Cotransfection Of Plasmid Research Articles

Increased production of orthoflavivirus single-round infectious particles produced in mammalian cells at a suboptimal culture temperature of 28°C

In the employment of serodiagnostic methods for the detection of orthoflavivirus infections, neutralization tests are known to be more accurate than measurements of antibody binding properties employing enzyme-linked immunosorbent assays. However, neutralization tests require infectious virus and laboratories with an appropriate level of biosafety. Single-round infectious particles (SRIPs), which encode a reporter gene instead of the viral structural protein genes, are replication incompetent and represent a safe and reliable alternative to the diagnosis of pathogenic viruses in neutralization tests. The orthoflavivirus SRIPs are produced by co-transfection of plasmids expressing virus-like particles and replicons into mammalian cell lines preferably with high transfection efficacy, such as HEK293T cells. However, certain orthoflavivirus SRIPs have limitations in their efficient expression at 37°C, which is the optimal temperature for mammalian cell growth, resulting in insufficient yields for neutralization tests. Here, we demonstrate that the production of orthoflavivirus SRIPs increases at the lower temperature of 28°C compared to 37°C. Moreover, infections with 28°C-cultured SRIPs in microneutralization tests were specifically inhibited in the presence of serum from mice infected with homologous viruses, suggesting that these SRIPs preserved their neutralizing epitopes for antibodies. Our method to produce high titer SRIPs is anticipated to promote efficient and safe SRIPs neutralization tests as a general serodiagnostic method for detecting virus-specific neutralizing antibodies against orthoflaviviruses.

The RRE-REV module has no effect on the packaging efficiency of cas9 and Gag proteins into nanomedic virus-like particles

Delivery of ribonucleoprotein complexes of Cas9 nuclease and guide RNA into target cells with virus-like particles (VLP) is one of the novel methods of genome editing, suitable for gene therapy of human diseases in the future. Efficiency of genome editing with VLPs depends on the packaging of Cas9 into VLPs, that is mediated by viral Gag protein. To increase the packaging of Cas9 into NanoMEDIC system VLPs plasmid constructs for expression of Cas9 and Gag were modified by the addition of HIV RRE (Rev response element), that is expected to increase the nuclear export of RRE-containing transcripts to cytosol via accessory protein Rev, as described for Vpr-Cas9-based VLP system. Here we found that Cas9 and Gag protein levels in the cell lysates are increased upon cotransfection of either Rev-expressing plasmid or empty control plasmid. Moreover, this effect does not depend on the presence of RRE in the transcript. On the top of that, we showed that AP21967-induced dimerization of FRB and FKBP12, but not the modification of plasmids with RRE and/or cotransfection of Rev-expressing plasmid, plays the major role in packaging of Cas9 into NanoMEDIC system VLPs. These data suggest that it is impractical to use the RRE-Rev module to enhance the packing of Cas9 nuclease into VLPs.

Isolation of Adeno-associated Viral Vectors Through a Single-step and Semi-automated Heparin Affinity Chromatography Protocol.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs. This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

CYP321A Subfamily P450s Contribute to the Detoxification of Phytochemicals and Pyrethroids in Spodoptera litura.

Although the induction of cytochrome P450 monooxygenases involved in insect detoxification has been well documented, the underlying regulatory mechanisms remain obscure. In Spodoptera litura, CYP321A subfamily members were effectively induced by exposure to flavone, xanthotoxin, curcumin, and λ-cyhalothrin, while knockdown of the CYP321A genes increased larval susceptibility to these xenobiotics. Homology modeling and molecular docking analyses showed that these four xenobiotics could stably bind to the CYP321A enzymes. Furthermore, two transcription factor genes, CncC and MafK, were significantly induced by the xenobiotics. Knockdown of CncC or MafK reduced the expression of four CYP321A genes and increased larval susceptibility to the xenobiotics. Dual-luciferase reporter assays showed that cotransfection of reporter plasmids carrying the CYP321A promoter with CncC and/or MafK-expressing constructs significantly magnified the promoter activity. These results indicate that the induction of CYP321A subfamily members conferring larval detoxification capability to xenobiotics is mediated by the activation of CncC and MafK.

CRISPR single base-editing: in silico predictions to variant clonal cell lines.

