BRASSINOSTEROID-INSENSITIVE 2 regulates salt stress tolerance in Arabidopsis by promoting AGL16 activity.

Salt stress is a negative environmental factors to affecting plants. Salinity inhibits seed germination and root growth, which reduces the biomass of agricultural plants. BRASSINOSTEROID-INSENSITIVE2 (BIN2) functions as a signalling hub to integrate the perception and transduction of plant growth and stress tolerance by the phosphorylation of target proteins. However, only a small number of target molecules have been discovered thus far. In this study, we present evidence that BIN2 controls the post-transcriptional activity of AGL16. BIN2 interacts and phosphorylates AGL16, which increases AGL16 stability and transcriptional activity. Genetic testing showed that the agl16 mutant can restore the reduction in the seed germination rate and primary root growth of the bin2-1 mutant, while the overexpression of AGL16 in the bin2-3bil1bil2 mutant reduced the salt tolerance compared with bin2-3bil1bil2 in response to salt stress. Taken together, our data identify a BIN2-AGL16 core protein module that is mediates the inhibition of seed germination and primary root growth under salt stress.

Transcriptome analysis provides insights into light condition effect on paclitaxel biosynthesis in yew saplings.

BACKGROUND: Taxus is a rare gymnosperm plant that is the sole producer of the anticancer drug paclitaxel. The growth and development of Taxus is affected by environmental factors such as light. However, little is known about how light conditions affect growth and metabolic processes, especially paclitaxel biosynthesis. RESULTS: In this study, we applied three different light conditions to Taxus chinensis young saplings and investigated the physiological response and gene expression. Our observations showed that exposure to high light led to oxidative stress, caused photoinhibition, and damaged the photosynthetic systems in T. chinensis. The paclitaxel content in T. chinensis leaves was significantly decreased after the light intensity increased. Transcriptomic analysis revealed that numerous genes involved in paclitaxel biosynthesis and phenylpropanoid metabolic pathways were downregulated under high light. We also analyzed the expression of JA signaling genes, bHLH, MYB, AP2/ERF transcription factors, and the CYP450 families that are potentially related to paclitaxel biosynthesis. We found that several CYP450s, MYB and AP2/ERF genes were induced by high light. These genes may play an important role in tolerance to excessive light or heat stress in T. chinensis. CONCLUSIONS: Our study elucidates the molecular mechanism of the effects of light conditions on the growth and development of T. chinensis and paclitaxel biosynthesis, thus facilitating the artificial regeneration of Taxus and enhancing paclitaxel production.

De novo transcriptome analysis provides insights into the salt tolerance of Podocarpus macrophyllus under salinity stress.

BACKGROUND: Soil salinization is causing ecosystem degradation and crop yield reduction worldwide, and elucidation of the mechanism of salt-tolerant plants to improve crop yield is highly significant. Podocarpus macrophyllus is an ancient gymnosperm species with a unique environmental adaptation strategy that may be attributed to its lengthy evolutionary process. The present study investigated the physiological and molecular responses of P. macrophyllus plants to salt stress by analyzing its photosynthetic system and antioxidant enzyme activity. We also analyzed the differentially expressed genes (DEGs) in P. macrophyllus under salt stress using RNA sequencing and de novo transcriptome assembly. RESULTS: Salt treatment significantly affected the photosynthetic system in P. macrophyllus seedlings, which decreased chlorophyll content, altered chloroplast ultrastructure, and reduced photosynthesis. The activities of antioxidant enzymes increased significantly following salt stress treatment. Transcriptome analysis showed that salt stress induced a large number of genes involved in multiple metabolic and biological regulation processes. The transcription levels of genes that mediate phytohormone transport or signaling were altered. K+ and Ca2+ transporter-encoding genes and the MYB transcription factor were upregulated under salt stress. However, the genes involved in cell wall biosynthesis and secondary metabolism were downregulated. CONCLUSION: Our research identified some important pathways and putative genes involved in salt tolerance in P. macrophyllus and provided clues for elucidating the mechanism of salt tolerance and the utilization of the salt tolerance genes of P. macrophyllus for crop improvement.

