Long term in vitro expansion of epithelial stem cells enabled by pharmacological inhibition of PAK1-ROCK-Myosin II and TGF-β signaling

Summary: Despite substantial self-renewal capability in vivo, epithelial stem and progenitor cells located in various tissues expand for a few passages in vitro in feeder-free condition before they succumb to growth arrest. Here, we describe the EpiX method, which utilizes small molecules that inhibit PAK1-ROCK-Myosin II and TGF-β signaling to achieve over one trillion-fold expansion of human epithelial stem and progenitor cells from skin, airway, mammary, and prostate glands in the absence of feeder cells. Transcriptomic and epigenomic studies show that this condition helps epithelial cells to overcome stresses for continuous proliferation. EpiX-expanded basal epithelial cells differentiate into mature epithelial cells consistent with their tissue origins. Whole-genome sequencing reveals that the cells retain remarkable genome integrity after extensive in vitro expansion without acquiring tumorigenicity. EpiX technology provides a solution to exploit the potential of tissue-resident epithelial stem and progenitor cells for regenerative medicine. : Zhang et al. screen a small-molecule collection and find that pharmacologic inhibition of TGF-β and PAK1-ROCK-Myosin II, in low calcium conditions, supports extended expansion of epithelial stem cells in 2D format. This approach enhances the potential of tissue-resident epithelial stem cells for cell therapy. Keywords: epithelial stem and progenitor cells, cell culture method, TGF-β, PAK1/ROCK/Myosin II, feeder-free, regenerative medicine, cell therap

Somatic mutation landscapes at single-molecule resolution.

Somatic mutations drive the development of cancer and may contribute to ageing and other diseases1,2. Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. Here, to overcome these limitations, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality. We used this single-molecule sensitivity to study somatic mutations in non-dividing cells across several tissues, comparing stem cells to differentiated cells and studying mutagenesis in the absence of cell division. Differentiated cells in blood and colon displayed remarkably similar mutation loads and signatures to their corresponding stem cells, despite mature blood cells having undergone considerably more divisions. We then characterized the mutational landscape of post-mitotic neurons and polyclonal smooth muscle, confirming that neurons accumulate somatic mutations at a constant rate throughout life without cell division, with similar rates to mitotically active tissues. Together, our results suggest that mutational processes that are independent of cell division are important contributors to somatic mutagenesis. We anticipate that the ability to reliably detect mutations in single DNA molecules could transform our understanding of somatic mutagenesis and enable non-invasive studies on large-scale cohorts

A pilot study on effectiveness of visual arts mediation in Cognitive Activation Therapy (CAT) on biological ageing and cognitive/functional status

