Search

Cardiomyocyte apoptosis is critical for the development of viral myocarditis (VMC), which is one of the leading causes of cardiac sudden death in young adults. Our previous studies have demonstrated that elevated calpain activity is involved in the pathogenesis of VMC. This study aimed to further explore the underlying mechanisms. Neonatal rat cardiomyocytes (NRCMs) and transgenic mice overexpressing calpastatin were infected with coxsackievirus B3 (CVB3) to establish a VMC model. Apoptosis was detected with flow cytometry, TUNEL staining, and western blotting. Cardiac function was measured using echocardiography. Mitochondrial function was measured using ATP assays, JC-1, and MitoSOX. Mitochondrial morphology was observed using MitoTracker staining and transmission electron microscopy. Colocalization of dynamin-related protein 1 (Drp-1) in mitochondria was examined using immunofluorescence. Phosphorylation levels of Drp-1 at Ser637 site were determined using western blotting analysis. We found that CVB3 infection impaired mitochondrial function as evidenced by increased mitochondrial ROS production, decreased ATP production and mitochondrial membrane potential, induced myocardial apoptosis and damage, and decreased myocardial function. These effects of CVB3 infection were attenuated by inhibition of calpain both by PD150606 treatment and calpastatin overexpression. Furthermore, CVB3-induced mitochondrial dysfunction was associated with the accumulation of Drp-1 in the outer membrane of mitochondria and subsequent increase in mitochondrial fission. Mechanistically, calpain cleaved and activated calcineurin A, which dephosphorylated Drp-1 at Ser637 site and promoted its accumulation in the mitochondria, leading to mitochondrial fission and dysfunction. In summary, calpain inhibition attenuated CVB3-induced myocarditis by reducing mitochondrial fission, thereby inhibiting cardiomyocyte apoptosis. Calpain is activated by CVB3 infection. Activated calpain cleaves calcineurin A and converts it to active form which could dephosphorylate Drp-1 at Ser637 site. Then, the active Drp-1 translocates from the cytoplasm to mitochondria and triggers excessive mitochondrial fission. Eventually, the balance of mitochondrial dynamics is broken, and apoptosis occurs.

Virus myocarditis (VMC) is a common cardiovascular disease and a major cause of sudden death in young adults. However, there is still a lack of effective treatments. Our previous studies found that calpain activation was involved in VMC pathogenesis. This study aims to explore the underlying mechanisms further. Neonatal rat cardiomyocytes (NRCMs) and transgenic mice overexpressing calpastatin (Tg-CAST), the endogenous calpain inhibitor, were used to establish VMC model. Hematoxylin and eosin and Masson staining revealed inflammatory cell infiltration and fibrosis. An ELISA array detected myocardial injury. Cardiac function was measured using echocardiography. CVB3 replication was assessed by capsid protein VP1. Apoptosis was measured by TUNEL staining, flow cytometry, and western blot. The endoplasmic reticulum (ER) stress-related proteins were detected by western blot. Our data showed that CVB3 infection resulted in cardiac injury, as evidenced by increased inflammatory responses and fibrosis, which induced myocardial apoptosis. Inhibiting calpain, both by PD150606 and calpastatin overexpression, could attenuate these effects. Furthermore, ER stress was activated during CVB3 infection. However, calpain inhibition could downregulate some ER stress-associated protein levels such as GRP78, pancreatic ER kinase-like ER kinase (PERK), and inositol-requiring enzyme-1α (IRE-1α), and ER stress-related apoptotic factors, during CVB3 infection. In conclusion, calpain inhibition attenuated CVB3-induced myocarditis by suppressing ER stress, thereby inhibiting cardiomyocyte apoptosis.

A new Trichoderma asperellum strain, TasT1, was isolated from the rhizosphere of Malus sieversii in Xinjiang Region of China. Its antifungal ability was evaluated by controlling root rot Fusarium oxysporum (Fox68) of M. sieversii. The results showed that TasT1 inhibited the growth of Fox68, with an inhibition rate of 66.7%. After treating Arabidopsis thaliana seedlings with different concentrations of TasT1 fermentation liquor, the root hair density of the seedlings changed significantly. Treatment with 60% fermentation liquor caused the root length to increase by 21%, and the fresh weight by 80%. Furthermore, TasT1 fermentation liquor improved the detoxification ability of A. thaliana seedlings against Fox68 toxin and greatly alleviated leaf wilting. Defense-related enzymes (e.g., peroxidase, POD) activity and malondialdehyde content of A. thaliana increased significantly. In addition, in the TasT1 + Fox68 group, the expression levels of PDF1.2B, PDF1.2C, and PR1 were upregulated by 5.2, 5.1, and 2.2 times respectively after treatment for 3 d. Meanwhile, we used GC–MS analysis of chemical component of TasT1 fermentation liquor and found that TasT1 fermentation liquor include 49 kinds of ingredients which contain a lot of antibacterial substances. Our findings suggest that TasT1 fermentation liquor could induce the accumulation of defense-related enzymes to improve disease resistance and growth of A. thaliana seedlings. TasT1 has the potential to control F. oxysporum (Fox68) and protect M. sieversii.

NAC (NAM, ATAF1/2 and CUC2) transcription factors play critical roles in plant development and abiotic stress responses, and aquaporins have diverse functions in environmental stress responses. In this study, we described the salt-induced transcriptional responses of ThNAC12 and ThPIP2;5 in Tamarix hispida, and their regulatory mechanisms in response to salt stress. Using yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays, we identified that ThNAC12 directly binds to the NAC recognition sequence (NACRS) of the ThPIP2;5 promoter and then activates the ThPIP2;5 expression. Subcellular localization and transcriptional activation assays demonstrated that ThNAC12 was a nuclear protein with a C-terminal transactivation domain. Compared with the corresponding control plants, transgenic plants overexpressing ThNAC12 exhibited enhanced salt tolerance and displayed increased reactive oxygen species (ROS) scavenging capability and antioxidant enzyme activity levels under salt stress. All results suggested that overexpression of ThNAC12 in plants enhanced salt tolerance through modulation of ROS scavenging via direct regulation of ThPIP2;5 expression in T. hispida.

The identification of genes conferring salt tolerance is important to reveal plant salt tolerance mechanisms. Here, we employed yeast expression system combined with high-throughput sequencing to identify genes conferring salt tolerance from Tamarix hispida Willd. A total of 1224 potential genes conferring salt tolerance were identified. Twenty-one genes were randomly selected for functional characterization using transient transformation in T. hispida and stable transformation in Arabidopsis thaliana (L.) Heynh. More than 90% of studied genes are found to confer tolerance to salt stress, indicating that the identified genes are reliable. More than 75% of the identified genes were highly expressed in roots rather than in leaves, suggesting roots play an important role in salt tolerance. The genes belonging to ‘response to stimulus’ were highly accumulated , and these accounted for 32% of the total identified genes. In addition, the processes of ‘protein translation’, ‘osmotic adjustment’, ‘scavenging of free radicals’, ‘photosynthesis, detoxification of cells’, ‘protection of cellular macromolecules’ and ‘maintenance of cellular pH’ play important roles in salt tolerance. This study provides useful information on the salt tolerance mechanism of T. hispida and offers a valuable resource for exploring genes used in salt tolerance breeding.

Determination of Cas9 cutting efficiency to the target sites is important for genome editing. However, this determination can only be made via an in vitro method, as the purification of Cas protein and synthesis of gRNA are necessary. Here, we developed an in vivo method, called transient CRISPR/Cas editing in plants (TCEP) to determine Cas9 cutting efficiency. The CRISPR/Cas vector for plant transformation mediated by Agrobacterium tumefaciens was constructed as normal. Using the transient transformation method we built, the Cas9 protein and gRNA were transiently expressed and formed a complex to cut its target sites, resulting in dynamic DNA breakage. The broken DNA was quantified using qPCR to measure the efficiencies of Cas9 cutting. We studied the Cas9 cutting efficiencies to different target sites in Betula platyphylla and Populus davidiana×P. bolleana plants using TCEP and an in vitro method. The results of TCEP were consistent with those of the in vitro method, suggesting that the TCEP method is reliable in determining cutting efficiency. Additionally, using the TCEP method, we showed that both heat and sonication treatment significantly improved CRISPR/Cas efficiency. Therefore, the TCEP method has broad application value and can not only be used to analyze the CRISPR/Cas efficiency but also to determine the factors involved in Cas9 cutting.

