SARS-CoV-2 NSP2 as a Potential Delivery Vehicle for Proteins.

The development of biomolecule delivery systems is essential for the treatment of various diseases such as cancer, immunological diseases, and metabolic disorders. For the first time, we found that SARS-CoV-2-encoded nonstructural protein 2 (NSP2) can be secreted from the cells, where it is synthesized. Brefeldin A and H89, inhibitors of ER/Golgi secretion pathways, did not inhibit NSP2 secretion. NSP2 is likely secreted via an unconventional secretory pathway. Moreover, both secreted and purified NSP2 proteins were able to traverse the plasma membrane barrier and enter both immortalized human umbilical vein endothelial cells and tumor cell lines. After entry, the NSP2 protein was localized in only the cytoplasm. Cytochalasin D, a potent inhibitor of actin polymerization, inhibited the entry of NSP2. NSP2 can carry other molecules into cells. Burkholderia lethal factor 1, a monomeric toxin from the intracellular pathogen Burkholderia pseudomallei, has demonstrated antitumor activity by targeting host eukaryotic initiation translation factor 4A. An NSP2-BLF1 fusion protein was translocated across the cellular membranes of Huh7 cells and mediated cell killing. By using different approaches, including protein purification, chemical inhibition, and cell imaging, we confirm that NSP2 is able to deliver heterologous proteins into cells. NSP2 can act as a potential delivery vehicle for proteins.

Human FcRn Is a Two-in-One Attachment-Uncoating Receptor for Echovirus 18.

Virus-receptor interactions determine viral host range and tissue tropism. CD55 and human neonatal Fc receptor (FcRn) were found to be the binding and uncoating receptors for some of the echovirus-related enterovirus species B serotypes in our previous study. Echovirus 18 (E18), as a member of enterovirus species B, is a significant causative agent of aseptic meningitis and viral encephalitis in children. However, it does not use CD55 as a critical host factor. We conducted CRISPR/Cas9 knockout screening to determine the receptors and entry mechanisms and identified FcRn working as a dual-function receptor for E18. Knockout of FCGRT and B2M, which encode the two subunits of FcRn, prevented infection by E18 and other echoviruses in the same physiological cluster. We then elucidated the underlying molecular mechanism of receptor recognition by E18 using cryogenic electron microscopy. The binding of the FCGRT subunit to the canyon region rotates the residues around the pocket, triggering the release of the pocket factor as observed for other enterovirus species B members. IMPORTANCE E18 is a member of enterovirus species B. As one of the most common enterovirus serotypes in nonpolio enterovirus detection, it easily infects children and causes various clinical symptoms. Aseptic meningitis and viral encephalitis are the most commonly reported syndromes associated with E18. No effective antiviral drugs or approved vaccines are available. Previous studies showed that CD55 and FcRn were the binding and uncoating receptors for some echoviruses. However, we found that CD55 is not the critical host factor for E18. Thus, we want to determine the receptors and elucidate the entry mechanism of E18. Our findings reveal that FcRn is a two-in-one attachment-uncoating receptor for E18.

TRIM26 is a critical host factor for HCV replication and contributes to host tropism.

Hepatitis C virus (HCV) remains a major human pathogen that requires better understanding of virus-host interactions. In this study, we performed a genome-wide CRISPR-Cas9 screening and identified TRIM26, an E3 ligase, as a critical HCV host factor. Deficiency of TRIM26 specifically impairs HCV genome replication. Mechanistic studies showed that TRIM26 interacts with HCV-encoded NS5B protein and mediates its K27-linked ubiquitination at residue K51, and thus promotes the NS5B-NS5A interaction. Moreover, mouse TRIM26 does not support HCV replication because of its unique six-amino acid insert that prevents its interaction with NS5B. Ectopic expression of human TRIM26 in a mouse hepatoma cell line that has been reconstituted with other essential HCV host factors promotes HCV infection. In conclusion, we identified TRIM26 as a host factor for HCV replication and a new determinant of host tropism. These results shed light on HCV-host interactions and may facilitate the development of an HCV animal model.

Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B.

