Phase-coherent sensing of the center-of-mass motion of trapped-ion crystals.

Trapped ions are sensitive detectors of weak forces and electric fields that excite ion motion. Here measurements of the center-of-mass motion of a trapped-ion crystal that are phase coherent with an applied weak external force are reported. These experiments are conducted far from the trap motional frequency on a two-dimensional trapped-ion crystal of approximately 100 ions, and determine the fundamental measurement imprecision of our protocol free from noise associated with the center-of-mass mode. The driven sinusoidal displacement of the crystal is detected by coupling the ion crystal motion to the internal spin degree of freedom of the ions using an oscillating spin-dependent optical dipole force. The resulting induced spin precession is proportional to the displacement amplitude of the crystal, and is measured with near-projection-noise-limited resolution. A 49 pm displacement is detected with a signal-to-noise ratio of 1 in a single experimental determination, which is an order-of-magnitude improvement over prior phase-incoherent experiments. This displacement amplitude is 40 times smaller than the zero-point fluctuations. With our repetition rate, an 8.4   pm / Hz displacement sensitivity is achieved, which implies 12   ( yN/ion ) / Hz and 77   ( µ V/m ) / Hz sensitivities to forces and electric fields, respectively. This displacement sensitivity, when applied on-resonance with the center-of-mass mode, indicates the possibility of weak force and electric field detection below 10-3 yN/ion and 1 nV/m, respectively.

Protein fingerprinting and quantification of ß-casein variants by ultra-performance liquid chromatography-high-resolution mass spectrometry.

Infant formulations are constantly evolving as novel protein ingredients are added to make them more closely mimic the protein profile of human milk; however, precise analytical methods for characterizing and quantifying the major milk proteins in such formulations are currently lacking. This article describes an ultra-performance liquid chromatography-high-resolution mass spectrometry method for intact proteins that can efficiently detect, identify, and characterize the major milk proteins and their proteoforms (phosphorylation status, degree of glycation, genetic variants among others) in ingredients and final products, with an emphasis on detecting and quantifying specific genetic variants of ß-casein in infant formulas. Method sensitivity allows detection of ß-casein A1 in A2-based infant formulas with a limit of detection of 2% (grams of ß-casein A1 per 100 g of total ß-casein). Protein glycation affects signal intensity in a linear fashion, which permits proteins to be quantified from their mass spectrometry signals after correction according to their measured glycation index. The method was validated for the quantification of ß-casein in infant formulas. Repeatability ranged from 2 to 3% and intermediate reproducibility from 5 to 9%. Calculated ß-casein amounts ranged between 77 and 110% of the values based on formulations and published protein profiles for milk. Altogether, this method can be used for general fingerprinting as well as specific characterization and quantification of individual major milk proteins in dairy-based ingredients and products.

Plasma Heating due to Cyclic Diffusion across a Separatrix.

We observe plasma heating due to collisional diffusion across a separatrix when a magnesium ion column in a Penning-Malmberg trap is cyclically pushed back and forth across a partial trapping barrier. The barrier is an externally applied axisymmetric "squeeze" potential, which creates a velocity separatrix between trapped and passing particles. Weak ion-ion collisions then cause separatrix crossings, leading to irreversible heating. The heating rate scales as the square root of the oscillation rate times the collision frequency and thus can be dominant for low-collisionality plasmas. The particle velocity distribution function is measured with coherent laser induced fluorescence and shows passing and trapped particles having an out-of-phase response to the forced plasma oscillations.

Trapped Particle Effects in the Parametric Instability of Near-Acoustic Plasma Waves.

Quantitative experiments on the parametric decay instability of near-acoustic plasma waves provide strong evidence that trapped particles reduce the instability threshold below fluid models. At low temperatures, the broad characteristics of the parametric instability are determined by the frequency detuning of the pump and daughter wave, and the wave-wave coupling strength, surprisingly consistent with cold fluid, three-wave theories. However, at higher temperatures, trapped particle effects dominate, and the pump wave becomes unstable at half the threshold pump wave amplitude with similar exponential growth rates as for a cold plasma.

Extensively hydrolysed infant formulas: Need for aligned definition of peptide size characteristics and standardisation of analytical methods.