The homeodomain-leucine zipper (HD-Zip) proteins play crucial roles in plant developmental and environmental responses. However, how they mediate gene expression to facilitate abiotic stress tolerance remains unknown. In the present study, we characterized BpHOX2 (encoding a HD-Zip I family protein) from birch (Betula platyphylla). BpHOX2 is predominately expressed in mature stems and leaves, but expressed at a low level in apical buds and roots, suggesting that it has tissue-specific characteristics. BpHOX2 expression was highly induced by osmotic and salt, but only slightly induced by abscisic acid. Overexpression of BpHOX2 markedly improved osmotic tolerance, while knockdown of BpHOX2 increased sensitivity to osmotic stress. BpHOX2 could induce the expression of pyrroline-5-carboxylate synthase, peroxidase, and superoxide dismutase genes to improve proline levels and the reactive oxygen species scavenging capability. Chromatin immunoprecipitation sequencing combined with RNA sequencing showed that BpHOX2 could bind to at least four cis-acting elements, including dehydration-responsive element "RCCGAC", Myb-p binding box "CCWACC," and two novel cis-acting elements with the sequences of "AAGAAG" and "TACGTG" (termed HBS1 and HBS2, respectively) to regulate gene expression. Our results suggested that BpHOX2 is a transcription factor that binds to different cis-acting elements to regulate gene expression, ultimately improving osmotic tolerance in birch.

Chromatin immunoprecipitation (ChIP) is the gold-standard method for detection of interactions between proteins and chromatin and is a powerful tool for identification of epigenetic modifications. Although ChIP protocols for plant species have been developed, many specific features of plants, especially woody plants, still hinder the efficiency of immunoprecipitation, resulting in inefficient ChIP enrichment and an active demand for a highly efficient ChIP protocol. In this study, using birch (Betula platyphylla) and Arabidopsis (Arabidopsis thaliana) as the research materials, we identified five factors closely associated with ChIP efficiency, including crosslinking, concentration of chromatin using centrifugal filters, use of a different immunoprecipitation buffer, rescue of DNA with proteinase K, and use of Suc to increase immunoprecipitation efficiency. Optimization of any these factors can significantly improve ChIP efficiency. Considering these factors together, we developed a robust ChIP protocol that achieved a 14-fold improvement in ChIP enrichment for birch and a >6-fold improvement for Arabidopsis compared to the standard ChIP method. As this ChIP method works well in both birch and Arabidopsis, it should also be suitable for other woody and herbaceous species. In addition, this ChIP method enables detection of low-abundance transcription factor-DNA interactions and may extend the application of ChIP in the plant kingdom.

The Japanese flying squid (Todarodes pacificus) is an important fishery resource in East Asia, including Japan, China, and Korea. Recent studies have suggested that it exhibits significant interannual variation with a close relationship with environmental variables, among which water temperature has an apparent influence on its spawning, hatching, and growth. Therefore, we need to understand how the Japanese flying squid in the Japan Sea (JS) will be affected by global warming. Based on particle tracking methods, we simulated migration and individual growth of Japanese flying squid from spawning to juveniles (182 days) in six cases: one for high-level catch years (1960–1966), one for low-level catch years (2008–2014), and four for the future, including two for 50 years (2056–2062) and two for 100 years from now (2104–2110) under the warming scenario of RCP2.6 and RCP8.5, respectively. The simulations showed that the number of squid increased with warming. The main reason for the increase was the warming of the JS, which increased the survival rate of autumn- and winter-spawning cohorts. In addition, the model also demonstrated the potential effects of warming on the physiological process of the early life stage of the Japanese flying squid.

Auxin response factors (ARFs) play an important role in modulating plant growth and development processes by regulating the expression of auxin-responsive genes. However, the modes of action of ARFs in birch (Betula platyphylla) remain largely unknown. In this study, fifteen ARF genes were identified in the birch (B. platyphylla) genome. Bioinformatics analysis revealed that the 15 BpARF genes were unevenly distributed on 7 chromosomes. The 15 BpARF proteins clustered into 6 groups, and all of them contained ARF and B3 motifs. The cis-acting elements present within the promoters of the BpARF genes were mostly related to stress resistance. Expression analysis revealed that most of the BpARF genes were significantly upregulated or downregulated in response to drought treatment in at least one organ. In particular, the expression of BpARF1 was significantly induced by drought stress. The function of BpARF1 was further studied via a transient transformation system. Under drought stress conditions, compared with vector control plants, BpARF1 RNA interference (RNAi)-inhibited plants presented reduced reactive oxygen species (ROS) accumulation, enhanced peroxide (POD) and superoxide dismutase (SOD) activities, increased ascorbic acid (AsA) and proline contents, and reduced electrolyte leakage and water loss rates. Conversely, BpARF1 overexpression plants displayed the opposite physiological changes. These results suggest that the silencing of BpARF1 can improve the drought tolerance of B. platyphylla.

Chronic tissue injury with fibrosis results in the disruption of tissue architecture, organ dysfunction, and eventual organ failure. Therefore, the development of effective antifibrotic drugs is urgently required. IMB-S7 is novel biphenyl compound derived from bifendate (biphenyldicarboxylate) that is used for the treatment of chronic hepatitis in China. In the current study we investigated the potential of IMB-S7 as an antihepatic fibrosis agent. In bile duct ligation (BDL) rat model, oral administration of IMB-S7 (400 mg· kg−1· d−1, for 14 days) significantly ameliorated BDL-induced liver necrosis, bile duct proliferation, and collagen accumulation. We then showed that IMB-S7 treatment markedly suppressed the TGF-β/Smad pathway in human hepatic stellate cell line LX2 and mouse primary HSCs, as well as in liver samples of BDL rats, thus inhibiting the transcription of most fibrogenesis-associated genes, including TGF-β1, COL1A1, and ACTA2. Furthermore, IMB-S7 treatment significantly suppressed the expression of integrin αv at the mRNA and protein levels in TGF-β-treated LX2 cells and liver samples of BDL rats. Using integrin αv overexpression and silencing, we demonstrated that integrin αv activity correlated positively with the activation of TGF-β/Smad pathway. Based on dual luciferase assay and DNA affinity precipitation assay, we revealed that IMB-S7 inactivated integrin αv through competitively inhibiting the binding of Sp1, a transcription factor, to the integrin αv (ITGAV) promoter (−173/−163 bp). These results suggest that IMB-S7 inhibits HSCs activation and liver fibrosis through Sp1-integrin αv signaling, and IMB-S7 may be a promising candidate to combat hepatic fibrosis in the future.

Background: Preventing suicide among adolescents is an urgent global public-health challenge, especially in Africa. Accordingly, the aim of this study was to examine the relationship between the early initiation (< 12 years old) of substance use (cigarette smoking, alcohol use, and drug use) and attempted suicide among in-school adolescents in seven African countries.Methods: Data on the early initiation of substance use and on attempted suicide among in-school adolescents over the previous 12 months in Benin, Liberia, Mauritius, Mozambique, Namibia, Seychelles, and the United Republic of Tanzania were collected from Global School-based Student Health Surveys and were pooled to determine the overall prevalence of these behaviors in adolescents. Univariate and multivariate logistic regressions were then performed to evaluate country-specific associations between the early initiation of substance use and attempted suicide in these adolescents, followed by meta-analyses to evaluate overall pooled associations.Results: In the abovementioned seven African low- or middle-income countries (LMICs), overall weighted prevalences of attempted suicide and early initiation of cigarette smoking, alcohol use, and drug use among in-school adolescents were 16.05, 7.76, 17.68, and 3.48%, respectively. Multivariate logistic regression analyses revealed that relative to non-smoking, the early initiation of smoking was significantly associated with attempted suicide in these adolescents [OR (95% CI) = 1.783 (1.219–2.348)]. Additionally, the relationship between early initiation of cigarette smoking and attempted suicide is mostly driven by a higher association in girls [OR (95% CI) = 1.867 (1.031–2.703)] than boys [OR (95% CI) = 1.392 (0.995−1.789)]. Moreover, relative to not using other drugs, the early and later initiation of other drug use were also significantly associated with attempted suicide in these adolescents [ORs (95% CIs) = 2.455 (1.701–3.208) and 1.548 (1.198–1.898)].Conclusion: Programs that can eliminate or decrease the early initiation of substance use among adolescents should be implemented in African LMICs to prevent subsequent suicide attempts, especially among adolescent girls.

