Evolution of molecular switches for regulation of transgene expression by clinically licensed gluconate.

Synthetic biology holds great promise to improve the safety and efficacy of future gene and engineered cell therapies by providing new means of endogenous or exogenous control of the embedded therapeutic programs. Here, we focused on gluconate as a clinically licensed small-molecule inducer and engineered gluconate-sensitive molecular switches to regulate transgene expression in human cell cultures and in mice. Several switch designs were assembled based on the gluconate-responsive transcriptional repressor GntR from Escherichia coli. Initially we assembled OFF- and ON-type switches by rewiring the native gluconate-dependent binding of GntR to target DNA sequences in mammalian cells. Then, we utilized the ability of GntR to dimerize in the presence of gluconate to activate gene expression from a split transcriptional activator. By means of random mutagenesis of GntR combined with phenotypic screening, we identified variants that significantly enhanced the functionality of the genetic devices, enabling the construction of robust two-input logic gates. We also demonstrated the potential utility of the synthetic switch in two in vivo settings, one employing implantation of alginate-encapsulated engineered cells and the other involving modification of host cells by DNA delivery. Then, as proof-of-concept, the gluconate-actuated genetic switch was connected to insulin secretion, and the components encoding gluconate-induced insulin production were introduced into type-1 diabetic mice as naked DNA via hydrodynamic tail vein injection. Normoglycemia was restored, thereby showcasing the suitability of oral gluconate to regulate in situ production of a therapeutic protein.

Design of programmable post-translational switch control platform for on-demand protein secretion in mammalian cells.

The development of novel strategies to program cellular behaviors is a central goal in synthetic biology, and post-translational control mediated by engineered protein circuits is a particularly attractive approach to achieve rapid protein secretion on demand. We have developed a programmable protease-mediated post-translational switch (POSH) control platform composed of a chimeric protein unit that consists of a protein of interest fused via a transmembrane domain to a cleavable ER-retention signal, together with two cytosolic inducer-sensitive split protease components. The protease components combine in the presence of the specific inducer to generate active protease, which cleaves the ER-retention signal, releasing the transmembrane-domain-linked protein for trafficking to the trans-Golgi region. A furin site placed downstream of the protein ensures cleavage and subsequent secretion of the desired protein. We show that stimuli ranging from plant-derived, clinically compatible chemicals to remotely controllable inducers such as light and electrostimulation can program protein secretion in various POSH-engineered designer mammalian cells. As proof-of-concept, an all-in-one POSH control plasmid encoding insulin and abscisic acid-activatable split protease units was hydrodynamically transfected into the liver of type-1 diabetic mice. Induction with abscisic acid attenuated glycemic excursions in glucose-tolerance tests. Increased blood levels of insulin were maintained for 12 days.

Controlling therapeutic protein expression via inhalation of a butter flavor molecule.

Precise control of the delivery of therapeutic proteins is critical for gene- and cell-based therapies, and expression should only be switched on in the presence of a specific trigger signal of appropriate magnitude. Focusing on the advantages of delivering the trigger by inhalation, we have developed a mammalian synthetic gene switch that enables regulation of transgene expression by exposure to the semi-volatile small molecule acetoin, a widely used, FDA-approved food flavor additive. The gene switch capitalizes on the bacterial regulatory protein AcoR fused to a mammalian transactivation domain, which binds to promoter regions with specific DNA sequences in the presence of acetoin and dose-dependently activates expression of downstream transgenes. Wild-type mice implanted with alginate-encapsulated cells transgenic for the acetoin gene switch showed a dose-dependent increase in blood levels of reporter protein in response to either administration of acetoin solution via oral gavage or longer exposure to acetoin aerosol generated by a commercial portable inhaler. Intake of typical acetoin-containing foods, such as butter, lychees and cheese, did not activate transgene expression. As a proof of concept, we show that blood glucose levels were normalized by acetoin aerosol inhalation in type-I diabetic mice implanted with acetoin-responsive insulin-producing cells. Delivery of trigger molecules using portable inhalers may facilitate regular administration of therapeutic proteins via next-generation cell-based therapies to treat chronic diseases for which frequent dosing is required.

