A5057198078- CRISPR and Genetic Engineering
- Nitric Oxide and Endothelin Effects
- Bacterial Genetics and Biotechnology
- RNA and protein synthesis mechanisms
- Renin-Angiotensin System Studies
- Heat shock proteins research
- Ion Transport and Channel Regulation
- Advanced biosensing and bioanalysis techniques
- Microbial Metabolic Engineering and Bioproduction
- Gene Regulatory Network Analysis
- Eicosanoids and Hypertension Pharmacology
- Molecular Communication and Nanonetworks
- ATP Synthase and ATPases Research
- Physiological and biochemical adaptations
- Caveolin-1 and cellular processes
- Receptor Mechanisms and Signaling
- Service-Oriented Architecture and Web Services
- HER2/EGFR in Cancer Research
- Electrochemical sensors and biosensors
- Cytokine Signaling Pathways and Interactions
- Collaborative Teaching and Inclusion
- Hemoglobin structure and function
- Teacher Education and Leadership Studies
- Parental Involvement in Education
- Diverse Education Studies and Reforms
Pathfinder International
2025
University of Washington
2018-2024
University of Trento
2014-2017
Yale University
1999-2007
Unisys (United States)
2004
University of Massachusetts Chan Medical School
2003
Vita-Salute San Raffaele University
2003
Harvard University
1999
Howard Hughes Medical Institute
1980
Abstract Previous efforts to control cellular behaviour have largely relied upon various forms of genetic engineering. Once the content a living cell is modified, that typically changes as well. However, other methods are possible. All cells sense and respond their environment. Therefore, artificial, non-living mimics could be engineered activate or repress already existing natural sensory pathways through chemical communication. Here we describe construction such system. The artificial...
Artificial cells capable of both sensing and sending chemical messages to bacteria have yet be built. Here we show that artificial are able sense synthesize quorum signaling molecules can chemically communicate with V. fischeri, harveyi, E. coli, P. aeruginosa. Activity was assessed by fluorescence, luminescence, RT-qPCR, RNA-seq. Two potential applications for this technology were demonstrated. First, the extent which could imitate natural quantified a type cellular Turing test. in response...
Methods to regulate gene expression programs in bacterial cells are limited by the absence of effective activators. To address this challenge, we have developed synthetic transcriptional activators E. coli linking activation domains programmable CRISPR-Cas DNA binding domains. Effective requires target sites situated a narrow region just upstream transcription start site, sharp contrast relatively flexible site requirements for eukaryotic cells. Together with existing tools CRISPRi...
Protein-protein interactions with the molecular chaperone hsp90 and phosphorylation on serine 1179 by protein kinase Akt leads to activation of endothelial nitric oxide synthase. However, interplay between these protein-protein remains be established. In present study, we show that vascular growth factor stimulates coordinated association hsp90, Akt, resultant eNOS. Characterization domains required bind eNOS, using yeast 2-hybrid, cell-based coprecipitation experiments, GST-fusion proteins,...
The balance of nitric oxide (·NO) and superoxide anion (O⨪2) plays an important role in vascular biology. association heat shock protein 90 (Hsp90) with endothelial nitric-oxide synthase (eNOS) is a critical step the mechanisms by which eNOS generates ·NO. As capable generating both ·NO O⨪2, we hypothesized that Hsp90 might also mediate eNOS-dependent O⨪2 production. To test this hypothesis, bovine coronary cells (BCEC) were pretreated geldanamycin (GA, 10 μg/ml; 17.8 μm) then stimulated...
Pathways controlling cell proliferation and survival require flexible adaptation to environmental stresses. These mechanisms are frequently exploited in cancer, allowing tumor cells thrive unfavorable milieus. Here, we show that Hsp90, a molecular chaperone is central the cellular stress response, associates with survivin, an apoptosis inhibitor essential regulator of mitosis. This interaction involves ATPase domain Hsp90 survivin baculovirus repeat. Global suppression function or targeted...
The activity of endothelial nitric-oxide synthase (eNOS) is regulated by its subcellular localization, phosphorylation and through interaction with different proteins. association eNOS caveolin-1 (Cav) believed to maintain in an inactive state; however, increased heat shock protein 90 (hsp90) observed following activation. In this study, we investigate the relationship between caveolin hsp90 as opposing regulatory proteins on function. Immunoprecipitation Cav-1 from bovine lung microvascular...
