SARS-CoV-2 nucleocapsid protein, rather than spike protein, triggers a cytokine storm originating from lung epithelial cells in patients with COVID-19.

PURPOSE: The aim of this study was to elucidate the factors associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that may initiate cytokine cascades and correlate the clinical characteristics of patients with coronavirus disease 2019 (COVID-19) with their serum cytokine profiles. METHODS: Recombinant baculoviruses displaying SARS-CoV-2 spike or nucleocapsid protein were constructed and transfected into A549 cells and THP-1-derived macrophages, to determine which protein initiate cytokine release. SARS-CoV-2-specific antibody titers and cytokine profiles of patients with COVID-19 were determined, and the results were associated with their clinical characteristics, such as development of pneumonia or length of hospital stay. RESULTS: The SARS-CoV-2 nucleocapsid protein, rather than the spike protein, triggers lung epithelial A549 cells to express IP-10, RANTES, IL-16, MIP-1α, basic FGF, eotaxin, IL-15, PDGF-BB, TRAIL, VEGF-A, and IL-5. Additionally, serum CTACK, basic FGF, GRO-α, IL-1α, IL-1RA, IL-2Rα, IL-9, IL-15, IL-16, IL-18, IP-10, M-CSF, MIF, MIG, RANTES, SCGF-ß, SDF-1α, TNF-α, TNF-ß, VEGF, PDGF-BB, TRAIL, ß-NGF, eotaxin, GM-CSF, IFN-α2, INF-γ, and MCP-1 levels were considerably increased in patients with COVID-19. Among them, patients with pneumonia had higher serum IP-10 and M-CSF levels than patients without. Patients requiring less than 3 weeks to show negative COVID-19 tests after contracting COVID-19 had higher serum IP-10 levels than the remaining patients. CONCLUSION: Our study revealed that nucleocapsid protein, lung epithelial cells, and IP-10 may be potential targets for the development of new strategies to prevent, or control, severe COVID-19.

Balloon Eustachian Tuboplasty Combined or Not with Myringotomy in Eustachian Tube Dysfunction.

BACKGROUND: Eustachian tube dysfunction (ETD) is a common disorder causing ear pressure, pain, and hearing loss. Balloon Eustachian tuboplasty (BET) is an emerging technique for dilating the Eustachian tube and treating ETD. Whether adding myringotomy improves BET efficacy is controversial. METHODS: This retrospective study included 95 ETD patients undergoing BET alone (n = 44) or BET with myringotomy (BET + M; n = 51) between June 2020 and August 2021 at a single medical center. The primary outcome was the change in ETDQ-7 symptom scores from baseline to 6 months after treatment. Secondary outcomes included audiometry, endoscopy, Valsalva maneuver, and complications. RESULTS: The ETDQ-7 scores improved significantly after treatment in both groups (p < 0.001), without significant between-group differences (p = 0.417). No significant differences occurred in the audiometry, endoscopy, and Valsalva results or in most complications between groups. One BET + M patient had a persistent tympanic membrane perforation. CONCLUSIONS: Both BET alone and BET + M effectively and safely improved the subjective and objective ETD outcomes. However, adding myringotomy did not further improve the outcomes over BET alone, while it incurred risks such as persistent perforation. BET alone may sufficiently treat ETD without requiring myringotomy in this cohort. Further randomized controlled trials should identify optimal candidates for BET alone versus combined approaches.

A CSF-1R-blocking antibody/IL-10 fusion protein increases anti-tumor immunity by effectuating tumor-resident CD8+ T cells.

Strategies to increase intratumoral concentrations of an anticancer agent are desirable to optimize its therapeutic potential when said agent is efficacious primarily within a tumor but also have significant systemic side effects. Here, we generate a bifunctional protein by fusing interleukin-10 (IL-10) to a colony-stimulating factor-1 receptor (CSF-1R)-blocking antibody. The fusion protein demonstrates significant antitumor activity in multiple cancer models, especially head and neck cancer. Moreover, this bifunctional protein not only leads to the anticipated reduction in tumor-associated macrophages but also triggers proliferation, activation, and metabolic reprogramming of CD8+ T cells. Furthermore, it extends the clonotype diversity of tumor-infiltrated T cells and shifts the tumor microenvironment (TME) to an immune-active state. This study suggests an efficient strategy for designing immunotherapeutic agents by fusing a potent immunostimulatory molecule to an antibody targeting TME-enriched factors.

Ferroptosis Signature Shapes the Immune Profiles to Enhance the Response to Immune Checkpoint Inhibitors in Head and Neck Cancer.