Renalase regulates renal tubular injury in diabetic nephropathy via the p38MAPK signaling pathway.

Diabetic nephropathy (DN) is an important complication of diabetes and the leading cause of end-stage renal disease globally. Renal tubular damage occurs to varying degrees in the early stages of DN prior to glomerular damage. Renalase (RNLS) is an amine oxidase, which is produced and secreted by the renal tubular epithelial cells. RNLS is reportedly closely related to renal tubular injury in acute and chronic kidney diseases. Herein, we aimed to evaluate the changes in tubular RNLS expression in DN and its correlation with DN-associated renal tubular injury. Conditional permanent renal tubular epithelial rat-cell line NRK-52E was transfected with pcDNA3-RNLS plasmid or administered recombinant rat RNLS protein and high glucose (HG) dose. A total of 22 adult Sprague-Dawley rats were randomly divided into the control (CON, n = 10) or diabetic nephrology (DN, n = 12) group. Random blood glucose levels of the rats were measured by sampling of the caudal vein weekly. After 8 weeks, the rat's body weight, 24-h urinary albumin concentration, and right kidney were evaluated. Our study suggested the decreased expression levels of RNLS in renal tissue and renal tubular epithelial cells in DN rats, accompanied by renal tubulointerstitial fibrosis, apoptosis of renal tubular epithelial cells, and activation of the p38MAPK signal pathway. Reversing the low RNLS expression can reduce the level of p38MAPK phosphorylation and delay renal tubular injury. Thus, the reduction of renal tubular RNLS expression in DN mediates tubulointerstitial fibrosis and cell apoptosis via the activation of the p38MAPK signal pathway. RNLS plays a key mediating role in DN-associated tubular injury via p38MAPK, which provides new therapeutic targets and a theoretical basis for early prevention and treatment of DN.

Wi-Fi-Based Indoor Localization and Navigation: A Robot-Aided Hybrid Deep Learning Approach.

Indoor localization and navigation have become an increasingly important problem in both industry and academia with the widespread use of mobile smart devices and the development of network techniques. The Wi-Fi-based technology shows great potential for applications due to the ubiquitous Wi-Fi infrastructure in public indoor environments. Most existing approaches use trilateration or machine learning methods to predict locations from a set of annotated Wi-Fi observations. However, annotated data are not always readily available. In this paper, we propose a robot-aided data collection strategy to obtain the limited but high-quality labeled data and a large amount of unlabeled data. Furthermore, we design two deep learning models based on a variational autoencoder for the localization and navigation tasks, respectively. To make full use of the collected data, a hybrid learning approach is developed to train the models by combining supervised, unsupervised and semi-supervised learning strategies. Extensive experiments suggest that our approach enables the models to learn effective knowledge from unlabeled data with incremental improvements, and it can achieve promising localization and navigation performance in a complex indoor environment with obstacles.

Prediction of surface roughness and the material removal rate in magnetorheological finishing.

Magnetorheological finishing (MRF) is a sub-aperture polishing process, which is often used to correct surface errors and remove sub-surface damage after grinding. A strong correlation exists between the material removal rate and surface roughness in MRF, but current theoretical models are incapable of predicting these two factors at the same time. In this paper, a theoretical model was developed to describe the material removal rate and surface quality after MRF in order to better understand the material removal mechanism of MRF and explain the relationship between surface roughness and material removal rate. Two modes of experiments (uniform polishing and fixed point polishing) were conducted on monocrystalline silicon to obtain the results of surface roughness and removal rate. The experimental results are highly consistent with the theoretical model calculated results. The theoretical model could be a reference for high-efficiency and ultra-smooth MRF process.

Self-calibration interferometric stitching test method for cylindrical surfaces.

