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Fluorescent RNAs (FRs) provide an attractive approach to visualizing RNAs in live cells. Although the color palette of FRs has been greatly expanded recently, a green FR with high cellular brightness and photostability is still highly desired. Here we develop a fluorogenic RNA aptamer, termed Okra, that can bind and activate the fluorophore ligand ACE to emit bright green fluorescence. Okra has an order of magnitude enhanced cellular brightness than currently available green FRs, allowing the robust imaging of messenger RNA in both live bacterial and mammalian cells. We further demonstrate the usefulness of Okra for time-resolved measurements of ACTBmRNA trafficking to stress granules, as well as live-cell dual-color superresolution imaging of RNA in combination with Pepper620, revealing nonuniform and distinct distributions of different RNAs throughout the granules. The favorable properties of Okra make it a versatile tool for the study of RNA dynamics and subcellular localization.

RNA molecules perform various crucial roles in diverse cellular processes, from translating genetic information to decoding the genome, regulating gene expression and catalyzing chemical reactions. RNA-binding proteins (RBPs) play an essential role in regulating the diverse behaviors and functions of RNA in live cells, but techniques for the spatiotemporal control of RBP activities and RNA functions are rarely reported yet highly desirable. We recently reported the development of LicV, a synthetic photoswitchable RBP that can bind to a specific RNA sequence in response to blue light irradiation. LicV has been used successfully for the optogenetic control of RNA localization, splicing, translation and stability, as well as for the photoswitchable regulation of transcription and genomic locus labeling. Compared to classical genetic or pharmacologic perturbations, LicV-based light-switchable effectors have the advantages of large dynamic range between dark and light conditions and submicron and millisecond spatiotemporal resolutions. In this protocol, we provide an easy, efficient and generalizable strategy for engineering photoswitchable RBPs for the spatiotemporal control of RNA metabolism. We also provide a detailed protocol for the conversion of a CRISPR–Cas system to optogenetic control. The protocols typically take 2–3 d, including transfection and results analysis. Most of this protocol is applicable to the development of novel LicV-based photoswitchable effectors for the optogenetic control of other RNA metabolisms and CRISPR–Cas functions.

The “design–build–test–learn” (DBTL) cycle has been adopted in rational high-throughput screening to obtain high-yield industrial strains. However, the mismatch between build and test slows the DBTL cycle due to the lack of high-throughput analytical technologies. In this study, a highly efficient, accurate, and noninvasive detection method of gentamicin (GM) was developed, which can provide timely feedback for the high-throughput screening of high-yield strains. First, a self-made tool was established to obtain data sets in 24-well plates based on the color of the cells. Subsequently, the random forest (RF) algorithm was found to have the highest prediction accuracy with an R2value of 0.98430 for the same batch. Finally, a stable genetically high-yield strain (998 U/mL) was successfully screened out from 3005 mutants, which was verified to improve the titer by 72.7% in a 5 L bioreactor. Moreover, the verified new data sets were updated on the model database in order to improve the learning ability of the DBTL cycle.

A new continuous-flow process for the enzymatic synthesis of optically pure γ-lactones, which are used as flavors and fragrances in the food and cosmetic industries, was developed in a three-dimensional microfluidic reactor. The microchannels (175 mm in length, 0.9 mm in depth, and 1.72 mL in volume) were carved precisely inside a single borosilicate glass (90 mm × 75 mm × 12 mm) with ultrafast femtosecond laser micromachining. The flow field analysis and reaction simulation showed that the mixing of substrates and enzymes was enhanced, allowing the adjustment of residence time in a wide window. SmCRV4, a carbonyl reductase with excellent catalytic activity and enantioselectivity toward γ/δ-keto acids, was employed for the asymmetric synthesis of various chiral lactones. 30 mmol/L (R)-γ-decalactone (3g) can be obtained in 26 s with a space-time yield (STY) up to 16,877 g L−1 d−1, which is 14.4 times higher than the highest STY of batch reaction reported previously. This continuous-flow process was applied to the synthesis of 6 chiral lactones. In addition, the scaled-up synthesis of 3gwas carried out in 6 cascade microreactors continuously for 6 h, demonstrating the feasibility and stability of the 3D continuous-flow process in enzymatic synthesis of optically pure compounds.

Near-infrared conjugated polymer nanoparticles (NIR-CPNs) have been widely used in in vivoimaging fields. However, most of them face the aggregation-induced fluorescence quenching (ACQ) dilemma and serious dye leakage behavior, which impedes the long-term monitoring of transplanted cells in vivo. In the present work, a novel strategy of sandwich-type encapsulation of the conjugated polymer interlayer in the crystalline SiO2core + shell (SSiO2@SPFTBT@CSiO2) is developed, which works well to avoid the ACQ problem by homogeneously dispersing poly((9,9-dioctylfluorene-2,7-diyl)-alt-(4,7-di(thiophene-2-yl)-2,1,3-benzothiadiazole)-5′,5″-diyl) (PFTBT) and suppressing intermolecular π–π stacking. Furthermore, the unparalleled nanostructure efficiently stabilizes nanoparticles and successfully achieves long-term biocompatibility without interfering the biological characteristics of stem cells, indicating the potential of SSiO2@SPFTBT@CSiO2in cell labeling. In addition, the fate of human umbilical cord mesenchymal stem cells (hucMSCs) in a mouse model with acute liver injury was disclosed. We found that the hucMSCs mainly migrated from the lungs to the injured liver and most transplanted hucMSCs were cleared up by the liver at 8 days post-injection. Revelation of the shuttle process and period will benefit in improving the clinical efficacy of hucMSCs, and the sandwich-type encapsulation strategy could also open a new avenue to obtain bright and robust NIR-CPNs for long-term fluorescence imaging.