Letter by Nutten et al. on article by Levin et al. (Levin ME, Blackhurst DM, Kirstein F, Kok D, van der Watt GF, Marais AD. Residual allergenicity of amino acid-based and extensively hydrolysed cow's milk formulas. S Afr Med J 2017;107(9):763-767. S Afr Med J 2017;107(3):258-263. https://doi.org/10.7196/SAMJ.2017.v107i9.12137); and response by Levin et al.

First Test of Long-Range Collisional Drag via Plasma Wave Damping.

This paper presents the first experimental confirmation of a new theory predicting enhanced drag due to long-range collisions in a magnetized plasma. The experiments measure damping of Langmuir waves in a multispecies pure ion plasma, which is dominated by interspecies collisional drag in certain regimes. The measured damping rates in these regimes exceed classical predictions of collisional drag damping by as much as an order of magnitude, but agree with the new theory.

Development and Application of Functionalized Protein Binders in Multicellular Organisms.

Protein-protein interactions are crucial for almost all biological processes. Studying such interactions in their native environment is critical but not easy to perform. Recently developed genetically encoded protein binders were shown to function inside living cells. These molecules offer a new, direct way to assess protein function, distribution and dynamics in vivo. A widely used protein binder scaffold are the so-called nanobodies, which are derived from the variable domain of camelid heavy-chain antibodies. Another commonly used scaffold, the DARPins, is based on Ankyrin repeats. In this review, we highlight how these binders can be functionalized in order to study proteins in vivo during the development of multicellular organisms. It is to be anticipated that many more applications for such synthetic protein binders will be developed in the near future.

Space charge frequency shifts of the cyclotron modes in multi-species ion plasmas.

Shifts of the cyclotron frequency away from the "bare" cyclotron frequency are observed to be proportional to the total ion density through the E × B rotation frequency, and to the relative concentration of each ion species, in quantitative agreement with analytic theory. These shifts are measured at small excitation amplitudes on the typical center-of-mass m = 1 mode, and also on cyclotron modes with m = 0 and m = 2 azimuthal dependence. The frequency spacing between these modes is proportional to the rotation frequency of the ion cloud, which is controlled and measured using a "rotating wall" and laser-induced fluorescence. These cylindrical ion plasmas consist of Mg(+) isotopes, with H3 O (+) and O2 (+) impurities. It is observed that the shift in the m = 1 cyclotron frequency is larger for the minority species (25)Mg(+) and (26)Mg(+), than for the majority species (24)Mg(+). A simple center-of-mass model is presented, which is in quantitative agreement with these results. It is also shown that this model interprets and expands the intensity dependent calibration equation, (M/q) = A/f + B/f (2) + CI/f (2).

Compounds from Sichuan and Melegueta peppers activate, covalently and non-covalently, TRPA1 and TRPV1 channels.

BACKGROUND AND PURPOSE: Oily extracts of Sichuan and Melegueta peppers evoke pungent sensations mediated by different alkylamides [mainly hydroxy-alpha-sanshool (alpha-SOH)] and hydroxyarylalkanones (6-shogaol and 6-paradol). We assessed how transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (TRPV1), two chemosensory ion channels, participate in these pungent sensations. EXPERIMENTAL APPROACH: The structure-activity relationships of these molecules on TRPA1 and TRPV1 was measured by testing natural and synthetic analogues using calcium and voltage imaging on dissociated dorsal root ganglia neurons and human embryonic kidney 293 cells expressing the wild-type channels or specific cysteine mutants using glutathione trapping as a model to probe TRPA1 activation. In addition, using Trpv1 knockout mice, the compounds' aversive responses were measured in a taste brief-access test. KEY RESULTS: For TRPA1 activation, the cis C6 double bond in the polyenic chain of alpha-SOH was critical, whereas no structural specificity was required for activation of TRPV1. Both 6-shogaol and 6-paradol were found to activate TRPV1 and TRPA1 channels, whereas linalool, an abundant terpene in Sichuan pepper, activated TRPA1 but not TRPV1 channels. Alkylamides and 6-shogaol act on TRPA1 by covalent bonding whereas none of these compounds activated TRPV1 through such interactions. Finally, TRPV1 mutant mice retained sensitivity to 6-shogaol but were not responsive to alpha-SOH. CONCLUSIONS AND IMPLICATIONS: The pungent nature of components of Sichuan and Melegueta peppers was mediated via interactions with TRPA1 and TRPV1 channels and may explain the aversive properties of these compounds.