Optimization of liquid media and biosafety assessment for algae-lysing bacterium NP23.

To control algal bloom caused by nutrient pollution, a wild-type algae-lysing bacterium was isolated from the Baiguishan reservoir in Henan province of China and identified as Enterobacter sp. strain NP23. Algal culture medium was optimized by applying a Placket-Burman design to obtain a high cell concentration of NP23. Three minerals (i.e., 0.6% KNO3, 0.001% MnSO4·H2O, and 0.3% K2HPO4) were found to be independent factors critical for obtaining the highest cell concentration of 10(13) CFU/mL, which was 10(4) times that of the control. In the algae-lysing experiment, the strain exhibited a high lysis rate for the 4 algae test species, namely, Chlorella vulgari, Scenedesmus, Microcystis wesenbergii, and Chlorella pyrenoidosa. Acute toxicity and mutagenicity tests showed that the bacterium NP23 had no toxic and mutagenic effects on fish, even in large doses such as 10(7) or 10(9) CFU/mL. Thus, Enterobacter sp. strain NP23 has strong potential application in the microbial algae-lysing project.

Effects of Enterobacter cloacae insecticidal protein on the Duox-ROS system and midgut bacterial community and function of Galleria mellonella larvae.

BACKGROUND: Enterobacter cloacae insecticidal proteins have been reported to kill Galleria mellonella larvae through affecting their midgut microbiome. However, the mechanisms involved remain unclear. Here we aim to investigate how the insecticidal proteins act on the midgut Duox-ROS system and microbial community of G. mellonella larvae. METHODS: Reverse transcription qPCR and fluorescence probes were utilized to assess the Duox expression levels and to evaluate quantitative changes of the ROS levels. Sequencing of the 16S rRNA gene sequences of the midgut bacteria of G. mellonella larvae was conducted for further analyses of bacterial diversity, composition, and abundance. RESULTS: After the injection of the insecticidal proteins, the Duox expression levels first increased within 28 h, then dramatically peaked at 36 h, and slowly decreased thereafter. Simultaneously, the ROS levels increased significantly at 36 h, peaked at 48 h, and rapidly declined to the normal level at 60 h. Responsive to the change of the ROS levels, the structure of the midgut microbial community was altered substantially, compared to that of the untreated larvae. The relative abundance of Enterobacteriaceae and other specific pathogenic bacteria increased significantly, whereas that of Lactobacillus decreased sharply. Importantly, notable shifts were observed in the crucial midgut predicted metabolic functions, including membrane transportation, carbohydrate metabolism, and amino acid metabolism. CONCLUSION: Insecticidal proteins of E. cloacae kill G. mellonella larvae mainly through generation of high oxidative stress, alterations of the midgut microbial community and function, and damage to the physiological functions. These findings provide insights into the inhibition mechanism of E. cloacae insecticidal proteins to G. mellonella larvae.

Sonographic evaluations of the skeletal muscles in patients with Pompe disease.

BACKGROUND: Pompe disease usually has muscle weakness due to glycogen accumulation. Heckmatt scale is commonly used to grade the pertinent findings of ultrasound. Nonetheless, it is difficult to detect subtle changes of the muscle. Besides, no ultrasonographic parameter has been proposed to predict the motor functions of Pompe disease. Therefore, we aimed to find out an ultrasonographic parameter that can quantify the muscle involvement and correlate with the motor functions in Pompe disease. METHODS: Eighteen patients with Pompe disease were enrolled. The echo heterogeneity index (standard deviation divided by mean echogenicity values by ImageJ analysis) and shear modulus were recorded from rectus femoris, biceps femoris, tibialis anterior, medial gastrocnemius, biceps brachii and triceps brachii muscles. Motor functions, including manual muscle strength, 6-min walk and four-limb stair climb tests were assessed. Correlations between ultrasonographic parameters and Heckmatt scale and motor functions were analyzed. RESULTS: The echo heterogeneity index, but not the shear modulus, was negatively correlated with the Heckmatt scale rating in all muscles. The echo heterogeneity indices of tibialis anterior (r = 0.698, p = 0.008) and medial gastrocnemius (r = 0.615, p = 0.025) muscles showed positive correlations with the walking distance. Besides, the echo heterogeneity indices of four lower limb muscles were negatively correlated with the duration of stair climbing. CONCLUSION: The echo heterogeneity index but not the shear modulus can be used to quantitatively describe the muscle involvement in Pompe disease. In addition, lower echo heterogeneity indices of lower limb muscles are associated with worse motor functions in these patients.

