Overexpression of cat2 restores antioxidant properties and production traits in degenerated strains of Volvariella volvacea.

Strain degeneration is an important factor hindering the development of the edible fungus industry. Strain degeneration is associated with the excessive accumulation of reactive oxygen species (ROS) in vivo. Catalase (CAT), an important antioxidant enzyme, can promote the clearance of ROS. In this study, the cat2 gene of Volvariella volvacea was first cloned into an overexpression plasmid via homologous recombination. Finally, through Agrobacterium-mediated transformation, this plasmid was inserted into degenerated strains of V. volvacea T19. The physiological properties, antioxidant properties, ROS content, matrix degradation activity, and cultivation properties of the transformants were tested. The results showed that the cloned cat2 gene was 99.94% similar to the reference sequence. Screening revealed that six positive transformants were successfully obtained. After the overexpression of cat2, the growth rate and biomass of the mycelium increased significantly in the transformant strains (versus the V. volvacea T19 degenerated strains). Moreover, the accumulation of superoxide radical (O2•-) and hydrogen peroxide (H2O2) was significantly reduced, and the activity of the enzymes CAT, superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPX) was significantly increased. Meanwhile, the expression of cat2, Mnsod1, Mnsod2, gpx, and gr was significantly upregulated, and the activity of eight matrix degradation-related enzymes was increased to varying degrees. More importantly, the overexpression of the cat2 gene promoted the regrowth of fruiting bodies in degenerated strains of V. volvacea T19. This study provides a new biotechnological strategy to control the degeneration of V. volvacea and other edible fungi.

Quantitative proteomics of the miR-301a/SOCS3/STAT3 axis reveals underlying autism and anxiety-like behavior.

Autism is a widespread neurodevelopmental disorder. Although the research on autism spectrum disorders has been increasing in the past decade, there is still no specific answer to its mechanism of action and treatment. As a pro-inflammatory microRNA, miR-301a is abnormally expressed in various psychiatric diseases including autism. Here, we show that miR-301a deletion and inhibition exhibited two distinct abnormal behavioral phenotypes in mice. We observed that miR-301a deletion in mice impaired learning/memory, and enhanced anxiety. On the contrary, miR-301a inhibition effectively reduced the maternal immune activation (MIA)-induced autism-like behaviors in mice. We further demonstrated that miR-301a bound to the 3'UTR region of the SOCS3, and that inhibition of miR-301a led to the upregulation of SOCS3 in hippocampus. The last result in the reduction of the inflammatory response by inhibiting phosphorylation of AKT and STAT3, and the expression level of IL-17A in poly(I:C)-induced autism-like features in mice. The obtained data revealed the miR-301a as a critical participant in partial behavior phenotypes, which may exhibit a divergent role between gene knockout and knockdown. Our findings ascertain that miR-301a negatively regulates SOCS3 in MIA-induced autism in mice and could present a new therapeutic target for ameliorating the behavioral abnormalities of autism.

Application fruit tree hole storage brick fertilizer is beneficial to increase the nitrogen utilization of grape under subsurface drip irrigation.

It is very important to promote plant growth and decrease the nitrogen leaching in soil, to improve nitrogen (N) utilization efficiency. In this experiment, we designed a new fertilization strategy, fruit tree hole storage brick (FTHSB) application under subsurface drip irrigation, to characterise the effects of FTHSB addition on N absorption and utilization in grapes. Three treatments were set in this study, including subsurface drip irrigation (CK) control, fruit tree hole storage brick A (T1) treatment, and fruit tree hole storage brick B (T2) treatment. Results showed that the pore number and size of FTHSB A were significantly higher than FTHSB B. Compared with CK, T1 and T2 treatments significantly increased the biomass of different organs of grape, N utilization and 15N content in the roots, stems and leaves, along with more prominent promotion at T1 treatment. When the soil depth was 15-30 cm, the FTHSB application significantly increased the soil 15N content. But when the soil depth was 30-45 cm, it reduced the soil 15N content greatly. T1 and T2 treatments obviously increased the activities of nitrite reductase (NR) and glutamine synthetase (GS) in grape leaves, also the urease activity(UR) in 30 cm of soil. Our findings suggest that FTHSB promoted plant N utilization by reducing N loss in soil and increasing the enzyme activity related to nitrogen metabolism. In addition, this study showed that FTHSB A application was more effective than FTHSB B in improving nitrogen utilization in grapes.

