Ultra High-Throughput Screening Assays for Inhibitors of TMPRSS2 and SPIKE Interaction

SARS-CoV-2, the coronavirus that causes the disease COVID- 19, was identified over three years ago, yet current small molecule therapies have limited usefulness and resistance to therapies and vaccines is inevitable. Ultra high-throughput screening (uHTS) assays for novel and repurposed inhibitors of a protein-protein interaction in the viral life cycle could be used to screen a vast number of compounds with a specific target of action. In particular, the interaction between viral SPIKE protein and human TMPRSS2 is an understudied, yet critical step in viral entry. Thus, we aim to create uHTS assays to rapidly and affordably identify inhibitors of the TMPRSS2 and SPIKE interaction for further biochemical studies and therapeutic development for SARS-CoV-2.We first sought to create a Time Resolved-Forster/Fluorescence Energy Transfer (TR-FRET) assay which uses lysates of cells with overexpressed SPIKE and TMPRSS2 and fluorescently labeled antibodies to detect interactions between these proteins. Initially, we developed and optimized this TR-FRET assay in a 384-well plate then miniaturized to a 1536-well plate. We conducted a pilot screen of compounds with known biological activity to test this assay's screening capabilities. To further narrow the hits from this TR-FRET screen, we developed an orthogonal uHTS Nanoluciferase Binary Technology (NanoBiT) assay to detect the interaction between tagged SPIKE and TMPRSS2 in live cells.With these two assays in hand, we expanded our TR-FRET screen to over 100 000 compounds and identified several that were also positive in the orthogonal NanoBiT assay. Four of these compounds were found to potentially interact with either SPIKE or TMPRSS2 from thermal shift experiments, providing support for their action as SPIKE and TMPRSS2 interaction inhibitors. Thus, we have developed TR-FRET and NanoBiT orthogonal uHTS assays which have allowed for the discovery of several possible repurposed and novel inhibitors of the SPIKE/ TMPRSS2 interaction. These uHTS assays can be employed as a model for future drug discovery efforts and the compounds identified may be used as exciting starting points for development of inhibitors of SARS-CoV-2. This research was supported in part by The Emory School of Medicine COVID Catalyst-I3 award, the NCI Emory Lung Cancer SPORE (SR, HF;P50CA217691) Career Enhancement Program (AI, P50CA217691), Emory initiative on Biological Discovery through Chemical Innovation (AI) and R01AI167356 (SS).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Novel bandpass filter for far UV-C emitting radiation sources.

Disinfection with far UV-C radiation (<230 nm) is an effective method to inactivate harmful microorganisms like the SARS-CoV2 virus. Due to the stronger absorption than regular UV-C radiation (254 nm) and hence limited penetration into human tissues, it has the promise of enabling disinfection in occupied spaces. The best far-UV sources so far are discharge lamps based on the KrCl* excimer discharge peaking at 222 nm, however they produce longer wavelength radiation as a by-product. In current KrCl* excimer lamps usually a dichroic filter is used to suppress these undesired longer wavelengths. A phosphor-based filter is an alternative which is cheaper and easier to apply. This paper describes the results of our exploration of this opportunity. Various compounds were synthesized and characterized to find a replacement for the dichroic filter. It was found that Bi3+-doped ortho-borates with the pseudo-vaterite crystal structure exhibit the best absorption spectrum i.e. high transmission around 222 nm and strong absorption in the 235-280 nm range. Y0.24Lu0.75Bi0.01BO3 showed the best absorption spectrum in the UV-C. To suppress the unwanted Bi3+ emission (UV-B), the excitation energy can be transferred to a co-dopant. Ho3+ turned out to be the best co-dopant, and Ho0.24Lu0.75Bi0.01BO3 appeared to be the best overall candidate for the phosphor filter material. A suitable formulation for a coating suspension containing this material was found, and quite homogeneous coatings were achieved. The efficiency of these filter layers was investigated and the results in terms of exposure limit increase i.e. gain factor vs. no filter were compared with the dichroic filter. We achieved a gain factor for the Ho3+ containing sample of up to 2.33, i.e. not as good as that of the dichroic filter (∼4.6), but a very relevant improvement, making Ho0.24Lu0.75Bi0.01BO3 an interesting material for a cost-effective filter for KrCl* far UV-C lamps.

