Oral colostrum priming shortens hospitalization without changing the immunomicrobial milieu.

OBJECTIVE: Oral colostrum priming (OCP) after birth in preterm infants is associated with improved weight gain and modification of the oral immunomicrobial environment. We hypothesized that OCP would modify salivary immune peptides and the oral microbiota in preterm infants. STUDY DESIGN: We conducted a prospective, randomized clinical trial to determine the effects of OCP on salivary immune peptide representation in preterm infants (<32 weeks completed gestation at birth). Saliva samples were collected before and after OCP. Salivary immune peptide representation was determined via mass spectroscopy. Oral microbiota representation was determined via sequencing of the 16S rRNA gene. RESULTS: Neonates who received OCP (n=48) had a 16-day reduction in the median length of hospitalization as compared with infants who did not receive OCP (n=51). No differences in salivary immune peptide sequence representation before OCP between groups were found. Longitudinal changes in peptides were detected (lysozyme C, immunoglobulin A, lactoferrin) but were limited to a single peptide difference (α-defensin 1) between primed and unprimed infants after OCP. We found no difference in microbial diversity between treatment groups at any time point, but diversity decreased significantly over time in both groups. OCP treatment marginally modified oral taxa with a decline in abundance of Streptococci in the OCP group at 30 days of life. CONCLUSIONS: OCP had neither an effect on the salivary peptides we examined nor on overall oral bacterial diversity and composition. Infants who received OCP had a reduced length of hospitalization and warrants further investigation.

Functional implications of dopamine D1 vs. D2 receptors: A 'prepare and select' model of the striatal direct vs. indirect pathways.

The functions of the D1- and D2-dopamine receptors in the basal ganglia have remained somewhat enigmatic, with a number of competing theories relating to the interactions of the 'direct' and 'indirect pathways'. Computational models have been good at simulating properties of the system, but are typically divorced from the underlying neural architecture. In this article we propose a new model which re-addresses response selection at the level of the basal ganglia. At the core of this response selection system the D1 DA receptor-expressing striatal pathways 'prepare' the set of possible appropriate responses. The D2DR-expressing striatal pathways then shape and 'select' from this initial response set framework. This article is part of a Special Issue entitled: Ventral Tegmentum & Dopamine.

Great expectorations: the potential of salivary 'omic' approaches in neonatal intensive care.

Among those that require critical care, preterm neonates have the greatest limitations on available blood or body fluids for clinical or research-based assessments. Recent technological advancements have improved our ability to detect genetic, proteomic and microbial material at the nanoscale level, making analyte and biomarker assessment from even the smallest quantities possible. Saliva is a unique body fluid that not only may be noninvasively and repeatedly obtained, but also contains multiple serum components, making it promising for noninvasive assessment of the newborn. The integration of high-throughput or 'omic' approaches on neonatal saliva holds great potential to improve diagnostic and prognostic accuracy for a wide range of developmental and pathological conditions affecting the vulnerable preterm neonatal population. Herein, we review the clinical applications and technical considerations regarding the integration of salivary 'omic' technology into the neonatal intensive care unit.

Translating cognition from animals to humans.

Many clinical disorders, whether neurological (e.g. Alzheimer's disease) or neuropsychiatric (e.g. schizophrenia and depression), exhibit cognitive symptoms that require pharmacological treatment. Cognition is multi-faceted and includes processes of perception, attention, working memory, long-term memory, executive function, language and social cognition. This article reviews how it is feasible to model many aspects of human cognition with the use of appropriate animal models and associated techniques, including the use of computer controlled tests (e.g. touch-screens), for optimising translation of experimental research to the clinic. When investigating clinical disorders, test batteries should aim to profile cognitive function in order to determine which aspects are impaired and which are preserved. In this review we have paid particular attention to the validation of translational methods; this may be done through the application of common theoretical principles, by comparing the effects of psychological manipulations and, wherever feasible, with the demonstration of homologous neural circuitry or equivalent pharmacological actions in the animal and human paradigms. Of particular importance is the use of 'back-translation' to ensure that the animal model has validity, for example, in predicting the effects of therapeutic drugs already found in human studies. It is made clear that the choice of appropriate behavioral tests is an important element of animal models of neuropsychiatric or neurological disorder; however, of course it is also important to select appropriate manipulations, whether genetic, neurodevelopmental, neurotoxic, or pharmacological, for simulating the neural substrates relevant to the disorders that lead to predictable behavioral and cognitive impairments, for optimising the testing of candidate compounds.

