Acid-induced Aggregation Research Articles

Interactions between xanthan gum and phenolic acids

The molecular and colloidal-level interactions between two major phenolic acids, gallic and caffeic acid, with a major food polysaccharide, xanthan gum, were studied in binary systems aiming to correlate the stability of the binary systems as a function of pH and xanthan-polyphenol concentrations. Global stability diagrams were built, acting as roadmaps for examining the phase separation regimes followed by the fluorimetry-based thermodynamics of the interactions. The effects of noncovalent interactions on the macroscopic behavior of the binary systems were studied, using shear and extensional rheometry. The collected data for caffeic acid – xanthan gum mixtures showed that the main interactions were pH-independent volume exclusions, while gallic acid interacts with xanthan gum, especially at pH 7 with other mechanisms as well, improving the colloidal dispersion stability. A combination of fluorimetry, extensional rheology and stability measurements highlight the effect of gallic acid-induced aggregation of xanthan gum, both in structuring and de-structuring the binary systems. The above provide a coherent framework of the physicochemical aspect of binary systems, shedding light on the role of xanthan gum in its oral functions, such as in inducing texture, in model complex systems containing phenolic acids.

Dynamic changes in the aggregation-depolymerization behavior of Ovomucin-Complex and its binding to urease during in vitro simulated gastric digestion

Ovomucin-Complex extracted from egg white is expected to have a barrier function similar to gastric mucin. In this study, the dynamic changes in structure, rheological properties and binding ability of Ovomucin-Complex during in vitro simulated gastric digestion were investigated. The results from HPLC and CLSM showed that extremely acidic pH (pH = 2.0) promoted Ovomucin-Complex to form aggregation. Acid-induced aggregation may hinder its binding to pepsin, thus rendering Ovomucin-Complex resistant to pepsin. Consequently, most of the polymer structure and weak gel properties of Ovomucin-Complex retained after simulated gastric digestion as verified by HPLC, CLSM and rheological measurement, although there was a small breakdown of the glycosidic bond as confirmed by the increased content of reducing sugar. The significantly reduced hydrophobic interactions of Ovomucin-Complex were observed under extremely acidic conditions and simulated gastric digestion compared with the native. Noticeably, the undigested Ovomucin-Complex after simulated gastric digestion showed a higher affinity (KD = 5.0 ± 3.2 nm) for urease - the key surface antigen of Helicobacter pylori. The interaction mechanism between Ovomucin-Complex and urease during gastric digestion deserves further studies. This finding provides a new insight to develop an artificial physical mucus barrier to reduce Helicobacter pylori infection.

Cationic polyelectrolytes prevent the aggregation of l-lactate dehydrogenase under unstable conditions

Unstructured biological macromolecules have attracted attention as protein aggregation inhibitors in living cells. Some are characterized by their free structural configuration, highly charged, and water-soluble. However, the importance of these properties in inhibiting protein aggregation remains unclear. In this study, we investigated the effect of charged poly (amino acids), which mimic these properties, on aggregation of l-lactate dehydrogenase (LDH) and compared their effects to monomeric amino acids and folded proteins. LDH was stable and active at a neutral pH (~7) but formed inactive aggregates at acidic pH (< 6). Adding cationic polyelectrolytes of poly-l-lysine and poly-l-arginine suppressed the acid-induced aggregation and inactivation of LDH under acidic pH values. Adding monomeric amino acids and cationic folded proteins also prevented LDH aggregation but with lower efficacy than cationic polyelectrolytes. These results indicate that unstructured polyelectrolytes effectively stabilize unstable enzymes because they interact flexibly and multivalently with them. Our findings provide a simple method for stabilizing enzymes under unstable conditions.

Pea soluble polysaccharide improves stability of acidic pea protein dispersions

Soluble pea polysaccharide (PPS) is an acidic extract from the by-product of pea protein processing. It was hypothesized that this polysaccharide, which is negatively charged, may be able to stabilize acidic protein dispersions at acidic pH. Model dispersions were prepared with PPS and commercial pea protein isolate (PPI), at ratios ranging between 0 and 1, and at pH 3.8, and at two protein concentrations, 1 and 2%. With increasing the ratio of PPS/PPI, there was a shift in the surface charge from +21 to −4 mV. Stable complexes were formed at ratios >0.25, as shown by an increase of unsedimented proteins, in spite of the low overall charge at these ratios. There was an effect of concentration, with 2% showing less stability than 1%. Transmission electron micrographs confirmed the presence of complexes, and the ability of PPS to limit the acid-induced aggregation of PPI at low pH.