The surface figure accuracy requirement of cylindrical surfaces widely used in rotors of gyroscope, spindles of ultra-precision machine tools and high-energy laser systems is nearly 0.1 µm. Cylindricity measuring instrument that obtains 1-D profile result cannot be utilized for deterministic figuring methods. Interferometric stitching test for cylindrical surfaces utilizes a CGH of which the system error will accumulated to unacceptable extent for large aperture/angular aperture that require many subapertures. To this end, a self-calibration interferometric stitching method for cylindrical surfaces is proposed. The mathematical model of cylindrical surface figure and the completeness condition of self-calibration stitching test of cylindrical surfaces were analyzed theoretically. The effects of shear/stitching motion error and the subapertures lattice on the self-calibration test results were analyzed. Further, a self-calibration interferometric stitching algorithm that can theoretically recover all the necessary components of the system error for testing cylindrical surfaces was proposed. Simulations and experiments on a shaft were conducted to validate the feasibility.

Ammonia-nitrogen removal from water with gC3N4-rGO-TiO2 Z-scheme system via photocatalytic nitrification-denitrification process.

Photocatalytic removal of NH3-N is expected to be an alternative to the biological method that accompanied with high energy consumption and secondary pollution. However, NH3-N is always oxidized into nitrate and nitrite during the photocatalytic processes, which also need to be removed from the water. Herein, the g-C3N4/rGO/TiO2 Z-scheme photocatalytic system was prepared and used for the NH3-N removal. The results showed the rate constant of NH3-N conversion on it was 0.705 h-1, 1.7 times as high as that on g-C3N4/TiO2, and most of the NH3-N were converted into gaseous products. And the experiment result indicated NH3-N and NO3- in water could enhance the removal of each other. According to the results, the main reaction mechanism is speculated as: ·OH radicals and ·O2- radicals were generated on TiO2 and oxidized the NH3-N into NO3-, and the latter was reduced into non-toxic N2 on the conduction band of g-C3N4. Finally, NH3-N removal performance for actual coking wastewater was investigated, and the stability of the photocatalyst was tested. This work provides some theoretical basis for the two-step degradation of pollutants by Z-scheme photocatalytic system.

Exosomes from donor-derived adipose mesenchymal stem cells prolong the survival of vascularized composite allografts.

Donor-derived adipose-derived mesenchymal stem cells (ADMSCs) dampen the alloimmune response and exosomes are reported to have biological activity similar to their parent cells. Here, we investigated the roles of exosomes from donor-derived ADMSCs (ADMSC-exo) in vascularized composite allotransplantation (VCA). Brown Norway-to-Lewis rat hindlimb transplantations were intravenously treated with either exosome from donor-derived ADMSCs or phosphate-buffered saline, combined with a short course of immunosuppression. We established that the treatment with ADMSC-exo prolongs the survival time of VCA grafts. Skin and muscle samples from ADMSC-exo-treated animals showed no histological signs of rejection, but samples from controls showed rejection of degree III. Comparing to the control group, a significant increase of donor cell chimerism, Tr1 and Treg, while a decrease of CD4+ T and Th1 cells were observed in the ADMSC-exo-treated group. Our findings imply that ADMSC-exo may be a valuable and safe treatment for extending VCA graft survival.

Smartphone-Flashlight-Mediated Remote Control of Rapid Insulin Secretion Restores Glucose Homeostasis in Experimental Type-1 Diabetes.

Emerging digital assessment of biomarkers by linking health-related data obtained from wearable electronic devices and embedded health and fitness sensors in smartphones is opening up the possibility of creating a continuous remote-monitoring platform for disease management. It is considered that the built-in flashlight of smartphones may be utilized to remotely program genetically engineered designer cells for on-demand delivery of protein-based therapeutics. Here, the authors present smartphone-induced insulin release in ß-cell line (iß-cell) technology for traceless light-triggered rapid insulin secretion, employing the light-activatable receptor melanopsin to induce calcium influx and membrane depolarization upon illumination. This iß-cell-based system enables repeated, reversible secretion of insulin within 15 min in response to light stimulation, with a high induction fold both in vitro and in vivo. It is shown that programmable percutaneous remote control of implanted microencapsulated iß-cells with a smartphone's flashlight rapidly reverses hyperglycemia in a mouse model of type-1 diabetes.

