Since the outbreak of COVID-19 in 2019, the 2019-nCovcoronavirus has appeared diverse mutational characteristics due to its ownflexible conformation. One multiple-mutant strain (Omicron) with surprisinglyinfective activity outburst, and affected the biological activities of currentdrugs and vaccines, making the epidemic significantly difficult to prevent andcontrol, and seriously threaten health around the world. Importunatelyexploration of mutant characteristics for novel coronavirus Omicron can supplystrong theoretical guidance for learning binding mechanism of mutant viruses.What’s more, full acknowledgement of key mutated-residues on Omicron strain canprovide new methodology of the novel pathogenic mechanism to human ACE2receptor, as well as the subsequent vaccine development.In this research, 3D structures of 32 single-point mutationsof 2019-nCov were firstly constructed, and 32-sites multiple-mutant Omicronwere finally obtained based one the wild-type virus by homology modeling method.One total number of 33 2019-nCov/ACE2 complex systems were acquired byprotein-protein docking, and optimized by using preliminary molecular dynamicsimulations. Binding free energies between each 2019-nCov mutation system andhuman ACE2 receptor were calculated, and corresponding binding patternsespecially the regions adjacent to mutation site were analyzed. The resultsindicated that one total number of 6 mutated sites on the Omicron strain playedcrucial role in improving binding capacities from 2019-nCov to ACE2 protein.Subsequently, we performed long-term molecular dynamic simulations andprotein-protein binding energy analysis for the selected 6 mutations. 3infected individuals, the mutants T478K, Q493R and G496S with lower bindingenergies -66.36, -67.98 and -67.09 kcal/mol also presents the high infectivity.These findings indicated that the 3 mutations T478K, Q493R and G496S play thecrucial roles in enhancing binding affinity of Omicron to human ACE2 protein.All these results illuminate important theoretical guidance for future virusdetection of the Omicron epidemic, drug research and vaccine development. Since the breakout of coronavirus disease 2019 (COVID-19) reported on December 8, 2019, millions of people have been infected and more than 3.3 million people were killed, making this epidemic difficult to prevent and dangerous to the human beings , and theIt is worth noting that the virus has shown multi-point mutational characteristics and can significantly affect the coronavirus activity function and even affect the current drug and vaccines’ function, significantly increasing the difficulty of epidemic prevention and control, and seriously threatening the human health and life safety of the world. One new variant of the novel coronavirus B.1.1 has been detected in South Africa, triggering rapid increase positive tested cases . PrelimiWorld Health Organization estimates that by mid-December, Omicron likely was in most countries in the world, whether they had detected it or not [What is more, one new mutant with >50 mutations (Omicron) was first reported in South Africa. Moreover, 60% of these mutations occur on the spike region that play critical roles in binding to human cells . Until Dt or not . The Omit or not . Varioust or not –23. All Herein, 35 structures (34 single-mutants affecting the spike protein and one multiple-mutant with 34 mutations) were constructed by homology modeling method. We then investigated the interaction patterns and binding affinities between ACE2 and all 36 mutants by in silico analysis. Results indicated that 15 single-point mutations including T95I, G142D, Δ211, S371L, S373P, K417N, G446S, S477N, E484A, Q498R, N501Y, Y505H, D796Y, Q954H, and N969K possess better binding energy higher than −55.18 kcal/mol, while 6 mutants  only possess lower than −60.00 kcal/mol and were selected for further binding mode analysis. Subsequently, >180-ns-long molecular dynamic simulations was operated and binding energy analysis for the 6 mutations indicated that T478K, Q493R, and G496S play the crucial roles in enhancing binding affinity of Omicron to human ACE2 protein.Multiple-mutant with 34 mutations on 2019-nCov-Spike region based on wild-type Omicron and ACE2 complex was obtained by homology modeling method. Homology models are useful to guide mutational experiments about the structure-functional relationship and reliable in predicting the conformation of the insertion or deletion. The primary structure sequence of Omicron was compared with the target protein (PDB ID: 6VXX) for sequence blasting, and select the protein most similar to the target protein as the template for homology module construction. A high sequence similarity of 95% existed between wild-type Spike and Omicron strains. Homology modeling construction was executed by using MODELLER module .Mutational process of the 3-dimensional multiple-mutant structures was performed using the PyMOL software . MoleculBased on the 200-ns molecular dynamic simulation, at least 3000 snapshots were extracted to obtain average structure for each protein-protein system from the equilibrium trajectory, and binding mode for each mutant was analyzed. To investigate how the mutations affect the binding modes of ACE2 to the 2019-nCoV-Spike, the root mean square deviation (RMSD)  of heavyThe variant Omicron owns 60 mutations compared with the original Wuhan variant Table . Among tThe Spike protein plays a critical role in identifying and binding the host cell surface receptors and mediating the fusion of viral envelope to the cell membrane. The spike protein is like a “key,” and the ACE2 receptor on the cell is like a “lock.” The key is locked for the virus to enter the cell. The main goal of developing the COVID-19 vaccine is also to prevent keys from opening locks to prevent the virus from infecting cells. Thus, learning mutation sites on Omicron spike proteins will supply an extremely important driving role for drug and vaccine development.Currently, multiple crystal structures of the 2019-nCov-Spike/ACE2 complex have been resolved in the RCSB PDB database. In this paper, structure of the SARS-nCov-2 spike glycoprotein (closed state) with RCSB PDB ID: 6VXX  was seleAs shown in Fig. The 3D structures of the wild-type 2019-nCov/ACE2 complex system was directly extracted from the crystal structure with PDB ID: 6VXX. Subsequently, single-point mutants based on the wild-type Spike protein were constructed by using the mutated wizard module on PyMOL software. A total of 34 single-point mutations were obtained.Mutation of an amino acid on protein often causes the variation of biological function. We firstly performed primary molecular simulations for 34 single-point mutant and 1 multiple-mutant. We used the molecular dynamic software Amber 16 to conduct structural optimization for each 2019-nCov-Spike/ACE2 mutant complex. A force of 1500 kcal/mol was applied to all the heavy atoms on the Spike and ACE2 proteins during the MD simulations. The root mean square deviation (RMSD) of the heavy atoms of each multiple-mutant was examined until the MD simulations reached equilibrium. Binding free energies of the 2019-nCoV-Spike/ACE2 mutants were extracted for differential analysis. Each positional optimization time was at least 6 ns per system. Binding energy between the ACE2 protein and the 2019-nCov-Spike mutation system was calculated by using the MMPBSA module based on the 6-ns molecular dynamic simulation, as shown in Table The binding free energy of wild-type 2019-nCov-Spike/ACE2 protein complex was −55.18 kcal/mol. Among the 34 single-point mutant systems, a total of 16 single-point mutations with energy higher than −55.18 kcal/mol were T95I, G142D, Δ211, S371L, S373P, K417N, G446S, S477N, E484A, Q498R, N501Y, Y505H, D796Y, Q954H, and N969K. This result indicated that not all mutations that appeared in Spike region on 2019-nCov virus can lead to increased binding capacity with human ACE2 protein. For 12 mutations, the energy is almost unchanged compared with that of the wild-type complex, indicating that these mutation sites do not affect much of the binding patterns between the virus and human beings. In addition, 6 complexes  own lower than −60.00 kcal/mol, accounting for 17.14%, indicating that in both mutations G496S and G339D, the binding energies of simulated system were −65.31 kcal/mol and −62.21 kcal/mol, respectively. Crucially, the multiple-mutant system with 32 mutations on the Spike protein has a minimum energy of −83.29 kcal/mol, which is completely consistent with the expected results, because the Omicron variant has an extremely strong infectious capacity.It can be seen from Fig. For the selected 6 single-point mutations  and 34-point mutation system, one 200-ns-long molecular dynamic simulations was conducted. Molecular dynamics were carried out by using the software Amber 16. Binding analysis and binding free energies for each system were calculated for protein-protein 2019-nCov-Spike/ACE2 complex after equilibrium phase based on the molecular dynamic trajectories.The root mean variance (RMSD) represents the dispersion of centroid coordinates means, which can reflect the structural changes of the protein. During molecular dynamic process, the RMSD trend between initial structure and each time are monitored in real time, and the molecular dynamic simulation is stopped until the RMSD value reached stable.As shown in Fig. 3 (ACE2) and 164 Å3 (Spike), which corresponds with the lowest binding free energies of −55.18 kcal/mol, whereas for 6 single-point mutants T478K, N440K, Q493R, S375F, G339D, and G496S, the contacting surface areas were 179 (ACE2)–237 Å3 (Spike), 180 (ACE2)–240 Å3 (Spike), 179(ACE2)–228 Å3 (Spike), 179 (ACE2)–226 Å3 (Spike), 186 (ACE2)–232 Å3 (Spike), 179 (ACE2)–237 Å3 (Spike), and 202 (ACE2)–260 Å3 (Spike). Interestingly, the six single-point mutants possessed the lower binding energies within −66.37 to approximately −67.99 kcal/mol. Residues of the single-point mutant 2019-nCov-Spike protein forms more intensive binding interface than that of wild-type virus. What is more, among all mutants, the Spike protein of Omicron has the lowest binding capacity to the human ACE2 at −85.80 kcal/mol. As shown in Fig. In order to elucidate the binding patterns of 2019-nCov-Spike to ACE2 protein in different mutants (6 single-point mutants and 34-mutations), we performed a comparative analysis of their structural difference especially the binding interfaces. Assessing the binding surface areas between these possible interfaces indicates that wild-type 2019-nCov-Spike wrap over ACE2 on the lowest level. The predicted interface here was consistent with the crystal structure of 2019-nCov-Spike/ACE2 (PDB ID 6VXX). According to the combined surface area values between 2019-nCov-Spike and human ACE2 protein, the wild-type mutant owns the minimum contacting surface area 206 ÅWe next investigated how the mutation sites influence the regional structure of 2019-nCov-Spike to the human ACE2 protein and analyzed the important residues of 7 mutants which disturb the physicochemical properties of binding interface. The 6 single-point mutants were divided into two categories according to the distance from binding interface: (1) mutation sites not directly form the binding interface ; (2) mutation sites formed direct interactions with ACE2 .As shown in Fig. Similar results were also observed for mutants T478K, Q493R, and G496S. These 3 mutation sites were all located at the binding interface, and direct structural perturbations on the protein structure were observed in all 3 mutants. In wild-type coronavirus, only normal hydrogen bonds were formed between Q493 (Spike) and E35-K31 (ACE2). In the mutant G496S, amino acid S496 of Spike region formed good hydrogen bonds with the adjacent residues K353 and D38 on ACE2. However, mutated residue R493 from the mutant Q493R formed stronger salt-bridging interaction with opposite E35 amino acid. The same situations were also detected in mutant T478K, as new polar interactions were formed between the mutated K478 and Q42-S19 on human ACE2 protein. The key point was that residues K478, R493, and S496 on 34-points’ multiple-mutant Omicron strain simultaneously formed the same interactions as shown in single-point mutants T478K, Q493R, and G496S. Thus, the new mutated residues K478, R493, and S496 formed intensive polar interactions which significantly affect the structural stability of different mutants. Thus, we can infer that these three amino acids K478, R493, and S496 are crucial for high viral infection rates for mutant Omicron strain.The binding interface between the novel coronavirus Omicron 2019-nCov-Spike and human receptor ACE2 plays an important role in the viral infection. Various mutations, especially those mutations on 2019-nCoV-Spike region, can result in dramatic changes in viral transmission. Structural investigations of mutations on Omicron strain that affect the binding affinity of 2019-nCoV-S1/ACE2 complex can supply significant roles in drug and vaccine study and development. In this paper, we constructed protein-protein complex of 34 single-point mutants participating in forming Omicron strain, and then optimized the mutant systems by using molecular dynamic simulations. Comparing with the binding free energies of wild-type coronavirus Spike/ACE2 complex , 6 single-point mutants G339D, S375F, N440K, T478K, Q493R, and G496S possessed relatively lower binding free energy values. Binding mode difference analysis of residues K478, R493, and S496 on 34-points’ multiple-mutant Omicron strain played the key roles in forming intensive polar interactions that maintain structural stability of mutants. Thus, we can infer that these three amino acids K478, R493, and S496 are crucial for high viral infection rates for mutant Omicron strain.


