Rtt105 stimulates Rad51-ssDNA assembly and orchestrates Rad51 and RPA actions to promote homologous recombination repair.

Homologous recombination (HR) is essential for the maintenance of genome stability. During HR, Replication Protein A (RPA) rapidly coats the 3'-tailed single-strand DNA (ssDNA) generated by end resection. Then, the ssDNA-bound RPA must be timely replaced by Rad51 recombinase to form Rad51 nucleoprotein filaments that drive homology search and HR repair. How cells regulate Rad51 assembly dynamics and coordinate RPA and Rad51 actions to ensure proper HR remains poorly understood. Here, we identified that Rtt105, a Ty1 transposon regulator, acts to stimulate Rad51 assembly and orchestrate RPA and Rad51 actions during HR. We found that Rtt105 interacts with Rad51 in vitro and in vivo and restrains the adenosine 5' triphosphate (ATP) hydrolysis activity of Rad51. We showed that Rtt105 directly stimulates dynamic Rad51-ssDNA assembly, strand exchange, and D-loop formation in vitro. Notably, we found that Rtt105 physically regulates the binding of Rad51 and RPA to ssDNA via different motifs and that both regulations are necessary and epistatic in promoting Rad51 nucleation, strand exchange, and HR repair. Consequently, disrupting either of the interactions impaired HR and conferred DNA damage sensitivity, underscoring the importance of Rtt105 in orchestrating the actions of Rad51 and RPA. Our work reveals additional layers of mechanisms regulating Rad51 filament dynamics and the coordination of HR.

Tool Wear Monitoring in Milling Based on Fine-Grained Image Classification of Machined Surface Images.

Cutting tool wear state assessment during the manufacturing process is extremely significant. The primary purpose of this study is to monitor tool wear to ensure timely tool change and avoid excessive tool wear or sudden tool breakage, which causes workpiece waste and could even damage the machine. Therefore, an intelligent system, that is efficient and precise, needs to be designed for addressing these problems. In our study, an end-to-end improved fine-grained image classification method is employed for workpiece surface-based tool wear monitoring, which is named efficient channel attention destruction and construction learning (ECADCL). The proposed method uses a feature extraction module to extract features from the input image and its corrupted images, and adversarial learning is used to avoid learning noise from corrupted images while extracting semantic features by reconstructing the corrupted images. Finally, a decision module predicts the label based on the learned features. Moreover, the feature extraction module combines a local cross-channel interaction attention mechanism without dimensionality reduction to characterize representative information. A milling dataset is conducted based on the machined surface images for monitoring tool wear conditions. The experimental results indicated that the proposed system can effectively assess the wear state of the tool.

Effects of cowpea (Vigna unguiculata) root mucilage on microbial community response and capacity for phenanthrene remediation.

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) is normally limited by their low solubility and poor bioavailability. Prior research suggests that biosurfactants are synthesized as intermediates during the production of mucilage at the root tip. To date the effects of mucilage on PAH degradation and microbial community response have not been directly examined. To address this question, our research compared 3 cowpea breeding lines (Vigna unguiculata) that differed in mucilage production for their effects on phenanthrene (PHE) degradation in soil. The High Performance Liquid Chromatography results indicated that the highest PHE degradation rate was achieved in soils planted with mucilage producing cowpea line C1, inoculated with Bradyrhizobium, leading to 91.6% PHE disappearance in 5 weeks. In root printing tests, strings treated with mucilage and bacteria produced larger clearing zones than those produced on mucilage treated strings with no bacteria or bacteria inoculated strings. Experiments with 14C-PHE and purified mucilage in soil slurry confirmed that the root mucilage significantly enhanced PHE mineralization (82.7%), which is 12% more than the control treatment without mucilage. The profiles of the PHE degraders generated by Denaturing gradient gel electrophoresis suggested that cowpea C1, producing a high amount of root mucilage, selectively enriched the PHE degrading bacteria population in rhizosphere. These findings indicate that root mucilage may play a significant role in enhancing PHE degradation and suggests that differences in mucilage production may be an important criterion for selection of the best plant species for use in phytoremediation of PAH contaminated soils.

