CRISETR: an efficient technology for multiplexed refactoring of biosynthetic gene clusters.

The efficient refactoring of natural product biosynthetic gene clusters (BGCs) for activating silent BGCs is a central challenge for the discovery of new bioactive natural products. Herein, we have developed a simple and robust CRISETR (CRISPR/Cas9 and RecET-mediated Refactoring) technique, combining clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 and RecET, for the multiplexed refactoring of natural product BGCs. By this approach, natural product BGCs can be refactored through the synergistic interaction between RecET-mediated efficient homologous recombination and the CRISPR/Cas9 system. We first performed a proof-of-concept validation of the ability of CRISETR, and CRISETR can achieve simultaneous replacement of four promoter sites and marker-free replacement of single promoter site in natural product BGCs. Subsequently, we applied CRISETR to the promoter engineering of the 74-kb daptomycin BGC containing a large number of direct repeat sequences for enhancing the heterologous production of daptomycin. We used combinatorial design to build multiple refactored daptomycin BGCs with diverse combinations of promoters different in transcriptional strengths, and the yield of daptomycin was improved 20.4-fold in heterologous host Streptomyces coelicolor A3(2). In general, CRISETR exhibits enhanced tolerance to repetitive sequences within gene clusters, enabling efficient refactoring of diverse and complex BGCs, which would greatly accelerate discovery of novel bioactive metabolites present in microorganism.

Transcriptomic and Physiological Studies Unveil that Brassinolide Maintains the Balance of Maize's Multiple Metabolisms under Low-Temperature Stress.

Low-temperature (LT) is one of the major abiotic stresses that restrict the growth and development of maize seedlings. Brassinolides (BRs) have been shown to enhance LT tolerance in several plant species; the physiological and molecular mechanisms by which BRs enhance maize tolerance are still unclear. Here, we characterized changes in the physiology and transcriptome of N192 and Ji853 seedlings at the three-leaf stage with or without 2 µM 2,4-epibrassinolide (EBR) application at 25 and 15 °C environments via high-performance liquid chromatography and RNA-Sequencing. Physiological analyses revealed that EBR increased the antioxidant enzyme activities, enhanced the cell membrane stability, decreased the malondialdehyde formation, and inhibited the reactive oxygen species (ROS) accumulation in maize seedlings under 15 °C stress; meanwhile, EBR also maintained hormone balance by increasing indole-3-acetic acid and gibberellin 3 contents and decreasing the abscisic acid level under stress. Transcriptome analysis revealed 332 differentially expressed genes (DEGs) enriched in ROS homeostasis, plant hormone signal transduction, and the mitogen-activated protein kinase (MAPK) cascade. These DEGs exhibited synergistic and antagonistic interactions, forming a complex LT tolerance network in maize. Additionally, weighted gene co-expression network analysis (WGCNA) revealed that 109 hub genes involved in LT stress regulation pathways were discovered from the four modules with the highest correlation with target traits. In conclusion, our findings provide new insights into the molecular mechanisms of exogenous BRs in enhancing LT tolerance of maize at the seedling stage, thus opening up possibilities for a breeding program of maize tolerance to LT stress.

Exogenous Melatonin Alleviates NaCl Injury by Influencing Stomatal Morphology, Photosynthetic Performance, and Antioxidant Balance in Maize.

