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Unlimited Possibility Group

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Mr 1e4 Zip 001 !EXCLUSIVE!

The kitchen has a checkered floor, a screen door leading to the laundry room, and a fridge with two cereal boxes on top of it, visible in "The Prank." The sink also has a garbage disposal large enough to put a DVD in, as shown in "The DVD." It has cabinets, brown and white cupboards, and a counter (which it shares with the dining room.)

Mr 1e4 Zip 001

The living room has sea dark blue patterned carpeting with tan wallpaper (but near the window, and the fourth wall, it changes to a dark brown wood.) The door entrance has a mat, and to the left of the window, there is a floor lamp with a tan and orange pattern for its shade. In the middle of the room, there is a brown plaid couch and a coffee table on top of a circular white and orange rug. Behind the couch are the stairs, which feature two paintings (the Wattersons and a unicorn.) There is also a door between the paintings that leads to Nicole and Richard's room, as seen in "The Man." On the fourth wall, there are two pairs of lights (with one having a pink tree-shaped handle.) There is a TV which is labeled "Soppy" near the screen, with a video game system, a purple jukebox, with circle speakers on both sides of the TV, and a bookshelf on the right. At the back of the living room, perpendicular to the stairs, is an arched doorway leading to the kitchen with a horseshoe lying above it. On the right of the doorway is an Art Deco painting of mountains, and the left side has a pink, wall-mounted touch tone telephone, and a pencil and a variety of notes hang beside it. Also on top of the door, there is a buzzer-like object.

Starting in season 2, the window lamp texture has changed to yellow with pink splatters on it, and a pair of shoes lie near it. Also, a light green TV book, a pencil, a glass, and a paper are added to the coffee table. On the left of the couch there is a red, floor based gooseneck lamp (which can sometimes be seen turned on.) There is also three pillows - One with a black coat of colorful patterns, the other with a wired circle on a tan coating, and the other on the right is a plain tan pillow. Also, the pink telephone near the kitchen entrance seems to have moved down a bit, and the said entrance has been raised higher.

The dining room is merged with the living room. It includes a table, chairs plus a high chair for Anais, and an entrance leading to the kitchen. Located in this room are paintings of a sad bunny clown on the wall shared with the kitchen and one of two strange looking white horses galloping located in the back most wall of the room. In "The Remote," a small picture of a white, fat creature is seen under the sad bunny clown photo. There is also a small table with a pink bowl sitting on top.

The bathroom is located on the second floor and it is across from the stairs. Walls are in part white and in part green. The floor is wooden with small carpet lying near a bathtub.The toilet is on the right and washbasin is on the left. There is also the bathtub with a curtain ahead. The mirror is hanging on the wall above the basin and the first aid kit is hanging above the lavatory. Next to the basin there is also a laundry basket and small trash can. The door to the bathroom was always opposite the bathtub, but starting in season 3, it is to the right of the bathtub. From "The Lie" onwards, a window is shown inside the shower.

This is the room where Gumball, Darwin, and Anais sleep. Darwin sleeps in the fishbowl (with a red "Please Knock" sticker on it) next to the bunk bed, where Gumball and Anais sleep in. Gumball sleeps on the bottom bunk while Anais, as shown in "The World," takes the top. Also in the room is a desk with a computer, along with posters, flags, and many other possessions of the children. The room is usually messy, which is rather typical of Gumball and Darwin.

In "The Date," Anais was seen in her own room. Not much was shown except a table, pale green walls, a poster of a horse, some dolls and a toy chest with a baseball glove on it. Anais said "Get out of my room," but this was most likely a writing error. To date, this room has never been mentioned again. Despite this, it appears in Fellowship of the Things as one of the rooms Gumball can visit. It is revealed later that Anais then sleeps in Gumball's room.

The attic made appearances in "The Poltergeist," "The Wand," "The Car," "The Treasure," "The Mirror," "The Check," "The Pest," "The Nest," "The Scam," "The Diet," "The Puppets,", "The Anybody," and The Ad." It is rather cluttered and a bit dusty.

Nicole and Richard's room has appeared in "The Poltergeist," "The Car," "The Treasure," "The Plan," "The Burden," "The Mirror," "The Man," "The Spoiler," "The Return," "The Routine," "The Origins," "The Deal," "The Worst," "The Singing," "The List," "The Ad," "The Web," "The Factory," and "The BFFs." It was mentioned briefly in "The Procrastinators." Like their children's room, this room also has a desktop computer, which Nicole uses as her home office. The furniture in the room appears to have a mid-century theme. The room is located near the front of the house on the first floor, and can be accessed from the living room, as confirmed by "The Man."

The basement was apparently previously unknown to the family, until its appearance in "The Nobody." It is located under the stairs and it is nondescript except for wall shelves and several small cardboard boxes. Rob lived in the room after escaping from the Void until Gumball and Darwin chased him out. The basement reappears in "The Gift," while Gumball and Darwin attempt to find a gift for Masami, In "The Detective," where Anais looks for Daisy the Donkey and finds Gumball. It also appears in "The Choices," "The Petals," and "The Rival." It is mentioned in "The Roots." The basement is full of items in "The Vase." In "The Petals," Leslie enters the basement by jumping through a grate. However, in "The Nest," this grate is seen to lead to a crawlspace underneath the house.