[Index components contents in honeysuckle change trend at different time in a day and different stubbles in a year].

The index components contents of different time and different stubbles in honeysuckle were measured by HPLC, and were analysis by using the method of SPSS. Results showed that the content of index ingredients of different time had differences, and firstly decreased, then increased with time. The content of index ingredients of different stubbles had significantly differences, and firstly decreased, then increased with time. The chlorogenic acid contents were 2.059%-3.593%. The luteolosid contents were 0.110%-0.171%. Results indicated that the best picking buds time is before seven o'clock in the morning and evening at before and after seven o'clock, the index component content is higher. Picking buds in spring and at autumn index component content is higher; Picking buds in summer index component content is low. The experiment provides theoretical support for quality control in the whole process of the honeysuckle harvested and comprehensive utilization of honeysuckle.

Olaparib combined with CDK12-IN-3 to promote genomic instability and cell death in ovarian cancer.

Large-scale phase III clinical trials of Olaparib have revealed benefits for ovarian cancer patients with BRCA gene mutations or homologous recombination deficiency (HRD). However, fewer than 50% of ovarian cancer patients have both BRCA mutations and HRD. Therefore, improving the effect of Olaparib in HR-proficient patients is of great clinical value. Here, a combination strategy comprising Olaparib and CDK12-IN-3 effectively inhibited the growth of HR-proficient ovarian cancer in cell line, patient-derived organoid (PDO), and mouse xenograft models. Furthermore, the combination strategy induced severe DNA double-strand break (DSB) formation, increased NHEJ activity in the G2 phase, and reduced HR activity in cancer cells. Mechanistically, the combination treatment impaired Ku80 poly(ADP-ribosyl)ation (PARylation) and phosphorylation, resulting in PARP1-Ku80 complex dissociation. After dissociation, Ku80 occupancy at DSBs and the resulting Ku80-primed NHEJ activity were increased. Owing to Ku80-mediated DNA end protection, MRE11 and Rad51 foci formation was inhibited after the combination treatment, suggesting that this treatment suppressed HR activity. Intriguingly, the combination strategy expedited cGAS nuclear relocalization, further suppressing HR and, conversely, increasing genomic instability. Moreover, the inhibitory effect on cell survival persisted after drug withdrawal. These findings provide a rationale for the clinical application of CDK12-IN-3 in combination with Olaparib.

She-Chuang-Si-Wu-Tang Alleviates Inflammation and Itching Symptoms in a Psoriasis Mouse Model by Regulating the Th17/IL-17 Axis via the STAT3/MAPK Pathways.

Purpose: Psoriasis is an immune-related disorder characterized by silver scales, epidermis thickness, and itching. She-Chuang-Si-Wu-Tang (SSWT), a traditional Chinese medicine decoction, has been used clinically for 400 years. Although it benefits psoriasis treatment, the mechanism of action is still unclear. This study explores SSWT's molecular mechanism in treating psoriasis through network pharmacology analysis and experiments. Methods: We identified relevant SSWT and psoriasis targets using network pharmacology and conducted SSWT quality control with high-performance liquid chromatography (HPLC). A mouse model of psoriasis was established using imiquimod (IMQ), with the drug administered continuously for seven days, spanning an eight-day period. During the experiment, we observed spontaneous scratching behaviors and assessed the Psoriasis Area and Severity Index (PASI) scores. At the conclusion of the experiment, we examined skin tissue pathology under an optical microscope and measured epidermal thickness. Additionally, we used enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to measure interleukin (IL)-23, IL-17A, IL-17F, and interferon (IFN)-γ levels in the mice's serum and their mRNA expression in the skin. Western blot analysis was conducted to assess protein levels related to signaling pathways. Results: Results indicate that SSWT may target IL-17 signaling pathways and T helper (Th) 17 cell differentiation, as predicted by network pharmacology. SSWT significantly improved the PASI and Baker scores, reduced epidermal thickness, and decreased spontaneous scratching in IMQ-induced mice. Additionally, SSWT treatment significantly lowered the concentrations of inflammatory factors in the serum and skin lesions, as well as mRNA expression levels, compared to the IMQ group. Furthermore, SSWT significantly inhibited the phosphorylation of both the signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) pathways. Conclusion: In summary, this study unveiled the potential anti-psoriatic mechanism of SSWT, offering new evidence for its clinical application.

