Lesions in a songbird vocal circuit increase variability in song syntax.

Complex skills like speech and dance are composed of ordered sequences of simpler elements, but the neuronal basis for the syntactic ordering of actions is poorly understood. Birdsong is a learned vocal behavior composed of syntactically ordered syllables, controlled in part by the songbird premotor nucleus HVC (proper name). Here, we test whether one of HVC's recurrent inputs, mMAN (medial magnocellular nucleus of the anterior nidopallium), contributes to sequencing in adult male Bengalese finches (Lonchura striata domestica). Bengalese finch song includes several patterns: (1) chunks, comprising stereotyped syllable sequences; (2) branch points, where a given syllable can be followed probabilistically by multiple syllables; and (3) repeat phrases, where individual syllables are repeated variable numbers of times. We found that following bilateral lesions of mMAN, acoustic structure of syllables remained largely intact, but sequencing became more variable, as evidenced by 'breaks' in previously stereotyped chunks, increased uncertainty at branch points, and increased variability in repeat numbers. Our results show that mMAN contributes to the variable sequencing of vocal elements in Bengalese finch song and demonstrate the influence of recurrent projections to HVC. Furthermore, they highlight the utility of species with complex syntax in investigating neuronal control of ordered sequences.

Dynamic top-down biasing implements rapid adaptive changes to individual movements.

Complex behaviors depend on the coordinated activity of neural ensembles in interconnected brain areas. The behavioral function of such coordination, often measured as co-fluctuations in neural activity across areas, is poorly understood. One hypothesis is that rapidly varying co-fluctuations may be a signature of moment-by-moment task-relevant influences of one area on another. We tested this possibility for error-corrective adaptation of birdsong, a form of motor learning which has been hypothesized to depend on the top-down influence of a higher-order area, LMAN (lateral magnocellular nucleus of the anterior nidopallium), in shaping moment-by-moment output from a primary motor area, RA (robust nucleus of the arcopallium). In paired recordings of LMAN and RA in singing birds, we discovered a neural signature of a top-down influence of LMAN on RA, quantified as an LMAN-leading co-fluctuation in activity between these areas. During learning, this co-fluctuation strengthened in a premotor temporal window linked to the specific movement, sequential context, and acoustic modification associated with learning. Moreover, transient perturbation of LMAN activity specifically within this premotor window caused rapid occlusion of pitch modifications, consistent with LMAN conveying a temporally localized motor-biasing signal. Combined, our results reveal a dynamic top-down influence of LMAN on RA that varies on the rapid timescale of individual movements and is flexibly linked to contexts associated with learning. This finding indicates that inter-area co-fluctuations can be a signature of dynamic top-down influences that support complex behavior and its adaptation.

Thiol and H2S-Mediated NO Generation from Nitrate at Copper(II).

Reduction of nitrate is an essential, yet challenging chemical task required to manage this relatively inert oxoanion in the environment and biology. We show that thiols, ubiquitous reductants in biology, convert nitrate to nitric oxide at a Cu(II) center under mild conditions. The ß-diketiminato complex [Cl2NNF6]Cu(κ2-O2NO) engages in O-atom transfer with various thiols (RSH) to form the corresponding copper(II) nitrite [CuII](κ2-O2N) and sulfenic acid (RSOH). The copper(II) nitrite further reacts with RSH to give S-nitrosothiols RSNO and [CuII]2(µ-OH)2 en route to NO formation via [CuII]-SR intermediates. The gasotransmitter H2S also reduces nitrate at copper(II) to generate NO, providing a lens into NO3-/H2S crosstalk. The interaction of thiols with nitrate at copper(II) releases a cascade of N- and S-based signaling molecules in biology.

Electrocatalytic Ammonia Oxidation by a Low-Coordinate Copper Complex.