Apple consumption affects cecal health by regulating 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HETE) levels through modifying the microbiota in rats.

Apples are rich in many nutrients and functional components. However, the mechanism of the effect of fresh apple consumption on rats remains unclear. In the present study, fresh apples (10 g kg-1) were added to the diet of Wistar rats, and changes in the microbiota and metabolite content of the cecum were analyzed after 28 days of feeding, and changes in the 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HETE) content and indicators related to inflammation, oxidative stress, and apoptosis were detected. Subsequently, a fecal microbiota transplantation (FMT) protocol was designed and carried out to verify the relationship between the microbiota and 12(S)-HETE, the cecal structure, and inflammatory factors. The results show that apple consumption significantly reduced the serum levels of alanine aminotransferase (ALT) and immunoglobulin G (IgG), altered the cecal histomorphology, and significantly upregulated the gene expression of claudin-1 and zonula occludens-1 (ZO-1), which encode tight junction proteins. Apple consumption also changed the structure of the cecal microbiota, increasing the abundance of some species (such as Shuttleworthia) and decreasing the abundance of others (such as Alphaproteobacteria). Metabolomic screening identified 64 significantly different metabolites. The FMT results showed that apple consumption reduced 12(S)-HETE metabolite levels in the cecal contents, improved the intestinal structure, and reduced the levels of proinflammatory factor expression by altering the cecal microbiota. In conclusion, this study provides further insight into the effects of apples on animals using rats as experimental animals. It provides basic data for future exploration of the mechanisms of the effect of apple consumption on humans.

Oxidative damage from repeated tissue isolation for subculturing causes degeneration in Volvariella volvacea.

The fungal fruiting body is the organized mycelium. Tissue isolation and mycelium succession are common methods of fungal species purification and rejuvenation in the production of edible mushrooms. However, repeated succession increases strain degeneration. In this study, we examined the effect of repeated tissue isolation from Volvariella volvacea fruitbodies on the occurrence of degeneration. The results showed that less than four times in succession improved production capacity, however, after 12 successions, the traits indicating strain degeneration were apparent. For instance, the density of aerophytic hyphae, hyphal growth rate and hyphal biomass were gradually reduced, while the hyphae branching was increased. Also, other degenerative traits such as prolonged production cycles and decreased biological efficiency became evident. In particular, after 19 successions, the strain degeneration became so severe no fruiting bodies were produces anymore. Meanwhile, with the increase in successions, the antioxidant enzyme activity decreased, reactive oxygen species (ROS) increased, the number of nuclei decreased, and the mitochondrial membrane potential decreased along with morphological changes in the mitochondria. This study showed that repeated tissue isolation increased oxidative damage in the succession strain due to the accumulation of ROS, causing cellular senescence, in turn, degeneration in V. volvacea strain.

Effect of Ward Noise Reduction Technology Combined with Music Therapy on Negative Emotions in Inpatients Undergoing Gastric Cancer Radiotherapy: A Retrospective Study.

Background: Gastric cancer is a common malignant tumour in clinics. Noise affects the condition of patients with gastric cancer to a certain extent. This study aims to explore an effective noise control measure. Methods and materials: This study retrospectively analysed the clinical data of 108 patients with gastric cancer who received radiotherapy in the oncology department of JiaoZhou Central Hospital from March 2021 to March 2022, and excluded eight patients who did not meet the inclusion criteria. The remaining patients were divided into a control group (CG, music therapy, n = 48) and a study group (SG, ward noise reduction technology + music therapy, n = 52) in accordance with different management modes. The key causes of noise in the ward that each patient thought were collected by questionnaire, and the ward noise, psychological state, and sleep changes in the two groups were observed under different management modes. Results: The noise was mainly from patients and their family members, call bell, monitors, treatment carts, medical staff and surrounding environment. After the management, SG had lower noise decibel values in daytime and nighttime and significantly lower scores of anxiety and depression than CG (P < 0.01). The sleep quality scores of the two groups after the management were lower than those before management (P < 0.001) and the sleep quality score of SG was significantly lower than that of CG (P < 0.01). Conclusions: Ward noise reduction technology combined with music therapy is an effective method to effectively reduce the ward noise and improve the clinical condition of patients.

