S309-CAR-NK cells bind the Omicron variants in vitro and reduce SARS-CoV-2 viral loads in humanized ACE2-NSG mice.

Recent progress on chimeric antigen receptor (CAR)-NK cells has shown promising results in treating CD19-positive lymphoid tumors with minimal toxicities [including graft versus host disease (GvHD) and cytokine release syndrome (CRS) in clinical trials. Nevertheless, the use of CAR-NK cells in combating viral infections has not yet been fully explored. Previous studies have shown that CAR-NK cells expressing S309 single-chain fragment variable (scFv), hereinafter S309-CAR-NK cells, can bind to SARS-CoV-2 wildtype pseudotyped virus (PV) and effectively kill cells expressing wild-type spike protein in vitro. In this study, we further demonstrate that the S309-CAR-NK cells can bind to different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants in vitro. We also show that S309-CAR-NK cells reduce virus loads in the NOD/SCID gamma (NSG) mice expressing the human angiotensin-converting enzyme 2 (hACE2) receptor challenged with SARS-CoV-2 wild-type (strain USA/WA1/2020). Our study demonstrates the potential use of S309-CAR-NK cells for inhibiting infection by SARS-CoV-2 and for the potential treatment of COVID-19 patients unresponsive to otherwise currently available therapeutics. IMPORTANCE: Chimeric antigen receptor (CAR)-NK cells can be "off-the-shelf" products that treat various diseases, including cancer, infections, and autoimmune diseases. In this study, we engineered natural killer (NK) cells to express S309 single-chain fragment variable (scFv), to target the Spike protein of SARS-CoV-2, hereinafter S309-CAR-NK cells. Our study shows that S309-CAR-NK cells are effective against different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants. The S309-CAR-NK cells can (i) directly bind to SARS-CoV-2 pseudotyped virus (PV), (ii) competitively bind to SARS-CoV-2 PV with 293T cells expressing the human angiotensin-converting enzyme 2 (hACE2) receptor (293T-hACE2 cells), (iii) specifically target and lyse A549 cells expressing the spike protein, and (iv) significantly reduce the viral loads of SARS-CoV-2 wild-type (strain USA/WA1/2020) in the lungs of NOD/SCID gamma (NSG) mice expressing hACE2 (hACE2-NSG mice). Altogether, the current study demonstrates the potential use of S309-CAR-NK immunotherapy as an alternative treatment for COVID-19 patients.

NSUN2-mediated m5C modification of HBV RNA positively regulates HBV replication.

Chronic hepatitis B virus (HBV) infection is a major cause of liver cirrhosis and liver cancer, despite strong prevention and treatment efforts. The study of the epigenetic modification of HBV has become a research hotspot, including the N6-methyladenosine (m6A) modification of HBV RNA, which plays complex roles in the HBV life cycle. In addition to m6A modification, 5-methylcytosine (m5C) is another major modification of eukaryotic mRNA. In this study, we explored the roles of m5C methyltransferase and demethyltransferase in the HBV life cycle. The results showed that m5C methyltransferase NSUN2 deficiency could negatively regulate the expression of HBV while m5C demethyltransferase TET2 deficiency positively regulates the expression of HBV. Subsequently, we combined both in vitro bisulfite sequencing and high-throughput bisulfite sequencing methods to determine the distribution and stoichiometry of m5C modification in HBV RNA. Two sites: C2017 and C131 with the highest-ranking methylation rates were identified, and mutations at these two sites could lead to the decreased expression and replication of HBV, while the mutation of the "fake" m5C site had no effect. Mechanistically, NSUN2-mediated m5C modification promotes the stability of HBV RNA. In addition, compared with wild-type HepG2-NTCP cells and primary human hepatocytes, the replication level of HBV after NSUN2 knockdown decreased, and the ability of the mutant virus to infect and replicate in wild-type HepG2-NTCP cells and PHHs was substantially impaired. Similar results were found in the experiments using C57BL/6JGpt-Nsun2+/- mice. Interestingly, we also found that HBV expression and core protein promoted the endogenous expression of NSUN2, which implied a positive feedback loop. In summary, our study provides an accurate and high-resolution m5C profile of HBV RNA and reveals that NSUN2-mediated m5C modification of HBV RNA positively regulates HBV replication by maintaining RNA stability.

