Adenylate cyclase activity of TIR1/AFB auxin receptors in plants.

The phytohormone auxin is the major coordinative signal in plant development1, mediating transcriptional reprogramming by a well-established canonical signalling pathway. TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) auxin receptors are F-box subunits of ubiquitin ligase complexes. In response to auxin, they associate with Aux/IAA transcriptional repressors and target them for degradation via ubiquitination2,3. Here we identify adenylate cyclase (AC) activity as an additional function of TIR1/AFB receptors across land plants. Auxin, together with Aux/IAAs, stimulates cAMP production. Three separate mutations in the AC motif of the TIR1 C-terminal region, all of which abolish the AC activity, each render TIR1 ineffective in mediating gravitropism and sustained auxin-induced root growth inhibition, and also affect auxin-induced transcriptional regulation. These results highlight the importance of TIR1/AFB AC activity in canonical auxin signalling. They also identify a unique phytohormone receptor cassette combining F-box and AC motifs, and the role of cAMP as a second messenger in plants.

The Comparative Effectiveness and Safety of Different Anticoagulation Strategies for Treatment of Left Atrial Appendage Thrombus in the Setting of Chronic Anticoagulation for Atrial Fibrillation or Flutter.

PURPOSE: To compare effectiveness of different treatments for atrial fibrillation (AF) patients who were scheduled for cardioversion (CV) or ablation (CA) presenting with left atrium appendage (LAA) thrombus despite chronic oral anticoagulation therapy (OAC). METHODS: This was a retrospective cohort study. We analyzed 2014-2019 medical records of patients scheduled for CV or CA of AF who were diagnosed with LAA thrombus despite optimal OAC and had a follow-up transesophageal echocardiogram (TOE). Changes in treatment were divided into the following groups: switch to a drug with different mechanism of action, switch to a drug with similar mechanism of action, initiation of combination therapy, or deliberate no change in treatment. Patients with contraindications to non-vitamin K antagonists were excluded from the analysis. RESULTS: We analyzed data of 129 patients comprising 181 cycles of treatment. The overall effectiveness of LAA thrombus dissolution was 51.9% regardless of the number of cycles and 42.6% for the first cycle of treatment. Any change of treatment was more effective than deliberate no change-OR 2.97 [95% CI: 1.07-8.25], P = 0.031, but no particular strategy seemed to be more effective than the other. Left atrium area (OR 0.908 [95% CI: 0.842-0.979]) and number of treatment cycles (OR 0.457 [95% CI: 0.239-0.872]) were both adversely related to thrombus resolution. There was one ischemic and three bleeding adverse events during the treatment. CONCLUSION: LAA thrombus resolution in patients already on OAC may require a change of previous OAC treatment but the overall effectiveness of dissolution seems to be about 50%.

BdGUCD1 and Cyclic GMP Are Required for Responses of Brachypodium distachyon to Fusarium pseudograminearum in the Mechanism Involving Jasmonate.

Guanosine 3',5'-cyclic monophosphate (cGMP) is an important signaling molecule in plants. cGMP and guanylyl cyclases (GCs), enzymes that catalyze the synthesis of cGMP from GTP, are involved in several physiological processes and responses to environmental factors, including pathogen infections. Using in vitro analysis, we demonstrated that recombinant BdGUCD1 is a protein with high guanylyl cyclase activity and lower adenylyl cyclase activity. In Brachypodium distachyon, infection by Fusarium pseudograminearum leads to changes in BdGUCD1 mRNA levels, as well as differences in endogenous cGMP levels. These observed changes may be related to alarm reactions induced by pathogen infection. As fluctuations in stress phytohormones after infection have been previously described, we performed experiments to determine the relationship between cyclic nucleotides and phytohormones. The results revealed that inhibition of cellular cGMP changes disrupts stress phytohormone content and responses to pathogen. The observations made here allow us to conclude that cGMP is an important element involved in the processes triggered as a result of infection and changes in its levels affect jasmonic acid. Therefore, stimuli-induced transient elevation of cGMP in plants may play beneficial roles in priming an optimized response, likely by triggering the mechanisms of feedback control.

