Characterization of metallothionein genes from Broussonetia papyrifera: metal binding and heavy metal tolerance mechanisms.

BACKGROUND: Broussonetia papyrifera is an economically significant tree with high utilization value, yet its cultivation is often constrained by soil contamination with heavy metals (HMs). Effective scientific cultivation management, which enhances the yield and quality of B. papyrifera, necessitates an understanding of its regulatory mechanisms in response to HM stress. RESULTS: Twelve Metallothionein (MT) genes were identified in B. papyrifera. Their open reading frames ranged from 186 to 372 bp, encoding proteins of 61 to 123 amino acids with molecular weights between 15,473.77 and 29,546.96 Da, and theoretical isoelectric points from 5.24 to 5.32. Phylogenetic analysis classified these BpMTs into three subclasses: MT1, MT2, and MT3, with MT2 containing seven members and MT3 only one. The expression of most BpMT genes was inducible by Cd, Mn, Cu, Zn, and abscisic acid (ABA) treatments, particularly BpMT2e, BpMT2d, BpMT2c, and BpMT1c, which showed significant responses and warrant further study. Yeast cells expressing these BpMT genes exhibited enhanced tolerance to Cd, Mn, Cu, and Zn stresses compared to control cells. Yeasts harboring BpMT1c, BpMT2e, and BpMT2d demonstrated higher accumulation of Cd, Cu, Mn, and Zn, suggesting a chelation and binding capacity of BpMTs towards HMs. Site-directed mutagenesis of cysteine (Cys) residues indicated that mutations in the C domain of type 1 BpMT led to increased sensitivity to HMs and reduced HM accumulation in yeast cells; While in type 2 BpMTs, the contribution of N and C domain to HMs' chelation possibly corelated to the quantity of Cys residues. CONCLUSION: The BpMT genes are crucial in responding to diverse HM stresses and are involved in ABA signaling. The Cys-rich domains of BpMTs are pivotal for HM tolerance and chelation. This study offers new insights into the structure-function relationships and metal-binding capabilities of type-1 and - 2 plant MTs, enhancing our understanding of their roles in plant adaptation to HM stresses.

ESRP1-mediated biogenesis of circPTPN12 inhibits hepatocellular carcinoma progression by PDLIM2/ NF-κB pathway.

BACKGROUND: Emerging evidence indicates the pivotal involvement of circular RNAs (circRNAs) in cancer initiation and progression. Understanding the functions and underlying mechanisms of circRNAs in tumor development holds promise for uncovering novel diagnostic indicators and therapeutic targets. In this study, our focus was to elucidate the function and regulatory mechanism of hsa-circ-0003764 in hepatocellular carcinoma (HCC). METHODS: A newly discovered hsa-circ-0003764 (circPTPN12) was identified from the circbase database. QRT-PCR analysis was utilized to assess the expression levels of hsa-circ-0003764 in both HCC tissues and cells. We conducted in vitro and in vivo experiments to examine the impact of circPTPN12 on the proliferation and apoptosis of HCC cells. Additionally, RNA-sequencing, RNA immunoprecipitation, biotin-coupled probe pull-down assays, and FISH were employed to confirm and establish the relationship between hsa-circ-0003764, PDLIM2, OTUD6B, P65, and ESRP1. RESULTS: In HCC, the downregulation of circPTPN12 was associated with an unfavorable prognosis. CircPTPN12 exhibited suppressive effects on the proliferation of HCC cells both in vitro and in vivo. Mechanistically, RNA sequencing assays unveiled the NF-κB signaling pathway as a targeted pathway of circPTPN12. Functionally, circPTPN12 was found to interact with the PDZ domain of PDLIM2, facilitating the ubiquitination of P65. Furthermore, circPTPN12 bolstered the assembly of the PDLIM2/OTUD6B complex by promoting the deubiquitination of PDLIM2. ESRP1 was identified to bind to pre-PTPN12, thereby fostering the generation of circPTPN12. CONCLUSIONS: Collectively, our findings indicate the involvement of circPTPN12 in modulating PDLIM2 function, influencing HCC progression. The identified ESRP1/circPTPN12/PDLIM2/NF-κB axis shows promise as a novel therapeutic target in the context of HCC.

Early identification of hepatocellular carcinoma patients at high-risk of recurrence using the ADV score: a multicenter retrospective study.

