Bacillus B2 promotes root growth and enhances phosphorus absorption in apple rootstocks by affecting MhMYB15.

Due to the chelation of phosphorus in the soil, it becomes unavailable for plant growth and development. The mechanisms by which phosphorus-solubilizing bacteria activate immobilized phosphorus to promote the growth and development of woody plants, as well as the intrinsic molecular mechanisms, are not clear. Through the analysis of microbial communities in the rhizosphere 16S V3-V4 and a homologous gene encoding microbial alkaline phosphomonoesterase (phoD) in phosphate-efficient (PE) and phosphate-inefficient apple rootstocks, it was found that PE significantly enriched beneficial rhizobacteria. The best phosphorus-solubilizing bacteria, Bacillus sp. strain 7DB1 (B2), was isolated, purified, and identified from the rhizosphere soil of PE rootstocks. Incubating with Bacillus B2 into the rhizosphere of apple rootstocks significantly increased the soluble phosphorus and flavonoid content in the rhizosphere soil. Simultaneously, this process stimulates the root development of the rootstocks and enhances plant phosphorus uptake. After root transcriptome sequencing, candidate transcription factor MhMYB15, responsive to Bacillus B2, was identified through heatmap and co-expression network analysis. Yeast one-hybrid, electrophoretic mobility shift assay, and LUC assay confirmed that MhMYB15 can directly bind to the promoter regions of downstream functional genes, including chalcone synthase MhCHS2 and phosphate transporter MhPHT1;15. Transgenic experiments with MhMYB15 revealed that RNAi-MhMYB15 silenced lines failed to induce an increase in flavonoid content and phosphorus levels in the roots under the treatment of Bacillus B2, and plant growth was slower than the control. In conclusion, MhMYB15 actively responds to Bacillus B2, regulating the accumulation of flavonoids and the uptake of phosphorus, thereby influencing plant growth and development.

Group-C/S1 bZIP heterodimers regulate MdIPT5b to negatively modulate drought tolerance in apple species.

Cytokinins play a central role in delaying senescence, reducing oxidative damage and maintaining plant growth during drought. This study showed that the ectopic expression of ProRE-deleted MdIPT5b, a key enzyme involved in cytokinin metabolism, increased the drought tolerance of transgenic Malus domestica (apple) callus and Solanum lycopersicum (tomato) seedlings by maintaining cytokinin homeostasis, and thus maintaining redox balance. Under restricted watering regimes, the yields of transgenic tomato plants were enhanced. Heterodimers of C/S1 bZIP are involved in the cytokinin-mediated drought response. The heterodimers bind the ProRE of MdIPT5b promoter, thus directly suppressing gene transcription. Single C/S1 bZIP members could not independently function as suppressors. However, specific paired members (heterodimers of MdbZIP80 with MdbZIP2 or with MdbZIP39) effectively suppressed transcription. The α-helical structure is essential for the heterodimerization of C/S1 bZIP members and for synergistic transcriptional suppression. As negative regulators of drought tolerance, suppressing either MdbZIP2 or MdbZIP39 alone does not improve the expression of MdIPT5b and did not increase the drought tolerance of transgenic apple callus. However, this could be achieved when they were co-suppressed. The suppression of MdbZIP80 alone could improve MdIPT5b expression and increase the drought tolerance of transgenic apple callus. However, these effects were reversed in response to the cosuppression of MdbZIP80 and MdIPT5b. Similar results were also observed during delayed dark-induced senescence in apple leaves. In conclusion, the apple C/S1 bZIP network (involving MdbZIP2, MdbZIP39 and MdbZIP80) directly suppressed the expression of MdIPT5b, thus negatively modulating drought tolerance and dark-induced senescence in a functionally redundant manner.

Quantitative Dot Blot (QDB) as a universal platform for absolute quantification of tissue biomarkers.

To circumvent the limitations associated with sandwich ELISA for tissue biomarker quantitation, Quantitative Dot Blot method (QDB) was proposed using antibodies clinically validated for immunohistochemistry (IHC), as this method requires only one primary antibody in the analysis. The protein levels of four breast cancer tissue biomarkers, including Estrogen Receptor (ER), Progesterone Receptor (PR), Ki67 and Her2, were absolutely quantitated successfully in 190 frozen breast tissue biopsies, and the results were further verified with provided IHC results. We propose QDB method as an alternative platform to Sandwich ELISA for absolute quantitation of tissue biomarkers with significantly reduced developing effort and time.

