Silver Nanoparticle-Decorated Personal Protective Equipment for Inhibiting Human Coronavirus Infectivity.

The Coronavirus disease 2019 (COVID-19) global outbreak and its continued growth and mutation into various forms emphasize the need for effective disinfectants to assist in the reduction of the virus's spread from individual to individuals and community to communities through various modes, including coughing, sneezing, touching of contaminated surfaces, and being in proximity of an unprotected infected person, to mention a few. The rapid development of reliable disinfecting materials or solutions and their incorporation in personal protective equipment is a critical need at the moment that will assist significantly in curbing the spread of the virus SARS-CoV-2, the cause of COVID-19 illness. Here, we present an in situ assembly of antiviral metal nanoparticles on a rigid surface and on commercial face masks made up of nonwoven and woven textiles. The results indicate a very high efficacy of 99.99% against a surrogate virus to SARS-CoV-2. Such a versatile and cost-effective approach using the blade-coating technique can be easily extended to the roll-to-roll manufacturing setting to expedite the efforts and mitigate the rapid spread of the virus.

The proteomic analysis of breast cell line exosomes reveals disease patterns and potential biomarkers.

Cancer cells release small extracellular vesicles, exosomes, that have been shown to contribute to various aspects of cancer development and progression. Differential analysis of exosomal proteomes from cancerous and non-tumorigenic breast cell lines can provide valuable information related to breast cancer progression and metastasis. Moreover, such a comparison can be explored to find potentially new protein biomarkers for early disease detection. In this study, exosomal proteomes of MDA-MB-231, a metastatic breast cancer cell line, and MCF-10A, a non-cancerous epithelial breast cell line, were identified by nano-liquid chromatography coupled to tandem mass spectrometry. We also tested three exosomes isolation methods (ExoQuick, Ultracentrifugation (UC), and Ultrafiltration-Ultracentrifugation) and detergents (n-dodecyl ß-D-maltoside, Triton X-100, and Digitonin) for solubilization of exosomal proteins and enhanced detection by mass spectrometry. A total of 1,107 exosomal proteins were identified in both cell lines, 726 of which were unique to the MDA-MB-231 breast cancer cell line. Among them, 87 proteins were predicted to be relevant to breast cancer and 16 proteins to cancer metastasis. Three exosomal membrane/surface proteins, glucose transporter 1 (GLUT-1), glypican 1 (GPC-1), and disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), were identified as potential breast cancer biomarkers and validated with Western blotting and high-resolution flow cytometry. We demonstrated that exosomes are a rich source of breast cancer-related proteins and surface biomarkers that may be used for disease diagnosis and prognosis.

Aptamer-facilitated cryoprotection of viruses.

Global vaccination and gene therapy programs have an urgent demand for stabilization of viral vectors at low temperature. We used a quadramer, a bridge-connected DNA tetra-aptamer to antivesicular stomatitis virus (VSV), as a viral cryoprotectant. Results showed that the tetravalent antivirus DNA aptamers protect viral activity during multiple freeze-thaw cycles, shield from neutralizing antibodies, and decrease aggregation of viral particles.

Carbohydrate-based ice recrystallization inhibitors increase infectivity and thermostability of viral vectors.

The inability of vaccines to retain sufficient thermostability has been an obstacle to global vaccination programs. To address this major limitation, we utilized carbohydrate-based ice recrystallization inhibitors (IRIs) to eliminate the cold chain and stabilize the potency of Vaccinia virus (VV), Vesicular Stomatitis virus (VSV) and Herpes virus-1 (HSV-1). The impact of these IRIs was tested on the potency of the viral vectors using a plaque forming unit assay following room temperature storage, cryopreservation with successive freeze-thaw cycles and lyophilization. Viral potency after storage with all three conditions demonstrated that N-octyl-gluconamide (NOGlc) recovered the infectivity of shelf stored VV, 5.6 Log10 PFU mL(-1) during 40 days, and HSV-1, 2.7 Log10 PFU mL(-1) during 9 days. Carbon-linked antifreeze glycoprotein analogue ornithine-glycine-glycine-galactose (OGG-Gal) increases the recovery of VV and VSV more than 1 Log10 PFU mL(-1) after 10 freeze-thaw cycles. In VSV, cryostorage with OGG-Gal maintains high infectivity and reduces temperature-induced aggregation of viral particles by 2 times that of the control. In total, OGG-Gal and NOGlc preserve virus potency during cryostorage. Remarkably, NOGlc has potential to eliminate the cold chain and permit room temperature storage of viral vectors.