Engineering single base edits using CRISPR technology including specific deaminases and single-guide RNA (sgRNA) is a rapidly evolving field. Different types of base edits can be constructed, with cytidine base editors (CBEs) facilitating transition of C-to-T variants, adenine base editors (ABEs) enabling transition of A-to-G variants, C-to-G transversion base editors (CGBEs) and recently adenine transversion editors (AYBE) that create A-to-C and A-to-T variants. The base-editing machine learning algorithm BE-Hive predicts which sgRNA and base editor combinations have the strongest likelihood of achieving desired base edits. We have used BE-Hive and TP53 mutation data from The Cancer Genome Atlas (TCGA) ovarian cancer cohort to predict which mutations can be engineered, or reverted to wild-type (WT) sequence, using CBEs, ABEs or CGBEs. We have developed and automated a ranking system to assist in selecting optimally designed sgRNA that considers the presence of a suitable protospacer adjacent motif (PAM), the frequency of predicted bystander edits, editing efficiency and target base change. We have generated single constructs containing ABE or CBE editing machinery, an sgRNA cloning backbone and an enhanced green fluorescent protein tag (EGFP), removing the need for co-transfection of multiple plasmids. We have tested our ranking system and new plasmid constructs to engineer the p53 mutants Y220C, R282W and R248Q into WT p53 cells and shown that these mutants cannot activate four p53 target genes, mimicking the behaviour of endogenous p53 mutations. This field will continue to rapidly progress, requiring new strategies such as we propose to ensure desired base-editing outcomes.

Enhancing CHO cell productivity through a dual selection system using Aspgand Gsin glutamine free medium.

The dominant method for generating Chinese hamster ovary (CHO) cell lines that produce high titers of biotherapeutic proteins utilizes selectable markers such as dihydrofolate reductase (Dhfr) or glutamine synthetase (Gs), alongside inhibitory compounds like methotrexateor methionine sulfoximine,respectively. Recent work has shown the importance of asparaginase (Aspg) for growth in media lacking glutamine-the selection medium for Gs-based selection systems. We generated a Gs/Aspg double knockout CHO cell line and evaluated its utility as a novel dual selectable system via co-transfectionof Gs-Enbrel and Aspg-Enbrel plasmids. Using the same selection conditions as the standard Gs system, the resulting cells from the Gs/Aspg dual selection showed substantially improved specific productivity and titer compared to the standard Gs selection method, however, with reduced growth rate and viability. Following adaptation in the selection medium, the cells improved viability and growth while still achieving ~5-fold higher specific productivity and ~3-fold higher titer than Gs selection alone. We anticipate that with further optimization of culture medium and selection conditions, this approach would serve as an effective addition to workflows for the industrial production of recombinant biotherapeutics.

TRIM26 inhibits hepatitis B virus replication by promoting HBx degradation and TRIM26 genetic polymorphism predicts PegIFNα treatment response of HBeAg‐positive chronic hepatitis B Patients

Hepatitis B virus (HBV) infection is a serious global health burden. TRIM26 has been reported to affect hepatitis C virus replication. To manifest the role of TRIM26 on HBV replication and explore if there are single-nucleotide polymorphisms (SNPs) in TRIM26 associated with response to pegylated interferon-alpha (PegIFNα) treatment in patients with chronic hepatitis B (CHB). We investigated the effect and mechanism of TRIM26 on HBV replication in vitro. The association between SNPs in TRIM26 and PegIFNα treatment response was evaluated in two independent cohorts including 238 and 707 patients with HBeAg-positive CHB. Knockdown of TRIM26 increased, while overexpression of TRIM26 inhibited, HBV replication. Co-immunoprecipitation assays and immunofluorescence showed that TRIM26 interacted and co-localised with HBx. Co-transfection of HBx-HIS and TRIM26-FLAG plasmids in Huh7 cells showed that TRIM26 inhibited the expression of HBx. Furthermore, TRIM26 inhibited HBV replication by mediating HBx ubiquitination degradation, and TRIM26 SPRY domain was responsible for the interaction and degradation of HBx. Besides, IFN increased TRIM26 expression. TRIM26 rs116806878 was associated with response to PegIFNα in two CHB cohorts. Moreover, a polygenic score integrating TRIM26 rs116806878, STAT4 rs7574865 and CFB rs12614 (previously reported to be associated with response to PegIFNα) was related to response to PegIFNα in CHB. TRIM26 inhibits HBV replication; IFN promotes TRIM26 expression. TRIM26 exerts an inhibitory effect on HBx by promoting ubiquitin-mediated degradation of HBx. Furthermore, TRIM26 rs116806878 is a potential predictive biomarker of response to PegIFNα in patients with CHB.

