests that our in-house BDNF protein can be used to differentiate SH-SY5Y neuroblastoma cells without the need for an endotoxin removal step.

Fluorescence Polarization Immunoassay of Human Lactoferrin in Milk Using Small Single-Domain Antibodies

Due to its unique structure and properties, human breast milk lactoferrin (hLF) has many nutritional and health-promoting functions in infants, including protection against inflammation and bacterial infections. The lack of LF in breastmilk or formula can result in the weakening of the infant’s immune system. Noncompetitive polarization fluorescence immunoassay (FPIA) is a promising method for hLF quantification in milk and dairy products, which does not require the separation of the bound and free protein and allows to avoid time-consuming sample preparation. The use of fluorescently labeled single-domain camelid antibodies (nanobodies) for protein recognition in FPIA makes it possible to quantify relatively large antigens, in particular, hLF. In this work, we used previously obtained fluorescein isothiocyanate (FITC)-conjugated anti-hLF5 and anti-hLF16 nanobodies, which selectively recognized two different human lactoferrin epitopes, but did not bind to goat lactoferrin. The kinetics of hLF interaction with the FITC-labeled nanobodies was studied. The dissociation constant (KD) for the anti-LF5 and anti-LF16 nanobodies was 3.2 ± 0.3 and 4.9 ± 0.4 nM, respectively, indicating the high-affinity binding of these nanobodies to hLF. We developed the FPIA protocol and determined the concentration of FITC-labeled anti-hLF5 and anti-hLF16 nanobodies that provided the optimal fluorescence signal and stable fluorescence polarization value. We also studied the dependence of fluorescence polarization on the hLF concentration in the noncompetitive FPIA with FITC-anti-hLF5 nanobody. The detection limit for hLF was 2.1 ± 0.2 µg/ml and the linear range for determining the hLF concentration was 3-10 µg/ml. FPIA is commonly used to assay low-molecular-weight substances; however, the use of fluorescently labeled nanobodies allows quantification of high-molecular-weight proteins. Here, we demonstrated that FPIA with fluorescently labeled nanobodies can be used for hLF quantification in milk.

Dimerization of the Free and Photosystem II-Associated PsbO Protein upon Irradiation with UV Light

Dimerization of the Free and Photosystem II-Associated PsbO Protein upon Irradiation with UV Light

Flavor compounds, free amino acids, and proteins in Agaricus bisporus mushroom powder

Mushrooms contain a remarkable amount of complete protein, indicating potential as a conventional protein alternative. Commercially available mushroom powder would be protein concentrate and isolate starting material, while valorizing all powder molecules is sustainable and economical. This study aimed to quantify taste-related compounds (five soluble sugars, five organic acids, and five 5′-nucleotides), 23 free amino acids, protein, and other proximate compositions in two A. bisporus mushroom powders. The most dominant sugar was mannitol (5.6 and 6.9% dry matter), followed by glucose and sucrose. The major acid was oxalic acid (0.30 and 0.48%), followed by acetic and malic. Among five nucleotides, 5′-GMP (umami taste, 0.08 and 0.11%) was predominant. Total free amino acids were 4.0 and 6.5%, of which Glu (umami taste, 1.1 and 1.4%) was dominant. Asp (umami taste, 0.27 and 0.67%) was lower. The mushroom powders contained all essential and sweet- and bitter-related amino acids along with around 20% protein; the protein included at least six different fractions per SDS-PAGE. The powders were also majorly comprised of carbohydrates, especially fiber. These results demonstrated taste-related compounds and the nutritional composition in mushroom powders, providing evidence for mushroom powder use as a starting material to develop mushroom protein concentrates and isolates.