Molecular catalysts for ammonia oxidation to dinitrogen represent enabling components to utilize ammonia as a fuel and/or source of hydrogen. Ammonia oxidation requires not only the breaking of multiple strong N-H bonds but also controlled N-N bond formation. We report a novel ß-diketiminato copper complex [iPr2NNF6]CuI-NH3 ([CuI]-NH3 (2)) as a robust electrocatalyst for NH3 oxidation in acetonitrile under homogeneous conditions. Complex 2 operates at a moderate overpotential (η = 700 mV) with a TOFmax = 940 h-1 as determined from CV data in 1.3 M NH3-MeCN solvent. Prolonged (>5 h) controlled potential electrolysis (CPE) reveals the stability and robustness of the catalyst under electrocatalytic conditions. Detailed mechanistic investigations indicate that electrochemical oxidation of [CuI]-NH3 forms {[CuII]-NH3}+ (4), which undergoes deprotonation by excess NH3 to form reactive copper(II)-amide ([CuII]-NH2, 6) unstable toward N-N bond formation to give the dinuclear hydrazine complex [CuI]2(µ-N2H4). Electrochemical studies reveal that the diammine complex [CuI](NH3)2 (7) forms at high ammonia concentration as part of the {[CuII](NH3)2}+/[CuI](NH3)2 redox couple that is electrocatalytically inactive. DFT analysis reveals a much higher thermodynamic barrier for deprotonation of the four-coordinate {[CuII](NH3)2}+ (8) by NH3 to give the copper(II) amide [CuII](NH2)(NH3) (9) (ΔG = 31.7 kcal/mol) as compared to deprotonation of the three-coordinate {[CuII]-NH3}+ by NH3 to provide the reactive three-coordinate parent amide [CuII]-NH2 (ΔG = 18.1 kcal/mol) susceptible to N-N coupling to form [CuI]2(µ-N2H4) (ΔG = -11.8 kcal/mol).

Lewis acid-assisted reduction of nitrite to nitric and nitrous oxides via the elusive nitrite radical dianion.

Reduction of nitrite anions (NO2-) to nitric oxide (NO), nitrous oxide (N2O) and ultimately dinitrogen (N2) takes place in a variety of environments, including in the soil as part of the biogeochemical nitrogen cycle and in acidified nuclear waste. Nitrite reduction typically takes place within the coordination sphere of a redox-active transition metal. Here we show that Lewis acid coordination can substantially modify the reduction potential of this polyoxoanion to allow for its reduction under non-aqueous conditions (-0.74 V versus NHE). Detailed characterization confirms the formation of the borane-capped radical nitrite dianion (NO22-), which features a N(II) oxidation state. Protonation of the nitrite dianion results in the facile loss of nitric oxide (NO), whereas its reaction with NO results in disproportionation to nitrous oxide (N2O) and nitrite (NO2-). This system connects three redox levels in the global nitrogen cycle and provides fundamental insights into the conversion of NO2- to NO.

NO Coupling at Copper to cis-Hyponitrite: N2O Formation via Protonation and H-Atom Transfer.

Copper nitrite reductases (CuNIRs) convert NO2- to NO as well as NO to N2O under high NO flux at a mononuclear type 2 Cu center. While model complexes illustrate N-N coupling from NO that results in symmetric trans-hyponitrite [CuII]-ONNO-[CuII] complexes, we report NO assembly at a single Cu site in the presence of an external reductant Cp*2M (M = Co, Fe) to give the first copper cis-hyponitrites [Cp*2M]{[CuII](κ2-O2N2)[CuI]}. Importantly, the κ1-N-bound [CuI] fragment may be easily removed by the addition of mild Lewis bases such as CNAr or pyridine to form the spectroscopically similar anion {[CuII](κ2-O2N2)}-. The addition of electrophiles such as H+ to these anionic copper(II) cis-hyponitrites leads to N2O generation with the formation of the dicopper(II)-bis-µ-hydroxide [CuII]2(µ-OH)2. One-electron oxidation of the {[CuII](κ2-O2N2)}- core turns on H-atom transfer reactivity, enabling the oxidation of 9,10-dihydroanthracene to anthracene with concomitant formation of N2O and [CuII]2(µ-OH)2. These studies illustrate both the reductive coupling of NO at a single copper center and a way to harness the strong oxidizing power of nitric oxide via the neutral cis-hyponitrite [Cu](κ2-O2N2).