openAgeing is one of the most significant phenomenons of the 21 st century with important consequences on all sectors of society. It’s also the greatest risk factor for the development of dementia, which is a complex clinic syndrome that gradually and irreversibly impairs cognitive functions and functional abilities. In this pilot study we analyse whether Cognitive Activation Therapy (CAT) with visual arts influences biological aging and cognition/functional. Twenty patients affected by major neurocognitive disorder in the mild to moderate phase of the disease (mainly Alzheimer's disease) were recruited at the Regional Center for the Study and Treatment of Cerebral Aging (CRIC). Patients aged between 47 and 85 years underwent a cycle of Cognitive Activation Therapy (CAT) mediated by the visual arts and a pre- and post-treatment neuropsychological and functional evaluation with 17 sessions of which 14 at the CRIC and 3 at the the Eremitani Civic Museums of Padua. The biological aging performed on blood samples at the beginning and at the end of the cognitive activation cycle mediated by the visual arts involved the evaluation of biological aging indicators: epigenetic age (DNAmAge) and telomere length. The preliminary statistical analysis of the data shows that the duration of the treatment, through the mediation of the visual arts in the use of a structured and standardized rehabilitation protocol (CAT), influences biological aging. The simple linear regression analysis showed a significant correlation (p=0.035) treatment days and an increased age acceleration DNAmAge (T1-T0). A multiple linear regression shows that the determinants in LTL elongation are being female and days of treatment. In particular, a prolonged duration of the rehabilitation protocol seems to significantly slow down biological aging. Standardizing this rehabilitation model would mean increasing the therapeutic options for people affected by neurocognitive disorders and identifying rehabilitation paradigms capable, not only of positively influencing cognition, but also of leveraging the person's potential, of creating social inclusion, modifying the social perception of disease and to combat stigma.Ageing is one of the most significant phenomenons of the 21 st century with important consequences on all sectors of society. It’s also the greatest risk factor for the development of dementia, which is a complex clinic syndrome that gradually and irreversibly impairs cognitive functions and functional abilities. In this pilot study we analyse whether Cognitive Activation Therapy (CAT) with visual arts influences biological aging and cognition/functional. Twenty patients affected by major neurocognitive disorder in the mild to moderate phase of the disease (mainly Alzheimer's disease) were recruited at the Regional Center for the Study and Treatment of Cerebral Aging (CRIC). Patients aged between 47 and 85 years underwent a cycle of Cognitive Activation Therapy (CAT) mediated by the visual arts and a pre- and post-treatment neuropsychological and functional evaluation with 17 sessions of which 14 at the CRIC and 3 at the the Eremitani Civic Museums of Padua. The biological aging performed on blood samples at the beginning and at the end of the cognitive activation cycle mediated by the visual arts involved the evaluation of biological aging indicators: epigenetic age (DNAmAge) and telomere length. The preliminary statistical analysis of the data shows that the duration of the treatment, through the mediation of the visual arts in the use of a structured and standardized rehabilitation protocol (CAT), influences biological aging. The simple linear regression analysis showed a significant correlation (p=0.035) treatment days and an increased age acceleration DNAmAge (T1-T0). A multiple linear regression shows that the determinants in LTL elongation are being female and days of treatment. In particular, a prolonged duration of the rehabilitation protocol seems to significantly slow down biological aging. Standardizing this rehabilitation model would mean increasing the therapeutic options for people affected by neurocognitive disorders and identifying rehabilitation paradigms capable, not only of positively influencing cognition, but also of leveraging the person's potential, of creating social inclusion, modifying the social perception of disease and to combat stigma

NASA Tech Briefs, September 2011

Topics covered include: Fused Reality for Enhanced Flight Test Capabilities; Thermography to Inspect Insulation of Large Cryogenic Tanks; Crush Test Abuse Stand; Test Generator for MATLAB Simulations; Dynamic Monitoring of Cleanroom Fallout Using an Air Particle Counter; Enhancement to Non-Contacting Stress Measurement of Blade Vibration Frequency; Positively Verifying Mating of Previously Unverifiable Flight Connectors; Radiation-Tolerant Intelligent Memory Stack - RTIMS; Ultra-Low-Dropout Linear Regulator; Excitation of a Parallel Plate Waveguide by an Array of Rectangular Waveguides; FPGA for Power Control of MSL Avionics; UAVSAR Active Electronically Scanned Array; Lockout/Tagout (LOTO) Simulator; Silicon Carbide Mounts for Fabry-Perot Interferometers; Measuring the In-Process Figure, Final Prescription, and System Alignment of Large; Optics and Segmented Mirrors Using Lidar Metrology; Fiber-Reinforced Reactive Nano-Epoxy Composites; Polymerization Initiated at the Sidewalls of Carbon Nanotubes; Metal-Matrix/Hollow-Ceramic-Sphere Composites; Piezoelectrically Enhanced Photocathodes; Iridium-Doped Ruthenium Oxide Catalyst for Oxygen Evolution; Improved Mo-Re VPS Alloys for High-Temperature Uses; Data Service Provider Cost Estimation Tool; Hybrid Power Management-Based Vehicle Architecture; Force Limit System; Levitated Duct Fan (LDF) Aircraft Auxiliary Generator; Compact, Two-Sided Structural Cold Plate Configuration; AN Fitting Reconditioning Tool; Active Response Gravity Offload System; Method and Apparatus for Forming Nanodroplets; Rapid Detection of the Varicella Zoster Virus in Saliva; Improved Devices for Collecting Sweat for Chemical Analysis; Phase-Controlled Magnetic Mirror for Wavefront Correction; and Frame-Transfer Gating Raman Spectroscopy for Time-Resolved Multiscalar Combustion Diagnostics