Current Trends in Liquid Biopsy Technology for Early Diagnosis of Metastatic Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC) is a disease with a wide variety of clinical progressions such as the rate of disease progression or the degree of metastasis. About 30% of ccRCC patients suffer from metastatic diseases, and about 30% develop metastasis after diagnosis. In the case of metastatic RCC, early prediction of the disease is important because of the poor prognosis, but ccRCC-specific molecular markers for clinical use are not available yet. As an alternative, liquid biopsy, which can find molecules released from tumor tissues in circulating blood and obtain information on metastatic dissemination and recurrence of ccRCC, is emerging. In this article, we will introduce molecules such as cell free DNA, cell free RNA, protein, and exosomes available as circulating biomarkers for liquid biopsy. We will also introduce some promising technologies that can compensate for the limitations of liquid biopsy.

Optimisation of a High-Throughput Model for Mucus Permeation and Nanoparticle Discrimination Using Biosimilar Mucus.

High-throughput permeation models are essential in drug development for timely screening of new drug and formulation candidates. Nevertheless, many current permeability assays fail to account for the presence of the gastrointestinal mucus layer. In this study, an optimised high-throughput mucus permeation model was developed employing a highly biorelevant mucus mimic. While mucus permeation is primarily conducted in a simple mucin solution, the complex chemistry, nanostructure and rheology of mucus is more accurately modelled by a synthetic biosimilar mucus (BSM) employing additional protein, lipid and rheology-modifying polymer components. Utilising BSM, equivalent permeation of various molecular weight fluorescein isothiocyanate-dextrans were observed, compared with native porcine jejunal mucus, confirming replication of the natural mucus permeation barrier. Furthermore, utilising synthetic BSM facilitated the analysis of free protein permeation which could not be quantified in native mucus due to concurrent proteolytic degradation. Additionally, BSM could differentiate between the permeation of poly (lactic-co-glycolic) acid nanoparticles (PLGA-NP) with varying surface chemistries (cationic, anionic and PEGylated), PEG coating density and size, which could not be achieved by a 5% mucin solution. This work confirms the importance of utilising highly biorelevant mucus mimics in permeation studies, and further development will provide an optimal method for high-throughput mucus permeation analysis.

SARS-CoV-2 Spike Protein-Activated Dendritic Cell-Derived Extracellular Vesicles Induce Antiviral Immunity in Mice.

The onset and spread of the SARS-CoV-2 virus have created an unprecedented universal crisis. Although vaccines have been developed against the parental SARS-CoV-2, outbreaks of the disease still occur through the appearance of different variants, suggesting a continuous need for improved and effective therapeutic strategies. Therefore, we developed a novel nanovesicle presenting Spike protein on the surface of the dendritic cell-derived extracellular vesicles (DEVs) for use as a potential vaccine platform against SARS-CoV-2. DEVs express peptide/MHC-I (pMHC-I) complexes, CCR-7, on their surface. The immunogenicity and efficacy of the Spike-activated DEVs were tested in mice and compared with free Spike protein. A 1/10 Spike equivalent dose of DEVs showed a superior potency in inducing anti-Spike IgG titers in blood of mice when compared to dendritic cells or free Spike protein treatment. Moreover, DEV-induced sera effectively reduced viral infection by 55-60% within 15 days of booster dose administration. Furthermore, a 1/10 Spike equivalent dose of DEV-treated mice was found to be equally effective in inducing CD19+CD38+ T-cells in the spleen and lymph node; CD8 cells in the bone marrow, spleen, and lymph node; and CD4+CD25+ T-cells in the spleen and lymph node after 90 days of treatment. Thus, our results support the immunogenic nature of DEVs, demonstrating that a low dose of DEVs induces antibodies to inhibit SARS-CoV-2 infection in vitro, therefore warranting further investigations.

Characterization of FcγRIa (CD64) as a Ligand Molecule for Site-Specific IgG1 Capture: A Side-By-Side Comparison with Protein A.

