With the outbreak of new respiratory viruses and high mortality rates of pulmonary diseases, physiologically relevant models of human respiratory system are urgently needed to study disease pathogenesis, drug efficacy, and pharmaceutics. In this paper, a 3D alveolar barrier model fabricated by printing four human alveolar cell lines, namely, type I and II alveolar cells (NCI-H1703 and NCI-H441), lung fibroblasts (MRC5), and lung microvascular endothelial cells (HULEC-5a) is presented. Automated high-resolution deposition of alveolar cells by drop-on-demand inkjet printing enables to fabricate a three-layered alveolar barrier model with an unprecedented thickness of ≈10 µm. The results show that the 3D structured model better recapitulate the structure, morphologies, and functions of the lung tissue, compared not only to a conventional 2D cell culture model, as expected, but also a 3D non-structured model of a homogeneous mixture of the alveolar cells and collagen. Finally, it is demonstrated that this thin multilayered model reproduce practical tissue-level responses to influenza infection. Drop-on-demand inkjet-printing is an enabling technology for customization, scalable manufacturing, and standardization of their size and growth, and it is believed that this 3D alveolar barrier model can be used as an alternative to traditional test models for pathological and pharmaceutical applications.

In the present study, we investigated the 3′ untranslated region (UTR) of the mouse core clock gene cryptochrome 1 (Cry1) at the post-transcriptional level, particularly its translational regulation. Interestingly, the 3′UTR of Cry1 mRNA decreased its mRNA levels but increased protein amounts. The 3′UTR is widely known to function as a cis-acting element of mRNA degradation. The 3′UTR also provides a binding site for microRNA and mainly suppresses translation of target mRNAs. We found that AU-rich element RNA binding protein 1 (AUF1) directly binds to the Cry1 3′UTR and regulates translation of Cry1 mRNA. AUF1 interacted with eukaryotic translation initiation factor 3 subunit B and also directly associated with ribosomal protein S3 or ribosomal protein S14, resulting in translation of Cry1 mRNA in a 3′UTR-dependent manner. Expression of cytoplasmic AUF1 and binding of AUF1 to the Cry1 3′UTR were parallel to the circadian CRY1 protein profile. Our results suggest that the 3′UTR of Cry1 is important for its rhythmic translation, and AUF1 bound to the 3′UTR facilitates interaction with the 5′ end of mRNA by interacting with translation initiation factors and recruiting the 40S ribosomal subunit to initiate translation of Cry1 mRNA.

Here, a new bioprinting process by combining drop-on-demand inkjet printing with a spray-coating technique, which enables the high-resolution, high-speed, and freeform fabrication of large-scale cell-laden hydrogel structures is reported. Hydrogel structures with various shapes and composed of different materials, including alginate, cellulose nanofiber, and fibrinogen, are fabricated using the inkjet-spray printing. To manufacture cell-friendly hydrogel structures with controllable stiffness, gelatine methacryloyl is saponified to stabilize jet formation and is subsequently mixed with sodium alginate to prepare blend inks. The hydrogels crosslinked from the blend inks are characterized by assessing physical properties including the microstructure and mechanical stiffness and cellular responses including the cell viability, metabolic activity, and functionality of human dermal fibroblasts within the hydrogel. Cell-laden hydrogel structures are generated on a large scale and collagen type I secretion and spreading of cells within the hydrogels are assessed. The results demonstrate that the inkjet-spray printing system will ensure the formation of a cell-laden hydrogel structure with high shape fidelity in a rapid and reliable manner. Ultimately, the proposed printing technique and the blend bioink to be used to fabricate 3D laminated large-scale tissue equivalents that potentially mimic the function of native tissues is expected.

