The PMN cell reporter system coupled with whole transcriptome readout allowed identification of a severity signature for sepsis that was highly accurate in two inde- pendent datasets. Current guidelines for diagnosing sep- sis and grading severity are based on multiple clinical parameters, which can be inaccurate and do not predict prognosis well . Although many potential biomarkers including cytokines, coagulation factors, and several others have been investigated for sepsis diagnosis and prognosis, none have proved reliable enough to enter routine clinical practice and so there is a need for better markers for risk stratification of septic patients to guide treatment and prognosis [17-19]. Elucidating the bio- logic mechanisms that differ among sepsis patients will help advance this field [12,20]. The promising perform- ance of the PMN transcriptomic reporter assay pre- sented here to stratify patients with sepsis by severity offers a novel and attractive platform for the develop- ment of biomarker signatures in sepsis. Similarly, the ability to select a gene panel that was specific to stimula- tion with plasma from severe sepsis patients shows the utility of this method to better understand the immuno- pathogenesis of sepsis.
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expression of E protein (Fig. 4C) and VLP production in the supernatant (Fig. 4D) were conﬁrmed, the prM-E region was subcloned into the lentivirus vector, generating pLenti-prM-E (Fig. 4E). Packaged lentiviral particles were used to transduce 293T cells and to generate a bulk-selected cell line and single clones (Fig. 4E). As described above for the C-prM-E cell lines, 6 single-cell clones were selected and characterized for ZIKV E protein expression (data not shown). Clones C4 and F4 showed the highest expres- sion levels by both immunoﬂuorescence assay (Fig. 4F) and ﬂow cytometry (Fig. 4G). Finally, we characterized the single-cell clones for production of VLPs in the superna- tants. For this purpose, supernatants from the 293T-prM-E cell lines were ultracentri- fuged through a glycerol cushion (49), the virus pellet was lysed, and the E protein was detected by Western blotting (Fig. 4H). Interestingly, the 293T-prM-E-F4 cell line showed signiﬁcantly more VLP production than the 293T-prM-E-Bulk and 293T-prM- E-C4 cell lines. Thus, we have generated cell lines that constitutively produce high levels of ZIKV prM-E VLPs in the supernatant and can be used for VLP-based vaccine studies. Production of VLPs from our stable cell lines can readily be scaled up for clinical trials. Immunization studies in mice. The lack of an approved vaccine for ZIKV amid the recent outbreaks and the association of ZIKV infections with severe congenital birth defects warrant development of a safe and efﬁcacious vaccine against the virus. For a vaccine to be available worldwide, especially in underdeveloped countries, it should be both easy to prepare and cost-effective. In this regard, stable cell lines constitutively producing ZIKV VLPs would be optimal, as they can readily be scaled up, with minimal scientiﬁc infrastructure, and can provide an economical alternative to other forms of vaccination. We hence tested the immunogenicity of the ZIKV VLPs in mice. Although prM-E VLPs
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children with MPP. The luciferase reporter assay were performed to confirm B7-H3 is the direct target of miR-29c. The gradual regulation in axis of miR-29c/ B7-H3/Th17 is confirmed in vitro, and the expression of miR-29c, sB7-H3, IL-17 during the acute and convales- cent phase is consistent with the result in vitro. The as- sociation between sB7-H3 and lactate dehydrogenase (LDH) was not statistically significant, but there was re- lated trends. LDH has been confirmed to be associated with the severity of MPP, and LDH is an indicated marker for steroid therapy for MPP [23, 24]. However, there was no association between miR-29c and sB7-H3 in children with MPP, it is probably because there are other miRNAs to regulate B7-H3 expression except for miR-29c [21 – 26]. It is interesting that the serum level of miR-29c expression was negatively correlated with the M. pneumoniae specific IgG and IgM level during the
Results: Recurrent LGG and glioblastoma (GBM) showed different transcriptome charac- teristics with less overlap of differentially expressed protein-coding genes (DEPs), lncRNAs (DELs) and miRNAs (DEMs) compared with primary samples. There were no overlapping gene in ontology (GO) terms related to GBM recurrence in the TCGA and CGGA databases, but there were overlaps associated with LGG recurrence. GO analysis and protein – protein interaction (PPI) network analysis identi ﬁ ed three core genes: TIMP1, COL1A1 and COL6A2. By hierarchical cluster analysis of them, LGGs could be clustered as Low_risk and High_risk group. The High_risk group with high expression of TIMP1, COL1A1, and COL6A2 showed worse prognosis. By coexpression networks analysis, competing endogen- ous RNA (ceRNA) network analysis, cell proliferation assay and luciferase reporter assay, we con ﬁ rmed that lncRNA HOXA-AS2 functioned as a ceRNA for miR-184 to regulate expression of COL6A2, which induced cell proliferation of low-grade glioma.