In Search of Monocot Phosphodiesterases: Identification of a Calmodulin Stimulated Phosphodiesterase from Brachypodium distachyon.

In plants, rapid and reversible biological responses to environmental cues may require complex cellular reprograming. This is enabled by signaling molecules such as the cyclic nucleotide monophosphates (cNMPs) cAMP and cGMP, as well as Ca2+. While the roles and synthesis of cAMP and cGMP in plants are increasingly well-characterized, the "off signal" afforded by cNMP-degrading enzymes, the phosphodiesterases (PDEs), is, however, poorly understood, particularly so in monocots. Here, we identified a candidate PDE from the monocot Brachypodium distachyon (BDPDE1) and showed that it can hydrolyze cNMPs to 5'NMPs but with a preference for cAMP over cGMP in vitro. Notably, the PDE activity was significantly enhanced by Ca2+ only in the presence of calmodulin (CaM), which interacts with BDPDE1, most likely at a predicted CaM-binding site. Finally, based on our biochemical, mutagenesis and structural analyses, we constructed a comprehensive amino acid consensus sequence extracted from the catalytic centers of annotated and/or experimentally validated PDEs across species to enable a broad application of this search motif for the identification of similar active sites in eukaryotes and prokaryotes.

In Vitro Characterization of Guanylyl Cyclase BdPepR2 from Brachypodium distachyon Identified through a Motif-Based Approach.

In recent years, cyclic guanosine 3',5'-cyclic monophosphate (cGMP) and guanylyl cyclases (GCs), which catalyze the formation of cGMP, were implicated in a growing number of plant processes, including plant growth and development and the responses to various stresses. To identify novel GCs in plants, an amino acid sequence of a catalytic motif with a conserved core was designed through bioinformatic analysis. In this report, we describe the performed analyses and consider the changes caused by the introduced modification within the GC catalytic motif, which eventually led to the description of a plasma membrane receptor of peptide signaling molecules-BdPepR2 in Brachypodium distachyon. Both in vitro GC activity studies and structural and docking analyses demonstrated that the protein could act as a GC and contains a highly conserved 14-aa GC catalytic center. However, we observed that in the case of BdPepR2, this catalytic center is altered where a methionine instead of the conserved lysine or arginine residues at position 14 of the motif, conferring higher catalytic activity than arginine and alanine, as confirmed through mutagenesis studies. This leads us to propose the expansion of the GC motif to cater for the identification of GCs in monocots. Additionally, we show that BdPepR2 also has in vitro kinase activity, which is modulated by cGMP.

Thermal conditions during neonatal anoxia affect the endogenous level of brain-derived neurotrophic factor.

Anoxia during delivery is a complication that can disturb infant brain development leading to various types of neurological disorders. Our studies have shown that increased body temperature of newborn rats of both sexes intensifies the postanoxic oxidative stress and prevents triggering the endogenous adaptive response such as HIF-1α activation. Currently, brain-derived neurotrophic factor-BDNF is considered to be a modulator of neuronal plasticity. In the developing brain, mature BDNF and its precursor exhibit prosurvival action through the TrkB receptor and proapoptotic functions binding to p75NTR , respectively. The aim of our experiments was to check the effects of body temperature on the postanoxic level of BDNF and on the expression of its receptors as well as on the marker of apoptosis-caspase-3 in the rat brain. Two-day-old Wistar Han rats (male/female ratio, 1:1) were exposed to anoxia in 100% nitrogen atmosphere for 10 min in different thermal conditions, which allowed them to regulate their rectal temperature at the following levels: normothermic-33°C; hyperthermic-37°C; and extremely hyperthermic-39°C. Thermal conditions during neonatal anoxia affected the level of proBDNF, BDNF as well as their receptors and caspase-3 in the forebrain. The increased BDNF protein level followed by decreased caspase-3 protein level was probably dependent on body temperature under anoxic conditions and was observed only in rats maintaining decreased body temperature. The positive effect of BDNF was not observed under hyperthermic conditions. Moreover, BDNF level changes correlated with body temperature probably affected the learning and spatial memory in juvenile rats.