Inhibitory Effect of Evodiamine on Psoriasis Lesions and Itching in Mice.

Purpose: This study seeks to investigate the effect of evodiamine on psoriasis and psoriatic pruritus. Methods: Imiquimod-induced psoriasiform dermatitis in mice was used as a model, and evodiamine was topically applied for seven days. The mice were observed daily for skin damage on the back, clinical score and their scratching behavior was recorded. Blood samples were collected on the final day of the experiment, and the serum levels of pruritus-associated inflammatory cytokines tumor necrosis factor (TNF) -α, interleukin (IL) -23, and IL-17A were measured using enzyme-linked immunosorbent assay. Histopathological changes were observed in Hematoxylin and Eosin-stained skin specimens. The expression levels of transient receptor potential vanilloid (TRPV) 1, TRPV3, TRPV4, and the pruritus-related mediators Substance P (SP), nerve growth factor (NGF), and calcitonin gene-related peptide (CGRP) in the skin lesions were analyzed using Western blot and qRT-PCR. The effect of evodiamine on the exploratory behavior, motor, and coordination abilities of mice was assessed using open field, suspension, and Rota-Rod experiments. Molecular docking was utilized to verify the binding of evodiamine to the residues of TRPV1, TRPV3, and TRPV4. Results: Evodiamine reduced pruritus and inhibited inflammation by decreasing the levels of inflammatory mediators TNF-α, IL-23, and IL-17A in the serum of Imiquimod-induced mice and attenuated the mRNA and protein expression levels of SP, NGF, CGRP, TRPV1, TRPV3, and TRPV4 in the skin. Conclusion: Evodiamine is an effective treatment for psoriasis and pruritus, due to its ability to inhibit immune inflammation and pruritic mediators.

Choroidal vascular changes in early-stage myopic maculopathy from deep learning choroidal analysis: a hospital-based SS-OCT study.

BACKGROUND: The objective of this study is to illustrate the changes in the choroidal vasculature in individuals with diffuse chorioretinal atrophy (DCA, early-stage myopic maculopathy) and investigate the association between them. METHODS: This study included 1418 highly myopic eyes from 720 participants aged 18 - 60 years from the Wenzhou High Myopia Cohort Study. These participants underwent comprehensive ophthalmic assessments. Myopic maculopathy classification followed the Meta-PM system, with pathological myopia defined as myopic maculopathy of DCA or severer. Eyes with myopic maculopathy categorized as no macular lesions (C0), tessellated fundus (C1), and DCA (C2) were enrolled in the analysis. Choroidal images were obtained from swept-source optical coherence tomography (SS-OCT), and the images were processed with a deep learning-based automatic segmentation algorithm and the Niblack auto-local threshold algorithm. RESULTS: DCA was detected in 247 eyes (17.4%). In comparison to eyes with C0, those with C2 exhibited significant reductions in choroidal thickness (ChT), luminal area (LA), and stromal area (SA) across all evaluated regions (all P < 0.001). An increase in choroidal vascular index (CVI) was observed in all regions, except for the nasal perifoveal (N2) and inferior perifoveal (I2) regions (all P < 0.01). Multivariable logistic regression analysis revealed a negative association between the presence of DCA and increases in choroidal LA and SA (odds ratio ≤ 0.099, P < 0.001). Multivariable linear regression analysis showed that the mean deviation of the visual field test was positively associated with LA and SA at the vertical meridian (B = 1.512, P < 0.001 for LA; B = 1.956, P < 0.001 for SA). Furthermore, the receiver operating characteristic curve analyses showed the optimal ChT to diagnose pathological myopia was 82.4 µm in the N2 region, the LA was 0.076 mm2 and the SA was 0.049 mm2, with area under the curves of 0.916, 0.908, and 0.895, respectively. CONCLUSIONS: The results of this study indicated that both the presence of DCA and visual function impairment were associated with reductions in choroidal perfusion and stromal components. Moreover, we established threshold values for choroidal parameters in diagnosing pathological myopia, offering valuable references for clinical diagnosis and management.

