Blockade of anti-dsDNA ameliorates systemic lupus erythematosus in MRL/Faslpr mice through ameliorating inflammation via the PKCδ-NLRC4 axis.

Anti-double-stranded DNA (anti-dsDNA) is closely associated with the inflammatory burden in the brain after ischemic stroke. Here, we studied the inflammatory cascade and investigated the mechanisms behind the pro-inflammatory role of dsDNA in systemic lupus erythematosus (SLE). The serum levels of interleukin-1beta (IL-1ß) and IL-6 in SLE patients and the corresponding controls were evaluated using ELISA, and the expression level of caspase-1 was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). We found that the serum levels of IL-1ß and IL-6 were increased in the SLE patients. The expression of caspase-1 was upregulated and positively correlated with the levels of pro-inflammatory factors. The level of anti-dsDNA was also elevated and positively correlated with the results for the mean fluorescence intensity (MFI) of caspase-1. Additionally, we evaluated the functions of PRKCD encoding protein kinase c delta (PKCδ) and NLRC4, in vivo, in MRL/Faslpr mice. We found that renal injury was aggravated, and the levels of pro-inflammatory factors were increased in the MRL/Faslpr mice. We also found that increased levels of NLRC4 in the mice exacerbated renal injury and increased the levels of pro-inflammatory factors, whereas inhibition of PKCδ had the opposite results. These findings provide unique perspectives on pathogenesis of SLE and indicate that inhibition of anti-dsDNA could attenuate renal inflammatory burden, representing a promising therapeutic opportunity for SLE.

Correction to: CD103-positive CSC exosome promotes EMT of clear cell renal cell carcinoma: role of remote MiR-19b-3p.

An amendment to this paper has been published and can be accessed via the original article.

CD103-positive CSC exosome promotes EMT of clear cell renal cell carcinoma: role of remote MiR-19b-3p.

BACKGROUND: Clear cell renal cell carcinoma (CCRCC) is characterized by a highly metastatic potential. The stromal communication between stem cells and cancer cells critically influences metastatic dissemination of cancer cells. METHODS: The effect of exosomes isolated from cancer stem cells (CSCs) of CCRCC patients on the progress of epithelial-mesenchymal transition (EMT) and lung metastasis of CCRCC cells were examined. RESULTS: CSCs exosomes promoted proliferation of CCRCC cells and accelerated the progress of EMT. Bioactive miR-19b-3p transmitted to cancer cells by CSC exosomes induced EMT via repressing the expression of PTEN. CSCs exosomes derived from CCRCC patients with lung metastasis produced the strongest promoting effect on EMT. Notably, CD103+ CSC exosomes were enriched in tumor cells and in lung as well, highlighting the organotropism conferred by CD103. In addition, CD103+ exosomes were increased in blood samples from CCRCC patients with lung metastasis. CONCLUSIONS: CSC exosomes transported miR-19b-3p into CCRCC cells and initiated EMT promoting metastasis. CD103+ acted to guide CSC exosomes to target cancer cells and organs, conferring the higher metastatic capacity of CCRCC to lungs, suggesting CD103+ exosomes as a potential metastatic diagnostic biomarker. ᅟ.

H5N1 influenza A virus with K193E and G225E double mutations in haemagglutinin is attenuated and immunogenic in mice.

Live-attenuated influenza vaccines (LAIVs) are now available for the prevention of influenza, with LAIV strains generally derived from serial passage in cultures or by reverse genetics (RG). The receptor-binding domain (RBD) in haemagglutinin (HA) of influenza virus is responsible for viral binding to the avian-type 2,3-α-linked or human-type 2,6-α-linked sialic acid receptor; however, the virulence determinants in the RBD of H5N1 virus remain largely unknown. In the present study, serial passage of H5N1 virus A/Vietnam/1194/2004 in Madin-Darby canine kidney cells resulted in the generation of adapted variants with large-plaque morphology, and genomic sequencing of selected variants revealed two specific amino acid substitutions (K193E and G225E) in the RBD. RG was used to generate H5N1 viruses containing either single or double substitutions in HA. The RG virus containing K193E and G225E mutations (rVN-K193E/G225E) demonstrated large-plaque morphology, enhanced replication and genetic stability after serial passage, without changing the receptor-binding preference. Importantly, in vivo virulence assessment demonstrated that rVN-K193E/G225E was significantly attenuated in mice. Microneutralization and haemagglutination inhibition assays demonstrated that immunization with rVN-K193E/G225E efficiently induced a robust antibody response against WT H5N1 virus in mice. Taken together, our experiments demonstrated that K193E and G225E mutations synergistically attenuated H5N1 virus without enhancing the receptor-binding avidity, and that the RG virus rVN-K193E/G225E represents a potential H5N1 LAIV strategy that deserves further development. These findings identify the RBD as a novel attenuation target for live vaccine development and highlight the complexity of RBD interactions.

