Spectral analysis of noise in the neonatal intensive care unit

Objective To perform spectral analysis of noise generated by equipments and activities in a level III neonatal intensive care unit (NICU) and measure the real time sequential hourly noise levels over a 15 day period. Methods Noise generated in the NICU by individual equipments and activities were recorded with a digital spectral sound analyzer to perform spectral analysis over 0.5–8 KHz. Sequential hourly noise level measurements in all the rooms of the NICU were done for 15 days using a digital sound pressure level meter. Independent sample t test and one way ANOVA were used to examine the statistical significance of the results. The study has a 90% power to detect at least 4 dB differences from the recommended maximum of 50 dB with 95 % confidence. Results The mean noise levels in the ventilator room and stable room were 19.99 dB (A) sound pressure level (SPL) and 11.81 dB (A) SPL higher than the maximum recommended of 50 dB (A) respectively (p < 0.001). The equipments generated 19.11 dB SPL higher than the recommended norms in 1–8 KHz spectrum. The activities generated 21.49 dB SPL higher than the recommended norms in 1–8 KHz spectrum (p< 0.001). The ventilator and nebulisers produced excess noise of 8.5 dB SPL at the 0.5 KHz spectrum.Conclusion Noise level in the NICU is unacceptably high. Spectral analysis of equipment and activity noise have shown noise predominantly in the 1–8 KHz spectrum. These levels warrant immediate implementation of noise reduction protocols as a standard of care in the NICU.

Peptide mimics for structural features in proteins. Crystal structures of three heptapeptide helices with a C-terminal 6-->1 hydrogen bond.

The crystal structure determination of three heptapeptides containing alpha-aminoisobutyryl (Aib) residues as a means of helix stabilization provides a high-resolution characterization of 6-->1 hydrogen-bonded conformations, reminiscent of helix-terminating structural features in proteins. The crystal parameters for the three peptides, Boc-Val-Aib-X-Aib-Ala-Aib-Y-OMe, where X and Y are Phe, Leu (I), Leu, Phe (II) and Leu, Leu (III) are: (I) space group P1, Z = 1, a = 9.903 A, b = 10.709 A, c = 11.969 A, alpha = 102.94 degrees, beta = 103.41 degrees, gamma = 92.72 degrees, R = 4.55%; (II) space group P21, Z = 2, a = 10.052 A, b = 17.653 A, c = 13.510 A, beta = 108.45 degrees, R = 4.49%; (III) space group P1, Z = 2 (two independent molecules IIIa and IIIb in the asymmetric unit), a = 10.833 A, b = 13.850 A, c = 16.928 A, alpha = 99.77 degrees, beta = 105.90 degrees, gamma = 90.64 degrees, R = 8.54%. In all cases the helices form 3(10)/alpha-helical (or 3(10)helical) structures, with helical columns formed by head-to-tail hydrogen bonding. The helices assemble in an all-parallel motif in crystals I and III and in an antiparallel motif in II. In the four crystallographically characterized molecules, I, II, IIIa and IIIb, Aib(6) adopts a left-handed helical (hL) conformation with positive phi, psi values, resulting in 6-->1 hydrogen-bond formation between Aib(2) CO and Leu(7)/Phe(7) NH groups. In addition a 4-->1 hydrogen bond is seen between Aib(3) CO and Aib(6) NH groups. This pattern of hydrogen bonding is often observed at the C-terminus of helices proteins, with the terminal pi-type turn being formed by four residues adopting the hRhRhRhL conformation.

The transient response of a two-terminal solion to a step-current input

The transition time associated with the time-variation of the voltage across a two-terminal diaphragm-less solion in response to a step-current stimulus has been studied experimentally. A theoretical analysis has also been made by solving the diffusion problem under the appropriate initial and boundary conditions. The behaviour of the theoretically predicted transition times is in agreement with the observed behaviour. The systems under study have been shown to be different from those used hitherto in thin-layer chronopotentiometry.

