Transcription Assays Research Articles

Accumulation of Water-Soluble Polysaccharides during Lychee Pulp Fermentation with Lactiplantibacillus plantarum Involves Endoglucanase Expression.

In the current work, lychee pulp was subjected to Lactiplantibacillus plantarum ATCC 14917 fermentation, leading to a substantial increase (2.32-2.67-fold) in water-soluble polysaccharides (WSP). Concurrently, a significant degradation occurred in water-insoluble polysaccharides (WISP) composed of glucose (28.73%), arabinose (28.25%), galacturonic acid (25.07%), and galactose (11.00%). To clarify polysaccharide conversion and its relevant mechanism, carbohydrate-active enzyme encoding sequences in the L. plantarum ATCC 14917 genome and structural alterations of two polysaccharide fractions were dissected. By integrating the transcriptional assay, prokaryotic expression, and enzymic hydrolysis, three endoglucanases were demonstrated to catalyze WISP degradation, leading to WSP accumulation during lychee pulp fermentation with L. plantarum ATCC 14917. Reductions in proportions of galactose and galacturonic acid in WSPs were partly attributed to the actions of multiple galactosidases. These findings provide an enzyme-based explanation for WSP accumulation during Lactobacillus fermentation and serve as a practical foundation for directional polysaccharide conversion.

AaMYB61-like and AabHLH137 jointly regulate anthocyanin biosynthesis in Actinidia arguta

BackgroundRed Actinidia arguta has recently become highly popular because of its red appearance resulting from anthocyanin accumulation, and has gradually become an important breeding direction. However, regulators involved in anthocyanin biosynthesis have not been fully characterized in A. arguta.ResultsHere, we demonstrated that a key R2R3-MYB transcription factor, AaMYB61-like, plays a crucial role in A. arguta anthocyanin biosynthesis. The RT-qPCR results revealed that transient overexpression of AaMYB61-like in A. arguta fruit at 90–100 DAFB significantly promoted anthocyanin biosynthesis, as did the gene expression levels of AaCHS, AaCHI, AaF3H, AaLDOX, and AaF3GT, whereas the result of VIGS revealed the opposite results in A. arguta fruit at 105–115 DAFB. A transcriptional activation assay indicated that AaMYB61-like exhibited transcriptional activation activity. Y1H and LUC assays revealed that AaMYB61-like activates the promoters of AaCHS, AaLDOX, and AaF3GT. In addition, AabHLH137 was found to be related to fruit color from the transcriptome data. We demonstrated that AaMYB61-like promotes anthocyanin biosynthesis by interacting with AabHLH137 via Y2H, BiFC, and Agrobacterium-mediated co-transformation.ConclusionsOur study not only reveals the functions of AaMYB61-like and AabHLH137 in anthocyanin regulation, but also broadly enriches color regulation theory, establishing a foundation for clarifying the molecular mechanism of fruit coloration in kiwifruit.

The obesogenic effects of Bisphenol A and its analogues are differentially regulated via PPARγ transactivation in mouse 3T3-L1 cells.

Exposure to environmental pollutants with obesogenic activity is being recognised as one of the contributing factors to the obesity epidemic. Bisphenol A (BPA) has been shown to stimulate adipogenesis in both human and mouse preadipocytes, to increase body weight and affect lipid metabolism in animal and epidemiological studies. Regulatory action and public concern has prompted industry to replace BPA with other structurally similar analogues that may have similar effects. In this study we investigated the effects of fifteen BPA analogues on adipogenesis in the mouse 3T3-L1 pre-adipocyte cell model in order to determine their adipogenic activity relative to BPA. 3T3-L1 cells were treated with increasing concentrations of BPA and replacements and mRNA expression of the mature adipocyte markers fatty acid binding protein 4 (Fabp4), perilipin (Plin) lipoprotein lipase (Lpl)and peroxisome proliferator-activated receptor (Ppar)γ and lipid accumulation were assessed. In addition, a luciferase reporter assay for PPARγ transactivation was employed to investigate mechanism of action. Our results show that BPC, BPS-MAE, BPS-MPE and TGSA, were the most adipogenic bisphenols, as shown by a robust increase in lipid accumulation and mRNA expression of adipogenic markers. BPS-MPE, BPC, BTUM, TGSA and D8 increased PPARγ transcriptional activity. Despite its ability to activate PPARγ in the transcriptional assay D8 did not affect adipogenesis in this cell model.