Fc γ receptors (FcγRs) are one of the structures that can initiate effector function for monoclonal antibodies. FcγRIa has the highest affinity toward IgG1-type monoclonal antibodies among all FcγRs. In this study, a comprehensive characterization was performed for FcγRIa as a potential affinity ligand for IgG1-type monoclonal antibody binding. The binding interactions were assessed with the SPR technique using different immobilization techniques such as EDC-NHS coupling, streptavidin-biotin interaction, and His-tagged FcγRIa capture. The His-tagged FcγRIa capture was the most convenient method based on assay repeatability. Next, a crude IgG1 sample and its fractions with different monomer contents obtained from protein A affinity chromatography were used to evaluate FcγRIa protein in terms of monoclonal antibody binding capacity. The samples were also compared with a protein A-immobilized chip (a frequently used affinity ligand) for IgG1 binding responses. The antibody binding capacity of the protein A-immobilized chip surface was significantly better than that of the FcγRIa-immobilized chip surface due to its 5 Ig binding domains. The antibody binding responses changed similarly with protein A depending on the monomer content of the sample. Finally, a different configuration was used to assess the binding affinity of free FcγRs (FcγRIa, FcγRIIa, and FcγRIIIa) to three different immobilized IgGs by immobilizing protein L to the chip surface. Unlike previous immobilization techniques tested where the FcγRIa was utilized as a ligand, nonimmobilized or free FcγRIa resulted in a significantly higher antibody binding response than free protein A. In this configuration, kinetics data of FcγRI revealed that the association rate (ka 50-80 × 105 M-1 s-1) increased in comparison to His capture method (1.9-2.4 × 105 M-1 s-1). In addition, the dissociation rate (kd 10-5 s-1) seemed slower over the His capture method (10-4 s-1) and provided stability on the chip surface during the dissociation phase. The KD values for FcγRIa were found in the picomolar range (2.1-10.33 pM from steady-state affinity analysis and 37.5-46.2 pM from kinetic analysis) for IgG1-type antibodies. FcγRIa possesses comparable ligand potential as well as protein A. Even though the protein A-immobilized surface bound more antibodies than the FcγRIa-captured surface, FcγRIa presented a significant antibody binding capacity in protein L configuration. The results suggest FcγRIa protein as a potential ligand for site-oriented immobilization of IgG1-type monoclonal antibodies, and it needs further performance investigation on different surfaces and interfaces for applications such as sensing and antibody purification.

Running for Your Life: Metabolic Effects of a 160.9/230 km Non-Stop Ultramarathon Race on Body Composition, Inflammation, Heart Function, and Nutritional Parameters.

Moderate endurance exercise leads to an improvement in cardiovascular performance, stress resilience, and blood function. However, the influence of chronic endurance exercise over several hours or days is still largely unclear. We examined the influence of a non-stop 160.9/230 km ultramarathon on body composition, stress/cardiac response, and nutrition parameters. Blood samples were drawn before (pre) and after the race (post) and analyzed for ghrelin, insulin, irisin, glucagon, cortisol, kynurenine, neopterin, and total antioxidant capacity. Additional measurements included heart function by echocardiography, nutrition questionnaires, and body impedance analyses. Of the 28 included ultra-runners (7f/21m), 16 participants dropped out during the race. The remaining 12 finishers (2f/10m) showed depletion of antioxidative capacities and increased inflammation/stress (neopterin/cortisol), while energy metabolism (insulin/glucagon/ghrelin) remained unchanged despite a high negative energy balance. Free fat mass, protein, and mineral content decreased and echocardiography revealed a lower stroke volume, left end diastolic volume, and ejection fraction post race. Optimizing nutrition (high-density protein-rich diet) during the race may attenuate the observed catabolic and inflammatory effects induced by ultramarathon running. As a rapidly growing discipline, new strategies for health prevention and extensive monitoring are needed to optimize the athletes' performance.

