The cellular representation of temperature across the somatosensory thalamus.

Although distinct thalamic nuclei encode sensory information for almost all sensory modalities, the existence of a thalamic representation of temperature with a role in thermal perception remains unclear. To address this, we performed high-density electrophysiological recordings across the entire forelimb somatosensory thalamus in awake mice, and identified an anterior and a posterior representation of temperature that spans three thalamic nuclei. We found that these parallel representations show fundamental differences in the cellular encoding of temperature which reflects their cortical output targets. While the anterior representation encodes cool only and the posterior both cool and warm; in both representations cool was more densely represented and showed shorter latency, more transient responses as compared to warm. Moreover, thalamic inactivation showed a major role in thermal perception. Our comprehensive dataset identifies the thalamus as a key structure in thermal processing and highlights a novel posterior pathway in the thalamic representation of warm and cool.

Movement is governed by rotational neural dynamics in spinal motor networks.

Although the generation of movements is a fundamental function of the nervous system, the underlying neural principles remain unclear. As flexor and extensor muscle activities alternate during rhythmic movements such as walking, it is often assumed that the responsible neural circuitry is similarly exhibiting alternating activity1. Here we present ensemble recordings of neurons in the lumbar spinal cord that indicate that, rather than alternating, the population is performing a low-dimensional 'rotation' in neural space, in which the neural activity is cycling through all phases continuously during the rhythmic behaviour. The radius of rotation correlates with the intended muscle force, and a perturbation of the low-dimensional trajectory can modify the motor behaviour. As existing models of spinal motor control do not offer an adequate explanation of rotation1,2, we propose a theory of neural generation of movements from which this and other unresolved issues, such as speed regulation, force control and multifunctionalism, are readily explained.

Structure and Dynamics of Cinnamycin-Lipid Complexes: Mechanisms of Selectivity for Phosphatidylethanolamine Lipids.

Cinnamycin is a lantibiotic peptide, which selectively binds to and permeabilizes membranes containing phosphatidylethanolamine (PE) lipids. As PE is a major component of many bacterial cell membranes, cinnamycin has considerable potential for destroying these. In this study, molecular dynamics simulations are used to elucidate the structure of a lipid-cinnamycin complex and the origin of selective lipid binding. The simulations reveal that cinnamycin selectively binds to PE by forming an extensive hydrogen-bonding network involving all three hydrogen atoms of the primary ammonium group of the PE head group. The substitution of a single hydrogen atom with a methyl group on the ammonium nitrogen destabilizes this hydrogen-bonding network. In addition to binding the primary ammonium group, cinnamycin also interacts with the phosphate group of the lipid through a previously uncharacterized phosphate-binding site formed by the backbone Phe10-Abu11-Phe12-Val13 moieties (Abu = 1-α-aminobutyric acid). In addition, hydroxylation of Asp15 at Cß plays a role in selective binding of PE due to its tight interaction with the charged amine of the lipid head group. The simulations reveal that the position and orientation of the peptide in the membrane depend on the type of lipid to which it binds, suggesting a reason for why cinnamycin selectively permeabilizes PE-containing membranes.

Decoupling of timescales reveals sparse convergent CPG network in the adult spinal cord.

During the generation of rhythmic movements, most spinal neurons receive an oscillatory synaptic drive. The neuronal architecture underlying this drive is unknown, and the corresponding network size and sparseness have not yet been addressed. If the input originates from a small central pattern generator (CPG) with dense divergent connectivity, it will induce correlated input to all receiving neurons, while sparse convergent wiring will induce a weak correlation, if any. Here, we use pairwise recordings of spinal neurons to measure synaptic correlations and thus infer the wiring architecture qualitatively. A strong correlation on a slow timescale implies functional relatedness and a common source, which will also cause correlation on fast timescale due to shared synaptic connections. However, we consistently find marginal coupling between slow and fast correlations regardless of neuronal identity. This suggests either sparse convergent connectivity or a CPG network with recurrent inhibition that actively decorrelates common input.

Molecular Modeling Investigation of the Interaction between Humicola insolens Cutinase and SDS Surfactant Suggests a Mechanism for Enzyme Inactivation.

