Characterization and Function of Glycans on the Spike Proteins of SARS-CoV-2 Variants of Concern.

SARS-CoV-2 variants of concern (VOCs) pose a great challenge to viral prevention and treatment owing to spike (S) protein mutations, which enhance their infectivity and capacity for immune evasion. However, whether these S protein mutations affect glycosylation patterns and thereby influence infectivity and immunogenicity remains unclear. In this study, four VOC S proteins-S-Alpha, S-Beta, S-Delta, and S-Omicron-were expressed and purified. Lectin microarrays were performed to characterize their glycosylation patterns. Several glycans were differentially expressed among the four VOC S proteins. Furthermore, the functional examination of glycans differentially expressed on S-Omicron revealed a higher expression of fucose-containing glycans, which modestly increased the binding of S-Omicron to angiotensin converting enzyme 2 (ACE2). A higher abundance of sialic acid and galactose-containing glycan was observed on S-Omicron, which significantly reduced its sensitivity against broad S protein-neutralizing antibodies. These findings contribute to the further understanding of SARS-CoV-2 infection mechanisms and provide novel glycan targets for emerging and future variants of SARS-CoV-2. IMPORTANCE Though glycosylation sites of SARS-CoV-2 S protein remain highly conserved, we confirmed that mutations in the Spike gene affect the S protein glycan expression pattern in different variants. More importantly, we found that glycans were differentially expressed on the S protein of the Omicron variant, enabling different forms of receptor binding and neutralization resistance. This study improves our understanding of SARS-CoV-2 glycomics and glycobiology and provides novel therapeutic and preventive strategies for SARS-CoV-2 VOCs.

Near-Infrared Luciferase Complementation Assay with Enhanced Bioluminescence for Studying Protein-Protein Interactions and Drug Evaluation Under Physiological Conditions.

Identification of protein-protein interactions (PPIs) that occur in various cellular processes helps to reveal their potential molecular mechanisms, and there is still an urgent need to develop the assays to explore PPIs in living subjects. Here, we reported a near-infrared split luciferase complementation assay (SLCA) with enhanced bioluminescence produced by cleaving a luciferase, Akaluc, for exploring and visualizing PPIs in living cells and live mice. Compared with the previously developed and widely used red SLCA based on split firefly luciferase (Fluc-SLCA), the signal intensities for PPI recognition in living cells and live mice of the Akaluc-SLCA increased by ∼3.79-fold and ∼18.06-fold in the measured condition, respectively. Additionally, the interactions between the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cellular RNA processing proteins were identified, and the drug evaluation assays were also performed in living cells using Akaluc-SLCA. This study provides a new tool in the near-infrared region for the identification of PPIs in living cells and in vivo and new information for the understanding and treatment of SARS-CoV-2.

Synthesis of Dual-Emitting CdZnSe/Mn:ZnS Quantum Dots for Sensing the pH Change in Live Cells.

Intracellular pH is an important regulator of cell function, and its subtle changes may greatly affect cell activities and cause diseases. Reliable imaging of intracellular pH remains a huge challenge. Dual-emitting Mn2+-doped quantum dots (QDs) can be directly used as a ratiometric fluorescent probe without further modification, but they displayed low performance in terms of photoluminescence, stability, and intensity ratio regulation. Here, we report intrinsic dual-emitting CdZnSe/Mn:ZnS QDs with high photoluminescence efficiency, good stability, and biocompatibility. The emission intensity ratio was selectively regulated by Mn2+ doping. Because of aggregation-induced quenching of QDs, the exciton emission of CdZnSe/Mn:ZnS QDs (471 nm) was sensitive to pH, while the Mn2+-doped emission (606 nm) was passive to pH, which was probably due to little self-quenching in Mn2+-doped emission caused by weak Mn-Mn coupling interaction. Dual-emitting CdZnSe/Mn:ZnS QDs exhibited excellent pH-responsiveness in the range of pH 4.0 to 12.0 and were used for pH imaging in live HeLa cells. When the pH value of HeLa cells changed from 5.0 to 9.0, the emission changed from red to blue. Furthermore, these dual-emitting CdZnSe/Mn:ZnS QDs can provide a versatile platform for biosensors and biological imaging.