Metabolomics profiling of cleidocranial dysplasia.

OBJECTIVES: Cleidocranial dysplasia (CCD) is a rare autosomal-dominantly inherited skeletal dysplasia that is predominantly associated with heterozygous mutations of RUNX2. However, no information is available regarding metabolic changes associated with CCD at present. MATERIALS AND METHODS: We analyzed members of a CCD family and checked for mutations in the RUNX2 coding sequence using the nucleotide BLAST program. The 3D protein structure of mutant RUNX2 was predicted by I-TASSER. Finally, we analyzed metabolites extracted from plasma using LC-MS/MS. RESULTS: We identified a novel mutation (c.1061insT) that generates a premature termination in the RUNX2 coding region, which, based on protein structure prediction models, likely alters the protein's function. Interestingly, metabolomics profiling indicated that 30 metabolites belonging to 13 metabolic pathways were significantly changed in the CCD patients compared to normal controls. CONCLUSIONS: The results highlight interesting correlations between a RUNX2 mutation, metabolic changes, and the clinical features in a family with CCD. The results also contribute to our understanding of the pathogenetic processes underlying this rare disorder. CLINICAL RELEVANCE: This study provides the first metabolomics profiling in CCD patients, expands our insights into the pathogenesis of the disorder, may help in diagnostics and its refinements, and may lead to novel therapeutic approaches to CCD.

Dynamic Evaluation of Lavage Efficacy in Upper Compartment of the Temporomandibular Joint.

PURPOSE: The aim of this study was to determine the most efficient temporomandibular joint (TMJ) lavage technique for complete irrigation and removal of synovial fluid. The corresponding total lavage fluid volume needed also was investigated. MATERIALS AND METHODS: Three-dimensional TMJ lavage models of the classic double- and single-needle techniques with modified cannula sizes (diameters, 2.4 and 0.8 mm) were generated based on a constructed upper compartment model. Models were integrated with 2-phase flow models to predict fractional fluid volume (α value) changes of lavage saline and synovial fluid within the upper compartment of the TMJ. Fluid flow diagrams, velocity vectors, and intra-articular pressure data were collected and compared among the models. Models were validated by clinical synovial fluid concentration analyses, with vitamin B12 used as an internal standard. RESULTS: In all 8 models, lavage fluid initially gathered around the inflow portal, with a stable mixture of synovial and lavage fluids eventually being established in the compartment. Use of the double-needle technique with a large inflow portal resulted in thorough lavage (α = 100%). When the single-needle or Shepard cannula technique was used, some areas within the upper compartment remained devoid of brisk flow. The 2.4-mm inflow model coupled with a 0.8-mm outflow portal resulted in a stably and persistently high intra-articular pressure (>2.7 × 104 Pa). A minimum volume of 109 mL of lavage fluid was necessary for complete replacement of synovial fluid by saline. CONCLUSIONS: When a 2.4-mm inflow portal needle was applied, a lavage rate of 100% was obtained with a minimum lavage volume of 109 mL. Using a small inflow portal could lead to inadequate flow, residual synovial fluid, and, ultimately, treatment failure.

MdARF3 switches the lateral root elongation to regulate dwarfing in apple plants.

Apple rootstock dwarfing and dense planting are common practices in apple farming. However, the dwarfing mechanisms are not understood. In our study, the expression of MdARF3 in the root system of dwarfing rootstock 'M9' was lower than in the vigorous rootstock from Malus micromalus due to the deletion of the WUSATAg element in the promoter of the 'M9' genotype. Notably, this deletion variation was significantly associated with dwarfing rootstocks. Subsequently, transgenic tobacco (Nicotiana tabacum) cv. Xanthi was generated with the ARF3 promoter from 'M9' and M. micromalus genotypes. The transgenic apple with 35S::MdARF3 was also obtained. The transgenic tobacco and apple with the highly expressed ARF3 had a longer root system and a higher plant height phenotype. Furthermore, the yeast one-hybrid, luciferase, electrophoretic mobility shift assays, and Chip-qPCR identified MdWOX4-1 in apples that interacted with the pMm-ARF3 promoter but not the pM9-ARF3 promoter. Notably, MdWOX4-1 significantly increased the transcriptional activity of MdARF3 and MdLBD16-2. However, MdARF3 significantly decreased the transcriptional activity of MdLBD16-2. Further analysis revealed that MdARF3 and MdLBD16-2 were temporally expressed during different stages of lateral root development. pMdLBD16-2 was mainly expressed during the early stage of lateral root development, which promoted lateral root production. On the contrary, pMmARF3 was expressed during the late stage of lateral root development to promote elongation. The findings in our study will shed light on the genetic causes of apple plant dwarfism and provide strategies for molecular breeding of dwarfing apple rootstocks.

