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Zhaoyang Guo,1,&ast; Yuanye Ma,1,&ast; Yaqing Wang,2 Hongfei Xiang,1 Shang-You Yang,3 Zhu Guo,1 Ronghuan Wang,1 Wujun Chen,4 Dongming Xing,4,5 Bohua Chen,1 Hao Tao,1 Xiaolin Wu1,4 1Department of Orthopedics, The Affiliated Hospital of Qingdao University, Qingdao, People’s Republic of China; 2Department of Nephrology, The First Affiliated Hospital of China Medical University, Shenyang, People’s Republic of China; 3School of Medicine-Wichita, University of Kansas, Wichita, KS, USA; 4Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao Cancer Institute, Qingdao, People’s Republic of China; 5School of Life Sciences, Tsinghua University, Beijing, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Xiaolin Wu; Hao Tao, Department of Orthopedics, The Affiliated Hospital of Qingdao University, Qingdao, People’s Republic of China, Email fyqs01@qdu.edu.cn; taohaob2008@163.comBackground: It is well established that discogenic low back pain (DLBP) is often caused by the inflammatory response during intervertebral disc degeneration (IDD). However, it remains unclear how inflammatory mediators such as Interleukin-6 (IL-6) are involved in discogenic pain caused by degeneration and intervertebral disc (IVD) metabolism. The purpose of this study is to study the relationship between IL-6 and Transmembrane protein 100 (TMEM100), and to analyze the different metabolites and metabolic pathways in various rat intervertebral discs by metabonomics.Methods: We established a rat model of IDD-DLBP by disc punctures and PBS infusion to examine the rat pain behaviors. Intervertebral disc tissues were harvested for molecular biology experiments to explore the relationship between IL-6 and TMEM100. High-resolution mass spectrometry (HRMS) was performed for untargeted metabolomics, and nuclear magnetic resonance spectroscopy metabolomics (MRS) for differential metabolites and metabolic pathways.Results: The results showed a significant decrease in vonFrey test, hot plate test and acetone test (P < 0.05). The expression of IL-6 and TMEM100 in DLBP model was significantly higher than that in sham control group and IDD discs without PBS infusion group (P < 0.05). There were 15 major contributing metabolites identified in the DLBP intervertebral discs through metabolomic studies, involving 6 major metabolic pathways. The main differential metabolites included nitric oxide (NO), ammonia, and lactic acid as the metabolic endpoints; and the differential metabolic pathways included the glycine-serine-threonine (Gly-Ser-Thr), which is gradually weakened with the progression of inflammation.Conclusion: The change of TMEM100 expression mediated by il-6 is related to the Gly-Ser-Thr metabolic axis and plays an important role in the relief of discogenic pain.Keywords: metabolomics, DLBP, IDD, IL-6, TMEM100, inflammatory mediators

Increasing studies have demonstrated that dysfunction of long noncoding RNAs (lncRNAs) plays critical roles in the development of human cancers. THAP9-AS1 has been reported to be dysregulated and associated with tumor progression in some cancers. However, the function and mechanism of THAP9-AS1 in osteosarcoma (OS) remain unclear. In the present study, we found that the expression of THAP9-AS1 was significantly upregulated in OS tissues and associated with the advanced stage of tumors and poor prognosis of patients. Blast comparison results showed that the SOCS3 promoter region and THAP9-AS1 had base complementary pairing binding sites. The interactions between THAP9-AS1, DNA methyltransferases (DNMTs), and SOCS3 were assessed by RIP and ChIP assays. The results of methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) validated that THAP9-AS1 enhanced the methylation level of the SOCS3 promoter. The mRNA levels of SOCS3 in OS cells could be reversed by the demethylation agent 5-aza-2 ′ -deoxycytidine. The mRNA expression of SOCS3 was downregulated in OS tissues and negatively correlated with THAP9-AS1 expression in tumors. Moreover, the western blot and immunofluorescence (IF) assay data showed that THAP9-AS1 activated the JAK2/STAT3 signaling pathway by upregulating p-JAK2 and p-STAT3 and the nuclear translocation of p-STAT3. Functionally, ectopic expression of THAP9-AS1 promoted cell proliferation, migration, and invasion and inhibited apoptosis, and this phenomenon could be reversed by SOCS3. Introduction of the JAK/STAT inhibitor AG490 partially abolished the stimulative effect of THAP9-AS1 on cellular processes. In addition, THAP9-AS1 decreased oxidative stress by reducing reactive oxygen species (ROS) and enhancing the mitochondrial membrane potential of OS cells via the SOCS3/JAK2/STAT3 pathway. Stable overexpression of THAP9-AS1 contributed to tumor growth and metastasis in vivo. In total, our findings suggested that upregulation of THAP9-AS1 might recruit DNMTs to epigenetically inhibit SOCS3, thereby activating the JAK2/STAT3 signaling pathway and oncogenesis of OS. These results provide novel insights for the understanding of OS progression.

