Transcriptomic Analysis of Mycobacterial Infected Macrophages Reveals a High MOI Specific Type I IFN Signaling.

Macrophage (MΦ) infection models are important tools for studying host-mycobacterial interactions. Although the multiplicity of infection (MOI) is an important experimental variable, the selection of MOI in mycobacterial infection experiments is largely empirical, without reference to solid experimental data. To provide relevant data, we used RNA-seq to analyze the gene expression profiles of MΦs 4 or 24 h after infection with Mycobacterium marinum (M. m) at MOIs ranging from 0.1 to 50. Analysis of differentially expressed genes (DEGs) showed that different MOIs are linked to distinct transcriptomic changes and only 10% of DEGs were shared by MΦ infected at all MOIs. KEGG pathway enrichment analysis revealed that type I interferon (IFN)-related pathways were inoculant dose-dependent and enriched only at high MOIs, whereas TNF pathways were inoculant dose-independent and enriched at all MOIs. Protein-protein interaction (PPI) network alignment showed that different MOIs had distinct key node genes. By fluorescence-activated cell sorting and follow-up RT-PCR analysis, we could separate infected MΦs from uninfected MΦs and found phagocytosis of mycobacteria to be the determinant factor for type I IFN production. The distinct transcriptional regulation of RAW264.7 MΦ genes at different MOIs was also seen with Mycobacterium tuberculosis (M.tb) infections and primary MΦ infection models. In summary, transcriptional profiling of mycobacterial infected MΦs revealed that different MOIs activate distinct immune pathways and the type I IFN pathway is activated only at high MOIs. This study should provide guidance for selecting the MOI most appropriate for different research questions.

TRIM14 is a mitochondrial adaptor that facilitates retinoic acid-inducible gene-I-like receptor-mediated innate immune response.

Innate immunity provides the first line of host defense against invading microbial pathogens. This defense involves retinoic acid-inducible gene-I-like receptors that detect viral RNA and activate the mitochondrial antiviral-signaling (MAVS) protein, an adaptor protein, leading to activation of the innate antiviral immune response. The mechanisms by which the MAVS signalosome assembles on mitochondria are only partially understood. Here, we identify tripartite motif 14 (TRIM14) as a mediator in the immune response against viral infection. TRIM14 localizes to the outer membrane of mitochondria and interacts with MAVS. Upon viral infection, TRIM14 undergoes Lys-63-linked polyubiquitination at Lys-365 and recruits NF-κB essential modulator to the MAVS signalosome, leading to the activation of both the IFN regulatory factor 3 and NF-κB pathways. Knockdown of TRIM14 disrupts the association between NF-κB essential modulator and MAVS and attenuates the antiviral response. Our results indicate that TRIM14 is a component of the mitochondrial antiviral immunity that facilitates the immune response mediated by retinoic acid-inducible gene-I-like receptors.

Histidine-tryptophan-ketoglutarate solution with added ebselen augments myocardial protection in neonatal porcine hearts undergoing ischemia/reperfusion.

Whether modified histidine-tryptophan-ketoglutarate (HTK) solution offers myocardial protection to newborn heart has not been documented. The purpose of this study was to compare myocardial protection using HTK added by ebselen with HTK in a piglet model of cardiopulmonary bypass (CPB). Fifteen piglets were randomly assigned to three groups: the control group (C group, n = 5), HTK solution group (HTK group, n = 5), and HTK added by 10 nM ebselen group (HTK+E group, n = 5). Animals in the two experimental groups were placed on hypothermic CPB, after which the ascending aorta had been clamped for 2 h. The control animals underwent normothermic CPB without cardiac arrest. Myocardial antioxidant activities, myocytes apoptosis and mitochondrial structures, as well as the release of cytochrome c and the expression of Bax, Bcl-2, and HSP72 protein in myocardium were measured. Increased myocardial superoxide dismutase (SOD) and Mn-SOD activities, decreased TUNEL-positive cells, and reduced release of cytochrome c were noted in the HTK+E group compared with those in the HTK group (P = 0.021, P = 0.020, P = 0.045, and P = 0.010, respectively). The Bax/Bcl-2 ratio in the HTK group was significantly higher than that in the C group (P = 0.024). The expression of HSP72 protein and mRNA in the HTK+E group was higher than that in the HTK group (P = 0.039 and P = 0.035, respectively). Mitochondrial score under electron microscope in the HTK+E group was lower than that in the HTK group (P = 0.047). Improved antioxidant defense, reduced myocytes apoptosis, and better preserved mitochondrial structure were observed in the HTK+E group. Ebselen added to HTK provides better myocardioprotection to HTK solution for the neonatal heart.

