Digital Protein Detection in Bulk Solutions.

Quick and accurate molecular diagnostics in protein detection can greatly benefit medicine in disease diagnosis and lead to positive patient outcomes. However, specialized equipment used in clinical laboratories often comes with trade-offs between operation and function serving a single role for very specific needs. For example, to achieve high analytical sensitivity and specificity, instruments such as high-performance liquid chromatography and/or liquid chromatography-mass spectrometry use a complex instrument design and require thorough training of the users. On the other hand, simple tests such as protein detection in urinary tract infection using dip-stick assays provide very quick results but suffer from poor analytical sensitivity. Here, we present an application study for the 3D particle counter technology, which is based on optical confocal detection in order to scan large sample volumes (0.5-3 mL) in glass cuvettes, that aims to close the gap between analytical sensitivity and turnover assay time and simplify protein detection by adopting bead-based immunoassays. Combining the 3D particle counter technology with bead-based immunoassays, a subpicomolar limit of detection-ranging from 119 to 346 fM-was achieved within 3.5-hour assay time for recombinant mouse interleukin 6 detection. As an alternative instrument to a flow cytometer, the 3D particle counter takes advantages of bead-based immunoassays and provides unique accessibility and flexibility for users.

A Protein Microarray-Based Respiratory Viral Antigen Testing Platform for COVID-19 Surveillance.

High-throughput and rapid screening testing is highly desirable to effectively combat the rapidly evolving COVID-19 pandemic co-presents with influenza and seasonal common cold epidemics. Here, we present a general workflow for iterative development and validation of an antibody-based microarray assay for the detection of a respiratory viral panel: (a) antibody screening to quickly identify optimal reagents and assay conditions, (b) immunofluorescence assay design including signal amplification for low viral titers, (c) assay characterization with recombinant proteins, inactivated viral samples and clinical samples, and (d) multiplexing to detect a panel of common respiratory viruses. Using RT-PCR-confirmed SARS-CoV-2 positive and negative pharyngeal swab samples, we demonstrated that the antibody microarray assay exhibited a clinical sensitivity and specificity of 77.2% and 100%, respectively, which are comparable to existing FDA-authorized antigen tests. Moreover, the microarray assay is correlated with RT-PCR cycle threshold (Ct) values and is particularly effective in identifying high viral titers. The multiplexed assay can selectively detect SARS-CoV-2 and influenza virus, which can be used to discriminate these viral infections that share similar symptoms. Such protein microarray technology is amenable for scale-up and automation and can be broadly applied as a both diagnostic and research tool.

Leveraging Allele-Specific Expression for Therapeutic Response Gene Discovery in Glioblastoma.

Glioblastoma is the most prevalent primary malignant brain tumor in adults and is characterized by poor prognosis and universal tumor recurrence. Effective glioblastoma treatments are lacking, in part due to somatic mutations and epigenetic reprogramming that alter gene expression and confer drug resistance. To investigate recurrently dysregulated genes in glioblastoma, we interrogated allele-specific expression (ASE), the difference in expression between two alleles of a gene, in glioblastoma stem cells (GSC) derived from 43 patients. A total of 118 genes were found with recurrent ASE preferentially in GSCs compared with normal tissues. These genes were enriched for apoptotic regulators, including schlafen family member 11 (SLFN11). Loss of SLFN11 gene expression was associated with aberrant promoter methylation and conferred resistance to chemotherapy and PARP inhibition. Conversely, low SLFN11 expression rendered GSCs susceptible to the oncolytic flavivirus Zika. This discovery effort based upon ASE revealed novel points of vulnerability in GSCs, suggesting a potential alternative treatment strategy for chemotherapy-resistant glioblastoma. SIGNIFICANCE: Assessing allele-specific expression reveals genes with recurrent cis-regulatory changes that are enriched in glioblastoma stem cells, including SLFN11, which modulates chemotherapy resistance and susceptibility to the oncolytic Zika virus.

A Clinical-Stage Cysteine Protease Inhibitor blocks SARS-CoV-2 Infection of Human and Monkey Cells.

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 µM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 µM. There was no toxicity to any of the host cell lines at 10-100 µM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.

A cysteine protease inhibitor blocks SARS-CoV-2 infection of human and monkey cells.

K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 µM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 µM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2 , differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.

Luminescent silicon nanoparticles for distinctive tracking of cellular targeting and trafficking.

Developing therapeutic nanoparticles that actively target disease cells or tissues by exploiting the binding specificity of receptors presented on the cell surface has extensively opened up biomedical applications for drug delivery and imaging. An ideal nanoparticle for biomedical applications is required to report confirmation of relevant targeting and the ultimate fate in a physiological environment for further verification, e.g. to adapt dosage or predict response. Herein, we demonstrate tracking of silicon nanoparticles through intrinsic photoluminescence (PL) during the course of cellular targeting and uptake. Time-resolved analysis of PL characteristics in cellular microenvironments provides dynamic information on the physiological conditions where the silicon nanoparticles are exposed. In particular, the PL lifetime of the silicon nanoparticles is in the order of microseconds, which is significantly longer than the nanosecond lifetimes exhibited by fluorescent molecules naturally presented in cells, thus allowing discrimination of the nanoparticles from the cellular background autofluorescence in time-gated imaging. The PL lifetime is a physically intensive property that reports the inherent characteristics of the nanoparticles regardless of surrounding noise. Furthermore, we investigate a unique means to inform the lifespan of the biodegradable silicon nanoparticles responsive to local microenvironment in the course of endocytosis. A multivalent strategy of nanoparticles for enhanced cell targeting is also demonstrated with complementary analysis of time-resolved PL emission imaging and fluorescence correlation spectroscopy. The result presents the promising potential of the photoluminescent silicon nanoparticles toward advanced cell targeting systems that simultaneously enable tracking of cellular trafficking and tissue microenvironment monitoring.

Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin αvß5 Axis.

Zika virus (ZIKV) causes microcephaly by killing neural precursor cells (NPCs) and other brain cells. ZIKV also displays therapeutic oncolytic activity against glioblastoma (GBM) stem cells (GSCs). Here we demonstrate that ZIKV preferentially infected and killed GSCs and stem-like cells in medulloblastoma and ependymoma in a SOX2-dependent manner. Targeting SOX2 severely attenuated ZIKV infection, in contrast to AXL. As mechanisms of SOX2-mediated ZIKV infection, we identified inverse expression of antiviral interferon response genes (ISGs) and positive correlation with integrin αv (ITGAV). ZIKV infection was disrupted by genetic targeting of ITGAV or its binding partner ITGB5 and by an antibody specific for integrin αvß5. ZIKV selectively eliminated GSCs from species-matched human mature cerebral organoids and GBM surgical specimens, which was reversed by integrin αvß5 inhibition. Collectively, our studies identify integrin αvß5 as a functional cancer stem cell marker essential for GBM maintenance and ZIKV infection, providing potential brain tumor therapy.

Mechanism of Action of Methotrexate Against Zika Virus.

Zika virus (ZIKV), which is associated with microcephaly in infants and Guillain-Barré syndrome, reemerged as a serious public health threat in Latin America in recent years. Previous high-throughput screening (HTS) campaigns have revealed several potential hit molecules against ZIKV, including methotrexate (MTX), which is clinically used as an anti-cancer chemotherapy and anti-rheumatoid agent. We studied the mechanism of action of MTX against ZIKV in relation to its inhibition of dihydrofolate reductase (DHFR) in vitro using Vero and human neural stem cells (hNSCs). As expected, an antiviral effect for MTX against ZIKV was observed, showing up to 10-fold decrease in virus titer during MTX treatment. We also observed that addition of leucovorin (a downstream metabolite of DHFR pathway) rescued the ZIKV replication impaired by MTX treatment in ZIKV-infected cells, explaining the antiviral effect of MTX through inhibition of DHFR. We also found that addition of adenosine to ZIKV-infected cells was able to rescue ZIKV replication inhibited by MTX, suggesting that restriction of de novo synthesis adenosine triphosphate (ATP) pools suppresses viral replication. These results confirm that the DHFR pathway can be targeted to inhibit replication of ZIKV, similar to other published results showing this effect in related flaviviruses.

Activity of Selected Nucleoside Analogue ProTides against Zika Virus in Human Neural Stem Cells.

Zika virus (ZIKV), an emerging flavivirus that causes neurodevelopmental impairment to fetuses and has been linked to Guillain-Barré syndrome continues to threaten global health due to the absence of targeted prophylaxis or treatment. Nucleoside analogues are good examples of efficient anti-viral inhibitors, and prodrug strategies using phosphate masking groups (ProTides) have been employed to improve the bioavailability of ribonucleoside analogues. Here, we synthesized and tested a small library of 13 ProTides against ZIKV in human neural stem cells. Strong activity was observed for 2'-C-methyluridine and 2'-C-ethynyluridine ProTides with an aryloxyl phosphoramidate masking group. Substitution of a 2-(methylthio) ethyl phosphoramidate for the aryloxyl phosphoramidate ProTide group of 2'-C-methyluridine completely abolished antiviral activity of the compound. The aryloxyl phosphoramidate ProTide of 2'-C-methyluridine outperformed the hepatitis C virus (HCV) drug sofosbuvir in suppression of viral titers and protection from cytopathic effect, while the former compound's triphosphate active metabolite was better incorporated by purified ZIKV NS5 polymerase over time. These findings suggest both a nucleobase and ProTide group bias for the anti-ZIKV activity of nucleoside analogue ProTides in a disease-relevant cell model.

Development and Validation of a Phenotypic High-Content Imaging Assay for Assessing the Antiviral Activity of Small-Molecule Inhibitors Targeting Zika Virus.

Zika virus (ZIKV) has been linked to the development of microcephaly in newborns, as well as Guillain-Barré syndrome. There are currently no drugs available to treat ZIKV infection, and accordingly, there is an unmet medical need for the discovery of new therapies. High-throughput drug screening efforts focusing on indirect readouts of cell viability are prone to a higher frequency of false positives in cases where the virus is viable in the cell but the cytopathic effect (CPE) is reduced or delayed. Here, we describe a fast and label-free phenotypic high-content imaging assay to detect cells affected by the virus-induced CPE using automated imaging and analysis. Protection from the CPE correlates with a decrease in viral antigen production, as observed by immunofluorescence. We trained our assay using a collection of nucleoside analogues with activity against ZIKV; the previously reported antiviral activities of 2'-C-methylribonucleosides and ribavirin against the Zika virus in Vero cells were confirmed using our developed method. To validate the ability of our assay to reveal new anti-ZIKV compounds, we profiled a novel library of 24 natural product derivatives and found compound 1 to be an inhibitor of the ZIKV-induced cytopathic effect; the activity of the compound was confirmed in human fetal neural stem cells (NSCs). The described technique can be easily leveraged as a primary screening assay for profiling of the activities of large compound libraries against ZIKV and can be expanded to other ZIKV strains and other cell lines displaying morphological changes upon ZIKV infection.