Maize (Zea mays L.) is sensitive to salt stress, especially during seed germination and seedling morphogenesis, which limits maize growth and productivity formation. As a novel recognized plant hormone, melatonin (MT) participates in multiple growth and developmental processes and mediates biotic/abiotic stress responses, yet the effects of salt stress on maize seedlings remain unclear. Herein, we investigated the effects of 150 µM exogenous MT on multiple phenotypes and physiologic metabolisms in three-leaf seedlings across eight maize inbred lines under 180 mM NaCl salt stress, including growth parameters, stomatal morphology, photosynthetic metabolisms, antioxidant enzyme activities, and reactive oxygen species (ROS). Meanwhile, the six gene expression levels controlling antioxidant enzyme activities and photosynthetic pigment biosynthesis in two materials with contrasting salt resistance were examined for all treatments to explore the possible molecular mechanism of exogenous MT alleviating salt injury in maize. The results showed that 150 µM exogenous MT application protected membrane integrity and reduced ROS accumulation by activating the antioxidant system in leaves of maize seedlings under salt stress, their relative conductivity and H2O2 level average reduced by 20.91% and 17.22%, while the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) averaged increased by 13.90%, 17.02%, 22.00%, and 14.24% relative to salt stress alone. The improvement of stomatal size and the deposition of photosynthetic pigments were more favorable to enhancing photosynthesis in leaves when these seedlings treated with MT application under salt stress, their stomatal size, chlorophyll content, and net photosynthetic rate averaged increased by 11.60%, 19.64%, and 27.62%. Additionally, Gene expression analysis showed that MT stimulation significantly increased the expression of antioxidant enzyme genes (Zm00001d009990, Zm00001d047479, Zm00001d014848, and Zm00001d007234) and photosynthetic pigment biosynthesis genes (Zm00001d011819 and Zm00001d017766) under salt stress. At the same time, 150 µM MT significantly promoted seedling growth and biomass accumulation. In conclusion, our study may unravel crucial evidence of the role of MT in maize seedlings against salt stress, which can provide a novel strategy for improving maize salt stress resistance.

Benzoxazinoids Biosynthetic Gene Cluster Identification and Expression Analysis in Maize under Biotic and Abiotic Stresses.

Benzoxazinoids (BXs) are unique bioactive metabolites with protective and allelopathic properties in maize in response to diverse stresses. The production of BXs involves the fine regulations of BXs biosynthetic gene cluster (BGC). However, little is known about whether and how the expression pattern of BGC members is impacted by biotic and abiotic stresses. Here, maize BGC was systemically investigated and 26 BGC gene members were identified on seven chromosomes, for which Bin 4.00-4.01/4.03-4.04/7.02 were the most enriched regions. All BX proteins were clearly divided into three classes and seven subclasses, and ten conserved motifs were further identified among these proteins. These proteins were localized in the subcellular compartments of chloroplast, endoplasmic reticulum, or cytoplasmic, where their catalytic activities were specifically executed. Three independent RNA-sequencing (RNA-Seq) analyses revealed that the expression profiles of the majority of BGC gene members were distinctly affected by multiple treatments, including light spectral quality, low-temperature, 24-epibrassinolide induction, and Asian corn borer infestation. Thirteen differentially expressed genes (DEGs) with high and specific expression levels were commonly detected among three RNA-Seq, as core conserved BGC members for regulating BXs biosynthesis under multiple abiotic/biotic stimulates. Moreover, the quantitative real-time PCR (qRT-PCR) verified that six core conserved genes in BGC were significantly differentially expressed in leaves of seedlings upon four treatments, which caused significant increases in 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) content under darkness and wound treatments, whereas a clear decrease in DIMBOA content was observed under low-temperature treatment. In conclusion, the changes in BX metabolites in maize were regulated by BGC gene members in multiple stress presences. Therefore, the identification of key genes associated with BX accumulation under biotic/abiotic stresses will provide valuable gene resources for breeding maize varieties with enhanced capability to adapt to environmental stresses.

Exogenous Serotonin (5-HT) Promotes Mesocotyl and Coleoptile Elongation in Maize Seedlings under Deep-Seeding Stress through Enhancing Auxin Accumulation and Inhibiting Lignin Formation.