Unveiling pathogenic mutations in BRCA1 and BRCA2 genes across head and neck squamous cell carcinoma patients via next generation sequencing.

Head and Neck Squamous Cell Carcinoma (HNSC) presents a formidable challenge in the field of oncology due to its aggressive nature and the limited therapeutic options available. In this study, our primary focus was on the Pakistani HNSC patient population, aiming to investigate germline oncogenic mutations within the BRCA1 and BRCA2 genes via Next Generation Sequencing (NGS) and explore their clinical implications. We sought to understand the functional consequences of these mutations via RT-qPCR and Immunohistochemistry (IHC) techniques. The key discovery of our research lies in the identification of three pathogenic mutations, including two within BRCA1 (p.Cys274Ter and p.Glu272Ter) and one within BRCA2 (p.Met1Val), among Pakistani HNSC patients. These mutations previously associated with an increased risk of various cancers. What sets our study apart is the uniqueness of these pathogenic mutations, absent in HNSC patients from other populations. This suggests a distinct genetic profile in Pakistani HNSC patients, possibly contributing to their susceptibility to this malignancy. Furthermore, our research revealed elevated expression levels of BRCA1 and BRCA2 genes in HNSC samples harboring pathogenic mutations, offering insights into mechanisms driving tumor progression in HNSC. Importantly, we identified significant enrichment of BRCA1/2 genes in pathways related to cancer development within the KEGG database. Finally, in our quest to explore therapeutic avenues, we systematically analyzed drugs targeting up-regulated and mutated BRCA1/2 genes, identifying promising candidates for tailored treatment modalities in HNSC. In conclusion, our study reveals the unique genetic profile of HNSC in Pakistani patients, featuring unique pathogenic mutations in BRCA1 and BRCA2 genes. These mutations offer promise as valuable diagnostic markers and potential therapeutic targets.

PKS5 Confers Cold Tolerance by Controlling Stomatal Movement and Regulating Cold-Responsive Genes in Arabidopsis.

Cold stress limits plant growth and development; however, the precise mechanisms underpinning plant acclimation to cold stress remain largely unknown. In this study, the Ser/Thr protein kinase SOS2-LIKE PROTEIN KINASE5 (PKS5) was shown to play a positive role in plant responses to cold stress. A PKS5 loss-of-function mutant (pks5-1) exhibited elevated sensitivity to cold stress, as well as a lower survival rate and increased ion leakage. Conversely, PKS5 gain-of-function mutants (pks5-3, pks5-4) were more tolerant to cold stress and exhibited higher survival rates and decreased ion leakage. Stomatal aperture analysis revealed that stomatal closure was slower during the first 25 min after cold exposure in pks5-1 compared to wild-type, whereas pks5-3 and pks5-4 displayed accelerated stomatal closure over the same time period. Further stomatal aperture analysis under an abscisic acid (ABA) treatment showed slower closure in pks5-1 and more rapid closure in pks5-3 and pks5-4. Finally, expression levels of cold-responsive genes were regulated by PKS5 under cold stress conditions, while cold stress and ABA treatment can regulate PKS5 expression. Taken together, these results suggest that PKS5 plays a positive role in short-term plant acclimation to cold stress by regulating stomatal aperture, possibly via ABA pathways, and in long-term acclimation by regulating cold-responsive genes.

Successive mycelial subculturing decreased lignocellulase activity and increased ROS accumulation in Volvariella volvacea.

