[Effects of Platycodonis Radix-Curcumae Rhizoma on oral nanoparticle uptake and in vitro inhibition against breast cancer metastasis].

This study combined the herbal pair Platycodonis Radix-Curcumae Rhizoma(PR-CR) possessing an inhibitory effect on tumor cell proliferation and metastasis with the active component of traditional Chinese medicine(TCM) silibinin-loaded nanoparticles(NPs) with a regulatory effect on tumor microenvironment based on the joint effect on tumor cells and tumor microenvironment to inhi-bit cell metastasis. The effects of PR-CR on the cellular uptake of NPs and in vitro inhibition against breast cancer proliferation and metastasis were investigated to provide an experimental basis for improving nanoparticle absorption and enhancing therapeutic effects. Silibinin-loaded lipid-polymer nanoparticles(LPNs) were prepared by the nanoprecipitation method and characterized by transmission electron microscopy. The NPs were spherical or quasi-spherical in shape with obvious core-shell structure. The mean particle size was 107.4 nm, Zeta potential was-27.53 mV. The cellular uptake assay was performed by in vitro Caco-2/E12 coculture cell model and confocal laser scanning microscopy(CLSM), and the results indicated that PR-CR could promote the uptake of NPs. Further, in situ intestinal absorption assay by the CLSM vertical scanning approach showed that PR-CR could promote the absorption of NPs in the enterocytes of mice. The inhibitory effect of NPs on the proliferation and migration of 4T1 cells was analyzed using 4T1 breast cancer cells and co-cultured 4T1/WML2 cells, respectively. The results of the CCK8 assay showed that PR-CR-containing NPs could enhance the inhibition against the proliferation of 4T1 breast cancer cells. The wound healing assay indicated that PR-CR-containing NPs enhanced the inhibition against the migration of 4T1 breast cancer cells. This study enriches the research on oral absorption of TCM NPs and also provides a new idea for utilizing the advantages of TCM to inhibit breast cancer metastasis.

Mast cells contribute to Enterovirus 71 infection-induced pulmonary edema in neonatal mice.

Enterovirus (EV) 71 infection has been widely acknowledged as the leading cause of severe hand, foot and mouth disease (HFMD), which may rapidly lead to fatal pulmonary edema. In this study, we established a mouse model for EV71 infection exhibiting high incidence of severe symptoms with pulmonary edema. Mast cells (MCs) accumulation, activation and allergic inflammation were found in the brains, lungs and skeletal muscle of mice after EV71 infection, especially in the lungs of mice. Levels of histamine, platelet-activating factor (PAF), interleukin (IL)-4, IL-5, IL-13, tumor necrosis factor-α (TNF-α), nitric oxide (NO), endocrine gland-derived vascular endothelial growth factor (EG-VEGF) and noradrenaline (NA) were increased in EV71-infected lungs. In addition, EV71 infection reduced the number of pulmonary T cells, dendritic cells (DCs) and monocytes, and increased the number of lung eosinophils, Tregs and MCs. MCs number and tryptase expression in target organs or tissues posed a trend towards an increase from control to severe mice. There were positive correlations between MCs number in the brains (r = 0.701, P = 0.003), lungs (r = 0.802, P < 0.0001), skeletal muscles (r = 0.737, P = 0.001) and mean clinical score. Thus, our results suggested that MCs contributed to the pulmonary edema during EV71 infection.

Pulmonary edema following central nervous system lesions induced by a non- mouse-adapted EV71 strain in neonatal BALB/c mice.