Previously, we found opposite patterns of expression in colon cancer for the genes encoding M1R and M3R. Likewise, M1R and M3R deficiency had opposite effects in a mouse colon cancer model. Based on these observations, we hypothesized that activating M1R might inhibit the growth of colon cancer cells. Here, we confirmed divergent expression of M1R and M3R protein in progressive colon cancer. Then, we discovered that in contrast to M3R activators, treating human colon cancer cells with M1R activators inhibited cell growth. The effects of M1R activation appeared to be mediated by a signaling pathway downstream of the receptor. Notably, M1R activation was more effective than conventional chemotherapy agents at inhibiting colon cancer cell growth and combining these agents augmented this action. We believe our findings support further investigation of selective M1R activators as treatments for advanced colon cancer.M3 muscarinic receptor (M3R) activation stimulates colon cancer cell proliferation, migration, and invasion; M3R expression is augmented in colon cancer and ablating M3R expression in mice attenuates colon neoplasia. Several lines of investigation suggest that in contrast to these pro-neoplastic effects of M3R, M1R plays an opposite role, protecting colon epithelial cells against neoplastic transformation. To pursue these intriguing findings, we examined the relative expression of M1R versus M3R in progressive stages of colon neoplasia and the effect of treating colon cancer cells with selective M1R agonists. We detected divergent expression of M1R and M3R in progressive colon neoplasia, from aberrant crypt foci to adenomas, primary colon cancers, and colon cancer metastases. Treating three human colon cancer cell lines with two selective M1R agonists, we found that in contrast to the effects of M3R activation, selective activation of M1R reversibly inhibited cell proliferation. Moreover, these effects were diminished by pre-incubating cells with a selective M1R inhibitor. Mechanistic insights were gained using selective chemical inhibitors of post-muscarinic receptor signaling molecules and immunoblotting to demonstrate M1R-dependent changes in the activation (phosphorylation) of key downstream kinases, EGFR, ERK1/2, and p38 MAPK. We did not detect a role for drug toxicity, cellular senescence, or apoptosis in mediating M1R agonist-induced attenuated cell proliferation. Lastly, adding M1R-selective agonists to colon cancer cells augmented the anti-proliferative effects of conventional chemotherapeutic agents. Collectively, these results suggest that selective M1R agonism for advanced colon cancer, alone or in combination with conventional chemotherapy, is a therapeutic strategy worth exploring.M In contrast, M2R and M4R (MReven), coupled to Gs, modulate protein kinase activity by altering cellular levels of cAMP. Notably, few studies have examined differences between the actions of muscarinic receptor subtypes within these two subfamilies.Members of the cholinergic muscarinic receptor family, expressed ubiquitously in all organs and tissues examined, play key roles in regulating important normal and neoplastic cell functions . Of the 3R selectively stimulates the expression and release of matrix metalloproteinase (MMP)1, MMP7, and other proteases that facilitate colon cancer cell migration and invasion. Treating human colon cancer cells with non-subtype-selective muscarinic agonists  stimulates cell proliferation, invasion, and intestinal tumor formation in murine colon cancer models. M3R overexpression by primary colon adenocarcinomas predicts metastatic disease and M3R-deficient mice and mice treated with a non-selective muscarinic receptor inhibitor (scopolamine) exhibit attenuated intestinal neoplasia  , reduced in colon cancer compared to a normal colon ] ] A. In conal colon A. Analys 0.044)] A. Moreov1R and M3R protein expression, we used subtype-selective antibodies for immunohistochemistry (IHC), an approach that also allowed us to compare receptor expression in normal and neoplastic areas within the same specimen. As recommended by Jositsch et al. to address the lack of subtype specificity of many anti-muscarinic receptor antibodies [1R and -M3R antibodies from Alomone Labs that were used previously for IHC by our lab [1R- and M3R-deficient, and dual M1R/M3R-deficient mice.For a variety of reasons, including post-transcriptional regulation, mRNA expression may not precisely reflect protein expression. It has been estimated that mRNA expression may account for only 40–60% of the variance in protein expression ,12,13,14tibodies , we used our lab , and who1R and M3R along the continuum from normal colonic mucosa to invasive cancer. Initially, we examined M1R and M3R staining in aberrant crypt foci (ACF), the first histological manifestation of colon neoplasia [APC gene mutations result in aberrant β-catenin signaling and exuberant colon neoplasia , thereby facilitating the acquisition and immunohistochemical comparison of M1R and M3R expression. Staining intensity in 1R and M3R expression levels were increased approximately three-fold in ACF compared to adjacent normal colon . As shown by representative images . In contrast to our findings in ACF, the intensity of M3R staining in adenomas was significantly greater than that for M1R staining . Nonetheless, the intensity of M3R staining in adenocarcinomas was again significantly greater than that for M1R staining  [CHRM1 and CHRM3 was not previously examined. Using qPCR, in HT-29 and H508 cells, we detected substantially higher relative expression of CHRM3 compared to CHRM1 mRNA  , their rRM1 mRNA A; H508 a studies A.1R muscarinic receptor subtype in regulating colon cancer cell proliferation, we employed previously validated orthosteric modulators of M1R activation. First, we tested the effects of McN-A-343, a M1R selective agonist developed over 60 years ago [3R at relatively higher levels than M1R , in tissues with limited expression of M4R, its use can be helpful to distinguish actions resulting from M1R agonism [1R agonism inhibited colon cancer cell proliferation, we tested the effects of another putative M1R selective agonist, xanomeline [50 xanomeline vs. McN-A-343 = 23.4 µM vs. 136.8 µM).To explore the role of the Mears ago , on well M1Rlow; A and who agonism ,28,29. Wnomeline ,31,32. A1R in human colon cancer cells selectively stimulates anti-proliferative cell signaling, not cell damage. Next, to test the reversibility of the effects of 0.3 and 1 mM McN-A-343 on cell proliferation, we incubated H508 cells with the M1R agonist for two days and then washed and re-incubated cells in culture medium without McN-A-343 for an additional three days. As shown in 1R agonist resumed proliferation, findings consistent with effects mediated by reversible M1R activation. These findings also supported the conclusion that the effects of the selective M1R agonist were not a consequence of cell toxicity.We used two approaches to exclude non-specific actions of these agents on colon cancer cell viability. First, to determine if cellular toxicity could account for the effects of McN-A-343 on cell proliferation, we measured the cellular release of lactate dehydrogenase (LDH), a commonly used indicator of cell membrane damage that is stable for long periods under our in vitro test conditions . As show1R (McN-A-343) agonist did not alter cleaved caspase-3 levels. Based on these findings, we concluded that neither induction of cellular senescence nor apoptosis explained the anti-proliferative effects we observed in cells treated with selective M1R agonists.We next performed experiments to explore the possibilities that stimulation of cellular senescence or apoptosis might explain the results observed with McN-A-343. To test for changes in cell senescence, we determined if treating H508 cells with McN-A-343 altered senescence associated-β-galactosidase staining, a validated marker of premature senescence ,36. Trea1R agonists were not idiosyncratic to H508 cells and to explore the effects of varying relative expression levels of M1R and M3R (1Rlow) and HCT116 (M1Rhigh) cells. As shown in 1R agonist in H508 and HT-29 human colon cancer cells; both cell lines are M1R low (50) was 7.89 µM for HCT116 cells, 136.8 µM for H508 cells, and 548.1 µM for HT-29 cells , a selective M1R antagonist [3R-predominant agonist. Then, we tested the effect of pretreating cells with a fixed concentration of VU on the dose–response curve for McN-A-343-induced inhibition of colon cancer cell proliferation. As shown in 50 with McN-A-343 alone was 136.8 µM versus 293.0 µM for McN plus VU; p = 0.0165). Moreover, as shown in p = 0.015). Collectively, these findings supported the concept that activating M1R inhibits colon cancer cell proliferation.Next, we determined if blocking Mtagonist , on carb1R activation, we used selective inhibitors for several kinases whose actions were previously implicated in post-muscarinic receptor signaling in colon cancer; we had previously validated the actions of these inhibitors in human colon cancer cells [p < 0.0001). As shown in p = 0.003). We previously showed that transactivation of EGFR occurs downstream of muscarinic receptor activation [To identify relevant signaling proteins downstream of Mer cells . First, tivation .As both ERK and p38 MAPK activation are downstream of EGFR activation , we purs1R agonist with currently used agents for colon cancer chemotherapy had therapeutic potential. Interestingly, we found that treating H508 human colon cancer cells with a submaximal dose of McN-A-343 (200 µM) in combination with either 100 µM 5-fluorouracil (5-FU)  E or 10 µl (5-FU) F potenti1 and M3 muscarinic receptors play in colon cancer progression, and the findings that they appear to be reciprocal regulators of colon cancer cell proliferation opens the door to therapeutic opportunities. CHRM3 and M3 muscarinic receptors are commonly over-expressed in colon cancer [Chrm3 or pharmacological inhibition of M3R activation in murine models of colon cancer attenuates tumor formation [CHRM1 mRNA expression is significantly reduced in human colon neoplasia [Chrm1 ablation in mouse models negates the anti-neoplastic effects of Chrm3 ablation [Chrm1/M1R expression appears to protect against colon cancer. This concept was again supported in the present work by our interrogation of a large public colon cancer database which confirmed that while CHRM3 levels were significantly elevated in cancer compared to those in normal colon tissue, CHRM1 levels were modestly, but significantly, reduced in cancer.The work presented here expands our understanding of the divergent roles Mormation . In conteoplasia  and Chrmablation ; that is1R and M3R protein expression in progressive stages of colon neoplasia. Notably, we observed that the relative expression of M3R compared to M1R increases with the progressive pre-metastatic development of colon cancer from ACF to adenomas to primary adenocarcinomas. Whereas M1R and