Methylphenidate (Ritalin) use and abuse

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Automation and scale-up of human induced pluripotent stem cell models of cardiovascular disease for drug screening

The global cost of heart failure is USD$45 billion and set to double in the next 15 years. The only method of treatment is heart transplant but demand far exceeds supply and is projected to increase. Meanwhile, global pharmaceutical development has been hindered by poor drug development success rates. Of the drugs that make it to phase I clinical trials, only 8 % pass phase III and existing drug screens do not always accurately predict or detect adverse cardiac events. Cardiotoxicity is the underlying reason for 26 % of safety related drug withdrawals between 1990-2006. Therefore, a source of human cardiomyocytes (CMs) is required to fill the need for regenerative medicine and drug screening applications. Differentiation of human pluripotent stem cells (hPSCs) to CMs is a viable solution to this bottleneck but the number of cells required is staggering; up to 5 million novel compounds are registered annually by pharmaceutical and academic institutions, while cell replacement studies in primates suggest that 10 billion CMs will be required per patient to repair the damaged myocardium post infarction. The objective of this thesis was to evaluate whether automated high throughput manufacture of hPSCs and CMs was possible, and to demonstrate that hPSC-CMs could be used in automated high throughput drug screening by carrying out assays in 384-well plates. This thesis started by carrying out three manual differentiation methods; an embryoid body (EB) based method and two monolayer methods. Batch variability in mouse embryonic fibroblast conditioned medium (MEF-CM) led to erratic and variable differentiation outcomes (as high as 94+/-0.3 % to as low as 25.6+/-39.7 % beating EBs per 96 well plate). Two monolayer methods, using defined media (mTeSR and E8) increased cell yields by up to 12-fold and 65-fold respectively and simplified the process technically. When these methods were automated, EB differentiation failed to generate spontaneously beating EBs, whereas both monolayer methods succeeded in generating spontaneously beating cardiomyocytes of purities >90 %. Finally, cryopreserved stocks of hiPSC-CMs produced by automation were used to evaluate whether cardiotoxicity from the anticancer drug doxorubicin could be decreased by co-treating with dexrazoxane (an existing doxorubicin cardio-protectant), carvedilol (a β-blocker), sildenafil (a vasoactive agent) and isoprenaline (a β-adrenoreceptor agonist). This was carried out in a real-time, fully automated assay setup to monitor induction of apoptosis by the marker propidium iodide using the Operetta confocal plate reader. The concentration of doxorubicin that led to 50 % hiPSC-CM death (TD50) was significantly reduced by co-treatment with dexrazoxane, carvedilol and sildenafil. Carvedilol showed the highest level of cardioprotection by increasing TD50 of doxorubicin by 7.5-fold. In contrast, isoprenaline reduced TD50 of doxorubicin, suggesting that isoprenaline would be contraindicated in patients undergoing doxorubicin treatment. Thus, this thesis demonstrated that automated differentiation of cardiomyocytes was technically feasible with capability of generating high yields (up to 39 million cells per flask) and high purity (>90 %) cardiomyocytes. Furthermore, this system was compatible with high content assays in 384-well plates for evaluating drug toxicity

N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells

© 2019, The Author(s), under exclusive licence to Springer Nature America, Inc. R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells1–4. Here we show that N6-methyladenosine (m6A) modification, contributing to different aspects of messenger RNA metabolism5,6, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that m6A-containing R-loops accumulate during G2/M and are depleted at G0/G1 phases of the cell cycle, and that the m6A reader promoting mRNA degradation, YTHDF2 (ref. 7), interacts with R-loop-enriched loci in dividing cells. Consequently, YTHDF2 knockout leads to increased R-loop levels, cell growth retardation and accumulation of γH2AX, a marker for DNA double-strand breaks, in mammalian cells. Our results suggest that m6A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability

Airborne S-Band SAR for forest biophysical retrieval in temperate mixed forests of the UK