As a type of immunogenic cell death, ferroptosis participates in the creation of immunoactive tumor microenvironments. However, knowledge of spatial location of tumor cells with ferroptosis signature in tumor environments and the role of ferroptotic stress in inducing the expression of immune-related molecules in cancer cells is limited. Here the spatial association of the transcriptomic signatures is demonstrated for ferroptosis and inflammation/immune activation located in the invasive front of head and neck squamous cell carcinoma (HNSCC). The association between ferroptosis signature and inflammation/immune activation is more prominent in HPV-negative HNSCC compared to HPV-positive ones. Ferroptotic stress induces PD-L1 expression through reactive oxygen species (ROS)-elicited NF-κB signaling pathway and calcium influx. Priming murine HNSCC with the ferroptosis inducer sensitizes tumors to anti-PD-L1 antibody treatment. A positive correlation between the ferroptosis signature and the active immune cell profile is shown in the HNSCC samples. This study reveals a subgroup of ferroptotic HNSCC with immune-active signatures and indicates the potential of priming HNSCC with ferroptosis inducers to increase the antitumor efficacy of immune checkpoint inhibitors.

Loss of cell-cell adhesion triggers cell migration through Rac1-dependent ROS generation.

Epithelial cells usually trigger their "migratory machinery" upon loss of adhesion to their neighbors. This default is important for both physiological (e.g., wound healing) and pathological (e.g., tumor metastasis) processes. However, the underlying mechanism for such a default remains unclear. In this study, we used the human head and neck squamous cell carcinoma (HNSCC) SAS cells as a model and found that loss of cell-cell adhesion induced reactive oxygen species (ROS) generation and vimentin expression, both of which were required for SAS cell migration upon loss of cell-cell adhesion. We demonstrated that Tiam1-mediated Rac1 activation was responsible for the ROS generation through NADPH-dependent oxidases. Moreover, the ROS-Src-STAT3 signaling pathway that led to vimentin expression was important for SAS cell migration. The activation of ROS, Src, and STAT3 was also detected in tumor biopsies from HNSCC patients. Notably, activated STAT3 was more abundant at the tumor invasive front and correlated with metastatic progression of HNSCC. Together, our results unveil a mechanism of how cells trigger their migration upon loss of cell-cell adhesion and highlight an important role of the ROS-Src-STAT3 signaling pathway in the progression of HNSCC.

Theory of magnetic-field effect on trions in two-dimensional materials.

In this work, we present a theoretical method to study the effect of magnetic field on trions in two-dimensional materials. The trion is modeled by a three-particle Schrödinger equation and the magnetic-field interaction is included by means of a vector potential in symmetric gauge. By using a coordinate transformation and a unitary transformation, the trion Hamiltonian can be converted into the sum of a translational term describing the Landau quantization for the trion center-of-mass motion, an internal term describing the trion binding, and a translational-internal coupling term depending linearly on the magnetic-field strength. The trion eigenenergy and wavefunction can then be calculated efficiently by using a variational method, and the quantum numbers of trions in magnetic fields can be assigned. The eigenenergies, binding energies, and correlation energies of three trion branches, which correspond to the ground-state trion and two excited-state trions solved from the trion Hamiltonian in zero magnetic field, are studied numerically in finite magnetic fields. The present method is applied to study the magnetic-field dependence of trion energy levels in hole-doped WSe2 monolayers. The binding energies and correlation energies of positive trions in WSe2 are investigated over a range of magnetic fields up to 25 T.

Twisted oxide lateral homostructures with conjunction tunability.

Epitaxial growth is of significant importance over the past decades, given it has been the key process of modern technology for delivering high-quality thin films. For conventional heteroepitaxy, the selection of proper single crystal substrates not only facilitates the integration of different materials but also fulfills interface and strain engineering upon a wide spectrum of functionalities. Nevertheless, the lattice structure, regularity and crystalline orientation are determined once a specific substrate is chosen. Here, we reveal the growth of twisted oxide lateral homostructure with controllable in-plane conjunctions. The twisted lateral homostructures with atomically sharp interfaces can be composed of epitaxial "blocks" with different crystalline orientations, ferroic orders and phases. We further demonstrate that this approach is universal for fabricating various complex systems, in which the unconventional physical properties can be artificially manipulated. Our results establish an efficient pathway towards twisted lateral homostructures, adding additional degrees of freedom to design epitaxial films.

Presence of Delocalized Ti 3d Electrons in Ultrathin Single-Crystal SrTiO3.

