Arabidopsis N6-methyladenosine methyltransferase FIONA1 regulates floral transition by affecting the splicing of FLC and the stability of floral activators SPL3 and SEP3.

N 6-methyladenosine (m6A) RNA methylation has been shown to play a crucial role in plant development and floral transition. Two recent studies have identified FIONA1 as an m6A methyltransferase that regulates the floral transition in Arabidopsis through influencing the stability of CONSTANS (CO), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), and FLOWERING LOCUS C (FLC). In this study, we confirmed that FIONA1 is an m6A methyltransferase that installs m6A marks in a small group of mRNAs. Furthermore, we show that, in addition to its role in influencing the stability of CO, SOC1, and FLC, FIONA1-mediated m6A methylation influences the splicing of FLC, a key floral repressor, and the stability of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 3 (SPL3) and SEPALLATA3 (SEP3), floral activators, which together play a vital role in floral transition in Arabidopsis. Our study confirms the function of FIONA1 as an m6A methyltransferase and suggests a close molecular link between FIONA1-mediated m6A methylation and the splicing of FLC and the destabilization of SPL3 and SEP3 in flowering time control.

ALKBH9C, a potential RNA m6 A demethylase, regulates the response of Arabidopsis to abiotic stresses and abscisic acid.

Although several studies have shown that AlkB homolog (ALKBH) proteins are potential RNA demethylases (referred to as 'erasers'), biological functions of only a few ALKBH proteins have been characterized to date. In this study, we determined the function of ALKBH9C (At4g36090) in seed germination and seedling growth of Arabidopsis thaliana in response to abiotic stress and abscisic acid (ABA). Seed germination of the alkbh9c mutant was delayed in response to salt, drought, cold and ABA. Moreover, seedling growth of the mutant was repressed under salt stress or ABA but enhanced under drought conditions. Notably, the stress-responsive phenotypes were associated with the altered expression of several m6 A-modified transcripts related to salt, drought or ABA response. Global m6 A levels were increased in the alkbh9c mutant, and ALKBH9C bound to m6 A-modified RNAs and had in vitro m6 A demethylase activity, suggesting its potential role as an m6 A eraser. The m6 A levels in several stress-responsive genes were increased in the alkbh9c mutant, and the stability of m6 A-modified transcripts was altered in the mutant. Collectively, our results suggest that m6 A eraser ALKBH9C is crucial for seed germination and seedling growth of Arabidopsis in response to abiotic stresses or ABA via affecting the stability of stress-responsive transcripts.

ECT12, an YTH-domain protein, is a potential mRNA m6A reader that affects abiotic stress responses by modulating mRNA stability in Arabidopsis.

N6-methyladenosine (m6A), the most abundant modification found in eukaryotic mRNAs, is interpreted by m6A "readers," thus playing a crucial role in regulating RNA metabolism. The YT521-B homology-domain (YTHD) proteins, also known as EVOLUTIONARILY CONSERVED C-TERMINAL REGION (ECT), are recognized as m6A reader proteins in plants and animals. Among the 13 potential YTHD family proteins in Arabidopsis thaliana, the functions of only a few members are known. In this study, we determined the function of ECT12 (YTH11) as a potential m6A reader that plays a crucial role in response to abiotic stresses. The loss-of-function ect12 mutants showed no noticeable developmental defects under normal conditions but displayed hypersensitivity to salt or dehydration stress. The salt- or dehydration-hypersensitive phenotypes were correlated with altered levels of several m6A-modified stress-responsive transcripts. Notably, the increased or decreased transcript levels were associated with each transcript's reduced or enhanced decay, respectively. Electrophoretic mobility shift and RNA-immunoprecipitation assays showed that ECT12 binds to m6A-modified RNAs both in vitro and in planta, suggesting its role as an m6A reader. Collectively, these results indicate that the potential m6A reader ECT12 regulates the stability of m6A-modified RNA transcripts, thereby facilitating the response of Arabidopsis to abiotic stresses.

FLK is an mRNA m6A reader that regulates floral transition by modulating the stability and splicing of FLC in Arabidopsis.