Dominant osteogenesis imperfecta with low bone turnover caused by a heterozygous SP7 variant.

Mutations in SP7 (encoding osterix) have been identified as a rare cause of recessive osteogenesis imperfecta ('OI type XII') and in one case of dominant juvenile Paget's disease. We present the first description of young adult siblings with OI due to a unique heterozygous mutation in SP7. The phenotype was characterized by fragility fractures (primarily of the long bone diaphyses), poor healing, scoliosis, and dental malocclusion. Both siblings had very low cortical volumetric bone mineral density on peripheral quantitative computed tomography of the radius (z-scores -6.6 and-6.7 at the diaphysis), porous cortices, and thin cortices at the radial metaphysis. Histomorphometry demonstrated thin cortices and low bone turnover with reduced osteoblast function. Both siblings were heterozygous for a missense variant affecting a highly conserved zinc finger domain of osterix (c.1019A>C; p.Glu340Ala) on DNA sequencing. Co-transfection of plasmids carrying the SP7 mutation with DLX5 and a luciferase reporter demonstrated that this variant impacted gene function (reduced transcription co-activation compared to wild-type SP7). The low cortical density and cortical porosity seen in our patients are consistent with previous reports of individuals with SP7 mutations. However, the low bone turnover in our patients contrasts with the high turnover state seen in previously reported patients with SP7 mutations. This report indicates that dominant variants in SP7 can give rise to OI. The predominant feature, low cortical density, is common in patients with other SP7 mutations, however other features appear to depend on the specific variant.

Transcriptome analysis of the post-larvae of giant freshwater prawn (Macrobrachium rosenbergii) after IAG gene knockdown with microRNA interference

The insulin-like androgenic gland hormone gene (IAG) of crustaceans plays pivotal roles in the regulation of sex differentiation. MicroRNAs (miRNAs) are a class of short, non-coding RNAs that function as post-transcriptional gene regulators. However, little information about the regulatory relationship between miRNA and Macrobrachium rosenbergii IAG (MrIAG) were exposed. In this study, we used the 3′ untranslated region (UTR) of MrIAG to predict potential target sites of miRNAs. The results showed that miR-184 has one target site in the 3′UTR of MrIAG. Dual-luciferase report assay in vitro confirmed that miR-184 can significantly down-regulate MrIAG expression. Besides, we constructed mutant plasmids of 3′UTR of MrIAG. The result displayed that after co-transfection of mutant plasmids and miR-184 agomir, the activity of luciferase was not affected compared to the control. These results indicated that miR-184 could directly regulate MrIAG. In addition, we found that overexpression of miR-184 in M. rosenbergii can lead to significant changes in the transcription level of genes. Compared with control group, we identified 1510 differentially expressed genes (DEGs) in the miR-184 injection group. Some DEGs were involved in sex differentiation, gonad development, growth and molting were found. qRT-PCR verification was performed on eight DEGs randomly, and the results showed that the expression level of sex-, growth-, and metabolism-related genes changed significantly after MrIAG gene knockdown. Collectively, findings from this study suggest that miR-184, by mediating IAG expression, may be involved in many physiological processes in M. rosenbergii. The current study lays a basic understanding for short-term silencing of MrIAG with miR-184, and facilitates miRNA function analysis in M. rosenbergii in future.