Radar backscatter from forest canopies is related to forest cover, canopy structure and aboveground biomass (AGB). The S-band frequency (3.1–3.3 GHz) lies between the longer L-band (1–2 GHz) and the shorter C-band (5–6 GHz) and has been insufficiently studied for forest applications due to limited data availability. In anticipation of the British built NovaSAR-S satellite mission, this study evaluates the benefits of polarimetric S-band SAR for forest biophysical properties. To understand the scattering mechanisms in forest canopies at S-band the Michigan Microwave Canopy Scattering (MIMICS-I) radiative transfer model was used. S-band backscatter was found to have high sensitivity to the forest canopy characteristics across all polarisations and incidence angles. This sensitivity originates from ground/trunk interaction as the dominant scattering mechanism related to broadleaved species for co-polarised mode and specific incidence angles. The study was carried out in the temperate mixed forest at Savernake Forest and Wytham Woods in southern England, where airborne S-band SAR imagery and field data are available from the recent AirSAR campaign. Field data from the test sites revealed wide ranges of forest parameters, including average canopy height (6–23 m), diameter at breast-height (7–42 cm), basal area (0.2–56 m2/ha), stem density (20–350 trees/ha) and woody biomass density (31–520 t/ha). S-band backscatter-biomass relationships suggest increasing backscatter sensitivity to forest AGB with least error between 90.63 and 99.39 t/ha and coefficient of determination (r2) between 0.42 and 0.47 for the co-polarised channel at 0.25 ha resolution. The conclusion is that S-band SAR data such as from NovaSAR-S is suitable for monitoring forest aboveground biomass less than 100 t/ha at 25 m resolution in low to medium incidence angle rang

Cardiomyocytes from human pluripotent stem cells: from laboratory curiosity to industrial biomedical platform

Cardiomyocytes from human pluripotent stem cells (hPSCs-CMs) could revolutionise biomedicine. Global burden of heart failure will soon reach USD $90bn, while unexpected cardiotoxicity underlies 28% of drug withdrawals. Advances in hPSC isolation, Cas9/CRISPR genome engineering and hPSC-CM differentiation have improved patient care, progressed drugs to clinic and opened a new era in safety pharmacology. Nevertheless, predictive cardiotoxicity using hPSC-CMs contrasts from failure to almost total success. Since this likely relates to cell immaturity, efforts are underway to use biochemical and biophysical cues to improve many of the ~ 30 structural and functional properties of hPSC-CMs towards those seen in adult CMs. Other developments needed for widespread hPSC-CM utility include subtype specification, cost reduction of large scale differentiation and elimination of the phenotyping bottleneck. This review will consider these factors in the evolution of hPSC-CM technologies, as well as their integration into high content industrial platforms that assess structure, mitochondrial function, electrophysiology, calcium transients and contractility. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel

The relationship between eruptive activity, flank collapse, and sea level at volcanic islands: A long-term (>1 Ma) record offshore Montserrat, Lesser Antilles

Hole U1395B, drilled southeast of Montserrat during Integrated Ocean Drilling Program Expedition 340, provides a long (>1 Ma) and detailed record of eruptive and mass-wasting events (>130 discrete events). This record can be used to explore the temporal evolution in volcanic activity and landslides at an arc volcano. Analysis of tephra fall and volcaniclastic turbidite deposits in the drill cores reveals three heightened periods of volcanic activity on the island of Montserrat (?930 ka to ?900 ka, ?810 ka to ?760 ka, and ?190 ka to ?120 ka) that coincide with periods of increased volcano instability and mass-wasting. The youngest of these periods marks the peak in activity at the Soufrière Hills volcano. The largest flank collapse of this volcano (?130 ka) occurred towards the end of this period, and two younger landslides also occurred during a period of relatively elevated volcanism. These three landslides represent the only large (>0.3 km3) flank collapses of the Soufrière Hills edifice, and their timing also coincides with periods of rapid sea-level rise (>5 m/ka). Available age data from other island arc volcanoes suggests a general correlation between the timing of large landslides and periods of rapid sea-level rise, but this is not observed for volcanoes in intra-plate ocean settings. We thus infer that rapid sea-level rise may modulate the timing of collapse at island arc volcanoes, but not in larger ocean-island settings

Reading and Ownership

First paragraph: ‘It is as easy to make sweeping statements about reading tastes as to indict a nation, and as pointless.’ This jocular remark by a librarian made in the Times in 1952 sums up the dangers and difficulties of writing the history of reading. As a field of study in the humanities it is still in its infancy and encompasses a range of different methodologies and theoretical approaches. Historians of reading are not solely interested in what people read, but also turn their attention to the why, where and how of the reading experience. Reading can be solitary, silent, secret, surreptitious; it can be oral, educative, enforced, or assertive of a collective identity. For what purposes are individuals reading? How do they actually use books and other textual material? What are the physical environments and spaces of reading? What social, educational, technological, commercial, legal, or ideological contexts underpin reading practices? Finding answers to these questions is compounded by the difficulty of locating and interpreting evidence. As Mary Hammond points out, ‘most reading acts in history remain unrecorded, unmarked or forgotten’. Available sources are wide but inchoate: diaries, letters and autobiographies; personal and oral testimonies; marginalia; and records of societies and reading groups all lend themselves more to the case-study approach than the historical survey. Statistics offer analysable data but have the effect of producing identikits rather than actual human beings. The twenty-first century affords further possibilities, and challenges, with its traces of digital reader activity, but the map is ever-changing

Schoolbooks and textbook publishing.