M3R expression was similar in a normal colon and the expression of both receptor subtypes increased equally in ACF, the ratio of M3R to M1R expression was significantly enhanced in adenomas and primary colon cancers. It is possible, although admittedly highly speculative, that increased M1R expression in colon ACF, adenomas, and cancers is a compensatory mechanism in reponse to greatly increased M3R expression and, thus, an anti-neoplastic effort on the part of cells to maintain homeostasis.Due to epigenetic and other effects, the functional component of gene expression, protein expression, may not mirror changes in mRNA levels ,42,43. T1R levels in lymph node and liver metastases, although increased compared to normal colon tissue, were not increased compared to primary cancers  to colon cancer cells, we detected no meaningful changes in LDH release, senescence-activated β-galactosidase activity, or cleaved caspase-3 generation. Nonetheless, we did observe a modest reduction in total caspase-3 levels in cells treated with the highest McN-A-343 concentrations. Although speculative, it is possible that McN-A-343 reduces expression of caspase-3 by a currently unclear mechanism. Moreover, our findings contrasted with those from investigators who observed that in HEK293 cells overexpressing M1R, treatment with carbachol induced apoptosis by an ERK-independent mechanism [1R expression, carbachol-induced cell death was observed in HEK293 cells expressing supra-physiological levels of M1R, a caveat highlighted by the authors [1R agonists and support the hypothesis that the anti-proliferative effects of M1R agonists derive from specific alterations in post-M1R signaling. This hypothesis was supported by the results of cell proliferation experiments that examined the effects of pre-treating cells with selective kinase inhibitors before adding an M1R agonist and detected M1R agonist-induced changes in kinase activation by immunoblotting for phosphorylated (activated) kinases.Several experiments provided evidence that the inhibitory effects of Mechanism . We ascr authors . Indeed,1R agonist-induced attenuation of colon cancer cell proliferation, we examined the effects of blocking key kinases in the muscarinic receptor signal transduction pathway [1R agonist-induced attenuation of cell proliferation; validated EGFR and p38 MAPK inhibitors significantly attenuated the actions of McN-A-343 on cell proliferation . Additionally, our experiments examining the effects of combining an M1R agonist with conventional colon cancer chemotherapeutics (1R agonism, alone or in combination with these agents, is a potential therapeutic strategy worthy of further investigation.In summary, we have demonstrated a novel anti-proliferative effect of Meoplasia  suggestsapeutics E,F sugge1R agonists to treat a host of neurological and psychiatric disorders, including schizophrenia and Alzheimer disease [1R-selective blood–brain barrier permeable agonist, with trospium, a non-selective muscarinic receptor inhibitor that does not cross the blood–brain barrier. The trospium moiety is intended to mitigate the off-target peripheral cholinergic side-effects of xanomeline, e.g., blurred vision, excess sweating and salivation, and urinary frequency. Marketed as KarXT by Karuna Therapeutics, this agent demonstrated an acceptable safety profile in Phase 2 clinical trials and is currently in Phase 3 trials for psychosis and schizophrenia [3R antagonism and M1R agonism may have great therapeutic promise to treat advanced colon cancer.Novel insights into the structural biology and mechanisms whereby muscarinic receptors regulate fundamental functions of the central and enteric nervous systems are driving drug development . There i disease ,48. For ophrenia ,50. Simi


Saccharomyces cerevisiae, the diverging H2A.Z C terminus is thought to provide the H2A.Z exclusive functions. To elucidate the roles of this H2A.Z C terminus genome-wide, we used derivatives in which the C terminus was replaced with the corresponding region of H2A (ZA protein), or the H2A region plus a transcriptional activating peptide (ZA-rII′), with the intent of regenerating the H2A.Z-dependent regulation globally. The distribution of these H2A.Z derivatives indicates that the H2A.Z C-terminal region is crucial for both maintaining the occupation level of H2A.Z and the proper positioning of targeted nucleosomes. Interestingly, the specific contribution on incorporation efficiency versus nucleosome positioning varies enormously depending on the locus analyzed. Specifically, the role of H2A.Z in global transcription regulation relies on its C-terminal region. Remarkably, however, this mostly involves genes without a H2A.Z nucleosome in the promoter. Lastly, we demonstrate that the main chaperone complex which deposits H2A.Z to gene regulatory region (SWR1-C) is necessary to localize all H2A.Z derivatives at their specific loci, indicating that the differential association of these derivatives is not due to impaired interaction with SWR1-C.The incorporation of histone variant H2A.Z into nucleosomes creates specialized chromatin domains that regulate DNA-templated processes, such as gene transcription. In IMPORTANCE We provide evidence that the Saccharomyces cerevisiae C-terminal region of histone variant H2A.Z can mediate its special function in performing gene regulation by interacting with effector proteins and chaperones. These functional interactions allow H2A.Z not only to incorporate to very specific gene regulatory regions, but also to facilitate the gene expression process. To achieve this, we used a chimeric protein which lacks the native H2A.Z C-terminal region but contains an acidic activating region, a module that is known to interact with components of chromatin-remodeling entities and/or transcription modulators. We reasoned that because this activating region can fulfill the role of the H2A.Z C-terminal region, at least in part, the role of the latter would be to interact with these activating region targets. The eukaryotic genome is organized as chromatin, of which the nucleosome is the basic unit. An individual nucleosome consists of an octameric histone core, formed by two H2A-H2B dimers and a H3-H4 tetramer, enveloped by 146 bp of DNA. This complex chromatin structure represents an obstacle to DNA-based processes, including gene transcription, DNA replication, and DNA repair. Therefore, for all of these transactions, nucleosomes must be remodeled in order to make DNA accessible. Several fundamental mechanisms can alter chromatin structure, including post-translational modifications of histones, which may modify their properties or interacting partners; ATP-dependent chromatin remodeling; and the replacement of replicative histones by histone variants which change the histone composition of nucleosomes. The latter mechanism creates specialized chromatin domains that can be permissive or not permissive to transcription  , 12, 14 9–29–Previous studies of the H2A.Z C-terminal region used either a complete replacement of the region by the corresponding region of H2A, H2A.Z C-terminal truncations of various lengths, or point mutations in the H2A.Z docking domain , 33–37. 33–33–Notwithstanding these studies, the involvement of the H2A.Z C-terminal docking domain has not been studied with regard to genome-wide localization and transcriptional control. Thus, in order to determine the role of the H2A.Z C-terminal docking domain in the functions of H2A.Z-containing nucleosomes, we used chimeric proteins derived from H2A.Z which bore modified C-terminal regions . Surprishtz1 cells. Previous data showed that the expression of the ZA chimera protein affects the expression of H2A.Z-regulated genes, namely, GAL1, GAL7, GAL10, and PUR5, and renders the cell more sensitive to genotoxic stress compared to H2A.Z-expressing cells  to map H2A.Z and the H2A.Z derivatives across the yeast genome. ChIP assays were performed using yeast strains bearing hemagglutinin (HA) epitopes on H2A.Z, ZA, and ZA-rII′. In each case, a ChIP for histone H3 (using an anti-H3 antibody) was hybridized together with the H2A.Z, ZA, or ZA-rII′ ChIPs to control for nucleosomal density. Genomic data visualization using the UCSC Genome Browser allowed us to determine that the H2A.Z, ZA, and ZA-rII′ proteins are distributed across the yeast genome in a non-random manner see 9–, 16, 36.2 ratio IP/H3) over these chromosome elements and the TSS of the respective gene classes using the Versatile Aggregate Profiler (VAP) tool , P values were well below 0.01, suggesting that all differences were significant. The results of these t tests are shown in Fig. S2. We conclude that the H2A.Z C-terminal docking domain has a role in the efficient recruitment of H2A.Z not only to promoters of protein-coding genes, but also to promoters of noncoding RNAs and on other chromosomal loci. Furthermore, its localization near centromeres, replication origins, and snoRNA genes is absolutely dependent on the C-terminal docking domain.H2A.Z is involved in several cellular processes and multiple groups have reported an association of H2A.Z with particular chromosome elements and other transcribed elements 9–, 32, 35.AP) tool . We founAP) tool  and B, wAP) tool  to E, suAP) tool  and D. IAP) tool . InteresAP) tool , but notAP) tool . We perfPHO5, PUR5, and GAL   . The enrP/Input) . The detP/Input) . HoweverP/Input) , indicat2 ratio ≥ 1.00) of H2A.Z, ZA, or ZA-rII′ at the TSS. We identified 2,240 genes that showed an enrichment of H2A.Z at the TSS and referred to them as “H2A.Z-associated genes.” These genes represent 44% of the yeast verified ORFs and are potentially transcriptionally regulated by H2A.Z. We also identified 1,128 ZA-rII′-associated genes (22% of verified ORFs) and 370 ZA-associated genes (7% of verified ORFs). Comparison of the pool of H2A.Z-associated genes with the ZA-associated genes showed that only 4% of ZA-associated genes are specific to this protein and 96% are in the pool of H2A.Z-associated genes  to analyze the effect of replacement of the H2A.Z C terminus on gene expression. We compared the transcriptomes of Δhtz1 cells (2-fold change ≥ 1.00 and P ≤ 0.05) were identified in the Δhtz1 strain compared to the WT, 530 differentially expressed genes in the ZA strain, and 427 differentially expressed genes in the ZA-rII′ strain  was plotted against ranked differential gene expression values (log2-fold change over H2A.Z strain)   and F, n strain) , 29. Thuswr1). We reasoned that if ZA, particularly ZA-rII′, functioned independently of SWR1-C, their association to gene regulatory regions would not be significantly affected by the loss of SWR1-C. swr1 cells. As expected, WT H2A.Z shows the strongest association to the TSS region of genes, followed by ZA-RII′ and then ZA. Importantly, and in all three cases, there is a loss of significant association of the H2A.Z derivatives to the TSS region of genes in the absence of the SWR1 complex. These results clearly demonstrate that