Recombinant Mitochondrial Transcription Factor A with N-terminal Mitochondrial Transduction Domain Increases Respiration and Mitochondrial Gene Expression in G11778A Leber's Hereditary Optic Neuropathy Cybrid Cells

Diseases involving mitochondrial defects usually manifest themselves in high-energy, post-mitotic tissues such as brain, retina, skeletal and cardiac muscle and frequently cause deficiencies in mitochondrial bioenergetics. We have developed a scalable procedure to produce recombinant human mitochondrial transcription factor A (TFAM) modified with an N-terminal protein transduction domain (PTD) and mitochondrial localization signal (MLS) that allow it to cross membranes and enter mitochondria through its "mitochondrial transduction domain" (MTD,=PTD+MLS). _In vitro_ studies in a classic mitochondrial disease cell model demonstrated that Alexa488-labeled MTD-TFAM rapidly entered the mitochondrial compartment. MTD-TFAM treatment of these cell lines reversibly increased oxygen consumption (respiration) rates 3-fold, levels of respiratory proteins and mitochondrial gene expression. _In vivo_ results demonstrated that respiration increased to lesser degrees in mitochondria from tissues of mice injected with MTD-TFAM. MTD-TFAM can alter mitochondrial bioenergetics and holds promise for treatment of mitochondrial diseases involving deficiencies of energy production

Design of Low Power Data Preserving Flip Flop Using MTCMOS Technique

In order to reduce overall power consumption, a well-known technique is to scale supply voltages. However, to maintain performance, device threshold voltages must scale as well, which will cause sub threshold leakage currents to increase exponentially. The sub threshold voltage has to affect the two parameters one is the delay and other one is the sub threshold leakage current. Smaller the threshold voltage smaller will be delay while larger will be the sub threshold current. Controlling sub threshold leakage has been explored significantly in the literature, especially in the context of reducing leakage currents in burst mode type circuits, where the system spends the majority of the time in an idle standby, or sleep, state where no computation is taking place. MTCMOS or multi-threshold CMOS has been proposed as a very effective technique for reducing leakage currents during the standby by state by utilizing high sleep devices to gate the power supplies of a low logic block. Although MTCMOS circuit techniques are effective for controlling leakage currents in combinational logic, a drawback is that it can cause internal nodes to float, and cannot be directly used in standard memory cells without corrupting stored data. As a result, several researchers have explored possible MTCMOS latch designs that can reduce leakage currents yet maintain state during the standby modes. In this work a data preserving flip flop with reduced leakage power is designed using MTCMOS technique in 90nm technology with the help of CADENCE tool. The simulation results have shown that the leakage power is reduced by 25.70% compared to CMOS flip flop

Epigenetic Modifications of the PGC-1α Promoter during Exercise Induced Expression in Mice

The transcriptional coactivator, PGC-1α, is known for its role in mitochondrial biogenesis. Although originally thought to exist as a single protein isoform, recent studies have identified additional promoters which produce multiple mRNA transcripts. One of these promoters (promoter B), approximately 13.7kb upstream of the canonical PGC-1α promoter (promoter A), yields alternative transcripts present at levels much lower than the canonical PGC-1α mRNA transcript. In skeletal muscle, exercise resulted in a substantial, rapid increase of mRNA of these alternative PGC-1α transcripts. Although the β2-adrenergic receptor was identified as a signaling pathway that activates transcription from PGC-1α promoter B, it is not yet known what molecular changes occur to facilitate PGC-1α promoter B activation following exercise. We sought to determine whether epigenetic modifications were involved in this exercise response in mouse skeletal muscle. We found that DNA hydroxymethylation correlated to increased basal mRNA levels from PGC-1α promoter A, but that DNA methylation appeared to play no role in the exercise-induced activation of PGC-1α promoter B. The level of the activating histone mark H3K4me3 increased with exercise 2–4 fold across PGC- 1α promoter B, but remained unaltered past the canonical PGC-1α transcriptional start site. Together, these data show that epigenetic modifications partially explain exercise-induced changes in the skeletal muscle mRNA levels of PGC-1α isoforms

Suppression of eukaryotic initiation factor 4E prevents chemotherapy-induced alopecia