In this chapter the author looks at the history of schoolbooks and textbook publishing. The nineteenth century saw a rise in the school book market in Britain due to the rise of formal schooling and public examinations. Although the 1870 Education and 1872 (Scotland) Education Acts made elementary education compulsory for childern between 5-13 years old, it was not until the end of the First World War that some sort form of secondary education became compulsory for all children

Automation and scale-up of human induced pluripotent stem cell models of cardiovascular disease for drug screening

The global cost of heart failure is USD$45 billion and set to double in the next 15 years. The only method of treatment is heart transplant but demand far exceeds supply and is projected to increase. Meanwhile, global pharmaceutical development has been hindered by poor drug development success rates. Of the drugs that make it to phase I clinical trials, only 8 % pass phase III and existing drug screens do not always accurately predict or detect adverse cardiac events. Cardiotoxicity is the underlying reason for 26 % of safety related drug withdrawals between 1990-2006. Therefore, a source of human cardiomyocytes (CMs) is required to fill the need for regenerative medicine and drug screening applications. Differentiation of human pluripotent stem cells (hPSCs) to CMs is a viable solution to this bottleneck but the number of cells required is staggering; up to 5 million novel compounds are registered annually by pharmaceutical and academic institutions, while cell replacement studies in primates suggest that 10 billion CMs will be required per patient to repair the damaged myocardium post infarction. The objective of this thesis was to evaluate whether automated high throughput manufacture of hPSCs and CMs was possible, and to demonstrate that hPSC-CMs could be used in automated high throughput drug screening by carrying out assays in 384-well plates. This thesis started by carrying out three manual differentiation methods; an embryoid body (EB) based method and two monolayer methods. Batch variability in mouse embryonic fibroblast conditioned medium (MEF-CM) led to erratic and variable differentiation outcomes (as high as 94+/-0.3 % to as low as 25.6+/-39.7 % beating EBs per 96 well plate). Two monolayer methods, using defined media (mTeSR and E8) increased cell yields by up to 12-fold and 65-fold respectively and simplified the process technically. When these methods were automated, EB differentiation failed to generate spontaneously beating EBs, whereas both monolayer methods succeeded in generating spontaneously beating cardiomyocytes of purities >90 %. Finally, cryopreserved stocks of hiPSC-CMs produced by automation were used to evaluate whether cardiotoxicity from the anticancer drug doxorubicin could be decreased by co-treating with dexrazoxane (an existing doxorubicin cardio-protectant), carvedilol (a β-blocker), sildenafil (a vasoactive agent) and isoprenaline (a β-adrenoreceptor agonist). This was carried out in a real-time, fully automated assay setup to monitor induction of apoptosis by the marker propidium iodide using the Operetta confocal plate reader. The concentration of doxorubicin that led to 50 % hiPSC-CM death (TD50) was significantly reduced by co-treatment with dexrazoxane, carvedilol and sildenafil. Carvedilol showed the highest level of cardioprotection by increasing TD50 of doxorubicin by 7.5-fold. In contrast, isoprenaline reduced TD50 of doxorubicin, suggesting that isoprenaline would be contraindicated in patients undergoing doxorubicin treatment. Thus, this thesis demonstrated that automated differentiation of cardiomyocytes was technically feasible with capability of generating high yields (up to 39 million cells per flask) and high purity (>90 %) cardiomyocytes. Furthermore, this system was compatible with high content assays in 384-well plates for evaluating drug toxicity

Current status of drug screening and disease modelling in human pluripotent stem cells

The emphasis in human pluripotent stem cell (hPSC) technologies has shifted from cell therapy to in vitro disease modelling and drug screening. This review examines why this shift has occurred, and how current technological limitations might be overcome to fully realise the potential of hPSCs. Details are provided for all disease-specific human induced pluripotent stem cell lines spanning a dozen dysfunctional organ systems. Phenotype and pharmacology have been examined in only 17 of 63 lines, primarily those that model neurological and cardiac conditions. Drug screening is most advanced in hPSC-cardiomyocytes. Responses for almost 60 agents include examples of how careful tests in hPSC-cardiomyocytes have improved on existing in vitro assays, and how these cells have been integrated into high throughput imaging and electrophysiology industrial platforms. Such successes will provide an incentive to overcome bottlenecks in hPSC technology such as improving cell maturity and industrial scalability whilst reducing cost