Prime-Pull Immunization of Mice with a BcfA-Adjuvanted Vaccine Elicits Sustained Mucosal Immunity That Prevents SARS-CoV-2 Infection and Pathology.

Vaccines against SARS-CoV-2 that induce mucosal immunity capable of preventing infection and disease remain urgently needed. In this study, we demonstrate the efficacy of Bordetella colonization factor A (BcfA), a novel bacteria-derived protein adjuvant, in SARS-CoV-2 spike-based prime-pull immunizations. We show that i.m. priming of mice with an aluminum hydroxide- and BcfA-adjuvanted spike subunit vaccine, followed by a BcfA-adjuvanted mucosal booster, generated Th17-polarized CD4+ tissue-resident memory T cells and neutralizing Abs. Immunization with this heterologous vaccine prevented weight loss following challenge with mouse-adapted SARS-CoV-2 (MA10) and reduced viral replication in the respiratory tract. Histopathology showed a strong leukocyte and polymorphonuclear cell infiltrate without epithelial damage in mice immunized with BcfA-containing vaccines. Importantly, neutralizing Abs and tissue-resident memory T cells were maintained until 3 mo postbooster. Viral load in the nose of mice challenged with the MA10 virus at this time point was significantly reduced compared with naive challenged mice and mice immunized with an aluminum hydroxide-adjuvanted vaccine. We show that vaccines adjuvanted with alum and BcfA, delivered through a heterologous prime-pull regimen, provide sustained protection against SARS-CoV-2 infection.

SARS-CoV-2 spreads through cell-to-cell transmission.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein, we provide evidence that SARS-CoV-2 spreads through cell-cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell-cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.

Cas13d knockdown of lung protease Ctsl prevents and treats SARS-CoV-2 infection.

SARS-CoV-2 entry into cells requires specific host proteases; however, no successful in vivo applications of host protease inhibitors have yet been reported for treatment of SARS-CoV-2 pathogenesis. Here we describe a chemically engineered nanosystem encapsulating CRISPR-Cas13d, developed to specifically target lung protease cathepsin L (Ctsl) messenger RNA to block SARS-CoV-2 infection in mice. We show that this nanosystem decreases lung Ctsl expression in normal mice efficiently, specifically and safely. We further show that this approach extends survival of mice lethally infected with SARS-CoV-2, correlating with decreased lung virus burden, reduced expression of proinflammatory cytokines/chemokines and diminished severity of pulmonary interstitial inflammation. Postinfection treatment by this nanosystem dramatically lowers the lung virus burden and alleviates virus-induced pathological changes. Our results indicate that targeting lung protease mRNA by Cas13d nanosystem represents a unique strategy for controlling SARS-CoV-2 infection and demonstrate that CRISPR can be used as a potential treatment for SARS-CoV-2 infection.

Inhibition of elastase enhances the adjuvanticity of alum and promotes anti-SARS-CoV-2 systemic and mucosal immunity.

Alum, used as an adjuvant in injected vaccines, promotes T helper 2 (Th2) and serum antibody (Ab) responses. However, it fails to induce secretory immunoglobulin (Ig) A (SIgA) in mucosal tissues and is poor in inducing Th1 and cell-mediated immunity. Alum stimulates interleukin 1 (IL-1) and the recruitment of myeloid cells, including neutrophils. We investigated whether neutrophil elastase regulates the adjuvanticity of alum, and whether a strategy targeting neutrophil elastase could improve responses to injected vaccines. Mice coadministered a pharmacological inhibitor of elastase, or lacking elastase, developed high-affinity serum IgG and IgA antibodies after immunization with alum-adsorbed protein vaccines, including the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). These mice also developed broader antigen-specific CD4+ T cell responses, including high Th1 and T follicular helper (Tfh) responses. Interestingly, in the absence of elastase activity, mucosal SIgA responses were induced after systemic immunization with alum as adjuvant. Importantly, lack or suppression of elastase activity enhanced the magnitude of anti-SARS-CoV-2 spike subunit 1 (S1) antibodies, and these antibodies reacted with the same epitopes of spike 1 protein as sera from COVID-19 patients. Therefore, suppression of neutrophil elastase could represent an attractive strategy for improving the efficacy of alum-based injected vaccines for the induction of broad immunity, including mucosal immunity.

