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Supplementary Files - Transcriptomic analysis of CPM-positive hiPSCs-derived liver progenitor cells in a microfluidic device shows zonation-like patterns.

Supplementary Files for the paper intitled Transcriptomic analysis of CPM-positive hiPSCs-derived liver progenitor cells in a microfluidic device shows zonation-like patterns.

opencc-zeroJul 2022View details →

dryad36/100

Impacts of development and adult sex on brain cell numbers in the Black Soldier Fly, Hermetia illucens L. (Diptera: Stratiomyidae)

The Black Soldier Fly (Hermetia illucens, Diptera: Stratiomyidae) has been introduced across the globe, with numerous industry applications predicated on its tremendous growth during the larval stage. However, basic research on H. illucens biology (for example, studies of their central nervous system) are lacking. Despite their small brain volumes, insects are capable of complex behaviors; understanding how these behaviors are completed with such a small amount of neural tissue requires understanding processing power (e.g. number of cells) within the brain. Brain cell counts have been completed in only a few insect species (mostly Hymenoptera), and almost exclusively in adults. This limits the taxonomic breadth of comparative analyses, as well as any conclusions about how development and body size growth may impact brain cell populations. Here, we present the first images and cell counts of the H. illucens brain at four time points across development (early, mid, and late larval stages, and both male and female adults) using immunohistochemistry and isotropic fractionation. To assess sexual dimorphism in adults, we quantified the number of cells in the central brain vs. optic lobes of males and females separately. To assess if increases in body size during development might independently affect different regions of the CNS, we quantified the larval ventral nerve cord and central brain separately at all three stages. Together, these data provide the first description of the nervous system of a popular, farmed invertebrate and the first study of brain cell numbers using IF across developmental stages in any insect.

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Data from: IFN-γ-independent control of M. tuberculosis requires CD4 T cell-derived GM-CSF and activation of HIF-1α

RNA sequencing dataset from "Van Dis et al., IFN-γ-independent control of M. tuberculosis requires CD4 T cell-derived GM-CSF and activation of HIF-1α. 2022" The prevailing model of protective immunity to tuberculosis is that CD4 T cells produce the cytokine IFN-γ to activate bactericidal mechanisms in infected macrophages. Although IFN-γ-independent CD4 T cell based control of M. tuberculosis infection has been demonstrated in vivo it is unclear whether CD4 T cells are capable of directly activating macrophages to control infection in the absence of IFN-γ. We developed a co-culture model using CD4 T cells isolated from the lungs of infected mice and M. tuberculosis-infected murine bone marrow-derived macrophages (BMDMs) to investigate mechanisms of CD4 dependent control of infection. This dataset represents RNA sequencing data from M. tuberculosis-infected wild-type and Ifngr-/- murine bone marrow-derived macrophages (BMDMs) after 24 hours of lung CD4 T cell co-culture. CD4 T cells induced differential regulation of 1825 genes in wild-type BMDMs and 1142 genes in Ifngr-/- BMDMs compared to untreated. Although wild-type and Ifngr-/- BMDMs infected with M. tuberculosis were transcriptionally very similar prior to activation, the transcriptome of these genotypes of macrophages diverged after CD4 T cell co-culture. This is likely due in part to the presence or absence of IFN-γ signaling as IFN-γ alone regulates the expression of >2500 genes during M. tuberculosis infection. Still, 769 genes were altered in an IFN-γ-independent manner, with >2-fold upregulation in both wild-type and Ifngr-/- BMDMs. We found no difference in macrophage polarization between untreated M. tuberculosis-infected wild-type and Ifngr-/- BMDMs, and no significant increase in the expression of genes associated with M2 macrophages after CD4 T cell co-culture in either genotype. However, there was significant upregulation of genes associated with M1 macrophages after CD4 T cell co-culture in both genotypes, with wild-type BMDMs slightly more polarized. Collectively, these results show comparable patterns of activation in wild-type and Ifngr-/- macrophage during CD4 T cell co-culture, indicating that CD4 T cells elicit significant polarizing, inflammatory and antimicrobial effects in M. tuberculosis-infected macrophages irrespective of IFN-γ signaling.

opencc-zeroJul 2022View details →

Molcular Dynamics Data for Therapeutic High Affinity T Cell Receptor Targeting a KRAS G12D Cancer Neoantigen

This folder contains the starting structures and input scripts required to simulate the wild-type and G12D KRAS peptide bound TCR-pHLA complexes, as performed in this study. Starting_Structures - This folder contains the amber parameter/topology files used to simulate each system (.prmtop) and the coordinates of the starting structure both as amber coordinate file (.rst) and PDB file (.pdb). MD_Inputs - This folder contains the amber MD inputs used to run the md simulations.  MMPBSA_inputs - This folder contains the input files for running MMPBSA with the MMPBSA.py script in amber. The mmpbsa.in script was used for calculating overall binding energy whereas the mmpbsa_decomp.in script was used for calculating the per-residue contribution to binding energy.