BACKGROUND: Postoperative recurrence is a vital reason for poor 5-year overall survival in hepatocellular carcinoma (HCC) patients. The ADV score is considered a parameter that can quantify HCC aggressiveness. This study aimed to identify HCC patients at high-risk of recurrence early using the ADV score. METHODS: The medical data of consecutive HCC patients undergoing hepatectomy from The First Affiliated Hospital of Nanjing Medical University (TFAHNJMU) and Nanjing Drum Tower Hospital (NJDTH) were retrospectively reviewed. Based on the status of microvascular invasion and the Edmondson-Steiner grade, HCC patients were divided into three groups: low-risk group (group 1: no risk factor exists), medium-risk group (group 2: one risk factor exists), and high-risk group (group 3: coexistence of two risk factors). In the training cohort (TFAHNJMU), the R package nnet was used to establish a multi-categorical unordered logistic regression model based on the ADV score to predict three risk groups. The Welch's T-test was used to compare differences in clinical variables in three predicted risk groups. NJDTH served as an external validation center. At last, the confusion matrix was developed using the R package caret to evaluate the diagnostic performance of the model. RESULTS: 350 and 405 patients from TFAHNJMU and NJDTH were included. HCC patients in different risk groups had significantly different liver function and inflammation levels. Density maps demonstrated that the ADV score could best differentiate between the three risk groups. The probability curve was plotted according to the predicted results of the multi-categorical unordered logistic regression model, and the best cut-off values of the ADV score were as follows: low-risk ≤ 3.4 log, 3.4 log < medium-risk ≤ 5.7 log, and high-risk > 5.7 log. The sensitivities of the ADV score predicting the high-risk group (group 3) were 70.2% (99/141) and 78.8% (63/80) in the training and external validation cohort, respectively. CONCLUSION: The ADV score might become a valuable marker for screening patients at high-risk of HCC recurrence with a cut-off value of 5.7 log, which might help surgeons, pathologists, and HCC patients make appropriate clinical decisions.

Development and validation of a gradient boosting machine to predict prognosis after liver resection for intrahepatic cholangiocarcinoma.

BACKGROUND: Accurate prognosis assessment is essential for surgically resected intrahepatic cholangiocarcinoma (ICC) while published prognostic tools are limited by modest performance. We therefore aimed to establish a novel model to predict survival in resected ICC based on readily-available clinical parameters using machine learning technique. METHODS: A gradient boosting machine (GBM) was trained and validated to predict the likelihood of cancer-specific survival (CSS) on data from a Chinese hospital-based database using nested cross-validation, and then tested on the Surveillance, Epidemiology, and End Results (SEER) database. The performance of GBM model was compared with that of proposed prognostic score and staging system. RESULTS: A total of 1050 ICC patients (401 from China and 649 from SEER) treated with resection were included. Seven covariates were identified and entered into the GBM model: age, tumor size, tumor number, vascular invasion, number of regional lymph node metastasis, histological grade, and type of surgery. The GBM model predicted CSS with C-Statistics ≥ 0.72 and outperformed proposed prognostic score or system across study cohorts, even in sub-cohort with missing data. Calibration plots of predicted probabilities against observed survival rates indicated excellent concordance. Decision curve analysis demonstrated that the model had high clinical utility. The GBM model was able to stratify 5-year CSS ranging from over 54% in low-risk subset to 0% in high-risk subset. CONCLUSIONS: We trained and validated a GBM model that allows a more accurate estimation of patient survival after resection compared with other prognostic indices. Such a model is readily integrated into a decision-support electronic health record system, and may improve therapeutic strategies for patients with resected ICC.

Effect of g-C3N4 on biodiversity and structure of bacterial community in sediment of Xiangjiang River under tetracycline pressure.

