Precise CRISPR/Cpf1 genome editing system in the Deinococcus radiodurans with superior DNA repair mechanisms.

Deinococcus radiodurans, with its high homologous recombination (HR) efficiency of double-stranded DNA breaks (DSBs), is a model organism for studying genome stability maintenance and an attractive microbe for industrial applications. Here, we developed an efficient CRISPR/Cpf1 genome editing system in D. radiodurans by evaluating and optimizing double-plasmid strategies and four Cas effector proteins from various organisms, which can precisely introduce different types of template-dependent mutagenesis without off-target toxicity. Furthermore, the role of DNA repair genes in determining editing efficiency in D. radiodurans was evaluated by introducing the CRISPR/Cpf1 system into 13 mutant strains lacking various DNA damage response and repair factors. In addition to the crucial role of RecA-dependent HR required for CRISPR/Cpf1 editing, D. radiodurans showed higher editing efficiency when lacking DdrB, the single-stranded DNA annealing (SSA) protein involved in the RecA-independent DSB repair pathway. This suggests a possible competition between HR and SSA pathways in the CRISPR editing of D. radiodurans. Moreover, off-target effects were observed during the genome editing of the pprI knockout strain, a master DNA damage response gene in Deinococcus species, which suggested that precise regulation of DNA damage response is critical for a high-fidelity genome editing system.

Porous Graphene Produced by Carbothermal Shock for Green Electromagnetic Interference Shielding in Both Microwave and Terahertz Bands.

The prospect of graphene-based shielding materials in the form of fillers is limited by the cumbersome preparation of graphene. Herein, defect-tunable porous graphene prepared by carbothermal shock using low-value sucrose as a precursor is proposed as an effective shielding filler. The resultant porous graphene exhibits 32.5 dB shielding efficiency (SE) and 2.5-18 GHz effective bandwidth at a mass loading of 20 wt%, competing with the shielding performance of graphene fillers prepared by other methods. Particularly, defect-rich graphene synthesized by increasing voltage and prolonging time shows increased electromagnetic (EM) wave absorption, echoing the current concept of green shielding. In addition, the strategy of controlling the discharge conditions to improve the absorption by the shield is developed in the terahertz band. The average SE and reflection loss of the samples in the THz band (0.2-1.2 THz) exhibit 40.7 and 15.9 dB at filler loading of 5 wt%, respectively, achieving effective shielding and absorption of THz waves. This work paves a new way for low-cost preparation of graphene for EM interference shielding fillers. Meanwhile, it supplies a reference for the shielding research of the upcoming applications integrating multiple EM bands (such as sixth-generation based integrated sensing and communication).

Probing the sORF-Encoded Peptides of Deinococcus radiodurans in Response to Extreme Stress.

Organisms have developed different mechanisms to respond to stresses. However, the roles of small ORF-encoded peptides (SEPs) in these regulatory systems remain elusive, which is partially because of the lack of comprehensive knowledge regarding these biomolecules. We chose the extremophile Deinococcus radiodurans R1 as a model species and conducted large-scale profiling of the SEPs related to the stress response. The integrated workflow consisting of multiple omics approaches for SEP identification was streamlined, and an SEPome of D. radiodurans containing 109 novel and high-confidence SEPs was drafted. Forty-four percent of these SEPs were predicted to function as antimicrobial peptides. Quantitative peptidomics analysis indicated that the expression of SEP068184 was upregulated upon oxidative treatment and gamma irradiation of the bacteria. SEP068184 was conserved in Deinococcus and exhibited negative regulation of oxidative stress resistance in a comparative phenotypic assay of its mutants. Further quantitative and interactive proteomics analyses suggested that SEP068184 might function through metabolic pathways and interact with cytoplasmic proteins. Collectively, our findings demonstrate that SEPs are involved in the regulation of oxidative resistance, and the SEPome dataset provides a rich resource for research on the molecular mechanisms of the response to extreme stress in organisms.

Proteogenomic discovery of sORF-encoded peptides associated with bacterial virulence in Yersinia pestis.

