Rice-produced classical swine fever virus glycoprotein E2 with herringbone-dimer design to enhance immune responses.

Pestiviruses, including classical swine fever virus, remain a concern for global animal health and are responsible for major economic losses of livestock worldwide. Despite high levels of vaccination, currently available commercial vaccines are limited by safety concerns, moderate efficacy, and required high doses. The development of new vaccines is therefore essential. Vaccine efforts should focus on optimizing antigen presentation to enhance immune responses. Here, we describe a simple herringbone-dimer strategy for efficient vaccine design, using the classical swine fever virus E2 expressed in a rice endosperm as an example. The expression of rE2 protein was identified, with the rE2 antigen accumulating to 480 mg/kg. Immunological assays in mice, rabbits, and pigs showed high antigenicity of rE2. Two immunizations with 284 ng of the rE2 vaccine or one shot with 5.12 µg provided effective protection in pigs without interference from pre-existing antibodies. Crystal structure and small-angle X-ray scattering results confirmed the stable herringbone dimeric conformation, which had two fully exposed duplex receptor binding domains. Our results demonstrated that rice endosperm is a promising platform for precise vaccine design, and this strategy can be universally applied to other Flaviviridae virus vaccines.

Inhibition of BET Family Proteins Suppresses African Swine Fever Virus Infection.

African swine fever (ASF), an acute, severe, highly contagious disease caused by African swine fever virus (ASFV) infection in domestic pigs and boars, has a mortality rate of up to 100%. Because effective vaccines and treatments for ASF are lacking, effective control of the spread of ASF remains a great challenge for the pig industry. Host epigenetic regulation is essential for the viral gene transcription. Bromodomain and extraterminal (BET) family proteins, including BRD2, BRD3, BRD4, and BRDT, are epigenetic "readers" critical for gene transcription regulation. Among these proteins, BRD4 recognizes acetylated histones via its two bromodomains (BD1 and BD2) and recruits transcription factors, thereby playing a pivotal role in transcriptional regulation and chromatin remodeling during viral infection. However, how BET/BRD4 regulates ASFV replication and gene transcription is unknown. Here, we randomly selected 12 representative BET family inhibitors and compared their effects on ASFV infection in pig primary alveolar macrophages (PAMs). These were found to inhibit viral infection by interfering viral replication. The four most effective inhibitors (ARV-825, ZL0580, I-BET-762, and PLX51107) were selected for further antiviral activity analysis. These BET/BRD4 inhibitors dose dependently decreased the ASFV titer, viral RNA transcription, and protein production in PAMs. Collectively, we report novel function of BET/BRD4 inhibitors in inducing suppression of ASFV infection, providing insights into the role of BET/BRD4 in the epigenetic regulation of ASFV and potential new strategies for ASF prevention and control. IMPORTANCE Due to the continuing spread of the ASFV in the world and the lack of commercial vaccines, the development of improved control strategies, including antiviral drugs, is urgently needed. BRD4 is an important epigenetic factor and has been commonly used for drug development for tumor treatment. Furthermore, the latest research showed that BET/BRD4 inhibition could suppress replication of virus. In this study, we first showed the inhibitory effect of agents targeting BET/BRD4 on ASFV infection with no significant host cytotoxicity. Then, we found four BET/BRD4 inhibitors that can inhibit ASFV replication, RNA transcription, and protein synthesis. Our findings support the hypothesis that BET/BRD4 can be considered as attractive host targets in antiviral drug discovery against ASFV.

Prognostic nomogram for predicting 5-year overall survival in Chinese patients with high-grade osteosarcoma.

This study aimed to construct a widely accepted prognostic nomogram in Chinese high-grade osteosarcoma (HOS) patients aged ≤ 30 years to provide insight into predicting 5-year overall survival (OS). Data from 503 consecutive HOS patients at our centre between 12/2012 and 05/2019 were retrospectively collected. Eighty-four clinical features and routine laboratory haematological and biochemical testing indicators of each patient at the time of diagnosis were collected. A prognostic nomogram model for predicting OS was constructed based on the Cox proportional hazards model. The performance was assessed by the concordance index (C-index), receiver operating characteristic curve and calibration curve. The utility was evaluated by decision curve analysis. The 5-year OS was 52.1% and 2.6% for the nonmetastatic and metastatic patients, respectively. The nomogram included nine important variables based on a multivariate analysis: tumour stage, surgical type, metastasis, preoperative neoadjuvant chemotherapy cycle, postoperative metastasis time, mean corpuscular volume, tumour-specific growth factor, gamma-glutamyl transferase and creatinine. The calibration curve showed that the nomogram was able to predict 5-year OS accurately. The C-index of the nomogram for OS prediction was 0.795 (range, 0.703-0.887). Moreover, the decision curve analysis curve also demonstrated the clinical benefit of this model. The nomogram provides an individualized risk estimate of the 5-year OS in patients with HOS aged ≤ 30 years in a Chinese population-based cohort.

