Verifying AXL and putative proteins as SARS-CoV-2 receptors by DnaE intein-based rapid cell-cell fusion assay.

As the understanding of the mechanisms of SARS-CoV-2 infection continues to grow, researchers have come to realize that ACE2 and TMPRSS2 receptors are not the only way for the virus to invade the host, and that there are many molecules that may serve as potential receptors or cofactors. The functionality of these numerous receptors, proposed by different research groups, demands a fast, simple, and accurate validation method. To address this issue, we here established a DnaE intein-based cell-cell fusion system, a key result of our study, which enables rapid simulation of SARS-CoV-2 host cell infection. This system allowed us to validate that proteins such as AXL function as SARS-CoV-2 spike protein receptors and synergize with ACE2 for cell invasion, and that proteins like NRP1 act as cofactors, facilitating ACE2-mediated syncytium formation. Our results also suggest that mutations in the NTD of the SARS-CoV-2 Delta variant spike protein show a preferential selection for Spike-AXL interaction over Spike-LDLRAD3. In summary, our system serves as a crucial tool for the rapid and comprehensive verification of potential receptors, screening of SARS-CoV-2-neutralizing antibodies, or targeted drugs, bearing substantial implications for translational clinical applications.

Gut microbiome changes in mouse, Mongolian gerbil, and hamster models following Clostridioides difficile challenge.

Introduction: Clostridioides difficile infection (CDI), as well as its etiology and pathogenesis, have been extensively investigated. However, the absence of suitable CDI animal models that reflect CDI symptoms and the associated gut microbiome changes in humans has limited research progress in this field. Thus, we aimed to investigate whether Mongolian gerbils, which present a range of human pathological conditions, can been used in studies on CDI. Methods: In this study, we infected Mongolian gerbils and two existing CDI model animals, mice and hamsters, with the hypervirulent ribotype 027 C. difficile strain, and comparatively analyzed changes in their gut microbiome composition via 16S rRNA gene sequencing. Methods: In this study, we infected Mongolian gerbils and two existing CDI model animals, mice and hamsters, with the hypervirulent ribotype 027 C. difficile strain, and comparatively analyzed changes in their gut microbiome composition via 16S rRNA gene sequencing. Results: The results obtained showed that C. difficile colonized the gastrointestinal tracts of the three rodents, and after the C. difficile challenge, C57BL/6J mice did not manifest CDI symptoms and their intestines showed no significant pathological changes. However, the hamsters showed explosive intestinal bleeding and inflammation and the Mongolian gerbils presented diarrhea as well as increased infiltration of inflammatory cells, mucus secretion, and epithelial cell shedding in their intestinal tissue. Further, intestinal microbiome analysis revealed significant differences with respect to intestinal flora abundance and diversity. Specifically, after C. difficile challenge, the Firmicutes/Bacteroidetes ratio decreased for C57BL/6J mice, but increased significantly for Mongolian gerbils and hamsters. Furthermore, the abundance of Proteobacteria increased in all three models, especially in hamsters, while that of Verrucomicrobia only increased significantly in C57BL/6J mice and Mongolian gerbils. Our results also indicated that differences in the relative abundances of Lactobacillaceae and Akkermansia were primarily responsible for the observed differences in response to C. difficile challenge. Conclusion: Based on the observed responses to C. difficile challenge, we concluded for the first time that the Mongolian gerbil could be used as an animal model for CDI. Additionally, the taxa identified in this study may be used as biomarkers for further studies on CDI and to improve understanding regarding changes in gut microbiome in CDI-related diseases.

Intestine epithelial cell-derived extracellular vesicles alleviate inflammation induced by Clostridioides difficile TcdB through the activity of TGF-ß1.

Background: Clostridioides difficile infection (CDI) has been primarily associated with the toxin B (TcdB), one of the three known protein toxins secreted by C. difficile, which can activate the intestinal immune system and lead to pathological damage. Even though the biological functions of intestine epithelial cell-derived extracellular vesicles (I-Evs) have been well documented, the role of I-Evs in the process of CDI is still unknown. Objectives: The protective effect of I-Evs against C. difficile TcdB was investigated both in cultured murine colon carcinoma MC38 cells and a mouse model used in this study. Results: Mouse I-Evs with mean diameter ranging from 100 to 200 nm and a density of 1.09-1.17 g/mL were obtained and confirmed containing the Ev-associated specific surface markers CD63 and TSG101 as well as high level of TGF-ß1. In MC38 cells, I-Evs were able to decrease the gene expression of IL-6, TNF-α, IL-1ß, and IL-22 induced by C. difficile TcdB, but to increase both the gene expression and protein levels of TGF-ß1. I-Evs treatment via intraperitoneal administration alleviates C. difficile TcdB-induced local colon inflammation in mice and increased their survival rate from 50% up to 80%. Furthermore, I-Evs induced an increase in the proportion of CD4+Foxp3+Tregs in vitro and in vivo through a TGF-ß1-dependent mechanism by activating the TGF-ß1 pathway and prompting phosphorylation of the downstream proteins Smad 2/3. Conclusion: For the first time, our study demonstrated that I-Evs originated from intestine epithelial cells can alleviate inflammation induced by C. difficile TcdB both in vitro and in vivo. Therefore, I-Evs might be potentially a novel endogenous candidate for effective treatment of CDI.