S1Fa-like transcription factors (TFs) are small molecular weight proteins that contain both nuclear localization and DNA binding domains. However, the functions of S1Fa-like TFs are poorly understood. In the present study, we identified the S1Fa-like TFs from the Populus trichocarpa genome, which revealed two S1Fa-like TF genes, PtS1Fa1 and PtS1Fa2. PtS1Fa1 and PtS1Fa2 expression was suppressed by drought and salt stress, and was also significantly altered by ABA, MeJA, or SA treatment. Both PtS1Fa1 and PtS1Fa2 are nuclear proteins. Transgenic P. trichocarpa plants overexpressing PtS1Fa1 and PtS1Fa2, respectively, were generated. The plants overexpressing PtS1Fa2 showed increased fresh weight, chlorophyll content, and root length and weight compared with those in wild-type (WT) P. trichocarpa under drought conditions. Meanwhile, these phenotype traits of plants overexpressing PtS1Fa1 were similar to those of WT plants. Furthermore, overexpression of PtS1Fa2 reduced the malondialdehyde (MDA) content, electrolyte leakage, H2O2 and O2- contents, and increased superoxide dismutase (SOD) and peroxidase (POD) activities. The expression of SOD and POD was also induced by PtS1Fa2. However, overexpression of PtS1Fa1 failed to affect any of these physiological parameters or SOD and POD gene expression. These results suggested that PtS1Fa2 plays a role in drought tolerance, and confers drought tolerance by increase antioxidant activity to reduce reactive oxygen species (ROS) accumulation.

MotivationData normalization is an essential step to reduce technical variation within and between arrays. Due to the different karyotypes and the effects of X chromosome inactivation, females and males exhibit distinct methylation patterns on sex chromosomes, thus it poses a significant challenge to normalise sex chromosome data without introducing bias. Currently, existing methods do not provide unbiased solutions to normalise sex chromosome data, usually, they just process autosomal and sex chromosomes indiscriminately.ResultsHere, we demonstrate that ignoring this sex difference will lead to introducing artificial sex bias, especially for thousands of autosomal CpGs. We present a novel two-step strategy (interpolatedXY) to address this issue, which is applicable to all quantile-based normalisation methods. By this new strategy, the autosomal CpGs are first normalised independently by conventional methods, such as funnorm or dasen; then the corrected methylation values of sex chromosome linked CpGs are estimated as the weighted average of their nearest neighbours on autosomes. The proposed two-step strategy can also be applied to other non-quantile-based normalisation methods, as well as other array-based data types. Moreover, we propose a useful concept: the sex explained fraction of variance, to quantitatively measure the normalisation effect.AvailabilityThe proposed methods are available by calling the function ‘adjustedDasen’ or ‘adjustedFunnorm’ in the latest wateRmelon package (https://github.com/schalkwyk/wateRmelon), with methods compatible with all the major workflows, including minfi.Contactxzhai@essex.ac.uk; lschal@essex.ac.ukSupplementary informationSupplementary data are available at...

Trichoderma can induce plant hormone signal pathways mediating plant defenses, resulting in broad-spectrum resistance to phytopathogens. Herein, Malus sieversii seedlings were treated with Trichoderma biofertilizer and/or Alternaria alternata f. sp. mali, and transcriptome analysis revealed significant differential expression. There was a high similarity between the transcriptome expression profiles of Trichoderma-induced and A. alternata-infected M. sieversii samples for genes related to jasmonic acid (JA), ethylene, and salicylic acid (SA) signaling pathways. Additionally, Trichoderma biofertilizer activated numerous disease-resistant genes (ERF, NAC, bHLH, and STK) and defense response genes (DRP, ABC, and HSP). Among transcription factors, members of the ERF family were the most differentially expressed (18 ERFs), indicating that they may be closely related to defense responses. Among ERFs, differential expression of MsERF105 was the most significant (upregulated 27.6-fold compared to controls). MsERF105 was heterologously expressed in PdPap poplar (Populus davidiana × Populus alba var. pyramidalis Louche), and following infection with A. alternata (Aal), transgenic PdPap-MsERF105s plants displayed lower malondialdehyde (downregulated 41.4%) and reactive oxygen species (ROSs) levels, and higher reductase activities, especially superoxide dismutase (SOD; upregulated 77.5% compared to PdPap-ROK2 plants). Furthermore, the lesion areas of PdPap-MsERF105s leaves were significantly smaller (0.2%) than those of PdPap-ROK2 leaves (∼26.0%), and the cell membrane integrity was superior for PdPap-MsERF105s leaves. Thus, MsERF105 enhanced the resistance of PaPap poplar to Aal, presumably because MsERF105 activates the expression of PR1 and PDF1.2. In conclusion, Trichoderma biofertilizer modulated the differential expression of numerous disease resistance genes and defense response genes in M. sieversii in response to pathogen attack, and MsERF105 played important roles in this process.

Background: Diabetic kidney disease (DKD) is a leading cause of chronic kidney disease in China. Tubular injury contributes to the progression of DKD. Our study was conducted to explore the differential gene expression profiles between kidneys from patients with DKD and kidney living donors (LDs).Methods: In total, seven DKD and eighteen LD gene expression profiles from the GSE104954 dataset were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were analyzed in R with the limma package. DEGs were uploaded to the g:Profiler online database to explore the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Ingenuity pathway analysis (IPA) was carried out using online IPA software. Weighted gene co-expression network analysis (WGCNA) was performed using the WGCNA R package. By integrating DEGs and genes from the top 1 phenotype-gene associated module, we determined the hub gene. We next tested the hub gene, VCAN, in the GSE30122 dataset. We also validated the versican levels in human kidney tissues, explored immune cell type enrichment using an online database xCell, and investigated the correlation between cell types and VCAN expression.Results: A total of 563 DEGs was identified. A large number of pathways were involved in the immune response process according to the results of GO, KEGG, and IPA. Using WGCNA, we selected the lightcyan module in which genes showed the strongest correlation with the phenotype and smallest P-value. We also identified VCAN as a hub gene by integrating DEG analysis and WGCNA. Versican expression was upregulated in human diabetic kidney tissue. Moreover, versican was speculated to play a role in immune injury according to the enrichment of functions and signaling pathways. VCAN transcript levels correlate with the assembly of immune cells in the kidney.Conclusion: Immune processes played an essential role in DKD tubulointerstitium injury. The hub gene VCAN contributed to this process.

Sex differences are known to play a role in disease etiology, progression and outcome. Previous studies have revealed autosomal epigenetic differences between males and females in some tissues, including differences in DNA methylation patterns. Here, we report for the first time an analysis of autosomal sex differences in DNAme using the Illumina EPIC array in human whole blood (n=1171). We identified 554 sex-associated differentially methylated CpG sites (saDMPs) with the majority found to be hypermethylated in females (70%). These saDMP’s are enriched in CpG islands and CpG shores and located preferentially at 5’UTRs, 3’UTRs and enhancers. Additionally, we identified 311 significant sex associated differentially methylated regions (saDMRs). Transcription factor binding site enrichment revealed enrichment of transcription factors related to critical developmental processes and sex determination such as SRY and SOX9. Our study reports a reliable catalogue of sex associated CpG sites and elucidates several characteristics of these sites.