Occurrence and reassortment of avian influenza A (H7N9) viruses derived from coinfected birds in China.

UNLABELLED: Over the course of two waves of infection, H7N9 avian influenza A virus has caused 436 human infections and claimed 170 lives in China as of July 2014. To investigate the prevalence and genetic diversity of H7N9, we surveyed avian influenza viruses in poultry in Jiangsu province within the outbreak epicenter. We found frequent occurrence of H7N9/H9N2 coinfection in chickens. Molecular clock phylogenetic analysis confirms coinfection by H7N9/H9N2 viruses and also reveals that the identity of the H7N9 outbreak lineage is confounded by ongoing reassortment between outbreak viruses and diverse H9N2 viruses in domestic birds. Experimental inoculation of a coinfected sample in cell culture yielded two reassortant H7N9 strains with polymerase segments from the original H9N2 strain. Ongoing reassortment between the H7N9 outbreak lineage and diverse H9N2 viruses may generate new strains with the potential to infect humans, highlighting the need for continued viral surveillance in poultry and humans. IMPORTANCE: We found frequent occurrence of H7N9/H9N2 coinfection in chickens. The H7N9 outbreak lineage is confounded by ongoing reassortment between H7N9 and H9N2 viruses. The importance of H9N2 viruses as the source of novel avian influenza virus infections in humans requires continuous attention.

Mouse lung-adapted mutation of E190G in hemagglutinin from H5N1 influenza virus contributes to attenuation in mice.

The highly pathogenic H5N1 avian influenza virus is one of the greatest influenza pandemic threats since 2003. The association of the receptor binding domain (RBD) with the virulence of influenza virus is rarely addressed, particularly of H5N1 influenza viruses. In this study, BALB/c mice were intranasally infected with A/Vietnam/1194/2004 (VN1194, H5N1). The mouse lung-adapted variants were isolated and the mutation of E190G (H3 numbering) in the RBD was recognized. The recombinant virus, rVN-E190G carrying E190G in hemagglutinin (HA) was designed and rescued using reverse genetics techniques. The receptor binding activity, growth curve and pathogenicity in mice of the rVN-E190G were investigated. Results demonstrated that rVN-E190G virus increased the binding avidity to α2,6 SA (sialic acid) and reduced the affinity to α2,3 SA, meanwhile weakened the viral replication in vitro. Moreover, the virulence assessment demonstrated that rVN-E190G was attenuated in mice. These results indicated that the mutation E190G in HA decreases H5N1 viral replication in vitro and significantly attenuates virulence in vivo. These findings identify one of the determinants in RBD which can be associated with H5N1 virulence in mice.

Simultaneous targeted analysis of five active compounds in licorice by ultra-fast liquid chromatography coupled to hybrid linear-ion trap tandem mass spectrometry.

An ultra-fast liquid chromatography with electrospray ionization tandem mass spectrometry (UFLC-ESI-MS/MS) method was developed for targeted analysis of 5 active compounds in licorice for the first time. The sample preparation procedure, chromatographic and mass spectrographic conditions were optimized. By using a Kinetex C18 100A column, the five compounds were separated within 8.0 min by gradient elution using methanol containing 0.1% acetic acid and 0.1% aqueous acetic acid. The precursor and product ions of the analytes were monitored on a hybrid quadrupole/linear ion trap mass spectrometer equipped with a turbo ion spray interface in negative ionization mode (ESI(-)) and were simultaneously characterized and quantified based on the multiple reaction monitoring-information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) mode. All standard calibration curves expressed satisfactory linearity (r ≥ 0.9954) within a relatively wide range. The precision was evaluated by intra- and inter-day tests, which revealed relative standard deviation (RSD) values within the ranges of 1.15-4.56% and 0.81-3.95%, respectively. The recovery assays for the quantified compounds were between 97.33 and 100.4% with RSD values less than 4.27%. The proposed method was demonstrated to be simple, rapid, specific and reliable and was successfully applied for identification and quantification of 5 active compounds in 10 batches of licorice. The results showed that the contents of the 5 compounds in licorice from different sources were widely varied.

Digital robot-assisted minimally invasive impacted tooth extraction: A case report.

