Faculty Bibliography

This protocol describes how to prepare rat liver rough microsomes that contain undegraded membrane-bound polysomes and can function very well in an in vitro translation system. It uses endogenous ribonuclease inhibitor in all steps, avoiding pelleting rough microsomes in all steps and sacrificing good recovery.

miRNAs have emerged as important regulators of lipoprotein metabolism. Work over the past few years has demonstrated that miRNAs control the expression of most of the genes associated with high-density lipoprotein (HDL) metabolism, including the ATP transporters, ABCA1 and ABCG1, and the scavenger receptor SRB1. These findings strongly suggest that miRNAs regulate HDL biogenesis, cellular cholesterol efflux and HDL cholesterol (HDL-C) uptake in the liver, thereby controlling all of the steps of reverse cholesterol transport. Recent work in animal models has demonstrated that manipulating miRNA levels including miR-33 can increase circulating HDL-C. Importantly, antagonizing miR-33 in vivo enhances the regression and reduces the progression of atherosclerosis. These findings support the idea of developing miRNA inhibitors for the treatment of dyslipidaemia and related cardiovascular disorders such as atherosclerosis. This review article focuses on how HDL metabolism is regulated by miRNAs and how antagonizing miRNA expression could be a potential therapy for treating cardiometabolic diseases.

Lewy bodies, a pathological hallmark of Parkinson's disease (PD), contain aggregated alpha-synuclein (alphaSyn), which is found in several modified forms and can be discovered phosphorylated, ubiquitinated and truncated. Aggregation-prone truncated species of alphaSyn caused by aberrant cleavage of this fibrillogenic protein are hypothesized to participate in its sequestration into inclusions subsequently leading to synaptic dysfunction and neuronal death. Here, we investigated the role of calpain cleavage of alphaSyn in vivo by generating two opposing mouse models. We crossed into human [A30P]alphaSyn transgenic (i) mice deficient for calpastatin, a calpain-specific inhibitor, thus enhancing calpain activity (SynCAST(-)) and (ii) mice overexpressing human calpastatin leading to reduced calpain activity (SynCAST(+)). As anticipated, a reduced calpain activity led to a decreased number of alphaSyn-positive aggregates, whereas loss of calpastatin led to increased truncation of alphaSyn in SynCAST(-). Furthermore, overexpression of calpastatin decreased astrogliosis and the calpain-dependent degradation of synaptic proteins, potentially ameliorating the observed neuropathology in [A30P]alphaSyn and SynCAST(+) mice. Overall, our data further support a crucial role of calpains, particularly of calpain 1, in the pathogenesis of PD and in disease-associated aggregation of alphaSyn, indicating a therapeutic potential of calpain inhibition in PD.

The Unfolded Protein Response of the endoplasmic reticulum (UPRER) controls proteostasis by adjusting the protein folding capacity of the ER to environmental and cell-intrinsic conditions. In metazoans, loss of proteostasis results in degenerative and proliferative diseases and cancers. The cellular and molecular mechanisms causing these phenotypes remain poorly understood. Here we show that the UPRER is a critical regulator of intestinal stem cell (ISC) quiescence in Drosophila melanogaster. We find that ISCs require activation of the UPRER for regenerative responses, but that a tissue-wide increase in ER stress triggers ISC hyperproliferation and epithelial dysplasia in aging animals. These effects are mediated by ISC-specific redox signaling through Jun-N-terminal Kinase (JNK) and the transcription factor CncC. Our results identify a signaling network of proteostatic and oxidative stress responses that regulates ISC function and regenerative homeostasis in the intestinal epithelium.

