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The flavoprotein Cytochrome P450 reductase (CPR) is the unique electron pathway from NADPH to Cytochrome P450 (CYPs). The conformational dynamics of human CPR in solution, which involves transitions from a "locked/closed" to an "unlocked/open" state, is crucial for electron transfer. To date, however, the factors guiding these changes remain unknown. By Site‐Directed Spin Labelling coupled to Electron Paramagnetic Resonance spectroscopy, we have incorporated a non‐canonical amino acid onto the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) domains of soluble human CPR, and labelled it with a specific nitroxide spin probe. Taking advantage of the endogenous FMN cofactor, we successfully measured for the first time, the distance distribution by DEER between the semiquinone state FMNH• and the nitroxide. The DEER data revealed a salt concentration‐dependent distance distribution, evidence of an "open" CPR conformation at high salt concentrations exceeding previous reports. We also conducted molecular dynamics simulations which unveiled a diverse ensemble of conformations for the "open" semiquinone state of the CPR at high salt concentration. This study unravels the conformational landscape of the one electron reduced state of CPR, which had never been studied before. [ABSTRACT FROM AUTHOR]

Bacterial genomes are a huge reservoir of genes encoding J‐domain protein co‐chaperones that recruit the molecular chaperone DnaK to assist protein substrates involved in survival, adaptation, or fitness. The atc operon of the aquatic mesophilic bacterium Shewanella oneidensis encodes the proteins AtcJ, AtcA, AtcB, and AtcC, and all of them, except AtcA, are required for growth at low temperatures. AtcJ is a short J‐domain protein that interacts with DnaK, but also with AtcC through its 21 amino acid C‐terminal domain. This interaction network is critical for cold growth. Here, we show that AtcJ represents a subfamily of short J‐domain proteins that (i) are found in several environmental, mostly aquatic, β‐ or ɣ‐proteobacteria and (ii) contain a conserved PX7W motif in their C‐terminal extension. Using a combination of NMR, biochemical and genetic approaches, we show that the hydrophobic nature of the tryptophan of the S. oneidensis AtcJ PX7W motif determines the strong AtcJ–AtcC interaction essential for cold growth. The AtcJ homologues are encoded by operons containing at least the S. oneidensis atcA, atcB, and atcC homologues. These findings suggest a conserved network of DnaK and Atc proteins necessary for low‐temperature growth and, given the variation in the atc operons, possibly for other biological functions. [ABSTRACT FROM AUTHOR]

Ireland has among the lowest rates of breastfeeding worldwide. Despite policies to support breastfeeding, breastfeeding initiation and exclusivity remain low in Ireland. Greater knowledge about support received in the maternity unit may—in part—shed light on why this is so. Our aim was to analyse women's experiences of the breastfeeding supports available in the early postnatal period in Ireland. We conducted an analysis of an open-ended question on a cross-sectional survey about breastfeeding support conducted in the Republic of Ireland in 2022. Participants were asked to provide comments about the breastfeeding support they received in the maternity unit or during your home birth. Data were analysed using Braun and Clarke's six-step Thematic Analysis Framework. There were 5,412 unique responses to the survey and 2,264 responses to the question of interest. Two themes were generated from the data: (i) 'Breastfeeding support in theory but not in practice.' Although breastfeeding was promoted by healthcare professionals antenatally, breastfeeding challenges were rarely mentioned. Participants then felt unsupported in overcoming challenges postnatally. (ii) 'Support was either inaccessible due to lack of staff/time, inadequate; i.e., unhelpful or non-specific, and/or physically inappropriate.' Most participants described receiving supports that were less than optimal in aiding them to establish breastfeeding. While many described difficulties in accessing supports, others found support to be 'non-specific,' 'rushed' and sometimes 'rough.' A lack of knowledge, time and support from healthcare professionals was frequently described, which was often recognised as a failing of the healthcare system. Women require practical, informative, and specific breastfeeding support. Barriers such as lack of time and trained staff in the maternity unit need to be addressed. [ABSTRACT FROM AUTHOR]