Real-time visualization of reconstituted transcription reveals RNA polymerase II activation mechanisms at single promoters.

RNA polymerase II (RNAPII) is regulated by sequence-specific transcription factors (TFs) and the pre-initiation complex (PIC): TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, Mediator. TFs and Mediator contain intrinsically-disordered regions (IDRs) and form phase-separated condensates, but how IDRs control RNAPII function remains poorly understood. Using purified PIC factors, we developed a Real-time In-vitro Fluorescence Transcription assay (RIFT) for second-by-second visualization of RNAPII transcription at hundreds of promoters simultaneously. We show rapid RNAPII activation is IDR-dependent, without condensate formation. For example, the MED1-IDR can functionally replace a native TF, activating RNAPII with similar (not identical) kinetics; however, MED1-IDR squelches transcription as a condensate, but activates as a single-protein. TFs and Mediator cooperatively activate RNAPII bursting and re-initiation and surprisingly, Mediator can drive TF-promoter recruitment, without TF-DNA binding. Collectively, RIFT addressed questions largely intractable with cell-based methods, yielding mechanistic insights about IDRs, condensates, enhancer-promoter communication, and RNAPII bursting that complement live-cell imaging data.

Melatonin induces resistance against Colletotrichum gloeosporioides in mango fruit via regulation of defense-related genes by MiWRKY45 transcription factor.

Anthracnose caused by Colletotrichum gloeosporioides is a major disease leading to postharvest loss of mango fruit. Melatonin (MT) is a natural bioactive molecule that has multiple physiological functions in plants. This study investigated the effect of exogenous MT on mango disease resistance against C. gloeosporioides and related molecular mechanism. MT treatment at 1 mmol L-1 limited the expansion of anthracnose in mango inoculated with C. gloeosporioides, which was associated with increased level of defense-related indexes, including activities of phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL) and peroxidase (POD), expression of MiPAL, Mi4CL and MiPOD and contents of total phenolics, flavonoids and lignin. RT-qPCR analysis of 15 MiWRKY members revealed that MiWRKY45 had the highest expression in response to MT + C. gloeosporioides. MiWRKY45 transcription factor was identified as a nucleus-localized transcriptional activator based on subcellular localization and transcriptional activation assays. MiWRKY45 bound to W-box motif and activated the expression of MiPAL, Mi4CL and MiPOD, as verified by DNA affinity purification-seq (DAP-seq), yeast one-hybrid (Y1H) and dual-luciferase reporter (DLR) assays. Transient transformation analysis revealed that MiWRKY45 positively regulated phenylpropanoid pathway, thereby enhancing mango resistance. These results suggest that MiWRKY45, as a positive regulator, is involved in MT-induced resistance against anthracnose in mangoes.

The P-type ATPase gene AHA5 is involved in proanthocyanidins accumulation in Medicago truncatula.