Interactions between Verticillium dahliae and cotton: pathogenic mechanism and cotton resistance mechanism to Verticillium wilt.

Cotton is widely grown in many countries around the world due to the huge economic value of the total natural fiber. Verticillium wilt, caused by the soil-borne pathogen Verticillium dahliae, is the most devastating disease that led to extensive yield losses and fiber quality reduction in cotton crops. Developing resistant cotton varieties through genetic engineering is an effective, economical, and durable strategy to control Verticillium wilt. However, there are few resistance gene resources in the currently planted cotton varieties, which has brought great challenges and difficulties for breeding through genetic engineering. Further revealing the molecular mechanism between V. dahliae and cotton interaction is crucial to discovering genes related to disease resistance. In this review, we elaborated on the pathogenic mechanism of V. dahliae and the resistance mechanism of cotton to Verticillium wilt. V. dahliae has evolved complex mechanisms to achieve pathogenicity in cotton, mainly including five aspects: (1) germination and growth of microsclerotia; (2) infection and successful colonization; (3) adaptation to the nutrient-deficient environment and competition of nutrients; (4) suppression and manipulation of cotton immune responses; (5) rapid reproduction and secretion of toxins. Cotton has evolved multiple physiological and biochemical responses to cope with V. dahliae infection, including modification of tissue structures, accumulation of antifungal substances, homeostasis of reactive oxygen species (ROS), induction of Ca2+ signaling, the mitogen-activated protein kinase (MAPK) cascades, hormone signaling, and PAMPs/effectors-triggered immune response (PTI/ETI). This review will provide an important reference for the breeding of new cotton germplasm resistant to Verticillium wilt through genetic engineering.

Effects of insecticidal proteins of Enterobacter cloacae NK on cellular immunity of Galleria mellonella larvae.

Enterobacter cloacae produces insecticidal proteins capable of causing toxicity in pests, but the insecticidal mechanisms of these proteins for insect control remain unclear. To elucidate the mechanisms, the purified insecticidal protein from E. cloacae NK was administered to Galleria mellonella larvae either by intraperitoneal injection or by feeding. The number of hemocytes, apoptosis in immune cells, and polyphenol oxidase (PO) activity of G. mellonella larvae were detected by hemocytometer, Annexin V-FITC/PI, and UV-vis spectrophotometer, respectively. With the extension of the invasion time of NK insecticidal protein, the number of hemocytes in G. mellonella larvae decreased significantly (p < 0.05), whereas the apoptosis rate of hemocytes increased. The activity of PO showed a trend of rising-peak-sharp decline and the melanization reaction was deepened simultaneously. Moreover, the phagocytosis and coating capabilities of hemocytes decreased, and the intraperitoneal injection method was more effective than the feeding method. Taking together, the insecticidal protein of E. cloacae NK inhibits and destroys the cellular immune response of G. mellonella larvae, which suggests an important role in killing the host insect.

A Novel Cancer Stemness-Related Signature for Predicting Prognosis in Patients with Colon Adenocarcinoma.