A safe and highly efficacious measles virus-based vaccine expressing SARS-CoV-2 stabilized prefusion spike.

The current pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights an urgent need to develop a safe, efficacious, and durable vaccine. Using a measles virus (rMeV) vaccine strain as the backbone, we developed a series of recombinant attenuated vaccine candidates expressing various forms of the SARS-CoV-2 spike (S) protein and its receptor binding domain (RBD) and evaluated their efficacy in cotton rat, IFNAR-/-mice, IFNAR-/--hCD46 mice, and golden Syrian hamsters. We found that rMeV expressing stabilized prefusion S protein (rMeV-preS) was more potent in inducing SARS-CoV-2-specific neutralizing antibodies than rMeV expressing full-length S protein (rMeV-S), while the rMeVs expressing different lengths of RBD (rMeV-RBD) were the least potent. Animals immunized with rMeV-preS produced higher levels of neutralizing antibody than found in convalescent sera from COVID-19 patients and a strong Th1-biased T cell response. The rMeV-preS also provided complete protection of hamsters from challenge with SARS-CoV-2, preventing replication in lungs and nasal turbinates, body weight loss, cytokine storm, and lung pathology. These data demonstrate that rMeV-preS is a safe and highly efficacious vaccine candidate, supporting its further development as a SARS-CoV-2 vaccine.

Extracellular Vesicles and Obesity.

Extracellular vesicles (EVs) are a group of vesicles with membrane structure released by cells, including exosomes, microvesicles, apoptotic bodies, and oncosomes. EVs are now recognized as important tools of cell-to-cell communication, allowing cells to exchange proteins, lipids, and genetic material to participate in physiological and pathological processes. It has been reported that EVs regulate host-pathogen interactions and participate in pathological processes of infectious disease, neurological diseases, cancer, cardiovascular diseases, etc., it also plays an important role in the process of growth and development. EVs have a bright future in clinical application. They can be used to monitor clinical status, therapeutic effect, and disease progression. At the same time, EVs have the potential to be developed as clinical drug delivery vectors due to their ability to deliver biomolecules. However, it is still unclear whether EVs are reliable and useful markers for the diagnosis or early detection of obesity, and whether they can be used as drug vectors for the treatment of obesity. In this review, we summarize the research progress of EVs and obesity. It is hoped that EVs may become a new target in the diagnosis and treatment of obesity.

Mapping the biodiversity conservation gaps in the East China sea.

Being one of the most productive China seas, the East China Sea is facing the challenge of unprecedented biodiversity loss and habitat degradation under the dual pressure of anthropogenic disturbance and climate change. Although marine protected areas (MPAs) are considered an effective conservation tool, it remains unclear whether existing MPAs adequately protect marine biodiversity. To investigate this issue, we first constructed a maximum entropy model to predict the distributions of 359 threatened species and identified its species richness hotspots in the East China Sea. Then we identified priority conservation areas (PCAs1) under different protection scenarios. Since the actual conservation in the East China Sea is far from the goals proposed by Convention on Biological Diversity, we calculated a more realistic conservation goal by quantifying the relationship between the percentage of protected areas in the East China Sea and the average proportion of habitats covered for all species. Finally, we mapped conservation gaps by comparing the PCAs under the proposed goal and existing MPAs. Our results showed that these threatened species were very heterogeneously distributed, and their abundance was highest at low latitudes and in nearshore areas. The identified PCAs were distributed mainly in nearshore areas, especially in the Yangtze River estuary and along the Taiwan Strait. Based on the current distribution of threatened species, we suggest a minimum conservation goal of 20.4% of the total area of the East China Sea. Only 8.8% of the recommended PCAs are currently within the existing MPAs. We recommend expanding the MPAs in six areas to achieve the minimum conservation target. Our findings provide a solid scientific reference and a reasonable short-term target for China to realize the vision of protecting 30% of its oceans by 2030.

pHisPred: a tool for the identification of histidine phosphorylation sites by integrating amino acid patterns and properties.