One of the largest commercial applications of enzymes and surfactants is as main components in modern detergents. The high concentration of surfactant compounds usually present in detergents can, however, negatively affect the enzymatic activity. To remedy this drawback, it is of great importance to characterize the interaction between the enzyme and the surfactant molecules at an atomistic resolution. The protein enzyme cutinase from the thermophilic and saprophytic fungus called Humicola insolens (HiC) is a promising candidate for use in detergents thanks to its hydrolase activity targeting mostly biopolyesters (e.g., cutin). HiC is, however, inhibited by low concentrations of sodium dodecyl sulfate (SDS), an ubiquitous surfactant. In this work, we investigate the interaction between HiC and SDS using molecular dynamics simulations. Simulations of HiC dissolved in different aqueous concentrations of SDS show the interaction between HiC and SDS monomers, as well as the formation and dynamics of SDS micelles on the surface of the enzyme. These results suggest a mechanism of cutinase inhibition by SDS, which involves the nucleation of aggregates of SDS molecules on hydrophobic patches on the cutinase surface. Notably, a primary binding site for monomeric SDS is identified near the active site of HiC constituting a possible nucleation point for micelles and leading to the blockage of the entrance to the enzymatic site. Detailed analysis of the simulations allow us to suggest a set of residues from the SDS binding site on HiC to probe as engineered mutations aimed at reducing SDS binding to HiC, thereby decreasing SDS inhibition of HiC.

Small-Molecule Positive Allosteric Modulators of the ß2-Adrenoceptor Isolated from DNA-Encoded Libraries.

Conventional drug discovery efforts at the ß2-adrenoceptor (ß2AR) have led to the development of ligands that bind almost exclusively to the receptor's hormone-binding orthosteric site. However, targeting the largely unexplored and evolutionarily unique allosteric sites has potential for developing more specific drugs with fewer side effects than orthosteric ligands. Using our recently developed approach for screening G protein-coupled receptors (GPCRs) with DNA-encoded small-molecule libraries, we have discovered and characterized the first ß2AR small-molecule positive allosteric modulators (PAMs)-compound (Cmpd)-6 [(R)-N-(4-amino-1-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-(N-isopropyl-N-methylsulfamoyl)-2-((4-methoxyphenyl)thio)benzamide] and its analogs. We used purified human ß2ARs, occupied by a high-affinity agonist, for the affinity-based screening of over 500 million distinct library compounds, which yielded Cmpd-6. It exhibits a low micro-molar affinity for the agonist-occupied ß2AR and displays positive cooperativity with orthosteric agonists, thereby enhancing their binding to the receptor and ability to stabilize its active state. Cmpd-6 is cooperative with G protein and ß-arrestin1 (a.k.a. arrestin2) to stabilize high-affinity, agonist-bound active states of the ß2AR and potentiates downstream cAMP production and receptor recruitment of ß-arrestin2 (a.k.a. arrestin3). Cmpd-6 is specific for the ß2AR compared with the closely related ß1AR. Structure-activity studies of select Cmpd-6 analogs defined the chemical groups that are critical for its biologic activity. We thus introduce the first small-molecule PAMs for the ß2AR, which may serve as a lead molecule for the development of novel therapeutics. The approach described in this work establishes a broadly applicable proof-of-concept strategy for affinity-based discovery of small-molecule allosteric compounds targeting unique conformational states of GPCRs.

How a short pore forming peptide spans the lipid membrane.

Many antimicrobial peptides function by forming pores in the plasma membrane of the target cells. Intriguingly, some of these peptides are very short, and thus, it is not known how they can span the membrane, or whether other mechanisms of cell disruption are dominant. Here, the conformation and orientation of the 14-residue peptaibol SPF-5506-A4 (SPF) are investigated in lipid environments by atomistic and coarse grained molecular dynamics (MD) simulations, circular dichroism, and nuclear magnetic resonance (NMR) experiments. The MD simulations show that SPF is inserted spontaneously in a transmembrane orientation in both 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers resulting in thinning of the bilayers near the peptides, which drives the peptide aggregation. Furthermore, the backbone conformation of the peptide in the bilayer bound state is different from that of the NMR model solved in small bicelles. These results demonstrate that mutual adaption between the peptides and the membrane is likely to be important for pore formation.

Bicelles and Other Membrane Mimics: Comparison of Structure, Properties, and Dynamics from MD Simulations.