Customized Luminescent Multiplexed Quick-Response Codes as Reliable Temperature Mobile Optical Sensors for eHealth and Internet of Things

The need to sense and track in real time through sustainable and multifunctional labels is exacerbated by the COVID-19 pandemic, where the simultaneous measurement of body temperature and the fast tracking of people is required. One of the big challenges is to develop effective low-cost systems that can promote healthcare provision everywhere and for that, smarter and personalized Internet of things (IoT) devices are a pathway in large exploration, toward cost reduction and sustainability. Using the concept of color-multiplexed quick response (QR) codes, customized smart labels formed by two independent layers and smart location patterns provide simultaneous tracking and multiple synchronous temperature reading with maximum sensitivity values of 8.5% K-1 in the physiological temperature range, overwhelming the state-of-the-art optical sensor for healthcare services provided electronically via the internet (eHealth) and mobile sensors (mHealth).

Bifunctional Au@Pt/Au nanoparticles as electrochemiluminescence signaling probes for SARS-CoV-2 detection.

A novel immunosensor based on electrochemiluminescence resonance energy transfer (ECL-RET) for the sensitive determination of N protein of the SARS-CoV-2 coronavirus is described. For this purpose, bifunctional core@shell nanoparticles composed of a Pt-coated Au core and finally decorated with small Au inlays (Au@Pt/Au NPs) have been synthesized to act as ECL acceptor, using [Ru (bpy)3]2+ as ECL donor. These nanoparticles are efficient signaling probes in the immunosensor developed. The proposed ECL-RET immunosensor has a wide linear response to the concentration of N protein of the SARS-CoV-2 coronavirus with a detection limit of 1.27 pg/mL. Moreover, it has a high stability and shows no response to other proteins related to different virus. The immunosensor has achieved the quantification of N protein of the SARS-CoV-2 coronavirus in saliva samples. Results are consistent with those provided by a commercial colorimetric ELISA kit. Therefore, the developed immunosensor provides a feasible and reliable tool for early and effective detection of the virus to protect the population.

Various charging Methods in Electric Vehicle Application: A Review

During the covid pandemic, air quality has improved due to prolonged lockdown conditions. Hence according to the international energy agency, about 22% of environmental pollution is contributed by the transportation sector. Electric vehicles help in reducing the contribution towards carbon emission and help in mitigating the fossil fuel crisis and also promotes sustainable transportation. To enhance the growth of electric vehicle, charging infrastructure and range anxiety issues in the long drive has to be resolved. This paper reviews the various charging methods available for an electric vehicle. Some charging methods are wired and wireless charging, solar-powered, battery-swapping, vehicle-to-grid and vehicle-to-vehicle charging. A comparative study of these methods is tabulated. Based on the limitation of each method the optimum charging method for a vehicle is adapted for a particular application. © 2023 IEEE.

Development of FRET and Stress Granule Dual-Based System to Screen for Viral 3C Protease Inhibitors.

3C proteases (3Cpros) of picornaviruses and 3C-like proteases (3CLpros) of coronaviruses and caliciviruses represent a group of structurally and functionally related viral proteases that play pleiotropic roles in supporting the viral life cycle and subverting host antiviral responses. The design and screening for 3C/3CLpro inhibitors may contribute to the development broad-spectrum antiviral therapeutics against viral diseases related to these three families. However, current screening strategies cannot simultaneously assess a compound's cytotoxicity and its impact on enzymatic activity and protease-mediated physiological processes. The viral induction of stress granules (SGs) in host cells acts as an important antiviral stress response by blocking viral translation and stimulating the host immune response. Most of these viruses have evolved 3C/3CLpro-mediated cleavage of SG core protein G3BP1 to counteract SG formation and disrupt the host defense. Yet, there are no SG-based strategies screening for 3C/3CLpro inhibitors. Here, we developed a fluorescence resonance energy transfer (FRET) and SG dual-based system to screen for 3C/3CLpro inhibitors in living cells. We took advantage of FRET to evaluate the protease activity of poliovirus (PV) 3Cpro and live-monitor cellular SG dynamics to cross-verify its effect on the host antiviral response. Our drug screen uncovered a novel role of Telaprevir and Trifluridine as inhibitors of PV 3Cpro. Moreover, Telaprevir and Trifluridine also modulated 3Cpro-mediated physiological processes, including the cleavage of host proteins, inhibition of the innate immune response, and consequent facilitation of viral replication. Taken together, the FRET and SG dual-based system exhibits a promising potential in the screening for inhibitors of viral proteases that cleave G3BP1.