As first demonstrated in the patient H.M., the hippocampus is critically involved in forming episodic memories, the recall of "what" happened "where" and "when." In rodents, the clearest functional correlate of hippocampal primary neurons is the place field: a cell fires predominantly when the animal is in a specific part of the environment, typically defined relative to the available visuospatial cues. However, rodents have relatively poor visual acuity. Furthermore, they are highly adept at navigating in total darkness. This raises the question of how other sensory modalities might contribute to a hippocampal representation of an environment. Rodents have a highly developed olfactory system, suggesting that cues such as odor trails may be important. To test this, we familiarized mice to a visually cued environment over a number of days while maintaining odor cues. During familiarization, self-generated odor cues unique to each animal were collected by re-using absorbent paperboard flooring from one session to the next. Visual and odor cues were then put in conflict by counter-rotating the recording arena and the flooring. Perhaps surprisingly, place fields seemed to follow the visual cue rotation exclusively, raising the question of whether olfactory cues have any influence at all on a hippocampal spatial representation. However, subsequent removal of the familiar, self-generated odor cues severely disrupted both long-term stability and rotation to visual cues in a novel environment. Our data suggest that odor cues, in the absence of additional rule learning, do not provide a discriminative spatial signal that anchors place fields. Such cues do, however, become integral to the context over time and exert a powerful influence on the stability of its hippocampal representation.

Stem cells are attractive candidates for the development of novel therapies, targeting indications that involve functional restoration of defective tissue. Although most stem cell therapies are new and highly experimental, there are clinics around the world that exploit vulnerable patients with the hope of offering supposed stem cell therapies, many of which operate without credible scientific merit, oversight, or other patient protection.We review the potential, as well as drawbacks, for incorporation of stem cells in cosmetic procedures. A review of FDA-approved indications and ongoing clinical trials with adipose stem cells is provided. Furthermore, a "snapshot" analysis of websites using the search terms "stem cell therapy" or "stem cell treatment" or "stem cell facelift" was performed.Despite the protective net cast by regulatory agencies such as the FDA and professional societies such as the American Society of Plastic Surgeons, we are witnessing worrying advertisements for procedures such as stem cell facelifts, stem cell breast augmentations, and even stem cell vaginal rejuvenation. The marketing and promotion of stem cell procedures in aesthetic surgery is not adequately supported by clinical evidence in the majority of cases.Stem cells offer tremendous potential, but the marketplace is saturated with unsubstantiated and sometimes fraudulent claims that may place patients at risk. With plastic surgeons at the forefront of stem cell-based regenerative medicine, it is critically important that we provide an example of a rigorous approach to research, data collection, and advertising of stem cell therapies.

OBJECTIVE: To examine the expression of ADAMTS-7 during the progression of osteoarthritis (OA), defining its role in the pathogenesis of OA, and elucidating the molecular events involved. METHODS: ADAMTS-7 expression in cartilage of a rat OA model was assayed using immunohistochemistry. Cartilage-specific ADAMTS-7 transgenic mice and ADAMTS-7 small interfering (si)RNA knockdown mice were generated and used to analyse OA progression in both spontaneous and surgically induced OA models. Cartilage degradation and OA was evaluated using Safranin-O staining, immunohistochemistry, ELISA and western blotting. In addition, mRNA expression of tumour necrosis factor (TNF)-alpha and metalloproteinases known to be involved in cartilage degeneration in OA was analysed. Furthermore, the transactivation of ADAMTS-7 by TNF-alpha and its downstream NF-kappaB signalling was measured using reporter gene assay. RESULTS: ADAMTS-7 expression was elevated during disease progression in the surgically induced rat OA model. Targeted overexpression of ADAMTS-7 in chondrocytes led to chondrodysplasia characterised by short-limbed dwarfism and a delay in endochondral ossification in 'young mice' and a spontaneous OA-like phenotype in 'aged' mice. In addition, overexpression of ADAMTS-7 led to exaggerated breakdown of cartilage and accelerated OA progression, while knockdown of ADAMTS-7 attenuated degradation of cartilage matrix and protected against OA development, in surgically induced OA models. ADAMTS-7 upregulated TNF-alpha and metalloproteinases associated with OA; in addition, TNF-alpha induced ADAMTS-7 through NF-kappaB signalling. CONCLUSIONS: ADAMTS-7 and TNF-alpha form a positive feedback loop in the regulation of cartilage degradation and OA progression, making them potential molecular targets for prevention and treatment of joint degenerative diseases, including OA.