BACKGROUND: Protein histidine phosphorylation (pHis) plays critical roles in prokaryotic signal transduction pathways and various eukaryotic cellular processes. It is estimated to account for 6-10% of the phosphoproteome, however only hundreds of pHis sites have been discovered to date. Due to the inherent disadvantages of experimental methods, it is an urgent task for developing efficient computational approaches to identify pHis sites. RESULTS: Here, we present a novel tool, pHisPred, for accurately identifying pHis sites from protein sequences. We manually collected the largest number of experimental validated pHis sites to build benchmark datasets. Using randomized tenfold CV, the weighted SVM-RBF model shows the best performance than other four commonly used classification models (LR, KNN, RF, and MLP). From ten thousands of features, 140 and 150 most informative features were individually selected out for eukaryotic and prokaryotic models. The average AUC and F1-score values of pHisPred were (0.81, 0.40) and (0.78, 0.46) for tenfold CV on the eukaryotic and prokaryotic training datasets, respectively. In addition, pHisPred significantly outperforms other tools on testing datasets, in particular on the eukaryotic one. CONCLUSION: We implemented a python program of pHisPred, which is freely available for non-commercial use at https://github.com/xiaofengsong/pHisPred . Moreover, users can use it to train new models with their own data.

A Methyltransferase-Defective Vesicular Stomatitis Virus-Based SARS-CoV-2 Vaccine Candidate Provides Complete Protection against SARS-CoV-2 Infection in Hamsters.

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic economic and health burdens. Although the worldwide SARS-CoV-2 vaccination campaign has begun, exploration of other vaccine candidates is needed due to uncertainties with the current approved vaccines, such as durability of protection, cross-protection against variant strains, and costs of long-term production and storage. In this study, we developed a methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidate. We generated mtdVSVs expressing SARS-CoV-2 full-length spike (S) protein, S1, or its receptor-binding domain (RBD). All of these recombinant viruses grew to high titers in mammalian cells despite high attenuation in cell culture. The SARS-CoV-2 S protein and its truncations were highly expressed by the mtdVSV vector. These mtdVSV-based vaccine candidates were completely attenuated in both immunocompetent and immunocompromised mice. Among these constructs, mtdVSV-S induced high levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) and Th1-biased T-cell immune responses in mice. In Syrian golden hamsters, the serum levels of SARS-CoV-2-specific NAbs triggered by mtdVSV-S were higher than the levels of NAbs in convalescent plasma from recovered COVID-19 patients. In addition, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 replication in lung and nasal turbinate tissues, cytokine storm, and lung pathology. Collectively, our data demonstrate that mtdVSV expressing SARS-CoV-2 S protein is a safe and highly efficacious vaccine candidate against SARS-CoV-2 infection. IMPORTANCE Viral mRNA cap methyltransferase (MTase) is essential for mRNA stability, protein translation, and innate immune evasion. Thus, viral mRNA cap MTase activity is an excellent target for development of live attenuated or live vectored vaccine candidates. Here, we developed a panel of MTase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidates expressing full-length S, S1, or several versions of the RBD. These mtdVSV-based vaccine candidates grew to high titers in cell culture and were completely attenuated in both immunocompetent and immunocompromised mice. Among these vaccine candidates, mtdVSV-S induces high levels of SARS-CoV-2-specific neutralizing antibodies (Nabs) and Th1-biased immune responses in mice. Syrian golden hamsters immunized with mtdVSV-S triggered SARS-CoV-2-specific NAbs at higher levels than those in convalescent plasma from recovered COVID-19 patients. Furthermore, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 challenge. Thus, mtdVSV is a safe and highly effective vector to deliver SARS-CoV-2 vaccine.