Cadmium (Cd) accumulation in maize grains is detrimental to human health. Developing maize varieties with low Cd content is important for safe consumption of maize grains. However, the key genes controlling maize grain Cd accumulation have not been cloned. Here, we identified one major locus for maize grain Cd accumulation (qCd1) using a genome-wide association study (GWAS) and bulked segregant RNA-seq analysis with a biparental segregating population of Jing724 (low-Cd line) and Mo17 (high-Cd line). The candidate gene ZmHMA3 was identified by fine mapping and encodes a tonoplast-localized heavy metal P-type ATPase transporter. An ethyl methane sulfonate mutant analysis and an allelism test confirmed that ZmHMA3 influences maize grain Cd accumulation. A transposon in intron 1 of ZmHMA3 is responsible for the abnormal amino acid sequence in Mo17. Based on the natural sequence variations in the ZmHMA3 gene of diverse maize lines, four PCR-based molecular markers were developed, and these were successfully used to distinguish five haplotypes with different grain Cd contents in the GWAS panel and to predict grain Cd contents of widely used maize inbred lines and hybrids. These molecular markers can be used to breed elite maize varieties with low grain Cd contents.

SummarySoil salinity is a major environmental constraint severely reducing plant growth and crop productivity worldwide. Knowledge of salt tolerance-related genes can facilitate improving crop salt tolerance and alleviating the threat of increasing saline area to world food security. Here, we identified a major locus SALT TOLERANCE 1 (qST1) conferring maize salt tolerance via bulked segregant RNA-Seq (BSR-Seq). qST1 encodes a plasma membrane Na+/H+ exchanger ZmSOS1 which is the ortholog of Arabidopsis thaliana SOS1 gene. In salt-sensitive variety D9H, the natural variation of 4-bp deletion in the coding sequence of ZmSOS1 gene was the causal allele for salt sensitivity. We identified two ethyl methanesulfonate-induced mutants, zmsos1-1 and zmsos1-2, which were sensitive to salt stress and can’t complement salt-sensitive variety under salt stress within an allelism test. Overexpression of ZmSOS1 enhanced maize seedling salt tolerance. ZmSOS1 can increase the salt tolerance of Arabidopsis sos1-1 mutant and can be activated by AtSOS2 and AtSOS3 in yeast cells, suggesting that ZmSOS1 confers salt tolerance through the conserved SOS signaling pathway in maize. The detrimental allele harboring the 4-bp deletion was rarely found in the natural population but appeared in an important heterotic group composed of x1132x-derived inbred lines which have been used widely for breeding dozens of hybrid varieties in China. The 4-bp deletion-based molecular marker has been successfully used to improve salt-sensitive varieties in a backcross and marker-assisted breeding program by screening and purging this deleterious allele.