Enterovirus B (EV-B), a major proportion of the genus Enterovirus in the family Picornaviridae, is the causative agent of severe human infectious diseases. Although cellular receptors for coxsackievirus B in EV-B have been identified, receptors mediating virus entry, especially the uncoating process of echovirus and other EV-B remain obscure. Here, we found that human neonatal Fc receptor (FcRn) is the uncoating receptor for major EV-B. FcRn binds to the virus particles in the "canyon" through its FCGRT subunit. By obtaining multiple cryo-electron microscopy structures at different stages of virus entry at atomic or near-atomic resolution, we deciphered the underlying mechanisms of enterovirus attachment and uncoating. These structures revealed that different from the attachment receptor CD55, binding of FcRn to the virions induces efficient release of "pocket factor" under acidic conditions and initiates the conformational changes in viral particle, providing a structural basis for understanding the mechanisms of enterovirus entry.

A computational study of the dual effect of intermittent and continuous administration of parathyroid hormone on bone remodeling.

Bone remodeling is a process known to be governed by constant interactions between osteoblast and osteoclast through complex pathway networks mediated by signaling factors. Experimental studies show that intermittent and continuous administration of PTH/PTHrP led to opposite outcomes in terms of bone mass. To investigate this dual effect of PTH/PTHrP, we develop a computational model based on a simplified signaling pathway network which includes relevant molecular effectors and cells. Multiple ordinary differential equations linking all considered components in the signaling pathway network through reaction kinetics are solved with dose values and patterns of injection from experiments as input. Modeling results show good agreement with experimental observations in that continuous injection of PTH/PTHrP generates catabolic effect on bone mass while intermittent injection yields anabolic effect. The signaling factors governing the interaction between osteoblast and osteoclast indeed play a key role in the dual effect of PTH/PTHrP. Furthermore, there appears to be an optimal interval for intermittent injection of PTH/PTHrP for yielding the most bone regeneration, and a synergistic outcome could be achieved by combining intermittent injection of PTH/PTHrP with application of a treatment (to mimic the filling of bone defects with polymeric scaffolds). This modeling work sheds valuable insights into the influence of temporal control of PTH/PTHrP on bone mass and presents a possible path toward bridging bioengineering approaches with clinical treatment strategies. STATEMENT OF SIGNIFICANCE: A computational model considering simplified signaling pathways containing crucial components of PTH, PTHrP, osteoblast precursor, osteoblast, osteoclast precursor, osteoclast, RANKL and IL-6 family cytokoines has been developed to study the dual effect of PTH/PTHrP on bone metabolism. The model takes the dose values and patterns of injection from experiments as input and yields predictions that convincingly match experimental measurements. This work highlights the importance of providing an optimal hormone treatment strategy for maintaining healthy bone metabolism. Moreover, the integrative approach of relying on experimental observations to find reasonable values for relevant modeling parameters has been proven to be powerful in advancing our understanding of biological interactions among cells and signaling factors.

Elucidating the Mechanisms of Two Unique Phenomena Governed by Particle-Particle Interaction under DEP: Tumbling Motion of Pearl Chains and Alignment of Ellipsoidal Particles.

Particle-particle interaction plays a crucial role in determining the movement and alignment of particles under dielectrophoresis (DEP). Previous research efforts focus on studying the mechanism governing the alignment of spherical particles with similar sizes in a static condition. Different approaches have been developed to simulate the alignment process of a given number of particles from several up to thousands depending on the applicability of the approaches. However, restricted by the simplification of electric field distribution and use of identical spherical particles, not much new understanding has been gained apart from the most common phenomenon of pearl chain formation. To enhance the understanding of particle-particle interaction, the movement of pearl chains under DEP in a flow condition was studied and a new type of tumbling motion with unknown mechanism was observed. For interactions among non-spherical particles, some preceding works have been done to simulate the alignment of ellipsoidal particles. Yet the modeling results do not match experimental observations. In this paper, the authors applied the newly developed volumetric polarization and integration (VPI) method to elucidate the underlying mechanism for the newly observed movement of pearl chains under DEP in a flow condition and explain the alignment patterns of ellipsoidal particles. The modeling results show satisfactory agreement with experimental observations, which proves the strength of the VPI method in explaining complicated DEP phenomena.

Electrocarving during Electrodeposition Growth.