Strontium titanate (STO), with a wide spectrum of emergent properties such as ferroelectricity and superconductivity, has received significant attention in the community of strongly correlated materials. In the strain-free STO film grown on the SrRuO3 buffer layer, the existing polar nanoregions can facilitate room-temperature ferroelectricity when the STO film thickness approaches 10 nm. Here we show that around this thickness scale, the freestanding STO films without the influence of a substrate show the tetragonal structure at room temperature, contrasting with the cubic structure seen in bulk form. The spectroscopic measurements reveal the modified Ti-O orbital hybridization that causes the Ti ion to deviate from its nominal 4+ valency (3d0 configuration) with excess delocalized 3d electrons. Additionally, the Ti ion in TiO6 octahedron exhibits an off-center displacement. The inherent symmetry lowering in ultrathin freestanding films offers an alternative way to achieve tunable electronic structures that are of paramount importance for future technological applications.

Sumoylation participates in the regulation of YB-1-mediated mismatch repair deficiency and alkylator tolerance.

Numerous reports indicate that enhanced expression of Y-box binding protein-1 (YB-1) in tumor cells is strongly associated with tumorigenesis, aggressiveness, drug resistance, as well as poor prognosis in several types of cancers, and YB-1 is considered to be an oncogene. The molecular mechanism contributing to the regulation of the biological activities of YB-1 remains obscure. Sumoylation, a post-translational modification involving the covalent conjugation of small ubiquitin-like modifier (SUMO) proteins to a target protein, plays key roles in the modulation of protein functions. In this study, our results revealed that YB-1 is sumoylated and that Lys26 is a critical residue for YB-1 sumoylation. Moreover, YB-1 was found to directly interact with SUMO proteins, and disruption of the SUMO-interacting motif (SIM) of YB-1 not only interfered with this interaction but also diminished YB-1 sumoylation. The subcellular localization, protein stability, and transcriptional regulatory activity of YB-1 were not significantly affected by sumoylation. However, decreased sumoylation disrupted the interaction between YB-1 and PCNA as well as YB-1-mediated inhibition of the MutSα/PCNA interaction and MutSα mismatch binding activity, indicating a functional role of YB-1 sumoylation in inducing DNA mismatch repair (MMR) deficiency and spontaneous mutations. The MMR machinery also recognizes alkylator-modified DNA adducts to signal for cell death. We further demonstrated that YB-1 sumoylation is crucial for the inhibition of SN1-type alkylator MNNG-induced cytotoxicity, G2/M-phase arrest, apoptosis, and the MMR-dependent DNA damage response. Collectively, these results provide molecular explanations for the impact of YB-1 sumoylation on MMR deficiency and alkylator tolerance, which may provide insight for designing therapeutic strategies for malignancies and alkylator-resistant cancers associated with YB-1 overexpression.

Variationally optimized orbital approach to trions in two-dimensional materials.

In this work, trions in two-dimensional (2D) space are studied by the variational method with trial wavefunctions being constructed by 2D slater-type orbitals. Via this method, trion energy levels and wavefunctions can be calculated efficiently with fairly good accuracy. We first apply this method to study trion energy levels in a 2D hydrogen-like system with respect to a wide range of mass ratios and screening lengths. We find that the ground-state trion is bound for the whole parameter range, and an excited-state trion with antisymmetric permutation of electrons with finite angular momentum is bound for large electron-hole mass ratios or long screening lengths. The binding energies of ground-state trions calculated by the present method agree well with those calculated by more sophisticated but computationally demanding methods. We then calculate trion binding energies in various monolayer transition metal dichalcogenides (TMDCs) by using this method with the inclusion of electron-hole exchange (EHX) interaction. For TMDCs, we found that the effect of EHX can be significant in determining the trion binding energy and the possible existence of stable excited-state trions.

Spectroscopic characterization of electronic structures of ultra-thin single crystal La0.7Sr0.3MnO3.

We have successfully fabricated high quality single crystalline La0.7Sr0.3MnO3 (LSMO) film in the freestanding form that can be transferred onto silicon wafer and copper mesh support. Using soft x-ray absorption (XAS) and resonant inelastic x-ray scattering (RIXS) spectroscopy in transmission and reflection geometries, we demonstrate that the x-ray emission from Mn 3s-2p core-to-core transition (3sPFY) seen in the RIXS maps can represent the bulk-like absorption signal with minimal self-absorption effect around the Mn L3-edge. Similar measurements were also performed on a reference LSMO film grown on the SrTiO3 substrate and the agreement between measurements substantiates the claim that the bulk electronic structures can be preserved even after the freestanding treatment process. The 3sPFY spectrum obtained from analyzing the RIXS maps offers a powerful way to probe the bulk electronic structures in thin films and heterostructures when recording the XAS spectra in the transmission mode is not available.

