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Inducing adventitious root (AR) formation in mature walnut species (Juglans L.) is challenging. However, the AR formation of mature trees can be improved by rejuvenation. In rejuvenated cuttings, exogenous indole-3-butyric acid (IBA) is essential for AR formation, and the underlying mechanism is still not well understood. Therefore, we utilized transcriptome sequencing to investigate the mechanism of IBA-induced AR formation. Our results revealed that, in comparison to the control group, IBA treatment (9 mmol · L-1) significantly increased the endogenous indole-3-acetic acid (IAA) content, leading to an enhanced rooting rate. We performed RNA sequencing to identify differentially expressed genes (DEGs) between the IBA-treated and control (CK) groups at 1, 2, 3, and 5 days after cutting (DAC). The results showed that, compared to the control cuttings, there were 1 539, 889, 785, and 984 up-regulated genes and 2 791, 2 936, 3 017, and 1 752 down-regulated genes, at 1, 2, 3, and 5 DAC, respectively. Analysis of RNA-seq data revealed that G-type ATP-binding cassette 36/37 (ABCG36/37) and ATP-binding cassette subfamily D 1 (ABCD1), associated with IBA transport, were down-regulated in the rejuvenation cuttings. In contrast, PIN-FORMED (PIN) and PINOID (PID), associated with auxin efflux, were up-regulated. We identified 49 auxin/indole-3-acetic acid (AUX/IAA)-encoding genes, including IAA1, IAA3, IAA5, IAA6, IAA8, IAA11, IAA12, IAA19, and IAA20, which were up-regulated at 1–5 DAC in the rejuvenated cuttings. This study highlights that the overexpression of JrWOX5/11 in poplar significantly enhance AR growth, as evidenced by increased root length, surface area, volume, and quantity. Moreover, the co-expression network analysis involving JrWOX11 and JrWOX5 in walnut cuttings elucidates complex genetic interactions, underscoring their pivotal role in the formation of AR. Our data supported the following molecular mechanism of IBA-induced adventitious root formation. Firstly, IBA is converted to free IAA in peroxisomes. Then, the highly concentrated IAA in the procambium and parenchyma cells induces WUSCHEL-related homeobox 11 (WOX11) expression at two days. Finally, WOX11 acts redundantly to up-regulate WOX5, initiating the development of root primordia cells.

JQ1 and GSK2801 are bromo domain inhibitors (BDI) known to exhibit enhanced anti-cancer activity when combined with other agents. However, the underlying molecular mechanisms behind such enhanced activity remain unclear. We used network-pharmacology approaches to understand the shared molecular mechanisms behind the enhanced activity of JQ1 and GSK2801 when used together to treat breast cancer (BC). The gene targets of JQ1 and GSK2801 were intersected with known BC-targets and their putative targets against BC were derived. The key genes were explored through gene-ontology-enrichment, Protein-Protein-Interaction (PPI) networking, survival analysis, and molecular modeling simulations. The genes, CTSB, MAPK14, MET, PSEN2 and STAT3, were found to be common targets for both drugs. In total, 49 biological processes, five molecular functions and 61 metabolic pathways were similarly enriched for JQ1 and GSK2801 BC targets among which several terms are related to cancer: IL-17, TNF and JAK-STAT signaling pathways. Survival analyses revealed that all five putative synergistic targets are significantly associated with survival in BC (log-rank p

The phenolic extracts of walnut cultivars with a red pellicle (HWE), a purple pellicle (ZWE) and a yellow pellicle (WWE) were compared. The total contents of phenolics, flavonoids and condensed tannins, as well as in vitro antioxidant activities (ABTS and DPPH) of the phenolic extracts were evaluated. The results showed that 25 phenolic compounds were identified in three colored walnuts. The HWE contained the highest contents of total phenolics and condensed tannins, while the ZWE contained the highest content of flavonoids. The DPPH and ABTS assays indicated that the phenolic extracts of colored-walnut pellicles showed a higher free radical scavenging ability than butylated hydroxytoluene (BHT) at the same concentration. Phenolic extracts of colored walnuts were also evaluated as natural antioxidants in walnut oil, substituting for BHT. The efficacy of phenolic extracts in walnut oil preservation was evaluated using accelerated storage (60 °C) for 12 days. After accelerated storage, walnut oil enriched with the phenolic extracts kept better qualities, with significantly lower peroxides, p-anisidine, and TOTOX values. The volatiles were measured and identified using the headspace solid phase micro-extraction gas chromatography to further assess oil quality. Aldehydes such as hexanal and 2-heptenal were identified as representative oxidative volatiles. Hexanal and 2-heptenal content indicated that adding phenolic extracts could prevent flavor deterioration of the oil. This study suggested that phenolic extracts of walnut pellicles could offer an effective alternative to synthetic antioxidants for walnut oil preservation.