Plague caused by Yersinia pestis is one of the deadliest diseases. However, many molecular mechanisms of bacterial virulence remain unclear. This study engaged in the discovery of small open reading frame (sORF)-encoded peptides (SEPs) in Y. pestis. An integrated proteogenomic pipeline was established, and an atlas containing 76 SEPs was described. Bioinformatic analysis indicated that 20% of these SEPs were secreted or localized to the transmembrane and that 33% contained functional domains. Two SEPs, named SEPs-yp1 and -yp2 and encoded in noncoding regions, were selected by comparative peptidomics analysis under host-specific environments and high-salinity stress. They displayed important roles in the regulation of antiphagocytic capability in a thorough functional assay. Remarkable attenuation of virulence in mice was observed in the SEP-deleted mutants. Further global proteomic analysis indicated that SEPs-yp1 and -yp2 affected the bacterial metabolic pathways, and SEP-yp1 was associated with the bacterial virulence by modulating the expression of key virulence factors of the Yersinia type III secretion system. Our study provides a rich resource for research on Y. pestis and plague, and the findings on SEP-yp1 and SEP-yp2 shed light on the molecular mechanism of bacterial virulence.

Diagnostic value of phosphatidylethanolamine binding protein 4 levels in patients receiving nursing interventions for advanced chronic kidney disease.

OBJECTIVE: To explore the diagnostic role of phosphatidylethanolamine binding protein 4 (PEBP4) in patients with chronic kidney disease (CKD) receiving nursing interventions. METHODS: ELISA was used to evaluate serum PEBP4 levels. Receiver-operating characteristic curve analysis was used to assess diagnostic accuracy. Spearman correlation analysis was used to assess the relationships between PEBP4 levels and biochemical indexes. RESULTS: Serum PEBP4 was high in CKD patients compared with healthy individuals. PEBP4 levels were positively correlated with pathological stage in CKD patients. PEBP4 had higher sensitivity for diagnosis of CKD than common indexes including blood urea nitrogen, creatinine and C-reactive protein. Among CKD patients treated with calcium channel blockers, serum PEBP4 levels declined notably and were associated with concentrations of K+, Na+, Cl- and Ca2+. Nursing interventions significantly decreased serum PEBP4 levels. A significant association between serum PEBP4 level and ionic concentration was observed in CKD patients receiving nursing interventions. CONCLUSIONS: This prospective study demonstrated that PEBP4 level might represent an effective diagnostic biomarker in CKD patients. PEBP4 also acted as a valuable care compliance factor for determining the necessity for nursing interventions. Nursing interventions restored ion channel function and subsequently resulted in decreased PEBP4 levels and proteinuria.

Cryopreservation of germline stem cells in American paddlefish (Polyodon spathula).

Most sturgeon and paddlefish are critically endangered; therefore, effective measures to conserve these genetic resources are required. Cryopreservation of gonad tissues containing germline stem cells could be an effective strategy for long term preservation and restoration of fish species using germ cell transplantation procedure. The aim of this study was to develop an optimal procedure for long-term cryopreservation of American paddlefish gonads using a slow-freezing method. Through optimization of permeating cryoprotectants, nonpermeating cryoprotectants, and supplementation of proteins, gonad tissues were frozen with a cryomedium containing 1.3 M dimethyl sulfoxide, 0.1 M trehalose, and 10 % fetal bovine serum at a cooling rate of -1 °C/min. This method was also successfully utilized for the cryopreservation of Yangtze sturgeon testes. Viability of gonadal cells isolated from frozen gonads was not different from cells isolated from fresh gonadal tissues, while the number of gonadal cells dissociated from frozen gonads was less. Germline stem cells dissociated from long-term (1 year) cryopreserved gonads were labeled with PKH26 fluorescent dye and intraperitoneally transplanted into larvae of Yangtze sturgeon. The colonization of transplanted germline stem cells was confirmed by the presence of PKH26-labeled donor germline stem cells and donor-derived mtDNA sequence in the recipient gonads, providing evidence that germline stem cells from sturgeon and paddlefish gonads that had been preserved for a long period maintained their functions. The results of present study indicate the procedures used are effective for long-term preservation of critically endangered species within the Acipenseriformes order which can later be regenerated using surrogate broodstock technology.

Assessment of Yangtze sturgeon as recipient for the production of American paddlefish gametes through spermatogonia transplantation.

The Chinese paddlefish (Psephurus gladius), one of the world's largest freshwater fish, was last seen alive in 2003; they are presumed now to be extinct. In fish, germ cell transplantation is currently known as one of the most powerful assisted reproductive technologies for the conservation of endangered species. In the event that a Chinese paddlefish is unexpectedly caught in the near future, we aimed to develop an experimental strategy to produce paddlefish gametes in the gonads of surrogate sturgeon. Spermatogonia were collected from the testes of 2.5-year-old immature male American paddlefish (Polyodon spathula), the species most closely related to the Chinese paddlefish, by Percoll gradient centrifugation, and transplanted into the peritoneal cavity of Yangtze sturgeon (Acipenser dabryanus) larvae at 7-8 days post-hatch. At two months post-transplantation, donor-derived spermatogonia had efficiently colonized in the recipient gonads and proliferated. A PCR analysis developed to detect xenogenic donor-derived mtDNA sequences in recipient gonads revealed that American paddlefish germ cells survived for at least seven months after transplantation in the gonads of Yangtze sturgeon recipients. These results show that the somatic microenvironment of Yangtze sturgeon gonads was able to support the colonization, proliferation, and survival of xenogeneic germ cells from a different taxonomic family. This study provides key information that could lead to future restoration of Chinese paddlefish using germ cell transplantation.

