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Heat-shock transcription factors (HSFs) are crucial for regulating plant responses to heat and various stresses, as well as for maintaining normal cellular functions and plant development. HSFA9 and HSFA2 are two of the Arabidopsis class A HSFs and their expressions are dramatically induced in response to heat shock (HS) stress among all 21 Arabidopsis HSFs. However, the detailed biological roles of their cooperation have not been fully characterized. In this study, we employed an integrated approach that combined bioinformatics, molecular genetics and computational analysis to identify and validate the molecular mechanism that controls seed longevity and thermotolerance in Arabidopsis. The acquisition of tolerance to deterioration was accompanied by a significant transcriptional switch that involved the induction of primary metabolism, reactive oxygen species and unfolded protein response, as well as the regulation of genes involved in response to dehydration, heat and hypoxia. In addition, the cis -regulatory motif analysis in normal stored and controlled deterioration treatment (CDT) seeds confirmed the CDT-repressed genes with heat-shock element (HSE) in their promoters. Using a yeast two-hybrid and molecular dynamic interaction assay, it is shown that HSFA9 acted as a potential regulator that can interact with HSFA2. Moreover, the knock-out mutants of both HSFA9 and HSFA2 displayed a significant reduction in seed longevity. These novel findings link HSF transcription factors with seed deterioration tolerance and longevity. [ABSTRACT FROM AUTHOR]

Background: Drought is one of the main consequences of global climate change and this problem is expected to intensify in the future. Resurrection plants evolved the ability to withstand the negative impact of long periods of almost complete desiccation and to recover at rewatering. In this respect, many physiological, transcriptomic, proteomic and genomic investigations have been performed in recent years, however, few epigenetic control studies have been performed on these valuable desiccation-tolerant plants so far. Results: In the present study, for the first time for resurrection plants we provide evidences about the differential chromatin accessibility of Haberlea rhodopensis during desiccation stress by ATAC-seq (Assay for Transposase Accessible Chromatin with high-throughput sequencing). Based on gene similarity between species, we used the available genome of the closely related resurrection plant Dorcoceras hygrometricum to identify approximately nine hundred transposase hypersensitive sites (THSs) in H. rhodopensis. The majority of them corresponds to proximal and distal regulatory elements of different genes involved in photosynthesis, carbon metabolism, synthesis of secondary metabolites, cell signalling and transcriptional regulation, cell growth, cell wall, stomata conditioning, chaperons, oxidative stress, autophagy and others. Various types of binding motifs recognized by several families of transcription factors have been enriched from the THSs found in different stages of drought. Further, we used the previously published RNA-seq data from H. rhodopensis to evaluate the expression of transcription factors putatively interacting with the enriched motifs, and the potential correlation between the identified THS and the expression of their corresponding genes. Conclusions: These results provide a blueprint for investigating the epigenetic regulation of desiccation tolerance in resurrection plant H. rhodopensis and comparative genomics between resurrection and non-resurrection species with available genome information. [ABSTRACT FROM AUTHOR]

Background: Drought is one of the main consequences of global climate change and this problem is expected to intensify in the future. Resurrection plants evolved the ability to withstand the negative impact of long periods of almost complete desiccation and to recover at rewatering. In this respect, many physiological, transcriptomic, proteomic and genomic investigations have been performed in recent years, however, few epigenetic control studies have been performed on these valuable desiccation-tolerant plants so far. Results: In the present study, for the first time for resurrection plants we provide evidences about the differential chromatin accessibility of Haberlea rhodopensis during desiccation stress by ATAC-seq (Assay for Transposase Accessible Chromatin with high-throughput sequencing). Based on gene similarity between species, we used the available genome of the closely related resurrection plant Dorcoceras hygrometricum to identify approximately nine hundred transposase hypersensitive sites (THSs) in H. rhodopensis. The majority of them corresponds to proximal and distal regulatory elements of different genes involved in photosynthesis, carbon metabolism, synthesis of secondary metabolites, cell signalling and transcriptional regulation, cell growth, cell wall, stomata conditioning, chaperons, oxidative stress, autophagy and others. Various types of binding motifs recognized by several families of transcription factors have been enriched from the THSs found in different stages of drought. Further, we used the previously published RNA-seq data from H. rhodopensis to evaluate the expression of transcription factors putatively interacting with the enriched motifs, and the potential correlation between the identified THS and the expression of their corresponding genes. Conclusions: These results provide a blueprint for investigating the epigenetic regulation of desiccation tolerance in resurrection plant H. rhodopensis and comparative genomics between resurrection and non-resurrection species with available genome information. [ABSTRACT FROM AUTHOR]

