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Graphical abstract By repeatedly selecting and pooling the most rapidly outgrowing clones during multiple single cell subcloning rounds, the time required for subcloning was reduced from 3 to 2 weeks and the cloning efficiency significantly increased. Transcriptome analyses of the resulting host cell lines revealed a diverse set of pathways differentially enriched in each host cell line treated, with the only shared DE pathway related to changes in extracellular matrix. This indicates that cells struggle with the lack of cell-to-cell communication in isolation during subcloning., Chinese Hamster Ovary (CHO) cells are the working horse of the pharmaceutical industry. To obtain high producing cell clones and to satisfy regulatory requirements single cell cloning is a necessary step in cell line development. However, it is also a tedious, labor intensive and expensive process. Here we show an easy way to enhance subclonability using subcloning by single cell sorting itself as the selection pressure, resulting in improved subcloning performance of three different host cell lines. These improvements in subclonability also lead to an enhanced cellular growth behavior during standard batch culture. RNA-seq was performed to shed light on the underlying mechanisms, showing that there is little overlap in differentially expressed genes or associated pathways between the cell lines, each finding their individual strategy for optimization. However, in all three cell lines pathways associated with the extracellular matrix were found to be enriched, indicating that cells struggle predominantly with their microenvironment and possibly lack of cell-to-cell contact. The observed small overlap may hint that there are multiple ways for a cell line to achieve a certain phenotype due to numerous genetic and subsequently metabolic redundancies.

Monoclonal antibodies (mAbs) are widely used in many fields due to their high specificity and ability to recognize a broad range of antigens. IL-17A can induce a rapid inflammatory response both alone and synergistically with other proinflammatory cytokines. Accumulating evidence suggests that therapeutic intervention of IL-17A signaling offers an attractive treatment option for autoimmune diseases and cancer. Here, we present a combinatorial approach for optimizing the affinity and thermostability of a novel anti-hIL-17A antibody. From a large naïve phage-displayed library, we isolated the anti-IL-17A mAb 7H9 that can neutralize the effects of recombinant human IL-17A. However, the modest neutralization potency and poor thermostability limit its therapeutic applications. In vitro affinity optimization was then used to generate 8D3 by using yeast-displayed random mutagenesis libraries. This resulted in four key amino acid changes and provided an approximately 15-fold potency increase in a cell-based neutralization assay. Complementarity-determining regions (CDRs) of 8D3 were further grafted onto the stable framework of the huFv 4D5 to improve thermostability. The resulting hybrid antibody 9NT/S has superior stabilization and affinities beyond its original antibody. Human fibrosarcoma cell-based assays and in vivo analyses in mice indicated that the anti-IL-17A antibody 9NT/S efficiently inhibited the secretion of IL-17A-induced proinflammatory cytokines. Therefore, this lead anti-IL-17A mAb might be used as a potential best-in-class candidate for treating IL-17A related diseases., Graphical abstract Anti-IL-17A antibodies were initially developed from a naïve fully human antibody library. The candidate was further affinity-matured by constructing a library of yeast-displayed single-chain Fv (scFv) mutants and thermostability-improved by CDR grafting. The lead anti-IL-17A mAb 9NT/S might be used as a potential best-in-class candidate for treating IL-17A related diseases.fx1

During signal transduction, the G protein, Gαq, binds and activates phospholipase C-β isozymes. Several diseases have been shown to manifest upon constitutively activating mutation of Gαq, such as uveal melanoma. Therefore, methods are needed to directly inhibit Gαq. Previously, we demonstrated that a peptide derived from a helix-turn-helix (HTH) region of PLC-β3 (residues 852–878) binds Gαq with low micromolar affinity and inhibits Gαq by competing with full-length PLC-β isozymes for binding. Since the HTH peptide is unstructured in the absence of Gαq, we hypothesized that embedding the HTH in a folded protein might stabilize the binding-competent conformation and further improve the potency of inhibition. Using the molecular modeling software Rosetta, we searched the Protein Data Bank for proteins with similar HTH structures near their surface. The candidate proteins were computationally docked against Gαq, and their surfaces were redesigned to stabilize this interaction. We then used yeast surface display to affinity mature the designs. The most potent design bound Gαq/i with high affinity in vitro (KD = 18 nM) and inhibited activation of PLC-β isozymes in HEK293 cells. We anticipate that our genetically encoded inhibitor will help interrogate the role of Gαq in healthy and disease model systems. Our work demonstrates that grafting interaction motifs into folded proteins is a powerful approach for generating inhibitors of protein–protein interactions.