The smallest near-infrared fluorescence complementation system for imaging protein-protein and RNA-protein interactions.

Bimolecular fluorescence complementation (BiFC) and its derivative molecular biosensor systems provide effective tools for visualizing biomolecular interactions. The introduction of red and near-infrared fluorescence emission proteins has expanded the spectrum of signal generating modules, enabling BiFC for in vivo imaging. However, the large size of the signal module of BiFC can hinder the interaction between proteins under investigation. In this study, we constructed the near-infrared BiFC and TriFC systems by splitting miRFP670nano, the smallest cyanobacteriochrome-evolved phytochrome available. The miRFP670nano-BiFC sensor system identified and enabled visualization of protein-protein interactions in living cells and live mice, and afforded a faster maturation rate and higher photostability and cellular stability when compared with those of reported near-infrared BiFC systems. We used the miRFP670nano-BiFC sensor system to identify interactions between the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cellular stress granule proteins in living cells and found that the N protein downregulated the expression level of granule protein G3BP1. With the advantages of small size and long wavelength emission of the signal module, the proposed molecular biosensor system should be suitable for various applications in cell imaging studies.

What happens to the gluteus medius in young and middle-aged patients with hip dysplasia?

BACKGROUND: Much research has focused on quantifying the bony characteristics of patients with developmental dysplasia of the hip (DDH). Far less attention, however, has been paid to muscle abnormalities around the hip such as those in the gluteus medius (GM). METHODS: We retrospectively examined clinical and imaging data, such as the age of onset and computed tomography (CT) findings, in 108 consecutive hips. Subjects for the control group were selected from our radiology database. Two readers independently evaluated the length (LGM), cross-sectional area (CSA), width (WGM), and thickness (TGM) of the GM and arm of GM (AGM) and angle of the GM activation (AOA) and bony parameters including the acetabulum-head index (AHI), lateral central edge angle (LCEA), acetabular index (AI), femoral offset (FO), and height of the rotation centre of femoral head (HCFH) among all cases using the imaging data. RESULTS: The patient group included 108 hips. The AGM, LGM, CSA, and TGM were lower in the DDH patients, while AOA was higher. However, there was no significant difference in the WGM between the two groups. Multiple linear regression analysis showed that AGM and AOA were independent factors affecting LCEA. The following regression equation was used: Y(LCEA) = 5.377 * X1 (AGM) - 0.310 * X2 (AOA) - 11.331. The mechanical characteristics of the GM and many bony parameters were significantly correlated (the AGM and AHI, LCEA, AI, FO, but not HCFH; AOA and AHI, LCEA, AI, but not FO or HCFH). The CSA was positively correlated with only HCFH. The rest were not statistical significance linear correlation. The multivariate regression results showed that the age of onset was positively correlated with AGM (r = 0.467). The regression equation used was Y = 9.0 * X (age of onset) - 11.4. CONCLUSION: We found difference in the morphological and mechanical characteristics of the GM between hips with DDH and hips of normal morphology. Of note, the mechanical characteristics of the GM were influenced by bony parameters in patients with DDH.

Diagnostic value of necrotic lesion boundary in bone collapse of femoral head osteonecrosis.

PURPOSE: Our research developed a novel approach to quantitatively evaluate the boundary of necrotic lesions in osteonecrosis of the femoral head (ONFH) and to explore its diagnostic value in predicting bone collapse of the femoral head. METHODS: A retrospective cross-sectional study was conducted in our institution, and 146 hips (121 cases) identified as ONFH were recruited. The anterior and lateral boundaries of each enrolled subject were measured in standard anteroposterior (AP) view and frog-leg (FL) view of plain radiographic images, the intact rate of which was then calculated and presented as the anteroposterior view intact ratio (APIR) and frog-leg view intact ratio (FLIR), respectively. Univariate and multivariate logistic regression analyses were performed to identify risk factors for collapse. A receiver operating characteristic (ROC) curve analysis was performed to determine the sensitivity, specificity and cutoff value of the APIR and FLIR. A Kaplan-Meier (K-M) analysis was applied to calculate the survival rate of the femoral head, and bone collapse of the femoral head was regarded as the endpoint. RESULTS: Femoral head collapse was observed in 61 hips during the follow-up period. Patients with or without femoral head collapse were categorized into the collapse group and non-collapse group, respectively. The mean follow-up time was 3.7 years (2-9) for the collapse group and 7.7 years (5-20) for the non-collapse group. Univariate and multivariate logistic regression analysis and ROC analysis showed that APIR (< 25.61%) and FLIR (< 24.43%) were significantly associated with femoral head collapse. The K-M survival curves indicated that the overall survival rate of APIR (≥ 25.61%) was 94.8% at 7.5 years and 76.6% at 10 years, while that of FLIR (≥ 24.43%) was 87.3% at 7.5 years and ten years. CONCLUSION: The present study demonstrates that APIR and FLIR are of high diagnostic value in the early and middle stages of ONFH. APIR and FLIR can be used to predict the occurrence of femoral head collapse in patients with JIC classification types B and C1. The measurement of these two parameters in plain radiography images may contribute to the selection of a proper hip preservation strategy.