Regulation of brain-derived neurotrophic factor-mediated transcription of the immediate early gene Arc by intracellular calcium and calmodulin.

The induction of the immediate early gene Arc is strongly implicated in synaptic plasticity. Although the role of ERK has been demonstrated, the regulation of Arc expression is largely unknown. In this study, we investigated the major signaling pathways underlying brain-derived neurotrophic factor (BDNF)-mediated Arc transcription in cultured cortical neurons. The BDNF-stimulated Arc transcription was regulated solely by the Ras-Raf-MAPK signaling through ERK, but not by phosphoinositide 3-kinase (PI3K) and PLC-gamma activities. Although it was demonstrated that BDNF might promote calcium entry through calcium channels and NMDA receptors, chelating extracellular calcium with EGTA failed to block Arc transcription. In contrast, chelating intracellular calcium ([Ca(2+)](i)) by BAPTA-AM abolished BDNF-mediated Arc up-regulation. Surprisingly, BAPTA-AM did not block ERK activation, indicating that [Ca(2+)](i) and Ras-Raf-MAPK are not coupled, and the activation of ERK alone is not sufficient to up-regulate Arc transcription. Moreover, we found that inhibition of calmodulin (CaM) by W13 blocked both Arc transcription and ERK activation, revealing a Ca(2+)-independent function of CaM. These data suggested novel functions of [Ca(2+)](i) and CaM in BDNF signaling. Comparison of the Arc transcription profiles between Ca(2+)-stimulated and BDNF-stimulated neurons demonstrated that the regulatory mechanisms were distinctively tailored to the complex features of neuronal activity. Specifically, PI3K and CaM-dependent protein kinase (CaMK) activity were required for Ca(2+)-stimulated Arc transcription through regulating ERK signaling. Such cross-talks between PI3K, CaMK, and ERK was absent in BDNF-stimulated neurons.

N-methyl-D-aspartate-stimulated ERK1/2 signaling and the transcriptional up-regulation of plasticity-related genes are developmentally regulated following in vitro neuronal maturation.

The general features of neuroplasticity are developmentally regulated. Although it has been hypothesized that the loss of plasticity in mature neurons may be due to synaptic saturation and functional reduction of N-methyl-D-aspartate receptors (NMDAR), the molecular mechanisms remain largely unknown. We examined the effects of NMDAR activation and KCl-mediated membrane depolarization on ERK1/2 signaling following in vitro maturation of cultured cortical neurons. Although NMDA stimulated a robust increase in intracellular calcium at both DIV (day in vitro) 3 and 14, the activation of ERK1/2 and cAMP responsive element-binding protein (CREB) was impaired at DIV 14. Specifically, the phosphorylation of ERK1/2 was stimulated by both NMDA and KCl at DIV 3. However, at DIV 14, NMDA- but not KCl-stimulated ERK1/2 and CREB phosphorylation was significantly diminished. Consistently, the NMDA-induced transcription of ERK/CREB-regulated genes Bdnf exon 4, Arc, and zif268 was significantly attenuated at DIV 14. Moreover, in comparison with 3 DIV neurons, the phosphorylated-ERK1/2 in 14 DIV neurons displayed a tremendous increase following maturation and was more susceptible to dephosphorylation. Blocking calcium channels by nifedipine or NMDAR by APV caused a more dramatic ERK dephosphorylation in 14 DIV neurons. We further demonstrate that the loss of plasticity-related signaling is unrelated to NMDA-induced cell death of the 14 DIV neurons. Taken together, these results suggest that the attenuation of certain aspects of neuroplasticity following maturation may be due to the reduction of NMDAR-mediated gene transcription and a saturation of ERK1/2 activity.

Alanine scanning mutagenesis of SP70 epitope in characterizing species­specific antibodies induced by enterovirus 71­based antigens.

