USP7 promotes IgA class switching through stabilizing RUNX3 for germline transcription activation.

Class switch recombination (CSR) diversifies the effector functions of antibodies and involves complex regulation of transcription and DNA damage repair. Here, we show that the deubiquitinase USP7 promotes CSR to immunoglobulin A (IgA) and suppresses unscheduled IgG switching in mature B cells independent of its role in DNA damage repair, but through modulating switch region germline transcription. USP7 depletion impairs Sα transcription, leading to abnormal activation of Sγ germline transcription and increased interaction with the CSR center via loop extrusion for unscheduled IgG switching. Rescue of Sα transcription by transforming growth factor ß (TGF-ß) in USP7-deleted cells suppresses Sγ germline transcription and prevents loop extrusion toward IgG CSR. Mechanistically, USP7 protects transcription factor RUNX3 from ubiquitination-mediated degradation to promote Sα germline transcription. Our study provides evidence for active transcription serving as an anchor to impede loop extrusion and reveals a functional interplay between USP7 and TGF-ß signaling in promoting RUNX3 expression for efficient IgA CSR.

Two-dimensional spatial coherence measurement of X-ray sources using aperture array mask.

Fourth-generation synchrotron radiation delivers x-ray sources with unprecedented coherence and brilliance, which enables the development of many advanced coherent techniques taking advantage of the inherent high coherence of the x-ray beams. Simple and accurate measurement of two-dimensional (2D) coherence is of utmost importance for the applications of these coherent experimental techniques. Here, we propose a novel approach based on diffraction of aperture array mask (AAM) to obtain accurate 2D spatial coherence with a single-shot measurement. We utilize a coherent mode decomposition algorithm to simulate the diffraction of AAM illuminated by Gaussian-Schell model beam and demonstrate that spatial coherence function of the incident light beam can be accurately and robustly retrieved. We expect that this new approach will be applied into transverse coherence measurements for the new-generation synchrotron radiation source and relevant coherent experimental techniques.

CT radiomics model for predicting the Ki-67 proliferation index of pure-solid non-small cell lung cancer: a multicenter study.

Purpose: This study aimed to explore the efficacy of the computed tomography (CT) radiomics model for predicting the Ki-67 proliferation index (PI) of pure-solid non-small cell lung cancer (NSCLC). Materials and methods: This retrospective study included pure-solid NSCLC patients from five centers. The radiomics features were extracted from thin-slice, non-enhanced CT images of the chest. The minimum redundancy maximum relevance (mRMR) and least absolute shrinkage and selection operator (LASSO) were used to reduce and select radiomics features. Logistic regression analysis was employed to build predictive models to determine Ki-67-high and Ki-67-low expression levels. Three prediction models were established: the clinical model, the radiomics model, and the nomogram model combining the radiomics signature and clinical features. The prediction efficiency of different models was evaluated using the area under the curve (AUC). Results: A total of 211 NSCLC patients with pure-solid nodules or masses were included in the study (N=117 for the training cohort, N=49 for the internal validation cohort, and N=45 for the external validation cohort). The AUC values for the clinical models in the training, internal validation, and external validation cohorts were 0.73 (95% CI: 0.64-0.82), 0.75 (95% CI:0.62-0.89), and 0.72 (95% CI: 0.57-0.86), respectively. The radiomics models showed good predictive ability in diagnosing Ki-67 expression levels in the training cohort (AUC, 0.81 [95% CI: 0.73-0.89]), internal validation cohort (AUC, 0.81 [95% CI: 0.69-0.93]) and external validation cohort (AUC, 0.78 [95% CI: 0.64-0.91]). Compared to the clinical and radiomics models, the nomogram combining both radiomics signatures and clinical features had relatively better diagnostic performance in all three cohorts, with the AUC of 0.83 (95% CI: 0.76-0.90), 0.83 (95% CI: 0.71-0.94), and 0.81 (95% CI: 0.68-0.93), respectively. Conclusion: The nomogram combining the radiomics signature and clinical features may be a potential non-invasive method for predicting Ki-67 expression levels in patients with pure-solid NSCLC.

[Construction of Nalm6-Cas9 Cell Line for Genome-Wide Translocation Sequencing].