BACKGROUND: Chemotherapy-induced hair loss (alopecia) (CIA) is one of the most feared side effects of chemotherapy among cancer patients. There is currently no pharmacological approach to minimize CIA, although one strategy that has been proposed involves protecting normal cells from chemotherapy by transiently inducing cell cycle arrest. Proof-of-concept for this approach, known as cyclotherapy, has been demonstrated in cell culture settings. METHODS: The eukaryotic initiation factor (eIF) 4E is a cap binding protein that stimulates ribosome recruitment to mRNA templates during the initiation phase of translation. Suppression of eIF4E is known to induce cell cycle arrest. Using a novel inducible and reversible transgenic mouse model that enables RNAi-mediated suppression of eIF4E in vivo, we assessed the consequences of temporal eIF4E suppression on CIA. RESULTS: Our results demonstrate that transient inhibition of eIF4E protects against cyclophosphamide-induced alopecia at the organismal level. At the cellular level, this protection is associated with an accumulation of cells in G1, reduced apoptotic indices, and was phenocopied using small molecule inhibitors targeting the process of translation initiation. CONCLUSIONS: Our data provide a rationale for exploring suppression of translation initiation as an approach to prevent or minimize cyclophosphamide-induced alopecia.1U01 CA168409 - NCI NIH HHS; P01 CA 87497 - NCI NIH HHS; P30 CA008748 - NCI NIH HHS; MOP-106530 - Canadian Institutes of Health Research; P01 CA013106 - NCI NIH HH

Mitochondrial DNA Copy Numbers in Pyramidal Neurons are Decreased and Mitochondrial Biogenesis Transcriptome Signaling is Disrupted in Alzheimer’s Disease Hippocampi

Alzheimer\u27s disease (AD) is the major cause of adult-onset dementia and is characterized in its pre-diagnostic stage by reduced cerebral cortical glucose metabolism and in later stages by reduced cortical oxygen uptake, implying reduced mitochondrial respiration. Using quantitative PCR we determined the mitochondrial DNA (mtDNA) gene copy numbers from multiple groups of 15 or 20 pyramidal neurons, GFAP(+) astrocytes and dentate granule neurons isolated using laser capture microdissection, and the relative expression of mitochondrial biogenesis (mitobiogenesis) genes in hippocampi from 10 AD and 9 control (CTL) cases. AD pyramidal but not dentate granule neurons had significantly reduced mtDNA copy numbers compared to CTL neurons. Pyramidal neuron mtDNA copy numbers in CTL, but not AD, positively correlated with cDNA levels of multiple mitobiogenesis genes. In CTL, but not in AD, hippocampal cDNA levels of PGC1α were positively correlated with multiple downstream mitobiogenesis factors. Mitochondrial DNA copy numbers in pyramidal neurons did not correlate with hippocampal Aβ1-42 levels. After 48 h exposure of H9 human neural stem cells to the neurotoxic fragment Aβ25-35, mtDNA copy numbers were not significantly altered. In summary, AD postmortem hippocampal pyramidal neurons have reduced mtDNA copy numbers. Mitochondrial biogenesis pathway signaling relationships are disrupted in AD, but are mostly preserved in CTL. Our findings implicate complex alterations of mitochondria-host cell relationships in AD

Clinico-Pathological study of Paediatric Liver Tumors.

