Engineering and evaluation of FXa bypassing agents that restore hemostasis following Apixaban associated bleeding.

Direct oral anticoagulants (DOACs) targeting activated factor Xa (FXa) are used to prevent or treat thromboembolic disorders. DOACs reversibly bind to FXa and inhibit its enzymatic activity. However, DOAC treatment carries the risk of anticoagulant-associated bleeding. Currently, only one specific agent, andexanet alfa, is approved to reverse the anticoagulant effects of FXa-targeting DOACs (FXaDOACs) and control life-threatening bleeding. However, because of its mechanism of action, andexanet alfa requires a cumbersome dosing schedule, and its use is associated with the risk of thrombosis. Here, we present the computational design, engineering, and evaluation of FXa-variants that exhibit anticoagulation reversal activity in the presence of FXaDOACs. Our designs demonstrate low DOAC binding affinity, retain FXa-enzymatic activity and reduce the DOAC-associated bleeding by restoring hemostasis in mice treated with apixaban. Importantly, the FXaDOACs reversal agents we designed, unlike andexanet alfa, do not inhibit TFPI, and consequently, may have a safer thrombogenic profile.

Investigation of thrombin concentration at the time of clot formation in simultaneous thrombin and fibrin generation assays.

Thrombin generation (TG) and fibrin clot formation represent the central process of blood coagulation. Up to 95% of thrombin is considered to be generated after the clot is formed. However, this was not investigated in depth. In this study, we conducted a quantitative analysis of the Thrombin at Clot Time (TCT) parameter in 5758 simultaneously recorded TG and clot formation assays using frozen plasma samples from commercial sources under various conditions of activation. These samples were supplemented with clotting factor concentrates, procoagulant lipid vesicles and a fluorogenic substrate and triggered with tissue factor (TF). We found that TCT is often close to a 10% of thrombin peak height (TPH) yet it can be larger or smaller depending on whether the sample has low or high TPH value. In general, the samples with high TPH are associated with elevated TCT. TCT appeared more sensitive to some procoagulant phenotypes than other commonly used parameters such as clotting time, TPH or Thrombin Production Rate (TPR). In a minority of cases, TCT were not predicted from TG parameters. For example, elevated TCT (above 15% of TPH) was associated with either very low or very high TPR values. We conclude that clotting and TG assays may provide complementary information about the plasma sample, and that the TCT parameter may serve as an additional marker for the procoagulant potential in plasma sample.

Efficiently Differentiating Agonists and Competitive Antagonists for Weak Allosteric Protein-Ligand Interactions with Linear Response Theory.

What makes an agonist and a competitive antagonist? In this work, we aim to answer this question by performing parallel tempering Monte Carlo simulations on the serotonin type 3A (5-HT3A) receptor. We use linear response theory to predict conformational changes in the 5-HT3A receptor active site after weak perturbations are applied to its allosteric binding sites. A covariance tensor is built from conformational sampling of its apo state, and a harmonic approximation allows us to substitute the calculation of ligand-induced forces with the binding site's displacement vector. Remarkably, our study demonstrates the feasibility of effectively discerning between agonists and competitive antagonists for multiple ligands, requiring computationally expensive calculations only once per protein.

Study of protonated dimers of cytosine, cytidine, and deoxycytidine using survival yield method and quantum mechanics calculations.