DNA Methylation Mechanism

‘Simplicity is the ultimate sophistication’- Leonardo Da Vinci The methylation that occurs simply by attaching one or more methyl molecules to a DNA molecule continues to confuse the scientific world by creating highly complex molecular arrangements. Research on methylation mechanisms have discovered that this simple biochemical event (which adapts to the changing micro/macro environment of the organism, to diseases and even cancerous processes) has shown that it is actually not as simple as it seems. In the last 50 years, our efforts to understand these mechanisms and use them to benefit human beings have continued. With this book called “DNA methylation mechanism”, in which we try to explain the effects on every stage of life, we hope that we have been able to create a resource book for everyone interested in this field, from students who are interested, to amateurs and professionals

Into the Wild: Parallel Transcriptomics of the Tsetse-Wigglesworthia Mutualism within Kenyan Populations

Tsetse flies (Diptera: Glossinidae) have medical significance as the obligate vectors of African trypanosomes. In addition, tsetse harbor a simple gut microbiota. A predominant gut microbiota member, the Gammaproteobacterium Wigglesworthia spp., has coevolved with tsetse for a significant portion of Glossina radiation proving critical to tsetse fitness. Although multiple roles have been described for Wigglesworthia within colony flies, little research has been dedicated towards functional characterization within wild tsetse. Here, dual RNA-Seq was performed to characterize the tsetse-Wigglesworthia symbiosis within flies captured in Nguruman, Kenya. A significant correlation in Gene Ontology (GO) distribution between tsetse and Wigglesworthia was observed, with homogeneous enrichment in metabolic and transport categories, likely supporting a hallmark of the symbiosis-bidirectional metabolic exchange. Within field flies, highly transcribed Wigglesworthia loci included those involved in B vitamin synthesis and in substrate translocation, including amino acid transporters and multidrug efflux pumps, providing a molecular means for interaction. The universal expression of several Wigglesworthiaand G. pallidipes orthologs, putatively involved in nutrient provisioning and resource allocation, was confirmed in sister tsetse species. These transcriptional profiles varied through host age and mating status likely addressing varying symbiont demands and also confirming their global importance within Glossina. This study, not only supports symbiont nutrient provisioning roles, but also serves as a foundation for insight into novel roles and molecular mechanisms associated with vector–microbiota interactions. The role of symbiont B vitamin provisioning towards impacting host epigenetics is discussed. Knowledge of vector–microbiota interactions may lead to the discovery of novel targets in pest control

Prognoses on DNA-based identification success rates of altered human remains using capillary electrophoresis and Next Generation Sequencing technologies

The DNA-based identification success of altered human remains relies on the condition of the collected tissue sample and the associated DNA quantity and quality. Due to tissue-specific differences in post-mortem DNA stability, sampling of the best-suited biological material is essential for successful and rapid identification. However, a large variety and partly contradicting recommendations on optimal material have been published so far. The observed insecurity in sampling strategies revealed the need for a broad and systematic approach in predicting short tandem repeat (STR) genotyping success rates in a wide range of tissue types. Therefore, the overarching aim of this thesis was to improve the DNA-based identification success of altered corpses by presenting novel recommendations and guidance for optimal tissue sampling according to the condition of the body. First, the current situation of identification processes in forensics casework was assessed by a retrospective study on the identification success of 402 altered human corpses over seven years (project I). The evaluation of medical as well as genetic reports revealed an increase in the examination of highly and profoundly decomposed corpses and challenges in molecular analyses of degraded and inhibited samples from altered remains. By comparing the number of successive and parallel PCR amplifications, the most unpredictable typing success and highest number of additional analyses were observed in muscle and bone samples. A comparison with previously published studies highlighted the challenges and insecurity in tissue sampling and the need for standardized guidelines. Furthermore, during project II, the reliability of novel DNA sequencing methods was assessed by validating the MiSeq FGx system for Next Generation Sequencing (NGS) of casework samples and optimizing the sequencing workflow for samples of altered remains. The extensive evaluation of sensitivity, concordance to currently used methods and reproducibility, among others, displayed the technology as robust and implementable in forensic routine casework. Additionally, the applicability of phenotype and biogeographic ancestry prediction was demonstrated in challenging samples of altered corpses. However, as the optimization results revealed, an additional PCR purification step, an increased pooling volume and a reduction of adapter volumes for DNA input concentrations ≥ 31.2 pg is recommended for sequencing highly degraded and inhibited samples. Finally, based on the outcomes of projects I and II, the multicentre study concludes with the presentation of novel recommendations on alteration-specific optimal tissue types for first-attempt identification of altered human remains in project III. By providing an easy and rapid scoring system, a precise assessment of the corpse alteration progress is enabled. Furthermore, the systematic approach included the comparison of DNA quantity, integrity and resulting STR profile completeness in an exceptional high number of 1698 DNA extracts from 949 samples of 19 different tissue types. Thereby, standard capillary electrophoresis as well as forthcoming NGS methods were used and the impact of DNA extraction methods was assessed. The final and first-time prognoses on genotyping success of a wide range of tissues separated for two DNA extraction methods (purifying and non-purifying) and two sets of STR loci (22 loci and 16 loci of the extended European Standard Set) provide guidance that improves the first-attempt DNA-based identification success of altered corpses