High-density electrode recordings reveal strong and specific connections between retinal ganglion cells and midbrain neurons.

This is the source file to be use for debugging problems on your own set-up. This files is a good example to both the repository provided for visual processing on the Kremow GitHub: https://github.com/KremkowLab/Axon-on-Neuropixels-in-Kilosort https://github.com/KremkowLab/tangential_recording

Live-cell imaging of LLC-PK1 cells microtubule dynamics

Stable LLC-PK1 cell line (ATCC:CL-101) was generated and provided by Michael W. Davidson [1,2]. Using a Zeiss Celldiscoverer 7 microscope with a 100X/1.47 NA oil-immersion objective (Plan-Apochromat, Zeiss) and a sCMOS sensor (Hamamatsu, ORCA-Fusion, C15440-20UP), cultured LLC-PK1 cells expressing mEmerald-EB3 were imaged with a 43 nm pixel size. Exposure time was 100 ms, with each frame being captured every two seconds. During acquisition, temperature and CO2 control was set to 37°C and 5%, respectively. mEmerald-EB3 was excited using a 488 nm laser at 1% power, with a FITC filter for fluorescence collection. Acquisition software ZEN 3.2 (blue edition) was used for the imaging protocol. 1. Rizzo, M. A., Davidson, M. W., & Piston, D. W. (2009). Fluorescent protein tracking and detection: fluorescent protein structure and color variants. Cold Spring Harbor Protocols, 2009(12), pdb-top63. 2. Huang, F., Hartwich, T., Rivera-Molina, F. et al. Video-rate nanoscopy using sCMOS camera–specific single-molecule localization algorithms. Nat Methods 10, 653–658 (2013).

PD1+CD8+ cells are an independent prognostic marker in patients with head and neck cancer

Data open: file with parametres used for the multivariate evaluation.

Volumetric imaging of fluorescently labeled BPAE cells

Using the Nanoimager-S microscope (ONI, Oxford Nanoimaging) with a sCMOS sensor (Hamamatsu, ORCA-Flash4.0 V2), FluoCells™ Prepared Slide #1 (Thermo, #F36924) were imaged with a 100X, 1.4 NA, oil-immersion objective (Olympus). DAPI, Alexa-488 and MitoTracker™ Red excitation was delivered by 405 nm, 473 nm and 561 nm lasers, respectively. Light was collected while using two emission filters (1: 525/50; 2: Band 1 575-616.5) and a Channel Splitter dichroic 561 LP. A 3D Z-stack of the sample was acquired for each channel. 22 frames were generated, each separated 50 nm from each other in Z.

opencc-by-4.0Jul 2024View details →

Overexpression of VEGF in dermal fibroblast cells accelerates the angiogenesis and wound healing function: in vitro and in vivo studies

Human dermal fibroblasts (Hu02) were transfected by pcDNA3.1(-)-VEGF vector. Following selecting fibroblast cells with hygromycin, recombinant cells were investigated in terms of VEGF expression by quantifying method. We used Real-Time PCR assay to quantitate gene expression of vascular endothelial growth factor from manipulated cells and represented VEGF overexpression. Reverse transcription was performed from 1 μg of total RNA transcribed to complementary DNA (cDNA) through 1 μL of random hexamer primer. The reactions were incubated at 70°C for 5 minutes. After that, 5X RT-buffer, dNTP, and RT-enzyme were added, and the mixtures were incubated at 42°C for 60 minutes and 70°C for 10 minutes. Reverse transcription was performed from 500 ng total RNA using the RT2 First Strand Kit (SA Biosciences). Quantitative real-time PCR was performed (Ampliqon, Denmark) with 40 cycles at 95 oC for 15 seconds and 60 oC for 60 seconds. The normalization and all the data analysis were performed according to RESR and Graph pad-Prism 8 software. For the normalization, it uses the housekeeping gene: β-Actin. Target gene signals normalized to housekeeping genes; 2^-deltaCt, where deltaCt = (Ct_Target − Ct_HKG)].