RATIONALE: Cytosine and its conjugates are prone to form protonated, triply-bonded dimers. Therefore, the nucleic-acid cytosine-rich sequence forms the four-stranded noncanonical secondary structure known as the intercalated motif (i-motif). This process has resulted in studies on cytosine protonated dimers. This communication focuses on the protonated dimers of cytosine and its nucleoside using the survival yield (SY) method and quantum mechanics calculations. METHODS: To obtain the precursor ion fragmentation curve, the plot of SY against Ecomδ , the product ion spectra of the protonated dimers were obtained using a Waters/Micromass Q-TOF Premier mass spectrometer. Quantum mechanics calculations were performed using GAUSSIAN 16, and full geometry optimizations and energy calculations were performed within the density functional theory framework at B3LYP/6-31G(d,p). RESULTS: The precursor ion fragmentation curve allowed the rating of the gas-phase stabilities of the analyzed protonated dimers. Substitution of sugar moiety at N1 cytosine atom decreased the gas-phase stabilities of the protonated dimers. The deoxycytidine dimer was found to be more stable than the cytidine dimer and cytidine-deoxycytidine dimer. Quantum chemical calculations indicated that cytosine aminohydroxy tautomer may be involved in the formation of protonated cytosine-cytosine nucleoside dimers but not in the formation of cytosine dimers. CONCLUSIONS: The results obtained for nucleoside dimers indicated that the SY method may reflect the i-motif stabilities observed under physiological conditions. Therefore, the analysis of other protonated dimers of variously substituted cytosine-cytosine nucleoside using the SY method may be important to study the effect of cytosine substitution on the i-motif stabilities. Cytosine tautomer containing C2-OH… N(2H)-C4 moiety may be involved in the formation of protonated cytosine-cytosine nucleoside dimers.

The MHC Associated Peptide Proteomics assay is a useful tool for the non-clinical assessment of immunogenicity.

The propensity of therapeutic proteins to elicit an immune response, poses a significant challenge in clinical development and safety of the patients. Assessment of immunogenicity is crucial to predict potential adverse events and design safer biologics. In this study, we employed MHC Associated Peptide Proteomics (MAPPS) to comprehensively evaluate the immunogenic potential of re-engineered variants of immunogenic FVIIa analog (Vatreptacog Alfa). Our finding revealed the correlation between the protein sequence affinity for MHCII and the number of peptides identified in a MAPPS assay and this further correlates with the reduced T-cell responses. Moreover, MAPPS enable the identification of "relevant" T cell epitopes and may contribute to the development of biologics with lower immunogenic potential.

Corrigendum to "Computational design of nanomolar-binding antibodies specific to multiple SARS-CoV-2 variants by engineering a specificity switch of antibody 80R using RosettaAntibodyDesign (RAbD) results in potential generalizable therapeutic antibodies for novel SARS-CoV-2 virus" [Heliyon 9(4) (April 2023) e15032].

[This corrects the article DOI: 10.1016/j.heliyon.2023.e15032.].

Relationships between Changes in Hematological Adaptations and Exercise Capacity in Olympic Rowers after a Period of Reduced Training Loads.

Endurance performance is positively associated with hematological adaptations; therefore, high total hemoglobin mass and intravascular volumes are commonly observed in high-level endurance athletes. However, it is still unclear whether the fluctuations in exercise capacity that typically occur in endurance athletes during the annual training cycle are directly associated with changes in hematological adaptations, which appear to be relatively stable during this time. To better understand this issue, a study was conducted with 10 Olympic rowers, who followed the same training program. Athletes underwent laboratory testing in the competitive and the general preparation phase of an annual training cycle (a 34% reduction in training volume). This included a graded exercise test on a rowing ergometer (GXT) and blood measurements of hemoglobin concentration (Hb), total hemoglobin mass (tHb-mass), plasma volume (PV), and blood volume (BV). Decreases in maximal values of power relative to body mass (p = 0.028), lactate concentration (p = 0.005), and heart rate (p = 0.017) in the GXT were registered. At the same time, absolute (p = 0.017) and relative (p = 0.005) PV decreased. Changes in PV (rS = 0.842, p = 0.002) and BV (rS = 0.818, p = 0.004), but not in tHb-mass (rS = 0.588, p = 0.074) and Hb (rS = -0.188, p = 0.602), were significantly correlated with changes in maximal power in the GXT. Our results indicate a close relationship between changes in intravascular volumes and maximal exercise capacity after a period of reduced training loads in elite endurance athletes.

