Understanding p300-transcription factor interactions using sequence variation and hybridization

The hypoxic response is central to cell function and plays a significant role in the growth and survival of solid tumours. HIF-1 regulates the hypoxic response by activating over 100 genes responsible for adaptation to hypoxia, making it a potential target for anticancer drug discovery. Although there is significant structural and mechanistic understanding of the interaction between HIF-1α and p300 alongside negative regulators of HIF-1α such as CITED2, there remains a need to further understand the sequence determinants of binding. In this work we use a combination of protein expression, chemical synthesis, fluorescence anisotropy and isothermal titration calorimetry for HIF-1α sequence variants and a HIF-1α- CITED hybrid sequence which we term CITIF. We show the HIF-1α sequence is highly tolerant to sequence variation through reduced enthalpic and less unfavourable entropic contributions, These data imply backbone as opposed to side chain interactions and ligand folding control the binding interaction and that sequence variations are tolerated as a result of adopting a more disordered bound interaction or fuzzy complex.

Epigenetic silencing of recombinant AAV genomes by NP220 and the HUSH complex

The single-stranded DNA genome of adeno-associated viruses (AAV) undergoes second-strand synthesis and transcription in the host cell nucleus. While wild-type AAV genomes are naturally silenced upon integration into the host genome, recombinant AAV (rAAV) genomes typically provide robust expression of transgenes persisting as extrachromosomal DNA or episomes. Episomal DNA associating with host histones are subject to epigenetic modifications, although the mechanisms underlying such are not well understood. Here, we provide evidence that the double-stranded DNA binding protein NP220, in association with the human silencing hub (HUSH) complex, mediates transcriptional silencing of single-stranded as well as self-complementary rAAV genomes. In cells lacking NP220 or other components of the HUSH complex, AAV genome transcript levels are increased and correlate with a marked reduction in repressive H3K9 histone methylation marks. We also provide evidence that the AAV capsid (serotype) can profoundly influence NP220-mediated mediated silencing of packaged genomes, indicating potential role(s) for capsid-genome or capsid-host factor interactions in regulating epigenetic silencing of rAAV genomes. Importance Recombinant AAV vectors can enable long term gene expression in a wide variety of tissues. However, transgene silencing has been reported in some human gene therapy clinical trials. Here, we demonstrate the human silencing hub (HUSH) complex can suppress transcript formation from rAAV vector genomes by epigenetic modification of associated host histones. Further, the AAV capsid appears to play an important role in this pathway. We postulate that modulation of epigenetic pathways could help improve rAAV expression.

Evaluating the Dominant Controls of Water Erosion in Three Dry Valley Types Using the RUSLE and Geodetector Method

The dry valley is a unique geographic phenomenon in Southwest China with severe water erosion. However, little is known regarding its dominant controls and the discrepancies between dry valley subtypes, leading to the poor management of water erosion. To solve these problems, the revised universal soil loss equation (RUSLE) and Geodetector method were used in a dry temperate (DT), dry warm (DW), and dry hot (DH) valley. Results indicated that dry valleys suffer severe water erosion with a value of 64.78, 43.85, and 33.81 t·ha−1·yr−1. The Geodetector method is proven to be an efficient tool to quantify the dominant factor of water erosion. It was established that land use types (LUT) have the closest relationship with water erosion. The controls for water erosion could be better explained by multi-factor interactions analysis, particularly for the combination of slope and LUT in DW (q = 0.71) and DH (q = 0.66). Additionally, regions at high risk of water erosion were characterized by steep slope (>30°) and low vegetation coverage (<50%) in DT, while the opposite is shown in DH. These findings could provide insight for guiding soil erosion management and ecological restoration strategies that balance economic and environmental sustainability.

Response Surface Designs Robust against Nuisance Factors

This paper discusses an algorithmic approach to constructing trend-free and orthogonally-blocked response surface designs. The constructed designs have the main effects, 2-factor interactions and second-order effects being orthogonal or near-orthogonal to the nuisance factors such as the time-trend or the blocking factors. The paper also provides a catalogue of (near-) trend-free Box–Behnkens designs and orthogonally blocked Box–Behnkens designs arranged in rows and columns.

