GFP Reporter Assay Research Articles

Involvement of Sirt1-FoxO3a-Bnip3 axis and autophagy mediated mitochondrial turnover in according protection to hyperglycemic NRK-52E cells by Berberine

Aberrant accumulation of dysfunctional mitochondria in renal cells during hyperglycemia signifies perturbed autophagy and mitochondrial turnover. This study aims to focus on the underlying mechanism involved in autophagy and mitophagy inducing efficacy of Berberine (isoquinoline alkaloid) in hyperglycemic NRK-52E cells. Berberine mediated protection to hyperglycemic cells prevented alteration in mitochondrial structure and function. Treatment with SRT-1720 (Sirt1 activator) enhanced autophagy, decreased apoptosis, upregulated expression of downstream moieties (FoxO3a and Bnip3) and ameliorated mitochondria related anomalies while nicotinamide (Sirt1 inhibitor) treatment exhibited reversal of the same. GFP reporter assay ascertained enhanced transcriptional activity of FoxO in Berberine-treated hyperglycemic cells, which was found to be correlated to increased expression of downstream protein Bnip3. Knocking down FoxO3a disrupted autophagy and stimulated apoptosis. N-acetyl-L-cysteine pre-treatment confirmed that generation of ROS intervened high glucose induced toxicity in NRK-52E cells. Berberine co-treatment resulted in differential expressions of key proteins involved in autophagy and mitophagy like LC3B, ATGs, Beclin1, Sirt1, Bnip3, FoxO3a and Parkin. Further, enhanced mitophagy in Berberine-treated cells was confirmed by transmission electron microscopy. Thus, our findings give evidence that the protection accorded by Berberine against hyperglycemia in renal proximal tubular cells (NRK-52E) involves instigation of Sirt1-FoxO3a-Bnip3 axis and autophagy mediated mitophagy induction.

Upstream open reading frame-introducing variants in patients with primary familial brain calcification.

More than 50% of patients with primary familial brain calcification (PFBC), a rare neurological disorder, remain genetically unexplained. While some causative genes are yet to be identified, variants in non-coding regions of known genes may represent a source of missed diagnoses. We hypothesized that 5'-Untranslated Region (UTR) variants introducing an AUG codon may initiate mRNA translation and result in a loss of function in some of the PFBC genes. After reannotation of exome sequencing data of 113 unrelated PFBC probands, we identified two upstream AUG-introducing variants in the 5'UTR of PDGFB. One, NM_002608.4:c.-373C>G, segregated with PFBC in the family. It was predicted to create an upstream open reading frame (ORF). The other one, NM_002608.4:c.-318C>T, was found in a simplex case. It was predicted to result in an ORF overlapping the natural ORF with a frameshift. In a GFP reporter assay, both variants were associated with a dramatic decrease in GFP levels, and, after restoring the reading frame with the GFP sequence, the c.-318C>T variant was associated with a strong initiation of translation as measured by western blotting. Overall, we found upstream AUG-introducing variants in the 5'UTR of PDGFB in 2/113 (1.7%) undiagnosed PFBC cases. Such variants thus represent a source of putative pathogenic variants.

Interaction between long noncoding RNA (lnc663) and microRNA (miR1128) regulates PDAT-like gene activity in bread wheat (Triticum aestivum L.)