Genome-wide identification of WOX family members in nine Rosaceae species and a functional analysis of MdWOX13-1 in drought resistance.

WUSCHEL-related homeobox (WOX) transcription factors (TFs) are important in plant development processes and evolutionary novelties. In this study, a genome-wide comprehensive analysis of WOX genes from nine Rosaceae species was carried out, and their potential roles in Malus were subsequently investigated. 125 WOXs in 9 Rosaceae species were identified and classified into three clades, i.e., the ancient, intermediate, and WUS clades. Prunus. domestica contained the most intra-genomic collinearity among the nine Rosaceae species. Additionally, the cis-elements in WOX gene family members were compared and classified into three categories, including phytohormone-responsive, plant growth and development, and abiotic and biotic stresses. Overexpression (OE) of MdWOX13-1 also increased the callus weight and enhanced ROS scavenging against drought stress. Furthermore, via yeast-one hybrid assay and LUC analyses, MdWOX13-1 could directly bind to the MdMnSOD promoter. Therefore, our results will facilitate further study of the WOX genes' function in the Rosaceae family.

MdbZIP74 negatively regulates osmotic tolerance and adaptability to moderate drought conditions of apple plants.

Drought is the most prominent threat to global agricultural production. The basic leucine zipper (bZIP) family is related to the response to a series of abiotic stress. In this case, apple calli and the seedlings of MdbZIP74-RNAi transgenic lines were obtained. Under osmotic stress and moderate drought conditions, the content of malondialdehyde, relative water content and other stress-related assays were measured. MdbZIP74 was found to negatively regulate the osmotic tolerance of apple callus. The growth of MdbZIP74-RNAi calli enhanced resistance without significant production loss. The silencing of MdbZIP74 contributes to redox balance and the adaptability of apple seedlings to moderate drought conditions. Four related differentially expressed genes in the biosynthesis of cytokinin and catabolic pathway were identified through a transcriptome analysis of MdbZIP74-RNAi seedlings under moderate drought conditions. MdLOG8 was further identified as the target of MdbZIP74 involved in the drought adaptability of apple plants using a dual experiment. Further confirmation showed MdLOG8 was maintained in the MdbZIP74-RNAi seedlings presumably acting as the growth regulator to enhance drought adaptability. It was concluded that the correct regulation of cytokinin level under moderate drought conditions maintains the redox balance and avoids the situation of plants surviving with the minimal resources.

MdGRF11-MdARF19-2 module acts as a positive regulator of drought resistance in apple rootstock.

14-3-3 proteins play an important role in the response of plants to drought resistance. In this study, 14-3-3 protein MdGRF11 was cloned from Malus xiaojinensis, and its positive regulation of drought resistance was verified using Orin calli and M. xiaojinensis plants. The transcription factor MdARF19-2 was further screened for interaction with this protein in vitro and in vivo. We also conducted experiments using Orin calli and found that the overexpression of MdARF19-2 decreased the level of reactive oxygen species (ROS) and increased the activity of enzymes that scavenge ROS in plant materials. This indicates that MdARF19-2 is a positive regulator in the drought resistance of plants. The drought tolerance was further improved by the overexpression of both MdGRF11 and MdARF19-2 in the calli. In addition, we examined several genes related to ROS scavenging with auxin response factor binding elements in their promoters and found that their level of expression was regulated by the MdGRF11-MdARF19-2 module. In conclusion, the enhancement of plant drought resistance by MdGRF11 could be owing to its accumulation at the protein level in response to drought, which then combined with MdARF19-2, affecting the expression of MdARF19-2 downstream genes. Thus, it scavenges ROS, which ultimately improves the resistance of plant to drought stress.

Genome-wide identification of apple PPI genes and a functional analysis of the response of MxPPI1 to Fe deficiency stress.

