Epigenetic Modulation in Parkinson’s Disease and Potential Treatment Therapies

In the recent past, huge emphasis has been given to the epigenetic alterations of the genes responsible for the cause of neurological disorders. Earlier, the scientists believed somatic changes and modifications in the genetic makeup of DNA to be the main cause of the neurodegenerative diseases. With the increase in understanding of the neural network and associated diseases, it was observed that alterations in the gene expression were not always originated by the change in the genetic sequence. For this reason, extensive research has been conducted to understand the role of epigenetics in the pathophysiology of several neurological disorders including Alzheimer’s disease, Parkinson’s disease and, Huntington’s disease. In a healthy person, the epigenetic modifications play a crucial role in maintaining the homeostasis of a cell by either up-regulating or down-regulating the genes. Therefore, improved understanding of these modifications may provide better insight about the diseases and may serve as potential therapeutic targets for their treatment. The present review describes various epigenetic modifications involved in the pathology of Parkinson’s Disease (PD) backed by multiple researches carried out to study the gene expression regulation related to the epigenetic alterations. Additionally, we will briefly go through the current scenario about the various treatment therapies including small molecules and multiple phytochemicals potent enough to reverse these alterations and the future directions for a better management of PD.

     

Evaluation of the potential for sexual reproduction in field populations of Cercospora beticola from USA

Cercospora leaf spot, caused by the hemibiotrophic fungal pathogen Cercospora beticola, is the most economically damaging foliar disease of sugarbeet worldwide. Although most C. beticola populations display characteristics reminiscent of sexual recombination, no teleomorph has been described. To assess whether populations in northern United States have characteristics consistent with sexual reproduction, 1024 isolates collected over a 3-y period were analyzed for frequency and distribution of mating type genes. After clone correction, an approximately equal distribution of mating types was found for each sampling year. Mating type frequency was also assessed in individual lesions. Lesions always consisted of isolates with a single mating type and microsatellite haplotype, but both mating types and up to five microsatellite haplotypes could be found on an individual leaf. The MAT1-1-1 and MAT1-2-1 genes were sequenced from 28 MAT1-1 and 28 MAT1-2 isolates, respectively. Three MAT1-1-1 nucleotide haplotypes were identified that encoded a single amino acid sequence. For MAT1-2-1, five nucleotide haplotypes were identified that encoded four protein variants. MAT1-1-1 and MAT1-2-1 gene expression analyses were conducted on plants inoculated with either or both mating types. MAT1-1-1 expression remained low, but MAT1-2-1 spiked during late stages of colonization. A segment of the MAT1-2-1 coding sequence was also found in MAT1-1 isolates. Taken together, these results suggest that C. beticola has the potential for sexual reproduction.     

Methodology for the formation of functional, cell-based cardiac pressure generation constructs in vitro

We have previously described a model to engineer three-dimensional (3-D) heart muscle in vitro. In the current study, we extend our model of 3-D heart muscle to engineer a functional cell-based cardiac pressure generating construct (CPGC). Tubular constructs were fabricated utilizing a phase separation method with chitosan as the scaffolding material. Primary cardiac cells isolated from rat hearts were plated on the surface of fibrin gels cast in 35 mm tissue culture dishes. CPGCs (N = 8) were formed by anchoring the tubular constructs to the center of the plate with primary cardiac cells seeded in fibrin gels wrapped around the tubular constructs. Intraluminal pressure measurements were evaluated with and without external electrical stimulation and histological evaluation performed. The fibrin gel spontaneously compacted due to the traction force of the cardiac cells. By 14 d after original cell plating, the cardiac cells had completely formed a monolayer around the tubular construct resulting in the formation of a cell-based CPGC. The spontaneous contractility of the CPGC was macroscopically visible and resulted in intraluminal pressure spikes of 0.08 mmHg. Upon electrical stimulation, the CPGCs generated twitch pressures of up to 0.05 mmHg. In addition, the CPGC constructs were electrically paced at frequencies of up to 3 Hz. Histological evaluation showed the presence of a continuous cell monolayer around the surface of the tubular construct. In this study, we describe a novel in vitro method to engineer functional cell-based CPGCs and demonstrate several physiological metrics of functional performance.     