Activation of hypoxia-inducible factor-1α in rat brain after perinatal anoxia: role of body temperature.

Transcriptional hypoxia-inducible factor-1α (HIF-1α) plays the fundamental role in adaptive processes in response to hypoxia. Specific HIF-1α target genes are involved in glycolysis, erythropoiesis and angiogenesis to promote survival. In our previous study we have demonstrated that naturally low body temperature of newborn rats protects them against damage due to perinatal hypoxia. Therefore, our experiments aimed at checking the effects of body temperature during simulated perinatal anoxia on subsequent changes of expression of HIF-1α and its specific target genes such as vascular endothelial growth factor (VEGF) and erythropoietin (EPO) in the rat brain. Two-day old Wistar rats were divided into three temperature groups: normothermic -33 °C, hyperthermic -37 °C and extremely hyperthermic -39 °C. The temperature was controlled 15 min before start and continued during 10 min of anoxia as well as for 2 h post-anoxia. HIF-1α was analysed by Western blot and immunofluorescence and mRNA levels of HIF-1α and its downstream genes (VEGF, EPO) were quantified by qRT-PCR. Thermal conditions during neonatal anoxia affected the hippocampal and neocortical level of HIF-1α protein. Physiological body temperature of newborn rats led to prominent accumulation of cerebral HIF-1α protein and significant upregulation of VEGF and EPO mRNA. In contrast, anoxia-induced HIF-1α activation at elevated body temperatures was less pronounced. Since HIF-1α and EPO have recently been regarded as promising therapeutical targets against brain lesions due to hypoxia/ischemia, presented data imply that in order to achieve a full effect of neuroprotection, the thermal conditions during and after the insult should be taken into consideration.

Fahr Syndrome - an Important Piece of a Puzzle in the Differential Diagnosis of Many Diseases.

Fahr syndrome is a rare neurodegenerative disorder characterized by symmetrical, bilateral calcifications in the basal ganglia, nucleus gyrus and cerebral cortex. The continuous advancement as well as widespread use of brain imaging have contributed to the increasing detection rates of such changes. Nevertheless, their etiology is understood only partially and the methods of causative treatment are limited. Due to various symptoms, Fahr syndrome may resemble diseases from the field of neurology, psychiatry, cardiology and even urology. This article provides an up-to-date review of the literature concerning Fahr syndrome in terms of clinical practice.

Transfusion-related acute lung injury in a young woman with ectopic pregnancy - case report and literature review.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a major cause of death associated with transfusion. Nevertheless it is still misdiagnosed and underreported. We present a case of this serious complication in order to make it more familiar to clinicians and indicate proper medical management, which is significant not only for the patients presenting TRALI symptoms but also for future recipients of blood components. CASE PRESENTATION: A 32-year-old white woman with in-vitro fertilization in anamnesis was admitted to Department of Obstetrics and Gynecology because of abdominal pain. Ultrasonography examination revealed pathological mass in left adnexa. The patient underwent laparoscopic left salpingectomy. The following day she reported progressive pain in lower abdomen with signs of peritoneal irritation. Emergency laparotomy was performed and active bleeding from fallopian tube was stopped. Four units of red blood cells concentrate and six units of fresh frozen plasma were transfused. Within two hours of surgery acute respiratory distress symptoms occurred, bilateral infiltrates were found on chest X-ray. The patient responded to supportive treatment (oxygen therapy, dexamethasone, diuretics) and her state improved within 12 hours. Serological diagnostics revealed anti-HLA antibodies in one donor which reacted with patient's granulocytes. Clinical picture and anti-leukocyte antibodies detected in blood component allowed to identify the immune-mediated TRALI. CONCLUSIONS: Transfusion-related acute lung injury is a life-threatening complication of transfusion which manifests as non-cardiogenic pulmonary edema. Each suspected case of this syndrome should be reported to blood center in order to confirm the diagnosis and implement preventive measures (exclusion of implicated donor from further blood donation).

[New strategies for assessing the risk of sudden cardiac death in hypertrophic cardiomyopathy]. / Nowe strategie oceny ryzyka naglego zgonu sercowego w przebiegu kardiomiopatii przerostowej.