THP-1 DNase I cell culture under confocal microscope

THP-1 DNase I cell imaged in suspension using a confocal microscope LSM780 Zeiss The cells have their nucleus, membrane, and mitochondria stained respectively using DAPI, Phalloidin, and TOMM20. The mitochondria signal is deconvoluted using Huygens SVI software. This is a high magnification cell culture data example for cell segmentation and mitochondria segmentation in 3D.

Additional movies for a manuscript titled 'Leaf epidermal cells respond to application and removal of mechanical force by distinct cytosolic calcium waves'

This repository provides 21 additional videos that highlight the spectrum of mechanically stimulated calcium responses observed in the leaf epidermis of Arabidopsis t. expressing the genetically encoded calcium indicator R-GECO1. These movies correspond to figures in a manuscript entitled 'Leaf epidermal cells respond to application and removal of mechanical force by distinct cytosolic calcium waves'. In each movie, a cantilever exerts approximately 5 mN force to the epidermis (where the cantilever looks like a shadow in the movies). Each movie contains a scale bar and the relative time (hr:min:sec). Placement of the cantilever generally occurs after approximately 1 minute.

Script and single cell RNA sequencing datasets of Biomphalaria glabrata hemocyte

The results of the gene and cell barcode counts (feature-barcode matrices) are available in the file "filtered_feature_bc_matrix_Naive". These data have been processed by cellRanger v3.1.0 and can be used with the script "scRNAseq_Biomphalaria_naive" which gathers all the analyses done for publication.

opencc-by-4.0Jun 2022View details →

TALON output for WTC11 cell lines

A range of long-read RNA-seq protocols is analyzed with TALON from WTC11 for the LRGASP challenge. We deposit our results here for further analysis.

opencc-by-4.0Dec 2021View details →

Rapid increase in transferrin receptor recycling promotes adhesion during T cell activation

This Dataset contains primary data used for the publication "Rapid increase in transferrin receptor recycling promotes adhesion during T cell activation" accepted for publication in BMC Biology on 15. July 2022 Abstract: Background T cell activation leads to increased expression of the receptor for the iron transporter transferrin (TfR) to provide iron required for the cell differentiation and clonal expansion that takes place during the days after encounter with a cognate antigen. However, T cells mobilise TfR to their surface within minutes after activation, although the reason and mechanism driving this process remain unclear. Results Here we show that T cells transiently increase endocytic uptake and recycling of TfR upon activation, thereby boosting their capacity to import iron. We demonstrate that increased TfR recycling is powered by a fast endocytic sorting pathway relying on the membrane proteins flotillins, Rab5 and Rab11a-positive endosomes. Our data further reveal that iron import is required for a non-canonical signalling pathway involving the kinases Zap70 and PAK, which controls adhesion of the integrin LFA-1 and eventually leads to conjugation with antigen-presenting cells. Conclusions Altogether, our data suggest that T cells boost their iron importing capacity immediately upon activation to promote adhesion to antigen-presenting cells.   Data are organised in compressed (.zip) folders entitled as the corresponding Figures in the publication. Programs we recommend to view the files are: .fcs files:    FlowJo software v10 (Tree Star, Ashland, OR, USA) .lsm and .czi files: ZEN 2012 SP1 and higher (Carl Zeiss, Jena, Germany) .lif files:       LAS X v3 (Leica Microsystems, Wetzlar, Germany) .pzfx files:   Prism v7 software (GraphPad, San Diego, CA, USA)

The effect of antidepressants on genes of endoplasmic reticulum stress in human astrocyte cell line.

Many central nervous system (CNS) diseases, including major depressive disorder (MDD), are underpinned by the unfolded protein response (UPR) activated under endoplasmic reticulum (ER) stress. New, more efficient, therapeutic options for MDD are needed to avoid adverse effects and drug resistance. Therefore, the aim of the work was to determine whether UPR signalling pathway activation in astrocytes may serve as a novel target for antidepressant drugs. Among the tested antidepressants (escitalopram, amitriptyline, S-ketamine), only S-ketamine induced the expression of most ER stress-responsive genes in astrocytes. <a href="https://doi.org/10.3390/pharmaceutics14040846">https://doi.org/10.3390/pharmaceutics14040846</a>

Supplementary Data for "The shaky foundations of simulating single-cell RNA sequencing data"

Supplementary Data for "The shaky foundations of simulating single-cell RNA sequencing data" See description.txt, Supplementary Text, Methods and https://github.com/HelenaLC/simulation-comparison for further description of the files available here.