Background DNA binding with one finger (Dof) proteins are plant-specific transcription factors playing vital roles in developmental processes and stress responses in plants. Nevertheless, the characterizations, expression patterns, and functions of the Dof family under drought stress (a key determinant of plant physiology and metabolic homeostasis) in woody plants remain unclear. Methods The birch (Betula platyphylla var. mandshuric) genome and plant TFDB database were used to identify Dof gene family members in birch plants. ClustalW2 of BioEdit v7.2.1, MEGA v7.0, ExPASy ProtParam tool, Subloc, TMHMM v2.0, GSDS v2.0, MEME, TBtools, KaKs Calculator v2.0, and PlantCARE were respectively used to align the BpDof sequences, build a phylogenetic tree, identify the physicochemical properties, analyze the chromosomal distribution and synteny, and identify the cis-elements in the promoter regions of the 26 BpDof genes. Additionally, the birch seedlings were exposed to PEG6000-simulated drought stress, and the expression patterns of the BpDof genes in different tissues were analyzed by qRT-PCR. The histochemical staining and the evaluation of physiological indexes were performed to assess the plant tolerance to drought with transient overexpression of BpDof4, BpDof11, and BpDof17 genes. SPSS software and ANOVA were used to conduct all statistical analyses and determine statistically significant differences between results. Results A total of 26 BpDof genes were identified in birch via whole-genome analysis. The conserved Dof domain with a C(x)2C(x)21C(x)2C zinc finger motif was present in all BpDof proteins. These birch BpDofs were classified into four groups (A to D) according to the phylogenetic analysis of Arabidopsis thaliana Dof genes. BpDof proteins within the same group mostly possessed similar motifs, as detected by conserved motif analysis. The exon–intron analysis revealed that the structures of BpDof genes differed, indicating probable gene gain and lose during the BpDof evolution. The chromosomal distribution and synteny analysis showed that the 26 BpDofs were unevenly distributed on 14 chromosomes, and seven duplication events among six chromosomes were found. Cis-acting elements were abundant in the promoter regions of the 26 BpDof genes. qRT-PCR revealed that the expression of the 26 BpDof genes was differentially regulated by drought stress among roots, stems, and leaves. Most BpDof genes responded to drought stress, and BpDof4, BpDof11, and BpDof17­ were significantly up-regulated. Therefore, plants overexpressing these three genes were generated to investigate drought stress tolerance. The BpDof4-, BpDof11-, and BpDof17­-overexpressing plants showed promoted reactive oxygen species (ROS) scavenging capabilities and less severe cell damage, suggesting that they conferred enhanced drought tolerance in birch. This study provided an in-depth insight into the structure, evolution, expression, and function of the Dof gene family in plants.

Allograft rejection is a common immunological feature in renal transplantation and is associated with reduced graft survival. A mouse renal allograft rejection model was induced and single-cell RNA sequencing (scRNA-seq) data of CD45+leukocytes in kidney allografts on days 7 (D7) and 15 (D15) after operation was analyzed to reveal a full immunological profiling. We identified 20 immune cell types among 10,921 leukocytes. Macrophages and CD8+T cells constituted the main populations on both timepoints. In the process from acute rejection (AR) towards chronic rejection (CR), the proportion of proliferating and naïve CD8+T cells dropped significantly. Both B cells and neutrophils decreased by about 3 folds. On the contrary, the proportion of macrophages and dendritic cells (DCs) increased significantly, especially by about a 4.5-fold increase in Ly6cloMrc1+macrophages and 2.6 folds increase in Ly6cloEar2+macrophages. Moreover, myeloid cells harbored the richest ligand and receptor (LR) pairs with other cells, particularly for chemokine ligands such as Cxcl9, Cxcl10, Cxcl16 and Yars. However, macrophages with weak response to interferon gamma (IFNg) contributed to rejection chronicization. To conclude, reduction in CD8 T cells, B cells and neutrophils while increasing in Ly6cloMrc1+macrophages and Ly6cloEar2+macrophages, may contribute significantly to the progress from AR towards CR.

Background Sex is an important covariate of epigenome-wide association studies due to its strong influence on DNA methylation patterns across numerous genomic positions. Nevertheless, many samples on the Gene Expression Omnibus (GEO) frequently lack a sex annotation or are incorrectly labelled. Considering the influence that sex imposes on DNA methylation patterns, it is necessary to ensure that methods for filtering poor samples and checking of sex assignment are accurate and widely applicable. Results Here we presented a novel method to predict sex using only DNA methylation beta values, which can be readily applied to almost all DNA methylation datasets of different formats (raw IDATs or text files with only signal intensities) uploaded to GEO. We identified 4345 significantly (p Conclusions This proposed sex classifier not only can be used for sex predictions but also applied to identify samples with sex chromosome aneuploidy, and it is freely and easily accessible by calling the ‘estimateSex’ function from the newest wateRmelon Bioconductor package (https://github.com/schalkwyk/wateRmelon).

Background Resistance to anti-tuberculosis (TB) drugs is a major issue in TB control, and demands the discovery of new drugs targeting the virulence factor ESX-1. Methods We first established a high-throughput screen (HTS) assay for the discovery of ESX-1 secretion inhibitors. The positive hits were then evaluated for the potency of diminishing the survival of virulent mycobacteria and reducing bacterial virulence. We further investigated the probability of inducing drug resistance and the underlying mechanism using mycobacterial protein fragment complementation. Results A robust HTS assay was developed to identify small molecules that inhibit ESX-1 secretion without impairing bacterial growth in vitro. A hit named IMB-BZ specifically inhibits the secretion of CFP-10 and reduces virulence in an ESX-1–dependent manner, therefore resulting in significant reduction in intracellular and in vivo survival of mycobacteria. Blocking the CFP-10–EccCb1 interaction directly or indirectly underlies the inhibitory effect of IMB-BZ on the secretion of CFP-10. Importantly, our finding shows that the ESX-1 inhibitors pose low risk of drug resistance development by mycobacteria in vitro as compared with traditional anti-TB drugs, and exhibit high potency against chronic mycobacterial infection. Conclusions Targeting ESX-1 may lead to the development of novel therapeutics for tuberculosis. IMB-BZ holds the potential for future development into a new anti-TB drug.

Background Chlorpyrifos (CPF) is one of the most widely used organophosphorus pesticides globally and can accumulate in the kidney. Researchers have confirmed the regulatory functions of long non-coding ribonucleic acid (lncRNA) in the kidney. However, very few studies have examined the effects of prenatal CPF exposure or lncRNA on kidney development. Methods High-throughput ribonucleic acid (RNA) sequencing was performed on embryonic kidneys obtained at E12.5, E14.5, E16.5, and E18.5 of prenatal CPF-exposed mice and the dimethyl sulfoxide (DMSO) control mice. A weighted gene co-expression network analysis (WGCNA) and a functional enrichment analysis were applied to construct a lncRNA-messenger ribonucleic acid (mRNA) network and screen targeted genes. These strategies were used to select the modules and genes correlated with prenatal CPF exposure in mouse kidney development. Results A gene ontology (GO) analysis revealed that the hub mRNAs linked to prenatal CPF exposure were mainly involved in the extracellular matrix and collagen degradation. Prss1, Prss2, and Prss3 were the most significantly upregulated mRNAs, and all had strong connections to lncRNAs Gm28760, Gm28139, and Gm26717. Additionally, we analyzed the lncRNA-mRNA network at different developmental kidney stages after prenatal CPF exposure. The results showed that kidney development was blocked at E12.5, which led to ectopic proximal tubule formation at E18.5. Conclusions In summary, the RNA-sequencing and weighted gene co-expression network analyses showed that molecular phenotype changes occur in kidney development in a prenatal CPF exposure mouse model.

New drugs with novel antibacterial targets for Gram-negative bacterial pathogens are desperately needed. The protein LpxC is a vital enzyme for the biosynthesis of lipid A, an outer membrane component of Gram-negative bacterial pathogens. The ACHN-975 molecule has high enzymatic inhibitory capacity against the infectious diseases, which are caused by multidrug-resistant bacteria, but clinical research was halted because of its inflammatory response in previous studies. In this work, the structure of the recombinant UDP-3-O-(R-3-hydroxymyristol)-N-acetylglucosamine deacetylase from Aquifex aeolicus in complex with ACHN-975 was determined to a resolution at 1.21 Å. According to the solved complex structure, ACHN-975 was docked into the AaLpxC’s active site, which occupied the site of AaLpxC substrate. Hydroxamate group of ACHN-975 forms five-valenced coordination with resides His74, His226, Asp230, and the long chain part of ACHN-975 containing the rigid alkynyl groups docked in further to interact with the hydrophobic area of AaLpxC. We employed isothermal titration calorimetry for the measurement of affinity between AaLpxC mutants and ACHN-975, and the results manifest the key residues (His74, Thr179, Tyr212, His226, Asp230 and His253) for interaction. The determined AaLpxC crystal structure in complex with ACHN-975 is expected to serve as a guidance and basis for the design and optimization of molecular structures of ACHN-975 analogues to develop novel drug candidates against Gram-negative bacteria.