Objective: This study investigated the clinical effects and applicability of minimally invasive impacted teeth extraction using digital robots. Methods: A marker was bonded to the non-surgical area before surgery. A Cone-Beam Computed Tomography (CBCT) scan was obtained and uploaded to the robot software to determine the drilling position of the ring drill. During the surgery, the robot arm automatically navigated to a predetermined position, and the ring drill removed part of the bone tissue and exposed and extracted the impacted teeth. Finally, the surgeon tightly sutured the wounds to the surgical area. Results: Three minimally invasive extractions of impacted teeth with robotic assistance were performed without complications. The surgical area showed good healing during the one-month follow-up examination. Conclusions: Digital robot-assisted minimally invasive extraction of impacted teeth is a highly feasible clinical procedure as it minimises trauma to the surgical area and protects the surrounding blood vessels and nerve bundles, making it a safe and valuable technique with significant potential for clinical application.

Metagenomic analysis of fever, thrombocytopenia and leukopenia syndrome (FTLS) in Henan Province, China: discovery of a new bunyavirus.

Since 2007, many cases of fever, thrombocytopenia and leukopenia syndrome (FTLS) have emerged in Henan Province, China. Patient reports of tick bites suggested that infection could contribute to FTLS. Many tick-transmitted microbial pathogens were tested for by PCR/RT-PCR and/or indirect immunofluorescence assay (IFA). However, only 8% (24/285) of samples collected from 2007 to 2010 tested positive for human granulocytic anaplasmosis (HGA), suggesting that other pathogens could be involved. Here, we used an unbiased metagenomic approach to screen and survey for microbes possibly associated with FTLS. BLASTx analysis of deduced protein sequences revealed that a novel bunyavirus (36% identity to Tehran virus, accession: HQ412604) was present only in sera from FTLS patients. A phylogenetic analysis further showed that, although closely related to Uukuniemi virus of the Phlebovirus genus, this virus was distinct. The candidate virus was examined for association with FTLS among samples collected from Henan province during 2007-2010. RT-PCR, viral cultures, and a seroepidemiologic survey were undertaken. RT-PCR results showed that 223 of 285 (78.24%) acute-phase serum samples contained viral RNA. Of 95 patients for whom paired acute and convalescent sera were available, 73 had serologic evidence of infection, with 52 seroconversions and 21 exhibiting a 4-fold increase in antibody titer to the virus. The new virus was isolated from patient acute-phase serum samples and named Henan Fever Virus (HNF virus). Whole-genome sequencing confirmed that the virus was a novel bunyavirus with genetic similarity to known bunyaviruses, and was most closely related to the Uukuniemi virus (34%, 24%, and 29% of maximum identity, respectively, for segment L, M, S at maximum query coverage). After the release of the GenBank sequences of SFTSV, we found that they were nearly identical (>99% identity). These results show that the novel bunyavirus (HNF virus) is strongly correlated with FTLS.

The first complete genomic characterization of an Amur virus isolate from China.

Amur virus (AMRV) is a member of the genus Hantavirus in the family Bunyaviridae. In this study, we determined for the first time the complete genome sequence of the AMRV H8205 strain, which was isolated from a patient with hemorrhagic fever with renal syndrome (HFRS) in China. The complete nucleotide sequence of the S segment of AMRV H8205 is 1699 nt long, with a 5' noncoding region (5'NC) of 36 nt, followed by a coding sequence of 1290 nt and a 3'NC of 373 nt. The complete sequence of the M segment is 3615 nt long, with a 5'NC of 40 nt, followed by a coding sequence of 3408 nt and a 3'NC of 167 nt. The complete sequence of the L segment is 6536 nt long, with a 5'NC of 37 nt, followed by a coding sequence of 6453 nt and a 3'NC of 40 nt. The major open reading frame (ORF) of each of the three segments (S, nt 37-1326; M, nt 41-3445; L, nt 38-6490) has a coding capacity of 430 aa, 1135 aa, 2151 aa, respectively. Phylogenetic analysis of the nucleotide sequences using the NJ method indicated that H8205 virus, together with the Amur strains isolated from Far-Eastern Russia and Korea, forms a well-supported lineage. Our results will provide insights into the genetic diversity of hantaviruses (HNTV).

The confirmation of three repeated sequence elements in the 3' untranslated region of Chikungunya virus.

In this study, the complete genomic nucleotide sequence of Chikungunya virus (CHIKV) strain S27 African prototype was determined and three 21 nucleotides repeated sequence elements (RSEs) at positions 11398-11418, 11533-11553, and 11620-11640 in the 3' untranslated region (3'UTR) were confirmed. In addition, the 3'UTRs of all CHIKV strains deposited in GenBank were analyzed. The results displayed that the majority of the CHIKV strains consisted of the three 21 nucleotides RSEs in the 3'UTRs, and the third RSE was the most conservative. The conservation of the three RSEs of 21 nucleotides within the 3'UTR of CHIKV genome may play an important role on the virus replication cycle.

