Human iPSC and CRISPR targeted gene knock-in strategy for studying the somatic TIE2L914F mutation in endothelial cells.

Induced pluripotent stem cell (iPSC) derived endothelial cells (iECs) have emerged as a promising tool for studying vascular biology and providing a platform for modelling various vascular diseases, including those with genetic origins. Currently, primary ECs are the main source for disease modelling in this field. However, they are difficult to edit and have a limited lifespan. To study the effects of targeted mutations on an endogenous level, we generated and characterized an iPSC derived model for venous malformations (VMs). CRISPR-Cas9 technology was used to generate a novel human iPSC line with an amino acid substitution L914F in the TIE2 receptor, known to cause VMs. This enabled us to study the differential effects of VM causative mutations in iECs in multiple in vitro models and assess their ability to form vessels in vivo. The analysis of TIE2 expression levels in TIE2L914F iECs showed a significantly lower expression of TIE2 on mRNA and protein level, which has not been observed before due to a lack of models with endogenous edited TIE2L914F and sparse patient data. Interestingly, the TIE2 pathway was still significantly upregulated and TIE2 showed high levels of phosphorylation. TIE2L914F iECs exhibited dysregulated angiogenesis markers and upregulated migration capability, while proliferation was not affected. Under shear stress TIE2L914F iECs showed reduced alignment in the flow direction and a larger cell area than TIE2WT iECs. In summary, we developed a novel TIE2L914F iPSC-derived iEC model and characterized it in multiple in vitro models. The model can be used in future work for drug screening for novel treatments for VMs.

Conformal Graphs as Twisted Partition Functions.

We show that a class of L-loop conformal ladder graphs are intimately related to twisted partition functions of free massive complex scalars in d=2L+1 dimensions. The graphs arise as four-point functions in certain two- and four-dimensional conformal fishnet models. The twisted thermal two-point function of the scalars becomes a generator of conformal ladder graphs for all loops. We argue that this correspondence is seeded by a system of two decoupled harmonic oscillators twisted by an imaginary chemical potential. We find a number of algebraic and differential relations among the conformal graphs that mirror the underlying free dynamics.

Highly sensitive and selective fluorescent "turn-on" sensor for Ag+ detection using MAPbBr3@PCN-221(Fe): An efficient Ag+-bridged energy transfer from perovskite to MOF.

Metal ion-induced water pollution is attracting increasing public attention. Perovskite quantum dots and metal-organic frameworks (MOFs), owing to their outstanding properties, hold promise as ideal probes for detecting metal ions. In this study, a composite material, MAPbBr3@PCN-221(Fe), was prepared by encapsulating MAPbBr3 quantum dots with PCN-221(Fe), demonstrating high chemical stability and good reusability. The composite material shows a sensitive fluorescence turn-on signal in the presence of silver ions. The fluorescence intensity of the composite material exhibits a linear relationship with the concentration of Ag+ in the solution, with a low detection limit of 8.68 µM. Moreover, the fluorescence signal exhibits a strong selectivity for Ag+, enabling the detection of Ag+ concentration. This fluorescence turn-on signal originates from the Ag+-bridged energy transfer from the conductive band of MAPbBr3 to the excited state of the MOF, which is directly proportional to the concentration of silver ions. Simultaneously, this finding may open up a new possibility in artificial controlled energy transfer from perovskite to MOF for future development.

An optimized filter design approach for enhancing imaging quality in industrial linear accelerator.

BACKGROUND: The polychromatic X-rays generated by a linear accelerator (Linac) often result in noticeable hardening artifacts in images, posing a significant challenge to accurate defect identification. To address this issue, a simple yet effective approach is to introduce filters at the radiation source outlet. However, current methods are often empirical, lacking scientifically sound metrics. OBJECTIVE: This study introduces an innovative filter design method that optimizes filter performance by balancing the impact of ray intensity and energy on image quality. MATERIALS AND METHODS: Firstly, different spectra under various materials and thicknesses of filters were obtained using GEometry ANd Tracking (Geant4) simulation. Subsequently, these spectra and their corresponding incident photon counts were used as input sources to generate different reconstructed images. By comprehensively comparing the intensity differences and noise in images of defective and non-defective regions, along with considering hardening indicators, the optimal filter was determined. RESULTS: The optimized filter was applied to a Linac-based X-ray computed tomography (CT) detection system designed for identifying defects in graphite materials within high-temperature gas-cooled reactor (HTR), with defect dimensions of 2 mm. After adding the filter, the hardening effect reduced by 22%, and the Defect Contrast Index (DCI) reached 3.226. CONCLUSION: The filter designed based on the parameters of Average Difference (AD) and Defect Contrast Index (DCI) can effectively improve the quality of defect images.