Prevention of Periprosthetic Joint Infection in Total Hip and Knee Replacement: One European Consensus.

Periprosthetic joint infection (PJI) is a devastating complication in total hip and knee replacement. Its prevention is key to decrease the incidence and avoid some consequences that seriously impact patients and health systems. In view of the variety of recommendations and guidelines, we decided to conduct an expert, peer-reviewed European consensus analysis about the pre-, intra-, and postoperative prevention of PJI. A multinational group of practicing orthopedic experts developed a series of 47 consensus statements in 6 main groups of intervention, and a 2-stage Delphi approach was launched with a threshold for agreement at 75% and for very high agreement at more than 90%. A total of 306 orthopedic surgeon responses were gathered from 9 countries. Consensus was reached for 42/47 statements, 31/47 of which achieved a very high consensus. Many preoperative actions gathered strong consensus, although areas like the use of alcoholic chlorhexidine or the timing of hair removal did not attain strong consensus, despite available evidence. Intra- and postoperative actions showed more variability regarding incise drapes, skin suturing techniques, and wound follow-up. This study confirms an important consensus among orthopedic surgeons across Europe in many areas well known to contribute to the prevention of PJI; however, there are still grounds for improvement.

Uncovering Redox Non-innocent Hydrogen-Bonding in Cu(I)-Diazene Complexes.

The life-sustaining reduction of N2 to NH3 is thermoneutral yet kinetically challenged by high-energy intermediates such as N2H2. Exploring intramolecular H-bonding as a potential strategy to stabilize diazene intermediates, we employ a series of [xHetTpCu]2(µ-N2H2) complexes that exhibit H-bonding between pendant aromatic N-heterocycles (xHet) such as pyridine and a bridging trans-N2H2 ligand at copper(I) centers. X-ray crystallography and IR spectroscopy clearly reveal H-bonding in [pyMeTpCu]2(µ-N2H2) while low-temperature 1H NMR studies coupled with DFT analysis reveals a dynamic equilibrium between two closely related, symmetric H-bonded structural motifs. Importantly, the xHet pendant negligibly influences the electronic structure of xHetTpCuI centers in xHetTpCu(CNAr2,6-Me2) complexes that lack H-bonding as judged by nearly indistinguishable ν(CN) frequencies (2113-2117 cm-1). Nonetheless, H-bonding in the corresponding [xHetTpCu]2(µ-N2H2) complexes results in marked changes in ν(NN) (1398-1419 cm-1) revealed through resonance Raman studies. Due to the closely matched N-H BDEs of N2H2 and the pyH0 cation radical, the aromatic N-heterocyclic pendants may encourage partial H-atom transfer (HAT) from N2H2 to xHet through redox-non-innocent H-bonding in [xHetTpCu]2(µ-N2H2). DFT studies reveal modest thermodynamic barriers for concerted transfer of both H-atoms of coordinated N2H2 to the xHet pendants to generate tautomeric [xHetHTpCu]2(µ-N2) complexes, identifying metal-assisted concerted dual HAT as a thermodynamically favorable pathway for N2/N2H2 interconversion.

Thionitrite and Perthionitrite in NO Signaling at Zinc.

NO and H2 S serve as signaling molecules in biology with intertwined reactivity. HSNO and HSSNO with their conjugate bases - SNO and - SSNO form in the reaction of H2 S with NO as well as S-nitrosothiols (RSNO) and nitrite (NO2- ) that serve as NO reservoirs. While HSNO and HSSNO are elusive, their conjugate bases form isolable zinc complexes Ph,Me TpZn(SNO) and Ph,Me TpZn(SSNO) supported by tris(pyrazolyl)borate ligands. Reaction of Na(15-C-5)SSNO with Ph,Me TpZn(ClO4 ) provides Ph,Me TpZn(SSNO) that undergoes S-atom removal by PEt3 to give Ph,Me TpZn(SNO) and S=PEt3 . Unexpectedly stable at room temperature, these Zn-SNO and Zn-SSNO complexes release NO upon heating. Ph,Me TpZn(SNO) and Ph,Me TpZn(SSNO) quickly react with acidic thiols such as C6 F5 SH to form N2 O and NO, respectively. Increasing the thiol basicity in p-substituted aromatic thiols 4-X ArSH in the reaction with Ph,Me TpZn(SNO) turns on competing S-nitrosation to form Ph,Me TpZn-SH and RSNO, the latter a known precursor for NO.