Computational design of nanomolar-binding antibodies specific to multiple SARS-CoV-2 variants by engineering a specificity switch of antibody 80R using RosettaAntibodyDesign (RAbD) results in potential generalizable therapeutic antibodies for novel SARS-CoV-2 virus.

The human infectious disease COVID-19 caused by the SARS-CoV-2 virus has become a major threat to global public health. Developing a vaccine is the preferred prophylactic response to epidemics and pandemics. However, for individuals who have contracted the disease, the rapid design of antibodies that can target the SARS-CoV-2 virus fulfils a critical need. Further, discovering antibodies that bind multiple variants of SARS-CoV-2 can aid in the development of rapid antigen tests (RATs) which are critical for the identification and isolation of individuals currently carrying COVID-19. Here we provide a proof-of-concept study for the computational design of high-affinity antibodies that bind to multiple variants of the SARS-CoV-2 spike protein using RosettaAntibodyDesign (RAbD). Well characterized antibodies that bind with high affinity to the SARS-CoV-1 (but not SARS-CoV-2) spike protein were used as templates and re-designed to bind the SARS-CoV-2 spike protein with high affinity, resulting in a specificity switch. A panel of designed antibodies were experimentally validated. One design bound to a broad range of variants of concern including the Omicron, Delta, Wuhan, and South African spike protein variants.

[The influence of working/learning remotely on the prevalence of musculoskeletal complaints in a group of university staff and students]. / Wplyw pracy/nauki zdalnej na wystepowanie dolegliwosci miesniowo-szkieletowych w grupie pracowników i studentów uczelni wyzszych.

BACKGROUND: The COVID-19 pandemic has led to a fundamental change in the lifestyle and the ways of learning and working patterns which in turn might lead to health consequences including musculoskeletal disorders. The aim of this study was to evaluate the conditions of e-learning and remote working and the impact of the learning/working modality on the occurrence of musculoskeletal symptoms among university students and workers in Poland. MATERIAL AND METHODS: This study covered 914 students and 451 employees who filled in an anonymous online questionnaire. The questions covered 2 periods: before the COVID-19 pandemic and during the period from October 2020 to June 2021 and were aimed at obtaining information about lifestyle (including physical activity, perceived stress and sleep pattern), ergonomic of computer workstations, the incidence and severity of musculoskeletal symptoms and headaches. RESULTS: During the outbreak, the severity of musculoskeletal complaints increased significantly in the teaching staff group (3.2±2.5 vs. 4.1±3.0 VAS pts), in the administrative staff group (3.1±2.5 vs. 4.0±3.1 VAS pts), and in the student group (2.8±2.4 vs. 3.5±2.8 VAS pts). The average level of burden and risk of musculoskeletal complaints was revealed by the assessment using the ROSA method, in all 3 study groups. CONCLUSIONS: In light of current results, it is very important to educate people on the rational use of new technology devices, including the appropriate design of computer workstations, planning breaks and time for recovery and physical activity. Med Pr. 2023;74(1):63-78.

Design and Synthesis of New α-hydroxy ß-fluoro/ß-trifluoromethyl and Unsaturated Phosphonates from Carbohydrate-Derived Building Blocks via Pudovik and Horner-Wadsworth-Emmons Reactions.

Herein, we present the application of fluorinated carbohydrate-derived building blocks for α-hydroxy ß-fluoro/ß-trifluoromethyl and unsaturated phosphonates synthesis. Pudovik and Horner-Wadsworth-Emmons reactions were applied to achieve this goal. The proposed pathway of the key reactions is supported by the experimental results, as well as quantum chemical calculations. The structure of the products was established by spectroscopic (1D, 2D NMR) and spectrometric (MS) techniques. Based on our data received, we claim that the progress of the Pudovik and HWE reactions is significantly influenced by the acidic protons present in the molecules as assessed by pKa values of the reagent.

