Developing a cardiomyocyte proliferation probe utilizing non-degradable CKAP2.

Currently, one of the biggest controversies in the field of cardiac regenerative medicine revolves around the ability of cardiomyocytes to proliferate. In contrast to the long-held hypothesis that the heart is terminally differentiated, post-mitotic organ, some studies have suggested that the heart is capable of undergoing limited regeneration following injury. Others have reported induction of cardiomyocyte proliferation following various treatments, mostly in vitro. Conventional tools such as BrdU labeling fail to distinguish between mitotic events and other phenomena such as endoreduplication or poly-nucleation, thus making it difficult to assess cardiomyocyte proliferation. The present study presents a novel and innovative way to unambiguously study cardiomyocyte proliferation by use of a cell division probe to identify cells that undergo mitosis. The system utilizes a mutant form of the mitotic regulator cytoskeleton-associated protein 2 (CKAP2). CKAP2 remains cytoplasmic during interphase, but translocates to the nucleus following mitotic cell division. Usually, wild-type CKAP2 is degraded via the ubiquitin-proteosome pathway following translocation to the nucleus; however, by mutation of a destruction motif, the protein persists in the daughter nuclei following cell division. Thus, this non-degradable mutant of CKAP2 (ndCKAP2) can be used to track mitotic events - ndCKAP2 should remain cytoplasmic in quiescent cells but appear nuclear in cells that have undergone mitosis. Here, the efficacy of ndCKAP2 as a cell division probe is demonstrated; in particular, ndCKAP2 is demonstrated to be easily visualizable, unambiguously track mitotic events, and present a relatively long half-life (and is therefore integrative in output). It is expected that future studios will utilize this molecular probe system to investigate cardiomyocyte proliferation in vivo under a variety of physiological and pathological conditions, and allow for assessment of various proposed clinical therapies

Gene dynamics of maturation in endogenous and pluripotent stem cell-derived cardiomyocytes

A primary limitation in the clinical application of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) is the failure of these cells to achieve full functional maturity. In vivo, cardiomyocytes undergo numerous adaptive changes during perinatal maturation. By contrast, PSC-CMs fail to fully undergo these developmental processes, instead remaining arrested at an embryonic stage of maturation. To date, however, the precise mechanisms by which directed differentiation differs from endogenous development, leading to consequent PSC-CM maturation arrest, are unknown. The advent of single cell RNA-sequencing (scRNA-seq) has offered great opportunities for studying CM maturation at single cell resolution. However, postnatal cardiac scRNA-seq has been limited owing to technical difficulties in the isolation of single CMs. Additionally, cross-study comparison is limited by dataset specific batch effects. In this dissertation, I first established large particle fluorescence-activated cell sorting (LP-FACS) for isolation of viable single adult CMs. I secondly developed transcriptomic entropy as a robust, batch effect-resistant approach to quantifying CM maturation. With these and other computational tools, I investigated gene expression trends in endogenous and PSC-derived CMs. I first generated an scRNA-seq reference of mouse in vivo CM maturation with extensive sampling of perinatal time periods. I subsequently generated isogenic embryonic stem cells and created an in vitro scRNA-seq reference of PSC-CM directed differentiation. Through computational analysis, I identified a perinatal iimaturation program in endogenous CMs that is poorly recapitulated in vitro. By comparison of these trajectories with previously published human datasets, I identified a network of nine transcription factors (TFs) whose targets are consistently dysregulated in PSC-CMs across species. Notably, I demonstrated that these TFs are only partially activated in common ex vivo approaches to engineer PSC-CM maturation. This dissertation represents the first direct comparison of CM maturation in vivo and in vitro at the single cell level. Moreover, the findings and tools developed here can be leveraged towards improving the clinical viability of PSC-CMs