BACKGROUND: Enterovirus (EV) infection has been a serious health issue in Asia-Pacific region. It has been indicated that the occurrence of fatal hand foot and mouth disease (HFMD) cases following EV71 infection is mainly attributed to pulmonary edema. However, the development of pulmonary disorders after EV71 infection remains largely unknown. To establish an EV71-infected animal model and further explore the underlying association of central nervous system (CNS) invasion with pulmonary edema, we isolated a clinical source EV71 strain (ZZ1350) from a severe case in Henan Province. METHODS: We evaluated the cytotoxicity of ZZ1350 strain and the susceptibility in 3-day-old BALB/c mice with intraperitoneal, intracerebral and intramuscular inoculation. Various histopathological and immunohistochemical techniques were applied to determine the target organs or tissue damage after infection. Correlation analysis was used to identify the relationship between CNS injury and pulmonary disorders. RESULTS: Our experimental results suggested that ZZ1350 (C4 subtype) had high cytotoxicity against African green monkey kidney (Vero) cells and human rhabdomyosarcoma (RD) cells and neonatal BALB/c mice were highly susceptible to the infection with ZZ1350 through three different inoculation routes (2 × 106 pfu/mouse) exhibiting severe neurological and respiratory symptoms that were similar to clinical observation. Viral replication was found in brain, spinal cord, skeletal muscle, lung, spleen, liver, heart of infected mice and these sections also showed histopathological changes. We found that brain histology score was positive correlated with lung histology score in total experimental mice and mice under the three inoculation routes (P < 0.05). At the same time, there were positive correlations between spinal cord score and lung score in total experimental mice and mice with intracerebral inoculation (P < 0.05). CONCLUSIONS: ZZ1350 strain is effective to establish animal model of EV71 infection with severe neurological and respiratory symptoms. The development of pulmonary disorders after EV71 infection is associated with severity of CNS damage.

Crystal structure of the CorA Mg2+ transporter.

The magnesium ion, Mg2+, is essential for myriad biochemical processes and remains the only major biological ion whose transport mechanisms remain unknown. The CorA family of magnesium transporters is the primary Mg2+ uptake system of most prokaryotes and a functional homologue of the eukaryotic mitochondrial magnesium transporter. Here we determine crystal structures of the full-length Thermotoga maritima CorA in an apparent closed state and its isolated cytoplasmic domain at 3.9 A and 1.85 A resolution, respectively. The transporter is a funnel-shaped homopentamer with two transmembrane helices per monomer. The channel is formed by an inner group of five helices and putatively gated by bulky hydrophobic residues. The large cytoplasmic domain forms a funnel whose wide mouth points into the cell and whose walls are formed by five long helices that are extensions of the transmembrane helices. The cytoplasmic neck of the pore is surrounded, on the outside of the funnel, by a ring of highly conserved positively charged residues. Two negatively charged helices in the cytoplasmic domain extend back towards the membrane on the outside of the funnel and abut the ring of positive charge. An apparent Mg2+ ion was bound between monomers at a conserved site in the cytoplasmic domain, suggesting a mechanism to link gating of the pore to the intracellular concentration of Mg2+.

A novel degradable tricalcium silicate/calcium polyphosphate/polyvinyl alcohol organic-inorganic composite cement for bone filling.

In this study, tricalcium silicate (C3S) calcium/polyphosphate/polyvinyl alcohol organic-inorganic self-setting composites were successfully designed. A variety of tests were conducted to characterize their self-setting properties, mechanical properties, degradation properties, and related biological properties. The composite bone cements showed a short setting time (5.5-37.5 min) with a 5:5-6:4 ratio of C3S/CPP to maintain a stable compressive strength (28 MPa). In addition, PVA effectively reduced the brittleness of the inorganic phase. Degradation experiments confirmed the sustainable surface degradation of bone cement. A maximum degradation rate of 49% was reached within 56 days, and the structure remained intact without collapse. Culturing MC3T3 cells with bone cement extracts revealed that the composite bone cements had excellent biological properties in vitro. The original extract showed a proliferation promotion effect on cells, whereas most of the other original extracts of degradable bone cements were toxic to the cells. Meanwhile, extracellular matrix mineralization and alkaline phosphatase expression showed remarkable effects on cell differentiation. In addition, a good level of adhesion of cells to the surfaces of materials was observed. Taken together, these results indicate that C3S/CPP/PVA composite bone cements have great potential in bone defect filling for fast curing.

Pathogenesis Study of Enterovirus 71 Using a Novel Human SCARB2 Knock-In Mouse Model.