Hypertrophic cardiomyopathy (HCM) is characterized by a primarily increased ventricular wall thickness, usually caused by the mutations in genes encoding sarcomere proteins. Thanks to the growing awareness in the medical community as well as advances in diagnostic techniques HCM can be diagnosed at earlier stages than ever before. However, in some cases the first symptom of this disease is a sudden cardiac death (SCD) and diagnosis remains unknown until post-mortem examination. Implantation of a cardioverterdefibrillator (ICD) provides the most effective method of SCD prevention. Nevertheless, due to a number of risks associated with surgery as well as the possession of such devices, predictive factors of serious ventricular arrhythmia in individual patients have been sought for many years. The aim of this review is to present the current strategies of risk assessment and prevention of SCD in patients with HCM.

Cutaneous adverse reactions of amiodarone.

Dermatological complications of amiodarone are commonly encountered problems in therapy. The incidence in the population of patients with prolonged use of amiodarone reaches nearly 75% according to various sources. Nevertheless, they are often misdiagnosed or overlooked. The aim of this review is to present the current state of knowledge about skin changes induced by amiodarone, including phototoxic and photoallergic reactions, as well as hyperpigmentation. In most cases, the adverse effects are reversible and disappear after discontinuation of the drug. Although the dermatological complications usually do not influence the outcome of the therapy and rarely cause discontinuation of treatment, they have a great impact on patient quality of life.

A SNF1-related protein kinase regulatory subunit functions as a molecular tuner.

Metabolic processes in prokaryotic and eukaryotic organisms are often modulated by kinases which are in turn, dependent on Ca2+ and the cyclic mononucleotides cAMP and cGMP. It has been established that some proteins have both kinase and cyclase activities and that active cyclases can be embedded within the kinase domains. Here, we identified phosphodiesterase (PDE) sites, enzymes that hydrolyse cAMP and cGMP, to AMP and GMP, respectively, in some of these proteins in addition to their kinase/cyclase twin-architecture. As an example, we tested the Arabidopsis thaliana KINγ, a subunit of the SnRK2 kinase, to demonstrate that all three enzymatic centres, adenylate cyclase (AC), guanylate cyclase (GC) and PDE, are catalytically active, capable of generating and hydrolysing cAMP and cGMP. These data imply that the signal output of the KINγ subunit modulates SnRK2, consequently affecting the downstream kinome. Finally, we propose a model where a single protein subunit, KINγ, is capable of regulating cyclic mononucleotide homeostasis, thereby tuning stimulus specific signal output.

Troponin T Assessment Allows for Identification of Mutation Carriers among Young Relatives of Patients with LMNA-Related Dilated Cardiomyopathy.

Background:LMNA-related dilated cardiomyopathy (LMNA-DCM) caused by mutations in the lamin A/C gene (LMNA) is one of the most common forms of hereditary DCM. Due to the high risk of mutation transmission to offspring and the high incidence of ventricular arrhythmia and sudden death even before the onset of heart failure symptoms, it is very important to identify LMNA-mutation carriers. However, many relatives of LMNA-DCM patients do not report to specialized centers for clinical or genetic screening. Therefore, an easily available tool to identify at-risk subjects is needed. Methods: We compared two cohorts of young, asymptomatic relatives of DCM patients who reported for screening: 29 LMNA mutation carriers and 43 individuals from the control group. Receiver operating characteristic (ROC) curves for potential indicators of mutation carriership status were analyzed. Results: PR interval, N-terminal pro-B-type natriuretic peptide (NT-proBNP), and high-sensitivity cardiac troponin T (hscTnT) serum levels were higher in the LMNA mutation carrier cohort. Neither group differed significantly with regard to creatinine concentration or left ventricular ejection fraction. The best mutation carriership discriminator was hscTnT level with an optimal cut-off value at 5.5 ng/L, for which sensitivity and specificity were 86% and 93%, respectively. The median hscTnT level was 11.0 ng/L in LMNA mutation carriers vs. <3.0 ng/L in the control group, p < 0.001. Conclusions: Wherever access to genetic testing is limited, LMNA mutation carriership status can be assessed reliably using the hscTnT assay. Among young symptomless relatives of LMNA-DCM patients, a hscTnT level >5.5 ng/L strongly suggests mutation carriers.