Strain degradation is a common problem in many artificially-cultivated edible mushrooms. As a fungus with poor tolerance to low-temperature, Volvariella volvacea cannot delay its degradation by long-term low temperature storage like other fungi, so its degradation is particularly severe, which hinders industrial applications. Periodic mycelial subculture is a common storage method for V. volvacea, but excessive subculturing can also lead to strain degeneration. After 20 months of continuous subculturing every 3 days, V. volvacea strains S1-S20 were obtained, and their characteristics throughout the subculture process were analyzed. With increasing number of subculture, the growth rate, mycelial biomass, the number of fruiting bodies and biological efficiency gradually decreased while the production cycle and the time to primordium formation was lengthened. Strains S13-S20, obtained after 13-20 months of mycelial subculturing, also lacked the ability to produce fruiting bodies during cultivation experiments. Determination of reactive oxygen species (ROS) content as well as enzyme activity showed that decreased lignocellulase activity, along with excessive accumulation of ROS, was concomitant with the subculture-associated degeneration of V. volvacea. Reverse transcription polymerase chain reaction (RT-PCR) was eventually used to analyze the gene expression for lignocellulase and antioxidant enzymes in subcultured V. volvacea strains, with the results found to be consistent with prior observations regarding enzyme activities. These findings could form the basis of further studies on the degeneration mechanism of V. volvacea and other fungi.

SQLE inhibition suppresses the development of pancreatic ductal adenocarcinoma and enhances its sensitivity to chemotherapeutic agents in vitro.

PURPOSE: In this study, we sought to explore the function of seven important enzymes (MSMO1, EBP, HMGCS1, IDI2, DHCR7, FDFT1, and SQLE) involved in cholesterol biosynthesis especially SQLE in PDAC therapy. METHODS AND RESULTS: The TCGA and Oncomine dataset were used to explore the expression of the seven enzymes in normal and cancerous pancreatic individual, and their anti-proliferation efficiency against PDAC cells was measured by cell viability assay. Expression level and prognostic values of SQLE were evaluated by western blot and Kaplan-Meier analysis. The influence of SQLE knockdown by shRNA in PDAC cells was assessed by transwell, colony formation and cell cycle analysis. RNA-seq and GSEA were utilized to investigate the potential mechanisms. The synergistic effect of SQLE inhibitor, terbinafine, combined with six chemotherapeutic drugs in PDAC cells was tested by CCK-8 method. We demonstrated that downregulation of those enzymes especially SQLE significantly suppressed PDAC cells survival. SQLE was upregulated in PDAC cell lines, and the elevated level of SQLE was correlated with poor prognosis in pancreatic cancer samples. SQLE knockdown could significantly inhibit the proliferation and migration of PDAC cells. Cell cycle was blocked in S phase after SQLE silencing. Mechanistically, GSEA analysis with RNA-seq data revealed that SQLE silencing negatively mediated mTORC1 and TNFα/NF-κB signaling pathways. Besides, SQLE inhibitor terbinafine enhanced chemotherapeutic sensitivity of the six compounds. CONCLUSIONS: This study demonstrated that SQLE was a novel target for PDAC therapy. The synergism role of SQLE inhibition and chemotherapy may be potential therapeutic strategy for pancreatic cancer treatment.

Effects of Temperature and Nitrogen Application on Carbon and Nitrogen Accumulation and Bacterial Community Composition in Apple Rhizosphere Soil.

Malus sieversii grows on the slopes of the Tianshan Mountains in Xinjiang where the difference in daily temperature is significant. In recent years, the rhizosphere soil health of Malus sieversii has been severely impacted by anthropogenic disturbance and pathogenic infestation. The soil nutrient content and soil microorganism diversity are the main components of soil health. Low temperature has negative effects on soil bacterial community structure by inhibiting the accumulation of carbon and nitrogen. However, the effects of temperature and nitrogen application on soil carbon and nitrogen accumulation and the bacterial community composition in the rhizosphere soil of Malus sieversii are unclear. We set two temperature levels, i.e., low temperature (L) and room temperature (R), combined with no nitrogen (N0) and nitrogen application (N1) to explore the response of plant carbon and nitrogen uptake, rhizosphere soil carbon and nitrogen accumulation and bacterial community composition to temperature and nitrogen fertilization. At the same temperature level, plant 13C abundance (P-Atom13C), plant 15N absolute abundance (P-Con15N), soil 15N abundance (S-Atom15N) and soil urease, protease and glutaminase activities were significantly higher under nitrogen application compared with the no-nitrogen application treatment. The bacterial community diversity and richness indices of the apple rhizosphere soil in the N1 treatment were higher than those in the N0 treatment. The relative abundances of Actinobacteria, Rhodopseudomonas, and Bradyrhizobium were higher in the LN1 treatment than in the LN0 treatment. Redundancy analysis (RDA) showed that plant 13C absolute abundance (P-Con13C) and plant 15N absolute abundance (P-Con15N) were the main factors affecting the soil bacterial community composition. In summary, Nitrogen application can alleviate the effects of low temperature stress on the soil bacterial community and is of benefit for the uptakes of carbon and nitrogen in Malus sieversii plants.