Gas-Phase Internal Ribose Residue Loss from Mg-ATP and Mg-ADP Complexes: Experimental and Theoretical Evidence for Phosphate-Mg-Adenine Interaction.

Gas-phase decompositions of magnesium complexes with adenosine-5'-triphosphate (ATP) and adenosine-5'-diphosphate (ADP) were studied by using electrospray ionization-collision-induced dissociation-tandem mass spectrometry, in the negative ion mode. The loss of internal ribose residue was observed and was found to occur directly from the [ADP-3H+Mg]- ion. The occurrence of this process indicates the presence of a strong phosphate-Mg-adenine interaction. The performed quantum mechanics calculations confirmed the occurrence of this interaction in the [ADP-3H+Mg]- ion, namely the presence of Mg-N7 bond and hydrogen bond between the phosphate oxygen atom and amino group. Although the finding concerns the gas phase, it indicates that phosphate-Mg-adenine interaction may be also of importance for biological processes. The loss of an internal ribose residue was also observed for calcium and zinc complexes with ATP/ADP as well as for magnesium complexes with guanosine-5'-triphosphate (GTP) or guanosine-5'-diphosphate (GDP). Therefore, it is reasonable to conclude that the presence of the phosphate-metal-nucleobase interaction is a feature of gas phase [NDP-3H+metal]- ion (NDP, nucleoside-5'-diphosphate) and may also be important for biological processes.

A structural homology approach to identify potential cross-reactive antibody responses following SARS-CoV-2 infection.

The emergence of the novel SARS-CoV-2 virus is the most important public-health issue of our time. Understanding the diverse clinical presentations of the ensuing disease, COVID-19, remains a critical unmet need. Here we present a comprehensive listing of the diverse clinical indications associated with COVID-19. We explore the theory that anti-SARS-CoV-2 antibodies could cross-react with endogenous human proteins driving some of the pathologies associated with COVID-19. We describe a novel computational approach to estimate structural homology between SARS-CoV-2 proteins and human proteins. Antibodies are more likely to interrogate 3D-structural epitopes than continuous linear epitopes. This computational workflow identified 346 human proteins containing a domain with high structural homology to a SARS-CoV-2 Wuhan strain protein. Of these, 102 proteins exhibit functions that could contribute to COVID-19 clinical pathologies. We present a testable hypothesis to delineate unexplained clinical observations vis-à-vis COVID-19 and a tool to evaluate the safety-risk profile of potential COVID-19 therapies.

Factor VIII-Fc Activates Natural Killer Cells via Fc-Mediated Interactions With CD16.

The most challenging complication associated with Factor VIII (FVIII) replacement therapy is the development of neutralizing anti-drug antibodies, or inhibitors, which occur in 23-35% of severe (FVIII level <1%) hemophilia A (HA) patients and are a serious hindrance to effective management of HA. Consequently, strategies that can either prevent anti-FVIII inhibitors from developing or "tolerize" individuals who develop such antibodies represent a clinically important unmet need. One intervention for patients with high-titer inhibitors is immune tolerance induction (ITI) therapy. Although ITI therapy is the only clinically proven strategy to eradicate anti-FVIII inhibitors, mechanisms of inhibitor reduction remain unknown. Factor VIII Fc-fusion (rFVIIIFc) is an enhanced half-life antihemophilic factor used in replacement therapy for HA. Fc-fusion is a successful protein bio-engineering platform technology. In addition to enhancement of plasma half-life via neonatal Fc receptor (FcRn) binding, other Fc-mediated interactions, including engagement with Fc gamma receptors (FcγR), may have immunological consequences. Several case reports and retrospective analyses suggest that rFVIIIFc offers superior outcomes with respect to ITI compared to other FVIII products. Previously we and others demonstrated rFVIIIFc interactions with activating FcγRIIIA/CD16. Here, we investigated if rFVIIIFc activates natural killer (NK) cells via CD16. We demonstrated rFVIIIFc signaling via CD16 independent of Von Willebrand Factor (VWF):FVIII complex formation. We established that rFVIIIFc potently activated NK cells in a CD16-dependent fashion resulting in IFNγ secretion and cytolytic perforin and granzyme B release. We also demonstrated an association between rFVIIIFc-mediated NK cell IFNγ secretion levels and the high-affinity (158V) CD16 genotype. Furthermore, we show that rFVIIIFc-activated CD16+ NK cells were able to lyse a B-cell clone (BO2C11) bearing an anti-FVIII B-cell receptor in an antibody-dependent cellular cytotoxicity (ADCC) assay. These in vitro findings provide an underlying molecular mechanism that may help explain clinical case reports and retrospective studies suggesting rFVIIIFc may be more effective in tolerizing HA patients with anti-FVIII inhibitors compared to FVIII not linked to Fc. Our in vitro findings suggest a potential use of Fc-fusion proteins acting via NK cells to target antigen-specific B-cells, in the management of unwanted immune responses directed against immunogenic self-antigens or therapeutic protein products.

