LDLR, LRP1, and Megalin redundantly participate in the uptake of Clostridium novyi alpha-toxin.

Clostridium novyi alpha-toxin (Tcnα) is a potent exotoxin that induces severe symptoms including gas gangrene, myositis, necrotic hepatitis, and sepsis. Tcnα binds to sulfated glycosaminoglycans (sGAG) for cell-surface attachment and utilizes low-density lipoprotein receptor (LDLR) for rapid entry. However, it was also shown that Tcnα may use alternative entry receptors other than LDLR. Here, we define that LRP1 and Megalin can also facilitate the cellular entry of Tcnα by employing reconstitutive LDLR family proteins. LDLR, LRP1, and Megalin recognize Tcnα via their ligand-binding domains (also known as LDL receptor type A repeats). Notably, LDLR and LRP1 have contrasting expression levels in many different cells, thus the dominant entry receptor for Tcnα could be cell-type dependent. These findings together increase our knowledge of the Tcnα actions and further help to understand the pathogenesis of C. novyi infection-associated diseases.

The Weak Small RNA-Binding Activity of the 2b Proteins of Subgroup II Cucumber Mosaic Virus Strains Is Insufficient for RNA Silencing Suppression.

The 2b proteins encoded by cucumber mosaic virus (CMV) subgroup I strains suppress RNA silencing primarily by competitively binding small RNAs (sRNAs) in the host cell cytoplasm. Interestingly, 2b proteins encoded by CMV subgroup II strains accumulate predominantly in nuclei. Here we determined that whereas the 2b protein (Fny2b) of subgroup IA strain Fny-CMV is highly effective in suppressing both sense RNA-induced and inverted repeat-induced posttranscriptional gene silencing, the 2b protein (LS2b) of the subgroup II strain LS-CMV was not as effective. Reducing nuclear accumulation of LS2b by mutating a residue in its nuclear localization sequence had no effect on RNA silencing suppressor activity, while attenuated viral symptoms. Electrophoretic mobility shift assays showed that the sRNA binding of LS2b was weaker and more selective than that of Fny2b. The domain determining the differential sRNA-binding ability was delimited to the putative helix α1 region. Moreover, LS2b mutants that completely lost suppressor activity still retained their weak sRNA-binding ability, suggesting that sRNA binding is not sufficient for LS2b to suppress RNA silencing. Considering the subgroup I strain-encoded 2b proteins that require sRNA-binding ability for the suppression of RNA silencing, we suggest that in addition to binding sRNA, the 2b proteins of subgroup II CMV strains would require extra biological activities to achieve RNA silencing inhibition.

UBQLN2-HSP70 axis reduces poly-Gly-Ala aggregates and alleviates behavioral defects in the C9ORF72 animal model.

Expansion of a hexanucleotide repeat GGGGCC (G4C2) in the intron of the C9ORF72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9-ALS/FTD). Transcripts carrying G4C2 repeat expansions generate neurotoxic dipeptide repeat (DPR) proteins, including poly-Gly-Ala (poly-GA), which tends to form protein aggregates. Here, we demonstrate that UBQLN2, another ALS/FTD risk factor, is recruited to reduce poly-GA aggregates and alleviate poly-GA-induced neurotoxicity. UBQLN2 could recognize HSP70 ubiquitination, which facilitates the UBQLN2-HSP70-GA complex formation and promotes poly-GA degradation. ALS/FTD-related UBQLN2 mutants fail to bind HSP70 and clear poly-GA aggregates. Disruption of the interaction between UBQLN2 and HSP70 inhibits poly-GA aggregation in C9-ALS/FTD iPSC-derived neurons. Finally, enhancing HSP70 by the chemical compound 17AAG at the adult stage mitigates behavioral defects in poly-GA animals. Our findings suggest a critical role of the UBQLN2-HSP70 axis in protein aggregate clearance in C9-ALS/FTD.

APC2CDH1 negatively regulates agrin signaling by promoting the ubiquitination and proteolytic degradation of DOK7.