Effect of streptomycin on the active force of bioengineered heart muscle in response to controlled stretch

In this study, we describe a bioreactor system to deliver controlled stretch protocols to bioengineered heart muscle (BEHMs) and test the system when streptomycin (an aminoglycoside antibiotic, which blocks stretch-activated channels) is either added to or excluded from the culture medium. Streptomycin is a very commonly used component of cell culture antibiotic-antimycotic media additives, so its effects on muscle development and functional response to mechanical signals in vitro is worthy of investigation. Our hypothesis is that BEHMs will not adapt to the applied mechanical stretch protocol when streptomycin is present in the culture medium, but will do so when streptomycin is excluded. Bioengineered heart muscles were formed by culturing primary neonatal cardiac myocytes in a fibrin gel using a method previously developed in our laboratory. A custom bioreactor system was designed using SolidWorks and structural components manufactured using fusion deposition modeling. We utilized a stretch protocol of 1 Hz, 10% strain for 7 d. BEHMs were stretched in the presence and absence of streptomycin. As controls, BEHMs were maintained in a cell culture incubator with and without streptomycin. The contractile properties of all BEHMs were evaluated to determine the active force. We were able to demonstrate compatibility of the bioreactor system with BEHMs and were able to stretch 58 constructs with zero incidence of failure. When the BEHMs were stretched in the absence of streptomycin, the active force increased from a mean value of 51.7 +/- 5.6 (N = 10) to 102.4 +/- 16.3 muN (N = 10), with p < 0.05. However, BEHMs that were stretched in the presence of streptomycin did not show any significant increase in active force generation. The average active force of BEHMs increased from a mean value of 57.6 +/- 10.2 (N = 10) to 91.4 +/- 19.8 muN (N = 10) when stretched in the presence of streptomycin. In this study, we demonstrate compatibility of the a bioreactor system with BEHMs, stability of the BEHMs in response to stretch protocols, and significant functional improvement in response to controlled stretch only when streptomycin is excluded from the culture medium, supporting our hypothesis.     

The interactions of metal cations and oxyanions with protein tyrosine phosphatase 1B

Protein tyrosine phosphatases are not considered to be metalloenzymes. Yet, they are inhibited by zinc cations and metal and non-metal oxyanions that are chemical analogues of phosphate, e.g. vanadate. Metal inhibition is generally not recognized as these enzymes are purified, supplied, and assayed with buffers containing chelating and reducing agents. We screened a series of cations and anions for their capacity to inhibit protein tyrosine phosphatase 1B and discuss the ensuing general issues with inhibition constants reported in the scientific literature. In contrast to zinc, which binds to the phosphocysteine intermediate in the closed conformation of protein tyrosine phosphatase 1B when the catalytic aspartate has moved into the active site, other divalent cations such as cadmium and copper may also bind to the enzyme in the open conformation. Inhibition by both anions and cations, conditions such as pH, the presence of metal ligands such as glutathione, and the existence of multiple conformational states of protein tyrosine phosphatases in the reaction cycle establish a complex pattern of inhibition of these important regulatory enzymes with implications for the physiology, pharmacology and toxicology of metal ions.     

Variable optimization for the formation of three-dimensional self-organized heart muscle