OBJECTIVE: To explore the cancer stemness features and develop a novel cancer stemness-related prognostic signature for colon adenocarcinoma (COAD). METHODS: We downloaded the mRNA expression data and clinical data of COAD from TCGA database and GEO database. Stemness index, mRNAsi, was utilized to investigate cancer stemness features. Weighted gene coexpression network analysis (WGCNA) was used to identify cancer stemness-related genes. Univariate and multivariate Cox regression analyses were applied to construct a prognostic risk cancer stemness-related signature. We then performed internal and external validation. The relationship between cancer stemness and COAD immune microenvironment was investigated. RESULTS: COAD patients with higher mRNAsi score or EREG-mRNAsi score have significant longer overall survival (OS). We identified 483 differently expressed genes (DEGs) between the high and low mRNAsi score groups. We developed a cancer stemness-related signature using fifteen genes (including RAB31, COL6A3, COL5A2, CCDC80, ADAM12, VGLL3, ECM2, POSTN, DPYSL3, PCDH7, CRISPLD2, COLEC12, NRP2, ISLR, and CCDC8) for prognosis prediction of COAD. Low-risk score was associated with significantly preferable OS in comparison with high-risk score, and the area under the ROC curve (AUC) for OS prediction was 0.705. The prognostic signature was an independent predictor for OS of COAD. Macrophages, mast cells, and T helper cells were the vital infiltration immune cells, and APC costimulation and type II IFN response were the vital immune pathways in COAD. CONCLUSIONS: We developed and validated a novel cancer stemness-related prognostic signature for COAD, which would contribute to understanding of molecular mechanism in COAD.

Toxicity of insecticidal proteins from entomopathogenic bacteria to Galleria mellonella larvae.

Entomopathogenic bacteria have great potential in insect control in the agricultural production because they produce a large variety of protein toxins that can kill their hosts by damaging the insect midgut. However, the mechanisms on how these toxins or specific insecticidal proteins act on insects are very diverse and elusive. Here we select Galleria mellonella larvae as the host to explore the effects of insecticidal proteins on the activities of three protective enzymes (SOD, POD, and CAT) and on the morphology of the midgut tissues. As a result, the activities of the three enzymes consistently increased and then decreased when the host was injected with the insecticidal proteins from the entomopathogenic bacterium Enterobacter cloacae. Moreover, the microscopy analysis showed that tissues, cells, and organelles of the host midgut are all diseased after uptake of the insecticidal proteins. Remarkably, the protein toxins contributed to the deformation of the midgut, blackening of the midgut surface, dissolution of cell membrane, shrinkage of cell nucleus, and chromatin condensation. Our findings will advance the explanation of G. mellonella pathogenesis caused by the insecticidal proteins.

Volume Matters in Ultrasound-Guided Perineural Dextrose Injection for Carpal Tunnel Syndrome: A Randomized, Double-Blinded, Three-Arm Trial.

Ultrasound-guided perineural dextrose injection (PDI) has been reported effective for carpal tunnel syndrome (CTS). Higher volume of injectate may reduce adhesion of median nerve from other tissues, but volume-dependent effects of PDI in CTS remain unknown. We aimed to investigate whether PDI with different injectate volumes had different effects for CTS participants. In this randomized, double-blinded, three-arm trial, 63 wrists diagnosed with CTS were randomized into three groups that received ultrasound-guided PDI with either 1, 2 or 4 ml of 5% dextrose water. All participants finished this study. Primary outcome as visual analog scale (VAS) and secondary outcomes including Boston Carpal Tunnel Questionnaire (BCTQ), Disability of the Arm, Shoulder and Hand score (QuickDASH), electrophysiological studies and cross-sectional area (CSA) of the median nerve at carpal tunnel inlet were assessed before and after PDI at the 1st, 4th, 12th and 24th weeks. For within-group analysis, all three groups (21 participants, each) revealed significant improvement from baseline in VAS, BCTQ and QuickDASH at the 1st, 4th, 12th and 24th weeks. For between-group analysis, 4 ml-group yielded better VAS reduction at the 4th and 12th weeks as well as improvement of BCTQ and QuickDASH at the 1st, 4th, and 12th weeks, compared to other groups. No significant between-group differences were observed in electrophysiological studies or median nerve CSA at any follow-up time points. There were no severe complications in this trial, and transient minor adverse effects occurred equally in the three groups. In conclusion, ultrasound-guided PDI with 4 ml of 5% dextrose provided better efficacy than with 1 and 2 ml based on symptom relief and functional improvement for CTS at the 1st, 4th, and 12th week post-injection, with no reports of severe adverse effects. There was no significant difference between the three groups at the 24th-week post-injection follow-up. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT03598322.

