Impact of N-Terminal Tags on De Novo Vimentin Intermediate Filament Assembly

Vimentin, a type III intermediate filament protein, is found in most cells along with microfilaments and microtubules. It has been shown that the head domain folds back to associate with the rod domain and this association is essential for filament assembly. The N-terminally tagged vimentin has been widely used to label the cytoskeleton in live cell imaging. Although there is previous evidence that EGFP tagged vimentin fails to form filaments but is able to integrate into a pre-existing network, no study has systematically investigated or established a molecular basis for this observation. To determine whether a tag would affect de novo filament assembly, we used vimentin fused at the N-terminus with two different sized tags, AcGFP (239 residues, 27 kDa) and 3 × FLAG (22 residues; 2.4 kDa) to assemble into filaments in two vimentin-deficient epithelial cells, MCF-7 and A431. We showed that regardless of tag size, N-terminally tagged vimentin aggregated into globules with a significant proportion co-aligning with β-catenin at cell–cell junctions. However, the tagged vimentin aggregates could form filaments upon adding untagged vimentin at a ratio of 1:1 or when introduced into cells containing pre-existing filaments. The resultant filament network containing a mixture of tagged and untagged vimentin was less stable compared to that formed by only untagged vimentin. The data suggest that placing a tag at the N-terminus may create steric hinderance in case of a large tag (AcGFP) or electrostatic repulsion in case of highly charged tag (3 × FLAG) perhaps inducing a conformational change, which deleteriously affects the association between head and rod domains. Taken together our results shows that a free N-terminus is essential for filament assembly as N-terminally tagged vimentin is not only incapable of forming filaments, but it also destabilises when integrated into a pre-existing network.     

Determination of IgG1 and IgG3 SARS-CoV-2 Spike Protein and Nucleocapsid Binding—Who Is Binding Who and Why?

The involvement of immunoglobulin (Ig) G3 in the humoral immune response to SARS-CoV-2 infection has been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) in COVID-19. The exact molecular mechanism is unknown, but it is thought to involve this IgG subtype’s differential ability to fix, complement and stimulate cytokine release. We examined the binding of convalescent patient antibodies to immobilized nucleocapsids and spike proteins by matrix-assisted laser desorption/ionization–time of flight (MALDI-ToF) mass spectrometry. IgG3 was a major immunoglobulin found in all samples. Differential analysis of the spectral signatures found for the nucleocapsid versus the spike protein demonstrated that the predominant humoral immune response to the nucleocapsid was IgG3, whilst for the spike protein it was IgG1. However, the spike protein displayed a strong affinity for IgG3 itself, as it would bind from control plasma samples, as well as from those previously infected with SARS-CoV-2, similar to the way protein G binds IgG1. Furthermore, detailed spectral analysis indicated that a mass shift consistent with hyper-glycosylation or glycation was a characteristic of the IgG3 captured by the spike protein.     

Sogatella furcifera Saliva Mucin-like Protein Is Required for Feeding and Induces Rice Defences

The white-backed planthopper (WBPH), Sogatella furcifera, is one of the most important piercing-sucking pests of rice (Oryza sativa) in Asia. Mucin-like salivary protein (SFMLP) is highly expressed in the salivary glands of WBPH, which plays an important role in WBPH feeding. In this study, WBPH injected with dsSFMLP had difficulty in sucking phloem sap from rice plants, which significantly reduced their food intake, weight, and survival. In contrast, the knockdown of the SFMLP gene had only a marginal effect on the survival of WBPH fed an artificial diet. Further studies showed that silencing SFMLP resulted in the short and single-branched salivary sheaths secretion and less formation of salivary flanges in rice. These data suggest that SFMLP is involved in the formation of the salivary sheath and is essential for feeding in WBPH. Overexpression of the SFMLP gene in rice plants promoted the feeding of WBPH, whereas silencing the gene in rice plants significantly decreased WBPH performance. Additionally, it was found that overexpression of SFMLP in rice plants elicited the signalling pathway of SA (salicylic acid) while suppressing JA (jasmonic acid); in contrast, silencing of the SFMLP gene in rice plants showed the opposite results. This study clarified the function of SFMLP in WBPH feeding as well as mediating rice defences.     