Sequence tracks of Ago2 Neural stem cells and differentiated neurons from single-cells- Related to Fig. 5

Single neural stem cells were isolated from the Hippocampus of newborn mice generated from a hybrid cross. Some of these cells were differentiated In vitro and either the NSC or differentiated neurons were lysed and underwent a reverse transcription. The newly formed cDNA was used as a template to amplify expressed Ago2 transcript which was then sent off for Sanger sequencing. A SNP located within the exon was used to determine whether the transcript from that cell was generated from the maternal or paternal allele.

opencc-zeroAug 2022View details →

dryad36/100

The role of cell-envelope synthesis for envelope growth and cytoplasmic density in Bacillus subtilis

All cells must increase their volumes in response to biomass growth to maintain intracellular mass density within physiologically permissive bounds. Here, we investigate the regulation of volume growth in the Gram-positive bacterium Bacillus subtilis. To increase volume, bacteria enzymatically expand their cell envelopes and insert new envelope material. First, we demonstrate that cell-volume growth is determined indirectly, by expanding their envelopes in proportion to mass growth, similarly to the Gram-negative Escherichia coli, despite their fundamentally different envelope structures. Next, we studied, which pathways might be responsible for robust surface-to-mass coupling: We found that both peptidoglycan synthesis and membrane synthesis are required for proper surface-to-mass coupling. However, surprisingly, neither pathway is solely rate-limiting, contrary to wide-spread belief, since envelope growth continues at a reduced rate upon complete inhibition of either process. To arrest cell-envelope growth completely, the simultaneous inhibition of both envelope-synthesis processes is required. Thus, we suggest that multiple envelope-synthesis pathways collectively confer an important aspect of volume regulation, the coordination between surface growth and biomass growth.

Variable allelic expression of imprinted genes at the Peg13, Trappc9, Ago2 cluster in single neural cells

Fig 1 5' RACE - Sequence tracks and analysis of alternative Trappc9 transcriptional start sites Fig 2 and suppl fig 3- Brain and Kidney tissue or Neural stem cells isolated from the Hippocampus region of newborn mice generated from a C57BL/6 (female) and a Cast/EiJ (male) cross (and its reciprocal cross) was used to determine allelic bias expression. A SNP located within an exon of Peg13, Trappc9, Ago2, Chrac1 and Kcnk9 was identified and amplified via pyrosequencing PCR with the percentage of SNP identification used to determine allele expression percentages. Additionally, Pyrorun sequences of reverse transcribed RNA from Trappc9 expression in different tissues. A hybrid cross between C57BL/6 and JF1 mouse was used to generate hybrid pups that were used to determine allele specificity of Trappc9 expression in Kidney and brain tissues. Figs 3, 4 & 5- Single neural stem cells were isolated from the Hippocampus of newborn mice generated from a hybrid cross. Some of these cells were differentiated In vitro and either the NSC or differentiated neurons were lysed and underwent a reverse transcription. The newly formed cDNA was used as a template to amplify expressed Peg13, Trappc9 or Ago2 transcripts which was then sent for Sanger sequencing. A SNP located within the exon was used to determine whether the transcript from that cell was generated from the maternal or paternal allele. Fig 6- Brain-specific regulatory elements were cloned into a pGL [Luc] vector containing a Trappc9 promoter. These newly generated plasmids were then transfected into either primary neuron or fibroblast cultures alongside a Renilla vector for normalization using Lipofectamine as a transfection reagent. After 48 hours the cells were lysed and analyzed using a Glomax illuminator to determine their impact on Luciferase expression compared to that of the pGL vector containing just the Trappc9 promoter. Additionally, Plasmid vectors that were used for transfection of primary neurons to determine the impact of brain-specific regulatory elements on transcription. Plasmids can be visualized using the free software pdraw.32 downloaded from http://acaclone.com/download/install.htm Suppl fig 2- Single Neural stem cells and in vitro differentiated neurons isolated from Mouse Hippocampus tissue underwent reverse transcription and a qPCR reaction intended to amplify cDNA of genes associated with specific neural cell types as a method of detecting cell fate and whether this had an impact on allele-specific expression and differential methylation. Suppl fig 4 & 5- DNA isolated from Neural stem cells was bisulfite converted for downstream identification of methyl group presence. CpG islands located at or near the promoters of the Peg13, Trappc9, Ago2, Chrac1 and Kcnk9 genes were amplified and cloned into a TOPO vector. The cloned segments were Sanger sequenced and compared to the original non-bisulfite converted sequence using the Quantification for methylation analysis (QUMA) tool to determine which CG dinucleotides were methylated and which weren't. Pyroruns determining methylation frequency at the CpG islands of these genes can be found in a separate upload on Zenodo.

Microchamber slide design for cell confinement during imaging- measurements

Spreadsheet of measurements of cells using CellProfiler.