Effect of solution condition on the binding behaviors of monoclonal antibody and fusion protein therapeutics in Protein A chromatography

Recent development in Protein A chromatography has adopted wash buffers with a wide range of pH to improve the removal of process-related impurities, e.g., host cell proteins (HCPs), from Fc-based target biological products. Thus, it is important to investigate the pH effect on the binding behaviors of target products of various properties on Protein A resins. Here we performed column breakthrough experiments for two monoclonal antibodies (mAbs) and one Fc-fusion protein on two Protein A resins from pH 4 to pH 10, and the experimental data was analyzed using a mechanistic model to obtain isotherm and mass transfer parameters. The two mAbs exhibited relatively high dynamic binding capacity (DBC) at 10% breakthrough of 43 – 67 g/L at pH 6–9 followed by a ∼30% decrease from pH 9 to pH 10; while the Fc-fusion protein reached the highest DBC at 10% breakthrough of 10 – 17 g/L at pH 5 and thereafter the capacity gradually reduced. Model analyses revealed that the two mAbs had higher maximum binding capacity by 2 – 5 fold but weaker binding affinity (12 – 64%) than the Fc-fusion protein from pH 5 to pH 9. For the three molecules, similar patterns of the pH impact were observed on the two Protein A resins with the Jetted A50 resin showing generally higher DBC and stronger binding affinity than the MabSelect SuRe LX resin. Additionally, an inverse relationship between the binding affinity and surface diffusivity was observed for both resins. Besides obtaining the isotherm parameters from the column breakthrough data, a direct batch equilibrium measurement showed comparable trend in these parameters with relatively more scattered values due to the inherent uncertainties to accurately determine the initial slope of the isotherm in highly favorable adsorption conditions. Finally, isothermal titration calorimetry (ITC) results revealed that the measured binding affinity using free Protein A ligand was stronger than that obtained from the breakthrough and isotherm results for the resin, possibly due to the reduced accessibility of the immobilized ligand on resin surface. Overall, this work can facilitate future Protein A ligand design and affinity chromatography process optimization for biomanufacturing.

Improvement of adzuki bean paste quality by Flavourzyme-mediated enzymatic hydrolysis

The primary aim of this study was to investigate the changes in the quality and flavor profiles of adzuki bean paste hydrolyzed by Flavourzyme. The texture analyzer and gas chromatography-mass spectrometry were used to evaluate the texture, and volatile organic compounds of adzuki bean paste. At the same time, rheometer, high-performance liquid chromatography (HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and size-exclusion chromatography (SEC) were used to evaluate the changes of adzuki bean paste viscoelasticity, free amino acids and protein. The results demonstrated that the appropriate amount (0.3%, adzuki bean basis) of Flavourzyme enhanced the quality of bean paste, resulting in denser taste and reduced hardness by 51.94% (from 2097.30 g to 1007.79 g). Flavourzyme had an obvious hydrolysis effect on the protein of adzuki bean. The results of SDS-PAGE indicated that low molecular weight proteins increased significantly with higher amount of Flavourzyme addition, which is consistent with the conclusions of SEC (≤12 kDa, increased from 26.12% to 88.65% with 0.3% Flavourzyme). The hydrolysis of protein might contribute to the reduced viscoelasticity and softer texture of adzuki bean paste. Furthermore, the addition of Flavourzyme produced more small peptides and amino acids. It could promote the Maillard reaction during bean paste frying. Thus Maillard-reaction-derived volatile compounds (furfural, maltol, 2-pentylfuran, etc.) were enhanced to improve the flavor of bean paste. The positive effects of appropriate amount of Flavourzyme on adzuki bean paste properties indicated the potential application of Flavourzyme on bean paste.