Gas-phase generation of dinuclear Au(I)-Au(II) complexes by laser desorption ionization mass spectrometry.

Alkali metal chloroaurates(III) were analysed by laser desorption ionization mass spectrometry. Among a number of generated gas-phase ionic clusters, the unusual ions [MAu2Cl5]- (were M stands for Na, K, Rb, Cs) were detected. The spectra of metastable ions and quantum mechanics calculations show the presence of unprecedented Au(I)-Au(II) interactions in the clusters.

Random Structure Searching with Orbital-Free Density Functional Theory.

The properties of a material depend on how its atoms are arranged, and predicting these arrangements from first principles is a longstanding challenge. Orbital-free density functional theory provides a quantum-mechanical model based solely on the electron density, not individual wave functions. The resulting speedups make it attractive for random structure searching, whereby random configurations of atoms are relaxed to local minima in the energy landscape. We use this strategy to map the low-energy crystal structures of Li, Na, Mg, and Al at zero pressure. For Li and Na, our searching finds numerous close-packed polytypes of almost-equal energy, consistent with previous efforts to understand their low-temperature forms. For Mg and Al, the searching identifies the expected ground state structures unambiguously, in addition to revealing other low-energy structures. This new role for orbital-free density functional theory-particularly as continued advances make it accurate for more of the periodic table-will expedite crystal structure prediction over wide ranges of compositions and pressures.

Chemical Changes of Wood Treated with Caffeine.

Earlier studies have revealed that wood treated with caffeine was effectively protected against decay fungi and molds. However, there is a need to establish how the caffeine molecule behaves after wood impregnation and how it can protect wood. The objective of the research was to characterize the interaction between caffeine and Scots pine (Pinus sylvestris L.) wood as well as to assess the stability of the alkaloid molecule in lignocellulosic material. For this purpose, an elementary analyzer was used to assess the nitrogen concentration in the treated wood. The results showed that caffeine is easily removed from the wood structure through large amounts of water. The changes occurring in the wood structure after impregnation were evaluated with regard to the results obtained by Fourier transform infrared (FTIR) spectroscopy of two model mixtures with caffeine and cellulose or lignin for the purpose of conducting a comparison with the spectrum of impregnated and non-impregnated samples. The observed changes in FTIR spectra involve the intensity of the C=O(6) caffeine carbonyl group and signals from guaiacyl units. It might indicate favorable interactions between caffeine and lignin. Additionally, molecular simulation of the caffeine's interaction with the guaiacyl ß-O-4 lignin model compound characteristic for the lignin structure using computational studies was performed. Consequently, all analyses confirmed that caffeine may interact with the methylene group derived from the aromatic rings of the guaiacyl group of lignin. In summary, scanning electron microscope (SEM) observations suggest that caffeine was accumulated in the lignin-rich areas of the primary walls.