Amylose, a starch subcomponent, can bind lipids within its helical groove and form an amylose-lipid complex, known as resistant starch type 5 (RS-5). RS contributes to lower glycaemic index of grain with health benefits. Unfortunately, genes involved in lipid biosynthesis in wheat grain remain elusive. Our study aims to characterize the lipid biosynthesis gene and its post-transcriptional regulation using the parent bread wheat variety ‘C 306’ and its EMS-induced mutant line ‘TAC 75’ varying in amylose content. Quantitative analyses of starch-bound lipids showed that ‘TAC 75’ has significantly higher lipid content in grains than ‘C 306’ variety. Furthermore, expression analyses revealed the higher expression of wheat phospholipid: diacylglycerol acyltransferase-like (PDAT-like) in the ‘TAC 75’ compared to the ‘C 306’. Overexpression and ectopic expression of TaPDAT in yeast and tobacco leaf confirmed its ability to accumulate lipids in vivo. Enzyme activity assay showed that TaPDAT catalyzes the triacylglycerol synthesis by acylating 1,2-diacylglycerol. Interestingly, the long non-coding RNA, lnc663, was upregulated with the TaPDAT gene, while the miRNA, miR1128, downregulated in the ‘TAC 75′, indicating a regulatory relationship. The GFP reporter assay confirmed that the lnc663 acts as a positive regulator, and the miR1128 as a negative regulator of the TaPDAT gene, which controls lipid accumulation in wheat grain. Our findings outline TaPDAT-mediated biosynthesis of lipid accumulation and reveal the molecular mechanism of the lnc663 and miR1128 mediated regulation of the TaPDAT gene in wheat grain.

Profiling the Pyrenophora tritici-repentis secretome: The Pf2 transcription factor regulates the secretion of the effector proteins ToxA and ToxB.

The global wheat disease tan spot is caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) which secretes necrotrophic effectors to facilitate host plant colonization. We previously reported a role of the Zn2 Cys6 binuclear cluster transcription factor Pf2 in the regulation of the Ptr effector ToxA. Here, we show that Pf2 is also a positive regulator of ToxB, via targeted deletion of PtrPf2 which resulted in reduced ToxB expression and defects in conidiation and pathogenicity. To further investigate the function of Ptr Pf2 in regulating protein secretion, the secretome profiles of two Δptrpf2 mutants of two Ptr races (races 1 and 5) were evaluated using a SWATH-mass spectrometry (MS) quantitative approach. Analysis of the secretomes of the Δptrpf2 mutants from in vitro culture filtrate identified more than 500 secreted proteins, with 25% unique to each race. Of the identified proteins, less than 6% were significantly differentially regulated by Ptr Pf2. Among the downregulated proteins were ToxA and ToxB, specific to race 1 and race 5 respectively, demonstrating the role of Ptr Pf2 as a positive regulator of both effectors. Significant motif sequences identified in both ToxA and ToxB putative promoter regions were further explored via GFP reporter assays.

Characterizing short germline-specific promoters with a range of expression levels in C. elegans.

A core tenet of synthetic biology is that well-characterized regulatory elements are essential for engineering biological systems. Here, we characterize the specificity and expression levels of 18 short (254 to 880 bp) candidate germline promoters using a single-copy gfp reporter assay in C. elegans . Six promoters resulted in ubiquitous expression, three did not drive detectable expression, and nine were germline-specific. Several promoters drove stronger germline expression than the commonly-used mex-5 promoter. The promoters range across expression levels and facilitate, for example, low expression of toxic transgenes or high expression of gene editing enzymes, and their compactness facilitates gene synthesis.

Expression of the Cyanobacterial FoF1 ATP Synthase Regulator AtpΘ Depends on Small DNA-Binding Proteins and Differential mRNA Stability.

