Investigation of the distribution of inguinal lymph nodes and delineation of the inguinal clinical target volume using 18F-FDG PET/CT.

OBJECTIVE: Radiotherapy is a crucial treatment modality for pelvic cancers, but uncertainties persist in defining the clinical target volume (CTV) for the inguinal lymphatic drainage region. Suboptimal CTV delineation may compromise treatment efficacy and result in subpar disease control. This study aimed to investigate and map the distribution of lymph node metastases (LNM) in the groin area to facilitate an improved and detailed CTV definition using 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT). METHODS: Inguinal LNM in patients with biopsy-proven pelvic malignancies were identified using 18F-FDG PET/CT scan. The longitudinally nearest axial plane was determined based on six typical bony landmarks, and the axial direction relative to the femoral artery of LNM was recorded. The distances from the LNM to the nearest edge of the femoral artery were measured on the axial plane. An optimal margin to cover 95% of LNM was estimated to develop contouring recommendations. RESULTS: In this study, 500 positive LNM were identified by 18F-FDG PET/CT among 185 patients with primary pelvic malignancies. Relative to the femoral artery, lymph nodes were distributed laterally (10:00-11:00, n = 35), anteriorly (12:00-1:00, n = 213), and medially (2:00-4: 00, n = 252). For CTV delineation, the recommended distances from the femoral artery on the SFH were lateral 19 mm, anterior 19 mm, and medial 25 mm; on the SGT were lateral 26 mm, anterior 20 mm, and medial 25 mm; on the SPS were lateral 28 mm, anterior 29 mm, and medial 26 mm; on the IPS were anterior 29 mm and medial 28 mm; on the IIT were anterior 27 mm and medial 27 mm; on the ILT were anterior 25 mm and medial 23 mm. Use interpolation to contour the area between six axial slices, including any radiographically suspicious LNM. CONCLUSIONS: Using 18F-FDG PET/CT, we investigated the distribution pattern of inguinal LNM and propose a more comprehensive guideline for inguinal CTV delineation.

Advances in injectable hydrogels for radiation-induced heart disease.

Radiological heart damage (RIHD) is damage caused by unavoidable irradiation of the heart during chest radiotherapy, with a long latency period and a progressively increasing proportion of delayed cardiac damage due to conventional doses of chest radiotherapy. There is a risk of inducing diseases such as acute/chronic pericarditis, myocarditis, delayed myocardial fibrosis and damage to the cardiac conduction system in humans, which can lead to myocardial infarction or even death in severe cases. This paper details the pathogenesis of RIHD and gives potential targets for treatment at the molecular and cellular level, avoiding the drawbacks of high invasiveness and immune rejection due to drug therapy, medical device implantation and heart transplantation. Injectable hydrogel therapy has emerged as a minimally invasive tissue engineering therapy to provide necessary mechanical support to the infarcted myocardium and to act as a carrier for various bioactive factors and cells to improve the cellular microenvironment in the infarcted area and induce myocardial tissue regeneration. Therefore, this paper combines bioactive factors and cellular therapeutic mechanisms with injectable hydrogels, presents recent advances in the treatment of cardiac injury after RIHD with different injectable gels, and summarizes the therapeutic potential of various types of injectable hydrogels as a potential solution.

Herbal combinations against COVID-19: A network pharmacology, molecular docking and dynamics study.

OBJECTIVE: The aim of this study is to identify molecules from traditional Chinese medicine (TCM) with potential activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants. METHODS: We applied the Apriori algorithm to identify important combinations of herbs in the TCM prescriptions for the treatment of coronavirus disease 2019 (COVID-19). Then, we explored the active components and core targets using network pharmacology. In addition, the molecular docking approach was performed to investigate the interaction of these components with the main structural and non-structural proteins, as well as the mutants. Furthermore, their stability in the binding pockets was further evaluated with the molecular dynamics approach. RESULTS: A combination of Amygdalus Communis Vas., Ephedra Herba and Scutellaria baicalensis Georgi was selected as the important herbal combination, and 11 main components and 20 core targets against COVID-19 were obtained. These components, including luteolin, naringenin, stigmasterol, baicalein, and so on, were the potentially active compounds against COVID-19. The binding affinity of these compounds with the potential targets was as high as the positive controls. Among them, baicalein could interfere with multiple targets simultaneously, and it also interfered with the interaction between spike protein and angiotensin-converting enzyme 2 receptor. Additionally, almost all the systems reached stability during dynamics simulation. CONCLUSION: The combination of A. communis, Ephedra Herba and S. baicalensis was the most important herbal combination for the treatment of COVID-19. Baicalein may be a potential candidate against SARS-CoV-2 and its variants. Please cite this article as: Song JB, Zhao LQ, Wen HP, Li YP. Herbal combinations against COVID-19: A network pharmacology, molecular docking and dynamics study. J Integr Med. 2023;21(6):593-604.

