Cobalt chloride-simulated hypoxia elongates primary cilia in immortalized human retina pigment epithelial-1 cells.

Primary cilia are microtubule-based organelles that are involved in sensing micro-environmental cues and regulating cellular homeostasis via triggering signaling cascades. Hypoxia is one of the most common environmental stresses that organ and tissue cells may often encounter during embryogenesis, cell differentiation, infection, inflammation, injury, cerebral and cardiac ischemia, or tumorigenesis. Although hypoxia has been reported to promote or inhibit primary ciliogenesis in different tissues or cultured cell lines, the role of hypoxia in ciliogenesis is controversial and still unclear. Here we investigated the primary cilia change under cobalt chloride (CoCl2)-simulated hypoxia in immortalized human retina pigment epithelial-1 (hTERT RPE-1) cells. We found CoCl2 treatment elongated primary cilia in a time- and dose-dependent manner. The prolonged cilia recovered back to near normal length when CoCl2 was washed out from the cell culture medium. Under CoCl2-simulated hypoxia, the protein expression levels of HIF-1/2α and acetylated-α-tubulin (cilia marker) were increased, while the protein expression level of Rabaptin-5 is decreased during hypoxia. Taken together, our results suggest that hypoxia may elongate primary cilia by downregulating Rabaptin-5 involved endocytosis. The coordination between endocytosis and ciliogenesis may be utilized by cells to adapt to hypoxia.

Pre-grafted Group on PE Surface by DBD Plasma and Its Influence on the Oxygen Permeation with Coated SiOx.

In this paper, we report on polyethylene (PE) film modified by atmospheric dielectric barrier discharge (DBD) plasma prior to the deposition of SiOx coating to improve its barrier properties. Three kinds of monomers: allylamine, acrylic acid, and ethanol, are used to modify the PE surface. For comparison, Ar and O2 plasma pre-treatments are also performed. It is found that with the addition of a monomer in the Ar DBD plasma, the grafted active groups on PE surfaces lead to dense, pinhole-free growth of the SiOx film. The oxygen transmission rate (OTR) decreases from 700 cc/m²·day·atm. for the pristine to ca. 70 cc/m²·day·atm. for the pretreatment-coated PE, which is more than a 10-fold reduction. The relationship between the grafted monomer and the great decrease of OTR is then explored via chemical composition by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and via morphology observation by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The results show that the grafted functional groups of -NH2, -COOH and -OH increase the surface energy and promote the nucleation of Si⁻O radicals on polymeric surfaces, and the formation of network and cage structures in SiOx film contributes to the significant improvement of OTR.

A highly efficient method for genomic deletion across diverse lengths in thermophilic Parageobacillus thermoglucosidasius.

Parageobacillus thermoglucosidasius is emerging as a highly promising thermophilic organism for metabolic engineering. The utilization of CRISPR-Cas technologies has facilitated programmable genetic manipulation in P. thermoglucosidasius. However, the absence of thermostable NHEJ enzymes limited the capability of the endogenous type I CRISPR-Cas system to generate a variety of extensive genomic deletions. Here, two thermophilic NHEJ enzymes were identified and combined with the endogenous type I CRISPR-Cas system to develop a genetic manipulation tool that can achieve long-range genomic deletion across various lengths. By optimizing this tool-through adjusting the expression level of NHEJ enzymes and leveraging our discovery of a negative correlation between GC content of the guide RNA (gRNA) and deletion efficacy-we streamlined a comprehensive gRNA selection manual for whole-genome editing, achieving a 100 % success rate in randomly selecting gRNAs. Notably, using just one gRNA, we achieved genomic deletions spanning diverse length, exceeding 200 kilobases. This tool will facilitate the genomic manipulation of P. thermoglucosidasius for both fundamental research and applied engineering studies, further unlocking its potential as a thermophilic cell factory.

High-yield porphyrin production through metabolic engineering and biocatalysis.

