Supplementary Figure S9. E-cadherin expression is determined in GC cells with drugs treatment.

Supplementary Figure S10. mIR-29c and catenin-ÃŽ' expression is determined in GC cells treated with imatinib.

Supplementary Figure S7. Catenin-ÃŽ' protein levels depict an increase in the GC tissues.

Legends for Supplementary Figures S1-S10.

Supplementary Figure S2. Correlations between miR-29s levels and histological grade of GCs.

Supplementary Figure S1. miR-29s expression analyses in GC cells.

Supplementary Figure S8. Correlation between mIR-29s expression and catenin-ÃŽ' in 66 cases of GCs.

Supplementary Figure S6. miR-29s and catenin-ÃŽ' expression depicts an inverse correlation in 20 cases of GC tissues.

Supplementary Figure S3. Enforced expression of miR-29s in MGC803 and HGC27 cells.

Supplementary Tables S1-S4. Comparison of miR-29s expression levels with clinic-pathological features in patients with primary gastric cancer (S1); miR-29s associated with disease-free survival of patients with GC in the training set (S2); Sequences of miR-29s, Ts-miRs, and catenin-ÃŽ' primers (S3); Immunohistochemistry reactions were quantitatively assessed using two methods (S4).

Cell-type biomarkers are useful in stem-cell manufacturing to monitor cell purity, quantity, and quality. However, the study on cell-type markers, specifically for stem cell manufacture, is limited. Emerging questions include which RNA transcripts can serve as biomarkers during stem cell culture and how to discover these biomarkers efficiently and precisely. We developed a scoring function system to identify RNA biomarkers with RNA-seq data for systems that have a limited number of cell types. We applied the method to two data sets, one for extracellular RNAs (ex-RNAs) and the other for intracellular microRNAs (miRNAs). The first data set has RNA-seq data of ex-RNAs from cell culture media for six different types of cells, including human embryonic stem cells. To get the RNA-seq data from intracellular miRNAs, we cultured three types of cells: human embryonic stem cells (H9), neural stem cells (NSC), hESC-derived endothelial cells (EC) and conducted small RNA-seq to their intracellular miRNAs. Using these data, we identified a set of ex-RNAs/smRNAs as candidates of biomarkers for different types of cells for cell manufacture. The validity of these findings was confirmed by the utilization of additional data sets and experimental procedures. We also used deep-learning-based prediction methods and simulated data to validate these discovered biomarkers.

Supplementary Figure S7. Catenin-ÃŽ' protein levels depict an increase in the GC tissues.

Supplementary Figure S5. The suppressive effect of miR-29c on GC formation.

Supplementary Materials and Methods. Description of additional methods and procedures used in the study.

Supplementary Figure S4. miR-29s inhibitors suppress the expression of endogenous miR-29s in GC cells.

Site-specific incorporation of distinct noncanonical amino acids (ncAAs) into proteins via genetic code expansion in mammalian cells represents a new avenue for protein engineering. Reassigning three TAGs with the same ncAA in mammalian cells has previously been achieved using translational machinery. However, simultaneous recoding of three nonsense codons with distinct ncAAs in mammalian cells remains a challenge due to low incorporation efficiencies. Here, three optimized aaRS/tRNA pairs (i.e., the E. coli-derived tyrosyl (EcTyr)/tRNAUUA , E. coli-derived leucyl (EcLeu)/tRNACUA , and Methanosarcina mazei pyrrolysyl (MmPyl)/tRNAUCA pairs) are screened for ncAA incorporation. Furthermore, introduced combinations of eukaryotic release factor 1 (eRF1) mutants (E55R, E55D, N129D, and Y125F) significantly improve the encoding efficiency of the three premature stop codons' sites from 0.78% to 11.6%. Thus, site-specific incorporation of three distinct ncAAs into a single protein is achieved in this study. This work markedly expands the potential for multiple site-specific protein modifications within mammalian cells, thereby facilitating new in vivo applications.

Vascular smooth muscle cells (VSMCs), which provides structural integrity and regulates the diameter of vasculature, are of great potential for modeling vascular-associated diseases and tissue engineering. Here, we presented a detailed comparison of differentiating human pluripotent stem cells (hPSCs) into VSMCs (hPSCs-VSMCs) in four different culture methods, including 2-dimensional (2D) culture, 3-dimensional (3D) PNIPAAm-PEG hydrogel culture, 3-dimensional (3D) alginate hydrogel culture, and transferring 3-dimensional alginate hydrogel culture to 2-dimensional (2D) culture. Both hydrogel-based culture methods could mimic in vivo microenvironment to protect cells from shear force, and avoid cells agglomeration, resulting in the extremely high culture efficiency (e.g., high viability, high purity and high yield) compared with 2D culture. We demonstrated hPSC-VSMCs produced from hydrogel-based culture methods had better contractile phenotypes and the potential of vasculature formation. The transcriptome analysis showed the hPSC-VSMCs derived from hydrogel-based culture methods displayed more upregulated genes in vasculature development, angiogenesis and blood vessel development, extracellular matrix compared with 2D culture. Taken together, hPSC-VSMCs produced from hydrogel-based culture system could be applied in various biomedical fields, and further indicated the suitable development of alginate hydrogel for industrial production by taking all aspects into consideration.

