Zinc mediates control of nitrogen fixation via transcription factor filamentation.

Plants adapt to fluctuating environmental conditions by adjusting their metabolism and gene expression to maintain fitness1. In legumes, nitrogen homeostasis is maintained by balancing nitrogen acquired from soil resources with nitrogen fixation by symbiotic bacteria in root nodules2-8. Here we show that zinc, an essential plant micronutrient, acts as an intracellular second messenger that connects environmental changes to transcription factor control of metabolic activity in root nodules. We identify a transcriptional regulator, FIXATION UNDER NITRATE (FUN), which acts as a sensor, with zinc controlling the transition between an inactive filamentous megastructure and an active transcriptional regulator. Lower zinc concentrations in the nodule, which we show occur in response to higher levels of soil nitrate, dissociates the filament and activates FUN. FUN then directly targets multiple pathways to initiate breakdown of the nodule. The zinc-dependent filamentation mechanism thus establishes a concentration readout to adapt nodule function to the environmental nitrogen conditions. In a wider perspective, these results have implications for understanding the roles of metal ions in integration of environmental signals with plant development and optimizing delivery of fixed nitrogen in legume crops.

Matrix-degrading protease ADAMTS-5 cleaves inter-α-inhibitor and releases active heavy chain 2 in synovial fluids from arthritic patients.

Destruction of the cartilage matrix in joints is an important feature of arthritis. Proteolytic degradation of cartilage glycoproteins can contribute to the loss of matrix integrity. Human inter-α-inhibitor (IαI), which stabilizes the extracellular matrix, is composed of the light-chain serine proteinase inhibitor bikunin and two homologous heavy chains (HC1 and HC2) covalently linked through chondroitin 4-sulfate. Inflammation promotes the transfer of HCs from chondroitin 4-sulfate to hyaluronan by tumor necrosis factor-stimulated gene-6 protein (TSG-6). This reaction generates a covalent complex between the heavy chains and hyaluronan that can promote leukocyte invasion. This study demonstrates that both IαI and the HC-hyaluronan complex are substrates for the extracellular matrix proteases ADAMTS-5 and matrix metalloprotease (MMP) -3, -7, and -13. The major cleavage sites for all four proteases are found in the C terminus of HC2. ADAMTS-5 and MMP-7 displayed the highest activity toward HC2. ADAMTS-5 degradation products were identified in mass spectrometric analysis of 29 of 33 arthropathic patients, indicating that ADAMTS-5 cleavage occurs in synovial fluid in arthritis. After cleavage, free HC2, together with TSG-6, is able to catalyze the transfer of heavy chains to hyaluronan. The release of extracellular matrix bound HC2 is likely to increase the mobility of the HC2/TSG-6 catalytic unit and consequently increase the rate of the HC transfer reaction. Ultimately, ADAMTS-5 cleavage of HC2 could alter the physiological and mechanical properties of the extracellular matrix and contribute to the progression of arthritis.

Reactive Center Loop Insertion in α-1-Antitrypsin Captured by Accelerated Molecular Dynamics Simulation.

Protease inhibition by metastable serine protease inhibitors (serpins) is mediated by one of the largest functional intradomain conformational changes known in biology. In this extensive structural rearrangement, protease-serpin complex formation triggers cleavage of the serpin reactive center loop (RCL), its subsequent insertion into central ß-sheet A, and covalent trapping of the target protease. In this study, we present the first detailed accelerated molecular dynamics simulation of the insertion of the fully cleaved RCL in α-1-antitrypsin (α1AT), the archetypal member of the family of human serpins. Our results reveal internal water pathways that allow the initial incorporation of side chains of RCL residues into the protein interior. We observed structural plasticity of the helix F (hF) element that blocks the RCL path in the native state, which is in excellent agreement with previous experimental reports. Furthermore, the simulation suggested a novel role of hF and the connected turn (thFs3A) as chaperones that support the insertion process by reducing the conformational space available to the RCL. Transient electrostatic interactions of RCL residues potentially fine-tune the serpin inhibitory activity. On the basis of our simulation, we generated the α1AT mutants K168E, E346K, and K168E/E346K and analyzed their inhibitory activity along with their intrinsic stability and heat-induced polymerization. Remarkably, the E346K mutation exhibited enhanced inhibitory activity along with an increased rate of premature structural collapse (polymerization), suggesting a significant role of E346 in the gatekeeping of the strain in the metastable native state.

Mutation-Induced Deamidation of Corneal Dystrophy-Related Transforming Growth Factor ß-Induced Protein.

Mutations in the transforming growth factor ß-induced protein (TGFBIp) cause phenotypically diverse corneal dystrophies, where protein aggregation in the cornea leads to severe visual impairment. Previous studies have shown a relationship between mutant-specific corneal dystrophy phenotypes and the thermodynamic stability of TGFBIp. Using liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance (NMR), we investigated correlations between the structural integrity of disease-related mutants of the fourth FAS1 domain (FAS1-4) and deamidation of TGFBIp residue Asn622. We observed a high rate of Asn622 deamidation in the A546D and A546D/P551Q FAS1-4 mutants that were both largely unstructured as determined by NMR. Conversely, the more structurally organized A546T and V624M FAS1-4 mutants had reduced deamidation rates, suggesting that a folded and stable FAS1-4 domain precludes Asn622 deamidation. Wild-type, R555Q, and R555W FAS1-4 mutants displayed very slow deamidation, which agrees with their similar and ordered NMR structures, where Asn622 is in a locked conformation. We confirmed the FAS1-4 mutational effect on deamidation rates in full-length TGFBIp mutants and found a similar ranking compared to that of the FAS1-4 domain alone. Consequently, the deamidation rate of Asn622 can be used to predict the structural effect of the many destabilizing and/or stabilizing mutations reported for TGFBIp. In addition, the deamidation of Asn622 may influence the pathophysiology of TGFBIp-induced corneal dystrophies.