Enterovirus 71 (EV71) can cause a severe hand-foot-mouth disease in children. However, the precise mechanism of EV71-associated disease, particularly the neuropathogenesis and pulmonary disorder, is still not fully understood because no suitable animal models are available. The human scavenger receptor class B, member 2 (hSCARB2), is a cellular receptor for EV71. Here, we generated a novel knock-in (KI) mouse model using the CRISPR/Cas9 system to insert the hSCARB2 gene into the mouse Rosa26 locus to study the pathogenesis of EV71. The hSCARB2 KI mice infected with clinical isolates of EV71 showed neurological symptoms, such as ataxia, paralysis, and death. Viral replication was detected in mainly astrocytes and a limited number of neurons and microglia, accompanied by gliosis. Vascular leakage and alveoli filled with erythrocytes were detected, suggesting that edema and hemorrhage, which are observed in human patients, also occurred in EV71-infected KI mice. In addition, proinflammatory cytokines and chemokines were significantly increased in the serum of infected KI mice. These pathological features of the KI mice after infection resembled those of EV71 encephalomyelitis in humans. Therefore, our KI mouse model is suitable to study the pathogenesis of EV71 and is of great significance for development of antiviral drugs and vaccines to treat or prevent EV71 infection.IMPORTANCE Enterovirus 71 (EV71) is associated with severe hand-foot-mouth disease. Recently, outbreaks of EV71 infection with high mortality have been reported in the Asia-Pacific region, posing a great challenge for global public health. To date, the precise mechanism of EV71-induced disease, particularly the neuropathogenesis and respiratory disorders, is still not fully understood because no suitable animal models are available. Human scavenger receptor class B, member 2 (hSCARB2), has been identified as a cellular receptor for EV71. Here, we introduce a novel CRISPR/Cas9-mediated hSCARB2 knock-in (KI) mouse model for the study of EV71 pathogenesis, which is of great significance for the development of antiviral drugs and vaccines.

Identification of immune and metabolic predictors of severe hand-foot-mouth disease.

Hand, foot and mouth disease (HFMD) is an infectious disease that affects mostly children. The children with HFMD also have other immune and metabolic disorders. However, the association of these disorders with the severity of HFMD has not yet been determined. In this study, we used a case-control study design to examine the correlation of immune and metabolic disorders with HFMD development in children. 406 mild and severe patients were recruited and divided into different subgroups based on the number of days from the initial onset time to hospitalization (1, 2, 3, 4, and ≥5 days). Logistic regression model was used to define the predictors of severe HFMD. We found that the patients from rural area (OR = 1.76, 95% CI [1.19~2.63], P = 0.005) or with body temperature of >39°C (OR = 2.14, 95% CI [1.12~4.12], P = 0.022) exhibited higher risk for severe symptoms. In addition, the risk increased with the rise of body temperature by using a Chis-quare trend test (P = 0.01). We also found that a decreased number of eosinophils was an predictor of severe HFMD at 1, 2, 3,and 4 days post infection (dpi). Decreased levels of Na+, Cl-, and creatine kinase were also predictors at 1 and ≥5 dpi. On the other hand, elevated level of globulin was a predictor for severe HFMD at 4 dpi and ≥5 dpi, and the increased number of neutrophils or increased level of alkaline phosphatase posed risk for severe HFMD at 3 and ≥5 dpi. Our results suggested that rural living, hyperpyrexia, changes in the immune system that include the numbers of eosinophils and neutrophils and the levels of IgG and globulin, and metabolic alterations, such as the levels of alkaline phosphatase, Na+, Cl-, and creatine kinase in peripheral blood are predictors of severe HFMD.

Innate Immunity Evasion by Enteroviruses Linked to Epidemic Hand-Foot-Mouth Disease.

Enterovirus (EV) infections are a major threat to global public health, and are responsible for mild respiratory illness, hand, foot, and mouth disease (HFMD), acute hemorrhagic conjunctivitis, aseptic meningitis, myocarditis, severe neonatal sepsis-like disease, and acute flaccid paralysis epidemic. Among them, HFMD is a common pediatric infectious disease caused by EVs of the family Picornaviridae including EV-A71, coxsackieviruses (CV)-A2, CV-A6, CV-A10, and CV-A16. Due to lack of vaccines and specific antiviral therapeutics, millions of children still suffer from HFMD. Innate immune system detects foreign invaders by means of a relatively limited number of sensors, such as pattern recognition receptors (PRRs) [e.g., retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), Toll-like receptors (TLRs), and NOD-like receptors (NLRs)] and even some secreted functional proteins. However, a range of research, highlighted in this review, suggest that EV-associated with HFMD have evolved different strategies to avoid detection by innate immunity via different proteases (e.g., 2A, 3C, 2C, and 3D). Ongoing efforts to better understand virus-host interactions that control innate immunity and then distill how that influences HFMD development promises to have real-world significance. In this review, we address this complex topic in nine sections including multiple proteins associated with PRR and type I interferon (IFN) signaling. Recognizing how EVs linked to HFMD evade host innate immune system, we also describe the interactions between them and, finally, suggest future directions to better inform drug development and public health.

Antiviral and Inflammatory Cellular Signaling Associated with Enterovirus 71 Infection.

