The unique regulation of iron-sulfur cluster biogenesis in a Gram-positive bacterium

Iron-sulfur clusters function as cofactors of a wide range of proteins, with diverse molecular roles in both prokaryotic and eukaryotic cells. Dedicated machineries assemble the clusters and deliver them to the final acceptor molecules in a tightly regulated process. In the prototypical Gram-negative bacterium Escherichia coli, the two existing iron-sulfur cluster assembly systems, iron-sulfur cluster (ISC) and sulfur assimilation (SUF) pathways, are closely interconnected. The ISC pathway regulator, IscR, is a transcription factor of the helix-turn-helix type that can coordinate a [2Fe-2S] cluster. Redox conditions and iron or sulfur availability modulate the ligation status of the labile IscR cluster, which in turn determines a switch in DNA sequence specificity of the regulator: cluster-containing IscR can bind to a family of gene promoters (type-1) whereas the clusterless form recognizes only a second group of sequences (type-2). However, iron-sulfur cluster biogenesis in Gram-positive bacteria is not so well characterized, and most organisms of this group display only one of the iron-sulfur cluster assembly systems. A notable exception is the unique Gram-positive dissimilatory metal reducing bacterium Thermincola potens, where genes from both systems could be identified, albeit with a diverging organization from that of Gram-negative bacteria. We demonstrated that one of these genes encodes a functional IscR homolog and is likely involved in the regulation of iron-sulfur cluster biogenesis in T. potens. Structural and biochemical characterization of T. potens and E. coli IscR revealed a strikingly similar architecture and unveiled an unforeseen conservation of the unique mechanism of sequence discrimination characteristic of this distinctive group of transcription regulators.We thank Jorge Vieira for help with Automatic Detection of Positively Selected Sites. We acknowledge the European Synchrotron Radiation Facility (ESRF) for provision of synchrotron radiation facilities and thank the ESRF staff for help with data collection. Microscale thermophoresis data collection was carried out at the Campus Science Support Facilities Protein Technologies Facility (www.csf.ac.at). This work was funded by Fundo Europeu de Desenvolvimento Regional through the Operational Competitiveness Programme-COMPETE and by national funds through Fundacao para a Ciencia e a Tecnologia under project FCOMP-01-0124-FEDER-028116 (PTDC/BBB - BEP/2127/2012) and PhD Fellowship SFRH/BD/66461/2009 (to J.A.S.). The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (Grant Agreement 283570)

Molecular fingerprints for a novel enzyme family in actinobacteria with glucosamine kinase activity