Abstract Background Triple-negative breast cancer (TNBC) constitutes 10–20% of breast cancers and is challenging to treat due to a lack of effective targeted therapies. Previous studies in TNBC cell lines showed in vitro growth inhibition when JQ1 or GSK2801 were administered alone, and enhanced activity when co-administered. Given their respective mechanisms of actions, we hypothesized the combinatorial effect could be due to the target genes affected. Hence the target genes were characterized for their expression in the TNBC cell lines to prove the combinatorial effect of JQ1 and GSK2801. Methods RNASeq data sets of TNBC cell lines (MDA-MB-231, HCC-1806 and SUM-159) were analyzed to identify the differentially expressed genes in single and combined treatments. The topmost downregulated genes were characterized for their downregulated expression in the TNBC cell lines treated with JQ1 and GSK2801 under different dose concentrations and combinations. The optimal lethal doses were determined by cytotoxicity assays. The inhibitory activity of the drugs was further characterized by molecular modelling studies. Results Global expression profiling of TNBC cell lines using RNASeq revealed different expression patterns when JQ1 and GSK2801 were co-administered. Functional enrichment analyses identified several metabolic pathways (i.e., systemic lupus erythematosus, PI3K-Akt, TNF, JAK-STAT, IL-17, MAPK, Rap1 and signaling pathways) enriched with upregulated and downregulated genes when combined JQ1 and GSK2801 treatment was administered. RNASeq identified downregulation of PTPRC, MUC19, RNA5-8S5, KCNB1, RMRP, KISS1 and TAGLN (validated by RT-qPCR) and upregulation of GPR146, SCARA5, HIST2H4A, CDRT4, AQP3, MSH5-SAPCD1, SENP3-EIF4A1, CTAGE4 and RNASEK-C17orf49 when cells received both drugs. In addition to differential gene regulation, molecular modelling predicted binding of JQ1 and GSK2801 with PTPRC, MUC19, KCNB1, TAGLN and KISS1 proteins, adding another mechanism by which JQ1 and GSK2801 could elicit changes in metabolism and proliferation. Conclusion JQ1-GSK2801 synergistically inhibits proliferation and results in selective gene regulation. Besides suggesting that combinatorial use could be useful therapeutics for the treatment of TNBC, the findings provide a glimpse into potential mechanisms of action for this combination therapy approach.

Lycium barbarum flavone is the main effective component of Chinese medicinal material Lycium barbarum, and it is also the main evaluation index of its quality. This paper investigated the effects of ethanol reflux extraction, high-speed shear-assisted ethanol extraction, high-speed shear-assisted DESs extraction, and ultrasound-assisted DESs extraction on the extraction amount of Lycium barbarum flavonoids. The best extraction method was selected, and the best extraction conditions were obtained by single factor experiments and orthogonal experiment. Based on the best extraction method, SIMCA 14.1 was used. The software analyzed the correlation between the ecological factors in different producing areas and the content of Lycium barbarum flavonoids. The results showed that the extraction efficiency of Lycium barbarum flavonoids with high-speed shearing assisted DESs was the best, the optimal extraction process was as follows: Speed 14000 r/min, material-to-liquid ratio 1:20 g/mL, extraction time 5 min, the average flavonoid extraction volume was 7.11 mg/g. The flavonoid content of Lycium barbarum from different origins in descending order was: Ningxia Yinchuan>Ningxia Zhongwei>Gansu Guazhou>Ningxia Guyuan>Xinjiang Jinghe>Ningxia Zhongning>Gansu Baiyin>Gansu Wuwei>Gansu Yumen>Xinjiang Altai>Xinjiang Yining>Qinghai Dulan>Qinghai Golmud>South Korea b>South Korea a. SIMCA 14.1 Software analysis showed that the positive regression coefficient value of Lycium barbarum flavonoids content and temperature and frost-free period was the largest, and the absolute value of negative regression coefficient with altitude was the largest. The most important ecological factors affecting the content of Lycium barbarum flavonoids were temperature and frost-free period.

Abstract Background Common walnut (Juglans regia L.) is one of the top four most consumed nuts in the world due to its health benefits and pleasant taste. Despite its economic importance, the evolutionary history and genetic control of its adaptation and agronomic traits remain largely unexplored. Results We report a comprehensive walnut genomic variation map based on whole-genome resequencing of 815 walnut accessions. Evolutionary analyses suggest that Chinese J. regia diverged from J. sigillata with extensive hybridizations after the split of the two species. In contrast to annual crops, the genetic diversity and heterozygous deleterious mutations of Chinese common walnut trees have continued to increase during the improvement process. Selective sweep analyses identify 902 genes uniquely selected in the improved common walnut compared to its progenitor population. Five major-effect loci are identified to be involved in walnut adaptations to temperature, precipitation, and altitude. Genome-wide association studies reveal 27 genomic loci responsible for 18 important agronomic traits, among which JrFAD2 and JrANR are the potentially major-effect causative genes controlling linoleic acid content and color of the endopleura of the nut, respectively. Conclusions The largest genomic resource for walnuts to date has been generated and explored in this study, unveiling their evolutionary history and cracking the genetic code for agronomic traits and environmental adaptation of this economically crucial crop tree.

Objective: Ewing Sarcoma Family of Tumors (ESFT) are a highly aggressive pediatric bone and soft tissue malignancy with poor outcomes in the refractory and recurrent setting. Over 90% of Ewing Sarcoma (ES) tumors are driven by the pathognomonic EWS-ETS chimeric transcripts and their corresponding oncoproteins. It has been suggested that the EWS-ETS oncogenic action can mediate microRNA (miRNA) processing. Importantly, small extracellular vesicles (sEVs), including those frequently referred to as exosomes have been shown to be highly enriched with tumor-derived small RNAs such as miRNAs. We hypothesized that ESFT-specific sEVs are enriched with certain miRNAs which could be utilized toward an exo-miRNA biomarker signature specific to this disease. Methods: We performed miRNAseq to compare both the exo-derived and cell-derived miRNA content from 8 ESFT, 2 osteosarcoma, 2 non-cancerous cell lines, and pediatric plasma samples. Results: We found that sEVs derived from ESFT cells contained nearly 2-fold more number of unique individual miRNAs as compared to non-ESFT samples. Quantitative analysis of the differential enrichment of sEV miRNAs resulted in the identification of 62 sEV-miRNAs (exo-miRNAs) with significant ( P < .05) enrichment variation between ESFT and non-ESFT sEV samples. To determine if we could utilize this miRNA signature to diagnose ESFT patients via a liquid biopsy, we analyzed the RNA content of total circulating sEVs isolated from 500 µL plasma from 5 pediatric ESFT patients, 2 pediatric osteosarcoma patients, 2 pediatric rhabdomyosarcoma patients, and 4 non-cancer pediatric controls. Pearson’s clustering of 60 of the 62 candidate exo-miRNAs correctly identified 80% (4 of 5) of pathology confirmed ESFT patients. Importantly, RNAseq analysis of tumor tissue from the 1 outlier, revealed a previously uncharacterized EWS-FLI1 translocation. Conclusions: Taken together, these findings support the development and validation of an exo-miRNA-based liquid biopsy to aid in the diagnosis and monitoring of ESFT.