Cleavage of interferon regulatory factor 7 by enterovirus 71 3C suppresses cellular responses.

Enterovirus 71 (EV71) is a positive-stranded RNA virus which is capable of inhibiting innate immunity. Among virus-encoded proteins, the 3C protein compromises the type I interferon (IFN-I) response mediated by retinoid acid-inducible gene-I (RIG-I) or Toll-like receptor 3 that activates interferon regulatory 3 (IRF3) and IRF7. In the present study, we report that enterovirus 71 downregulates IRF7 through the 3C protein, which inhibits the function of IRF7. When expressed in mammalian cells, the 3C protein mediates cleavage of IRF7 rather than that of IRF3. This process is insensitive to inhibitors of caspase, proteasome, lysosome, and autophagy. H40D substitution in the 3C active site abolishes its activity, whereas R84Q or V154S substitution in the RNA binding motif has no effect. Furthermore, 3C-mediated cleavage occurs at the Q189-S190 junction within the constitutive activation domain of IRF7, resulting in two cleaved IRF7 fragments that are incapable of activating IFN expression. Ectopic expression of wild-type IRF7 limits EV71 replication. On the other hand, expression of the amino-terminal domain of IRF7 enhances EV71 infection, which correlates with its ability to interact with and inhibit IRF3. These results suggest that control of IRF7 by the 3C protein may represent a viral mechanism to escape cellular responses.

Integrated single-cell transcriptome and T cell receptor profiling reveals defects of T cell exhaustion in pulmonary tuberculosis.

Tuberculosis-affected lungs with chronic inflammation harbor abundant immunosuppressive immune cells but the nature of such inflammation is unclear. Dysfunction in T cell exhaustion, while implicated in chronic inflammatory diseases, remains unexplored in tuberculosis. Given that immunotherapy targeting exhaustion checkpoints exacerbates tuberculosis, we speculate that T cell exhaustion is dysfunctional in tuberculosis. Using integrated single-cell RNA sequencing and T cell receptor profiling we reported defects in exhaustion responses within inflamed tuberculosis-affected lungs. Tuberculosis lungs demonstrated significantly reduced levels of exhausted CD8+ T cells and exhibited diminished expression of exhaustion-related transcripts among clonally expanded CD4+ and CD8+ T cells. Additionally, clonal expansion of CD4+ and CD8+ T cells bearing T cell receptors specific for CMV was observed. Expanded CD8+ T cells expressed the cytolytic marker GZMK. Hence, inflamed tuberculosis-affected lungs displayed dysfunction in T cell exhaustion. Our findings likely hold implications for understanding the reactivation of tuberculosis observed in patients undergoing immunotherapy targeting the exhaustion checkpoint.

Ulinastatin as a neuroprotective and anti-inflammatory agent in infant piglets model undergoing surgery on hypothermic low-flow cardiopulmonary bypass.

OBJECTIVE: Infants are potentially more susceptible to brain injury mediated via cell death attributed to cardiopulmonary bypass (CPB) especially with prolonged hypothermic low flow (HLF). We hypothesized that a human urinary protease inhibitor (ulinastatin), by its anti-inflammatory effect, would reduce central nervous system (CNS) injury during HLF. METHODS: Fifteen general-type infant piglets were randomized to ulinastatin group (group U, n = 5), control group (group C, n = 5), and sham operation group (group S, n = 5). Routine CPB was established after median thoracotomy in group U and C under anesthesia. When the temperature of infant piglets dropped down to 25 °C, low-flow CPB (50 ml·kg(-1) ·min(-1) ) was instituted. After 120 min of aortic cross-clamping and 20- to 30-min rewarming, the aortic cross-clamp was removed and finally the piglet was weaned from CPB. Five thousand units per killogram of ulinastatin and equivalently normal saline were, respectively, given at the beginning of and at aortic declamping in group U and group C. group S just received sham median thoracotomy. Venous blood samples were taken immediately after anesthesia induction in all three groups, 5- and 120-min post CPB in both group U and C, respectively; plasma markers of inflammation and CNS injury were compared. Pathology results of hippocampus were observed by light microscopy. RESULTS: Statistically significant differences between group C and U were noted in the expression of inflammatory markers such as IL-10, TNF-α and neuron-specific enolase at 120-min post CPB. Brain injuries were observed in both groups (index cases and controls) and were milder in group U. CONCLUSIONS: In our study, HLF CPB on infant piglets resulted in brain injury, and ulinastatin might reduce the extent of such injury.

