Genetically engineered human cortical spheroid models of tuberous sclerosis.

Tuberous sclerosis complex (TSC) is a multisystem developmental disorder caused by mutations in the TSC1 or TSC2 genes, whose protein products are negative regulators of mechanistic target of rapamycin complex 1 signaling. Hallmark pathologies of TSC are cortical tubers-regions of dysmorphic, disorganized neurons and glia in the cortex that are linked to epileptogenesis. To determine the developmental origin of tuber cells, we established human cellular models of TSC by CRISPR-Cas9-mediated gene editing of TSC1 or TSC2 in human pluripotent stem cells (hPSCs). Using heterozygous TSC2 hPSCs with a conditional mutation in the functional allele, we show that mosaic biallelic inactivation during neural progenitor expansion is necessary for the formation of dysplastic cells and increased glia production in three-dimensional cortical spheroids. Our findings provide support for the second-hit model of cortical tuber formation and suggest that variable developmental timing of somatic mutations could contribute to the heterogeneity in the neurological presentation of TSC

The Role of Pot1 in Telomere Protection and Maintenance

POT1 is a single stranded telomeric DNA binding protein implicated in telomere length regulation in human cells. To address the role of POT1 in telomere protection we used RNAi in human cells. We confirmed that POT1 acts as a negative regulator of telomerase and showed that POT1 is required to protect telomeres. Reduced levels of POT1 elicited a strong telomere DNA damage response and a growth arrest in primary cells; the 3’ telomeric overhang shortened and the sequence of the 5’ terminus changed from its precise sequence (ATC-5’) to a randomized ending. In order to determine the phenotype of complete POT1 loss, we used conditional gene deletion in the mouse. Unexpectedly, we identified two POT1 orthologs in the mouse and rat genomes, whereas other mammals have one. As both proteins (POT1a and POT1b) localized to telomeres based on IF and ChIP, we targeted both genes for Cremediated deletion. POT1a/b double-knockout (DKO) cells exhibited a telomeric DNA damage signal and senescence. DKO cells also displayed a novel telomere dysfunction phenotype, extensive endoreduplication. However, POT1a/b were largely dispensable for repression of telomere fusions, which is a prominent outcome of inhibition of another telomere binding protein, TRF2. Previous structural analysis of POT1 and its binding partner TPP1 predict that they act interdependently. Consistent with this, we found that TPP1 is essential for the telomeric function of both mouse POT1 proteins. Single knockouts and complementation experiments revealed that POT1a and POT1b have distinct functions. POT1a was found to be primarily responsible for repression of the DNA damage signal at telomeres, while POT1b had a unique role in repressing an activity that creates extended telomeric overhangs. POT1b KO cells showed accelerated telomere shortening indicating that POT1b controls exonucleolytic degradation of the C-rich strand. The enhanced telomere shortening of POT1b KO cells explains our finding that POT1b KO mice display aging phenotypes reminiscent of late generation telomerase knockout mice. My results argue that a gene duplication event gave rise to two functionally distinct POT1 proteins in rodents. Such a divergence is unprecedented in chromosome biology and has implications for modeling telomere biology and telomere-related diseasestates in the mouse