TIM-mediated inhibition of HIV-1 release is antagonized by Nef but potentiated by SERINC proteins.

The T cell Ig and mucin domain (TIM) proteins inhibit release of HIV-1 and other enveloped viruses by interacting with cell- and virion-associated phosphatidylserine (PS). Here, we show that the Nef proteins of HIV-1 and other lentiviruses antagonize TIM-mediated restriction. TIM-1 more potently inhibits the release of Nef-deficient relative to Nef-expressing HIV-1, and ectopic expression of Nef relieves restriction. HIV-1 Nef does not down-regulate the overall level of TIM-1 expression, but promotes its internalization from the plasma membrane and sequesters its expression in intracellular compartments. Notably, Nef mutants defective in modulating membrane protein endocytic trafficking are incapable of antagonizing TIM-mediated inhibition of HIV-1 release. Intriguingly, depletion of SERINC3 or SERINC5 proteins in human peripheral blood mononuclear cells (PBMCs) attenuates TIM-1 restriction of HIV-1 release, in particular that of Nef-deficient viruses. In contrast, coexpression of SERINC3 or SERINC5 increases the expression of TIM-1 on the plasma membrane and potentiates TIM-mediated inhibition of HIV-1 production. Pulse-chase metabolic labeling reveals that the half-life of TIM-1 is extended by SERINC5 from <2 to ∼6 hours, suggesting that SERINC5 stabilizes the expression of TIM-1. Consistent with a role for SERINC protein in potentiating TIM-1 restriction, we find that MLV glycoGag and EIAV S2 proteins, which, like Nef, antagonize SERINC-mediated diminishment of HIV-1 infectivity, also effectively counteract TIM-mediated inhibition of HIV-1 release. Collectively, our work reveals a role of Nef in antagonizing TIM-1 and highlights the complex interplay between Nef and HIV-1 restriction by TIMs and SERINCs.

An account of in silico identification tools of secreted effector proteins in bacteria and future challenges.

Bacterial pathogens secrete numerous effector proteins via six secretion systems, type I to type VI secretion systems, to adapt to new environments or to promote virulence by bacterium-host interactions. Many computational approaches have been used in the identification of effector proteins before the subsequent experimental verification because they tolerate laborious biological procedures and are genome scale, automated and highly efficient. Prevalent examples include machine learning methods and statistical techniques. In this article, we summarize the computational progress toward predicting secreted effector proteins in bacteria, with an opening of an introduction of features that are used to discriminate effectors from non-effectors. The mechanism, contribution and deficiency of previous developed detection tools are presented, which are further benchmarked based on a curated testing data set. According to the results of benchmarking, potential improvements of the prediction performance are discussed, which include (1) more informative features for discriminating the effectors from non-effectors; (2) the construction of comprehensive training data set of the machine learning algorithms; (3) the advancement of reliable prediction methods and (4) a better interpretation of the mechanisms behind the molecular processes. The future of in silico identification of bacterial secreted effectors includes both opportunities and challenges.

Increasement of Cd adsorption capacity of rice stubble from being alive until death in a modified rice-fish system.

Soil heavy metal contamination is an increasingly urgent problem throughout the world. Phytoremediation is a cost-effective and ecologically friendly in situ method for the remediation of heavy metal contaminated soils. Rice has the potential for use in soil remediation due to its high biomass production, however, risks related to food safety and low accumulation potential exist. Therefore, in the current study, rice stubble was used as the adsorbent in a modified rice-fish system (MRFS) to assess its accumulation capacity in a model paddy field dosed with 0-40.0 mg kg-1 Cd. The weighted mean concentration (WMC) of Cd in rice stubble increased from 0.498 to 36.365 mg kg-1 to 1.038-71.180 mg kg-1 from 0 to 60 days post-harvest (dph), and the corresponding increment rate was 107.68%, 117.42%, 157.77% and 95.73%, respectively. Sixty-days post-harvest, removal rate of Cd from contaminated soils was 1.11-1.40%, which was greater than that of the Cd-hyperaccumulator Thlaspi caerulescens. The WMC of the heavy metals Cd, Zn, Pb, Cr and Cu in rice stubble increased 51.11-97.50%, and removal rate was 1.93-2.66%. Overall, rice stubble had a high capacity of heavy metal accumulation, mainly benefiting from the synthesis effects of MRFS and the changes of accumulation mechanism within the plant from being alive until death. Notably, this study also provides a new idea for in situ, herbage-based phytoremediation of heavy metal-contaminated soils.