[Effectiveness and safety of tranexamic acid in patients receiving on-pump coronary artery bypass grafting without clopidogrel and aspirin cessation].

OBJECTIVE: To evaluate the effectiveness and safty of tranexamic acid in patients receiving on-pump coronary artery bypass grafting (CABG) without clopidogrel and aspirin cessation. METHODS: The current study is a prospective, randomized and placebo-control trial. A total of 116 patients receiving selective on-pump CABG with their last ingestion of clopidogrle and aspirin within 7 days preoperatively were recruited. Despite 6 patients withdrawal their consent, the rest 110 were randomized to receive tranexamic acid or placebo. The tranexamic acid regimen was a bolus of 10 mg/kg followed by a maintenance of 10 mg·kg(-1)·h(-1) throughout the surgery. The primary outcome was the volume of allogeneic erythrocyte transfused perioperatively. RESULTS: Baseline characteristics were comparable between the groups. In patients receiving tranexamic acid and placebo respectively, the volume of allogeneic erythrocyte transfused was 4.0 (7.5) units and 6.0(6.0) units (W = 1021, P < 0.01). In these 2 groups respectively, blood loss was 930 (750) ml and 1210 (910) ml (W = 1042, P < 0.01), the incidence of major bleeding was 50.9% and 76.4% (χ(2) = 7.70, P < 0.01), the incidence of reoperation was 0 and 9.1% (χ(2) = 5.24, P = 0.02); the volume of plasma transfused was 400 (600) ml and 600 (650) ml (W = 1072, P = 0.01), the exposure of plasma was 60.0% and 85.5% (χ(2) = 8.98, P < 0.01) and the exposure to any allogeneic blood products was 85.5% and 98.2% (χ(2) = 5.93, P = 0.01). Perioperative mortality, morbidity and the incidence of adverse events were balanced between the groups without statistical significance. CONCLUSION: Tranexamic acid reduced significantly postoperative bleeding and transfusion in patients receiving on-pump CABG without clopidogrel and aspirin cessation.

Enterovirus 71 induces degradation of TRIM38, a potential E3 ubiquitin ligase.

BACKGROUND: The tripartite motif (TRIM) proteins are a family of more than 70 members in human. However, only a few of them have been well studied. The TRIM proteins contain the conserved RING, B-box, coiled-coil, and SPRY domains, most of which are involved in protein ubiquitination. TRIM38 is a member of the TRIM protein family, which we studied in more detail here as its functions are largely unknown. RESULTS: Our study shows that, similar to other TRIM family members, TRIM38 is localized in the cytoplasm. TRIM38 increases ubiquitination of other cellular proteins and catalyzes self-ubiquitination. TRIM38 also promotes K63- and K48-linked ubiquitination of cellular proteins. An intact RING domain is important for the functions of TRIM38. In addition, enterovirus 71 infection induces TRIM38 degradation. CONCLUSIONS: Our observations demonstrate that TRIM38 has E3 ubiquitin ligase activity and can be degraded during virus infection. These findings may provide insight into innate immune signaling pathways.

Lysosome-associated membrane glycoprotein 3 is involved in influenza A virus replication in human lung epithelial (A549) cells.

