Future therapeutic applications of differentiated human embryonic stem cells (hESC) carry a risk of teratoma formation by contaminating undifferentiated hESC. We generated 10 monoclonal antibodies (mAbs) against surface antigens of undifferentiated hESC, showing strong reactivity against undifferentiated, but not differentiated hESC. The mAbs did not cross react with mouse fibroblasts and showed weak to no reactivity against human embryonal carcinoma cells. Notably, one antibody (mAb 84) is cytotoxic to undifferentiated hESC and NCCIT cells in a concentration-dependent, complementindependent manner. mAb 84 induced cell death of undifferentiated, but not differentiated hESC within 30 minutes of incubation, and immunoprecipitation of the mAb-antigen complex revealed that the antigen is podocalyxin-like protein-1. Importantly, we observed absence of tumor formation when hESC and NCCIT cells were treated with mAb 84 prior to transplantation into severe combined immunodeficiency mice. Our data indicate that mAb 84 may be useful in eliminating residual hESC from differentiated cells populations for clinical applications.

ObjectiveTumour heterogeneity represents a major obstacle to accurate diagnosis and treatment in gastric adenocarcinoma (GA). Here, we report a systematic transcriptional atlas to delineate molecular and cellular heterogeneity in GA using single-cell RNA sequencing (scRNA-seq).DesignWe performed unbiased transcriptome-wide scRNA-seq analysis on 27 677 cells from 9 tumour and 3 non-tumour samples. Analysis results were validated using large-scale histological assays and bulk transcriptomic datasets.ResultsOur integrative analysis of tumour cells identified five cell subgroups with distinct expression profiles. A panel of differentiation-related genes reveals a high diversity of differentiation degrees within and between tumours. Low differentiation degrees can predict poor prognosis in GA. Among them, three subgroups exhibited different differentiation grade which corresponded well to histopathological features of Lauren’s subtypes. Interestingly, the other two subgroups displayed unique transcriptome features. One subgroup expressing chief-cell markers (eg, LIPF and PGC) and RNF43 with Wnt/β-catenin signalling pathway activated is consistent with the previously described entity fundic gland-type GA (chief cell-predominant, GA-FG-CCP). We further confirmed the presence of GA-FG-CCP in two public bulk datasets using transcriptomic profiles and histological images. The other subgroup specifically expressed immune-related signature genes (eg, LY6K and major histocompatibility complex class II) with the infection of Epstein-Barr virus. In addition, we also analysed non-malignant epithelium and provided molecular evidences for potential transition from gastric chief cells into MUC6+TFF2+ spasmolytic polypeptide expressing metaplasia.ConclusionAltogether, our study offers valuable resource for deciphering gastric tumour heterogeneity, which will provide assistance for precision diagnosis and prognosis.

Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.