Gastric aggregation of micellar casein powders induced by high hydrostatic pressure: Effect of serum Ca2+ level

Micellar casein (MC) has a unique gastric colloidal behavior in response to Ca2+ cross-linking, and its aggregation properties are closely related to pepsin and gastric acid. In this study, MC with different levels of colloidal calcium phosphate (CCP) was obtained by high hydrostatic pressure (HHP) at different pressures, followed by spray drying to obtain the powders. Different amounts of calcium chloride (exogenous Ca2+) were added to MC powders prior to in vitro simulated digestion to investigate the effect of exogenous serum Ca2+ levels on the aggregation behavior and the structure change of curds generated in gastric tract. The results revealed that HHP induced the emergence of more Ca2+-binding sites, thus Ca2+ was more likely to bind to MC matrix with low CCP levels. Meanwhile, high serum Ca2+ level provided more opportunities to form aggregates. The Highest pressure (500 MPa) with the highest Ca2+ level (5 mM) caused the lowest solubility aggregates, which were only 30% at the end of gastric digestion (120 min), half of the control sample (0 MPa with 0.15 mM Ca2+). The results of wide-angle X-ray scattering / small-angle X-ray scattering suggested that both pepsin and gastric acid-induced aggregation via Ca2+ as a bridge. For pepsin, Ca2+ cross-linked between para-κ-casein; For gastric acid, Ca2+ recombined phosphorylation sites and caused cross-linking of casein subunits.

Daboxin P, a phospholipase A2 of Indian Daboia russelii venom, modulates thrombin-mediated platelet aggregation.

Daboxin P, reported earlier from the venom of Daboia russellii, disturbs the blood coagulation cascade by targeting factor X and factor Xa. The present study exhibits that Daboxin P also inhibits platelet aggregation induced by various agonists. The thrombin-induced platelet aggregation was inhibited maximum whereas inhibition of collagen-induced platelet aggregation was found to be 50% and no inhibition of adenosine diphosphate (ADP)and arachidonic acid-induced aggregation was observed. Daboxin P dose-dependently inhibited the thrombin-induced platelet aggregation with Anti-Aggregation 50 (AD50 ) dose of 55.166 nM and also reduced the thrombin-mediated calcium influx. In-silico interaction studies suggested that Daboxin P binds to thrombin and blocks its interaction with its receptor on the platelet surface. Quenching of thrombin's emission spectrum by Daboxin P and electrophoretic profiles of pull-down assay further reveals the binding between Daboxin P and thrombin. Thus, the present study demonstrates that Daboxin P inhibits thrombin-induced platelet aggregation by binding to thrombin.

Aqueous Extract of Malus domestica Inhibits Human Platelet Aggregation Induced by Multiple Agonists

Cardiovascular diseases such as hypertension, myocardial infarction, and atherosclerosis are the major cause of morbidity and mortality in humankind and not only afflict industrialized nations but also affect developing countries such as Pakistan. We attempted to screen one of the most common fruits, Malus domestica (MD or apple), used in Pakistan for its potential cardio-protective effects. The crude aqueous extract (MD-Aq) was prepared and investigated for inhibitory activity against several important cardiovascular drug targets. Activities of antioxidant enzymes such as glutathione peroxidase (GPx) and superoxide dismutase (SOD) were studied, as well as total antioxidant status (TAS). Calcium channel blocking activity was measured using guinea pig ileum in an isolated tissue bath setup. Platelet aggregation studies were carried out using a dual-channel aggregometer. MD-Aq showed dose-dependent improvement in the GPX activity. The highest SOD levels were observed with 10 mg/ml and were 185 U/L compared to 174 U/L for saline, which means no significant effect by MD-Aq. TAS levels were highest at 10 mg/ml of MD-Aq, reaching 1.75 mmol/L of plasma. At 10 mg/ml, close to 90% inhibition of KCl-induced opening of calcium channels was observed, compared with the effect of 1mM Verapamil. At each dose of 1, 5, and 10 mg/ml, a significantly higher arachidonic acid-induced inhibition of platelet aggregation was observed as compared to the saline effect. MD-Aq effects on platelet-activating factor-induced platelet aggregation were less potent. We concluded that MD-Aq could elevate GPX and TAS levels, is inactive at SOD, and is excellent as a calcium channel blocker, a more potent blocker of arachidonic acid-induced aggregation than platelet-activating factor-induced aggregation. These activities can help treat various cardiovascular diseases.