Specificity of FGF signaling in cell migration in Drosophila.

We wanted to investigate the relationship between receptor tyrosine kinase (RTK) activated signaling pathways and the induction of cell migration. Using Drosophila tracheal and mesodermal cell migration as model systems, we find that the intracellular domain of the fibroblast growth factor receptors (FGFRs) Breathless (Btl) and Heartless (Htl) can be functionally replaced by the intracellular domains of Torso (Tor) and epidermal growth factor receptor (EGFR). These hybrid receptors can also rescue cell migration in the absence of Downstream of FGFR (Dof), a cytoplasmic protein essential for FGF signaling. These results demonstrate that tracheal and mesodermal cells respond during a specific time window to a receptor tyrosine kinase (RTK) signal with directed migration, independent of the presence or absence of Dof. We discuss our findings in the light of the recent findings that RTKs generate a generic signal that is interpreted in responding cells according to their developmental history.

A HOX complex, a repressor element and a 50 bp sequence confer regional specificity to a DPP-responsive enhancer.

A central theme during development and homeostasis is the generation of cell type-specific responses to the action of a limited number of extant signaling cascades triggered by extracellular ligands. The molecular mechanisms by which information from such signals are integrated in responding cells in a cell-type specific manner remain poorly understood. We have undertaken a detailed characterization of an enhancer that is regulated by DPP signaling and by the homeotic protein Labial and its partners, Extradenticle and Homothorax. The expression driven by this enhancer (lab550) and numerous deletions and point mutants thereof was studied in wild-type and mutant Drosophila embryos as well as in cultured cells. We find that the lab550 enhancer is composed of two elements, a Homeotic Response Element (HOMRE) and a DPP Response Element (DPPRE) that synergize. None of these two elements can reproduce the expression of lab550, either with regard to expression level or with regard to spatial restriction. The isolated DPPRE of lab550 responds extremely weakly to DPP. Interestingly, we found that the inducibility of this DPPRE is weak because it is tuned down by the action of a repressor element. This repressor element and an additional 50 bp element appear to be crucial for the cooperation of the HOMRE and the DPPRE, and might tightly link the DPP response to the homeotic input. The cooperation between the different elements of the enhancer leads to the segmentally restricted activity of lab550 in the endoderm and provides a mechanism to create specific responses to DPP signaling with the help of a HOX protein complex.

Nuclear interpretation of Dpp signaling in Drosophila.

Signaling by Decapentaplegic (Dpp), a member of the TGFbeta superfamily of signaling molecules similar to vertebrate BMP2 and BMP4, has been implicated in many developmental processes in Drosophila melanogaster. Notably, Dpp acts as a long-range morphogen during imaginal disc growth and patterning. Genetic approaches led to the identification of a number of gene products that constitute the core signaling pathway. In addition to the ligand-activated heteromeric receptor complex and the signal-transducing intracellular Smad proteins, Dpp signaling requires two nuclear proteins, Schnurri (Shn) and Brinker (Brk), to prime cells for Dpp responsiveness. A complex interplay between the nuclear factors involved in Dpp signaling appears to control the transcriptional readout of the Dpp morphogen gradient. It remains to be seen whether similar molecular mechanisms operate in the nucleus in vertebrate systems.

Purification, cloning, and characterization of a second arylalkylamine N-acetyltransferase from Drosophila melanogaster.

In insects, amine acetylation by the enzyme arylalkylamine N-acetyltransferase (AANAT) is involved in melatonin formation, sclerotization, and neurotransmitter inactivation. This wide spectrum of activities suggests that several AANAT enzymes are present. We recently purified a protein fraction with AANAT activity from Drosophila melanogaster and cloned the corresponding gene, aaNAT1. Following the same strategy, we now report the purification of an additional AANAT from D. melanogaster, AANAT2, and the cloning of the corresponding cDNA. The isolated protein differs from AANAT1a and AANAT1b in its molecular weight and isoelectric point. The AANAT2 shares about 30% identity with the products of the aaNAT1 gene. The enzyme does not follow one-site Michaelis-Menten kinetics when assayed with various concentrations of the arylalkylamine tryptamine and a constant concentration (0.5 mM) of the cofactor acetyl coenzyme A. The data can be interpreted in terms of an enzyme with two kinetic regimes (K(m1) = 7.2 microM, K(m2) = 0.6 mM, and v(max2) = 2.7 v(max1)) that are governed by binding of the substrate to a regulatory site (K(r) = 6.2 mM). These findings demonstrate the presence of a second expressed gene encoding an AANAT in D. melanogaster. Northern blot analysis revealed no diurnal variation of aaNAT2 transcription, similar to the results obtained for aaNAT1a and aaNAT1b.