MicroRNA miR-124-3p suppresses proliferation and epithelial–mesenchymal transition of hepatocellular carcinoma via ARRDC1 (arrestin domain containing 1)

ABSTRACT Hepatocellular carcinoma (HCC) is responsible for high morbidity and mortality worldwide. Increasing evidence suggests that microRNAs intensively participate in HCC development and progression. In the current study, we aimed to explore the impact of miR-124-3p in the proliferation and epithelial–mesenchymal transition (EMT) of HCC. The RT-qPCR assay was employed to determine miR-124-3p expression in human HCC specimens and cell lines. Luciferase assay was used to validate the miR-124-3p target gene. Western Blot and RT-qPCR were performed to study the effects of miR-124-3p modulation on ARRDC1 (Arrestin Domain Containing 1) mRNA and protein expressions. MTT assay, wound healing assay, EdU assay, and Transwell assay were utilized to verify the impact of miR-144-3p modulation on HCC proliferation and EMT via ARRDC1. We found that MiR-124-3p expression downregulates in HCC. Overexpression of miR-124-3p reduced the HCC cell proliferation and EMT. Meanwhile, we determined that the expression of ARRDC1 is increased in HCC, and miR-124-3p directly binds the 3ʹUTR of ARRDC1 and inhibits its expression at mRNA and protein level, suggesting that miR-124-3p was capable of negatively modulating ARRDC1. Besides, cotransfection of ARRDC1-overexpression plasmid and miR-124-3p mimics increased the cell proliferation and EMT as compared to miR-124-3p mimics. Our study concluded that miR-124-3p directly binds the 3ʹUTR of ARRDC1 and exerts anti-tumorous effects by inhibiting the HCC proliferation and EMT. Therefore, miR-124-3p/ARRDC1 axis may serve as a novel therapeutic target to inhibit HCC growth and metastasis.

Circular RNA circCHFR downregulation protects against oxidized low-density lipoprotein-induced endothelial injury via regulation of microRNA-15b-5p/growth arrest and DNA damage inducible gamma

ABSTRACT Atherosclerosis is the leading cause of coronary heart disease. In recent years, circ_0029589 (circCHFR) has been found to be associated with atherosclerosis development. However, the molecular mechanism of circCHFR action in atherosclerosis development is unknown. This study was aimed to investigate the function and action mechanism of circCHFR in atherosclerosis development. An atherosclerosis cell model was created by exposing human vascular endothelial cells (HUVECs) to oxidized low-density lipoprotein. The expression of circCHFR, microRNA(miR)-15b-5p, growth arrest and DNA damage inducible gamma (GADD45G), and their associated proteins was evaluated using quantitative reverse transcription-polymerase chain reaction and Western blotting. Additionally, cell viability, apoptosis, and cytokine levels were determined using Cell Counting Kit-8 (CCK8) assay, flow cytometry, and enzyme-linked immunosorbent assay, respectively. circCHFR expression was upregulated in patients with atherosclerosis and oxidized low-density lipoprotein (ox-LDL)-exposed HUVECs, whereas miR-15b-5p expression was downregulated. circCHFR silencing significantly improved viability and reduced apoptosis of HUVECs. In addition, the pro-apoptotic protein Bax and atherosclerosis-associated cytokines (interleukin-1β, interleukin-6, and tumor necrosis factor-α) were significantly downregulated, whereas the anti-apoptotic protein Bcl-2 was upregulated. Further, we discovered that circCHFR serves as a molecular sponge of miR-15b-5p. GADD45G was found to be an important target of miR-15b-5p; miR-15b-5p mimic inhibited GADD45G expression, reduced apoptosis and proinflammatory cytokine secretion, and improved cell survival. However, these effects of miR-15b-5p on (ox-LDL) induced HUVECs were reversed with GADD45G plasmid co-transfection. In conclusion, circCHFR promotes atherosclerosis progression via the miR-15b-5p/GADD45G axis and may be an important target for atherosclerosis treatment.

MiR-211-5p inhibits the proliferation, migration, invasion, and induces apoptosis of human hypertrophic scar fibroblasts by regulating TGFβR2 expression.

