Improved osteogenic vector for non-viral gene therapy

Therapeutic compensation of deficient bone regeneration is a challenging task and a topic of on-going search for novel treatment strategies. One promising approach for improvement involves non-viral gene delivery using the bone morphogenetic protein-2 (BMP-2) gene to provide transient, local and sustained expression of the growth factor. However, since efficiency of non-viral gene delivery is low, this study focused on the improvement of a BMP-2 gene expression system, aiming for compensation of poor transfection efficiency. First, the native BMP-2 gene sequence was modified by codon optimisation and altered by inserting a highly truncated artificial intron (96 bp). Transfection of multiple cell lines and rat adipose-derived mesenchymal stem cells with plasmids harbouring the improved BMP-2 sequence led to a several fold increased expression rate and subsequent osteogenic differentiation. Additionally, comparing expression kinetics of elongation factor 1 alpha (EF1α) promoter with a state of the art CMV promoter revealed significantly higher BMP-2 expression when under the influence of the EF1α promoter. Results obtained by quantification of bone markers as well as osteogenic assays showed reduced sensitivity to promoter silencing effects of the EF1α promoter in rat adipose-derived mesenchymal stem cells. Finally, screening of several protein secretion signals using either luciferase or BMP-2 as reporter protein revealed no superior candidates for potential replacement of the native BMP-2 secretion signal. Taken together, by enhancing the exogenous BMP-2 expression system, low transfection efficiencies in therapeutic applications can be compensated, making safe non-viral systems even more suitable for tissue regeneration approaches

The False Idea of Human Nature’s Duality in \u3cem\u3eStrange Case of Dr. Jekyll and Mr. Hyde\u3c/em\u3e

Dr. Jekyll and Mr. Hyde. The mention of these two people will evoke a specific thought in most people’s minds: good and evil; light and dark; normal and deformed. However, the truth isn’t nearly so simple. Humans are free, and therefore a mixture of good and evil; accordingly, both Dr. Jekyll and Mr. Hyde are neither solely good nor evil. Due to the stifling sexual and social repression in Victorian society, Jekyll became convinced that he could somehow purge himself of the evil inside him by splitting his soul; this, however, had far graver consequences than he suspected. Due to his experiment, Jekyll caused a tragic amount of human suffering and even death, culminating in his own complete destruction. What he failed to understand and what I propose in this paper is that human nature can never be truly dual; all parts of a human’s soul are intrinsically tied to each other, and attempts to split them result in psychological catastrophe

Experimental Designs for Precision Health & Medicine

Randomized trials investigating the efficacy of a single treatment compared to a placebo,quantified by the average treatment effect (ATE), have long been the “gold standard” inevidence-based medicine (EBM). While profoundly successful, these trials provide guidancethat is better suited for regulatory rather than clinical decision making because the ATEdoesn’t consider the characteristics of individual patients. Precision medicine attempts tobridge this gap through a data-driven approach to the long-standing medical practice oftailoring treatment according to patient characteristics. Experimental designs can offer hopeto provide evidence supporting causally-valid decision making without sacrificing EBM’srigor by replacing the need for strong assumptions with randomization. In this dissertation,we detail the design of two such trials, the Biomarkers for Evaluating Spine Treatments (BEST)and the North Carolina Works for Health (NCW4H) studies, and present preliminary workon a third experimental design. We first present the statistical design of the BEST trial, a two-stage sequential multipleassignment randomized trial (SMART) comparing four active treatments that will inform aprecision medicine approach to chronic lower back pain (cLBP). We extend the minimiza-tion method for balancing covariates across treatments to accommodate contraindications,propose an analog for “power” when the primary aim is to estimate a dynamic treatmentregime (DTR), and conduct sample size simulations. We then present the NCW4H study investigating a multi-level intervention (MLI) toreduce chronic disease risks in socioeconomically disadvantaged, unemployed populations.Existing designs for MLIs focus on individually randomized factorial designs, which may beinfeasible due to logistical or other concerns, and cluster-randomized experiments whichcan pose identifiability challenges and be less efficient. We propose a design that combinesindividual-level randomization with a stepped wedge design at the cluster level. We derive aGEE-based power formula and provide an R package for sample size calculation. Finally, we present a sequential design for when the goal is to estimate a DTR and theexpected treatment response is a linear function of a patient’s characteristics . We proposeremoving suboptimal treatments based on changes in the value function thereby implicitlyaddressing potential subgroups with higher treatment response.Doctor of Philosoph

Multilevel Intervention Stepped Wedge Designs (MLI-SWDs)

Multilevel interventions (MLIs) hold promise for reducing health inequities by intervening at multiple types of social determinants of health consistent with the socioecological model of health. In spite of their potential, methodological challenges related to study design compounded by a lack of tools for sample size calculation inhibit their development. We help address this gap by proposing the Multilevel Intervention Stepped Wedge Design (MLI-SWD), a hybrid experimental design which combines cluster-level (CL) randomization using a Stepped Wedge design (SWD) with independent individual-level (IL) randomization. The MLI-SWD is suitable for MLIs where the IL intervention has a low risk of interference between individuals in the same cluster, and it enables estimation of the component IL and CL treatment effects, their interaction, and the combined intervention effect. The MLI-SWD accommodates cross-sectional and cohort designs as well as both incomplete (clusters are not observed in every study period) and complete observation patterns. We adapt recent work using generalized estimating equations for SWD sample size calculation to the multilevel setting and provide an R package for power and sample size calculation. Furthermore, motivated by our experiences with the ongoing NC Works 4 Health study, we consider how to apply the MLI-SWD when individuals join clusters over the course of the study. This situation arises when unemployment MLIs include IL interventions that are delivered while the individual is unemployed. This extension requires carefully considering whether the study interventions will satisfy additional causal assumptions but could permit randomization in new settings

An embryonic stem cell–like gene expression signature in poorly differentiated aggressive human tumors

Cancer cells possess traits reminiscent of those ascribed to normal stem cells. It is unclear, however, whether these phenotypic similarities reflect the activity of common molecular pathways. Here, we analyze the enrichment patterns of gene sets associated with embryonic stem (ES) cell identity in the expression profiles of various human tumor types. We find that histologically poorly differentiated tumors show preferential overexpression of genes normally enriched in ES cells, combined with preferential repression of Polycomb-regulated genes. Moreover, activation targets of Nanog, Oct4, Sox2 and c-Myc are more frequently overexpressed in poorly differentiated tumors than in well-differentiated tumors. In breast cancers, this ES-like signature is associated with high-grade estrogen receptor (ER)-negative tumors, often of the basal-like subtype, and with poor clinical outcome. The ES signature is also present in poorly differentiated glioblastomas and bladder carcinomas. We identify a subset of ES cell-associated transcription regulators that are highly expressed in poorly differentiated tumors. Our results reveal a previously unknown link between genes associated with ES cell identity and the histopathological traits of tumors and support the possibility that these genes contribute to stem cell–like phenotypes shown by many tumors

Enantioselective nickel-catalyzed intramolecular allylic alkenylations enabled by reversible alkenylnickel E/Z isomerization

Enantioselective nickel-catalyzed arylative cyclizations of substrates containing a Z-allylic phosphate tethered to an alkyne are described. These reactions give multisubstituted chiral aza- and carbocycles, and are initiated by the addition of an arylboronic acid to the alkyne, followed by cyclization of the resulting alkenylnickel species onto the allylic phosphate. The reversible E/Z isomerization of the alkenylnickel species is essential for the success of the reactions