Critical patients with the coronavirus disease 2019 (COVID-19), even those whose nucleic acid test results had turned negative and those receiving maximal medical support, have been noted to progress ...to irreversible fatal respiratory failure. Lung transplantation (LT) as the sole therapy for end-stage pulmonary fibrosis related to acute respiratory distress syndrome has been considered as the ultimate rescue therapy for these patients.
From February 10 to March 10, 2020, three male patients were urgently assessed and listed for transplantation. After conducting a full ethical review and after obtaining assent from the family of the patients, we performed three LT procedures for COVID-19 patients with illness durations of more than one month and extremely high sequential organ failure assessment scores.
Two of the three recipients survived post-LT and started participating in a rehabilitation program. Pearls of the LT team collaboration and perioperative logistics were summarized and continually improved. The pathological results of the explanted lungs were concordant with the critical clinical manifestation, and provided insight towards better understanding of the disease. Government health affair systems, virology detection tools, and modern communication technology all play key roles towards the survival of the patients and their rehabilitation.
LT can be performed in end-stage patients with respiratory failure due to COVID-19-related pulmonary fibrosis. If confirmed positive-turned-negative virology status without organ dysfunction that could contraindicate LT, LT provided the final option for these patients to avoid certain death, with proper protection of transplant surgeons and medical staffs. By ensuring instant seamless care for both patients and medical teams, the goal of reducing the mortality rate and salvaging the lives of patients with COVID-19 can be attained.
Arabidopsis mutants produced by constitutive overexpression of the CRISPR/Cas9 genome editing system are usually mosaics in the T1 generation. In this study, we used egg cell-specific promoters to ...drive the expression of Cas9 and obtained non-mosaic T1 mutants for multiple target genes with high efficiency. Comparisons of 12 combinations of eight promoters and two terminators found that the efficiency of the egg cell-specific promoter-controlled CRISPR/Cas9 system depended on the presence of a suitable terminator, and the composite promoter generated by fusing two egg cell-specific promoters resulted in much higher efficiency of mutation in the T1 generation compared with the single promoters.
Long non‐coding RNAs (lncRNAs) serve critical roles in the pathogenesis of various cancers, including lung adenocarcinoma (LUAD). Herein, in this study, we aimed to investigate the biological and ...clinical significance of lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) in LUAD. It was observed that DGCR5 was upregulated in LUAD tissues and LUAD cell lines. Inhibition of DGCR5 can prevent LUAD progression via playing anti‐apoptosis roles. Both mRNA expression and protein levels of BCL‐2 were increased by DGCR5 downregulation while reversely BAX was increased. Additionally, a novel microRNA target of DGCR5, hsa‐mir‐22‐3p was identified through bioinformatics search and confirmed by dual‐luciferase reporter system. Gain and loss‐of‐function studies were performed to verify whether DGCR5 exerts its biological functions through regulating hsa‐mir‐22‐3p in vitro. Overexpression of DGCR5 was able to reverse the tumor inhibitory effect of hsa‐mir‐22‐3p mimics. Furthermore, in vivo tests tumor xenografts were established to detect the function of DGCR5 in LUAD tumorigenesis. Downregulated DGCR5 expression was greatly associated with smaller tumor size, implying a favorable prognosis of LUAD patients. Taken these together, DGCR5 could be considered as a prognostic biomarker and therapeutic target in LUAD diagnosis and treatment.
Our findings in LUAD cell lines and xenografts support the use of DGCR5 as an oncogene to promote LUAD development. This is the first reporting of a possible mechanism for a crosstalk between DGCR and hsa‐mir‐22‐3p crosstalk in LUAD. These results suggest DGCR5 as a potential target for LUAD treatment.
To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with ...additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required.
We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation.
We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.
