Characterization of a molecular switch system that regulates gene expression in mammalian cells through a small molecule

Abstract Background Molecular switch systems that activate gene expression by a small molecule are effective technologies that are widely used in applied biological research. Nuclear receptors are valuable candidates for these regulation systems due to their functional role as ligand activated transcription factors. Previously, our group engineered a variant of the retinoid × receptor to be responsive to the synthetic compound, LG335, but not responsive to its natural ligand, 9-cis-retinoic acid. Results This work focuses on characterizing a molecular switch system that quantitatively controls transgene expression. This system is composed of an orthogonal ligand/nuclear receptor pair, LG335 and GRQCIMFI, along with an artificial promoter controlling expression of a target transgene. GRQCIMFI is composed of the fusion of the DNA binding domain of the yeast transcription factor, Gal4, and a retinoid × receptor variant. The variant consists of the following mutations: Q275C, I310M, and F313I in the ligand binding domain. When introduced into mammalian cell culture, the switch shows luciferase activity at concentrations as low as 100 nM of LG335 with a 6.3 ± 1.7-fold induction ratio. The developed one-component system activates transgene expression when introduced transiently or virally. Conclusions We have successfully shown that this system can induce tightly controlled transgene expression and can be used for transient transfections or retroviral transductions in mammalian cell culture. Further characterization is needed for gene therapy applications.</p

Association of ultra-processed food intake with risk of inflammatory bowel disease: Prospective cohort study

Objective: To evaluate the relation between intake of ultra-processed food and risk of inflammatory bowel disease (IBD).Design: Prospective cohort study.Setting: 21 low, middle, and high income countries across seven geographical regions (Europe and North America, South America, Africa, Middle East, south Asia, South East Asia, and China).Participants: 116 087 adults aged 35-70 years with at least one cycle of follow-up and complete baseline food frequency questionnaire (FFQ) data (country specific validated FFQs were used to document baseline dietary intake). Participants were followed prospectively at least every three years.Main outcome measures: The main outcome was development of IBD, including Crohn\u27s disease or ulcerative colitis. Associations between ultra-processed food intake and risk of IBD were assessed using Cox proportional hazard multivariable models. Results are presented as hazard ratios with 95% confidence intervals.Results: Participants were enrolled in the study between 2003 and 2016. During the median follow-up of 9.7 years (interquartile range 8.9-11.2 years), 467 participants developed incident IBD (90 with Crohn\u27s disease and 377 with ulcerative colitis). After adjustment for potential confounding factors, higher intake of ultra-processed food was associated with a higher risk of incident IBD (hazard ratio 1.82, 95% confidence interval 1.22 to 2.72 for ≥5 servings/day and 1.67, 1.18 to 2.37 for 1-4 servings/day compared with \u3c1 serving/day, P=0.006 for trend). Different subgroups of ultra-processed food, including soft drinks, refined sweetened foods, salty snacks, and processed meat, each were associated with higher hazard ratios for IBD. Results were consistent for Crohn\u27s disease and ulcerative colitis with low heterogeneity. Intakes of white meat, red meat, dairy, starch, and fruit, vegetables, and legumes were not associated with incident IBD.Conclusions: Higher intake of ultra-processed food was positively associated with risk of IBD. Further studies are needed to identify the contributory factors within ultra-processed foods.Study registration: ClinicalTrials.gov NCT03225586

Engineering Protein Molecular Switches To Regulate Gene Expression with Small Molecules

Small molecule dependent molecular switches that control gene expression are important tool in understanding biological cellular processes and for regulating gene therapy. Nuclear receptors are ligand activated transcription factors that have been engineered to selectively respond to synthetic ligands and used as regulators of gene expression. In this work the retinoid X receptor (RXR), has been used to develop an inducible molecular switch with a near drug like compound LG335. Three RXR variants (Q275C; I310M; F313I), (I268A; I310A; F313A; L436F), (I268V; A272V; I310M; F313S; L436M) were created via site-directed mutagenesis and a structure based approach, such that they preferentially bind to the synthetic ligand LG335 and not its natural ligand, 9-cis retinoic acid. These variants show reverse ligand specificity as designed and have an EC50 for LG335 of 80 nM, 30 nM, 180 nM, respectively. The ligand binding domains of the RXR variants were fused to a yeast transcription factor Gal4 DNA binding domain. This modified chimeric fusion protein showed reverse response element specificity as designed and recognized the Gal4 response element instead of the RXR response element. The modified RXR protein did not heterodimerize with wild type RXR or with other nuclear receptor such as retinoic acid receptor. These RXR-based molecular switches were tested in retroviral vectors using firefly luciferase and green fluorescence protein and they maintain their inducible behavior with LG335. These experiments demonstrate the orthogonality of RXR variants and their possible use in regulating gene therapy.Ph.D.Committee Chair: Donald F. Doyle; Committee Member: C. David Sherrill ; Committee Member: H.Trent Spencer; Committee Member: Joseph M. LeDoux; Committee Member: Nicholas V. Hu

COVID-19-Associated Mucormycosis: A Matter of Concern Amid the SARS-CoV-2 Pandemic