differential association of H2A.Z derivatives to chromatin regions cannot be attributed to a problem with their association to the main H2A.Z chaperone complex.One important issue that needed to be addressed was whether or not the differential association of H2A.Z derivatives to chromatin would mean that they would no longer depend on physical association to SWR1-C, an important H2A.Z chaperone, and would therefore function through a different chromatin-depositing factor. To address this, we performed ChIP-seq experiments using HA-tagged versions of H2A.Z, ZA, and ZA-RII′ in wild-type cells or cells lacking SWR1 , and one which additionally contained an acidic activation domain (ZA-rII′ protein). We performed ChIP-chip on these two variant proteins and compared the results of the chimaera proteins with those of WT H2A.Z. First of all, the overall localization of the WT H2A.Z reassessed here coincides almost perfectly with previous data 9–, 14, 29.As expected , ZA protThe changes in localization patterns of H2A.Z derivatives over RNA Pol III-transcribed tRNA genes are remarkably similar to Pol II promoters; that is, the replacement of the H2A.Z C-terminal region caused a reduced occupancy of H2A.Z derivatives at the +1 nucleosome at tRNA promoters and the rII′ addition complements this loss almost back to WT levels.HZAD loci are large areas encompassing at least several nucleosomes that are enriched in H2A.Z. Again, the ZA derivative is almost completely lost from these areas and the rII′ peptide does rescue this loss somewhat ; thus, tWhile snoRNA genes are transcribed by RNA polymerase II, there was clearly a different effect upon the replacement of the H2A.Z C-terminal region. First, the reduction in occupancy at the +1 nucleosome was similar for both H2A.Z derivatives, thus with no recovery for the ZA-rII′ protein. Second, the ZA- and ZA-rII′-nucleosomes appeared in two distinct peaks on either side of the TSS compared to one strong enrichment peak upstream of the TSS for the WT H2A.Z . At presThere was clearly a different distribution of the H2A.Z derivatives in the chromatin surrounding centromeres and replication origins compared to WT H2A.Z –B. For eSWR1 (Δswr1), the binding of all H2A.Z chimeras was nearly abolished, suggesting that ZA and ZA-RII′ work primarily through SWR1-C for their genomic localization. This is also in line with a recent study by Brewis et al.  was used in this study. The Δeviously , 33. Briof H2A.Z . The ZA-600) of 0.6 and fixed with 1% formaldehyde for 10 min. Next, 350 μL of sonicated whole-cell extract was incubated with either anti-HA  or anti-H3  antibody coupled to magnetic beads  overnight at 4°C with agitation. Immunoprecipitated DNA was used for genome-wide location analysis.ChIP experiments were performed in duplicates as previously described , 9. BrieFor the ChIP-chip, H2A.Z, ZA, and ZA-rII′ ChIP DNA, labeled with Cy5, was hybridized in competition with Cy3-labeled H3 ChIP or input DNA. The microarrays used for location analysis have been described previously  and wereFor the ChIP-seq, ChIP experiments were performed in duplicates as previously described . For antGSE156492 (enter token gzozeeimrrqlrqd into the box).The ChIP-chip data were normalized using the Limma Loess method, and replicates were combined using a weighted average method as described previously . The datSaccharomyces cerevisiae R64-1-1 reference genome using Burrows-Wheeler Aligner (BWA) v0.7.17 (https://bitbucket.org/mugqic/genpipes/src/master/pipelines/chipseq/) as follows:Reads were analyzed using the GenPipes ChIP-seq pipeline Macs2 callpeak -fix-bimodal -gsize 9725684.0 -nolambdaHomer annotatePeaks.pl -gsize 9725684.0 -cons -CpGHomer findMotifsGenome.plhttps://bitbucket.org/labjacquespe/vap_interface/downloads/vap-2.0.0-alpha3.dmgDownload VAP from http://ftp.ensembl.org/pub/release-77/gtf/saccharomyces_cerevisiae/Saccharomyces_cerevisiae.R64-1-1.77.gtf.gz)Annotation GTF from Saccharomyces_cerevisiae R64 downloaded from Ensembl release 77 -10(inter)-25(ref)-6(inter)-6(down)Windows to smooth: 6# dataset/graph: allChIP-seq reads were analyzed using the GenPipes ChIP-seq pipeline . Briefly v0.7.17 . Reads a v0.7.17 . Next, H v0.7.17 . Peak cahtz1 strain and on Δhtz1 strains with an integrated copy of the HTZ1 gene or the ZA or ZA-rII′ constructions. Total RNA extraction was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research) according to the manufacturer’s specifications. Illumina sequencing libraries were prepared using 3′-B and 5′-A adaptors, as well as IGA-PCR-PE-F, TruSeq-MPEX-R, DSN-TruSeq-F, and DSNTruSeq-R primers. The trimmed reads were aligned using Tophat37 onto a genome composed of the S. cerevisiae S288C genome (sacCer3). DESeq2 was used to identify the differentially expressed genes. Yeast genes showing a differential expression with an adjusted P value of <0.05 and having an absolute log2-fold change of >1.00 were considered differentially expressed. The Gene Ontology term enrichment analysis for differentially expressed genes was investigated using Metascape (https://metascape.org/gp/index.html#/main/step1). All data sets for the RNA-seq experiment were deposited into NCBI GEO database under accession no. PRJNA657613.The RNA-seq analysis was performed in triplicates on a Δhttp://www.ncbi.nlm.nih.gov/geo/) and are accessible under accession no. GSE156492 and PRJNA657613.The raw reads and processed files from the ChIP-chip, ChIP-seq, and RNA-seq experiments have been deposited in the NCBI Gene Expression Omnibus (GEO) database (


Upon encounter of full-length spike antigen, CoVAX_MNS-induced CD4+ T cells were responsible for accelerated CD8+ T cell and IgG Ab responses specific to the incoming spike, irrespective of its sarbecovirus origin. Finally, both vaccines elicited partial protection against a lethal SARS-CoV-2 challenge in human angiotensin-converting enzyme 2–transgenic mice. These results support clinical testing of these universal sarbecovirus vaccines for pandemic preparedness.SARS-CoV-2 is the third zoonotic coronavirus to cause a major outbreak in humans in recent years, and many more SARS-like coronaviruses with pandemic potential are circulating in several animal species. Vaccines inducing T cell immunity against broadly conserved viral antigens may protect against hospitalization and death caused by outbreaks of such viruses. We report the design and preclinical testing of 2 T cell–based pan-sarbecovirus vaccines, based on conserved regions within viral proteins of sarbecovirus isolates of human and other carrier animals, like bats and pangolins. One vaccine (CoVAX_ORF1ab) encoded antigens derived from nonstructural proteins, and the other (CoVAX_MNS) encoded antigens from structural proteins. Both multiantigen DNA vaccines contained a large set of antigens shared across sarbecoviruses and were rich in predicted and experimentally validated human T cell epitopes. In mice, the multiantigen vaccines generated both CD8  Within a year after the start of the SARS-CoV-2 outbreak, the first COVID-19 vaccines received Emergency Use Authorization from the FDA. The mRNA- and adenovirus-based vaccines were highly effective against infection, infection spreading, disease, and death caused by the original SARS-CoV-2 Wuhan-Hu-1 strain and its D614G variant –8. As a Sarbecovirus subgenus, share the greatest sequence similarity, and both viruses rely on the ACE2 receptor to enter cells. Few Abs raised by SARS-CoV-1 infection or vaccination neutralize SARS-CoV-2, and the Abs raised by SARS-CoV-2 infection or vaccination have limited activity against SARS-CoV-1 , together representing a cross section of all sarbecovirus branches , nucleoprotein (N), or S or from the nonstructural ORF1ab proteins of the SARS-CoV-2 Wuhan-Hu-1 sequence NC_045512.2), based on sequence conservation and HLA class I binding. To this end, the aa sequences of 16 sarbecoviruses  and 1 for M, N, and S antigens (CoVAX_MNS). Two versions of each vaccine were produced, 1 lacking and 1 containing 3 C-terminal S–derived CD8b-restricted reporter CD8+ T cell epitope were tracked in blood using tetramer staining. Both the CoVAX_MNS . Up to 0.9% of CD8+ T cells produced TNF and IFN-γ in response to 1 of these peptides (TGYHFREL), predicted to bind H-2Kb . CD4+ T cells in bulk cultures from vaccinated, but not control, mice upregulated CD40L and produced TNF specifically in response to CoVAX_MNS-derived shared S peptide VQIDRLITGRLQSLQTYVTQQLIRAAEIRA , CD8+ T cells with this specificity were approximately 5-fold more frequent in the blood of CoVAX_MNS compared with control mock- or CoVAX_ORF1ab-vaccinated mice (+ T cells nor Abs were detected (data not shown). For both types of responses, depletion of CD4+ T cells prior to S exposure eliminated this CoVAX_MNS effect . Thus, T cell responses against shared sarbecovirus antigens induced by vaccination accelerated CD8+ T and B cell responses specific to a diverse array of incoming sarbecovirus S proteins.Because the CoVAX_MNS antigens are shared across sarbecoviruses by design, vaccine-induced T cell responses to conserved epitopes would be expected to improve not only CD8cination . Compare+ T cell , 20-fold+ T cell , and 50-+ T cell  Ab respo+ T cell . At this+ T cell responses to the conserved M and ORF1ab antigens ; the other, antigens from structural proteins . Both DNA vaccines contained a large set of antigens shared across sarbecoviruses and rich in predicted and validated human T cell epitopes. Injected intradermally into C57BL/6 mice without adjuvant, the multiantigen vaccines generated both CD8The shared antigenic domains were selected for inclusion based on strict criteria regarding aa sequence identity to ensure that all T cell epitopes would be identical across sarbecoviruses. First and foremost, this reduces the risk that the fraction of vaccine-induced T cells contributing to protection will diverge among sarbecoviruses. Second, this reduces the risk that the “original antigenic sin” phenomenon , 63 reduA*01:01, A*02:01, A*03:01, A*11:01), A24 , B07-B35 , B08-B18-B39 (B*08:01), B15-B40 (B*40:01), and B44   . In addiB*44:03) , CoVAX_Oulations . In shor+ T cells accelerated the generation of class-switched IgG Abs specific for the incoming S. In vitro, these CD4+ T cells upregulated CD40L and produced TNF upon S peptide stimulation, demonstrating that vaccination elicited S-specific T helper type 1 (TH1) cells, which explains class switching to IgG2c. The most likely scenario is that upon encountering the S, the S-specific CD4+ T cells, including TH1 and, potentially, T follicular helper cells, are recruited to lymph nodes and promote a germinal center response, which involves interaction with naive, S-specific B cells to promote their activation and differentiation into Ab-secreting plasmablasts presenting S epitopes to CD4+ and CD8+ T cells in lymph nodes. This effect on CD8+ T cell responses is also likely to occur with CoVAX_ORF1ab. Thus, when a vaccinated individual is infected with a sarbecovirus, vaccine-induced T cells specific for conserved antigens will not only attack infected cells directly but also accelerate the activation of new T and B cells recognizing nonconserved epitopes. These accelerated CD8+ T and Ab responses specific for the incoming sarbecovirus may well form a crucial part of the protection afforded by this type of vaccine. Similarly, our data suggest that individuals vaccinated with this type of pan-sarbecovirus vaccine will demonstrate improved responses to conventional S-based vaccines directed against any sarbecovirus.When mice were exposed to S antigen after vaccination, CoVAX_MNS-induced CD4mablasts . Similar+ T cell as well as conserved sarbecovirus CD8+ T cell epitope peptides from N, M, and the S2 part of the S proteins known to bind to multiple class I and class II HLA alleles  based on the following 2 criteria: shared and immunogenic.