Mosaic analysis of stem cell function and wound healing in the mouse corneal epithelium

<p>Abstract</p> <p>Background</p> <p>The mouse corneal epithelium is a continuously renewing 5–6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age.</p> <p>Results</p> <p>The initial pattern of corneal epithelial patches in <it>XLacZ</it>^+/-X-inactivation mosaics was replaced after birth by radial stripes, indicating activation of LSCs. Stripe patterns (clockwise, anticlockwise or midline) were independent between paired eyes. Wound healing in organ culture was analysed by mosaic analysis of <it>XLacZ</it>^+/-eyes or time-lapse imaging of GFP mosaics. Both central and peripheral wounds healed clonally, with cells moving in from all around the wound circumference without significant cell mixing, to reconstitute striping patterns. Mosaic analysis revealed that wounds can heal asymmetrically. Healing of peripheral wounds produced stripe patterns that mimicked some aberrant striping patterns observed in unwounded corneas. Quantitative analysis provided no evidence for an uneven distribution of LSC clones but showed that corrected corneal epithelial stripe numbers declined with age (implying declining LSC function) but stabilised after 39 weeks.</p> <p>Conclusion</p> <p>Striping patterns, produced by centripetal movement, are defined independently and stochastically in individual eyes. Little cell mixing occurs during the initial phase of wound healing and the direction of cell movement is determined by the position of the wound and not by population pressure from the limbus. LSC function declines with age and this may reflect reduced LSCs numbers, more quiescent LSCs or a reduced ability of older stem cells to maintain tissue homeostasis. The later plateau of LSC function might indicate the minimum LSC function that is sufficient for corneal epithelial maintenance. Quantitative and temporal mosaic analyses provide new possibilities for studying stem cell function, tissue maintenance and repair.</p

Computer simulation of neutral drift among limbal epithelial stem cells of mosaic mice

Acknowledgements We thank Graham West for writing the software that made this study possible and Ronnie Grant for help with some of the figures. Disclosure of potential conflicts of interest The authors indicate no potential conflicts of interest. Funding information This work was supported by the UK Biotechnology and Biological Sciences Research Council (grants BB/J015172/1 and BB/J015237/1).Peer reviewedPublisher PD

Application of Impedance-Based Techniques in Hepatology Research

There are a variety of end-point assays and techniques available to monitor hepatic cell cultures and study toxicity within in vitro models. These commonly focus on one aspect of cell metabolism and are often destructive to cells. Impedance-based cellular assays (IBCAs) assess biological functions of cell populations in real-time by measuring electrical impedance, which is the resistance to alternating current caused by the dielectric properties of proliferating of cells. While the uses of IBCA have been widely reported for a number of tissues, specific uses in the study of hepatic cell cultures have not been reported to date. IBCA monitors cellular behaviour throughout experimentation non-invasively without labelling or damage to cell cultures. The data extrapolated from IBCA can be correlated to biological events happening within the cell and therefore may inform drug toxicity studies or other applications within hepatic research. Because tight junctions comprise the blood/biliary barrier in hepatocytes, there are major consequences when these junctions are disrupted, as many pathologies centre around the bile canaliculi and flow of bile out of the liver. The application of IBCA in hepatology provides a unique opportunity to assess cellular polarity and patency of tight junctions, vital to maintaining normal hepatic function. Here, we describe how IBCAs have been applied to measuring the effect of viral infection, drug toxicity/IC50, cholangiopathies, cancer metastasis and monitoring of the gut-liver axis. We also highlight key areas of research where IBCAs could be used in future applications within the field of hepatology

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling

Gene expression analysis by quantitative real-time polymerase chain reaction (RT-qPCR) is routinely used in biomedical studies. The reproducibility and reliability of the data fundamentally depends on experimental design and data interpretation. Despite the wide application of this assay, there is significant variation in the validation process of gene expression data from research laboratories. Since the validity of results depends on appropriate normalisation, it is crucial to select appropriate reference gene(s), where transcription of the selected gene is unaffected by experimental setting. In this study we have applied geNorm technology to investigate the transcription of 12 &lsquo;housekeeping&rsquo; genes for use in the normalisation of RT-qPCR data acquired using a widely accepted HepaRG hepatic cell line in studies examining models of pre-clinical drug testing. geNorm data identified a number of genes unaffected by specific drug treatments and showed that different genes remained invariant in response to different drug treatments, whereas the transcription of &lsquo;classical&rsquo; reference genes such as GAPDH (glyceralde- hyde-3-phosphate dehydrogenase) was altered by drug treatment. Comparing data normalised using the reference genes identified by geNorm with normalisation using classical housekeeping genes demonstrated substantial differences in the final results. In light of cell therapy application, RT-qPCR analyses has to be carefully evaluated to accurately interpret data obtained from dynamic cellular models undergoing sequential stages of phenotypic change