Risk factors and survival analysis for central nervous system complications after allogeneic hematopoietic stem cell transplantation. / å¼‚åŸºå› é€ è¡€å¹²ç»†èƒžç§»æ¤åŽä¸­æž¢ç¥žç»ç³»ç»Ÿå¹¶å‘ç—‡çš„å±é™©å› ç´ åŠç”Ÿå­˜åˆ†æž.

OBJECTIVES: To investigate the risk factors as well as their impact on patients' survival of central nervous system (CNS) complications following allogeneic hematopoietic stem cell transplantation (HSCT). METHODS: All relevant clinical data from a total of 323 patients, who underwent allogeneic HSCT in Xiangya Hospital of Central South University from September 2016 to September 2019, were retrospectively reviewed in this study. The complications' occurrence time, common symptoms and some other clinical data of the patients who developed CNS complications were analyzed descriptively. The risk factors for CNS complications following allogeneic HSCT were analyzed through univariate and multivariate analysis. And the survival analysis was conducted as well. RESULTS: Among the 323 patients who underwent allogeneic HSCT, 32 patients developed CNS complications. These complications occurred in these patients at a median of 32 (range from -1 to 584) d after transplantation. Common symptoms were disturbance of consciousness (78.1%), convulsion (59.4%), and headache (12.5%). Univariate analysis showed that there were significant differences in neutrophil engraftment, platelet (PLT) engraftment, serum cytomegalovirus (CMV) DNA positive, combined with acute graft-versus-host disease (aGVHD), donor selection (P=0.011, P<0.001, P=0.006, P<0.001 or P=0.035, respectively). Multivariate analysis showed that the delay or the failure of PLT engraftment (P<0.001) and combined with aGVHD (P<0.001) were the risk factors for CNS complications. Survival analysis showed that the 1-year overall survival rate (OS) and 2-year OS were significantly lower in patients who developed CNS complications than in the non-CNS complication group (55%±9% vs 89%±2%, 37%±11% vs 85%±3%; both P<0.001). The 1-year disease-free survival rate (DFS) and 2-year DFS were significantly lower in patients who developed CNS complications than in the non-CNS complication group (55%±9% vs 88%±2%, 29%±11% vs 83%±3%; both P<0.001). The 1-year transplantation-related mortality (TRM) and 2-year TRM were significantly higher in patients who developed CNS complications than those in the non-CNS complication group (45%±9% vs 8%±2%, 58%±11% vs 11%±2%; both P<0.001). CONCLUSIONS: The delay or the failure of PLT engraftment and combined with aGVHD are the risk factors for CNS complications. The facts indicate that we should prevent CNS complications when patients who underwent allogeneic HSCT with the delay or the failure of PLT engraftment or aGVHD. Compared with non-CNS complication group, patients who developed CNS complications usually have poor prognosis.

Identification and Characterization of a Ribose 2'-O-Methyltransferase Encoded by the Ronivirus Branch of Nidovirales.