BACKGROUND: Influenza A virus mutates rapidly, rendering antiviral therapies and vaccines directed against virus-encoded targets ineffective. Knowledge of the host factors and molecular pathways exploited by influenza virus will provide further targets for novel antiviral strategies. However, the critical host factors involved in influenza virus infection have not been fully defined. RESULTS: We demonstrated that LAMP3, a member of lysosome-associated membrane glycoprotein (LAMP) family, was significantly induced in human lung epithelial (A549) cells upon influenza A virus infection. Knockdown of LAMP3 expression by RNA interference attenuated production of viral nucleoprotein (NP) as well as virus titers. Confocal microscopy results demonstrated that viral NP is colocalized within LAMP3 positive vesicles at early stages of virus infection. Furthermore, knockdown of LAMP3 expression led to a reduction in nuclear accumulation of viral NP and impeded virus replication. CONCLUSIONS: LAMP3 is an influenza A virus inducible gene, and plays an important role in viral post-entry steps. Our observations may provide insights into the mechanism of influenza virus replication and potential targets for novel anti-influenza therapeutics.

Rotavirus nonstructural protein 1 antagonizes innate immune response by interacting with retinoic acid inducible gene I.

BACKGROUND: The nonstructural protein 1 (NSP1) of rotavirus has been reported to block interferon (IFN) signaling by mediating proteasome-dependent degradation of IFN-regulatory factors (IRFs) and (or) the ß-transducin repeat containing protein (ß-TrCP). However, in addition to these targets, NSP1 may subvert innate immune responses via other mechanisms. RESULTS: The NSP1 of rotavirus OSU strain as well as the IRF3 binding domain truncated NSP1 of rotavirus SA11 strain are unable to degrade IRFs, but can still inhibit host IFN response, indicating that NSP1 may target alternative host factor(s) other than IRFs. Overexpression of NSP1 can block IFN-ß promoter activation induced by the retinoic acid inducible gene I (RIG-I), but does not inhibit IFN-ß activation induced by the mitochondrial antiviral-signaling protein (MAVS), indicating that NSP1 may target RIG-I. Immunoprecipitation experiments show that NSP1 interacts with RIG-I independent of IRF3 binding domain. In addition, NSP1 induces down-regulation of RIG-I in a proteasome-independent way. CONCLUSIONS: Our findings demonstrate that inhibition of RIG-I mediated type I IFN responses by NSP1 may contribute to the immune evasion of rotavirus.

Cyclophilin A interacts with influenza A virus M1 protein and impairs the early stage of the viral replication.

Influenza A virus matrix protein (M1) is the most abundant conservative protein that regulates the replication, assembly and budding of the viral particles upon infection. Several host cell factors have been determined to interact with M1 possibly in regulating influenza virus replication. By yeast two-hybrid screening, the isomerase cyclophilin A (CypA) was identified to interact with the M1 protein. CypA specifically interacted with M1 both in vitro and in vivo. The mutagenesis results showed CypA bound to the functional middle (M) domain of M1. The depletion of endogenous CypA by RNA interference resulted in the increase of influenza virus infectivity while overexpression of CypA caused decreasing the infectivity in affected cells. The immunofluorescence assays indicated that overexpressed CypA deduced the infectivity and inhibited the translocation of M1 protein into the nucleus while did not affect nucleoprotein entering the nucleus. Further studies indicated that overexpression of CypA significantly increased M1 self-association. Western blot with purified virions confirmed that CypA was encapsidated within the virus particle. These results together indicated that CypA interacted with the M1 protein and affected the early stage of the viral replication.

Zinc transporter SLC39A7 relieves zinc deficiency to suppress alternative macrophage activation and impairment of phagocytosis.

Macrophages are key phagocytic cells and play an important role in eliminating external microorganisms and endogenous danger signals. Dysregulation in macrophage functions have been reported in patients with asthma. Zinc homeostasis is critical in maintaining macrophage functions. The solute carrier (SLC) protein SLC39A7, a Zn2+ importer, has recently been linked to asthma. However, the roles of SLC39A7 in macrophage phagocytosis are not well understood. Here we found that phagocytosis efficiency was significantly decreased in SLC39A7-knockdown THP-1 cells, however the phagocytosis capability could be reversed with zinc supplementation. SLC39A7 deficiency skewed macrophages towards alternative activation, as indicated by increased expression of M2 activation marker CD206 and decreased expression of M1 activation marker NOS2. Consistent to this result, SLC39A7-knockdown cells produced reduced amounts of proinflammatory cytokines TNF- and IL-6. Furthermore, the mRNA level of receptor Clec4e previously known to be involved in phagocytosis of BCG was significantly reduced in SLC39A7 knockdown cells. Importantly, all these defects due to SLC39A7 deficiency could be reversed by zinc supplementation. Thus, zinc transporter SLC39A7 provide support for phagocytosis and classical macrophage activation.