Late embryogenesis abundant group3 protein (DrLEA3) is involved in antioxidation in the extremophilic bacterium Deinococcus radiodurans.

Deinococcus radiodurans is able to survive under extreme conditions, including high doses of ionizing radiation, desiccation and oxidative stress. In addition to enhanced DNA repair capabilities, an effective antioxidation system plays an important role in its robustness. Previous studies have linked the radiation resistance of D. radiodurans to its prolonged desiccation tolerance phenotype, which both cause DNA damage. In the current study, we investigated the roles of dr_1172 in D. radiodurans, the gene encoding a typical group 3 LEA protein (DrLEA3) conserved within Deinococcus species. In addition to the increased transcriptional level under oxidative stress, the inactivation of dr_1172-sensitized cells to H2O2 treatments and the reduced cellular antioxidation activities suggested that dr_1172 is involved in the cellular defense against oxidative stress. Moreover, DrLEA3 was enriched at the cell membrane and bound to various types of metal ions. Cells devoid of DrLEA3 showed a decreased intracellular Mn/Fe concentration ratio, indicating that DrLEA3 also plays a role in maintaining metal ion homeostasis in vivo.

Succinylome Analysis Reveals the Involvement of Lysine Succinylation in the Extreme Resistance of Deinococcus radiodurans.

Increasing evidence shows that the succinylation of lysine residues mainly regulates enzymes involved in the carbon metabolism pathway, in both prokaryotic and eukaryotic cells. Deinococcus radiodurans is one of the most radioresistant organisms on earth and is famous for its robust resistance. A major goal in the current study of protein succinylation is to explore its function in D. radiodurans. High-resolution LC-MS/MS is used for qualitative proteomics to perform a global succinylation analysis of D. radiodurans and 492 succinylation sites in 270 proteins are identified. These proteins are involved in a variety of biological processes and pathways. It is found that the enzymes involved in nucleic acid binding/processing are enriched in D. radiodurans compared with their previously reported levels in other bacteria. The mutagenesis studies confirm that succinylation regulates the enzymatic activities of species-specific proteins PprI and DdrB, which belong to the radiation-desiccation response regulon. Together, these results provide insight into the role of lysine succinylation in the extreme resistance of D. radiodurans.

Molybdenum Disulfide Quantum Dots Prepared by Bipolar-Electrode Electrochemical Scissoring.

A convenient bipolar-electrode (BPE) electrochemical method was engineered to produce molybdenum disulfide (MoS2) quantum dots (QDs) using pure phosphate buffer (PBS) as the electrolyte and the MoS2 powder as the precursor. Meanwhile, the corresponding by-product precipitate was studied, in which MoS2 nanosheets were observed. The BPE design would not be restricted by the shape and size of the MoS2 precursor. It could lead to the defect generation and 2H → 1T phase variation of the MoS2, resulting in the formation of nanosheets and finally the QDs. The as-prepared MoS2 QDs exhibited high photoluminescence (PL) quantum yield of 13.9% and average lateral size of 4.4 ± 0.2 nm, respectively. Their excellent PL property, low cytotoxicity, and good aqueous dispersion offer promising applicability in PL staining and cell imaging. Meanwhile, the as-obtained byproduct containing the nanosheets could be used as an effective electromagnetic wave (EMW) absorber. The minimum reflection loss (RL) value was -54.13 dB at the thickness of 3.3 mm. The corresponding bandwidth with efficient attenuation (<-10 dB) was up to 7.04 GHz (8.8-15.84 GHz). The as-obtained EMW performance was far superior over most previously reported MoS2-based nanomaterials.

Facile Preparation of Snowflake-Like MnO2 @NiCo2 O4 Composites for Highly Efficient Electromagnetic Wave Absorption.