UNLABELLED: The order Nidovirales currently comprises four virus families: Arteriviridae, Coronaviridae (divided into the subfamilies Coronavirinae and Torovirinae), Roniviridae, and the recently recognized Mesoniviridae RNA cap formation and methylation have been best studied for coronaviruses, with emphasis on the identification and characterization of two virus-encoded methyltransferases (MTases) involved in RNA capping, a guanine-N7-MTase and a ribose-2'-O-MTase. Although bioinformatics analyses suggest that these MTases may also be encoded by other nidoviruses with large genomes, such as toroviruses and roniviruses, no experimental evidence has been reported thus far. In this study, we show that a ronivirus, gill-associated virus (GAV), encodes the 2'-O-MTase activity, although we could not detect 2'-O-MTase activity for the homologous protein of a torovirus, equine torovirus, which is more closely related to coronaviruses. Like the coronavirus 2'-O-MTase, the roniviral 2'-O-MTase harbors a catalytic K-D-K-E tetrad that is conserved among 2'-O-MTases and can target only the N7-methylated cap structure of adenylate-primed RNA substrates. However, in contrast with the coronavirus protein, roniviral 2'-O-MTase does not require a protein cofactor for stimulation of its activity and differs in its preference for several biochemical parameters, such as reaction temperature and pH. Furthermore, the ronivirus 2'-O-MTase can be targeted by MTase inhibitors. These results extend our current understanding of nidovirus RNA cap formation and methylation beyond the coronavirus family. IMPORTANCE: Methylation of the 5'-cap structure of viral RNAs plays important roles in genome replication and evasion of innate recognition of viral RNAs by cellular sensors. It is known that coronavirus nsp14 acts as an N7-(guanine)-methyltransferase (MTase) and nsp16 as a 2'-O-MTase, which are involved in the modification of RNA cap structure. However, these enzymatic activities have not been shown for any other nidoviruses beyond coronaviruses in the order Nidovirales In this study, we identified a 2'-O-methyltransferase encoded by ronivirus that shows common and unique features in comparison with that of coronaviruses. Ronivirus 2'-O-MTase does not need a protein cofactor for MTase activity, whereas coronavirus nsp16 needs the stimulating factor nsp10 for its full activity. The conserved K-D-K-E catalytic tetrad is identified in ronivirus 2'-O-MTase. These results extend our understanding of nidovirus RNA capping and methylation beyond coronaviruses and also strengthen the evolutionary and functional links between roniviruses and coronaviruses.

Comparative Transcriptomic Analysis Reveals Candidate Genes and Pathways Involved in Larval Settlement of the Barnacle Megabalanus volcano.

Megabalanus barnacle is one of the model organisms for marine biofouling research. However, further elucidation of molecular mechanisms underlying larval settlement has been hindered due to the lack of genomic information thus far. In the present study, cDNA libraries were constructed for cyprids, the key stage for larval settlement, and adults of Megabalanus volcano. After high-throughput sequencing and de novo assembly, 42,620 unigenes were obtained with a N50 value of 1532 bp. These unigenes were annotated by blasting against the NCBI non-redundant (nr), Swiss-Prot, Cluster of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Finally, 19,522, 15,691, 14,459, and 10,914 unigenes were identified correspondingly. There were 22,158 differentially expressed genes (DEGs) identified between two stages. Compared with the cyprid stage, 8241 unigenes were down-regulated and 13,917 unigenes were up-regulated at the adult stage. The neuroactive ligand-receptor interaction pathway (ko04080) was significantly enriched by KEGG enrichment analysis of the DEGs, suggesting that it possibly involved in larval settlement. Potential functions of three conserved allatostatin neuropeptide-receptor pairs and two light-sensitive opsin proteins were further characterized, indicating that they might regulate attachment and metamorphosis at cyprid stage. These results provided a deeper insight into the molecular mechanisms underlying larval settlement of barnacles.

Coronavirus nsp10/nsp16 Methyltransferase Can Be Targeted by nsp10-Derived Peptide In Vitro and In Vivo To Reduce Replication and Pathogenesis.