Phosphorus (P) deficiency is a major factor constraining plant growth and yield production worldwide because it is essential for living cells and organisms. To avoid disadvantageous genetic correlations between growth duration and root system, traits are critical in developing oilseed rape cultivars that use P efficiently. To understand the genetic basis underlying the correlations between root system traits and two levels of P availability in oilseed, we investigated 6091 different expression genes in the shoot and root tissues of the cultivar Eyou Changjia under low and normal P conditions. Subsequently, the Illumina Brassica 60 K single-nucleotide polymorphism (SNP) arrays were used in a diverse and representative core collection of 405 cultivated oilseed rape accessions, and nearly 13,340 functional SNPs with high polymorphisms were genotyped at the threshold interval of candidate genes. Four root system traits were investigated in the oilseed rape accessions in both low and normal P environments. A total of 224 suggestive association loci were identified for root system traits under low and normal P conditions. Eleven were co-located with the interval of quantitative trait loci (QTLs) for the same traits detected in previous studies. Mutation of co-located SNPs with favourable alleles leads to gene sequence mutations or gene expression changes that result in different phenotypes. The interaction between multiple optimal loci and the effect of QTL aggregation of combined haplotype types with C(A3) and A(A7) formed optimal haplotypes and could improve our understanding of molecular mechanisms underlying P efficiency in oilseed rape. [ABSTRACT FROM AUTHOR]

Abstract: Transforming growth factor-beta receptor II (TGF-βRII) is an attractive target for anti-scarring therapy in wound healing because it attenuates excessive TGF-β which has pleiotropic effects on the immune system. In the present study, the cDNA of rabbit TGF-βRII (rTGF-βRII) was amplified from rabbit peripheral blood by RT-PCR. The open reading frame of rTGF-βRII encodes a protein consisting of 567 amino acids, which contains a predicted transmembrane domain and a Serine/Threonine protein kinase domain similar to other identified mammalian TGF-βRIIs. The amino acid sequences of the biologically active, soluble rTGF-βRII and mouse, rat, human and chicken counterparts are 81%, 81%, 89% and 61%, respectively, identical. Recombinant soluble rTGF-βRII (rsTGF-βRII) fused with His tag was efficiently expressed in Escherichia coli BL21 (DE3) expression host strain. This fusion protein''s molecular weight of ∼19kDa was identified by SDS-PAGE and Western blotting. In vitro, purified rsTGF-βRII was able to inhibit the proliferation of keloid rabbit fibroblasts and decrease the level of collagen. These findings indicate that rTGF-βRII plays an important role in inhibiting the proliferation of keloid rabbit fibroblasts and provides the basis for investigations on the role of TGF-βRII in this important domestic species. [Copyright &y& Elsevier]

Disruption of epidermal permeability barrier induces an increase in proinflammatory cytokine expression and release, stimulation of epidermal lipid and DNA synthesis, and expression of antimicrobial peptides. Although alterations in epidermal function in the aged skin are known, whether the epidermal transcriptomic responses to barrier disruption differ between aged and young mice remains unknown. Here, we performed RNA sequencing of the epidermis in 2‐month‐ vs. 20‐month‐old mice following barrier disruption with repeated tape‐stripping. At baseline condition, the epidermis of 20‐month‐old mice displayed an upregulation of inflammation‐associated genes and down‐regulation of epidermal structure‐ and development‐related genes in comparison to 2‐month‐old mice. Barrier disruption upregulated expression levels of 327 genes and downregulated 209 genes in 2‐month‐old mice. In 20‐month‐old mice, the numbers of upregulated and down‐regulated genes were 537 and 299, respectively. In comparison to young mice, the prominently upregulated genes in the 20‐month‐old mice were associated with the IL‐17 signalling pathway, while downregulated genes were mainly involved in the cytokine–cytokine receptor interaction pathway. These results indicate that inflammation‐associated signalling pathways are upregulated, while epidermal structure‐ and development‐related genes are downregulated in the epidermis of aged mice, with further aggravation following barrier disruption, suggesting the importance of improving epidermal function in the elderly. [ABSTRACT FROM AUTHOR]