The enterovirus 71 (EV71) SP70 epitope, derived from amino acids 208­222 of VP1, is a neutralizing epitope. The present study aimed to assess the inter­species differences of the antibodies induced by EV71­based antigens in responses to SP70 mutant peptides. BALB/c mice and Lou/C rats were immunized with EV71 SP70. Monoclonal antibodies (Mabs) were produced by hybridoma clones. Serum polyclonal antibodies (Pabs) were produced from BALB/c mice and New Zealand white rabbits immunized with recombinant EV71 VP1 (rEV71­VP1) protein or inactivated EV71. Micro­neutralization and immunofluorescence assays were used to evaluate the capacity of the antibodies to bind to EV71. Reactivity of Mabs and Pabs to mutated SP70 were determined by alanine scanning mutagenesis. Furthermore, serums from EV71­infected patients were collected to examine the affinity of SP70 antibody in the serum to mutated SP70, using competitive ELISA. The binding affinity of mouse Mabs to the SP70 epitope was increased by alanine substitution at sites of 210, 212, 213, 214, and 221. The binding affinity of rat Mabs to the SP70 epitope was increased by alanine substitution at sites 210, 217, 219, and 221. Mouse serum Pabs elicited by inactivated EV71 bound wild­type SP70, but lost affinity for mutated peptides. Conversely, rabbit serum Pabs elicited by inactivated EV71 robustly recognized SP70 mutants. Mouse serum Pabs elicited by rEV71­VP1 presented the same trend as mouse Mabs. Mutations at sites 214, 215, and 217 led to loss of recognition by rabbit Pabs elicited by rEV71­VP1, while most mutations did not influence antibody binding. Compared with the wild­type, mutations at the sites 209, 219 and 221 of SP70 lead to increased affinity with the serum antibodies produced by the EV71­infected patients. Antibody responses triggered by inactivated EV71, rEV71­VP1 and EV71 SP70 differed among species in neutralizing capacity and affinity for SP70 mutant peptides.

Serine phosphorylation differentially affects RhoA binding to effectors: implications to NGF-induced neurite outgrowth.

Activation of RhoA prevents NGF-induced outgrowth and causes retraction of neurites in neuronal cells, including PC12 cells. Despite its inhibitory effect on neurite outgrowth, NGF activates GTP loading of and effector binding to RhoA, setting up an apparent contradiction. According to the molecular switch hypothesis of GTPase function GTP-loading of RhoA should be sufficient to activate its effectors uniformly. However, when monitoring NGF-induced binding of GTP-RhoA to multiple targets, we noted differential interactions with its effectors. We found that NGF elicits a protein kinase A-mediated phosphorylation of RhoA on serine(188), which renders it unable to bind to Rho-associated kinase (ROK), whereas it retains the ability to interact with other RhoA targets including rhotekin, mDia-1 and PKN. We show in vitro and in vivo that phosphorylation of serine(188) represents an additional switch, capable of directing signals among effector pathways. In the context of PC12 cell differentiation, NGF-induced phosphorylation of RhoA on serine(188) prevents it from interacting with ROK, which would otherwise block neurite outgrowth. Transfection of RhoA(S188A) mutant into PC12 cells prevents NGF-induced neurite outgrowth, just like constitutively activated RhoA(14V) does, indicating the requirement of this phosphorylation site. Replacement of serine(188) with the phosphomimetic glutamate residue in RhoA(V14/S188E) selectively impairs interaction with ROK and when transfected into PC12 cells restores NGF-induced neurite outgrowth. Therefore, phosphorylation of serine(188) may serve as a novel secondary switch of RhoA capable of overriding GTP-binding-elicited effector activation to a subset of targets such as ROK, which interact with the C-terminus of RhoA.

[Prediction and Identification of Immunodominant Linear B Cell Epitopes in the Nucleocapsid Protein of SFTSV].

In order to identify immunodominant linear B cell epitopes in the nucleocapsid protein N of severe fever with thrombocytopenia syndrome virus(SFTSV),bioinformatics programs were used to analyze antigenicity, hydrophilicity and surface probability of the amino acid sequence and predict possible linear B cell epitopes. PyMOL software was used to analyze the distribution of linear B cell epitopes in nucleocapsid protein N based on its crystal structure. Corresponding peptides were synthesized and examined in peptide enzyme-linked immunosorbent assay(Peptide-ELISA)individually to check whether they reacted with sera from SFTSV-infected patients. As a result, a total of six potential linear B cell epitopes were predicted as the following: A(40-KKLKETGGDDWVKDTK-55), B(71-ASGKMSNSGSKRL-83), C(94-ERAETRL-100),D(135-LKVENYPP-142),E(157-GVSEATT-163)and F(184-KMRGASKTEVYNSFRDP-200).All epitopes were located on the surface of the nucleocapsid protein N and contained flexible loops. Each of the six synthetic peptides reacted positively with sera from SFTSV-infected patients and were identified as immunodominant linear B cell epitopes. Linear regression analysis showed a positive correlation between each peptide-ELISA and commercialized N protein-based EIA. In this study, immunodominant linear B cell epitopes from the nucleocapsid protein N of SFTSV were successfully predicted and confirmed. These findings may help to establish the molecule basis of specific antigenicity and disease diagnosis.