Pendulum Mode Thermal Noise in Advanced Interferometers: A comparison of Fused Silica Fibers and Ribbons in the Presence of Surface Loss

The use of fused-silica ribbons as suspensions in gravitational wave interferometers can result in significant improvements in pendulum mode thermal noise. Surface loss sets a lower bound to the level of noise achievable, at what level depends on the dissipation depth and other physical parameters. For LIGO II, the high breaking strength of pristine fused silica filaments, the correct choice of ribbon aspect ratio (to minimize thermoelastic damping), and low dissipation depth combined with the other achievable parameters can reduce the pendulum mode thermal noise in a ribbon suspension well below the radiation pressure noise. Despite producing higher levels of pendulum mode thermal noise, cylindrical fiber suspensions provide an acceptable alternative for LIGO II, should unforeseen problems with ribbon suspensions arise.Comment: Submitted to Physics Letters A (Dec. 14, 1999). Resubmitted to Physics Letters A (Apr. 3, 2000) after internal (LSC) review process. PACS - 04.80.Nn, 95.55.Ym, 05.40.C

Transition from organogenesis to stem cell maintenance in the mouse adrenal cortex

Mice showing mosaic expression of an appropriate marker gene that is activated during development provide simple tools for investigating cell lineages. We used the mosaic β-galactosidase staining patterns in adrenal cortices of 21OH/LacZ transgenic mice to study both organogenesis and maintenance of the adult tissue. Randomly orientated mosaic patterns present in embryonic day 14.5 (E14.5) adrenals changed progressively during the perinatal period from discrete spots, via patches and radial arrays, to radial stripes, which first emerged between postnatal days 0 and 7 (P0 and P7). The mosaic radial stripe pattern was fully established by P21 and remained unchanged throughout the adult period (8–52 weeks). The mouse adrenal gland grew continuously between E14.5 and P21, including the period during which stripes emerge. Ki67-positive, proliferative cells in the adrenal cortex were mainly localized to the outer cell layers between E18.5 and P3. By P10, cell proliferation had increased, and the proliferative region had expanded but was still mainly confined to the outer cortex. Correlation of changes in mosaic patterns in 21OH/LacZ adrenal cortices with the locations of adrenocortical cell proliferation suggest that the radial stripes arise by edge-biased growth during the perinatal period, even if they are maintained by stem cells in adults. The stability of the adult stripe pattern suggests that stem cell function is unchanged between 8 and 52 weeks

Investigating Effects of Tulathromycin Metaphylaxis on the Fecal Resistome and Microbiome of Commercial Feedlot Cattle Early in the Feeding Period

The objective was to examine effects of treating commercial beef feedlot cattle with therapeutic doses of tulathromycin, a macrolide antimicrobial drug, on changes in the fecal resistome and microbiome using shotgun metagenomic sequencing. Two pens of cattle were used, with all cattle in one pen receiving metaphylaxis treatment (800 mg subcutaneous tulathromycin) at arrival to the feedlot, and all cattle in the other pen remaining unexposed to parenteral antibiotics throughout the study period. Fecal samples were collected from 15 selected cattle in each group just prior to treatment (Day 1), and again 11 days later (Day 11). Shotgun sequencing was performed on isolated metagenomic DNA, and reads were aligned to a resistance and a taxonomic database to identify alignments to antimicrobial resistance (AMR) gene accessions and microbiome content. Overall, we identified AMR genes accessions encompassing 9 classes of AMR drugs and encoding 24 unique AMR mechanisms. Statistical analysis was used to identify differences in the resistome and microbiome between the untreated and treated groups at both timepoints, as well as over time. Based on composition and ordination analyses, the resistome and microbiome were not significantly different between the two groups on Day 1 or on Day 11. However, both the resistome and microbiome changed significantly between these two sampling dates. These results indicate that the transition into the feedlot—and associated changes in diet, geography, conspecific exposure, and environment—may exert a greater influence over the fecal resistome and microbiome of feedlot cattle than common metaphylactic antimicrobial drug treatment

Improvement to the Prediction of Fuel Cost Distributions Using ARIMA Model

Availability of a validated, realistic fuel cost model is a prerequisite to the development and validation of new optimization methods and control tools. This paper uses an autoregressive integrated moving average (ARIMA) model with historical fuel cost data in development of a three-step-ahead fuel cost distribution prediction. First, the data features of Form EIA-923 are explored and the natural gas fuel costs of Texas generating facilities are used to develop and validate the forecasting algorithm for the Texas example. Furthermore, the spot price associated with the natural gas hub in Texas is utilized to enhance the fuel cost prediction. The forecasted data is fit to a normal distribution and the Kullback-Leibler divergence is employed to evaluate the difference between the real fuel cost distributions and the estimated distributions. The comparative evaluation suggests the proposed forecasting algorithm is effective in general and is worth pursuing further.Comment: Accepted by IEEE PES 2018 General Meetin