Functional analysis of conserved amino acid residues in the C-terminus of ACC synthase

Abstract only availableEthylene is an important plant hormone that regulates growth, development, and stress response. Synthesis of Ethylene from its immediate precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), is catalyzed by ACC oxidase. ACC is produced from S-Adenosy1-L-Methionine (SAM) in a reaction catalyzed by ACC synthase (ACS). ACS is the rate limiting enzyme of ethylene biosynthesis. Selected isoforms of ACS are substrates of MPK6 and MPK3, the two Arabidopsis stress-responsive mitogen-activated protein kinases (MAPKs). Phosphorylation of ACS6 by MPK6 stabilizes the ACS protein, thus, elevating the levels of cellular ACS activity and ethylene production. Expression of ACS6DDD, a gain-of-function ACS6 mutant that mimics the phosphorylated form of ACS6, shows constitutive ethylene production and ethylene-induced phenotypes. Analysis of Arabidopsis ACS6 and its orthologs from other species in the database revealed conserved charged amino acids (AAs) in addition to the MAPK phosphorylation sites in their C-termini. We hypothesized that these conserved residues may be involved in the regulation of ACS stability. We used site-directed mutagenesis to mutate the conserved residues to Ala, Ile, or Leu in the ACS6WT or ACS6DDD background using the polymerase chain reaction (PCR). Mutation was confirmed by DNA sequencing. ACS6 mutant gene was transformed into Arabidopsis plants. The stability of ACS6 protein was tested in vivo to determine if the mutation enhances or diminishes its stability. Ethylene production was used as an output reading and the levels of ACS6 protein were determined by immunoblot analysis. Mutation of positively charged AAs makes the ACS6 protein more stable, whereas the mutation of the negatively charged AAs which are close to the phosphorylation sites destabilizes it. Interestingly, deletion of the C-terminus stabilizes the ACS6 protein, suggesting that C-terminus is required for ACS6 degradation. We observed ethylene-regulated morphologies like short hairy main roots and epinastic leaves in ethylene-overproducing seedlings.MU Monsanto Undergraduate Research Fellowshi

Centerline Extraction for Image Segmentation Using Gradient and Direction Vector Flow Active Contours

In this paper, we propose a fast centerline extraction method to be used for gradient and direction vector flow of active contours. The gradient and direction vector flow is a recently reported active contour model capable of significantly improving the image segmentation performance especially for complex object shape, by seamlessly integrating gradient vector flow and prior directional information. Since the prior directional information is provided by manual line drawing, it can be inconvenient for inexperienced users who might have difficulty in finding the best place to draw the directional lines to achieve the best segmentation performance. This paper describes a method to overcome this problem by automatically extracting centerlines to guide the users for providing the right directional information. Experimental results on synthetic and real images demonstrate the feasibility of the proposed method

Functional analysis of MPK3 and MPK6, two mitogen-activated protine kinases in Arabidopsis thaliana

Abstract only availableMitogen-activated protein kinase (MAPK) cascades are major pathways involved in the transduction of extracellular signals into intracellular responses. A MAPK cascade consists of three kinases; MAPK, MAPK kinase (MAPKK or MEK) and MAPKK kinase (MAPKKK or MEKK). MAPKKK is at the top of this three-tier cascade. Upon its activation by a receptor/sensor, MAPKKK phosphorylates MAPKK, which in turn phosphorylates MAPK and activates it. The activated MAPK can then phosphorylate other protein kinases or be translocated to the nucleus where it can phosphorylate transcription factors and activate gene expression. About 20 MAPKs were identified in the fully sequenced Arabidopsis genome. To study the function of MPK3 and MPK6, the two most closely related MAPKs in Arabidopsis, we isolated the corresponding T-DNA mutants from mutant libraries generated at Wisconsin Arabidopsis Knockout Facility and Salk Institute Genomic Analysis Laboratory. No morphological or developmental phenotypes were observed in the MPK3-/- and MPK6-/- single mutants. In order to determine if MPK3 and MPK6 have overlapping functions, we crossed the two single mutants (MPK3-/- and MPK6-/-) to generate double mutants. Among the 172 F2 plants that we genotyped, no double homozygous (MPK3-/-/MPK6-/-) mutant plants was identified, indicating that this genotype is lethal. We further observed that plants with the MPK3+/-/MPK6-/- genotype are a little smaller and sterile. Reciprocal back cross to wild type plants demonstrated that MPK3+/-/MPK6-/- plants are female sterile. The resilience of the pollens from such plants is still under investigation. In contrast to MPK3+/-/MPK6-/- plants, MPK3-/-/MPK6+/- plants are fertile and apparently normal. Together with the normal phenotype of MPK3-/- and MPK6-/- single mutants, we conclude that MPK3 and MPK6 perform overlapping but not identical roles in the reproduction and development of Arabidopsis thaliana.EXPRESS Progra

Functional analysis of MAP kinases in Arabidopsis thaliana: Fully rescuing the mpk3/mpk6 mutant phenotypes