Neuromuscular junctions (NMJs) are peripheral synapses between motoneurons and skeletal muscle fibers that are critical for the control of muscle contraction. Dysfunction of these synapses has been implicated in congenital myasthenic syndrome (CMS). In vertebrates, agrin-LRP4-MuSK signaling plays a critical role in acetylcholine receptor (AChR) clustering and NMJ formation. The adaptor protein DOK7 is the downstream substrate of MuSK and also a cytoplasmic activator of MuSK. The role of DOK7 in the promotion of AChR clustering and the mechanisms involved have been well studied; however, the negative regulation of DOK7 after MuSK activation remains unknown. Anaphase-promoting complex 2 (APC2), the core subunit of APC/C E3 ligase complex, was originally believed to regulate cell-cycle transitions. Here, we show that APC2 is enriched at post-synapse of NMJs in postmitotic myotubes. In response to agrin stimulation, APC2 negatively regulates AChR clustering by promoting the ubiquitination of DOK7 at lysine 243 for its proteolytic degradation, which relies on MuSK kinase activity and the phosphorylation of tyrosine 106 in DOK7. Thus, this study provides a mechanism whereby agrin signaling is negatively regulated as part of vertebrate NMJ homeostasis.

Loss of mitochondrial protein CHCHD10 in skeletal muscle causes neuromuscular junction impairment.

The neuromuscular junction (NMJ) is a synapse between motoneurons and skeletal muscles to control motor behavior. Acetylcholine receptors (AChRs) are restricted at the synaptic region for proper neurotransmission. Mutations in the mitochondrial CHCHD10 protein have been identified in multiple neuromuscular disorders; however, the physiological roles of CHCHD10 at NMJs remain elusive. Here, we report that CHCHD10 is highly expressed at the postsynapse of NMJs in skeletal muscles. Muscle conditional knockout CHCHD10 mice showed motor defects, abnormal neuromuscular transmission and NMJ structure. Mechanistically, we found that mitochondrial CHCHD10 is required for ATP production, which facilitates AChR expression and promotes agrin-induced AChR clustering. Importantly, ATP could effectively rescue the reduction of AChR clusters in the CHCHD10-ablated muscles. Our study elucidates a novel physiological role of CHCHD10 at the peripheral synapse. It suggests that mitochondria dysfunction contributes to neuromuscular pathogenesis.

Nuclear-cytoplasmic partitioning of cucumber mosaic virus protein 2b determines the balance between its roles as a virulence determinant and an RNA-silencing suppressor.

UNLABELLED: The Cucumber Mosaic Virus (CMV) 2b protein is an RNA-silencing suppressor that plays roles in CMV accumulation and virulence. The 2b proteins of subgroup IA CMV strains partition between the nucleus and cytoplasm, but the biological significance of this is uncertain. We fused an additional nuclear localization signal (NLS) to the 2b protein of subgroup IA strain Fny-CMV to create 2b-NLS and tested its effects on subcellular distribution, silencing, and virulence. The additional NLS enhanced 2b protein nuclear and nucleolar accumulation, but nuclear and nucleolar enrichment correlated with markedly diminished silencing suppressor activity in patch assays and abolished 2b protein-mediated disruption of microRNA activity in transgenic Arabidopsis. Nucleus/nucleolus-localized 2b protein possesses at least some ability to inhibit antiviral silencing, but this was not sufficient to prevent recovery from disease in younger, developing leaves in Arabidopsis. However, enhanced nuclear and nucleolar accumulation of 2b increased virulence and accelerated symptom appearance in older leaves. Experiments with Arabidopsis lines carrying mutant Dicer-like alleles demonstrated that compromised suppressor activity explained the diminished ability of 2b-NLS to enhance virus accumulation. Remarkably, the increased virulence that 2b-NLS engendered was unrelated to effects on microRNA- or short interfering RNA-regulated host functions. Thus, although nucleus- and nucleolus-localized 2b protein is less efficient at silencing suppression than cytoplasm-localized 2b, it enhances CMV virulence. We propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus. IMPORTANCE: In this work, the main finding is that nucleus/nucleolus-localized 2b protein is strongly associated with CMV virulence, which is independent of its effect on small RNA pathways. Moreover, this work supports the contention that the silencing suppressor activity of CMV 2b protein is predominantly exerted by that portion of the 2b protein residing in the cytoplasm. Thus, we propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus.

Using a viral vector to reveal the role of microRNA159 in disease symptom induction by a severe strain of Cucumber mosaic virus.