LifeLogging: personal big data

We have recently observed a convergence of technologies to foster the emergence of lifelogging as a mainstream activity. Computer storage has become significantly cheaper, and advancements in sensing technology allows for the efficient sensing of personal activities, locations and the environment. This is best seen in the growing popularity of the quantified self movement, in which life activities are tracked using wearable sensors in the hope of better understanding human performance in a variety of tasks. This review aims to provide a comprehensive summary of lifelogging, to cover its research history, current technologies, and applications. Thus far, most of the lifelogging research has focused predominantly on visual lifelogging in order to capture life details of life activities, hence we maintain this focus in this review. However, we also reflect on the challenges lifelogging poses to an information retrieval scientist. This review is a suitable reference for those seeking a information retrieval scientist’s perspective on lifelogging and the quantified self

Proactive software rejuvenation solution for web enviroments on virtualized platforms

The availability of the Information Technologies for everything, from everywhere, at all times is a growing requirement. We use information Technologies from common and social tasks to critical tasks like managing nuclear power plants or even the International Space Station (ISS). However, the availability of IT infrastructures is still a huge challenge nowadays. In a quick look around news, we can find reports of corporate outage, affecting millions of users and impacting on the revenue and image of the companies. It is well known that, currently, computer system outages are more often due to software faults, than hardware faults. Several studies have reported that one of the causes of unplanned software outages is the software aging phenomenon. This term refers to the accumulation of errors, usually causing resource contention, during long running application executions, like web applications, which normally cause applications/systems to hang or crash. Gradual performance degradation could also accompany software aging phenomena. The software aging phenomena are often related to memory bloating/ leaks, unterminated threads, data corruption, unreleased file-locks or overruns. We can find several examples of software aging in the industry. The work presented in this thesis aims to offer a proactive and predictive software rejuvenation solution for Internet Services against software aging caused by resource exhaustion. To this end, we first present a threshold based proactive rejuvenation to avoid the consequences of software aging. This first approach has some limitations, but the most important of them it is the need to know a priori the resource or resources involved in the crash and the critical condition values. Moreover, we need some expertise to fix the threshold value to trigger the rejuvenation action. Due to these limitations, we have evaluated the use of Machine Learning to overcome the weaknesses of our first approach to obtain a proactive and predictive solution. Finally, the current and increasing tendency to use virtualization technologies to improve the resource utilization has made traditional data centers turn into virtualized data centers or platforms. We have used a Mathematical Programming approach to virtual machine allocation and migration to optimize the resources, accepting as many services as possible on the platform while at the same time, guaranteeing the availability (via our software rejuvenation proposal) of the services deployed against the software aging phenomena. The thesis is supported by an exhaustive experimental evaluation that proves the effectiveness and feasibility of our proposals for current systems