Non‐coding RNAs can exert significant roles various cancers, including NSCLC. Previously, we indicated that lncRNA DGCR5 can promote lung cancer progression through inhibiting hsa‐mir‐22‐3p. In our ...current study, we investigated the role of DGCR5 in cancer stem cell‐like properties of NSCLC. CSCs have been recognized as the frequent cause of tumor metastasis, tumor recurrence, and chemotherapy resistance. Here, lung cancer stem cells were successfully enriched from the parental NSCLC A549, H460, and H1299 cells by using tumor sphere formation assays and side population (SP) assays. We observed that DGCR5 was up‐regulated in the enriched CSCs of NSCLC. DGCR5 can inhibit the stemness of NSLCL while overexpression of DGCR5 promoted CSC‐like traits. In addition, miR‐330‐5p was predicted as target of DGCR5 and the correlation between them was validated by dual‐luciferase reporter assay, RIP assay, and RNA pull‐down assay. Meanwhile, it was found that miR‐330‐5p was decreased in CSCs of NSCLC. miR‐330‐5p mimics repressed the stemness while miR‐330‐5p inhibition enhanced CSC‐like properties by targeting CD44. Taken these together, DGCR5 can act as a crucial regulator of CSCs in NSCLC by modulating miR‐330‐5p/CD44 axis.
This is the first reporting of a possible mechanism for an interaction between DGCR and miR‐330‐5p in lung CSCs. We exhibited that DGCR5 silence can inhibit CSC‐like phenotypes in NSLCL by sponging miR‐330‐5p and increasing CD44 expression. These data suggested a possibility of developing DGCR5 as a potential target for NSCLC.
•The multi-scale optimization strategy can serve as a conceptual material paradigm.•A giant Wrec of 5.02 J·cm−3 and a high η of ~ 90% can be obtained in the 0.5NBT-0.5SST ceramic.•The underlying ...mechanism of high energy storage properties has been visualized.
Dielectric ceramic materials with high power density and fast charge–discharge speed have attracted increasing attention in recent years. However, their mutually restricted energy density and efficiency as well as unsatisfactory temperature stability have been the main obstacles for their practical applications. Herein, a high recoverable energy density of 5.02 J·cm−3 and a high efficiency of ~ 90% can be obtained under 422 kV·cm−1 in the Sr0.85Sm0.1TiO3 (SST)-modified Na0.5Bi0.5TiO3 (NBT) ceramics via composition design and domain engineering strategy, and the excellent stability of energy storage properties in frequency (1–100 Hz) and temperature (20–180 °C) were also observed at 250 kV·cm−1 in the 0.50NBT-0.50SST ceramics, which are attributed to the improved breakdown strength (Eb) and the enhanced relaxation behavior. The increased band gap width and refined grain size are responsible for the significantly enhanced Eb of Na0.5Bi0.5TiO3-based solid solution, being confirmed by ultraviolet and visible (UV–vis) absorption spectra as well as scanning electron microscopy. The generation of polar nanoregions as demonstrated by piezoresponse force microscopy and transmission electron microscopy results in a negligible remanent polarization and thermally stable polarization-field response. It is worth noting that the energy density will be further greatly optimized due to the improvement of Eb if this ceramic composite is made into multilayer ceramic capacitor as a dielectric layer. Moreover, a large power density of 188.6 MW·cm−3 and a fast discharge speed of 70 ns can also be achieved in the optimized composition. The results show that the multi-scale optimization strategy is an effective way to realize excellent comprehensive energy storage performances in the Na0.5Bi0.5TiO3 based ceramics.
To fully exploit the microbial genome resources, a high-throughput experimental platform is needed to associate genes with phenotypes at the genome level. We present here a novel method that enables ...investigation of the cellular consequences of repressing individual transcripts based on the CRISPR interference (CRISPRi) pooled screening in bacteria. We identify rules for guide RNA library design to handle the unique structure of prokaryotic genomes by tiling screening and construct an E. coli genome-scale guide RNA library (~60,000 members) accordingly. We show that CRISPRi outperforms transposon sequencing, the benchmark method in the microbial functional genomics field, when similar library sizes are used or gene length is short. This tool is also effective for mapping phenotypes to non-coding RNAs (ncRNAs), as elucidated by a comprehensive tRNA-fitness map constructed here. Our results establish CRISPRi pooled screening as a powerful tool for mapping complex prokaryotic genetic networks in a precise and high-throughput manner.