Bacteria regulate their metabolism to adapt and survive adverse conditions, in particular to stressful downshifts in nutrient availability. These shifts trigger the so-called stringent response, coordinated by the signaling molecules guanosine tetra and pentaphosphate collectively referred to as (p)ppGpp. In Escherichia coli, accumulation of theses alarmones depends on the (p) ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT. A tight regulation of these intracellular activities is therefore crucial to rapidly adjust the (p)ppGpp levels in response to environmental stresses but also to avoid toxic consequences of (p)ppGpp overaccumulation. In this study, we show that the small protein NirD restrains RelA-dependent accumulation of (p)ppGpp and can inhibit the stringent response in E. coli. Mechanistically, our in vivo and in vitro studies reveal that NirD directly binds the catalytic domains of RelA to balance (p) ppGpp accumulation. Finally, we show that NirD can control RelA activity by directly inhibiting the rate of (p)ppGpp synthesis. [ABSTRACT FROM AUTHOR]

The FrzCD chemoreceptor from the gliding bacterium Myxococcus xanthus forms cytoplasmic clusters that occupy a large central region of the cell body also occupied by the nucleoid. In this work, we show that FrzCD directly binds to the nucleoid with its N-terminal positively charged tail and recruits active signaling complexes at this location. The FrzCD binding to the nucleoid occur in a DNA-sequence independent manner and leads to the formation of multiple distributed clusters that explore constrained areas. This organization might be required for cooperative interactions between clustered receptors as observed in membrane-bound chemosensory arrays. [ABSTRACT FROM AUTHOR]

The chemical attack of ore by ferric iron and/or sulfuric acid releases valuable metals. The products of these reactions are recycled by iron and sulfur oxidizing microorganisms. These acidophilic chemolithotrophic prokaryotes, among which Acidithiobacillus ferrooxidans, grow at the expense of the energy released from the oxidation of ferrous iron and/or inorganic sulfur compounds (ISCs). In At. ferrooxidans, it has been shown that the expression of the genes encoding the proteins involved in these respiratory pathways is dependent on the electron donor and that the genes involved in iron oxidation are expressed before those responsible for ISCs oxidation when both iron and sulfur are present. Since the redox potential increases during iron oxidation but remains stable during sulfur oxidation, we have put forward the hypothesis that the global redox responding two components system RegB/RegA is involved in this regulation. To understand the mechanism of this system and its role in the regulation of the aerobic respiratory pathways in At. ferrooxidans, the binding of different forms of RegA (DNA binding domain, wild-type, unphosphorylated and phosphorylated-like forms of RegA) on the regulatory region of different genes/operons involved in ferrous iron and ISC oxidation has been analyzed. We have shown that the four RegA forms are able to bind specifically the upstream region of these genes. Interestingly, the phosphorylation of RegA did not change its affinity for its cognate DNA. The transcriptional start site of these genes/operons has been determined. In most cases, the RegA binding site(s) was (were) located upstream from the -35 (or -24) box suggesting that RegA does not interfere with the RNA polymerase binding. Based on the results presented in this report, the role of the RegB/RegA systemin the regulation of the ferrous iron and ISC oxidation pathways in At. ferrooxidans is discussed. [ABSTRACT FROM AUTHOR]

The article presents the discussion on nurses responsible for assisting patients in end-of-life decision-making and goals of care conversations including undergraduate nursing students to developing competency in advance care planning (ACP).

Human Galectin-3 is found in the nucleus, the cytoplasm and at the cell surface. This lectin is constituted of two domains: an unfolded N-terminal domain and a C-terminal Carbohydrate Recognition Domain (CRD). There are still uncertainties about the relationship between the quaternary structure of Galectin-3 and its carbohydrate binding properties. Two types of self-association have been described for this lectin: a C-type self-association and a N-type self-association. Herein, we have analyzed Galectin-3 oligomerization by Dynamic Light Scattering using both the recombinant CRD and the full length lectin. Our results proved that LNnT induces N-type self-association of full length Galectin-3. Moreover, from Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance experiments, we observed no significant specificity or affinity variations for carbohydrates related to the presence of the N-terminal domain of Galectin-3. NMR mapping clearly established that the N-terminal domain interacts with the CRD. We propose that LNnT induces a release of the N-terminal domain resulting in the glycan-dependent self-association of Galectin-3 through N-terminal domain interactions. [ABSTRACT FROM AUTHOR]