Benziodarone and 6-hydroxybenziodarone are potent and selective inhibitors of transthyretin amyloidogenesis

Transthyretin amyloidosis is a progressive systemic disorder that is caused by the amyloid deposition of transthyretin in various organs. Stabilization of the native transthyretin is an effective strategy for the treatment of transthyretin amyloidosis. In this study we demonstrate that the clinically used uricosuric agent benziodarone is highly effective to stabilize the tetrameric structure of transthyretin. An acid-induced aggregation assay showed that benziodarone had strong inhibitory activity similar to that of tafamidis, which is currently used as a therapeutic agent for transthyretin amyloidosis. Moreover, a possible metabolite, 6-hydroxybenziodarone, retained the strong amyloid inhibitory activity of benziodarone. An ex vivo competitive binding assay using a fluorogenic probe showed that benziodarone and 6-hydroxybenziodarone were highly potent for selective binding to transthyretin in human plasma. An X-ray crystal structure analysis revealed that the halogenated hydroxyphenyl ring was located at the entrance of the thyroxine binding channel of transthyretin and that the benzofuran ring was located in the inner channel. These studies suggest that benziodarone and 6-hydroxybenziodarone would potentially be effective against transthyretin amyloidosis.

Effects of Walnut Bark Extract on the Human Platelet Aggregation, Adhesion, and Plasmatic Coagulation In Vitro.

Thrombosis is the formation of a clot within a blood vessel. Antithrombotic drugs are used for treating thrombosis, which can be the cause of hemorrhage. Currently, there is a need to discover novel antithrombotic drugs. Walnut is widely used to treat a wide range of health complaints. In this study, walnut bark extract was tested in hemostasis parameters: platelets adhesion, aggregation, and plasmatic coagulation in human blood. The crude aqueous extract of walnut bark was prepared by infusion and tested in vitro on hemostasis. Through blood collection from healthy volunteer donors, we studied different parameters of the primary hemostasis: platelet adhesion on the collagen-coated surface under flow, ADP, collagen, thrombin, and arachidonic acid-induced platelet aggregation, and of the secondary hemostasis by measuring prothrombin time (PT) and activated partial thromboplastin (APTT) parameters. All experiments are realized in the absence and presence of the extract and repeated at least twice. The obtained data showed that the extract (1 and 2 mg/mL) significantly (p < 0.001) reduced the activated platelet adhesion on the collagen-coated surface. In the same way, the effect of the extract on platelet aggregation seems to depend on its concentration and on the nature of the agonist. The strongest inhibition of aggregation was observed in the case of collagen at 1 mg/mL, while there was no observed effect on arachidonic acid-induced aggregation. Moreover, the extract (1 mg/mL) affects the extrinsic, intrinsic, and common pathways of the human blood coagulation cascade by extending significantly (p < 0.001), both PT and APTT times. This study provides evidence that walnut bark extract, by its antiadhesive, antiaggregant, and anticoagulant activities, could be considered as a serious source of biological compounds for the prevention and treatment of thrombosis.

A novel colorimetric biosensor for rapid detection of dengue virus upon acid-induced aggregation of colloidal gold.

The dengue virus, once transmitted to people through a mosquito bite, causes an infectious disease called dengue fever. Dengue fever can develop into two fatal syndromes, namely dengue shock syndrome and dengue hemorrhagic fever. The existing strategies for detecting dengue infection mainly employ serological immunoassays and a real time PCR technique. Along with the positive features of efficiency, accuracy, and reproducibility, these procedures are limited by being time-consuming, costly, requiring special equipment for analysis, and unable to be carried out at the point-of-care level. Herein, we developed a colorimetric nanosensor for detecting dengue virus in clinical samples that is rapid, accurate, sensitive, and less expensive. The sensing platform relies on the specific binding between the DNA-conjugated AuNPs and genomic RNA of dengue, which results in the DNA-RNA heteroduplex structure formation that turns the gold colloid's ruby red color to blue due to the nano-aggregation in an acidic environment, which can be detected by the naked eye or measuring the absorbance. The DNA probe was designed to bind to a genomic RNA conserved region recognized in all four dengue serotypes. Dengue virus serotype 1 was utilized as a framework for virus detection; the designed nanosensor exhibited great specificity and selectivity, with the detection limit of ∼1 pg μL-1 (∼1.66 × 106 RNA copies per reaction) and time of analysis of about 1 h including the RNA extraction step. The proposed colorimetric nanosensor offers an alternative tool for specific and highly sensitive detection of dengue that eliminates the requirement for thermal cycling and primer sets in PCR-based assays.