Iron (Fe) deficiency affects plant growth and development. The proton pump interactor (PPI) in plants responds to multiple abiotic stresses, although it has not been well characterized under Fe deficiency stress. In this study, we systematically identified and analyzed the PPI gene family in apple. Three PPI candidate genes were found, and they contained 318-1349 amino acids and 3-7 introns. Under Fe deficiency stress, we analyzed the expression of all the PPI genes in roots of apple rootstock Malus xiaojinensis. Expression of the gene MD11G1247800, designated PPI1, is obviously induced by Fe deficiency treatment in M. xiaojinensis. We first cloned MxPPI1 from M. xiaojinensis and determined its subcellular localization, which indicated that it is localized in the cell membrane and nucleus in tobacco. We found that the level of expression of the MxPPI1 protein increased significantly under Fe deficiency stress in apple calli. Moreover, overexpressing MxPPI1 in apple calli enhanced the activities of ferric chelate reductase and H+-ATPase, H+ secretion, MxHA2 gene expression and total Fe content when compared with the wild type calli. We further found that MxPPI1 interacted with MxHA2 using bimolecular fluorescence complementation and luciferase complementation assays. Overall, we demonstrated that MxPPI1 interacts with MxHA2 to enhance the activity of H+-ATPase to regulate Fe absorption in M. xiaojinensis.

Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method.

Developing immunoassay for absolute quantitation of protein biomarkers in Formalin Fixed Paraffin Embedded (FFPE) samples promises improved objectivity, consistency and accuracy in daily clinical practice. The feasibility of Quantitative Dot Blot (QDB) method for this purpose was explored in this study. We were able to measure HER2 protein levels using 0.5 µg/sample total protein lysate extracted from 2 × 5 µm FFPE slices absolutely and quantitatively using QDB method in 332 breast cancer FFPE samples. HER2 levels measured using two clinically validated antibodies for immunohistochemistry respectively were highly correlated (r = 0.963). We also achieved area under the curve (AUC) at 0.9998 ± 0.0002 (p < 0.0001, n = 224) with IHC analysis, and 0.9942 ± 0.0031 (p < 0.0001, n = 319) with combined results from IHC and Fluorescence in situ hybridization (FISH) analyses when analyzed with Receiver Operative Characteristics analysis (ROC) respectively. When the results were converted dichotomously with optimized cutoffs from ROC analyses, we achieved 99.5% concordance with IHC; and 96.9% with combined results from both IHC and FISH analyses. Therefore, we were able to demonstrate QDB method as the first immunoassay platform for absolute quantitation of protein biomarkers in FFPE samples to meet the need of daily clinical practice, especially for local laboratories or laboratories in developing countries.

Novel drug delivery system based on hollow mesoporous magnetic nanoparticles for head and neck cancers--targeted therapy in vitro and in vivo.

Magnetic targeting delivery of anti-cancer drug with controlled drug release function has been recognized as a promising strategy for pursuit of the increased chemotherapeutic efficacy and reduced adverse effects. Superparamagnetic nano-carrier is proved to be an efficient manner for superficial tumor therapy like head and neck cancers. The anti-tumor effect of chemotherapy drug can be enhanced by combining with external magnet. Herein, we reported the fabrication and functionalization of biocompatible and superparamagnetic hollow mesoporous nanoparticles with magnetic targeting. The nanoparticles drug delivery system was constructed by surface-engineering polyacrylic acid (PAA) onto the superparamagnetic nanoparticles which can load bleomycin (BLM) both in the mesoporous structure and via bonding with PAA. The drug was targeted and retained to the focal area under the magnetic field with the nano-carriers, and released sustainably. Detailed investigations demonstrated that PAA-functionalized magnetite nanoparticles loading BLM could stimulate tumor cells to apoptosis locally. The drug loaded and delivery system endowed the anticancer drug with targeting capability in vitro and suppressed the growth of tumor in vivo. The present targeted drug delivery system is a rather simple method without sophisticated chemistry or materials engineering and is promising in contributing to the progress of nanotherapeutics toward efficient head and neck cancer treatment.