One-dimensional DOSY.

A new NMR experiment for correlating diffusion coefficients and chemical shifts is presented. This experiment provides the same information as the conventional DOSY experiment, but only requires a single dimension because a nonuniform magnetic field gradient is used to encode the diffusion information into the lineshapes of the peaks in the chemical shift dimension. By fitting the resulting lineshapes, the diffusion coefficient for each peak in the spectrum can be extracted. Using this experiment, a qualitative DOSY spectrum can be generated using the results from a single one-dimensional experiment. Quantitative results can be determined with the use of reference experiments.

Palladium catalysis for the synthesis of hydrophobic C-6 and C-2 aryl 2'-deoxynucleosides. Comparison of C-C versus C-N bond formation as well as C-6 versus C-2 reactivity.

Suzuki-Miyaura cross-coupling of haloaromatic compounds with arylboronic acids provides a simple entry to biaryl systems. Despite its ease, to date, there are no detailed investigations of this procedure for deoxynucleoside modification. As shown in this study, a wide variety of C-6 arylpurine 2'-deoxyriboside (C-6 aryl 2'-deoxynebularine analogues) and C-2 aryl 2'-deoxyinosine analogues can be conveniently prepared via the Pd-mediated cross-coupling of arylboronic acids with the C-6 halonucleosides, 6-bromo- or 6-chloro-9[2-deoxy-3,5-bis-O-(tert-butyldimethylsilyl)-beta-D-erythro-pentofuranosyl]purine (1 and 2), and the C-2 halonucleoside, 2-bromo-O(6)-benzyl-3',5'-bis-O-(tert-butyldimethylsilyl)-2'-deoxyinosine (3). Although bromonucleoside 1 proved to be a good substrate for the Pd-catalyzed Suzuki-Miyaura cross-couplings, we have noted that for several C-6 arylations, the chloronucleoside 2 provides superior coupling yields. Also described in this study is a detailed evaluation of catalytic systems that led to optimal product recoveries. Finally, a comparison of the C-C and C-N bond-forming reactions of deoxynucleosides is also reported. On the basis of this comparison, we provide evidence that C-N bond formation at the C-6 position, leading to N-aryl 2'-deoxyadenosine analogues, is more sensitive to the ligand used, whereas C-C bond-forming reactions at the same position are not. In contrast to the ligand dependency exhibited in C-N bond formation at the C-6 position, comparable reactions at the C-2 position of purine deoxynucleosides proceed with less sensitivity to the ligand used.

Measurement of convection and temperature profiles in liquid samples.

This paper describes a method for measuring the rate of convective flow in a liquid sample used for high-resolution NMR. The measurement is straightforward and achieves a clean separation of convection from other effects such as diffusion and relaxation. Convection results from temperature gradients within the sample, and it is shown how these can be measured with the aid of a simple chemical shift imaging experiment of a sample whose spectrum shows a strong and well characterized temperature dependence. The use of these two methods is illustrated by showing how the rate of convection and the temperature profile depend on the solvent, temperature, and gas flow rate of the temperature regulating system. It is also shown that broadband (13)C decoupling results in significant temperature gradients and associated convection.

Backbone dynamics of chymotrypsin inhibitor 2: effect of breaking the active site bond and its implications for the mechanism of inhibition of serine proteases.