Plant heat shock transcription factors (HSFs) are involved in heat and other abiotic stress responses. However, their functions in salt tolerance are little known. In this study, we characterized the function of a HSF from Arabidopsis, AtHSFA7b, in salt tolerance. AtHSFA7b is a nuclear protein with transactivation activity. ChIP-seq combined with an RNA-seq assay indicated that AtHSFA7b preferentially binds to a novel cis-acting element, termed the E-box-like motif, to regulate gene expression; it also binds to the heat shock element motif. Under salt conditions, AtHSFA7b regulates its target genes to mediate serial physiological changes, including maintaining cellular ion homeostasis, reducing water loss rate, decreasing reactive oxygen species accumulation, and adjusting osmotic potential, which ultimately leads to improved salt tolerance. Additionally, most cellulose synthase-like (CSL) and cellulose synthase (CESA) family genes were inhibited by AtHSFA7b; some of them were randomly selected for salt tolerance characterization, and they were mainly found to negatively modulate salt tolerance. By contrast, some transcription factors (TFs) were induced by AtHSFA7b; among them, we randomly identified six TFs that positively regulate salt tolerance. Thus, AtHSFA7b serves as a transactivator that positively mediates salinity tolerance mainly through binding to the E-box-like motif to regulate gene expression., The heat shock transcription factor AtHSFA7b binds to an E-box-like cis-acting element to control ion homeostasis, stomatal aperture, ROS accumulation, osmotic potential, and cellulose biosynthesis, which ultimately mediates salt tolerance.

The manipulation of bile acid (BA) homeostasis by blocking the ileal apical Na(+)-dependent bile salt transporter (ASBT/SLC10A2) may have therapeutic effects in nonalcoholic fatty liver disease. We developed a novel ASBT inhibitor, an N-(3,4-o-dichlorophenyl)−2-(3-trifluoromethoxy) benzamide derivative referred to as IMB17–15, and investigated its therapeutic effects and the molecular mechanisms underlying the effects. Syrian golden hamsters were challenged with high-fat diet (HFD) to induce NAFLD and were subsequently administered 400 mg/kg IMB17–15 by gavage daily for 21 days. Serum, liver, and fecal samples were collected for further analysis. Plasma concentration-time profiles of IMB17–15 were also constructed. The human hepatocyte cell line HL-7702 was treated with Oleic acid (OA) with or without IMB17–15. Western blotting and real-time PCR were used to study the molecular mechanisms of IMB17–15. We found that IMB17–15 inhibited ASBT and subsequently suppressed ileal farnesoid X receptor (FXR) and FXR-activated fibroblast growth factor15/19 (FGF15/19) expression, which reduced the hepatic phosphorylated extracellular regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) levels and upregulated the cholesterol 7α-hydroxylase (CYP7A1) activity. Additionally, IMB17–15 stimulated adenosine monophosphate (AMP)-activated protein kinase (AMPKα) phosphorylation and enhanced peroxisome proliferator activated receptor α (PPARα) expression and thus promoted triglyceride (TG) oxidation and high-density lipoprotein cholesterol (HDL-c) metabolism through an ASBT-independent mechanism. In conclusion, a novel ASBT inhibitor known as IMB17–15 protected hamsters against HFD-induced NFALD by manipulating BA and lipid homeostasis. IMB17–15 also reduced lipid deposition in human hepatic cell lines, indicating that it may be useful as a therapy for NAFLD patients.

This study investigates the biocontrol potential of Trichoderma asperellum mutants against Rhizoctonia solani, Alternaria alternata, and Fusarium oxysporum and growth promotion of Populus davidiana × P. alba var. pyramidalis (PdPap poplar) seedlings. A T-DNA insertion mutant library of T. asperellum was constructed using Agrobacterium tumefaciens-mediated transformation. Sixty-five positive transformants (T1–T65) were obtained. Growth rates of the mutants T39 and T45 were the same, 39.68% faster than the WT. In toxin tolerance tests, only T39 had greater tolerance to A. alternata fermentation broth than the WT, but mutant T45 had the same tolerance as the WT to all fermentation broths. Furthermore, T39 and T45 had a greater antagonistic ability than the WT strain against R. solani and A. alternata. The inhibition rate of the mutants T39 and T45 against A. alternata are 73.92% and 80.76%, respectively, and 63.51% and 63.74%, respectively. Furthermore, the three strains increased the activities of superoxide dismutases, peroxidase, catalase (CAT) and phenylalanine ammonia lyase (PAL) in PdPap seedling leaves. CAT and PAL activity in the PdPap seedling leaves was 11.25 and 5.50 times higher, respectively, in the presence of T39 than in the control group and 12 and 6.35 times higher, respectively, in the presence of T45 than in the control group. All three strains promoted seedling growth and the root and stem development, especially mutant T45. Mutants T39 and T45 reduced the incidence of pathogenic fungi in poplar and stimulated poplar seedling growth.

MYB plays vital roles in regulating proliferation and differentiation of hematopoietic progenitor cells, dysregulation of MYB has been implicated in the pathogenesis of leukemia. Although the transcription of MYB has been well studied, its detailed underlying regulatory mechanisms still remain elusive. Here, we detected the long-range interaction between the upstream regions, −34k and −88k, and the MYB promoter in K562, U937, and HL-60 cells using circularized chromosome conformation capture (4C) assay, which declined when MYB was downregulated during chemical-induced differentiation. The enrichment of enhancer markers, H3K4me1 and H3K27ac, and enhancer activity at the −34k and −88k regions were confirmed by ChIP-qPCR and luciferase assay respectively. ChIP-qPCR showed the dynamic binding of GATA1, TAL1, and CCAAT/enhancer-binding protein (C/EBPβ) at −34k and −88k during differentiation of K562 cells. Epigenome editing by a CRISPR-Cas9-based method showed that H3K27ac at −34k enhanced TF binding and MYB expression, while DNA methylation inhibited MYB expression. Taken together, our data revealed that enhancer elements at −34k are required for MYB expression, TF binding, and epigenetic modification are closely involved in this process in human myeloid leukemia cells.

Betula L. (birch) is a pioneer hardwood tree species with ecological, economic, and evolutionary importance in the Northern Hemisphere. We sequenced the Betula platyphylla genome and assembled the sequences into 14 chromosomes. The Betula genome lacks evidence of recent whole-genome duplication and has the same paleoploidy level as Vitis vinifera and Prunus mume. Phylogenetic analysis of lignin pathway genes coupled with tissue-specific expression patterns provided clues for understanding the formation of higher ratios of syringyl to guaiacyl lignin observed in Betula species. Our transcriptome analysis of leaf tissues under a time-series cold stress experiment revealed the presence of the MEKK1–MKK2–MPK4 cascade and six additional mitogen-activated protein kinases that can be linked to a gene regulatory network involving many transcription factors and cold tolerance genes. Our genomic and transcriptome analyses provide insight into the structures, features, and evolution of the B. platyphylla genome. The chromosome-level genome and gene resources of B. platyphylla obtained in this study will facilitate the identification of important and essential genes governing important traits of trees and genetic improvement of B. platyphylla.

The TabZIP15 gene encoding a 396 amino acid (aa) polypeptide in the fungus Trichoderma asperellum ACCC30536 was cloned and characterised. The protein includes a basic region motif (NR-x2-QR-x2-R) and has a pillar-like structure. The 25 basic region/leucine zipper transcription factors (TFs) identified in the T. asperellum genome were divided into YAP (14 TFs), ATF2 (5), GCN4 (2), Zip1 (2), BRLZ (1) and u1 (1) subfamilies based on conserved domains. T. asperellum was cultured in minimal media (MM) control, C-Hungry and N-Hungry medium (to simulate nutrient competition and interaction with pathogens, respectively), and differential expression analysis showed that 14 TabZIP genes (including TabZIP15) were significantly altered under both conditions; TabZIP23 responded strongly to N-Hungry media and TabZIP24 responded strongly to C-Hungry media. However, only YAP genes TabZIP15, TabZIP12 and TabZIP2 were significantly upregulated under both conditions, and expression levels of TabZIP15 were highest. T. asperellum was also cultured in the presence of five fungal pathogenic toxins, and RT-qPCR results showed that TabZIP15 was significantly upregulated in four of the five toxin stress conditions (MM + Rhizoctonia solani, MM + Fusarium oxysporum, MM + Alternaria alternata and MM + Cytospora chrysosperma).

Agrobacterium tumefaciens-mediated gene transfer is the most commonly used method for plant genetic engineering. However, during the period of A. tumefaciens culture, the effects of Agrobacterium culture before inoculation on genetic transformation are poorly understood. In the present study, we investigated the factors that affect the genetic transformation efficiency during Agrobacterium culture using Tamarix hispida as transgenic plant material. Agrobacterium treatment with spermidine (Spe), azacitidine (5-AzaC), dithiothreitol (DTT), or acetosyringone (AS) alone all significantly improved the efficiency of T-DNA transfer. Treatment with 5-AzaC reduced DNA methylation in Agrobacterium to induce the expression of virulence (vir) family genes, including virA, virB1, virC1, virD2, virD4 virE2, and virG. Spe treatment significantly induced the expression of all the studied genes, including virA, virB1, virC1, virD1, virD2, virD4, virE2, and virG. DTT treatment decreased reactive oxygen species accumulation. AS treatment activated the expression of the genes virA, virB1, virC1, virD1, virD2, virD4 and virG. All these effects resulted in increased T-DNA transfer. Additionally, combined Spe, 5-AzaC, DTT, and AS treatment improve Agrobacterium infection to a greater extent compared with their use alone, increasing T-DNA transfer by more than 8-fold relative to no treatment. Therefore, to improve genetic transformation, pretreatment of Agrobacterium during the culture period is important for improving genetic transformation efficiency.