Landscape Analysis of Quercetin: A Potential Candidate Against SARS-CoV-2

Fruit, vegetables, and green tea contain quercetin (a flavonoid). Some of the diet's most signifi-cant sources of quercetin are apples, onions, tomatoes, broccoli, and green tea. Antioxidant, anticancer, anti-inflammatory, antimicrobial, antibacterial, and anti-viral effects have been studied of quercetin. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, ribonucleic acid (RNA) polymer-ase, and other essential viral life-cycle enzymes are all prevented from entering the body by quercetin. Despite extensive in vitro and in vivo investigations on the immune-modulating effects of quercetin and vitamin C treatment. 3-methyl-quercetin has been shown to bind to essential proteins necessary to convert minus-strand RNA into positive-strand RNAs, preventing the replication of viral RNA in the cytoplasm. Quercetin has been identified as a potential SARS-CoV-2 3C-like protease (3CLpro) suppressor in recent molecular docking studies and in silico assessment of herbal medicines. It has been demonstrated that quercetin increases the expression of heme oxygenase-1 through the nuclear factor erythroid-related factor 2 (Nrf2) signal network. Inhibition of heme oxygenase-1 may increase bilirubin synthesis, an endoge-nous antioxidant that defends cells. When human gingival fibroblast (HGF) cells were exposed to lipo-polysaccharide (LPS), inflammatory cytokine production was inhibited. The magnesium (Mg+2) cation complexation improves quercetin free radical scavenging capacity, preventing oxidant loss and cell death. The main objective of this paper is to provide an overview of the pharmacological effects of quercetin, its protective role against SARS-CoV-2 infection, and any potential molecular processes.Copyright © 2022 Bentham Science Publishers.

Carbon Dots Conjugated Antibody as an Effective FRET-Based Biosensor for Progesterone Hormone Screening.

In this work, carbon dots (CDs) were synthesized by a one-step hydrothermal method using citric acid and ethylene diamine, and covalently functionalized with antibodies for the sensing of progesterone hormone. The structural and morphological analysis reveals that the synthesized CDs are of average size (diameter 8-10 nm) and the surface functionalities are confirmed by XPS, XRD and FT-IR. Further graphene oxide (GO) is used as a quencher due to the fluorescence resonance energy transfer (FRET) mechanism, whereas the presence of the analyte progesterone turns on the fluorescence because of displacement of GO from the surface of CDs effectively inhibiting FRET efficiency due to the increased distance between donor and acceptor moieties. The linear curve is obtained with different progesterone concentrations with 13.8 nM detection limits (R2 = 0.974). The proposed optical method demonstrated high selectivity performance in the presence of structurally resembling interfering compounds. The PL intensity increased linearly with the increased progesterone concentration range (10-900 nM) under the optimal experimental parameters. The developed level-free immunosensor has emerged as a potential platform for simplified progesterone analysis due to the high selectivity performance and good recovery in different samples of spiked water.

Detection of COVID-19 DNA using Förster Resonance Energy Transfer between upconversion and gold nanoparticles

FRET is ascribed to the spectral overlapping of upconversion luminescence and the absorption of AuNPs. This experiment enables early-stage coronavirus detection. The results show a sensitivity of 100 fM for the detection of COVID-19 DNA. © 2022 The Author(s)

Rapid point-of-care detection of SARS-CoV-2 RNA with smartphone-based upconversion luminescence diagnostics.