The novel ZmR1

Objective To study the distribution of sympathetic nerves of the ligamentum flavum (LF), confirm its existence by histological observation and nuclear magnetic resonance spectroscopy, and analyze the relationship between sympathetic nerve fibers and the biomechanical structure of the LF. Methods Randomly controlled scientific research selected 15 cases of posterior surgery in the affiliated hospital of Qingdao University from January 2013 to December 2019. The average age was 67.5 ± 14.5 years old, eight males and seven females. The LF specimens (completely separated fresh tissue) of different segments (C3‐7) were taken during the operation. Two pages of LF specimens on the left and right sides of the same segment are randomly allocated by the pairing method for formalin fixation and cryopreservation in liquid nitrogen. LF specimens extracted from seven other adult cadaver specimens (average age at death of about 56.8 ± 4.0 years, three males and four females) were used as a control group; together with formalin‐ fixed specimens obtained during surgery, 3D slices were given layer by layer. The distribution of sympathetic nerves in different parts of the LF was analyzed by glyoxylic acid‐induced biological monoamine fluorescent technique (SPG) and hematoxylin–eosin (HE) staining. Fifteen liquid nitrogen storage specimens were divided into the back of the LF and the spinal canal through frozen sections, and were analyzed by nuclear magnetic resonance spectroscopy‐hydrogen spectrum (1H ‐NMR) for neurotransmitters and neurometabolites. Results There were type C sympathetic nerve fibers in the LF, which were divided into linear shape (α) and wave shape (β). Experimental group (χ2 = 1.705, P > 0.05) and control group (χ2 = 0.879, P > 0.05) can detect no difference in fluorescence units. Nerve fiber transmitter metabolites choline (Cho), creator (Cr), γ‐aminobutyric acid (GABA) also indicate that the sympathetic nerve is present in the LF. LF sympathetic nerve fibers were mainly distributed in the proximal spinal canal surface, nerve fibers on the medial belt (area II) were fewer than the lateral belt (area I) (W = 210, P, Sympathetic nerve fibers distributed on ligamentum flavum. Their distribution may be correlated with histological and mechanical characteristics of ligamentum flavum. It may also be the anatomical basis of cervical vertigo.

The maize stalk is an important mechanical supporting tissue. The stalk fracture resistance is closely related to lodging resistance, and thus the yield. In this study, we showed that the basal zone (BZ) was more fragile than the middle zone (MZ) of the stalk internode before tasseling. In order to clarify the relationship between the different zones and fragile resistance between the internodes, we systematically analyzed the phenotypic, metabolomic and transcriptomic differences. The results indicated that the BZ zone had lower stalk strength, which corresponded to the results of less lignin, cellulose and hemicellulose than that of the MZ. The 27 highly enriched metabolites and 4430 highly expressed genes in the BZ mainly participated in pentose phosphate, and in ribosome and sterol synthesis pathways, respectively. In addition, the BZ had higher vascular bundles density but smaller size compared with the MZ. By contrast, the 28 highly enriched known metabolites and 4438 highly expressed genes in the MZ were mainly involved in lignin synthesis, and secondary metabolites synthesis, respectively, especially the phenylpropanoid synthesis. The results provide a deeper understanding of the relationship between development and fracture differences in stalk, and may facilitate the improvement of field management practice to reduce lodging.

SummaryCadmium (Cd) accumulation in maize grains is detrimental to human health. Developing maize varieties with low-Cd contents via marker-assisted selection is important for ensuring the production of maize grains safe for consumption. However, the key gene controlling maize grain Cd accumulation has not been cloned. In this study, we identified two major loci for maize grain Cd accumulation (qCd1 and qCd2) on chromosome 2 during a genome-wide association study (GWAS). The qCd1 locus was analyzed by bulked segregant RNA-seq and fine mapping with a biparental segregating population of Jing724 (low-Cd line) and Mo17 (high-Cd line). The ZmCd1 candidate gene in the qCd1 locus encodes a vacuolar membrane-localized heavy metal P-type ATPase transporter, ZmHMA3, which is orthologous to the tonoplast Cd transporter OsHMA3. Genomic DNA sequence and transcript analyses suggested that a transposon in intron 1 of ZmCd1 is responsible for the abnormal amino acid sequence in Mo17. An EMS mutant analysis and an allelism test confirmed ZmCd1 influences maize grain Cd accumulation. The natural variations in ZmCd1 were used to develop four PCR-based molecular markers, which revealed five ZmCd1 haplotypes in the GWAS population. The molecular markers were also used to predict the grain Cd contents in commonly cultivated maize germplasms in China. The predicted Cd contents for 36 inbred lines and 13 hybrids were consistent with the measured Cd contents. Furthermore, several low-Cd elite inbred lines and hybrids were identified, including Jing2416, MC01, Jingnonke728, and Jingke968. Therefore, the molecular markers developed in this study are applicable for molecular breeding and developing maize varieties with low grain Cd contents.