The intraviral protein-protein interaction of SARS-CoV-2 reveals the key role of N protein in virus-like particle assembly.

Intraviral protein-protein interactions (PPIs) of SARS-CoV-2 in host cells may provide useful information for deep understanding of virology of SARS-CoV-2. In this study, 22 of 55 interactions of the structural and accessory proteins of SARS-CoV-2 were identified by biomolecular fluorescence complementation (BiFC) assay. The nucleocapsid (N) protein was found to have the most interactions among the structural and accessory proteins of SARS-CoV-2, and also specifically interacted with the putative packaging signal (PS) of SARS-CoV-2. We also demonstrated that the PS core containing PS576 RNA bears a functional PS, important for the assembly of the viral RNA into virus like particles (VLPs), and the packaging of SARS-CoV-2 RNA was N dependent.

SARS-CoV-2 spike protein receptor-binding domain N-glycans facilitate viral internalization in respiratory epithelial cells.

N-glycosylation plays an important role in the pathogenesis of viral infections. However, the role of SARS-CoV-2 RBD N-glycosylation in viral entry remains elusive. In this study, we expressed and purified N331 and N343 N-glycosite mutants of SARS-CoV-2 RBD. We found that de-glycosylation at N331 and N343 drastically reduces the RBD binding to ACE2. More importantly, based on qualitative and quantitative virology research methods, we show that the mutation of RBD N-glycosites interfered with SARS-CoV-2 internalization rather than attachment potentially by decreasing RBD binding to the receptors. Also, the double N-glycosites mutant (N331 + N343) showed significantly increased sensitivity against the designated RBD neutralizing antibodies. Taken together, these results suggest that N-glycosylation of SARS-CoV-2 RBD is not only critical for viral internalization into respiratory epithelial cells but also shields the virus from neutralization. It may provide new insights into the biological process of early-stage SARS-CoV-2 infection with potential therapeutic implications.

Long noncoding RNA HOTAIR interacts with Y-Box Protein-1 (YBX1) to regulate cell proliferation.

HOTAIR is a long noncoding RNA (lncRNA) which serves as an important factor regulating diverse processes linked with cancer development. Here, we used comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) to explore the HOTAIR-protein interactome. We were able to identify 348 proteins interacting with HOTAIR, allowing us to establish a heavily interconnected HOTAIR-protein interaction network. We further developed a novel near-infrared fluorescent protein (iRFP)-trimolecular fluorescence complementation (TriFC) system to assess the interaction between HOTAIR and its interacting proteins. Then, we determined that HOTAIR specifically binds to YBX1, promotes YBX1 nuclear translocation, and stimulates the PI3K/Akt and ERK/RSK signaling pathways. We further demonstrated that HOTAIR exerts its effects on cell proliferation, at least in part, through the regulation of two YBX1 downstream targets phosphoenolpyruvate carboxykinase 2 (PCK2) and platelet derived growth factor receptor ß. Our findings revealed a novel mechanism, whereby an lncRNA is able to regulate cell proliferation via altering intracellular protein localization. Moreover, the imaging tools developed herein have excellent potential for future in vivo imaging of lncRNA-protein interaction.

Nanoscale Imaging of RNA-Protein Interactions with a Photoactivatable Trimolecular Fluorescence Complementation System.