A Fast Route Towards Freestanding Single-Crystalline Oxide Thin Films by Using YBa2Cu3O7-x as a Sacrificial Layer.

Researchers have long been seeking multifunctional materials that can be adopted for next-generation nanoelectronics, and which, hopefully, are compatible with current semiconductor processing for further integration. Along this vein, complex oxides have gained numerous attention due to their versatile functionalities. Despite the fact that unbounded potential of complex oxides has been examined over the past years, one of the major challenges lies in the direct integration of these functional oxides onto existing devices or targeted substrates that are inherently incompatible in terms of oxide growth. To fulfill this goal, freestanding processes have been proposed, in which wet etching of inserted sacrificial layers is regarded as one of the most efficient ways to obtain epitaxial high-quality thin films. In this study, we propose using an alternative oxide, YBa2Cu3O7 (YCBO), as a sacrificial layer, which can be easily dissolved in light hydrochloric acid in a more efficient way, while protecting selected complex oxides intact. The high epitaxial quality of the selected complex oxide before and after freestanding process using YBCO as a sacrificial layer is comprehensively studied via a combination of atomic force microscopy, X-ray diffraction, transmission electron microscopy, and electrical transports. This approach enables direct integration of complex oxides with arbitrary substrates and devices and is expected to offer a faster route towards the development of low-dimensional quantum materials.

Polysaccharide-containing fraction from Artemisia argyi inhibits tumor cell-induced platelet aggregation by blocking interaction of podoplanin with C-type lectin-like receptor 2.

Tumor cell-induced platelet aggregation (TCIPA) is a mechanism that involves the protection of tumor cells in the circulation and the promotion of tumor cell invasion and metastases. The C-type lectin-like receptor 2 (CLEC-2) that binds podoplanin (PDPN) is on the platelet surface and facilitates the TCIPA. Selective blockage of the PDPN-mediated platelet-tumor cell interaction is thereby a plausible strategy for inhibiting metastases. In a search for antagonists of PDPN- and tumor cell-induced platelet aggregation, traditional Chinese medicines were screened and it was found that the water extract of Artemisia argyi leaves selectively inhibited the PDPN-induced platelet aggregation. Bioactivity-guided fractionation analysis was performed for defining a polysaccharide-containing fraction (AAWAP) characterized by inhibition of PDPN activity and tumor cell-induced platelet aggregation. The pharmacological effects of AAWAP on PDPN-activated CLEC-2 signaling were determined by using Western blot and alpha screening analyses. AAWAP was non-toxic to the cells and platelets and it suppressed PDPN- and tumor cell-induced platelet aggregation by irreversibly blocking the interaction between PDPN and CLEC-2 in a dose-dependent manner. These findings indicate that AAWAP is an antagonist of the PDPN-CLEC-2 interaction. This action by AAWAP may result in the prevention of tumor cell metastases, and if so, could become an effective pharmacological agent in treating cancer patients.

Multiview Contouring for Breast Tumor on Magnetic Resonance Imaging.

The shape and contour of the lesion are shown to be effective features for physicians to identify breast tumor as benign or malignant. The region of the lesion is usually manually created by the physician according to their clinical experience; therefore, contouring tumors on breast magnetic resonance imaging (MRI) is difficult and time-consuming. For this purpose, an automatic contouring method for breast tumors was developed for less burden in the analysis and to decrease the observed bias to help in making decisions clinically. In this study, a multiview segmentation method for detecting and contouring breast tumors in MRI was represented. The preprocessing of the proposed method reduces any amount of noises but preserves the shape and contrast of the breast tumor. The two-dimensional (2D) level-set segmentation method extracts contours of breast tumors from the transverse, coronal, and sagittal planes. The obtained contours are further utilized to generate appropriate three-dimensional (3D) contours. Twenty breast tumor cases were evaluated and the simulation results show that the proposed contouring method was an efficient method for delineating 3D contours of breast tumors in MRI.

Elucidation of the TMab-6 Monoclonal Antibody Epitope Against Telomerase Reverse Transcriptase.

Telomerase reverse transcriptase (TERT) and mutations of the TERT promoter are significant in the pathogenesis of 1p/19q-codeleted oligodendrogliomas and isocitrate dehydrogenase gene wild-type glioblastomas, as well as melanomas and squamous cell carcinomas. We previously developed an antihuman TERT monoclonal antibody (mAb), TMab-6, which is applicable in immunohistochemistry for human tissues. However, the binding epitope of TMab-6 against TERT is yet to be elucidated. In this study, enzyme-linked immunosorbent assay and immunohistochemistry were utilized for investigating the epitope of TMab-6. The findings revealed that the critical epitope of TMab-6 is the TERT sequence PSTSRPPRPWD; Thr310 and Ser311 of TERT are especially significant amino acids for TMab-6 recognition.