The subcellular localization of endothelial nitric-oxide synthase (eNOS) is critical for optimal coupling extracellular stimulation to nitric oxide production. Because eNOS activated by Akt-dependent phosphorylation produce (NO), we determined the distribution phosphorylated on serine 1179 using a variety methodologies. Based sucrose gradient fractionation, phosphorylated-eNOS (P-eNOS) was found in both caveolin-1-enriched membranes and intracellular domains. Co-transfection with Akt cells...
Abstract In bacterial systems, CRISPR-Cas transcriptional activation (CRISPRa) has the potential to dramatically expand our ability regulate gene expression, but we lack predictive rules for designing effective gRNA target sites. Here, identify multiple features of promoters that impose stringent requirements on CRISPRa Notably, observe narrow, 2–4 base windows sites with a periodicity corresponding one helical turn DNA, spanning ~40 bases and centered ~80 upstream TSS. However, also two...
The 894G→T polymorphism within exon 7 of the human endothelial nitric-oxide synthase (eNOS) gene codes for glutamate or aspartate, respectively, at residue 298 and has been associated with several diseases cardiovascular origin. A recent report indicates that Asp298-eNOS (E298D) is cleaved intracellularly to 100- 35-kDa fragments, suggesting a mechanism reduced function. Here we have documented precise cleavage site E298D variant as unique aspartyl-prolyl (Asp298–Pro299) bond not seen in...
The heterogeneous localization of endothelial nitricoxide synthase (eNOS) on the Golgi complex versus plasma membrane has made it difficult to dissect regulation each pool enzyme. Here, we generated fusion proteins that specifically target or cytoplasmic aspects and have assessed eNOS activation. Plasma membrane-targeted constructs were constitutively active, phosphorylated, responsive transmembrane calcium fluxes, yet insensitive further activation by Akt-mediated phosphorylation. In...
Engineering metabolism to efficiently produce chemicals from multi-step pathways requires optimizing multi-gene expression programs achieve enzyme balance. CRISPR-Cas transcriptional control systems are emerging as important tools for programming expression, but poor predictability of guide RNA folding can disrupt control. Here, we correlate efficacy modified RNAs (scRNAs) CRISPR activation (CRISPRa) in E. coli with a computational kinetic parameter describing scRNA rate into the active...
Methods for implementing dynamically‐controlled multi‐gene programs could expand capabilities to engineer metabolism efficiently producing high‐value compounds. This work explores whether CRISPRi repression can be tuned in E. coli through the regulated expression of machinery. When dCas9 is not limiting, variations sgRNA alone lead levels ranging from 5‐ 300‐fold. Titrating over a 2.5‐fold range results 16‐fold changes reporter gene expression. Many different classes genetic controllers...
Objectives— Heat-shock protein 90 (Hsp90) coordinates the regulation of diverse signaling proteins. We try to develop a new tool explore regulatory functions Hsp90 in endothelial cells (ECs) instead existing chemical approaches. Methods and Results— designed dominant-negative construct by site-direct mutagenesis residue Asp-88 Asn (D88N-Hsp90) based on structure ATP/ADP-binding site. Recombinant wild-type binds ATP-Sepharose beads manner inhibited ATP or 17-AAG, specific inhibitor for Hsp90,...
Abstract Targeting RNA molecules that encode for undruggable proteins involved in cancer is an emerging strategy small molecule drug development. While a handful of compounds have entered the clinic, progress has been slow due to longstanding challenges identifying are both functional and selective. Wayfinder Biosciences developed RNA-based sensor technology addresses these bottlenecks deployed it discover bind structured elements MYC mRNA, which deregulated majority human cancers. The 5’...
An increase in the association of heat shock protein 90 (HSP90) with endothelial nitric oxide (NO) synthase (eNOS) is well recognized for increasing NO (NO*) production. Despite progress this field, mechanisms by which HSP90 modulates eNOS remain unclear due, part, to fact that geldanamycin (GA) redox cycles generate superoxide anion (O(2)(-*) and inhibiting GA or radicicol (RAD) destabilizes tyrosine kinases rely on chaperone maturation. In report, we determine extent these side effects...
We investigated the molecular mechanisms of sodium vanadate (vanadate)-induced nitric oxide (NO) production. Exposure bovine lung microvascular cells (BLMVEC) to increased release biologically active NO in endothelium/smooth muscle cocultures, as measured by accumulation its surrogate marker, cGMP. This was sensitive synthase (NOS) inhibition and greater than that observed with ionomycin. Although calcium chelators (BAPTA, EGTA) inhibited basal ionomycin-induced production, they failed...