Serotonin (5-HT), an indoleamine compound, has been known to mediate many physiological responses of plants under environmental stress. The deep-seeding (≥20 cm) of maize seeds is an important cultivation strategy to ensure seedling emergence and survival under drought stress. However, the role of 5-HT in maize deep-seeding tolerance remains unexplored. Understanding the mechanisms and evaluating the optimal concentration of 5-HT in alleviating deep-seeding stress could benefit maize production. In this study, two maize inbred lines were treated with or without 5-HT at both sowing depths of 20 cm and 3 cm, respectively. The effects of different concentrations of 5-HT on the growth phenotypes, physiological metabolism, and gene expression of two maize inbred lines were examined at the sowing depths of 20 cm and 3 cm. Compared to the normal seedling depth of 3 cm, the elongation of the mesocotyl (average elongation 3.70 cm) and coleoptile (average elongation 0.58 cm), secretion of indole-3-acetic acid (IAA; average increased 3.73 and 0.63 ng g-1 FW), and hydrogen peroxide (H2O2; average increased 1.95 and 0.63 µM g-1 FW) in the mesocotyl and coleoptile were increased under 20 cm stress, with a concomitant decrease in lignin synthesis (average decreased 0.48 and 0.53 A280 g-1). Under 20 cm deep-seeding stress, the addition of 5-HT activated the expression of multiple genes of IAA biosynthesis and signal transduction, including Zm00001d049601, Zm00001d039346, Zm00001d026530, and Zm00001d049659, and it also stimulated IAA production in both the mesocotyl and coleoptile of maize seedlings. On the contrary, 5-HT suppressed the expression of genes for lignin biosynthesis (Zm00001d016471, Zm00001d005998, Zm00001d032152, and Zm00001d053554) and retarded the accumulation of H2O2 and lignin, resulting in the elongation of the mesocotyl and coleoptile of maize seedlings. A comprehensive evaluation analysis showed that the optimum concentration of 5-HT in relieving deep-seeding stress was 2.5 mg/L for both inbred lines, and 5-HT therefore could improve the seedling emergence rate and alleviate deep-seeding stress in maize seedlings. These findings could provide a novel strategy for improving maize deep-seeding tolerance, thus enhancing yield potential under drought and water stress.

Long-term outcomes of kidney transplantation from expanded criteria donors with Chinese novel donation policy: donation after citizens' death.

INTRODUCTION: The Chinese Government initiated the Donation after Citizens' Death policy in 2010. To now, it has been a major source of organs for transplant. Since it is still a young policy, corresponding clinical evidence is still urgently needed for its improvement. Compared to kidneys donated by SCD (standard criteria donor), increasing the use of ECD (expanded criteria donor) derived kidneys is a way to expand the donor pool but is also a result of the aging demography of China. This study is based on the data of kidney transplantation in our center with the Donation after Citizens' Death policy, aiming to provide a reference for the clinical use of ECD kidneys. METHOD: A retrospective study enrolled 415 kidney transplants derived from 211 donors performed between October 2011 and October 2019. A total of 311 (74.9%) organs were donated from 159 (75.4%) SCDs, and the remaining 104 (25.1%) were from 52 (24.6%) ECDs. The log-rank test was used to compare the difference in survival and postoperative complications. The Chi-square test was used to compare the occurrence of postoperative complications and postoperative renal function. The Cox regression analysis was used for risk factor screening. RESULT: Analysis showed that grafts from ECD were poorer in survival (P = 0.013), while their recipients had comparable (P = 0.16) survival. Moreover, it also was an independent risk factor for graft loss (HR 2.27, P = 0.044). There were significantly more AR occurrences in the ECD group compared with SCD group (25.0% vs. 15.8%, P = 0.004), but no significant difference was found in infection (51.9% vs. 47.6%, P = 0.497) and DGF (26.0% vs. 21.9%, P = 0.419) between them. Similarly, fewer recipients in the ECD group were free from AR within 1 year after transplantation (P = 0.040), with no statistical difference in all-cause infection prevalence in 1 year (P = 0.168). The eGFR in the ECD group was significantly worse than that in the SCD group at 3 months, 6 months, 1 year, 3 years, and the highest value posttransplant (all < 0.05), but no difference at 5 years posttransplant. Besides, results showed cardiac arrest (uncontrolled vs. controlled, HR 2.49, P = 0.049), HLA mismatch (4-6 loci vs. 0-3 loci, HR 3.61, P = 0.039), and AR occurrence (HR 2.91, P = 0.006) were demonstrated to be independent risk factors for graft loss. CONCLUSION: The ECD-derived kidney was worse than the SCD-derived kidney in terms of graft survival and AR occurrence, and trend to an inferior renal function postoperative. However, the recipient survival, DGF occurrence, and all-cause infection occurrence were similar.