Modified aptamers as reagents to characterize recombinant human erythropoietin products.

Reliable and reproducible monitoring of the conformational state of therapeutic protein products remains an unmet technological need. This need is amplified by the increasing number of biosimilars entering the drug development pipeline as many branded biologics are reaching the end of their market exclusivity period. Availability of methods to better characterize protein conformation may improve detection of counterfit and unlicensed therapeutic proteins. In this study, we report the use of a set of modified DNA aptamers with enhanced chemical diversity to probe the conformational state of 12 recombinant human erythropoietin (rHuEPO) therapeutic protein products; one FDA-licensed rHuEPO originator biological product, three rHuEPO products that are approved for marketing in the US or EU as biosimilars, and eight rHuEPO products that are not approved for marketing in the US or EU. We show that several of these modified aptamers are able to distinguish rHuEPO reference products or approved biosimilars from non-licensed rHuEPO products on the basis of differences in binding kinetics and equilibrium affinity constants. These reagents exhibit sensitivity to the conformational integrity of various forms of rHuEPO and as such represent powerful, simple-to-use analytical tools to monitor the conformational integrity of therapeutic-proteins during manufacture and to screen for and identify both substandard and counterfeit products.

Mitigation of T-cell dependent immunogenicity by reengineering factor VIIa analogue.

Vatreptacog alfa (VA), a recombinant activated human factor VII (rFVIIa) variant with 3 amino acid substitutions, was developed to provide increased procoagulant activity in hemophilia patients with inhibitors to factor VIII or factor IX. In phase 3 clinical trials, changes introduced during the bioengineering of VA resulted in the development of undesired anti-drug antibodies in some patients, leading to the termination of a potentially promising therapeutic protein product. Here, we use preclinical biomarkers associated with clinical immunogenicity to validate our deimmunization strategy applied to this bioengineered rFVIIa analog. The reengineered rFVIIa analog variants retained increased intrinsic thrombin generation activity but did not elicit T-cell responses in peripheral blood mononuclear cells isolated from 50 HLA typed subjects representing the human population. Our algorithm, rational immunogenicity determination, offers a broadly applicable deimmunizing strategy for bioengineered proteins.

Peptides identified on monocyte-derived dendritic cells: a marker for clinical immunogenicity to FVIII products.

The immunogenicity of protein therapeutics is an important safety and efficacy concern during drug development and regulation. Strategies to identify individuals and subpopulations at risk for an undesirable immune response represent an important unmet need. The major histocompatibility complex (MHC)-associated peptide proteomics (MAPPs) assay directly identifies the presence of peptides derived from a specific protein therapeutic on a donor's MHC class II (MHC-II) proteins. We applied this technique to address several questions related to the use of factor VIII (FVIII) replacement therapy in the treatment of hemophilia A (HA). Although >12 FVIII therapeutics are marketed, most fall into 3 categories: (i) human plasma-derived FVIII (pdFVIII), (ii) full-length (FL)-recombinant FVIII (rFVIII; FL-rFVIII), and (iii) B-domain-deleted rFVIII. Here, we investigated whether there are differences between the FVIII peptides found on the MHC-II proteins of the same individual when incubated with these 3 classes. Based on several observational studies and a prospective, randomized, clinical trial showing that the originally approved rFVIII products may be more immunogenic than the pdFVIII products containing von Willebrand factor (VWF) in molar excess, it has been hypothesized that the pdFVIII molecules yield/present fewer peptides (ie, potential T-cell epitopes). We have experimentally tested this hypothesis and found that dendritic cells from HA patients and healthy donors present fewer FVIII peptides when administered pdFVIII vs FL-rFVIII, despite both containing the same molar VWF excess. Our results support the hypothesis that synthesis of pdFVIII under physiological conditions could result in reduced heterogeneity and/or subtle differences in structure/conformation which, in turn, may result in reduced FVIII proteolytic processing relative to FL-rFVIII.