Tag: FACS

Exploring the Latest Advances in Transcriptomics

Since the idea of genetic regulation was first postulated back in the 1950s, our scientific understanding of the transcriptome has deepened greatly.1 Looking into the transcriptome of cells and tissues has helped scientists to understand the biological processes that drive both health and disease; however, the complex and occasionally mysterious…

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A high-resolution transcriptomic and spatial atlas of cell types in the whole mouse brain

Mouse breeding and husbandry All experimental procedures related to the use of mice were approved by the Institutional Animal Care and Use Committee of the AIBS, in accordance with NIH guidelines. Mice were housed in a room with temperature (21–22 °C) and humidity (40–51%) control within the vivarium of the AIBS…

Continue Reading A high-resolution transcriptomic and spatial atlas of cell types in the whole mouse brain

Single-cell analysis of chromatin accessibility in the adult mouse brain

Tissue preparation and nucleus isolation All experimental procedures using live animals were approved by the SALK Institute Animal Care and Use Committee under protocol number 18-00006. Adult C57BL/6J male mice were purchased from Jackson Laboratories. Brains were extracted from 56–63-day-old mice and sectioned into 600 µm coronal sections along the anterior–posterior…

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Atg7 senses ATP levels and regulates AKT1-PDCD4 phosphorylation-ubiquitination axis to promote survival during metabolic stress

Atg7 interacts with PDCD4 and negatively regulates PDCD4 protein levels Previous studies have demonstrated the role of Atg7 in maintaining metabolic homeostasis by initiating autophagy and arresting the cell cycle17,18,22. Herein, the function of Atg7 was more comprehensively investigated and it was determined whether Atg7 reduces protein synthesis to save…

Continue Reading Atg7 senses ATP levels and regulates AKT1-PDCD4 phosphorylation-ubiquitination axis to promote survival during metabolic stress

Phenotypic profiling of solute carriers characterizes serine transport in cancer

Cell culture All cell lines used in this study were cultured at 37 °C in 5% CO2 in a humidified incubator. Human cell lines were authenticated by STR profiling using Promega GenePrint 10 and tested for Mycoplasma using Mycoalert (Lonza). Other than HCT116 p21−/− (a gift of B. Vogelstein60) all cell…

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The 6 Biggest Stories in Medicine in 2023

It’s so hard to avoid spoilers online. Take, for example, right now. Welcome back to our 7th edition of This Year in Medicine, our annual end-of-year recap series that highlights the greatest stories, challenges, moments and innovations in health care. On every Monday, Wednesday and Friday this December, our team…

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RHEX-RNAseq – a tagmentation-based, rRNA blocked, random hexamer primed RNAseq method for generating stranded RNAseq libraries directly from very low numbers of lysed cells.

Gustafsson C, Hauenstein J, Frengen N, Krstic A, Luc S, Månsson R BMC Genomics 24 (1) 205 [2023-04-17; online 2023-04-17] RNA sequencing has become the mainstay for studies of gene expression. Still, analysis of rare cells with random hexamer priming – to allow analysis of a broader range of transcripts…

Continue Reading RHEX-RNAseq – a tagmentation-based, rRNA blocked, random hexamer primed RNAseq method for generating stranded RNAseq libraries directly from very low numbers of lysed cells.

ESRP1 controls biogenesis and function of a large abundant multiexon circRNA | Nucleic Acids Research

Abstract While the majority of circRNAs are formed from infrequent back-splicing of exons from protein coding genes, some can be produced at quite high level and in a regulated manner. We describe the regulation, biogenesis and function of circDOCK1(2–27), a large, abundant circular RNA that is highly regulated during epithelial-mesenchymal…

Continue Reading ESRP1 controls biogenesis and function of a large abundant multiexon circRNA | Nucleic Acids Research

Genome editing approaches with CRISPR/Cas9: the association of NOX4 expression in breast cancer patients and effectiveness evaluation of different strategies of CRISPR/Cas9 to knockout Nox4 in cancer cells | BMC Cancer

A. Bioinformatics analyzes Search strategy and study selection From April to June 2021, we read relevant and related publications in Medline, EMBASE, Web of Science, and Wanfang. The search term “NOX4” have been used. The articles that fit specified criteria were all added at once: (1) Participants were split into…

Continue Reading Genome editing approaches with CRISPR/Cas9: the association of NOX4 expression in breast cancer patients and effectiveness evaluation of different strategies of CRISPR/Cas9 to knockout Nox4 in cancer cells | BMC Cancer

A Trimodal Approach to Interrogate Breast Cancer

Sponsored content brought to you by Along with advances in single-cell sequencing and multiomic analysis, researchers are able to more precisely decode the stages of human cancers—from precursor to development of invasive cancer—to more accurately inform therapeutic approaches. With more than 2 million global cases of breast cancer diagnosed annually,…

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Immune-privileged tissues formed from immunologically cloaked mouse embryonic stem cells survive long term in allogeneic hosts

Mice C57BL/6N (strain 005304), C3H/HeJ (strain 000659), FVB/NJ (strain 001800), BALB/cJ (strain 000651) and NSG mice (stock 005557) were purchased from the Jackson Laboratory. CD-1 (stock 022) mice were purchased from Charles River. Mice (6–20-week-old) of each strain/background were used for teratoma assays. Mice were housed in a pathogen-free facility…

Continue Reading Immune-privileged tissues formed from immunologically cloaked mouse embryonic stem cells survive long term in allogeneic hosts

High-throughput screening of genetic and cellular drivers of syncytium formation induced by the spike protein of SARS-CoV-2

Plasmid construction All the constructs used in this study were generated with standard cloning strategies, including PCR, overlapping PCR, oligo annealing, digestion and ligation. Primers were purchased from Genewiz. The plasmid sequence was verified by Sanger sequencing. The pCAG-spike(D614G)-GFP11-mCherry plasmid was modified from Addgene plasmid 158761. Briefly, GFP11 and mCherry…

Continue Reading High-throughput screening of genetic and cellular drivers of syncytium formation induced by the spike protein of SARS-CoV-2

Decline of DNA damage response along with myogenic differentiation

Introduction Proper functioning of all living organisms depends on the faithful maintenance and transmission of genomic information stored in the molecule of DNA. However, DNA integrity is continuously challenged by a variety of endogenous and exogenous agents causing DNA lesions which have a critical impact on cellular activities and homeostasis….