The ETHYLENE INSENSITIVE 3-LIKE (EIL) transcription factor (TF) family plays a key role in the ethylene signal transduction pathway. However, it is still unknown how EIL proteins mediate salt tolerance in Tamarix hispida. Here, we cloned and functionally characterized the TF ThEIL1 from T. hispida. ThEIL1 is a nuclear protein possessing transcriptional activation activity. In addition to binding to the PERE motif (GGATTCAA), a novel EIL protein-DNA interaction was identified, in which ThEIL1 binds to GCC-box (AGCCGCC). Gain- and loss-of-function analysis in T. hispida showed that ThEIL1 regulates the expression of trehalose-6-phosphate synthase (TPS), pyrroline-5-carboxylate synthetase (P5CS), and trehalose-6-phosphate phosphatase (TPP) to mediate the biosynthesis of proline and trehalose, respectively, thereby elevating osmotic potential. Additionally, ThEIL1 controls the activities of peroxidase (POD) and superoxide dismutase (SOD) by regulating the expression of genes encoding SODs and PODs, leading to increased reactive oxygen species scavenging capability. Moreover, overexpression of ThEIL1 in Arabidopsis significantly increased root length and fresh weight under salt treatment conditions. Furthermore, the physiological traits of T. hispida were consistent with those of Arabidopsis stably overexpressing ThEIL1. Thus, ThEIL1 serves as a TF that mediates salt tolerance by elevating osmotic potential and improving ROS scavenging capability.

Due to non-response to chemotherapy, incomplete surgical resection, and resistance to checkpoint inhibitors, breast cancer with bone metastasis is notoriously difficult to cure. Therefore, the development of novel, efficient strategies to tackle bone metastasis of breast cancer is urgently needed. Chemokines, which induce directed migration of immune cells and act as guide molecules between diverse cells and tissues, are small proteins indispensable in immunity. These complex chemokine networks play pro-tumor roles or anti-tumor roles when produced by breast cancer cells in the tumor microenvironment. Additionally, chemokines have diverse roles when secreted by various immune cells in the tumor microenvironment of breast cancer, which can be roughly divided into immunosuppressive effects and immunostimulatory effects. Recently, targeting chemokine networks has been shown to have potential for use in treatment of metastatic malignancies, including bone-metastatic breast cancer. In this review, we focus on the role of chemokines networks in the biology of breast cancer and metastasis to the bone. We also discuss the therapeutic opportunities and future prospects of targeting chemokine networks, in combination with other current standard therapies, for the treatment of bone-metastatic breast cancer.

The inhibition of enhancer of zeste homolog 2 (EZH2) has been suggested to be synthetic lethal with polybromo-1 (PBRM1) deficiency, rendering EZH2 to be an attractive target for the treatment of PBRM1 frequently mutated cancers. In the current study, we combined computational and biochemical approaches to establish an efficient system for the screening and validation of synthetic lethal inhibitors from a large pool of chemical compounds. Five putative EZH2 inhibitors were identified through structure-based virtual screening from 47,737 chemical compounds and analyzed with molecular dynamics. The efficacy of these compounds against EZH2 was tested using PBRM1 deficient and wide-type cell lines. The compound L501-1669 selectively inhibited the proliferation of PBRM1-deficient cells and down-regulated the tri-methylation of histone H3 at Lysine 27 (H3K27me3). Importantly, we also observed an increase in apoptotic activities in L501-1669 treated PBRM1-deficient cells. Taken together, our results demonstrate that L501-1669 is a selective EZH2 inhibitor with promising application in the targeted therapy of PBRM1-deficient cancers.

Chromatin immunoprecipitation (ChIP) is the gold-standard method to detect the interactions between proteins and chromatin, and is a powerful tool to identify epigenetic modifications. Although ChIP protocols for plant species have been developed, many specific features of plants, especially woody plants, still hinder the efficiency of immunoprecipitation, resulting inefficient ChIP enrichment. There is an active demand for a highly efficient ChIP protocol. In the present study, we employed Betula platyphylla (birch) and Arabidopsis thaliana as the research materials, and five factors closely associated with ChIP efficiency were identified, including crosslinking, chromatin concentration using centrifugal filter, using new immunoprecipitation buffer, rescue DNA with proteinase K, and using sucrose to increase immunoprecipitation efficiency. Optimization of any these factors can significantly improve ChIP efficiency. Considering these factors together, a robust ChIP protocol was developed, for which the average fold enrichments were 16.88 and 6.43 fold of that gained using standard ChIP in birch and Arabidopsis, respectively. As this built ChIP method works well in both birch and Arabidopsis, it should be also suitable for other woody and herbaceous species. In addition, this ChIP method make it is possible to detect low-abundance TF-DNA interactions, and may extend the application of ChIP in plant kingdom.One sentence summaryBuilding a ChIP method that increases fold enrichment of birch by 16 folds in average and is adapted for both woody and herbaceous plants.

[Image: see text] Flexible heterocyclic moieties as the P2 ligands of HIV-1 protease inhibitors may be adapted to the minimally distorted active site of mutations easily and enhance activity against DRV-resistant HIV-1 variants. Herein, the design, synthesis, and biological evaluation of a new series of inhibitors containing morpholine derivatives as the P2 ligands were described, among which, carbamate inhibitor 23a and carbamido inhibitor 27a exhibited almost 4- and 2-fold superior activity with enzyme K(i) of 0.092 nM and 0.21 nM, as well as antiviral IC(50) values of 0.41 nM and 0.95 nM, respectively, compared to DRV. Besides, they exhibited excellent activity with inhibition of 94% and 91%, respectively. Furthermore, they also showed appreciable antiviral activity against DRV-resistant HIV-1 variants.

A series of potent HIV-1 protease inhibitors, containing diverse piperidine analogues as the P2-ligands, 4-substituted phenylsulfonamides as the P2'-ligands and a hydrophobic cyclopropyl group as the P1'-ligand, were designed, synthesized and evaluated in this work. Among these twenty-four target compounds, many of them exhibited excellent activity against HIV-1 protease with half maximal inhibitory concentration (IC50) values below 20 nM. Particularly, compound 22a containing a (R)-piperidine-3-carboxamide as the P2-ligand and a 4-methoxylphenylsulfonamide as the P2'-ligand exhibited the most effective inhibitory activity with an IC50 value of 3.61 nM. More importantly, 22a exhibited activity with inhibition of 42% and 26% against wild-type and Darunavir (DRV)-resistant HIV-1 variants, respectively. Additionally, the molecular docking of 22a with HIV-1 protease provided insight into the ligand-binding properties, which was of great value for further study.

Selecting appropriate reference genes is vital to normalize gene expression analysis in birch (Betula platyphylla) under different abiotic stress conditions using quantitative real-time reverse transcription PCR (qRT-PCR). In this study, 11 candidate birch reference genes (ACT, TUA, TUB, TEF, 18S rRNA, EF1α, GAPDH, UBC, YLS8, SAND, and CDPK) were selected to evaluate the stability of their expression in different tissues and under different abiotic stress conditions. Three statistical algorithms (GeNorm, NormFinder, and BestKeeper) were used to analyze the stability of the 11 candidate reference genes to identify the most appropriate one. The results indicated that EF-1α was the most stable reference gene in different birch tissues, ACT was the most stable reference gene for normal conditions, ACT and TEF were the most stable reference genes for salt stress treatment, TUB was the most stable reference gene for osmotic stress treatment, and ACT was the most appropriate choice in all samples of birch. In conclusion, the most appropriate reference genes varied among different experimental conditions. However, in this study, ACT was the optimum reference gene in all experimental groups, except in the different tissues group. GAPDH was the least stable candidate reference gene in all experimental conditions. In addition, three stress-induced genes (BpGRAS1, BpGRAS16, and BpGRAS19) were chosen to verify the stability of the selected reference genes in different tissues and under salt stress. This study laid the foundation for the selection of appropriate reference gene(s) for future gene expression pattern studies in birch.