Imaging RNA-protein interaction in the cellular space with single molecule sensitivity is attractive for studying gene expression and regulation, but remains a challenge. In this study, we reported a photoactivatable trimolecular fluorescence complementation (TriFC) system based on fluorescent protein, mIrisFP, to identify and visualize RNA-protein interactions in living mammalian cells. We also combined this TriFC system with photoactivated localization microscopy (PALM), named the TriFC-PALM technique, which allowed us to image the RNA-protein interactions with single molecule sensitivity. Using this TriFC-PALM technique, we identified the actin-bundling protein, FSCN1, specifically interacting with the HOX Transcript Antisense RNA (HOTAIR). The TriFC-PALM imaging acquired a higher resolution compared with the traditional method of total internal reflection (TIRF) imaging. The TriFC-PALM thus provides a useful tool for imaging and identifying the RNA-protein interactions inside cells at the nanometer scale.

Dual-Fluorescence Labeling Pseudovirus for Real-Time Imaging of Single SARS-CoV-2 Entry in Respiratory Epithelial Cells.

The pseudovirus strategy makes studies of highly pathogenic viruses feasible without the restriction of high-level biosafety facility, thus greatly contributing to virology and is used in the research studies of SARS-CoV-2. Here, we generated a dual-color pseudo-SARS-CoV-2 virus using a human immunodeficiency virus-1 pseudovirus production system and the SARS-CoV-2 spike (S) glycoprotein, of which the membrane was labeled with a lipophilic dye (DiO) and the genomic RNA-related viral protein R (Vpr) of the viral core was fused with mCherry. With this dual-color labeling strategy, not only the movement of the whole virus but also the fate of the labeled components can be traced. The pseudovirions were applied to track the viral entry at a single-particle level in four types of the human respiratory cells: nasal epithelial cells (HNEpC), pulmonary alveolar epithelial cells (HPAEpiC), bronchial epithelial cells (BEP-2D), and oral epithelial cells (HOEC). Pseudo-SARS-CoV-2 entered into the host cell and released the viral core into the cytoplasm, which clearly indicates that the host entry mainly occurred through endocytosis. The infection efficiency was found to be correlated with the expression of the known receptor of SARS-CoV-2, angiotensin-converting 2 (ACE2) on the host cell surface. We believe that the dual-color fluorescently labeled pseudovirus system created in this study can be applied as a useful tool for many purposes in SARS-CoV-2/COVID-19.

Construction and applications of SARS-CoV-2 pseudoviruses: a mini review.

The ongoing coronavirus disease 2019 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed a serious threat to global public health and social stability. There is an urgent need for understanding the nature and infection mechanism of the virus. Owing to its high infectivity and pathogenicity and lack of effective treatments, live SARS-CoV-2 has to be handled in biosafety level 3 laboratories, which has impeded research into SARS-CoV-2 and the development of vaccines and therapeutics. Pseudotyped viruses that lack certain gene sequences of the virulent virus are safer and can be investigated in biosafety level 2 laboratories, providing a useful virological tool for the study of SARS-CoV-2. In this review, we will discuss the construction of SARS-CoV-2 pseudoviruses based on different packaging systems, current applications, limitations, and further explorations.

A tandem near-infrared fluorescence complementation system with enhanced fluorescence for imaging protein-protein interactions in vivo.

Bimolecular fluorescence complementation (BiFC) is an effective tool for visualizing protein-protein interactions (PPIs). However, a BiFC system with long wavelength and high fluorescence intensity is yet to be developed for in vivo imaging. In this study, we constructed a tandem near-infrared BiFC (tBiFC) system by splitting a near-infrared phytochrome, IFP2.0. This system allows the identification and visualization of PPIs in live cells and living mice. The photophysical properties of the complementary fluorescence of the tBiFC system were similar to those of its parent protein IFP2.0, but the intensity was twice that of a single-copy IFP2.0-based BiFC system. Compared with previously reported near infrared BiFC systems-iRFP-BiFC and IFP1.4-BiFC-the signal intensity of the tBiFC system increased by ~1.48- and ~400-fold for weak PPIs in living cells, and ~1.51- and ~8-fold for strong PPIs, respectively. When applied to imaging PPIs in live mice, the complementary fluorescence intensity of the tBiFC system was also significantly higher than that of the other near-infrared BiFC systems. The use of this bright phytochrome in a tandem arrangement constitutes a powerful tool for imaging PPIs in the near infrared region.