Estrogen, muscle damage, and the repeated bout effect

PURPOSE: The aim of this study was to investigate estrogen’s effect on markers of muscle damage and the repeated bout effect in women following multiple downhill runs. METHODS: Thirteen moderately trained females (VO2 max: 36-47 ml/kg/min), 18-35 years old, and who used hormonal contraception participated in the study. They completed two 40 min downhill runs (-10% grade) at 65-70% VO2 max during either the third week of hormone use (low estrogen group (LE), n=7) or the placebo week (high estrogen group (HE), n=6). Trials were separated by four weeks. Creatine kinase (CK) activity, pressure tolerance (front thigh, shin, and calf), and circumferences (thigh and calf) were measured before (pre) and at 24 hr, 48 hr, and 72 hr after exercise. Muscle soreness of the front thigh, shin and calf was reported immediately (0 hr) and at 24 hr, 48 hr, and 72 hr after exercise. Knee extension and ankle dorsiflexion peak torque were measured pre and 72 hr postexercise. RESULTS: A run effect was found in front thigh (P = 0.009) and calf (P = 0.02) pressure tolerance such that DH1 was lower than DH2. Soreness of the front thigh was lower following DH2 compared to DH1 (P = 0.05). A group x time interaction was observed for shin soreness such that soreness in LE was higher at 24 hr and 48 hr compared to 0 hr (P \u3c 0.001; P \u3c 0.001, respectively) and 72 hr (P \u3c 0.01; P = 0.04, respectively). Following DH1, CK activity was higher at 24 hr (P \u3c 0.001), 48 hr (P = 0.04), and 72 hr (P = 0.03) compared to pre-exercise. CK activity was lower at 24 hr following DH2 (P \u3c 0.001) compared to 24 hr following DH1. No significant changes were noted for shin pressure tolerance, calf soreness, circumference, or peak torque measurements. CONCLUSION: Women with lower estrogen levels may show a greater response in markers of muscle damage following the initial bout of exercise as evidenced by increased soreness in the shin. The RBE was observed as evidenced by a less robust response to pressure tolerance, soreness and CK activity following DH2, with no group differences in the RBE response. Higher estrogen levels at the time of exercise may mitigate markers of muscle damage following an initial bout of eccentric exercise but does not seem to influence the repeated bout effect

Spatial and Temporal Distribution of Fungicides Applied to Creeping Bentgrass

Turf managers often rely on fungicides to limit damage caused by root diseases. Since fungicides do not move basipetally, they are effective only when fungitoxic concentrations are delivered to the rhizosphere. This research focused on the distribution of modern fungicides in verdure, thatch, sand, and roots of creeping bentgrass (Agrostis stolonifera L. var. palustris (Huds.) Farw.) maintained as a putting green. Fungicides (azoxystrobin (methyl (E)-2-[2-[6-(2-cyanophenoxy)pyrimidin-4-yloxy]phenyl]-3-methoxyacrylate), propiconazole (1,2,4-Triazole, 1-((2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl)methyl), pyraclostrobin (carbamic acid, [2-[[[1-(4-chlorophenyl)-1H-pyrazol-3-yl]oxy]methyl]phenyl]methoxy-,methyl ester), and thiophanate-methyl (dimethyl 4,4\u27-o-phenylenebis[3-thioallophanate]) were applied to replicate field plots in a water volume of 815 L ha-1. Plots were sampled over time (0, 3, 7, 10, 14, 17, 21 days after application) by extracting cores measuring 2 cm diameter by 3.8 cm deep. Cores were separated into verdure/thatch, sand, and roots before quantitative determination (liquid chromatography, triple quadrupole mass spectrometry) of fungicide residues. Fungicide residues in verdure/thatch declined steadily with time and support previously reported results describing fungicide depletion. Fungicides were detected in roots and sand within 5 hours of application, although at very low (1-15 ppm) concentrations. Residues in roots and sand remained at low levels throughout the experiment. Fungicides differed with respect to amounts recovered per turfgrass component

Written emotional expression: Effects on weight loss and maintenance

The consequences of the current obesity epidemic are taking a serious toll on the health and the economics of our society. The prevention and treatment of obesity remain a challenge for researchers and society. Over the past 15-20 years behavioral treatments have been developed which have been successful in helping people lose weight, but individuals struggle to maintain this weight loss. Several studies provide evidence that emotional and psychological factors may play an important role in weight loss and maintenance. However, most behavioral treatment approaches do not adequately address the role of emotional factors. Interventions specifically designed to help individuals who are overweight or obese better cope with emotional distress may attenuate the negative effects of distress on weight loss and maintenance through improved emotional self-regulatory processes. This study examined the effects of expressive writing on weight regain, stress, and emotional eating among individuals who have recently lost weight. Men and women (N = 64) who recently lost weight were recruited and randomized to either an emotional expressive (n = 31) or to a placebo control writing intervention (n = 33). Baseline assessments were completed followed by writing sessions that took place once weekly across four consecutive weeks. Follow-up assessments of weight, stress and emotional eating were completed at the end of the final writing session, and again at 1 and 3 months. Results from this study indicated that there was no support for expressive writing to impact the rate of weight regain among individuals who have recently lost weight. However, there was partial support for expressive writing to decrease stress levels. No support was found, however, for expressive writing to decrease emotional eating. The results from this study provide poor support for the beneficial effects of written emotional disclosure on buffering weight regain and decreasing emotional eating following weight loss treatment. There is, however, minimal evidence that expressive writing may produce favorable effects on stress levels in individuals who are attempting to maintain weight loss. Implications of these findings and ideas for future research to improve weight loss and maintenance outcomes are discussed

Cancer-associated TERT promoter mutations abrogate telomerase silencing.