Pseudomurein endoisopeptidases PeiW and PeiP, two moderately related members of a novel family of proteases produced in Methanothermobacter strains.

Sequence comparison of pseudomurein endoisopeptidases PeiW encoded by the defective prophage PsiM100 of Methanothermobacter wolfeii, and PeiP encoded by phage PsiM2 of Methanothermobacter marburgensis, revealed that the two enzymes share only limited similarity. Their amino acid sequences comprise an N-terminal domain characterized by the presence of direct repeats and a C-terminal domain with a catalytic triad C-H-D as in thiol proteases and animal transglutaminases. Both PeiW and PeiP catalyze the in vitro lysis of M. marburgensis cells under reducing conditions and exhibit characteristics of metal-activated peptidases. Optimal temperature and pH were determined to be 63 degrees C and 6.4 for His-tagged PeiP and 71 degrees C and 6.4 for His-tagged PeiW, respectively. Database search results suggest that PeiW and PeiP are the first two experimentally identified members of a novel family of proteases in a superfamily of archaeal, bacterial, and eukaryotic protein homologs of animal transglutaminases.

Regulation of brain-derived neurotrophic factor exon IV transcription through calcium responsive elements in cortical neurons.

Activity-dependent transcription of brain-derived neurotrophic factor (BDNF) has been studied as an important model to elucidate the mechanisms underlying numerous aspects of neuroplasticity. It has been extensively emphasized that Ca(2+) influx through different routes may have significantly different effects on BDNF transcription. Here, we examined the regulatory property of the major calcium responsive elements (CaRE) in BDNF promoter IV in cultured rat cortical neurons. BDNF promoter IV, as well as CaRE1 and CaRE3, was significantly activated by Ca(2+) influx through L-type voltage-gated calcium channel (L-VGCC) or NMDA receptor (NMDAR). However, the L-VGCC- and NMDAR-mediated activation of CaRE was differentially regulated by different Ca(2+)-stimulated protein kinases. Specifically, PKA, CaMKI, and CaMKIV activity were required for L-VGCC-, but not NMDAR-mediated CaRE1 activation. CaMKI activity was required for NMDAR- but not L-VGCC-mediated CaRE3 activation. Surprisingly, the activation of CaRF, a previously identified transcription factor for CaRE1, was stimulated via L-VGCC but not NMDAR, and required MEK, PI3K and CaMKII activity. These results suggest a new working model that activity-dependent BDNF IV up-regulation may be coordinately mediated by CaRE1 and CaRE3 activity, which show different responses to Ca(2+)-stimulated kinases. Our data also explain how the individual cis-element in BDNF promoter is distinctively coupled to different Ca(2+) routes.

A novel strategy for specifically down-regulating individual Rho GTPase activity in tumor cells.

The Rho family GTPases RhoA, RhoB, and RhoC regulate the actin cytoskeleton, cell movement, and cell growth. Unlike Ras, up-regulation or overexpression of these GDP/GTP binding molecular switches, but not activating point mutations, has been associated with human cancer. Although they share over 85% sequence identity, RhoA, RhoB, and RhoC appear to play distinct roles in cell transformation and metastasis. In NIH 3T3 cells, RhoA or RhoB overexpression causes transformation whereas RhoC increases the cell migration rate. To specifically target RhoA, RhoB, or RhoC function, we have generated a set of chimeric molecules by fusing the RhoGAP domain of p190, a GTPase-activating protein that accelerates the intrinsic GTPase activity of all three Rho GTPases, with the C-terminal hypervariable sequences of RhoA, RhoB, or RhoC. The p190-Rho chimeras were active as GTPase-activating proteins toward RhoA in vitro, co-localized with the respective active Rho proteins, and specifically down-regulated Rho protein activities in cells depending on which Rho GTPase sequences were included in the chimeras. In particular, the p190-RhoA-C chimera specifically inhibited RhoA-induced transformation whereas p190-RhoC-C specifically reversed the migration phenotype induced by the active RhoC. In human mammary epithelial-RhoC breast cancer cells, p190-RhoC-C, but not p190-RhoA-C or p190-RhoB-C, reversed the anchorage-independent growth and invasion phenotypes caused by RhoC overexpression. In the highly metastatic A375-M human melanoma cells, p190-RhoC-C specifically reversed migration, and invasion phenotypes attributed to RhoC up-regulation. Thus, we have developed a novel strategy utilizing RhoGAP-Rho chimeras to specifically down-regulate individual Rho activity and demonstrate that this approach may be applied to multiple human tumor cells to reverse the growth and/or invasion phenotypes associated with disregulation of a distinct subtype of Rho GTPase.