Exosomal MicroRNA Signatures in Central Nervous System Diseases

During the last decade there has been a growing interest in studying extracellular vesicles, in particular exosomes and their miRNA contents. Exosomes are released by almost all cell types. They are packed with specific information, stable against degradation processes, are small and flexible enough to cross the blood-brain barrier (BBB), and are readily found in biological fluids including blood. MicroRNAs (miRNAs) are involved in nearly every cellular process and play a regulatory role in central nervous system (CNS) associated diseases. Accordingly, exosomal miRNAs could be ideal biomarkers to measure CNS disease activity and treatment response. In this thesis, the aim was to establish a robust protocol to investigate whether the differential expression of serum exosomal miRNA can be used as a biomarker for the accurate diagnosis of the CNS diseases multiple sclerosis (MS) and glioblastoma multiforme (GBM), as well as for the monitoring of disease progression and treatment response. Exosomes were purified from serum and their RNA contents profiled using highthroughput sequencing. In my first study, I profiled exosome–associated miRNAs in serum samples from MS patients and identified distinct biomarkers for the diagnosis of MS and identification of the disease subtype. In my second study, I investigated the effect of treatment in MS patients. I hypothesised that the deregulation of serum exosomal miRNAs is associated with the efficacy of therapy and is predictive of MS activity phases. Finally, I studied serum exosomal miRNA profiles to discover diagnostic biomarkers for GBM, and to demonstrate the applicability of my protocol to other neurological diseases. Taken together, my results demonstrate the exceptional utility of serum exosomal miRNA profiles as a blood-based biomarker to diagnose the CNS associated diseases, using a robust and easily reproducible protocol

Approaches to in vitro tissue regeneration with application for human disease modeling and drug development

Reliable in vitro human disease models that capture the complexity of in vivo tissue behaviors are crucial to gain mechanistic insights into human disease and enable the development of treatments that are effective across broad patient populations. The integration of stem cell technologies, tissue engineering, emerging biomaterials strategies and microfabrication processes, as well as computational and systems biology approaches, is enabling new tools to generate reliable in vitro systems to study the molecular basis of human disease and facilitate drug development. In this review, we discuss these recently developed tools and emphasize opportunities and challenges involved in combining these technologies toward regenerative science.National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant 5R01EB010246-02)National Center for Advancing Translational Sciences (U.S.) (Grant 1UH2TR000496)United States. Defense Advanced Research Projects Agency (Cooperative Agreement W911NF-12-2-0039

Biomechanical effects of lumbar fusion surgery on adjacent segments using musculoskeletal models of the intact, degenerated and fused spine

ABSTRACT: Adjacent segment disorders are prevalent in patients following a spinal fusion surgery. Postoperative alterations in the adjacent segment biomechanics play a role in the etiology of these conditions. While experimental approaches fail to directly quantify spinal loads, previous modeling studies have numerous shortcomings when simulating the complex structures of the spine and the pre/postoperative mechanobiology of the patient. The biomechanical effects of the L4–L5 fusion surgery on muscle forces and adjacent segment kinetics (compression, shear, and moment) were investigated using a validated musculoskeletal model. The model was driven by in vivo kinematics for both preoperative (intact or severely degenerated L4–L5) and postoperative conditions while accounting for muscle atrophies. Results indicated marked changes in the kinetics of adjacent L3–L4 and L5–S1 segments (e.g., by up to 115% and 73% in shear loads and passive moments, respectively) that depended on the preoperative L4–L5 disc condition, postoperative lumbopelvic kinematics and, to a lesser extent, postoperative changes in the L4–L5 segmental lordosis and muscle injuries. Upper adjacent segment was more affected post-fusion than the lower one. While these findings identify risk factors for adjacent segment disorders, they indicate that surgical and postoperative rehabilitation interventions should focus on the preservation/restoration of patient's normal segmental kinematics

Aag-initiated base excision repair promotes ischemia reperfusion injury in liver, brain, and kidney