The well-developed root system enables plant survival under various environmental stresses. WUSCHEL-RELATED HOMEOBOX GENE 11 (WOX11) plays a critical role in adventitious root formation and development in rice, Arabidopsis, and easy-to-root tree poplar. However, in difficult-to-root trees, the knowledge of WOX11 during adventitious root formation and development remains scarce. In this study, the JrWOX11 gene was isolated from a difficult-to-root tree walnut and heterologously expressed in the “84K” poplar. The results showed that JrWOX11 contained a similar structure and sequence to the homologous genes in rice, Arabidopsis, and poplar, but had different numbers and types of motifs and cis-elements. JrWOX11 lacked the motif GGAIQY compared to that in easy-to-root trees. In addition, JrWOX11 expression was induced by ABA, PEG, and NaCl treatments. Overexpression of JrWOX11 in poplar promoted root initiation and significantly increased adventitious root (ARs) number, lateral roots (LRs) number, and root hair (RH) length. Furthermore, the aboveground biomass was notably increased under NaCl and PEG treatments in transgenic plants. When NaCl and PEG were removed, the survival rate, aerial shoot development, and de novo root organogenesis were also markedly enhanced in transgenic shoot cuttings. The study provides valuable information on the differences between JrWOX11 and the homologous genes in rice, Arabidopsis, and poplar, and supports the critical role of JrWOX11 in the formation of AR and tolerance to salt and osmotic stresses.

Background: It is important to identify when two exposures impact a molecular marker (e.g., a gene’s expression) in similar ways, for example, to learn that a new drug has a similar effect to an existing drug. Currently, statistically robust approaches for making comparisons of equivalence of effect sizes obtained from two independently run treatment vs. control comparisons have not been developed.Results: Here, we propose two approaches for evaluating the question of equivalence between effect sizes of two independent studies: a bootstrap test of the Equivalent Change Index (ECI), which we previously developed, and performing Two One-Sided t-Tests (TOST) on the difference in log-fold changes directly. The ECI of a gene is computed by taking the ratio of the effect size estimates obtained from the two different studies, weighted by the maximum of the two p-values and giving it a sign indicating if the effects are in the same or opposite directions, whereas TOST is a test of whether the difference in log-fold changes lies outside a region of equivalence. We used a series of simulation studies to compare the two tests on the basis of sensitivity, specificity, balanced accuracy, and F1-score. We found that TOST is not efficient for identifying equivalently changed gene expression values (F1-score = 0) because it is too conservative, while the ECI bootstrap test shows good performance (F1-score = 0.95). Furthermore, applying the ECI bootstrap test and TOST to publicly available microarray expression data from pancreatic cancer showed that, while TOST was not able to identify any equivalently or inversely changed genes, the ECI bootstrap test identified genes associated with pancreatic cancer. Additionally, when investigating publicly available RNAseq data of smoking vs. vaping, no equivalently changed genes were identified by TOST, but ECI bootstrap test identified genes associated with smoking.Conclusion: A bootstrap test of the ECI is a promising new statistical approach for determining if two diverse studies show similarity in the differential expression of genes and can help to identify genes which are similarly influenced by a specific treatment or exposure. The R package for the ECI bootstrap test is available at https://github.com/Hecate08/ECIbootstrap.

MicroRNAs are non-coding RNAs that regulate gene expression post-transcriptionally. In the placenta, the master regulator of foetal growth and development, microRNAs shape the basic processes of trophoblast biology and specific microRNA have been associated with foetal growth. To comprehensively assess the role of microRNAs in placental function and foetal development, we have performed small RNA sequencing to profile placental microRNAs from two independent mother-infant cohorts: the Rhode Island Child Health Study (n = 225) and the New Hampshire Birth Cohort Study (n = 317). We modelled microRNA counts on infant birthweight percentile (BWP) in each cohort, while accounting for race, sex, parity, and technical factors, using negative binomial generalized linear models. We identified microRNAs that were differentially expressed (DEmiRs) with BWP at false discovery rate (FDR) less than 0.05 in both cohorts. hsa-miR-532-5p (miR-532) was positively associated with BWP in both cohorts. By integrating parallel whole transcriptome and small RNA sequencing in the RICHS cohort, we identified putative targets of miR-532. These targets are enriched for pathways involved in adipogenesis, adipocytokine signalling, energy metabolism, and hypoxia response, and included Leptin, which we further demonstrated to have a decreasing expression with increasing BWP, particularly in male infants. Overall, we have shown a robust and reproducible association of miR-532 with BWP, which could influence BWP through regulation of adipocytokines Leptin and Adiponectin.