Accurate COVID-19 screening via molecular technologies is still hampered by bulky instrumentation, complicated procedure, high cost, lengthy testing time, and the need for specialized personnel. Herein, we develop point-of-care upconversion luminescence diagnostics (PULD), and a streamlined smartphone-based portable platform facilitated by a ready-to-use assay for rapid SARS-CoV-2 nucleocapsid (N) gene testing. With the complementary oligo-modified upconversion nanoprobes and gold nanoprobes specifically hybridized with the target N gene, the luminescence resonance energy transfer effect leads to a quenching of fluorescence intensity that can be detected by the easy-to-use diagnostic system. A remarkable detection limit of 11.46 fM is achieved in this diagnostic platform without the need of target amplification, demonstrating high sensitivity and signal-to-noise ratio of the assay. The capability of the developed PULD is further assessed by probing 9 RT-qPCR-validated SARS-CoV-2 variant clinical samples (B.1.1.529/Omicron) within 20 min, producing reliable diagnostic results consistent with those obtained from a standard fluorescence spectrometer. Importantly, PULD is capable of identifying the positive COVID-19 samples with superior sensitivity and specificity, making it a promising front-line tool for rapid, high-throughput screening and infection control of COVID-19 or other infectious diseases.

Detection of COVID-19 DNA using Förster Resonance Energy Transfer between upconversion and gold nanoparticles

FRET is ascribed to the spectral overlapping of upconversion luminescence and the absorption of AuNPs. This experiment enables early-stage coronavirus detection. The results show a sensitivity of 100 fM for the detection of COVID-19 DNA. © 2022 The Author(s)

Is Economic Planning Our Future?

The article is devoted to the topic of economic planning at the state level. The historical excursus shows the origins of the planned economy elements on the example of three countries: Japan, India and France, as well as the evolution of its planned policy in the 20th century. The article provides a rationale for the relevance of planning, including as a tool to overcome market failures. The ESG agenda, in particular the issue of reducing the negative anthropological impact on the environment, is seen as one of the drivers for strengthening the role of planning.

Systematic identification of chemical components in Fufang Shuanghua oral liquid and screening of potential active components against SARS-CoV-2 protease.

Coronavirus disease (COVID-19) caused by SARS-COV-2 infection has been widely prevalent in many countries and has become a common challenge facing mankind. Traditional Chinese medicine (TCM) has played a prominent role in this pandemic, and especially TCM with the function of "heat-clearing and detoxifying" has shown an excellent role in anti-virus. Fufang Shuanghua oral liquid (FFSH) has been used to treat the corresponding symptoms of influenza such as fever, nasal congestion, runny nose, sore throat, and upper respiratory tract infections in clinic, which are typical symptoms of COVID-19. The content of chlorogenic acid, andrographolide and dehydrated andrographolide as the quality control components of FFSH is not less than 1.0 mg/mL, 60 µg/mL and 60 µg/mL respectively. In this study, UPLC-Q-TOF-MS/MS was employed to describe the chemical profile of FFSH. Virtual screening and fluorescence resonance energy transfer (FRET) were used to screen the effective components of FFSH acting on SARS-CoV-2 main protease (Mpro). As a result, 214 compounds in FFSH were identified or preliminarily characterized by UPLC-Q-TOF-MS/MS, and 61 active ingredients with potential inhibitory effects on Mpro were selected through receptor-based and ligand-based virtual screening. In particular, quercetin, forsythoside A, and linoleic acid showed a good inhibitory effect on Mpro in FRET evaluation with IC50 values of 26.15 µM, 22.26 µM and 47.09 µM respectively, and had a strong binding affinity with the receptor Mpro (6LU7) in molecular docking. CYS145 and HIS41 were the main amino acid residues affected by small molecules in the protein binding domain. In brief, we characterized, for the first time, 214 chemical components in FFSH, and three of them, including quercetin, forsythoside A and linoleic acid, were screened out to exert beneficial anti-COVID-19 effects through CYS145 and HIS41 sites, which may provide a new research strategy for TCM to develop new therapeutic drugs against COVID-19.

A highly selective and sensitive competitive aptasensor capable of quantifying thrombin elevated with side effects in severe COVID-19.