Aptamer-facilitated Protection of Oncolytic Virus from Neutralizing Antibodies.

Oncolytic viruses promise to significantly improve current cancer treatments through their tumor-selective replication and multimodal attack against cancer cells. However, one of the biggest setbacks for oncolytic virus therapy is the intravenous delivery of the virus, as it can be cleared from the bloodstream by neutralizing antibodies before it reaches the tumor cells. We have selected DNA aptamers against an oncolytic virus, vesicular stomatitis virus, using a competitive binding approach, as well as against the antigen binding fragment (Fab) of antivesicular stomatitis virus polyclonal antibodies, in order to shield the virus from nAbs and enhance its in vivo survival. We used flow cytometry to identify these aptamers and evaluated their efficiency to shield vesicular stomatitis virus in a cell-based plaque forming assay. These oligonucleotides were then modified to obtain multivalent binders, which led to a decrease of viral aggregation, an increase in its infectivity and an increase in its stability in serum. The aptamers were also incubated in nondiluted serum, showing their effectiveness under conditions mimicking those in vivo. With this approach, we were able to increase viral infectivity by more than 70% in the presence of neutralizing antibodies. Thus, this method has the potential to enhance the delivery of vesicular stomatitis virus through the bloodstream without compromising the patient's immune system.

Four-way junction formation promoting ultrasensitive electrochemical detection of microRNA.

MicroRNAs (miRNAs) represent a class of biomarkers that are frequently deregulated in cancer cells and have shown a great promise for cancer classification and prognosis. Here, we endeavored to develop a DNA four-way junction based electrochemical sensor (4J-SENS) for ultrasensitive miRNA analysis. The developed sensor can be operated within the dynamic range from 10 aM to 1 fM and detect as low as 2 aM of miR-122 (∼36 molecules per sample), without PCR amplification. Furthermore, the 4J-SENS was employed to profile endogenouse hsa-miR-122 in healthy human and chronic lymphocyitc leukemia (CLL) patient serum, and the results were validated by qPCR analysis.

Three-mode electrochemical sensing of ultralow microRNA levels.

MicroRNAs (miRNAs) are an emerging class of biomarkers that are frequently deregulated in cancer cells and have shown great promise for cancer classification and prognosis. In this work, we developed a three-mode electrochemical sensor for detection and quantitation of ultralow levels of miRNAs in a wide dynamic range of measured concentrations. The sensor facilitates three detection modalities based on hybridization (H-SENS), p19 protein binding (P-SENS), and protein displacement (D-SENS). The combined three-mode sensor (HPD-SENS) identifies as low as 5 aM or 90 molecules of miRNA per 30 µL of sample without PCR amplification, and can be operated within the dynamic range from 10 aM to 1 µM. The HPD sensor is made on a commercially available gold nanoparticles-modified electrode and is suitable for analyzing multiple miRNAs on a single electrode. This three-mode sensor exhibits high selectivity and specificity and was used for sequential analysis of miR-32 and miR-122 on one electrode. In addition, the H-SENS can recognize miRNAs with different A/U and G/C content and distinguish between a fully matched miRNA and a miRNA comprising either a terminal or a middle single base mutation. Furthermore, the H- and P-SENS were successfully employed for direct detection and profiling of three endogenous miRNAs, including hsa-miR-21, hsa-miR-32, and hsa-miR-122 in human serum, and the sensor results were validated by qPCR.

Anti-Fab aptamers for shielding virus from neutralizing antibodies.

Oncolytic viruses are promising therapeutics that can selectively replicate in and kill tumor cells. However, repetitive administration of viruses provokes the generation of neutralizing antibodies (nAbs) that can diminish their anticancer effect. In this work, we selected DNA aptamers against the antigen binding fragment (Fab) of antivesicular stomatitis virus polyclonal antibodies to shield the virus from nAbs and enhance its in vivo survival. For the first time, we used flow cytometry and electrochemical immunosensing to identify aptamers targeting the Fab region of antibodies. We evaluated the aptamers in a cell-based infection assay and found that several aptamer clones provide more than 50% shielding of VSV from nAbs and thus have the potential to enhance the delivery of VSV without compromising the patient's immune system. In addition, we developed a bifunctional label-free electrochemical immunosensor for the quantitation of aptamer-mediated degree of shielding and the amount of vesicular stomatitis virus (VSV) particles. Electrochemical impedance spectroscopy was employed to interrogate the level of VSV in a linear range from 5 × 10(4) to 5 × 10(6) PFU mL(-1) with a detection limit of 10(4) PFU mL(-1).