Proanthocyanidins (PAs) are the second most abundant plant phenolic natural products. The proton membrane H+-ATPase (AHA) is required for PA transportation in vacuoles, but it remains unclear which AHA gene(s) encode tonoplast proton pump in M. truncatula. Here, we identified three Tnt1 mutant lines of MtAHA5, resulting in PAs deficit in seeds. MtAHA5 was preferentially expressed in developing seeds, exhibiting its highest transcript levels at early stages. Although MtAHA3, MtAHA4, and MtAHA9 shared similar transcript patterns with MtAHA5 and other structural genes involved in PA biosynthesis, their mutant lines did not exhibit any PA-deficit phenotypes. Subcellular localization analysis demonstrated that MtAHA5 is targeted to the tonoplast in tobacco leaves; conversely, MtAHA3 and MtAHA9 are localized to the cytoplasm, suggesting that MtAHA5 acts as a tonoplast proton pump but not MtAHA3 or MtAHA9. Further genetic analyses revealed that MtAHA5 could complement the PA-deficit phenotype in mtaha5 mutants and ataha10 mutants. Transient transcription assays indicated that MtAHA5 is activated by the MBW complex to regulate the PA accumulation. Collectively, our findings suggest that MtAHA5 serves as a tonoplast proton pump to generate the driving force for MATE1-mediated transport of PA precursors into vacuoles.

The White Clover Single-Copy Nuclear Gene TrNAC002 Promotes Growth and Confers Drought Resistance in Plants Through Flavonoid Synthesis

White clover (Trifolium repens) is vulnerable to drought stress. In response to abiotic stress, plants are regulated by NAC transcription factors. The NAC in white clover has not been thoroughly documented until recently. We have identified one white clover NAC transcription factor called TrNAC002. TrNAC002’s coding sequence is localized to specific regions on the 3P and 5O chromosomes of white clover and is part of a single-copy nuclear gene. Subcellular localization demonstrates that TrNAC002 is located in the nucleus, while the transcriptional activity assay indicates its transcriptional activity. Arabidopsis plants overexpressing TrNAC002 (OE) exhibit enlarged leaves and increased lateral root growth compared to the wild type (WT). Additionally, the expression levels of the shoot apical meristem (SAM), WUSCHEL (WUS), DNA-binding protein (DBP), and auxin-induced in root cultures3 (AIR3) genes are significantly higher in OE as compared to WT. These findings imply that TrNAC002 could promote vegetative growth by increasing the expression of these genes. Under natural drought stress, OE can survive in dry soil for a longer period of time than WT. Furthermore, OE exhibits a lower level of reactive oxygen species (ROS) level and a higher content of flavonoids than WT. This is also positively correlated with an increased flavonoid content. In white clover, the expression of TrNAC002, chalcone synthase (CHS), and chalcone isomerase (CHI) in leaves demonstrates significant upregulation after drought stress and ABA treatment, as does the flavonoid content. However, the pTRV-VIGS experiment suggests that pTRV2-TrNAC002 white clover shrinks compared to the Mock and Water controls. Additionally, pTRV2-TrNAC002 white clover displays a statistically higher malondialdehyde (MDA) content than the Mock and Water controls, and a significantly lower level of total antioxidant activities, flavonoid content, CHS and CHI relative expression than that of the Mock and Water controls. These findings indicate that TrNAC002 responds to drought and modulates flavonoid biosynthesis in white clover. This study is the first to suggest that TrNAC002 likely responds to drought via ABA and enhances plant drought resistance by synthesizing flavonoids.

Method for rapid detection of recombinant protein E of West Nile virus

It was noted that the detection time of West Nile virus protein E by standard methods – immunoenzyme assay for West Nile virus antigen, antibody seroconversion, polymerase chain reaction with reverse transcription, virus isolation and neutralization assay, is at least one hour. West Nile virus (genus Flavivirus) belongs to the Japanese encephalitis antigenic complex of the family Flaviviridae and is capable of causing West Nile fever or severe West Nile disease. To increase the detection rate of recombinant protein E of West Nile virus, an express detection method using the developed promising biosensor-based analytical device was proposed. The biosensor is based on a field-effect transistor fabricated by optical lithography using silicon-on-insulator technology. The biosensor design was modernized – the topology of the crystal was changed (one ground electrode was formed in the center, around which 20 field-effect transistors are located), and the crystal surface was additionally covered with a hafnium oxide layer to stabilize the electrical characteristics. Protein detection by means of the biosensor is based on the measurement of current amplitude in the source-to-source circuit of the biosensor with monoclonal antibodies immobilized on the surface of its gate in response to the appearance of the antigen – recombinant protein E of West Nile virus – in the analyzed sample. It was experimentally established that the biosensor is capable of detecting protein concentration of 10 pg/μl. It is necessary to continue further studies to determine the error of measured concentrations and statistical reliability of the results obtained by the biosensor.