In transgenic Arabidopsis (Arabidopsis thaliana), expression of the Cucumber mosaic virus (CMV) 2b silencing suppressor protein from the severe subgroup IA strain Fny disrupted microRNA (miRNA)-regulated development but orthologs from mild subgroup II strains (Q and LS) did not, explaining strain-specific differences in symptom severity. However, it is unknown which miRNAs affected by Fny2b critically affect viral symptoms. Observations that Fny2b-transgenic plants phenocopy microRNA159ab (mir159ab) mutant plants and that Fny2b altered miR159ab-regulated transcript levels suggested a role for miR159ab in elicitation of severe symptoms by Fny-CMV. Using restoration of the normal phenotype in transgenic plants expressing an artificial miRNA as a proof of concept, we developed a LS-CMV-based vector to express sequences mimicking miRNA targets. Expressing a miR159 target mimic sequence using LS-CMV depleted miR159 and induced symptoms resembling those of Fny-CMV. Suppression of Fny-CMV-induced symptoms in plants harboring mutant alleles for the miR159ab targets MYB domain protein33 (MYB33) and MYB65 confirmed the importance of this miRNA in pathogenesis. This study demonstrates the utility of a viral vector to express miRNA target mimics to facilitate functional studies of miRNAs in plants.

Fully human broadly neutralizing monoclonal antibodies against influenza A viruses generated from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient.

Whether the 2009 pandemic H1N1 influenza vaccine can induce heterosubtypic cross-protective anti-hemagglutinin (HA) neutralizing antibodies is an important issue. We obtained a panel of fully human monoclonal antibodies from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient. Most of the monoclonal antibodies targeted the HA protein but not the HA1 fragment. Among the analyzed antibodies, seven mAbs exhibited neutralizing activity against several influenza A viruses of different subtypes. The conserved linear epitope targeted by the neutralizing mAbs (FIEGGWTGMVDGWYGYHH) is part of the fusion peptide on HA2. Our work suggests that a heterosubtypic neutralizing antibody response primarily targeting the HA stem region exists in recipients of the 2009 pandemic H1N1 influenza vaccine. The HA stem region contains various conserved neutralizing epitopes with the fusion peptide as an important one. This work may aid in the design of a universal influenza A virus vaccine.

Functional screening for miRNAs targeting Smad4 identified miR-199a as a negative regulator of TGF-ß signalling pathway.

The transforming growth factor-ß (TGF-ß) signalling pathway participates in various biological processes. Dysregulation of Smad4, a central cellular transducer of TGF-ß signalling, is implicated in a wide range of human diseases and developmental disorders. However, the mechanisms underlying Smad4 dysregulation are not fully understood. Using a functional screening approach based on luciferase reporter assays, we identified 39 microRNAs (miRNAs) as potential regulators of Smad4 from an expression library of 388 human miRNAs. The screening was supported by bioinformatic analysis, as 24 of 39 identified miRNAs were also predicted to target Smad4. MiR-199a, one of the identified miRNAs, was inversely correlated with Smad4 expression in various human cancer cell lines and gastric cancer tissues, and repressed Smad4 expression and blocked canonical TGF-ß transcriptional responses in cell lines. These effects were dependent on the presence of a conserved, but not perfect seed paired, miR-199a-binding site in the Smad4 3'-untranslated region (UTR). Overexpression of miR-199a significantly inhibited the ability of TGF-ß to induce gastric cancer cell growth arrest and apoptosis in vitro, and promoted anchorage-independent growth in soft agar, suggesting that miR-199a plays an oncogenic role in human gastric tumourigenesis. In conclusion, our functional screening uncovers multiple miRNAs that regulate the cellular responsiveness to TGF-ß signalling and reveals important roles of miR-199a in gastric cancer by directly targeting Smad4.

Novel sinomenine derivative 1032 improves immune suppression in experimental autoimmune encephalomyelitis.