This study investigated a new approach for synthesizing Bacillus subtilisbiofilm-supported Mn-Ce/Zeolite catalysts for the degradation of gaseous toluene. Four different metal oxide nano-catalysts (ZMn, ZMnCe-10%, ZMnCe-20%, and ZMnCe-30%) were synthesized with varying ratios of manganese (Mn) and cerium (Ce) on zeolite nanoparticles. TEM, SEM, XRD, BET, XPS, and EDX mapping were used to examine these four samples, as well as simple zeolite. Based on these analyses, the catalytic activity of the prepared samples ZMn, ZMnCe-10%, ZMnCe-20%, and ZMnCe-30% for the complete oxidation of toluene and toluene intermediate products were tested with Non-thermal plasma (NTP) technology in a dielectric barrier discharge (DBD) reactor. Among all, ZMnCe-20% showed the highest toluene degradation efficiency (89%) at low concentrations (200 ppm) and humidity (>50%). Later, highly efficient and hydrophobic nano-biocatalysts were prepared by combining B. subtilisbiofilm wild-type (WT) and engineered B. subtilisbiofilm EPS with ZMnCe-20% catalyst. EPS is the main component found in biofilm matrix and plays a key role in influencing properties such as biofilm stability, electron transfer, surface roughness and hydrophobicity. Compared to WT B. subtilisbiofilm, EPS overexpressed B. subtilisbiofilm showed stronger growth and development on ZMnCe-20% nanocatalyst. Moreover, the NTP system packed with ZMnCe-20%/biofilm (EPS+) nano-biocatalyst exhibited the highest toluene degradation activity (99%) with (83%) CO2selectivity, (up to 50%) reduction in NOx concentration and complete ozone decomposition at (250 ppm) toluene concentrations and increased humidity (>90%). High-energy electrons generated in the NTP system break the C-H and C-C bond between the rings of the toluene molecule, forming several byproducts which are later reacted with active radical species such as O•, OH•, and O3and further converted into final degradation products (CO2and H2O). The results demonstrated successful biofilm development and growth on the ZMnCe-20% catalyst with advanced features such as superhydrophobicity, H2O resistance, improved surface roughness, and electron generation. In short, the study's approach combines bioengineering and material science to develop sustainable nano-biocatalysts for removing VOCs in industrial and environmental settings.

In the production of l-lactic acid, the residual sugar in fermentation broth would lead to increased sugar content in its commercial products, bringing about lactic acid deterioration because of Maillard reactions and thus shortening their shelf life. In this work, the residual sugar components in l-lactic acid industrial fermentation broth with agricultural biomass-derived sugar were determined by means of high-performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD). A feasible off-line sample clean-up procedure consisting of sodium oxalate extraction, ethanol extraction and ion-exchange column extraction was developed to remove calcium ions, proteins, anion acids and organic acids before analysis, and their removal efficiencies were 100, 98.74, 97.99 and 99.41%, respectively. Under the optimized analytical conditions, the seventeen sugars, including four monosaccharides, eight disaccharides and five oligosaccharides, have been completely separated in one experimental run on a Dionex Carbopac PA 200 column with sodium hydroxide and sodium acetate as eluents. The method was validated, with good linearity (R2> 0.99), high precision (RSDs < 5.47%), and sensitive detection limits (0.35–44.61 μg L−1); recoveries were >86.95% for short chain carbohydrates (degree of polymerization, DP < 4), while recoveries ranged from 81.87 to 61.41% for DP4 to DP7. The method was successfully applied for analysis of the residual sugar in lactic acid broth with a salt content as high as 120 g L−1calcium lactate, using different kinds of starch (corn starch, corn flour and cassava starch) hydrolysates as carbon sources. Isomaltose (47.97–60.43%), sucrose (17.50–29.73%) and trehalose (3.05–7.51%) were the main components of residual sugar in these broths.

Sertoli cells (SCs), which are recognized as the “nurse cells” of the testis due to their important biofunctions, have been used in cotransplantation with neurons in cell therapy. However, it is not clear whether SCs influence neuronal communication and survival. In this study, we showed that approximately 60% of cortical neural stem cells (NSCs) cocultured with SCs differentiated into mature neurons. In addition, the neurite outgrowth and neuronal survival rates were significantly enhanced in the coculture system compared with differentiated neurons induced by a differentiation medium. The neuroleukin (NLK) secretion of SCs was also identified at the RNA and protein level, and the roles of NLK in neuromorphology and physiological regulation were systematically investigated for the first time. These results not only highlight the significance of paracrine regulation of NSCs by SCs but also confirm the role NLK plays in the differentiation and survival of NSCs. Finally, we proposed a possible hypothesis for the mechanism of NLK in the growth and survival of SC-induced neurons based on Western blotting results, which is that NLK secreted by SCs activates the Ras/Raf/MEK/Erk, Jak/Stat, and PI3K/Akt pathways, but not the NF-κB pathway, in neurons resulting in their growth and survival.

Abstract: The effects of different culture conditions on the expression level of human interferon-α2a (IFN-α2a) by using recombinant yeast were investigated in a 2.6-Ljar fermentor. Appropriate supplement of glucose and the maintenance of residual glucose at a low level resulted in the reduction of ethanol formation and enhancement of the bioactivity of IFN-α2a to 4.9106 from 3.1106 IU/mL. When adenine was added evenlly for 10–20 h of fermentation into the basal culture medium at a speed of 2 μg/mL of medium/h, OD600 was greatly increased to 24, and the protein increased to 276 mg/L. The content of ethanol generated was also reduced tremendously during the process and as a result, 1.3107 IU/mL of biologic activity was achieved. In the expression phase, pH had an important impact on expression level, which should be controlled at 5.5