The transient response of a two-terminal solion to a stepcrrent input

The transition time associated with the time-variation of the voltage across a two-terminal diaphragm-less solion in response to a step-current stimulus has been studied experimentally. A theoretical analysis has also been made by solving the diffusion problem under the appropriate initial and boundary conditions. The behaviour of the theoretically predicted transition times is in agreement with the observed behaviour. The systems under study have been shown to be different from those used hitherto in thin-layer chronopotentiometry.

The Critical Role of N- and C-Terminal Contact in Protein Stability and Folding of a Family 10 Xylanase under Extreme Conditions

Background: Stabilization strategies adopted by proteins under extreme conditions are very complex and involve various kinds of interactions. Recent studies have shown that a large proportion of proteins have their N- and C-terminal elements in close contact and suggested they play a role in protein folding and stability. However, the biological significance of this contact remains elusive. Methodology: In the present study, we investigate the role of N- and C-terminal residue interaction using a family 10 xylanase (BSX) with a TIM-barrel structure that shows stability under high temperature,alkali pH, and protease and SDS treatment. Based on crystal structure,an aromatic cluster was identified that involves Phe4, Trp6 and Tyr343 holding the Nand C-terminus together; this is a unique and important feature of this protein that might be crucial for folding and stabilityunder poly-extreme conditions. Conclusion: A series of mutants was created to disrupt this aromatic cluster formation and study the loss of stability and function under given conditions. While the deletions of Phe4 resulted in loss of stability, removal of Trp6 and Tyr343 affected in vivo folding and activity. Alanine substitution with Phe4, Trp6 and Tyr343 drastically decreased stability under all parameters studied. Importantly,substitution of Phe4 with Trp increased stability in SDS treatment.Mass spectrometry results of limited proteolysis further demonstrated that the Arg344 residue is highly susceptible to trypsin digestion in sensitive mutants such as DF4, W6A and Y343A, suggesting again that disruption of the Phe4-Trp6-Tyr343 (F-W-Y) cluster destabilizes the N-and C-terminal interaction. Our results underscore the importance of N- and C-terminal contact through aromatic interactions in protein folding and stability under extreme conditions, and these results may be useful to improve the stability of other proteins under suboptimal conditions.

Time Scale Separated Nonlinear Partial Integrated Guidance and Control of Interceptors in the Terminal Phase

The paper proposes a time scale separated partial integrated guidance and control of an interceptor for engaging high speed targets in the terminal phase. In this two loop design, the outer loop is an optimal control formulation based on nonlinear model predictive spread control philosophies. It gives the commanded pitch and yaw rates whereas necessary roll-rate command is generated from a roll-stabilization loop. The inner loop tracks the outer loop commands using the dynamicinversion philosophy. However, unlike conventional designs, in both the loops the Six degree of freedom (Six-DOF) interceptor model is used directly. This intelligent manipulation preserves the inherent time scale separation property between the translational and rotational dynamics, and hence overcomes the deficiency of current IGC designs, while preserving its benefits. Six-DOF simulation studies have been carried out accounting for three dimensional engagement geometry. Different comparison studies were also conducted to measure the performance of the algorithm.

Solution structure of O-glycosylated C-terminal leucine zipper domain of human salivary mucin (MUC7)

Solution structures of a 23 residue glycopeptide II (KIS* RFLLYMKNLLNRIIDDMVEQ, where * denotes the glycan Gal-beta-(1-3)-alpha-GalNAc) and its deglycosylated counterpart I derived from the C-terminal leucine zipper domain of low molecular weight human salivary mucin (MUC7) were studied using CD, NMR spectroscopy and molecular modeling. The peptide I was synthesized using the Fmoc chemistry following the conventional procedure and the glycopeptide II was synthesized incorporating the O-glycosylated building block (N alpha-Fmoc-Ser-[Ac-4,-beta-D-Gal-(1,3)-Ac(2)alpha-D-GalN(3)]-OPfp) at the appropriate position in stepwise assembly of peptide chain. Solution structures of these glycosylated and nonglycosylated peptides were studied in water and in the presence of 50% of an organic cosolvent, trifluoroethanol (TFE) using circular dichroism (CD), and in 50% TFE using two-dimensional proton nuclear magnetic resonance (2D H-1 NMR) spectroscopy. CD spectra in aqueous medium indicate that the apopeptide I adapts, mostly, a beta-sheet conformation whereas the glycopeptide II assumes helical structure. This transition in the secondary structure, upon glycosylation, demonstrates that the carbohydrate moiety exerts significant effect on the peptide backbone conformation. However, in 50% TFE both the peptides show pronounced helical structure. Sequential and medium range NOEs, C alpha H chemical shift perturbations, (3)J(NH:C alpha H) couplings and deuterium exchange rates of the amide proton resonances in water containing 50% TFE indicate that the peptide I adapts alpha-helical structure from Ile2-Val21 and the glycopeptide II adapts alpha-helical structure from Ser3-Glu22. The observation of continuous stretch of helix in both the peptides as observed by both NMR and CD spectroscopy strongly suggests that the C-terminal domain of MUC7 with heptad repeats of leucines or methionine residues may be stabilized by dimeric leucine zipper motif. The results reported herein may be invaluable in understanding the aggregation (or dimerization) of MUC7 glycoprotein which would eventually have implications in determining its structure-function relationship.