MRI features of pituitary adenoma apoplexy and their relationship with hypoxia, proliferation, and pathology.

OBJECTIVE: We aim to study the MRI features of pituitary adenoma (PA) apoplexy and their relationship with hypoxia, proliferation, and pathology. METHODS: Sixty-seven patients with MRI signs of PA apoplexy were selected. According to the MRI signs, they were divided into the parenchymal group and the cystic group. The parenchymal group had a low signal area on T2WI without cyst >2 mm and this area was not significantly enhanced on the corresponding TW1 enhancement. The cystic group had a cyst >2 mm on T2WI, and the cyst showed liquid stratification on T2WI or high signal on T1WI. The relative T1WI (rT1WI) enhancement value and relative T2WI (rT2WI) value of non-apoplexy areas were measured. Protein levels of hypoxia-inducible factor-1 (HIF-1α), pyruvate dehydrogenase kinase 1 (PDK1), and Ki67 were detected with immunohistochemistry and Western blot. Nuclear morphology was observed with HE staining. RESULTS: The rT1WI enhancement average value, rT2WI average value, Ki67 protein expression level, and the number of abnormal nuclear morphology of non-apoplexy lesions in the parenchymal group were significantly lower than those in the cystic group. The protein expression levels of HIF-1α and PDK1 in the parenchymal group were significantly higher than those in the cystic group. HIF-1α protein was positively correlated with PDK1 but negatively correlated with Ki67. CONCLUSION: When there is PA apoplexy, the ischemia and hypoxia of the cystic group are lesser than those of the parenchymal group, but the proliferation is stronger.

A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes.

Media and feed optimization have fueled many-fold improvements in mammalian biopharmaceutical production, but genome editing offers an emerging avenue for further enhancing cell metabolism and bioproduction. However, the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we present a comprehensive pooled CRISPR screen for CHO cell metabolism, including ~16,000 gRNAs against ~2500 metabolic enzymes and regulators. Using this screen, we identified a glutamine response network in CHO cells. Glutamine is particularly important since it is often over-fed to drive increased TCA cycle flux, but toxic ammonia may accumulate. With the screen we found one orphan glutamine-responsive gene with no clear connection to our network. Knockout of this novel and poorly characterized lipase, Abhd11, substantially increased growth in glutamine-free media by altering the regulation of the TCA cycle. Thus, the screen provides an invaluable targeted platform to comprehensively study genes involved in any metabolic trait, and elucidate novel regulators of metabolism.

Awakening dormant glycosyltransferases in CHO cells with CRISPRa.

Chinese hamster ovary (CHO) cells are the preferred workhorse for the biopharmaceutical industry, and CRISPR/Cas9 has proven powerful for generating targeted gene perturbations in CHO cells. Here, we expand the CRISPR engineering toolbox with CRISPR activation (CRISPRa) to increase transcription of endogenous genes. We successfully increased transcription of Mgat3 and St6gal1, and verified their activity on a functional level by subsequently detecting that the appropriate glycan structures were produced. This study demonstrates that CRISPRa can make targeted alterations of CHO cells for desired phenotypes.

The Correlative Factors and Probable Mechanism of Epidural Fluid Collection After Cranioplasty.