OBJECTIVE: In order to conduct high-throughput genome-wide translocation sequencing based on CRISPR/Cas9, Nalm6-cas9 monoclonal cell line expressing Cas9 protein was constructed by lentivirus transduction. METHODS: Lentiviral vectors LentiCas9-Blast, pSPAX2, and pMD2.G were used to co-transfect HEK293T cells to obtain recombinant lentivirus. After Nalm6 cells were infected with the recombinant lentivirus, the cells were screened by Blasticidin, and multiple monoclonal cell lines expressing Cas9 protein were obtained by limited dilution. Western blot was used to detect the expression level of Cas9 protein in monoclonal cell lines, and cell count analysis was used to detect the proliferation activity of monoclonal cell lines. LentiCRISPRV2GFP-Δcas9, LentiCRISPRV2GFP-Δcas9-AF4, LentiCRISPRV2GFP-Δ cas9-MLL plasmids were constructed, and transfected with pSPAX2 and pMD2.G, respectively. T vector cloning was used to detect the function of Cas9 protein in Nalm6-Cas9 monoclonal cell line infected with virus. RESULTS: Western blot showed that Nalm6-Cas9_1-6 monoclonal cell line had high expression of Cas9 protein. Cell count analysis showed that high expression of Cas9 protein in Nalm6-Cas9_1-6 monoclonal cell line did not affect cell proliferation activity. The Nalm6-Cas9_1-6 monoclonal cell line had high cleavage activity, and the editing efficiency of AF4 and MLL genes was more than 90% which was determined by T vector cloning. CONCLUSION: Nalm6-Cas9_1-6 monoclonal cell line stably expressing highly active Cas9 protein was obtained, which provided a basis for exploring the translocation of MLL in therapy-related leukemias based on CRISPR/Cas9 genome-wide high-throughput genome-wide translocation sequencing.

The Qc5 Allele Increases Wheat Bread-Making Quality by Regulating SPA and SPR.

Common wheat (Triticum aestivum L.) is an important food crop with a unique processing quality. The Q gene positively regulates the processing quality of wheat, but the underlying mechanism remains unclear. Here, a new Q allele (Qc5) responsible for compact spikes and good bread performance was identified. Compared with the Q allele widely distributed in modern common wheat cultivars, Qc5 had a missense mutation outside the miRNA172-binding site. This missense mutation led to a more compact messenger RNA (mRNA) secondary structure around the miRNA172-binding region, resulting in increased Qc5 expression during the spike development stage and a consequent increase in spike density. Furthermore, this missense mutation weakened the physical interaction between Qc5 and storage protein activator (SPA) in seeds and suppressed the expression of storage protein repressor (SPR). These changes increased the grain protein content and improved the bread-making quality of wheat. In conclusion, a missense mutation increases Q expression because of the resulting highly folded mRNA secondary structure around the miRNA172-binding site. Furthermore, this mutation improves the bread-making quality of wheat by repressing the expression of SPR and influencing the physical interaction between Q and SPA. These findings provide new insights into the miRNA172-directed regulation of gene expression, with implications for wheat breeding.

A new inoculation method of sulfur autotrophic denitrification reactor for accelerated start-up and better low-temperature adaption.

Elemental sulfur (S0) autotrophic denitrification (SAD) has been proved feasible for nitrate removal from aquatic environments. The long start-up period up to weeks of the SAD reactor impedes its industrial application. To accelerate the start-up process, this study employed S0 powder packed sequencing batch reactor operated for 10 days to obtain a seed biofilm, which was inoculated into a regular S0 flake packed bed reactor afterwards. Merely two days after inoculation, the reactor inoculated with seed biofilm was well started up and outperformed the control reactor, which was inoculated with regular anaerobic sludge and operated for more than 10 days, delivering much increased denitrification rate of 126 ± 0.68 mg N/(L·d) and a high nitrate removal efficiency of 93.0%. Batch tests during the start-up period showed that the seed biofilm developed well on S0 flakes and delivered improved nitrate removal performance than the control. Extracellular polymeric substance (EPS) analysis revealed an abundant content of protein in tightly bound EPS in the biofilm developed from the seed biofilm, which was recognized as a major contributor to facilitate the biofilm's attachment and growth onto S0 flakes. After operating under moderate temperature, the reactors were tested at a reduced temperature of 15 °C. Results indicated that the reactor inoculated with seed biofilm showed stronger adaptation ability towards low temperature and sustained better denitrification performance than the control, which was attributed to increased protein content in tightly bound EPS produced by the microbes against low-temperature. Determination of the microbial communities in tested reactors when the whole experiment was closing found that sulfur-related genera were dominating in the packed-bed reactor inculcated with seed biofilm, which played an important role in the S0-based denitrification process.