Decoding the trajectory of antibiotic resistance genes in saline and alkaline soils: Insights from different fertilization regimes.

The soil salinity and alkalinity play an important role in the occurrence and proliferation of antibiotic resistance genes (ARGs). Yet, little is known the underlying mechanism by which soil salinity and alkalinity affect antibiotic resistance evolution. Here we investigated the ARGs variation in soil salinity and alkalinity environments created by different fertilization, and explored the biological mechanisms that salinity and alkalinity alter the evolutionary paradigm of antibiotic resistance. The results showed the soil treated by organic fertilizer exhibited a low salinity, neutral level (TSD 239.20 μS/cm, pH 7.17). The ARG abundance in the OF treatment was the highest, keeping an average of 67.83 TPM. Beside the effect of direct input of organic fertilizer at the beginning, it was important to note that, ARGs abundance during planting showed significant correlations with pH and electric conductivity. We observed that changes in microbial survival strategies under different salinity and alkalinity conditions further affected ARG hosts abundance. Indoor experiments demonstrated that there was a survival trade-off between the growth of resistant bacteria and the evolution of antibiotic resistance in salinity and alkalinity environments. Meta-genomic and Meta-transcriptomic analysis consistently demonstrated bacterial antibiotic resistance was primarily associated with pyruvate, energy and lipid metabolic pathways. The functional gene related to salinity and alkalinity, like cysH, cysK, plsB and plsC showed negative correlations with MDR. Prokaryotic transcription assays validated these relations. This study well explains the prevalence of soil ARGs after different fertilization regimes and will give a deeper understanding for the effect of soil salinity and alkalinity on antibiotic resistance evolution.

Multiple factors regulate the expression of sufCDSUB in Streptococcus mutans.

The sufCDSUB gene cluster, encoding the sole iron-sulfur (Fe-S) cluster assembly system in S. mutans, was recently shown to be up-regulated in response to oxidative stressors and Fe limitation. In this study, luciferase reporter fusion assays, electrophoretic gel mobility shift assays (EMSA) and in vitro transcription assays (IVT) were used to dissect the cis- and trans-acting factors that regulate the expression of sufCDSUB. Results showed deletion of perR, for the only Fur-family transcriptional regulator in S. mutans, resulted in >5-fold increases in luciferase activity under the control of the sufCDSUB promoter (P<0.01), as compared to the parent strain, UA159 when the reporter strains were grown in medium with no supplemental iron. Site-directed mutagenesis of a PerR-box in the promoter region led to elevation of the reporter activity by >1.6-fold (P<0.01). In an EMSA, recombinant PerR (rPerR) was shown to bind to the cognate sufCDSUB promoter leading to mobility retardation. On the other hand, the reporter activity was increased by >84-fold (P<0.001) in response to the addition of cysteine at 4 mM to the culture medium. Deletion of cysR, for a LysR-type of transcriptional regulator, led to reduction of the reporter activity by >11.6-fold (P<0.001). Addition of recombinant CysR (rCysR) to an EMSA caused mobility shift of the sufCDSUB promoter probe, indicative of rCysR-promoter interaction, and rCysR was shown to enhance sufC transcription under the direction of sufCDSUB promoter in vitro. These results suggest that multiple factors are involved in the regulation of sufCDSUB expression in response to environmental cues, including cysteine and Fe availability, consistent with the important role of sufCDSUB in S. mutans physiology.

