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For each cluster of the standard trajectory, calculate the probability of observing a given gene (i.e. the fraction of counts in that cluster from the gene). For genes that are not detected add 1e-07 total counts.

For each cell in the direct programming trajectory, calculate the log-likelihood that it was drawn from each of these clusters. This log-likelihood is from the multinomial distribution function using the probabilities obtained in step 2.

4.Identify and tally the maximum likelihood assignments of all direct programming cells. Normalize raw assignments so that they sum to 100 (giving the percentage). Plot the percentage of direct programming cells assigned to each standard protocol state.

Cell-cycle activity can be estimated from a cell cycle associated gene expression signature; populations that express higher average levels of cell cycle genes are most likely cycling at higher frequency than a population with lower level expression of these genes. In Figure 4b and Figure 4—figure supplement 1 we performed an analysis in this spirit to determine which parts of the DP and SP differentiation trajectories appeared to be proliferative, and to estimate where cells are exiting the cell cycle. We computed a proliferation score that was the aggregate expression of a panel of 21 cell cycle related genes: Aurka, Top2a, Ccna2, Ccnd1, Ccnd2, Ccnd3, Ccne1, Ccne2, Ccnb1, Cdk4, Cdk6, Cdk2, Cdk1, Cdkn2b, Cdkn2a, Cdkn2c, Cdkn2d, Cdkn1a, Cdkn1b, Cdkn1c, Mcm6, Cdc20, Plk1, and Pcna. We also computed a cell cycle exit score on the basis of the aggregate expression of a panel of 4 tumor suppressor genes that inhibit the cell cycle: Cdkn1c, Cdkn1b, Cdkn1a, and Cdkn2d. In Figure 4—figure supplement 1 we show the expression of representative individual genes from this score; in general cell cycle genes were correlated with each other in their expression over cells, as were cell cycle exit genes.

We were interested to convert a bound on the population expression of Olig2, obtained by qPCR, into a bound on the lifetime of a hypothetical rare subpopulation that expressed appreciable levels of this transcript. To proceed, we noted that in SP, Olig2 is expressed 5-fold higher than Gapdh. By contrast, qPCR indicated that in DP, Olig2 was expressed 10-fold lower than Gapdh. Since we took qPCR measurements on every day during differentiation, we next reasoned that the maximum possible number of these intermediate cells would exist if they were spread uniformly across our timecourse (otherwise there would be a spike in their number, increasing the chances of their detection). This is unrealistic, but sets a conservative upper bound. Since the total differentiation protocol took 11 days, any Olig2+intermediate that expressed the gene at the levels seen in SP (five fold higher than Gapdh) must thus exist for less than 11 days/10/5, or less than 0.2 s. To estimate what this lifetime would be for an Olig2+population that expressed just one molecule per cell, we conservatively allowed for Gapdh expression levels of 1000 molecules per cell (smRNA-FISH studies have estimated~200–500 Gapdh copies per cell). The maximum fraction of Olig2+cells expressing one molecule per cell at any timepoint in DP is correspondingly 0.1% (1000/10*100%). Our lifetime calculation becomes 11 days/10, or less than 15 min. Since the timescales of mRNA production and degradation are typically on the order of hours, we therefore concluded that an Olig2+subpopulation must not exist during DP.

How does the transcriptional state of motor neurons produced by both protocols compare to that of motor neurons in vivo? To answer this question we leveraged the ability of single-cell RNA sequencing to compare cell states even within populations that are not pure (also see below for functional comparisons of the phenotypes). We performed three analyses. First, we computed the cosine similarity between the centroids of each cell state in both protocols and primary motor neurons ( ICONS LEA SPORT GUM FOOTWEAR Lowtops amp; sneakers Le Coq Sportif FnbU53FVJw
). The specific steps of this analysis were as follows:

Second, we compared single cells from the DP and SP terminal states to pMN reference single cells. The goal of this analysis was to detect whether heterogeneity within the DP and SP terminal states correlated with similarity to pMNs. To gain a broad understanding of the relationship between the DP and SP trajectories to our pMN reference, we initially projected pMN single cells into the PCA-space used in the visualization from Goji Black Tassel Earrings The Fashion Bible LgmIwvxKwt
( Figure 5C–i ). The projection was performed by multiplying the pMN counts matrix (after quality filtering cells and z-score normalizing genes) by the PC-loadings matrix that was built on the DP and SP cells as described above. The combined coordinates of all cells in this space were then used as input to SPRING, which builds and visualizes a kNN graph. To generate a more refined visualization of how the terminal populations were related to each other we next constructed a new PC-space on EMN and LMN DP and SP cells, combined with pMNs, following the same steps of SPRING visualization described above ( Figure 5C–ii ). This differs from the original projection in that the second PC-space was constructed to reflect variation within only the mature populations of cells (EMN, LMN and pMN). It thus has higher sensitivity to detect subtle differences between MN subpopulations, because the PC-space does not contain dimensions that describe unrelated process that occur in the process of becoming a neuron from an mES transcriptional state, and thus only introduce noise into the comparison of terminal states. To quantify how similar individual cells were to the pMN reference we performed nearest neighbor analysis on the distance matrix underlying this second SPRING visualization. For each DP or SP terminal state, we asked of every cell whether it had a pMN cell among its 50 most similar cells, and found the fraction of cells with similarity to pMNs. The number of nearest neighbors chosen, in this case 50, sets the size of the region in which we required a pMN cell to fall in order for a cell to be classified as similar to pMN. 50 cells equated to roughly 2% of the total cells in this visualization, and so provided an appropriate and stringent threshold.