Reduced imine cage (RCC3) was covalently bonded to the surface of silica spheres, and then the secondary amine group of the molecular cage was embedded in non-polar C10 for modification to prepare a novel RCC3-C10@silica HPLC stationary phase with multiple separation functions. Through infrared spectroscopy, thermogravimetric analysis and nitrogen adsorption–desorption characterization, it was confirmed that RCC3-C10 was successfully bonded to the surface of silica spheres. The resolution of RCC3-C10@silica in reversed-phase separation mode is as high as 2.95, 3.73, 3.27 and 4.09 for p-phenethyl alcohol, 1-phenyl-2-propanol, p-methylphenethyl alcohol and 1-phenyl-1-propanol, indicating that the stationary phase has excellent chiral resolution performance. In reversed-phase and hydrophilic separation modes, RCC3-C10@silica realized the separation and analysis of a total of 70 compounds in 8 classes of Tanaka mixtures, alkylbenzene rings, polyphenyl rings, phenols, anilines, sulfonamides, nucleosides and flavonoids, and the analysis of a variety of chiral and achiral complex mixtures have been completed at the same time. Compared with the traditional C18 commercial column, RCC3-C10@silica exhibits better chromatographic separation selectivity, aromatic selectivity and polar selectivity. The multifunctional separation mechanism exhibited by the stationary phase originates from various synergistic effects such as hydrophobic interaction, π-π interaction, hydrogen bonding and steric interaction provided by RCC3 and C10 groups. This work provides flexible selectivity and application prospects for novel multi-separation functional chromatographic columns.

Juglans regia L. and J. sigillata D. have a long cultivation history in China. With the purpose of detecting interspecific and intraspecific genetic variation, 35 clonal cultivars of J. regia, J. sigillata, J. sigillata×J. regia and J. hopeiensis Hu. (J. mandshurica Maxim. ×J. regia) were selected from a broad geographic range and applied in amplified-fragment length polymorphism (AFLP) analysis. Clear-cut AFLP profiles were produced using 9 EcoR I/Mse I primer combinations. As for J. regia, J. sigillata and their hybrids, the percentages of polymorphic bands (PPBs) averaged 93.86%, 93.94% and 60.46%; the observed number of alleles (Na) averaged 1.5370, 1.6389 and 1.2593; the effective number of alleles (Ne) averaged 1.2582, 1.2543 and 1.1646; Nei's gene diversity (H) scored 0.1560, 0.1556 and 0.0951; Shannon’s information index (I) averaged 0.2402, 0.2459 and 0.1414, respectively. These results implied a moderate level of genetic diversity within the investigated species. The total gene diversity (Ht = 0.1596) can be distributed into intra- (Hs = 0.1356) and inter- (Dst = 0.0240) species gene diversity. The proportion of the distributed genetic diversity among populations (Gst) was 0.1505, indicating that only 15% of the total gene diversity was attributed to differences between species. The factorial correspondence analysis (FCA) and unweighted pair-group method of arithmetic averages (UPGMA) analysis revealed a close genetic distance between these two species.

Walnut is one of the world’s four largest nuts. Currently, the bottleneck in walnut breeding is the production of resistant variants. Soil salinization is a global problem, and the use of salt-tolerant rootstocks is a basic strategy to overcome the challenge of sustained walnut production. Providing a scientific basis for the selection of walnut salt-tolerant rootstocks is possible by studying the physiological and biochemical response characteristics and salt tolerance variations of different walnut genotypes under salt stress. In the present study, seedlings of four genotypes of walnut rootstocks, including J1 (Juglans hindsii), J2 (J. mandshurica), J3 (J. regia × J. mandshurica), and J4 (J. regia × J. hindsii), were employed as test materials to conduct a 28-day pot experiment under NaCl stress with five NaCl concentrations (0, 50, 100, 200, and 300 mmol/L). Under different NaCl treatment concentrations, seedling morphology, growth indices, chlorophyll content, photosynthetic parameters, relative electrical conductivity (REC), malondialdehyde (MDA), proline (Pro), soluble sugar (SS), and the activity of superoxide dismutase (SOD) and peroxidase (POD) in the leaves were examined. Salt stress altered the morphological characteristics and growth indices of seedlings from four genotypes to varying degrees. In addition, according to the analysis of physiological and biochemical data, salt stress had a considerable impact on both the physiological and biochemical processes of seedlings. Salt stress decreased the chlorophyll content and photosynthetic parameters of four genotypes, the REC, MDA content, Pro content, and SS content of each genotype increased by different degrees, and the enzymatic activities showed different trends. The salt tolerance of rootstocks was evaluated thoroughly using principal component analysis and membership function analysis based on the 16 parameters. The results of a comprehensive evaluation of salt tolerance showed that the order of salt tolerance of the four genotypes was J4 > J1 > J3 > J2, which corresponded to the order of the morphological symptoms of salt injury. In conclusion, J4 has strong salt tolerance and is an important germplasm resource for walnut salt-tolerant rootstock breeding.