The increased interest in studying membrane proteins has led to the development of new membrane mimics such as bicelles and nanodiscs. However, only limited knowledge is available of how these membrane mimics are affected by embedded proteins and how well they mimic a lipid bilayer. Herein, we present molecular dynamics simulations to elucidate structural and dynamic properties of small bicelles and compare them to a large alignable bicelle, a small nanodisc, and a lipid bilayer. Properties such as lipid packing and properties related to embedding both an α-helical peptide and a transmembrane protein are investigated. The small bicelles are found to be very dynamic and mainly assume a prolate shape substantiating that small bicelles cannot be regarded as well-defined disclike structures. However, addition of a peptide results in an increased tendency to form disc-shaped bicelles. The small bicelles and the nanodiscs show increased peptide solvation and difference in peptide orientation compared to embedding in a bilayer. The large bicelle imitated a bilayer well with respect to both curvature and peptide solvation, although peripheral binding of short tailed lipids to the embedded proteins is observed, which could hinder ligand binding or multimer formation.

Divisive gain modulation of motoneurons by inhibition optimizes muscular control.

When using muscles, the precision with which force is delivered is as important as the delivery of force itself. Force is regulated by both the number of recruited motoneurons and their spike frequency. While it is known that the recruitment is ordered to reduce variability in force, it remains unclear whether the motoneuron gain, i.e., the slope of the transformation between synaptic input and spiking output, is also modulated to reduce variability in force. To address this issue, we use turtle hindlimb scratching as a model for fine motor control, since this behavior involves precise limb movement to rub the location of somatic nuisance touch. We recorded intracellularly from motoneurons in a reduced preparation where the limbs were removed to increase mechanical stability and the motor nerve activity served as a surrogate for muscle force. We found that not only is the gain of motoneurons regulated on a subsecond timescale, it is also adjusted to minimize variability. The modulation is likely achieved via an expansive nonlinearity between spike rate and membrane potential with inhibition having a divisive influence. These findings reveal a versatile mechanism of modulating neuronal sensitivity and suggest that such modulation is fundamentally linked to optimization.

Lipid dynamics studied by calculation of 31P solid-state NMR spectra using ensembles from molecular dynamics simulations.

We present a method to calculate (31)P solid-state NMR spectra based on the dynamic input from extended molecular dynamics (MD) simulations. The dynamic information confered by MD simulations is much more comprehensive than the information provided by traditional NMR dynamics models based on, for example, order parameters. Therefore, valuable insight into the dynamics of biomolecules may be achieved by the present method. We have applied this method to study the dynamics of lipid bilayers containing the antimicrobial peptide alamethicin, and we show that the calculated (31)P spectra obtained with input from MD simulations are in agreement with experiments under a large range of different sample conditions, including vesicles and oriented samples with and without peptides. We find that the changes in the (31)P spectra upon addition of peptide stem from lipids with reduced diffusion due to peptide-lipid interactions.

Premotor spinal network with balanced excitation and inhibition during motor patterns has high resilience to structural division.

Direct measurements of synaptic inhibition (I) and excitation (E) to spinal motoneurons can provide an important insight into the organization of premotor networks. Such measurements of flexor motoneurons participating in motor patterns in turtles have recently demonstrated strong concurrent E and I as well as stochastic membrane potentials and irregular spiking in the adult turtle spinal cord. These findings represent a departure from the widespread acceptance of feedforward reciprocal rate models for spinal motor function. The apparent discrepancy has been reviewed as an experimental artifact caused by the distortion of local networks in the transected turtle spinal cord. We tested this assumption in the current study by performing experiments to assess the integrity of motor functions in the intact spinal cord and the cord transected at segments D9/D10. Excitatory and inhibitory synaptic inputs to motoneurons were estimated during rhythmic motor activity and demonstrated primarily intense inputs that consisted of qualitatively similar mixed E/I before and after the transection. To understand this high functional resilience, we used mathematical modeling of networks with recurrent connectivity that could potentially explain the balanced E/I. Both experimental and modeling data support the concept of a locally balanced premotor network consisting of recurrent E/I connectivity, in addition to the well known reciprocal network activity. The multifaceted synaptic connections provide spinal networks with a remarkable ability to remain functional after structural divisions.