Photocatalysts have been widely prepared and used in wastewater treatment. Although the influence of photocatalyst application on survival and activity of organisms has been examined, its impact on composition and diversity of microbial community is not fully understood. In this study, the impact of photocatalyst g-C3N4 (Graphitic carbon nitride) on microbial communities in riverbed sediments polluted by antibiotic tetracycline (TC) was investigated. The sediment samples collected from the Xiangjiang River of China were exposed to different concentrations of TC, g-C3N4 and TC/g-C3N4 and the bacterial community were analyzed by Illumina sequencing. The results showed that the dominant bacterial phyla were Acidobacteriota, Proteobacteria, Actinobacteriota, and Chloroflexi in the study site. When compared to the control treatments, the application of TC, g-C3N4 and TC/g-C3N4 exhibited distinguishable effects on bacterial community structure in sediments. The presence of TC had greater influence on bacterial composition, while g-C3N4 and TC/g-C3N4 had less influence on bacteria. The diversity and richness of microorganisms in sediment increased under g-C3N4 application and reached the highest values when g-C3N4 was 75 mg/kg. The photocatalyst g-C3N4 restored bacterial community diversity affected by TC, reduced the TC residues in aquatic environment, and eliminated the side effects of TC application in sediments. Our study indicated that g-C3N4 was an environmentally friendly photocatalyst with lightly negative effects on microbial community in riverbed sediments, and could be used for effective remediation of TC-contaminated environments.

Walnut ethylene response factor JrERF2-2 interact with JrWRKY7 to regulate the GSTs in plant drought tolerance.

Juglans regia is a world-famous woody oil plant, whose yield and quality are affected by drought stress. Ethylene-responsive factors (ERFs) play vital role in plant stress response. In current study, to comprehend the walnut molecular mechanism of drought stress response, an ERF transcription factor was clarified from J. regia (JrERF2-2) and its potential function mechanism to drought was clarified. The results showed that JrERF2-2 could be induced significantly by drought. The transgenic Arabidopsis over-expression of JrERF2-2 displayed enhanced growth, antioxidant enzyme vitalities, reactive oxygen species scavenging and proline produce under drought stress. Especial the glutathione-S-transferase (GST) activity and most GST genes' transcription were elevated obviously. Yeast one-hybrid (Y1H) and co-transient expression (CTE) methods revealed that JrERF2-2 could recognize JrGST4, JrGST6, JrGST7, JrGST8, and JrGSTF8 by binding to GCC-box, and recognize JrGST11, JrGST12, and JrGSTN2 by binding to DRE motif. Meanwhile, the binding activity was strengthened by drought stress. Moreover, JrERF2-2 could interact with JrWRKY7 to promote plant drought tolerance; JrWRKY7 could also distinguish JrGST4, JrGST7, JrGST8, JrGST11, JrGST12, and JrGSTF8 via binding to W-Box motif. These results suggested that JrERF2-2 could effectively improve plant drought tolerance through interacting with JrWRKY7 to control the expression of GSTs. JrERF2-2 is a useful plant representative gene for drought response in molecular breeding.

Streptomyces phaeolivaceus sp. nov. and Streptomyces broussonetiae sp. nov., isolated from the leaves and rhizosphere soil of Broussonetia papyrifera.

Two novel actinobacteria, designated strains GY16T and T44T, were isolated from the leaves and rhizosphere soil of Broussonetia papyrifera, respectively. A polyphasic approach was used for determining their taxonomic position. Results of 16S rRNA gene sequence analysis indicated that strain GY16T exhibited highest similarities to Streptomyces cinereoruber subsp. fructofermentans CGMCC 4.1593T (98.82 %), Streptomyces deccanensis KCTC 19241T (98.76 %), Streptomyces scabiei NRRL B-16523T (98.69 %), Streptomyces europaeiscabiei KACC 20186T (98.69 %) and Streptomyces rishiriensis NBRC 13407T (98.69 %), and strain T44T showed 99.2, 99.1, 99.1 and <98.7 % sequence similarities to Streptomyces filipinensis CGMCC 4.1452T, Streptomyces achromogenes subsp. achromogenes DSM 40028T, Streptomyces durhamensis DSM 40539T and other Streptomyces species, respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain GY16T formed an independent subclade, which indicated that strain GY16T should belong to a potential novel species; and strain T44T was closely related to S. filipinensis CGMCC 4.1452T, S. achromogenes subsp. achromogenes DSM 40028T, S. durhamensis DSM 40539T and S. yokosukanensis DSM 40224T. However, the multilocus sequence analysis evolutionary distance, average nucleotide identity and DNA-DNA hybridization values between closely related relatives were far from the species-level thresholds. In addition, phenotypic and chemotaxonomic characteristics further confirmed that strains GY16T and T44T belonged to two distinct species. Based on these results, it is concluded that the isolated strains represent novel species within the genus Streptomyces, for which the names Streptomyces phaeolivaceus sp. nov. (type strain GY16T=CICC 24807T=KCTC 49326T) and Streptomyces broussonetiae sp. nov. (type strain T44T=CICC 24819T=JCM 33918T) are proposed.