[Role of inflammation in etiology of atrial fibrillation--is lone arrhythmia really alone]. / Etiologia zapalna migotania przedsionków--samoistne migotanie przedsionków nie zawsze jest samoistne.

We present the case study of recurrent AF episodes initially considered as a lone arrhythmia. Due to elevated C-reactive protein and fibrinogen serum levels with absence of other potential risk factors of arrhythmia and diseases underlying the genesis of inflammatory response we consider the plays a prominent role in etiology in perpetuation and maintenance of AF. Additional use of atorvastatin to a conventional therapy leads to significant reduction of C-reactive protein and fibrinogen and improves effect of long-term sinus rhythm maintenance.

Twin cyclic mononucleotide cyclase and phosphodiesterase domain architecture as a common feature in complex plant proteins.

The majority of proteins in both prokaryote and eukaryote proteomes consist of two or more functional centers, which allows for intramolecular tuning of protein functions. Such architecture, as opposed to animal orthologs, applies to the plant cyclases (CNC) and phosphodiesterases (PDEs), the vast majority of which are part of larger multifunctional proteins. In plants, until recently, only two cases of combinations of CNC-PDE in one protein were reported. Here we propose that in plants, multifunctional proteins in which the PDE motif has been identified, the presence of the additional CNC center is common. Searching the Arabidopsis thaliana proteome with a combined PDE-CNC motif allowed the creation of a database of proteins with both activities. One such example is methylenetetrahydrofolate dehydrogenase, in which we determined the activities of adenylate cyclase (AC) and PDE. Based on biochemical and mutagenesis analyses we assessed the impact of the AC and PDE catalytic centers on the dehydrogenase activity. This allowed us to propose additional regulatory mechanism that govern folate metabolism by cAMP. It is therefore conceivable that the combined CNC-PDE architecture is a common regulatory configuration, where control of the level of cyclic nucleotides (cNMP) influences other catalytic activities of the protein.

A quality of life, clinical and biochemical improvements after catheter ablation of persistent arrhythmia in patients with structural heart disease and arrhythmia-mediated cardiomyopathy.

BACKGROUND: Arrhythmia-mediated cardiomyopathy (AMC) is an essential clinical situation that is commonly underdiagnosed. Successful arrhythmia control leads to improvement in health-related quality of life (HRQoL) and heart failure (HF) symptoms in patients with structural heart disease (SHD). AIMS: The study aimed to evaluate the impact of catheter ablation (CA) of persistent arrhythmia on HRQoL, biochemical and clinical parameters HF in patients with SHD and AMC. METHODS: Patients with SHD, on optimal medical treatment, with persistent arrhythmia and strong suspicion of AMC, scheduled for CA were prospectively enrolled. Study procedures included: HRQoL measurement (the Minnesota Living with Heart Failure Questionnaire [MLHFQ] and the EuroQol Research Foundation [EQ-5D-3L] questionnaire), biomarkers (N-terminal pro-B-type natriuretic peptide [NT-proBNP], troponin T [TnT], matrix metalloproteinase-9 [MMP-9], soluble suppression of tumorigenesis-2 [sST2], tissue inhibitor of matrix metalloproteinase-1 [TIMP-1]), transthoracic echocardiography and clinical assessment. RESULTS: At 6 months, 30/35 (86%) patients were free of persistent arrhythmia. Patients who underwent successful CA had a significant improvement in HRQoL: MLHFQ (median [interquartile range, IQR], -22 [-28; -11]; P <0.001), EQ5D-3L score (mean [standard deviation], 21.8 (16.8); P <0.001); EQ5D-3L index (median [IQR], 0.09 (0.05; 0.18); P <0.001). A significant decrease in injury bio markers was observed: NT-proBNP (median [IQR], -414 [-1397; -318] pg/ml; P <0.001), TnT (median [IQR], -2.27 (-8.52; 0.55) ng/l; P <0.01) but not in fibrosis biomarkers: (median [IQR], sST2: 2.20 [-5.4; 4.3] ng/ml; P = 0.741, MMP-9: 34 [-376; 283] ng/ml; P = 0.881, TIMP-1: 11.1 [-17.1; 31.9] ng/ml; P = 0.215). There was a significant increase of left ventricular ejection fraction (LVEF) (mean [SD], 9.8 [5.9] %; P <0.01). CONCLUSIONS: Successful CA significantly improved clinical status, LVEF, and HRQoL of patients with SHD and AMC.