N6-methyladenosine (m6A), which is added, removed, and interpreted by m6A writers, erasers, and readers, respectively, is the most abundant modification in eukaryotic mRNAs. The m6A marks play a pivotal role in the regulation of floral transition in plants. FLOWERING LOCUS K (FLK), an RNA-binding protein harboring K-homology (KH) motifs, is known to regulate floral transition by repressing the levels of a key floral repressor FLOWERING LOCUS C (FLC) in Arabidopsis. However, the molecular mechanism underlying FLK-mediated FLC regulation remains unclear. In this study, we identified FLK as a novel mRNA m6A reader protein that directly binds the m6A site in the 3'-untranslated region of FLC transcripts to repressing FLC levels by reducing its stability and splicing. Importantly, FLK binding of FLC transcripts was abolished in vir-1, an m6A writer mutant, and the late-flowering phenotype of the flk mutant could not be rescued by genetic complementation using the mutant FLKm gene, in which the m6A reader encoding function was eliminated, indicating that FLK binds and regulates FLC expression in an m6A-dependent manner. Collectively, our study has addressed a long-standing question of how FLK regulates FLC transcript levels and established a molecular link between the FLK-mediated recognition of m6A modifications on FLC transcripts and floral transition in Arabidopsis.

Molecular intercommunication between the complement and coagulation systems.

The complement system as well as the coagulation system has fundamental clinical implications in the context of life-threatening tissue injury and inflammation. Associations between both cascades have been proposed, but the precise molecular mechanisms remain unknown. The current study reports multiple links for various factors of the coagulation and fibrinolysis cascades with the central complement components C3 and C5 in vitro and ex vivo. Thrombin, human coagulation factors (F) XIa, Xa, and IXa, and plasmin were all found to effectively cleave C3 and C5. Mass spectrometric analyses identified the cleavage products as C3a and C5a, displaying identical molecular weights as the native anaphylatoxins C3a and C5a. Cleavage products also exhibited robust chemoattraction of human mast cells and neutrophils, respectively. Enzymatic activity for C3 cleavage by the investigated clotting and fibrinolysis factors is defined in the following order: FXa > plasmin > thrombin > FIXa > FXIa > control. Furthermore, FXa-induced cleavage of C3 was significantly suppressed in the presence of the selective FXa inhibitors fondaparinux and enoxaparin in a concentration-dependent manner. Addition of FXa to human serum or plasma activated complement ex vivo, represented by the generation of C3a, C5a, and the terminal complement complex, and decreased complement hemolytic serum activity that defines exact serum concentration that results in complement-mediated lysis of 50% of sensitized sheep erythrocytes. Furthermore, in plasma from patients with multiple injuries (n = 12), a very early appearance and correlation of coagulation (thrombin-antithrombin complexes) and the complement activation product C5a was found. The present data suggest that coagulation/fibrinolysis proteases may act as natural C3 and C5 convertases, generating biologically active anaphylatoxins, linking both cascades via multiple direct interactions in terms of a complex serine protease system.

Varicella zoster viral infection complicating into necrotizing fasciitis: A case report.

Necrotizing fasciitis is a rare complication of varicella-zoster viral infection in adults, occurring due to a secondary bacterial infection. A 35-year-old female healthy patient had post-varicella zoster infection with NSAID use as a possible risk factor. She was diagnosed early by clinical and laboratory parameters.

Pyrosequencing-based strategy for a successful SNP detection in two hypervariable regions: HV-I/HV-II of the human mitochondrial displacement loop.

Forensic analysis of mitochondrial displacement loop (D-loop) sequences using Sanger sequencing or SNP detection by minisequencing is well established. Pyrosequencing has become an important alternative because it enables high-throughput analysis and the quantification of individual mitochondrial DNAs (mtDNAs) in samples originating from more than one individual. DNA typing of the mitochondrial D-loop region is usually the method of choice if STR analysis fails because of trace amounts of DNA and/or extensive degradation. The main aim of the present work was to optimize the efficiency of pyrosequencing. To do this, 31 SNPs within the hypervariable regions I and II of the D-loop of human mtDNA were simultaneously analyzed. As a novel approach, we applied two sets of amplification primers for the multiplexing assay. These went in combination with four sequencing primers for pyrosequencing. This method was compared with conventional sequencing of mtDNA from blood and biological trace materials.