In vitro analysis of a competitive inhibition model for T7 RNA polymerase biosensors

T7 RNA polymerase (T7 RNAP) biosensors, in which T7 RNAP transcribes some reporter gene or signal in response to external stimuli, have wide applications in synthetic biology and metabolic engineering. We adapted a biochemical reaction network model and used an in vitro transcription assay to determine network parameters for different T7 RNAP constructs. Under conditions where template DNA is limiting, the EC50 values of native and engineered T7 RNAPs ranged from 33 nM (29–37 95 % c.i.) to 570 nM (258–714 95 % c.i.) (wild-type T7 RNAP). The measured EC50 values were largely insensitive to free magnesium, pH, or other buffer conditions. Many biosensor configurations use a split RNAP construct, where the C-terminal (CT7) and N-terminal T7 (NT7) are fused to proximity induced dimerization modules. We used proteolysis and ion exchange chromatography to prepare a CT7 (80 kDa) product. The impact of free CT7 on T7 RNAP transcriptional activity was well described by a competitive inhibition model, with an inhibitory constant KI = 23 nM (18–28 95 % c.i.) of the sensor. These model parameters will be useful for forward modeling and design of T7 RNAP-based genetic circuits.

Reporter Gene Assays to Measure FOXO-Specific Transcriptional Activity.

The forkhead box O (FOXO) family of transcription factors translates environmental cues into precise gene expression patterns maintaining cellular equilibrium while influencing critical determinations of cell destiny and differentiation. FOXO proteins exert their effects through specific consensus binding to promoter sites within target genes. Notably, among the array of techniques available for assessing the transcriptional activity of FOXO factors, the utilization of luciferase-based reporters emerges as particularly distinctive. Luciferase, an enzyme sourced from bioluminescent organisms, instigates the oxidation of luciferin, culminating in the generation of oxyluciferin accompanied by discernible luminescence, a quantifiable event readily gauged using a luminometer. The adoption of luciferase activity as a measure in transcriptional assays is widespread due to its numerous advantages including simplicity, remarkable reproducibility, and high sensitivity. Moreover, the continuous advancements witnessed in luciferase-based vectors and measurement reagents bestow notable flexibility upon this methodology. Luciferase-based reporters offer a powerful tool for uncovering constituents within the signaling pathways governing FOXO factor function. Furthermore, these assays are also suitable for evaluating the efficacy of FOXO-targeting agents, whether they be inhibitors or activators. Here, we present a comprehensive, step-by-step elucidation of a commonly employed assay, adeptly quantifying the potential of small molecular compounds to amplify FOXO-specific transcriptional activity in U2OS cells.

ETV2 transcriptionally activates Rig1 gene expression and promotes reprogramming of the endothelial lineage.

ETV2 is an essential transcription factor as Etv2 null murine embryos lack all vasculature, blood and are lethal early during embryogenesis. Previous studies have established that ETV2 functions as a pioneer factor and directly reprograms fibroblasts to endothelial cells. However, the underlying molecular mechanisms regulating this reprogramming process remain incompletely defined. In the present study, we examined the ETV2-RIG1 cascade as regulators that govern ETV2-mediated reprogramming. Mouse embryonic fibroblasts (MEFs) harboring an inducible ETV2 expression system were used to overexpress ETV2 and reprogram these somatic cells to the endothelial lineage. Single-cell RNA-seq from reprogrammed fibroblasts defined the induction of the transcriptional network involved in Rig1-like receptor signaling pathways. Studies using ChIP-seq, electrophoretic mobility shift assays, and transcriptional assays demonstrated that ETV2 was a direct upstream activator of Rig1 gene expression. We further demonstrated that the knockdown of Rig1 and separately, Nfκb1 using shRNA significantly reduced the efficiency of endothelial cell reprogramming. These results highlight that ETV2 reprograms fibroblasts to endothelial cells by directly activating RIG1. These findings extend our current understanding of the molecular mechanisms underlying ETV2-mediated reprogramming and will be important in the design of revascularization strategies for the treatment of ischemic tissues such as ischemic heart disease.