Snowflake-like MnO2 @NiCo2 O4 composites were successfully fabricated by employing crossed snowflake-like MnO2 nanorods as cores and one-dimensional (1D) NiCo2 O4 nanoneedles as shells. Impressively, the MnO2 @NiCo2 O4 composites exhibited a highly efficient electromagnetic wave (EMW) absorbing capability, and the minimum reflection loss (RL) value reached -58.4 dB at 6.8 GHz for a thickness of 4.0 mm. The reasons for the improved EMW absorption capability of snowflake-like MnO2 @NiCo2 O4 composites were analyzed. The unique core-shell structure, good impedance matching, and high dielectric loss were all found to be important contributors. Moreover, the interfacial polarization mainly stemmed from the heterostructure, a microcurrent generated from the 1D MnO2 nanorods and NiCo2 O4 nanoneedles under alternating electromagnetic fields, and the synergistic effect from the different components were all beneficial to improve the EMW absorption performance. These results demonstrated that snowflake-like MnO2 @NiCo2 O4 composites could be utilized as promising materials for practical EMW absorbing applications.

Ultra-efficient electromagnetic wave absorption with ethanol-thermally treated two-dimensional Nb2CTx nanosheets.

Nb2CTx, an emerging type of MXene, should be a promising electromagnetic wave (EMW) absorbing material to overcome the EMW pollution nowadays due to its unique layered structure and extremely thin monolayer thickness, but was lack of systematic study till now. Meanwhile, Nb2CTx nanosheets obtained upon HF etching of Nb2AlC MAX was unfortunately found with limited absorption performance due to its mainly dielectric loss mechanism herein. Therefore, the Nb2CTx nanosheets were further treated with solvothermal strategy in various solvents. As a result, the absorption performance of the as-treated Nb2CTx nanosheets could be significantly improved, while the ones in ethanol showed much more superior absorption capability, especially in the low-frequency band (2.0-4.0 GHz). The minimum reflection loss value could reach -52.2 dB at 3.93 GHz with the thickness of only 2.90 mm, indicating more than 99.999% EMW was absorbed. These should be due to the multi-loss mechanism including dielectric, interfacial, and multiple reflection ones resulting from the enlarged interlayer spacing, and increased surface functional groups on the Nb2CTx nanosheets upon the ethanol-based solvothermal treatment.

Characterization and role of a 2',3'-cyclic phosphodiesterase from Deinococcus radiodurans.

OBJECTIVES: A 2',3'-cyclic phosphodiesterase gene (drCPDase) has been characterized from Deinococcus radiodurans and is involved in the robust resistance of this organism. RESULTS: Cells lacking 2',3'-cyclic phosphodiesterase gene (drCPDase) showed modest growth defects and displayed increased sensitivities to high doses of various DNA-damaging agents including ionizing radiation, mitomycin C, UV and H2O2. The transcriptional level of drCPDase increased after H2O2 treatment. Additional nucleotide monophosphate partially recovered the phenotype of drCPDase knockout cells. Complementation of E. coli with drCPDase resulted in enhanced H2O2 resistance. CONCLUSIONS: The 2',3'-cyclic phosphodiesterase (drCPDase) contributes to the extreme resistance of D. radiodurans and is presumably involved in damaged nucleotide detoxification.

Crystal structure of the RNA 2',3'-cyclic phosphodiesterase from Deinococcus radiodurans.

2',3'-Cyclic phosphodiesterase (CPDase) homologues have been found in all domains of life and are involved in diverse RNA and nucleotide metabolisms. The CPDase from Deinococcus radiodurans was crystallized and the crystals diffracted to 1.6 Šresolution, which is the highest resolution currently known for a CPDase structure. Structural comparisons revealed that the enzyme is in an open conformation in the absence of substrate. Nevertheless, the active site is well formed, and the representative motifs interact with sulfate ion, which suggests a conserved catalytic mechanism.

Structural insights into catalysis and dimerization enhanced exonuclease activity of RNase J.

RNase J is a conserved ribonuclease that belongs to the ß-CASP family of nucleases. It possesses both endo- and exo-ribonuclease activities, which play a key role in pre-rRNA maturation and mRNA decay. Here we report high-resolution crystal structures of Deinococcus radiodurans RNase J complexed with RNA or uridine 5'-monophosphate in the presence of manganese ions. Biochemical and structural studies revealed that RNase J uses zinc ions for two-metal-ion catalysis. One residue conserved among RNase J orthologues (motif B) forms specific electrostatic interactions with the scissile phosphate of the RNA that is critical for the catalysis and product stabilization. The additional manganese ion, which is coordinated by conserved residues at the dimer interface, is critical for RNase J dimerization and exonuclease activity. The structures may also shed light on the mechanism of RNase J exo- and endonucleolytic activity switch.