Inflammation is accompanied by the release of highly reactive oxygen and nitrogen species (RONS) that damage DNA, among other cellular molecules. Base excision repair (BER) is initiated by DNA glycosylases and is crucial in repairing RONS-induced DNA damage; the alkyladenine DNA glycosylase (Aag/Mpg) excises several DNA base lesions induced by the inflammation-associated RONS release that accompanies ischemia reperfusion (I/R). Using mouse I/R models we demonstrate that Aag[superscript −/−] mice are significantly protected against, rather than sensitized to, I/R injury, and that such protection is observed across three different organs. Following I/R in liver, kidney, and brain, Aag[superscript −/−] mice display decreased hepatocyte death, cerebral infarction, and renal injury relative to wild-type. We infer that in wild-type mice, Aag excises damaged DNA bases to generate potentially toxic abasic sites that in turn generate highly toxic DNA strand breaks that trigger poly(ADP-ribose) polymerase (Parp) hyperactivation, cellular bioenergetics failure, and necrosis; indeed, steady-state levels of abasic sites and nuclear PAR polymers were significantly more elevated in wild-type vs. Aag[superscript −/−] liver after I/R. This increase in PAR polymers was accompanied by depletion of intracellular NAD and ATP levels plus the translocation and extracellular release of the high-mobility group box 1 (Hmgb1) nuclear protein, activating the sterile inflammatory response. We thus demonstrate the detrimental effects of Aag-initiated BER during I/R and sterile inflammation, and present a novel target for controlling I/R-induced injury.National Institutes of Health (U.S.) (Grant R01-CA055042)National Institutes of Health (U.S.) (Grant R01-CA149261)National Institutes of Health (U.S.) (Grant P30-ES02109)Ellison Medical Foundatio

An enhanced CRISPR repressor for targeted mammalian gene regulation.

The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor, but inefficiencies in target-gene silencing have limited its utility. Here we describe an improved Cas9 repressor based on the C-terminal fusion of a rationally designed bipartite repressor domain, KRAB-MeCP2, to nuclease-dead Cas9. We demonstrate the system's superiority in silencing coding and noncoding genes, simultaneously repressing a series of target genes, improving the results of single and dual guide RNA library screens, and enabling new architectures of synthetic genetic circuits

Metabolite profiling and pharmacokinetic evaluation of hydrocortisone in a perfused 3D human liver bioreactor

Endotoxin lipopolysaccharide (LPS) is known to cause liver injury primarily involving inflammatory cells such as Kupffer cells, but few in vitro culture models are applicable for investigation of inflammatory effects on drug metabolism. We have developed a 3D human microphysiological hepatocyte-Kupffer-cell coculture system and evaluated the anti-inflammatory effect of glucocorticoids on liver cultures. LPS was introduced to the cultures to elicit an inflammatory response and assessed by the release of pro-inflammatory cytokines, IL6 and TNFα. A sensitive and specific RP-UHPLC-QTOF-MS method was used to evaluate hydrocortisone disappearance and metabolism at near physiological levels. For this, the systems were dosed with 100 nM hydrocortisone and circulated for two days; hydrocortisone was depleted to approximately 30 nM, with first-order kinetics. Phase I metabolites, including tetrahydrocortisone and dihydrocortisol, accounted for 8-10 % of the loss, and 45-52 % was phase II metabolites, including glucuronides of tetrahydrocortisol and tetrahydrocortisone. Pharmacokinetic parameters, i.e., half-life (t1/2), rate of elimination (kel), clearance (CL), and area under the curve (AUC), were 23.03 h, 0.03 h-1, 6.6x10-5 L. h-1 and 1.03 mg/L*h respectively. The ability of the bioreactor to predict the in vivo clearance of hydrocortisone was characterized and the obtained intrinsic clearance values correlated with human data. This system offers a physiologically-relevant tool for investigating hepatic function in an inflamed liver. Endotoxin lipopolysaccharide (LPS) is known to cause liver injury primarily involving inflammatory cells such as Kupffer cells, but few in vitro culture models are applicable for investigation of inflammatory effects on drug metabolism. We have developed a 3D human microphysiological hepatocyte-Kupffer-cell coculture system and evaluated the anti-inflammatory effect of glucocorticoids on liver cultures. LPS was introduced to the cultures to elicit an inflammatory response and assessed by the release of pro-inflammatory cytokines, IL6 and TNFα. A sensitive and specific RP-UHPLC-QTOF-MS method was used to evaluate hydrocortisone disappearance and metabolism at near physiological levels. For this, the systems were dosed with 100 nM hydrocortisone and circulated for two days; hydrocortisone was depleted to approximately 30 nM, with first-order kinetics. Phase I metabolites, including tetrahydrocortisone and dihydrocortisol, accounted for 8-10 % of the loss, and 45-52 % was phase II metabolites, including glucuronides of tetrahydrocortisol and tetrahydrocortisone. Pharmacokinetic parameters, i.e., half-life (t[subscript 1/2]), rate of elimination (k[subscript el]), clearance (CL), and area under the curve (AUC), were 23.03 h, 0.03 h[superscript -1], 6.6x10[superscript -5] L. h-1 and 1.03 mg/L*h respectively. The ability of the bioreactor to predict the in vivo clearance of hydrocortisone was characterized and the obtained intrinsic clearance values correlated with human data. This system offers a physiologically-relevant tool for investigating hepatic function in an inflamed liver.United States. Defense Advanced Research Projects Agency (DARPA-BAA-11-73 Microphysiological Systems W911NF-12-2-0039)National Institutes of Health (U.S.) (5-UH2-TR000496)Massachusetts Institute of Technology. Center for Environmental Health Sciences (P30-ES002109