Highlights: [•] Polyproline motif included in ErbB2 C-terminal domain. [•] Fyn SH3 binds ErbB2 PPII motif. [•] ErbB2 C-terminal domain interacts with a Src kinase. [Copyright &y& Elsevier]

Nuclear inelastic scattering of 57Fe labeled [NiFe] hydrogenase is shown to give information on different states of the enzyme. It was thus possible to detect and assign FeCO and FeCN bending and stretching vibrations of the active site outside the spectral range of the FeS cluster normal modes. [ABSTRACT FROM AUTHOR]

Blick ins Zentrum: Die Methode der inelastischen kernresonanten Streuung (NRVS) wurde genutzt, um 57Fe ‐ markierte [NiFe] ‐ Hydrogenase in unterschiedlichen Zuständen des Enzyms zu untersuchen. Damit gelang es, die Fe ‐ CO ‐ und Fe ‐ CN ‐ Beuge ‐ und ‐ Streckschwingung außerhalb des Spektralbereichs der Normalmoden der Fe ‐ S ‐ Cluster zu detektieren und zuzuordnen (siehe Spektrum). [ABSTRACT FROM AUTHOR]

S100A1 is a member of the S100 family of small ubiquitous Ca2+-binding proteins, which participates in the regulation of cell differentiation, motility, and survival. It exists as homo- or heterodimers. S100A1 has also been shown to bind Zn2+, but the molecular mechanisms of this binding are not yet known. In this work, using ESI-MS and ITC, we demonstrate that S100A1 can coordinate 4 zinc ions per monomer, with two high affinity (KD~4 and 770 nm) and two low affinity sites. Using competitive binding experiments between Ca2+ and Zn2+ and QM/MM molecular modeling we conclude that Zn2+ high affinity sites are located in the EF-hand motifs of S100A1. In addition, two lower affinity sites can bind Zn2+ even when the EF-hands are saturated by Ca2+, resulting in a 2Ca2+:S100A1:2Zn2+ conformer. Finally, we show that, in contrast to calcium, an excess of Zn2+ produces a destabilizing effect on S100A1 structure and leads to its aggregation. We also determined a higher affinity to Ca2+ (KD~0.16 and 24 μm) than was previously reported for S100A1, which would allow this protein to function as a Ca2+/Zn2+-sensor both inside and outside cells, participating in diverse signaling pathways under normal and pathological conditions. [ABSTRACT FROM AUTHOR]

The amoeba-infecting Mimivirus is the largest known double-stranded DNA virus, with a 400 nm particle size, comparable to that of mycoplasma. The complete sequence of its 1.2 Mbp genome has recently been determined [Raoult et al. (2004), Science, 306, 1344-1350] and revealed numerous genes that were not expected to be found in a virus, such as genes encoding translation components, including 4-amino-acyl tRNA synthetases and homologues to various translation initiation, elongation and termination factors. A comprehensive structural and functional study of these Mimivirus gene products was initiated, as they may hold important clues about the origin of DNA viruses. Here, the first preliminary crystallographic and functional results obtained on one of these targets, Mimivirus TyrRS, are reported. Preliminary phasing was obtained using an original combination of homology modelling and normal mode analysis. Experimental evidence that Mimivirus tyrosyl tRNA synthetase recombinant gene product does indeed activate tyrosine is also presented. [ABSTRACT FROM AUTHOR]

The article explains why registered nurses (RN) thrive to pursue a Bachelor of Science in Nursing (BSN) program. According to the American Nurses Association, due to the changing healthcare environment, competencies associated with baccalaureate-level education are needed. It discusses a pilot study which designed using Ajzen's theory to examine the planned behavior of a nurse.