Optimisation and Characterisation of Novel Angiotensin-Converting Enzyme Inhibitory Peptides Prepared by Double Enzymatic Hydrolysis from Agaricus bisporus Scraps.

Food-derived hypotensive peptides have attracted attention in the field of active peptide research in recent years. In this study, based on ACE inhibition rate and using the Box-Behnken central combination design principle to optimise the process of ACE inhibitor peptides prepared by double-enzyme hydrolysis. The amino acid sequences of ACE inhibitor peptides were determined by liquid chromatography mass spectrometry (LC-MS/MS), and their binding to ACE was studied by molecular docking. The optimal processing conditions were 1:1 alkaline protease: compound protease, pH was 8.43, enzymolysis temperature was 44.32 °C, and enzymolysis time was 3.52 h. Under these conditions, the ACE inhibition rate reached 65.12%, and the inhibition rate after separation and purification was 80.68% (IC50 = 0.9 mg/mL). Three novel peptides with ACE inhibitory activity were detected by LC-MS/MS, with sequences LVYP (Leu-Val-Tyr-Pro), VYPW(Val-Tyr-Pro-Trp) and YPWT(Tyr-Pro-Trp-Thr). Molecular docking revealed that the three novel peptides all established hydrogen bonds with the S1(Tyr523, Glu384, Ala354) and S2 (His353) pockets of ACE. Among them, LVYP, VYPW and YPWT, respectively, formed eleven hydrogen bonds, six hydrogen bonds and nine hydrogen bonds with ACE. The study revealed that these peptides have the potential for the development of novel ACE inhibitor drugs and provide a new avenue for high-value utilisation of mushrooms scraps.

Natural compound library screening identifies Sanguinarine chloride for the treatment of SCLC by upregulating CDKN1A.

OBJECTIVES: Small cell lung cancer (SCLC) is notorious for aggressive malignancy without effective treatment, and most patients eventually develop tumor progression with a poor prognosis. There is an urgent need for discovering novel antitumor agents or therapeutic strategies for SCLC. MATERIALS AND METHODS: We performed a screening method based on CCK-8 assay to screen 640 natural compounds for SCLC. The effects of Sanguinarine chloride on SCLC cell proliferation, colony formation, cell cycle, apoptosis, migration and invasion were determined. RNA-seq and bioinformatics analysis was performed to investigate the anti-SCLC mechanism of Sanguinarine chloride. Publicly available datasets and samples were analyzed to investigate the expression level of CDKN1A and its clinical significance. Loss of functional cancer cell models were constructed by shRNA-mediated silencing. Quantitative RT-PCR and Western blot were used to measure gene and protein expression. Immunohistochemistry staining was performed to detect the expression of CDKN1A, Ki67, and Cleaved caspase 3 in xenograft tissues. RESULTS: We identified Sanguinarine chloride as a potential inhibitor of SCLC, which inhibited cell proliferation, colony formation, cell cycle, cell migration and invasion, and promoted apoptosis of SCLC cells. Sanguinarine chloride played an important role in anti-SCLC by upregulating the expression of CDKN1A. Furthermore, Sanguinarine chloride in combination with panobinostat, or THZ1, or gemcitabine, or (+)-JQ-1 increased the anti-SCLC effect compared with either agent alone treatment. CONCLUSIONS: Our findings identified Sanguinarine chloride as a potential inhibitor of SCLC by upregulating the expression of CDKN1A. Sanguinarine chloride in combination with chemotherapy compounds exhibited strong synergism anti-SCLC properties, which could be further clinically explored for the treatment of SCLC.