Lack of phosphorus (P) is a major environmental factor affecting rapeseed (Brassica napus. L) root growth and development. For breeding purposes, it is crucial to identify the molecular mechanisms underlying root system architecture traits that confer low-P tolerance in rapeseed. Natural variations in the glycine-rich protein gene BnGRP1 were analysed in the natural population of 400 rapeseed cultivars under low-P stress through genome-wide association study and transcriptome analysis. Based on 11 single nucleotide polymorphism mutations in the BnGRP1 sequence, 10 haplotypes (Hap) were formed. Compared with the other types, the cultivar BnGRP1 Hap1 in the panel demonstrated the longest root length and heaviest root weight. BnGRP1 Hap1 overexpression in rapeseed led to enhanced low-P tolerance. CRISPR/Cas9-derived BnGRP1 Hap4 knockout mutations in rapeseed can lead to sensitivity to low-P stress. Furthermore, BnGRP1 Hap1 influences the expression of the phosphate transporter 1 gene (PHT1) associated with P absorption. Overall, the findings of this study highlight new insights into the mechanisms of GRP1 enhancement of low-P tolerance in rapeseed. [ABSTRACT FROM AUTHOR]

Background: 'Conditioning' [ischemic preconditioning (IPC), ischemic postconditioning (IPO) and remote ischemic preconditioning (RIPC)] the heart to render it more resistant to an episode of acute myocardial ischemia-reperfusion (I/R) injury is an endogenous cardioprotective strategy. There are several mechanisms proposed for 'conditioning', such as endogenous mediators or cytoprotective proteins. In recent reports, microRNAs (miRNAs) were involved in controlling the expression of myocardial ischemia-related genes. Some studies have demonstrated that cardiac miRNA-1 and miRNA-21 were significantly increased by late IPC with an increase in their target proteins [endothelial nitric oxide synthase and heat shock protein 70 (HSP70)], but their expression levels in 'conditioning' strategies are currently unknown. Methods: In the current study, Langendorff-perfused Sprague-Dawley rat hearts were randomly assigned to one of four groups [control group (CON group, n = 12), IPC group (n = 12), IPO group (n = 12) and RIPC group (n = 12)]. Cardiac function was digitalized and analyzed. The expression of miRNA-1 and miRNA-21 was detected by real-time reverse transcription polymerase chain reaction. The expression of HSP70, programmed cell death protein 4 (PDCD4), B-cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2-associated X protein (Bax) was detected by Western blot. Cardiac infarct size and myocardial apoptosis were determined using the 2,3,5-triphenyltetrazolium chloride assay and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, respectively. Results: The results revealed that miRNA-1 (233 ± 45%) and miRNA-21 (356 ± 33%) expression was up-regulated in the IPC group, but the expression of miRNA-1 was down-regulated in the RIPC (61 ± 16%) group and IPO group (61 ± 13%). The expression of PDCD4 [IPC (74 ± 11%), RIPC (81 ± 16%), IPO (83 ± 12%)], HSP70 [IPC (74 ± 5%), RIPC (81 ± 6%), IPO (67 ± 11%)] and Bax [IPC (27 ± 6%), RIPC (21 ± 3%), IPO (27 ± 4%)] was down-regulated in the conditioning groups compared with the CON group [PDCD4 (130 ± 11%), HSP70 (121 ± 11%) and Bax (63 ± 8%)]. In the conditioning hearts, infarct size [IPC (31.7 ± 4.1%), RIPC (29.6 ± 6.19%) and IPO (32.8 ± 4.71%)] and myocardial apoptosis [IPC (15.2 ± 4.21%), RIPC (17.2 ± 1.92%) and IPO (15.6 ± 4.04%)] were significantly decreased compared with the CON group (infarct size: 51.77 ± 4.3%, myocardial apoptosis: 32.8 ± 3.96%). Conclusion: We concluded that miRNA-1 and miRNA-21 expression differed in IPC, RIPC and IPO groups, and their target proteins were not inversely correlated with the miRNAs in all the conditioning groups, which revealed that the miRNAs were regulated but complicated by the different conditioning protocols. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