Lipids associated with plant-bacteria interaction identified using a metabolomics approach in an Arabidopsis thaliana model.

Background: Systemic acquired resistance (SAR) protects plants against a wide variety of pathogens. In recent decades, numerous studies have focused on the induction of SAR, but its molecular mechanisms remain largely unknown. Methods: We used a metabolomics approach based on ultra-high-performance liquid chromatographic (UPLC) and mass spectrometric (MS) techniques to identify SAR-related lipid metabolites in an Arabidopsis thaliana model. Multiple statistical analyses were used to identify the differentially regulated metabolites. Results: Numerous lipids were implicated as potential factors in both plant basal resistance and SAR; these include species of phosphatidic acid (PA), monogalactosyldiacylglycerol (MGDG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and triacylglycerol (TG). Conclusions: Our findings indicate that lipids accumulated in both local and systemic leaves, while other lipids only accumulated in local leaves or in systemic leaves. PA (16:0_18:2), PE (34:5) and PE (16:0_18:2) had higher levels in both local leaves inoculated with Psm ES4326 or Psm avrRpm1 and systemic leaves of the plants locally infected with Psm avrRpm1 or Psm ES4326. PC (32:5) had high levels in leaves inoculated with Psm ES4326. Other differentially regulated metabolites, including PA (18:2_18:2), PA (16:0_18:3), PA (18:3_18:2), PE (16:0_18:3), PE (16:1_16:1), PE (34:4) and TGs showed higher levels in systemic leaves of the plants locally infected with Psm avrRpm1 or Psm ES4326. These findings will help direct future studies on the molecular mechanisms of SAR.

Study on the established customized limits for the daily quality assurance procedure.

Since there are no corresponding specification limits for some new daily quality assurance (QA) items in the TG-142, it is a compromise that the specification limits used in the monthly or annual QA procedures are used for the daily QA procedure in work. But there is no basis for whether this is feasible. The purpose of this article is to analyze QA results using SPC to determine the tolerance limits at our institution, and to present the usefulness of the analysis method using SPC. The data of three groups daily QA processes performed with Daily QA3 in three years were analyzed using statistical process control (SPC). For calculating capability indices (Cp, Cpk, Cpm and Cpmk) of processes, the appropriate number of calculation points was analyzed firstly. Then, in calculating the capability indices for output, limits ±3% of the daily QA in the TG-142 were used as the specification limit, while for flatness and symmetry, an annual QA limits of ±1% was used. For putting forward measures to solve the problem, customized tolerance and action limits were established for each process. And the process control charts calculated using data measured by the five therapists and a medical physicist were compared. At least six to eight weeks of control daily check data points (i.e. 30-40 points) should be used for calculating the individuals and moving range (I-MR) control chart to ensure the stability of control lines. Process capability indices of output were all ≥1, some were up to 3-4. While for symmetry, some processes failed to meet the requirements that capability indices were < 1. For different processes of the same daily QA items, the calculated customized limits were quite different. The range of upper control line (UCL) and lower control line (LCL) was smaller for output and the CL was closer to the target value of 0 for flatness and symmetry in the I-MR control chart calculated using data measured by one staff. For different quality control processes without management by the SPC method at our institution, calculated tolerance and action limits of the same measurement item were quite different. And in most measurement items, the specification limits used in the monthly or annual QA procedures in the TG-142 are not suitable to the daily QA procedure. So the analysis method using SPC is useful and necessary.