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Case presentation: A 50-year-old woman and her 27-year-old daughter were followed up because of hypoparathyroidism. They had bilateral sensorineural deafness. Abdominal computed tomography scanning revealed renal dysplasia in the mother, but no renal anomaly in the daughter. Direct sequencing of GATA3 gene revealed a novel heterozygous missense mutation at codon 299 (p.R299Q) in exon 4. This mutation is located at the junction between the 2 zinc fingers. The structure prediction showed that it caused a conformation change in this junction area, affecting the spatial position of the zinc fingers. Additionally, a more marked conformation change was observed in the N-terminal zinc finger region compared to that in the C-terminal region. Functional analysis of this mutant protein using an in vitro luciferase reporter assay system confirmed that the mutation abolished the enhancing effects of wild-type GATA3 on the promoter activity of the consensus GATA responsive element and that of human PTH gene.
Previous studies have reported that many microRNAs could bind to sites located in the 3 ′ -UTR region of target genes but only a few miRNAs could bind to the 5 ′ -UTR or CDS of target genes [33–35]. miRNA regulates gene expression at the post-transcriptional and translational levels [36, 37]. A previous study, conducted using Bom- byx mori, demonstrated that let-7 plays a fundamental role in molting in arthropods . Our results indicate that let-7 sequences are conserved among ticks. For this reason, the complete AamECR gene sequence and the partial HaaECR-A2 gene sequence were selected for analysis of the binding sites using RNAhybrid software, and our results indicated that these sequences have the same binding sites in the 3 ′ -UTR, suggesting that the let-7 binding sites in HaaECR are located in the 3 ′ -UTR. For this reason, the 3 ′ -UTR of HaaECR was used in the dual luciferase reporter assay. However, because the bio- informatics analysis indicated that the multiple cloning site (MCS) of the pmirGLO reporter vector interacted with let-7, the sequence that did not interact with let-7 was cloned into the pmirGLO reporter vector to create the recombinant plasmid designated RP-NC. The result of the dual luciferase reporter assay showed that the
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Abstract: Osteosarcoma is a primary bone malignancy, and it usually occurs in children and adolescents under 20 years old. Osteosarcoma is highly malignant, and the tumor cells will migrate to brain, lung, kidney, and other tis- sues. The mechanisms of osteosarcoma tumor genesis have not yet been fully studied. Some groups reported ac- tivation of Kras and abnormal expression of several miRNAs during osteosarcoma tumor progression. In this study, we focused on the regulatory relationship between miR-143 and Kras and its effects on osteosarcoma cell prolifera- tion. Dual-luciferase reporter assay was performed to determine the regulatory relationship between miR-143 and Kras using wild type or mutated Kras 3’UTR inserted reporter vectors; Kras expression was detected by RT-qPCR and western blot after miR-143 mimic or miR-143 inhibitor transfection; CCK8 assay was used to check the role of miR-143 on osteosarcoma cell proliferation under different miR-143 expression levels. Dual-luciferase reporter assay showed that miR-143 targets the 3’UTR of Kras. High expression of miR-143 decreases Kras protein to 63%; while inhibited miR-143 expression elevates Kras protein to 1.67 fold. CCK8 assay revealed that increased miR-143 expression inhibits osteosarcoma cell proliferation. Kras is a target of miR-143; miR-143 inhibits osteosarcoma cell proliferation by repression of Kras.
Plasmids were transfected into an HEK293 or SKNSH cell line in 24-well plates using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufac- turer’s protocol manufacturer. At 30 hours after trans- fection, cells were lysed and the luciferase activities were measured and normalized against Renilla luciferase using the Dual-Luciferase Reporter Assay System (Pro- mega, Madison, WI, USA). Transfection of each plasmid construct was conducted in quadruplicate in each re- porter gene experiment, and the reporter gene experi- ment was repeated three times. The ratio of firefly luciferase to Renilla luciferase was used to represent the normalized reporter gene activity of each construct. Comparison of reporter gene activity among different expression constructs was conducted using one-way analysis of variance (ANOVA) with post-hoc Tukey test. P < 0.05 was considered statistically significant.