Pseudonocardia broussonetiae sp. nov., an endophytic actinomycete isolated from the roots of Broussonetia papyrifera.

A novel endophytic actinomycete, designated strain Gen 01T, was isolated from the roots of Broussonetia papyrifera and characterized by using a polyphasic approach. The predominant cellular fatty acids were iso-C16 : 0, summed feature 3, iso H-C16 : 1, C16 : 0 and iso-C14 : 0. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannosides, phospholipids of unknown structure containing glucosamine inositol, phosphatidylinositol and unidentified phospholipids. The major menaquinone was MK-8 (H4). The DNA G+C content of the genome sequence, consisting of 7 177 725 bp, was 74.5 mol%. Phylogenetic analysis of the full-length 16S rRNA gene sequences showed that strain Gen 01T belongs to the genus Pseudonocardia with the highest sequence similarity to Pseudonocardia petroleophila CGMCC 4.1532T (98.9 %) and lower than 98.7 % similarity to other species of the genus Pseudonocardia with validly published names. The average nucleotide identity and digital DNA-DNAhybridization values between strain Gen 01T and P. petroleophila CGMCC 4.1532T were 84.6 and 30.9 %, respectively. Furthermore, the morphological, physiological and biochemical characteristics were sufficient to categorize strain Gen 01T as being distinct from P. petroleophila CGMCC 4.1532T. Consequently, based on phenotypic and genotypic characteristics, strain Gen 01T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia broussonetiae sp. nov. is proposed. The type strain is Gen 01T (=CICC 24820T=JCM 33840T).

Physiological responses of Broussonetia papyrifera to manganese stress, a candidate plant for phytoremediation.

Effective phytoremediation of Mn contaminated soil requires the selection of a species with good manganese tolerance. Broussonetia papyrifera is an important economic plant and pioneer species, it could be well adapted to drought and saline-alkali environment. In order to understand the effect of Mn stress on B. papyrifera, the effects of different concentrations of Mn (0-50 mmol/L) stress on the growth, morphology, Mn tolerance and physiological indexes of the plant were explored. The results showed that the biomass, surface area, length, root volume, tips, forks, and crossings of B. papyrifera reached the maximum at the Mn concentration of 1 mmol/L. Mn content in the tissue and TF in plants increased with the increase of concentration, while the BCF increased first and then decreased, and the maximum BCF was 0.154 at 10 mmol/L. The accumulation of Mn lead to cell membrane lipid peroxidation, which increased toxic substances in plants, resulting in the increase of MDA and PRO, and affected the synthesis of chlorophyll. However, B. papyrifera could effectively alleviate oxidative stress by increasing the activities of antioxidant enzymes (SOD, POD, CAT), protein and soluble sugar. The results suggested that B. papyrifera had a good oxidative stress mechanism to Mn stress and could be used as candidates for remediation of pollution in mining areas.

New insight into the molecular basis of cadmium stress responses of wild paper mulberry plant by transcriptome analysis.

BACKGROUND: Heavy metal contamination is becoming a limitation to the utilization of soil and the distribution of vegetation. In particular, cadmium (Cd) pollution has had a serious impact on the food chain. Broussonetia papyrifera is a widely distributed pioneer tree species of heavy metal contaminated areas with important economic value. However, little is known about the genomic background of the Cd-tolerance mechanism in B. papyrifera. RESULTS: The CdCl2 responsive physiology was evaluated and proved to be involved in antioxidase activity and active oxygen species (ROS) accumulation. The leaf and root transcriptomes derived from B. papyrifera grown under normal and CdCl2 stress conditions were systematically investigated using the Illumina HiSeq method. A total of 180,678,660 bp (27.1 GB) clean reads were assembled into 589,487 high-quality unigenes, of which 256,025 (43.43% of the total) and 250,251 (42.45% of the total) were aligned in Gene Ontology (GO) and Protein family (Pfam), respectively. A total of 24,414 differentially expressed genes (DEGs) were GO-annotated into 53, 23, 55, and 60 terms from the transcriptomes of root and leaf tissues under Cd stress and control conditions. A total of 117,547 Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO)-annotated DEGs were enriched in at least 47 KEGG pathway terms among the four comparisons. Many genes encoding important transcription factors (e.g., auxin/indole-3-acetic acid (AUX/IAA), basic helix-loop-helix (bHLH), DNA-binding one zinc finger (Dof), and MYB) and proteins involved in plant-pathogen interactions, phenylpropanoid biosynthesis, plant hormone signal transduction, oxidative phosphorylation, carbon fixation, peroxisomes, flavonoid biosynthesis, and glutathione metabolism, among others, were substantially upregulated under CdCl2 stress. CONCLUSIONS: These genes represent important candidates for studying Cd-response mechanisms and molecular biology of B. papyrifera and related species. Our findings provide a genomic sequence resource for functional genetic assignments in B. papyrifera, which will help elucidate the molecular mechanisms of its Cd-stress responses and facilitate the bioremediation of heavy metal contaminated areas via breeding of new stress-tolerant cultivars.