The F-box E3 ubiquitin ligase AtSDR is involved in salt and drought stress responses in Arabidopsis.

F-box protein genes have been shown to play vital roles in plant development and stress respones. In Arabidopsis, there are more than 600 F-box proteins, and most of their functions are unclear. The present study shows that the F-box (SKP1-Cullin/CDC53-F-box) gene At5g15710 (Salt and Drought Responsiveness, SDR) is involved in abiotic stress responses in Arabidopsis. SDR is expressed in all tissues of Arabidopsis and is upregulated by salt and heat stresses and ABA treatment but downregulated by drought stress. Subcellular localization analysis shows that the SDR protein colocalizes with the nucleus. 35S:AntiSDR plants are hypersensitive to salt stress, but 35S:SDR plants display a salt-tolerant phenotype. Furthermore, 35S:SDR plants are hypersensitive to drought stress, while 35S:AntiSDR plants are significantly more drought tolerant. Overall, our results suggest that SDR is involved in salt and drought stress responses in Arabidopsis.

Roles of Endogenous Melatonin in Resistance to Botrytis cinerea Infection in an Arabidopsis Model.

Melatonin is an important bioactive molecule in plants. Two synthetases, N-acetylserotonin methyltransferase (ASMT) and serotonin N-acetyltransferase (SNAT) are involved in the final two steps of melatonin synthesis. Melatonin participates in responses to a variety of biotic and abiotic stresses in plants, but few studies have addressed the roles of endogenous melatonin in pathogen resistance. We investigated the role of endogenous melatonin in resistance to Botrytis cinerea infection in an Arabidopsis thaliana model system. Plant lines that overexpressed ASMT or SNAT through genetic manipulation showed upregulated expression of resistance genes PR1 and PR5, transcription factor gene WRKY33, and jasmonic acid (JA) defense pathway marker gene PDF1.2, and downregulated transcription factor gene MYC2 in JA signaling pathway. Higher melatonin content also enhanced the activity of antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD), increased JA content, reduced plant disease symptoms, and reduced lesion size in leaves. These findings indicate that endogenous melatonin enhances plant resistance to B. cinerea infection. In contrast, ASMT and SNAT gene silencing lines showed opposite results and were more susceptible to B. cinerea. Thus, it can be demonstrated that melatonin functions as an effective regulator of plant stress resistance at the genetic level. A schematic model is presented for its role in resistance to B. cinerea infection. Our findings also helped to elucidate the associated signal transduction pathways and interactions between melatonin and other plant hormones.

Distribution and Dynamic Changes in Matrix Metalloproteinase (MMP)-2, MMP-9, and Collagen in an In Stent Restenosis Process.

OBJECTIVE: The aim of this study was to observe the spatial distribution and dynamic changes of matrix metalloproteinase (MMP)-2, MMP-9, and collagen in in stent restenosis (ISR) and to explore their influence on ISR. METHODS: Sixty Z type stents were implanted into the common iliac arteries of minipigs, which were divided into 10 groups (six in each group) according to euthanasia time (6 hours, and 1, 3, 7, 14, 28, 56, 84, 168, and 336 days). After the samples were harvested, haematoxylin and eosin staining, immunohistochemical staining, Western blotting, and Picrosirius red staining were performed for all groups. RESULTS: ISR occurred in all six minipigs in the 56 day group (percentage diameter stenosis range 71.6%-79.2%, mean ± standard deviation 75.6% ± 2.5%). The percentage diameter stenosis decreased to 38.3% ± 2.7% at 336 days (p < .001). Immunohistochemical staining showed that MMP-2 and MMP-9 were strongly stained near the internal elastic lamina or in the damaged parts of the intima, around the struts and neointimal lumen surface in the ISR process. The expression of MMP-2 and MMP-9 at 56 days was significantly lower compared with their peaks (seven days and one day [p < .001; p = .002], respectively). At 56 days, the collagen content reached its maximum (mean integrated optical density range 0.73-0.92, mean ± standard deviation 0.82 ± 0.09). From the 14 day group to the 336 day group, mature collagen in neointima was correlated negatively with MMP-2 (γ(36) = -0.816; p < .001) and MMP-9 expression (γ(36) = -0.853; p < .001). During the neointimal regression period, new collagen in neointima was positively correlated with MMP-2 (γ(24) = 0.683; p < .001) and MMP-9 (γ(24) = 0.873; p < .001). CONCLUSION: This study has demonstrated the spatial distribution of and dynamic changes in MMP-2, MMP-9, and collagen in ISR by simulating the process of neointima from generation to regression after stent implantation. When ISR occurred, MMP-2 and MMP-9 expression decreased and collagen content reached its maximum, which might contribute to ISR.