This study investigated the efficiency and safety of hypofractionated radiotherapy (HFR) combined with paclitaxel chemotherapy for the treatment of postsurgery tracheoesophageal groove lymph node (TGLN) metastasis in patients with esophageal cancer (EC). Fifty-three EC patients with TGLN metastasis after surgery admitted to the Yancheng Third People’s Hospital from January 2013 to June 2015 were included in this study. They were randomly divided into the HFR group (n = 25) and conventional fractioned radiotherapy (CFR) group (n = 28) based on the random grouping method. Patients in the HFR group received treatment with radiation of 60 Gy (5 fractions per week, total 20 fractions) combined with paclitaxel chemotherapy at a dose of 50 mg once per week for 4 weeks. Patients in the CFR group received radiation of 60 Gy (5 fractions per week, total 30 fractions) combined with paclitaxel chemotherapy at a dose of 50 mg once per week for 6 weeks. The adverse events and treatment outcomes in these two groups were analyzed. It was found that there was no significant difference in the incidence of radiation esophagitis in the HFR group compared with that of the CFR group (grades 3–4, 44.0 vs. 25.0%; P = 0.149). There was no statistical difference in the incidence of radiation pneumonitis between these two groups (grades 3–4, 16.0 vs. 7.1%; P = 0.314). No statistical difference was noticed in complete response (CR), partial response (PR), and no response (NR) between these two groups. The median overall survival (OS) in the HRF group was significantly longer compared with that of the CRF group (24.2 months (95% CI, 16.2–32.1 months) vs. 11.8 months (95% CI, 9.2–14.4 months); P = 0.024). Our results indicated that the combination of HFR and chemotherapy improved the prognosis of EC patients with TGLN metastasis with no increased adverse events.

Abstract Background Observations of precocious (early bearing) genotypes of walnut (Juglans regia L.) under natural conditions encouraged us to study the origin and genetic control of these fascinating traits. Results In this study, the self-fertility, progeny performance, and simple sequence repeat (SSR) locus variation of iron walnut (Juglans sigillata Dode), an ecotype of J. regia, were investigated. The average self-pollinated fruit set rate of J. sigillata cv. ‘Dapao’ (DP) was 7.0% annually from 1979 to 1982. The average germination rate of self-pollinated seeds was 45.2% during the 4-year period. Most progeny had inbreeding depression. Nine representative self-pollinated progeny (SP1–SP9), with special or typical traits of DP, were selected. SP1–SP4 were precocious because they initiated flowers as early as 2 years after germination, compared to the 7–10-yr period that is typical of DP. SP9 had not flowered since 1980. Twelve SSR markers were used to analyze the SP and DP. The genome of SP had a tendency toward high levels of homozygosity. The high levels of homozygosity reported in 18 additional precocious walnut genotypes complemented the results of this study. Conclusions These results provide evidence of precocious phenotypes and genomes with high levels of homozygosity that might be generated from self-pollinating walnut. This suggests that self-pollination might facilitate the generation of unique homozygous parents for subsequent use in walnut-breeding programs. The results also indicate that more attention should be focused on adequate management of precocious walnut to avoid early depression in the production of nuts.

Assessing DNA methylation profiles in human blood has become a major focus of epidemiologic inquiry. Understanding variability in CpG-specific DNA methylation over moderate periods of time is a critical first step in identifying CpG sites that are candidates for DNA methylation-based etiologic, diagnostic and prognostic predictors of pathogenesis. Using the Illumina MethylationEPIC [850K] BeadArray, DNA methylation was profiled in paired whole blood samples collected approximately 1 year apart from 35 healthy women enrolled in the Nurses Study II cohort. The median intraclass correlation coefficient (ICC) across all CpG loci was 0.19 [Interquartile Range (IQR) 0.00–0.50]; 74.8% of ICCs were in the low range (0–0.5), 16.9% in the mid-range of ICCs (0.5–0.8), and 8.3% in the high-range of ICCs (0.8–1). ICCs were similar for CpG probes on the 450K Illumina array (median 0.17) and the new probes added to the 850K array (median 0.21). ICCs for CpG loci on the sex chromosomes and known metastable epialleles were high (median 0.71, 0.97, respectively), and ICCs among methylation quantitative trait loci (mQTL) CpGs were significantly higher as compared to non-mQTL CpGs (median 0.73, 0.16, respectively, P

Abstract Background Juglans is a difficult-to-root tree. In the present study, we successfully rejuvenated stock plants by grafting and then burying them horizontally. Results Rooting rates of rejuvenated shoots were 98.1% 20 days after cutting. We recorded spatial and temporal variation in endogenous indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellin A3 (GA3) and zeatin-riboside (ZR) under root induction. The four types of endohormones were mainly confined to the phloem sieve and companion cells (S&Cs) at the base of either rejuvenated or mature soft shoots. IAA and ABA levels were higher in rejuvenated shoots than in mature shoots, whereas the opposite was true for GA3 and ZR. During rooting induction, GA3 was the first hormone to be observed outside phloem S&Cs, followed by IAA, ABA and ZR. In rejuvenating soft shoots, IAA accumulated in the cross-sectional areas of the cambium and phloem, where root primordia were evident. Conclusions The improvement in the rooting ability that was evident after rejuvenation most likely results a transformation of the plant to a juvenile form, from elevated IAA levels in phloem S&Cs and from a promotion of all four endohormones outside phloem S&Cs, in particular, from an accumulation of IAA in the cross-sectional areas of the cambium and phloem.

Paradox is one of the most important rootstock resources in USA and China walnut industry. In this study, we characterized the complete chloroplast genome of Paradox (Juglans major × J. regia) based on next generation sequencing. The circular complete chloroplast genome was 160,324 bp in length, containing a large single-copy (LSC) region of 89,852 bp, and a small single-copy (SSC) region of 18,410 bp. These two regions were separated by a pair of inverted repeat regions (IRa and IRb) of 26,031 bp each. A total of 131 functional genes were encoded, consisted of 87 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. The overall GC content of the chloroplast genome was 36.1% and the GC contents of the LSC, SSC, and IR regions were 33.7, 29.9, and 42.6%, respectively. The phylogenetic analysis by Neighbor-Joining showed that Paradox was relatively closely related to J. major compared to other species of Juglans genus. This complete chloroplast genome will provide valuable insight into evolution, molecular breeding, and phylogenetic analysis of Juglans species.