INTRODUCTION : Primary neoplasms of the liver occur rarely during childhood and constitute only 0.3-2% of all pediatric tumors. However, they comprise a variety of entities including benign and malignant epithelial, as well as mesenchymal tumors, the most common of these being hepatoblastoma. Other malignant liver tumors are quite rare and include biliary rhabdomyosarcoma, angiosarcoma, rhabdoid tumor, and undifferentiated sarcoma. The commonly seen benign liver tumors in children are infantile hemangioma, mesenchymal hamartoma, and focal nodular hyperplasia. Rare benign tumors are hepatic adenoma and teratoma. Clinical presentation, especially in young children is relatively uniform with abdominal enlargement and a painless tumor, and often specific symptoms develop late. Prerequisites for clinical diagnosis are a comprehensive laboratory workup and good quality imaging mainly with ultrasound, as well as CT and/or MRI scans. Histological diagnosis is essential for differential diagnosis. Major progress has been achieved during the last decades in the treatment of malignant liver tumors in children, both in chemotherapy and in surgical management. Surgery is the mainstay of treatment for all benign and malignant liver tumors. Prognosis nowadays usually is good in all benign tumors and hepatoblastoma, as well as in some other rare malignancies, but dismal in hepatocellular carcinoma and other chemotherapy non sensitive malignant tumors. The future in the treatment of children with advanced malignant liver tumors lies in primary liver transplantation. AIM : 1. To study the incidence and age distribution of various liver tumors in children. 2. To study the mode of presentation and clinical profile of both benign and malignant liver tumors. 3. To study the role of tumor markers and imaging techniques in the preoperative workup of these tumors. 4. To study the role of primary liver resections in achieving disease free survival. 5. To assess the effectiveness of adjuvant and neoadjuvant chemotherapy in downstaging the disease. 6. To study the outcome based on morbidity and mortality profile of the children treated within the period of study. 7. To identify pitfalls (if any) in the management protocol to improve the long term survival. 8. To suggest guidelines for future management. MATERIALS AND METHODS : This study on liver tumors in children is based on 34 patients admitted to the Institute of Child Health and hospital for children [ICH & HC], Egmore during the period from January 2005 to December 2009. The study includes 24 boys and 10 girls between the age group 6 days to 9 years. Data collected for each patient had been recorded in the enclosed chart. CONCLUSION : Surgery of liver has a special point in paediatric surgical practice because Hepatic tumors are major disorders affecting children with high morbidity and mortality rates. This study covers 34 patients over a 5 year period and compares well with other established centers in India and around the world. The results are not as abysmal as previously thought of. Experience of the surgeon, familiarity to complex anatomy, strict adherence to anatomical planes, advances in anaesthesia, surgical technique, use of newer surgical tools (like CUSA, Intraoperative ultrasound, staplers etc) and chemotherapy have led to a significant improvement in the prognosis of children with hepatoblastoma. Outcomes for hepatocellular carcinoma remain poor. Anatomical resection becomes more comfortable in malignant tumors which are initially treated by preoperative chemotherapy. Liver transplantation is useful in patients with unresectable tumors. Long term goal of the centers undertaking major hepatic resections for liver malignancies in children must be pre tuned to liver transplantation, which will become the gold standard in the management of liver tumors in children in the future. Continued cooperation of multiinstitutional pediatric cancer study groups will be required to achieve additional advances in the treatment of malignant liver neoplasms

Investigations on CPI Centric Worst Case Execution Time Analysis

Estimating program worst case execution time (WCET) is an important problem in the domain of real-time systems and embedded systems that are deadline-centric. If WCET of a program is found to exceed the deadline, it is either recoded or the target architecture is modified to meet the deadline. Predominantly, there exist three broad approaches to estimate WCET- static WCET analysis, hybrid measurement based analysis and statistical WCET analysis. Though measurement based analyzers benefit from knowledge of run-time behavior, amount of instrumentation remains a concern. This thesis proposes a CPI-centric WCET analyzer that estimates WCET as a product of worst case instruction count (IC) estimated using static analysis and worst case cycles per instruction (CPI) computed using a function of measured CPI. In many programs, it is observed that IC and CPI values are correlated. Five different kinds of correlation are found. This correlation enables us to optimize WCET from the product of worst case IC and worst case CPI to a product of worst case IC and corresponding CPI. A prime advantage of viewing time in terms of CPI, enables us to make use of program phase behavior. In many programs, CPI varies in phases during execution. Within each phase, the variation is homogeneous and lies within a few percent of the mean. Coefficient of variation of CPI across phases is much greater than within a phase. Using this observation, we estimate program WCET in terms of its phases. Due to the nature of variation of CPI within a phase in such programs, we can use a simple probabilistic inequality- Chebyshev inequality, to compute bounds of CPI within a desired probability. In some programs that execute many paths depending on if-conditions, CPI variation is observed to be high. The thesis proposes a PC signature that is a low cost way of profiling path information which is used to isolate points of high CPI variation and divides a phase into smaller sub-phases of lower CPI variation. Chebyshev inequality is applied to sub-phases resulting in much tighter bounds. Provision to divide a phase into smaller sub-phases based on allowable variance of CPI within a sub-phase also exists. The proposed technique is implemented on simulators and on a native platform. Other advantages of phases in the context of timing analysis are also presented that include parallelized WCET analysis and estimation of remaining worst case execution time for a particular program run