Major Facilitator Superfamily Transporter Gene FgMFS1 Is Essential for Fusarium graminearum to Deal with Salicylic Acid Stress and for Its Pathogenicity towards Wheat.

Wheat is a major staple food crop worldwide, due to its total yield and unique processing quality. Its grain yield and quality are threatened by Fusarium head blight (FHB), which is mainly caused by Fusarium graminearum. Salicylic acid (SA) has a strong and toxic effect on F. graminearum and is a hopeful target for sustainable control of FHB. F. graminearum is capable of efficientdealing with SA stress. However, the underlying mechanisms remain unclear. Here, we characterized FgMFS1 (FGSG_03725), a major facilitator superfamily (MFS) transporter gene in F. graminearum. FgMFS1 was highly expressed during infection and was upregulated by SA. The predicted three-dimensional structure of the FgMFS1 protein was consistent with the schematic for the antiporter. The subcellular localization experiment indicated that FgMFS1 was usually expressed in the vacuole of hyphae, but was alternatively distributed in the cell membrane under SA treatment, indicating an element of F. graminearum in response to SA. ΔFgMFS1 (loss of function mutant of FgMFS1) showed enhanced sensitivity to SA, less pathogenicity towards wheat, and reduced DON production under SA stress. Re-introduction of a functional FgMFS1 gene into ∆FgMFS1 recovered the mutant phenotypes. Wheat spikes inoculated with ΔFgMFS1 accumulated more SA when compared to those inoculated with the wild-type strain. Ecotopic expression of FgMFS1 in yeast enhanced its tolerance to SA as expected, further demonstrating that FgMFS1 functions as an SA exporter. In conclusion, FgMFS1 encodes an SA exporter in F. graminearum, which is critical for its response to wheat endogenous SA and pathogenicity towards wheat.

Artificial electrochemically active biofilm for improved sensing performance and quickly devising of water quality early warning biosensors.

A major challenge for devising an electrochemically active biofilm (EAB)-based biosensor for real-time water quality early-warning is the formation of EAB that requires several days to weeks. Besides the onerous and time-consuming preparation process, the naturally formed EABs are intensively concerned as they can hardly deliver repeatable electrical signals even at identical experimental conditions. To address these concerns, this study employed sodium alginate as immobilization agent to encapsulate Shewanella oneidensis MR-1 and prepared EAB for devising a biosensor in a short period of less than 1 h. The artificial EAB were found capable of delivering highly consistent electrical signals with each other when fed with the same samples. Morphology and bioelectrochemical properties of the artificial EAB were investigated to provide interpretations for these findings. Different concentrations of bacteria and alginate in forming the EAB were investigated for their effects on the biosensor's sensitivity. Results suggested that lower concentration of bacteria would be beneficial until it increased to 0.06 (OD660). Concentration of sodium alginate affected the sensitivity as well and 1% was found an optimum amount to serve in the formation of EAB. A long-term operation of the biosensor with artificial EAB for 110 h was performed. Clear warning signals for incoming toxicants were observed over random signal fluctuations. All results suggested that the artificial EAB electrode would support a rapid devised and highly sensitivity biosensor.

Utilization of Elemental Sulfur in Constructed Wetlands Amended with Granular Activated Carbon for High-Rate Nitrogen Removal.