Porphyromonas gingivalis lipopolysaccharide activates platelet Cdc42 and promotes platelet spreading and thrombosis.

BACKGROUND: Periodontitis confers an increased risk for cardiovascular diseases, including thrombosis. However, the molecular mechanisms that potentially link periodontitis with thrombosis are undefined. Here we test the hypothesis that Gram-negative periodontal infection promotes pathological platelet activation and amplifies shape change. We focus specifically on lipopolysaccharide (LPS) signaling to platelets. METHODS: Platelets were isolated from blood samples and allowed to spread on coverslips in the presence or absence of LPS purified from the periodontal pathogen Porphyromonas gingivalis. Platelets were fixed and stained with Alexa-488-phalloidin to label the actin cytoskeleton. The degree of platelet spreading and shape change was quantified by confocal microscopy. In a translational pilot study, blood samples were obtained from human subjects exhibiting generalized severe periodontitis (SP) or healthy periodontium (HP). Rotational thromboelastometry was used to quantify the rate of clot formation via the intrinsic coagulation pathway. RESULTS: LPS-treated platelets exhibited significantly (P < 0.05) greater spreading and higher numbers of actin-rich filopodia (cell extensions) than controls. We also found that LPS stimulation of platelets promoted the activation of Cdc42, the small GTPase responsible for filopodia formation. Exposure of whole blood samples to LPS significantly (P < 0.05) reduced clotting times. Blood from SP patients clotted significantly (P < 0.05) more rapidly and exhibited shorter partial thromboplastin times compared with HP controls. CONCLUSIONS: This is the first study to suggest a mechanism by which LPS stimulation drives Cdc42 activation and platelet spreading. These data are consistent with the notion that periodontitis promotes accelerated clot formation and an increased risk of thrombosis.

In vitro germination and biochemical profiling of Brassica napus in response to biosynthesised zinc nanoparticles.

With the progression of nanotechnology, the use of nanoparticles (NPs) in consumer products has increased dramatically and green synthesis is one of the cheapest and eco-friendly methods to obtain non-hazardous NPs. In the current research zinc (Zn) NPs synthesis was carried out by using the fresh and healthy leaves of Mentha arvensis L. followed by characterisation through ultraviolet (UV)-visible spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM). UV-visible spectroscopy confirmed the green synthesis of ZnNPs, while XRD confirmed the size of NPs, which was 30-70 nm. SEM shows that the shape of ZnNPs was irregular. The effects of green synthesised NPs on two different varieties of Brassica napus were evaluated. Exposure to ZnNPs (5, 15, and 25 mg/l-1) caused a significant increase in root and shoot length of B. napus. The application of NPs significantly improved plant germination and triggered the production of secondary metabolite and antioxidant enzymes. ZnNPs showed a significant increase in chlorophyll, superoxide dismutase, total flavonoid content (TFC) and antioxidant enzymes while total phenolic content was decreased when TFC increased. Thus, it has been concluded from the current study that ZnNPs may possibly trigger the production of antioxidant enzymes and various biochemical compounds.

Interaction between the coagulation and complement system.

The complement system as a main column of innate immunity and the coagulation system as a main column in hemostasis undergo massive activation early after injury. Interactions between the two cascades have often been proposed but the precise molecular pathways of this interplay are still in the dark. To elucidate the mechanisms involved, the effects of various coagulation factors on complement activation and generation of anaphylatoxins were investigated and summarized in the light of the latest literature. Own in vitro findings suggest, that the coagulation factors FXa, FXIa and plasmin may cleave both C5 and C3, and robustly generate C5a and C3a (as detected by immunoblotting and ELISA). The produced anaphylatoxins were found to be biologically active as shown by a dose-dependent chemotactic response of neutrophils and HMC-1 cells, respectively. Thrombin did not only cleave C5 (Huber-Lang et al. 2006) but also in vitro-generated C3a when incubated with native C3. The plasmin-induced cleavage activity could be dose-dependently blocked by the serine protease inhibitor aprotinin and leupeptine. These findings suggest that various serine proteases belonging to the coagulation system are able to activate the complement cascade independently of the established pathways. Moreover, functional C5a and C3a are generated, both of which are known to be crucially involved in the inflammatory response.

