Postmitotic Specification of Drosophila Insulinergic Neurons from Pioneer Neurons

Insulin and related peptides play important and conserved functions in growth and metabolism. Although Drosophila has proved useful for the genetic analysis of insulin functions, little is known about the transcription factors and cell lineages involved in insulin production. Within the embryonic central nervous system, the MP2 neuroblast divides once to generate a dMP2 neuron that initially functions as a pioneer, guiding the axons of other later-born embryonic neurons. Later during development, dMP2 neurons in anterior segments undergo apoptosis but their posterior counterparts persist. We show here that surviving posterior dMP2 neurons no longer function in axonal scaffolding but differentiate into neuroendocrine cells that express insulin-like peptide 7 (Ilp7) and innervate the hindgut. We find that the postmitotic transition from pioneer to insulin-producing neuron is a multistep process requiring retrograde bone morphogenetic protein (BMP) signalling and four transcription factors: Abdominal-B, Hb9, Fork Head, and Dimmed. These five inputs contribute in a partially overlapping manner to combinatorial codes for dMP2 apoptosis, survival, and insulinergic differentiation. Ectopic reconstitution of this code is sufficient to activate Ilp7 expression in other postmitotic neurons. These studies reveal striking similarities between the transcription factors regulating insulin expression in insect neurons and mammalian pancreatic β-cells

Expression of the Pupal Determinant broad during Metamorphic and Neotenic Development of the Strepsipteran Xenos vesparum Rossi

Derived members of the endoparasitic order Strepsiptera have acquired an extreme form of sexual dimorphism whereby males undergo metamorphosis and exist as free-living adults while females remain larviform, reaching sexual maturity within their hosts. Expression of the transcription factor, broad (br) has been shown to be required for pupal development in insects in which both sexes progress through metamorphosis. A surge of br expression appears in the last larval instar, as the epidermis begins pupal development. Here we ask if br is also up-regulated in the last larval instar of male Xenos vesparum Rossi (Stylopidae), and whether such expression is lost in neotenic larviform females. We clone three isoforms of br from X. vesparum (Xv′br), and show that they share greatest similarity to the Z1, Z3 and Z4 isoforms of other insect species. By monitoring Xv′br expression throughout development, we detect elevated levels of total br expression and the Xv′Z1, Xv′Z3, and Xv′Z4 isoforms in the last larval instar of males, but not females. By focusing on Xv′br expression in individual samples, we show that the levels of Xv′BTB and Xv′Z3 in the last larval instar of males are bimodal, with some males expressing 3X greater levels of Xv′br than fourth instar femlaes. Taken together, these data suggest that neoteny (and endoparasitism) in females of Strepsiptera Stylopidia could be linked to the suppression of pupal determination. Our work identifies a difference in metamorphic gene expression that is associated with neoteny, and thus provides insights into the relationship between metamorphic and neotenic development. © 2014 Erezyilmaz et al

Structural basis for native agonist and synthetic inhibitor recognition by the Pseudomonas aeruginosa quorum sensing regulator PqsR (MvfR)

Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4-quinolones including the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone), its precursor 2-heptyl-4- hydroxyquinoline (HHQ) and their C9 congeners, 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS) and 2-nonyl-4-hydroxyquinoline (NHQ). These drive the autoinduction of AQ biosynthesis and the up-regulation of key virulence determinants as a function of bacterial population density. Consequently, PqsR constitutes a potential target for novel antibacterial agents which attenuate infection through the blockade of virulence. Here we present the crystal structures of the PqsR co-inducer binding domain (CBD) and a complex with the native agonist NHQ. We show that the structure of the PqsR CBD has an unusually large ligand-binding pocket in which a native AQ agonist is stabilized entirely by hydrophobic interactions. Through a ligand-based design strategy we synthesized and evaluated a series of 50 AQ and novel quinazolinone (QZN) analogues and measured the impact on AQ biosynthesis, virulence gene expression and biofilm development. The simple exchange of two isosteres (OH for NH2) switches a QZN agonist to an antagonist with a concomitant impact on the induction of bacterial virulence factor production. We also determined the complex crystal structure of a QZN antagonist bound to PqsR revealing a similar orientation in the ligand binding pocket to the native agonist NHQ. This structure represents the first description of an LTTR-antagonist complex. Overall these studies present novel insights into LTTR ligand binding and ligand-based drug design and provide a chemical scaffold for further anti-P. aeruginosa virulence drug development by targeting the AQ receptor PqsR

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Role of the subesophageal zone in sensorimotor control of orientation in Drosophila larva