Morphological and molecular characterization of the new aethaloid species Didymium yulii.

Five specimens, initially presumed to be Fuligo septica or Mucilago crustacea, were collected from Jilin Province and the Inner Mongolia Autonomous Region in China, but all five turned out to represent a new aethaloid species, Didymium yulii. This new species is characterized by pseudocapillitia without capillitia and an aethalioid fruiting body, features that are morphologically distinct from those of any other species of Didymium. To assess the phylogenetic relationships between D. yulii and other members of Didymium and in the Didymiaceae, sequences from two nonoverlapping targeted portions of nuc 18S rDNA (~450 bp and ~1050 bp) and translation elongation factor 1-alpha (tef1) were obtained and analyzed. The results indicate that D. yulii forms a single clade separate from other species of Didymium and the clade that contains M. crustacea, which strongly supports the identification of the five specimens as a new species.

Increased Organic Fertilizer and Reduced Chemical Fertilizer Increased Fungal Diversity and the Abundance of Beneficial Fungi on the Grape Berry Surface in Arid Areas.

Fertilizer practices can significantly impact the fruit quality and microbial diversity of the orchards. The fungi on the surface of fruits are essential for fruit storability and safety. However, it is not clear whether fertilization affects the fungal diversity and community structure on the surface of grape berries. Here, grape quality and the fungal diversity on the surface of grapes harvested from three fertilizer treatments were analyzed shortly after grape picking (T0) and following 8 days of storage (T1). The study involved three treatments: (1) common chemical fertilizer for 2 years (CH); (2) increased organic fertilizer and reduced chemical fertilizer for 1 year (A.O); and (3) increased organic fertilizer and reduced chemical fertilizer for 2 years (B.O). The application of increased organic fertilizer and reduced chemical fertilizer increased the soluble solids content (SSC) of the grape berries and decreased the pH of the grape juice. A total of 827,947 high-quality fungal sequences were recovered and assigned to 527 operational taxonomic units. Members of the Ascomycota phylum were dominant in all samples and accounted for 94.41% of the total number of detected sequences, followed by the Basidiomycota (5.05%), and unidentified fungi (0.54%). Alpha and beta diversity analyses revealed significantly different fungal populations in the three fertilizer treatments over the two time periods. The fungal diversity and richness on the grape berry surface in the B.O and A.O treatments were higher than those in the CH treatment. Among the detected fungi, the B.O treatments were mainly Pichia, Aureobasidium, and Candida genera, while the CH treatments were Botrytis, Aspergillus, and Penicillium. Moreover, significant differences were revealed between the two assessment times (T0 and T1). The samples from the T0 timepoint had higher fungal richness and diversity than the samples from T1 timepoint. Increasing organic fertilizer usage in grape management could improve grape quality and went on to increase the fungal diversity, as well as the relative abundance (RA) of beneficial fungi on grape berry surfaces. The correlation analysis suggested that the pH of the grape juice was significantly negatively correlated with fungal diversity parameters.

Mechanism and Regulation of Silique Dehiscence, Which Affects Oil Seed Production.

Silique dehiscence is an important physiological process during natural growth that enables mature seeds to be released from plants, which then undergo reproduction and ensure the survival of future generations. In agricultural production, the time and degree of silique dehiscence affect the harvest time and processing of crops. Premature silique dehiscence leads to seeds being shed before harvest, resulting in substantial reductions to yields. Conversely, late silique dehiscence is not conducive to harvesting, and grain weight and oil content will be reduced due to the respiratory needs of seeds. In this paper, the mechanisms and regulation of silique dehiscence, and its application in agricultural production is reviewed.

Increased organic fertilizer application and reduced chemical fertilizer application affect the soil properties and bacterial communities of grape rhizosphere soil.