The β2-Subunit (AMOG) of Human Na+, K+-ATPase Is a Homophilic Adhesion Molecule

The β₂ subunit of Na⁺, K⁺-ATPase was originally identified as the adhesion molecule on glia (AMOG) that mediates the adhesion of astrocytes to neurons in the central nervous system and that is implicated in the regulation of neurite outgrowth and neuronal migration. While β₁ isoform have been shown to trans-interact in a species-specific mode with the β₁ subunit on the epithelial neighboring cell, the β₂ subunit has been shown to act as a recognition molecule on the glia. Nevertheless, none of the works have identified the binding partner of β₂ or described its adhesion mechanism. Until now, the interactions pronounced for β₂/AMOG are heterophilic cis-interactions. In the present report we designed experiments that would clarify whether β₂ is a cell–cell homophilic adhesion molecule. For this purpose, we performed protein docking analysis, cell–cell aggregation, and protein–protein interaction assays. We observed that the glycosylated extracellular domain of β₂/AMOG can make an energetically stable trans-interacting dimer. We show that CHO (Chinese Hamster Ovary) fibroblasts transfected with the human β₂ subunit become more adhesive and make large aggregates. The treatment with Tunicamycin in vivo reduced cell aggregation, suggesting the participation of N-glycans in that process. Protein–protein interaction assay in vivo with MDCK (Madin-Darby canine kidney) or CHO cells expressing a recombinant β₂ subunit show that the β₂ subunits on the cell surface of the transfected cell lines interact with each other. Overall, our results suggest that the human β₂ subunit can form trans-dimers between neighboring cells when expressed in non-astrocytic cells, such as fibroblasts (CHO) and epithelial cells (MDCK).     

C-Reactive Protein Velocity (CRPv) as a New Biomarker for the Early Detection of Acute Infection/Inflammation

C-reactive protein (CRP) is considered a biomarker of infection/inflammation. It is a commonly used tool for early detection of infection in the emergency room or as a point-of-care test and especially for differentiating between bacterial and viral infections, affecting decisions of admission and initiation of antibiotic treatments. As C-reactive protein is part of a dynamic and continuous inflammatory process, a single CRP measurement, especially at low concentrations, may erroneously lead to a wrong classification of an infection as viral over bacterial and delay appropriate antibiotic treatment. In the present review, we introduce the concept of C-reactive protein dynamics, measuring the velocity of C-reactive protein elevation, as a tool to increase this biomarker’s diagnostic ability. We review the studies that helped define new metrics such as estimated C-reactive protein velocity (velocity of C-reactive protein elevation from symptoms’ onset to first C-reactive protein measurement) and the measured C-reactive protein velocity (velocity between sequential C-reactive protein measurements) and the use of these metrics in different clinical scenarios. We also discuss future research directions for this novel metric.     

八钢型材厂电气隔离保护技术的应用

八钢型材厂运用的电气隔离保护技术,在不同工况下的实际应用。     

Non-Cytokine Protein Profile of the Mesenchymal Stem Cell Secretome That Regulates the Androgen Production Pathway

Polycystic ovary syndrome (PCOS) is the most common endocrine and metabolic disorder in reproductive-aged women, and it typically involves elevated androgen levels. Recently, it has been reported that human bone marrow mesenchymal stem cells (hBM-MSCs) can regulate androgen synthesis pathways. However, the details of the mechanism are still unclear. hBM-MSC-derived secreted factors (the secretome) are promising sources of cell-based therapy as they consist of various types of proteins. It is thus important to know which proteins interact with disease-implicated biomolecules. This work aimed to investigate which secretome components contain the key factor that inhibits testosterone synthesis. In this study, we fractionated hBM-MSC-conditioned media into three fractions based on their molecular weights and found that, of the three fractions, one had the ability to inhibit the androgen-producing genes efficiently. We also analyzed the components of this fraction and established a protein profile of the hBM-MSC secretome, which was shown to inhibit androgen synthesis. Our study describes a set of protein components present in the hBM-MSC secretome that can be used therapeutically to treat PCOS by regulating androgen production for the first time.     

Transduction of Brain Neurons in Juvenile Chum Salmon (Oncorhynchus keta) with Recombinant Adeno-Associated Hippocampal Virus Injected into the Cerebellum during Long-Term Monitoring

Corpus cerebelli in juvenile chum salmon is a multiprojective region of the brain connected via afferent and efferent projections with the higher regions of the brainstem and synencephalon, as well as with multiprojection regions of the medulla oblongata and spinal cord. During the postembryonic development of the cerebellum in chum salmon, Oncorhynchus keta, the lateral part of the juvenile cerebellum gives rise to the caudomedial part of the definitive cerebellum, which is consistent with the data reported for zebrafish and mouse cerebellum. Thus, the topographic organization of the cerebellum and its efferents are similar between fish (chum salmon and zebrafish) and mammals, including mice and humans. The distributions of recombinant adeno-associated viral vectors (rAAVs) after an injection of the base vector into the cerebellum have shown highly specific patterns of transgene expression in bipolar neurons in the latero-caudal lobe of the juvenile chum tectum opticum. The distribution of rAAVs in the dorsal thalamus, epithalamus, nucleus rotundus, and pretectal complex indicates the targeted distribution of the transgene via the thalamo-cerebellar projections. The detection of GFP expression in the cells of the epiphysis and posterior tubercle of juvenile chum salmon is associated with the transgene’s distribution and with the cerebrospinal fluid flow, the brain ventricles and its outer surface. The direct delivery of the rAAV into the central nervous system by intracerebroventricular administration allows it to spread widely in the brain. Thus, the presence of special projection areas in the juvenile chum salmon cerebellum, as well as outside it, and the identification of the transgene’s expression in them confirm the potential ability of rAAVs to distribute in both intracerebellar and afferent and efferent extracerebellar projections of the cerebellum.