One of the major challenges encountered in engineering complex tissues in vitro is to increase levels of complexity at the micron scale in 3D structures. Here, a strategy to create self‐organized 3D collagen microstructures by 2D micropatterning of fibroblasts is developed. Drop‐on‐demand inkjet printing is used to pattern fibroblast cells on a collagen substrate in pre‐designed patterns and with controlled density. It is found that cell‐to‐ECM interaction promotes cellular self‐organization of 3D microstructures on collagen hydrogel, whereas the formation of 3D microstructure is inhibited by disruption of actin polymerization. Using this phenomena, the controlled sizes and morphologies of the 3D collagen microstructures is demonstrated by manipulating the designs of cell patterns and the density of cells. Finally, this technique is applied to build a human skin model with papillary microstructures at the dermo‐epidermal junction. This approach to create 3D cell‐laden collagen microstructures by cell patterning provides a simple and powerful way to mimic the structures and functions of complex tissues and organs, and can make a contribution to reduce the gap between the human body and in vitro tissue models.

Rhythmic arylalkylamine N-acetyltransferase (AANAT) synthesis is a prominent circadian-controlled response that occurs in most mammals. AANAT is the core enzyme in melatonin production; because melatonin participates in many physiological processes, the regulation of AANAT is an important research topic. In this study, we focused on the role of heterogeneous ribonucleoprotein R (hnRNP R) in the translation of AANAT. A novel RNA-binding protein hnRNP R widely interacted with the 5' untranslated region (UTR) of AANAT mRNA and contributed to translation through an internal ribosomal entry site (IRES). Fine-tuning of AANAT protein synthesis occurred in response to knockdown and overexpression of hnRNP R. Nocturnal elevation of AANAT protein was dependent on the rhythmic changes of hnRNP R, whose levels are elevated in the pineal gland during nighttime. Increases in hnRNP R additionally improved AANAT production in rat pinealocytes under norepinephrine (NE) treatment. These results suggest that cap-independent translation of AANAT mRNA plays a role in the rhythmic synthesis of melatonin through the recruitment of translational machinery to hnRNP R-bound AANAT mRNA.

Quantitative probing of Cu(2+) ions naturally present in single living cells is realized by developing a quantum-dot-embedded nanowire-waveguide probe. The intracellular Cu(2+) ion concentration is quantified by direct monitoring of photoluminescence quenching during the insertion of the nanowire in a living neuron. The measured intracellular Cu(2+) ion concentration is 3.34 ± 1.04 × 10(-6) m (mean ± s.e.m.) in single hippocampal neurons.

Nuclear factor, interleukin 3, regulated (Nfil3, also known as E4 Promoter-Binding Protein 4 (E4BP4)) protein is a transcription factor that binds to DNA and generally represses target gene expression. In the circadian clock system, Nfil3 binds to a D-box element residing in the promoter of clock genes and contributes to their robust oscillation. Here, we show that the 5′-untranslated region (5′-UTR) of Nfil3 mRNA contains an internal ribosome entry site (IRES) and that IRES-mediated translation occurs in a phase-dependent manner. We demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) binds to a specific region of Nfil3 mRNA and regulates IRES-mediated translation. Knockdown of hnRNP A1 almost completely abolishes protein oscillation without affecting mRNA oscillation. Moreover, we observe that intracellular calcium levels, which are closely related to bone formation, depend on Nfil3 levels in osteoblast cell lines. We suggest that the 5′-UTR mediated cap-independent translation of Nfil3 mRNA contributes to the rhythmic expression of Nfil3 by interacting with the RNA binding protein hnRNP A1. These data provide new evidence that the posttranscriptional regulation of clock gene expression is important during bone metabolism.

The algal cell immobilization is a commonly used technique for treatment of waste water, production of useful metabolites and management of stock culture. However, control over the size of immobilized droplets, the population of microbes, and production rate in current techniques need to be improved. Here, we use drop-on-demand inkjet printing to immobilize spores of the alga Ecklonia cava within alginate microparticles for the first time. Microparticles with immobilized spores were generated by printing alginate-spore suspensions into a calcium chloride solution. We demonstrate that the inkjet technique can control the number of spores in an ejected droplet in the range of 0.23 to 1.87 by varying spore densities in bioink. After the printing-based spore encapsulation, we observe initial sprouting and continuous growth of thallus until 45 days of culture. Our study suggest that inkjet printing has a great potential to immobilize algae, and that the ability to control the number of encapsulated spores and their microenvironments can facilitate research into microscopic interactions of encapsulated spores.