The QS regulator AphBvp promotes expression of the AHPND PirAvp and PirBvp toxins and may enhance virulence under acidic conditions

Shrimp acute hepatopancreatic necrosis disease (AHPND) is one of the most devastating diseases to impact the global shrimp farming industry, with a mortality rate of 70 %–100 %. The key virulence factors are a pair of Photorhabdus insect-related (Pir)-like toxins, PirAvp and PirBvp. In this study, by using an in vitro transcription and translation assay, we first confirmed that the quorum sensing transcriptional regulator AphBvp could trigger the expression of its downstream genes after binding to the AphBvp binding sequence in the promoter region of the pirAvp/pirBvp operon. Next, we showed that AphBvp was essential for the expression of these toxins by using an aphBvp-deletion mutant (ΔaphBvp) derived from the AHPND-causing Vibrio parahaemolyticus. Lastly, we discovered that the expression levels of PirAvp and PirBvp were up-regulated under acidic conditions (pH 4.5), and further showed that an acidic environment promoted the binding of AphBvp to the pirABvp promoter. We speculate that this was because the acidic environment favored the formation of AphBvp tetramers, which is important for binding to DNA. Taken together, these findings improve our understanding of the gene regulatory mechanisms of pirAvp and pirBvp, and suggest that the pH value of the environment might affect the virulence of AHPND-causing V. parahaemolyticus.

Genome-Wide Identification of GRAS Gene Family in Cunninghamia lanceolata and Expression Pattern Analysis of ClDELLA Protein Under Abiotic Stresses.

The Chinese fir (Cunninghamia lanceolata) is a significant species utilized in afforestation efforts in southern China. It is distinguished by its rapid growth and adaptability to diverse environmental conditions. The GRAS gene family comprises a group of plant-specific transcription factors that play a pivotal role in plant growth and development, response to adversity, and hormone regulatory networks. However, the exploration of the GRAS family in gymnosperm Chinese fir has not yet begun. In this study, a total of 43 GRAS genes were identified in the whole genome of Chinese fir, and a phylogenetic analysis classified them into nine distinct subfamilies. Gene structure analysis revealed that the majority of ClGRAS genes lacked introns. It is notable that among these proteins, both ClGAI and ClGRA possess distinctive DELLA structural domains. Cis-acting element analysis revealed that nearly all ClGRAS genes contained light-responsive elements, while hormone-responsive elements, environmental-responsive elements (low-temperature- or defense-responsive elements), and meristem-organization-related elements were also identified. Based on transcriptome data and RT-qPCR expression patterns, we analyzed the expression of ClGAI and ClRGA genes across different developmental stages, hormones, and three abiotic stresses. Subcellular localization analysis demonstrated that ClGAI and ClRGA were localized to the nucleus. Transcriptional activation assays showed that both genes have self-activating activity. In conclusion, the results of this study indicate that the ClGRAS gene family is involved in the response of Chinese fir to environmental stress. Further research on the ClDELLA genes provides valuable information for exploring the potential regulatory network of DELLA proteins in Chinese fir.

Abstract 4136316: BMP10 as novel marker for right atrial dilatation and pressure in precapillary pulmonary hypertension