Spontaneously Myopic Guinea Pig: Model of Early Pathologic Myopia.

Purpose: To evaluate whether pigmented guinea pigs with spontaneous myopia present characteristic changes of pathologic myopia. Methods: The fundus images of guinea pigs (3 weeks old) were graded according to fundus tessellation (FT) degree. Biometric parameters, including refraction, vitreous chamber depth (VCD), and axial length (AL), were measured at ages 21 and 43 days. Some of these animals were divided into three groups: hyperopic without FT (H w/o FT), myopic without FT (M w/o FT), and myopic with FT (M w/ FT). The horizontal and vertical radii of curvature of posterior sclera (RP-H and RP-V, respectively) and the radii of curvature and arc lengths of superior sclera (RS and LS, respectively), inferior sclera (RI and LI, respectively), nasal sclera (RN and LN, respectively), and temporal sclera (RT and LT) were evaluated by Fuji. Results: The fundi were graded as type A or type B (both without FT), type C (mild FT), or type D (severe FT). The prevalence of FT was correlated with myopic refraction, longer VCD, and longer AL. Eyes of M w/FT animals had shorter RP-H and RP-V, longer RS and RT, and longer LS and LT than eyes of H w/o FT or M w/o FT animals. Refractions shifted toward hyperopia in eyes lacking FT, but not in eyes having FT. The changes in VCD were consistent with the changes in refraction. This relatively myopic shift in refraction and shortening of VCD were found only in myopic eyes with FT, but not in myopic eyes without FT. Conclusions: Spontaneously myopic guinea pig eyes have a high prevalence of FT. Myopic eyes with FT presented characteristic signs of pathologic myopia.

Technology-Facilitated Abuse Against Women From Culturally and Linguistically Diverse Backgrounds: A Scoping Review of the Literature.

Women from culturally and linguistically diverse (CALD) backgrounds are particularly vulnerable to domestic and family violence, including technology-facilitated abuse. Often CALD women depend on technology to connect with support networks in their home country. Technology-facilitated abuse can be devastating and isolating. There is limited comprehensive knowledge of how technology-facilitated abuse is experienced by CALD women. This scoping review addresses this gap by exploring and analysing the available literature on technology-facilitated abuse amongst CALD women in the context of domestic and family violence. Employing a scoping review methodology, a total of nine studies were identified from a database search and other sources (including snowball, web search, and search verification processes). Studies were included if they contained the following three elements: (1) a focus on technology-facilitated abuse, (2) the inclusion of CALD women's experiences, and (3) a context of domestic and family violence (DFV). This review firstly maps the methodologies and characteristics of the studies. Second, the most common types of technology-facilitated abuse that disproportionally affect CALD women are identified together with culturally related help-seeking barriers. Areas for future research are discussed along with suggestions for improving practises and policies for prevention and intervention.

1-[4-(4-Chloro-but-oxy)-2-hy-droxy-phen-yl]ethanone.

In the title compound, C(12)H(15)ClO(3), the eth-oxy group is nearly coplanar with the benzene ring, making a dihedral angle of 9.03 (4)°, and is involved in an intra-molecular O-H⋯O hydrogen bond to the neighbouring hy-droxy group.

Non-coding RNA-mediated autophagy in cancer: A protumor or antitumor factor?

Autophagy, usually referred to as macroautophagy, is a cytoprotective behavior that helps cells, especially cancer cells, escape crises. However, the role of autophagy in cancer remains controversial. The induction of autophagy is favorable for tumor growth, as it can degrade damaged cell components accumulated during nutrient deficiency, chemotherapy, or other stresses in a timely manner. Whereas the antitumor effect of autophagy might be closely related to its crosstalk with metabolism, immunomodulation, and other pathways. Recent studies have verified that lncRNAs and circRNAs modulate autophagy in carcinogenesis, cancer cells proliferation, apoptosis, metastasis, and chemoresistance via multiple mechanisms. A comprehensive understanding of the regulatory relationships between ncRNAs and autophagy in cancer might resolve chemoresistance and also offer intervention strategies for cancer therapy. This review systematically displays the regulatory effects of lncRNAs and circRNAs on autophagy in the contexts of cancer initiation, progression, and resistance to chemo- or radiotherapy and provides a novel insight into cancer therapy.