Currently, an inactivated vaccine has been widely used with encouraging results as a prophylactic agent against COVID-19 infection, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants. However, in vitro SARS-CoV-2 vaccine-specific immune features remain elusive, hindering the promotion of a third dose of the vaccine. Here, we present a detailed in vitro immune cellular response and large-scale multi-omics analysis for peripheral blood mononuclear cells (PBMCs) from participants vaccinated with CoronaVac (Sinovac Life Sciences, Beijing, China) and recovered participants from COVID-19. The mean titers of SARS-CoV-2 serum-neutralizing antibodies were significantly increased after the boosting immunization (Day 45) compared to the unimmunized state. We observed that type-1 helper T cells (Th1) tended to dominate after the first dose of vaccine, while humoral immune responses became dominant after the second dose due to the activation of type-2 helper T cell (Th2), memory B cells, and plasmablasts. T follicular helper cells (Tfh) involved in antibody production were activated after the first dose and were maintained for the observed time points. Single-cell RNA sequencing of PBMCs revealed specific changes in cell compositions and gene expression in immunized participants. Multi-omics analysis also demonstrated that CoronaVac-specific serum proteins, plasma metabolites, and plasma lipid changes were skewed to those changes in convalescent patients. Collectively, we provide a comprehensive understanding of CoronaVac-specific in vitro immune features.

Transplanting cell cultured brown adipocytes (BAs) represents a promising approach to prevent and treat obesity (OB) and its associated metabolic disorders, including type 2 diabetes mellitus (T2DM). However, transplanted BAs have a very low survival rate in vivo. The enzymatic dissociation during the harvest of fully differentiated BAs also loses significant cells. There is a critical need for novel methods that can avoid cell death during cell preparation, transplantation, and in vivo. Here, we reported that preparing BAs as injectable microtissues could overcome the problem. We found that 3D culture promoted BA differentiation and UCP-1 expression, and the optimal initial cell aggregate size was 100 μm. The microtissues could be produced at large scales via 3D suspension assisted with a PEG hydrogel and could be cryopreserved. Fabricated microtissues could survive in vivo for long term. They alleviated body weight and fat gain and improved glucose tolerance and insulin sensitivity in high-fat diet (HFD)-induced OB and T2DM mice. Transplanted microtissues impacted multiple organs, secreted protein factors, and influenced the secretion of endogenous adipokines. To our best knowledge, this is the first report on fabricating human BA microtissues and showing their safety and efficacy in T2DM mice. The proposal of transplanting fabricated BA microtissues, the microtissue fabrication method, and the demonstration of efficacy in T2DM mice are all new. Our results show that engineered 3D human BA microtissues have considerable advantages in product scalability, storage, purity, safety, dosage, survival, and efficacy.

The frequent emergence of drug resistance during the treatment of influenza A virus (IAV) infections highlights a need for effective antiviral countermeasures. Here, we present an antiviral method that utilizes unnatural amino acid-engineered drug-resistant (UAA-DR) virus. The engineered virus is generated through genetic code expansion to combat emerging drug-resistant viruses. The UAA-DR virus has unnatural amino acids incorporated into its drug-resistant protein and its polymerase complex for replication control. The engineered virus can undergo genomic segment reassortment with normal virus and produce sterilized progenies due to artificial amber codons in the viral genome. We validate in vitro that UAA-DR can provide a broad-spectrum antiviral strategy for several H1N1 strains, different DR-IAV strains, multidrug-resistant (MDR) strains, and even antigenically distant influenza strains (e.g., H3N2). Moreover, a minimum dose of neuraminidase (NA) inhibitors for influenza virus can further enhance the sterilizing effect when combating inhibitor-resistant strains, partly due to the promoted superinfection of unnatural amino acid-modified virus in cellular and animal models. We also exploited the engineered virus to achieve adjustable efficacy after external UAA administration, for mitigating DR virus infection on transgenic mice harboring the pair, and to have substantial elements of the genetic code expansion technology, which further demonstrated the safety and feasibility of the strategy. We anticipate that the use of the UAA-engineered DR virion, which is a novel antiviral agent, could be extended to combat emerging drug-resistant influenza virus and other segmented RNA viruses.