Predictors of malignancy in pancreatic head mass: a prospective study

A prospective study of patients presented with pancreatic head mass was conducted in a tertiary care referral hospital, Manipal, India from May 2006 to November 2008. The study population was divided into malignant and benign groups based on the final histopathology report. A univariate and multivariate analysis of potential predictive factors for malignancy were conducted. Results: A total of 102 patients with pancreatic head mass were included in the study after fulfilling the inclusion/exclusion criteria. 78 were malignant and 24 were benign. There was significant weight loss (p<0.001) and high mean bilirubin levels (p=0.002) in the malignant group. Mean CA 19-9 was significantly higher in the malignant group (290.7 vs. 30.3 U/ml; p<0.001). Sensitivity and specificity of CA 19-9 for detecting malignancy in pancreatic head mass at a cut off of 35U/ml was 86% and 79% respectively. CA 19-9 positivity rate was higher with increasing cut off values of 100, 200 and 300U/ml but such high levels occurred in fewer patients. All the non-jaundiced patients (100%) with raised CA 19-9 levels were found to be malignant compared to 86% malignancy in jaundiced patients. In multivariate analysis, a combination of weight loss > 10% of body weight and bilirubin > 3 mg/dl and CA 19-9 > 35U/ml had specificity and positive predictive value of 100% for predicting malignancy in pancreatic head mass. Conclusion: The presence of weight loss and jaundice and raised CA 19-9 levels together in a patient with pancreatic head mass can be predictive of malignancy. A very high CA 19-9 level can be an indicator of malignancy in a pancreatic head mass. A raised CA 19-9 level may be more predictive of malignancy in non-jaundiced patients than in jaundiced patients.Key words: Pancreatic Carcinoma, CA 19-9 Antige

Development of a Molecular Probe for Measuring Cardiomyocyte Proliferation

Currently, one of the biggest controversies in the field of cardiac regenerative medicine revolves around the ability of cardiomyocytes to proliferate. In contrast to the long-held hypothesis that the heart is a terminally differentiated, post-mitotic organ, some studies have suggested that the heart is capable of undergoing limited regeneration following injury. Others have reported induction of cardiomyocyte proliferation following various treatments mostly in vitro. Conventional tools such as BrdU labeling fail to distinguish between mitotic events and other phenomena such as endoreduplication or poly-nucleation, thus making it difficult to assess cardiomyocyte proliferation. The present study presents an innovative approach to unambiguously study cardiomyocyte proliferation by use of a cell division probe to identify cells that undergo mitosis. The system utilizes a mutant form of the mitotic regulator cytoskeletonassociated protein 2 (CKAP2). CKAP2 remains cytoplasmic during interphase, but translocates to the nucleus following mitotic cell division. Usually, wildtype CKAP2 is degraded via the ubiquitin-proteosome pathway following translocation to the nucleus; however, by mutation of a destruction motif, the protein persists in the daughter nuclei following cell division. Thus, this non-degradable mutant of CKAP2 (ndCKAP2) can be used to track mitotic events ndCKAP2 should remain cytoplasmic in quiescent cells but appear nuclear in cells that have undergone mitosis. Here we show the efficacy of ndCKAP2 as an in vitro cell division probe. NIH 3T3 and HEK 293 cells were transfected with GFP-ndCKAP2. Following transfection, cells were arrested at the G0 phase through serum starvation. Synchronous re-entry into cell cycle was controlled by re-supplying cells with serum-containing media. Proliferation status and localization of ndCKAP2 were studied with epifluorescence and time-lapse microscopy as well as flow cytometry. We also propose an in vivo cardiomyocyte-specific tetracycline-regulated expression system for ndCKAP2. This system will allow for an investigation of cardiomyocyte proliferation in vivo under a variety of physiological and pathological conditions, and allow for assessment of various proposed clinical therapies

Gene dynamics of maturation in endogenous and pluripotent stem cell-derived cardiomyocytes

A primary limitation in the clinical application of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) is the failure of these cells to achieve full functional maturity. In vivo, cardiomyocytes undergo numerous adaptive changes during perinatal maturation. By contrast, PSC-CMs fail to fully undergo these developmental processes, instead remaining arrested at an embryonic stage of maturation. To date, however, the precise mechanisms by which directed differentiation differs from endogenous development, leading to consequent PSC-CM maturation arrest, are unknown. The advent of single cell RNA-sequencing (scRNA-seq) has offered great opportunities for studying CM maturation at single cell resolution. However, postnatal cardiac scRNA-seq has been limited owing to technical difficulties in the isolation of single CMs. Additionally, cross-study comparison is limited by dataset specific batch effects. In this dissertation, I first established large particle fluorescence-activated cell sorting (LP-FACS) for isolation of viable single adult CMs. I secondly developed transcriptomic entropy as a robust, batch effect-resistant approach to quantifying CM maturation. With these and other computational tools, I investigated gene expression trends in endogenous and PSC-derived CMs. I first generated an scRNA-seq reference of mouse in vivo CM maturation with extensive sampling of perinatal time periods. I subsequently generated isogenic embryonic stem cells and created an in vitro scRNA-seq reference of PSC-CM directed differentiation. Through computational analysis, I identified a perinatal iimaturation program in endogenous CMs that is poorly recapitulated in vitro. By comparison of these trajectories with previously published human datasets, I identified a network of nine transcription factors (TFs) whose targets are consistently dysregulated in PSC-CMs across species. Notably, I demonstrated that these TFs are only partially activated in common ex vivo approaches to engineer PSC-CM maturation. This dissertation represents the first direct comparison of CM maturation in vivo and in vitro at the single cell level. Moreover, the findings and tools developed here can be leveraged towards improving the clinical viability of PSC-CMs