BackgroundHypertrophic-scar (HS) is the most common pathological healing phenomenon after trauma, especially after deep burns. We aimed to investigate the expression and role of microRNA-211-5p (miR-211-5p) in HS and explore its underlying mechanism.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-211-5p in 15 cases of HS tissues and normal skin tissues, as well as its expression in human hypertrophic scar fibroblasts (hHSFs) and normal fibroblasts. At the same time, the cell counting kit-8 (CCK-8), scratch test, cell invasion test, and flow cytometry were used to determine cell proliferation, migration, invasion, and apoptosis, respectively. Western blot assay was used to determine the expression of proteins. TargetScan was performed to predict the potential binding sites between miR-211-5p and TGFβR2, which was then verified by western blotting and luciferase reporter gene experiments. Also, co-transfection of plasmids that overexpress miR-211-5p and TGFβR2 were used to observe the reversal effect of miR-211-5p.ResultsThe level of miR-211-5p in HS tissues and hHSFs cells was significantly down-regulated (both P<0.05). The TGFβR2/Smad3 signaling pathway was activated (both P<0.05). Furthermore, the overexpression of miR-211-5p could inhibit the proliferation (P<0.05), migration (P<0.05), and invasion (P<0.05) of hHSFs cells, and induce their apoptosis (P<0.05), and could also regulate the expression of related proteins (all P<0.05). Moreover, the overexpression of miR-211-5p could also inhibit the accumulation of ECM and the activation of the TGF-βR2/Smad3 pathway (all P<0.05), while the opposite effect (all P<0.05) was observed when the level of miR-211-5p was interfered with. Finally, it was confirmed that miR-211-5p could target TGFβR2 (all P<0.05), and when hHSFs cells simultaneously overexpressed miR-211-5p and TGFβR2, the promotion effect of TGFβR2 on cells was reversed by miR-211-5p (all P<0.05).ConclusionsmiR-211-5p can inhibit the activation of the TGF-βR2/Smad3 signaling pathway by targeting TGFβR2, thereby suppressing the proliferation, migration, invasion, and ECM production of hHSFs, and inducing their apoptosis, suggesting that miR-211-5p can become a potential target for the treatment of HS.

Upregulated UCA1 contributes to oxaliplatin resistance of hepatocellular carcinoma through inhibition of miR-138-5p and activation of AKT/mTOR signaling pathway.

Hepatocellular carcinoma (HCC) inevitably developed oxaliplatin (OXA) resistance after long‐term treatment, but the mechanism remains unclear. Here, we found that LncRNA UCA1 was upregulated in most of OXA‐resistant HCC tissues and cells (HepG2/OXA and SMMC‐7721/OXA). Follow‐up analysis and online Kaplan–Meier Plotter revealed that HCC patients with high UCA1 level had a shorter survival compared with those with low expression. Overexpression of UCA1 increased OXA IC50 in HepG2 and SMMC‐7721 cells, whereas knockdown of UCA1 decreased OXA IC50 in resistant counterparts. Moreover, dual luciferase reporter assay showed that co‐transfection of UCA1‐WT plasmid with miR‐138‐5p mimics enhanced fluorescence signals, whereas co‐transfection of UCA1‐Mut plasmid and miR‐138‐5p mimics did not induce any changes. Consistently, UCA1 levels in HepG2/OXA and SMMC‐7721/OXA cells were downregulated after transfected with miR‐138‐5p mimics. UCA1 silencing or transfection of miR‐138‐5p mmics inhibited the activation of AKT and mTOR in HepG2/OXA and SMMC‐7721/OXA cells, whereas UCA1 overexpression increased the phosphorylated AKT and mTOR levels in parental counterparts. Rapamycin or miR‐138‐5p mimics similarly suppressed the activation of AKT and mTOR, whereas UCA1 overexpression exert opposite roles. Interestingly, administration of rapamycin or miR‐138‐5p mimics apparently antagonized the effects of UCA1 on AKT and mTOR activation. Besides, depletion of UCA1 triggered more dramatic regression of HepG2 xenografts than that of HepG2/OXA xenografts with OXA treatment and impaired the p‐AKT and p‐mTOR levels in vivo. In conclusion, our findings provide the evidence that UCA1 may contribute to OXA resistance via miR‐138‐5p‐mediated AK /mTOR activation, suggesting that UCA1 is a potential therapeutic target for HCC.