Mechanism of O-Atom Transfer from Nitrite: Nitric Oxide Release at Copper(II).

Nitric oxide (NO) is a key signaling molecule in health and disease. While nitrite acts as a reservoir of NO activity, mechanisms for NO release require further understanding. A series of electronically varied ß-diketiminatocopper(II) nitrite complexes [CuII](κ2-O2N) react with a range of electronically tuned triarylphosphines PArZ3 that release NO with the formation of O═PArZ3. Second-order rate constants are largest for electron-poor copper(II) nitrite and electron-rich phosphine pairs. Computational analysis reveals a transition-state structure energetically matched with experimentally determined activation barriers. The production of NO follows a pathway that involves nitrite isomerization at CuII from κ2-O2N to κ1-NO2 followed by O-atom transfer (OAT) to form O═PArZ3 and [CuI]-NO that releases NO upon PArZ3 binding at CuI to form [CuI]-PArZ3. These findings illustrate important mechanistic considerations involved in NO formation from nitrite via OAT.

Copper(ii) ketimides in sp3 C-H amination.

Commercially available benzophenone imine (HN[double bond, length as m-dash]CPh2) reacts with ß-diketiminato copper(ii) tert-butoxide complexes [CuII]-O t Bu to form isolable copper(ii) ketimides [CuII]-N[double bond, length as m-dash]CPh2. Structural characterization of the three coordinate copper(ii) ketimide [Me3NN]Cu-N[double bond, length as m-dash]CPh2 reveals a short Cu-Nketimide distance (1.700(2) Å) with a nearly linear Cu-N-C linkage (178.9(2)°). Copper(ii) ketimides [CuII]-N[double bond, length as m-dash]CPh2 readily capture alkyl radicals R˙ (PhCH(˙)Me and Cy˙) to form the corresponding R-N[double bond, length as m-dash]CPh2 products in a process that competes with N-N coupling of copper(ii) ketimides [CuII]-N[double bond, length as m-dash]CPh2 to form the azine Ph2C[double bond, length as m-dash]N-N[double bond, length as m-dash]CPh2. Copper(ii) ketimides [CuII]-N[double bond, length as m-dash]CAr2 serve as intermediates in catalytic sp3 C-H amination of substrates R-H with ketimines HN[double bond, length as m-dash]CAr2 and t BuOO t Bu as oxidant to form N-alkyl ketimines R-N[double bond, length as m-dash]CAr2. This protocol enables the use of unactivated sp3 C-H bonds to give R-N[double bond, length as m-dash]CAr2 products easily converted to primary amines R-NH2 via simple acidic deprotection.

Quantification of Ni-N-O Bond Angles and NO Activation by X-ray Emission Spectroscopy.

A series of ß-diketiminate Ni-NO complexes with a range of NO binding modes and oxidation states were studied by X-ray emission spectroscopy (XES). The results demonstrate that XES can directly probe and distinguish end-on vs side-on NO coordination modes as well as one-electron NO reduction. Density functional theory (DFT) calculations show that the transition from the NO 2s2s σ* orbital has higher intensity for end-on NO coordination than for side-on NO coordination, whereas the 2s2s σ orbital has lower intensity. XES calculations in which the Ni-N-O bond angle was fixed over the range from 80° to 176° suggest that differences in NO coordination angles of ∼10° could be experimentally distinguished. Calculations of Cu nitrite reductase (NiR) demonstrate the utility of XES for characterizing NO intermediates in metalloenzymes. This work shows the capability of XES to distinguish NO coordination modes and oxidation states at Ni and highlights applications in quantifying small molecule activation in enzymes.