Unfolded Von Willebrand Factor Interacts with Protein S and Limits Its Anticoagulant Activity

Background: The critical plasma anticoagulant protein S (PS) circulates in two pools: free (anticoagulant) or bound to complement component 4-binding protein (C4BP) (antiinflammatory). Acquired free PS deficiency occurs in patients with severe viral infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and is associated with increased risk of thrombosis. von Willebrand Factor (VWF) is a large multimeric protein , which unfolds under shear, exposing protein binding sites. Elevation of VWF antigen and activity are associated with infection-induced vascular endothelial activation, including during SARS-CoV-2 infection. Here, we identified a shear-dependent association between VWF and PS, and assessed the effect of VWF on PS anticoagulant activity. Methods: This study was approved by the University of Kentucky Institutional Review Board. Citrated plasma was collected from consenting adults, including 28 SARS-CoV-2+ inpatients, 49 SARS-CoV-2+ outpatients, and 31 healthy controls. Results: Initial studies in our SARS-CoV-2+ patient cohort revealed that free PS, a measure of the anticoagulant pool, was similarly reduced in both inpatients (p=0.037) and outpatients (p=0.008) compared to controls, while total PS (p=0.964) was unchanged. This indicated an increase in a plasma PS-binding protein. However, known abundant PS-binding proteins C4BPβ and protein C, an anticoagulant that functions with PS, were unchanged (p=0.647 and p=0.185, respectively), and Mer, a macrophage receptor that binds PS, was only mildly increased (p=0.042). Mass spectrometric and immunoblotting analyses revealed that PS interacts with VWF in pooled donor plasmas and that this interaction is enhanced >10,000,000-fold by shear-induced unfolding of VWF. Plasma VWF was elevated 3.8-fold in inpatients (p<0.0001) compared to outpatients and controls, suggesting endothelial activation, though no change in multimer distribution was apparent (n=4). We hypothesize that shear-induced unfolding of VWF exposes a binding site, which interacts either directly or indirectly with PS and contributes to acquired free PS deficiency. Consistent with this hypothesis, sheared-VWF dose-dependently inhibited free, but not total, PS antigen measurements, when measuring either purified proteins or plasma. We next assessed the functional consequences of the VWF/PS interaction using purified proteins. Sheared-VWF dose dependently reversed FXa inhibition by the PS/Tissue Factor Pathway Inhibitor-α complex, in a chromogenic FXa activity assay. Conversely, Activated Protein C (APC) cofactor function was unaffected, either on phospholipid vesicles or on the surface of activated platelets, as measured either by the cleavage of factor Va heavy chain by western blot and by the rate of thrombin activation. Finally, to assess PS function in patients, we measured plasma thrombin generation. Despite anticoagulation, plasma thrombin generation in inpatient samples was comparable to controls (Endogenous Thrombin Potential (ETP): p=0.257; Lag Time: p=0.095), suggesting a significant hypercoagulability, possibly exacerbated by PS deficiency. Consistent with this, free PS negatively correlated with plasma thrombin generation parameters measured with thrombomodulin (TM) supplementation (r=-0.443, p=0.0008), which is required for sensitivity to the APC/PS pathway; ETP ratio (ETP with TM/ ETP without TM) in inpatients was higher compared to controls indicating less APC/PS activity (p=0.009); and these patients had profoundly elevated D-dimer (p<0.0001). Conclusion: We propose a novel mechanism of acquired free PS deficiency, by which pathological shear forces unfold VWF, which interacts with PS, and at least partially blocks its anticoagulant function. It is possible that unfolded VWF further limits PS anticoagulant activity in vivo by sequestering it from APC, factor IXa, or other interacting partners. We anticipate that this mechanism also contributes to acquired free PS deficiency in other inflammatory conditions in which VWF is similarly unfolded.

Value of Inpatient Inherited Thrombophilia Testing Following Acute VTE: A 3-Year Retrospective Study