“Shared” refers to identical in the sarbecoviruses listed in A*01:01, A*02:01, A*03:01, A*11:01, A*23:01, A*24:02) or HLA-B  alleles  or HLA-B  alleles  epitope VNFNFNGL  epitope (K)CYGVSATKL (E. coli cultures and purified using Nucleobond Xtra Maxi EF columns (MACHEREY-NAGEL) according to the manufacturer’s instructions. For vaccination, plasmids were column-purified twice, each time using a fresh column. Flow cytometry and Western blot analysis demonstrated that 293T cells (ATCC) transfected with CoVAX_MNS or CoVAX_ORF1ab, indeed, expressed HA-tagged proteins of the correct size (data not shown).Two plasmid DNA vaccines were designed, 1 containing M, N, and S antigens  under control of the cytokeratin 18 (K18) promoter , were obFor each DNA vaccination, 50 μg of DNA, dissolved in 30 μL of a sterile buffer , was injected intradermally at the base of the tail using a 0.5 mL U-100 Insulin 29G Micro-Fine needle . Mock-vaccinated mice received 30 μL of 0.9% NaCl, 0.09 mM Tris at pH 8.0, and 0.009 mM EDTA at pH 8.0.Peripheral blood from the tail vein was collected in heparin tubes (Sarstedt). Splenocytes were obtained by mincing the tissue through 70 μm nylon cell strainers . Blood cells and splenocytes were depleted of erythrocytes using ammonium chloride lysis buffer.For tetramer staining, cells were washed with PBA  and incubated for 30 minutes at room temperature (RT) with tetramers, followed by another 30 minutes on ice in the presence of anti-CD8α.For ICS, splenocytes were cultured with peptide-loaded D1  cells foAb details are listed in 2SO4, and within 5 minutes, the plates were measured with a microplate reader  at 450 nm.ELISAs were performed to determine Ab titers in sera. Nunc MaxiSorp ELISA plates (Merck) were coated with 1 μg/mL spike S1 plus S2ECD-His recombinant protein  in ELISA coating buffer (BioLegend) overnight at 4°C. Plates were washed 5 times and blocked with 1% BSA in PBS with 0.05% Tween 20  for 1 hour at RT. Plates were washed and incubated with serial dilutions of mouse sera and incubated for 1 hour at RT. Plates were again washed and then incubated with a 1:4,000 dilution of HRP-conjugated anti–mouse IgG secondary Ab  and incubated for 1 hour at RT. To develop the plates, 50 μL of 3′3′,5′,5′-tetramethylbenzidine  was added to each well and incubated for 5 minutes at RT. The reaction was stopped by the addition of 50 μL 1 M H3 PFU of SARS-CoV-2 in a total volume of 50 μL DMEM. Mouse weight and clinical discomfort were monitored daily. Euthanasia criteria were weight loss of greater than 20% of BW compared with the prestudy weight and a moribund state. All experiments with SARS-CoV-2 were performed in the Biosafety Level 3 laboratories at the LUMC.Clinical isolate SARS-CoV-2/human/NLD/Leiden-0008/2020  was used for the SARS-CoV-2 infection of mice. The next-generation sequencing data of this virus isolate are available under GenBank accession number MT705206.1 and show 1 mutation in the Leiden-0008 virus S protein compared with the Wuhan S protein resulting in Asp>Gly substitution at position 614 (D614G). In addition, several nonsilent (C12846U and C18928U) and silent mutations  in other genes were found. Isolate Leiden-0008 was propagated and titrated in Vero-E6 cells . K18-hACE2tg mice were anesthetized with isoflurane gas and infected i.n. with 5 × 10P values are depicted in the figures as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.002, and ****P ≤ 0.0004.Statistical analyses were performed using GraphPad Prism (version 9.4.1), using the statistical tests mentioned in the figure legends. Multiplicity-adjusted All animal experiments were performed in accordance with Dutch Animal Ethical Committee guidelines and were approved by the Animal Welfare body of LUMC , and performed according to the recommendations and guidelines set by LUMC and by the Dutch Experiments on Animals Act.Values for all data points in graphs are reported in the JVB and MGMC were responsible for antigen selection, GCZ was responsible for vaccine design, cloning, and production. MGMC, INP, DV, WYL, SKM, and AAL performed experiments. MGMC, JVB, GCZ, and FO interpreted experiment results. MK and RA provided essential advice. FO supervised the project. JVB wrote the manuscript. On these grounds, and given that MGMC performed the large majority of the experiments, we consider the contributions of JVB and MGMC of comparable value and shared first authorships justified. Because JVB had a greater intellectual contribution to the project and wrote the manuscript, his name precedes that of MGMC on the author list.


The aberrant expression of BAFF is related with the autoimmune diseases development like Systemic Lupus Erythematosus (SLE) for promoting self-reactive B cells survival. BAFF functions are exerted through its receptors BAFF-R (BR3), transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA) that are reported to have differential expression on B cells in SLE. Recently, atypical B cells that express CD11c have been associated with SLE because they are prone to develop into antibody-secreting cells, however the relationship with BAFF remains unclear. This study aims to analyze the BAFF system expression on CXCR5Forty-five SLE patients and 15 healthy subjects (HS) were included. Flow cytometry was used to evaluate the expression of the receptors in the B cell subpopulations. Enzyme-linked immunosorbent assay (ELISA) was performed to quantify the soluble levels of BAFF (sBAFF) and IL-21.- CD11c+ atypical B cell subpopulations DN2, aNAV, SWM and USM B cells in SLE patients compared to HS. SLE patients had increased expression of membrane BAFF (mBAFF) and BCMA receptor in classic B cell subsets . Also, the CXCR5+ CD11c- DN1, resting naïve (rNAV), SWM and USM B cell subsets showed higher mBAFF expression in SLE. CXCR5- CD11c+ atypical B cell subpopulations DN2, SWM and USM B cells showed strong correlations with the expression of BAFF receptors. The atypical B cells DN2 in SLE showed significant decreased expression of TACI, which correlated with higher IL-21 levels. Also, lower expression of TACI in atypical B cell DN2 was associated with high disease activity.We found increased frequency of CXCR5- CD11c+ atypical B cell subsets in SLE patients. Decreased TACI expression on atypical B cells DN2 correlated with high disease activity in SLE patients supporting the immunoregulatory role of TACI in autoimmunity.These results suggest a participation of the BAFF system in CXCR5 Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by hyperactive B cells and autoantibody production . BAFF B–4. BAFF - IgD- CXCR5- CD11c+) and activated naïve (aNAV) B cells (CD27- IgD+ CXCR5- CD11c+). aNAV B cells are precursors of DN2 B cells and differentiate into PC through IL-21, IFN-γ and TLR7 stimulation  were isolated from fresh peripheral blood with Histopaque-1077  by density gradient, and stained using standard flow cytometry methodology with the antibodies described in the Serum was separated from the total blood of each subject at the time they were involved in the study and were stored at -20°C. Serum cytokine levels of BAFF and IL-21 were quantified in SLE patients and HS by Enzyme-Linked immunosorbent Assay (ELISA) according to the manufacturer’s instructions. Soluble BAFF was measured with Quantikine Human BAFF/BLyS/TNFSF13B  and IL-21 was measured with MAX Deluxe Set Human IL-21 .post hoc. Correlations were calculated with Spearman test. Statistical significance was represented as follows: ns was p>0.05, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.Data were analyzed using GraphPad Prism v.9. Categorical variables are presented as absolute values. Continued variables are presented as medians with 25th-75th percentiles. Normality tests were performed, and data didn’t have normal distribution. Statistical differences between for 2 groups were calculated with Mann-Whitney U test and for ≥3 groups with Kruskal-Wallis’s test and Dunn’s test as Forty-five SLE patients and 15 healthy subjects were included in this study, all females with a median age of 36 (27-46) years for SLE and 32 (21-50) for HS. The clinical features of SLE patients are described in - IgD-), naïve B cells NAV (CD27- IgD+), switched memory B cells SWM (CD27+ IgD-) and unswitched memory B cells USM (CD27+ IgD+) (+ CD11c-) and DN2 B cells (CXCR5- CD11c+). The NAV B cells were separated into resting naïve (rNAV) (CXCR5+ CD11c-) and activated naïve (aNAV) (CXCR5- CD11c+). Also, the memory B cells were sub-stratified using the CXCR5 and CD11c expression according to that reported by Sanz et al., 2019, in SWM (CXCR5+ CD11c-) and SWM (CXCR5- CD11c+); USM (CXCR5+ CD11c-) and USM (CXCR5- CD11c+)  B cells were decreased in SLE patients compared with HS  , rNAV , CXCR5+ CD11c- SWM  and CXCR5+ CD11c- USM  , aNAV , CXCR5- CD11c+ SWM  and CXCR5- CD11c+ USM  . Also, the frequency of BCMA positive B cells were elevated in SLE patients  , rNAV , CXCR5+ CD11c- SWM  and CXCR5+ CD11c- USM   compared to HS  of SLE patients  and aNAV  (- CD11c+) and USM (CXCR5- CD11c+) B cells , soluble levels of IL-21 were higher in SLE patients compared to HS  , DN B cells  and DN1 B cells  (MexSLEDAI score was negatively correlated with the expression (MFI) of TACI in DN2 B cells  and BCMA in aNAV B cells   in SLE patients. This decreased USM frequency was reported to be related with the increase of DN B cells (CD27- IgD-) after TLR9 stimulation . ABCs have been described previously by the expression of CD11c and T-bet . This elevated expression of mBAFF could be a consequence of B cells activation related to SLE pathogenesis. The expression of membrane-bound BAFF (mBAFF) is null in resting B cells; however, in vitro studies have shown that B cells can express mBAFF when they are activated by TLR9 through MyD88  as well as in all the CXCR5gh MyD88 , 25. B cgh MyD88  and its gh MyD88 . CXCR5- + T-bet+), a lower BR3 expression has been previously reported (- CD23-) that have similar expression of BR3 with the follicular B cells and have the same capacity to bind BAFF but doesn’t rely on the BAFF/BR3 axis to survive . The elevated expression of BCMA