Enterovirus 71 (EV71) infection has become a major threat to global public health, especially in infants and young children. Epidemiological studies have indicated that EV71 infection is responsible for severe and even fatal cases of hand, foot, and mouth disease (HFMD). Accumulated evidence indicates that EV71 infection triggers a plethora of interactive signaling pathways, resulting in host immune evasion and inflammatory response. This review mainly covers the effects of EV71 infection on major antiviral and inflammatory cellular signal pathways. EV71 can activate cellular signaling networks including multiple cell surface and intracellular receptors, intracellular kinases, calcium flux, and transcription factors that regulate antiviral innate immunity and inflammatory response. Cellular signaling plays a critical role in the regulation of host innate immune and inflammatory pathogenesis. Elucidation of antiviral and inflammatory cellular signaling pathways initiated by EV71 will not only help uncover the potential mechanisms of EV71 infection-induced pathogenesis, but will also provide clues for the design of therapeutic strategies against EV71 infection.

Involvement of the renin-angiotensin system in the progression of severe hand-foot-and-mouth disease.

BACKGROUND: Hand-foot-and-mouth disease (HFMD) is generally considered as a mild exanthematous disease to infants and young children worldwide. HFMD cases are usually mild and self-limiting but for few cases leads to complicated severe clinical outcomes, and even death. Previous studies have indicated that serum Ang II levels in patients with H7N9 infection were related to the severity of infection. However, the mechanisms underlying the pathogenesis of severe HFMD remain unclear. This study was undertaken to clarify the role of the renin-angiotensin system (RAS) in the progression of severe HFMD. METHODS: In the present study, 162 children including HFMD patients and healthy controls were recruited. The data was analyzed by time-series fashion. Concentrations of angiotensin II (Ang II) and noradrenaline (NA) in serum of patients were measured with ELISA. We established a mouse model for enterovirus 71 (EV71) infection and determined concentrations of Ang II, NA in tissue lysates at 3, 5 and 7 days post infection (dpi). RESULTS: The concentrations of Ang II and NA in serum of the HFMD patients with mild or severe symptoms were significantly higher than that in healthy controls. Additionally, the concentrations of Ang II and NA in serum of severe cases were significantly higher than those mild cases and the increased concentrations of Ang II and NA showed the same time trend during the progression of HFMD in the severe cases. Furthermore, the concentrations of Ang II and NA in target organs of EV71-infected mice including brains, skeletal muscle, and lungs were increased with the progression of EV71 infection in mice. Histopathological alterations were observed in the brains, skeletal muscle and lungs of EV71-infected mice. CONCLUSION: Our study suggested that activation of the RAS is implicated in the pathogenesis of severe HFMD.

Characterization of Critical Functions of Long Non-Coding RNAs and mRNAs in Rhabdomyosarcoma Cells and Mouse Skeletal Muscle Infected by Enterovirus 71 Using RNA-Seq.

Enterovirus 71 (EV71) is the main pathogen of severe hand-foot-mouth disease (HFMD). Long non-coding RNAs (lncRNAs) are recognized as pivotal factors during the pathogenesis of viral infection. However, the critical functions of lncRNAs in EV71⁻host interactions have not been characterized. Here, for the first time, we performed global transcriptome analysis of lncRNA and mRNA expression profiles in EV71-infected human rhabdomyosarcoma (RD) cells and skeletal muscle of mice using second-generation sequencing. In our study, a total of 3801 novel lncRNAs were identified. In addition, 23 lncRNAs and 372 mRNAs exhibited remarkable differences in expression levels between infected and uninfected RD cells, while 104 lncRNAs and 2647 mRNAs were differentially expressed in infected skeletal muscle from neonatal mice. Comprehensive bioinformatics analysis included target gene prediction, lncRNA­mRNA co-expression network construction, as well as gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis mainly focused on differentially-expressed genes (DEGs). Our results suggest that lncRNAs may participate in EV71 infection-induced pathogenesis through regulating immune responses, protein binding, cellular component biogenesis and metabolism. The present study provides novel insights into the functions of lncRNAs and the possible pathogenic mechanism following EV71 infection.

Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability.

Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter-repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.

Involvement of inducible nitric oxide synthase and mitochondrial dysfunction in the pathogenesis of enterovirus 71 infection.