Shape- and size-controlled synthesis of micro/nanostructures is of fundamental importance in many applications of physics and chemistry. Wet chemical growth methods have achieved shape- and size-controlled synthesis of colloidal nanocrystals of various compositions. Compared with wet chemical methods, electrochemical deposition (ECD) yields micro/nanostructures affixed to a substrate, but the resulting structures are poorly controlled. Herein, the controllable electrochemical fabrication of well-defined silver-oxide clathrate micro/nanostructures is realized by intentionally manipulating the previously neglected electrocarving process during electrodeposition growth (MEDEG). Most importantly, the dominance of the electrocarving and the electrodeposition growth process can be immediately manipulated by varying the deposition voltage and/or the composition of the electrolyte. Unique delta-wing-, arrowhead-, and butterfly-like silver-oxide clathrate structures are created using the MEDEG method. MEDEG complements the capability of ECD for controllable synthesis of micro/nanostructures of various materials directly on a substrate. The study details the mechanisms that may enable MEDEG to become a competitive alternative to traditional wet chemical methods in the controllable synthesis of micro/nanostructures. This understanding of MEDEG should motivate applications in fields which demand well-defined micro/nanostructures affixed to a substrate.

Elucidating the mechanism governing cell rotation under DEP using the volumetric polarization and integration method.

Cell rotation can be achieved by utilizing rotating electric fields through which torques are generated due to phase difference between the dipole moment of cells and the external electric field. While reports of cell rotation under non-rotating electrical fields, such as dielectrophoresis (DEP), are abound, the underlying mechanism is not fully understood. Because of this, contradicting arguments remain regarding if a single cell can rotate under conventional DEP. What's more, the current prevailing DEP theory is not adequate for identifying the cause for such disagreements. In this work we applied our recently developed Volumetric Polarization and Integration (VPI) method to investigate the possible causes for cell rotation under conventional DEP. Three-dimensional (3D) computer models dealing with a cell in a DEP environment were developed to quantify the force and torque imparted on the cell by the external DEP field using COMSOL Multiphysics software. Modeling results suggest that eccentric inclusions with low conductivity inside the cell will generate torques (either in clockwise or counter-clockwise directions) sufficient to cause cell rotation under DEP. For validation of modeling predictions, experiments with rat adipose stem cells containing large lipid droplets were conducted. Good agreement between our modeling and experimental results suggests that the VPI method is powerful in elucidating the underlying mechanisms governing the complicated DEP phenomena.

Mussel-inspired fabrication of porous anodic alumina nanochannels and a graphene oxide interfacial ionic rectification device.

A mussel-inspired new interfacial ionic rectification device is fabricated using porous anodic alumina nanochannels and graphene oxide via dopamine polymerization. In addition, the rectification mechanism is elucidated based on the principles of physics and thermodynamics via finite element simulation and the results show that the interface distance and surface charge play a vital role in ion transport.

Kaposi Sarcoma Herpesvirus (KSHV) Latency-Associated Nuclear Antigen (LANA) recruits components of the MRN (Mre11-Rad50-NBS1) repair complex to modulate an innate immune signaling pathway and viral latency.

Kaposi Sarcoma Herpesvirus (KSHV), a γ2-herpesvirus and class 1 carcinogen, is responsible for at least three human malignancies: Kaposi Sarcoma (KS), Primary Effusion Lymphoma (PEL) and Multicentric Castleman's Disease (MCD). Its major nuclear latency protein, LANA, is indispensable for the maintenance and replication of latent viral DNA in infected cells. Although LANA is mainly a nuclear protein, cytoplasmic isoforms of LANA exist and can act as antagonists of the cytoplasmic DNA sensor, cGAS. Here, we show that cytosolic LANA also recruits members of the MRN (Mre11-Rad50-NBS1) repair complex in the cytosol and thereby inhibits their recently reported role in the sensing of cytoplasmic DNA and activation of the NF-κB pathway. Inhibition of NF-κB activation by cytoplasmic LANA is accompanied by increased lytic replication in KSHV-infected cells, suggesting that MRN-dependent NF-κB activation contributes to KSHV latency. Cytoplasmic LANA may therefore support the activation of KSHV lytic replication in part by counteracting the activation of NF-κB in response to cytoplasmic DNA. This would complement the recently described role of cytoplasmic LANA in blocking an interferon response triggered by cGAS and thereby promoting lytic reactivation. Our findings highlight a second point at which cytoplasmic LANA interferes with the innate immune response, as well as the importance of the recently discovered role of cytoplasmic MRN complex members as innate sensors of cytoplasmic DNA for the control of KSHV replication.