Actinobacteria have long been the main source of antibiotics, secondary metabolites with tightly controlled biosynthesis by environmental and physiological factors. Phosphorylation of exogenous glucosamine has been suggested as a mechanism for incorporation of this extracellular material into secondary metabolite biosynthesis, but experimental evidence of specific glucosamine kinases in Actinobacteria is lacking. Here, we present the molecular fingerprints for the identification of a unique family of actinobacterial glucosamine kinases. Structural and biochemical studies on a distinctive kinase from the soil bacterium Streptacidiphilus jiangxiensis unveiled its preference for glucosamine and provided structural evidence of a phosphoryl transfer to this substrate. Conservation of glucosamine-contacting residues across a large number of uncharacterized actinobacterial proteins unveiled a specific glucosamine binding sequence motif. This family of kinases and their genetic context may represent the missing link for the incorporation of environmental glucosamine into the antibiotic biosynthesis pathways in Actinobacteria and can be explored to enhance antibiotic production. IMPORTANCE The discovery of novel enzymes involved in antibiotic biosynthesis pathways is currently a topic of utmost importance. The high levels of antibiotic resistance detected worldwide threaten our ability to combat infections and other 20th-century medical achievements, namely, organ transplantation or cancer chemotherapy. We have identified and characterized a unique family of enzymes capable of phosphorylating glucosamine to glucosamine-6-phosphate, a crucial molecule directly involved in the activation of antibiotic production pathways in Actinobacteria, nature’s main source of antimicrobials. The consensus sequence identified for these glucosamine kinases will help establish a molecular fingerprint to reveal yet-uncharacterized sequences in antibiotic producers, which should have an important impact in biotechnological and biomedical applications, including the enhancement and optimization of antibiotic production.We acknowledge the European Synchrotron Radiation Facility (Grenoble, France) for provision of synchrotron radiation facilities and thank their staff for help with data collection. Part of these experiments were performed at beamline BL13-XALOC of ALBA Synchrotron (Cerdanyola del Vallès, Spain), with the collaboration of ALBA staff and CALIPSOplus (grant 730872) funding. The support of the X-ray Crystallography Scientific Platform of i3S (Porto, Portugal) is also acknowledged. We thank Pedro Lamosa from CERMAX, ITQB-NOVA (Oeiras, Portugal), for acquiring and interpreting the NMR data. This work was supported by the Structured Program on Bioengineered Therapies for Infectious Diseases and Tissue Regeneration (Norte-01-0145-FEDER-‏000‏012), funded by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through Fundo Europeu de Desenvolvimento Regional (FEDER) and by FEDER through the COMPETE 2020-Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT-Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of project Institute for Research and Innovation in Health Sciences (POCI-01-0145-FEDER-007274) and also by grants UID/NEU/04539/2019 and POCI-01-0145-FEDER-029221. D.N.-C. acknowledges the European Regional Development Fund (CENTRO-01-0145-FEDER-000012-ealthyAging2020) for a research fellowship and FCT for Ph‏D f‏ellowship SFRH/BD/117777/2016

Magnesium lactate in the treatment of Gitelman syndrome: patient-reported outcomes.

BACKGROUND: Gitelman syndrome (GS) is a rare recessively inherited renal tubulopathy associated with renal potassium (K) and magnesium (Mg) loss. It requires lifelong K and Mg supplementation at high doses that are at best unpalatable and at worst, intolerable. In particular, gastrointestinal side effects often limit full therapeutic usage. METHODS: We report here the analysis of a cohort of 28 adult patients with genetically proven GS who attend our specialist tubular disorders clinic, in whom we initiated the use of a modified-release Mg preparation (slow-release Mg lactate) and who were surveyed by questionnaire. RESULTS: Twenty-five patients (89%) preferred the new treatment regimen. Of these 25, 17 (68%) regarded their symptom burden as improved and seven reported no worsening. Of the 25 who were not Mg-treatment naïve, 13 (59%) patients reported fewer side effects, 7 (32%) described them as the same and only 2 (9%) considered side effects to be worse. Five were able to increase their dose without ill-effect. Overall, biochemistry improved in 91% of the 23 patients switched from therapy with other preparations who chose to continue the modified-release Mg preparation. Eleven (48%) improved both their Mg and K mean levels, 3 (13%) improved Mg levels only and in 7 cases (30%), K levels alone rose. CONCLUSIONS: Patient-reported and biochemical outcomes using modified-release Mg supplements were very favourable, and patient choice should play a large part in choosing Mg supplements with GS patients.This work was supported by the Wellcome Trust and the NIHR Cambridge Biomedical Research Centre, and contains data that were presented in abstract form at ASN Kidney week 2014.This is the final version of the article. It first appeared from Oxford University Press via https://doi.org/10.1093/ndt/gfw01

Crystallization and preliminary X-ray diffraction analysis of a novel trypsin inhibitor from seeds of Copaifera langsdorffii

A novel trypsin inhibitor isolated from seeds of Copaifera langsdorffii was purified to homogeneity and crystallized. Crystals suitable for X-ray analysis were grown using the hanging-drop vapour-diffusion method at 291 K in sodium acetate buffer at pH values near 4.3 using PEG 4000 as precipitant. The crystals presented symmetry compatible with the space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 58.71, c = 93.75 Angstrom, and diffracted to 1.83 Angstrom resolution at the synchrotron source.5791316131

How can latent trajectories of back pain be translated into defined subgroups?