The inflammatory tumor microenvironment has been implicated as a major player fueling tumor progression and an enabling characteristic of cancer, proline, glutamic acid, and leucine-rich protein 1 (PELP1) is a novel nuclear receptor coregulator that signals across diverse signaling networks, and its expression is altered in several cancers. However, investigations to find the role of PELP1 in inflammation-driven oncogenesis are limited. Molecular studies here, utilizing macrophage cell lines and animal models upon stimulation with lipopolysaccharide (LPS) or necrotic cells, showed that PELP1 is an inflammation-inducible gene. Studies on the PELP1 promoter and its mutant identified potential binding of c-Rel, an NF-κB transcription factor subunit, to PELP1 promoter upon LPS stimulation in macrophages. Recruitment of c-Rel onto the PELP1 promoter was validated by chromatin immunoprecipitation, further confirming LPS mediated PELP1 expression through c-Rel–specific transcriptional regulation. Macrophages that overexpress PELP1 induces granulocyte–macrophage colony-stimulating factor secretion, which mediates cancer progression in a paracrine manner. Results from preclinical studies with normal–inflammatory–tumor progression models demonstrated a progressive increase in the PELP1 expression, supporting this link between inflammation and cancer. In addition, animal studies demonstrated the connection of PELP1 in inflammation-directed cancer progression. Taken together, our findings provide the first report on c-Rel–specific transcriptional regulation of PELP1 in inflammation and possible granulocyte–macrophage colony-stimulating factor–mediated transformation potential of activated macrophages on epithelial cells in the inflammatory tumor microenvironment, reiterating the link between PELP1 and inflammation-induced oncogenesis. Understanding the regulatory mechanisms of PELP1 may help in designing better therapeutics to cure various inflammation-associated malignancies.

Background & Aims aISCs (aISCs) are sensitive to acute insults including chemotherapy and irradiation. Regeneration after aISC depletion has primarily been explored in irradiation (IR). However, the cellular origin of epithelial regeneration after doxorubicin (DXR), a common chemotherapeutic, is poorly understood. Methods We monitored DXR’s effect on aISCs by enumerating Lgr5-eGFP+ and Olfm4+ crypts, cleaved caspase-3 (CASP3+) immunofluorescence, and time-lapse organoid imaging. Lineage tracing from previously identified regenerative cell populations (Bmi1+, Hopx+, Dll1+, and Defa6+) was performed with DXR damage. Lineage tracing from aISCs was compared with lineage tracing from early progeny cells (transit-amplifying cells arising from aISCs 1 day predamage) in the context of DXR and IR. We compared stem cell and DNA damage response (DDR) transcripts in isolated aISCs and early progeny cells 6 and 24 hours after DXR. Results Epithelial regeneration after DXR primarily arose from early progeny cells generated by aISCs. Early progeny cells upregulated stem cell gene expression and lacked apoptosis induction (6 hours DXR: 2.5% of CASP3+ cells, p, Graphical abstract

Background & Aims The enteric nervous system (ENS) regulates gastrointestinal function via different subtypes of neurons, organized into fine-tuned neural circuits. It is not clear how cell diversity is created within the embryonic ENS; information required for development of cell-based therapies and models of enteric neuropathies. We aimed to identify proteins that regulate ENS differentiation and network formation. Methods We generated and compared RNA expression profiles of the entire ENS, ENS progenitor cells, and non-ENS gut cells of mice, collected at embryonic days 11.5 and 15.5, when different subtypes of neurons are formed. Gastrointestinal tissues from R26ReYFP reporter mice crossed to Sox10-CreERT2 or Wnt1-Cre mice were dissected and the 6 populations of cells were isolated by flow cytometry. We used histochemistry to map differentially expressed proteins in mouse and human gut tissues at different stages of development, in different regions. We examined enteric neuronal diversity and gastric function in Wnt1-Cre x Sox6fl/fl mice, which do not express the Sox6 gene in the ENS. Results We identified 147 transcription and signaling factors that varied in spatial and temporal expression during development of the mouse ENS. Of the factors also analyzed in human ENS, most were conserved. We uncovered 16 signaling pathways (such as fibroblast growth factor and Eph/ephrin pathways). Transcription factors were grouped according to their specific expression in enteric progenitor cells (such as MEF2C), enteric neurons (such as SOX4), or neuron subpopulations (such as SATB1 and SOX6). Lack of SOX6 in the ENS reduced the numbers of gastric dopamine neurons and delayed gastric emptying. Conclusions Using transcriptome and histochemical analyses of the developing mouse and human ENS, we mapped expression patterns of transcription and signaling factors. Further studies of these candidate determinants might elucidate the mechanisms by which enteric stem cells differentiate into neuronal subtypes and form distinct connectivity patterns during ENS development. We found expression of SOX6 to be required for development of gastric dopamine neurons., Graphical abstract