Development of an immunochromatographic strip for rapid detection of H7 subtype avian influenza viruses.

BACKGROUND: H7N9 avian influenza virus (AIV) including highly and low pathogenic viruses have been detected in China since 2013. H7N9 AIV has a high mortality rate after infection in humans, and most human cases have close contacted with poultry in the live poultry market. Therefore, it is necessary to develop a rapid point-of-care testing (POCT) technique for H7N9 AIV detection. METHODS: The H7N9 AIV was inactivated and purified, and was used as the antigen to immunize BALB/c. Twelve H7-HA specific monoclonal antibodies (McAbs) were produced through the hybridoma technique. The McAb 10A8 was conjugated with colloid gold as detecting antibody; McAb 9B6 was dispensed on the nitrocellulose membran as the capture test line and the Goat-anti mouse IgG antibody was dispensed as control line respectively. The immunochromatographic strip was prepared. RESULTS: The analysis of ELISA and virus neutralization test showed that the obtained McAbs specifically recognized H7 HA. Based on the prepared strip, the detection of H7 AIV was achieved within 10 min. No cross-reaction occurred between H7 AIVs and other tested viruses. The detection limit of the strip for H7 was 2.4 log10EID50/0.1 mL for chicken swab samples. CONCLUSION: The McAbs were specific for H7 and the immunochromatographic strip developed in this study was convenient, rapid and reliable for the detection of H7 AIV. The strip could provide an effective method for the rapid and early detection of H7 AIV.

Transcriptome Sequencing reveals the expressed profiles of mRNA and ncRNAs and regulate network via ceRNA mediated molecular mechanism of lung adenocarcinoma bone metastasis in Xuanwei.

BACKGROUND: The most ordinary subtype of lung cancer is lung adenocarcinoma (LuAC), which is characterized by strong metastatic ability. And LuAC rates in Xuanwei leads to the poor prognosis and high death rate. In this study, we systematically explored the molecular mechanism of LuAC bone metastasis in Xuanwei by transcriptome sequencing. METHODS: RNA Sequencing was conducted to explore the noncoding RNAs (ncRNAs) expression profiles in primary LuAC and LuAC bone metastasis. We identified differentially expressed mRNAs (DEmRNAs), miRNAs (DEmiRNAs), lncRNAs (DElncRNAs) and circRNAs (DEcircRNAs). Bioinformatics analyses the possible relationships and functions of the LuAC bone metastasis-related competing endogenous RNA (ceRNA). And qRT-PCR was performed to evaluate the expression of these differently expressed genes in serum. RESULTS: A total of 2,141 DEmRNAs, 43 DEmiRNAs, 136 DElncRNAs and 706 DEcircRNAs were identified in the Xuanwei patients with primary LuAC vs. LuAC bone metastasis, respectively. The circRNA/miRNA/mRNA and lncRNA/miRNA/mRNA networks of LuAC in Xuanwei with bone metastasis were built, and the gene expression mechanisms regulated by ncRNAs were unveiled via the ceRNA regulatory networks. We observe that lncRNA (ADAMTS9-AS2, TEX41, DLEU2, LINC00152)-miR-223-3p-SCARB1 and hsa_circ_0000053-miR-196a-5p/miR-196b-5p-HOXA5 ceRNA networks might play an important role in bone metastasis of Xuanwei LuAC. CONCLUSIONS: We comprehensively identified ceRNA regulatory networks of LuAC in Xuanwei with bone metastasis as well as revealed the contribution of different ncRNAs expression profiles. Our data demonstrate the association between mRNAs and ncRNAs in the metastasis mechanism of LuAC in Xuanwei with bone metastasis.

