Sensitive electrochemical detection of enrofloxacin in eggs based on carboxylated multi-walled carbon nanotubes-reduced graphene oxide nanocomposites: Molecularlyimprintedrecognition versus direct electrocatalytic oxidation.

A sensitive electrochemical method for detecting enrofloxacin was proposed using carboxylated multi-walled carbon nanotubes-reduced graphene oxide (MWCNT-COOH-RGO) nanocomposites. The MWCNT-COOH-RGO nanocomposites were firstly electrodeposited on a bare electrode, followed by electropolymerization of molecularly imprinted polymers. Enrofloxacin was determined by the mechanisms of direct electrocatalytic oxidation and molecularly imprinted recognition, respectively. Under the optimized conditions, a response range of 5.0×10-7 M to 5.5×10-5 M and limit of detection (LOD) of 2.3×10-7 M were obtained by direct electrocatalytic oxidation of enrofloxacin using chronoamperometry. By contrast, the response range of 1.0×10-10 M to 5.0×10-5 M and LOD of 2.5×10-11 M were achieved by molecularly imprinted recognition of enrofloxacin using square-wave voltammetry. Moreover, the proposed method exhibited good repeatability, stability and selectivity, and could be used for enrofloxacin detection in egg samples with satisfactory results.

Highly sensitive detection of fluoride based on poly(3-aminophenylboronic acid)-reduced graphene oxide multilayer modified electrode.

A novel electrochemical method for detecting fluoride was developed based on gold electrode modified by layer-by-layer (LBL) assembly of poly(3-aminophenylboronic acid)-reduced graphene oxide (PAPBA-RGO) multilayers. The PAPBA-RGO multilayer-modified gold electrode was constructed by using alternating LBL assembly of RGO and PAPBA on a bare gold electrode by one-step electrodeposition. Fluoride was electrochemically determined based on the proposed modified electrode by evaluating the changes in peak current for potassium ferricyanide reduction caused by the conjunction of fluoride and boronic acid groups of PAPBA. The results indicated that the peak current for potassium ferricyanide reduction obviously decreased with the increasing fluoride concentration. The response range of our method for fluoride was 1 × 10-8 to 1 × 10-1 M with a detection limit of 6 × 10-10 M and high sensitivity and selectivity. This method was applied to detect fluoride in tap water, rice, apple, and edible fungi samples.

Prognostic Value and Potential Regulatory Mechanism of H19 in Stomach Adenocarcinoma.

The first lncRNA discovered, H19, has been found to participate in the regulation of diverse biological processes, including the pathogenesis of stomach adenocarcinoma. In addition to its oncogenic function in tumor formation, a high level of H19 in tumor tissues has also been reported to be an indicator for poor prognosis. However, although many previous works have investigated the level of H19 as an independent indicator for prognosis, the real value of H19 in predicting survival has rarely been evaluated. In this study, we established a prognostic model and nomogram for stomach adenocarcinoma by combining the expression level of H19 with traditional indices, which showed the value of H19 in predicting the survival rates of patients. In addition, we investigated the mechanism underlying the correlation of the H19 level in cancer tissue with poor prognosis in patients. Our results showed that H19 could function as ceRNA by sponging five miRNAs, which may promote the progression of cancer.

Molecular cloning, characterization, and functional analysis of the uncharacterized C11orf96 gene.

BACKGROUND: The mammalian genome encodes millions of proteins. Although many proteins have been discovered and identified, a large part of proteins encoded by genes are yet to be discovered or fully characterized. In the present study, we successfully identified a host protein C11orf96 that was significantly upregulated after viral infection. RESULTS: First, we successfully cloned the coding sequence (CDS) region of the cat, human, and mouse C11orf96 gene. The CDS region of the C11orf96 gene is 372 bp long, encodes 124 amino acids, and is relatively conserved in different mammals. From bioinformatics analysis, we found that C11orf96 is rich in Ser and has multiple predicted phosphorylation sites. Moreover, protein interaction prediction analysis revealed that the protein is associated with several transmembrane family proteins and zinc finger proteins. Subsequently, we found that C11orf96 is strictly distributed in the cytoplasm. According to the tissue distribution characteristics, C11orf96 is distributed in all tissues and organs, with the highest expression levels in the kidney. These results indicate that C11orf96 may play a specific biological role in the kidney. CONCLUSIONS: Summarizing, these data lay the foundation for studying the biological functions of C11orf96 and for exploring its role in viral replication.