Porphyrins and their derivatives find extensive applications in medicine, food, energy and materials. In this study, we produced porphyrin compounds by combining Rhodobacter sphaeroides as an efficient cell factory with enzymatic catalysis. Genome-wide CRISPRi-based screening in R. sphaeroides identifies hemN as a target for improved coproporphyrin III (CPIII) production, and exploiting phosphorylation of PrrA further improves the production of bioactive CPIII to 16.5 g L-1 by fed-batch fermentation. Subsequent screening and engineering high-activity metal chelatases and coproheme decarboxylase results in the synthesis of various metalloporphyrins, including heme and the anti-tumor agent zincphyrin. After pilot-scale fermentation (200 L) and setting up the purification process for CPIII (purity >95%), we scaled up the production of heme and zincphyrin through enzymatic catalysis in a 5-L bioreactor, with CPIII achieving respective enzyme conversion rates of 63% and 98% and yielding 10.8 g L-1 and 21.3 g L-1, respectively. Our strategy offers a solution for high-yield bioproduction of heme and other valuable porphyrins with substantial industrial and medical applications.

A thermostable type I-B CRISPR-Cas system for orthogonal and multiplexed genetic engineering.

Thermophilic cell factories have remarkably broad potential for industrial applications, but are limited by a lack of genetic manipulation tools and recalcitrance to transformation. Here, we identify a thermophilic type I-B CRISPR-Cas system from Parageobacillus thermoglucosidasius and find it displays highly efficient transcriptional repression or DNA cleavage activity that can be switched by adjusting crRNA length to less than or greater than 26 bp, respectively, without ablating Cas3 nuclease. We then develop an orthogonal tool for genome editing and transcriptional repression using this type I-B system in both thermophile and mesophile hosts. Empowered by this tool, we design a strategy to screen the genome-scale targets involved in transformation efficiency and established dynamically controlled supercompetent P. thermoglucosidasius cells with high efficiency ( ~ 108 CFU/µg DNA) by temporal multiplexed repression. We also demonstrate the construction of thermophilic riboflavin cell factory with hitherto highest titers in high temperature fermentation by genome-scale identification and combinatorial manipulation of multiple targets. This work enables diverse high-efficiency genetic manipulation in P. thermoglucosidasius and facilitates the engineering of thermophilic cell factories.

[Determination of electron density in atmospheric pressure radio frequency dielectric barrier discharges by Stark broadening].

The use of high frequency power to generate plasma at atmospheric pressure is a relatively new development. An apparatus of atmospheric pressure radio frequency dielectric barrier discharge was constructed. Plasma emission based measurement of electron density in discharge columns from Stark broadening Ar is discribed. The spacial profile of electron density was studied. In the middle of the discharge column, as the input power increases from 138 to 248 W, the electron density rises from 4.038 x 10(21) m(-3) to 4.75 x 10(21) m(-3).

High-throughput and reliable acquisition of in vivo turnover number fuels precise metabolic engineering.

As synthetic biology enters the era of quantitative biology, mathematical information such as kinetic parameters of enzymes can offer us an accurate knowledge of metabolism and growth of cells, and further guidance on precision metabolic engineering. k cat , termed the turnover number, is a basic parameter of enzymes that describes the maximum number of substrates converted to products each active site per unit time. It reflects enzyme activity and is essential for quantitative understanding of biosystems. Usually, the k cat values are measured in vitro, thus may not be able to reflect the enzyme activity in vivo. In this case, Davidi et al. defined a surrogate k m a x v i v o (k app ) for k cat and developed a high throughput method to acquire k m a x v i v o from omics data. Heckmann et al. and Chen et al. proved that the surrogate parameter can be a good embodiment of the physiological state of enzymes and exhibit superior performance for enzyme-constrained metabolic model to the default one. These breakthroughs will fuel the development of system and synthetic biology.

An Ionically Conductive, Self-Powered and Stable Organogel for Pressure Sensing.