Application of Taxol Nanomicelles with Lyp-1 Target in Targeted Therapy of Colon Cancer

As a new type of non-viral gene drug carrier, paclitaxel with Lyp-1 target has unique transmembrane ability due to its unique structure. In this paper, amino acids and surfactants are used to disperse SWCNTs in water, and non-covalent interactions are used to adsorb paclitaxel to the surface of SWCNTs. DSPE-PEG-Maleimide is then connected to NGR to achieve active targeting. To investigate the effect of NGR-SWCNTs-Paclitaxel on isolated cells, and to observe the antitumor effect of NGR-SWCNTs-Paclitaxel on S180 colon cancer mice in vivo, we provide theoretical and experimental basis for targeted cancer treatment. The luciferase activity test results showed that mi R-218 mimics had no significant effect on the intensity of the blank reporter plasmid group and p MIR-REPORT/UTR mutant luciferase activity, but in mi R-218 mimics and p MIR-REPORT/UTR Luciferase activity decreased after co-transfection of wild-type plasmids into cells. The validation results of the luciferase activity analysis system showed that mi R-218 was able to bind to Sp13’UTR. Overexpression of mi R-218 can significantly reduce the expression level of Sp1 protein but has no significant effect on Sp1 m RNA level, indicating that mi R-218 can target the regulation of Sp1 expression at the translation level.

MiR-1249-5p regulates the osteogenic differentiation of ADSCs by targeting PDX1

BackgroundOsteoporosis (OP) is an age-related systemic bone disease. MicroRNAs (miRNAs) are involved in the regulation of osteogenic differentiation. The purpose of this study was to explore the role and mechanism of miR-1249-5p for promoting osteogenic differentiation of adipose-derived stem cells (ADSCs).MethodsGSE74209 dataset was retrieved from NCBI Gene Expression Omnibus (GEO) database and performed bioinformatic analyses. OP tissue and healthy control tissues were obtained and used for RT-PCR analyses. ADSCs were incubated with miR-1249-5p mimic, inhibitor and corresponding negative control (NC), alkaline phosphatase (ALP) staining, and Alizarin Red Staining (ARS) were then performed to assess the role of miR-1249-5p for osteogenesis of ADSCs. Targetscan online website and dual-luciferase reporter assay were performed to verify that the 3′-UTR of PDX1 mRNA is a direct target of miR-1249-5p. RT-PCR and western blot were also performed to identify the mechanism of miR-1249-5p for osteogenesis of ADSCs.ResultsA total of 170 differentially expressed miRNAs were selected, among which, 75 miRNAs were downregulated and 95 miRNAs were upregulated. Moreover, miR-1249-5p was decreased in OP patients, while showed a gradual increase with the extension of induction time. miR-1249-5p mimic significantly increased osteogenic differentiation capacity and p-PI3K and p-Akt protein levels. Luciferase activity in ADSCs co-transfected of miR-1249-5p mimic with PDX1-WT reporter plasmids was remarkably decreased, but there was no obvious change in miR-1249-5p mimic with PDX1-MUT reporter plasmids co-transfection group. Overexpression PDX1 could partially reverse the promotion effects of miR-1249-5p on osteogenesis of ADSCs.ConclusionIn conclusion, miR-1249-5p promotes osteogenic differentiation of ADSCs by targeting PDX1 through the PI3K/Akt signaling pathway.

Evidence for the extracellular delivery of influenza NS1 protein

We constructed a reporter influenza A/Puerto Rico/8/1934 virus expressing truncated 124aa N-terminal NS1 protein fused to a luciferase reporter sequence (NanoLuc) without signal peptide. The reproduction activity of the vector correlated well with the luminescent activity in the lysates of infected cell cultures or mouse respiratory organ suspensions. Surprisingly, we found that luciferase enzymatic activity was present not only in the intracellular compartments but also in cell culture supernatants as well as in the sera or bronchiolar lavages of infected mice. This fact allowed us to formulate a working hypothesis about the extracellular delivery mechanism of the NS1 protein. To test this idea, we conducted co-transfection experiments in Vero cells with different combinations of plasmids encoding influenza genomic segments and chimeric NS1-NanoLuc encoding plasmid. We found that the emergence of the luciferase reporter in the extracellular compartment was promoted by the formation of the ribonucleoprotein complex (RNP) from the co-transfection of plasmids expressing PB1, PB2, PA, and NP proteins. Therefore, influenza NS1 protein may be delivered to the extracellular compartment together with the nascent RNP complexes during the maturation of virus particles.