A risk predictor of restenosis after superficial femoral artery stent implantation: relevance of mean platelet volume.

BACKGROUND: To investigate the relationship between an increase in the pre- and post-operative mean platelet volume (MPV) and superficial femoral artery in-stent restenosis (ISR) rate. METHODS AND RESULTS: We recruited patients that underwent superficial femoral artery stenting for lower extremity arteriosclerosis obliterans at our hospital from March 2015 to March 2018. All patients gave venous blood three days before and following implantation. Doppler ultrasound, computed tomography angiography or digital subtraction angiography were used for regular follow-up examination. Logistic regression was used to identify predictors of ISR after superficial femoral artery stenting. We enrolled 173 patients, of which 34 (19.6%) were determined as having ISR for a mean of 8.9 ± 2.7 months (3-12 months). Neutrophil count, neutrophil ratio, lymphocyte ratio and platelet count pre-implantation, and platelet count and MPV after stent implantation, and the pre- and post-operative mean platelet volume difference (MPVD) and mean platelet volume difference ratio (MPVDR) were all statistically different when comparing the ISR and non-restenosis groups (p < 0.05). A positive correlation was found for post-operative MPV and presence of ISR (r = 0.58; P < 0.001). A MPVD not less than 1.5 fL was associated with an odds ratio of 9.17 (95% CI [3.76 to 22.35]; P < 0.001) for presence of ISR. A MPVDR of not less than 17.9% was associated with an odds ratio of 7.68 (95% CI [3.19 to 18.49]; P < 0.001) for occurrence of ISR. CONCLUSIONS: An increase in pre- and post-operative MPV was correlated with the occurrence of superficial femoral artery ISR.

Rhubarb-Aconite Decoction (RAD) Drug-Containing Serum Alleviated Endotoxin-Induced Oxidative Stress Injury and Inflammatory Response in Caco-2 Cells In Vitro.

Rhubarb-Aconite Decoction (RAD), a famous Chinese medicine prescription, has been widely used for treating intestinal injury. However, the effect of RAD on intestinal epithelial cells is unclear. The aim of this study was to investigate the effects of RAD drug-containing serum on the oxidative stress injury and inflammatory response induced by endotoxin (ET) in Caco-2 cells in vitro. Lipid peroxide malondialdehyde (MDA), lactate dehydrogenase (LDH), caspase-11, tumor necrosis factor-α(TNF-α), interleukin-3(IL-3), and cytokeratin (CK)18, adenosine triphosphate (ATP) activity, and intracellular free calcium ion levels were measured. The results showed that ET triggered the activation of caspase-11 and the massive release of TNF-α, increased the inhibitory rate of cell growth, MDA, and LDH expressions in Caco-2 cells. Moreover, RAD drug-containing serum could inhibit caspase-11 activation, decrease the release of TNF-α and IL-3, reduce intracellular free calcium ion, and enhance CK 18 expression and ATP activity. These novel findings demonstrated that ET-induced oxidative stress injury and inflammatory response of Caco-2 cells were improved by RAD drug-containing serum, indicating that RAD may be a good choice for the treatment of intestinal injury.

Effects of a Matrix Metalloproteinase Inhibitor-Eluting Stent on In-Stent Restenosis.