UNLABELLED: The 5' cap structures of eukaryotic mRNAs are important for RNA stability and protein translation. Many viruses that replicate in the cytoplasm of eukaryotes have evolved 2'-O-methyltransferases (2'-O-MTase) to autonomously modify their mRNAs and carry a cap-1 structure (m7GpppNm) at the 5' end, thereby facilitating viral replication and escaping innate immune recognition in host cells. Previous studies showed that the 2'-O-MTase activity of severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 16 (nsp16) needs to be activated by nsp10, whereas nsp16 of feline coronavirus (FCoV) alone possesses 2'-O-MTase activity (E. Decroly et al., J Virol 82:8071-8084, 2008, http://dx.doi.org/10.1128/JVI.00407-08; M. Bouvet et al., PLoS Pathog 6:e1000863, 2010, http://dx.doi.org/10.1371/journal.ppat.1000863; E. Decroly et al., PLoS Pathog 7:e1002059, 2011, http://dx.doi.org/10.1371/journal.ppat.1002059; Y. Chen et al., PLoS Pathog 7:e1002294, 2011, http://dx.doi.org/10.1371/journal.ppat.1002294) . In this study, we demonstrate that stimulation of nsp16 2'-O-MTase activity by nsp10 is a universal and conserved mechanism in coronaviruses, including FCoV, and that nsp10 is functionally interchangeable in the stimulation of nsp16 of different coronaviruses. Based on our current and previous studies, we designed a peptide (TP29) from the sequence of the interaction interface of mouse hepatitis virus (MHV) nsp10 and demonstrated that the peptide inhibits the 2'-O-MTase activity of different coronaviruses in biochemical assays and the viral replication in MHV infection and SARS-CoV replicon models. Interestingly, the peptide TP29 exerted robust inhibitory effects in vivo in MHV-infected mice by impairing MHV virulence and pathogenesis through suppressing virus replication and enhancing type I interferon production at an early stage of infection. Therefore, as a proof of principle, the current results indicate that coronavirus 2'-O-MTase activity can be targeted in vitro and in vivo. IMPORTANCE: Coronaviruses are important pathogens of animals and human with high zoonotic potential. SARS-CoV encodes the 2'-O-MTase that is composed of the catalytic subunit nsp16 and the stimulatory subunit nsp10 and plays an important role in virus genome replication and evasion from innate immunity. Our current results demonstrate that stimulation of nsp16 2'-O-MTase activity by nsp10 is a common mechanism for coronaviruses, and nsp10 is functionally interchangeable in the stimulation of nsp16 among different coronaviruses, which underlies the rationale for developing inhibitory peptides. We demonstrate that a peptide derived from the nsp16-interacting domain of MHV nsp10 could inhibit 2'-O-MTase activity of different coronaviruses in vitro and viral replication of MHV and SARS-CoV replicon in cell culture, and it could strongly inhibit virus replication and pathogenesis in MHV-infected mice. This work makes it possible to develop broad-spectrum peptide inhibitors by targeting the nsp16/nsp10 2'-O-MTase of coronaviruses.

Leptin Genes in Blunt Snout Bream: Cloning, Phylogeny and Expression Correlated to Gonads Development.

To investigate the leptin related genes expression patterns and their possible function during the gonadal development in fish, the cDNA and genomic sequences of leptin, leptin receptor (leptinR), and leptin receptor overlapping transcript like-1 (leprotl1) were cloned and their expression levels were quantified in the different gonadal development stages of Megalobrama amblycephala. The results showed that the full length cDNA sequences of leptin, leptinR and leprotl1 were 953, 3432 and 1676 bp, coding 168, 1082, and 131 amino acid polypeptides, and the genomic sequences were 1836, 28,528 and 5480 bp, which respectively had 3, 15 and 4 exons, respectively. The phylogenetic analysis revealed that three genes were relatively conserved in fish species. Quantitative real-time PCR results showed that the three genes were ubiquitously expressed in all examined tissues during the different gonadal development stages. The leptin and leptinR took part in the onset of puberty, especially in female M. amblycephala, by increasing the expression levels in brain during the stage I to III of ovary. The expression levels of leptin and leptinR had significant differences between male and female in hypothalamic-pituitary-gonadal (HPG) axis tissues (p < 0.05). The leptinR had the same variation tendency with leptin, but the opposite changes of expression levels were found in leprotl1, which may resist the expression of leptinR for inhibiting the function of leptin in target organ. These findings revealed details about the possible role of these genes in regulating gonadal maturation in fish species.