In wild strains of Bacillus, a handful of extracellular natural products act as signals that can regulate multicellular behavior, but relatively little is known about molecular mechanisms' detail. We proposed a previously unreported molecular mechanism for triggering multicellularity in B. velezensis Bs916 by an endogenous cyclic lipopeptide, bacillomycin L. The genome-wide effect on gene expression was caused by the disruption of bacillomycin L gene cluster, and 100 µg/mL bacillomycin L was revealed by quantitative transcriptomics. A total of 878 differentially expressed genes among Bs916, Δbl, and Δbl + 100BL were identified and grouped into 9 functional categories. The transcription levels of 40 candidate genes were further evaluated by RT-qPCR analysis. The expression of eight candidate genes regulated by bacillomycin L in a dose-dependent manner was revealed by LacZ fusion experiment. Although the addition of bacillomycin L could not completely restore the expression levels of the differentially regulated genes in △bl, our results strongly suggest that bacillomycin L acts as a tuning signal of swarming motility and complex biofilm formation by indirectly regulating the expression levels of some two-component systems (TCSs) connector genes, particularly including several Raps that potentially regulate the phosphorylation levels of three major regulators ComA, DegU, and Spo0A. Key points • Proposed model for bacillomycin L regulation in B. velezensis Bs916. • Bacillomycin L can act as an extracellular signal to regulate the phosphorylation levels of three major regulators, ComA, DegU, and Spo0A and control the multicellular processes of vegetative growth, competent, motility, matrix production, sporulation, and autolysis. [ABSTRACT FROM AUTHOR]

Locillomycins are cyclic lipononapeptides assembled by a nonlinear hexamodular NRPS and have strong antibacterial activity. In this study, we genetically engineered Bacillus velezensis FZB42 as a surrogate host for the heterologous expression of the loc gene cluster for locillomycins. The fosmid N13 containing whole loc gene cluster was screened from the B. velezensis 916 genomic library. Subsequently, a spectinomycin resistance cassette, and the cassette fused with an IPTG inducible promoter Pspac, was introduced in the fosmid N13 using λ Red recombination system, respectively. The resulting fosmids, designated N13+Spec and N13+PSSpec, were used for the transformation of B. velezensis FZB42 to obtain derivative strains FZBNPLOC and FZBPSLOC. RT-PCR and qRT-PCR results revealed the efficient heterologous expression of the loc gene cluster in both derivative strains. Particularly, there was positive correlation between the derivative FZBPSLOC strain and the enhanced production of locillomycins upon addition of the inducer IPTG with the highest production of locillomycins at 15-fold more than that of B. velezensis 916. This overproduction of locillomycins was also related to the enhancement of antibacterial activity against methicillin-resistant Staphylococcus aureus, and exhibited moderate changes in its hemolytic activity. Together our findings demonstrate that the nonlinear hexamodular NRPS, encoded by the loc gene cluster from B. velezensis 916, is sufficient for the biosynthesis of cyclic lipononapeptide locillomycins in the surrogate host B. velezensis FZB42. Moreover, the FZBPSLOC strain will also be useful for further development of novel locillomycins derivatives with improved antibacterial activity. [ABSTRACT FROM AUTHOR]