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closely related ORF2p molecule C-terminal sequences (hu- man, mouse, and rat) to identify potential individual amino acids that could be important to retrotransposition. We rea- soned that these two approaches in parallel could aid in advancing our understanding of this under-studied area of the ORF2p. To investigate the importance of the C-terminal ORF2p amino acid sequence to retrotransposition, we used the Alu retrotransposition reporter assay in conjunction with truncated ORF2, and ORF2 with mutations in conserved areas of the extreme C terminus of the molecule. We chose to assess Alu mobilization because it relies on the function of the ORF2p and does not require ORF1p, simplifying the ex- perimental design and interpretation of results. Our ﬁndings demonstrate that the unannotated C-terminal portion of the ORF2p is important to Alu retrotransposition, with the excep- tion of the last 13 amino acids. Additionally, we identiﬁed a Y residue (Y1180) that is critical to Alu retrotransposition driven by either the ORF2p alone or the full-length L1. In contrast to Alu retrotransposition, a Y1180A mutation improved L1 mo- bilization in HeLa cells. The data presented here demonstrate that the Y1180 residue is not required for cDNA synthesis by the ORF2p molecule RT, but may be involved via an unknown mechanism in the coordinated function of ORF1p and ORF2p during retrotransposition.
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The expression of P-glycoprotein, cyclin D1, p21 and p27 was detected in the gastric cancer cells using real-time PCR (Figure 3) and western blot (Figure 4). Down-regulation of miR-27a could significantly decrease the expression of P- glycoprotein and cyclin D1, and up-regulate the expression of p21. To evaluate whether cyclin D1 was a genuine target of miR-27a, luciferase reporter assay was performed. As shown in Figure 5, co-transfection of increasing amounts of antagomirs of miR-27a with cyclin D1 reporter gene led to significantly decrease in cyclin D1 promoter activity, suggesting that miR-27a might target cyclin D1.
PARI, an element of the homologous recombination pathway of DNA repair,is involved in the regulation of cell cycle and carcinogenesis in pancreatic cancer. However, little is known about the function and regulatory mechanism of PARI in other cancers. In the present study, we evaluated the expression of PARI in gastric cancer (GC) by immunohistochemical analysis in a tissue microarray and characterized its functions using in vitro assays and in vivo animal models. We found higher expression of PARI protein was shown in GC tissues compared with related adjacent normal gastric mucosa tissues. Knockdown of PARI by RNA inference decreased cell proliferation, migration, and invasion of GC cells in vitro, as well as reduced the xenograft tumor growth and lung metastasis formation in vivo. Quantitative real-time PCR and western blot results revealed that PARI expression was activated by a well-known oncogene FOXM1 and positively correlated with FOXM1 expression at mRNA level in 38 paired of GC samples. Luciferase reporter assay and chromatin immunoprecipitation assay further demonstrated that FOXM1 directly regulated PARI transcription by binding to the specific sequences of PARI promoter. In addition, PARI knockdown blocked the effect of FOXM1 on GC cell migration. Taken together, our results suggest that PARI plays potential oncogenic roles and functions as a transcriptional target and effector of FOXM1 in GC development.
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Additional file 1: Figure S1. Data unavailability is a persistent feature of the MAVE literature. We compiled a list of 159 publications that contained at least one new deep mutational scanning or massively parallel reporter assay dataset and manually inspected the publication ’ s supplementary data and methods to determine whether counts or scores for the assayed variants were present. Refer to https://github.com/ VariantEffect/MaveReferences for the full table. This figure was generated from release v0.1.1. Of the 159 total publications, 91 (57%) provide scores or counts. Figure S2. UML (Unified Markup Language) diagram of the complete MaveDB schema in PDF format. The diagram was generated using the Django Extensions package and visualized using Graphviz.