Effect of ulinastatin on post-operative blood loss and allogeneic transfusion in patients receiving cardiac surgery with cardiopulmonary bypass: a prospective randomized controlled study with 10-year follow-up.

BACKGROUND: Major bleeding and allogeneic transfusion leads to negative outcomes in patients receiving cardiac surgery with cardiopulmonary bypass (CPB). Ulinastatin, a urine trypsin inhibitor, relieves systemic inflammation and improves coagulation profiles with however sparse evidence of its effects on blood loss and allogeneic transfusion in this specific population. METHODS: In this prospective randomized controlled trial, 426 consecutive patients receiving open heart surgery with CPB were randomly assigned into three groups to receive ulinastatin (group U, n = 142), tranexamic acid (group T, n = 143) or normal saline (group C, n = 141). The primary outcome was the total volume of post-operative bleeding and the secondary outcome included the volume and exposure of allogeneic transfusion, the incidence of stroke, post-operative myocardial infarction, renal failure, respiratory failure and all-cause mortality. A ten-year follow-up was carried on to evaluate long-term safety. RESULTS: Compared with placebo, ulinastatin significantly reduced the volume of post-operative blood loss within 24 h (688.39 ± 393.55 ml vs 854.33 ± 434.03 ml MD - 165.95 ml, 95%CI - 262.88 ml to - 69.01 ml, p < 0.001) and the volume of allogeneic erythrocyte transfusion (2.57 ± 3.15 unit vs 3.73 ± 4.21 unit, MD-1.16 unit, 95%CI - 2.06 units to - 0.26 units, p = 0.002). The bleeding and transfusion outcomes were comparable between the ulinastatin group and the tranexamic acid group. In-hospital outcomes and 10-year follow-up showed no statistical difference in mortality and major morbidity among groups. CONCLUSIONS: Ulinastatin reduced post-operative blood loss and allogeneic erythrocyte transfusion in heart surgery with CPB. The mortality and major morbidity was comparable among the groups shown by the 10-year follow-up. TRIAL REGISTRATION: The trial was retrospectively registered on February 2, 2010. TRIAL REGISTRATION NUMBER: https://www.clinicaltrials.gov Identifier: NCT01060189.

Effects of mixed amendments on the phytoavailability of Cd in contaminated paddy soil under a rice-rape rotation system.

A field experiment was performed to study the effects of mixed amendments, namely lime + organic fertilizer (LO), lime + organic fertilizer + calcium-magnesium phosphate fertilizer (LOC), lime + organic fertilizer + sepiolite (LOS), and lime + organic fertilizer + calcium-magnesium phosphate fertilizer + sepiolite (LOCS), on the availability and uptake of Cd from contaminated paddy soil under a rice-rape (Oryza sativa L. and Brassica napus L.) rotation system. The results showed that the grain yields of rice and rape with mixed amendment-treated were slightly influenced, in that the soil pH significantly increased while the DTPA-extractable Cd content of the soil and Cd uptake by the rice and rape were significantly reduced. The uptake of Cd by brown rice decreased significantly (p < 0.05), by 55.9-59.3% and 69.6-75.5% in the 2016 and 2017 crops, respectively, compared with that of the control (CK). The Cd uptake by rapeseeds during the 2017 season observably (p < 0.05) decreased by 38.2 and 29.6% under LO and LOC treatments, respectively. The Cd concentrations in rapeseeds were 0.11-0.18 mg kg-1 under all the treatments except LOCS treatment, which is lower than the National Standard of Pollutants in Food of China (GB 2762-2017, 0.2 mg kg-1). From both economic and food safety standpoints, rape is recommended for Cd-contaminated soil because it has a low Cd accumulation ability. The results showed that the rice-rape rotation combined with LO or LOC application was useful for reducing the Cd content in both rice and rape in Cd-contaminated soil and the effects could be sustained at least for three crop seasons.

Atmospheric bulk deposition of heavy metal(loid)s in central south China: Fluxes, influencing factors and implication for paddy soils.