Interferometric stitching method for testing cylindrical surfaces with large apertures.

Cylindrical surfaces widely used in high-energy laser systems can have nearly semi-meter-scale dimensions, and aperture angles can exceed R/3. State-of-the-art interferometric stitching test methods involve stitching only along the arc direction, and the reported dimensions of ∼50 × 50 mm2 are far smaller than those required in high-energy laser systems. To rectify this limitation, an interferometric stitching method for cylindrical surfaces with large apertures is proposed. Moreover, a subaperture stitching algorithm that can stitch along both the linear and arc directions is developed. An interferometric stitching workstation equipped with a six-axis motion stage and a series of computer-generated holograms is established, where cylindrical surfaces with R/# values as large as R/0.5 and apertures up to 700 mm can be tested based on the theoretical analysis. A convex cylindrical surface with a 350 × 380 mm2 aperture is tested to validate the proposed method's feasibility in enlarging the testable aperture of cylindrical surfaces significantly from Ф50 mm to Ф700 mm, thereby promoting the application of large cylindrical surfaces in high-energy laser systems.

Programmable and printable Bacillus subtilis biofilms as engineered living materials.

Bacterial biofilms can be programmed to produce living materials with self-healing and evolvable functionalities. However, the wider use of artificial biofilms has been hindered by limitations on processability and functional protein secretion capacity. We describe a highly flexible and tunable living functional materials platform based on the TasA amyloid machinery of the bacterium Bacillus subtilis. We demonstrate that genetically programmable TasA fusion proteins harboring diverse functional proteins or domains can be secreted and can assemble into diverse extracellular nano-architectures with tunable physicochemical properties. Our engineered biofilms have the viscoelastic behaviors of hydrogels and can be precisely fabricated into microstructures having a diversity of three-dimensional (3D) shapes using 3D printing and microencapsulation techniques. Notably, these long-lasting and environmentally responsive fabricated living materials remain alive, self-regenerative, and functional. This new tunable platform offers previously unattainable properties for a variety of living functional materials having potential applications in biomaterials, biotechnology, and biomedicine.

The common risk factors for progression and mortality in COVID-19 patients: a meta-analysis.

Coronavirus disease 2019 (COVID-19), defined by the World Health Organization (WHO), has affected more than 50 million patients worldwide and caused a global public health emergency. Therefore, there is a recognized need to identify risk factors for COVID-19 severity and mortality. A systematic search of electronic databases (PubMed, Embase and Cochrane Library) for studies published before September 29, 2020, was performed. Studies that investigated risk factors for progression and mortality in COVID-19 patients were included. A total 344,431 participants from 34 studies were included in this meta-analysis. Regarding comorbidities, cerebrovascular disease (CVD), chronic kidney disease (CKD), coronary heart disease (CHD), and malignancy were associated with an increased risk of progression and mortality in COVID-19 patients. Regarding clinical manifestations, sputum production was associated with a dramatically increased risk of progression and mortality. Hemoptysis was a risk factor for death in COVID-19 patients. In laboratory examinations, increased neutrophil count, decreased lymphocyte count, decreased platelet count, increased C-reactive protein (CRP), coinfection with bacteria or fungi, increased alanine aminotransferase (ALT) and creatine kinase (CK), increased N-terminal pronatriuretic peptide (NT-proBNP), and bilateral pneumonia in CT/X-ray were significantly more frequent in the severe group compared with the non-severe group. Moreover, the proportion of patients with increased CRP and total bilirubin (TBIL) was also significantly higher in the deceased group than in the survival group. CVD, CKD, sputum production, increased neutrophil count, decreased lymphocyte count, decreased platelet count, increased CRP, coinfection with bacteria or fungi, increased ALT and CK, increased NT-proBNP, and bilateral pneumonia in CT/X-ray were associated with an increased risk of progression in COVID-19 patients. Moreover, the proportion of patients with increased sputum production, hemoptysis, CRP and TBIL was also significantly higher in the deceased group.

LncRNA ZEB1-AS1 regulates hepatocellular carcinoma progression by targeting miR-23c.