OBJECTIVE: Epidural fluid collection (EFC) is one of the postoperative complications of cranioplasty and is easily ignored. Not only the predictive factors of EFC formation are unknown, the pathologic mechanisms are also unknown. We determined to analyze the predictive factors and the mechanism of EFC formation. METHODS: A total of 340 patients underwent cranioplasty were retrospectively analyzed in this study. A series of factors were compared in the EFC and none-EFC groups and farther compared in the progress epidural fluid collection (PEFC) and none-PEFC subgroups to determine the predictive factors. The t test, χ test, and logistic regression analysis were used in statistical analysis. RESULTS: The rate of EFC formation was 34.41%, and the size of skull defect, preoperative volume of collapse, intraoperative dura suspending, a pre- or intraoperative ventriculoperitoneal shunt (V-P shunt), and an postoperative air bubble in epidural space were predictive factors for EFC formation. Furthermore, the incidence of PEFC was 10.29%, the size of skull defects and intraoperative dura suspending were predictive factors for PEFC formation. The protein ratio and lactate dehydrogenase (LDH) ratio of effusion to serum were >0.5 and 0.6, respectively. There was no adverse prognosis. CONCLUSIONS: Although EFC can be treated with conservative therapy, we need to emphasize EFC incidence and development. As neurosurgeons, it is necessary to analyze the preoperative predictive factors of EFC, pay attention to the intraoperative details such as dura suspending to prevent PEFC formation, and the early intervention should be performed in the postoperative.

CRISPR/Cas9-based genome editing for simultaneous interference with gene expression and protein stability.

Interference with genes is the foundation of reverse genetics and is key to manipulation of living cells for biomedical and biotechnological applications. However, classical genetic knockout and transcriptional knockdown technologies have different drawbacks and offer no control over existing protein levels. Here, we describe an efficient genome editing approach that affects specific protein abundances by changing the rates of both RNA synthesis and protein degradation, based on the two cross-kingdom control mechanisms CRISPRi and the N-end rule for protein stability. In addition, our approach demonstrates that CRISPRi efficiency is dependent on endogenous gene expression levels. The method has broad applications in e.g. study of essential genes and antibiotics discovery.

BE-FLARE: a fluorescent reporter of base editing activity reveals editing characteristics of APOBEC3A and APOBEC3B.

BACKGROUND: Base Editing is a precise genome editing method that uses a deaminase-Cas9 fusion protein to mutate cytidine to thymidine in target DNA in situ without the generation of a double-strand break. However, the efficient enrichment of genetically modified cells using this technique is limited by the ability to detect such events. RESULTS: We have developed a Base Editing FLuorescent Activity REporter (BE-FLARE), which allows for the enrichment of cells that have undergone editing of target loci based on a fluorescence shift from BFP to GFP. We used BE-FLARE to evaluate the editing efficiency of APOBEC3A and APOBEC3B family members as alternatives deaminase domains to the rat APOBEC1 domain used in base editor 3 (BE3). We identified human APOBEC3A and APOBEC3B as highly efficient cytidine deaminases for base editing applications with unique properties. CONCLUSIONS: Using BE-FLARE to report on the efficiency and precision of editing events, we outline workflows for the accelerated generation of genetically engineered cell models and the discovery of alternative base editors.

Enhanced protein and biochemical production using CRISPRi-based growth switches.

Production of proteins and biochemicals in microbial cell factories is often limited by carbon and energy spent on excess biomass formation. To address this issue, we developed several genetic growth switches based on CRISPR interference technology. We demonstrate that growth of Escherichia coli can be controlled by repressing the DNA replication machinery, by targeting dnaA and oriC, or by blocking nucleotide synthesis through pyrF or thyA. This way, total GFP-protein production could be increased by up to 2.2-fold. Single-cell dynamic tracking in microfluidic systems was used to confirm functionality of the growth switches. Decoupling of growth from production of biochemicals was demonstrated for mevalonate, a precursor for isoprenoid compounds. Mass yield of mevalonate was increased by 41%, and production was maintained for more than 45h after activation of the pyrF-based growth switch. The developed methods represent a promising approach for increasing production yield and titer for proteins and biochemicals.

Delivery of Transferrin-Conjugated Polysaccharide Nanoparticles in 9L Gliosacoma Cells.

To investigate the possibility of drug targeting via the transferrin receptor-mediated pathway, iron-saturated transferrin was conjugated with chitosan (Tr-chitosan) and complexed with doxorubicin-conjugated methoxy poly(ethylene glycol)-b-dextran succinate (DEX-DOX). DEX-DOX nanoparticles have spherical morphologies with less than 150 nm particle sizes. When Tr-chitosan was complexed with DEX-DOX nanoparticles (TR nanoparticle), particle sizes were increased to higher than 200 nm. Viability of 9L cells with treatment of doxorubicin (DOX) or DEX-DOX nanoparticle was dose-dependently decreased regardless of transferrin receptor blocking. However, cytotoxicity of TR nanoparticles was reduced by blocking of transferrin receptor. Flow cytometric analysis and confocal microscopic observation showed that fluorescence intensity of tumor cells with treatment of TR nanoparticles was significantly decreased by blocking of transferring receptor while DEX-DOX nanoparticles were not affected by blocking of transferring receptor. These results indicated that TR nanoparticles are promising candidates for brain tumor drug delivery.