Gel-based ionic conductors are promising candidates for flexible electronics, serving as stretchable sensors or electrodes. However, most of them suffer from a short operating life, low conductivity and rely on an external power supply, limiting their practical application. Herein, we report a stable organogel ionic conductor with high conductivity and self-powering ability. Briefly, lithium trifluoromethanesulfonate, as a conductive salt, provides high conductivity and the poly(1,1-difluoroethylene) layers, as a self-powering system, supply stable energy output under the influence of pressure. Moreover, the proposed conductors withstand long-term and multi-cycle durability tests. The prepared auxiliary training device can withstand the impact of a basketball and detect the impact force, showing potential in passive sensing during practical applications.

Optimization of shaped pulses for radio frequency excitation in NMR logging.

The radio frequency (RF) excitation pulse of the nuclear magnetic resonance (NMR) logging tool can realize slice measurement by designing shaped pulses. In the case of a certain main magnetic field, the accuracy of the shaped pulse design has a very important impact on the signal-to-noise ratio (SNR) of the NMR signal and the measurement of the short relaxation signal. Hard pulse excitation will produce an undesirable infinite number of side lobes that may perturb the spins in unwanted regions. Soft pulse can achieve selective excitation and has a better slice profile and shorter energy release time while it is not conducive to the measurement of short relaxation signals. This article focuses on the design of shaped pulses in extreme downhole environments and analyzes the characteristics of the three shaped pulses in the two cases of equivalent bandwidth and equivalent pulse duration. At the same time, a kind of RF-shaped pulse transmitting circuit with phase difference control is realized. According to the pulse type optimization strategy, the appropriate shaped pulse is selected. When echo spacing (TE) >0.6 ms, the SNR can be increased to more than 12%. When TE is small, it will automatically switch to the hard pulse mode, which is good for short relaxation measurement.

Engineering thermophilic Geobacillus thermoglucosidasius for riboflavin production.

The potential advantages for fermentation production of chemicals at high temperatures are attractive, such as promoting the rate of biochemical reactions, reducing the risk of contamination and the energy consumption for fermenter cooling. In this work, we de novo engineered the thermophile Geobacillus thermoglucosidasius to produce riboflavin, since this bacterium can ferment diverse carbohydrates at an optimal temperature of 60°C with a high growth rate. We first introduced a heterogeneous riboflavin biosynthetic gene cluster and enabled the strain to produce detectable riboflavin (28.7 mg l-1 ). Then, with the aid of an improved gene replacement method, we preformed metabolic engineering in this strain, including replacement of ribCGtg with a mutant allele to weaken the consumption of riboflavin, manipulation of purine pathway to enhance precursor supply, deletion of ccpNGtg to tune central carbon catabolism towards riboflavin production and elimination of the lactate dehydrogenase gene to block the dominating product lactic acid. Finally, the engineered strain could produce riboflavin with the titre of 1034.5 mg l-1 after 12-h fermentation in a mineral salt medium, indicating G. thermoglucosidasius is a promising host to develop high-temperature cell factory of riboflavin production. This is the first demonstration of riboflavin production in thermophilic bacteria at an elevated temperature.

Integrating PCR-free amplification and synergistic sensing for ultrasensitive and rapid CRISPR/Cas12a-based SARS-CoV-2 antigen detection.

Antigen detection provides particularly valuable information for medical diagnoses; however, the current detection methods are less sensitive and accurate than nucleic acid analysis. The combination of CRISPR/Cas12a and aptamers provides a new detection paradigm, but sensitive sensing and stable amplification in antigen detection remain challenging. Here, we present a PCR-free multiple trigger dsDNA tandem-based signal amplification strategy and a de novo designed dual aptamer synergistic sensing strategy. Integration of these two strategies endowed the CRISPR/Cas12a and aptamer-based method with ultra-sensitive, fast, and stable antigen detection. In a demonstration of this method, the limit of detection was at the single virus level (0.17 fM, approximately two copies/µL) in SARS-CoV-2 antigen nucleocapsid protein analysis of saliva or serum samples. The entire procedure required only 20 min. Given our system's simplicity and modular setup, we believe that it could be adapted reasonably easily for general applications in CRISPR/Cas12a-aptamer-based detection.

Inside-out azimuthally selective NMR tool using array coil and capacitive decoupling.