on B cells have been reported in previous studies. Also, this elevated expression of BCMA could be related with TLR9 stimulation (- CD11c+ atypical B cells with no differences between SLE and HS. This increased expression of BCMA on CD11c+ B cells could be associated with the capacity of BCMA to promote Ag-presentation through JNK-axis activation (+ B cells because of their interrelationship with IL-21, nevertheless, it has been hypothesized that CD11c+ B cells can stimulate Tfh cells through strong Ag-presentation (In addition, our work showed that SLE patients had an increased expression of BCMA on rNAV, CXCR5mulation , 33. In tivation . T follientation , 36. Alt- CD11c+ atypical B cells DN2 and aNAV, showed decreased expression of TACI in SLE patients compared with HS. This finding is interesting because of the paradoxical functions of TACI in B cells (+ B cells with Tfh-like cells could be mediated by ICOS-ICOSL because TACI deficiency can increase ICOSL expression on GC B cells in mice (- CD11c+ atypical B cells DN2 and aNAV in SLE could be responsible in part for loss of regulation of these populations, highlighting the BAFF role in the SLE pathogenesis.Furthermore, we found an increased expression of TACI on rNAV B cells of SLE patients. However, the CXCR5 B cells . It’s kn B cells , but the B cells . In addi B cells  that pro in mice , 41. The in mice . Althoug in mice . Also, i in mice . We hypoin vivo.Finally, levels of sBAFF were positively correlated with aNAV frequency, whereas the expression of BR3 on aNAV B cells was negatively correlated with sBAFF levels. This correspond with previous reports where the decreased expression of BR3 was correlated with elevated levels of sBAFF , 28, 30.+ CD11c- SWM, DN and DN1 B cells. The above highlights the regulatory role of TACI in B cells homeostasis, which appears to be affected in the autoimmunity context.We previously have reported that the decreased expression of TACI and BCMA on B cells was related with the disease activity in SLE . In this- CD11c+ atypical B cell subsets in SLE patients. Decreased TACI expression on DN2 B cells correlated with high disease activity in SLE patients supporting the immunoregulatory role of TACI in autoimmunity.These results suggest a participation of the BAFF system in CXCR5The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by The ethics in research committee of the Hospital General de Occidente, Guadalajara, Mexico. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.JG, DS-C and CP-S contributed with conception of the study. JG, IR-F, PO-L and CP-S contributed with the design of the experiments. MM-R and NS-F contributed with patients’ inclusion. JG and IR-F done data acquisition. JG, DS-C, NE-G and PO-L contributed with data analysis and interpretation. JG wrote the first draft of the manuscript. DS-C, AC, JM-V and CP-S wrote sections of the manuscript. All authors contributed to the article and approved the submitted version.


Mn/Fe PSACs can reduce the depletion of reactive oxygen species (ROS) by consuming glutathione (GSH). Here, we demonstrated the Mn/Fe PSACs-mediated synergistic antitumor efficacy among in vitro and in vivo experiments. This study proposes new promising single-atom nanozymes with highly efficient biocatalytic sites and synergistic therapeutic effects, which will give birth to abundant inspirations in ROS-related biological applications in broad biomedical fields.Tumor microenvironment (TME)-induced nanocatalytic therapy is a trending strategy for tumor-targeting therapy, but the low catalytic efficiency remains to limit its therapeutic effect. The single-atom catalysts (SACs) appear as a novel type of nanozymes that possesses incredible catalytic activity. Here, we developed PEGylated manganese/iron-based SACs (Mn/Fe PSACs) by coordinating single-atom Mn/Fe to nitrogen atoms in hollow zeolitic imidazolate frameworks (ZIFs). Mn/Fe PSACs catalyze cellular hydrogen peroxide (H The generation of •OH was characterized by a characteristic quadruple signal (1:2:2:1) of DMPO-OH using a spin trapper 5,5-Dimethyl-1-pyrroline N-oxide (DMPO)  E. This rntration F. The KMy in TME . Moreovey in TME G and o-py in TME . SimilarAs previously mentioned, POD-like nanozymes generated ROS could be depleted by GSH presented in excess in the TME ,18,20. M2 generation ability in cancer cells was also detected by using [Ru(dpp)3]Cl2, an intracellular O2 level indicator with red fluorescence. The fluorescence in cells treated with Mn/Fe PSACs plus H2O2 was much weaker than that of the PBS control, H2O2, and Mn/Fe PSACs groups due to the increased intracellular O2 level based on Mn/Fe PSACs-induced single atom-based catalysis  with green fluorescence. As shown in2O2 exhibited the strongest green fluorescence due to the admirable POD-mimetic catalytic performance of Mn/Fe PSACs, which could catalyze the decomposition of H2O2 into ROS, while the green signals were negligible in PBS, H2O2, and Mn/Fe PSACs groups.Next, the cellular uptake behavior of Mn/Fe PSACs was monitored after labeling NPs with Cy5.5. CLSM showed that Mn/Fe PSACs could be internalized after 2 h of incubation and increased within 6 h A. The inatalysis C. These 2O2 greatly reduced cell viability (97.5%) compared to PBS, H2O2, and single Mn/Fe PSACs treated groups, which was consistent with living/dead staining experiments. To explore the concentration-dependent manner effect of nanozyme-induced cancer cell death, we investigated cell viability by varying the concentration  of Mn/Fe PSACs. In 2O2, and Mn/Fe PSACs groups, HepG2 cells co-cultured with the Mn/Fe PSACs plus H2O2 showed stronger green fluorescence from JC-1 monomers and negligible red fluorescence from JC-1 aggregates. This result confirmed that Mn/Fe PSACs could induce cancer cell apoptosis with the existence of H2O2 by generating toxic ROS to damage the mitochondrial membrane.The apoptosis of HepG2 cells treated with different treatments were visualized by co-staining with calcein-AM  and PI . As shown in −1 s−1, which was higher than that of clinically Gd-based contrast agents . Thereafter, we utilize those samples to perform a T1-weighted MRI in a tumor-bearing mouse model. Before and after (24 h distribution) Mn/Fe PSAC (15 mg/kg) i.v. injection. The MRI signal intensity of Mn/Fe PSACs in the tumor area was significantly enhanced. Quantitative MRI signals within Mn/Fe PSACs-treated tumor sites further corroborate these findings  of the Mn/Fe PSAC solutions with different Mn concentrations were evaluated using 3.0 T clinical MRI equipment. T1-weighted magnetic resonance images of Mn/Fe PSACs showed concentration-dependent signal enhancement D. The cofindings E. To confindings F. The inalf-life G, indica2O2 into O2 in the presence of Mn/Fe PSACs.To test the activation of the nanoreactors specific to tumor sites, we first monitored ROS levels in living mice using CLSM. After 24 h post-injection of Mn/Fe PSACs and control groups, the ROS probe (DCFH-DA) was intravenously injected, and the green fluorescence intensity in the tumor was monitored using dorsal window chamber models due to the ROS-responsive green fluorescence conduction characteristics of the probe. As shown inSubsequently, we explored the antitumor effects of the combined enzymatic properties of Mn/Fe PSACs. The treatment procedure was as follows: Mn/Fe PSACs were injected into tumor-bearing BALB/c nude mice at the dose of 15 mg/kg. Interestingly, Mn/Fe PSACs significantly inhibited tumor growth during the treatment period for 14 days C. At the2O2 in the TME to efficiently generate ROS to kill cancer cells. The CAT-like function of Mn/Fe PSACs catalyzed the decomposition of H2O2 and generated a large amount of O2 in cancer cells. Subsequently, the OXD-like function mediated electron transfer to O2, which generates large amounts of cytotoxic •O2−, thereby inducing apoptosis. The POD-like activity of Mn/Fe PSACs catalyzed •OH generation via H2O2 decomposition. Upon internalization by tumor cells, the catalytic activity of Mn/Fe PSACs was significantly elevated by local weakly acidic conditions. In vitro and in vivo acute toxicity evaluation experiments proved that Mn/Fe PSACs had good biosafety and could be used safely for intravenous administration. Moreover, O2 and ROS had been demonstrated to be abundantly produced in tumor cells. In vivo and in vitro cancer treatment results showed that Mn/Fe PSACs could effectively kill tumor cells, thus possessing excellent CDT therapeutic ability. Taken together, Mn/Fe PSACs can serve as a tumor-specific activatable nanomedicine against liver cancer with significant potential for clinical translation.In conclusion, dual POD-like and GSH-like nanozyme-Mn/Fe PSACs were synthesized for catalytic antitumor therapy. The doping of Fe and Mn single atoms significantly enhanced the POD-like catalytic activity and GSH-depleting function. This multifunctional single-atom enzyme catalyzes a cascade of reactions that harnesses the high levels of H


The incorporation of chemotherapy into the neoadjuvant setting has confers benefit in non–small-cell lung cancer (NSCLC) . Further. As shown in Fig. Single-cell RNA sequencing of samples from a total of 11 SCLC patients  were analyzed . QualityAdditionally, we analyzed and compared the TME in paratumoral tissues between SCLC patients with and without NAC treatment, which was consistent with the milieu in tumoral tissues between non-NAC-treated and NAC-treated SCLC patients  and the number of genes. B Principal analyses of the top 2000 variant genes using RunPCA. C Visualization of clustering using DimPlot. D Stratification and cell-type identification of single cells from 11 SCLC cases. Different types of cells were grouped into distinctive cell clusters. E. The infiltration of subpopulation of immune cells in tumor tissues in 2 NAC-treated SCLC patients and 9 non-NAC-treated SCLC patients prior to surgical resection.Additional file 2: Figure S2. A tSNE of different immune cells in para-tumoral tissue with each cell color coded for the associated cell type. B tSNE of different immune cells in para-tumoral tissue with each cell color coded for the treatment type. C. The infiltration of subpopulation of immune cells in para-tumoral tissues in 2 NAC-treated SCLC patients and 9 non-NAC-treated SCLC patients prior to surgical resection.Additional file 3: Figure S3. A The dots plots and gating strategies to determine the lymphocyte were shown. B The dots plots and gating strategies to determine the macrophages were shown C Comparison of TNF-α + CD8 + T cell infiltration in pre-NAC and post-NAC PBMC of SCLC, as detected by flow cytometry. D Comparison of γ-IFN + CD8 + T cell infiltration in pre-NAC and post-NAC PBMC of SCLC, as detected by flow cytometry.Additional file 4: Table S1. The baseline characteristics of 8 SCLC patients.Additional file 5: Table S2. Staining used for multiplex immunofluorescence.Additional file 6: Table S3. Antibodies for flow cytometry staining.Additional file 7: Methods.