Thyroxine metabolite-derived 3-iodothyronamine (T1AM) and synthetic analogs as efficient suppressors of transthyretin amyloidosis

Aggregation and fibrillization of transthyretin (TTR) is a fatal pathogenic process that can cause cardiomyopathic and polyneuropathic diseases in humans. Although several therapeutic strategies have been designed to prevent and treat related pathological events, there is still an urgent need to develop better strategies to improve potency and wider applicability. Here, we present our study demonstrating that 3-iodothyronamine (T1AM) and selected thyronamine-like compounds can effectively prevent TTR aggregation. T1AM is one of the thyroid hormone (TH) metabolites, and T1AM and its analogs, such as SG2, SG6, and SG12, are notable molecules for their beneficial activities against metabolic disorders and neurodegeneration. Using nuclear magnetic resonance (NMR) spectroscopy and biochemical analysis, we confirmed that T1AM analogs could bind to and suppress acid-induced aggregation of TTR. In addition, we employed computational approaches to further understand the detailed mechanisms of the interaction between T1AM analogs and TTR. This study demonstrates that T1AM analogs, whose beneficial effects against several pathological processes have already been proven, may have additional benefits against TTR aggregation and fibrillization. Moreover, we believe that our work provides invaluable insights to enhance the pleiotropic activity of T1AM and structurally related analogs, relevant for their therapeutic potential, with particular reference to the ability to prevent TTR aggregation.

Can Isoquinoline Alkaloids Affect Platelet Aggregation in Whole Human Blood?

Isoquinoline alkaloids have multiple biological activities, which might be associated with positive pharmacological effects as well as negative adverse reactions. As bleeding was suggested to be a side effect of the isoquinoline alkaloid berberine, we decided to ascertain if different isoquinoline alkaloids could influence hemocoagulation through the inhibition of either platelet aggregation or blood coagulation. Initially, a total of 14 compounds were screened for antiplatelet activity in whole human blood by impedance aggregometry. Eight of them demonstrated an antiplatelet effect against arachidonic acid-induced aggregation. Papaverine and bulbocapnine were the most potent compounds with biologically relevant IC50 values of 26.9 ± 12.2 μM and 30.7 ± 5.4 μM, respectively. Further testing with the same approach confirmed their antiplatelet effects by employing the most physiologically relevant inducer of platelet aggregation, collagen, and demonstrated that bulbocapnine acted at the level of thromboxane receptors. None of the alkaloids tested had an effect on blood coagulation measured by a mechanical coagulometer. In conclusion, the observed antiplatelet effects of isoquinoline alkaloids were found mostly at quite high concentrations, which means that their clinical impact is most likely low. Bulbocapnine was an exception. It proved to be a promising antiplatelet molecule, which may have biologically relevant effects.

Photothermal conversion performance and acid-induced aggregation of PLNP-Bi2S3 composite nanoplatforms.

Poly(sodium 4-styrenesulfonate) (PSS) molecule modified PLNP-Bi2S3 composite nanoplatforms were constructed by using polyvinylpyrrolidone (PVP) modified Bi2S3 nanoparticles (∼4.6 nm) as a photothermal agent and hexadecyl trimethyl ammonium bromide (CTAB) coated Zn2Ga2.98Ge0.75O8:Cr0.023+ (ZGGO:Cr3+@CTAB) persistent luminescence nanoparticles (PLNPs) through electrostatic adsorption. It is found that the above composite nanoplatforms have excellent laser-irradiation thermal stability and good photothermal conversion performance. The measured photothermal conversion efficiency is ∼44%, which is higher than that (∼37%) of the PLNP-GNR (gold nanorod) composite nanoplatforms. Meanwhile, PSS modified PLNP-Bi2S3 composite nanoplatforms exhibited good solution dispersibility in blood and normal tissue environments. While reaching tumor sites, the above composite nanoplatforms can be rapidly accumulated in cancer cells with acidic environments. This pH-responsive acid-induced aggregation can be ascribed to the chemical reaction induced by the protonation of PSS modified PLNP-Bi2S3 composite nanoplatforms with a negatively charged surface in the acidic environments. Our results suggest that PSS modified PLNP-Bi2S3 composite nanoplatforms might be applied to precision diagnosis and therapy of deep-tissue tumors.