The depositions of heavy metal(loid)s (HMs) were measured in an urban agglomeration of China to investigate the fluxes, influencing factors, sources, and potential effects of these HMs. Our results showed that the deposition fluxes of As and Cd were higher in this area than in other regions. In the area, 59.63% of the total deposition fluxes of the Cr, Cu, Ni, Pb and Zn were observed in the wet season (March to July). Lower total fluxes of HMs were observed at the rural site. Principal component analysis (PCA) results showed that the As, Cd, Pb, and Zn might originate from the same anthropogenic sources, including traffic and industrial sources, and that the Cr, Cu, and Ni might come from natural sources. Correlation analysis and redundancy analysis (RDA) showed that rainfall, wind speed, and PMs were critical factors influencing the atmospheric bulk deposition of HMs. For the paddy soil, the input fluxes of HMs by deposition, accounted for 38.66-84.57% (except for Cr) of the total input fluxes. The prediction indicated that the accumulation of HMs in surface soil will notably increase over the next decades due to the influence of atmospheric deposition.

[Distribution and Accumulation of Cadmium in Paddy Soil and Rice Affected by Pollutant Sources Control and Improvement Measures].

The objective of this study was to determine the effect of five scenarios on the accumulation of Cd in the soil-rice system, including the return of straw to the field and the lack of the return, atmospheric deposition control, use of clean water for irrigation, and the use of lime. For the field experiments, three typical paddies were selected and divided into five plots (5 m×6 m) in Xiangtan, Zhuzhou, and Liling in the Hunan province from April to October 2016. The results showed that the application of lime can increase pH by 0.87, while the available Cd concentration in the soil was decreased by 33.7%. The accumulations of Cd in roots, stems, and brown rice were decreased by 47.9%, 46.7%, and 54.8%, respectively, with a decrease in the corresponding bioconcentration factors. Irrigating with clean water and liming tended to increase the soil pH by 0.44 and 0.49, respectively, while the available Cd concentration in the soil was decreased by 18.2% and 14.5%, respectively. The Cd concentrations in roots, stems, and brown rice were decreased by 32.6%, 24.2%, and 18.0%, and 17.6%, 11.3%, and 25.4% with decreased bioconcentration factors under both treatments (irrigating with clean water and liming). The available Cd concentration in the soil was increased by 6.1% and the Cd accumulation in the rice plants also increased with the return of straw to the soil. The bioconcentration factors of the rice plants were also increased when the paddy straw was returned to the fields. The results showed that the measures, such as the use of lime, atmospheric deposition control, use of clean water for irrigation, and lack of the return of straw to the paddy soil, should be helpful for the safe production of brown rice. The possible long-term risks associated with returning straw to the paddy field should be evaluated scientifically.

Cloning and characterization of a novel feline IFN-omega.

The interferons (IFNs) are a large family of multifunctional secreted protein involved in antiviral defense, cell growth regulation, and immune activation. The human IFNs are used worldwide as antiviral drugs. Here, we present cDNAs encoding 13 novel feline IFN-omega (FeIFN-omega) subtypes that share 95%-99% amino acid sequence identity. FeIFN-omega2 and FeIFN-omega4 have seven additional amino acids at position 109 that are not present in other subtypes. Sequence identity of the present FeIFN proteins encoded by the 13 subtypes is approximately 57% compared with human IFN-omega (HuIFN-omega). All 13 FeIFN-omega subtypes were expressed in Escherichia coli using a periplasmic expression system. The antiviral activity of each product was evaluated in vitro. In addition, subtype FeIFN-omega2 was cytoplasm expressed in E. coli and secretion expressed in Pichia pastoris. The purified mature recombinant protein demonstrated significant antiviral activity on both homologous and heterologous animal cells in vitro.

Ulinastatin Protects against Acute Kidney Injury in Infant Piglets Model Undergoing Surgery on Hypothermic Low-Flow Cardiopulmonary Bypass.