The walnut JrVHAG1 gene is involved in cadmium stress response through ABA-signal pathway and MYB transcription regulation.

BACKGROUND: Vacuolar H+-ATPase (V-ATPase) is a vital protein complex involved in abiotic stress response in plants. The G subunit of Juglans regia (JrVHAG1) was previously identified as a drought tolerance-related gene involved in the ABA (abscisic acid)-signal pathway. Heavy metal stress is becoming a major detriment for plant growth, development, and production. In order to understand the role of JrVHAG1, the potential function mechanism of JrVHAG1 exposed to CdCl2 stress was confirmed in this study. RESULTS: Transcription of JrVHAG1 was induced by ABA and increased to 58.89-fold (roots) and 7.38-fold (leaves) and by CdCl2 to 2.65- (roots) and 11.42-fold (leaves) relative to control, respectively. Moreover, when treated simultaneously with ABA and CdCl2 (ABA+CdCl2), JrVHAG1 was up-regulated to 110.13- as well as 165.42-fold relative to control in the roots and leaves, accordingly. Compared to the wild type (WT) Arabidopsis plants, the transgenic plants with overexpression of JrVHAG1 (G2, G6, and G9) exhibited increased seed germination rate, biomass accumulation, proline content, and activities of superoxide dismutase (SOD) and peroxidase (POD) under ABA, CdCl2, and ABA+CdCl2 treatments. In contrast, the reactive oxygen species (ROS) staining, malondialdehyde (MDA) content, hydrogen dioxide (H2O2) content, as well as electrolyte leakage (EL) rates of transgenic seedlings were all lower than those of WT exposed to ABA, CdCl2 and ABA+CdCl2 stresses. Furthermore, a 1200 bp promoter fragment of JrVHAG1 was isolated by analyzing the genome of J. regia, in which the cis-elements were identified. This JrVHAG1 promoter fragment showed expression activity that was enhanced significantly when subjected to the above treatments. Yeast one-hybrid assay and transient expression analysis demonstrated that JrMYB2 specifically bound to the MYBCORE motif and shared similar expression patterns with JrVHAG1 under ABA, CdCl2 and ABA+CdCl2 stress conditions. CONCLUSIONS: Our results suggested that the JrVHAG1 gene functions as a CdCl2 stress response regulator by participating in ABA-signal pathway and MYB transcription regulation network. JrVHAG1 gene is a useful candidate gene for heavy metal stress tolerance in plant molecular breeding.

The walnut transcription factor JrGRAS2 contributes to high temperature stress tolerance involving in Dof transcriptional regulation and HSP protein expression.