The heating furnace is an essential oilfield facility for surface gathering, treatment, and transportation, so the energy consumption level of an oilfield is directly affected by its operational efficiency. In this paper, the thermal efficiency, exhaust gas temperature, external surface temperature, excess air coefficient, and load rate of a heating furnace are taken as energy efficiency evaluation indexes. By improving game theory, the objective and subjective weights are combined to determine the final weights of each index. On this basis, the grey TOPSIS method is used to establish the energy efficiency evaluation model of an oilfield heating furnace, which is to comprehensively evaluate the energy consumption of the heating furnace by calculating the closeness degree between its actual and ideal operational states. Finally, the effectiveness of the energy efficiency evaluation model is verified by taking an actual oilfield as an example, the results show that the weight sequence of the indexes is thermal efficiency, exhaust gas temperature, external surface temperature, load rate, and excess air coefficient. In addition, the relative closeness of the heating furnace is mostly concentrated between 0.5 and 0.7, which shows that the efficiency is low. The weak link of energy consumption is analyzed, and the corresponding improvement measures are put forward.

Additional file 1: Figure S1. The construction process of JING724A1 and the F2:3 population. (A) The development of JING724A1. (B) The construction of the F2:3 population using JING724 and JING724A1. Figure S2. Measurement of stalk fracture angle. Figure S3. The increased ratio of specific cell wall components in JING724A1 compared to JING724. Table S1. Overview of sequencing results. Table S2. Summary of SNPs and InDels for the parents and pools. Table S3. Four KASP makers and the significance with the stalk fracture angle. Table S4. The expression level of 12 genes. Table S5. The important SNPs and InDels for the two candidate genes. Table S6. Sequences of two KASP markers for the two candidate genes.

The impacts of low temperature occasionally encountered at higher latitude regions on maize seed germination present significant threats to yield and cultivation. Exploring the association of antioxidant system with low temperature (LT) germination could support the breeding strategies for better responding to LT disturbance. In this study, we have examined the germination rate and growth potential of a set of elite maize inbred accessions under LT and normal temperature (NT) conditions in the field. These accessions were found to have variable germination rate and growth potential when grown at LT, whereas the difference is not significant under NT. Physiological study revealed lower hydrogen peroxide content in LT tolerant accessions when compared with sensitive ones. LT-tolerant and LT-sensitive lines maintained similar content of ascorbate (AsA) and glutathione (GSH), whereas the reduced substrate content of which were significantly higher in LT-tolerant accessions. Consistently, activities of ascorbate peroxidase and dehydroascorbate reductase, the enzyme components that responsible for the AsA-GSH recycling, were much higher in LT-tolerant lines. Transcription profile revealed the increased expression of ZmVTC2 gene in LT-tolerant inbred line, which was rate limited step in AsA biosynthesis. These data indicates that the coordinated improvement of AsA biosynthesis and AsA-GSH recycling increase the pool size of the total antioxidants, which ameliorate LT-induced oxidative stress during maize seed germination.

