HOUSE DUST MITE ALLERGENS: FROM NATIVE EXTRACTS TO MODIFIED PREPARATIONS

The growth of allergic diseases dictates the need to develop new forms of therapeutic allergens with high immunogenic and low allergenic activity. For many years, our laboratory has been developing drugs for the diagnosis and treatment of house dust mites (HDM) allergies. The purpose of this study is to summarize the results of the five-year development of therapeutic preparations of HDM allergens. During this period, we obtained the following forms of therapeutic allergens: a granular sublingual dosage form of a mixed allergen from Dermatophagoides pteronyssinus (Der.p) and Dermatophagoides farinaе (Der.f) and a succinylated monomeric HDM allergoid Der.p. The physicochemical and immunobiological properties of the obtained preparations were studied by methods: electrophoresis in PAGE in the presence of SDS-sodium, micropoint immunoblot, ELISA, inhibition of the binding reaction of allergen-specific IgE in the sera of patients. The research results showed that the obtained preparations have a reduced allergenic and increased immunogenic activity in comparison with native extracts. The created forms of mite allergens can be further used to treat patients sensitized to HDM of the genus Dermatophagoides.

Common cardiac medications potently inhibit ACE2 binding to the SARS-CoV-2 Spike, and block virus penetration and infectivity in human lung cells

To initiate SARS-CoV-2 infection, the Receptor Binding Domain (RBD) on the viral spike protein must first bind to the host receptor ACE2 protein on pulmonary and other ACE2-expressing cells. We hypothesized that cardiac glycoside drugs might block the binding reaction between ACE2 and the Spike (S) protein, and thus block viral penetration into target cells. To test this hypothesis we developed a biochemical assay for ACE2:Spike binding, and tested cardiac glycosides as inhibitors of binding. Here we report that ouabain, digitoxin, and digoxin, as well as sugar-free derivatives digitoxigenin and digoxigenin, are high-affinity competitive inhibitors of ACE2 binding to the Original [D614] S1 and the α/β/γ [D614G] S1 proteins. These drugs also inhibit ACE2 binding to the Original RBD, as well as to RBD proteins containing the β [E484K], Mink [Y453F] and α/β/γ [N501Y] mutations. As hypothesized, we also found that ouabain, digitoxin and digoxin blocked penetration by SARS-CoV-2 Spike-pseudotyped virus into human lung cells, and infectivity by native SARS-CoV-2. These data indicate that cardiac glycosides may block viral penetration into the target cell by first inhibiting ACE2:RBD binding. Clinical concentrations of ouabain and digitoxin are relatively safe for short term use for subjects with normal hearts. It has therefore not escaped our attention that these common cardiac medications could be deployed worldwide as inexpensive repurposed drugs for anti-COVID-19 therapy.

Agar binding reaction as a method for diagnosing cancer

In 1959, a message appeared from Hungarian researchers Ksab, Toro, Kiss and Janikovsky (Med.ref.j., II, 1959, 1) about a new method for diagnosing cancer, based on the fact that mucopolysaccharides with high molecular weight, the latter are formed during the destruction of the barrier "hyaluronic acid - heparin", which can be identified using agar.

A Review: Application and Implementation of Optic Fibre Sensors for Gas Detection

At the present time, there are major concerns regarding global warming and the possible catastrophic influence of greenhouse gases on climate change has spurred the research community to investigate and develop new gas-sensing methods and devices for remote and continuous sensing. Furthermore, there are a myriad of workplaces, such as petrochemical and pharmacological industries, where reliable remote gas tests are needed so that operatives have a safe working environment. The authors have concentrated their efforts on optical fibre sensing of gases, as we became aware of their increasing range of applications. Optical fibre gas sensors are capable of remote sensing, working in various environments, and have the potential to outperform conventional metal oxide semiconductor (MOS) gas sensors. Researchers are studying a number of configurations and mechanisms to detect specific gases and ways to enhance their performances. Evidence is growing that optical fibre gas sensors are superior in a number of ways, and are likely to replace MOS gas sensors in some application areas. All sensors use a transducer to produce chemical selectivity by means of an overlay coating material that yields a binding reaction. A number of different structural designs have been, and are, under investigation. Examples include tilted Bragg gratings and long period gratings embedded in optical fibres, as well as surface plasmon resonance and intra-cavity absorption. The authors believe that a review of optical fibre gas sensing is now timely and appropriate, as it will assist current researchers and encourage research into new photonic methods and techniques.