Production and characterization of bacterial cellulose obtained by Gluconacetobacter xylinus utilizing the by-products from Baijiu production.

Bacterial cellulose (BC) has extensive application prospects in many fields in view of its unique characteristics. However, the large-scale applications of BC are severely limited because of relatively low BC productivity and high cost of culture medium. Herein, the distiller's grain enzymatic hydrolysate (DEH) and yellow water were successfully combined as an effective substitute (the best distiller's grains-yellow water medium, BDY medium) for traditional Hestrin-Schramm medium (HS medium) for BC production by Gluconacetobacter xylinus through the response surface methodology. The BC production in BDY medium was significantly enhanced to 7.42 g/l with BC conversion yield of 42.4% after 7 days static cultivation, which was 3.72-fold and 3.37-fold higher than that in HS medium, respectively. The structure and properties of BC membranes produced in HS and BDY medium were evaluated by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), field emission scanning electron microscopy (FE-SEM), thermogravimetric analysis (TGA) and hydrophilicity analysis. There was no significant difference between BC samples produced in the HS and BDY medium, indicating that BDY, as abundant and inexpensive substrates, can effectively replace HS medium to enhance BC production. The employment of distiller's grains and yellow water to BC production not only is conducive to achieve industrial production of BC, but also can effectively realize the recycling of waste from Baijiu distillery.

Protective effects of mesenchymal stromal cells on adriamycin-induced minimal change nephrotic syndrome in rats and possible mechanisms.

BACKGROUND AIMS: Minimal change nephrotic syndrome is the most frequent cause of nephrotic syndrome in childhood. Current treatment regimes, which include glucocorticoid hormones and immunosuppressive therapy, are effective and have fast response. However, because of the side effects, long treatment course, poor patient compliance and relapse, novel approaches for the disease are highly desired. METHODS: The adriamycin-induced nephrotic rat model was established. Rats were allocated to a model group, a prednisone group or mesenchymal stromal cell (MSC) group. Clinical parameters in each treatment group were determined at 2 weeks, 4 weeks and 8 weeks. The messenger RNA (mRNA) levels of synaptopodin, p21 and monocyte chemoattractant protein-1 were determined through the use of quantitative real-time-polymerase chain reaction. Protein levels were determined by means of Western blot or enzyme-linked immunosorbent assay. Podocytes were isolated and apoptotic rate after adriamycin with or without MSC treatment was analyzed by means of flow cytometry. RESULTS: MSC intervention improved renal function as assessed by urinary protein, blood creatinine and triglyceride levels. MSC intervention reduced adriamycin-induced renal tissue damage visualized by immunohistochemistry and light and electron microscopic analysis and reduced adriamycin-induced podocyte apoptosis. After MSC intervention, mRNA and protein levels of synaptopodin and p21 in renal cortex were significantly increased. MSCs also restored synaptopodin mRNA and protein expression in isolated podocytes. In addition, monocyte chemoattractant protein-1 mRNA in renal cortex and protein level in serum of the MSC treatment group were significantly decreased compared with that in the adriamycin-induced nephropathy model group. CONCLUSIONS: Our data indicate that MSCs could protect rats from adriamycin-induced minimal change nephrotic syndrome, and the protective effects of MSCs are mediated through multiple actions.

BMSCs reduce rat granulosa cell apoptosis induced by cisplatin and perimenopause.