Enterovirus71 (EV71) is recognized as the main causative agent of severe hand, foot and mouth disease (HFMD). However, the pathogenesis of EV71 infection has not been well characterized. Clinical evidence indicated that inducible nitric oxide synthase (iNOS) induction in the lung of HFMD patients contributes to the severe symptoms of pulmonary edema. In the present study, we recruited 142 subjects including HFMD patients and controls, and serum level of nitric oxide (NO) was determined. Next, cellular and animal model were used to further investigate the roles of iNOS and mitochondria damage during EV71 infection. Serum NO level in HFMD patients with mild or severe symptoms was higher than that in controls, and there was a trend towards an increase in the serum NO level of severe cases relative to mild cases. EV71 infection caused apoptosis and increased levels of NO, iNOS, superoxide dismutase (SOD) activity and malondialdehyde (MDA), and degraded mitochondrial membrane potential (ΔΨm) in vitro. Pathological alterations of mitochondrial morphology were observed in vitro and in vivo. Furthermore, the expression of iNOS levels in target organs including brain, spinal cord, skeletal muscle, lung and heart were increased with the progression of the pathogenesis of EV71 infection in mice. Taken together, iNOS and mitochondrial damage participate in the pathogenesis of EV71 infection.

Time series analysis of hand-foot-mouth disease hospitalization in Zhengzhou: establishment of forecasting models using climate variables as predictors.

BACKGROUND: Large-scale outbreaks of hand-foot-mouth disease (HFMD) have occurred frequently and caused neurological sequelae in mainland China since 2008. Prediction of the activity of HFMD epidemics a few weeks ahead is useful in taking preventive measures for efficient HFMD control. METHODS: Samples obtained from children hospitalized with HFMD in Zhengzhou, Henan, China, were examined for the existence of pathogens with reverse-transcriptase polymerase chain reaction (RT-PCR) from 2008 to 2012. Seasonal Autoregressive Integrated Moving Average (SARIMA) models for the weekly number of HFMD, Human enterovirus 71(HEV71) and CoxsackievirusA16 (CoxA16) associated HFMD were developed and validated. Cross correlation between the number of HFMD hospitalizations and climatic variables was computed to identify significant variables to be included as external factors. Time series modeling was carried out using multivariate SARIMA models when there was significant predictor meteorological variable. RESULTS: 2932 samples from the patients hospitalized with HFMD, 748 were detected with HEV71, 527 with CoxA16 and 787 with other enterovirus (other EV) from January 2008 to June 2012. Average atmospheric temperature (T{avg}) lagged at 2 or 3 weeks were identified as significant predictors for the number of HFMD and the pathogens. SARIMA(0,1,0)(1,0,0)52 associated with T{avg} at lag 2 (T{avg}-Lag 2) weeks, SARIMA(0,1,2)(1,0,0)52 with T{avg}-Lag 2 weeks and SARIMA(0,1,1)(1,1,0)52 with T{avg}-Lag 3 weeks were developed and validated for description and predication the weekly number of HFMD, HEV71-associated HFMD, and Cox A16-associated HFMD hospitalizations. CONCLUSION: Seasonal pattern of certain HFMD pathogens can be associated by meteorological factors. The SARIMA model including climatic variables could be used as an early and reliable monitoring system to predict annual HFMD epidemics.

Serum inflammatory cytokine levels correlate with hand-foot-mouth disease severity: a nested serial case-control study.

BACKGROUND: Hand-food-mouth disease (HFMD) cases can be fatal. These cases develop rapidly, and it is important to predict the severity of HFMD from mild to fatal and to identify risk factors for mild HFMD. The objective of this study was to correlate the levels of serum inflammatory cytokines with HFMD severity. METHODS: This study was designed as a nested serial case-control study. The data collected included general information, clinical symptoms and signs, laboratory findings and serum cytokine levels. RESULTS: The levels of IL-4, IL-6, IL-10, TNF-α and IFN-γ in patients with severe HFMD were significantly higher than in mild patients during the 2nd to 5th day after disease onset. The levels of IL-4, IL-6, IL-10 and IFN-γ increased from the 2nd day to the 4th day and later decreased. The levels of TNF-α were high on the first two days and subsequently decreased. The changes of IL-10, TNF-α and IFN-γ in the controls were similar for all cases. The levels of IL-4, IL-6 and IL-17 in the controls were not significantly different with the progression of HFMD. CONCLUSIONS: Our findings indicate that the IL-4, IL-6, IL-10, TNF-α and IFN-γ levels correlate with HFMD severity.