Dogs in the Wroclaw Stronghold, 2nd Half of the 10th-1st Half of the 13th Century (Lower Silesia, Poland)-An Zooarchaeological Overview.

This article pertains to the issue of early medieval dogs (10th-mid-13th century) from the territory of Poland and Central Europe. The study is based on dog remains from the Wroclaw Cathedral Island (Ostrów Tumski), one of the most important administrative centres of early medieval Poland, the capital of a secular principality and the seat of diocese authorities. The main morphological and functional types of dogs living in Wroclaw and other parts of Poland were characterized on that basis. It has been concluded that the roles and perceptions of dogs were very ambiguous. On the one hand, they were hunting companionship for the elite and were considered a symbol of devotion and loyalty. On the other hand, dogs symbolised disgrace. In everyday life, these animals were sometimes abused, their skin was sometimes tanned and their bones modified into tools, and in exceptional cases, dogs were even eaten.

A tandem motif-based and structural approach can identify hidden functional phosphodiesterases.

Cyclic nucleotide monophosphates (cNMPs) are increasingly recognized as essential signaling molecules governing many physiological and developmental processes in prokaryotes and eukaryotes. Degradation of cNMPs is as important as their generation because it offers the capability for transient and dynamic cellular level regulation but unlike their generating enzymes, the degrading enzymes, cyclic nucleotide phosphodiesterases (PDEs) are somewhat elusive in higher plants. Based on sequence analysis and structural properties of canonical PDE catalytic centers, we have developed a consensus sequence search motif and used it to identify candidate PDEs. One of these is an Arabidopsis thaliana K+-Uptake Permease (AtKUP5). Structural and molecular docking analysis revealed that the identified PDE domain occupies the C-terminal of this protein forming a solvent-exposed distinctive pocket that can spatially accommodate the cyclic adenosine monophosphate (cAMP) substrate and importantly, cAMP assumes a binding pose that is favorable for interactions with the key amino acids in the consensus motif. PDE activity was confirmed by the sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Notably, this activity was stimulated by the Ca2+/CaM complex, the binding of which to the PDE center was confirmed by surface plasmon resonance (SPR). Since AtKUP5 also has adenylate cyclase (AC) activity that is essential for K+ transport, we propose that this dual moonlighting AC-PDE architecture, offers modulatory roles that afford intricate intramolecular regulation of cAMP levels thereby enabling fine-tuning of cAMP signaling in K+ homeostasis.

Functional Crypto-Adenylate Cyclases Operate in Complex Plant Proteins.

Adenylyl cyclases (ACs) and their catalytic product cAMP are regulatory components of many plant responses. Here, we show that an amino acid search motif based on annotated adenylate cyclases (ACs) identifies 12 unique Arabidopsis thaliana candidate ACs, four of which have a role in the biosynthesis of the stress hormone abscisic acid (ABA). One of these, the 9-cis-epoxycarotenoid dioxygenase (NCED3 and At3g14440), was identified by sequence and structural analysis as a putative AC and then tested experimentally with two different methods. Given that the in vitro activity is low (fmoles cAMP pmol-1 protein min-1), but highly reproducible, we term the enzyme a crypto-AC. Our results are consistent with a role for ACs with low activities in multi-domain moonlighting proteins that have at least one other distinct molecular function, such as catalysis or ion channel activation. We propose that crypto-ACs be examined from the perspective that considers their low activities as an innate feature of regulatory ACs embedded within multi-domain moonlighting proteins. It is therefore conceivable that crypto-ACs form integral components of complex plant proteins participating in intra-molecular regulatory mechanisms, and in this case, potentially linking cAMP to ABA synthesis.