Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics

FilmArray, an Automated Nested Multiplex PCR System for Multi-Pathogen Detection: Development and Application to Respiratory Tract Infection

The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the “FilmArray”, which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon

Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons

Genotyping by high-resolution melting analysis of small amplicons is homogeneous and simple. However, this approach can be limited by physical and chemical components of the system that contribute to intersample melting variation. It is challenging for this method to distinguish homozygous G::C from C::G or A::T from T::A base-pair neutral variants, which comprise ∼16% of all human single nucleotide polymorphisms (SNPs). We used internal oligonucleotide calibrators and custom analysis software to improve small amplicon (42–86 bp) genotyping on the LightScanner®. Three G/C (PAH c.1155C>G, CHK2 c.1-3850G>C and candidate gene BX647987 c.261+22,290C>G) and three T/A (CPS1 c.3405-29A>T, OTC c.299-8T>A and MSH2 c.1511-9A>T) human single nucleotide variants were analyzed. Calibration improved homozygote genotyping accuracy from 91.7 to 99.7% across 1105 amplicons from 141 samples for five of the six targets. The average Tm standard deviations of these targets decreased from 0.067°C before calibration to 0.022°C after calibration. We were unable to generate a small amplicon that could discriminate the BX647987 c.261+22,290C>G (rs1869458) SNP, despite reducing standard deviations from 0.086°C to 0.032°C. Two of the sites contained symmetric nearest neighbors adjacent to the SNPs. Unexpectedly, we were able to distinguish these homozygotes by Tm even though current nearest neighbor models predict that the two homozygous alleles would be identical

A Survey of Air-to-Ground Propagation Channel Modeling for Unmanned Aerial Vehicles

In recent years, there has been a dramatic increase in the use of unmanned aerial vehicles (UAVs), particularly for small UAVs, due to their affordable prices, ease of availability, and ease of operability. Existing and future applications of UAVs include remote surveillance and monitoring, relief operations, package delivery, and communication backhaul infrastructure. Additionally, UAVs are envisioned as an important component of 5G wireless technology and beyond. The unique application scenarios for UAVs necessitate accurate air-to-ground (AG) propagation channel models for designing and evaluating UAV communication links for control/non-payload as well as payload data transmissions. These AG propagation models have not been investigated in detail when compared to terrestrial propagation models. In this paper, a comprehensive survey is provided on available AG channel measurement campaigns, large and small scale fading channel models, their limitations, and future research directions for UAV communication scenarios

Construction and Performance of Large-Area Triple-GEM Prototypes for Future Upgrades of the CMS Forward Muon System

At present, part of the forward RPC muon system of the CMS detector at the CERN LHC remains uninstrumented in the high-\eta region. An international collaboration is investigating the possibility of covering the 1.6 < |\eta| < 2.4 region of the muon endcaps with large-area triple-GEM detectors. Given their good spatial resolution, high rate capability, and radiation hardness, these micro-pattern gas detectors are an appealing option for simultaneously enhancing muon tracking and triggering capabilities in a future upgrade of the CMS detector. A general overview of this feasibility study will be presented. The design and construction of small (10\times10 cm2) and full-size trapezoidal (1\times0.5 m2) triple-GEM prototypes will be described. During detector assembly, different techniques for stretching the GEM foils were tested. Results from measurements with x-rays and from test beam campaigns at the CERN SPS will be shown for the small and large prototypes. Preliminary simulation studies on the expected muon reconstruction and trigger performances of this proposed upgraded muon system will be reported.Comment: 7 pages, 25 figures, submitted for publication in conference record of the 2011 IEEE Nuclear Science Symposium, Valencia, Spai

Scheduling of grid tied battery energy storage system participating in frequency response services and energy arbitrage

Battery energy storage systems (BESS) are widely used to smooth power fluctuations and maintain the voltage and frequency of the power feeder at a desired level. T he National Grid Electricity Transmission (NGET), the primary electricity transmission network operator in the UK, has introduced various frequency response servic es that are designed to provide a real - time response to deviations in the grid frequency. In this study , a control algorithm is developed which generates a charge/discharge power output with respect to deviations in the grid frequency and the requisite service specifications. Using historical UK electricity prices, a new balancing service scheduling approach has also been developed to maximize energy arbitrage revenue by layering different types of balancing services throughout the day . Simulation result s show that the proposed algorithm delivers both dynamic and non - dynamic firm frequency response (FFR) and also enhanced frequency response (EFR) to NGET specifications while generating arbitrage revenue as well as service availability payment s in the balancing market. A comparative study is also presented to compare the yearly arbitrage revenue obtained from the work presented in this paper and a previous reference study . Finall y, exper imental results of a grid - tied 2MW /1MWh BESS have been used for verification purposes

Greater aortic inflammation and calcification in abdominal aortic aneurysmal disease than atherosclerosis: a prospective matched cohort study

Funder: British Heart Foundation; FundRef: http://dx.doi.org/10.13039/501100000274Objective: Using combined positron emission tomography and CT (PET-CT), we measured aortic inflammation and calcification in patients with abdominal aortic aneurysms (AAA), and compared them with matched controls with atherosclerosis. Methods: We prospectively recruited 63 patients (mean age 76.1±6.8 years) with asymptomatic aneurysm disease (mean size 4.33±0.73 cm) and 19 age-and-sex-matched patients with confirmed atherosclerosis but no aneurysm. Inflammation and calcification were assessed using combined 18F-FDG PET-CT and quantified using tissue-to-background ratios (TBRs) and Agatston scores. Results: In patients with AAA, 18F-FDG uptake was higher within the aneurysm than in other regions of the aorta (mean TBRmax2.23±0.46 vs 2.12±0.46, p=0.02). Compared with atherosclerotic control subjects, both aneurysmal and non-aneurysmal aortae showed higher 18F-FDG accumulation (total aorta mean TBRmax2.16±0.51 vs 1.70±0.22, p=0.001; AAA mean TBRmax2.23±0.45 vs 1.68±0.21, p<0.0001). Aneurysms containing intraluminal thrombus demonstrated lower 18F-FDG uptake within their walls than those without (mean TBRmax2.14±0.43 vs 2.43±0.45, p=0.018), with thrombus itself showing low tracer uptake (mean TBRmax thrombus 1.30±0.48 vs aneurysm wall 2.23±0.46, p<0.0001). Calcification in the aneurysmal segment was higher than both non-aneurysmal segments in patients with aneurysm (Agatston 4918 (2901–8008) vs 1017 (139–2226), p<0.0001) and equivalent regions in control patients (442 (304-920) vs 166 (80-374) Agatston units per cm, p=0.0042). Conclusions: The entire aorta is more inflamed in patients with aneurysm than in those with atherosclerosis, perhaps suggesting a generalised inflammatory aortopathy in patients with aneurysm. Calcification was prominent within the aneurysmal sac, with the remainder of the aorta being relatively spared. The presence of intraluminal thrombus, itself metabolically relatively inert, was associated with lower levels of inflammation in the adjacent aneurysmal wall