Kinetics of Eosin-5-maleimide Binding to Red Blood Cells

Introduction/Objective The binding of eosin-5-maleimide (EMA) to red blood cells in different pH conditions has not been well-investigated. The current standard protocol uses phosphate buffered saline at pH 7.0 to 7.5 for the binding reaction. The reaction typically uses a sixty-minute incubation. However, the effect of pH on EMA binding remains to be determined, nor have the reaction times at different pH conditions. This study utilizes optimal pH conditions of 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 to characterize EMA binding. The two parameters, time, and pH were investigated to extensively describe the binding kinetics of eosin-5-maleimide. Methods/Case Report The working EMA solutions in phosphate buffered saline at different pHs (specifically 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5) were incubated in the dark with 1 µL of washed red blood cells and 25 µL of working EMA solution. The red blood cells and working EMA solution were vigorously mixed before the specified incubation time. The binding was recorded at specified time points, namely 1, 2, 4, 8, 16, 32, and 64 minutes for each pH condition. The binding reaction was terminated by washing three times with 0.1% bovine serum albumin (BSA) in PBS and centrifuged at 400 g for 5 minutes. The red blood cells were then resuspended with 500 µL of PBS and ran in the BD FACSLyric™ flow cytometer. Results (if a Case Study enter NA) The current EMA binding protocol utilizes a. neutral pH of 7.0 to 7.5 recorded at 64 min. Based on our data, better discrimination of hereditary spherocytosis red blood cells from normal red blood cells may be obtained at a more basic pH of 8.5. Thus, a statistical t-test was performed for the comparison between the current setup and the apparent optimal pH conditions at shorter reaction times of 16 min and 32min at pH 8.5. Conclusion The resulting data suggest that enhanced discrimination of HS red blood cells can be achieved in a more basic pH. This was evidently observed at pH 8.5, where the highest percentage decrease is recorded, and the most significant difference compared to the other pH conditions. In conclusion, the study identifies that better discrimination of hereditary spherocytosis red blood cells from normal red blood cells can be determined using pH 8.5 with a shorter incubation time of 32 min. The pH 8.5 at 16 min condition could also be considered if the clinical batch size allows precise incubation of 15 min.

Cryo-EM as a powerful tool for drug discovery: recent structural based studies of SARS-CoV-2

The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has arisen as a global pandemic affecting the respiratory system showing acute respiratory distress syndrome (ARDS). However, there is no targeted therapeutic agent yet and due to the growing cases of infections and the rising death tolls, discovery of the possible drug is the need of the hour. In general, the study for discovering therapeutic agent for SARS-CoV-2 is largely focused on large-scale screening with fragment-based drug discovery (FBDD). With the recent advancement in cryo-electron microscopy (Cryo-EM), it has become one of the widely used tools in structural biology. It is effective in investigating the structure of numerous proteins in high-resolution and also had an intense influence on drug discovery, determining the binding reaction and regulation of known drugs as well as leading the design and development of new drug candidates. Here, we review the application of cryo-EM in a structure-based drug design (SBDD) and in silico screening of the recently acquired FBDD in SARS-CoV-2. Such insights will help deliver better understanding in the procurement of the effective remedial solution for this pandemic.

Complement binding reaction in gonads transplant. I. M. Shustrov, G. A. Vasiliev (Moscow Medical Zh., 1926, No. 4)

Research carried out in this direction by I. M. Shustrov G. A. Vasiliev (Moscow. Med. Zh., 1926, No. 4)

SARS-CoV-2 spike protein-induced platelet activation: Mechanism for virus and vaccine-induced thrombotic thrombocytopenia

Covid-19 pandemic stimulated an extremely fast development of effective vaccines. Recent studies found platelet-activating antibodies against platelet factor 4 (PF4) in both clinically ill Covid-19 patients and vaccine-induced thrombotic thrombocytopenia (VITT) patients. Here, we use various tools to identify the binding reaction of the SARS-CoV-2 spike glycoprotein (SP) with PF4 that results in immunogenic platelet-activating PF4/SP complexes. This binding is evidenced by an increase in mass, optical intensity, and stable binding force observed by quartz crystal microbalance, enzyme immune assay, and force spectroscopy, respectively. The SP induced an increase in the size of PF4 and switched the surface zeta potential of the PF4 from positive to negative values as evaluated by dynamic light scattering. The SP-induced platelet aggregation was identified by functional assay and flow cytometry but in a concentration-dependent manner. Our results indicated that the formed PF4/SP complexes can, on one hand, trigger the formation of PF4-antibodies and on the other hand mediate/activate platelets followed by inducing thrombotic events, which is the mechanism for excessive procoagulant activity observed in Covid-19 patients. With vector-based vaccines, we suggest that soluble SP are produced during the transcription process, forming antigenic PF4/SP complexes that result in a high rate of clotting effects in vaccinated individuals with Ad26.COV2.S and ChAdOx1nCoV-19 vaccines. An additional consideration of PF4/SP complexes in the current guidelines for the diagnosis of VITT will improve the treatment in patients. Our results serve a high demand to develop an effective method to treat Covid-19 patients and improve the safety for Covid-19 vaccination.

VEGF Detection via Simplified FLISA Using a 3D Microfluidic Disk Platform

Fluorescence-linked immunosorbent assay (FLISA) is a commonly used, quantitative technique for detecting biochemical changes based on antigen–antibody binding reactions using a well-plate platform. As the manufacturing technology of microfluidic system evolves, FLISA can be implemented onto microfluidic disk platforms which allows the detection of trace biochemical reactions with high resolutions. Herein, we propose a novel microfluidic system comprising a disk with a three-dimensional incubation chamber, which can reduce the amount of the reagents to 1/10 and the required time for the entire process to less than an hour. The incubation process achieves an antigen–antibody binding reaction as well as the binding of fluorogenic substrates to target proteins. The FLISA protocol in the 3D incubation chamber necessitates performing the antibody-conjugated microbeads’ movement during each step in order to ensure sufficient binding reactions. Vascular endothelial growth factor as concentration with ng mL−1 is detected sequentially using a benchtop process employing this 3D microfluidic disk. The 3D microfluidic disk works without requiring manual intervention or additional procedures for liquid control. During the incubation process, microbead movement is controlled by centrifugal force from the rotating disk and the sedimentation by gravitational force at the tilted floor of the chamber.