Nucleolin interacts with the rabbit hemorrhagic disease virus replicase RdRp, nonstructural proteins p16 and p23, playing a role in virus replication.

Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family and cannot be propagated in vitro, which has impeded the progress of investigating its replication mechanism. Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells. Here, aided by this replicon system and using two-step affinity purification, we purified the RHDV replicase and identified its associated host factors. We identified rabbit nucleolin (NCL) as a physical link, which mediating the interaction between other RNA-dependent RNA polymerase (RdRp)-related host proteins and the viral replicase RdRp. We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 â€‹cells, respectively. NCL was identified to directly interact with RHDV RdRp, p16, and p23. Furthermore, NCL knockdown severely impaired the binding of RdRp to RdRp-related host factors. Collectively, these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins.

Decreased inhibition of exosomal miRNAs on SARS-CoV-2 replication underlies poor outcomes in elderly people and diabetic patients.

Elderly people and patients with comorbidities are at higher risk of COVID-19 infection, resulting in severe complications and high mortality. However, the underlying mechanisms are unclear. In this study, we investigate whether miRNAs in serum exosomes can exert antiviral functions and affect the response to COVID-19 in the elderly and people with diabetes. First, we identified four miRNAs (miR-7-5p, miR-24-3p, miR-145-5p and miR-223-3p) through high-throughput sequencing and quantitative real-time PCR analysis, that are remarkably decreased in the elderly and diabetic groups. We further demonstrated that these miRNAs, either in the exosome or in the free form, can directly inhibit S protein expression and SARS-CoV-2 replication. Serum exosomes from young people can inhibit SARS-CoV-2 replication and S protein expression, while the inhibitory effect is markedly decreased in the elderly and diabetic patients. Moreover, three out of the four circulating miRNAs are significantly increased in the serum of healthy volunteers after 8-weeks' continuous physical exercise. Serum exosomes isolated from these volunteers also showed stronger inhibitory effects on S protein expression and SARS-CoV-2 replication. Our study demonstrates for the first time that circulating exosomal miRNAs can directly inhibit SARS-CoV-2 replication and may provide a possible explanation for the difference in response to COVID-19 between young people and the elderly or people with comorbidities.

Zinc finger antiviral protein (ZAP) inhibits small ruminant morbillivirus replication in vitro.

Small ruminant morbillivirus (SRMV) is a highly contagious and economically important viral disease of small domestic and wild ruminants. Difficulty with its stable proliferation in ovis aries-derived cells has led to a relative lag in the study of its natural immunity and pathogenesis. Here we report the antiviral properties of ZAP against SRMV, a single-stranded negative-stranded RNA virus of the genus Morbillivirus. ZAP expression was significantly induced in sheep endometrial epithelial cells following SRMV infection. ZAP inhibited SRMV replication in cells after infection, while its overexpression in Vero-SLAM cells significantly increased their resistance to SRMV replication. The ZAP protein co-localized with SRMV RNA in the cytoplasm and ZAP-responsive elements were mapped to the 5' untranslated region of SRMV nucleocapsid, phosphoprotein, matrix, and fusion. In summary, ZAP confers resistance to SRMV infection by directly targeting viral RNA and inhibiting viral replication. Our findings further extend the ranges of viral targets of ZAP and help elucidate the mechanism of SRMV replication.

Caprine MAVS Is a RIG-I Interacting Type I Interferon Inducer Downregulated by Peste des Petits Ruminants Virus Infection.

The mitochondrial antiviral-signaling protein (MAVS, also known as VISA, IPS-1, or CARDIF) plays an essential role in the type I interferon (IFN) response and in retinoic acid-inducible gene I (RIG-I) mediated antiviral innate immunity in mammals. In this study, the caprine MAVS gene (caMAVS, 1566 bp) was identified and cloned. The caMAVS shares the highest amino acid similarity (98.1%) with the predicted sheep MAVS. Confocal microscopy analysis of partial deletion mutants of caMAVS revealed that the transmembrane and the so-called Non-Characterized domains are indispensable for intracellular localization to mitochondria. Overexpression of caMAVS in caprine endometrial epithelial cells up-regulated the mRNA levels of caprine interferon-stimulated genes. We concluded that caprine MAVS mediates the activation of the type I IFN pathway. We further demonstrated that both the CARD-like domain and the transmembrane domain of caMAVS were essential for the activation of the IFN-ß promotor. The interaction between caMAVS and caprine RIG-I and the vital role of the CARD and NC domain in this interaction was demonstrated by co-immunoprecipitation. Upon infection with the Peste des Petits Ruminants Virus (PPRV, genus Morbillivirus), the level of MAVS was greatly reduced. This reduction was prevented by the addition of the proteasome inhibitor MG132. Moreover, we found that viral protein V could interact and colocalize with MAVS. Together, we identified caMAVS as a RIG-I interactive protein involved in the activation of type I IFN pathways in caprine cells and as a target for PPRV immune evasion.