Ultrafast Giant Photostriction of Epitaxial Strontium Iridate Film with Superior Endurance.

Photostriction, optical stimulus driven mechanical deformation in materials, provides a solution toward next-generation technology. Here, the giant photostriction (∼2% change of lattice) of epitaxial strontium iridate (SrIrO3) films under illumination at room temperature is revealed via power-dependent Raman scattering, which is significantly larger as compared to conventional inorganic materials. The time scale and mechanism of this giant photostriction in SrIrO3 are further studied through time-resolved transient reflectivity measurements. The main mechanism is determined to be the electron-phonon coupling. In addition, we find that such an exotic behavior happens within few picoseconds and remains up to 107 cyclic on/off operations. The observation of giant photostriction in SrIrO3 films with superior endurance promises the advance of shape responsive solids that are sensitive to environmental stimuli, which could be widely utilized for multifunctional optoelectronics and optomechanical devices.

Anti-podocalyxin antibody exerts antitumor effects via antibody-dependent cellular cytotoxicity in mouse xenograft models of oral squamous cell carcinoma.

Podocalyxin (PODXL) overexpression is associated with progression, metastasis, and poor outcomes in cancers. We recently produced the novel anti-PODXL monoclonal antibody (mAb) PcMab-47 (IgG1, kappa). Herein, we engineered PcMab-47 into 47-mG2a, a mouse IgG2a-type mAb, to add antibody-dependent cellular cytotoxicity (ADCC). We further developed 47-mG2a-f, a core fucose-deficient type of 47-mG2a to augment its ADCC. Immunohistochemical analysis of oral cancer tissues using PcMab-47 and 47-mG2a revealed that the latter stained oral squamous cell carcinoma (OSCC) cells in a cytoplasmic pattern at a much lower concentration. PcMab-47 and 47-mG2a detected PODXL in 163/201 (81.1%) and in 197/201 (98.0%) OSCC samples, respectively. 47-mG2a-f also detected PODXL in OSCCs at a similar frequency as 47-mG2a. In vitro analysis revealed that both 47-mG2a and 47-mG2a-f exhibited strong complement-dependent cytotoxicity (CDC) against CHO/hPODXL cells. In contrast, 47-mG2a-f exhibited much stronger ADCC than 47-mG2a against OSCC cells, indicating that ADCC and CDC of those anti-PODXL mAbs depend on target cells. In vivo analysis revealed that both 47-mG2a and 47-mG2a-f exerted antitumor activity in CHO/hPODXL xenograft models at a dose of 100 µg or 500 µg/mouse/week administered twice. 47-mG2a-f, but not 47-mG2a, exerted antitumor activity in SAS and HSC-2 xenograft models at a dose of 100 µg/mouse/week administered three times. Although both 47-mG2a and 47-mG2a-f exerted antitumor activity in HSC-2 xenograft models at a dose of 500 µg/mouse/week administered twice, 47-mG2a-f also showed higher antitumor activity than 47-mG2a. These results suggested that a core fucose-deficient anti-PODXL mAb could be useful for antibody-based therapy against PODXL-expressing OSCCs.

Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134.

The epidermal growth factor receptor (EGFR) is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb), which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7%) to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375-394 amino acids of EGFR) neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377-RGDSFTHTPP-386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments.

Epitope Mapping of Monoclonal Antibody PMab-48 Against Dog Podoplanin.

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed on normal renal podocytes, pulmonary type I alveolar cells, and lymphatic endothelial cells. Increased expression of PDPN in cancers is associated with poor prognosis and hematogenous metastasis through interactions with C-type lectin-like receptor 2 (CLEC-2) on platelets. We previously reported a novel PMab-48 antibody, which is an anti-dog PDPN (dPDPN) monoclonal antibody (mAb) recognizing PDPN expressed in lymphatic endothelial cells. However, the binding epitope of PMab-48 is yet to be clarified. In this study, an enzyme-linked immunosorbent assay and flow cytometry were used to investigate epitopes of PMab-48. The results revealed that the critical epitope of PMab-48 comprises Asp29, Asp30, Ile31, Ile32, and Pro33 of dPDPN.

Monoclonal Antibody L1Mab-13 Detected Human PD-L1 in Lung Cancers.

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L1Mab-13 (IgG1, kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses. L1Mab-13 specifically reacted hPD-L1 of hPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (human fibroblast) in flow cytometry and Western blot. Furthermore, L1Mab-13 reacted with lung cancer cell lines (EBC-1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L1Mab-13, is very useful for detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.