Progress in the chemotherapeutic treatment of osteosarcoma.

Osteosarcoma (OS) is the most common type of primary bone tumor in children and adolescents and has been associated with a high degree of malignancy, early metastasis, rapid progression and poor prognosis. However, the use of adjuvant chemotherapy improves the prognosis of patients with OS. OS chemotherapy is based primarily on the use of adriamycin, cisplatin (DDP), methotrexate (MTX), ifosfamide (IFO), epirubicin (EPI) and other drugs. Previous studies have revealed that the survival rate for patients with OS appears to have plateaued: 5-year survival rates remain close to 60%, even with the use of combined chemotherapy. The most limiting factors include complications and fatal toxicity associated with chemotherapy agents, particularly high-dose MTX (HD-MTX), for which high toxicity and great individual variation in responses have been observed. Docetaxel (TXT) is a representative member of the relatively recently developed taxane class of drugs, which function to inhibit OS cell proliferation and induce apoptosis. Recently, more clinical studies have reported that TXT combined with gemcitabine (GEM) is effective in the treatment of OS (relapse/refractory and progressive), providing evidence in support of potential novel treatment strategies for this patient population. However, there is still no global consensus on this type of chemotherapy approach. The present review summarizes current studies surrounding progress in the chemotherapeutic treatment of OS and discusses the advantages and potential feasibility of TXT+GEM in the treatment of OS.

Does intensified chemotherapy increase survival outcomes of osteosarcoma patients? A meta-analysis.

STUDY DESIGN: Meta-analysis. BACKGROUND: Although some new insights have been offered for clinical and scientific relevance, minor progress has been made in osteosarcoma treatment after a dramatic survival improvement in the late 1980s with the addition of chemotherapy to surgery. Intensified chemotherapy strategies have been suggested to increase the survival rate of patients with osteosarcoma. We performed this study to access whether intensified chemotherapy strategiesincreased survival outcomes of osteosarcoma patients compared with conventional chemotherapy strategies. METHODS: MEDLINE/PubMed, EMBASE, BIOSIS Previews, and Cochrane Library were searched from database set up to October2016. Randomized controlled trials (RCTs) and comparative clinical trials (CCTs) on intensified versus conventional chemotherapy strategies for osteosarcoma patients met the inclusion criteria, and the methodological quality standard were retrieved and reviewed. Data on participant characteristics, interventions, follow-up period, and outcomes were extracted from the included studies and analyzed by Review Manager 5.3. RESULTS: 12 studies (8 RCTs and 4CCT) involving 4112 patients were selected. There were no significant differences between intensified and conventional chemotherapy strategies group in 3-year event-free survival (OR, 1.01; 95% CI, [0.74-1.37]; P = 0.97), 5-year event-free survival (OR, 1.00; 95% CI, [0.86-1.17]; P = 0.97), and 5-year overall survival (OR, 1.04; 95% CI, [0.87-1.26]; P = 0.64), and good histologic response to preoperative chemotherapy (OR, 1.12; 95% CI, [0.78-1.60]; P = 0.55). Pooled analysis of local recurrence rate showed that local recurrence rate was significantly decreased in the intensified group compared with that in the conventional group (OR, 0.60; 95% CI, [0.42-0.85]; P = 0.004). CONCLUSIONS: Intensified chemotherapy might not be a preferred treatment for all of the osteosarcoma patients.

Advances in limb salvage treatment of osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor; its standard treatment includes neoadjuvant chemotherapy combined with surgery. Neoadjuvant chemotherapy has significantly improved the 5-year survival and limb salvage rates in osteosarcoma since the 1870s. The survival rate of patients with limb salvage was not inferior to that of amputees, and therefore, limb salvage has become the main surgical option for patients with osteosarcoma. The 5-year survival rate for osteosarcoma has plateaued. However, new advances in limb salvage therapy in osteosarcoma, including adjuvant chemotherapy, ablation techniques, bone transport techniques, and computer navigation techniques, are now available. This report summarizes the recent advances in limb salvage therapy for osteosarcoma over the past decade.

Comparative proteomic and bioinformatic analysis of Theileria luwenshuni and Theileria uilenbergi.