ABSTRACTFoF1 ATP synthases produce ATP, the universal biological energy source. ATP synthase complexes on cyanobacterial thylakoid membranes use proton gradients generated either by photosynthesis or respiration. AtpΘ is an ATP synthase regulator in cyanobacteria which is encoded by the gene atpT. AtpΘ prevents the hydrolysis of ATP (reverse reaction) that otherwise would occur under unfavorable conditions. In the cyanobacterium Synechocystis sp. PCC 6803, AtpΘ is expressed maximum in darkness but at very low levels under optimum phototrophic growth conditions or in the presence of glucose. DNA coimmunoprecipitation experiments followed by mass spectrometry identified the binding of the two transcriptional regulators cyAbrB1 and cyAbrB2 to the promoter and the histone-like protein HU to the 5’UTR of atpT. Analyses of nucleotide substitutions in the promoter and GFP reporter assays identified a functionally relevant sequence motif resembling the HLR1 element bound by the RpaB transcription factor. Electrophoretic mobility shift assays confirmed interaction of cyAbrB1, cyAbrB2, and RpaB with the promoter DNA. However, overall the effect of transcriptional regulation was comparatively low. In contrast, atpT transcript stabilities differed dramatically, half-lives were 1.6 min in the light, 33 min in the dark and substantial changes were observed if glucose or DCMU were added. These findings show that transcriptional control of atpT involves nucleoid-associated DNA-binding proteins, positive regulation through RpaB, while the major effect on the condition-dependent regulation of atpT expression is mediated by controlling mRNA stability, which is related to the cellular redox and energy status.IMPORTANCE FoF1 ATP synthases produce ATP, the universal biological energy source. Under unfavorable conditions, ATP synthases can operate in a futile reverse reaction, pumping protons while ATP is used up. Cyanobacteria perform plant-like photosynthesis, but they cannot use the same mechanism as plant chloroplasts to inhibit ATP synthases during the night because respiratory and photosynthetic complexes are both located in the same membrane system. AtpΘ is a small protein encoded by the gene atpT in cyanobacteria that can prevent the ATP synthase reverse reaction (ATPase activity). Here we found that three transcription factors contribute to the regulation of atpT expression. However, the control of mRNA stability was identified as the major regulatory process governing atpT expression. Thus, it is the interplay between transcriptional and posttranscriptional regulation that position the AtpΘ-based regulatory mechanism within the context of the cellular redox and energy balance.

γ-secretase inhibitor disturbs the morphological development of differentiating neurons through affecting Notch/miR-342-5p

During neurodevelopment, differentiation of neural stem/progenitor cells (NSPCs) into neurons are regulated by many factors including Notch signaling pathway. Herein, we report the effect of a Notch signaling blocker, i.e. γ -secretase inhibitor (GSI), on this differentiating process, especially on the morphological development. NSPCs were cultured and induced to differentiate with or without GSI. The neurite outgrowth was impeded by GSI application and the expression of a Notch signaling downstream effector miR-342-5p increased with the downregulated expression of Notch effectors Hes1 and Hes5. Upregulated expression of miR-342-5p in differentiating NSPCs could shorten the neurite length of progeny neurons, which was similar to the effect of GSI. To avoid the possible influence from astrocytes into neurons, we directly applied cultured neurons, on which GSI could shorten the processes and RBP-J knockdown could also reduce the neurite length. Similarly, transfection of miR-342-5p mimics or inhibitors into PC12 cells led to shorter or longer processes of cells compared with control ones. Furthermore, in differentiating NSPCs, GSI-induced shorter neurites could be partially rescued by miR-342-5p inhibitors, and STAT3 was one of the possible targets of miR-342-5p during this differentiating process as indicated by results of Western Blot test, luciferase reporter assay and GFP reporter assay. To further demonstrate the role of STAT3, it was introduced into GSI-treated neurons and the GSI-affected neurites could also be partially rescued. In conclusion, GSI could influence the morphological development of neurons and the possible mechanism involved Notch/miR-342-5p and STAT3. These results would be informative for future therapeutic research.

Identification of Helicoverpa armigera promoters for biotechnological applications

Helicoverpa armigera and Helicoverpa zea are highly polyphagous major agricultural pests with a global distribution. Their control is based on insecticides, however, new, effective, and environmentally friendly control tools are required to be developed and validated. In an effort to facilitate the development of advanced biotechnological tools in these species that will take advantage of new powerful molecular biology techniques like CRISPR/Cas9, we used available transcriptomic data and literature resources, in order to identify RNA polymerase II and III promoters active in RP-HzGUT-AW1(MG), a midgut derived cell line from Helicoverpa zea. Following functional analysis in insect cell lines, four RNA polymerase II promoters from the genes HaLabial, HaTsp-2A, HaPtx-I and HaCaudal were found to exhibit high transcriptional activity in vitro. The HaTsp-2A promoter did not exhibit any activity in the non-midgut derived cell lines Sf-9 and Hi-5 despite high sequence conservation among Lepidoptera, suggesting that it may function in a gut specific manner. Furthermore, considering the utility of RNA polymerase III U6 promoters in methodologies such as RNAi and CRISPR/Cas9, we identified and evaluated four different U6 promoters of H. armigera. In vitro experiments based on luciferase and GFP reporter assays, as well as in vivo experiments targeting an essential gene of Helicoverpa, indicate that these U6 promoters are functional and can be used to experimentally silence or knockout target genes through the expression of shRNAs and sgRNAs respectively. Taking our findings together, we provide a set of promoters useful for the genetic manipulation of Helicoverpa species, that can be used in various applications in the context of agricultural biotechnology.