Mutations in the human telomerase reverse transcriptase (TERT) promoter are the most frequent non-coding mutations in cancer, but their molecular mechanism in tumorigenesis has not been established. We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease. Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity. However, upon differentiation into somatic cells, which normally silence telomerase, cells with TERT promoter mutations failed to silence TERT expression, resulting in increased telomerase activity and aberrantly long telomeres. Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations. These data establish that TERT promoter mutations can promote immortalization and tumorigenesis of incipient cancer cells

Induced Pluripotent Stem Cells Meet Genome Editing

It is extremely rare for a single experiment to be so impactful and timely that it shapes and forecasts the experiments of the next decade. Here, we review how two such experiments - the generation of human induced pluripotent stem cells (iPSCs) and the development of CRISPR/Cas9 technology - have fundamentally reshaped our approach to biomedical research, stem cell biology, and human genetics. We will also highlight the previous knowledge that iPSC and CRISPR/Cas9 technologies were built on as this groundwork demonstrated the need for solutions and the benefits that these technologies provided and set the stage for their success.National Institutes of Health (U.S.) (Grant 1R01NS088538-01)National Institutes of Health (U.S.) (Grant 2R01MH104610-15

4D cell biology: big data image analytics and lattice light-sheet imaging reveal dynamics of clathrin-mediated endocytosis in stem cell-derived intestinal organoids.

New methods in stem cell 3D organoid tissue culture, advanced imaging, and big data image analytics now allow tissue-scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high-content imaging data. We expressed fluorescent protein fusions of clathrin and dynamin2 at endogenous levels in genome-edited human embryonic stem cells, which were differentiated into hESC-derived intestinal epithelial organoids. Lattice light-sheet imaging with adaptive optics (AO-LLSM) allowed us to image large volumes of these organoids (70 × 60 × 40 µm xyz) at 5.7 s/frame. We developed an open-source data analysis package termed pyLattice to process the resulting large (∼60 Gb) movie data sets and to track clathrin-mediated endocytosis (CME) events. CME tracks could be recorded from ∼35 cells at a time, resulting in ∼4000 processed tracks per movie. On the basis of their localization in the organoid, we classified CME tracks into apical, lateral, and basal events and found that CME dynamics is similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO-LLSM makes possible quantitative high temporal and spatial resolution analysis of subcellular events within tissues

A Drug-Inducible System for Direct Reprogramming of Human Somatic Cells to Pluripotency

SummaryCurrent approaches to reprogram human somatic cells to pluripotent iPSCs utilize viral transduction of different combinations of transcription factors. These protocols are highly inefficient because only a small fraction of cells carry the appropriate number and stoichiometry of proviral insertions to initiate the reprogramming process. Here we have generated genetically homogeneous “secondary” somatic cells, which carry the reprogramming factors as defined doxycycline (DOX)-inducible transgenes. These cells were obtained by infecting fibroblasts with DOX-inducible lentiviruses, isolating “primary” iPSCs in the presence of the drug, and finally differentiating to “secondary” fibroblasts. When “secondary” fibroblast lines were cultured in the presence of DOX without further viral infection, up to 2% of the cells were reprogrammed to pluripotent “secondary” human iPSCs. This system will facilitate the characterization of the reprogramming process and provides a unique platform for genetic or chemical screens to enhance reprogramming or replace individual factors

Genetic engineering of human ES and iPS cells using TALE nucleases

Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator–like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).National Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant RO1-CA087869)National Institutes of Health (U.S.) (Grant RO1-HD045022)Howard Hughes Medical Institut