BACKGROUND: GRAS transcription factor (TF) family is unique and numerous in higher plants with diverse functions that involving in plant growth and development processes, such as gibberellin (GA) signal transduction, root development, root nodule formation, and mycorrhiza formation. Walnut tree is exposed to various environmental stimulus that causing concern about its resistance mechanism. In order to understand the molecular mechanism of walnut to adversity response, a GRAS TF (JrGRAS2) was cloned and characterized from Juglans regia in this study. RESULTS: A 1500 bp promoter fragment of JrGRAS2 was identified from the genome of J. regia, in which the cis-elements were screened. This JrGRAS2 promoter displayed expression activity that was enhanced significantly by high temperature (HT) stress. Yeast one-hybrid assay, transient expression and chromatin immunoprecipitation (Chip)-PCR analysis revealed that JrDof3 could specifically bind to the DOFCOREZM motif and share similar expression patterns with JrGRAS2 under HT stress. The transcription of JrGRAS2 was induced by HT stress and up-regulated to 6.73-~11.96-fold in the leaf and 2.53-~4.50-fold in the root to control, respectively. JrGRAS2 was overexpressed in Arabidopsis, three lines with much high expression level of JrGRAS2 (S3, S7, and S8) were selected for HT stress tolerance analysis. Compared to the wild type (WT) Arabidopsis, S3, S7, and S8 exhibited enhanced seed germination rate, fresh weight accumulation, and activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and glutathione-S-transferase (GST) under HT stress. In contrast, the Evans blue staining, electrolyte leakage (EL) rates, hydrogen dioxide (H2O2) and malondialdehyde (MDA) content of transgenic seedlings were all lower than those of WT exposed to HT stress. Furthermore, the expression of heat shock proteins (HSPs) in S3, S7, and S8 was significant higher than those in WT plants. The similar results were obtained in JrGRAS2 transient overexpression walnut lines under normal and HT stress conditions. CONCLUSIONS: Our results suggested that JrDof3 TF contributes to improve the HT stress response of JrGRAS2, which could effectively control the expression of HSPs to enhance HT stress tolerance. JrGRAS2 is an useful candidate gene for heat response in plant molecular breeding.

Characterization of a vacuolar H+-ATPase G subunit gene from Juglans regia (JrVHAG1) involved in mannitol-induced osmotic stress tolerance.

KEY MESSAGE: JrVHAG1 is an important candidate gene for plant osmotic tolerance regulation. Vacuolar H+-ATPase (V-ATPase) is important for plant responses to abiotic stress; the G subunit is a vital part of V-ATPase. In this study, a G subunit of V-ATPase was cloned from Juglans regia (JrVHAG1) and functionally characterized. JrVHAG1 transcription was induced by mannitol that increasing 17.88-fold in the root at 12 h and 19.16-fold in the leaf at 96 h compared to that under control conditions. JrVHAG1 was overexpressed in Arabidopsis and three lines (G2, G6, and G9) with highest expression levels were selected for analysis. The results showed that under normal conditions, the transgenic and wild-type (WT) plants displayed similar germination, biomass accumulation, reactive oxygen species (ROS) level, and physiological index. However, when treated with mannitol, the fresh weight, root length, water-holding ability, and V-ATPase, superoxide dismutase, and peroxidase activity of G2, G6, and G9 were significantly higher than those of WT. In contrast, the ROS and cell damage levels of the transgenic seedlings were lower than those of WT. Furthermore, the transcription levels of V-ATPase subunits, ABF, DREB, and NAC transcription factors (TFs), all of which are factors of ABA signaling pathway, were much higher in JrVHAG1 transgenic plants than those in WT. The positive induction of JrVHAG1 gene under abscisic acid (ABA) treatments in root and leaf tissues indicates that overexpression of JrVHAG1 improves plant tolerance to osmotic stress relating to the ABA signaling pathway, which is transcriptionally activated by ABF, DREB, and NAC TFs, and correlated to ROS scavenging and V-ATPase activity.

In planta characterization of a tau class glutathione S-transferase gene from Juglans regia (JrGSTTau1) involved in chilling tolerance.

KEY MESSAGE: JrGSTTau1 is an important candidate gene for plant chilling tolerance regulation. A tau subfamily glutathione S-transferase (GST) gene from Juglans regia (JrGSTTau1, GeneBank No.: KT351091) was cloned and functionally characterized. JrGSTTau1 was induced by 16, 12, 10, 8, and 6 °C stresses. The transiently transformed J. regia showed much greater GST, glutathione peroxidase (GPX), superoxide dismutase (SOD), and peroxidase (POD) activities and lower H2O2, malondialdehyde (MDA), reactive oxygen species (ROS), and electrolyte leakage (EL) rate than prokII (empty vector control) and RNAi::JrGSTTau1 under cold stress, indicating that JrGSTTau1 may be involved in chilling tolerance. To further confirm the role of JrGSTTau1, JrGSTTau1 was heterologously expressed in tobacco, transgenic Line5, Line9, and Line12 were chosen for analysis. The germinations of WT, Line5, Line9, and Line12 were similar, but the fresh weight, primary root length, and total chlorophyll content (tcc) of the transgenic lines were significantly higher than those of WT under cold stress. When cultivated in soil, the GST and SOD activities of transgenic tobacco were significantly higher than those of WT; however, the MDA and H2O2 contents of WT were on average 1.47- and 1.96-fold higher than those of Line5, Line9, and Line12 under 16 °C. The DAB, Evans blue, and PI staining further confirmed these results. Furthermore, the abundances of NtGST, MnSOD, NtMAPK9, and CDPK15 were elevated in 35S::JrGSTTau1 tobacco compared with WT. These results suggested that JrGSTTau1 improves the plant chilling tolerance involved in protecting enzymes, ROS scavenging, and stress-related genes, indicating that JrGSTTau1 is a candidate gene for the potential application in molecular breeding to enhance plant abiotic stress tolerance.