The backbone dynamics of uniformly 15N-labeled chymotrypsin inhibitor 2 (CI2) and of the complex formed by the association of two fragments consisting of residues 20-59 and 60-83 have been studied. A data set consisting of 15N longitudinal (T1) and transverse (T1 rho) relaxation times and (1H)-15N NOE enhancements has been measured for all backbone NH groups in both proteins. Information on internal motions has been extracted from these data using the model-free approach to determine order parameters (S2) and effective internal correlation times (tau e). The data indicate that most of the backbone of CI2 is highly constrained (S2 approximately 0.9) with the exception of residues in the binding loop (residues 54-64), which have slightly lower order parameters. Most of the residues in the CI2(20-59).(60-83) complex are also highly constrained (S2 approximately 0.9). However, the loss of the covalent bond between Met59 and Glu60 leads to a large increase in the mobility of residues in the loop region. The residues in the first half of the loop region have significantly lower order parameters than those in the second half of the loop. This observation suggests that the NH2 group that is released on cleavage of the scissile bond remains anchored in its original position, inhibiting the attack of water on the acyl-enzyme that is formed between the protease and the cleaved inhibitor. More importantly, the NH2 group is optimally placed for reversing the formation of the acyl-enzyme so that the equilibrium between the cleaved and uncleaved inhibitor, bound to the protease, greatly favors the uncleaved complex.

Minimisation of sensitivity losses due to the use of gradient pulses in triple-resonance NMR of proteins.

The use of pulsed field gradients in multiple-pulse NMR experiments has many advantages, including the possibility of obtaining excellent water suppression without the need for selective presaturation. In such gradient experiments the water magnetization is dephased deliberately; exchange between the saturated protons of the solvent water and the NH protons of a protein transfers this saturation to the protein. As the solvent is in large excess and relaxes relatively slowly, the result is a reduction in the sensitivity of the experiment due to the fact that the NH proton magnetization is only partially recovered. These effects can be avoided by ensuring that the water magnetization remains intact and is returned to the +z-axis at the start of data acquisition. General procedures for achieving this aim in any triple-resonance experiment are outlined and two specific examples are given. Experimental results confirm the sensitivity advantage of the modified sequences.

The solution structure and dynamics of the DNA-binding domain of HMG-D from Drosophila melanogaster.

BACKGROUND: The HMG-box is a conserved DNA-binding motif that has been identified in many high mobility group (HMG) proteins. HMG-D is a non-histone chromosomal protein from Drosophila melanogaster that is closely related to the mammalian HMG-box proteins HMG-1 and HMG-2. Previous structures determined for an HMG-box domain from rat and hamster exhibit the same global topology, but differ significantly in detail. It has been suggested that these differences may arise from hinge motions which allow the protein to adapt to the shape of its target DNA. RESULTS: We present the solution structure of HMG-D determined by NMR spectroscopy to an overall precision of 0.85 A root mean squared deviation (rmsd) for the backbone atoms. The protein consists of an extended amino-terminal region and three alpha-helices that fold into a characteristic 'L' shape. The central core region of the molecule is highly stable and maintains an angle of approximately 80 degrees between the axes of helices 2 and 3. The backbone dynamics determined from 15N NMR relaxation measurements show a high correlation with the mean residue rmsd determined from the calculated structures. CONCLUSIONS: The structure determined for the HMG-box motif from HMG-D is essentially identical to the structure determined for the B-domain of mammalian HMG-1. Since these proteins have significantly different sequences our results indicate that the global fold and the mode of interaction with DNA are also likely to be conserved in all eukaryotes.

Nuclear magnetic resonance relaxation studies of poly(hydroxybutyrate) in whole cells and in artificial granules.

The physical state of poly(hydroxybutyrate) (PHB) in whole cells and in the form of artificial biomimetic granules has been probed using 13C nuclear magnetic resonance (NMR) spectroscopy. Studies on varying concentrations of whole cells of Alcaligenes eutrophus show that changes in the line widths of PHB in whole cells do not correlate with changes in transverse relaxation times. Solid-state magic-angle spinning NMR studies demonstrate that the line broadening results from a reduction in the static field homogeneity rather than from intrinsic properties of the PHB within the cells. Transverse and longitudinal relaxation times of PHB in whole cells and in artificial granules are similar, indicating similarities in structure and mobility.

Reactive oxygen species produced in metal-catalyzed oxidation of bis(trifluoromethyl)disulfide and protection by ZE.