Assessment of the acute subjective psychedelic experience: A review of patient-reported outcome measures in clinical research on classical psychedelics.

BACKGROUND: The classical psychedelics psilocybin, peyote, ayahuasca/ N, N-dimethyltryptamine, and lysergic acid diethylamide can temporarily produce altered states of consciousness, characterized by changes in sensory perception, thought, mood, and the sense of self-reality and meaning. It is important to have reliable instruments for quantifying these altered states in trials, due to a plausible link between the acute subjective experience and treatment outcome. METHODS: We conducted a review of outcome measures applied in research on classical psychedelics to assess one or more dimensions of the acute subjective psychedelic experience. Three relevant databases were searched electronically. Two reviewers independently conducted article selection and data extraction regarding the instruments, dimensions, geography, population, and psychedelic substance investigated in the included studies. We identified the five most utilized instruments for the most recent 6 years, as well as the five most utilized instruments for each psychedelic. RESULTS: We included 93 papers, which reported on 93 unique trials and utilized 17 different rating scales. Of these, the most utilized were the Five-Dimensional Altered States of Consciousness Questionnaire, visual analog or Likert scales specially developed for the trials, the Hallucinogen Rating Scale, the States of Consciousness Questionnaire, and the Abnormer Psychischer Zustand. DISCUSSION: Considerable variability was found in the instruments utilized in clinical trials on classical psychedelics. We advise and encourage the development of a core outcome set for psychedelic research to enable altered state comparisons across compounds, participants, and settings. We further advise that instruments be designed to assess the "setting" of a psychedelic experience.

Risk of bias in randomized clinical trials on psychedelic medicine: A systematic review.

BACKGROUND: The classical psychedelics, psilocybin, peyote, ayahuasca/N,N-dimethyltryptamine, and lysergic acid diethylamide are considered promising new treatments for psychiatric illnesses, such as depression, anxiety, addiction, and obsessive-compulsive disorders. However, their profound and characteristic subjective effects raise concern for distinctive biases in randomized clinical trials. METHODS: We performed a systematic literature search to identify all clinical trials on classical psychedelics with patient populations to examine descriptive data and determine the risk of bias. Two independent reviewers searched three databases (PubMed, Embase, and APA PsycNet) and extracted information on study design, study population, use of active or inactive placebo, dropouts, evaluation of blinding of intervention, and reporting of expectancy and therapeutic alliance. RESULTS: We included 10 papers reporting on 10 unique trials. The trials generally included populations that were predominantly white and highly educated. The trials had small samples and considerable dropout. Blinding was either unsuccessful or not reported regardless of type of placebo. Few trials published protocols, statistical analysis plans (SAPs), and outcomes relating to psychotherapy fidelity. All trials but one were rated as high risk of bias. CONCLUSION: Successful blinding of intervention is a significant challenge in this field. To better accommodate this, we suggest that future trials use a parallel-group design and utilize an active placebo on a psychedelic-naïve population. Future trials should publish trial protocol and SAPs, use clinician-rated outcomes accessed by a blinded rater, evaluate blinding of intervention, and consider measuring expectancy and therapeutic fidelity.

ITIH4 acts as a protease inhibitor by a novel inhibitory mechanism.

Inter-α-inhibitor heavy chain 4 (ITIH4) is a poorly characterized plasma protein that is proteolytically processed in multiple pathological conditions. However, no biological function of ITIH4 has been identified. Here, we show that ITIH4 is cleaved by several human proteases within a protease-susceptible region, enabling ITIH4 to function as a protease inhibitor. This is exemplified by its inhibition of mannan-binding lectin-associated serine protease-1 (MASP-1), MASP-2, and plasma kallikrein, which are key proteases for intravascular host defense. Mechanistically, ITIH4 acts as bait that, upon cleavage, forms a noncovalent, inhibitory complex with the executing protease that depends on the ITIH4 von Willebrand factor A domain. ITIH4 inhibits the MASPs by sterically preventing larger protein substrates from accessing their active sites, which remain accessible and fully functional toward small substrates. Thus, we demonstrate that ITIH4 functions as a protease inhibitor by a previously undescribed inhibitory mechanism.

Determination of cell mass and polymyxin using multi-wavelength fluorescence.

Multi-wavelength fluorescence was applied for on-line monitoring of cell mass and the antibiotic polymyxin B in Bacillus polymyxa cultivations. By varying the phosphate and nitrogen content of the medium different polymyxin-cell mass ratios could be obtained. Using this strategy, it was possible to investigate if multi-wavelength fluorescence is able to give independent prediction of the two parameters. Partial least square (PLS) regression was applied to establish mathematical relationships between off-line determined cell mass and polymyxin concentrations and on-line collected fluorescence data. For polymyxin one universal PLS model, with a correlation of 0.95 and a root mean square error of cross validation (RMSECV) of 35 mgl(-1), could be constructed. However, correlation between fluorescence and cell mass dry weight could not be established including data from all three types of cultivations. For data from each type of cultivation, separate models with high correlation and low RMSECV values were built. A large variation in cellular composition as a result of the different levels of nitrogen and phosphorus in the cultivations was the probable reason to the necessity of building three models. The results of the present investigation indicate that production of polymyxin is concomitantly regulated by phosphate and nitrogen as the highest polymyxin yield on cell mass, 0.17+/-0.01 gg(-1), was reached in the cultivations where both nitrogen and phosphate concentrations were kept low.