Conventional microbial cell cultivation techniques are typically labor intensive, low throughput, and poorlyparallelized, rendering them inefficient. The development of automated, modular microbial ...cell micro‐cultivation systems, particularly those employing droplet microfluidics, have gained attention for their high‐throughput, highly paralellized and efficient cultivation capabilities. Here, we report the development of a microbial microdroplet culture system (MMC), which is an integrated platform for automated, high‐throughput cultivation and adaptive evolution of microorganisms. We demonstrated that the MMC yielded both accurate and reproducible results for the manipulation and detection of droplets. The superior performance of MMC for microbial cell cultivation was validated by comparing the growth curves of six microbial strains grown in MMC, conventional shake flasks or well plates. The highest incipient growth rate for all six microbial strains was achieved by using MMC. We also conducted an 18‐day process of adaptive evolution of methanol‐essential Escherichia coli strain in MMC and obtained two strains exhibiting higher growth rates compared with the parent strain. Our study demonstrates the power of MMC to provide an efficient and reliable approach for automated, high‐throughput microbial cultivation and adaptive evolution.
An integrated platform called microbial microdroplet culture system (MMC), which can perform various accurate and reproducible operations on microliter droplets, is developed for automated, high‐throughput cultivation and adaptive evolution of multiple microorganisms. Here, Jian and Guo validate the superior performance of MMC for microbial cultivation by comparing the growth rates of six microbial strains grown in MMC, conventional shake flasks or well plates. The authors also demonstrate its efficient power for adaptive evolution using a methanol–essential Escherichia coli strain.
Tea plants Camellia sinensis (L.) O. Kuntze are an important leaf-type crop that are widely used for the production of non-alcoholic beverages in the world. Exposure to excessive amounts of heavy ...metals adversely affects the quality and yield of tea leaves. To analyze the molecular responses of tea plants to heavy metals, a reliable quantification of gene expression is important and of major importance herein is the normalization of the measured expression levels for the target genes. Ideally, stably expressed reference genes should be evaluated in all experimental systems. In this study, 12 candidate reference genes (i.e., 18S rRNA, Actin, CYP, EF-1α, eIF-4α, GAPDH, MON1, PP2AA3, TBP, TIP41, TUA, and UBC) were cloned from tea plants, and the stability of their expression was examined systematically in 60 samples exposed to diverse heavy metals (i.e., manganese, aluminum, copper, iron, and zinc). Three Excel-based algorithms (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of these genes. PP2AA3 and 18S rRNA were the most stably expressed genes, even though their expression profiles exhibited some variability. Moreover, commonly used reference genes (i.e., GAPDH and TBP) were the least appropriate reference genes for most samples. To further validate the suitability of the analyzed reference genes, the expression level of a phytochelatin synthase gene (i.e., CsPCS1) was determined using the putative reference genes for data normalizations. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in tea plants.
Pancreatic cancer (PC) represents a relatively rare but severe malignancy worldwide. Accumulated studies have emphasized the potential of long noncoding RNA (lncRNA) as therapeutic strategies for ...several human cancers. Thus, we aimed to investigate whether a novel non-coding RNA regulatory circuitry involved in PC. Aberrantly expressed lncRNAs and mRNAs were screened out of microarray database. Following the determination of RNA expression, PANC-1 and BxPC-3 PC cells were adopted, after which the expression of miR-330-5p, PAX8 and LINC00958 were subsequently altered. RNA crosstalk was validated by dual-luciferase reporter gene assay. In order to detect whether LINC00958 could act as ceRNA to competitively sponge miR-330-5p and regulate PAX8, subcellular location of LINC00958 and interaction between LINC00958 and miR-330-5p were measured by FISH and RNA pull down respectively. The epithelial mesenchymal transition (EMT) process, cell invasion, and tumor growth were determined in vitro and in vivo. LINC00958 and PAX8 were up-regulated, while miR-330-5p was down-regulated during PC. LINC00958 mainly expressed in the cytoplasm and LINC00958 competitively sponged miR-330-5p. Upregulated miR-330-5p or downregulated PAX8 inhibited the EMT process as well as the invasion and metastasis ability of the PC cells. Moreover, the results indicated that miR-330-5p negatively targeted PAX8, and LINC00958 ultimately showcasing its ability to bind to miR-330-5p through its interaction with AGO2. Therefore, silencing of LINC00958 may bind to miR-330-5p to inhibit PAX8 in a competitive fashion, thereby preventing the progression of PC.
•This study explores the effects of LINC00958 on PC via the miR-330-5p/PAX8 axis.•LINC00958 and PAX8 are highly expressed, yet miR-330-5p down-regulated in PC cells.•PAX8 is a target gene of miR-330-5p.•LINC00958 promotes PAX8 by binding with miR-330-5p in PC.•Down-regulated LINC00958 inhibits EMT, invasion, and metastasis of PC cells.
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