To investigate the role of granular activated carbon (GAC) on nitrogen removal performance of elemental sulfur-based constructed wetlands (S0-based CWs), three systems were constructed according to the different configurations in the functional layer, namely S-CW (S0 added in the functional layer), CSC-CW (GAC, S0 and GAC placed in layers in the functional layer) and SC-CW (S0 and GAC mixed evenly in the functional layer). In CSC-CW and SC-CW, the volumetric ratio of S0:GAC was 9:1. Three CWs were operated under four different hydraulic retention times (HRTs) ranged from 48 h to 6 h. Over the experiment, total inorganic nitrogen (TIN) removal rates of the three CWs were 3.1 - 23.6 g m-2 d-1, 3.5 - 24.1 g m-2 d-1 and 3.4 - 11.5 g m-2 d-1, respectively; CSC-CW remained high TIN removal efficiency (from 74.7 ± 20.2 % to 93.4 ± 1.9 %) while SC-CW had significant lower values when HRT = 6 h (29.8 ± 30.1 %). Mass balance and high-throughput sequencing analysis revealed that mixotrophic denitrification at the sulfur layer and simultaneous nitrification-denitrification (SND) at the rhizosphere played the major role in N removal from CSC-CW (> 95 %). GAC addition facilitated the growth of Iris pseudacorus with the final fresh weight increased from 33.9 gFW ind-1 to 82.3 gFW ind-1 in CSC-CW and 82.7 gFW ind-1 in SC-CW. This study optimizes the practical application of S0-based CWs amended with GAC for N removal from carbon-limited wastewater.

Benzoic and salicylic acid are the signaling molecules of Chlorella cells for improving cell growth.

Cell-to-cell communication regulates microalgae production via signaling molecules (SMs), but few microalgal SM species are known. Here, we document two new microalgae SMs, benzoic acid (BA) and salicylic acid (SA). Initially, crude SMs were extracted from a microalgae culture in which microalgae grew on heterotrophic-enriched phosphorus nutrition. The extracted SMs enhanced Chlorella growth by ∼72%, promoted nutrient uptake, and up-regulated the mitogen-activated protein-kinase signaling cascade. Fourier transform infrared and nuclear magnetic resonance analyses identified the putative SMs was aromatic carboxylic acids. BA and SA were identified using high-resolution mass spectrometry. BA and SA addition increased cell growth by ∼75% and ∼25%; and improved ATP production by ∼35% and ∼20%. Transcriptomic analysis showed that BA and SA were biosynthesized via CoA-dependent, non-oxidative pathway. The SMs upregulated TCA-cycle enzymes, which promoted carbon assimilation and activated DNA-replicating enzyme, so that accelerated cell division. This study identified two new SMs for microalgae cell communication and provides means to identify other SMs.

Using easy-to-biodegrade co-substrate to eliminate microcystin toxic on electrochemically active bacteria and enhance bioelectricity generation from cyanobacteria biomass.

Cyanobacterial biomass is a promising natural resource for power generation, through the reactions bio-catalyzed by electrochemically active bacteria (EAB). However, the major limitation is the involvement of Microcystin-LR (MC-LR) in inhibiting EAB activation. In this work, toxic M. aeruginosa biomass was employed as analyte of a microbial fuel cell (MFC), and sodium acetate was applied as easy-to-biodegrade co-substrate to alleviate the MC-LR stress on EAB survival. The running stability was continuously enhanced with the increment of co-substrate concentration. The sufficient co-substrate supply (6.0 mM) eliminated the negative effects of MC-LR on the cyanobacteria biomass fed-MFC performance; it contributed 12.7% extension on the electric cyclic terms and caused the productions of the power density which was comparable and even 3.8% higher than its corresponding control (MFC treated with acetate alone). The co-substrate addition also increased coulombic efficiency by 60.1%, microcystin-LR removal efficiency increased by 64.7%, and diversified the microbial community with more species able to biodegrade the MC-LR, bio-transforming the metabolites and EAB. Microcystin-degrading bacteria, such as Sphingopyxis sp., Burkholderia-Paraburkholderia, and Bacillus sp., were remarkably increased, and EAB, including Shewanella sp., Desulfovibrio desulfuricans, Aeromonas hydrophila, were also much more enriched in co-substrate use protocol. Therefore, this study verified a co-substrate strategy for simultaneously eliminating MC-LR toxin and enhancing bioelectricity generation from cyanobacterial biomass via an MFC.

Organic carbon coupling with sulfur reducer boosts sulfur based denitrification by Thiobacillus denitrificans.