Development of a High Yielding Bioprocess for a Pre-fusion RSV Subunit Vaccine

Respiratory syncytial virus (RSV) is a highly contagious virus causing severe infection in infants and the elderly. Various approaches are being used to develop an effective RSV vaccine. The RSV fusion (F) subunit, particularly the cleaved trimeric pre-fusion F, is one of the most promising vaccine candidates under development. The pre-fusion conformation elicits the majority of neutralizing antibodies during natural infection. However, this pre-fusion conformation is metastable and prone to conversion to a post-fusion conformation, thus hindering the potential of this construct as a vaccine antigen. The Vaccine Research Center (VRC) at the National Institutes of Health (NIH) designed a structurally stabilized pre-fusion F glycoprotein, DS-Cav1, that showed high immunogenicity and induced a neutralizing response in animal studies. To advance this candidate to clinical manufacturing, a production process that maintained product quality (i.e. a cleaved trimer with pre-fusion conformation) and delivered high protein expression levels was required. This report describes the development of the vaccine candidate including vector design and cell culture process development to meet these challenges. Co-transfection of individual plasmids to express DS-Cav1 and furin (for DS-Cav1 cleavage and activation) demonstrated a superior protein product expression and pre-fusion conformation compared to co-expression with a double gene vector. A top clone was selected based on these measurements. Protein expression levels were further increased by seeding density optimization and a biphasic hypothermia temperature downshift. The combined efforts led to a high-yield fed-batch production of approximately 1,500 mg/L (or up to 15,000 doses per liter) at harvest. The process was scaled up and demonstrated to be reproducible at 50 L-scale for toxicity and Phase I clinical trial use. Preliminary phase I data indicate the pre-fusion antigen has a promising efficacy (Crank et al., 2019).

Rapid Development of an Effective Newcastle Disease Virus Vaccine Candidate by Attenuation of a Genotype VII Velogenic Isolate Using a Simple Infectious Cloning System.

Genotype-matched vaccines provide ideal protection against infection caused by new Newcastle disease virus (NDV) genotypes or variants even in the vaccinated chickens. In this study, we report a protocol for attenuation and rapid development of a velogenic NDV isolate as an effective vaccine candidate, using a simple and reliable infectious cloning platform. Based on DHN3, a genotype VII velogenic NDV isolate, recombinant rDHN3 was rescued by co-transfection of plasmids expressing the genomic RNA, NDV proteins NP, P and L, and the T7 polymerase without using a helper virus. Subsequently, an attenuated strain rDHN3-mF was produced by substitution of residues from amino acids 112 to 117 in the DHN3 F protein with the corresponding sequence from the LaSota strain. Both rDHN3 and rDHN3-mF are genetically stable during propagation in cell culture and chicken embryos. Further characterization through determination of EID50, MDT and clinical assessments confirmed that rDHN3 is velogenic and rDHN3-mF lentogenic. Vaccination of one-week-old SPF chicks with inactivated rDHN3-mF produced much higher anti-DHN3 antibody response and better protection against live DHN3 challenge than did the commercial LaSota vaccine, providing 100% protection and much earlier viral clearance. This attenuated NDV isolate would merit further development into a vaccine product.

Effect of circular RNA hsa_circ_0008898 on oral squamous cell carcinoma and its mechanism