Risk factors and biomarkers of non-alcoholic fatty liver disease: an observational cross-sectional population survey.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a major public health burden in China, and its prevalence is increasing. This study aimed to determine the risk factors and biomarkers of NAFLD. DESIGN: An observational cross-sectional primary survey. SETTING: Central China. PARTICIPANTS: The study included 1479 participants aged over 18 and below 80 years, not currently being treated for cancer or infectious disease or no surgery in the previous year, and no history of cancer or an infectious disease. Participants underwent clinical examination, metabolomic assay and anthropometric assessment. Univariate and logistic regression analyses were used to evaluate associations between covariates and NAFLD. MAIN OUTCOME MEASURES: Risk factors and metabolic biomarkers including sex, body mass index, hypertension, body fat ratio, blood triglycerides, blood fasting glucose, liver enzyme elevation, uric acid and oleic acid-hydroxy oleic acid (OAHOA). RESULTS: Data from the 447 participants (mean age 44.3±11.9 years) were analysed, and the prevalence of NAFLD was 24.7%. Male sex (OR 3.484, 95% CI 2.028 to 5.988), body mass index ≥24 kg/m2 (OR 8.494, 95% CI 5.581 to 12.928), body fat ratio (≥25 for women, ≥20 for men) (OR 1.833, 95% CI 1.286 to 2.756), triglycerides ≥1.7 mmol/L (OR 1.340, 95% CI 1.006 to 1.785), fasting glucose ≥6.1 mmol/L (OR 3.324, 95% CI 1.888 to 5.850), blood pressure ≥140/90 mm Hg or antihypertensive drug treatment (OR 1.451, 95% CI 1.069 to 1.970), uric acid (≥357 µmol/L for women, ≥416 µmol/L for men) (OR 2.755, 95% CI 2.009 to 3.778) and OAHOA (<5 nmol/L) (OR 1.340, 95% CI 1.006 to 1.785) were independent predictors of NAFLD (all P<0.05). These results were verified by all 1479 participants. CONCLUSIONS: NAFLD was common among the study participants. In particular, NAFLD was correlated with uric acid. We identified OAHOA as a novel marker of NAFLD prevalence. It provides a reference on the prevention of NAFLD and related metabolic diseases with the rapid urbanisation, technological advancement and population ageing in China over the recent decades.

A novel three-dimensional smile analysis based on dynamic evaluation of facial curve contour.

The influence of three-dimensional facial contour and dynamic evaluation decoding on factors of smile esthetics is essential for facial beauty improvement. However, the kinematic features of the facial smile contour and the contribution from the soft tissue and underlying skeleton are uncharted. Here, the cheekbone-maxilla contour and nasolabial fold were combined into a "smile contour" delineating the overall facial topography emerges prominently in smiling. We screened out the stable and unstable points on the smile contour using facial motion capture and curve fitting, before analyzing the correlation between soft tissue coordinates and hard tissue counterparts of the screened points. Our finding suggests that the mouth corner region was the most mobile area characterizing smile expression, while the other areas remained relatively stable. Therefore, the perioral area should be evaluated dynamically while the static assessment outcome of other parts of the smile contour contribute partially to their dynamic esthetics. Moreover, different from the end piece, morphologies of the zygomatic area and the superior part of the nasolabial crease were determined largely by the skeleton in rest, implying the latter can be altered by orthopedic or orthodontic correction and the former better improved by cosmetic procedures to improve the beauty of smile.

Bleomycin loaded magnetite nanoparticles functionalized by polyacrylic acid as a new antitumoral drug delivery system.

OBJECTIVE: To prepare, characterize, and analyze the release behavior of bleomycin-loaded magnetite nanoparticles (BLM-MNPs) coated with polyacrylic acid (PAA) as a new drug delivery system that can be specifically distributed in the tumor site. METHODS: BLM-MNPs coated with PAA were prepared using a solvothermal approach. The particles were characterized using scanning electron microscope (SEM), vibrating sample magnetometer (VSM), and Fourier transform infrared spectroscopy (FTIR). The loading and release behaviors of BLM-MNPs were examined by a mathematical formula and in vitro release profile at pH 7.5. RESULTS: The sphere Fe3O4 nanoparticles with the size of approximately 30 nm exhibit a saturation magnetization of 87 emu/g. The noncoordinated carboxylate groups of PAA confer on the good dispersibility in the aqueous solution and lead to a good loading efficiency of BLM reaching 50% or higher. Approximately 98% of immobilized BLM could be released within 24 h, of which 22.4% was released in the first hour and then the remaining was released slowly and quantitatively in the next 23 hours. CONCLUSION: BLM-MNPs were prepared and characterized successfully. The particles show high saturation magnetization, high drug loading capacity, and favorable release property, which could contribute to the specific delivery and controllable release of BLM, and the BLM-MNPs could be a potential candidate for the development of treating solid tumors.