Increasing organic fertilizer application can improve the sustainability of soil productivity, but the effects of increased organic fertilizer application with reduced chemical fertilizer application over different time periods on chemical properties and bacterial community of grape rhizosphere soil in an arid region are not clear. In this study, three years of fixed-point field tests were used to compare the effects of various fertilization treatments on the soil properties and bacterial community in the grape rhizosphere. The results showed that (1) T1 and T2 significantly increased SOM, AN, AP and AK contents in grape rhizosphere soil. TN, TP and TK contents in grape leaves of T2 were the highest of those in five fertilization treatments. (2) The abundances of Proteobacteria and Bacteroidetes phyla and especially of Arthrobacter, Pseudomonas, Nitrosopira and Bacillus genera were higher in T2 than in the other samples. (3) SOM, AP and AN contents in soil were the main factors affecting soil bacterial community and mineral element contents in grape leaves and roots according to an RDA analysis. In summary, the application of organic fertilizer with reduced chemical fertilizer for two years had the greatest impact on the soil properties and bacterial community of the grape rhizosphere soil.

An improved protein lipid overlay assay for studying lipid-protein interactions.

BACKGROUND: Lipids perform multiple functions in the cell, and lipid-protein interactions play a key role in metabolism. Although various techniques have been developed to study lipid-protein interactions, the interacting protein partners that bind to most lipids remain unknown. The protein lipid overlay (PLO) assay has revealed numerous lipid-protein interactions, but its application suffers from unresolved technical issues. RESULTS: Herein, we found that blocking proteins may interfere with interactions between lipids and their binding proteins if a separate blocking step is carried out before the incubation step in the PLO assay. To overcome this, we modified the PLO assay by combining an incubation step alongside the blocking step. Verification experiments included phosphatidylinositol-3-phosphate (PI3P) and its commercially available interacting protein G302, C18:1, C18:2, C18:3 and the Arabidopsis plasma membrane H+-ATPase (PM H+-ATPase) AHA2 C-terminus, phosphatidylglycerol (PG) and AtROP6, and phosphatidylserine (PS) and the AHA2 C-terminus. The lipid-protein binding signal in the classical PLO (CPLO) assay was weak and not reproducible, but the modified PLO (MPLO) assay displayed significantly improved sensitivity and reproducibility. CONCLUSIONS: This work identified a limitation of the CPLO assay, and both sensitivity and reproducibility were improved in the modified assay, which could prove to be more effective for investigating lipid-protein interactions.

Genome-wide identification of the NPR1-like gene family in Brassica napus and functional characterization of BnaNPR1 in resistance to Sclerotinia sclerotiorum.

KEY MESSAGE: The BnaNPR1-like gene family was identified in B. napus, and it was revealed that repression of BnaNPR1 significantly reduces resistance toS. sclerotiorum, intensifies ROS accumulation, and changes the expression of genes associated with SA and JA/ET signaling in response to this pathogen. The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) and related NPR1-like genes play an important role in regulating plant defense. Oilseed rape (Brassica napus L.) is an important oilseed crop; however, little is known about the B. napus (Bna) NPR1-like gene family. Here, a total of 19 BnaNPR1-like genes were identified in the B. napus genome, and then named according to their respective best match in Arabidopsis thaliana (At), which led to the determination of B. napus homologs of every AtNPR1-like gene. Analysis of important protein domains and functional motifs indicated the conservation and variation among these homologs. Phylogenetic analysis of these BnaNPR1-like proteins and their Arabidopsis homologs revealed six distinct sub-clades, consequently indicating that their name classification totally conformed to their phylogenetic relationships. Further, B. napus transcriptomic data showed that the expression of three BnaNPR1s was significantly down-regulated in response to infection with Sclerotinia sclerotiorum, the most important pathogen of this crop, whereas BnaNPR2/3/4/5/6s did not show the expression differences in general. Further, we generated B. napus BnaNPR1-RNAi lines to interpret the effect of the down-regulated expression of BnaNPR1s on resistance to S. sclerotiorum. The results showed that BnaNPR1-RNAi significantly decreased this resistance. Further experiments revealed that BnaNPR1-RNAi intensified ROS production and changed defense responses in the interaction of plants with this pathogen. These results indicated that S. sclerotiorum might use BnaNPR1 to regulate specific physiological processes of B. napus, such as ROS production and SA defense response, for the infection.