Effect of population density on reproduction in Microtus fortis under laboratory conditions.

Between December 2011 and March 2012, the reproductive characteristics of Microtus fortis reared in the laboratory at different population densities were assessed. In all, 258 male and female voles were randomly divided into 4 groups and reared at densities of 2, 4, 6, and 8 animals per cage (sex ratio: 1:1). The results showed that the pregnancy rate (χ2 = 21.671, df = 3, P < 0.001) and first farrowing interval (F = 12.355, df = 3, P < 0.001) were significantly different among the different population density groups, but the mean litter size (mean ± SD) was not (F = 2.669, df = 3, P > 0.05). In particular, the reproductive index and sex hormone levels showed a significant difference among the different density groups studied.

Morphological variation and expressed sequence tags-simple sequence repeats-based genetic diversity of Aspergillus cristatus in Chinese dark tea.

Introduction: Aspergillus cristatus is a homothallic fungus that is used in the natural fermentation process of Chinese Fuzhuan tea and has been linked to the production of bioactive components. However, not much is known about the variations present in the fungus. To understand the variation of the dominant microorganism, A. cristatus, within dark tea, the present study investigated the genetic and morphological diversity of 70 A. cristatus collected across six provinces of China. Methods: Expressed sequence tags-simple sequence repeats (EST-SSR) loci for A. cristatus were identified and corresponding primers were developed. Subsequently, 15 specimens were selected for PCR amplification. Results: The phylogenetic tree obtained revealed four distinct clusters with a genetic similarity coefficient of 0.983, corresponding to previously identified morphological groups. Five strains (A1, A11, B1, D1, and JH1805) with considerable differences in EST-SSR results were selected for further physiological variation investigation. Microstructural examinations revealed no apparent differentiation among the representative strains. However, colony morphology under a range of culture media varied substantially between strains, as did the extracellular enzymatic activity (cellulase, pectinase, protease, and polyphenol oxidase); the data indicate that there are differences in physiological metabolic capacity among A. cristatus strains. Discussion: Notably, JH1805, B1, and A11 exhibited higher enzymatic activity, indicating their potential application in the production of genetically improved strains. The findings provide valuable insights into species identification, genetic diversity determination, and marker-assisted breeding strategies for A. cristatus.

PIN1 promotes the metastasis of cholangiocarcinoma cells by RACK1-mediated phosphorylation of ANXA2.

BACKGROUND: Cholangiocarcinoma (CCA), a primary hepatobiliary malignancy, is characterized by a poor prognosis and a lack of effective treatments. Therefore, the need to explore novel therapeutic approaches is urgent. While the role of Peptidylprolyl Cis/Trans Isomerase, NIMA-Interacting 1 (PIN1) has been extensively studied in various tumor types, its involvement in CCA remains poorly understood. METHODS: In this study, we employed tissue microarray (TMA), reverse transcription-polymerase chain reaction (RT-PCR), and The Cancer Genome Atlas (TCGA) database to assess the expression of PIN1. Through in vitro and in vivo functional experiments, we investigated the impact of PIN1 on the adhesion and metastasis of CCA. Additionally, we explored downstream molecular pathways using RNA-seq, western blotting, co-immunoprecipitation, immunofluorescence, and mass spectrometry techniques. RESULTS: Our findings revealed a negative correlation between PIN1 overexpression and prognosis in CCA tissues. Furthermore, high PIN1 expression promoted CCA cell proliferation and migration. Mechanistically, PIN1 functioned as an oncogene by regulating ANXA2 phosphorylation, thereby promoting CCA adhesion. Notably, the interaction between PIN1 and ANXA2 was facilitated by RACK1. Importantly, pharmacological inhibition of PIN1 using the FDA-approved drug all-trans retinoic acid (ATRA) effectively suppressed the metastatic potential of CCA cells in a nude mouse lung metastasis model. CONCLUSION: Overall, our study emphasizes the critical role of the PIN1/RACK1/ANXA2 complex in CCA growth and functionality, highlighting the potential of targeting PIN1 as a promising therapeutic strategy for CCA.