Immunohistochemical profile of the dural tail in intracranial meningiomas.

OBJECTIVE: The dural tail sign was first described as a thin, tapering rim of dural enhancement, in continuity with meningiomas on enhanced T1-weighted magnetic resonance (MR) images. However, the exact nature of the dural tail is still unclear. This study investigated the immunohistochemical (IHC) characteristics of the dural tail in intracranial meningiomas and the correlation between clinicopathological profiles and tumor invasion of the dural tail. METHODS: The study group consisted of 36 patients of meningioma with the dural tail noted on MR imaging and in pathological findings, and 18 patients of meningioma without the dural tail as the control group. IHC staining of tumor masses and dural tails for vascular endothelial growth factor (VEGF), epithelial membrane antigen, CD34, Ki-67, and vimentin were performed. RESULTS: The data showed that 61.1 % (22/36) of cases in the study group revealed tumor invasion of dural tail, and 55.6 % (30/54) of all the cases demonstrated dura mater invasion in all the samples. The dura mater invasion was significantly positively related to invasion of the dural tail in the study group (p = 0.009). IHC staining detected higher expression of VEGF and CD34 in the dural tail than in the main tumor mass. CONCLUSIONS: Considering the high proportion of patients with tumor invasion into the dural tail, we tried to perform wide resection of the dural tail during intracranial meningioma surgery. Furthermore, VEGF was strongly expressed in tumor cells that invaded into the dural tail, and hence VEGF can be used as a marker to differentiate tumor cells from normal meningeal cells in the dural tail.

IDNet: Information Decomposition Network for Fast Panoptic Segmentation.

Traditional CNN-based pipelines for panoptic segmentation decompose the task into two subtasks, i.e., instance segmentation and semantic segmentation. In this way, they extract information with multiple branches, perform two subtasks separately and finally fuse the results. However, excessive feature extraction and complicated processes make them time-consuming. We propose IDNet to decompose panoptic segmentation at information level. IDNet only extracts two kinds of information and directly completes panoptic segmentation task, saving the efforts to extract extra information and to fuse subtasks. By decomposing panoptic segmentation into category information and location information and recomposing them with a serial pipeline, the process for panoptic segmentation is simplified greatly and unified with regard to stuff and things. We also adopt two correction losses specially designed for our serial pipeline, guaranteeing the overall predicting performance. As a result, IDNet strikes a better balance between effectiveness and efficiency, achieving the fastest inference speed of 24.2 FPS at a resolution of 800×1333 on a Tesla V100 GPU and a PQ of 43.8, which is comparable in one-stage CNN-based methods. The code will be released at https://github.com/AronLin/IDNet.

Water-stable perovskite CsPb2Br5/CdSe quantum dot-based photoelectrochemical sensors for the sensitive determination of dopamine.

A heterojunction of CdSe quantum dots in situ grown on the perovskite CsPb2Br5 (CsPb2Br5/CdSe) for water-stable photoelectrochemical (PEC) sensing was simply synthesized using the hot-injection method. Due to the inherent built-in electric field and the matching band structure between CsPb2Br5 and CdSe, the CsPb2Br5/CdSe p-n heterojunction demonstrates enhanced photoelectrochemical properties. Accelerated interfacial charge transfer and increased electron-hole pair separation enable hydrolysis-resistant CsPb2Br5/CdSe sensors to exhibit heightened sensitivity with an ultra-low detection limit (0.0124 µM) and a wide linear range (0.4-303.9 µM) in subsequent dopamine detection. Moreover, the CsPb2Br5/CdSe sensors show excellent anti-interference ability, as well as remarkable stability and reproducibility in water solvent. It is noteworthy that this work is conducted in an aqueous environment, which provides an inspiring and convenient way for photoelectric and photoelectrocatalysis applications based on water-resistant perovskites.

Corrigendum: Active substances of myxobacteria against plant diseases and their action mechanisms.

[This corrects the article DOI: 10.3389/fmicb.2023.1294854.].