Objective: To investigate the effect and molecular mechanism of hsa_circ_0008898 on the cell proliferation, migration, invasion and tumor formation of oral squamous cell carcinoma (OSCC). Methods: Quantitative real-time PCR (qPCR) and Western blotting were used to detect the expression of hsa_circ_0008898, miR-197-5p and ras homolog gene family member A (RHOA) in OSCC tissues, adjacent tissues, OSCC cells and human normal oral keratinocytes (NOK). CAL27 and SCC-25 cells were transfected with si-hsa_circ_0008898#1 (knockdown group 1), si-hsa_circ_0008898#2 (knockdown group 2), hsa_circ_0008898 (circ overexpression group) and blank plasmid (circ blank group), respectively. Then miR-197-5p inhibitor (inhibition group) and blank plasmid (inhibition control group) were transfected into hsa_circ_0008898 knockdown cells (knockdown group 1). CAL27 and SCC-25 cells were transfected with miR-197-5p mimics (miR overexpression group) and blank plasmid (miR blank group), and then transfected hsa_circ_0008898 vector (co-transfection group 1), RHOA vector (co-transfection group 2) and blank plasmid (co-transfection control group) in cells overexpressing miR-197-5p. Cell counting kit 8 (CCK-8), colony formation, Transwell and scratch test were used to detect cell proliferation, cloning ability, cell cycle distribution, cell invasion and migration ability. Ten nude mice were equally divided into two groups, with 5 mice in each group. SCC-25 cells transfected with blank plasmid (control group) and SCC-25 cells transfected with sh-hsa_circ_0008898 (knockout group) were subcutaneously injected into the armpit. The volume and mass of the tumor were measured. Results: The expressions of hsa_circ_0008898 (2.89±0.72) and RHOA (2.62±0.21) in OSCC tissues were significantly higher than those in para-carcinoma tissues (1.00±0.48, 1.00±0.11, respectively), while the expression of miR-197-5p in OSCC tissues （0.46±0.24） was significantly lower than that in para-carcinoma tissues (1.00±0.42) (P<0.05). Compared with NOK, the expression of hsa_circ_0008898 and RHOA in CAL27 and SCC-25 cells increased significantly, while the expression of miR-197-5p decreased (P<0.05). Compared with circ blank group, the cell viability, colony formation, scratch healing rate and invasive cell number of CAL27 and SCC-25 cells in knockdown group 1 and group 2 were significantly decreased, while the proportion of cells in G1 phase was significantly increased (P<0.05). Compared with inhibition control group, the cell viability, colony formation, scratch healing rate and invasive cell number of CAL27 and SCC-25 in inhibition group were significantly increased, while the proportion of cells in G1 phase was significantly decreased in inhibition group (P<0.05). Compared with miR blank group, the cell viability, colony formation, scratch healing rate and invasive cell number of CAL27 and SCC-25 in miR overexpression group were significantly decreased, while the proportion of cells in G1 phase was significantly increased in miR overexpression group (P<0.05). Compared with co-transfection control group, the cell viability, colony formation, migration area and invasive cell number of CAL27 and SCC-25 in co-transfection group2 were significantly increased, while the proportion of cells in G1 phase was significantly decreased in co-transfection group 2 (P<0.05). The volume and mass of transplanted tumor in knockout group ［(660.4±67.8) mm(3 )and (0.60±0.06) g, respectively］ were significantly lower than those in control group ［(1 210.4±198.9) mm(3) and (1.00±0.12) g, respectively］. Conclusions: Knockdown of hsa_circ_0008898 inhibited OSCC cells proliferation, cloning, migration and invasion and induced cell cycle arrest in vitro by regulating the miR-197-5p/RHOA. Additionally, Knockdown of hsa_circ_0008898 also inhibited tumor formation of OSCC cells in vivo.

Hepatitis B virus X gene mutants emerge during antiviral therapy and increase cccDNA levels to compensate for replication suppression.

Hepatitis B virus (HBV) X gene (HBx) mutants can develop during the natural course of chronic HBV infection. However, little is known about whether the emergence of HBx mutants during long-term antiviral therapy is an adaptation of HBV to antiviral stress. This study was to identify HBx mutants that emerged in patients experiencing Lamivudine resistance or suboptimal treatment. Forty-six Lamivudine-resistant patients and 46 patients with suboptimal treatment responses to Entecavir were enrolled in this study. HBx mutants were identified by sequence analysis and their roles in the HBV replication cycle were characterized. We show that deletion/truncation/insertion mutations were only detected in the Lamivudine resistance group, while synonymous mutations were found in both groups. Follow-up analyses revealed that five patients in the Lamivudine group developed hepatocellular carcinoma, while patients in the Entecavir group did not. These mutants were characterized by a significant decrease in transactivation of the pre-S1 promoter, and varying effects on transactivation of the X promoter. Co-transfection of HBx-mutant plasmid and HBV replication-competent clone into HepG2 cells resulted in increased nuclear-to-cytoplamic HBV core antigen, HBV-DNA ratios, and nuclear covalently closed circular DNA (cccDNA). Antiviral drug sensitivity assays revealed that these mutants exhibited a compensatory effect to counteract antiviral drug suppression, resulting in elevated secretory HBV-DNA levels. Our study demonstrates that HBx mutants can emerge during Lamivudine or Entecavir therapy. These mutants exhibit altered transactivation of the HBV pre-S1 and X promoters, leading to increased cccDNA levels to compensate for replication suppression.