An easy operating pathogen microarray (EOPM) platform for rapid screening of vertebrate pathogens.

BACKGROUND: Infectious diseases emerge frequently in China, partly because of its large and highly mobile population. Therefore, a rapid and cost-effective pathogen screening method with broad coverage is required for prevention and control of infectious diseases. The availability of a large number of microbial genome sequences generated by conventional Sanger sequencing and next generation sequencing has enabled the development of a high-throughput high-density microarray platform for rapid large-scale screening of vertebrate pathogens. METHODS: An easy operating pathogen microarray (EOPM) was designed to detect almost all known pathogens and related species based on their genomic sequences. For effective identification of pathogens from EOPM data, a statistical enrichment algorithm has been proposed, and further implemented in a user-friendly web-based interface. RESULTS: Using multiple probes designed to specifically detect a microbial genus or species, EOPM can correctly identify known pathogens at the species or genus level in blinded testing. Despite a lower sensitivity than PCR, EOPM is sufficiently sensitive to detect the predominant pathogens causing clinical symptoms. During application in two recent clinical infectious disease outbreaks in China, EOPM successfully identified the responsible pathogens. CONCLUSIONS: EOPM is an effective surveillance platform for infectious diseases, and can play an important role in infectious disease control.

Dishevelled-2 silencing reduces androgen-dependent prostate tumor cell proliferation and migration and expression of Wnt-3a and matrix metalloproteinases.

To identify Dishevelled-2 (Dvl2) is a prostate cancer-associated gene and analyze the effects on the growth and invasive capacity of human prostate cancer (PCa) cells. Dvl2 mRNA expression was measured in PCa cell lines and tissue samples, by real-time reverse transcription PCR (qRT-PCR). Immunohistochemistry was used to examine the distribution of Dvl2 in PCa specimens. Silencing Dvl2 in LNCaP cells, proliferation was measured by the CCK-8 assay, cell motility and invasiveness by scratch wound and transwell migration assays, and Wnt-3a, AR, and matrix metalloproteinase (MMP) expression by western blotting. Dvl2 was overexpressed in LNCaP cells compared with the AI PCa lines DU-145 and PC-3, as well as in the majority of PCa tissue specimens examined by qRT-PCR (14/27, 51.9 %). Dvl2 expression was low in all 10 BPH specimens, weakly positive in 26/104 AD PCa specimens (23.8 %), positive in 60/104 AD PCa specimens (55 %), and strongly positive in all 5 AI PCa specimens. Dvl2 expression was significantly correlated with combined Gleason score (p = 0.02), lymph node metastasis (p = 0.005), and TNM stage (p = 0.015). Silencing of Dvl2 mRNA expression significantly reduced LNCaP cell proliferation, motility, invasiveness and Wnt-3a, AR, MMP-2, and MMP-9 expression. Dvl2 may increase PCa growth and metastasis potential, possibly by upregulating Wnt-3a, AR, and MMP expression. Silencing Dvl2 expression may be an effective treatment strategy for PCa.

[Pathogenicity of tick-borne encephalitis virus to monocytes].

OBJECTIVE: To investigate the pathogenicity of tick-borne encephalitis virus (TBEV) to monocytes and its proliferation characteristics. METHODS: After being infected with TBEV, the cytopathic effect of monocyte cell line THP-1 was observed and viral titers were evaluated. Cell culture supernatants at different time points after infection were collected and the nucleic acids of TBE virus and the infection percentage were tested by using Real-time RT-PCR and fluorescence-activated cell sorting (FACS); the survival rate of THP-1 after TBEV infection was detected by Dimethylthiazol-carboxymethoxyp- henyl-Sulfophenyl-2H-tetrazolium inner salt (MTS). RESULTS: Real-time RT-PCR and cytopathic assay both demonstrated that TBE virus could replicate in monocytes. The virus particles could be detected and visualized by FACS. In addition, monocyte viability was significantly decreased 5 days after infection with TEBV. CONCLUSION: TBEV can efficiently replicate and proliferate inTHP-1 cells, indicating that monocytes may play an important role in the process of TBEV spreading to various tissues and organs after infection.

[Status of pesticide registration and residue analysis for traditional Chinese medicine in China].

The present paper outlined pesticide registration status for traditional Chinese medicines (TCMs) and summarized the characteristics of pesticide contamination in different regions of some widely used TCMs by retrieving last 10 years' literatures. At present, the problems of pesticide residues for TCM include less pesticide registrations, widespread high-residue organochlorine pesticides contamination, pesticide abuse, irregular GAP bases and imperfect pesticide limit standards, etc. According to the current situation, we should adopt some control measures to strengthen the quality control of TCMs so as to ensure the safety of TCMs and related products.