To protect critical injury from blood clots with side effects in severe COVID-19, a highly selective and sensitive biosensor was developed for the quantification of trace levels of thrombin using the combination of a DNA aptamer (TBA) of thrombin and a complementary strand of TBA. TBA rapidly binds with thrombin, whereas it slowly binds with the complementary strand to form a double stranded DNA (dsDNA). SFC green intercalated into dsDNA cannot emit light in 1,1'-oxalyldiimidazole chemiluminescence (ODI-CL) reaction because high-energy intermediates formed from ODI-CL reaction cannot transfer energy to SFC trapped in dsDNA. However, SFC freely existing with the formation of G-quadruplex from the reaction of thrombin and TBA emits bright chemiluminescence because the high-energy intermediates can transfer energy to SFC (or camel) in solution. Thus, the brightness of light emitted in ODI-CL reaction was proportionally enhanced with the increase of thrombin in a sample due to the increase of G-quadruplex and reduction of dsDNA. The limit of detection (LOD) of the label free aptasensor operated with good linear calibration curve (10-320 mU/ml) was as low as 3 mU/ml (or 43 pM). Also, the biosensor was quantified trace levels of thrombin with good accuracy, precision, and reliability.

Rapid and Highly Sensitive Detection of Target DNA Related to COVID-19 Virus With a Fluorescent Bio-conjugated Probe via a FRET Mechanism.

A novel cyanine 3 (Cy3)-based bio-conjugated sensor has been developed to detect target DNA or extracted RNA from COVID -19 samples using the fluorescence resonance energy transfer (FRET) experiment. A special sequence of the COVID -19 genome was selected as a complementary DNA (target DNA) part. The opposite chain of this target sequence was designed in 2 parts; one part was attached to the Cy3 organic dye (capture DNA or Cy3- DNA), and the other part was attached to the BHQ2 molecule (quencher DNA or BHQ2- DNA). The Cy3 molecule acts as a donor pair, and BHQ2 acts as an acceptor pair in the FRET experiment. The capture DNA and quencher DNA can form a sandwiched complex in the presence of target DNA. The formation of the entitled sandwiched hybrid causes the decrement of emission intensity of the Cy3 donor in bio-conjugated Cy3-DNA via energy transfer from Cy3 (as a donor) to BHQ2 (as an acceptor). Indeed, in the presence of non-complementary DNA, the pairing of DNA strands does not occur, the FRET phenomenon does not exist, and therefore fluorescence intensity of Cy3 does not decrease. Moreover, this biosensor was successfully applied to analyze real samples containing extracted RNA of COVID -19 prepared for the reverse transcriptase-polymerase chain reaction (RT-PCR) test, and the results were promising.

A smartphone-based platform for ratiometric visualization of SARS-CoV-2 via an oligonucleotide probe.

COVID-19 necessitates the development of reliable and convenient diagnostic tools. In this work, a facile 3D-printed smartphone platform was constructed that achieved reliable visual detection of SARS-CoV-2 by eliminating the effect of ambient light and fixing the camera position relative to the sample. The oligonucleotide probe is modified with orange-red-emitting TAMRA working as an internal standard and green-emitting FAM serving as a sensitive sensing agent. Under 365-nm UV excitation, the emission wavelengths of TAMRA and FAM are 580 nm and 518 nm, respectively. When the probes interact with the targets, the green fluorescence gradually restores while the orange-red fluorescence remains stable. Thus, a striking color transition from orange-red to green could be observed by the naked eye. The detection limit of SARS-CoV-2 nucleic acid is 0.23 nM, and the entire process of color change could be completed in 25 min. Furthermore, the RGB value analysis of the sample solution was conducted using a smartphone for reliable and reproducible discrimination of SARS-CoV-2. The proposed smartphone platform might establish a general method for visual detection of SARS-CoV-2 nucleic acid as well as other virus-related diseases.

[Discovery of SARS-CoV-2 main protease inhibitors using an optimized FRET-based high-throughput screening assay].

For rapid discovery of novel SARS-CoV-2 main protease (Mpro) inhibitors, an optimized fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) assay was developed. The recombinant Mpro was expressed in Escherichia coli Rosetta (DE3) cells and the specific activity of purified Mpro was assessed by a FERT assay using a fluorescently labeled substrate. Subsequently, the reaction buffer, working concentration of Mpro, incubation temperature and length, and DMSO tolerance were systematically optimized. The Mpro was solubly expressed in E. coli cells and exhibited an expected enzymatic activity (40 000 U/mg) in a FRET assay. Through these systematic optimizations, we selected 0.4 µmol/L Mpro and 5 µmol/L FRET substrate as the optimal working concentrations in this FRET screening assay, and a high Z' factor of 0.79 was achieved. More importantly, the addition of reducing reagent 1, 4-dithiothreitol in reaction buffer is necessary to faithfully assess the reliability of the screening assay. Using this assay, plumbagin (PLB) and ginkgolic acid (GA) were identified as potential Mpro inhibitors in vitro from a natural product library. In summary, we developed an optimized FRET-based HTS assay for the discovery of Mpro inhibitors, and PLB and GA could serve as the promissing lead compounds to generate more potent antiviral agents targeting SARS-CoV-2 Mpro.