CO and HCHO Sensing by Single Au Atom-Decorated WS2 Monolayer for Diagnosis of Thermal Aging Faults in the Dry-Type Reactor: A First-Principles Study.

CO and HCHO are the main pyrolysis gases in long-term running dry-type reactors, and thus the diagnosis of thermal insulation faults inside such devices can be realized by sensing these gases. In this paper, a single Au atom-decorated WS2 (Au-WS2) monolayer is proposed as an original sensing material for CO or HCHO detection to evaluate the operation status of dry-type reactors. It was found that the Au atom prefers to be adsorbed at the top of the S atom of the pristine WS2 monolayer, wherein the binding force is calculated as -3.12 eV. The Au-WS2 monolayer behaves by chemisorption upon the introduction of CO and HCHO molecules, with the adsorption energies of -0.82 and -1.01 eV, respectively. The charge density difference was used to analyze the charge-transfer and bonding behaviors in the gas adsorptions, and the analysis of density of state as well as band structure indicate gas-sensing mechanisms. As calculated, the sensing responses of the Au-WS2 monolayer upon CO and HCHO molecule introduction were 58.7% and -74.4%, with recovery times of 0.01 s and 11.86 s, respectively. These findings reveal the favorable potential of the Au-WS2 monolayer to be a reusable and room-temperature sensing candidate for CO and HCHO detections. Moreover, the work function of the Au-WS2 monolayer was decreased by 13.0% after the adsorption of CO molecules, while it increased by 1.2% after the adsorption of HCHO molecules, which implies its possibility to be a work-function-based gas sensor for CO detection. This theoretical report paves the way for further investigations into WS2-based gas sensors in some other fields, and it is our hope that our findings can stimulate more reports on novel gas-sensing materials for application in evaluating the operation conditions of dry-type reactors.

Effect of Laser-Textured Cu Foil with Deep Ablation on Si Anode Performance in Li-Ion Batteries.

Si is a highly promising anode material due to its superior theoretical capacity of up to 3579 mAh/g. However, it is worth noting that Si anodes experience significant volume expansion (>300%) during charging and discharging. Due to the weak adhesion between the anode coating and the smooth Cu foil current collector, the volume-expanded Si anode easily peels off, thus damaging anode cycling performance. In the present study, a femtosecond laser with a wavelength of 515 nm is used to texture Cu foils with a hierarchical microstructure and nanostructure. The peeling and cracking phenomenon in the Si anode are successfully reduced, demonstrating that volume expansion is effectively mitigated, which is attributed to the high specific surface area of the nanostructure and the protection of the deep-ablated microgrooves. Moreover, the hierarchical structure reduces interfacial resistance to promote electron transfer. The Si anode achieves improved cycling stability and rate capability, and the influence of structural features on the aforementioned performance is studied. The Si anode on the 20 µm-thick Cu current collector with a groove density of 75% and a depth of 15 µm exhibits a capacity of 1182 mAh/g after 300 cycles at 1 C and shows a high-rate capacity of 684 mAh/g at 3 C.

Active substances of myxobacteria against plant diseases and their action mechanisms.

Myxobacteria have a complex life cycle and unique social behavior, and obtain nutrients by preying on bacteria and fungi in soil. Chitinase, ß-1,3 glucanase and ß-1,6 glucanase produced by myxobacteria can degrade the glycosidic bond of cell wall of some plant pathogenic fungi, resulting in a perforated structure in the cell wall. In addition, isooctanol produced by myxobacteria can lead to the accumulation of intracellular reactive oxygen species in some pathogenic fungi and induce cell apoptosis. Myxobacteria can also perforate the cell wall of some plant pathogenic oomycetes by ß-1,3 glucanase, reduce the content of intracellular soluble protein and protective enzyme activity, affect the permeability of oomycete cell membrane, and aggravate the oxidative damage of pathogen cells. Small molecule compounds such as diisobutyl phthalate and myxovirescin produced by myxobacteria can inhibit the formation of biofilm and lipoprotein of bacteria, and cystobactamids can inhibit the activity of DNA gyrase, thus changing the permeability of bacterial cell membrane. Myxobacteria, as a new natural compound resource bank, can control plant pathogenic fungi, oomycetes and bacteria by producing carbohydrate active enzymes and small molecular compounds, so it has great potential in plant disease control.