High-Dose Irradiation Inhibits Motility and Induces Autophagy in Caenorhabditis elegans

Radiation damages many cellular components and disrupts cellular functions, and was previously reported to impair locomotion in the model organism Caenorhabditis elegans. However, the response to even higher doses is not clear. First, to investigate the effects of high-dose radiation on the locomotion of C. elegans, we investigated the dose range that reduces whole-body locomotion or leads to death. Irradiation was performed in the range of 0–6 kGy. In the crawling analysis, motility decreased after irradiation in a dose-dependent manner. Exposure to 6 kGy of radiation affected crawling on agar immediately and caused the complete loss of motility. Both γ-rays and carbon-ion beams significantly reduced crawling motility at 3 kGy. Next, swimming in buffer was measured as a motility index to assess the response over time after irradiation and motility similarly decreased. However, swimming partially recovered 6 h after irradiation with 3 kGy of γ-rays. To examine the possibility of a recovery mechanism, in situ GFP reporter assay of the autophagy-related gene lgg-1 was performed. The fluorescence intensity was stronger in the anterior half of the body 7 h after irradiation with 3 kGy of γ-rays. GFP::LGG-1 induction was observed in the pharynx, neurons along the body, and the intestine. Furthermore, worms were exposed to region-specific radiation with carbon-ion microbeams and the trajectory of crawling was measured by image processing. Motility was lower after anterior-half body irradiation than after posterior-half body irradiation. This further supported that the anterior half of the body is important in the locomotory response to radiation.

Chemical Screen Identifies Diverse and Novel Histone Deacetylase Inhibitors as Repressors of NUT Function: Implications for NUT Carcinoma Pathogenesis and Treatment.

NUT carcinoma (NC), characterized most commonly by the BRD4-NUTM1 fusion, is a rare, aggressive variant of squamous carcinoma with no effective treatment. BRD4-NUT drives growth and maintains the poorly differentiated state of NC by activating pro-growth genes such as MYC, through the formation of massive, hyperacetylated, superenhancer-like domains termed megadomains. BRD4-NUT-mediated hyperacetylation of chromatin is facilitated by the chromatin-targeting tandem bromodomains of BRD4, combined with NUT, which recruits the histone acetyltransferase, p300. Here, we developed a high-throughput small-molecule screen to identify inhibitors of transcriptional activation by NUT. In this dCAS9-based GFP-reporter assay, the strongest hits were diverse histone deacetylase (HDAC) inhibitors. Two structurally unrelated HDAC inhibitors, panobinostat and the novel compound, IRBM6, both repressed growth and induced differentiation of NC cells in proportion to their inhibition of NUT transcriptional activity. These two compounds repressed transcription of megadomain-associated oncogenic genes, such as MYC and SOX2, while upregulating pro-differentiation, non-megadomain-associated genes, including JUN, FOS, and key cell-cycle regulators, such as CDKN1A. The transcriptional changes correlate with depletion of BRD4-NUT from megadomains, and redistribution of the p300/CBP-associated chromatin acetylation mark, H3K27ac, away from megadomains toward regular enhancer regions previously populated by H3K27ac. In NC xenograft models, we demonstrated that suppression of tumor growth by panobinostat was comparable with that of bromodomain inhibition, and when combined they improved both survival and growth suppression. IMPLICATIONS: The findings provide mechanistic and preclinical rationale for the use of HDAC inhibitors, alone or combined with other agents, in the treatment of NUT carcinoma.

Comprehensive optimization of a reporter assay toolbox for three distinct CRISPR-Cas systems.