BACKGROUND: Inherited thrombophilia testing following venous thromboembolism (VTE) is of limited utility and, performed in the setting of an acute thrombotic episode, is especially unreliable. Studies suggest that inpatient thrombophilia workups, often performed in the context of acute VTE or stroke, lead to high rates of false positives and can result in inappropriate long-term management decisions. Therefore, various professional societies advise against such testing, especially in acute care settings. These include Choosing Wisely recommendations from the American Society of Hematology, who recommend against routine testing following first episode of provoked VTE, and the American Society for Clinical Pathology, who advise against ordering these tests during an active clotting event. However, these studies continue to be ordered regularly, increasing the overall cost of health care with limited clinical benefit. METHODS: We carried out a retrospective chart review of all inpatient inherited thrombophilia tests ordered between January 1st, 2019 and December 31st, 2021 at Thomas Jefferson University Hospitals, including its 3 primary hospitals in Philadelphia, PA. The goal was to determine indications for testing, rate of positivity, and whether positive results changed patients’ clinical management. Tests included in the study were for the following conditions: Factor V Leiden (FVL); prothrombin G20210A variant (PTG20210A); Protein C, S, and antithrombin deficiency; hyperhomocysteinemia; and plasminogen activator inhibitor-1 (PAI-1) elevation. The study included 152 patients, among whom 574 unique tests were ordered. Tests sent for non-thrombotic indications or in patients with a known factor deficiency were excluded. RESULTS: Patient demographics, clinical presentations, and VTE risk factors are summarized in Table 1. Median age of the patients was 49 years (IQR 34-62) and 53.9% were under age 50. 93 patients (61.2%) were female. VTE was associated with a major transient risk factor or cancer in 44.7% of patients. Conditions for which testing was ordered included FVL (24.7%), PTG20210A (20.4%) Protein S deficiency (16.5%), Protein C deficiency (14.8%), antithrombin deficiency (13.0%), hyperhomocysteinemia (10.3%), and PAI-1 elevation (0.2%). Overall, 81.9% of tests were normal. Among unique tests, MTHFR analysis was most frequently abnormal (71.4% of ordered tests), followed by antithrombin (44.0%), Protein C antigen (27.3%), total Protein S (26.7%), Protein C function (25.7%), and free protein S (20.0%). Certain tests, including antithrombin activity, Protein C antigen and function, and total Protein S, have reference ranges in our lab that start well above 50%, so results that fell below normal but >60% were termed "borderline positive” and considered to be likely false positives. Among individual patients, 62/152 (40.8%) had at least one abnormal result and 47/152 (30.9%) had at least one positive result not considered borderline. There was no significant difference in rate of having at least one non-borderline positive result between patients with unprovoked VTE (27/84, 32.1%) and those with a major transient risk factor, estrogen use/pregnancy, or cancer (20/68, 29.4%; χ2, P=.717). There was also no difference in positive test rate between patients age < 50 years (24/82, 29.3%) and those ≥ 50 years (23/70, 32.9%; P=.633) (Figure 1). All charts were reviewed for inpatient and outpatient documentation to determine short- and long-term clinical decision-making. In 2/152 (1.3%) patients, documentation supported a definitive change in management because of a positive thrombophilia test result. These included a 20-year-old woman with large PE while on a combined oral contraceptive and a 33-year-old man with provoked DVT following severe spinal cord injury (resulting in tetraplegia) who were continued on long-term anticoagulation for Protein S deficiency and double heterozygosity for FVL and PTG20210A, respectively. Based on query of the hospital chargemaster, all tests during the study period totaled $264,913 in charges. CONCLUSION: Our single-institution study found that following acute VTE, inpatient testing for inherited thrombophilias was frequently done in patients with provoked events and resulted in many abnormal tests likely comprising false positives, rarely changed clinical management, and was associated with high costs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Base Editing for Therapeutic Induction of Fetal Hemoglobin in β-Thalassemia