BACKGROUND: & Aims: Subsets of leukocytes synergize with regenerative growth factors to promote hepatic regeneration. gammadeltaTau cells are early responders to inflammation-induced injury in a number of contexts. We investigated the role of gammadeltaTau cells in hepatic regeneration using mice with disruptions in Tcrd (encodes the T cell receptor delta chain) and Clec7a (encodes C-type lectin domain family 7 member a, also known as DECTIN1). METHODS: We performed partial hepatectomies on wild-type C57BL/6, CD45.1, Tcrd-/-, or Clec7a-/- mice. Cells were isolated from livers of patients and mice via mechanical and enzymatic digestion. gammadeltaTau cells were purified by fluorescence-activated cell sorting. RESULTS: In mice, partial hepatectomy upregulated expression of CCL20 and ligands of Dectin-1, associated with recruitment and activation of gammadeltaTau cells and their increased production of interleukin (IL)17 family cytokines. Recruited gammadeltaTau cells induced production of IL6 by antigen-presenting cells and suppressed expression of interferon gamma by natural killer T cells, promoting hepatocyte proliferation. Absence of IL17-producing gammadeltaTau cells or deletion of Dectin-1 prevented development of regenerative phenotypes in subsets of innate immune cells. This slowed liver regeneration and was associated with reduced expression of regenerative growth factors and cell cycle regulators. Conversely, exogenous administration of IL17 family cytokines or Dectin-1 ligands promoted regeneration. More broadly, we found that gammadeltaTau cells are required for inflammatory responses mediated by IL17 and Dectin-1. CONCLUSIONS: gammadeltaT cells regulate hepatic regeneration by producing IL22 and IL17, which have direct mitogenic effects on hepatocytes and promote a regenerative phenotype in hepatic leukocytes, respectively. Dectin-1 ligation is required for gammadeltaT cells to promote hepatic regeneration.

BACKGROUND: Animal spinal cord injury (SCI) models have proved invaluable in better understanding the mechanisms involved in traumatic SCI and evaluating the effectiveness of experimental therapeutic interventions. Over the past 25 years, substantial gains have been made in developing consistent, reproducible and reliable animal SCI models. STUDY DESIGN: Review. OBJECTIVE: The objective of this review was to consolidate current knowledge on SCI models and introduce newer paradigms that are currently being developed. RESULTS: SCI models are categorized based on the mechanism of injury into contusion, compression, distraction, dislocation, transection or chemical models. Contusion devices inflict a transient, acute injury to the spinal cord using a weight-drop technique, electromagnetic impactor or air pressure. Compression devices compress the cord at specific force and duration to cause SCI. Distraction SCI devices inflict graded injury by controlled stretching of the cord. Mechanical displacement of the vertebrae is utilized to produce dislocation-type SCI. Surgical transection of the cord, partial or complete, is particularly useful in regenerative medicine. Finally, chemically induced SCI replicates select components of the secondary injury cascade. Although rodents remain the most commonly used species and are best suited for preliminary SCI studies, large animal and nonhuman primate experiments better approximate human SCI. CONCLUSION: All SCI models aim to replicate SCI in humans as closely as possible. Given the recent improvements in commonly used models and development of newer paradigms, much progress is anticipated in the coming years.

Embryonic heart formation requires the production of an appropriate number of cardiomyocytes; likewise, cardiac regeneration following injury relies upon the recovery of lost cardiomyocytes. The basic helix-loop-helix (bHLH) transcription factor Hand2 has been implicated in promoting cardiomyocyte formation. It is unclear, however, whether Hand2 plays an instructive or permissive role during this process. Here, we find that overexpression of hand2 in the early zebrafish embryo is able to enhance cardiomyocyte production, resulting in an enlarged heart with a striking increase in the size of the outflow tract. Our evidence indicates that these increases are dependent on the interactions of Hand2 in multimeric complexes and are independent of direct DNA binding by Hand2. Proliferation assays reveal that hand2 can impact cardiomyocyte production by promoting division of late-differentiating cardiac progenitors within the second heart field. Additionally, our data suggest that hand2 can influence cardiomyocyte production by altering the patterning of the anterior lateral plate mesoderm, potentially favoring formation of the first heart field at the expense of hematopoietic and vascular lineages. The potency of hand2 during embryonic cardiogenesis suggested that hand2 could also impact cardiac regeneration in adult zebrafish; indeed, we find that overexpression of hand2 can augment the regenerative proliferation of cardiomyocytes in response to injury. Together, our studies demonstrate that hand2 can drive cardiomyocyte production in multiple contexts and through multiple mechanisms. These results contribute to our understanding of the potential origins of congenital heart disease and inform future strategies in regenerative medicine.