The antihypertensive effect of carteolol, a new potent nonselective beta-adrenergic antagonist, was investigated in a double-blind, parallel study of 35 patients with mild-to-moderate, essential hypertension whose blood pressures were not adequately controlled with a diuretic. Patients were randomly assigned to receive placebo, carteolol 5 mg, or carteolol 20 mg once a day in addition to hydrochlorothiazide 50 mg for six weeks. Thirty-four patients completed the study: 11 patients received placebo (group 1), 12 patients received carteolol 5 mg (group 2), and 11 patients received carteolol 20 mg (group 3). After six weeks of treatment, the two groups that received carteolol had significant reductions in systolic (SBP) and diastolic (DBP) blood pressure from baseline in both the supine and standing positions. In group 2, mean SBP decreases for supine and standing positions were 11 ± 2.1 and 11 ± 1.9 mm Hg, respectively (P < .01) and 8 ± 1.2 and 9 ± 1.3 mm Hg, respectively, for DBP (P < .01); whereas in group 3, values were 8 ± 2.8 and 12 ± 3.0 mm Hg for SBP, in 5 ± 1.9 and 9 ± 3.1 mm Hg, respectively, for DBP (P < .01). The hypotensive effect was associated with slight but significant decreases in heart rate (P < .01 in group 2 and P < .05 in group 3). Reductions in SBP and DBP with the 20-mg dose were not significantly different from those with the 5-mg dose. With both carteolol regimens, the fall in mean blood pressure did not correlate with the initial level; but in the group taking the 5-mg dose, it did correlate significantly with the decrease in heart rate (r = .72, P < .01). Side effects attributable to carteolol were infrequent and did not persist during continued therapy. It is concluded that carteolol, when combined with hydrochlorothiazide, is effective in reducing blood pressure in patients with mild to moderate hypertension. [ABSTRACT FROM AUTHOR]

Chemosensory systems are highly organized signaling pathways that allow bacteria to adapt to environmental changes. The Frz chemosensory system from M. xanthus possesses two CheW-like proteins, FrzA (the core CheW) and FrzB. We found that FrzB does not interact with FrzE (the cognate CheA) as it lacks the amino acid region responsible for this interaction. FrzB, instead, acts upstream of FrzCD in the regulation of M. xanthus chemotaxis behaviors and activates the Frz pathway by allowing the formation and distribution of multiple chemosensory clusters on the nucleoid. These results, together, show that the lack of the CheA-interacting region in FrzB confers new functions to this small protein. Author summary: Chemosensory systems are signaling complexes that are widespread in bacteria and allow the modulation of different cellular functions, such as taxis and development, in response to the environment. We show that the Myxococcus xanthus FrzB is a divergent CheW lacking the region involved in the interaction with the histidine kinase FrzE. Instead, it acts upstream of FrzCD to allow the formation of multiple distributed Frz chemosensory arrays at the nucleoid. The loss of the CheA-interacting region in FrzB might have been selected to confer plasticity to nucleoid-associated chemosensory systems. By unraveling a new accessory protein and its function, this work opens new insights into the knowledge of the regulatory potentials of bacterial chemosensory systems. [ABSTRACT FROM AUTHOR]