Synergistic interaction between C5a and NOD2 signaling in the regulation of chemokine expression in RAW 264.7 macrophages.

The innate immune response is a complex process involving multiple pathogen-recognition receptors, including toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors. Complement is also a critical component of innate immunity. While complement is known to interact with TLR-mediated signals, the interactions between NOD-like receptors and complement are not well understood. Here we report a synergistic interaction between C5a and Nod2 signaling in RAW 264.7 macrophages. Long-term treatment with muramyl dipeptide (MDP), a NOD2 ligand, enhanced C5a-mediated expression of chemokine mRNAs in RAW 264.7 cells. This response was dependent on NOD2 expression and was associated with a decrease in expression of C5L2, a receptor for C5a which acts as a negative modulator of C5a receptor (C5aR) activity. MDP amplified C5a-mediated phosphorylation of p38 MAPK. Treatment of RAW264.7 cells with an inhibitor of p38 attenuated the synergistic effects of C5a on MDP-primed cells on MIP-2, but not MCP-1, mRNA. In contrast, inhibition of AKT prevented C5a stimulation of MCP-1, but not MIP-2, mRNA, in MDP-primed cells. Taken together, these data demonstrated a synergistic interaction between C5a and NOD2 in the regulation of chemokine expression in macrophages, associated with a down-regulation of C5L2, a negative regulator of C5a receptor activity.

Alteration of complement hemolytic activity in different trauma and sepsis models.

Complement activation is involved in various diseases in which innate immunity plays a crucial role. However, its pathophysiological relevance is not clearly understood. Experimental models have been widely used to characterize the role of complement activation under different pathological conditions, such as hypoxemia, ischemia and reperfusion, tissue damage, and polymicrobial invasion. Screening of the complement status and function is, however, strongly dependent on the laboratory-specific techniques being used to sample and measure complement, making it difficult to compare the results found in different laboratories. Therefore, we evaluated complement function by measuring complement hemolytic activity (CH50) in various animal models of isolated ischemia reperfusion (I/R: kidney, liver, gut), hemorrhagic traumatic shock (HTS), endotoxic shock (LPS), and sepsis (CLP). Complement activation was less pronounced in isolated models of ischemia and reperfusion, whereas a strong complement response was observed early after HTS, CLP, and LPS. In summary, CH50 is a well-established, quick, and cost-effective screening method of complement function. However, because we obtained different results in clinically relevant animal models, further differentiation using specific complement factor analysis is necessary.

Early expression changes of complement regulatory proteins and C5A receptor (CD88) on leukocytes after multiple injury in humans.

As a crucial element of innate immunity, the complement cascade becomes activated after severe trauma. Regulation of the complement cascade and protection against complement-mediated tissue destruction is provided by a selection of soluble and membrane-bound complement regulatory proteins (CRegs). To date, the leukocyte expression profile of CRegs in multiple injured patients is unknown. In the present study, expression of CRegs and the C5a receptor (CD88) was analyzed on neutrophils, monocytes, and lymphocytes by flow cytometry. Whole blood samples were obtained from healthy volunteers (n = 16) or multiple injured patients (n = 12) on admission in the emergency department and 4, 12, 24, 120, and 240 h after trauma. The content of CRegs and CD88 on leukocytes was significantly altered posttrauma: CD55 (decay accelerating factor) displayed a time-dependent, elevated expression pattern on neutrophils and monocytes, but not on lymphocytes. CD59 (membrane attack complex inhibitor) expression was significantly increased on neutrophils and monocytes at the time of admission and after 5 to 10 days in lymphocytes. CD46 (membrane cofactor protein) was significantly down-regulated in all three cell types posttrauma. CD35 (complement receptor 1) expression on neutrophils was initially decreased, whereas monocytes presented a significant increase in CD35 expression. CD35 on lymphocyte remained unchanged throughout the observation period. CD88 expression was considerably reduced on leukocytes between 0 and 240 h after injury. CD59, CD46, and CD88 expression values on neutrophils reversely correlated with severity of injury. In summary, expression profiles of CRegs and CD88 on leukocytes are specifically altered after polytrauma in humans, indicating a trauma-induced "complementopathy."