Aquaporin (AQP) proteins constitute a large family of protein channels that facilitate the transport of water and small neutral solutes through biological membranes, which is important for plants to combat abiotic stress. However, the precise functions of AQP genes involved in abiotic stress are not completely understood in plants. In this study, ThPIP2;5 , an AQP gene of the PIP2 subgroup, was cloned and characterized from Tamarix hispida . Cellular localization assays showed that ThPIP2;5 was localized to the plasma membrane. Transgenic Arabidopsis plants overexpressing ThPIP2;5 displayed improved seed germination rates and increased root growth and fresh weight gain under salt and osmotic stresses, indicating that ThPIP2;5 can improve abiotic stress tolerance. ThPIP2;5- overexpressing and RNAi-silencing T. hispida plants were generated using the transient transformation method and selected for gain- and loss-of ThPIP2;5 analyses, respectively. Overexpression of ThPIP2;5 in transgenic Tamarix and Arabidopsis plants increased ROS-scavenging capability, activities of antioxidant enzymes and proline contents; decreased malondialdehyde (MDA) and H 2 O 2 contents; and reduced electrolyte leakage rates compared to equivalent controls under salt and osmotic stresses. Together, these data suggested that ThPIP2;5 plays an important physiological role in abiotic stress tolerance in transgenic plants by reducing ROS accumulation and membrane damage and increasing the activities of antioxidants.

The transcription factor BplMYB46 has been identified as a regulator of abiotic stress responses and promoter of secondary wall deposition in Betula platyphylla. To investigate the downstream targets of BplMYB46, the expression profiles of genes in stems from BplMYB46-overexpressing (OE) and BplMYB46-silencing (SE) plants were studied. In OE stems, 952 genes were upregulated, and 1469 were downregulated in comparison to SE stems. In a KEGG pathway enrichment analysis of differentially expressed genes (DEGs), 1387 differentially expressed genes were annotated for 117 metabolic pathways. DEGs were abundant for metabolic pathway, secondary metabolite biosynthesis, plant hormone signal transduction, phenylpropanoid and flavonoid biosynthesis. DEGs were implicated lignin or cellulose biosynthesis, cell wall modification, xylem development, disease resistance, stress responses, and anthocyanin biosynthesis. These results suggested that BplMYB46 regulates cell wall development and stress resistance by affecting the expression of these genes. Our study further elucidates the mechanism by which BplMYB46 mediates abiotic stress responses and secondary cell wall biosynthesis in birch.

Research and regulation of tree growth at molecular level is a new trend in the development of forest biotechnology. However, as the limitations of wood as a research material, it is difficult to operate and compare the growth cycle in the laboratory, which greatly hinders study of the molecular biology of forest trees. Arabidopsis thaliana is the most important model plant in botanical research and is an excellent material for plant molecular biology research. Recent studies have shown that herbaceous Arabidopsis can not only provide genetic resources for tree molecular breeding but also can be used as an auxiliary means of wood molecular biology research and even can be directly used as an experimental model for the study of the molecular mechanism of tree development. This paper mainly discusses the establishment process of the tree molecular gene system platform under the background of the Internet of Things and studies the molecular genome of Arabidopsis, combining the characteristics of the tree itself and using the diversity of the frequency of different bases in the gene fragment to cluster the tree molecular genome, which is a molecular biology research and provides an important way of thinking.

Peroxiredoxins (Prxs) are ubiquitous thiol-specific antioxidant enzymes that are critically involved in cell defense and protect cells from oxidative damage. In this study, a putative Type II Prx (ThPrx1) was identified and characterized from Tamarix hispida. The expression of ThPrx1 is highly induced in response to hydrogen peroxide (H2O2) and methyl viologen (MV) stresses. When expressed ectopically, ThPrx1 showed enhanced tolerance against oxidative stress in yeast and Arabidopsis. In addition, transgenic Arabidopsis plants overexpressing ThPrx1 displayed improved seedling survival rates and increased root growth and fresh weight gain under H2O2 and MV treatments. Moreover, transgenic Arabidopsis plants showed decreased accumulation of H2O2, superoxide (O2•−) and malondialdehyde (MDA), increased superoxide dismutase (SOD) activity compared to wild-type (WT) plants under oxidative stress. Moreover, transgenic plants maintained higher photosynthesis efficiency and lower electrolyte leakage rates than that of WT plants under stress conditions. These results clearly indicated that ThPrx1 plays an important role in cellular redox homeostasis under stress conditions, leading to the maintenance of membrane integrity and increased tolerance to oxidative stress.

Ethylene-Responsive Factors (ERFs) are plant-specific transcription factors (TFs) involved in multiple biological processes, especially in abiotic stress tolerance. However, the ERFs from woody halophytes that are involved in salt stress have been little studied. In the present investigation, we characterized a subfamily member of ERF TFs from Tamarix hispida, ThCRF1, which responds to salt stress. ThCRF1 is a nuclear protein that binds to the motifs including TTG, DRE and GCC-box. Transient transformation was performed to generate T. hispida overexpressing ThCRF1 and RNA interference (RNAi)-silenced ThCRF1 to analyze its function using gain- and loss-of-function methods. Overexpression of ThCRF1 in T. hispida significantly improved tolerance to salt-shock-induced stress; by contrast, RNAi-silence of ThCRF1 significantly decreased tolerance to salt-shock-induced stress. Further experiments showed that ThCRF1 induces the expression of genes including those encoding pyrroline-5-carboxylate synthetase (P5CS), trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), superoxide dismutase (SOD) and peroxidase (POD), which lead to enhanced proline and trehalose levels and increased SOD and POD activities. These results were further confirmed by studying transgenic Arabidopsis plants overexpressing ThCRF1. Therefore, the results suggested that ThCRF1 improves tolerance to salt-shock-induced stress by enhancing trehalose and proline biosynthesis to adjust the osmotic potential, and by improving SOD and POD activities to increase reactive oxygen species scavenging capability.

5-aminolevulinic acid mediated PDT (5-ALA-PDT) is an approved therapeutic procedure for treating carcinomas of the cervix. However, when employed as a monotherapy, 5-ALA-PDT could not produce satisfactory results toward large and deep tumors. Therefore, developing a method to improve the efficacy of 5-ALA-PDT becomes important. In this study, we demonstrate an enhanced antitumor effect of 5-ALA-PDT by the modulation of mitochondrial morphology. The mitochondria in the cells were regulated into tubular mitochondria or fragmented mitochondria through over expression of Drp1 or Mfn2. Then these cells were treated with identical dose of 5-ALA-PDT. Our results suggest that HeLa cells predominantly containing fragmented mitochondria were more sensitive to 5-ALA-PDT than the cells predominantly containing tubular mitochondria. The morphology of mitochondria changed as the cell cycle progressed, with tubular mitochondria predominantly exhibited in the S phase and uniformly fragmented mitochondria predominantly displayed in the M phase. Paclitaxel significantly increased the population of M-phase cells, while 5-fluorouracil significantly increased the population of S-phase cells in xenograft tumors. Furthermore, low-dose paclitaxel significantly increased the antitumor effects of PDT. However, 5-fluorouracil didn't improve the antitumor effects of PDT. These results demonstrated an enhanced antitumor effect of 5-ALA-PDT from the modulation of mitochondrial morphology. We anticipate that our results will provide an insight for selecting potential chemotherapeutic agents to combine with PDT for tumor treatment.

Upon the basis of both possible ligand-binding site interactions and the uniformity of key residues in active sites, a novel class of HIV-1 PR/RT dual inhibitors was designed and evaluated. Cinnamic acids or phenylpropionic acids with more flexible chain and smaller steric hindrance were introduced into the inhibitors, giving rise to significant improvement in HIV-1 RT inhibitory activity by one or two orders of magnitude, with comparable or even improved potency against PR at the same time, compared with coumarin anologues in our previous studies. Among these inhibitors, 38d displayed a 19-fold improvement in anti-PR activity with IC50 value of 0.081 nM compared to the control DRV. In addition, inhibitor 38c exhibited an excellent anti-RT IC50 value of 0.43 μM, only a 4.7-fold less potent activity than the control EFV. More significantly, the disparate ratio between HIV-1 PR and RT inhibition became more reasonable with ratio of 1: 10.4, just as 37b. Furthermore, the assays on HIV-1 late stage and early stage supported the rationality of designing dual inhibitors. The SAR data as well as molecular modeling studies provided new insight for further optimization of more potent HIV-1 PR/RT dual inhibitors.