Direct transcriptional control of the Dpp target omb by the DNA binding protein Brinker.

The gradient morphogen Decapentaplegic (Dpp) organizes pattern by inducing the transcription of different target genes at distinct threshold concentrations during Drosophila development. An important, albeit indirect, mode by which Dpp controls the spatial extent of its targets is via the graded downregulation of brinker, whose product in turn negatively regulates the expression of these targets. Here we report the molecular dissection of the cis-regulatory sequences of optomotor-blind (omb), a Dpp target gene in the wing. We identify a minimal 284 bp Dpp response element and demonstrate that it is subject to Brinker (Brk) repression. Using this omb wing enhancer, we show that Brk is a sequence-specific DNA binding protein. Mutations in the high-affinity Brk binding site abolish responsiveness of this omb enhancer to Brk and also compromise the input of an unknown transcriptional activator. Our results therefore identify Brk as a novel transcription factor antagonizing Dpp signalling by directly binding target genes and repressing their expression.

Genetic control of branching morphogenesis during Drosophila tracheal development.

Branching morphogenesis is a widely used strategy to increase the surface area of a given organ. A number of tissues undergo branching morphogenesis during development, including the lung, kidney, vascular system and numerous glands. Until recently, very little has been known about the genetic principles underlying the branching process and about the molecules participating in organ specification and branch formation. The tracheal system of insects represents one of the best-characterised branched organs. The tracheal network provides air to most tissues and its development during embryogenesis has been studied intensively at the morphological and genetic level. More than 30 genes have been identified and ordered into sequential steps controlling branching morphogenesis. These studies have revealed a number of important principles that might be conserved in other systems.

Schnurri mediates Dpp-dependent repression of brinker transcription.

Signalling by Decapentaplegic (Dpp), a member of the TGFbeta superfamily of signalling molecules, controls many aspects of Drosophila development by activating and repressing target genes. Several essential components of the Dpp signalling pathway have been identified, including the Dpp receptors Punt and Thick veins (Tkv) as well as the cytoplasmic mediators Mad and Medea. For target genes to be activated, Dpp signalling must suppress transcription of a repressor encoded by the brinker (brk) gene. Here we show that Schnurri (Shn), a large zinc-finger protein, is essential for Dpp-mediated repression of brk transcription; in contrast, Shn is not required for target-gene activation. Thus, the Dpp signalling pathway bifurcates, downstream of the signal-mediating SMAD proteins, into a Shn-dependent pathway leading to brk repression and a Shn-independent pathway leading to gene activation. The existence of several Shn-like proteins in vertebrates and the observation that Brk functions in BMP signalling in Xenopus indicates that a similar regulatory cascade may be conserved in higher organisms.

Expression of the blistered/DSRF gene is controlled by different morphogens during Drosophila trachea and wing development.

The Drosophila serum response factor (DSRF) is expressed in the precursors of the terminal tracheal cells and in the future intervein territories of the third instar wing imaginal disc. Dissection of the DSRF regulatory region reveals that a single enhancer element, which is under the control of the fibroblast growth factor (FGF)-receptor signalling pathway, is sufficient to induce DSRF expression in the terminal tracheal cells. In contrast, two separate enhancers direct expression in distinct intervein sectors of the wing imaginal disc. One element is active in the central intervein sector and is induced by the Hedgehog signalling pathway. The other element is under the control of Decapentaplegic and is active in two separate territories, which roughly correspond to the intervein sectors flanking the central sector. Hence, each of the three characterized enhancers constitutes a molecular link between a specific territory induced by a morphogen signal and the localized expression of a gene required for the final differentiation of this territory.