Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 Detection

The fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays to allow infection diagnosis and prevention of transmission. We aimed to develop a molecular beacon (MB)-based detection assay for SARS-CoV-2, designed to detect the ORF1ab and S genes, proposing a two-stage COVID-19 testing strategy. The novelty of this work lies in the design and optimization of two MBs for detection of SARS-CoV-2, namely, concentration, fluorescence plateaus of hybridization, reaction temperature and real-time results. We also identify putative G-quadruplex (G4) regions in the genome of SARS-CoV-2. A total of 458 nasopharyngeal and throat swab samples (426 positive and 32 negative) were tested with the MB assay and the fluorescence levels compared with the cycle threshold (Ct) values obtained from a commercial RT-PCR test in terms of test duration, sensitivity, and specificity. Our results show that the samples with higher fluorescence levels correspond to those with low Ct values, suggesting a correlation between viral load and increased MB fluorescence. The proposed assay represents a fast (total duration of 2 h 20 min including amplification and fluorescence reading stages) and simple way of detecting SARS-CoV-2 in clinical samples from the upper respiratory tract.

Evaluation of in vitro antiplasmodial effect of extracts from seven medicinal plants used to treat malaria in Côte d'Ivoire.

Introduction: Plasmodium falciparum strains had been increasingly resistant to commonly used molecules including artemisinin. It is therefore urges to find new therapeutic alternatives. Methods: In this study, the antiplasmodial activity of 21 extracts obtained from seven plants of the Anthocleista djalonensis, Cochlospermum planchonii, Harungana madagascariensis, Hoslundia opposita, Mangifera indica, Margaritaria discoidea and Pericopsis laxiflora of the Ivorian pharmacopoeia was evaluated on the chloroquine sensitive (NF54) and multi-resistant (K1) reference strains and on clinical isolates as well. The technique used was the microtiter method based on fluorescence reading with SYBR Green. Results: The aqueous extract of the bark of H. madagascariensis and methanolic extracts of P. laxiflora showed the best antiplasmodial activity with IC50 values of 6.16 µg/mL and 7.44 µg/mL, respectively. On the other hand, extracts of M. indica showed a very moderate activity with IC50 values between 15 µg/mL and 50 µg/mL (5<IC50<50 µg/mL) on the same strains of P. falciparum. Only the aqueous extract of A. djalonensis had IC50 values greater than 50 µg/mL. The phytochemical analysis showed a strong presence of polyphenols and alkaloids in extracts with a cumulative rate of 90.47% and 95.23%, respectively. Conclusion: The results obtained were also justified by the composition of these plants, which have several secondary metabolites involved in the treatment of malaria. The antiplasmodial properties of these plants could partially justify their use in malaria treatment. Further studies on these extracts are needed to manufacture a stable galenic formulation for the development of an improved traditional medicine.

PCL19-114: Development of Locally Deliverable Polymeric Microspheres With Environmentally Specific Release for the Improvement of Cancer Therapy

Background: Cancer therapy has made incredible leaps in extending patient lifespans. Unfortunately, most therapy is delivered via a systemic route and often suffers from dose-limiting systemic toxicities, resulting in cessation of therapy or decline in quality of life. Nanotechnology efforts to improve tolerability of systemic agents have suffered limited market viability due to problems such as rapid clearance, circulation instability, and low tumor site accumulation. While local delivery allows for overcoming the aforementioned hurdles, pre-set formulations and nonspecific release pose alternate issues. To approach these challenges, we aimed to investigate our previously developed solid nanoparticles for delivery of protein-based therapy capable of addressing a tumor-specific profile and with selective release within the tumor environment due to elevated thrombin levels. Methods: Poly(ethylene glycol) diacrylate microspheres [PEGDAmS] were prepared using previously described polymerization within a reverse phase emulsion. Compounding patient-specific therapy was evaluated using fluorophore labeled glutathione-S-transferase (GST) and GST-green fluorescent protein (GFP) fusion proteins. Protein loading was analyzed using fluorescence reading and concurrent gel densitometry. Loaded PEGDAmS were delivered into the frontal lobes of Sprague-Dawley rats through a syringe and analyzed using fluorescence microscopy after sacrifice at 48 hours. Results: The addition of 0.2% tween20 surfactant to polymerization reduced PEGDAmS diameter (38.3±30.7 μm to 8.6±6.5 μm) and allowed for minimally invasive delivery to the cranial cavity through a 26S gauge needle. Compounding a tumor-specific regimen is achievable as premixed protein solutions exhibited solution-representative loading on PEGDAmS. Additionally, preloaded vehicles did not demonstrate appreciable cross contamination over 7 days when incubated in close proximity, demonstrating the feasibility of concurrent delivery of various preloaded formulations. Loaded PEGDAmS exhibited environment specific delivery with extensive release of thrombin cleavable GST-GFP in plasma but greatly reduced in anticoagulated plasma. Conclusion: We have shown the ability to locally administer a delivery system with modular therapy-loading and responsiveness to the tumor microenvironment. Further investigation is warranted to explore local delivery of GST-tagged oncology-relevant biologic agents in the presence of a tumor in vivo.

Adhesion Performance of Amino Groups (ε-NH2) of Soybean Glycinin Proteins

Amino groups of a protein have been considered as a major factor contributing to adhesion strength and water absorption. Positively charged amino groups (−NH2+) of soybean glycinin protein were replaced with negative charged carboxyl groups (−COO−) by using succinylation modification. The amino groups also were replaced with hydrophobic methyl groups (−CH3) by using methylation modification. Adhesion strength and water resistance were reduced when the −NH2 groups were substituted with −COOH groups at pH 7.6. But adhesive performance at the same pH environment was improved when the −NH2 group was replaced with −CH3, which suggests that hydrophobic groups of protein contribute to adhesion and that hydrophilic groups reduce adhesive strength. Both methylation and succinylation modifications caused the fluorescence reading (λmax) of the protein samples to shift to a higher wavelength. Native glycinin protein λmax was 344.84 nm. Methylated glycinin at 6% substitute degree (SD) had a λmax of 350.92 nm, and at 8% SD of succinylated glycinin λmax was 354.92 nm. The native glycinin has a denaturation temperature (Tp) of 165.7 °C and enthalpy (ΔH) of 16.3 J/g of glycinin. The Tp of methylated glycinin was 152 °C and ΔH was 13.9 J/g of glycinin at 4% SD. At 6% SD, Tp was 147.4 °C and the ΔH was 11.3 J/g of glycinin. The Tp for succinylated glycinin was 165.6 °C and the ΔH was −2.5 J/g of glycinin when SD = 3%. At 8% SD, the Tp was 173.6 °C and ΔH was −4.8 J/g of glycinin (SD = 8%).