BACKGROUND: It has been reported that long-chain non-coding RNA (lncRNA) zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) is an oncogene in various cancers, including hepatocellular carcinoma (HCC). We investigated the role and mechanism of ZEB1-AS1 as a competitive endogenous RNA (ceRNA) combined with miR-23c in HCC cell proliferation and invasion. METHODS: QRT-PCR was used to detect ZEB1-AS1 and miR-23c expressions in HCC tissues and cells. The dual luciferase reporter assay detected the targeted regulation of miR-23c and ZEB1-AS1. We also performed the correlation analysis of their expression in HCC tissues by the Spearman's correlation analysis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the proliferation of hepatoma cells. Cell invasion was assessed by the Transwell assay. RESULTS: QRT-PCR results indicated ZEB1-AS1 was upregulated and miR-23c was downregulated in HCC tissues and cell lines. ZEB1-AS1 knockdown hampered the proliferation and invasion of HCC cells. Dual luciferase reporter assay showed that miR-23c is a target of ZEB1-AS1, and ZEB1-AS1 was significantly negatively correlated with the miR-23c expression in HCC tissues. The results of MTT and Transwell assay showed that miR-23c inhibition restored the inhibitory effect of ZEB1-AS1 knockdown on HCC cells proliferation and invasion. CONCLUSIONS: As a ceRNA, lncRNA ZEB1-AS1 may play a vital role in inhibiting HCC progression through miR-23c, which will provide new clues and theoretical basis for the HCC diagnosis and treatment.

Laser energy absorption prediction of silicon substrate surface from a mid- and high-spatial frequency error.

Additional laser energy absorption of optical elements limits the further development of high-energy laser systems. In engineering, inexpensive and precise absorption test technology is essential. We attempt to predict energy absorption via surface spatial error value based on the roughness-induced absorption (RIA) theory. However, the absorption coefficients cannot match roughness values measured with an atomic force microscope or white light interferometer. We find three influencing factors and optimize the definition of RIA to spatial error-induced absorption (SEIA). SEIA is proportional to δ2 of a mid- and high-spatial frequency error in a certain frequency range. This range depends on laser diameter, wavelength, and coating. Excluding the absorption induced by fabrication defects, the total absorption can be classified into SEIA and background absorption (BGA). BGA is decided by material and process technology, which can be obtained by calculations. The sum of SEIA and BGA is predictable because both can be estimated. The substrate absorption of high-energy optics can be semi-quantificationally predicted. SEIA provides a new angle to research element-absorbed laser energy for high-power laser technologies.

Patterned Amyloid Materials Integrating Robustness and Genetically Programmable Functionality.

The precise manipulation, localization, and assembly of biological and bioinspired molecules into organized structures have greatly promoted material science and bionanotechnology. Further technological innovation calls for new patternable soft materials with the long-sought qualities of environmental tolerance and functional flexibility. Here, we report a patterned amyloid material (PAM) platform for producing hierarchically ordered structures that integrate these material attributes. This platform, combining soft lithography with generic amyloid monomer inks (consisting of genetically engineered biofilm proteins dissolved in hexafluoroisopropanol), along with methanol-assisted curing, enables the spatially controlled deposition and in situ reassembly of amyloid monomers. The resulting patterned structures exhibit spectacular chemical and thermal stability and mechanical robustness under harsh conditions. The PAMs can be programmed for a vast array of multilevel functionalities, including anchoring nanoparticles, enabling diverse fluorescent protein arrays, and serving as self-supporting porous sheets for cellular growth. This PAM platform will not only drive innovation in biomanufacturing but also broaden the applications of patterned soft architectures in optics, electronics, biocatalysis, analytical regents, cell engineering, medicine, and other areas.

Long non-coding RNA LINC00152 promotes cell growth and invasion of papillary thyroid carcinoma by regulating the miR-497/BDNF axis.