BACKGROUND The aim of this study was to compare changes in the extracellular matrix after implantation of a stent that elutes a matrix metalloproteinase (MMP) inhibitor (GM6001); and to determine the effects of the GM6001-eluting stent upon prevention of in-stent restenosis (ISR). MATERIAL AND METHODS We included 48 Guangxi Bama mini-pigs in this study. A GM6001-eluting stent was placed in one iliac artery and a stent that did not elute GM6001 was placed in the contralateral iliac artery. The iliac arteries were removed at 6 hours as well as 1, 7, 14, 56, 84, and 336 days after stent placement. Arteries were analyzed for morphometry, gelatinase content, different phenotypes of vascular smooth muscle cells (VSMCs), collagen content, apoptotic rate, and cell density. RESULTS The vascular lumen areas of the GM6001 group were significantly increased and the neointimal areas were significantly reduced compared with the control group from the 7 days to the 336 days. In the 2 groups, expression of MMP-2 and MMP-9 peaked simultaneously, but GM6001-eluting stents inhibited expression of MMP-2 and MMP-9 in the vascular media and neointima (especially around the struts) significantly. In the GM6001 group, expression of tissue inhibitor of matrix metalloproteinase (TIMP)-1, TIMP-2, myosin heavy chain 10 (MYH-10, marker of the proliferative phenotype of VSMCs), collagen content, percentage of apoptotic cells, and cell density were also decreased significantly compared with those in the control group. CONCLUSIONS Use of GM6001-eluting stents resulted in persistent and potent inhibition of intimal hyperplasia, an increase in luminal area, and no obvious thrombosis in the arteries of the mini-pigs.

High Nighttime Temperature Induces Antioxidant Molecule Perturbations in Heat-Sensitive and Heat-Tolerant Coisogenic Rice ( Oryza sativa) Strains.

Global warming-associated increases in temperature, particularly at nighttime, are detrimental to rice yield and quality. Metabolomic profiling was used to examine and compare the short-term extreme high nighttime temperature-induced molecular perturbations in rice ( Oryza sativa) coisogenic strains with contrasting heat-tolerances at the first stage of seed ripening. Compared to the heat-sensitive strain, antioxidant molecules were higher in abundance in the heat-tolerant strain, whereas the abundances of molecules involved in photosynthesis, nucleotide catabolism, and the S-adenosylmethionine (SAM) cycle varied only slightly. Thus, we proposed that the high abundance of antioxidant molecules in the heat-tolerant strain alleviated cellular oxidative stress, which protected photosynthesis, nucleotide catabolism, and the SAM cycle, leading to good grain filling.

Identification of Cd-responsive RNA helicase genes and expression of a putative BnRH 24 mediated by miR158 in canola (Brassica napus).

RNA helicases play crucial roles in RNA splicing, transport, editing and degradation, protein translation initiation and siRNA-mediated gene silencing. However, knowledge about their functionality in rapeseed (Brassica napus) is rare. In the study, we identified and annotated 271 RNA helicase genes from B. napus using bioinformatics and high-throughput RNA-sequencing (RNA-seq). Three subfamilies DEAD-box, DEAH-box, or DExD/H-box have been identified. One hundred and ninety-five RNA helicases were confirmed by RNA-seq and 49 were identified to differentially respond to cadmium (Cd) stress (> 1.5 fold change, p < 0.05). As an example, we functionally specified BnaA04g26450D encoding a BnRH24 under Cd exposure. BnRH24 is a constitutive gene expressing throughout the life span. Using our previously generated degradome datasets, we found that BnRH24 can be cleaved by miR158, suggesting that BnRH24 is a target of miR158 in B. napus. The mature miR158 was induced, while BnRH24 was repressed in B. napus under Cd stress. The contrasting expression pattern of B. napus miR158 and BnRH24 under the normal and Cd would support the post-transcriptional regulation of BnRH24 by miR158. Ectopic expression of BnRH24 in Arabidopsis revealed that the transgenic lines showed more sensitivity to Cd toxicity by reducing root elongation, fresh mass production, chlorophyll accumulation and increasing oxidative products such as O2-., H2O2 and thiobarbituric acid reactive substances (TBARS), indicating that the controlling the level of BnRH24 by miR158 may be required for Cd tolerance in plants.

Gadd45a gene silencing by RNAi promotes cell proliferation and inhibits apoptosis and senescence in skin squamous cell carcinoma through the p53 signaling pathway.