Baphicacanthus cusia (Nees) Bremek is an herb widely used for the clinical treatment of colds, fever, and influenza in Traditional Chinese Medicine. The roots, stems and leaves can be used as natural medicine, in which indigo and indirubin are two main active ingredients. In this study, quantification of indigo, indirubin, indican and adenosine among various tissues of B. cusia was conducted using HPLC-DAD. Leaves have significantly higher contents than stems and roots (380.66, 315.15, 20,978.26, 4323.15 μg/g in leaves, 306.36, 71.71, 3,056.78, 139.45 μg/g in stems, and 9.31, 7.82, 170.45, 197.48 μg/g in roots, respectively). De novo transcriptome sequencing of B. cusia was performed for the first time. The sequencing yielded 137,216,248, 122,837,394 and 140,240,688 clean reads from leaves, stems and roots respectively, which were assembled into 51,381 unique sequences. A total of 33,317 unigenes could be annotated using the databases of Nr, Swiss-Prot, KEGG and KOG. These analyses provided a detailed view of the enzymes involved in indican backbone biosynthesis, such as cytochrome P450, UDP-glycosyltransferase, glucosidase and tryptophan synthase. Analysis results showed that tryptophan synthase was the candidate gene involved in the tissue-specific biosynthesis of indican. We also detected sixteen types of simple sequence repeats in RNA-Seq data for use in future molecular mark assisted breeding studies. The results will be helpful in further analysis of B. cusia functional genomics, especially in increasing biosynthesis of indican through biotechnological approaches and metabolic regulation. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress and mitochondrial dysfunction play a pivotal role in the pathological process of sepsis-induced acute lung injury (ALI). Quercetin has been proved to exert anti-inflammation in ALI. This study aimed to explore the protection mechanism of quercetin against sepsis-induced ALI via suppressing ER stress and mitochondrial dysfunction. Cecal ligation and puncture (CLP) mouse model was established to mimic sepsis, and LPS was used to stimulate murine lung epithelial (MLE-12) cells. We observed that quercetin ameliorated pulmonary pathological lesion and oxidative damage in sepsis-induced mice. In LPS-stimulated MLE-12 cells, quercetin could inhibit the level of ER stress as evidenced by decreased mRNA expression of PDI, CHOP, GRP78, ATF6, PERK, IRE1α and improve mitochondrial function, as presented by increased MMP, SOD level and reduced production of ROS, MDA. Meanwhile, transcriptome analysis revealed that quercetin upregulated SIRT1/AMPK mRNA expression. Furthermore, we used siRNA to knockdown SIRT1 in MLE-12 cells, and we found that SIRT1 knockdown could abrogate the quercetin-elicited antioxidation in vitro. Therefore, quercetin could protect against sepsis-induced ALI by suppressing oxidative stress-mediated ER stress and mitochondrial dysfunction via induction of the SIRT1/AMPK pathways. • The present study linked the anti-inflammatory and antioxidant of quercetin to endoplasmic reticulum (ER) stress and mitochondrial dysfunction. • In this study, we focused on the protective effects of quercetin on sepsis-induced ER stress and mitochondrial dysfunction via activating the SIRT1/AMPK pathway and its potential therapeutic effects on sepsis-induced ALI. I feel that we provided the evidence that sepsis destroys alveolar epithelial cells by causing ER stress and mitochondrial dysfunction leading to acute lung injury. Notably, quercetin mitigated ER stress and mitochondrial dysfunction through the SIRT1/AMPK pathways both in vivo and in vitro. • To further explore the underlying mechanism of quercetin on ALI, we performed a transcriptome study by RNA-seq. Transcriptome analysis revealed that quercetin markedly elevated the mRNA level of SIRT1 and AMPK. [ABSTRACT FROM AUTHOR]

Aplastic anemia (AA) is a bone marrow failure syndrome with high morbidity and mortality. Bone marrow (BM)-mesenchymal stem cells (MSCs) are the main components of the BM microenvironment, and dysregulation of BM-MSC adipogenic differentiation is a pathologic hallmark of AA. MicroRNAs (miRNAs/miRs) are crucial regulators of multiple pathological processes such as AA. However, the role of miR-30a-5p in the modulation of BM-MSC adipogenic differentiation in AA remains unclear. The present study aimed to explore the effect of miR-30a-5p on AA BM-MSC adipogenic differentiation and the underlying mechanism. The levels of miR-30a-5p expression and family with sequence similarity 13, member A (FAM13A) mRNA expression in BM-MSCs were quantified using reverse transcription-quantitative (RT-q) PCR. The mRNA expression levels of adipogenesis-associated factors [fatty acid-binding protein 4 (FABP4), lipoprotein lipase (LPL), perilipin-1 (PLIN1), peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα)] were analyzed using RT-qPCR. Lipid droplet accumulation was evaluated using Oil Red O staining in BM-MSCs. The interaction between miR-30a-5p and the FAM13A 3′-untranslated region was identified by TargetScan, and a dual-luciferase reporter assay was used to confirm the interaction. The expression levels of FAM13A and Wnt/β-catenin pathway-related proteins were examined via western blotting. The results showed that miR-30a-5p expression levels were significantly elevated in BM-MSCs from patients with AA compared with those in control subjects (iron deficiency anemia). miR-30a-5p expression levels were also significantly increased in adipose-induced BM-MSCs in a time-dependent manner. miR-30a-5p significantly promoted AA BM-MSC adipogenic differentiation, and significantly enhanced the mRNA expression levels of FABP4, LPL, PLIN1, PPARγ and C/EBPα as well as lipid droplet accumulation. miR-30a-5p was also demonstrated to target FAM13A in AA BM-MSCs. FAM13A significantly reduced BM-MSC adipogenic differentiation by activating the Wnt/β-catenin signaling pathway. In conclusion, miR-30a-5p was demonstrated to serve a role in AA BM-MSC adipogenic differentiation by targeting the FAM13A/Wnt/β-catenin signaling pathway. These findings suggest that miR-30a-5p may be a therapeutic target for AA. [ABSTRACT FROM AUTHOR]