Theileria is an obligatory intraerythrocytic protozoan parasite that causes economic losses to the cattle, sheep and goats industry. However, very little information is available on the genomes, transcriptomes, and proteomes of the ovine parasites, Theileria luwenshuni and Theileria uilenbergi. Differences in protein expression between these species were investigated to better understand their biology. Parasites were digested with trypsin, and the resulting peptides labeled with isobaric tags for relative and absolute quantification, followed by LC-MS/MS. More than 670 proteins, classified into categories primarily related to cellular process (29.78%), metabolic process (28.80%), localization (5.22%) and biological regulation (5.00%), were identified. Seventy-one proteins were differentially expressed; T. luwenshuni had 39 proteins more highly expressed than in T. uilenbergi, whereas T. uilenbergi had 32 that were more highly expressed. Several proteins related to parasite virulence and invasion (cysteine proteinase, histone deacetylase, pyruvate kinase, small nuclear ribonucleoprotein and orotate phosphoribosyltransferase) were differentially expressed. Real-time quantitative PCR validated protein expression changes at the transcript level. This is the first report on protein expression for the two most economically important Theileria species in China, and our findings may provide novel opportunities for ovine and caprine theileriosis control.

A comparison of loop-mediated isothermal amplification (LAMP) with other surveillance tools for Echinococcus granulosus diagnosis in canine definitive hosts.

BACKGROUND: Cystic echinococcosis is highly prevalent in northwest China. A cost-effective, easy to operate diagnostic tool with high sensitivity and specificity would greatly facilitate the monitoring of Echinococcus infections in canine definitive hosts. METHODS: The primers used in the LAMP assay were based on the mitochondrial nad5 gene of E. granulosus sensu stricto (E. granulosus s.s., or E.g.s.s.) and were designed using Primer Explorer V4 software. The developed LAMP assay was compared with a conventional PCR method, copro-ELISA and microscopy, using the faeces of dogs experimentally infected with E.g.s.s., and field-collected faeces of domestic dogs including 190 from Qinghai province highly endemic for E.g.s.s. and 30 controls from an area in Gansu, where a domestic dog de-worming program was in operation. RESULTS: The positivity rates obtained for the field-collected faecal samples were 12.6%, 1.6% and 2.1% by the LAMP, PCR and copro-ELISA assays, respectively. All samples obtained from the control dogs were negative. Compared with the conventional PCR, the LAMP assay provided 88.8% specificity and 100% sensitivity. The higher sensitivity of the LAMP method was also shown by the fact that it could detect the presence of laboratory challenge dog infections of E. granulsous s.s. four days earlier than the PCR method. Three copro-samples shown positive by the commercial copro-ELISA were all negative by LAMP, PCR and microscopy, which suggests these samples may have originated from another infection rather than E. granulsous s.s., possibly E. shiquicus or E. Canadensis, which is also present in China. CONCLUSIONS: We have developed a potentially useful surveillance tool for determining the prevalence of canine E. granulosus s.s. infections in the field. The LAMP assay may lead to a more cost-effective and practicable way of tracking Echinococcus infections in canids, especially when combined with the copro-ELISA.

Loop-mediated isothermal amplification (LAMP) assay for the identification of Echinococcus multilocularis infections in canine definitive hosts.

BACKGROUND: Alveolar echinococcosis, caused by the metacestode larval stage of Echinococcus multilocularis, is a zoonosis of public health significance and is highly prevalent in northwest China. To effectively monitor its transmission, we developed a new rapid and cheap diagnostic assay, based on loop-mediated isothermal amplification (LAMP), to identify canine definitive hosts infected with E. multilocularis. METHODS: The primers used in the LAMP assay were based on the mitochondrial nad5 gene of E. multilocularis and were designed using Primer Explorer V4 software. The developed LAMP assay was compared with a conventional PCR assay, using DNA extracted from the feces of dogs experimentally infected with E. multilocularis, on 189 dog fecal samples collected from three E. multilocularis-endemic regions in Qinghai province, the People's Republic of China, and 30 negative control copro-samples from dogs from an area in Gansu province that had been subjected to an intensive de-worming program. Light microscopy was also used to examine the experimentally obtained and field collected dog copro-samples for the presence of E. multilocularis eggs. RESULTS: The E. multilocularis-positivity rates obtained for the field-collected fecal samples were 16.4% and 5.3% by the LAMP and PCR assays, respectively, and all samples obtained from the control dogs were negative. The LAMP assay was able to detect E. multilocularis DNA in the feces of experimentally infected dogs at 12 days post-infection, whereas the PCR assay was positive on the 17th day and eggs were first detectable by light microscopy at day 44 post-challenge. CONCLUSION: The earlier specific detection of an E. multilocularis infection in dog copro-samples indicates that the LAMP assay we developed is a realistic alternative method for the field surveillance of canines in echinococcosis-endemic areas.