Skin squamous cell carcinoma (SCC) is generally considered as nonaggressive lesions and mainly caused by ultraviolet (UV) radiation. Gadd45a is a key component protecting skin against UV-induced tumors. For that, the study aims to investigate the mechanism of Gadd45a gene silencing on cell proliferation, apoptosis, and senescence in nude mice with skin SCC through the p53 signaling pathway. Healthy nude mice was collected as the normal group and 40 nude mouse models of skin SCC were successfully established as the model group, which were sub-divided into five groups. The incidence, size, and weight of SCC tumor of nude mice were observed. The mRNA expression of Gadd45a, Cyclin B1, MMP-2, Bcl-2, and Bax were determined by RT-qPCR. Cell viability, cell cycle and apoptosis, cell senescence were detected by MTT assay, flow cytometry, and ß-galactosidase staining, respectively. The levels of inflammatory factors and vascular endothelial growth factor (VEGF) were detected by using ELISA. The protein expression rate of mutant p53 was detected by immunohistochemistry. Mice transfected with siGadd45a showed increased tumor incidence, size, and weight. Cells transfected with siGadd45a showed decrease in expression of Gadd45a and Bax; and increase in expression of Cyclin B1, MMP-2, and Bcl-2, expression of mutant p53, IL-1α, IL-1ß, IL-6, TNF-α, and VEGF. Cell apoptosis and senescence were inhibited, while cell viability and proliferation were promoted after siGadd45a treatment. The results reveal that Gadd45a silencing increases tumor cell proliferation and reduces apoptosis and senescence through the p53 signaling pathway in skin SCC.

Dai-Huang-Fu-Zi-Tang alleviates pulmonary and intestinal injury with severe acute pancreatitis via regulating aquaporins in rats.

BACKGROUND: Dai-Huang-Fu-Zi-Tang (DHFZT) is a famous traditional Chinese prescription with intestinal obstruction, acute pancreatitis and cholecystalgia for thousands of years. Our previous work found that DHFZT could act against pulmonary and intestinal pathological injury in rats with severe acute pancreatitis (SAP). But the underlying mechanism has not been fully elucidated. The aim of present study was to investigate whether DHFZT could relieve pulmonary and intestinal injury by regulating aquaporins after SAP induced by sodium taurocholate in rats. METHODS: Forty of SD rats were used for dose dependant experiments of DHFZT.Accurate-mass Time-of-flight liquid chromatography-mass spectrometry was used for qualitative screening of chemical compositions of DHFZT. Twenty-four rats were randomly divided into 3 groups: sham group (n = 8), model group (SAP, n = 8), DHFZT group (SAP with DHFZT treatment, n = 8). SAP models were established by retrograde injections of 5% sodium taurocholate solutions into rat pancreaticobiliary ducts. Blood samples were taken at 0, 12, 24, 48 h post-operation for detecting serum amylase, lipase, endotoxin, TNF-α, IL-6 and IL-10. Protein expression and location of aquaporin (AQP)1, 5, 8 and 9 were assessed by immunohistochemistry, western blot and immunofluorescence respectively. RESULTS: The study showed that 27 kinds of chemical composition were identified, including 10 kinds in positive ion mode and 17 kinds in negative ion mode. The results showed that AQP1, AQP5 of lung, and AQP1, AQP5, AQP8 of intestine in model group were significantly lower than that of sham group (P < 0.05), and which were obviously reversed by treatment with DHFZT. In addition, protein levels of pro-inflammatory cytokines such as TNF-α, IL-6 and endotoxin in peripheral blood were significantly suppressed by DHFZT, and that anti-inflammatory cytokine like IL-10 was just opposite. Finally, we also noted that DHFZT reduced serum levels of amylase, lipase and endotoxin, and also improved edema and pathological scores of lung and intestine after SAP. CONCLUSIONS: DHFZT ameliorated the pulmonary and intestinal edema and injury induced by SAP via the upregulation of different AQPs in lung and intestine, and suppressed TNF-α, IL-6 expression and enhanced IL-10 expression.