Three-Coordinate Copper(II) Alkynyl Complex in C-C Bond Formation: The Sesquicentennial of the Glaser Coupling.

Copper(II) alkynyl species are proposed as key intermediates in numerous Cu-catalyzed C-C coupling reactions. Supported by a ß-diketiminate ligand, the three-coordinate copper(II) alkynyl [CuII]-C≡CAr (Ar = 2,6-Cl2C6H3) forms upon reaction of the alkyne H-C≡CAr with the copper(II) tert-butoxide complex [CuII]-OtBu. In solution, this [CuII]-C≡CAr species cleanly transforms to the Glaser coupling product ArC≡C-C≡CAr and [CuI](solvent). Addition of nucleophiles R'C≡C-Li (R' = aryl, silyl) and Ph-Li to [CuII]-C≡CAr affords the corresponding Csp-Csp and Csp-Csp2 coupled products RC≡C-C≡CAr and Ph-C≡CAr with concomitant generation of [CuI](solvent) and {[CuI]-C≡CAr}-, respectively. Supported by density functional theory (DFT) calculations, redox disproportionation forms [CuIII](C≡CAr)(R) species that reductively eliminate R-C≡CAr products. [CuII]-C≡CAr also captures the trityl radical Ph3C· to give Ph3C-C≡CAr. Radical capture represents the key Csp-Csp3 bond-forming step in the copper-catalyzed C-H functionalization of benzylic substrates R-H with alkynes H-C≡CR' (R' = (hetero)aryl, silyl) that provide Csp-Csp3 coupled products R-C≡CR via radical relay with tBuOOtBu as oxidant.

Lewis Acid Coordination Redirects S-Nitrosothiol Signaling Output.

S-Nitrosothiols (RSNOs) serve as air-stable reservoirs for nitric oxide in biology. While copper enzymes promote NO release from RSNOs by serving as Lewis acids for intramolecular electron-transfer, redox-innocent Lewis acids separate these two functions to reveal the effect of coordination on structure and reactivity. The synthetic Lewis acid B(C6 F5 )3 coordinates to the RSNO oxygen atom, leading to profound changes in the RSNO electronic structure and reactivity. Although RSNOs possess relatively negative reduction potentials, B(C6 F5 )3 coordination increases their reduction potential by over 1 V into the physiologically accessible +0.1 V vs. NHE. Outer-sphere chemical reduction gives the Lewis acid stabilized hyponitrite dianion trans-[LA-O-N=N-O-LA]2- [LA=B(C6 F5 )3 ], which releases N2 O upon acidification. Mechanistic and computational studies support initial reduction to the [RSNO-B(C6 F5 )3 ] radical anion, which is susceptible to N-N coupling prior to loss of RSSR.

Tris(pyrazolyl)borate Copper Hydroxide Complexes Featuring Tunable Intramolecular H-Bonding.

A modular synthesis provides access to a series of new tris(pyrazolyl)borate ligands XpyMeTpK that possess a single functionalized pendant pyridyl (py) or pyrimidyl (pyd) arm designed to engage in tunable intramolecular H-bonding to metal-bound functionalities. To illustrate such H-bonding interactions, a series of [XpyMeTpCu]2(µ-OH)2 (6a-6e) complexes were synthesized from the corresponding XpyMeTpCu-OAc (5a-5e) complexes. Single crystal X-ray structures of three new dinuclear [XpyMeTpCu]2(µ-OH)2 complexes reveal H-bonding between the pendant heterocycle and bridging hydroxide ligands while the donor arm engages the copper center in an unusual monomeric DMAPMeTpCu-OH complex. Vibrational studies (IR) of each bridging hydroxide complex reveal reduced νOH frequencies that tracks with the H-bond accepting ability of the pendant arm. Reversible protonation studies that interconvert [XpyMeTpCu]2(µ-OH)2 and [XpyMeTpCu(OH2)]OTf species indicate that the acidity of the corresponding aquo ligand decreases with increasing H-bond accepting ability of the pendant arm.

Discrete Evaluative and Premotor Circuits Enable Vocal Learning in Songbirds.