Long intergenic non-coding RNA 152 (LINC00152) was reported to be tightly linked to tumorigenesis and progression in multiple cancers. However, its biological role and modulatory mechanism in papillary thyroid carcinoma (PTC) has not been elucidated. In this study, we determined the expression levels of LINC00152 in PTC tissues and cell lines by quantitative real time polymerase chain reaction (qRT-PCR). Cell proliferation, colony formation, migration, and invasion were measured by a Cell Counting Kit-8 assay, colony formation analysis, wound healing, and transwell invasion assay, respectively. A luciferase reporter assay and qRT-PCR were used to determine whether LINC00152 interacts with miR-497 directly. We established a xenograft mouse model to examine the underlying molecular mechanism and effect of LINC00152 on tumor growth in vivo. We found that LINC00152 expression was significantly increased in PTC tissues and derived cell lines. LINC00152 knockdown significantly inhibited proliferation, colony formation, migration, and invasion in vitro, and impaired tumor growth in vivo. We revealed that LINC00152 functioned as a competing endogenous RNA to the miR-497 sponge, downregulating its downstream target brain-derived neurotrophic factor (BDNF), which is an oncogene in thyroid cancer. These findings suggest that LINC00152 is responsible for PTC cell proliferation and invasion and exerts its function by regulating the miR-497/BDNF axis.

Intelligence enhancement of the adaptive wavefront interferometer.

The adaptive wavefront interferometer (AWI) we have reported recently is utilized to test in-process surfaces with severe surface figure error which is beyond dynamic range of conventional interferometers [S. Xue, S. Chen, Z. Fan, and D. Zhai, Opt. Express26, 21910 (2018).]. However, it shows low intelligence when Monte-Carlo simulation is conducted to apply AWI on various surface figure error. In some simulation cases, the unresolvable fringes keep still or cannot be turned into completely resolvable fringes. To troubleshoot this issue, we studied AWIs in a general framework of global optimization for the first time. Under this framework, we explained that three optimization issues contribute to the poor performance of AWI. On this basis, we proposed a machine vision and genetic algorithm combined method (MV-GA) to control AWI to realize efficient and robust tests of various surface figure error. Monte-Carlo simulation and experiment verify the robustness has been greatly enhanced.

Adaptive null interferometric test using spatial light modulator for free-form surfaces.

We report a method of using a liquid-crystal spatial light modulator (LC-SLM) as reconfigurable multi-level interferogram-type computer generated holograms (ICGHs) to perform dynamic null tests for aspheric and free-form surfaces. With the proposed multi-level ICGHs encoding method, amplitude and accuracy of the applicable aberration of LC-SLMs are both suitable for interferometric test. No other equipment is required to monitor the dynamic phase of LC-SLM for guaranteeing test accuracy. Moreover, complicated phase response calibration of the LC-SLM is not required. Besides being used in collimated beams, the LC-SLM is demonstrated for the first time to be used in divergent beams; hence, concave surfaces with apertures larger than that of the LC-SLMs can be tested. For realizing practical tests, the calibration of inherit wavefront distortion of the LC-SLM, diffraction orders isolation, and alignment are analyzed in detail. Two free-form surfaces with about 20 µm departure from flat and spherical surfaces are successfully measured in collimated beam and divergent beam, respectively. Cross tests are provided to verify the test accuracy.

Flexible interferometric null testing for concave free-form surfaces using a hybrid refractive and diffractive variable null.

Free-form surfaces have been applied in a wide range of modern optical systems. As a supporting technique for fabricating free-form surfaces, the interferometric null method for testing the surface figure error has very limited flexibility. In this Letter, we report a flexible interferometric null test method which can test free-form surfaces with a very broad departure varying range. In the presented flexible null method, a hybrid refractive and diffractive variable null (HRDVN) is utilized as the flexible null. The HRDVN has superb aberration types adaptability, amplitude adaptability, and moderate phase generating accuracy. A flexible interferometric null testing setup was established using the HRDVN. Its superb adaptive capacity and moderate test accuracy were successfully demonstrated by measuring a free-form surface with rotationally symmetric departure of 173.486λ (λ=632.8 nm) and non-rotationally symmetric departure of 23.786λ.