Neural control of vocalization: Respiratory and emotional influences

Previous research has shown that a region of the midbrain, the periaqueductal gray matter (PAG), is critical for vocalization. In this review, we describe the results of previous investigations in which we sought to find out how PAG neurons integrate the activity and precise timing of respiratory, laryngeal, and oral muscle activity for natural-sounding vocalization using the technique of excitatory amino acid microinjections in cats. In these studies, all surgical procedures were carried out under deep anaesthesia. In the precollicular decerebrate cat two general types of vocalization, classified as voiced and unvoiced, could be evoked by exciting neurons in the lateral part of the intermediate part of the PAG. The patterns of evoked electromyographic activity were strikingly similar to previously reported patterns of human muscle activity. Coordinated patterns of activity were evoked with just-threshold excitation leading to the conclusion that patterned muscle activity corresponding to the major categories of voiced and voiceless sound production are represented in the PAG. In a parallel series of human and animal experiments, we also determined that the speech and vocalization respiratory patterns are integrated and coordinated with afferent signals related to lung volume. These data have led to the proposal of a new hypothesis for the neural control of vocalization: that the PAG is a crucial brain site for mammalian voice production, not only in the production of emotional or involuntary sounds, but also as a generator of specific respiratory and laryngeal motor patterns essential for human speech and song     

Partitioning the Vertices of a Graph into Two Total Dominating Sets

A total dominating set in a graph G is a set S of vertices of G such that every vertex in G is adjacent to a vertex of S. We study graphs whose vertex set can be partitioned into two total dominating sets. In particular, we develop several sufficient conditions for a graph to have a vertex partition into two total dominating sets. We also show that with the exception of the cycle on five vertices, every selfcomplementary graph with minimum degree at least two has such a partition.     

Conformational behavior of pyrazine-bridged and mixed-bridged cavitands: a general model for solvent effects on thermal "vase-kite" switching

The controllable switching of suitably bridged resorcin[4]arene cavitands between a "vase" conformation, with a cavity capable of guest inclusion, and a "kite" conformation, featuring an extended flattened surface, provides the basis for ongoing developments of dynamic molecular receptors, sensors, and molecular machines. This paper describes the synthesis, X-ray crystallographic characterization, and NMR analysis of the "vase-kite" switching behavior of a fully pyrazine-bridged cavitand and five other mixed-bridged quinoxaline-bridged cavitands with one methylene, phosphonate, or phosphate bridge. The pyrazine-bridged resorcin[4]arene cavitand displayed an unexpectedly high preference for the kite conformation in nonpolar solvents, relative to the quinoxaline-bridged analogue. This observation led to extensive solvent-dependent switching studies that provide a detailed picture of how solvent affects the thermal vase-kite equilibration. As for any thermodynamic process in the liquid phase, the conformational equilibrium is affected by how the solvent stabilizes the two individual states. Suitably sized solvents (benzene and derivatives) solvate the cavity of the vase form and reduce the propensity for the vase-to-kite transition. Correspondingly, the kite geometry becomes preferred in bulky solvents such as mesitylene, incapable of penetrating the vase cavity. As proposed earlier by Cram, the kite form is preferred at low temperatures due to the more favorable enthalpy of solvation of the enlarged surface. Furthermore, the kite conformation is more preferred in solvents with substantial hydrogen-bonding acidity: weak hydrogen-bonding interactions between the mildly basic quinoxaline and pyrazine nitrogen atoms and solvent molecules are more efficient in the open kite than in the closed vase form. Vase-to-kite conversion is entirely absent in dipolar aprotic solvents lacking any H-bonding acidity. Thermal vase-kite switching requires fully quinoxaline- or pyrazine-bridged cavitands, whereas pH-controlled switching is also applicable to systems incorporating only two or three such bridges.     