Chemotaxis is a powerful paradigm to investigate how nervous systems represent and integrate changes in sensory signals to direct navigational decisions. In the Drosophila melanogaster larva, chemotaxis mainly consists of an alternation of distinct behavioral modes: runs and directed turns. During locomotion, turns are triggered by the integration of temporal changes in the intensity of the stimulus. Upon completion of a turning maneuver, the direction of motion is typically realigned toward the odor gradient. While the anatomy of the peripheral olfactory circuits and the locomotor system of the larva are reasonably well documented, the neural circuits connecting the sensory neurons to the motor neurons remain unknown. We combined a loss-of-function behavioral screen with optogenetics-based clonal gain-of-function manipulations to identify neurons that are necessary and sufficient for the initiation of reorientation maneuvers in odor gradients. Our results indicate that a small subset of neurons residing in the subesophageal zone controls the rate of transition from runs to turns-a premotor function compatible with previous observations made in other invertebrates. After having shown that this function pertains to the processing of inputs from different sensory modalities (olfaction, vision, thermosensation), we conclude that the subesophageal zone operates as a general premotor center that regulates the selection of different behavioral programs based on the integration of sensory stimuli. The present analysis paves the way for a systematic investigation of the neural computations underlying action selection in a miniature brain amenable to genetic manipulations.M.L. acknowledges funding from the Spanish Ministry of Science and Innovation (MICINN, BFU2008-00362, BFU2009-07757-E/BMC, and BFU2011-26208), the EMBL/CRG Systems Biology Program, and a Marie Curie Reintegration Grant (PIRG02-GA-2007-224791). J.W.T. was supported by the Howard Hughes Medical Institute. I.T. acknowledges funding from the Marie Curie FP7 Programme through FLiACT (ITN

Restarting and timing of oral anticoagulation after traumatic intracranial hemorrhage: a review and summary of ongoing and planned prospective randomized clinical trials.

Anticoagulant-associated traumatic intracranial hemorrhage (tICrH) is a devastating injury with high morbidity and mortality. For survivors, treating clinicians face the dilemma of restarting oral anticoagulation with scarce evidence to guide them. Thromboembolic risk is high from the bleeding event, patients' high baseline risks, that is, the pre-existing indication for anticoagulation, and the risk of immobility after the bleeding episode. This must be balanced with potentially devastating hematoma expansion or new hemorrhagic lesions. Retrospective evidence and expert opinion support restarting oral anticoagulants in most patients with tICrH, but timing is uncertain. Researchers have failed to make clear distinctions between tICrH and spontaneous intracranial hemorrhage (sICrH), which have differing natural histories. While both appear to benefit from restarting, sICrH has a higher rebleeding risk and similar or lower thrombotic risk. Clinical equipoise on restarting is also divergent. In sICrH, equipoise is centered on whether to restart. In tICrH, it is centered on when. Several prospective randomized clinical trials are ongoing or about to start to examine the risk-benefit of restarting. Most of them are restricted to patients with sICrH, with antiplatelet control groups. Most are also restricted to direct oral anticoagulants (DOACs), as they are associated with a lower overall risk of ICrH. There is some overlap with tICrH via subdural hematoma, and one trial is specific to restart timing with DOACs in only traumatic cases. This is a narrative review of the current evidence for restarting anticoagulation and restart timing after tICrH along with a summary of the ongoing and planned clinical trials

Activation and inhibition of PqsR in <i>P. aeruginosa</i> by the 2-alkyl-4(1<i>H</i>)-quinolones.

<p>*EC₅₀ determined in a <i>P. aeruginosa</i> Δ<i>pqsA</i> CTX::<i>pqsA'-lux</i> strain; **EC₅₀ determined in a <i>P. aeruginosa</i> Δ<i>pqsAH</i> CTX::<i>pqsA'</i>-lux strain; – no activity.</p

Response of PqsR^CBD ligand binding site mutants to AQs.

<p>(<b>A</b>) PqsR-6His is functional in <i>P. aeruginosa</i>. The <i>pqsR</i> gene with or without a 6His tag on pME6032 was introduced into a <i>P. aeruginosa pqsR</i> deletion mutant (Δ<i>pqsR</i>) containing a chromosomal miniCTX::<i>pqsA'-lux</i> reporter gene fusion. Relative PqsR activity was determined as a % of the maximum bioluminescence produced by the miniCTX::<i>pqsA'-lux</i> reporter fusion in a wild type <i>P. aeruginosa</i> PAO1 background carrying the empty pME6032 vector; (<b>B</b>) Light output from the <i>P. aeruginosa</i> Δ<i>pqsR</i> miniCTX::<i>pqsA'-lux</i> strain transformed with one of each of the 13 site-specific mutations introduced into the PqsR^CBD ligand-binding pocket. Bioluminescence is presented as % of <i>pqsA</i> promoter activity with respect to PAO1 Δ<i>pqsR</i> miniCTX::<i>pqsA'-lux</i> expressing the PqsR-6His protein (WT); (<b>C</b>) Western blot analysis confirming expression of each of the PqsR-6His mutant proteins; (<b>D</b>) Light output from the <i>P. aeruginosa</i> Δ<i>pqsA</i> Δ<i>pqsH</i> Δ<i>pqsR</i> miniCTX::<i>pqsA'-lux</i> strain transformed with either the gene coding for PqsR-6His or one of the 13 site-specific mutants and supplemented with either HHQ or PQS (40 µM). Bioluminescence is presented as % of <i>pqsA</i> promoter activity with respect to the <i>P. aeruginosa</i> Δ<i>pqsA</i> Δ<i>pqsH</i> Δ<i>pqsR</i> miniCTX::<i>pqsA'-lux</i> strain expressing PqsR-6His.</p