BACKGROUND: The objective of this study was to evaluate the effect of bone marrow mesenchymal stem cells (BMSCs) on the apoptosis of granulosa cells (GCs) in rats. RESULTS: Cisplatin increased GC apoptosis from 0.59% to 13.04% in the control and cisplatin treatment groups, respectively, which was significantly reduced upon co-culture with BMSCs to 4.84%. Cisplatin treatment increased p21 and bax and decreased c-myc mRNA expression, which was reversed upon co-culture with BMSCs. As compared to young rats, increased apoptosis was observed in the perimenopausal rats (P < 0.001). After 3 months, the apoptosis rate in the BMSC group was significantly lower than that of the control group (P = 0.007). CONCLUSIONS: BMSC therapy may protect against GC apoptosis induced by cisplatin and perimenopause. Further studies are necessary to evaluate therapeutic efficacy of BMSCs.

Photoelastic Stress Field Recovery Using Deep Convolutional Neural Network.

Recent work has shown that deep convolutional neural network is capable of solving inverse problems in computational imaging, and recovering the stress field of the loaded object from the photoelastic fringe pattern can also be regarded as an inverse problem solving process. However, the formation of the fringe pattern is affected by the geometry of the specimen and experimental configuration. When the loaded object produces complex fringe distribution, the traditional stress analysis methods still face difficulty in unwrapping. In this study, a deep convolutional neural network based on the encoder-decoder structure is proposed, which can accurately decode stress distribution information from complex photoelastic fringe images generated under different experimental configurations. The proposed method is validated on a synthetic dataset, and the quality of stress distribution images generated by the network model is evaluated using mean squared error (MSE), structural similarity index measure (SSIM), peak signal-to-noise ratio (PSNR), and other evaluation indexes. The results show that the proposed stress recovery network can achieve an average performance of more than 0.99 on the SSIM.

Label-free proteomic dissection on dptP-deletion mutant uncovers dptP involvement in strain growth and daptomycin tolerance of Streptomyces roseosporus.

Daptomycin (DAP) is a novel microbial lipopeptide antibiotic synthesized by the DAP biosynthetic gene cluster dpt of Streptomyces roseosporus (S. roseosporus). DptP gene locates upstream of dpt and confers DAP resistance to Streptomyces ambofaciens (S. ambofaciens). So far, the biological functions of dptP gene for S. roseosporus growth are still completely uncovered. We performed label-free quantification proteomic dissections with loss- and gain-of-function experiments to decipher dptP-involved functions. Deletion of dptP gene activated energy metabolism and metabolism of secondary metabolites pathways and enhanced the transcription levels and protein abundance of key members of the dpt cluster. Whereas dptP deletion inhibited transport/signal transduction and drug resistance pathways and protein abundance of cell division-relative proteins, subsequently decreased mycelia cell growth rate. S. roseosporus strain with dptP deletion was more sensitive to DAP treatment compared to the wild type. In contrast, overexpression of dptP gene decreased transcription levels of DAP biosynthetic genes and enhanced growth rate of Streptomcyes strain upon elevated culture temperature and DAP supplementation. Taken together, dptP gene contributes to Streptomcyes primary growth under elevated temperature and DAP treatment, whereas it plays negative roles on metabolism of secondary metabolites and transcription of DAP biosynthetic genes.

Evaluation of electrode-sample contact impedance under different curing humidity conditions during measurement of AC impedance of cement-based materials.

In this study, a simple method was proposed to calculate electrode-sample contact impedance in the cases of two-point and four-point measurements. The results indicated that when using the saturated calcium hydroxide solution (SCH) as conductive medium, the contact impedance in the four-point measurement is negligible for the impedance range of cement-based materials. The SCH can be used as a reference for correction of the contact impedance. A reasonable combination of curing humidity and different conductive media is recommended for the two-point measurement, which is suitable for testing the ACIS of cement-based materials. In a case of contact impedance not being precisely known, it is highly recommended that a four-point measurement with two different ratios of the length of the sample and the center spacing of the voltage electrodes (L/a) should be conducted to evaluate the effect of the contact impedance following the procedure proposed in this study.

Measurement of wind field data in Southeast China.