Xylanase secreted by Trichoderma asperellum ACCC30536 can stimulate the systemic resistance of host plants against pathogenic fungi. Following T. asperellum conidia co-culture with Populus davidiana × P. alba var. pyramidalis Louche (PdPap) seedlings, the expression of xylanases TasXyn29.4 and TasXyn24.2 in T. asperellum were upregulated, peaking at 12 h, by 106 (26.74) and 10.1 (23.34)-fold compared with the control, respectively. However, the expression of TasXyn24.4 and TasXyn24.0 was not detected. When recombinant xylanases rTasXyn29.4 and rTasXyn24.2 were heterologously expressed in Pichia pastoris GS115, their activities reached 18.9 IU/mL and 20.4 IU/mL, respectively. In PdPap seedlings induced by rTasXyn29.4 and rTasXyn24.2, the auxin and jasmonic acid signaling pathways were activated to promote growth and enhance resistance against pathogens. PdPap seedlings treated with both xylanases showed increased methyl jasmonate contents at 12 hpi, reaching 122 % (127 μg/g) compared with the control. However, neither of the xylanases could induce the salicylic acid signaling pathway in PdPap seedlings. Meanwhile, both xylanases could enhance the antioxidant ability of PdPap seedlings by improving their catalase activity. Both xylanases significantly induced systemic resistance of PdPap seedlings against Alternaria alternata, Rhizoctonia solani, and Fusarium oxysporum. However, the xylanases could only be sensed by the roots of the PdPap seedlings, not the leaves. In summary, rTasXyn29.4 and rTasXyn24.2 from T. asperellum ACCC30536 promoted growth and induced systemic resistance of PdPap seedlings, which endowed the PdPap seedlings broad-spectrum resistance to phytopathogens.

Heat shock transcription factors (HSFs) play critical roles in several types of environmental stresses. However, the detailed regulatory mechanisms in response to salt stress are still largely unknown. In this study, we examined the salt-induced transcriptional responses of ThHSFA1-ThWRKY4 in Tamarix hispida and their functions and regulatory mechanisms in salt tolerance. ThHSFA1 protein acts as an upstream regulator that can directly activate ThWRKY4 expression by binding to the heat shock element (HSE) of the ThWRKY4 promoter using yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays. ThHSFA1 and ThWRKY4 expression was significantly induced by salt stress and abscisic acid (ABA) treatment in the roots and leaves of T. hispida. ThHSFA1 is a nuclear-localized protein with transactivation activity at the C-terminus. Compared to nontransgenic plants, transgenic plants overexpressing ThHSFA1 displayed enhanced salt tolerance and exhibited reduced reactive oxygen species (ROS) levels and increased antioxidant enzyme activity levels under salt stress. Therefore, we further concluded that ThHSFA1 mediated the regulation of ThWRKY4 in response to salt stress in T. hispida.

As a biocontrol agent, Trichoderma can ameliorate fungal phytopathogen infection of plants by inducing plant resistance, promoting plant growth, and improving the soil environment. Trichoderma rossicum T7 (TroT7) and Trichoderma harzianum T2 (ThaT2) can significantly inhibit the growth of Alternaria alternata, Fusarium spp., and Cytospora mandshurica, with inhibition rates ranging from 34% to 58%. Therefore, spores of TroT7 and ThaT2 were mixed as a biofertilizer (mixTroTha). Soil irrigated with mixTroTha had a decreased population of phytopathogens. Following pretreatment with mixTroTha, the leaves of two-month-old Malus sieversii seedlings infected by A. alternata had improved cell membrane permeability and decreased numbers of disease spots (by 16.67%, in the laboratory). The leaves of three-year-old M. sieversii seedlings also had 6.71% fewer disease spots (in the field). The mechanism might involve the increased jasmonic acid content, which upregulated the expression of PDF1.2 (encoding plant defensin 1.2), by 12.4-fold at 72 h compared with the control. In addition, following Trichoderma colonization of the apple tree rhizosphere, the fungal communities changed significantly: 12 of 14 pathogenic genera showed a lower species abundance, and 4 of 5 biocontrol genera had a higher species abundance. However, the bacterial communities did not change significantly. Moreover, exposure to mixTroTha promoted the growth of lateral roots of three-year-old M. sieversii seedlings, and also accelerated soil metabolic processes, further increasing the contents of available nutrients in soil. Thus, mixTroTha could mediate M. sieversii seedling resistance to A. alternata via diverse methods.

Protein kinase B (PknB) is one of the Mycobacterium tuberculosis serine/threonine protein kinases and has an essential role in sustaining mycobacterial growth. Here, we identified and characterized a novel small molecule compound named IMB-YH-8 that inhibited PknB and served as anti-mycobacteria lead compound. IMB-YH-8 inhibited PknB auto-phosphorylation and the phosphorylation of GarA by PknB in a dose-dependent manner. The compound did not inhibit human Akt1 or other serine/threonine kinases in M. tuberculosis except for the highly homologous PknA. IMB-YH-8 bound to PknB with a moderate affinity. Molecular docking revealed that IMB-YH-8 interacts with the catalytic domain of PknB. Observations of electron microscopy showed that IMB-YH-8 changed the morphology of H37Rv and disrupted the cell wall. The differential transcriptional response of M. tuberculosis to IMB-YH-8 revealed changes in SigH regulatory pathways modulated by PknB. Notably IMB-YH-8 not only potently inhibited drug-sensitive and multidrug-resistant clinical isolates but also exhibited a dose dependent inhibition of intracellular M. tuberculosis. Taken together, these in vitro data demonstrate that IMB-YH-8 is a novel inhibitor of PknB, which potently prevents growth of M. tuberculosis. It is as yet unclear whether inhibition of PknA contributes to the anti-tubercular action of IMB-YH-8.

Our study found that birch employs different physiological pathways to tolerance salt stress in roots and leaves, and the genes closely correlated with these physiological changes were identified. Birches are fast-growing woody plants that are adapted to adverse environments, and are widely distributed from north Europe to northeast Asia. However, the salt stress tolerance mechanism of birch has little been studied. Here, we investigated the physiological and molecular response of white birch (Betula Platyphylla) to salt stress. Long-term salt stress inhibited photosynthetic activity, and decreased stomatal conductance of birch. Abscisic acid was induced in birch during the early salt stress period, and Ca2+ level was increased slowly but maintained at a higher level for a long time. Under salt conditions, the salt-overly-sensitive pathway was activated in birch roots; reactive oxygen species (ROS) was highly accumulated, and superoxide dismutase is the main ROS scavenger in roots, while peroxidase is the main ROS scavenger in leaves. Proline plays a role in salt tolerance in both roots and leaves; however, soluble sugars and trehalose also have roles in salt stress tolerance, but mainly in leaves. Additionally, the genes that might have essential roles in controlling some of these physiological changes were identified, which represent good candidate genes to characterize the salt tolerance mechanism of birch. This study increased our understanding of the salt tolerance mechanism of birch plants.

Identification of the upstream regulators of a gene is important to characterize the transcriptional pathway and the function of the gene. Previously, we found that a zinc finger protein (ThZFP1) is involved in abiotic stress tolerance of Tamarix hispida. In the present study, we further investigated the transcriptional pathway of ThZFP1. Dof motifs are abundant in the ThZFP1 promoter; therefore, we used them to screen for transcriptional regulators of ThZFP1. A Dof protein, ThDof1.4, binds to the Dof motif specifically, and was hypothesized as the upstream regulator of ThZFP1. Further study showed that overexpression of ThDof1.4 in T. hispida activated the expression of GUS controlled by the ThZFP1 promoter. In T. hispida, transient overexpression of ThDof1.4 increased the transcripts of ThZFP1; conversely, transient RNAi-silencing of ThDof1.4 reduced the expression of ThZFP1. Chromatin immunoprecipitation indicated that ThDof1.4 binds to the ThZFP1 promoter. Additionally, ThDof1.4 and ThZFP1 share similar expression patterns in response to salt or drought stress. Furthermore, like ThZFP1, ThDof1.4 could increase the proline level and enhance ROS scavenging capability to improve salt and osmotic stress tolerance. Together, these results suggested that ThDof1.4 and ThZFP1 form a transcriptional regulatory cascade involved in abiotic stress resistance in T. hispida.