The clustered, regularly interspaced, short palindromic repeats‐associated DNA nuclease (CRISPR‐Cas) protein system allows programmable gene editing through inducing double‐strand breaks. Reporter assays for DNA cleavage and DNA repair events play an important role in advancing the CRISPR technology and improving our understanding of the underlying molecular mechanisms. Here, we developed a series of reporter assays to probe mechanisms of action of various editing processes, including nonhomologous DNA end joining, homology‐directed repair and single‐strand annealing. With special target design, the reporter assays as an optimized toolbox can be used to take advantage of three distinct CRISPR‐Cas systems (Streptococcus pyogenes Cas9, Staphylococcus aureus Cas9 and Francisella novicida U112 Cpf1) and two different reporters (GFP and Gaussia luciferase). We further validated the Gaussia reporter assays using a series of small molecules, including NU7441, RI‐1 and Mirin, and showcased the use of a GFP reporter assay as an effective tool for enrichment of cells with edited genome.

VraCP regulates cell wall metabolism and antibiotic resistance in vancomycin-intermediate Staphylococcus aureus strain Mu50.

ObjectivesVancomycin-intermediate Staphylococcus aureus (VISA) is increasingly being reported. Previous studies have shown that vraC and vraP may be involved in vancomycin resistance, although the molecular mechanism remains elusive.MethodsThe vraC (SAV0577), vraP (SAV0578) and vraCP mutants were constructed in Mu50 by allelic replacement. Some common VISA phenotypes were assessed in mutants, such as, susceptibility to the cell wall-associated antibiotics, cell wall thickness, autolysis activity and growth rate. RT-qPCR was performed to reveal the differential genes associated with these phenotypes. The binding abilities of VraC and VraCP to the promoters of target genes were determined by electrophoretic mobility shift assay (EMSA).ResultsVraP forms a stable complex with VraC to preserve their own stability. The vraC, vraP and vraCP mutants exhibited increased susceptibility to the cell wall-associated antibiotics and thinner cell walls compared with the WT strain. Consistent with these phenotypes, RT-qPCR revealed downregulated transcription of glyS, sgtB, ddl and alr2, which are involved in cell wall biosynthesis. Moreover, the transcription of cell wall hydrolysis genes, including sceD, lytM and isaA, was significantly downregulated, supporting the finding that mutants exhibited reduced autolysis rates. EMSA confirmed that both VraC and VraCP can directly bind to the sceD, lytM and isaA promoter regions containing the consensus sequence (5′-TTGTAAN2AN3TGTAA-3′), which is crucial for the binding of VraCP with target genes. GFP-reporter assays further revealed VraC and VraCP can enhance promoter activity of sceD to positively regulate its expression.Conclusions vraCP plays a significant role in cell wall metabolism and antibiotic resistance in Mu50.

Design and evaluation of small interfering RNAs for the treatment of Severe Acute Respiratory Syndrome-Coronavirus-2

SARS-CoV-2 is the virus responsible for the COVID-19 pandemic. As the 2019 coronavirus disease continues to spread, it will be useful to have as many effective treatment options as possible. This research has the potential to create a siRNA treatment that is safe, effective, and practical in design and administration; 192 siRNAs were designed to target conserved regions of the SARS-CoV-2 genome. The first aim of this study is to confirm, via sequence analysis, that these target sites have remained highly conserved over the course of the pandemic. Multiple sequence alignments were generated for the first half of 30,312 full SARS-CoV-2 genomes, which were averaged and compared with the Wuhan-Hu-1 reference genome. Most target sites maintained a very high level of conservation, suggesting that potential repressor siRNAs could be effective in a majority of infected individuals. To evaluate the efficacy of the 192 test siRNAs, we cloned sections of the SARS-CoV-2 RNA genome into GFP fusion genes. Some of these constructs were transfected in different conditions to set up a screening assay based on GFP expression. Preliminary data on the setup of this GFP reporter assay show that the M, N, E, ORF8, and ORF10 constructs produced a good GFP signal, whereas the S, F1, F2 and F3 constructs did not produce a sufficiently strong GFP signal to detect above background. In a preliminary experiment, we evaluated siRNAs targeting the M, N, and E open reading frames and found some to be efficacious. Future directions for this project include generating alignments of the second half of the SARS-CoV-2 genome for a complete sequence conservation estimate, and cell metabolism assays for supplementing visual observations of siRNA toxicity, optimization of GFP readout, and screening of all designed siRNAs.