The osmium-silicon triple bond: synthesis, characterization, and reactivity of an osmium silylyne complex.

The first silylyne complex of a metal beyond group 6, [Cp*((i)Pr3P)(H)Os≡Si(Trip)][HB(C6F5)3], was prepared by a new synthetic route involving hydride abstraction from silicon. NMR and DFT computations support the presence of a silylyne ligand, and NBO and ETS-NOCV analysis revealed the nature of this Os-Si interaction as a triple bond consisting of a covalent σ bond and two strong π back-donations. The discovery of this complex allowed observations of the first cycloadditions involving a silylyne complex, and terminal alkynes are shown to react via C-H bond additions across the Os≡Si bond.

Ecological function zoning based on ecosystem service bundles and trade-offs: a study of Dongjiang Lake Basin, China.

Although the research framework of ecological function zoning is complex and diverse, there are not many spatially continuous zoning results, which can be effectively applied to watershed management practices. Ecosystem service bundles and trade-offs can identify interactions among multiple ecosystem services, and achieve better social-ecosystem management when applying to ecological function zoning. Taking the Dongjiang Lake Basin, China, as research area, the study used the InVEST model to investigate the trade-offs and synergies of ecosystem services at township and grid scales, respectively. Then, the study conducted ecological function zoning based on the bundles and trade-off intensity among ecosystem services. The results showed that food production showed extremely significant trade-offs with other services in the two scales, in which the trade-off intensity between food production and water purification was the largest, and the water areas were the hotspots of trade-off intensity. Based on the ecosystem service bundles at the township, combined with the trade-off intensity, the watershed was finally divided into four ecological functional zones, namely, agricultural product supply area (southern part in the study area), economic forestry area (northeast regions in the study area), water supply area (western areas of the study area), and forest conservation area (northern areas in the study area), accounting for 29.27%, 14.63%, 17.07%, and 39.03%, respectively. The study contributed to the ecological function maintenance and sustainable development in Dongjiang Lake Basin and provided an important reference in ecological zoning.

RP11-40C6.2 Inactivates Hippo Signaling by Attenuating YAP1 Ubiquitylation in Hepatitis B Virus-associated Hepatocellular Carcinoma.

Background and Aims: Chronic hepatitis caused by hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma (HCC). We investigated the roles of oncogenic HBV infection-associated long noncoding RNAs in HCC. Methods: Bioinformatics analysis of data from the Cancer Genome Atlas (TCGA) was performed to screen potential oncogenic HBV-related lncRNAs. Next, we assessed their expression in clinical samples and investigated their correlation with clinical characteristics. The detailed oncogenic effects were analyzed by performing in vitro and in vivo studies. Results: RP11-40C6.2, an HBV infection-related lncRNA, was identified by analysis of the TCGA-Liver Hepatocellular Carcinoma database. Gene Set Enrichment Analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes revealed a strong association of RP11-40C6.2 with the Hippo signaling pathway. RP11-40C6.2 was overexpressed in HCC patients with HBV infection compared to those without HBV infection. RP11-40C6.2 transcription showed a positive association with HBV-X protein (HBx), but not HBV core protein (HBc) expression, both of which are carcinogenic proteins. Luciferase gene reporter and ChIP assays revealed that YAP1/TAZ/TEADs complex enhanced RP11-40C6.2 transcription by binding to its promoter area. RP11-40C6.2 showed oncogenic characteristics in HCC cell lines and in animal models that were mediated via activation of YAP1. In vitro ubiquitylation assay revealed that RP11-40C6.2 can promote the stabilization of YAP1 by stopping phosphorylation at its s127 residue and further stopping its degradation through binding to 14-3-3. Conclusions: RP11-40C6.2 is an HBV infection-related lncRNA that exerts its oncogenic effects by targeting the Hippo signaling pathway.