Abstract Tau is an intrinsically disordered microtubule-associated protein that is implicated in several neurodegenerative disorders called tauopathies. In these diseases, Tau is found in the form of intracellular inclusions that consist of aggregated paired helical filaments (PHFs) in neurons. Given the importance of this irreversible PHF formation in neurodegenerative disease, Tau aggregation has been extensively studied. Several different factors, such as mutations or post translational modifications, have been shown to influence the formation of late-stage non-reversible Tau aggregates. It was recently shown that zinc ions accelerated heparin-induced oligomerization of Tau constructs. Indeed, in vitro studies of PHFs have usually been performed in the presence of additional co-factors, such as heparin, in order to accelerate their formation. Using turbidimetry, we investigated the impact of zinc ions on Tau in the absence of heparin and found that zinc is able to induce a temperature-dependent reversible oligomerization of Tau. The obtained oligomers were not amyloid-like and dissociated instantly following zinc chelation or a temperature decrease. Finally, a combination of isothermal titration calorimetry and dynamic light scattering experiments showed zinc binding to a high-affinity binding site and three low-affinity sites on Tau, accompanied by a change in Tau folding. Altogether, our findings stress the importance of zinc in Tau oligomerization. This newly identified Zn-induced oligomerization mechanism may be a part of a pathway different of and concurrent to Tau aggregation cascade leading to PHF formation. Graphical Abstract Unlabelled Image Highlights • Tau aggregation mechanism is still poorly understood. • Zinc ions induce fast and reversible oligomerization of Tau without heparin. • Tau has 2 types of zinc-binding sites. • Zinc ions binding to tau induces structural changes of tau. • New oligomerization mechanism might be concurrent to PHF formation. [ABSTRACT FROM AUTHOR]

The active site of [NiFe] hydrogenase is an excellent catalyst for hydrogen conversion with an intriguing ability to recover from exposure to dioxygen. In their Communication on page 724 ff., W. Lubitz, S. P. Cramer, and co‐workers use the synchrotron technique of nuclear resonance vibrational spectroscopy to identify normal modes associated with active‐site FeCN and FeCO motion. [ABSTRACT FROM AUTHOR]

Das aktive Zentrum der [NiFe] ‐ Hydrogenase ist ein exzellenter Katalysator der Wasserstoffumsetzung mit der faszinierenden Fähigkeit, sich nach Sauerstoffexposition zurückzubilden. W. Lubitz, S. P. Cramer et al. nutzen in ihrer Zuschrift auf S. 752 ff. die Synchrotrontechnik der kernresonanten inelastischen Streuung, um Normalmoden der Fe ‐ CN ‐ und Fe ‐ CO ‐ Bewegung im aktiven Zentrum zu identifizieren. [ABSTRACT FROM AUTHOR]

Tau is a naturally disordered microtubule associated protein which forms intraneuronal aggregates in several neurodegenerative diseases including Alzheimer's disease (AD). It was reported that zinc interaction with tau protein can trigger its aggregation. Recently we identified three zinc binding sites located in the N-terminal part, repeat region and the C-terminal part of tau. Here we characterized zinc binding to each of the three sites using isothermal titration calorimetry (ITC) and determined the impact of each site on aggregation using dynamic light scattering (DLS) assays. First, we confirmed the presence of three zinc binding sites on tau and determined the thermodynamic parameters of binding of zinc to these sites. We found a high-affinity zinc binding site located in the repeat region of tau and two N- and C-terminus binding sites with a lower binding constant for zinc. Second, we showed that tau aggregation necessitates zinc binding to the high affinity site in the R2R3 region, while LLPS necessitates zinc binding to any two binding sites. With regard to the role of zinc ions in the aggregation of proteins in neurodegenerative diseases, these findings bring new insights to the understanding of the aggregation mechanism of tau protein induced by zinc. • Zinc is known to trigger tau aggregation and droplet formation. • Three zinc-binding sites have been identified in a NMR study. • Zinc binding site located in the R2R3 domain is crucial for tau aggregation. • Zinc binding to only one site does not induce tau aggregation nor LLPS. • Any two of three existing zinc-binding sites are necessary to induce droplets. [ABSTRACT FROM AUTHOR]

The genetic organization of the aioBA operon, encoding the arsenite oxidase of the moderately acidophilic and facultative chemoautotrophic bacterium Thiomonas arsenitoxydans, is different from that of the aioBA operon in the other arsenite oxidizers, in that it encodes AioF, a metalloprotein belonging to the ArsR/SmtB family. AioF is stabilized by arsenite, arsenate, or antimonite but not molybdate. Arsenic is tightly attached to AioF, likely by cysteine residues. When loaded with arsenite or arsenate, AioF is able to bind specifically to the regulatory region of the aio operon at two distinct positions. In Thiomonas arsenitoxydans, the promoters of aioX and aioB are convergent, suggesting that transcriptional interference occurs. These results indicate that the regulation of the aioBA operon is more complex in Thiomonas arsenitoxydans than in the other aioBA containing arsenite oxidizers and that the arsenic binding protein AioF is involved in this regulation. On the basis of these data, a model to explain the tight control of aioBA expression by arsenic in Thiomonas arsenitoxydans is proposed. [ABSTRACT FROM AUTHOR]