DNA interactions and in vitro anticancer evaluations of pyridine-benzimidazole-based Cu complexes.

Copper is an essential element and has redox potential, thus copper complexes have been developed rapidly with the hope of curing cancer. To further develop anticancer agents and investigate their anticancer mechanisms, two Cu complexes, [Cu(bpbb)0.5·Cl·SCN]·(CH3OH) (1) and [Cu2(bpbb)·Br3·(OH)] n (2), were synthesized and characterized using 4,4'-bis((2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)methyl)biphenyl (bpbb), with associated Cu(ii) salts. Complex 1 is a binuclear structure, whereas 2 is a one-dimensional complex. Compared with 2, complex 1 exhibited potent in vitro cytotoxicity toward four cell lines (HCT116, BGC823, HT29, and SMMC7721), and was most effective against HCT116 cells. Therefore, further in-depth investigation was carried out using complex 1. Absorption spectral titration experiments, ethidium bromide displacement assays, and circular dichroism spectroscopic studies suggested that complex 1 binds strongly to DNA by intercalation. Complex 1 exhibited a clear concentration-dependent pBR322 DNA cleavage activity. Inductively coupled plasma mass spectrometry testing implied that complex 1 could enter cells and that DNA was one important target. Cellular level assays suggested that complex 1 activates the generation of intracellular reactive oxygen species, causing DNA damage, promoting cell cycle arrest and mitochondria dysfunction, and inducing cellular apoptosis.

Cytotoxicity, dual-targeting apoptosis induction evaluation of multinuclear cu complexes based on pyrazine-benzimidazole derivative.

To investigate the cytotoxicity and mechanism of action of multinuclear Cu complexes against tumor cell lines, two complexes, Cu6(bpbib)4Br8 (1) and Cu2(bpbib)2(BF4)2Cl2 (2) (bpbib = 1,4-bis((2-(pyrazin-2-yl)-1H-benzo[d]imidazol-1-yl)methyl)benzene) were synthesized and characterized. Both Cu complexes showed high selectivity toward cancer and not normal cells, and the SMMC7721 cell line showed most sensitivity toward both complexes. Complex 1 exhibited more potent cytotoxicity and enhanced cellular uptake, and therefore, was comprehensively investigated. Complex 1 exhibited dual effects in the inhibition of tumor cell proliferation of SMMC7721 cells, causing nuclear DNA damage and mitochondrial dysfunction involving simultaneous reactive oxygen species (ROS) generation and Ca2+ increase. DNA binding studies suggest that intercalation might be the most probable binding mode. Fluorescence spectrometry also detected a medium affinity of complex 1 to bovine serum albumin (BSA) at distinct temperatures and resulted in BSA fluorescence static quenching.

Two symbiotic bacteria of the entomopathogenic nematode Heterorhabditis spp. against Galleria mellonella.

The entomopathogenic nematode Heterorhabditis spp. is considered a promising agent in the biocontrol of injurious insects of agriculture. However, different symbiotic bacteria associated with the nematode usually have different specificity and virulence toward their own host. In this study, two symbiotic bacteria, LY2W and NK, were isolated from the intestinal canals of two entomopathogenic nematode Heterorhabditis megidis 90 (PDSj1 and PDSj2) from Galleria mellonela, separately. To determine their species classification, we carried out some investigations on morphology, culture, biochemistry, especially 16S rDNA sequence analyses. As a result, both of them belong to Enterobacter spp., showing the closest relatedness with Enterobacter gergoviae (LY2W) and Enterobacter cloacae (NK), respectively. Moreover, the toxicity to Galleria mellonella was examined using both the metabolites and washed cells (primary and secondary) of these two strains. The results indicated both metabolites and cells of the primary-type bacteria could cause high mortalities (up to 97%) to Galleria mellonella, while those of the primary-type bacteria only killed 20%. These findings would provide new symbiotic bacteria and further references for biological control of the agricultural pest.