Novel N-substituted indole hydrazones as potential antiplatelet agents: synthesis, biological evaluations, and molecular docking studies.

Background and purpose:Antiplatelet agents can diminish the chance of coronary heart diseases due to the prevention of unusual clotting in the arteries by inhibiting platelet aggregation and avoiding the formation of a blood clot. This mechanism can help to prevent ischemic stroke likewise. To improve the activity of these drugs and reduce their side effects, further studies are required.Experimental approach:Based on the previous studies representing the promising antiplatelet activity of indole hydrazones, a series of their homologs containing twenty-one compounds were prepared in two steps. First, alkylation reaction on the nitrogen of the indole ring, and second, chiff base formation by condensation of a primary amine and N-substituted indole-3 carbaldehyde. Consequently, their platelet anti-aggregation activity was evaluated based on the Born turbidimetric method.Findings/Results:Most of the compounds exhibited noticeable activity against platelet aggregation induced by arachidonic acid. Amongst them, two compounds 2e and 2f showed higher activity with IC50 values that made comparable to indomethacin and acetylsalicylic acid as standard drugs and had no toxicity on platelets.Conclusion and implications:The synthesized compounds exhibited promising activity against arachidonic acid-induced aggregation; however, none of them showed noticeable antiplatelet activity induced by adenosine di-phosphate. Chemical structure comparison of the prepared derivatives indicated the existence of a lipophilic medium-sized group on the phenyl ring increased their activity. In addition, the docking studies confirmed this hydrophobic interaction in the lipophilic pocket of cyclooxygenase-1 enzyme suggesting that hydrophobicity of this region plays a pivotal role in the anti-platelet activity of these compounds. To prove this finding, the enzymatic evaluation with the target enzyme is required.

State of platelet-plasma hemostasis in patients with hypertension and non-alcoholic fatty liver disease in condition of hypercholesterolemia

Abstract Non-alcoholic fatty liver disease (NAFLD) is a marker of metabolic and dyslipidemic disorders. Given the prevalence of atherogenic dyslipidemias and their proven effect on the development of thrombotic cardiovascular complications in patients with NAFLD, it is important to understand the role of hemostatic blood activity. Aim of the study To increase the effectiveness of early diagnosis of thrombophilic blood changes in patients with hypertension (HT) combined with non-alcoholic fatty liver disease by determining the state of plasma hemostasis in conditions of hypercholesterolemia and concomitant statin therapy. Materials and methods 152 patients were examined. Groups of patients: I – 46 patients with stage II GC, II – 54 patients with NAFLD without GC, group III – 52 patients with stage II GC with concomitant NAFLD. The analysis of spontaneous and induced platelet aggregation, indicators of coagulation, anticoagulant and fibrinolytic hemostasis. Results The degree of spontaneous aggregation was significantly higher in patients with hypercholesterolemia – by 32.4% (p&amp;lt;0.05). The level of fibrinogen was higher by 13.5% (p&amp;lt;0.05) due to hypercholesterolemia, at the same time there was a decrease in antithrombin (AT) III by 8.7% (p&amp;lt;0.05) in patients with high cholesterol. Patients with comorbid HT and NAFLD on statin therapy had a 16.5% (p&amp;lt;0.05) lower degree of spontaneous aggregation than patients who did not receive this treatment. In the NAFLD group, patients receiving statins had a significantly lower degree of arachidonic acid-induced aggregation against patients without lipid-lowering therapy (by 54.0%, p&amp;lt;0.001). However, the analysis of the general population of the examined revealed a decrease in the degree of collagen-induced aggregation by 38.7% (p&amp;lt;0.05) in a subgroup of patients receiving treatment. We observed a 23.4% (p&amp;lt;0.001) shortening of prothrombin time in the NAFLD group and a 16.0% (p&amp;lt;0.05) shortening in the combined course of HT and NAFLD. In the absence of statin therapy in the NAFLD group, there was a significant decrease in INR – by 9.7% (p&amp;lt;0.05) – compared with patients receiving lipid-lowering therapy. A decrease in thrombin time by 12.2% (p&amp;lt;0.05) was observed in the subgroup receiving statins among patients with NAFLD. In the general cohort, the use of statins increased the activity of AT III by 10.7% (p&amp;lt;0.01), but in the NAFLD group this difference was more significant – by 14.3% (p&amp;lt;0.001) AT III was more active in patients who received lipid-lowering therapy. Conclusion The results of the analysis showed that patients with hypercholesterolemia have procoagulant and prothrombogenic activity of blood. But statin treatment decrease platelet aggregation, blood coagulation potential and increased activity of anticoagulant hemostasis, which complements the mechanism of prophylactic effect of HMG CoA reductase inhibitor (statin). Funding Acknowledgement Type of funding sources: None.