LDAP (Lightweight Directory Access Protocol) is a technology that can provide directory services to a range of applications. Directory service, a critical part of distributed computing, is the central point where network services, security services, and applications can form an integrated distributed computing environment. The simplicity of LDAP enables users to store and retrieve data easily from the directory. Nevertheless, as the use of directory services becomes more widespread, directories will need to scale to support millions of entries and millions of user requests with subsecond predictable performance. LDAP directories can be implemented using various storage mechanisms such as flat files, b-trees, or databases. This paper discusses an implementation of LDAP that uses the IBM DATABASE 2 Trademark relational database as the data store and query engine to meet the directory service requirements. Performance analysis is provided to show that a relational database can be used to successfully meet the performance and scale needs of an LDAP directory while remaining secure and competitive with other vendor implementations. [ABSTRACT FROM AUTHOR]

The interaction of pancreatic lipase-related protein 2 (PLRP2) with various micelles containing phospholipids was investigated using pHstat enzyme activity measurements, differential light scattering, size exclusion chromatography (SEC) and transmission IR spectroscopy. Various micelles of 1,2-dipalmitoyl- sn -glycero-3-phosphocholine (DPPC) and lysophosphatidylcholine were prepared with either bile salts (sodium taurodeoxycholate or glycodeoxycholate) or Triton X-100, which are substrate-dispersing agents commonly used for measuring phospholipase activities. PLRP2 displayed a high activity on all phospholipid-bile salt micelles, but was totally inactive on phospholipid-Triton X-100 micelles. These findings clearly differentiate PLRP2 from secreted pancreatic phospholipase A2 which is highly active on both types of micelles. Using an inactive variant of PLRP2, SEC experiments allowed identifying two populations of PLRP2-DPPC-bile salt complexes corresponding to a high molecular weight 1:1 PLRP2-micelle association and to a low molecular weight association of PLRP2 with few monomers of DPPC/bile salts. IR spectroscopy analysis showed how DPPC-bile salt micelles differ from DPPC-Triton X-100 micelles by a higher fluidity of acyl chains and higher hydration/H-bonding of the interfacial carbonyl region. The presence of bile salts allowed observing changes in the IR spectrum of DPPC upon addition of PLRP2 (higher rigidity of acyl chains, dehydration of the interfacial carbonyl region), while no change was observed with Triton X-100. The differences between these surfactants and their impact on substrate recognition by PLRP2 are discussed, as well as the mechanism by which high and low molecular weight PLRP2-DPPC-bile salt complexes may be involved in the overall process of DPPC hydrolysis. [ABSTRACT FROM AUTHOR]

Mimivirus, a virus infecting Acanthamoeba, is the prototype of the Mimiviridae, the latest addition to the nucleocytoplasmic large DNA viruses. The Mimivirus genome encodes close to 1000 proteins, many of them never before encountered in a virus, such as four amino-acyl tRNA synthetases. To explore the physiology of this exceptional virus and identify the genes involved in the building of its characteristic intracytoplasmic "virion factory," we coupled electron microscopy observations with the massively parallel pyrosequencing of the polyadenylated RNA fractions of Acanthamoeba castellanii cells at various time post-infection. We generated 633,346 reads, of which 322,904 correspond to Mimivirus transcripts. This first application of deep mRNA sequencing (454 Life Sciences [Roche] FLX) to a large DNA virus allowed the precise delineation of the 5' and 3' extremities of Mimivirus mRNAs and revealed 75 new transcripts including several noncoding RNAs. Mimivirus genes are expressed across a wide dynamic range, in a finely regulated manner broadly described by three main temporal classes: early, intermediate, and late. This RNA-seq study confirmed the AAAATTGA sequence as an early promoter element, as well as the presence of palindromes at most of the polyadenylation sites. It also revealed a new promoter element correlating with late gene expression, which is also prominent in Sputnik, the recently described Mimivirus "virophage." These results-validated genome-wide by the hybridization of total RNA extracted from infected Acanthamoeba cells on a tiling array (Agilent)-will constitute the foundation on which to build subsequent functional studies of the Mimivirus/Acanthamoeba system. [ABSTRACT FROM AUTHOR]