Sinomenine (SIN) is an alkaloid isolated from the Chinese medicinal plant Sinomenium acutum. It is widely used as an immunosuppressive drug for treating autoimmune diseases. Due to its poor efficiency, the large-dose treatment presents some side effects and limits its further applications. In this study, we used chemical modification to improve the therapeutic effect of SIN in vitro and in vivo. A new derivative of sinomenine, named 1032, demonstrates significantly improved immunosuppressive activity over that of its parent natural compound (SIN). In an experimental autoimmune encephalomyelitis (EAE) model, 1032 significantly reduced encephalitogenic T cell responses and induced amelioration of EAE, which outcome was related to its selective inhibitory effect on the production of IL-17. By contrast, SIN treatment only led to a moderate alleviation of EAE severity and the expression level of IL-17 was not significantly reduced. Furthermore, 1032 exhibited suppression of Th17, but not Treg, cell differentiation, a result probably related to its inhibitory effect on IkappaB-alpha degradation as well as on IL-6 and TNF-alpha secretion in BMDCs. We speculate that 1032 as a novel anti-inflammatory agent may target DC to block IL-6 production, which in turn would terminate Th17 cell development. Thus, SIN derivative 1032 presents considerable potential in new drug development for treating autoimmune and inflammatory disease.

[Investigation of emission characteristics for light duty vehicles with a portable emission measurement system].

Emission from 7 typical light-duty vehicles under actual driving conditions was monitored using a portable emission measurement system to gather data for characterization of the real world vehicle emission in Shenzhen, including the effects of driving modes on vehicle emission, comparison of fuel consumption based emission factors (g x L(-1) with mileage based emission factors (g x km(-1)), and the average emission factors of the monitored vehicles. The acceleration and deceleration modes accounted for 66.7% of total travel time, 80.3% of traveling distance and 74.6%-79.2% of vehicle emission; the acceleration mode contributed more than other driving modes. The fuel based emission factors were less dependent on the driving speed; they may be utilized in building macro-scale vehicle emission inventory with smaller sensitivity to the vehicle driving conditions. The effect of vehicle technology on vehicle emission was significant; the emission factors of CO, HC and NO(x) of carbureted vehicles were 19.9-20.5, 5.6-26.1 and 1.8-2.0 times the more advanced vehicles of Euro II, respectively. Using the ECE + EUDC driving cycle would not produce the desired real-world emission rates of light duty vehicles in a typical Chinese city.

[The relationship between the parotid glands function and the dose-volume effect in nasopharyngeal carcinoma patients with intensity-modulated radiation therapy].

OBJECTIVE: To study the preservation of parotid glands function and relationship between parotid glands function and dose-volume histogram (DVH) in nasopharyngeal carcinoma (NPC) patients treated by intensity modulated radiation therapy (IMRT). METHODS: From August 2002 to December 2004, the excretion index (EI) and uptake index (UI) of parotids in 48 NPC patients underwent radical IMRT was examined by ECT at the beginning, the end of and the 3 months after radiotherapy. The relationship between parotid function (EI and UI) and DVH were analyzed. RESULTS: The mean doses to the contralateral parotid and ipsilateral parotid were 22.8 +/- 4.5 Gy and 31.9 +/- 4.1 Gy, respectively. The symptom of xerostomia was mild at the end of radiotherapy. ECT showed EI of contralateral parotid were 0.35 +/- 0.25, 0.31 +/- 0.24 and 0.33 +/- 0.22 at the beginning, the end of and 3 months after radiotherapy (RT), respectively. UI were 7.12 +/- 3.56, 5.81 +/- 2.25 and 5.72 +/- 2.81 at the same intervals. This shows no statistical difference. The EI and UI of ipsilateral parotid at the completion of radiotherapy declined significantly (0.21 +/- 0.16 and 4.87 +/- 2.45, respectively) compared with those of pre-treatment (0.36 +/- 0.27 and 8.02 +/- 3.89, respectively) (P < 0.05). DVH showed: at the end of RT, the EI was significant difference between mean dose < 26 Gy and > or = 26 Gy group (P = 0.009) and decreased significantly in the group of V25 (the percentages of parotid volume irradiated with < 25 Gy) > or = 50% compared with the group of V25 < 50% (P < 0.01). The UIs were no significant difference in two groups (P > 0.05). CONCLUSION: 26 Gy is a threshold dose for the preservation of parotid glands function. There is also a threshold volume irradiated for the preservation of the parotid glands function. Based on the precondition of assuring significant dose to the target volume (PTV), we should reduce the irradiated volume and dose to parotid glands as possible as we can so as to preserve its function.