Juglans cathayensis var. formosana (Hayata) is a precious Juglans tree endemic to China. In this study, we reported its complete chloroplast genome assembled from Illumina pair-end sequencing data. The completed plastome was 159,967 bp in length, including a large-single copy (LSC) region of 89,320 bp, a small-single copy (SSC) region of 18,175 bp, and a pair of inverted repeat (IR) regions of 26,236 bp. The overall GC content of the genome was 36.12%, and the corresponding values of the LSC, SSC, and IR regions were 33.71, 29.79, and 42.43%, respectively. A total of 131 genes were annotated, including 87 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. Phylogenetic analysis strongly supported that J. cathayensis var. formosana and J. cathayensis formed a sister clade to J. mandshurica and J. hopeiensis. The newly characterized complete chloroplast genome of J. cathayensis var. formosana will provide essential data for further investigation of this precious species.

Walnuts (Juglans, Juglandaceae) are known throughout the world as economically important trees that provide fat, protein, vitamins, and minerals as a food source, and produce high-quality timber. We have amended the purpose section to say “However,” the omics resources are limited, which hampered the elucidation of molecular mechanisms resulting in their economically important traits (such as yield, fertility alternation, oil synthesis, and wood formation). To enrich the omics database of walnut, there is great need for analyses of its genomic and transcriptomic characteristics. In this study, we reported for the first time of the transcriptome landscape of six important organs or tissues in walnut interspecies hybrid using next-generation sequencing technology. Over 338 million clean reads were obtained. This yielded 74,072 unigenes with an average length of 782.71 bp. To develop an understanding of gene functions and regulatory pathways, 66,355 of the unigenes were identified as homologs of annotated genes and classified into three general categories with 61 functional subcategories. 2,288 out of 2,549 unmapped unigenes had at least one BLAST hit against the public databases. A total of 1,237 transcription factor-encoding genes (TFs) and 2,297 tissue-specific unigenes were identified. Interestingly, in the new shoot between an adult seedling and a grafted tree, the expression of 9,494 unigenes were significantly different, among which 4,388 were up-regulated and 5,106 were down-regulated. Of these, 195, 177, 232, 75, 114, and 68 unigenes were related to transcription factors, cell wall, defense response, transport, plant hormone biosynthesis, and other cambial activity-related functions, respectively. The obtained sequences and putative functional data constitute a resource for future functional analyses in walnut and other woody plants. These findings will be useful in further studies addressing the molecular mechanisms underlying grafting-related cambial activity.

ABSTRACT Elizabethkingia anophelis is an emerging global multidrug-resistant opportunistic pathogen. We assessed the diversity among 13 complete genomes and 23 draft genomes of E. anophelis strains derived from various environmental settings and human infections from different geographic regions around the world from 1950s to the present. Putative integrative and conjugative elements (ICEs) were identified in 31/36 (86.1%) strains in the study. A total of 52 putative ICEs (including eight degenerated elements lacking integrases) were identified and categorized into three types based on the architecture of the conjugation module and the phylogeny of the relaxase, coupling protein, TraG, and TraJ protein sequences. The type II and III ICEs were found to integrate adjacent to tRNA genes, while type I ICEs integrate into intergenic regions or into a gene. The ICEs carry various cargo genes, including transcription regulator genes and genes conferring antibiotic resistance. The adaptive immune CRISPR-Cas system was found in nine strains, including five strains in which CRISPR-Cas machinery and ICEs coexist at different locations on the same chromosome. One ICE-derived spacer was present in the CRISPR locus in one strain. ICE distribution in the strains showed no geographic or temporal patterns. The ICEs in E. anophelis differ in architecture and sequence from CTnDOT, a well-studied ICE prevalent in Bacteroides spp. The categorization of ICEs will facilitate further investigations of the impact of ICE on virulence, genome epidemiology, and adaptive genomics of E. anophelis. IMPORTANCE Elizabethkingia anophelis is an opportunistic human pathogen, and the genetic diversity between strains from around the world becomes apparent as more genomes are sequenced. Genome comparison identified three types of putative ICEs in 31 of 36 strains. The diversity of ICEs suggests that they had different origins. One of the ICEs was discovered previously from a large E. anophelis outbreak in Wisconsin in the United States; this ICE has integrated into the mutY gene of the outbreak strain, creating a mutator phenotype. Similar to ICEs found in many bacterial species, ICEs in E. anophelis carry various cargo genes that enable recipients to resist antibiotics and adapt to various ecological niches. The adaptive immune CRISPR-Cas system is present in nine of 36 strains. An ICE-derived spacer was found in the CRISPR locus in a strain that has no ICE, suggesting a past encounter and effective defense against ICE.

Traffic flow modeling plays an important role in routing，MAC algorithm and protocol designs in vehicular Ad Hoc networks （VANET）. An accurate traffic flow model is crucial to traffic management of an intelligent transportation system （ITS）and information safety in a VANET. Based on Shanghai’s traffic flow data，the performance of the two different models was compared using auto regressive（AR）model and back-propagation（BP） network model，and the corresponding prediction result was given. Research finds that both of the two models can efficiently predict the traffic data，but they have different prediction accuracy for the data of different periods. The research result will provide support for future research on ITS and VANET.

Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is one of the pressing contemporary public health challenges. Investigations into the genomic structure of SARS-CoV-2 may inform ongoing vaccine development efforts and/or provide insights into vaccine efficacy to fight against COVID-19. Evolutionary analysis of 540 genomes spanning 20 different countries/territories was conducted and revealed an increase in the genomic divergence across successive generations. The ancestor of the phylogeny was found to be the isolate from the 2019/2020 Wuhan outbreak. Its transmission was outlined across 20 countries/territories as per genomic similarity. Our results demonstrate faster evolving variations in the genomic structure of SARS-CoV-2 when compared to the isolates from early stages of the pandemic. Genomic alterations were predominantly located and mapped onto the reported vaccine candidates of structural genes, which are the main targets for vaccine candidates. S protein showed 34, N protein 25, E protein 2, and M protein 3 amino acid variations in 246 genomes among 540. Among identified mutations, 23 in S protein, 1 in E, 2 from M, and 7 from N protein were mapped with the reported vaccine candidates explaining the possible implications on universal vaccines. Hence, potential target regions for vaccines would be ideally chosen from the structural regions of the genome that lack high variation. The increasing variations in the genome of SARS-CoV-2 together with our observations in structural genes have important implications for the efficacy of a successful universal vaccine against SARS-CoV-2.

Although the effects of fertilization and microbiota on plant growth have been widely studied, our understanding of the chemical fertilizers to alter soil chemical and microbiological properties in woody plants is still limited. The aim of the present study is to investigate the impact of long-term application of chemical fertilizers on chemical and microbiological properties of root-associated soils of walnut trees. The results show that soil organic matter (OM), pHkcl, total nitrogen (TN), nitrate-nitrogen (NO3−), and total phosphorus (TP) contents were significantly higher in non-fertilized soil than after chemical fertilization. The long-term fertilization led to excessive ammonium-nitrogen (NH4+) and available phosphorus (AP) residues in the cultivated soil, among which NH4+ resulted in soil acidification and changes in bacterial community structure, while AP reduced fungal diversity. The naturally grown walnut trees led to an enrichment in beneficial bacteria such as Burkholderia, Nitrospira, Pseudomonas, and Candidatus_Solibacter, as well as fungi, including Trichoderma, Lophiostoma, Phomopsis, Ilyonectria, Purpureocillium, Cylindrocladiella, Hyalorbilia, Chaetomium, and Trichoglossum. The presence of these bacterial and fungal genera that have been associated with nutrient mobilization and plant growth was likely related to the higher soil OM, TN, NO3−, and TP contents in the non-fertilized plots. These findings highlight that reduced chemical fertilizers and organic cultivation with beneficial microbiota could be used to improve economic efficiency and benefit the environment in sustainable agriculture.

Abstract Background The mosquito gut harbors a variety of bacteria that are dynamically associated with mosquitoes in various contexts. However, little is known about bacterial factors that affect bacterial inhabitation in the gut microbial community. Enterobacter sp. Ag1 is a predominant Gram negative bacterium in the mosquito midgut. Methods In a mutant library that was generated using transposon Tn5-mediated mutagenesis, a mutant was identified, in which the gene waaL was disrupted by the Tn5 insertion. The waaL encodes O antigen ligase, which is required for the attachment of O antigen to the outer core oligosaccharide of the lipopolysaccharide (LPS). Results The waaL − mutation caused the O antigen repeat missing in the LPS. The normal LPS structure was restored when the mutant was complemented with a plasmid containing waaL gene. The waaL − mutation did not affect bacterial proliferation in LB culture, the mutant cells grew at a rate the same as the wildtype (wt) cells. However, when waaL − strain were co-cultured with the wt strain or complemented strain, the mutant cells proliferated with a slower rate, indicating that the mutants were less competitive than wt cells in a community setting. Similarly, in a co-feeding assay, when fluorescently tagged wt strain and waaL − strain were orally co-introduced into the gut of Anopheles stephensi mosquitoes, the mutant cells were less prevalent in both sugar-fed and blood-fed guts. The data suggest that the mutation compromised the bacterial inhabitation in the gut community. Besides, the mutant was more sensitive to oxidative stress, demonstrated by lower survival rate upon exposure to 20 mM H2O2. Conclusion Lack of the O antigen structure in LPS of Enterobacter compromised the effective growth in co-culture and co-feeding assays. In addition, O-antigen was involved in protection against oxidative stress. The findings suggest that intact LPS is crucial for the bacteria to steadily stay in the gut microbial community.

Development of more sensitive nuclear DNA markers for identification of species, particularly closely allied taxa has been a challenging task that has attracted interest from scientists in fields of biotechnological development and genetic diversity detection. In this study, the sequence of the ubiquitin ligase gene (UBE3) region of nuclear DNA was tested for applicability and efficacy in revealing genetic diversity of walnut resources, with an emphasis on inter- and intra-specific levels. Analysis on genetic relationship among the taxa was conducted with the neighbor-joining (NJ) method. The number of variable bases in the UBE3 region was 20 sites. All nine taxa (species/variety/cultivars) were distinguished using the UBE3 sequence. In addition, each taxon was characterized molecularly with a unique nucleotide molecular formula using ten variable base sites derived from the nuclear DNA UBE3 gene sequence. This study presents a good complementary methodology for developing new DNA markers for identification of genus Juglans.