Virtuosic motor performance requires the ability to evaluate and modify individual gestures within a complex motor sequence. Where and how the evaluative and premotor circuits operate within the brain to enable such temporally precise learning is poorly understood. Songbirds can learn to modify individual syllables within their complex vocal sequences, providing a system for elucidating the underlying evaluative and premotor circuits. We combined behavioral and optogenetic methods to identify 2 afferents to the ventral tegmental area (VTA) that serve evaluative roles in syllable-specific learning and to establish that downstream cortico-basal ganglia circuits serve a learning role that is only premotor. Furthermore, song performance-contingent optogenetic stimulation of either VTA afferent was sufficient to drive syllable-specific learning, and these learning effects were of opposite valence. Finally, functional, anatomical, and molecular studies support the idea that these evaluative afferents bidirectionally modulate VTA dopamine neurons to enable temporally precise vocal learning.

Regulation of subcellular dendritic synapse specificity by axon guidance cues.

Neural circuit assembly occurs with subcellular precision, yet the mechanisms underlying this precision remain largely unknown. Subcellular synaptic specificity could be achieved by molecularly distinct subcellular domains that locally regulate synapse formation, or by axon guidance cues restricting access to one of several acceptable targets. We address these models using two Drosophila neurons: the dbd sensory neuron and the A08a interneuron. In wild-type larvae, dbd synapses with the A08a medial dendrite but not the A08a lateral dendrite. dbd-specific overexpression of the guidance receptors Unc-5 or Robo-2 results in lateralization of the dbd axon, which forms anatomical and functional monosynaptic connections with the A08a lateral dendrite. We conclude that axon guidance cues, not molecularly distinct dendritic arbors, are a major determinant of dbd-A08a subcellular synapse specificity.

Establishing Expert, Multi-Disciplinary, Peer-Reviewed Consensus to Lead a Paradigm Shift in Optimal Blood Glucose Management.

INTRODUCTION: The National Health Service (NHS) in the UK appears unclear on how blood glucose monitoring (BGM) should be used to support diabetes patient care and empowerment, and local interpretation of NICE guidance on the availability of devices varies widely. An expert group of clinicians and commissioners considered BGM in terms of access, guidance, resources, data integration, patient education, and patient choice. METHODS: The group generated a series of questions on BGM into a 38-statement questionnaire using Delphi methodology. This was circulated to clinicians involved in diabetes management across the UK, receiving 222 responses. RESULTS: From the questionnaire, 35 of the 38 statement responses showed > 66% consensus, with 26 of these achieving > 90% agreement. CONCLUSION: The expert group reviewed the responses and made recommendations based on the clear professional consensus demonstrated. These included the need to use new technology and data integration and that wider factors, including patient choice rather than cost alone, should inform formulary inclusion of BGM equipment. FUNDING: LifeScan U.K. Ltd.

Celestial navigation in Drosophila.

Many casual observers typecast Drosophila melanogaster as a stationary pest that lurks around fruit and wine. However, the omnipresent fruit fly, which thrives even in desert habitats, likely established and maintained its cosmopolitan status via migration over large spatial scales. To perform long-distance dispersal, flies must actively maintain a straight compass heading through the use of external orientation cues, such as those derived from the sky. In this Review, we address how D. melanogaster accomplishes long-distance navigation using celestial cues. We focus on behavioral and physiological studies indicating that fruit flies can navigate both to a pattern of linearly polarized light and to the position of the sun - the same cues utilized by more heralded insect navigators such as monarch butterflies and desert ants. In both cases, fruit flies perform menotaxis, selecting seemingly arbitrary headings that they then maintain over time. We discuss how the fly's nervous system detects and processes this sensory information to direct the steering maneuvers that underlie navigation. In particular, we highlight recent findings that compass neurons in the central complex, a set of midline neuropils, are essential for navigation. Taken together, these results suggest that fruit flies share an ancient, latent capacity for celestial navigation with other insects. Furthermore, they illustrate the potential of D. melanogaster to help us to elucidate both the cellular basis of navigation and mechanisms of directed dispersal on a landscape scale.