Low Expression of RILPL2 Predicts Poor Prognosis and Correlates With Immune Infiltration in Endometrial Carcinoma.

Increasing numbers of biomarkers have been identified in various cancers. However, biomarkers associated with endometrial carcinoma (EC) remain largely to be explored. In the current research, we downloaded the RNA-seq data and corresponding clinicopathological features from the Cancer Genome Atlas (TCGA) database. We conducted an expression analysis, which resulted in RILPL2 as a novel diagnostic biomarker in EC. The dysregulation of RILPL2 in EC was also validated in multiple datasets. The correlations between clinical features and RILPL2 expression were assessed by logistic regression analysis. Then, Kaplan-Meier analysis, univariate and multivariate Cox regression analysis were performed to estimate prognostic values of RILPL2 in the TCGA cohort, which revealed that increased level of RILPL2 was remarkably associated with better prognosis and could act as an independent prognostic biomarker in patients with EC. Moreover, correlation analysis of RILPL2 and tumor-infiltrating immune cells (TIICs) indicated that RILPL2 might play a critical role in regulating immune cell infiltration in EC and is related to immune response. Besides, high methylation level was a significant cause of low RILPL2 expression in EC. Subsequently, weighted gene co-expression network analysis (WGCNA) and enrichment analysis were conducted to explore the RILPL2-involved underlying oncogenic mechanisms, and the results indicated that RILPL2 mainly regulated cell cycle. In conclusion, our findings provided evidence that downregulation of RILPL2 in EC is an indicator of adverse prognosis and RILPL2 may act as a promising target for the therapeutics of EC.

Identification of an eleven-miRNA signature to predict the prognosis of endometrial cancer.

Endometrial cancer (EC) is the most common gynecological malignancy. Recent studies have uncovered miRNA acted a striking role in predicting the prognosis of multiple tumors. Over 500 EC samples were selected from the Cancer Genome Atlas (TCGA) database. Univariate, LASSO and multivariate Cox regression analysis were employed to screen out the prognosis-involved miRNAs. Kaplan-Meier (K-M) and time-dependent receiver operation characteristic (ROC) curves were conducted to reveal survival analysis and assess the accuracy of the signature. The independence of the model was verified via univariate and multivariate Cox regression analysis. Besides, qRT-PCR was conducted to testified the expression of 11 miRNAs in 16 paired tissues. A total of 514 specimens were randomly divided into the training set and the testing set, then an 11 miRNAs-based signature were determined which divided the patients into high-risk group and low-risk group. The survival was markedly different and the ROC curve exhibited a precise prediction. Meanwhile, the univariate and multivariate Cox regression analysis verified the miRNAs-based model was an independent indicator of EC. Moreove, the prediction ability of this model with clinicopathological features was more efficient. Finally, functional enrichment analysis demonstrated these miRNAs were associated with the occurrence and progression of cancer. Additionally, hsa-mir-216b, hsa-mir-363, hsa-mir-940 and hsa-mir-1301 were highly expressed in EC tissues in contrast to normal tissues through qRT-PCR. Importantly, the eleven-miRNA signature was full of robust ability to predict the prognosis of EC.

CSMD3 is Associated with Tumor Mutation Burden and Immune Infiltration in Ovarian Cancer Patients.