OBJECTIVE: Infants are more vulnerable to kidney injuries induced by inflammatory response syndrome and ischemia-reperfusion injury following cardiopulmonary bypass especially with prolonged hypothermic low-flow (HLF). This study aims to evaluate the protective role of ulinastatin, an anti-inflammatory agent, against acute kidney injuries in infant piglets model undergoing surgery on HLF cardiopulmonary bypass. METHODS: Eighteen general-type infant piglets were randomly separated into the ulinastatin group (Group U, n = 6), the control group (Group C, n = 6), and the sham operation group (Group S, n = 6), and anaesthetized. The groups U and C received following experimental procedure: median thoracotomy, routine CPB and HLF, and finally weaned from CPB. The group S only underwent sham median thoracotomy. Ulinastatin at a dose of 5,000 units/kg body weight and a certain volume of saline were administrated to animals of the groups U and C at the beginning of CPB and at aortic declamping, respectively. Venous blood samples were collected at 3 different time points: after anesthesia induction in all experimental groups, 5 minutes, and 120 minutes after CPB in the Groups U and C. Markers for inflammation and acute kidney injury were tested in the collected plasma. N-acetyl-ß-D-glucosaminidase (NAG) from urine, markers of oxidative stress injury and TUNEL-positive cells in kidney tissues were also detected. RESULTS: The expressions of plasma inflammatory markers and acute kidney injury markers increased both in Group U and Group C at 5 min and 120 min after CPB. Also, numbers of TUNEL-positive cells and oxidative stress markers in kidney rose in both groups. At the time point of 120-min after CPB, compared with the Group C, some plasma inflammatory and acute kidney injury markers as well as TUNEL-positive cells and oxidative stress markers in kidney were significantly reduced in the Group U. Histologic analyses showed that HLF promoted acute tubular necrosis and dilatation. CONCLUSIONS: HLF cardiopulmonary bypass surgery could intensify systemic inflammatory responses and oxidative stress on infant piglets, thus causing acute kidney injury. Ulinastatin might reduce such inflammatory response and oxidative stress and the extent of kidney injury.

Fragile X mental retardation protein stimulates ribonucleoprotein assembly of influenza A virus.

The ribonucleoprotein (RNP) of the influenza A virus is responsible for the transcription and replication of viral RNA in the nucleus. These processes require interplay between host factors and RNP components. Here, we report that the Fragile X mental retardation protein (FMRP) targets influenza virus RNA synthesis machinery and facilitates virus replication both in cell culture and in mice. We demonstrate that FMRP transiently associates with viral RNP and stimulates viral RNP assembly through RNA-mediated interaction with the nucleoprotein. Furthermore, the KH2 domain of FMRP mediates its association with the nucleoprotein. A point mutation (I304N) in the KH2 domain, identified from a Fragile X syndrome patient, disrupts the FMRP-nucleoprotein association and abolishes the ability of FMRP to participate in viral RNP assembly. We conclude that FMRP is a critical host factor used by influenza viruses to facilitate viral RNP assembly. Our observation reveals a mechanism of influenza virus RNA synthesis and provides insights into FMRP functions.

Identification of rabies virus mimotopes screened from a phage display peptide library with purified dog anti-rabies virus serum IgG.

The rabies virus glycoprotein (G) is a key protein for both virus infectivity and eliciting protective immunity as an antigen. What is more, the nucleoprotein (N) is also a significant rabies virus antigen. In this study, purified anti-rabies virus IgG from dogs immunized with the standard CVS-11 strain was used to screen the Ph.D.-12™ Phage Display Peptide Library for peptides that correspond to or mimic native G and N epitopes. In contrast to previous reports that use monoclonal antibodies or human anti-rabies virus serum, this study describes the first use of dog serum to screen for epitopes. After three rounds of biopanning, selected phage clones were identified by plaque screening, western blotting (WB), and ELISA. Positive phage clones were sequenced, and their amino acid sequences were deduced. Alignment of the peptide sequences to G and N indicated that the epitope peptides matched well with G amino acids at positions 34-42, 198-200, 226-264, 296-371, and 330-343, as well as to N amino acids at positions 22-168 (N-terminal) and 262-450 (C-terminal), confirming that the sequences were indeed mimicking epitopes. Thirty percent of the selected clones matched reported antigenic regions located at sites II and III of the glycoprotein. Two sequences, LEPKGRYDDPWT and ATRYDDIWASTA, that have no homology to the known antigenic sites of either the G or N exhibited a common RYDD-W-T motif that is highly homologous to the amino acid residues at positions 126-141 of the G. This finding indicates that this motif may be a new potential RABV G B cell epitope. Amino acids 126-141 containing the RYDD-W-T motif may become a novel key epitope region and allow the development of a rabies vaccine or diagnostic reagents for the treatment of rabies.