Abstract only availableMitogen Activated Protein Kinase (MAPK) cascades are three-stage modules involved in signal transduction. MAPKs function at the lower tier of these cascades and they phosphorylate transcription factors and other protein kinases upon activation, ultimately leading to cellular responses. Twenty genes coding for MAPKs were identified in the fully sequenced Arabidopsis genome. MPK3 and MPK6 are the most closely related. Analysis of T-DNA insertional lines revealed no phenotype in the mpk3 and mpk6 single mutants; however, female sterility is observed in MPK3+/-/MPK6-/- plants and embryo lethality results from knocking out both genes. This indicates overlapping function of MPK3 and MPK6. To better understand the function of these two kinases, an attempt was made to rescue these phenotypes by introducing a Dexamethasone (DEX) inducible: MPK6 transgene. This construct led to only partial rescue of the lethal double mutants, and no signs of fertility were evident in MPK3+/-/MPK6-/- plants. In an attempt to attain complete rescue of these phenotypes, new MPK3 and MPK6 constructs were engineered with the following features: • Transgenes regulated by endogenous promoters were used in order to maintain normal cell/tissue specific expression of the protein, which may be essential for normal plant function. • The transgene products were tagged with Yellow Florescent Protein and Green Florescent Protein in order to ascertain their expression patterns. • Genomic DNA, as opposed to complementary DNA, was used as the coding regions in order to ensure the presence of introns, which may be significant for gene function. Currently, T1 generation transgenic plants have been isolated and transgenic lines with good expression of the transgene proteins, in vivo, will be identified by Western Blot analysis. Indication of a full rescue will be verified in the T2 generation. Failure to observe completely rescued lines may indicate protein tag interference, and untagged constructs will then be attempted.MU Monsanto Undergraduate Research Fellowshi

High mobility group box 1 promotes radioresistance in esophageal squamous cell carcinoma cell lines by modulating autophagy

Resistance to radiotherapy results in relapse and treatment failure in locally advanced esophageal squamous cell carcinoma (ESCC). High mobility group box 1 (HMGB1) is reported to be associated with the radioresistance in bladder and breast cancer. However, the role of HMGB1 in the radiotherapy response in ESCC has not been fully elucidated. Here, we investigated the role of HMGB1 to radioresistance in ESCC clinical samples and cell lines. We found that HMGB1 expression was associated with tumor recurrence after postoperative radiotherapy in locally advanced ESCC patients. HMGB1 knockdown in ESCC cells resulted in increased radiosensitivity both in vitro and in vivo. Autophagy level was found depressed in HMGB1 inhibition cells and activation of autophagy brought back cell's radioresistance. Our results demonstrate that HMGB1 activate autophagy and consequently promote radioresistance. HMGB1 may be used as a predictor of poor response to radiotherapy in ESCC patients. Our finding also highlights the importance of the utility of HMGB1 in ESCC radiosensitization.Peer reviewe

Cardiovascular Response of Aged Outpatients With Systemic Diseases During Tooth Extraction: A Single-Center Retrospective Observational Study

BackgroundAged people are maintaining many natural teeth due to improved oral health. However, compromised general health and poor oral hygiene habits at earlier ages resulted in poor status of preserved teeth. Therefore, tooth extraction is required in many aged people. More knowledge is needed because there are many risk factors during the surgery in frail aged adults. The aim of this study was to evaluate the cardiovascular response of such a population during tooth extraction and analyze risk factors to provide clinical guidance.MethodsA retrospective study was performed on aged patients with systemic diseases who underwent tooth extraction. Data regarding demographic profiles and cardiovascular parameters of heart rate and blood pressure were collected preoperative, when local anesthesia was administered, at the beginning of tooth extraction, 5 min after tooth extraction, and postoperative. The effects of risk factors, including age, sex, and systemic diseases on these parameters were analyzed with a multilevel model.ResultsHeart rate (HR), systolic blood pressure (SBP), and diastolic blood pressure (DBP) of aged patients increased significantly when performing local anesthesia and tooth extraction. During the operation, the older patients (β = 2.011, P = 0.005) and the diabetics (β = 3.902, P < 0.0001) were associated with higher SBP, while those with more tooth extractions exhibited higher HR (β = 0.893, P = 0.007). Women patients showed both significantly elevated HR (β = 1.687, P < 0.0001) and SBP (β = 2.268, P < 0.0001). However, for coronary artery disease patients, HR (β = −2.747, P < 0.0001) and blood pressure [SBP (β = −4.094, P < 0.0001) and DBP (β = −0.87, P = 0.016)] were markedly lower than those of patients without a diagnosis of coronary artery disease.ConclusionCardiovascular response of aged outpatients with systemic diseases during tooth extraction is quite significant. Age, sex, systemic diseases, and the number of tooth extraction could be risk factors closely associated with cardiovascular response. The findings might provide safety guidance for dentists on tooth extraction in this population

Up-Regulation of Mitochondrial Activity and Acquirement of Brown Adipose Tissue-Like Property in the White Adipose Tissue of Fsp27 Deficient Mice