Maize is a globally important crop that was a classic model plant for genetic studies. Here, we report a 2.2 Gb draft genome sequence of an elite maize line, HuangZaoSi (HZS). Hybrids bred from HZS-improved lines (HILs) are planted in more than 60% of maize fields in China. Proteome clustering of six completed sequenced maize genomes show that 638 proteins fall into 264 HZS-specific gene families with the majority of contributions from tandem duplication events. Resequencing and comparative analysis of 40 HZS-related lines reveals the breeding history of HILs. More than 60% of identified selective sweeps were clustered in identity-by-descent conserved regions, and yield-related genes/QTLs were enriched in HZS characteristic selected regions. Furthermore, we demonstrated that HZS-specific family genes were not uniformly distributed in the genome but enriched in improvement/function-related genomic regions. This study provides an important and novel resource for maize genome research and expands our knowledge on the breadth of genomic variation and improvement history of maize.

We isolated a novel lytic phage of Ralstonia solanacearum, GP4. The GP4 phage has a latent period of ~ 2 h at its optimal multiplicity of infection and is stable at temperatures ranging from 40–70 °C. GP4 lysed 16 strains of R. solanacearum belonging to phylotype IV. High-throughput sequencing revealed that GP4 has a linear dsDNA genome that consists of 61,129 bp, contains 83 open reading frames, and encodes a tRNA for cysteine. The GP4 genome has low similarity to other phage sequences in the GenBank database. Phylogenetic analysis indicated that GP4 can be taxonomically classified as a member of the Bcep22-like subfamily of the family Podoviridae.

Maize (Zea mays L.) is the largest food crops in China with the plangting area and total yield of 37.076 million hectares and 215.67 million tons respectively in 2014. The technology of cross breeding was the primary method to cultivate new maize varieties and promote the yield level. In recent years, more and more agriculturalists discovered the existence of endophyte in maize and their close relationship with soil environmental adaption which affect the production of maize. In this study, the seeds of six different maize varieties which were self-developed and cultivated from capital city of China “Beijing” and extensively planted in China were collected, this is the first time to acquire all of the “Beijing” hybrid maize to investigate their endopytes. We clarified eight species exists in all the varieties and the relative abundance of top three species including Pantoea agglomerans, Enterobacter cloacae and Aeribacillus pallidus taken about 60 % of the whole endophyte. Besides these, we also discovered the correlations between the endophytic bacteria which might affect the growth of maize. On the other hand, the distributions of E. cloacae and A. pallidus between maize varieties with different male parent were apparently different. So we deduced the endophyte affect the environmental adaptation of different maize varieties and the results showed the light on the future maize variety cultivation from the angle of endophyte.

Enterococcus faecalis is one of the main bacteria in the human and animal intestine but is also classed as an opportunistic pathogen. During normal growth, E. faecalis produces natural antibiotics and is conducive to human health. As ectopic parasites, E. faecalis is capable of causing infective endocarditis, neonatal sepsis, bloodstream infections, bacteremia, and intraabdominal infections. With the incidence of antibiotic resistance reaching crisis point, it is imperative to find alternative treatments for multidrug-resistant infections. Using phage for pathogen control is a promising treatment option to combat bacterial resistance. In this study, a lytic phage, designated vB_EfaP_IME195, was isolated from hospital sewage using a clinical multidrug-resistant Enterococcus faecalis strain as an indicator. The one-step growth curve with the optimal multiplicity of infection of (MOI) 0.01 revealed a latent period of ~ 30 min and a burst size of ~ 120 plaque-forming units (pfu) per cell. Transmission electron microscopy showed that the phage belongs to the family Podoviridae. Phage vB_EfaP_IME195 has a linear, double-stranded DNA genome of 18,607 bp with a G + C content of 33% and 27 coding sequences (GenBank accession no. KT932700). Run-off sequencing experiments showed that the phage has a unique 59-bp inverted repeat sequences at the terminal ends. BLASTn analysis revealed that vB_EfaP_IME195 shares 92% identity (93% genome coverage) with unpublished E. faecalis phage Idefix. This study reported a novel E. faecalis phage with unique genome termini containing inverted repeats. The isolation and characterization of this novel lytic E. faecalis phage provides the basis for the development of new therapeutic agents like phage cocktails for multidrug-resistant E. faecalis infection, and its unique genomic feature would also provide valuable knowledge and insight for further phage genome analysis.