Digital techniques and trends for seed phenotyping using optical sensors.

BACKGROUND: The breeding of high-quality, high-yield, and disease-resistant varieties is closely related to food security. The investigation of breeding results relies on the evaluation of seed phenotype, which is a key step in the process of breeding. In the global digitalization trend, digital technology based on optical sensors can perform the digitization of seed phenotype in a non-contact, high throughput way, thus significantly improving breeding efficiency. AIM OF REVIEW: This paper provides a comprehensive overview of the principles, characteristics, data processing methods, and bottlenecks associated with three digital technique types based on optical sensors: spectroscopy, digital imaging, and three-dimensional (3D) reconstruction techniques. In addition, the applicability and adaptability of digital techniques based on the optical sensors of maize seed phenotype traits, namely external visible phenotype (EVP) and internal invisible phenotype (IIP), are investigated. Furthermore, trends in future equipment, platform, phenotype data, and processing algorithms are discussed. This review offers conceptual and practical support for seed phenotype digitization based on optical sensors, which will provide reference and guidance for future research. KEY SCIENTIFIC CONCEPTS OF REVIEW: The digital techniques based on optical sensors can perform non-contact and high-throughput seed phenotype evaluation. Due to the distinct characteristics of optical sensors, matching suitable digital techniques according to seed phenotype traits can greatly reduce resource loss, and promote the efficiency of seed evaluation as well as breeding decision-making. Future research in phenotype equipment and platform, phenotype data, and processing algorithms will make digital techniques better meet the demands of seed phenotype evaluation, and promote automatic, integrated, and intelligent evaluation of seed phenotype, further helping to lessen the gap between digital techniques and seed phenotyping.

Enhancer RNA promotes resistance to radiotherapy in bone-metastatic prostate cancer by m6A modification.

Rationale: Prostate cancer metastasizes to the bone with the highest frequency and exhibits high resistance to 177Lu-prostate-specific membrane antigen (PSMA) radioligand therapy. Little is known about bone metastatic prostate cancer (mPCa) resistance to radiation. Methods: We filtered the metastatic eRNA using RNA-seq, MeRIP-seq, RT-qPCR and bioinformation. Western blot, RT-qPCR, CLIP, co-IP and RNA pull-down assays were used for RNA/protein interaction, RNA or protein expression examination. MTS assay was used to determine cell viability in vitro, xenograft assay was used to examine the tumor growth in mice. Results: In this study, we screened and identified bone-specific N6 adenosine methylation (m6A) on enhancer RNA (eRNA) that played a post-transcriptional functional role in bone mPCa and was correlated with radiotherapy (RT) resistance. Further data demonstrated that RNA-binding protein KHSRP recognized both m6A at eRNA and m6Am at 5'-UTR of mRNA to block RNA degradation from exoribonuclease XRN2. Depletion of the MLXIPe/KHSRP/PSMD9 regulatory complex inhibited tumor growth and RT sensitization of bone mPCa xenograft in vitro and in vivo. Conclusions: Our findings indicate that a bone-specific m6A-modified eRNA plays a vital role in regulating mPCa progression and RT resistance and might be a novel specific predictor for cancer RT.

Comprehensive mapping of West Nile virus (WNV)- and Japanese encephalitis virus serocomplex-specific linear B-cell epitopes from WNV non-structural protein 1.

West Nile virus (WNV) non-structural protein 1 (NS1) elicits protective immune responses during infection of animals. WNV NS1-specific antibody responses can provide the basis for serological diagnostic reagents, so the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the conservation of these sites among the Japanese encephalitis virus (JEV) serocomplex members also needs to be defined. The present study describes the mapping of linear B-cell epitopes in WNV NS1. We screened eight NS1-specific mAbs and antisera (polyclonal antibodies; pAbs) from mice immunized with recombinant NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. The screen using mAbs identified four WNV-specific (including Kunjin virus) epitopes, located at aa 21-36, 101-116, 191-206 and 261-276 in WNV NS1. However, using pAbs, only three WNV-specific epitopes were identified, located at positions 101-116, 191-206 and 231-246. Two of these epitopes (aa 21-36 and 261-276) had different reactivity with mAbs and pAbs. The knowledge and reagents generated in this study have potential applications in differential diagnostics and epitope-based marker vaccine development for WNV and viruses of the JEV serocomplex.

Development of an ELISA-array for simultaneous detection of five encephalitis viruses.

Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.