Mucormycosis is an invasive fungal infection caused by fungi belonging to order Mucorales. Recently, with the increase in COVID-19 infections, mucormycosis infections have become a matter of concern globally, because of the high morbidity and mortality rates associated with them. Due to the association of mucormycosis with COVID-19 disease, it has been termed COVID-19-associated mucormycosis (CAM). In the present review, we focus on mucormycosis incidence, pathophysiology, risk factors, immune dysfunction, interactions of Mucorales with endothelial cells, and the possible role of iron in Mucorales growth. We review the limitations associated with current diagnostic procedures and the requirement for more specific, cost-effective, convenient, and sensitive assays, such as PCR-based assays and monoclonal antibody-based assays for the effective diagnosis of mucormycosis. We discuss the current treatment options involving antifungal drug therapies, adjunctive therapy, surgical treatment, and their limitations. We also review the importance of nutraceuticals-based therapy for the prevention as well as treatment of mucormycosis. Our review also highlights the need to explore the potential of novel immunotherapeutics, which include antibody-based therapy, cytokine-based therapy, and combination/synergistic antifungal therapy, as treatment options for mucormycosis. In summary, this review provides a complete overview of COVID-19-associated mucormycosis, addressing the current research gaps and future developments required in the field

Finding Homes for Orphan Cytochrome P450s: CYP4V2 and CYP4F22 in Disease States

Genetic analyses have identified a wide spectrum of mutations in the CYP4V2 gene from patients suffering from Bietti’s crystalline corneoretinal dystrophy, and mutations in the CYP4F22 gene have been linked to lamellar ichthyosis. These strong gene–disease associations will be better understood if we can elucidate the substrate specificity of the heretofore “orphan” P450s and unravel the biochemical pathways that go awry in disease states. The complex biotransformations that underlie eicosanoid signaling, however, pose great challenges for the enzymologist seeking to assign specific metabolic roles to these members of the CYP4 family. Inductive reasoning and modeling are crucial tools for designing the experiments that will define disease progression in terms of CYP function

Association of ultra-processed food intake with risk of inflammatory bowel disease: prospective cohort study

OBJECTIVE: To evaluate the relation between intake of ultra-processed food and risk of inflammatory bowel disease (IBD). DESIGN: Prospective cohort study. SETTING: 21 low, middle, and high income countries across seven geographical regions (Europe and North America, South America, Africa, Middle East, south Asia, South East Asia, and China). PARTICIPANTS: 116 087 adults aged 35-70 years with at least one cycle of follow-up and complete baseline food frequency questionnaire (FFQ) data (country specific validated FFQs were used to document baseline dietary intake). Participants were followed prospectively at least every three years. MAIN OUTCOME MEASURES: The main outcome was development of IBD, including Crohn\u27s disease or ulcerative colitis. Associations between ultra-processed food intake and risk of IBD were assessed using Cox proportional hazard multivariable models. Results are presented as hazard ratios with 95% confidence intervals. RESULTS: Participants were enrolled in the study between 2003 and 2016. During the median follow-up of 9.7 years (interquartile range 8.9-11.2 years), 467 participants developed incident IBD (90 with Crohn\u27s disease and 377 with ulcerative colitis). After adjustment for potential confounding factors, higher intake of ultra-processed food was associated with a higher risk of incident IBD (hazard ratio 1.82, 95% confidence interval 1.22 to 2.72 for ≥5 servings/day and 1.67, 1.18 to 2.37 for 1-4 servings/day compared with/day, P=0.006 for trend). Different subgroups of ultra-processed food, including soft drinks, refined sweetened foods, salty snacks, and processed meat, each were associated with higher hazard ratios for IBD. Results were consistent for Crohn\u27s disease and ulcerative colitis with low heterogeneity. Intakes of white meat, red meat, dairy, starch, and fruit, vegetables, and legumes were not associated with incident IBD. CONCLUSIONS: Higher intake of ultra-processed food was positively associated with risk of IBD. Further studies are needed to identify the contributory factors within ultra-processed foods. STUDY REGISTRATION: ClinicalTrials.gov NCT03225586

Overall survival in the OlympiA phase III trial of adjuvant olaparib in patients with germline pathogenic variants in BRCA1/2 and high-risk, early breast cancer

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Effects of once-weekly exenatide on cardiovascular outcomes in type 2 diabetes

BACKGROUND: The cardiovascular effects of adding once-weekly treatment with exenatide to usual care in patients with type 2 diabetes are unknown. METHODS: We randomly assigned patients with type 2 diabetes, with or without previous cardiovascular disease, to receive subcutaneous injections of extended-release exenatide at a dose of 2 mg or matching placebo once weekly. The primary composite outcome was the first occurrence of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke. The coprimary hypotheses were that exenatide, administered once weekly, would be noninferior to placebo with respect to safety and superior to placebo with respect to efficacy. RESULTS: In all, 14,752 patients (of whom 10,782 [73.1%] had previous cardiovascular disease) were followed for a median of 3.2 years (interquartile range, 2.2 to 4.4). A primary composite outcome event occurred in 839 of 7356 patients (11.4%; 3.7 events per 100 person-years) in the exenatide group and in 905 of 7396 patients (12.2%; 4.0 events per 100 person-years) in the placebo group (hazard ratio, 0.91; 95% confidence interval [CI], 0.83 to 1.00), with the intention-to-treat analysis indicating that exenatide, administered once weekly, was noninferior to placebo with respect to safety (P<0.001 for noninferiority) but was not superior to placebo with respect to efficacy (P=0.06 for superiority). The rates of death from cardiovascular causes, fatal or nonfatal myocardial infarction, fatal or nonfatal stroke, hospitalization for heart failure, and hospitalization for acute coronary syndrome, and the incidence of acute pancreatitis, pancreatic cancer, medullary thyroid carcinoma, and serious adverse events did not differ significantly between the two groups. CONCLUSIONS: Among patients with type 2 diabetes with or without previous cardiovascular disease, the incidence of major adverse cardiovascular events did not differ significantly between patients who received exenatide and those who received placebo