A Review on Segmentation of Knee Articular Cartilage: from Conventional Methods Towards Deep Learning

In this paper, we review the state-of-the-art approaches for knee articular cartilage segmentation from conventional techniques to deep learning (DL) based techniques. Knee articular cartilage segmentation on magnetic resonance (MR) images is of great importance in early diagnosis of osteoarthritis (OA). Besides, segmentation allows estimating the articular cartilage loss rate which is utilised in clinical practice for assessing the disease progression and morphological changes. Topics covered include various image processing algorithms and major features of different segmentation techniques, feature computations and the performance evaluation metrics. This paper is intended to provide researchers with a broad overview of the currently existing methods in the field, as well as to highlight the shortcomings and potential considerations in the application at clinical practice. The survey showed that the state-of-the-art techniques based on DL outperforms the other segmentation methods. The analysis of the existing methods reveals that integration of DL-based algorithms with other traditional model-based approaches have achieved the best results (mean Dice similarity cofficient (DSC) between 85:8% and 90%)

CRISPR transcriptional repression devices and layered circuits in mammalian cells

A key obstacle to creating sophisticated genetic circuits has been the lack of scalable device libraries. Here we present a modular transcriptional repression architecture based on clustered regularly interspaced palindromic repeats (CRISPR) system and examine approaches for regulated expression of guide RNAs in human cells. Subsequently we demonstrate that CRISPR regulatory devices can be layered to create functional cascaded circuits, which provide a valuable toolbox for engineering purposes.National Institutes of Health (U.S.) (Grant 5R01CA155320-04)National Institutes of Health (U.S.) (Grant P50 GM098792)Korea (South). Ministry of Science, Information and Communication Technolgy. Intelligent Synthetic Biology Center of Global Frontier Project (2013M3A6A8073557

Fluid shear stress modulation of hepatocyte like cell function

Freshly isolated human adult hepatocytes are considered to be the gold standard tool for in vitro studies. However, primary hepatocyte scarcity, cell cycle arrest and the rapid loss of cell phenotype limit their widespread deployment. Human embryonic stem cells and induced pluripotent stem cells provide renewable sources of hepatocyte-like cells (HLCs). Despite the use of various differentiation methodologies, HLCs like primary human hepatocytes exhibit unstable phenotype in culture. It has been shown that the functional capacity can be improved by adding back elements of human physiology, such as cell co-culture or through the use of natural and/or synthetic surfaces. In this study, the effect of fluid shear stress on HLC performance was investigated. We studied two important liver functions, cytochrome P450 drug metabolism and serum protein secretion, in static cultures and those exposed to fluid shear stress. Our study demonstrates that fluid shear stress improved Cyp1A2 activity by approximately fivefold. This was paralleled by an approximate ninefold increase in sensitivity to a drug, primarily metabolised by Cyp2D6. In addition to metabolic capacity, fluid shear stress also improved hepatocyte phenotype with an approximate fourfold reduction in the secretion of a foetal marker, alpha-fetoprotein. We believe these studies highlight the importance of introducing physiologic cues in cell-based models to improve somatic cell phenotype

Bioreactor technologies to support liver function in vitro

Liver is a central nexus integrating metabolic and immunologic homeostasis in the human body, and the direct or indirect target of most molecular therapeutics. A wide spectrum of therapeutic and technological needs drives efforts to capture liver physiology and pathophysiology in vitro, ranging from prediction of metabolism and toxicity of small molecule drugs, to understanding off-target effects of proteins, nucleic acid therapies, and targeted therapeutics, to serving as disease models for drug development. Here we provide perspective on the evolving landscape of bioreactor-based models to meet old and new challenges in drug discovery and development, emphasizing design challenges in maintaining long-term liver-specific function and how emerging technologies in biomaterials and microdevices are providing new experimental models.National Institutes of Health (U.S.) (R01 EB010246)National Institutes of Health (U.S.) (P50-GM068762-08)National Institutes of Health (U.S.) (R01-ES015241)National Institutes of Health (U.S.) (P30-ES002109)5UH2TR000496-02National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems (CBET-0939511)United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039