Human SIRT2 and SIRT3 deacetylases function in DNA homologous recombinational repair.

SIRT2 and SIRT3 protein deacetylases maintain genome integrity and stability. However, their mechanisms for maintaining the genome remain unclear. To examine the roles of SIRT2 and SIRT3 in DSB repair, I-SceI-based GFP reporter assays for HR, single-strand annealing (SSA) and nonhomologous end joining (NHEJ) repair were performed under SIRT2- or SIRT3-depleted conditions. SIRT2 or SIRT3 depletion inhibited HR repair equally to RAD52 depletion, but did not affect SSA and NHEJ repairs. SIRT2 or SIRT3 depletion disturbed the recruitment of RAD51 to DSB sites, an essential step for RAD51-dependent HR repair, but not directly through RAD52 deacetylation. SIRT2 or SIRT3 depletion decreased the colocalization of γH2AX foci with RPA1, and thus, they might be involved in initiating DSB end resection for the recruitment of RAD51 to DSB sites at an early step in HR repair. These results show the novel underlying mechanism of the SIRT2 and SIRT3 functions in HR for genome stability.

Abstract PO-23: Somatic hypermutation is perturbed in ABC-DLBCL lymphoma cell lines expressing high levels of activation-induced deaminase

Abstract Somatic hypermutation (SHM) is a B cell-specific process that supports the affinity maturation of antibodies. The reaction targets the immunoglobulin loci (“on-target" activity) and is initiated by activation-induced cytidine deaminase (AID). AID is known to be able to mutate the genome more broadly (“off-target” activity) and hence was found to be linked to various types of B-cell malignancies. However, the mechanism by which AID is recruited and exerts its function is partially known. To unravel this puzzle, we have analyzed two types of diffuse large B-cell lymphoma (DLBCL) cell lines: germinal B cell like (GCB-DLBCL), in which AID is expressed and introduces mutations, and activated B cell like (ABC-DLBCL), where AID levels are typically increased but paradoxically, SHM is much reduced. To begin to accomplish this goal, we have taken advantage of a Diversification Activator (DIVAC)-GFP reporter assay to measure targeted SHM activity. We first confirmed that AID is expressed in ABC-DLBCL cell lines at levels up to fourfold higher than in GCB-DLBCL cell lines by RT-qPCR. In contrast to the high levels of AID seen in ABC-DLBCL cells, we have observed that SHM is reduced by as much as 10-fold in ABC versus GCB lines using the DIVAC–GFP assay. As this assay should provide a good surrogate for SHM activity at Ig loci, our data support the possibility that the Ig-locus-specific SHM targeting program is defective in ABC-DLBCL. To search for factors that might explain the differential activity of the SHM program in GCB versus ABC cells, we performed RNA-seq using cell lines from each group. Initial analysis revealed that expression of two cell surface markers, CD27 and CD38, is highly correlated with the high mutation phenotype. Flow cytometry analysis for these two markers along with the pan B cell marker CD20 revealed various subpopulations in some DLBCL lines. Isolation, characterization, and measurement of SHM activity using the DIVAC-GFP assay of these subpopulations further strengthened the correlation between CD27/CD28 expression and SHM activity. These analyses also uncovered SHM-permissive subpopulations in ABC lines and SHM-resistant subpopulations in GCB lines. This observation explains residual SHM activity in ABC lines and argues for heterogeneity in the activity of the SHM program in DLBCL cell lines and perhaps within tumor populations in patients. Future studies will include deletion and introduction of specific factors from GCB or ABC lines to validate phenotype and identify regulators of the SHM program. Finally, sequence-analysis will be done to quantify mutations with the broad goal of understanding how AID targeting and mis-targeting are regulated. Citation Format: Huseyin Saribasak, Ravi Dinesh, Lizhen Wu, David G. Schatz. Somatic hypermutation is perturbed in ABC-DLBCL lymphoma cell lines expressing high levels of activation-induced deaminase [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-23.