β-thalassemia, one of the most common inherited genetic disorders worldwide, is caused by more than 300 different mutations in the β-globin locus (HBB), all of which result in quantitative deficiency of β-globin protein with severe anemia and reduced or absent HbA (α2β2). Additionally, free α-globin protein forms toxic intracellular inclusions resulting in hemolysis of mature red blood cells, maturation arrest and apoptosis of erythroid precursors. The only approved cure for severe β-thalassemia is allogeneic hematopoietic stem cell transplantation. However, many patients lack ideal histocompatible donors and immune complications such as graft rejection and graft-versus-host disease can occur. These problems can be avoided by "gene therapies” in which the patient's own hematopoietic stem cells (HSCs) are isolated, genetically modified ex vivo and reinfused after administration of bone marrow conditioning to facilitate engraftment of the modified cells. Most β-thalassemia mutations can be treated by genetic manipulations that de-repress fetal-expressed γ-globin genes (HBG1 and HBG2) to induce fetal hemoglobin production (HbF, α2γ2) postnatally. Thus, Cas9 disruption of an erythroid enhancer in the γ-globin repressor gene BCL11A shows promising results in an ongoing clinical trial. However, multiple other strategies to induce HbF exist and the best one is not known. Compared to conventional Cas9 nuclease, base editors offer theoretical advantages, including the ability to create precise base pair substitutions without requiring genotoxic double strand DNA breaks. We used the adenine base editor ABE8e to generate the naturally occurring γ-globin -175 A>G variant, which causes hereditary persistence of fetal hemoglobin (HPFH), a benign genetic condition that alleviates co-inherited β-thalassemia. We edited human CD34+ hematopoietic stem and progenitor cells (HSPCs) from two individuals with severe transfusion dependent β-thalassemia by electroporating ribonucleoprotein (RNP) complexes of ABE8e-NG protein + single guide RNA (sgRNA) designed to install the γ-globin -175 A>G variant, followed by in vitro erythroid differentiation. On-target editing rates were 48-53% for donor 1 (n=3 replicates) and 22.9% for donor 2 (n=1). Analysis of burst forming unit erythroid (BFU-E) colonies revealed at least one on-target edit in 78% of donor 1 HSPCs and 63% of those from donor 2. Reverse phase HPLC of erythroid progeny showed increased β-like/α globin protein ratios: 0.92 ± 0.04 vs 0.53 ± 0.03 in unedited controls (p<0.0001) for donor 1, and 0.8 vs 0.5 in unedited controls for donor 2. Quantitative RT-PCR showed 6-8-fold increased γ-globin mRNA in the ABE-treated β-thalassemia cells. Ion exchange HPLC analysis of erythroid cell lysates showed reductions in free α-globin levels after ABE treatment: from 21.5 ± 2% of total globin protein in unedited cells to undetectable levels after editing in donor 1 and from 18% to 2.5% for donor 2. Maturation to the reticulocyte stage was also increased in base edited cells: 14.6 ± 2% vs 3.3 ± 1% in unedited controls for donor 1 (P<0.0001); and 26% vs 6.9% in unedited controls for donor 2. Reduction in apoptosis (37.4 ± 2.2% vs 23.6 ± 1.5% in unedited control cells, p=0.0003) and reactive oxygen species measured by DCFDA (MFI: 690 ± 97 vs 266 ± 9 in unedited control cells, p=0.009) was also seen in the ABE edited groups. To study editing outcomes in vivo, CD34+ HSPCs from donor 2 were treated with RNP and transplanted into immunodeficient NBSGW mice. After 16-18 weeks, human donor-derived cells were isolated from mouse recipient bone marrow and analyzed (n=4 mice per group). On-target editing frequencies in CD235a+ erythroblasts and CD34+ hematopoietic stem/progenitors were 17.5 ± 5% and 19.5 ±3%, respectively, compared to 22.9% in the input CD34+ cells. Similar to what we observed after in vitro differentiation, base edited erythroid cells generated in vivo exhibited improved globin chain balance with β-like/α globin protein ratios of 0.86 ± 0.12 vs 0.69 ± 0.04 in unedited controls, (p=0.04), reduced apoptosis (16.8 ± 4.2 % vs 26.6 ± 2.9 % in unedited control cells, p=0.004) and enhanced maturation into enucleated reticulocytes (14.4 ± 4.7 % vs 2.87 ± 1.4 % in unedited control cells, p=0.005). Our study provides proof of concept that ABE8e generation of the γ-globin -175 A>G HPFH variant in β-thalassemic HSCs represents a new therapeutic approach for β-thalassemia.