In vivo self-assembled small RNAs as a new generation of RNAi therapeutics.

RNAi therapy has undergone two stages of development, direct injection of synthetic siRNAs and delivery with artificial vehicles or conjugated ligands; both have not solved the problem of efficient in vivo siRNA delivery. Here, we present a proof-of-principle strategy that reprogrammes host liver with genetic circuits to direct the synthesis and self-assembly of siRNAs into secretory exosomes and facilitate the in vivo delivery of siRNAs through circulating exosomes. By combination of different genetic circuit modules, in vivo assembled siRNAs are systematically distributed to multiple tissues or targeted to specific tissues (e.g., brain), inducing potent target gene silencing in these tissues. The therapeutic value of our strategy is demonstrated by programmed silencing of critical targets associated with various diseases, including EGFR/KRAS in lung cancer, EGFR/TNC in glioblastoma and PTP1B in obesity. Overall, our strategy represents a next generation RNAi therapeutics, which makes RNAi therapy feasible.

RPS5 interacts with the rabbit hemorrhagic disease virus 3' extremities region and plays a role in virus replication.

Rabbit hemorrhagic disease virus (RHDV), a member of Caliciviridae family, causes a highly contagious disease in rabbits. The RHDV replication mechanism is poorly understood due to the lack of a suitable culture system in vitro. This study identified RHDV 5' and 3' extremities (Ex) RNA binding proteins from the rabbit kidney cell line RK-13 based on a pull-down assay by applying a tRNA scaffold streptavidin aptamer. Using mass spectrometry (MS), several host proteins were discovered which interact with RHDV 5' and 3' Ex RNA. The ribosomal protein S5 (RPS5) was shown to interact with RHDV 3' Ex RNA directly by RNA-pulldown and confocal microscopy. To further investigate the role of RPS5 in RHDV replication, small interfering RNAs for RPS5 and RPS5 eukaryotic expression plasmids were used to change the expression level of RPS5 in RK-13 cells and the results showed that the RHDV replication and translation levels were positively correlated with the expression level of RPS5. It was also verified that RPS5 promoted RHDV replication by constructing RPS5 stable overexpression cell lines and RPS5 knockdown cell lines. In summary, it has been identified that RPS5 interacted with the RHDV 3' Ex RNA region and played a role in virus replication. These results will help to understand the mechanism of RHDV replication.

Construction and immunogenicity of novel bivalent virus-like particles bearing VP60 genes of classic RHDV(GI.1) and RHDV2(GI.2).

Rabbit hemorrhagic disease (RHD) is an acute, inflammatory, septic, and devastating infectious disease caused by Rabbit hemorrhagic disease virus (RHDV), which poses a serious threat to the rabbit industry. RHDV2 (GI.2/RHDVb), a recently reported new variant could cause RHD in wild populations, but also RHDV-vaccinated rabbits. For now, both RHDV and RHDV2 are the main causes of RHD. To develop a new subunit vaccine that could protect rabbits against both classic RHDV and RHDV2 infections, we constructed a recombinant baculovirus (Bac-classic RHDV VP60-RHDV2 VP60) containing the VP60 genes of classic RHDV and RHDV2. Both VP60 genes were well expressed simultaneously in Spodoptera frugiperda cells (Sf9) after infection with the recombinant baculovirus. Transmission electron microscopy showed that the recombinant VP60 self-assembled into virus-like particles (VLPs). The antigenicity and immunogenicity of the bivalent VLPs vaccine were examined with animal experiments. Our results demonstrated that both the humoral and cellular immune responses were efficiently induced in rabbits by a subunit vaccine based on the recombinant baculovirus. In addition, all rabbits immunized with the bivalent VLPs vaccine survived after challenged with classic RHDV, and showed no clinical signs of RHD, whereas all the rabbits in the negative control group died from classic RHDV infection and showed typical clinical signs of RHD. In summary, our results indicated that the recombinant baculovirus carrying two VP60 genes is a candidate construct from which to develop a bivalent VLPs vaccine against both classic RHDV and RHDV2 infections.