Effect of Metal Nanoparticle Aggregate Structure on the Thermodynamics of Oxidative Dissolution.

Here, we compare the electrochemical oxidation potential of 15 nm diameter citrate-stabilized Au NPs aggregated by acid (low pH) to those aggregated by tetrakis(hydroxymethyl) phosphonium chloride (THPC). For acid-induced aggregation, the solution changes to a blue-violet color, the localized surface plasmon resonance (LSPR) band of Au NPs at 520 nm decreases along with an increase in absorbance at higher wavelengths (600-800 nm), and the peak oxidation potential (Ep) in anodic stripping voltammetry (ASV) obtained in bromide has a positive shift by as large as 200 mV. For THPC-induced aggregation (Au/THPC mole ratio = 62.5), the solution changes to a blue color as the LSPR band at 520 nm decreases and a new distinct peak at 700 nm appears, but the Ep does not exhibit a positive shift. Scanning transmission electron microscopy (STEM) images reveal that acid-induced aggregates are three-dimensional with strongly fused Au NP-Au NP contacts, while THPC-induced aggregates are linear or two-dimensional with ∼1 nm separation between Au NPs. The surface area-to-volume ratio (SA/V) decreases for acid-aggregated Au NPs due to strong Au NP-Au NP contacts, which leads to lower surface free energy and a higher Ep. The SA/V does not change for THPC-aggregated Au NPs since space remains between them and their SA is fully accessible. These findings show that metal NP oxidative stability, as determined by ASV, is highly sensitive to the details of the aggregate structure.

Assessing Whey Protein Sources, Dispersion Preparation Method and Enrichment of Thermomechanically Stabilized Whey Protein Pectin Complexes for Technical Scale Production

Whey protein pectin complexes can be applied to replace fat in food products, e.g., pudding and yogurt, contributing to creaminess while adding a source of protein and fiber. Production of these complexes is usually conducted on the laboratory scale in small batches. Recently, a process using a scraped-surface heat exchanger (SSHE) has been employed; however, dispersion preparation time, feasibility of using different whey protein sources and enrichment of the complexes for subsequent drying have not been assessed. Preparing whey protein pectin dispersions by solid mixing of pectin and whey protein powders resulted in larger complexes than powders dispersed separately and subsequently mixed after a hydration time. Dispersions without hydration of the mixed dispersions before thermomechanical treatment had the largest particle sizes. The targeted particle size of d90,3 < 10 µm, an important predictor for creaminess, was obtained for five of the six tested whey protein sources. Dispersions of complexes prepared using whey protein powders had larger particles, with less particle volume in the submicron range, than those prepared using whey protein concentrates. Efficiency of complex enrichment via acid-induced aggregation and subsequent centrifugation was assessed by yield and purity of protein in the pellet and pectin in the supernatant.

The state of platelets and their functional activity in patients with arterial hypertension in combination with non-alcoholic fatty liver disease