In view of shortcomings of complicated operating, inconvenient maintenance and incidental misoperation existed in mine auxiliary car operating control system based on single-chip microcomputer or integrated circuit, a design scheme of mine auxiliary shaft car operating control system based on PLC was proposed. Structure, control method, control process and core interlock relationships of the system were introduced. Wellhead and downhole work station of the system were separately installed a set of car operating PLC, and they use PLC to receive position signals and garage control signals to control running of car operating equipments. The practical operation showed that the performance of the system is stable and reliable, which ensures safety production of coal mine.

In view of problems of high cost and inconvenient installation by use of piezoelectric force sensor and impact force sensor to measure impact frequency, a low-frequency impact frequency measuring circuit with resistance strain force sensor was designed. According to working principle of resistance strain force sensor, the circuit firstly uses amplifying circuit to amplify output signal of the sensor, then uses voltage comparing circuit to generate square wave signal, and uses monostable trigger circuit to make shaping for the square wave signal at last, so as to get standard square wave signal. The actual application shows that the circuit has stable running with higher measuring precision.

A simple and convenient method was developed for the preparation of hindered tertiary amines via direct reductive amination of ketones with secondary aryl amines, using trichlorosilane as reducing agent and tetramethylethylenediamine (TMEDA) as organic Lewis base activator. A broad range of ketones were reacted with N-methylaniline to afford the corresponding tertiary amine products in high yield. An open transition model was proposed for the reductive step.

The prioritization of capability gaps for weapon system of systems is the basis for design and capability planning in the system of systems development process. In order to address input information uncertainties, the prioritization of capability gaps is computed in two steps using the conditional evidential network method. First, we evaluated the belief distribution of degree of required satisfaction for capabilities, and then calculated the reverse conditional belief function between capability hierarchies. We also provided verification for the feasibility and effectiveness of the proposed method through a prioritization of capability gaps calculation using an example of a spatial-navigation-and-positioning system of systems.

Elizabethkingia anophelis is a dominant bacterial species in the gut ecosystem of the malaria vector mosquito Anopheles gambiae. We recently sequenced the genomes of two strains of E. anophelis, R26T and Ag1, isolated from different strains of A. gambiae. The two bacterial strains are identical with a few exceptions. Phylogenetically, Elizabethkingia is closer to Chryseobacterium and Riemerella than to Flavobacterium. In line with other Bacteroidetes known to utilize various polymers in their ecological niches, the E. anophelis genome contains numerous TonB dependent transporters with various substrate specificities. In addition, several genes belonging to the polysaccharide utilization system and the glycoside hydrolase family were identified that could potentially be of benefit for the mosquito carbohydrate metabolism. In agreement with previous reports of broad antibiotic resistance in E. anophelis, a large number of genes encoding efflux pumps and β-lactamases are present in the genome. The component genes of resistance-nodulation-division type efflux pumps were found to be syntenic and conserved in different taxa of Bacteroidetes. The bacterium also displays hemolytic activity and encodes several hemolysins that may participate in the digestion of erythrocytes in the mosquito gut. At the same time, the OxyR regulon and antioxidant genes could provide defense against the oxidative stress that is associated with blood digestion. The genome annotation and comparative genomic analysis revealed functional characteristics associated with the symbiotic relationship with the mosquito host.

Resistance to TKI treatment is a major obstacle in effective treatment of NSCLC. Besides EGFR mutation status, the mechanisms involved are largely unknown. Some evidence supports a role for microRNA 21 in modulating drug sensitivity of chemotherapy but its role in NSCLC TKI resistance still remains unexplored. This study aimed to investigate whether NSCLC miR-21 mediated resistance to TKIs also results from Pten targeting. Here, we show miR-21 promotes cancer by negatively regulating Pten expression in human NSCLC tissues: high miR-21 expression levels were associated with shorter DFS in 47 NSCLC patients; high miR-21/low Pten expression levels indicated a poor TKI clinical response and shorter overall survival in another 46 NSCLC patients undergoing TKI treatment. In vitro assays showed that miR-21 was up-regulated concomitantly to down-regulation of Pten in pc-9/GR cells in comparison with pc-9 cells. Moreover, over-expression of miR-21 significantly decreased gefitinib sensitivity by down-regulating Pten expression and activating Akt and ERK pathways in pc-9 cells, while miR-21 knockdown dramatically restored gefitinib sensitivity of pc-9/GR cells by up-regulation of Pten expression and inactivation of AKT and ERK pathways, in vivo and in vitro. We propose alteration of miR-21/Pten expression as a novel mechanism for TKI resistance in NSCLC cancer. Our findings provide a new basis for using miR 21/Pten-based therapeutic strategies to reverse gefitinib resistance in NSCLC.

BACKGROUND: Recent evidence indicates that microRNAs (miRNAs) can function as tumor suppressors and oncogenes. Single nucleotide polymorphisms (SNPs) at miRNA genes can influence the maturation of miRNAs or miRNA-mediated transcriptional regulation. Our objective was to investigate the association of SNPs in deregulated miRNAs with clinical outcome in patients with non-small cell lung cancer (NSCLC) in a Chinese population. METHODS: Deregulated miRNAs in NSCLC and their SNPs were identified through public databases. A single SNP, rs895819 in pre-miR-27a, was found suitable for selection. TaqMan assays were performed for genotyping and to assess the effect on the overall survival (OS) and chemotherapy response in 576 NSCLC patients. RESULTS: Log-rank test and Cox regression analysis indicated that the G allele of rs895819 was associated with shorter survival and increased risk of death in NSCLC [dominant model: 22.0 vs. 46.0 months, P