Ring-borylated 15-electron and 17-electron ansa-chromocene complexes, their physical properties and molecular structures

A detailed study of the thermal decomposition of the zwitterionic, ring-borylated ansa-chromocene hydrido carbonyl complex [Cr(CO)H{Me(4)C(2)(C(5)H(4))[C(5)H(3)B(C(6)F(5))(3)]}] (2) is described. This complex is formed in the reaction between [Cr(CO){Me(4)C(2)(C(5)H(4))(2)}] (1) and B(C(6)F(5))(3) in toluene at -78 degrees C. Above -25 degrees C, 2 decomposes to a 50:50 mixture of the low-spin, 17e Cr(III) complexes [Cr(CO){Me(4)C(2)(C(5)H(4))[C(5)H(3)B(C(6)F(5))(3)]}] (3b) and [Cr(CO){Me(4)C(2)(C(5)H(4))(2)}][HB(C(6)F(5))(3)] (4). Carbon monoxide elimination from 3 b generates high-spin, 15 e [Cr{Me(4)C(2)(C(5)H(4))[C(5)H(3)B(C(6)F(5))(3)]}] (3a), which coordinates two other electron-donating ligands, such as xylyl isocyanide, PMe3, and PPh(2)Me to form the low-spin, 17 e electron complexes 3c, 3d, and 3e, respectively. High-spin, 15 e [Cr{Me(4)C(2)(C(5)H(4))(2)}][HB(C(6)F(5))(3)] (5) is generated by heating 3 b in toluene at 100 degrees C and periodically removing the evolved CO. Efforts to isolate more than a few X-ray quality crystals of 5 were thwarted by its tendency to form an insoluble precipitate (6) with the same molecular formula. Heating the solution of 5 at 120 degrees C results in its partial conversion (ca. 28 %) to 3a, thereby allowing the formation of 3a in yields as high as 74 % from the reaction between 1 and B(C(6)F(5))(3). The X-ray crystal structures of 3 b-e and 5 are described. Cyclic voltammetry measurements on 3 a-e reveal a dramatic reduction in the redox potentials of the complexes relative to their non-borylated analogues. DFT calculations show that this is due primarily to electrostatic stabilization of the oxidized species by the negatively charged borylate group. EPR and 19F NMR spectroscopy allow 3a to be distinguished from its Lewis base adducts 3 b-e and reveal the relative affinities of different Lewis bases for the chromium.     

Fragmentation and reaction processes in cluster-surface collisions

We review the processes which have been observed from collisions between alkali-halide clusters and solid surfaces. Soft impact of nanocrystalline Na_nF _n?1 ⁺ clusters against solid surfaces causes them to cleave along the lowest energy (100) plane. At higher collision energies (E_i>1 eV/atom), an evaporative cascade occurs which is characteristic of a transformation of the nanocrystal to a molten state. Efficient F^? transfer from the cluster to the surface can occur for the larger clusters (>60 atoms) scattering from Si(111), in direct competition with the cleaving channel at low energies. In this regime, strong bonds can form between the F^? and silicon surface. The reaction probability increases with cluster size indicating that an impact-initiated shock wave is needed to enhance the reactive process.     

Eliminating false positive C4 sugar tests on New Zealand Manuka honey

Carbon isotope analyses (δ¹³C) of some New Zealand Manuka honeys show that they often fail the internationally recognised Association of Official Analytical Chemists sugar test (AOAC method 998.12) which detects added C₄ sugar, although these honeys are from unadulterated sources. Failure of these high value products is detrimental to the New Zealand honey industry, not only in lost export revenue, but also in brand and market reputation damage. The standard AOAC test compares the carbon isotope value of the whole honey and corresponding protein isolated from the same honey. Differences between whole honey and protein δ¹³C values should not be greater than +1.0‰, as it indicates the possibility of adulteration with syrups or sugars from C₄ plants such as high fructose corn syrup or cane sugar. We have determined that during the standard AOAC method, pollen and other insoluble components are isolated with the flocculated protein. These non‐protein components have isotope values which are considerably different from those of the pure protein, and can shift the apparent δ¹³C value of protein further away from the δ¹³C value of the whole honey, giving a false positive result for added C₄ sugar. To eliminate a false positive C₄ sugar test for Manuka honey, prior removal of pollen and other insoluble material from the honey is necessary to ensure that only the pure protein is isolated. This will enable a true comparison between whole honey and protein δ¹³C isotopes. Furthermore, we strongly suggest this modification to the AOAC method be universally adopted for all honey C₄ sugar tests. Copyright © 2010 John Wiley & Sons, Ltd.     