DNA Clasping by Mycobacterial HU: The C-Terminal Region of HupB Mediates Increased Specificity of DNA Binding

Background: HU a small, basic, histone like protein is a major component of the bacterial nucleoid. E. coli has two subunits of HU coded by hupA and hupB genes whereas Mycobacterium tuberculosis (Mtb) has only one subunit of HU coded by ORF Rv2986c (hupB gene). One noticeable feature regarding Mtb HupB, based on sequence alignment of HU orthologs from different bacteria, was that HupB(Mtb) bears at its C-terminal end, a highly basic extension and this prompted an examination of its role in Mtb HupB function. Methodology/Principal Findings: With this objective two clones of Mtb HupB were generated; one expressing full length HupB protein (HupB(Mtb)) and another which expresses only the N terminal region (first 95 amino acid) of hupB (HupB(MtbN)). Gel retardation assays revealed that HupBMtbN is almost like E. coli HU (heat stable nucleoid protein) in terms of its DNA binding, with a binding constant (K-d) for linear dsDNA greater than 1000 nM, a value comparable to that obtained for the HU alpha alpha and HU alpha beta forms. However CTR (C-terminal Region) of HupB(Mtb) imparts greater specificity in DNA binding. HupB(Mtb) protein binds more strongly to supercoiled plasmid DNA than to linear DNA, also this binding is very stable as it provides DNase I protection even up to 5 minutes. Similar results were obtained when the abilities of both proteins to mediate protection against DNA strand cleavage by hydroxyl radicals generated by the Fenton's reaction, were compared. It was also observed that both the proteins have DNA binding preference for A: T rich DNA which may occur at the regulatory regions of ORFs and the oriC region of Mtb. Conclusions/Significance: These data thus point that HupB(Mtb) may participate in chromosome organization in-vivo, it may also play a passive, possibly an architectural role.

Crystallization and preliminary X-ray studies of the C-terminal domain of Mycobacterium tuberculosis LexA

The C-terminal domain of Mycobacterium tuberculosis LexA has been crystallized in two different forms. The form 1 and form 2 crystals belonged to space groups P3(1)21 and P3(1), respectively. Form 1 contains one domain in the asymmetric unit, while form 2 contains six crystallographically independent domains. The structures have been solved by molecular replacement.

Conformations of the amino terminal tetrapeptide of emerimicins and antiamoebin in solution and in the solid state

The conformations of Boc-l-Phe-(AiB)3-OH (1) and Boc-l-Phe-(Aib)3-OMe (2) which correspond to the amino terminal sequence of the emerimicins and antiamoebins have been studied in solution using 270 MHz 1H n.m.r. In dimethyl sulphoxide solution both peptides show the presence of two strongly solvent shielded Aib NH groups, consistent with a consecutive β-turn conformation, involving the Aib(3) and Aib(4) NH groups in intramolecular 4 → I hydrogen bonds. This folded conformation is maintained for 2 in chloroform solution. Nuclear Overhauser effect studies provide evidence for a Type II Phe-Aib β-turn. An X-ray diffraction study of Boc-(d,l)-Phe-(Aib)3-OH establishes a single type III(III′) β-turn conformation with Aib(2)-Aib(3) as the corner residues. A single intramolecular 4 → I hydrogen bond between Phe(I) CO and Aib(4) NH groups is observed in the crystal. The solution conformation may incorporate a consecutive type II-III′ structure for the Phe(1)-Aib(2)-Aib(3) segment, with the initial type II β-turn being destabilized by intermolecular interactions in the solid state.