Prevent the degradation of algicidal ability in Scenedesmus-lysing bacteria using optimized cryopreservation.

With the anthropogenic nutrient loading increasing, the frequency and impacts of harmful algal blooms (HABs) have intensified in recent years. To biocontrol HABs, many corresponding algal-lysing bacteria have been exploited successively. However, there are few studies on an effective algal-lysing culture collection to prevent cells from death and particularly the degradation of algicidal ability to their hosts. An optimized cryopreservation was developed and experiments on the validation of this method on preventing algicidal degradation and effects of this optimized cryopreservation on the survival rate of Scenedesmus-lysing bacterium, Enterobacter NP23, isolated from Scenedesmus sp. community, China, on the algicidal dynamic of Scenedesmus wuhanensis was investigated. The optimized cryoprotectant composition consists of 30.0 g/L gelatin, 48.5 g/L sucrose, and 28.4 g/L glycerol, respectively. Using this approach, the survival rate of NP23 cells can still maintain above 90 % and the algal-lysing rate only decline 4 % after the 18-month cryoprotection. Moreover, the 16 generations' passage experiment showed a significant (p < 0.05) genetic stability of algicidal capacity after 18 months. The growth dynamic of S. wuhanensis was investigated in a 5-L bioreactor during 132 h in the absence or presence of NP23. As a result, NP23 has a significant (p < 0.05) inhibition to S. wuhanensis growth when injected into algal culture in the exponential phase at 60th hour. In addition, S. wuhanensis culture initially with NP23 exhibited a slow growth, performing a prolonged lag phase without a clear stationary phase and then rapidly decreased. Our findings, combined with the capacity of preventing the degradation of algicidal ability collectively suggest that the use of this opitimized cryopreservation may be a promising strategy for maintaining algicidal cells.

Primitive neuroectodermal tumor of the prostate: Case report from China.

Peripheral primitive neuroectodermal tumor (PNET) in the prostate is one of the most aggressive tumors and a rare, uncommon clinical disease entity with a very poor prognosis. We reported a case of PNET in the prostate of a 49-year-old man and diagnosed through a biopsy. The patient underwent chemotherapy followed by adjuvant external radiation therapy without cystoprostatectomy as recommended, and 2 years later there is no sign of recurrence or distant metastasis. The patient had a good recovery and satisfactory outcomes in the follow-up. The successful treatment of PNET in the prostate in our case without surgery will provide a good therapeutic regime for reference until now.

Two Novel Algicidal Isolates Kill Chlorella pyrenoidosa by Inhibiting their Host Antioxidase Activities.

In the biocontrol of harmful algal blooms, there has been considerable interest about the role of algicidal bacteria in algicidal activity. In this experiment, two novel algicidal bacteria (strains NP23 and AM11) against Chlorella pyrenoidosa were isolated from the Baiguishan reservoir in China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains NP23 and AM11 belonged to Enterobacter cloacae and Gibberella moniliformis, respectively. To further understand the algicidal activities, five parameters including the chlorophyll a content, cell survival rate, superoxide dismutase (SOD) peroxide dismutase (POD), and catalase (CAT) were tested in the C. pyrenoidosa cells after inoculation with the algicidal bacteria Enterobacter cloacae NP23 and Gibberella moniliformis AM11. As a result, the growth of the treated C. pyrenoidosa was significantly restrained with a great decline of chlorophyll a content. Meanwhile, three antioxidase activities of the treated C. pyrenoidosa were initially stimulated from day 1 to day 3 but then dramatically inhibited at low level. These results induced that the oxidative imbalance (i.e., inhibition of antioxidase activities) caused by algicidal bacteria could be the killing agent of the C. pyrenoidosa cells.