Sensitive and reliable evaluation of single-cut sgRNAs to restore dystrophin by a GFP-reporter assay.

Most Duchenne muscular dystrophy (DMD) cases are caused by deletions or duplications of one or more exons that disrupt the reading frame of DMD mRNA. Restoring the reading frame allows the production of partially functional dystrophin proteins, and result in less severe symptoms. Antisense oligonucleotide mediated exon skipping has been approved for DMD, but this strategy needs repeated treatment. CRISPR/Cas9 can also restore dystrophin reading frame. Although recent in vivo studies showed the efficacy of the single-cut reframing/exon skipping strategy, methods to find the most efficient single-cut sgRNAs for a specific mutation are lacking. Here we show that the insertion/deletion (INDEL) generating efficiency and the INDEL profiles both contribute to the reading frame restoring efficiency of a single-cut sgRNA, thus assays only examining INDEL frequency are not able to find the best sgRNAs. We therefore developed a GFP-reporter assay to evaluate single-cut reframing efficiency, reporting the combined effects of both aspects. We show that the GFP-reporter assay can reliably predict the performance of sgRNAs in myoblasts. This GFP-reporter assay makes it possible to efficiently and reliably find the most efficient single-cut sgRNA for restoring dystrophin expression.

Protein expression profiling identifies differential modulation of homologous recombination by platinum-based antitumor agents.

Oxaliplatin and satraplatin demonstrate activity against cisplatin-resistant tumor cells. Although the two platinum analogs are structurally-related, oxaliplatin is more active. Therefore, studies focusing on protein expression profiling were undertaken to identify the molecular mechanism for the difference in antitumor activity. We included cisplatin as reference and DAP as a Pt(IV)-prodrug of oxaliplatin to offset Pt(IV) status of satraplatin, and utilized A2780, cisplatin-resistant 2780CP/Cl-16, U2OS, and HCT-116 tumor cells in the investigation. Protein expressions following drug exposures were examined by reverse-phase protein array and ingenuity pathway analysis. Cell cycle was assessed by flow cytometry, cytotoxicity by growth inhibition assay, and homologous recombination (HR) by a GFP reporter assay. Clustering analysis paired oxaliplatin with DAP and, surprisingly, satraplatin with cisplatin. This correlated with differential upregulation of p53/p21 pathway, with S and G2/M arrests by cisplatin and satraplatin in contrast to G1 arrest by oxaliplatin and DAP. Moreover, Rad51 and BRCA1 were severely downregulated by oxaliplatin and DAP, but not cisplatin and satraplatin. As a result, HR was inhibited only by oxaliplatin and DAP and this also contributed to their greater drug activity over cisplatin and satraplatin. Oxaliplatin and DAP robustly activate p53 and p21, which downregulate HR proteins to enhance drug activity. More significantly, since oxaliplatin induces a BRCAness state, it may have potential against BRCA-proficient cancers. Satraplatin, on the other hand, resembled cisplatin in its protein expression profile, which indicates that small changes in chemical structure can substantially alter signal transduction pathways to modulate drug activity.