T0) and 1-year follow-up (T1). Patients were then divided according to cognitive status into MSA with normal cognition (MSA-NC) and MSA with mild cognitive impairment (MCI). At T1, we divided the sample according to worsening/non worsening of cognitive status compared to baseline evaluation. Logistic regression analysis showed that age  and T1w/T2w value in the left putamen  were significant predictors of global cognitive status at T0, explaining 65% of the variance. Logistic regression analysis showed that ∆-values of WM density in the cerebellum/brainstem  significantly predicted cognitive worsening at T1, explaining 64% of the variance. Our results suggest a role for the putamen and cerebellum in the cognitive changes of MSA, probably due to their connections with the cortex. The putaminal T1w/T2w ratio may deserve further studies as a marker of cognitive impairment in MSA.The exact pathophysiology of cognitive impairment in multiple system atrophy (MSA) is unclear. In our longitudinal study, we aimed to analyze (I) the relationships between cognitive functions and some subcortical structures, such as putamen and cerebellum assessed by voxel-based morphometry (VBM) and T1-weighted/T2-weighted (T1w/T2w) ratio, and (II) the neuroimaging predictors of the progression of cognitive deficits. Twenty-six patients with MSA underwent a comprehensive neuropsychological battery, motor examination, and brain MRI at baseline (The online version contains supplementary material available at 10.1007/s12311-022-01455-8. Multiple system atrophy (MSA) is a sporadic, progressive neurodegenerative disease characterized by parkinsonian features, cerebellar ataxia, autonomic failure, urogenital dysfunction and corticospinal disorders. The two subtypes of MSA are MSA with predominant parkinsonism (MSA-P) and MSA with predominant cerebellar ataxia (MSA-C) .Cognitive impairment, characterized by prevalent executive dysfunctions, is an integral feature of the disease, and it presents largely variable frequency among studies between 33 and 83%) . Kim et 3 and 83%On the other hand, a subcortical pathogenesis of cognitive impairment has been proposed for MSA, since MSA patients with cognitive impairment showed only focal volume reduction in the left dorsolateral prefrontal cortex compared with MSA with normal cognition.In a previous study , investiThe exact pathophysiology of cognitive dysfunction in MSA remains unclear. Since a model of subcortical cognitive impairment has been proposed for MSA , we asseT0) and at follow-up (T1), 12 months later. The local Ethics Committee approved the study, and all patients signed informed consent.Twenty-six patients with a diagnosis of probable MSA, of which 14 with the parkinsonian variant (MSA-P) and 12 with the cerebellar variant (MSA-C), according to current criteria . Spearman’s correlations between ∆-value of neuropsychological tests and ∆-MRI data were conducted. We also assessed the same correlations in worsening sub-group separately. The Mann–Whitney’s U test was used to compare ∆-MRI values between worsening and non worsening patients. Where necessary, we applied correction for multiple comparisons. A logistic regression analysis with Bootstrap method was performed with worsening or nonworsening as the dependent variable, p < 0.05 deemed significant, to evaluate a possible effect of ∆-MRI data on cognitive status. In addition to the ∆-MRI data, the age of the subjects was also included as an independent variable.Changes in neuropsychological scores and MRI data between T0 and between neuropsychological changes and MRI changes, ∆ (T1-T0), were analyzed also in MSA-C and MSA-P, separately. Due to the small sample, Mann–Whitney’s test and logistic regression were not performed in MSA-C and MSA-P separately.Finally, Spearman’s correlations between neuropsychological tests and MRI data at All statistical analyses were performed by SPSS-20 .T0, CA was directly related to GM density in the cerebellum/brainstem , WM density in the cerebellum/brainstem , and T1w/T2w (WM) in the cerebellum/brainstem . There was a negative correlation between GM density in the right putamen and TMT-A . After correction for multiple comparisons, only correlation between T1w/T2w (WM) in the cerebellum/brainstem and CA was retained (Table At N = 6) and NC (N = 20) (p > 0.05).We found no significant differences in MRI parameters between patients with MCI  and T1w/T2w value in the Left putamen  were significant predictors of global cognitive status at T0, explaining 65% of the variance (R2 = 0.65).Logistic regression analysis showed that age . There was a negative correlation between ∆-values of the T1w/T2w (WM) cerebellum/brainstem and TMT-A  , and between ∆-values of the GM density in the Left putamen and non-words repetitions E.N.P.A. test . There was a positive correlation between ∆-values of the GM density in the right putamen and word repetition E.N.P.A. test . Moreover, a trend towards significance was found for the following correlation: ∆-values of the T1w/T2w in the cerebellum/brainstem GM and CA  (Table At 1) Table . In the N = 21) and nonworsening (N = 5) (p > 0.05).We found no significant differences on ∆-MRI parameters between patients with cognitive worsening  significantly predicted cognitive worsening at T1, explaining 64% of the variance (R2 = 0.64).Logistic regression analysis showed that ∆-values of WM density in the cerebellum/brainstem (p = 0.04), between ∆-values of WM density in the cerebellum/brainstem and BJLO , between ∆-values of T1w/T2w in the Right putamen and phonological fluency , and between ∆-values of T1w/T2w in the Left putamen and phonological fluency .Finally, in MSA-P, significant correlations were found between ∆-values of GM density in the cerebellum/brainstem and 15-RAWLT- immediate recall , ∆-values of GM density right putamen and Prosa test , ∆-values of GM density in the left putamen and non-words repetition E.N.P.A. test , ∆-values of T1w/T2w in the cerebellum/brainstem GM and both ROCF recall , and TMT-A .In MSA-C, significant correlations were found between ∆-values of GM density in the cerebellum/brainstem and nonword repetition E.N.P.A. test .As for subtype analysis, no correlations survived the correction for multiple comparisons Below is the link to the electronic supplementary material.