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Chromatin priming elements direct tissue-specific gene activity before hematopoietic specification

Introduction The development of multicellular organisms requires the activation of different gene batteries which specify the identity of each individual cell type. Such shifts in cellular identity are driven by shifts in the gene regulatory network (GRN) consisting of transcription factors (TFs) binding to the enhancers and promoters of their…

Continue Reading Chromatin priming elements direct tissue-specific gene activity before hematopoietic specification

Megakaryocytes possess a STING pathway that is transferred to platelets to potentiate activation

Introduction Host defence against infection relies on two important systems: the immune and the coagulation systems. Platelets are anucleate cells produced by megakaryocytes and are responsible for blood clotting and haemostasis (Lefrancais et al, 2017). They are also very abundant and play unexpected roles in immune responses (Maouia et al,…

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Elevated stress response marks deeply quiescent reserve cells of gastric chief cells

Generation of inducible H2b-GFP knock-in mice Generation of inducible H2b-GFP knock-in mice was performed by using CRISPR/Cas943. In brief, a mixture containing a sgRosa26-1 crRNA43 (8.7 ng/μl, Fasmac, Japan), a tracrRNA (14.3 ng/μl, Fasmac, Japan), a single strand oligo donor nucleotide (ssODN) composed of 5′ arm, adenovirus splicing acceptor, SV40 pA, TRE3G…

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A single pseudouridine on rRNA regulates ribosome structure and function in the mammalian parasite Trypanosoma brucei

Cell growth and transfections Procyclic form (PCF) T. brucei, strain 29-1354, which carries integrated genes for the T7 polymerase and the tetracycline repressor, was grown in SDM-79 medium supplemented with 10% fetal calf serum, in the presence of 50 μg/ml hygromycin. Cells were grown in the presence of 15 μg/ml G418 for…

Continue Reading A single pseudouridine on rRNA regulates ribosome structure and function in the mammalian parasite Trypanosoma brucei

Single-cell RNAseq analysis of spinal locomotor circuitry in larval zebrafish

. 2023 Nov 17:12:RP89338. doi: 10.7554/eLife.89338. Affiliations Expand Affiliation 1 Vollum Institute, Oregon Health & Science University, Portland, United States. Item in Clipboard Jimmy J Kelly et al. Elife. 2023. Show details Display options Display options Format AbstractPubMedPMID . 2023 Nov 17:12:RP89338. doi: 10.7554/eLife.89338. Affiliation 1 Vollum Institute, Oregon Health &…

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11-100test | EpCAM antibody [VU-1D9] (PerCP-Cy5.5) Clinisciences

Application Note *Optimal dilutions/concentrations should be determined by the researcher. Application Recommended Dilution FACS 4 μl reagent / 100 μl of whole blood or 10⁶ cells in a suspension Not tested in other applications. Calculated MW 35 kDa. ( Note ) Product Note This antibody recognizes an epitope within EGF-like…

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Xtandi Gains Approval for Nonmetastatic Castration-Sensitive Prostate Cancer

The Food and Drug Administration (FDA) has approved the supplemental New Drug Application (sNDA) for Xtandi® (enzalutamide) for the treatment of patients with nonmetastatic castration-sensitive prostate cancer (nmCSPC) with biochemical recurrence at high risk for metastasis (high-risk BCR).  The approval was based on data from the phase 3 EMBARK trial…

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RStudio Lab Week 7 – Logistic Regression and Model Building – RStudio Lab Week 7: Logistic

RStudio Lab Week 7: Logistic Regression and Model Building Data Part 1: Logistic Regression The logreg.csv is a data set from a study of depression. The objective of this analysis is to use the depression diagnosis of 150 individuals (cases) and assess its association with the sex of the respondent…

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Circular extrachromosomal DNA promotes tumor heterogeneity in high-risk medulloblastoma

Statistical methods Statistical tests, test statistics and P values are indicated where appropriate in the main text. Categorical associations were established using the chi-squared test of independence if n > 5 for all categories and Fisherʼs exact test otherwise. For both tests, the Python package scipy.stats v1.5.3 implementation was used64. Multiple hypothesis corrections…

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FAT10 is phosphorylated by IKK{beta} to inhibit the antiviral type-I interferon response

Introduction The innate immune system represents the host´s first-line defense against viral infections (Koyama et al, 2008). In this context, IFN-I are produced by all nucleated cells and represent the principal cytokines that counteract viral replication (Ivashkiv & Donlin, 2014). In fact, several receptors of the infected cell can recognize…

Continue Reading FAT10 is phosphorylated by IKK{beta} to inhibit the antiviral type-I interferon response

CloneMedia CHO Growth A

Limiting dilution or fluorescence-activated cell sorting (FACS) is traditionally used to clone CHO cells. These methods can be inefficient as they may require additional rounds of sub-cloning to obtain a high probability of monoclonality. The CloneMedia CHO Growth A is a viscous semi-solid media that immobilizes single CHO cells, allowing…

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An FcRn-targeted mucosal vaccine against SARS-CoV-2 infection and transmission

Mice and Golden Syrian hamsters All research experiments in this study comply with all relevant ethical regulations. The Institutional Animal Care and Use Committee approved the animal protocol at the University of Maryland (#R-APR-20-18 and #R-MAR-21-19). The animals were acclimatized at the animal facility for 4–6 days before initiating experiments….

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Phase 3 LYNX-1 trial data show phentolamine ophthalmic solution improves low-contrast night vision

Investigators concluded both that the active treatment significantly improved low-contrast night and that the drug had a favorable safety profile. Image credit: ©Drobot Dean – stock.adobe.com Reviewed by P. Dee G. Stephenson, MD, FACS P. Dee G. Stephenson, MD, FACS, and colleagues reported that 0.75% phentolamine ophthalmic solution (Ryzumvi, Ocuphire…

Continue Reading Phase 3 LYNX-1 trial data show phentolamine ophthalmic solution improves low-contrast night vision

Epigenetic regulation during cancer transitions across 11 tumour types

Specimen data All samples for MM, OV, BRCA, PDAC, UCEC, CRC, CESC/AD, SKCM and HNSCC, as well as 2 NATs for GBM and 1 NAT for ccRCC were collected with informed consent in concordance with Institutional Review Board (IRB) approval at the School of Medicine at Washington University in St…

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How An Unexpected Discovery in a University Pond Changed the DNA Rulebook

Accidental discoveries have led to numerous breakthroughs and innovations throughout history; the antibiotic penicillin, the microwave and even the drug Viagra are all products of chance discoveries.  Serendipity in science often challenges laws or rules that we have previously regarded as established and absolute. It serves as a tool for…

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TET2 modulates spatial relocalization of heterochromatin in aged hematopoietic stem and progenitor cells

Animal models Animal studies were approved by the Institutional Animal Care Use Committee of the Institute of Biosciences and Technology, Texas A&M University (AUP 2020-0195). Most mouse strains bear a C57BL/6 genetic background unless otherwise noted. All the animals were housed in a certified animal facility with a standard dark/light…

Continue Reading TET2 modulates spatial relocalization of heterochromatin in aged hematopoietic stem and progenitor cells

DNA-binding protein PfAP2-P regulates parasite pathogenesis during malaria parasite blood stages

Parasite culture, maintenance, synchronization and transfection The DiCre-expressing P. falciparum clone II3 (ref. 10) was maintained in human A+ erythrocytes at 37 °C in Roswell Park Memorial Institute 1640 medium containing AlbumaxII (Invitrogen) supplemented with 2 mM l-glutamine. Parasites were either synchronized by sorbitol treatment or by purifying mature schizont stages using…

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GoM DE: interpreting structure in sequence count data with differential expression analysis allowing for grades of membership | Genome Biology

Models for single-cell ATAC-seq data In single-cell ATAC-seq data, \(x_{ij}\) is the number of unique reads mapping to peak or region j in cell i. Although \(x_{ij}\) can take non-negative integer values, it is common to “binarize” the accessibility data (e.g., [19, 74, 133,134,135]), meaning that \(x_{ij} = 1\) when…

Continue Reading GoM DE: interpreting structure in sequence count data with differential expression analysis allowing for grades of membership | Genome Biology

Inactive S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in human stem cell model of Parkinson’s disease

Cloning of CRISPR/sgRNA lentiviral constructs with fluorescent selection markers A tetracycline-inducible promoter (TRE3G) was used to control the expression of S. aureus dCas9 in a lentiviral vector. To facilitate selection of cells by FACS, pHR:TRE3G-SadCas9-2xKRAB-p2a-tdTomato (Addgene ID #209298) was subcloned from a pHR:TRE3G-SadCas9-2xKRAB-p2a-zeo (A gift from Professor Stanley Qi), where zeocin…

Continue Reading Inactive S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in human stem cell model of Parkinson’s disease

Neuron Navigator 1 (Nav1) regulates the response to cocaine in mice

Mouse strains All mouse procedures were approved by the Institutional Animal Care and Use Committees at Binghamton or Stanford University; and were conducted in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals, Eighth Edition. All mice were originally obtained from Jackson Laboratories, and…

Continue Reading Neuron Navigator 1 (Nav1) regulates the response to cocaine in mice

Research Associate / Postdoc (m/f/x) job with TECHNISCHE UNIVERSITAT DRESDEN (TU DRESDEN)

The Center for Regenerative Therapies Dresden (CRTD), an institute of the Center for Molecular and Cellular Bioengineering (CMCB), is a research center at the TUD Dresden University of Technology with currently over 20 research groups. The research focus of CRTD lies in regeneration and stem cell research from fundamental research…

Continue Reading Research Associate / Postdoc (m/f/x) job with TECHNISCHE UNIVERSITAT DRESDEN (TU DRESDEN)

Stanford Scientists Uncover New Indicators of Health, Disease, and Aging

The human lipidome, encompassing all the body’s lipids, is gaining attention for its role in human physiology, particularly its direct influence by diet and gut microbes, and its potential in disease intervention, especially in conditions like Type 2 diabetes. A recent study dives deep into the lipidome, revealing its association…

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Characterizing the regulatory Fas (CD95) epitope critical for agonist antibody targeting and CAR-T bystander function in ovarian cancer

Mice strains Six- to eight-week-old (age), 20–25-g (weight), both male and female (sex) mice were used for tumor xenografts generation and in vivo efficacy studies as described in the text (see Table 3). Athymic Nude (Envigo) Foxn1nu/Foxn1+ or NOD.Cg Prkdcscid Il2rgtm1Wjl/SzJ also called NSG mice were used. All animal procedures were…

Continue Reading Characterizing the regulatory Fas (CD95) epitope critical for agonist antibody targeting and CAR-T bystander function in ovarian cancer

EOGT enables residual Notch signaling in mouse intestinal cells lacking POFUT1

Single cell RNAseq bioinformatics Bioinformatic trajectory analysis of scRNAseq data obtained from Epcam+CD45− C57Bl6/J adult intestinal crypt cells and deposited as GSE188339 was performed as described35. Primers and antibodies Primer sequences are given in Supplementary Table 1 and antibodies are given in Supplementary Table 2. Generation of Chinese hamster ovary…

Continue Reading EOGT enables residual Notch signaling in mouse intestinal cells lacking POFUT1

CITE-seq data visualization

Hello, I have produced quite some single cell CITE-seq data at the single cell level, using the 10x genomics platform and analyzed in Seurat. I am at the moment trying to visualize the protein markers from the CITEseq against to each other in a way that would resemble how we…

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Global Single Cell Genomics Market Research Report 2023 with Executive and Consultant Guides – Forecasts to 2027 – ResearchAndMarkets.com

DUBLIN–(BUSINESS WIRE)–The “Single Cell Genomics Markets. Forecasts by Technology, Product, Workflow, User and Country with Executive and Consultant Guides. 2023 to 2027” report has been added to ResearchAndMarkets.com‘s offering. Discover the opportunities, navigate potential pitfalls, and gain insights into growth projections and the ultimate market size. This report is your…

Continue Reading Global Single Cell Genomics Market Research Report 2023 with Executive and Consultant Guides – Forecasts to 2027 – ResearchAndMarkets.com

Rapid discovery of high-affinity antibodies via massively parallel sequencing, ribosome display and affinity screening

Construct design To transcribe and translate sequenced DNA clusters on an Illumina flow cell, our DNA constructs contained the following elements: a P5 adaptor, followed by a 28 nt unique barcode, a 27 nt unstructured spacer (5p UNS v2), a ribosome binding site, start codon, protein coding region, TolAk short linker, 2×…

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Global Single Cell Genomics Market Research Report

Dublin, Oct. 06, 2023 (GLOBE NEWSWIRE) — The “Single Cell Genomics Markets. Forecasts by Technology, Product, Workflow, User and Country with Executive and Consultant Guides. 2023 to 2027” report has been added to ResearchAndMarkets.com‘s offering. Discover the opportunities, navigate potential pitfalls, and gain insights into growth projections and the ultimate…

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OmniAb, Inc. hiring Senior Bioinformatics Scientist in Emeryville, California, United States

We are seeking a Senior Bioinformatics Scientist to support our ongoing antibody discovery and genetic engineering efforts. The role includes refining custom tools and algorithms to facilitate identification of therapeutic leads from OmniAb immune repertoires and applying cutting edge computational biology approaches to all stages of antibody discovery, from antigen…

Continue Reading OmniAb, Inc. hiring Senior Bioinformatics Scientist in Emeryville, California, United States

Mitochondrial DNA damage triggers spread of Parkinson’s disease-like pathology

Lack of IFNβ/IFNAR signaling causes mtDNA oxidization and mutation in a hotspot in complex I respiratory chain subunits mimicking PD brain pathology We analyzed transcriptomic datasets from sPD patients [28] to identify molecular pathways related to the disease pathology. Dysregulated oxidative phosphorylation (OXPHOS) emerged as the top-ranked pathway in sPDD, patients with…

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Researchers find mitochondrial DNA damage triggers spread of Parkinson’s disease-like pathology

Complex I respiratory chain subunits are altered in PD patients and PD models (Ifnb-/-/Ifnar1-/-) due to mtDNA damages. A Enrichment plot of KEGG-OXPHOS from microarray data comparing sporadic Parkinson’s disease with dementia (sPDD/N = 13) and healthy controls (HC/N = 14). B Immunoblot showing loss of ND4 and ND5 proteins in the prefrontal cortex…

Continue Reading Researchers find mitochondrial DNA damage triggers spread of Parkinson’s disease-like pathology

Selection and enrichment of microbial species with an increased lignocellulolytic phenotype from a native soil microbiome by activity-based probing

Soil incubation and cell extraction Approximately 3 kg of unmanaged marginal soil (pH 8) was collected from the Pacific Northwest National Laboratory field site in Prosser, Washington (46° 15′ 04″ N and 119° 43′ 43″ W) [23], homogenized, sieved (4 mm mesh size), and stored at 4 °C until further processing. Probe specific…

Continue Reading Selection and enrichment of microbial species with an increased lignocellulolytic phenotype from a native soil microbiome by activity-based probing

Role of YAP in hematopoietic differentiation and erythroid lineage specification of human-induced pluripotent stem cells | Stem Cell Research & Therapy

Culture of human-induced pluripotent stem cells (hiPSCs) Human iPSC lines were cultured in Matrigel-coated plates in the Nutristem medium (Corning, USA). Cells were passaged every 5 days by treating with Versene (Thermo Fisher Scientific, USA) for 3–5 min, transferred to freshly prepared Matrigel-coated plates, and cultured under hypoxic conditions with 5% CO2…

Continue Reading Role of YAP in hematopoietic differentiation and erythroid lineage specification of human-induced pluripotent stem cells | Stem Cell Research & Therapy

CITE-seq reveals inhibition of NF-kB pathway in B cells from vitamin D-treated multiple sclerosis patients

Abstract Vitamin D deficiency is a recognized risk factor for multiple sclerosis (MS) and has been associated with disease activity and progression. Vitamin D treatment has emerged as potentially protective, despite conflicting results from randomized controlled trials. Here, we used single-cell RNA-sequencing (scRNA-seq) combined with barcoded antibodies targeting surface markers…

Continue Reading CITE-seq reveals inhibition of NF-kB pathway in B cells from vitamin D-treated multiple sclerosis patients

Assembloid CRISPR screens reveal impact of disease genes in human neurodevelopment

Culture of hiPS cells The hiPS cell lines used in this study were validated as previously described51. The SEC61B-mEGFP hiPS cell line (AICS-0059 cl.36) was developed at the Allen Institute for Cell Science (www.allencell.org/cell-catalog.html) and is distributed through Coriell43. The CAG::Cas9 cell line was genetically engineered in the laboratory (the…

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Phase 3 Trial Data Highlights Efficacy of OnabotulinutoxinA in Platysma Prominence

Last week, Allergan Aesthetics announced positive topline results from the second of its phase 3 studies exploring the use of onabotulinumtoxinA (BOTOX Cosmetic) in the indication of platysma prominence. According to Allergan, results of the study were consistent with the first of the trials, of which results were shared in…

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KCNQ potassium channels modulate Wnt activity in gastro-oesophageal adenocarcinomas

Introduction The KCNQ (potassium voltage-gated channel subfamily Q) family of ion channels encode potassium transporters (1). KCNQ proteins typically repolarise the plasma membrane of a cell after depolarisation by allowing the export of potassium ions, and are therefore involved in wide-ranging biological functions including cardiac action potentials (2), neural excitability…

Continue Reading KCNQ potassium channels modulate Wnt activity in gastro-oesophageal adenocarcinomas

A computationally designed antigen eliciting broad humoral responses against SARS-CoV-2 and related sarbecoviruses

Phylogenetic analysis Protein sequences of spike proteins were downloaded from the NCBI virus database for all the known sarbecoviruses (June 2020). A multiple sequence alignment was generated using MUSCLE36. The resulting multiple sequence alignment was pruned to the RBD region, filtered at 95% sequence identity and used as input for…

Continue Reading A computationally designed antigen eliciting broad humoral responses against SARS-CoV-2 and related sarbecoviruses

Split gene-editing tool offers greater precision

Chemically inducible split ABE (sABE) with tightly regulated deaminase activity. a Schematic of the EYFP fluorescence. An A-to-G conversion on the highlighted adenine on the antisense strand of the dysfunctional EYFP* gene can restore the expression of functional EYFP protein. b Schematic of the sABE. In the absence of rapamycin…

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Single-cell screening of cerebral organoids to identify developmental defects in autism

In a recent study published in Nature, researchers develop the clustered regularly interspaced short palindromic repeats (CRISPR)-human organoids-single-cell ribonucleic acid (RNA) sequencing (CHOOSE) system to identify developmental brain defects in autism. Study: Single-cell brain organoid screening identifies developmental defects in autism. Image Credit: Yurchanka Siarhei / Shutterstock.com Diagnosing autism Autism spectrum disorder (ASD) is…

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Mismatch repair deficiency is not sufficient to elicit tumor immunogenicity

Mice All animal use was approved by the Department of Comparative Medicine at the Massachusetts Institute of Technology (MIT) and the Institutional Animal Care and Use Committee under protocol no. 0714-076-17. Mice were housed with a 12-h light/12-h dark cycle with temperatures in the range 20–22 °C and 30–70% humidity. KrasLSL-G12D…

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FACS (Fluorescence-activated cell sorting NOT cyTOF),Automating the removal of dead and duplicated cells

FACS (Fluorescence-activated cell sorting NOT cyTOF),Automating the removal of dead and duplicated cells 0 I have a large FACS project involving hundreds of files. So, it is not practical or suitable to manually gate these in Flowjo etc and to remove dead and duplicated cells. I have found many different…

Continue Reading FACS (Fluorescence-activated cell sorting NOT cyTOF),Automating the removal of dead and duplicated cells

Single-cell brain organoid screening identifies developmental defects in autism

Stem cell and cerebral organoid culture conditions Feeder-free hES cells or iPS cells were cultured on hES cell-qualified Matrigel (Corning, catalogue no. 354277)-coated plates with Essential8 stem cell medium supplemented with bovine serum albumin (BSA). H9 embryonic stem cells were obtained from WiCell. Cells were maintained in a 5% CO2 incubator at 37 °C….

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Exosomal circEZH2_005, an intestinal injury biomarker, alleviates intestinal ischemia/reperfusion injury by mediating Gprc5a signaling

Human plasma collection Fifty patients who were assigned for CPB surgery between May 2021 and February 2022 at the Department of Cardiac Surgery, Southern Medical University Nanfang Hospital (Guangzhou, China) were recruited given their increased risk of intestinal I/R injury. In patients undergoing cardiac surgery, CPB is potentially responsible for…

Continue Reading Exosomal circEZH2_005, an intestinal injury biomarker, alleviates intestinal ischemia/reperfusion injury by mediating Gprc5a signaling

Symmetric inheritance of parental histones governs epigenome maintenance and embryonic stem cell identity

The research in this study was conducted under the ethical approval of the Danish Regulatory Authority under project license 2018-15-0201-01520. Cell culture and differentiation assays WT, MCM2-2A, MCM2-R and POLE4-KO mouse ESCs used in this study were derived from the male, E14JU cell line with a 129/Ola background81. Genome editing…

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Generation of inactivated IL2RG and RAG1 monkeys with severe combined immunodeficiency using base editing

Animals The gene targeting experiment was conducted on cynomolgus monkeys, which served as the experimental subjects. These monkeys were housed at Guangdong LANDAU Biotechnology Co. Ltd and TOPGENE Biotechnology Co. Ltd, where they received unrestricted access to water and were provided with a standard diet in accordance with standard care…

Continue Reading Generation of inactivated IL2RG and RAG1 monkeys with severe combined immunodeficiency using base editing

Epitope editing enables targeted immunotherapy of acute myeloid leukaemia

Sleeping Beauty transgene overexpression Bidirectional Sleeping Beauty plasmids encoding the codon-optimized cDNA of the gene of interest (FLT3, KIT, CD123) or their mutated variants were cloned downstream of an EEF1A1 promoter. The same construct included a reporter cassette comprising a fluorescent protein (mCherry or mTagBFP2 for FLT3 and KIT, respectively)…

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Non-cell-autonomous cancer progression from chromosomal instability

Cell culture IMR90, 4T1, CT26, RAW264.7 and B16F10 cell lines were purchased from the American Type Culture Collection and cultured in MEM (IMR90), DMEM (B16F10, RAW264.7) or RPMI (4T1, IMR90, CT26) supplemented with 10% FBS in the presence of penicillin (50 U ml−1) and streptomycin (50 μg ml−1). All cells were found to be…

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CircRNA-CIRH1A Promotes the Development of Osteosarcoma by Regulating PI3K/AKT and JAK2/STAT3 Signaling Pathways

Tissue Collection The study was approved by the Medical Ethics Committee at the Fuyang Traditional Chinese Medicine Orthopedics Hospital (2019-F027). OS tumor tissues and adjacent normal tissues were collected from 40 patients diagnosed with OS at the Fuyang Traditional Chinese Medicine Orthopedics Hospital from June 2018 to Dec 2019. Patient…

Continue Reading CircRNA-CIRH1A Promotes the Development of Osteosarcoma by Regulating PI3K/AKT and JAK2/STAT3 Signaling Pathways

Senior Research Associate/Associate Scientist, Cell Biology – Cambridge, Cambridgeshire, United Kingdom – CS Genetics Limited

Description Our Company CS Genetics is a privately-held genomics-technology company based in Cambridge, UK and San Diego, California. We are a nimble, highly-capable team focused on delivering genuine step-change innovation in genomic tools for research and clinical applications. We have developed a disruptive platform for single-cell genomics which leverages instrument-free,…

Continue Reading Senior Research Associate/Associate Scientist, Cell Biology – Cambridge, Cambridgeshire, United Kingdom – CS Genetics Limited

CRISPR/Cas9 generation of knock-out iPSCs

May 23, 2023 MANUSCRIPT CITATION: Dou D, Smith EM, Evans CS, Boecker CA, Holzbaur EL, Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes. Cell reports 42(5). doi: 10.1016/j.celrep.2023.112448 License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,…

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Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia

This protocol describes the isolation of double-negative thymocytes from the mouse thymus followed by retroviral transduction and co-culture on the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4) for further functional analysis. Transducing immature thymocytes with retroviral vectors and culturing these cells on OP9 DL4 stromal cells is…

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Ulcerative Colitis Treatment by CLP loaded with NLRP3 siRNA

Introduction Since the 20th century, tThe incidence of ulcerative colitis (UC) has been increasing over time, negatively affecting peoples’ health.1,2 UC is a multifactorial induced inflammatory bowel disease (IBD) characterized by chronic recurrent and remitting mucosal inflammation.3,4 It originates in the rectum and then extends to the proximal segments of…

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Transient naive reprogramming corrects hiPS cells functionally and epigenetically

Cell culture All cell lines used and derived by different approaches in this study are listed in Supplementary Table 1. Detailed information about the experimental design, materials and reagents is presented in the Reporting Summary. Primary human adult dermal fibroblasts (HDFa) from three different female donors were obtained from Gibco…

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In vivo screening characterizes chromatin factor functions during normal and malignant hematopoiesis

Mouse models C57BL/6J (strain 000664, The Jackson Laboratory) and B6J.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*/-EGFP)Fezh/J (strain no. 026179, The Jackson Laboratory) were used for all experimental procedures. The Npm1c/Flt3-ITD/Cas9 model has been extensively described previously31,57,58. The maximal tumor size allowed by the Home Office license for this project and authorized by the Animal Welfare Ethical…

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Dynamic thresholding and tissue dissociation optimization for CITE-seq identifies differential surface protein abundance in metastatic melanoma

Workflow overview CITE-seq and cell hashing were performed on liquid and solid tissue biopsies (Fig. 1a, b). Experimentally, cells from 17 samples (Supplementary Data 2) were hashed and stained with a panel of 97 antibodies (Supplementary Data 2) covering key as well as exploratory immuno-oncology markers resulting in 57,261 cells after preprocessing and…

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Molecular reshaping of phage-displayed Interleukin-2 at beta chain receptor interface to obtain potent super-agonists with improved developability profiles

Soft-randomization of phage-displayed hIL-2 beta chain interface revealed hotspots modulating the interaction Soft-randomization was chosen for an initial mutational scanning at hIL-2/hIL-2R beta interface, focused on solvent-exposed (>20%) hIL-2 residues close to the beta chain (<5 Å). A library of 7 × 107 members was obtained by Kunkel mutagenesis15 with spiked mutagenic oligonucleotides…

Continue Reading Molecular reshaping of phage-displayed Interleukin-2 at beta chain receptor interface to obtain potent super-agonists with improved developability profiles

Computational optimization of antibody humanness and stability by systematic energy-based ranking

Raybould, M. I. J. et al. Thera-SAbDab: the therapeutic structural antibody database. Nucleic Acids Res. 48, D383–D388 (2020). Article  CAS  PubMed  Google Scholar  Schroff, R. W., Foon, K. A., Beatty, S. M., Oldham, R. K. & Morgan, A. C. Jr Human anti-murine immunoglobulin responses in patients receiving monoclonal antibody therapy….

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Neoantigen discovery sheds light on cancer immunogenicity

In a recent article published in Nature Communications, researchers reported a proteogenomic approach to investigate fresh solid melanoma tumor material. The approach combines liquid chromatography-tandem mass spectrometry (LC-MS/MS) of immuno-precipitated human leukocyte antigen class I (pHLA-I) peptides with whole exome sequencing (WES) for the discovery and validation of neoantigen candidates at the…

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Understanding plant pathogen interactions using spatial and single-cell technologies

Plants can be infected by diverse pathogenic organisms that modulate host cells to enable their growth and dissemination. Pathogens deploy a variety of manipulation strategies that can vary during the course of infection. These strategies include plant defense suppression as well as deployment of toxins and degradative enzymes to facilitate…

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Effects of N-acetylcysteine on CG8005 gene-mediated proliferation and apoptosis of Drosophila S2 embryonic cells

Cell lines Drosophila S2 embryonic cells were obtained from Drosophila Genomics Resource Center. Reagents and instruments The reagents and equipment information used in this study are as follows: DMEM (Gibco, USA), Fetal Bovine Serum (Bioind, Israel), LipofectamineTM2000 (Invitrogen, USA), opti-MEM (Gibco, USA), Small Interfering RNA (Suzhou Gema Gene), N-acetyl-l-cysteine (Shanghai…

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Proteogenomic analysis reveals RNA as a source for tumor-agnostic neoantigen identification

Human study The study was approved by the institutional review boards (Ethics Commission of the Medical Faculty of Technical University Munich (protocol 193/17S) and Ethics Committee of the Medical Faculty of Heidelberg University (protocol S-206/2011)) and all patients provided written informed consent under these protocols. The study was conducted in…

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SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response

SF3B1 MUT cells show selective sensitivity to PARP inhibitors To identify candidate therapeutic targets for cancers with SF3B1 hotspot mutations, we utilized the leukemia K562K700E (SF3B1K700E) and parental (SF3B1WT) isogenic cells1, to model one of the most prevalent SF3B1 hotspot mutations seen in patients8,19,20 (Fig. 1a,b and Supplementary Fig. 1a)….

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Multiparametric senescent cell phenotyping reveals targets of senolytic therapy in the aged murine skeleton

Development and validation of a senescence CyTOF antibody panel We constructed and validated a comprehensive CyTOF antibody panel to include markers for both cell identity and senescent phenotype (Table 1). A defining characteristic of senescent cells is expression of cell cycle inhibitors, in particular p16 or p2129, so we carefully validated…

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Transcriptomic analysis of neutrophil apoptosis induced by large B-cell lymphoma

introduce Difuse large B-cell lymphoma is the most common subtype of non-Hodgkin lymphoma, accounting for 30 percent of new cases in NHL each year. Although approximately 50-60% of human patients are cured by R-CHOP immunochemotherapy, more than 30% of patients are refractory to these regimens or relapse after remission, and…

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Development of a mammalian cell-based ZZ display system for IgG quantification | BMC Biotechnology

Cell culture CHO cells (a kind gift provided by Prof. Tom K. Hei, Columbia University, USA) were cultured in DMEM/F12 medium (Gibco, USA) containing 10% fetal bovine serum (FBS, Bio-west, USA), 100 U/mL penicillin and 100 µg/mL streptomycin (Beyotime, China), at 37 °C in a 5% CO2 incubator. Vector construction To capture…

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Cell Therapy Technologies Market Surges as Life-Changing Treatments Gain Momentum

PRESS RELEASE Published July 25, 2023 “Thermo Fisher Scientific offers a complete and integrated portfolio of solutions and services for clinical science, laboratory research, and analysis. It operates in four business segments—Life Sciences Solutions, Analytical Instruments, Specialty Diagnostics, and Laboratory Products and Services.” global cell therapy technologies market is projected…

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Decreased left heart flow in fetal lambs causes left heart hypoplasia and pro-fibrotic tissue remodeling

Coil implantation in fetal lambs We have complied with all relevant ethical regulations for animal testing. All procedures followed the Canadian Council on Animal Care guidelines and were approved by the University of Western Ontario Council on Animal Care (protocol 2010-257). Time-dated pregnant Dorset × Rideau Arcott ewes (gestational age 76 days,…

Continue Reading Decreased left heart flow in fetal lambs causes left heart hypoplasia and pro-fibrotic tissue remodeling

Antiviral HIV-1 SERINC restriction factors disrupt virus membrane asymmetry

Constructs and expression of human SERINC proteins SERINC3 The gene that encodes human SERINC3 (Genscript-OHu02717D) was inserted upstream of a thrombin protease cleavable linker (LVPRGS) and Strep II epitope (WSHPQFEK) in a modified pFastBac vector by In-Fusion cloning (Clontech). Mutagenesis using the QuikChange Site-Directed Mutagenesis kit (Agilent) was performed to…

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Heterologous Protein Production in CHO Cells Methods and Protocols

00000nam a22000005i 4500 001     978-1-4939-6972-2 003     DE-He213 005     20200709043250.0 007     cr nn 008mamaa 008     170510s2017 xxu| s |||| 0|eng d 020 a 9781493969722 9 978-1-4939-6972-2 024 7 a 10.1007/978-1-4939-6972-2 2 doi 050 4 a QD431-431.7 072 7 a PSBC 2…

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PLSCR1 identified as a potent anti-SARS-CoV-2 factor in COVID-19 immunity

In a recent study published in Nature, researchers identified the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) function of phospholipid scramblase 1 (PLSCR1), a cell-autonomous restriction factor induced by interferon-gamma (IFNγ). Its identification will help enhance the understanding of protective immunity in the human IFN response. The study used genome-wide…

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Spatially resolved multiomics of human cardiac niches

Research ethics for donor tissues All heart tissue samples were obtained from transplant donors after Research Ethics Committee approval and written informed consent from donor families as previously described2. The following ethics approvals for donors of additional heart tissue were obtained: D8 and A61 (REC reference 15/EE/0152, East of England…

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Overcoming the challenges of limiting dilution

Therapeutic proteins play a crucial role in the biological pharmaceutical sector, serving as treatment options for diseases such as diabetes, cancer, and anemia.   In recombinant protein development, a primary objective is establishing highly efficient monoclonal cell lines capable of producing substantial quantities of the desired protein. Chinese hamster ovary…

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nf-core/marsseq

Introduction nf-core/marsseq is a bioinformatics single-cell preprocessing pipeline for MARS-seq v2.0 experiments. MARS-seq is a plate-based technique that can be combined with FACS in order to study rare populations of cells. On top of the pre-existing pipeline, we have developed an RNA velocity workflow that can be used to study…

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How to automate cloning for CRISPR workflows

The process of gene editing involves the transformation of a substantial number of cells, followed by the isolation of individual cells from the larger population to establish clonal colonies.   Due to the significant number of gene edits required in modern research and the labor-intensive workflow, there is a pressing…

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How to accelerate the generation of single-cell clones

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), genome engineering of cell lines, facilitate target discovery. It provides an excellent disease model for preclinical research, particularly in chimeric antigen receptor T-cell therapy. The primary challenge in cell engineering remains the generation of single-cell clones. Current approaches range from manual limiting dilution…

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Reprogramming method does not impact the neuronal differentiation potential of 16p11.2 deletion patient iPSCs

Abstract A major impediment to the actualization of the induced pluripotent stem cell (iPSC)-based personalized medicine revolution is the lack of widely accepted standard operating procedures (SOPs) across different groups and institutions. The various methods employed can include choice of starting materials, reprogramming agents, and culture conditions, with each of…

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Key considerations before commencing cell line development

Cell line development (CLD) is a critical, early step when entering the development phase of a biotherapeutic. Access to specialist skills and experience, either in-house or remotely, and to multiple technologies and equipment can prove invaluable for the timely and successful completion of this stage. Here, Dr Alison Porter addresses…

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Isolation and Transcriptome Analysis of Plant Cell Types

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis. Transcriptome analysis on single cell or cell types can detect the subtle…

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CAR T cells as micropharmacies against solid cancers: Combining effector T-cell mediated cell death with vascular targeting in a one-step engineering process

General Lab Operation The assays were performed by experienced individuals throughout the course of the study. The study was performed using established laboratory protocols covering the processing, freezing, storage and thawing of cells as well as the staining procedure, data acquisition and gating strategy. Additional data beyond the data found…

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Glutamine synthetase (GS) knockout (KO) using CRISPR/Cpf1 diversely enhances selection efficiency of CHO cells expressing therapeutic antibodies

Identification and expression of GS genes in CHO cells Database analysis (see Supplementary information 1 indicated that the endogenous GS genes in CHO-K1 include two genes on chromosome 5 (GS5) and chromosome 1 (GS1), for which in-house RNAseq data and previous publication, listed in Supplementary Table S1, showed high and low…

Continue Reading Glutamine synthetase (GS) knockout (KO) using CRISPR/Cpf1 diversely enhances selection efficiency of CHO cells expressing therapeutic antibodies

Synergetic enrichment of aggrecan in nucleus pulposus cells by scAAV6-shRNA-mediated knockdown of aggrecanase-1 and aggrecanase-2

Degenerative disk disease (DDD) that aggravates structural deterioration of intervertebral disks (IVDs) can be accompanied by painful inflammation and immunopathological progressions. Current surgical or pharmacological therapies cannot repair the structure and function of IVDs. Nucleus pulposus (NP) cells are crucial for the preservation or restoration of IVDs by balancing the…

Continue Reading Synergetic enrichment of aggrecan in nucleus pulposus cells by scAAV6-shRNA-mediated knockdown of aggrecanase-1 and aggrecanase-2

Engineered circular guide RNAs boost CRISPR/Cas12a- and CRISPR/Cas13d-based DNA and RNA editing | Genome Biology

Plasmid construction For U6+27 gRNA-expressing constructs, the DNA sequences including the hU6+27 promoter, a gRNA scaffold, and a gRNA insert site were synthesized and cloned into the pBluSKM vector. For Pre gRNA-expressing constructs, a second gRNA scaffold was inserted downstream of the gRNA insert site in the U6+27 gRNA-expressing constructs….

Continue Reading Engineered circular guide RNAs boost CRISPR/Cas12a- and CRISPR/Cas13d-based DNA and RNA editing | Genome Biology

Single-cell quantification of ribosome occupancy in early mouse development

PDMS chip fabrication Moulds were 3D-printed by Proto Labs with WaterShed XC 11122 at high resolution (Supplementary Fig. 1). Reusable moulds were assembled by taping 3D-printed molds to glass slides (5″ × 4″; Ted Pella). Sylgard 184 PDMS monomer and curing agent (4019862, Ellsworth Adhesives) were mixed at a 10:1…

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Magnetic Beads Market to grow at a CAGR of 11.68% from 2022 to 2027; Rising use of solid-phase magnetic beads technology to be a major trend in the market.

NEW YORK, June 21, 2023 /PRNewswire/ — The global magnetic beads market size is estimated to increase by USD 1,672.62 million from 2022 to 2027. The market’s growth momentum will progress at a CAGR of 11.68% during the forecast period. The growing usage of solid-phase magnetic beads technology is the primary trend…

Continue Reading Magnetic Beads Market to grow at a CAGR of 11.68% from 2022 to 2027; Rising use of solid-phase magnetic beads technology to be a major trend in the market.

Adipogenic and SWAT cells separate from a common progenitor in human brown and white adipose depots

Human samples Human adipogenic progenitor cells were isolated from the stromal vascular fraction of adipose tissue samples on the day they were obtained (surgery or biopsy) from four regions: (1) visceral adipose tissue (obtained during gallbladder surgery); (2) perirenal adipose tissue (obtained during nephrectomy surgery); (3) abdominal subcutaneous adipose tissue…

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Prioritization and functional validation of target genes from single-cell transcriptomics studies

List of congruent tip TEC markers The list of congruent tip TEC markers was extracted from a publicly available lung tumor study8. In brief, to identify congruent tip cell markers across freshly isolated human and murine TECs, as well as cultured human TECs, all genes that were most highly expressed…

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