Third, we performed differential gene expression analysis, comparing the most mature motor neuron states from each protocol with primary HB9+motor neurons ( Womens Alyssa Closed Toe Heels Kurt Geiger EPP2pT
). This analysis was performed as described above.

1.Combine all cell states from both protocols, and from HB9+E13.5 primary tissue, extract PV-genes (as described above), and z-score normalize their expression.

The public comment period for the following draft chapters has been closed. The department is currently analyzing the comments received and will be making changes to the chapters as appropriate. Once all the changes are complete, the department will publish the finalized ROA document on this site.

Finalized chapters

The public comment period closed for the Great Northwest and Upper Lake Michigan Coastal regions on October 27th. These chapters have now been finalized by the department. You can view the finalized chapters at the links below:

General Information

The goals of the Recreation Opportunities Analysis are to:

Our analysis of recreation opportunities in Wisconsin will develop eight regional reports. These reports will describe existing recreation opportunities, future needs and priorities, and the potential role of state-owned lands in helping meet these priorities. The reports will provide guidance to the DNR's master planning process on a wide variety of recreation needs, including opportunities for incorporating motorized access.

The DNR will start by engaging representatives of statewide stakeholder groups from a variety of recreation interests. This group will help the DNR develop a set of recreation goals and objectives and develop criteria to identify appropriate locations for recreational activities. The criteria will include attributes and conditions needed to provide a high-quality recreational experience, identify the potential impacts of those activities and determine appropriate geographic distribution of opportunities.

Next, the DNR will engage regional representatives from recreation interest groups, recreation providers, business and environmental groups and others. For purposes of this study, the DNR will use eight regions [PDF] developed by previous recreation studies. These regional teams will generate information on existing recreation opportunities, future needs and initial guidance on how DNR properties could help meet these needs. The public will have multiple opportunities to provide input in the process.

This analysis is being conducted concurrently with a legislatively-mandated DNR project to inventory and map all roads on department properties. The results of this road inventory project will help inform the Recreation Opportunities Analysis.

Also concurrent with this analysis is the development of the Oper weHwg3dM

The department will be using the results of the Recreation Opportunities Analysis to help inform the development of department master plans, including an amendment to the recreation portion of the Womens Divina Shoulder Bag Mario Valentino Zge1xcAqrP

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March 31, 2018 April 28, 2019

Fans have served as accessories of fashion and utility since antiquity but reached their peak production and use in eighteenth-century Europe. Made from and embellished by precious materials such as ivory, mother-of-pearl, and silver and gold leaf, eighteenth-century fans also featured designs that reflected the spirit of their times. Fans addressed current events as well as themes of broad interest, including biblical and mythological tales and romanticized domestic and pastoral vignettes. explores this quintessential period of fan production through a selection of examples from the permanent collection.

Image: "The Noble Wedding" (detail), 1715–1725. Italy. Vellum, paper, mother-of-pearl, metal, and jewel; opaque watercolor and carved, incised, and gilded sticks and guards; rivet; 11 in. (27.9 cm) length, 18 5/8 in. (47.3 cm) width (open). Fine Arts Museums of San Francisco, Gift of Mrs. Reginald Rives, 1978.10.5a

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After Nicolas Lancret (French, 1690–1743), 'The Dancer Camargo', reproduction in the 18th-century style,ca. 1900 (after painting,ca. 1730). France. Silk, ivory, metal, and jewel; oil-painted and imitation-lacquered (Vernis Martin) sticks, guards, and leaf; grosgrain ribbon; rivet; 7 in. (17.8 cm) length, 12 1/2 in. (31.8 cm) width (open). Fine Arts Museums of San Francisco, Gift of Mrs. Jane Houston and Mr. and Mrs. John Dixon Lannom, 1998.125.1

'Diana and the Maidens', 1740–1750. Netherlands or Germany. Paper, ivory, mother-of-pearl, and metal; opaque watercolor and carved, incised, and gilded sticks and guards; rivet; 11 1/4 in. (28.6 cm) length, 19 3/4 in. (50.2 cm) width (open). Fine Arts Museums of San Francisco, Gift of Mr. and Mrs. Floyd F. Reighley, 1956.82a

"Shepherd and Shepherdess", 1730–1740. Italy. Paper, ivory, mother-of-pearl, metal, and jewel; opaque watercolor and carved, incised, gilded, and applied sticks and guards; rivet; 10 1/4 in. (26 cm) length, 17 1/2 in. (44.5 cm) width (open). Fine Arts Museums of San Francisco, Gift of Mrs. Beatrice Greenough, 1964.89




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