Altered Fruit and Seed Development of Transgenic Rapeseed (Brassica napus) Over-Expressing MicroRNA394.

Fruit and seed development in plants is a complex biological process mainly involved in input and biosynthesis of many storage compounds such as proteins and oils. Although the basic biochemical pathways for production of the storage metabolites in plants are well characterized, their regulatory mechanisms are not fully understood. In this study, we functionally identified rapeseed (Brassica napus) miR394 with its target gene Brassica napus leaf curling responsiveness (BnLCR) to dissect a role of miR394 during the fruit and seed development. Transgenic rapeseed plants over-expressing miR394 under the control of the cauliflower mosaic virus 35S promoter were generated. miR394 over-expression plants exhibited a delayed flowering time and enlarged size of plants, leaf blade, pods and seed body, but developed seeds with higher contents of protein and glucosinolates (GLS) and lower levels of oil accumulation as compared to wild-type. Over-expression of miR394 altered the fatty acid (FA) composition by increasing several FA species such as C16:0 and C18:0 and unsaturated species of C20:1 and C22:1 but lowering C18:3. This change was accompanied by induction of genes coding for transcription factors of FA synthesis including leafy cotyledon1 (BnLEC1), BnLEC2, and FUSCA3 (FUS3). Because the phytohormone auxin plays a crucial role in fruit development and seed patterning, the DR5-GUS reporter was used for monitoring the auxin response in Arabidopsis siliques and demonstrated that the DR5 gene was strongly expressed. These results suggest that BnmiR394 is involved in rapeseed fruit and seed development.

The F-box family genes as key elements in response to salt, heavy mental, and drought stresses in Medicago truncatula.

F-box protein is a subunit of Skp1-Rbx1-Cul1-F-box protein (SCF) complex with typically conserved F-box motifs of approximately 40 amino acids and is one of the largest protein families in eukaryotes. F-box proteins play critical roles in selective and specific protein degradation through the 26S proteasome. In this study, we bioinformatically identified 972 putative F-box proteins from Medicago truncatula genome. Our analysis showed that in addition to the conserved motif, the F-box proteins have several other functional domains in their C-terminal regions (e.g., LRRs, Kelch, FBA, and PP2), some of which were found to be M. truncatula species-specific. By phylogenetic analysis of the F-box motifs, these proteins can be classified into three major families, and each family can be further grouped into more subgroups. Analysis of the genomic distribution of F-box genes on M. truncatula chromosomes revealed that the evolutional expansion of F-box genes in M. truncatula was probably due to localized gene duplications. To investigate the possible response of the F-box genes to abiotic stresses, both publicly available and customer-prepared microarrays were analyzed. Most of the F-box protein genes can be responding to salt and heavy metal stresses. Real-time PCR analysis confirmed that some of the F-box protein genes containing heat, drought, salicylic acid, and abscisic acid responsive cis-elements were able to respond to the abiotic stresses.

miR394 and LCR are involved in Arabidopsis salt and drought stress responses in an abscisic acid-dependent manner.