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The role of PA in host adaptation is not as well character- ized as the role of PB2. However, a recent study indicates the PA mutation T97I increases polymerase activity of an avirulent avian virus in a reporter gene assay, as well as virulence in a mouse model (28). In our study, we have shown that multiple residues, especially 85I, 186S, and 336M in Cal PA, contribute to the enhancement of avian polymerase activity in mammalian cells, which is essential for mammalian host adaptation. The addition of each mutation to Nan PA significantly increased Nan polymerase activity (Fig. 3). However, individual muta- tions in Cal PA at positions 85, 186, or 336 to Nan residues only slightly decreased Cal polymerase activity, supporting the idea that multiple residues in Cal PA contribute to enhanced Cal polymerase activity in mammalian cells. PA possesses the en- donuclease activity required for the “cap-snatching” mecha- nism (4, 35), and the crystal structures of two domains of PA have been determined (4, 11, 24, 35). The amino acids that increased polymerase activity, 85I, 186S, and 336M, are all surface exposed on their domains. 85I and 186S are located in the domain containing the endonuclease active site. While 186S is located on the opposite side of the structure from the endonuclease active site, it is part of a proposed bipartite nuclear localization signal, residues 124 to 139 and 186 to 247 (23, 35). The residue 85I is located on the surface adjacent to the endonuclease site. The branched, hydrophobic isoleucine
miRNAs are a type of short, non-coding RNA that post-transcriptionally regulate gene expression. miRNAs function by guiding the RNA-induced silencing complex (RISC) to target mRNAs, dictated by base pairing to tar- get sites primarily in their 3′-untranslated regions (UTRs). miRNAs can downregulate the expression of several to hundreds of target genes by repressing transla- tion and/or destabilizing target mRNAs [8,9]. miRNAs are required for normal skeletal muscle development in mice , and several miRNAs have been shown to be dynamically regulated during hypertrophy , acute ex- ercise , regeneration after injury , and in the re- modeling that occurs in response to genetic muscle disease [14-17]. In particular, miR-206 is expressed spe- cifically in skeletal muscle, and is highly expressed in re- generating fibers . miR-206 has been shown to promote terminal differentiation of myoblasts by regulat- ing the expression of genes including connexin43 , utrophin , pax3 , pax7  and DNA polymerase α . Many of these studies rely on data obtained from transgenic mouse studies, and ex vivo sample analysis - both currently invaluable to the study of miRNAs. The study of miRNA regulation in skeletal muscle, however, would benefit from a system that enabled rapid, repro- ducible, longitudinal in vivo reporter assays, at a fraction of the cost of transgenic mouse production and analysis, and with fewer animals needed than for ex vivo studies.
anti-HCV status or modify the sensitivity to anti-HCV agents. Thus, the robust replication of this reporter replicon may be essential to detect the anti-HCV effect with higher sensitivity and accuracy. Second, in this reporter system, we did not use the established replicon-hosting cells. Instead, we used the transient-transfection method. Replicon-hosting cells were se- lected to be sufficiently permissive for replicon replication (2). These cells, known as permissive cells, were expected to have disruptions in their antiviral systems, such as IFN signaling pathways. Thus, the study of established replicon-hosting cells to detect antiviral activities may lead to false conclusions. Be- sides, cell-derived ribavirin resistance was identified in Huh7 cells recently (7). Long-term cultivation with ribavirin may select the cells with these characteristics. To overcome this disadvantage, we used the transient-transfection assay for the reporter replicon system and used normal Huh7 cells. Third, we used a luciferase assay to quantify the reporter replicon replication in this study. Some of the previous data regarding anti-HCV effects with replicon systems were determined by colony-forming efficiency or replicon titer that was quantified by real-time detection PCR. Drawbacks of the assay for co- lony-forming efficiency are that it is affected by the condition of transfected cells and it consumes a lot of time. A disadvantage of real-time detection PCR is that it might be affected by degraded RNA fragments. We introduced the luciferase gene (instead of a neomycin resistance gene) into the replicon con- struct (Fig. 1); this allowed us to estimate the replicon RNA replication by measuring the luciferase activity. This reorga- nized replicon construct not only improved the accuracy and sensitivity of this system but also made replicon replication easy to measure. Finally, in this study, the luciferase activity data were adjusted by the luciferase activity 4 h after transfec- tion and by the viable cell count (determined by MTS assay). Thus, we obtained more accurate and reliable data by avoiding the variations caused by transfection efficiency and cell seeding or the cytotoxic effects of anti-HCV agents and could detect the intracellular anti-HCV effects of IFN and ribavirin.