Identification of Epigallocatechin-3-Gallate (EGCG) from Green Tea Using Mass Spectrometry

In an era where humanity is reinstating its lost hope and expectation on natural products, green tea occupies quite a position for what it has proven to be, in its endeavors for human welfare and health. Epigallocatechin-3-gallate (EGCG) is the key to the vast biological activities of green tea. Green tea is no longer in the backdrop; it has emerged as the most viral, trending bioactive molecule when it comes to health benefits for human beings. This review focuses on the use of various analytical techniques for the analysis of EGCG. That which has been achieved so far, in terms of in vitro, pure component analysis, as well as those spikes in biological fluids and those in vivo in animal and human samples, was surveyed and presented. The use of MS-based techniques for the analysis of EGCG is elaborately reviewed and the need for improvising the applications is explained. The review emphasizes that there is plenty of room to explore matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) applications in this subject area

Surgeons’ Views on Shared Decision-Making

Purpose: Shared decision-making (SDM) has a significant role in surgical encounters, where decisions are influenced by both clinician and patient preferences. Herein, we sought to explore surgeons’ practices and beliefs about SDM. Methods: We performed a qualitative study consisting of semi-structured individual interviews with 18 surgeons from private practice and academic surgery practices in Baltimore, Maryland. We purposively sampled participants to maximize diversity of practice type (academic vs private), surgical specialty, gender, and experience level. Interview topics included benefits and challenges to patient involvement in decision-making, communicating uncertainty to patients, and use of decision aids. Interviews were audio-recorded and transcribed. Transcripts were analyzed using content analysis to identify themes. Results: Surgeons were supportive of patients being involved in decision-making, particularly in cases with uncertainty about treatment options. However, surgeons identified SDM as being more appropriate for patients whom surgeons perceived as interested in decision-making involvement and for decisions in which surgeons did not have strong preferences. Additionally, surgeons reported typically presenting only a subset of available options, remaining confident in their ability to filter less suitable options based on intuitive risk assessments. Surgeons differed in their approach to making recommendations, with some guiding patients towards what they saw as the correct or optimal decision while others sought to maintain neutrality and support of the patients’ chosen decision. Conclusions: Many surgeons do not believe SDM is universally optimal for every surgical decision. They instead use assessments of patient disposition or potential clinical uncertainty to guide their perceived appropriateness of using SDM

Optimization of microwave-assisted extraction of bioactive polyphenolic compounds from Marsilea quadrifolia L. using RSM and ANFIS modelling

Extraction of bioactive compounds, rich in plant secondary metabolites as a form of polyphenolic compounds has gained utmost important in the food and pharmaceutical industries due to their antioxidant properties. Microwave-assisted extraction (MAE) was utilized for maximum extraction of bioactive polyphenolic compounds from Marsilea quadrifolia L. with consuming less toxic solvent. A central composite rotatable design (CCRD) based on response surface methodology (RSM) and adaptive neuro-fuzzy inference system (ANFIS) were followed to design and optimize the experimental parameters to get highest yield of bioactive polyphenolic compounds from M. quadrifolia L. The quantitative effects of experimental parameters such as methanol concentration (X1), microwave power (X2), irradiation temperature (X3) and irradiation time (X4) were investigated to obtain the maximum yields of total phenolic (TPC), total flavonoid contents (TFC) and antioxidant properties. The optimum conditions were observed at methanol concentration (X1= 87.5 %), microwave power (X2= 25 %), irradiation temperature (X3= 60 ºC) and irradiation time (X4= 15 min). Under these conditions, the highest yields of TPC (y1)= 693.28 mg gallic acid equivalents (GAE)/g), TFC (y2)= 84.86 mg rutin equivalents (RU)/g), % DPPHsc (y3)= 81.06 %, %ABTSsc (y4)= 71.34% and FRAP (y5)= 68.09 μg mol (Fe (II)/g) has been attained. Further, the experimental results were highly acknowledged with predicted values of RSM and ANFIS. The analysis of LC-ESI-MS spectrum confirmed 6 major bioactive compounds, namely, Betasitosterol, Tridecyliodide, 2,3,7,8 tetracholorodibenzofuaran, Chlorogenic acid, Pentachlorophenylacetate and Triacontyl hexacosanoate in the optimized extract of M. quadrifolia L. The optimized extract can be used as an alternative of synthetic antioxidants for product manufacturing in food and pharmaceutical industries