BACKGROUND: MicroRNAs (miRNAs) are a class of short, endogenous non-coding small RNAs that have ability to base pair with their target mRNAs to induce their degradation in plants. miR394a/b are conserved small RNAs and its target gene LCR (LEAF CURLING RESPONSIVENESS) encodes an F-box protein (SKP1-Cullin/CDC53-F-box) but whether miR394a/b and its target gene LCR are involved in regulation of plant response to abscisic acid (ABA) and abiotic stresses is unknown. RESULTS: Mature miR394 and precursor miR394a/b are shown to be slightly induced by ABA. By contrast, LCR expression is depressed by ABA. Analysis of LCR and its promoter (pLCR::GUS) revealed that LCR is expressed at all development stages. MIR394a/b over-expression (35S::MIR394a/b) and lcr (LCR loss of function) mutant plants are hypersensitive to salt stress, but LCR over-expressing (35S::m5LCR) plants display the salt-tolerant phenotype. Both 35S::MIR394a/b and lcr plants are highly tolerant to severe drought stress compared with wild-type, but 35S::m5LCR plants are susceptible to water deficiency. Over-expression of MIR394a/b led to ABA hypersensitivity and ABA-associated phenotypes, whereas 35S::m5LCR plants show ABA resistance phenotypes. Moreover, 35S::MIR394a/b plants accumulated higher levels of ABA-induced hydrogen peroxide and superoxide anion radicals than wild-type and 35S::m5LCR plants. Expressions of ABA- and stress-responsive genes, ABI3, ABI4, ABI5, ABF3, and ABF4 are up-regulated in MIR394a/b over-expressing plants but down-regulated in 35S::m5LCR plants. Over-expression of MIR394a in abi4-1 or abi5-1 background resulted in loss of ABA-sensitivity in 35S::MIR394a plants. CONCLUSIONS: The silencing of LCR mRNA by miR394 is essential to maintain a certain phenotype favorable for the adaptive response to abiotic stresses. The contrasting phenotypes of salt and drought responses may be mediated by a functional balance between miR394 and LCR. If the balance is perturbed in case of the abiotic stress, an identical phenotype related to the stress response occurs, resulting in either ABA sensitive or insensitive response. Thus, miR394-regulated LCR abundance may allow plants to fine-tune their responses to ABA and abiotic stress.

AtHO1 is involved in iron homeostasis in an NO-dependent manner.

AtHO1 (HY1) encodes heme oxygenase-1 in Arabidopsis, catalyzing cleavage of heme to biliverdin with the release of iron and carbon monoxide (CO). Our previous study showed that CO as an endogenous component is able to improve plant adaptation to iron deficiency. Here, we performed a genetic study to identify further the putative role of AtHO1 in the iron deficiency response. Iron deficiency induced AtHO1 expression at the transcriptional and translational levels. Evidence has been provided that overexpression of AtHO1 could confer plant tolerance to iron deficiency by improving expression of AtFIT, AtFRO2 and AtIRT1, the activity of ferric-chelate reductase (FCR) and iron accumulation. In contrast, RNA interference with AtHO1 expression in 35S::AntiHO1 plants and the AtHO1 loss-of-function (hy1 mutant) resulted in adverse phenotypes. In 35S::AtHO1 transgenic lines, a higher level of CO and water-soluble iron, and a lower level of heme were identified, suggesting that AtHO1-regulated iron homeostasis was possibly through the catabolism of heme to produce CO and free iron. Because nitric oxide (NO) is known to regulate iron homeostasis in plants, the connection between AtHO1 expression and NO action was examined. AtHO1-overexpressing plants generated more NO, whereas knock-down of AtHO1 expression reduced the level of NO in plants. The NO scavenger cPTIO [2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylini dazoline-1-oxyl-3-oxide] caused a decrease in AtHO1-induced FCR activity. Under both iron-sufficient and -deficient conditions, administration of the NO donor sodium nitroprusside induced FCR activity in the hy1 plants. These results suggest that AtHO1 is involved in iron homeostasis in an NO-dependent manner.

miR395 is involved in detoxification of cadmium in Brassica napus.

The toxic metal cadmium (Cd) constitutes one of the major inorganic contaminants in environments. microRNAs (miRNAs) are a class of endogenous non-coding small RNAs. miR395 is conserved and regulates sulfate assimilation and distribution in higher plants, but whether it is involved in detoxification of Cd in plants has not been described. In this study, transgenic rapeseed (Brassica napus) over-expressing miR395 was identified under Cd stress. miR395-over-expressing plants showed a lower degree of Cd-induced oxidative stress than wild type. By contrast, chlorophyll, glutathione and non-protein thiols contents were higher in the transformants than wild type. Determination of growth response showed that 35S::MIR395 plants accumulated higher levels of biomass and sulfur than wild type under Cd exposure. miR395 transgenic plants had higher levels of Cd in plants, particularly at the high supply of Cd in the medium, but they tended to repress Cd translocation from roots to shoots. Simultaneously, expression of metal-tolerance genes such as BnPCS1, BnHO1 and Sultr1;1 was up-regulated under Cd stress, and the expression of the genes was more pronounced in 35S::MIR395 plants than in wild type. These results suggest that miR395 would be involved in detoxification of Cd in B. napus.