BackgroundRibosomes are essential ribonucleoprotein complexes that are engaged in translation and thus indispensable for growth. Arabidopsis thaliana ribosomes are composed of 80 distinct ribosomal proteins (RPs), each of which is encoded by two to seven highly similar paralogous genes. Little information is available on how RP genes respond to a shortage of essential mineral nutrients such as phosphate (Pi) or iron (Fe). In the present study, the expression of RP genes and the differential accumulation of RPs upon Pi or Fe deficiency in Arabidopsis roots were comprehensively analyzed.ResultsComparison of 3,106 Pi-responsive genes with 3,296 Fe-responsive genes revealed an overlap of 579 genes that were differentially expressed under both conditions in Arabidopsis roots. Gene ontology (GO) analysis revealed that these 579 genes were mainly associated with abiotic stress responses. Among the 247 RP genes retrieved from the TAIR10 release of the Arabidopsis genome (98 small subunit RP genes, 143 large subunit RP genes and six ribosome-related genes), seven RP genes were not detected in Arabidopsis roots by RNA sequencing under control conditions. Transcripts from 20 and 100 RP genes showed low and medium abundance, respectively; 120 RP genes were highly expressed in Arabidopsis roots. As anticipated, gene ontology (GO) analysis indicated that most RP genes were related to translation and ribosome assembly, but some of the highly expressed RP genes were also involved in the responses to cold, UV-B, and salt stress. Only three RP genes derived from three ‘sets’ of paralogous genes were differentially expressed between Pi-sufficient and Pi-deficient roots, all of which were induced by Pi starvation. In Fe-deficient plants, 81 RP genes from 51 ’sets’ of paralagous RP genes were significantly down-regulated in response to Fe deficiency. The biological processes ’translation’ (GO: 0006412), ’ribosome biogenesis (GO: 0042254), and ’response to salt (GO: 0009651), cold (GO: 0009409), and UV-B stresses (GO: 0071493)’ were enriched in this subset of RP genes. At the protein level, 21 and two RPs accumulated differentially under Pi- and Fe-deficient conditions, respectively. Neither the differentially expressed RP genes nor the differentially expressed RPs showed any overlap between the two growth types.ConclusionsIn the present study three and 81 differentially expressed RP genes were identified under Pi and Fe deficiency, respectively. At protein level, 21 and two RP proteins were differentially accumulated under Pi- and Fe-deficient conditions. Our study shows that the expression of paralogous genes encoding RPs was regulated in a stress-specific manner in Arabidopsis roots, presumably resulting in an altered composition of ribosomes and biased translation. These findings may aid in uncovering an unexplored mechanism by which plants adapt to changing environmental conditions.

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Centrosome cohesion, mostly regarded as a proteinaceous linker between parental centrioles, ensures the interphase centrosome(s) to function as a single microtubule-organizing center. Impairment of centrosome cohesion leads to the splitting of centrosomes. Although the list of cohesion proteins is growing, the precise composition and regulation of centrosome cohesion are still largely unknown. In this study, we identify that the novel centriolar protein, Centlein, localizes to the proximal ends of the centrioles and directly interacts with both C-Nap1 and Cep68. Moreover, Centlein complexes with C-Nap1 and Cep68 at the proximal ends of centrioles during interphase and functions as a molecular link between C-Nap1 and Cep68. Depletion of Centlein impairs recruitment of Cep68 to the centrosomes, in turn, results in centrosome splitting. Both Centlein and Cep68 are novel Nek2A substrates. Collectively, our data bring to light centrosome cohesion maintained by the novel complex of C-Nap1-Centlein-Cep68.

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O-linked-N-acetylglucosamine (O-GlcNAc), a post translational modification, has emerged as an important cue in controlling key cell mechanisms. Here, we investigate O-GlcNAc's role in the maintenance and differentiation of human pluripotent stem cells (hPSC). We reveal that protein expression of O-GlcNAc transferase and hydrolase both decreases during hPSC differentiation. Upregulating O-GlcNAc with O-GlcNAc hydrolase inhibitors has no significant effect on either the maintenance of pluripotency in hPSC culture, or the loss of pluripotency in differentiating hPSC. However, in spontaneously differentiating hPSC, excess O-GlcNAc alters the expression of specific lineage markers: decrease of ectoderm markers (PAX6 by 53-88%, MSX1 by 26-49%) and increase of adipose-related mesoderm markers (PPARγ by 28-100%, C/EBPα by 46-135%). All other lineage markers tested (cardiac, visceral-endoderm, trophectoderm) remain minimally affected by upregulated O-GlcNAc. Interestingly, we also show that excess O-GlcNAc triggers a feedback mechanism that increases O-GlcNAc hydrolase expression by 29-91%. To the best of our knowledge, this is the first report demonstrating that excess O-GlcNAc does not affect hPSC pluripotency in undifferentiated maintenance cultures; instead, it restricts the hPSC differentiation towards specific cell lineages. These data will be useful for developing targeted differentiation protocols and aid in understanding the effects of O-GlcNAc on hPSC differentiation.