Introduction: Precapillary pulmonary hypertension (precPH) leads to increased right atrial (RA) stretch and pressure. The right atrium is the major source of bone morphogenetic protein 10 (BMP10) in adults. Aim: This study aims to investigate BMP10 in relation to RA dilatation and pressure overload. Methods: We studied BMP10 mRNA in fresh RA tissue of N=5 Chronic Thromboembolic Pulmonary Hypertension (CTEPH) patients and N=9 controls and protein expression in paraffin-embedded RA tissue of N=4 Pulmonary Arterial Hypertension (PAH) patients and N=6 controls. We prospectively included 48 precPH patients (N=22 idiopathic PAH, N=14 hereditary PAH and N=12 CTEPH, and N=16 controls. To study BMP10-induced transcriptional activity, we assayed serum samples in human microvascular endothelial cells (HMEC) with a BMP-responsive transcriptional luciferase assay. BMP10 activity was calculated by subtracting BMP activity after incubation with antibodies for Alk1Fc and bone morphogenetic protein 9 (BMP9). We assessed correlations between BMP10 activity and RA dilatation (RA max volume&gt;79 ml/m 2 for male or &gt;69 ml/m 2 for female) and right ventricular (RV) dilatation (RV end-diastolic volume&gt;108 ml/m 2 for male and &gt;96 ml/m 2 for female). In a cohort of CTEPH patients that underwent pulmonary endarterectomy (PEA) we assessed the effect of pressure unloading on BMP10 activity. Normality of data was checked and statistical differences between groups were tested using an independent sample t-test or Wilcoxon rank-sum test. Results: BMP10 mRNA, protein and downstream activity were higher in the precPH RA tissue (Figure 1A-F). High systemic BMP10 activity was associated with RA dilatation and high RA pressure, but not RV dilatation in precPH (Figure 2A-C). Finally, pressure unloading after PEA in CTEPH patients results in a reduction in BMP10 activity (figure 2D). Conclusions: Local RA BMP10 expression and activity is increased in precPH. Increased systemic BMP10 activity was related to RA dilatation and pressure. Pressure unloading of the right heart leads to a reduction of systemic BMP10 activity. Future studies are needed to determine whether increased BMP10 release is an adaptative mechanism or can be used as therapeutic target.

TGF-β receptor-specific NanoBRET Target Engagement in living cells for high-throughput kinase inhibitor screens

Targeting transforming growth factor-β (TGF-β) receptors is a promising pharmacological approach to normalize aberrant signaling in genetic and non-genetic TGF-β associated diseases including fibrosis, cancer, cardiovascular and musculoskeletal disorders. To identify novel TGF-β receptor kinase inhibitors, methods like in vitro kinase assays, western blot or transcriptional reporter assays are often used for screening purposes. While these methods may have certain advantages, the lack of integration of key features such as receptor specificity, high-throughput capability, and cellular context resemblance remains a major disadvantage. This deficiency could ultimately hinder the translation of study outcomes into later (clinical) stages of drug development. In this study, we introduce an adjusted and optimized live cell NanoBRET Target Engagement (TE)-based method to identify TGF-β receptor specific kinase inhibitors. This comprehensive toolkit contains various TGF-β type I and type II receptors, with corresponding nanoBRET tracers, and disease-related cell lines, including novel non-commercially available materials. The nanoBRET capacity and kinase inhibitory window can be significantly enhanced for functional measurements when stable expression cell lines and substantially low tracer concentrations are used. In addition, this system can be tailored to study TGF-β associated genetic disorders and possibly be used to screen for disease-specific therapeutics. Therefore, the use of this optimized, live cell, antibody-independent nanoBRET Target Engagement assay is highly encouraged for future high-throughput compound screens targeting TGF-β/BMP receptors.

Abstract 4119913: Aging-Associate Peptide Medin Induces Proinflammatory Activation in Human Brain Vascular Smooth Muscle Cells

Background: Medin is one of the most common amyloidogenic proteins and accumulates in the vasculature with aging. Vascular medin accumulation is associated with Alzheimer's disease, vascular dementia and aortic aneurysms. In vitro , medin has been shown to induce endothelial proinflammatory activation and in ex vivo human cerebral arterial tissue, medin induces both endothelial and smooth muscle dysfunction. The role of medin in vascular smooth muscle (VSMC) activation remains unknown. Aim: The aim of the study is to determine using gene transcription assay whether medin induces VSMC activation and phenotypic transformation. Methods: Human brain vascular smooth muscle cells (HBVSMCs, passages 6-10) were exposed to medin at doses observed in human tissues (0, 0.5, 1 and 5 υM) for 20 hours and reverse transcription polymerase chain reaction (rtPCR) was used to quantify gene expression of proinflammatory factors (interleukin (IL)-6, IL-8, IL-1b) and structural and enzyme proteins associated with VSMC phenotypic transformation (ACTA2, MYH11 and NOX4). In separate experiments, HBVSMCs were exposed to vehicle, medin 5 υM without or with small molecule NFκB inhibitor RO106-9920 (10 υM) or NLGM1 nanoliposome (70% phosphatidylcholine, 25% cholesterol, 5% monosialoganglioside, 100 υg/mL, agent shown to reverse medin-induced endothelial dysfunction and proinflammatory activation). Results: Medin induced a dose-dependent increase in gene expression of IL-6, IL-8 and IL-1b but did not alter gene expression of ACTA2, MYH11 and NOX4 (Fig. 1). Co-treatment of medin with RO106-9920 and NLGM1 showed a trend (not statistically significant) of reduced IL-6 and IL-8 (Fig. 2). Conclusions: Physiologic doses of medin induced pro-inflammatory activation of HBVSMCs but did not affect gene expression of structural proteins (ACTA2 and MYH11) and enzyme (NOX4) involved in VSMC phenotypic transformation. Although the mechanisms behind this effect remains to be explored, results suggest that medin may be an important mediator of aging-associated vascular inflammation.