Abstract Background According to modern concepts of hypertension (HT) and obesity, the hemostatic system in these diseases is characterized by prothrombogenic changes. Since the liver is the site of the formation of many factors of hemostasis, the concomitant non-alcoholic fatty liver disease (NAFLD), which often accompanies the course of both of these diseases, is an actual problem. Purpose To improve the efficiency of early diagnosis of thrombophilic blood changes in hypertensive patients with concomitant obesity and NAFLD by determining the state of platelets and their functional activity. Materials and methods We examined 167 patients (80 men and 87 women). The average patient age 55.5 [47.0; 61.0] years. Patients were divided into three groups: I - 46 patients with hypertension without NAFLD, II - 54 patients with NAFLD without hypertension, III group - 52 hypertensive patients with NAFLD. The control group consisted of 15 healthy subjects matched for age and sex. Platelet count and mean platelet volume (MPV) was performed on an automated analyzer, spontaneous and induced platelet aggregation was evaluated by laser aggregometer. Results A significant increase in the degree of spontaneous aggregation of platelet has been found in patients in all groups compared to the control group: I group - 2.2-fold increasing in aggregation (p&amp;lt;0.05), II group to 4.2-fold (p&amp;lt;0.05), III group had increasing by 4.1-fold (p&amp;lt;0.05). ADP-induced platelet aggregation was the same in I group and control, but it was 39% higher (p&amp;lt;0.001) in II cohort and by 22.6% (p&amp;lt;0.01) in III group. Adrenalin-induced platelet activity increased in all groups versus control: I group – 2.1-fold (p&amp;lt;0.001), II group – 2.3-fold (p&amp;lt;0.001), III group – 1.6-fold (p&amp;lt;0.01) elevation. Arachidonic acid-induced aggregation elevated by 64,2% (p&amp;lt;0.001) in I group and decreased by 56.3% (p&amp;lt;0.01) in II and by 43% (p&amp;lt;0.05) III cohorts. Collagen-induced activity had not significant difference between groups. MPV was increased in both groups with NAFLD by 5.9, but in II group significance level was higher - p&amp;lt;0.01 than in group III - p&amp;lt;0.001. MPV had not significant changes in I group versus control. Conclusion Spontaneous platelet aggregation is increased in hypertensive patients and it is significantly enhanced in combination with NAFLD. Thus in patients with isolated NAFLD also observed a statistically significant increase in spontaneous aggregation of platelets. That is possibly explained by an increase in MPV which can be considered as one of the NAFLD risk factors thrombophilic changes in primary hemostasis. An analysis of induced platelet aggregation showed that patients with NAFLD may have a lower sensitivity to acetylsalicylic acid therapy. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Bogomolets National Medical University

Enhanced radiotherapy using photothermal therapy based on dual-sensitizer of gold nanoparticles with acid-induced aggregation

The damaged DNA strands caused by radiotherapy (RT) can repair by themselves. A gold nanoparticles (GNPs) system with acid-induced aggregation was developed into a dual sensitizer owing to its high radioactive rays attenuation ability and enhanced photothermal heating efficiency after GNPs aggregation to achieve a combination therapy of RT and photothermal therapy (PTT). In this combination therapy, the formed GNP aggregates firstly showed a higher sensitize enhancement ratio (SER) value (1.52). Importantly, the self-repair of damaged DNA strands was inhibited by mild PTT through down-regulating the expression of DNA repair protein, thus resulting in a much higher SER value (1.68). Anti-tumor studies further demonstrated that this combination therapy exhibited ideal anti-tumor efficacy. Furthermore, the imaging signals of GNPs in computed tomography and photoacoustic were significantly improved following the GNPs aggregation. Therefore, a dual sensitizer with multimodal imaging was successfully developed and can be further applied as a new anti-tumor therapy.

Engineering a human IgG2 antibody stable at low pH.

IgG2 subclass antibodies have unique properties that include low effector function and a rigid hinge region. Although some IgG2 subclasses have been clinically tested and approved for therapeutic use, they have a higher propensity than IgG1 for aggregation, which can curtail or abolish their biological activity and enhance their immunogenicity. In this regard, acid-induced aggregation of monoclonal antibodies during purification and virus inactivation must be prevented. In the present study, we replaced the constant domain of IgG2 with that of IgG1, using anti-2,4-dinitrophenol (DNP) IgG2 as a model antibody, and investigated whether that would confer greater stability. While the anti-DNP IgG2 antibody showed significant aggregation at low pH, this was reduced for the IgG2 antibody containing the IgG1 CH2 domain. Substituting three amino acids within the CH2 domain-namely, F300Y, V309L, and T339A (IgG2_YLA)-reduced aggregation at low pH and increased CH2 transition temperature, as determined by differential scanning calorimetric analysis. IgG2_YLA exhibited similar antigen-binding capacity to IgG2, low affinity for FcγRIIIa, and low binding ability to C1q. The same YLA substitution also reduced the aggregation of panitumumab, another IgG2 antibody, at low pH. Our engineered human IgG2 antibody showed reduced aggregation during bioprocessing and provides a basis for designing improved IgG2 antibodies for therapeutic applications.