BACKGROUND: Globally, ovarian cancer (OC), the deadliest gynecologic malignancy, remains a major cause of mortality, with a rising number of cases in many low- and middle-income countries. Immunotherapy has been proven to be promising for OC. There is increasing awareness of the vital role that tumor mutation burden (TMB) plays in predicting the efficacy of immunotherapy. Women with a family history of OC are at higher risk of the disease due to gene mutations. However, whether these gene mutations are related to immune response and TMB remains to be explored. METHODS: Our present work analyzed genetic mutation data of OC patients obtained from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) cohorts, and we identified 11 frequently mutated genes, namely, APOB, CSMD3, DST, FAT3, FLG, HMCN1, MUC16, RYR1, TP53, TTN, and USH2A, in accordance with the overlap of two databases. RESULTS: A statistically higher TMB was detected by whole-exome sequencing in patients with OC with CSMD3 mutation than in those with mutations in the other frequently mutated genes. Prognosis analysis performed with patients from the TCGA cohort revealed that those with CSMD3 mutation had an overall survival (OS) that was inferior to that of those with wild-type CSMD3. Gene set enrichment analysis (GSEA) and CIBERSORT analysis indicated that OC samples with CSMD3 mutations had significant involvement of pathways related to the immune response. CONCLUSION: In summary, we found that CSMD3 mutation is highly correlated with increased TMB and poor clinical prognosis and that it might function as a biomarker for predicting prognosis and choosing an immunotherapy regimen.

Coping Strategies and Suicidality: A Cross-Sectional Study From China.

Background and Objective: Suicide is a leading cause of death in young people. Suicidal thoughts and behaviors can be triggered by life and study stresses; therefore, it is important to understand the role of coping strategies. The current study analyzed the link between different coping strategies and suicidality in university students in China. Methods: A cross-sectional study of 2,074 undergraduate students from China used a stratified-clustered-random sampling method (response rate 94.4%). The Suicidal Behaviors Questionnaire-Revised Scale was used to identify suicidal risks, while the Brief COPE scale was used to measure different coping strategies. Univariate and multivariate logistic regression analyses were utilized to examine coping strategies and suicidality. Results: A negative association of some coping skills (active coping and positive reframing) with suicidality and a positive association of some other coping skills (self-distraction, substance abuse, behavioral disengagement, venting, and self-blame) with suicidality were observed after adjusting for sociodemographic and mental health variables. Conclusions: Training and supporting young people to identify and apply adaptive coping strategies to deal with life stress could help to reduce suicidal ideation and behavior.

Antipruritic Effect of Ethyl Acetate Extract from Fructus cnidii in Mice with 2,4-Dinitrofluorobenzene-Induced Atopic Dermatitis.

Atopic dermatitis (AD) is a common inflammatory skin disease characterized by intense pruritus and skin lesions. The exact cause of AD is not yet known and the available therapeutic strategies for AD are limited. Fructus cnidii is commonly used in traditional Chinese medicine as an herb for treating chronic itch. However, the mechanism underlying the antipruritic effects of Fructus cnidii is not well understood. In the present study, we investigated the antipruritic effect of locally administered ethyl acetate extract from Fructus cnidii (EAEFC) to 2,4-dinitrofluorobenzene- (DNFB-) induced AD in a mouse model. The scratching behavior, skin thickness, dermatitis score, weight, blood immunoglobulin E (IgE) level, and itch-related cytokine levels were subsequently monitored and evaluated. Results showed that EAEFC treatment attenuated the DNFB-induced AD-like symptoms by alleviating the skin lesions and decreasing the dermatitis score. Hematoxylin and eosin (H&E) and toluidine blue (TB) staining analyses demonstrated that EAEFC mitigated the DNFB-induced increase in skin thickness and prevented the infiltration of mast cells. Behavioral tests showed that EAEFC decreased the DNFB-induced acute and chronic scratching behaviors. Furthermore, EAEFC reduced the levels of itch-related cytokines, such as thymic stromal lymphopoietin (TSLP), interleukin- (IL-) 17, IL-33, and IL-31, and the DNFB-induced boost in serum IgE. Collectively, these results suggest that EAEFC is a potential therapeutic candidate for the treatment of chronic itch in AD.

Zinc Resistance Mechanisms of P1B-type ATPases in Sinorhizobium meliloti CCNWSX0020.