Classic dendritic cells (cDCs) play a central role in the immune system by processing and presenting antigens to activate T cells, and consist of two major subsets: CD141+ cDC (cDC1) and CD1c+ cDC (cDC2). A population of migratory precursor cells, the pre-cDCs, is the immediate precursors to both cDC subsets. Previous studies showed that there were two pre-committed pre-cDC subpopulations. However, the key molecular drivers of pre-commitment in human pre-cDCs were not investigated.

To identify the key molecular drivers for pre-commitment in human pre-cDCs, we performed single cell RNA sequencing (RNA-Seq) of two cDC subsets and pre-cDCs, and bulk RNA-Seq of pre-cDCs and cDCs from human peripheral blood. We found that pre-DC subpopulations cannot be separated by either variable genes within pre-cDCs or differentially expressed genes between cDC1 and cDC2. In contrast, they were separated by 16 transcription factors that are themselves differentially expressed or have regulated targets enriched in the differentially expressed genes between bulk cDC1 and cDC2, with one subpopulation close to cDC1 and the other close to cDC2. More importantly, these two pre-cDC sub-populations are correlated with ratio of IRF8 to IRF4 expression level more than their individual expression level. We also verified these findings using three recently published datasets.

In this study, we demonstrate that single cell transcriptome profiling can reveal pre-cDCs differentiation map, and our results suggest the concept that combinatorial dose of transcription factors determines cell differentiation fate.

The hematopoietic system is one of the best model systems for the study of lineage differentiation and diversification. One type of hematopoietic cells, dendritic cells (DCs), plays a central regulatory role in the immune system. It detects pathogen-associated molecular pattern (PAMP) signal or danger-associated molecular pattern (DAMP) signal, captures antigens from self or invaders, processes and presents them to activate T cells, and sustains the memory adaptive immune response and Tregs, thereby bridging innate and adaptive immune response [1]. The functional complexity of DCs is reflected in their diversity. Conventional dendritic cells (cDCs) in humans that activate T cells are comprised of two subsets with distinct phenotypes and functions, namely, CD141+ cDCs (cDC1) and CD1c+ cDCs (cDC2). Specifically, human cDC1 and their equivalent in mouse, CD8+ cDCs, specialize in activating CD8 T cell response [2], whereas human cDC2 and their murine counterpart CD11b+ cDCs, specialize in activating CD4 T cell response [3]. Both cDC1 and cDC2 descend from migratory dendritic cell precursor cells, or pre-cDCs, which arise from common dendritic cell progenitor (CDP) in the bone marrow and egress to the periphery where they terminally differentiate into cDCs [4]. Several phenotypically distinct pre-cDC cell populations have been identified in human peripheral blood with potential to produce cDC1 and cDC2 [5,6,7,8].

Single cell RNA sequencing (scRNA-Seq) studies not only led to the identification of a new pre-cDC population in human blood [7], but also revealed that seemingly homogeneous pre-cDCs in mouse and human were heterogeneous and led to the discovery that pre-cDCs have two subpopulations pre-committed to cDC1 and cDC2, respectively [5, 6, 9]. However, the identification and characterization of the pre-cDC subpopulations in these studies were not based on scRNA-Seq data, but mainly from prospective isolation of cells, followed by bulk RNA-Seq. It remains unclear whether we can use scRNA-Seq data directly to separate and identify pre-committed subpopulations. Additionally, the transcriptional program that drives pre-cDC pre-commitment is not well characterized.

Here we report a single cell transcriptomics study of pre-cDCs to investigate core transcriptional program underlying cDC1 and cDC2 lineages. We used the Fluidigm C1 platform to sequence mixed pre-cDCs, cDC1, and cDC2 cells, and developed a computational approach to find master regulator transcription factors that drive the pre-cDC differentiation process. We ask 1) whether single-cell transcriptome profiling can distinguish two cDC subsets and their immediate precursor pre-cDCs; 2) whether pre-cDCs are composed of distinct committed subpopulations that resemble terminally differentiated cDC cells; and 3) which transcription factors drive the terminal differentiation of pre-cDCs. To answer these questions, we implemented a workflow as shown in Additional file 1: Figure S1 Specifically, we used multidimensional scaling (MDS) to infer cell types based on scRNA-Seq data and remove outliers. We then identified genes with highly variable expression in pre-cDC population and showed that they can be used to separate cDC1 and cDC2, indicating the heterogeneity of pre-cDCs is associated with cDC commitment. Finally, we identified transcription factors that are differentially expressed between bulk cDC1 and cDC2 (indicated in red and blue) or have targets enriched among differentially expressed genes (indicated in olive), and showed that these are potential master regulators that underline pre-cDC heterogeneity and drive differentiation. 041b061a72


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