Maruca vitrata. These two genes belong to G protein-coupled receptors (GPCRs) and have differences in the C-terminus but share a 7-transmembrane region and GPCR family 1 signature. These isoforms were expressed in all developmental stages and adult tissues. MviPBANR-C had the highest expression level in pheromone glands among the examined tissues. Through in vitro heterologous expression in HeLa cell lines, only MviPBANR-C-transfected cells responded to MviPBAN (≥5 µM MviPBAN), inducing Ca2+ influx. Sex pheromone production and mating behavior were investigated using gas chromatography and a bioassay after MviPBANR-C suppression by RNA interference, which resulted in the major sex pheromone component, E10E12-16:Ald, being quantitatively reduced compared to the control, thereby decreasing the mating rate. Our findings indicate that MviPBANR-C is involved in the signal transduction of sex pheromone biosynthesis in M. vitrata and that the C-terminal tail plays an important role in its function.Insect sex pheromones are volatile chemicals that induce mating behavior between conspecific individuals. In moths, sex pheromone biosynthesis is initiated when pheromone biosynthesis-activating neuropeptide (PBAN) synthesized in the suboesophageal ganglion binds to its receptor on the epithelial cell membrane of the pheromone gland. To investigate the function of PBAN receptor (PBANR), we identified two PBANR isoforms, MviPBANR-B and MviPBANR-C, in the pheromone glands of  In moths, the females’ ability to attract conspecific males is important for successful reproduction. Female moths secrete species-specific volatile compounds known as sex pheromones, which induce mating behavior ,2. Sex p2 in the C-terminal, which is essential for their activity . Mv. Mv24]. The deduced amino acid sequences of MviPBANR-B and -C were compared to those of other lepidopteran PBANRs. The protein sequences were aligned with ClustalW using MegAlign software (version 7.1.0). A phylogenetic tree was constructed with maximum likelihood, and bootstrap analysis with 1000 replications was performed using Molecular Evolutionary Genetic Analysis software (version X) .−ΔΔCT method [To examine the expression pattern of MviPBANR-B and -C in each developmental stage  and adult tissues , total RNA extraction and cDNA synthesis were performed. Quantitative real-time PCR (qRT-PCR) was performed with TOPreal™ qPCR 2× PreMIX (SYBR Green with low ROX)  and specific primers . The folT method .M. vitrata. Total RNA extraction and cDNA synthesis were performed in the same manner as described above. A PCR was performed with 2× TOPsimple™ DyeMIX-HOT  and specific primers containing KpnI and HindIII sequences  were cultured on a flux-bottom confocal dish for 24 h using a Fluo-4 directTM calcium assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, the transfected cells were incubated with Fluo-4 direct calcium assay reagent solution diluted in culture medium (1:1) at 37 °C for 40 min. The alteration of calcium localization following injections of various concentrations of PBAN or 2 μM ionomycin (positive control) was detected every second for 1 min. Time-lapse imaging of calcium signals was taken using a Nikon AX confocal microscope .The expression vectors, pcMviPBANR-B and -C, were transfected into human cervical cancer (HeLa) cell lines using lipofectamine 2000 . The HeLa cell lines were purchased from Korean Cell Line Bank , and were cultured in RPMI-1640  containing 4.5 g/L D-glucose, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate, 10% FBS , 100 U/mL penicillin, and 100 g/mL streptomycin (Gibco) The cultured cells were maintained at 37 °C in a humidified atmosphere of 5% COM. vitrata, total RNA extraction, and cDNA synthesis were performed every 4 h, starting 2 h before the photophase (1 a.m.), as described above. qRT-PCR was performed with TOPreal™ qPCR 2× PreMIX (SYBR Green with low ROX)  and specific primers  fragments were amplified with a specific primer set containing a T7 promoter region at both ends  in a 1:1 ratio and incubated at 25 °C for 30 min. dsRNAs (200 ng) were injected into the female pupae 1 d before eclosion. Enhanced green fluorescent protein (EGFP)-specific dsRNAs were also injected as a negative control. EGFP-specific dsRNAs containing the T7 promoter region at both ends were amplified using specific primers  [To investigate the function of MviPBANR-C on sex pheromone biosynthesis in -NH2-3′)  and placTo examine the effect of MviPBANR-C on mating behavior, suppression of PBANR-C by RNAi was performed as described above. The mating behavior of 10 adult male and female pairs was examined, one pair at a time, independently in an acrylic breeding box  for 3 d. The mating behavior was determined by the presence of the fertilized eggs. The mating rate was defined as the ratio of fertilizing egg-laying pairs to the total examined mating ones. Experiments were performed independently and repeated in triplicate.t-tests were used to analyze statistically significant differences using the Sigmaplot software .ANOVA and M. vitrata [In our previous study, we identified two PBANR isoforms, MviPBANR-B and -C, from the transcriptome of the pheromone glands of  vitrata . In the M. vitrata formed a clade with Ostrinia nubilalis in the Crambidae family  and no Ca2+ influx was observed in cells treated with MviPBAN  was measured using GC on the second day after the injection. During MviPBANR-C suppression, the amount of E10E12-16:Ald decreased by two-thirds compared to that of the controls  and expressed in all developmental stages and tissues. Among them, the expression level of MbrPBANR-C was the highest in the pheromone glands of two-day-old females [M. vitrata were consistent with that of the MbrPBANR isoforms. MviPBANR-C showed higher expression levels than that of MviPBANR-B in the pheromone glands of two-day-old virgin females  and Ca2+ influx in response to SliPBAN [B. mori, MAPK is known to be involved in prothoracicotropic hormone-induced ecdysone biosynthesis [M. vitrata.In addition, PBANR of octuidae . As repoarmigera , MviPBANarmigera , and PBAarmigera . MviPBAN SliPBAN . In B. mynthesis ,42,43. B2+ plays an important role in the cellular signaling cascade caused by ligand-GPCR interactions [2+ influx via heterologous expression [2+ influx in the PBANR-expressed cells induced by PBAN is quantitatively different among PBANR isoforms. Ca2+ influx induced by HarPBAN was observed only in sf9 cells expressing HarPBANR-B and HarPBANR-C in H. armigera [O. nubilalis, Ca2+ influx was observed only in sf9 cells expressing OnuPBANR-C in response to OnuPBAN [2+ influx was observed only in cells expressing MviPBANR-C   , which c(5 p.m.) . These r binding ,47. Howe binding ,36.M. vitrata, and only two MviPBANR isoforms were identified from the pheromone glands. Among these isoforms, only MviPBANR-C induced Ca2+ influx by responding to MviPBAN. MviPBANR-C is involved in sex pheromone biosynthesis in M. vitrata by interacting with MviPBAN, which affects sex pheromone production and mating behavior. These results suggest that MviPBANR-C is a potential pest control factor.In this study, we performed a functional analysis of MviPBANR isoforms involved in signal transduction of pheromone biosynthesis in


Escherichia, several monophyletic clades other than the traditionally defined species have been identified. Of these, cryptic clade I (C-I) appears to represent a subspecies of E. coli, but due to the difficulty in distinguishing it from E. coli sensu stricto, the population structure and virulence potential of C-I are unclear.Within the genus n = 465), including a Shiga toxin 2a (Stx2a)-producing isolate from a patient with bloody diarrhoea identified by the retrospective analyses using a C-I-specific detection system. Through genomic analysis of 804 isolates from the cryptic clades, including these C-I strains, we revealed their global population structures and the marked accumulation of virulence genes and antimicrobial resistance genes in C-I. In particular, half of the C-I strains contained hallmark virulence genes of Stx-producing E. coli (STEC) and/or enterotoxigenic E. coli (ETEC). We also found the host-specific distributions of virulence genes, which suggests bovines as the potential source of human infections caused by STEC- and STEC/ETEC hybrid-type C-I strains, as is known in STEC.We defined a set of true C-I strains (Our findings demonstrate the emergence of human intestinal pathogens in C-I lineage. To better understand the features of C-I strains and their infections, extensive surveillance and larger population studies of C-I strains are needed. The C-I-specific detection system developed in this study will be a powerful tool for screening and identifying C-I strains.The online version contains supplementary material available at 10.1186/s12915-023-01584-4. Escherichia cryptic clade I (C-I) is one of the six cryptic clades proposed in the genus Escherichia . The genome sequences of 17 C-II strains [To identify strains belonging to C-I, C-II (“Ew”), Er (C-III/IV), Em (C-V) and other cryptic clades in the 170,489 n levels   . SNP sitE. albertii, E. fergusonii and E. coli using Roary [Through a pangenome analysis of 792 genomes of C-I, C-II (“Ew”), Er (C-III/C-IV), Em (C-V), C-VI and C-VIII strains and the reference strains of ng Roary  . Libraries were prepared using the Nextera XT DNA Sample Preparation Kit  or the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB) and sequenced on the Illumina MiSeq platform to generate 300-bp paired-end reads. Genome assembly of the Illumina sequence reads was performed using the Platanus_B assembler .The four finished C-I genomes  were obtained by mixed assembly of MiSeq and MinION  reads using the Unicycler pipeline version 0.4.4 with the default settings . LibrariE. coli hallmark virulence genes were performed using an in-house database . The result was considered positive when all genes of a given CF were conserved (> 90% identity and > 80% coverage). AMR genes were identified using ABRicate version 1.0.1 (https://github.com/tseemann/abricate) with the default settings using the CARD (http://www.mgc.ac.cn/VFs) databases.In silico serotyping was conducted by using ECTyper version 1.0.0 with the default settings . The ideg at 4 °C for 10 min. The Stx2 concentration in each cell lysate was determined using a previously described sandwich ELISA [Bacterial cells were inoculated into 40 ml of lysogeny broth and grown to mid-log phase at 37 °C with shaking. MMC was then added to the culture at a final concentration of 500 ng/ml. At 5 h after MMC addition, 100 µl of the culture was collected and immediately subjected to sonication using a Bioruptor . The soluble fractions of each cell lysate were obtained via centrifugation at 14,000 × ch ELISA  using RIch ELISA .t test was used to examine the differences in the average number of AMR genes between strains.All statistical analyses were performed using R version 3.6.1 . The prop test was conducted to examine the differences in the proportion of strains containing no AMR gene between nonintestinal pathogenic strains and intestinal pathogenic strains . The Additional file 1: Figure S1. Core gene-based phylogenomic tree of the strains in EnteroBase that were suspected to belong to Escherichia cryptic clades and recently defined Escherichia species. Figure S2. Agarose gel electrophoresis analysis of the products obtained by C-I detection PCR. Figure S3. Continents, sources and serotype distributions of strains belonging to C-I, other cryptic clades and recently defined Escherichia species. Figure S4. Summary of the prevalence of virulence genes in C-I, other cryptic clades and recently defined Escherichia species. Figure S5. Distribution of AMR genes in STEC-type, ETEC-type, STEC/ETEC hybrid-type and nonintestinal pathogenic C-I strains. Figure S6. Summary of the prevalence of AMR genes in C-I. Figure S7. Distribution of AMR genes in C-I, other cryptic clades and recently defined Escherichia species. Figure S8. Summary of the prevalence of AMR genes in C-I strains from humans and livestock animals. Figure S9. Distribution of AMR genes in STEC-type, ETEC-type, STEC/ETEC hybrid-type and nonintestinal pathogenic C-I strains. Figure S10. Comparison of genes in the K88-like CF loci with those in the K88 CS locus. Figure S11. Circular map of the plasmid encoding multiple AMR genes and the sepA gene in the C-I strain 10290.Additional file 2: Table S1. Characteristics of strains used in this study. Table S2. Length of chromosome and plasmids in cryptic clade I strains in which the genome sequence was compldetely determined. Table S3. Alignment coverages of the completely sequenced C-I phages and plasmids encoding major virulence genes with the scaffold sequences of the C-I strains. Table S4. List of the nucleotide sequences of the major virulence genes used in this study. Table S5. List of the nucleotide sequences of E. coli virulence genes used in this study.Additional file 3: Supplementary Text. Colonization factors in C-I strains and structural comparisons of the Stx and LT phages and the virulence plasmids in C-I strains.