The Sinorhizobium meliloti (S. meliloti) strain CCNWSX0020 displayed tolerance to high levels exposures of multiple metals and growth promotion of legume plants grown in metal-contaminated soil. However, the mechanism of metal-resistant strain remains unknown. We used five P1B-ATPases deletions by designating as ∆copA1b, ∆fixI1, ∆copA3, ∆zntA and ∆nia, respectively to investigate the role of P1B-ATPases in heavy metal resistance of S. meliloti. The ∆copA1b and ∆zntA mutants were sensitive to zinc (Zn), cadmium (Cd) and lead (Pb) in different degree, whereas the other mutants had no significant influence on the metal resistance. Moreover, the expression of zntA was induced by Zn, Cd and Pb whereas copA1b was induced by copper (Cu) and silver (Ag). This two deletions could led to the increased intracellular concentrations of Zn, Pb and Cd, but not of Cu. Complementation of ∆copA1b and ∆zntA mutants showed a restoration of tolerance to Zn, Cd and Pb to a certain extent. Taken together, the results suggest an important role of copA1b and zntA in Zn homeostasis and Cd and Pb detoxification in S. meliloti CCNWSX0020.

Purification and characterization of a novel phloretin-2'-O-glycosyltransferase favoring phloridzin biosynthesis.

Phloretin-2'-O-glycosyltransferase (P2'GT) catalyzes the last glycosylation step in the biosynthesis of phloridzin that contributes to the flavor, color and health benefits of apples and processed apple products. In this work, a novel P2'GT of Malus x domestica (MdP2'GT) with a specific activity of 46.82 µkat/Kg protein toward phloretin and uridine diphosphate glucose (UDPG) at an optimal temperature of 30 °C and pH 8.0 was purified from the engineered Pichia pastoris broth to homogeneity by anion exchange chromatography, His-Trap affinity chromatography and gel filtration. The purified MdP2'GT was low N-glycosylated and secreted as a stable dimer with a molecular mass of 70.7 kDa in its native form. Importantly, MdP2'GT also exhibited activity towards quercetin and adenosine diphosphate glucose (ADPG), kaempferol and UDPG, quercetin and UDP-galactose, isoliquiritigenin and UDPG, and luteolin and UDPG, producing only one isoquercitrin, astragalin, hyperoside, isoliquiritin, or cynaroside, respectively. This broad spectrum of activities make MdP2'GT a promising biocatalyst for the industrial preparation of the corresponding polyphenol glycosides, preferably for their subsequent isolation and purification. Besides, MdP2'GT displayed the lowest Km and the highest kcat/Km for phloretin and UDPG compared to all previously reported P2'GTs, making MdP2'GT favor phloridzin synthesis the most.

Functional characterization of a csoR-cueA divergon in Bradyrhizobium liaoningense CCNWSX0360, involved in copper, zinc and cadmium cotolerance.

Random mutagenesis in a symbiotic nitrogen-fixing Bradyrhizobium liaoningense CCNWSX0360 (Bln0360) using Tn5 identified five copper (Cu) resistance-related genes. They were functionally sorted into three groups: transmembrane transport (cueA and tolC); oxidation (copA); and protection of the membrane barrier (lptE and ctpA). The gene cueA, together with the upstream csoR (Cu-sensitive operon repressor), constituted a csoR-cueA divergon which plays a crucial role in Cu homeostasis. Deletion of cueA decreased the Cu tolerance of cells, and complementation of this mutant restored comparable Cu resistance to that of the wild-type. Transcriptional and fusion expression analysis demonstrated that csoR-cueA divergon was up-regulated by both the monovalent Cu+ and divalent Zn2+/Cd2+, and negatively regulated by transcriptional repressor CsoR, via a bidirectional promoter. Deletion of csoR renders the cell hyper-resistant to Cu, Zn and Cd. Although predicted to encode a Cu transporting P-type ATPase (CueA), cueA also conferred resistance to zinc and cadmium; two putative N-MBDs (N-terminal metal binding domains) of CueA were required for the Cu/Zn/Cd tolerance. Moreover, cueA is needed for nodulation competitiveness of B. liaoningense in Cu rich conditions. Together, the results demonstrated a crucial role for the csoR-cueA divergon as a component of the multiple-metal resistance machinery in B. liaoningense.