Fsp27, a member of the Cide family proteins, was shown to localize to lipid droplet and promote lipid storage in adipocytes. We aimed to understand the biological role of Fsp27 in regulating adipose tissue differentiation, insulin sensitivity and energy balance. Fsp27−/− mice and Fsp27/lep double deficient mice were generated and we examined the adiposity, whole body metabolism, BAT and WAT morphology, insulin sensitivity, mitochondrial activity, and gene expression changes in these mouse strains. Furthermore, we isolated mouse embryonic fibroblasts (MEFs) from wildtype and Fsp27−/− mice, followed by their differentiation into adipocytes in vitro. We found that Fsp27 is expressed in both brown adipose tissue (BAT) and white adipose tissue (WAT) and its levels were significantly elevated in the WAT and liver of leptin-deficient ob/ob mice. Fsp27−/− mice had increased energy expenditure, lower levels of plasma triglycerides and free fatty acids. Furthermore, Fsp27−/− and Fsp27/lep double-deficient mice are resistant to diet-induced obesity and display increased insulin sensitivity. Moreover, white adipocytes in Fsp27−/− mice have reduced triglycerides accumulation and smaller lipid droplets, while levels of mitochondrial proteins, mitochondrial size and activity are dramatically increased. We further demonstrated that BAT-specific genes and key metabolic controlling factors such as FoxC2, PPAR and PGC1α were all markedly upregulated. In contrast, factors inhibiting BAT differentiation such as Rb, p107 and RIP140 were down-regulated in the WAT of Fsp27−/− mice. Remarkably, Fsp27−/− MEFs differentiated in vitro show many brown adipocyte characteristics in the presence of the thyroid hormone triiodothyronine (T3). Our data thus suggest that Fsp27 acts as a novel regulator in vivo to control WAT identity, mitochondrial activity and insulin sensitivity

Screening and fermentation medium optimization of a strain favorable to Rice–fish Coculture

Rice–fish coculture (RF) is a small ecosystem in which microorganisms are widely distributed in the fish, water environment, soil, and plants. In order to study the positive effects of microorganisms on common carp and rice in the RF ecosystem, a total of 18 strains with growth-promoting ability were screened from common carp (Cyprinus carpio) gut contents, among which three strains had the ability to produce both DDP-IV inhibitors and IAA. The strain with the strongest combined ability, FYN-22, was identified physiologically, biochemically, and by 16S rRNA, and it was initially identified as Bacillus licheniformis. As the number of metabolites secreted by the strain under natural conditions is not sufficient for production, the FYN-22 fermentation medium formulation was optimized by means of one-factor-at-a-time (OFAT) experiments and response surface methodology (RSM). The results showed that, under the conditions of a soluble starch concentration of 10.961 g/l, yeast concentration of 2.366 g/l, NH4Cl concentration of 1.881 g/l, and FeCl3 concentration of 0.850 g/l, the actual measured number of FYN-22 spores in the fermentation broth was 1.913 × 109 CFU/ml, which was 2.575-fold improvement over the pre-optimization value. The optimized fermentation solution was used for the immersion operation of rice seeds, and, after 14 days of incubation in hydroponic boxes, the FYN-22 strain was found to have a highly significant enhancement of 48.31% (p < 0.01) on the above-ground part of rice, and different degrees of effect on root length, fresh weight, and dry weight (16.73, 17.80, and 21.97%, respectively; p < 0.05). This study may provide new insights into the fermentation process of Bacillus licheniformis FYN-22 and its further utilization in RF systems

Knowledge atlas of antibody-drug conjugates on CiteSpace and clinical trial visualization analysis

ObjectiveAntibody-drugs conjugates (ADCs) are novel drugs with highly targeted and tumor-killing abilities and developing rapidly. This study aimed to evaluate drug discovery and clinical trials of and explore the hotspots and frontiers from 2012 to 2022 using bibliometric methods.MethodsPublications on ADCs were retrieved between 2012 and 2022 from Web of Science (WoS) and analyzed with CiteSpace 6.1.R2 software for the time, region, journals, institutions, etc. Clinical trials were downloaded from clinical trial.org and visualized with Excel software.ResultsA total of 696 publications were obtained and 187 drug trials were retrieved. Since 2012, research on ADCs has increased year by year. Since 2020, ADC-related research has increased dramatically, with the number of relevant annual publications exceeding 100 for the first time. The United States is the most authoritative and superior country and region in the field of ADCs. The University of Texas MD Anderson Cancer Center is the most authoritative institution in this field. Research on ADCs includes two clinical trials and one review, which are the most influential references. Clinical trials of ADCs are currently focused on phase I and phase II. Comprehensive statistics and analysis of the published literature and clinical trials in the field of ADCs, have shown that the most studied drug is brentuximab vedotin (BV), the most popular target is human epidermal growth factor receptor 2 (HER2), and breast cancer may become the main trend and hotspot for ADCs indications in recent years.ConclusionAntibody-drug conjugates have become the focus of targeted therapies in the field of oncology. The innovation of technology and combination application strategy will become the main trend and hotspots in the future