Background: Similar types of trajectory patterns have been identified by Latent Class Analyses (LCA) across multiple low back pain (LBP) cohorts, but these patterns are impractical to apply to new cohorts or individual patients. It would be useful to be able to identify trajectory subgroups from descriptive definitions, as a way to apply the same definitions of mutually exclusive subgroups across populations. In this study, we investigated if the course trajectories of two LBP cohorts fitted with previously suggested trajectory subgroup definitions, how distinctly different these subgroups were, and if the subgroup definitions matched with LCA-derived patterns. Methods: Weekly measures of LBP intensity and frequency during 1 year were available from two clinical cohorts. We applied definitions of 16 possible trajectory subgroups to these observations and calculated the prevalence of the subgroups. The probability of belonging to each of eight LCA-derived patterns was determined within each subgroup. LBP intensity and frequency were described within subgroups and the subgroups of 'fluctuating' and 'episodic' LBP were compared on clinical characteristics. Results: All of 1077 observed trajectories fitted with the defined subgroups. 'Severe episodic LBP' was the most frequent pattern in both cohorts and 'ongoing LBP' was almost non-existing. There was a clear relationship between the defined trajectory subgroups and LCA-derived trajectory patterns, as in most subgroups, all patients had high probabilities of belonging to only one or two of the LCA patterns. The characteristics of the six defined subgroups with minor LBP were very similar. 'Fluctuating LBP' subgroups were significantly more distressed, had more intense leg pain, higher levels of activity limitation, and more negative expectations about future LBP than 'episodic LBP' subgroups. Conclusion: Previously suggested definitions of LBP trajectory subgroups could be readily applied to patients' observed data resulting in subgroups that matched well with LCA-derived trajectory patterns. We suggest that the number of trajectory subgroups can be reduced by merging some subgroups with minor LBP. Stable levels of LBP were almost not observed and we suggest that minor fluctuations in pain intensity might be conceptualised as 'ongoing LBP'. Lastly, we found clear support for distinguishing between fluctuating and episodic LBP

M1 muscarinic allosteric modulators slow prion neurodegeneration and restore memory loss

This is the final version of the article. Available from American Society for Clinical Investigation via the DOI in this record.The current frontline symptomatic treatment for Alzheimer’s disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR–selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD.ABT, AC, and PMS received funding from a Wellcome Trust Collaborative Award (201529/Z/16/Z). ABT, SJB, AJB, and TMH were funded through a Medical Research Council programme leader grant provided by the MRC Toxicology Unit. CCF, LMB, AJM, and HES were funded by the Eli Lilly Company. JMB received funding through a Lilly Research Award Program (LRAP) grant (Eli Lilly). RP received funding from the Marie Curie grant “Extrabrain” (European Commission). AC is a senior principal research fellow and PMS a principal research fellow of the National Health and Medical Research Council of Australia. Tissue samples were from Randy Woltjer at the Oregon Alzheimer’s Disease Center. The Oregon Alzheimer’s Disease Center is supported by NIH grant P30AG008017

Acute kidney disease and renal recovery : consensus report of the Acute Disease Quality Initiative (ADQI) 16 Workgroup

Consensus definitions have been reached for both acute kidney injury (AKI) and chronic kidney disease (CKD) and these definitions are now routinely used in research and clinical practice. The KDIGO guideline defines AKI as an abrupt decrease in kidney function occurring over 7 days or less, whereas CKD is defined by the persistence of kidney disease for a period of > 90 days. AKI and CKD are increasingly recognized as related entities and in some instances probably represent a continuum of the disease process. For patients in whom pathophysiologic processes are ongoing, the term acute kidney disease (AKD) has been proposed to define the course of disease after AKI; however, definitions of AKD and strategies for the management of patients with AKD are not currently available. In this consensus statement, the Acute Disease Quality Initiative (ADQI) proposes definitions, staging criteria for AKD, and strategies for the management of affected patients. We also make recommendations for areas of future research, which aim to improve understanding of the underlying processes and improve outcomes for patients with AKD