Four Gram-stain positive bacterial strains, designated as 4R1(T), 4R9, 4L13 and 4L18, isolated from seeds of hybrid maize (Zea mays L., Jingke 968), were investigated using a polyphasic taxonomic approach. The cells were found to be facultatively aerobic, motile, spore-forming and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates should be recognised as a species of the genus Paenibacillus, with two close neighbours being Paenibacillus nicotianae YIM h-19(T) (98.41 % similarity) and Paenibacillus hordei RH-N24(T) (98.37 %). The DNA G+C content of strain 4R1(T) was determined to be 51.6 mol %. Its polar lipid profile was found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified lipid. The predominant respiratory quinone was identified as MK-7 and the major fatty acids were found to be anteiso-C15:0, anteiso-C12:0, anteiso-C13:0 and anteiso-C11:0. Strains 4R1(T), 4R9, 4L13 and 4L18 were clearly distinguished from the reference type strains using phylogenetic analysis, DNA-DNA hybridization and a range of physiological and biochemical characteristics. It is evident from the genotypic and phenotypic data that strains 4R1(T), 4R9, 4L13 and 4L18 represent a novel species of the genus Paenibacillus, for which the name Paenibacillus chinensis sp. nov. is proposed. The type strain is 4R1(T) (=KCTC 33672(T) = CICC 23864(T)).

Endophytes are related to the health and growth of plants. In this study, the endophytic bacterial strain Paenibacillus sp. 5 L8 was isolated from the seeds of maize (Zea mays L., Jingke 968). Strain 5 L8 was identified as Paenibacillus polymyxa by polyphasic taxonomy based on morphological and physiological methods, and 16S rRNA gene phylogenetic analysis. Via antagonistic testing against the common plant pathogens Fusarium graminearum, Bipolaris maydis, Bipolaris sorokiniana, Cochliobolus heterostrophus, Aspergillus aculeatus, Phomopsis chimonanthi and Verticillium dahliae, strain 5 L8 showed comprehensive and good antagonistic activity. The draft genome of Paenibacillus sp. 5 L8 was sequenced by Illumina HiSeq 2000, and three coding sequences (CDSs) for the glucanase gene were annotated and correlated to antagonistic activity. Using specific primers to amplify the biocontrol gene in the glucanase family, the β-1,3-1,4-glucanase gene (gluB) was found. This study supplies a basis for further study of industrial research and application.

Four Gram-stain-positive bacterial strains, designated 6R2T, 6R18, 3T2 and 3T10, isolated from seeds of hybrid maize (Zea mays L., Jingke 968) were investigated using a polyphasic taxonomic approach. Cells were aerobic, motile, spore-forming and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolates may represent a novel species of the genus Paenibacillus, the four closest neighbours being Paenibacillus lautus NRRL NRS-666T (97.1 % similarity), Paenibacillus glucanolyticus DSM 5162T (97.0 %), Paenibacillus lactis MB 1871T (97.0 %) and Paenibacillus chibensis JCM 9905T (96.8 %). The DNA G+C content of strain 6R2T was 51.8 mol%. Its polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The predominant respiratory quinone was menaquinone 7 (MK-7) and the major fatty acids were anteiso-C15 : 0 and iso-C14 : 0. Strains 6R2T, 6R18, 3T2 and 3T10 were clearly distinguished from the above type strains using phylogenetic analysis, DNA–DNA hybridization, and a range of physiological and biochemical characteristics. It is evident from the genotypic and phenotypic data that strains 6R2T, 6R18, 3T2 and 3T10 represent a novel species of the genus Paenibacillus, for which the name Paenibacillus zeae sp. nov. is proposed. The type strain is 6R2T ( = KCTC 33674T = CICC 23860T).