Elucidating the DEP phenomena using a volumetric polarization approach with consideration of the electric double layer.

Dielectrophoretic (DEP) phenomena have been explored to great success for various applications like particle sorting and separation. To elucidate the underlying mechanism and quantify the DEP force experienced by particles, the point-dipole and Maxwell Stress Tensor (MST) methods are commonly used. However, both methods exhibit their own limitations. For example, the point-dipole method is unable to fully capture the essence of particle-particle interactions and the MST method is not suitable for particles of non-homogeneous property. Moreover, both methods fare poorly when it comes to explaining DEP phenomena such as the dependence of crossover frequency on medium conductivity. To address these limitations, the authors have developed a new method, termed volumetric-integration method, with the aid of computational implementation, to reexamine the DEP phenomena, elucidate the governing mechanism, and quantify the DEP force. The effect of an electric double layer (EDL) on particles' crossover behavior is dealt with through consideration of the EDL structure along with surface ionic/molecular adsorption, unlike in other methods, where the EDL is accounted for through simply assigning a surface conductance value to the particles. For validation, by comparing with literature experimental data, the authors show that the new method can quantify the DEP force on not only homogeneous particles but also non-homogeneous ones, and predict particle-particle interactions fairly accurately. Moreover, the authors also show that the predicted dependence of crossover frequency on medium conductivity and particle size agrees very well with experimental measurements.

Seeking Convergence to advance Biomaterials Science and Translation by Chinese Association for Biomaterials.

The Chinese Association for Biomaterials (CAB) organized a CAB Day Symposium with a theme of "Seeking Convergence to Advance Biomaterials Science and Translation."Image 1.

A Capacitance-Based Sensor for Hemodialysis Cannulation Training: A Proof-of-Concept Study.

In this work, we present a novel capacitance-based sensor for teaching hemodialysis cannulation skills. Initial results from a pilot experiment suggest that data from the sensor could be used to indicate puncture events in tissue-like materials.

Cytoplasmic isoforms of Kaposi sarcoma herpesvirus LANA recruit and antagonize the innate immune DNA sensor cGAS.

The latency-associated nuclear antigen (LANA) of Kaposi sarcoma herpesvirus (KSHV) is mainly localized and functions in the nucleus of latently infected cells, playing a pivotal role in the replication and maintenance of latent viral episomal DNA. In addition, N-terminally truncated cytoplasmic isoforms of LANA, resulting from internal translation initiation, have been reported, but their function is unknown. Using coimmunoprecipitation and MS, we found the cGMP-AMP synthase (cGAS), an innate immune DNA sensor, to be a cellular interaction partner of cytoplasmic LANA isoforms. By directly binding to cGAS, LANA, and particularly, a cytoplasmic isoform, inhibit the cGAS-STING-dependent phosphorylation of TBK1 and IRF3 and thereby antagonize the cGAS-mediated restriction of KSHV lytic replication. We hypothesize that cytoplasmic forms of LANA, whose expression increases during lytic replication, inhibit cGAS to promote the reactivation of the KSHV from latency. This observation points to a novel function of the cytoplasmic isoforms of LANA during lytic replication and extends the function of LANA from its role during latency to the lytic replication cycle.

Detecting and identifying small molecules in a nanopore flux capacitor.

A new method of molecular detection in a metallic-semiconductor nanopore was developed and evaluated with experimental and computational methods. Measurements were made of the charging potential of the electrical double layer (EDL) capacitance as charge-carrying small molecules translocated the nanopore. Signals in the charging potential were found to be correlated to the physical properties of analyte molecules. From the measured signals, we were able to distinguish molecules with different valence charge or similar valence charge but different size. The relative magnitude of the signals from different analytes was consistent over a wide range of experimental conditions, suggesting that the detected signals are likely due to single molecules. Computational modeling of the nanopore system indicated that the double layer potential signal may be described in terms of disruption of the EDL structure due to the size and charge of the analyte molecule, in agreement with Huckel and Debye's analysis of the electrical atmosphere of electrolyte solutions.

Active current gating in electrically biased conical nanopores.