A monoclonal antibody recognizing the C-terminal region of chicken egg white riboflavin carrier protein terminates early pregnancy in mice

In order to identify the functionally relevant epitopes on chicken riboflavin carrier protein, we have raised monoclonal antibodies to the vitamin carrier. One of these, 6B2C12, was found to interact specifically with a synthetic oligopeptide corresponding to the C-terminal 17 amino acid residues of the chicken egg white riboflavin carrier protein, which is missing in part in the egg yolk riboflavin carrier protein. This epitope is conserved through evolution in mammals including humans. Administration of the ascites fluid of 6B2C12 to pregnant mice intraperitoneally, resulted in the termination of pregnancy indicating that this epitope is involved in or closely associated with the transplacental transport of the vitamin from the maternal circulation to the growing fetus.

A novel approach to design of cis-acting DNA structural element for regulation of gene expression in vivo

Taking advantage of the degeneracy of the genetic code we have developed a novel approach to introduce, within a gene, DNA sequences capable of adopting unusual structures and to investigate the role of such sequences in regulation of gene expression in vivo. We used a computer program that generates alternative codon sequences for the same amino-acid sequence to convert a stretch of nucleotides into an inverted-repeat sequence with the potential to adopt cruciform structure. This approach was used to replace a 51-base-pair EcoRI-HindIII segment in the N-terminal region of the beta-galactosidase gene in plasmid pUC19 with a 51-bp synthetic oligonucleotide sequence with the potential to adopt a cruciform structure with 18 bp in the stem region. In selecting the 51-bp sequence, care was taken to include those codons that are preferred in E. coli. E. coli DH5-alpha cells harbouring the plasmid containing the redesigned sequence showed drastic reduction in expression of the beta-galactosidase gene compared to cells harbouring the plasmid with the native sequence. This approach demonstrates the possibility of introducing DNA secondary-structure elements to alter regulation of gene expression in vivo.

Diproline Templates as Folding Nuclei in Designed Peptides. Conformational Analysis of Synthetic Peptide Helices Containing Amino Terminal Pro-Pro Segments

The effect of N-terminal diproline segments in nucleating helical folding in designed peptides has been studied in two model sequences Piv-Pro-Pro-Aib-Leu-Aib-Phe-OMe (1) and Boc-Aib-Pro-Pro-Aib-Val-Ala-Phe-OMe (2). The structure of 1 in crystals, determined by X-ray diffraction, reveals a helical (RR) conformation for the segment residues 2 to 5, stabilized by one 4 -> 1 hydrogen bond and two 5 -> 1 interactions. The N-terminus residue, Pro(1) adopts a polyproline II (P-II) conformation. NMR studies in three different solvent systems support a conformation similar to that observed in crystals. In the apolar solvent CDCl3, NOE data favor the population of both completely helical and partially unfolded structures. In the former, the Pro-Pro segment adopts an alpha(R)-alpha(R) conformation, whereas in the latter, a P-II-alpha(R) structure is established. The conformational equilibrium shifts in favor of the P-II-alpha(R) structure in solvents like methanol and DMSO. A significant population of the Pro(1)- Pro(2) cis conformer is also observed. The NMR results are consistent with the population of at least three conformational states about Pro- Pro segment: trans alpha(R)-alpha(R), trans P-II-alpha(R) and cis P-II-alpha(R). Of these, the two trans conformers are in rapid dynamic exchange on the NMR time scale, whereas the interconversion between cis and trans form is slow. Similar results are obtained with peptide 2. Analysis of 462 diproline segments in protein crystal structures reveals 25 examples of the alpha(R)-alpha(R) conformation followed by a helix. Modeling and energy minimization studies suggest that both P-II-alpha(R) and alpha(R)-alpha(R) conformations have very similar energies in the model hexapeptide 1