Cardiac tissue-engineering research is focused on the development of functional three-dimensional (3D) heart muscle in vitro. These models allow the detailed study of critical events in organogenesis, such as the establishment of cell–cell communication and construction and modification of the extracellular matrix. We have previously described a model for 3D heart muscle, termed cardioids, formed by the spontaneous delamination of a cohesive monolayer of primary cells in the absence of any synthetic scaffolding material. In an earlier publication, we have shown that, upon electrical stimulation, cardioids generate a twitch force in the range of 200–300 μN, generate a specific force (twitch force normalized to total cross-sectional area) of 2–4 kN/m², and can be electrically paced at frequencies of up to 10 Hz without any notable fatigue. We have two objectives for the current study: model development and model optimization. Our model development efforts are focused on providing additional characterization of the cardioid model. In this study, we show for the first time that cardioids show a pattern of gene expression comparable to that of cells cultured in two dimensions on tissue culture plastic and normal mammalian heart muscle. Compared with primary cardiac cells cultured on tissue culture plastic, the expression of α-myosin heavy chain (MHC), β-MHC, SERCA2, and phospholamban was significantly higher in cardioids. Our second objective, model optimization, is focused on evaluating the effect of several cell culture variables on cardioid formation and function. Specifically, we looked at the effect of plating density (1.0–4.0 × 10⁶ cells per cardioid), concentration of two adhesion proteins (laminin at 0.2–2.0 μg/cm² and fibronectin at 1–10 μg/cm²), myocyte purity (using preplating times of 15 and 60 min), and ascorbic acid stimulation (1–100 μl/ml). For our optimization studies, we utilized twitch force in response to electrical stimulation as our endpoint metric. Based on these studies, we found that cardioids formed with a plating density in the range 3–4 × 10⁶ cells per cardioid generated the maximum twitch force, whereas increasing the surface adhesion protein (using either laminin or fibronectin) and increasing the myocyte purity both resulted in a decrease in twitch force. In addition, increasing the ascorbic acid concentration resulted in an increase in the baseline force of cardioids, which was recorded in the absence of electrical stimulation. Based on the model development studies, we have shown that cardioids do indeed exhibit a gene expression pattern similar to normal mammalian heart muscle. This provides further validity for the cardioid model. Based on the model optimization studies, we have identified specific cell culture regimes which support cardioid formation and function. These results are specific to the cardioid model; however, they may be translated and applied to other tissue-engineering models. Collectively, the work described in this study provides insight into the formation of functional 3D heart muscle and the effect of several cell culture variables on tissue formation and function.     

Effect of thyroid hormone on the contractility of self-organized heart muscle

Tissue-engineered heart muscle may provide an alternative treatment modality for end-stage congestive heart failure. We have previously described a method to engineer contractile heart muscle in vitro (termed cardioids). This study describes a method to improve the contractile properties of cardioids utilizing thyroid hormone (T3) stimulation. Cardioids were engineered by promoting the self-organization of primary neonatal cardiac cells into a contractile tissue construct. Cardioids were maintained in standard cell culture media supplemented with varying concentrations of T3 in the range 1-5ng/ml. The contractile properties of the cardioids were evaluated 48h after formation. Stimulation with T3 resulted in an increase in the specific force of cardioids from an average value of 0.52 +/- 0.16kPa (N = 6) for control cardioids to 2.42 +/- 0.29kPa (N = 6) for cardioids stimulated with 3ng/ml T3. In addition, there was also an increase in the rate of contraction and relaxation in response to T3 stimulation. Cardioids that were stimulation with T3 exhibited improved pacing characteristics in response to electrical pacing at 1-5Hz and an increase in the degree of spontaneous contractility. Changes in the gene expression of SERCA2, phospholamban, alpha-myosin heavy chain, and beta-myosin heavy chain correlated with the changes in contractile properties. This study demonstrates the modulation of the contractile properties of tissue-engineered heart muscle using T3 stimulation.     

Cardiac cells implanted into a cylindrical,vascularized chamber in vivo: pressure generation and morphology

We have previously described a model to implant dissociated cells into a cylindrical, vascularized bed in vivo to promote the formation of functional cardiac muscle constructs. We now investigate the cellular organization and the ability of the constructs to generate intra-luminal pressure. Primary cardiac cells were isolated from hearts of 2–3 day old rats, suspended in fibrin gel and inserted into the lumen of silicone tubing. The silicone tubing was then implanted around the femoral vessels in the groin region of recipient animals. After 3 weeks, the constructs were harvested, placed in an in vitro bath and cannulated via the incorporated femoral artery with a pressure transducer for evaluation of intra-luminal pressure dynamics. Histological evaluation showed the presence of a concentric ring of cardiac cells surrounding the femoral vessels. There was also a significant amount of collagen present around cardiac cells. In addition, we observed a significant amount of neovascularization of the explanted constructs. Electron microscopy showed the presence of longitudinally aligned fibers with a large number of gap junctions. Upon electrical stimulation of a single pulse (7 V, 1.2 ms), the constructs generated an intra-luminal pressure of 1.19 ± 0.45 mmHg (n = 6). In addition, we were able to electrically pace the constructs at frequencies of 0.5–5 Hz. A Starling behavior of the inverse relation between baseline pressure and twitch pressure was observed. Cardiac cells implanted for 3 weeks into the cylindrical vascularized bed formed a tissue construct that demonstrated many of the contractile properties and morphology expected of functioning cardiac tissues.     

Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction

Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly.