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Internship Report for Summer-Pranav Sahasrabudhe
No full textIn the last report we touched upon the basics of Single-Cell technology, its working and methods
used to analyze the data. It was mentioned in the future development section that the focus
would be to evaluate R packages in order to provide additional utilities and biological insights
compared to the standard Seurat [1] workflow for other major steps of single-cell RNA-seq
analysis, including clustering, cell type annotation and additional downstream analysis beyond
differential expression (i.e. Pseudotime analysis, enrichment analysis, receptor-ligand
enrichment, etc.) In the present report, we focus on utilities provided through packages like SC3
[2], scPred [3] for clustering and annotations respectively
YAP, but Not RSPO-LGR4/5, Signaling in Biliary Epithelial Cells Promotes a Ductular Reaction in Response to Liver Injury
No full textThe liver has an intrinsically high capacity to regenerate, involving several discrete cellular niches. Oval cells in a ductular reaction (DR) transiently expand around the portal vein following damage to support liver regeneration. However, the regulatory mechanisms controlling a DR are poorly understood. Here, we performed a CRISPR loss-of-function (LOF) oval cell expansion screen in liver organoids, hit validation in a DR in vivo model, and oval cell single-cell RNA sequencing (scRNA-Seq) combined with histological analyses rendering a detailed picture of regulatory pathways controlling the DR. Our scRNA-Seq analysis further revealed two distinct clusters of EPCAM+ oval cells and the signalling networks determining their cellular identity. We clarified the role of LGR4/5-mediated Wnt/β-Catenin signaling during a DR, show that AXIN2/LGR5 are essential for hepatocyte dedifferentiation enabling their regenerative potential, and linked hepatocyte-mediated liver regeneration to DR resolution. Our data highlight a spatio-temporal requirement of signalling pathways in controlling regulatory mechanisms of a DR and liver regeneration
Inhibition of the transcription factor ROR-γ reduces pathogenic Th17 cells in acetylcholine receptor antibody positive myasthenia gravis
No full textIL-17 producing CD4 T cells (Th17) cells increase significantly with disease severity in myasthenia gravis (MG) patients. To suppress the generation of Th17 cells, we examined the effect of inhibiting retinoic acid receptor-related-orphan-receptor-C (RORγ), a Th17-specific transcription factor critical for differentiation. RORγ inhibition profoundly reduced Th17 cell frequencies, including IFN-γ and IL-17 co-producing pathogenic Th17 cells. Other T helper subsets were not affected. In parallel, CD8 T cell subsets producing IL-17 and IL-17/IFN-γ were increased in MG patients and inhibited by the RORγ inhibitor. These findings provide rationale for exploration of targeted Th17 therapies, including ROR-γ inhibitors, to treat MG patients
Evaluation of Therapeutics for Severely Debilitating or Life-Threatening Diseases or Conditions: Defining Scope to Enable Global Guidance Development
No full textA significant regulatory gap exists to facilitate global development of therapeutics for nononcology severely debilitating or life-threatening diseases or conditions (SDLTs). In a 2017 publication, a streamlined approach to the development of treatments for SDLTs was proposed to facilitate earlier and continued patient access to new, potentially beneficial therapeutics.1 However, a major hindrance to broad adoption of this streamlined approach has been the lack of universally accepted, objective criteria to define SDLTs. This article serves to extend the 2017 publication by further addressing the challenge of defining SDLT scope in order to stimulate broader discussion and facilitate development of regional and ultimately international guidelines on the development of therapeutics for SDLTs. Using case examples, we describe key attributes of SDLTs and provide criteria for consideration of an SDLT scope definition
Immune cell landscaping reveals a protective role for regulatory T cells during kidney injury and fibrosis
No full textAcute kidney injury (AKI) and chronic kidney diseases are associated with high mortality and morbidity. Although the underlying mechanisms determining the transition from acute to chronic injury are not completely understood, immune-mediated processes are critical in renal injury. We have performed a comparison of 2 mouse models leading to either kidney regeneration or fibrosis. Using global gene expression profiling we could identify immune-related pathways accounting for the majority of the observed transcriptional changes during fibrosis. Unbiased examination of the immune cell composition, using single-cell RNA sequencing, revealed major changes in tissue-resident macrophages and T cells. Following injury, there was a marked increase in tissue-resident IL-33R+ and IL-2Ra+ regulatory T cells (Tregs). Expansion of this population before injury protected the kidney from injury and fibrosis. Transcriptional profiling of Tregs showed a differential upregulation of regenerative and proangiogenic pathways during regeneration, whereas in the fibrotic environment they expressed markers of hyperactivation and fibrosis. Our data point to a hitherto underappreciated plasticity in Treg function within the same tissue, dictated by environmental cues. Overall, we provide a detailed cellular and molecular characterization of the immunological changes during kidney injury, regeneration, and fibrosis
Synthesis and Solid State Conformation of Tetrapeptide Amides Containing two Aib and two (Me)Phe Residues – Use of Enantiomerically Pure 2-Benzyl-2-methyl-2H-azirin-3-amines as (Me)Phe-Synthons
No full textA series of tetrapeptide amides containing two aminoisobutyric acids (Aib) and two -methylphenylalanine ((Me)Phe) units were prepared via the ‘azirine/oxazolone method’. New 2-benzyl-2-methyl-2H-azirine-3-amines have been used for the selective introduction of (S)- and (R)-(Me)Phe, respectively. The solid-state conformations of five tetrapeptide amides were determined by X-ray crystallography. In all cases, two -turns stabilize 310-helical conformations and it was confirmed that, in contrast to proteinogenic amino acids, the configuration of (Me)Phe does not determine the screw-sense of the helix
A dual role of Irf1 in maintaining epithelial identity but also enabling EMT and metastasis formation of breast cancer cells.
No full textAn epithelial to mesenchymal transition (EMT) is an embryonic dedifferentiation program which is aberrantly activated in cancer cells to acquire cellular plasticity. This plasticity increases the ability of breast cancer cells to invade into surrounding tissue, to seed metastasis at distant sites and to resist to chemotherapy. In this study, we have observed a higher expression of interferon-related factors in basal-like and claudin-low subtypes of breast cancer in patients, known to be associated with EMT. Notably, Irf1 exerts essential functions during the EMT process, yet it is also required for the maintenance of an epithelial differentiation status of mammary gland epithelial cells: RNAi-mediated ablation of Irf1 in mammary epithelial cells results in the expression of mesenchymal factors and Smad transcriptional activity. Conversely, ablation of Irf1 during TGFβ-induced EMT prevents a mesenchymal transition and stabilizes the expression of E-cadherin. In the basal-like murine breast cancer cell line 4T1, RNAi-mediated ablation of Irf1 reduces colony formation and cell migration in vitro and shedding of circulating tumor cells and metastasis formation in vivo. This context-dependent dual role of Irf1 in the regulation of epithelial-mesenchymal plasticity provides important new insights into the functional contribution and therapeutic potential of interferon-regulated factors in breast cancer
SUMO ylated PRC 1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin
No full textChromatin integrity is essential for cellular homeostasis. Polycombgroup proteins modulate chromatin states and transcriptionallyrepress developmental genes to maintain cell identity. They alsorepress repetitive sequences such as major satellites and consti-tute an alternative state of pericentromeric constitutive hete-rochromatin at paternal chromosomes (pat-PCH) in mouse pre-implantation embryos. Remarkably, pat-PCH contains the histoneH3.3 variant, which is absent from canonical PCH at maternal chro-mosomes, which is marked by histone H3 lysine 9 trimethylation(H3K9me3), HP1, and ATRX proteins. Here, we show that SUMO2-modified CBX2-containing Polycomb Repressive Complex 1 (PRC1)recruits the H3.3-specific chaperone DAXX to pat-PCH, enablingH3.3 incorporation at these loci. Deficiency of Daxx or PRC1 compo-nents Ring1 and Rnf2 abrogates H3.3 incorporation, induces chro-matin decompaction and breakage at PCH of exclusively paternalchromosomes, and causes their mis-segregation. Complementationassays show that DAXX-mediated H3.3 deposition is required forchromosome stability in early embryos. DAXX also regulates repres-sion of PRC1 target genes during oogenesis and early embryogene-sis. The study identifies a novel critical role for Polycomb inensuring heterochromatin integrity and chromosome stability inmouse early development
Predicting Bioavailability of Monoclonal Antibodies after Subcutaneous Administration: Open Innovation Challenge
No full textDespite the increasing trend towards subcutaneous (SC) delivery of monoclonal antibodies (mAbs), factors influencing the SC bioavailability of mAbs remain poorly understood. To address critical knowledge gaps and issues during development of SC dosage forms for mAbs, the SC Drug Delivery and Development Consortium was convened in 2018 as a pre-competitive collaboration of recognized industry experts. One of the Consortium’s eight problem statements highlights the challenges of predicting human bioavailability of subcutaneously administered mAbs due to a lack of reliable in vitro and preclinical in vivo predictive models. In this paper, we assess the current landscape in SC bioavailability prediction for mAbs and discuss the gaps and opportunities associated with bioavailability models for biotherapeutics. We also issue an open challenge to industry and academia, encouraging the development of reliable models to enable SC bioavailability prediction of therapeutic large molecules in humans and improve translation from preclinical species