We observed that the ionic current through a gold/silicon nitride (Si3N4) nanopore could be modulated and gated by electrically biasing the gold layer. Rather than employing chemical modification to alter device behavior, we achieved control of conductance directly by electrically biasing the gold portion of the nanopore. By stepping through a range of bias potentials under a constant trans-pore electric field, we observed a gating phenomenon in the trans-pore current response in a variety of solutions including potassium chloride (KCl), sodium chloride (NaCl), and potassium iodide (KI). A computational model with a conical nanopore was developed to examine the effect of the Gouy-Chapman-Stern electrical double layer along with nanopore geometry, work function potentials, and applied electrical bias on the ionic current. The numerical results indicated that the observed modulation and gating behavior was due to dynamic reorganization of the electrical double layer in response to changes in the electrical bias. Specifically, in the conducting state, the nanopore conductance (both numerical and experimental) is linearly proportional to the applied bias due to accumulation of charge in the diffuse layer. The gating effect occurs due to the asymmetric charge distribution in the fluid induced by the distribution of potentials at the nanopore surface. Time dependent changes in current due to restructuring of the electrical double layer occur when the electrostatic bias is instantaneously changed. The nanopore device demonstrates direct external control over nanopore behavior via modulation of the electrical double layer by electrostatic biasing.

Hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p inhibitors can reduce the cytotoxicity of Ebola virus glycoprotein in vitro.

Ebola virus (EBOV) causes a highly lethal hemorrhagic fever syndrome in humans and has been associated with mortality rates of up to 91% in Zaire, the most lethal strain. Though the viral envelope glycoprotein (GP) mediates widespread inflammation and cellular damage, these changes have mainly focused on alterations at the protein level, the role of microRNAs (miRNAs) in the molecular pathogenesis underlying this lethal disease is not fully understood. Here, we report that the mi-RNAs hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p were induced in human umbilical vein endothelial cells (HUVECs) following expression of EBOV GP. Among the proteins encoded by predicted targets of these miRNAs, the adhesion-related molecules tissue factor pathway inhibitor (TFPI), dystroglycan1 (DAG1) and the caspase 8 and FADD-like apoptosis regulator (CFLAR) were significantly downregulated in EBOV GP-expressing HUVECs. Moreover, inhibition of hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p, or overexpression of TFPI, DAG1 and CFLAR rescued the cell viability that was induced by EBOV GP. Our results provide a novel molecular basis for EBOV pathogenesis and may contribute to the development of strategies to protect against future EBOV pandemics.

The effects of the electrical double layer on giant ionic currents through single-walled carbon nanotubes.

We developed a computational model for investigating the cause for the high ionic current through a single-walled carbon nanotube nanofluidic device by considering the electrical double layer at a solid-liquid interface. With this model, we were able to examine the influence of the Gouy-Chapman-Stern electrical double layer and the solution concentration on the ionic conductance in the device. Results showed that the conductance-concentration relationship predicted from our model agreed well with experimental observation. Moreover, our model showed that the compact layer thickness increased with the increase of the bulk solution concentration, reducing the internal volume of the nanotube channel available for fluid transport. Fluid within the channel had an enhanced concentration and a net charge which increased the electroosmotic and electrophoretic transport properties of the device, increasing the total ionic conductance of the system.

Inhibition of SARS pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans.

It has been reported that lactoferrin (LF) participates in the host immune response against Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) invasion by enhancing NK cell activity and stimulating neutrophil aggregation and adhesion. We further investigated the role of LF in the entry of SARS pseudovirus into HEK293E/ACE2-Myc cells. Our results reveal that LF inhibits SARS pseudovirus infection in a dose-dependent manner. Further analysis suggested that LF was able to block the binding of spike protein to host cells at 4°C, indicating that LF exerted its inhibitory function at the viral attachment stage. However, LF did not disrupt the interaction of spike protein with angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV. Previous studies have shown that LF colocalizes with the widely distributed cell-surface heparan sulfate proteoglycans (HSPGs). Our experiments have also confirmed this conclusion. Treatment of the cells with heparinase or exogenous heparin prevented binding of spike protein to host cells and inhibited SARS pseudovirus infection, demonstrating that HSPGs provide the binding sites for SARS-CoV invasion at the early attachment phase. Taken together, our results suggest that, in addition to ACE2, HSPGs are essential cell-surface molecules involved in SARS-CoV cell entry. LF may play a protective role in host defense against SARS-CoV infection through binding to HSPGs and blocking the preliminary interaction between SARS-CoV and host cells. Our findings may provide further understanding of SARS-CoV pathogenesis and aid in treatment of this deadly disease.