We have discovered a protein with an amino acid composition exceptionally rich in glycine and cysteine residues in the giant virus mimivirus. This small 6 kDa protein is among the most abundant proteins in the icosahedral 0.75 μm viral particles; it has no predicted function but is probably essential for infection. The aerobically purified red-brownish protein overproduced in Escherichia coli contained both iron and inorganic sulfide. UV/vis, EPR, and Mössbauer studies revealed that the viral protein, coined GciS, accommodated two distinct Fe-S clusters: a diamagnetic S = 0 [2Fe-2S] 2+ cluster and a paramagnetic S = 5/2 linear [3Fe-4S] 1+ cluster, a geometry rarely stabilized in native proteins. Orthologs of mimivirus GciS were identified within all clades of Megavirinae , a Mimiviridae subfamily infecting Acanthamoeba , including the distantly related tupanviruses, and displayed the same spectroscopic features. Thus, these glycine/cysteine-rich proteins form a new family of viral Fe-S proteins sharing unique Fe-S cluster binding properties.

Chaperone proteins are essential in all living cells to ensure protein homeostasis. Hsp90 is a major adenosine triphosphate (ATP)-dependent chaperone highly conserved from bacteria to eukaryotes. Recent studies have shown that bacterial Hsp90 is essential in some bacteria in stress conditions and that it participates in the virulence of pathogenic bacteria. In vitro, bacterial Hsp90 directly interacts and collaborates with the Hsp70 chaperone DnaK to reactivate model substrate proteins; however, it is still unknown whether this collaboration is relevant in vivo with physiological substrates. Here, we used site-directed mutagenesis on Hsp90 to impair DnaK binding, thereby uncoupling the chaperone activities. We tested the mutants in vivo in two bacterial models in which Hsp90 has known physiological functions. We found that the Hsp90 point mutants were defective to support (1) growth under heat stress and activation of an essential Hsp90 client in the aquatic bacterium Shewanella oneidensis and (2) biosynthesis of the colibactin toxin involved in the virulence of pathogenic Escherichia coli . Our study therefore demonstrates the essentiality of the direct collaboration between Hsp90 and DnaK in vivo in bacteria to support client folding. It also suggests that this collaboration already functional in bacteria has served as an evolutionary basis for a more complex Hsp70-Hsp90 collaboration found in eukaryotes.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited., Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19., Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020., Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108)., Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%)., Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively., Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions., Trial Registration: ClinicalTrials.gov Identifier: NCT02735707.

Local activation and long-range inhibition are mechanisms conserved in self-organizing systems leading to biological patterns. A number of them involve the production by the developing cell of an inhibitory morphogen, but how this cell becomes immune to self-inhibition is rather unknown. Under combined nitrogen starvation, the multicellular cyanobacterium Nostoc PCC 7120 develops nitrogen-fixing heterocysts with a pattern of one heterocyst every 10-12 vegetative cells. Cell differentiation is regulated by HetR which activates the synthesis of its own inhibitory morphogens, diffusion of which establishes the differentiation pattern. Here, we show that HetR interacts with HetL at the same interface as PatS, and that this interaction is necessary to suppress inhibition and to differentiate heterocysts. hetL expression is induced under nitrogen-starvation and is activated by HetR, suggesting that HetL provides immunity to the heterocyst. This protective mechanism might be conserved in other differentiating cyanobacteria as HetL homologues are spread across the phylum., Competing Interests: XX, VR, DB, SC, BD, AL No competing interests declared, (© 2020, Xu et al.)