Infection of small ruminants and their red blood cells with Theileria annulata schizonts.

Theileria annulata, the causative agent of tropical theileriosis, is a protozoan parasite that also causes lymphoproliferative diseases in cattle. In vivo, parasitized cells undergo clonal expansion and infiltrate both the lymphoid and non-lymphoid tissues of the infected host. To determine whether the small ruminants and their red blood cells (RBCs) were invaded by T. annulata schizonts or not, T. annulata schizonts were used to infect bovine, ovine and caprine RBCs in vitro, and sheep and goats in vivo. The results showed that the schizonts infected bovine, ovine and caprine RBCs in vitro, but not sheep and goats, which showed only an increase in body temperature and no development of piroplasms. To our knowledge, this is the first report of infection of small ruminants and their RBCs by T. annulata schizonts.

Development of an enzyme linked immunosorbent assay and an immunochromatographic assay for detection of organophosphorus pesticides in different agricultural products.

OBJECTIVE: Organophosphorus (OP) pesticides are considered hazardous substances because of their high toxicity to nontarget species and their persistence in the environment and agricultural products. Therefore, it is important to develop a rapid, sensitive, and economical method for detecting OP pesticides and their residues in food and the environment. METHODS: A broad, selective monoclonal antibody (MAb) for organophosphorus pesticides was produced. Based on the MAb, an enzyme linked immunosorbent assay (ELISA) and an immunochromatography assay (ICA) for detecting OP pesticides in different agricultural products were developed using a binding inhibition format on microtiter plates and a membrane strip, respectively. RESULTS: Under the optimized conditions, the IC(50) values of the ELISA ranged from 3.7 to 162.2 ng mL(-1) for the 8 OP pesticides. The matrix interferences of Apple, Chinese cabbage, and greengrocery were removed by 40-fold dilution, the recoveries from spiked samples ranged from 79.1% to 118.1%. The IC(50) values of ICA for the 8 OP pesticides ranged from 11.8 to 470.4 ng mL(-1). The matrix interference was removed from the Chinese cabbage and Apple samples with 5-fold dilution, and the interference was removed from the greengrocery samples with 20-fold dilution. The recoveries from the spiked samples ranged between 70.6 and 131.9%. The established ELISA and ICA were specific selectivity for the 8 OP pesticides. CONCLUSIONS: The established ELISA is a sensitive screening method for the detection of OP pesticides, but the ELISA detection method depends on a laboratory platform and requires a relative long assay time and several steps operation. The established ICA is very useful as a screening method for the quantitative, semi-quantitative or qualitative detection of OP pesticides in agricultural products, and it has advantages over ELISA methods with regard to factors such as the testing procedure, testing time, and matrix interferences, among others.

Development of an immunochromatographic strip for rapid detection of antibodies against classical swine fever virus.

The genes encoding the Erns and E2 antigen epitopes of classical swine fever virus (CSFV) were expressed as a chimeric protein in Escherichia coli BL21 by pET expression system. The antigenicity of the expressed protein CnC2 was identified by indirect enzyme-linked immunoabsorbant assay (ELISA) and immunoblot with anti-CSFV antibodies. Based on the CnC2 protein, an immunochromatographic strip was developed to evaluate the antibody titer of serum samples from swine vaccinated with CSFV vaccine rapidly. The chimeric protein used as a detector was labeled with colloidal gold. Staphylococcal protein A (SPA) and anti-CnC2 monoclonal antibodies (mAbs) were blotted onto the nitrocellulose membrane as the test and control lines, respectively. The strip assay could be performed within 5min, which did not require any special equipment or skills. Through testing sera against various strains of CSFV, the sensitivity of the strip was determined to be 97.0% (65/67) and the specificity was 100% (98/98). The strip results were consistent with those of the existing commercial ELISA kit, and their correlation coefficient was 0.935. In conclusion, the immunochromatographic strip was an acceptable method for surveying CSFV-antibody titers in pigs.