Inside-out nuclear magnetic resonance (NMR) is a unique technique for investigating large in-situ objects outside of tools, to provide pore structure and pore-bearing fluids properties. However, in borehole, objects towards azimuthal orientations pose different properties, referred to as azimuthal spatial heterogeneity. This may lead to ambiguous evaluations by utilizing present inside-out NMR measurement, which hardly resolves azimuthal information and loses the location information of oil/gas. In this paper, we for the first time design and construct an innovative tool to investigate the heterogeneity of large in-situ samples. The most key component, array coil, which performs with azimuthal selection, measurement consistency and interactive isolation, configured in this novel tool to capture heterogeneity information. Whereas, strong coupling between neighboring coil elements largely decrease the coil sensitivity. Capacitive decoupling network is bridged into adjacent ports without segmenting coils to be decoupled and could be easily adjusted by electrical relays. The coil model and numerical simulation are firstly given to demonstrate the array coil configuration, B1 field map and mutual coupling effects on coil sensitivity. Capacitive network is then introduced to be theoretically and practically analyzed to minimize coupling effects. Simulation and experimental results demonstrate that these coil elements have excellent consistency and independence to feasibly acquire the azimuthal NMR data.

New magnet array design for downhole NMR azimuthal measurement.

In low-field NMR, depth information and radial profile information of downhole formation can be easily acquired with the help of static gradient magnetic field produced by permanent magnets, called downhole NMR imaging. Based on the hypothesis that the formation is homogeneous, average signals detected by centralized or decentralized sensors can provide enough information for petrophysical parameters. In fact, the inhomogeneity of formation may have serious impact on description of the characteristics of formation and oil/gas location which is rarely studied in NMR well-logging. To improve this, we design and implement a new quadrupolar magnet array aimed at achieving azimuthal measurement in this paper. A new quadrupolar magnet array is consisted of four bread-shaped magnets combined with additional small hexangular magnets to produce enough strength and high homogeneity of static field along with circumferential direction at deeper DOI (depth of investigation). Azimuthal measurements are achieved by using coil array combined with quadrupolar magnet array.

A new side-looking downhole magnetic resonance imaging tool.

In general, only the depth information can be acquired using the centralized downhole NMR tools. The radial profile information is equally important to the depth. Improving the pad tools, also called side-looking tools, is the appropriate direction for solving this problem. The side-looking downhole measurement can provide depth and radially resolved information of the reservoir. In this research a new side-looking tool which includes main magnets and pre-polarized magnets has been designed and built. The pre-polarized magnets in both sides are used to adjust the homogeneity of magnetic field along the length direction of the instrument and polarize the samples when the tool is moving up and down along the borehole with a speed up to 500 m/h. A winding coil with several frequencies corresponding to different depths has been designed to match the static magnetic field. The sensitive region of this tool is about one-third of a hollow cylinder at every frequency which gives a side-looking image of the borehole wall. We have demonstrated that this new side-looking tool behaves well with an echo time short to 0.25 ms, which ensures the richness and accuracy of the measurements. Such a new side-looking tool is suitable for the detection of unconventional reservoirs.

The Antibacterial Polyamide 6-ZnO Hierarchical Nanofibers Fabricated by Atomic Layer Deposition and Hydrothermal Growth.

In this paper, we report the combination of atomic layer deposition (ALD) with hydrothermal techniques to deposit ZnO on electrospun polyamide 6 (PA 6) nanofiber (NF) surface in the purpose of antibacterial application. The micro- and nanostructures of the hierarchical fibers are characterized by field emission scanning electron microscopy (FE-SEM), high-resolution transmission electron microscopy (HRTEM), and scanning transmission electron microscopy (STEM). We find that NFs can grow into "water lily"- and "caterpillar"-like shapes, which depend on the number of ALD cycles and the hydrothermal reaction period. It is believed that the thickness of ZnO seed layer by ALD process and the period in hydrothermal reaction have the same importance in crystalline growth and hierarchical fiber formation. The tests of antibacterial activity demonstrate that the ZnO/PA 6 core-shell composite fabricated by the combination of ALD with hydrothermal are markedly efficient in suppressing bacteria survivorship.