SUN-718 Reciprocal Regulation of miR-375 and ICER in Pancreatic Beta Cells

MicroRNA-375 (miR-375) is overexpressed in people with type 2 diabetes (T2D) and has been linked to decreased insulin secretion and beta cell proliferation. Investigation into the transcription factor inducible cAMP early repressor (ICER) as an intermediate regulator of miR-375 was proposed because both are regulated by the cAMP pathway. This overexpression of miR-375 in T2D led us to hypothesize that beta cells with elevated and reduced levels of miR-375 will result in decreased and increased glucose-stimulated insulin secretion (GSIS), respectively. Results showed that when miR-375 was overexpressed, GSIS decreased by 61% when compared to a control in 25 mM glucose. Results showed that when miR-375 was inhibited, GSIS increased 6% when compared to a control in 25 mM glucose. In human islets, we found that inhibiting miR-375 led to an average 19% increase in GSIS, though due to the variability of human tissue these data were not significant (N=5). To investigate ICER’s binding affinity to the miR-375 promoter, a luciferase reporter assay was conducted. HEK-293 (human embryonic kidney) cells that were transfected with a luciferase reporter plasmid containing a cAMP recognition element (CRE) and a plasmid driving the overexpression of ICER had a 75% decrease when compared to our control (P<0.05). When transiently-expressed ICER was knocked down via siRNA, promoter activity increased by 13.1-fold (P< 0.05). Using a chromatin immunoprecipitation assay we found that an ICER antibody pulled down the rat miR-375 promoter an average of 13-fold compared with a control antibody (N=2). Additionally, because of a sequence alignment showing possible binding of miR-375 to the human ICER transcript we hypothesize that the two are in a negative feedback loop and can regulate each other’s expression. To investigate the double negative feedback loop a plasmid was constructed containing the GFP reporter gene and either the human or rodent ICER 3’UTR predicted miR-375 binding site. The GFP reporter assay was conducted to determine if miR-375 binds to ICER’s microRNA recognition element (MRE) in a species-specific way. In our GFP reporter experiment, data shows there is a 50% reporter gene decrease between our negative control and the human ICER MRE (N=4, P=0.017). Understanding microRNA gene regulation in the pancreas may have important implications for patients with T2D. MiR-375 may have the ability to interact with human ICER in a double negative feedback loop in the cAMP second messenger pathway, which will further clarify cellular mechanisms to potentially improve T2D drugs.

BRD4 inhibition exerts anti-viral activity through DNA damage-dependent innate immune responses.

Chromatin dynamics regulated by epigenetic modification is crucial in genome stability and gene expression. Various epigenetic mechanisms have been identified in the pathogenesis of human diseases. Here, we examined the effects of ten epigenetic agents on pseudorabies virus (PRV) infection by using GFP-reporter assays. Inhibitors of bromodomain protein 4 (BRD4), which receives much more attention in cancer than viral infection, was found to exhibit substantial anti-viral activity against PRV as well as a range of DNA and RNA viruses. We further demonstrated that BRD4 inhibition boosted a robust innate immune response. BRD4 inhibition also de-compacted chromatin structure and induced the DNA damage response, thereby triggering the activation of cGAS-mediated innate immunity and increasing host resistance to viral infection both in vitro and in vivo. Mechanistically, the inhibitory effect of BRD4 inhibition on viral infection was mainly attributed to the attenuation of viral attachment. Our findings reveal a unique mechanism through which BRD4 inhibition restrains viral infection and points to its potent therapeutic value for viral infectious diseases.

Activity of Cinnamaldehyde on Quorum Sensing and Biofilm Susceptibility to Antibiotics in Pseudomonas aeruginosa.

Quorum sensing (QS) plays an important role during infection for the opportunistic human pathogen Pseudomonas aeruginosa. Quorum sensing inhibition (QSI) can disrupt this initial event of infection without killing bacterial cells, and thus QS inhibitors have been suggested as novel approaches for anti-infective therapy. Cinnamaldehyde (CAD) is a P. aeruginosa biofilm inhibitor and disperser of preformed biofilms. In this study, the combined use of CAD and colistin (COL) revealed a synergistic activity, but this was not the case for CAD combined with carbenicillin, tobramycin (TOB), or erythromycin in checkerboard assays for P. aeruginosa. CAD demonstrated QSI activity by repression of the expression of lasB, rhlA and pqsA in GFP reporter assays. Approximately 70% reduction in GFP production was observed with the highest CAD concentration tested in all the QS reporter strains. TOB also showed strong QSI when combined with CAD in reporter assays. Combination treatments revealed an additive activity of CAD with COL and TOB in biofilm inhibition (75.2% and 83.9%, respectively) and preformed biofilm dispersion (~90% for both) when compared to the individual treatments. Therefore, a proposed method to mitigate P. aeruginosa infection is a combination therapy of CAD with COL or CAD with TOB as alternatives to current individual drug therapies.