Automated multi-attribute method sample preparation using high-throughput buffer exchange tips

Rationale

The multi-attribute method (MAM) has become a valuable mass spectrometry (MS)-based tool that can identify and quantify the site-specific product attributes and purity information for biotherapeutics such as monoclonal antibodies (mAbs) and fusion molecules in recent years. As we expand the use of the MAM at various stages of drug development, it is critical to enhance the sample preparation throughput without additional chemical modifications and variability.

Methods

In this study, a fully automated MAM sample preparation protocol is presented, where rapid desalting in less than 15 minutes is achieved using miniaturized size-exclusion chromatography columns in pipette tips on an automated liquid handler. The peptide samples were analyzed using an electrospray ionization (ESI) orbitrap mass spectrometer coupled to an ultra-high-performance liquid chromatography (UHPLC) system with a dual column switching system.

Results

No significant change was observed in product attributes and their quantities compared with manual, low-artifact sample preparation. The sample recovery using the buffer exchange tips was greatly enhanced over the manual spin cartridges while still demonstrating excellent reproducibility for a wide variety of starting sample concentrations. Unlike a plate desalting system, the individual columns provide flexibility in the number of samples prepared at a time and sample locations within plates.

Conclusions

This automated protocol enables the preparation of up to 96 samples with less “at-bench” time than the manual preparation of a smaller batch of samples, thereby greatly facilitating support of process development and the use of the MAM in quality control.

     

Human Group IIA Phospholipase A2—Three Decades on from Its Discovery

Phospholipase A₂ (PLA₂) enzymes were first recognized as an enzyme activity class in 1961. The secreted (sPLA₂) enzymes were the first of the five major classes of human PLA₂s to be identified and now number nine catalytically-active structurally homologous proteins. The best-studied of these, group IIA sPLA₂, has a clear role in the physiological response to infection and minor injury and acts as an amplifier of pathological inflammation. The enzyme has been a target for anti-inflammatory drug development in multiple disorders where chronic inflammation is a driver of pathology since its cloning in 1989. Despite intensive effort, no clinically approved medicines targeting the enzyme activity have yet been developed. This review catalogues the major discoveries in the human group IIA sPLA₂ field, focusing on features of enzyme function that may explain this lack of success and discusses future research that may assist in realizing the potential benefit of targeting this enzyme. Functionally-selective inhibitors together with isoform-selective inhibitors are necessary to limit the apparent toxicity of previous drugs. There is also a need to define the relevance of the catalytic function of hGIIA to human inflammatory pathology relative to its recently-discovered catalysis-independent function.     

A General Chemistry Experiment to Predict and Measure Functional-Group Stretching Frequencies

An experiment suitable for first-year students is reported. In this activity, students use a molecular modeling program to compute infrared spectra for a series of molecules. From the data obtained, students generate a group frequency chart and use it to identify unknowns. This provides students with an introduction to vibrational spectroscopy and the use of molecular modeling.     

Toward a microchip-based solid-phase extraction method for isolation of nucleic acids

A silica-based solid-phase extraction system suitable for incorporation into a microchip platform (nu-total analytical system; nu-TAS) would find utility in a variety of genetic analysis protocols, including DNA sequencing. The extraction procedure utilized is based on adsorption of the DNA onto bare silica. The procedure involves three steps: (i) DNA adsorption in the presence of a chaotropic salt, (ii) removal of contaminants with an alcohol/water solution, and (iii) elution of the adsorbed DNA in a small volume of buffer suitable for polymerase chain reaction (PCR) amplification. Multiple approaches for incorporation of this protocol into a microchip were examined with regard to extraction efficiency, reproducibility, stability, and the potential to provide PCR-amplifiable DNA. These included packing microchannels with silica beads only, generating a continuous silica network via sol-gel chemistry, and combinations of these. The optimal approach was found to involve immobilizing silica beads packed into the channel using a sol-gel network. This method allowed for successful extraction and elution of nanogram quantities of DNA in less than 25 min, with the DNA obtained in the elution buffer fraction. Evaluation of the eluted DNA indicated that it was of suitable quality for subsequent amplification by PCR.