Sulfur autotrophic denitrification utilizes elemental sulfur as the electron donor for nitrate removal from aquatic environments. Organic carbon could stimulate the conversion of sulfur and facilitates the S0-based denitrification process in the mix-trophic. In this study, the co-cultured system of sulfur reducer (Geobacter sulfurreducens) and Thiobacillus denitrificans was used to investigate that how organic carbon could boost the S0-based denitrification. The results showed that the rate of S0-based denitrification was improved with C/N ratio of 0.13 and this improvement continued even after the acetate was exhausted. Sulfur probe test and Raman analysis suggested that reduced sulfur species (Sx2-) were formed with the addition of organic carbon. The Sx2- could recombine with element sulfur and the bioavailability of S0 would be improved, as a result, the rate of S0-based denitrification increased as well. Nitrate reduction rate could further increase with the C/N ratio of 0.88, but it would decrease significantly when the C/N ratio increased to 1.50 as the high concentration of generated S2-. Our results provided explanations that why organic carbon addition would improve the bioavailability of S0 which could further promote the S0-dominant denitrification process.

High-rate nitrogen removal from carbon limited wastewater using sulfur-based constructed wetland: Impact of sulfur sources.

This study aims to explore the application of sulfur-based constructed wetlands (CWs) for effective nitrogen (N) removal from wastewater. Two solid sulfur sources namely elemental sulfur (S0) and pyrite (FeS2) were used as substrates in two CWs, i.e. S-CW and P-CW, respectively. The CWs were vegetated with a common wetland plant Iris pseudacorus, and were operated to investigate the effects of hydraulic retention time (HRT) and temperature on N removal. The use of S0 resulted in the highest denitrification rate (19.0 ± 7.5 g m-2 d-1), whereas up to 20 times slower total inorganic nitrogen (TIN) removal was observed with FeS2. Different sulfur sources had negligible effects on the growth of I. pseudacorus, but the element contents (e.g., N, S, and P) within the plant tissues were different. Iris roots in S-CW had higher S content compared with those in P-CW, which resulted in the difference in shoots colors. The characteristics of rhizospheric microbial communities were closely related to the sulfur and nitrogen sources. Briefly, denitrifying and sulfur-oxidizing genera (e.g., Denitratisoma, Sulfurimonas, Thiobacillus) were dominating in the S-CW, suggesting the occurrence of both autotrophic and heterotrophic denitrification processes in the wetland. On the other hand, nitrifying bacteria were more abundant (e.g. Nitrospira, Piscinibacter) in the P-CW. S0 layer and rhizosphere accounted for 99.3% of nitrogen removal and the former part most likely played important roles with a decrease in HRT. Low temperature strongly affected the rate and efficiency of denitrification but recovered to 49.2 ± 25.8% when added with 30 mg L-1 sodium acetate. This study broadens the applications of sulfur-based CWs and provides a promising management strategy for denitrification at low temperatures.

Phosphorus supply via a fed-batch strategy improves lipid heterotrophic production of Chlorella regularis.

Intracellular phosphorus (P) accumulation can improve microalgal growth and lipid synthesis. However, large excess of P causes cell poisoning. This study utilized a P-fed-batch strategy to investigate its potential to improve the utilization of the excessive P, while avoiding toxic side effects. This strategy contributed to a more complete utilization of the intracellularly stored P, which enhanced the microalgae biomass by 10-15% by upregulating the brassinosteroid growth hormone gene at a P-fed-batch frequency of 2-8. Furthermore, the lipid content increased by 4-16% via upregulation of lipid synthesis-related genes. As a result, the P-fed-batch strategy significantly increased the lipid production by 13-19%. The content of saturated fatty acid increased by ~ 100%, implying improved combustibility and oxidative stability. This is the first study of this P-fed-batch strategy and provides a new concept for the complete utilization of excessive P.

A breeding strategy targeting the secondary gene pool of bread wheat: introgression from a synthetic hexaploid wheat.