Comparative proteomic analysis of senescence in the freshwater cladoceran Daphnia pulex.

Cladocera are small freshwater crustaceans that have attracted considerable attention in recent years. They are commonly used for studying senescence. In this study, we used LC-MS/MS with eight-plex iTRAQ to perform a comparative proteomic analysis of senescence in Daphnia pulex. Of 3076 primordial proteins, 2325 were credible (the remaining were low-confidence proteins) and 247 significantly differentially expressed proteins (DEPs). Of the latter, 87, 91, and 69 DEPs were identified in the Day 15 vs. Day 5, Day 20 vs. Day 5, and Day 25 vs. Day 5 groups, respectively. Gene ontology enrichment analysis showed that oxidative damage may be the main cause of senescence in D. pulex. Using Kyoto Encyclopedia of Genes and Genomes pathway analysis, we found that the peroxisome pathway played an important role in aging. Our results suggest that D. pulex alleviates excessive oxidative damage by altering key enzymes involved in carbohydrate and protein metabolism.

Genetically Encoded, Photostable Indicators to Image Dynamic Zn2+ Secretion of Pancreatic Islets.

As an essential element for living organisms, zinc (Zn2+) exerts its biological functions both intracellularly and extracellularly. Previous studies have reported a number of genetically encoded Zn2+ indicators (GEZIs), which have been widely used to monitor Zn2+ in the cytosol and intracellular organelles. However, it is challenging to localize existing GEZIs to the extracellular space to detect secreted Zn2+. Herein, we report two photostable, green fluorescent protein (GFP) based indicators, ZIBG1 and ZIBG2, which respond to Zn2+ selectively and have affinities suited for detecting Zn2+ secretion from intracellular vesicles. In particular, ZIBG2 can be effectively targeted to the extracellular side of plasma membrane. We applied cell surface-localized ZIBG2 to monitor glucose-induced dynamic Zn2+ secretion from mouse insulinoma MIN6 cells and primary mouse and human pancreatic islets. Because Zn2+ is co-released with insulin from ß-cells, the fluorescence of cell surface-localized ZIBG2 was shown to be a strong indicator for the functional potency of islets. Our work here has thus expanded the use of GEZIs to image dynamic Zn2+ secretion in live tissue. Because it is convenient to use genetically encoded indicators for expression over extended periods and for in vivo delivery, we envision future applications of ZIBG2 in development of induced ß-cells or islets to advance cell replacement therapies for diabetes and in direct imaging of Zn2+ secretion dynamics in vivo.

Assessing the ecological health of the Chongming Dongtan Nature Reserve, China, using different benthic biotic indices.

To assess the ecological health of the Chongming Dongtan Nature Reserve (CDNR), univariate and multimetric indices-AZTI's Marine Biotic Index (AMBI), multivariate AMBI (M-AMBI), abundance-biomass comparison curves, taxonomic diversity, Shannon-Wiener index (H') (log2), and Benthic Opportunistic Polychaetes Amphipods index (BOPA) - were used to translate the macrobenthic community into classifications of ecological health in 2016 and 2017. Based on the results of the various indicators, the overall habitat status of the CDNR ranged from good to high status. Sites 4 and 5, which fell in the remaining tidal flats following reclamation activities, were classified as disturbed, whereas the degree of disturbance at site 3 was low. The results indicate that the CDNR is under some form of human disturbance, which includes reclamation and animal (mainly cattle) grazing. H' (log2) and M-AMBI index may be more suitable for assessing ecological quality in intertidal zones, including the CDNR.

Effects of microplastics on the innate immunity and intestinal microflora of juvenile Eriocheir sinensis.