The data presented in this article are the wind measurements acquired from a tower in Southeast China during typhoon Nesat (1709#) and typhoon Haitang (1710#). Three 3D ultrasonic anemometers Wind Master Pro were utilized to obtain 3D wind data. The anemometer works well with wind speed range of 0-65 m/s and wind angle range of 0-360°. Three direction wind speeds and wind angles were recorded per every 0.1 s. The present research analyzed wind characteristics based on recorded data. In this article, the detailed test set-up and data pre-processing methodology for the wind characteristics analysis are provided.

Expression levels of Notch1 and Delta-like 4 in peripheral lymphocytes and their relationship with T helper 17 (Th17) cells in renal transplant recipients.

OBJECTIVE: This study aimed to investigate the role of the Notch1/Delta-like 4 signaling pathway and its relationship with T helper 17 (Th17) cells in the peripheral transplantation immune of renal transplant recipients. METHODS: Fifty-two kidney transplant recipients in our hospital were selected and divided into the acute rejection group (AR), renal tubular necrosis (ATN) group, and stable renal function group, according to their postoperative recovery. Flow cytometry was used to detect the expression of Notch1 and Delta-like 4 in peripheral lymphocytes and the presence of Th17 cells in the kidney of transplant recipients. RESULTS: The expression levels of Notch1 and Delta-like 4 and level of Th17 cells among the three groups before surgery and at postoperative day 1 showed no significant differences (P>0.05). At 3, 7, and 14d after surgery, these three factors in the AR group were significantly higher than in the stable renal function group (P<0.01) and ATN group (P<0.01), where the levels in the latter two groups were similar. Upon the occurrence of acute rejection, the Notch1 and Delta-like 4 expression and Th17 cell ratio were significantly increased (P<0.01) but gradually decreased after anti-rejection therapy. Notch1 and Delta-like 4 were significantly positively correlated with Th17 cells (r=0.893, P<0.01 and r=0.893, P<0.01, respectively). CONCLUSION: The detection of Notch1 and Delta-like 4 expression in peripheral blood lymphocytes of renal transplant recipients can serve as a positive indicator for evaluating the diagnosis and treatment efficacy of the AR reaction.

Prognostic factors for renal allograft survival in patients with immunoglobulin A nephropathy: a case control study.

The renal allograft survival rates of patients with immunoglobulin A nephropathy (IgAN), and patients with or without other glomerular diseases, have yet to be fully elucidated. In this study, the clinicopathological factors associated with long-term allograft survival for the prognosis of renal allograft recipients with IgAN were examined. All patients enrolled in this study were diagnosed with IgAN following clinical and pathological examinations. Patients underwent renal graft biopsy and were hospitalized at the Fuzhou General Hospital between June, 2004 and December, 2010. Common demographic and clinical indicators were recorded in patients who had graft loss and in those who had functional renal grafts. Forty-two of the 202 biopsy specimens (20.8%) met the diagnostic criteria for IgAN and were divided into two groups, the graft loss group (n=17) and the functional graft group (n=25). Patients were followed up for 1-257 months after kidney transplantation. The mean patient age was 40.6 ± 9.3 years at the time of renal graft biopsy. Examination results indicated concomitant proteinuria and hematuria in 25 patients (59.5%) and proteinuria alone in six patients (14.3%). Graft loss occurred in 17 patients during the follow-up period. Comparison of the graft loss and the functional graft groups indicated that patients in the graft loss group were more likely to have proteinuria (P=0.047), high creatinine levels at the time of biopsy (P=0.009), low glomerular filtration rates (P=0.013), low serum total protein (P=0.01), a high Banff score (P=0.001), extensive glomerulosclerosis (P=0.002), a greater likelihood of crescent formation (P=0.01), severe tubular atrophy (P=0.013) and more extensive interstitial fibrosis (P=0.033). However, the two groups showed no significant differences in blood pressure, hematuria, BUN, UA, Hb, TG and CHO levels. The allograft survival rate of patients with IgAN was identified to be similar to that of patients with and without other glomerular diseases.