Bis(trifluoromethyl)disulfide (TFD), used as an industrial fumigant, was found to generate a thiyl free radical as seen by EPR/spin trapping. Oxygen appears to be an absolute requirement for radical production. The results obtained in this investigation implicate the production of thiyl and reactive oxygen species (ROS), superoxide radical anion and hydroxyl radicals, during TFD autoxidation. The rate of production of these free radical intermediates was found to increase in the presence of iron(III) and copper(II). In addition, the metal ion chelator DETAPAC and ROS scavengers ethanol, mannitol, and PEG-SOD/catalase were found to inhibit free radical production. Reactive oxygen species were not formed when a high-potency zinc plus antioxidant, ZE caps, was present. These results provide support for the pro-oxidation of TFD and a protective role for zinc.

Improving solvent suppression in jump-return NOESY experiments.

The problems associated with solvent suppression in jump-return NOESY spectra and in particular the difficulties experienced with using short mixing times are examined. It is shown that the degree of water suppression depends critically on the extent of radiation damping of the water magnetisation during the mixing time of the NOESY sequence. A new jump-return NOESY sequence is proposed which incorporates field gradients and which achieves good levels of water suppression for all values of the mixing time, and for all increments of the NOESY experiment.

Autoionization reaction of phosgene (OCCl2) studied by electron paramagnetic resonance/spin trapping techniques.

The reaction of phosgene with nitrone spin traps was investigated using electron paramagnetic resonance (EPR)/spin trapping techniques. Evidence for the intermediacy of a carbamoyl monochloride intermediate was obtained. Isotopic substitution of 13C-phosgene was employed to verify the hyperfine coupling constant assignments. The implications of these observations on pulmonary damage caused by inhalation of phosgene are mentioned.

Partially folded conformation of the (30-51) intermediate in the disulphide folding pathway of bovine pancreatic trypsin inhibitor. 1H and 15N resonance assignments and determination of backbone dynamics from 15N relaxation measurements.

An analogue of the important folding intermediate of BPTI with only the disulphide bond between Cys30 and Cys51 has been characterized by 1H and 15N NMR techniques. In particular, the dynamics of the polypeptide backbone were characterized using (1H)-15N NOE and 15N T1 and T2 relaxation data. The intermediate is partially folded, with part of the polypeptide chain stably folded and the remainder flexible or unfolded. The folded portion consists of the major elements of native-like secondary structure interacting through the hydrophobic core of the molecule. The 15N relaxation data show that the N-terminal 15 residues are very flexible, and the (1H, 1H) NOESY data show that these residues have no NOE interactions with the remainder of the molecule. The segment of residues 37 to 41 is also flexible. These observations explain why during folding this intermediate most readily forms any of the possible disulphide bonds between Cys5, Cys14 and Cys38, including the non-native 5-14 and 5-38 bonds. The native-like folded portion of the molecule limits the possible disulphide bonds that can be formed to those in the remainder of the polypeptide chain. Also, forming the non-native disulphide bonds need not involve any disruption of that folded structure, as the Cys residues involved are in flexible regions of the molecule.

Effect of addition of hyaluronidase to bupivacaine during axillary brachial plexus block.

We have studied in 22 patients the effect of adding hyaluronidase to bupivacaine during axillary brachial plexus block (BPB) in a double-blind design. Patients received BPB using bupivacaine 2 mg kg-1 with adrenaline 1 in 200,000, either with or without hyaluronidase 3000 iu, in a volume of 0.5 ml per 2.54 cm of the patient's height. The use of hyaluronidase did not increase the speed of onset of anaesthesia or reduce the incidence of inadequate nerve block. Hyaluronidase produced a significant reduction in the duration of anaesthesia. Changes in grip strength and skin temperature were useful in assessing the onset and progress of BPB.

Protamine--the need to determine the dose. Comparison of a simple protamine titration method with an empirical dose regimen for reversal of heparinisation following cardiopulmonary bypass.

A simple method of protamine titration using the Hemochron system was compared with an empirical dose protocol for reversal of heparinisation following cardiopulmonary bypass in 40 patients undergoing elective myocardial revascularisation. Protamine titration revealed a wide range for protamine requirement and resulted in a significant reduction in protamine dose compared with the empirical dose protocol (p less than 0.01). Heparin reversal was assessed as adequate in all patients. The titration technique was easy and straightforward to use in the operating theatre.