Context-specific regulation of lysosomal lipolysis through network-level diverting of transcription factor interactions

Plasticity in multicellular organisms involves signaling pathways converting contexts—either natural environmental challenges or laboratory perturbations—into context-specific changes in gene expression. Congruently, the interactions between the signaling molecules and transcription factors (TF) regulating these responses are also context specific. However, when a target gene responds across contexts, the upstream TF identified in one context is often inferred to regulate it across contexts. Reconciling these stable TF–target gene pair inferences with the context-specific nature of homeostatic responses is therefore needed. The induction of the Caenorhabditis elegans genes lipl-3 and lipl-4 is observed in many genetic contexts and is essential to survival during fasting. We find DAF-16/FOXO mediating lipl-4 induction in all contexts tested; hence, lipl-4 regulation seems context independent and compatible with across-context inferences. In contrast, DAF-16–mediated regulation of lipl-3 is context specific. DAF-16 reduces the induction of lipl-3 during fasting, yet it promotes it during oxidative stress. Through discrete dynamic modeling and genetic epistasis, we define that DAF-16 represses HLH-30/TFEB—the main TF activating lipl-3 during fasting. Contrastingly, DAF-16 activates the stress-responsive TF HSF-1 during oxidative stress, which promotes C. elegans survival through induction of lipl-3. Furthermore, the TF MXL-3 contributes to the dominance of HSF-1 at the expense of HLH-30 during oxidative stress but not during fasting. This study shows how context-specific diverting of functional interactions within a molecular network allows cells to specifically respond to a large number of contexts with a limited number of molecular players, a mode of transcriptional regulation we name “contextualized transcription.”

Elucidating the Role of Nrf2 Factor Interactions in Various Disorders of Human System and It’s Proteomic Approaches: A Novel Study

Nuclear factor erythroid 2-related factor 2 (Nrf2), which is also known as nuclear factor erythroid-derived-like-2, is a transcription factor which is encoded by the NFE2L2 gene. It is a basic leucine zipper (bZIP) protein which coordinates the basal and stress-inducible activation of a vast array of cytoprotective genes. It modulates a cellular antioxidant response program and plays a major role in the protection against oxidants and electrophiles; extracellular and intracellular oxidant/electrophiles have great contributions to the damages in cellular macromolecules such as proteins, lipids or DNA. Keap1 protein which is a regulator of Nrf2, is a highly redox-sensitive member of BTB-Kelch family assembling with Cul3 protein to form a Cullin-RING E3 ligase complex for Nrf2 degradation. Thus, this factor is a regulator of many processes of life and it’s signalling system (Nrf2-KEAP-1-ARE pathway) has been found to participate in various ocular or eye diseases and even other systemic diseases such as respiratory disease, chronic diseases or cancer. In microbial infections, the host oxidative stress response may lead to the production of cytoprotective molecules, which in turn induces the activation of cellular Nrf2 factor. The crystallins or eye lens proteins, (?B-crystallin being one of them) may possibly interact with Nrf2 factor and regulate oxidative stress, but it is yet to be deciphered. Proteomic studies may provide valuable information, regarding such detailed protein interactions and their pathways especially in case of diseases or infections in the upcoming days.

Experimental Comparison of Straight Flanging and Rotary Die Bending Based on Springback

Springback in sheet bending is a well-defined phenomenon; however, variation of springback is difficult to control causing quality problems in especially mass-produced goods such as home appliances. As an alternative to straight flanging, the rotary die bending process offers reduced springback as well as reduced geometric variation; however, there is little knowledge in the literature. The effects of process parameters on the springback behavior of straight flanging and rotary die bending as applied to home appliance side panels are investigated experimentally. For each flange bending method, effects of die radius, punch-die clearance, rolling direction, flange length, and material supplier on springback are tested on EN DC01 carbon and SAE 430 stainless steel sheets. A full factorial experimental design was applied to investigate the factor interactions as well as the main effects using ANOVA. In both methods, die radius was the most dominant factor on springback, clearance being the second, and the inevitable material property variations being the third one. Nevertheless, in rotary die bending, springback values were smaller with significantly less scatter compared to straight flanging. Consequently, rotary die bending is a much more preferable process especially in mass production performed with narrow profit margins.

Adeno-Associated Virus (AAV) Gene Delivery: Dissecting Molecular Interactions Upon Cell Entry

Human gene therapy has advanced from twentieth-century conception to twenty-first-century reality. The recombinant Adeno-Associated Virus (rAAV) is a major gene therapy vector. Research continues to improve rAAV safety and efficacy using a variety of AAV capsid modification strategies. Significant factors influencing rAAV transduction efficiency include neutralizing antibodies, attachment factor interactions and receptor binding. Advances in understanding the molecular interactions during rAAV cell entry combined with improved capsid modulation strategies will help guide the design and engineering of safer and more efficient rAAV gene therapy vectors.