Modeling the Self-Assembly and Stability of DHPC Micelles Using Atomic Resolution and Coarse Grained MD Simulations.

Membrane mimics such as micelles and bicelles are widely used in experiments involving membrane proteins. With the aim of being able to carry out molecular dynamics simulations in environments comparable to experimental conditions, we set out to test the ability of both coarse grained and atomistic resolution force fields to model the experimentally observed behavior of the lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), which is a widely used lipid for biophysical characterization of membrane proteins. It becomes clear from our results that a satisfactory modeling of DHPC aggregates in solution poses different demands to the force field than do the modeling of bilayers. First, the representation of the short tailed lipid DHPC in the coarse grained force field MARTINI is assessed with the intend of successfully self-assemble micelles with structural characteristics comparable to experimental data. Then, the use of the recently presented polarizable water model in MARTINI is shown to be essential for producing micelles that are structurally in accordance with experiments. For the atomistic representations of DHPC micelles in solution the GROMOS96 force field with lipid parameters by A. Kukol fails to maintain stable micelles, whereas the most recent CHARMM36 lipid parameters and GROMOS96 with the so-called Berger lipid parameters both succeed in this regard.

Rapid implementation of an integrated large-scale HIV counseling and testing, malaria, and diarrhea prevention campaign in rural Kenya.

BACKGROUND: Integrated disease prevention in low resource settings can increase coverage, equity and efficiency in controlling high burden infectious diseases. A public-private partnership with the Ministry of Health, CDC, Vestergaard Frandsen and CHF International implemented a one-week integrated multi-disease prevention campaign. METHOD: Residents of Lurambi, Western Kenya were eligible for participation. The aim was to offer services to at least 80% of those aged 15-49. 31 temporary sites in strategically dispersed locations offered: HIV counseling and testing, 60 male condoms, an insecticide-treated bednet, a household water filter for women or an individual filter for men, and for those testing positive, a 3-month supply of cotrimoxazole and referral for follow-up care and treatment. FINDINGS: Over 7 days, 47,311 people attended the campaign with a 96% uptake of the multi-disease preventive package. Of these, 99.7% were tested for HIV (87% in the target 15-49 age group); 80% had previously never tested. 4% of those tested were positive, 61% were women (5% of women and 3% of men), 6% had median CD4 counts of 541 cell/µL (IQR; 356, 754). 386 certified counselors attended to an average 17 participants per day, consistent with recommended national figures for mass campaigns. Among women, HIV infection varied by age, and was more likely with an ended marriage (e.g. widowed vs. never married, OR.3.91; 95% CI. 2.87-5.34), and lack of occupation. In men, quantitatively stronger relationships were found (e.g. widowed vs. never married, OR.7.0; 95% CI. 3.5-13.9). Always using condoms with a non-steady partner was more common among HIV-infected women participants who knew their status compared to those who did not (OR.5.4 95% CI. 2.3-12.8). CONCLUSION: Through integrated campaigns it is feasible to efficiently cover large proportions of eligible adults in rural underserved communities with multiple disease preventive services simultaneously achieving various national and international health development goals.

Synthesis and characterization of cruciform-conjugated molecules based on tetrathiafulvalene.

Stepwise acetylenic scaffolding provides cruciform-conjugated molecules with vertically disposed pi-systems, one being an extended tetrathiafulvene (TTF) unit. Two synthetic routes for a cruciform dimer incorporating a total of two extended TTFs was developed, employing either an oxidative Hay coupling reaction as the final dimerization step or as one of the initial steps. Both routes take advantage of ready access to new mono- and diethynylbenzene building blocks incorporating a variety of other functionalitites. The redox and optical properties of the cruciform molecules were investigated by cyclic and differential pulse voltammetries and UV-vis absorption spectroscopy. The access to multiple redox states renders the molecules attractive candidates as wires, or possibly transistors, for molecular electronics. Generation of a quinoid structure by oxidation is supported by density functional theory calculations.

Synthesis of oligo(phenyleneethynylene)-tetrathiafulvalene cruciforms for molecular electronics.

[reaction: see text] Novel oligo(phenyleneethynylene) (OPE)-tetrathiafulvalene (TTF) cruciform molecules containing thiol end-groups have been prepared and characterized. These redox-active molecules are interesting for future applications as molecular wires/transistors for molecular electronics.