Infection control methods. Running a sample through the flow cytometer may create an aerosol. Therefore, the flow cytom- eter was kept under a biosafety hood in a BSL3 facility. Preliminary experiments with nonpathogenic mycobacteria revealed no con- tamination outside the biosafety hood using this approach. All FIG 3 Flow cytometry (FACS) analysis of 3 representative clinical sputum samples. Flow cytometry plots showing side scatter (SSC) (y axis) plotted against green fluorescence protein (Log GFP) (x axis). Columns represent sputum from 3 patients. Column 1 is sputum from a patient who did not have TB (true negative). Column 2 is sputum from a patient with drug-susceptible TB (drug-susceptible TB). Column 3 is sputum from a patient with multidrug-resistant TB (MDR-TB). Row A, sputum alone; Row B, sputum ⫹ reporter phage; Row C, sputum ⫹ reporter phage ⫹ RIF. FACS gate is the red dashed line. For the true negative, there is no increase in gated events after inoculation with reporter phage (1B) because there is no viable M. tuberculosis in sputum. For the drug-susceptible TB patient, fluorescent events increase after the addition of reporter phage (2B) but decrease with RIF (2C) indicating RIF-susceptibility. For the MDR-TB patient, there is an increase in gated events after reporter phage (3B) and no substantial decrease after RIF (3C) indicating RIF resistance.
Establishment of a new reporter plasmid containing the mutant form of the GRE within the U3 region of the BLV-LTR Previously, we established a reporter cell line, CC81-BLU3G, and used it to measure the cell-to-cell infectivity of BLV-in- fected cells; the cell line harbors a reporter plasmid, pBLU3-EGFP, which contains the BLV-LTR U3 region linked to the EGFP gene . To develop more sensitive as- says for BLV infectivity, we tried to establish more sensitive fluorescence-based reporter cell lines. A previous report shows that the GRE in the LTR U3 region is involved in translational activity independent of tax activation . We considered that reporter cells containing a reporter plasmid harboring a GRE-mutated LTR-U3 promoter will be more sensitive due to reduced background fluorescence. Hence, we constructed a new reporter plasmid (pBLU3 GREM -EGFP)
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Since the β isoform was expressed almost exclusively in the brain, the promoter activity of the β CaM kinase II gene was also examined. The structure of the 5’ flanking region of the β CaM kinase II gene and one of the luciferase-reporters (pGL3.2) is shown in Fig. 1D. Fragments possessing a common 3’ end (+78 bp) containing sequences of 3.2, 0.57, 0.32, 0.22, 0.12, 0.06, and 0.03 kbp 5’ upstream from the transcription initiation site were cloned upstream of the luciferase reporter gene in a promoterless pGL2-basic vector to make respective pGL constructs. The luciferase activity of these reporters was analyzed in neuronal cells, NG108-15,
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B16F10 cells were seeded at a density of 5 × 10 4 cells per well in 6-well culture plates and incubated for 24 h. They were first treated with GAE for 2 h and then stim- ulated with 50 nM α-MSH for an additional 72 h. HEMa-DP cells were treated with GAE (10 and 20 μg/ mL) and incubated for 5 days. The treated cells were washed twice using PBS and lysed with lysis buffer. Cell lysates were clarified by centrifugation at 5,000 rpm for 15 min at 4 °C. Enzyme activity was normalized to pro- tein concentration, as determined by the Bradford assay. The cellular Tyrosinase and L-DOPA solution (0.1 M in sodium phosphate buffer) reaction were performed at 37 °C for 1 h. The absorbance at 450 nm was measured using the aforementioned spectrophotometer to moni- tor the production of dopachrome, corrected for auto-oxidation of L-DOPA.
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The purified PCR fragments were employed in equimolar concentrations for a ligation PCR reaction using the primer overhangs (underlined in the primer sequences) for hybridization and the primers mOCPs and CMVas for subsequent amplification. The generated element was cloned into the BglII/HindIII digested pCDNA3 vector. The mOCP fragment from the first mOCPs1/mOCPas reaction was cloned into the BglII/HindIII digested pcDNA3 vector. The reporter genes EYFPHis and MetLuc were then ligated into the multiple cloning site of the intermediate pcDNA3-CMVE/mOCP plasmid to produce the reporter plasmids pCMVE/mOCP-EYFPHis and pCMVE/mOCP-MetLuc (Figure 1, A, B). The unenhanced control reporter plasmids pmOCP-EYFPHis (Figure 1, C) and pmOCP-MetLuc (Figure 1, D) were produced by ligation of EYFPHis or MetLuc into the multiple cloning site of the mOCP containing pCDNA3 intermediate plasmid. The EYFPHis fragment was created by HindIII/EcoRI digestion of the pEYFP (Clontech, Palo Alto, CA, USA) derivate pEYFPHis. The MetLuc fragment was generated by HindIII/XbaI or HindIII/NotI restriction digest of the pMetLuc reporter plasmid purchased from Clontech. All designed plasmids were verified by control restriction digests and sequenced (data not shown).
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