KEY MESSAGE: Introgressing one-eighth of synthetic hexaploid wheat genome through a double top-cross plus a two-phase selection is an effective strategy to develop high-yielding wheat varieties. The continued expansion of the world population and the likely onset of climate change combine to form a major crop breeding challenge. Genetic advances in most crop species to date have largely relied on recombination and reassortment within a relatively narrow gene pool. Here, we demonstrate an efficient wheat breeding strategy for improving yield potentials by introgression of multiple genomic regions of de novo synthesized wheat. The method relies on an initial double top-cross (DTC), in which one parent is synthetic hexaploid wheat (SHW), followed by a two-phase selection procedure. A genotypic analysis of three varieties (Shumai 580, Shumai 969 and Shumai 830) released from this program showed that each harbors a unique set of genomic regions inherited from the SHW parent. The first two varieties were generated from very small populations, whereas the third used a more conventional scale of selection since one of bread wheat parents was a pre-breeding material. The three varieties had remarkably enhanced yield potential compared to those developed by conventional breeding. A widely accepted consensus among crop breeders holds that introducing unadapted germplasm, such as landraces, as parents into a breeding program is a risky proposition, since the size of the breeding population required to overcome linkage drag becomes too daunting. However, the success of the proposed DTC strategy has demonstrated that novel variation harbored by SHWs can be accessed in a straightforward, effective manner. The strategy is in principle generalizable to any allopolyploid crop species where the identity of the progenitor species is known.

Hormesis effects of phosphorus on the viability of Chlorella regularis cells under nitrogen limitation.

BACKGROUND: Phosphorus (P) is an essential element of microalgae, which is either required for anabolism or for energy metabolism. When employing a nitrogen limitation strategy to trigger microalgal intracellular lipid accumulation, P supplementation was always simultaneously applied to compensate for the accompanied growth inhibition. RESULTS: This study identified that P exerts hormesis effects on microalgae. Slight excess of P (≤ 45 mg L-1) under nitrogen limitation condition stimulated the cell growth of Chlorella regularis and achieved a 10.2% biomass production increase. This also improved mitochondrial activity by 25.0% compared to control (P = 5.4 mg L-1). The lipid productivity reached 354.38 mg (L d)-1, which increased by 39.3% compared to control. Such an improvement was caused by the intracellularly stored polyphosphate energy pool. However, large excess of P (250 mg L-1) inhibited the cell growth by 38.8% and mitochondrial activity decreased by 71.3%. C. regularis cells showed obvious poisoning status, such as enlarged size, plasmolysis, deformation of cell walls, and disorganization of organelles. This is probably because the over-accumulated P protonated the amide-N and disrupted membrane permeability. CONCLUSIONS: These results provide new insight into the roles of P in microalgae lipid production: P does not always play a positive role under nitrogen limitation conditions.

Genetic mapping of a major QTL promoting homoeologous chromosome pairing in a wheat landrace.

KEY MESSAGE: Common wheat landrace Kaixian-luohanmai carries a gene(s) that promotes homoeologous chromosome pairing. A major QTL responsible for this effect was mapped to chromosome arm 3AL. Polyhaploid hybrids of a Chinese common wheat landrace Kaixian-luohanmai (KL) and related species show increased levels of chromosome pairing. Over 90% of that pairing is between homoeologous arms of wheat chromosomes, with a very strong preference for pairing between homoeologs from genomes A and D. Wheat-rye pairing was also observed at low frequency. Two mapping populations were created from the hybrids of KL with two wheat genotypes top crossed to rye. Mean chiasmata numbers per plant were used as phenotypic data. Wheat 660 K and 15 K SNP arrays, DArT markers and SSR markers were used for genotyping of the top-cross ABDR hybrids. One major QTL, named QPh.sicau-3A, for increased homoeologous pairing was detected on chromosome arm 3AL, and it was responsible for ca. 16% of the total variation. This QTL was located in the interval 696-725 Mb in the Chinese Spring reference genome. SNP markers closely linked with QPh.sicau-3A were converted to KASP markers and validated for marker-assisted selection.

Bright and efficient quantum dot light-emitting diodes with double light-emitting layers.

We demonstrate high brightness and efficient light-emitting diodes (QLEDs) with 60-nm-thick double quantum dot light-emitting layers (D-EMLs) based on poly(p-phenylene benzobisoxazole) precursors. This structure distributes the charge balance by blocking electrons. The D-EML QLEDs exhibit significant improvement in brightness, efficiency, and stability. The external quantum efficiency and luminance of D-EML QLEDs show 170% and 48% enhancement compared with a single light-emitting layer, respectively. The efficiency roll-off of D-EML QLEDs is only 16% of that of the S-EML up to 10 V.