Peripubertal exposure to oxyfluorfen, a diphenyl herbicide, delays pubertal development in the male rat by antagonizing androgen receptor activity.

We recently identified the herbicide oxyfluorfen as an inhibitor of iodide uptake by the sodium iodide symporter (NIS), a key step in thyroid hormone synthesis, using in vitro assays. We also observed a suppression of serum T4 and T3 in juvenile rats exposed orally to oxyfluorfen for 4-8 days. The purpose of the present study was to further evaluate effects of an extended 31-day oral exposure using a male pubertal rat study (15 to 500 mg/kg). Oxyfluorfen delayed puberty at all doses (1.3 -3.5 days) suppressing ventral prostate at 62.5 mg/kg and above and seminal vesicle weights at 31.25 mg/kg and above with no effect on testosterone or luteinizing hormone. Serum T4 and T3 were suppressed by all doses up to 80%, with a linear increase in serum TSH. Based on delayed puberty without changes in testosterone, we hypothesized that oxyfluorfen interferes with androgen receptor (AR) function. Results from our Hershberger study, with oxyfluorfen (62.5 and 125 mg/kg) co-treated with testosterone proprionate (TP , 1 mg/kg) for 10 days showed 3 of 5 of the androgenic tissue weights were suppressed compared to TP alone indicating AR antagonism. We next confirmed this effect in an in vitro AR transcriptional activation reporter assay (0-20 μM) with 125 pM 5αDH-11-ketotestosterone and found concentration-dependent inhibition of AR luminescence activity (EC50 1.75 µM) without cytotoxicity. Here, we confirmed the endocrine disrupting effects of oxyfluorfen using both in vitro and in vivo evaluations of the thyroid hormone and AR pathways. This abstract doesn't necessarily reflect U.S. EPA policy.

NusG-dependent RNA polymerase pausing is a common feature of riboswitch regulatory mechanisms.

Transcription by RNA polymerase is punctuated by transient pausing events. Pausing provides time for RNA folding and binding of regulatory factors to the paused elongation complex. We previously identified 1600 NusG-dependent pauses throughout the Bacillus subtilis genome, with ∼20% localized to 5' leader regions, suggesting a regulatory role for these pauses. We examined pauses associated with known riboswitches to determine whether pausing is a common feature of these mechanisms. NusG-dependent pauses in the fmnP, tenA, mgtE, lysP and mtnK riboswitches were in strategic positions preceding the critical decision between the formation of alternative antiterminator or terminator structures, which is a critical feature of transcription attenuation mechanisms. In vitro transcription assays demonstrated that pausing increased the frequency of termination in the presence of the cognate ligand. NusG-dependent pausing also reduced the ligand concentration required for efficient termination. In vivo expression studies with transcriptional fusions confirmed that NusG-dependent pausing is a critical component of each riboswitch mechanism. Our results indicate that pausing enables cells to sense a broader range of ligand concentrations for fine-tuning riboswitch attenuation mechanisms.