Implementation of a coplanar-waveguide chip for the measurement of EM wave absorption spectrum of SARS-Cov-2 virus

Currently, aerosol is considered as the major route for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. A safe sterilization method with an excellent penetration capability and ability to sterilize free spaces is urgently needed. Previously it has been experimentally demonstrated that microwave-based sterilization can effectively inactivate the H3N2 Influenza A virus through the structure-resonant energy transfer (SRET) effect with a radiation field intensity following the IEEE standard. In order to utilize the same mechanism to inactivate the SARS-CoV-2 virus, firstly, the structural resonant frequencies with electromagnetic (EM) waves have to be identified. In this paper we report our design and implementation of a spectrum measurement chip utilizing the coplanar waveguide with pre-printed mask. With the mask, the repeatability of the insertion loss measurement can be well-controlled. Our microwave absorption spectra results revealed that the coplanar-waveguide chip can identify the resonant microwave frequencies of difference viruses, including the SARS-CoV-2 viruses, highlighting the potential applications for not only the virus detection but also the safe and non-thermal sterilization of public spaces. During the presentation, we will also report the resonant EM wave frequencies of various corona viruses monitored by the aforementioned chip. Copyright © 2022 SPIE.

SARS-CoV-2 VARIANTS INCREASE KINETIC STABILITY of OPEN SPIKE CONFORMATIONS

Background: SARS-CoV-2 variants of concern harbor mutations in the Spike (S) glycoprotein that confer more efficient transmission and dampen the efficacy of COVID-19 vaccines and antibody therapies. S mediates virus entry and is the primary target for antibody responses, with structural studies of soluble S variants revealing an increased propensity towards conformations accessible to the human Angiotensin-Converting Enzyme 2 (hACE2) receptor. However, real-time observations of conformational dynamics that govern the structural equilibriums of the S variants have been lacking. Methods: Here, we report single-molecule Förster Resonance Energy Transfer (smFRET) studies of S variants of concern containing critical mutations, including D614G and E484K, in the context of virus particles. Results: Investigated variants were shown by smFRET to predominantly occupy more open hACE2-accessible conformations, agreeing with predictions from structures of soluble trimers. Additionally, S variants exhibited decelerated transitions from hACE2-accessible/bound states. Conclusion: Here, we provide the real-time dimension to distinct structures of Spikes in the context of virus particles and present the first experimental evidence of increased stability of Spike variants. Our finding of increased S kinetic stability in the open conformation provides a new perspective on SARS-CoV-2 adaptation to the human population.

Wireless Powered Sensor Network Based Covid Dissemination Quantifier

The increase in the frequency of Covid-19 is the latest concern to world health, and there is a vast quantity of unimaginably horrific Covid-19 disinformation spreading online. The proposed paper measures vital signs like heart rate, temperature and blood pressure without direct contact with patients. It is very important, to maintain contactless visits to avoid the spread. As a result, it is easier to keep track of the needed parameters. Brought the notion of telemedicine or e-medicine. Wireless rechargeable sensor networks have widely utilised wireless power transfer systems to transfer energy (WRSNs). These methods try to extend the life of a network by transferring electricity to end devices. The incurred charging delay to replenish the sensor nodes is considered one of the major issues in wireless sensor networks using these wireless techniques (WSNs). This sensor for the parameter exchangers regulates the body temperature, which is monitored using a simple device, the vital priority is given to tracking the heart rate and respiratory rate of the patients. Patient data are monitored from the doctor's side;our proposed system is also provided to show the same with nearby Covid-19 patient's identity for a self-safeguard. The main objective of our work is to self-guard unaffected and also the Covid-19 patients, with the help of a proposed system, and also share the details with the doctor. © 2022 IEEE.