The effects of microplastic exposure on the non-specific immune responses and intestinal microflora remain unclear. In this study, juveniles of the Chinese mitten crab (Eriocheir sinensis) were exposed to different concentrations of microplastics (0, 0.04, 0.4, 4, and 40 mg/L) for 7, 14, and 21 days to explore their effects. Under microplastic-induced stress, the contents or activities of most immune-related factors [haemocyanin (Hc), alkaline phosphatase (AKP), phenoloxidase (PO), lysozyme (LSZ), and acid phosphatase (ACP)] decreased after an initial increase in the low-dose or short exposure times in the haemolymph and hepatopancreas. The trends in Hc and LSZ gene expression were consistent with the corresponding changes in enzyme activities. Moreover, the haemocyte expression of caspase and MyD88 in the groups with microplastic-induced stress was higher than that in the control group, whereas the expression levels in the hepatopancreas were first increased and then decreased. Furthermore, the relative abundance of Firmicutes and Bacteroidetes decreased following exposure to 40 mg/L microplastics, whereas that of Fusobacteria and Proteobacteria increased. These results indicate that microplastics affect immune enzyme activity and immune-related gene expression and change the diversity and composition of the intestinal microflora in E. sinensis.

Changes in ultrastructure of gonads and external morphology during aging in the parthenogenetic cladoceran Daphnia pulex.

The water flea Daphnia pulex, a small crustacean that lives in freshwater ponds, undergoes parthenogenesis and gamogenesis according to environmental conditions. In D. pulex, different morphological characteristics can be observed using a microscope during aging. In this study, we recorded the growth, reproduction, and survival indicators of parthenogenetic D. pulex females to analyze the morphological changes that occur during senescence. We observed the growth and development of the parthenogenetic females on the 7th, 14th, 21st, and 28th days by using scanning electron microscopy and ultrastructure of the gonads on the 7th, 14th and 21st days with transmission electron microscopy. We found that body length and reproductive capacity in the parthenogenetic individuals first increased and then decreased with aging, and the survival rate decreased gradually. The depression of the brood pouch on the back of the parthenogenetic females implied a decline in gonadal function and gradual aging. During senescence, the number of lipid droplets in the gonads of the parthenogenetic females decreased, and the integrity of the mitochondria and peroxisome was destroyed. The results of our study revealed the morphological characteristics of senescence in D. pulex.

ATP-Independent Bioluminescent Reporter Variants To Improve in Vivo Imaging.

Coelenterazine (CTZ)-utilizing marine luciferases and their derivatives have attracted significant attention because of their ATP-independency, fast enzymatic turnover, and high bioluminescence brightness. However, marine luciferases typically emit blue photons and their substrates, including CTZ and the recently developed diphenylterazine (DTZ), have poor water solubility, hindering their in vivo applications. Herein, we report a family of pyridyl CTZ and DTZ analogs that exhibit spectrally shifted emission and improved water solubility. Through directed evolution, we engineered a LumiLuc luciferase with broad substrate specificity. In the presence of corresponding pyridyl substrates (i.e., pyCTZ, 6pyDTZ, or 8pyDTZ), LumiLuc generates highly bright blue, teal, or yellow bioluminescence. We compared our LumiLuc-8pyDTZ pair with several benchmark reporters in a tumor xenograft mouse model. Our new pair, which does not need organic cosolvents for in vivo administration, surpasses other reporters by detecting early tumors. We further fused LumiLuc to a red fluorescent protein, resulting in a LumiScarlet reporter with further red-shifted emission and enhanced tissue penetration. LumiScarlet-8pyDTZ was comparable to Akaluc-AkaLumine, the brightest ATP-dependent luciferase-luciferin pair, for detecting cells in deep tissues of mice. In summary, we have engineered a new family of ATP-independent bioluminescent reporters, which will have broad applications because of their ATP-independency, excellent biocompatibility, and superior in vivo sensitivity.

Identification of Factors Complicating Bioluminescence Imaging.

In vivo bioluminescence imaging (BLI) has become a standard, non-invasive imaging modality for following gene expression or the fate of proteins and cells in living animals. Currently, bioluminescent reporters used in laboratories are mostly derivatives of two major luciferase families: ATP-dependent insect luciferases and ATP-independent marine luciferases. Inconsistent results of experiments using different bioluminescent reporters, such as Akaluc and Antareas2, have been reported. Herein, we re-examined the inconsistency in several experimental settings and identified the factors, such as ATP dependency, stability in serum, and molecular sizes of luciferases, that contributed to the observed differences. We expect this study will make the research community aware of these factors and facilitate more accurate interpretation of BLI data by considering the nature of each bioluminescent reporter.